A Sustainable Future
To highlight the vital role microbiology plays in delivering on the UN Sustainable Development Goals (SDGs), we have created a collection of must-read research on three critical aspects of the SDGs: antimicrobial resistance, soil health, and the circular economy.
Collection Contents
1 - 100 of 107 results
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Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes
More LessLinezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA-LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 27 publicly available optrA-positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010–2017) could be phylogenetically related (3–126 SNPs difference). optrA was located on the chromosome within a Tn6674-like element (n=10) or on medium-size plasmids (30–60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn6674) or plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted into the same ∆radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012–2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.
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Repurposing bioactive compounds for treating multidrug-resistant pathogens
More LessIntroduction. Antimicrobial development is being outpaced by the rising rate of antimicrobial resistance in the developing and industrialized world. Drug repurposing, where novel antibacterial functions can be found for known molecular entities, reduces drug development costs, reduces regulatory hurdles, and increases rate of success.
Aim. We sought to characterize the antimicrobial properties of five known bioactives (DMAQ-B1, carboplatin, oxaliplatin, CD437 and PSB-069) that were discovered in a high-throughput phenotypic screen for hits that extend Caenorhabditis elegans survival during exposure to Pseudomonas aeruginosa PA14.
Methodology. c.f.u. assays, biofilm staining and fluorescence microscopy were used to assay the compounds' effect on various virulence determinants. Checkerboard assays were used to assess synergy between compounds and conventional antimicrobials. C. elegans-based assays were used to test whether the compounds could also rescue against Enterococcus faecalis and Staphyloccus aureus. Finally, toxicity was assessed in C. elegans and mammalian cells.
Results. Four of the compounds rescued C. elegans from a second bacterial pathogen and two of them (DMAQ-B1, a naturally occurring insulin mimetic, and CD437, an agonist of the retinoic acid receptor) rescued against all three. The platinum complexes displayed increased antimicrobial activity against P. aeruginosa . Of the molecules tested, only CD437 showed slight synergy with ampicillin. The two most effective compounds, DMAQ-B1 and CD437, showed toxicity to mammalian cells.
Conclusion. Although these compounds' potential for repurposing is limited by their toxicity, our results contribute to this growing field and provide a simple road map for using C. elegans for preliminary testing of known bioactive compounds with predicted antimicrobial activity.
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Hydroxyethoxy phenyl butanone, a new cosmetic preservative, does not cause bacterial cross-resistance to antimicrobials
More LessIntroduction. Biocide-induced cross-resistance to antimicrobials in bacteria has been described and is a concern for regulators. We have recently reported on a new protocol to predict the propensity of biocide to induce phenotypic resistance in bacteria.
Aim. To measure bacterial propensity to develop antimicrobial resistance following exposure to a new cosmetic preservative developed by L’Oréal R and I.
Methodology. Well-established antimicrobials including triclosan (TRI) and benzalkonium chloride (BZC) and a new molecule hydroxyethoxy phenyl butanone (HEPB) were investigated for their antimicrobial efficacy, effect on bacterial growth, and their potential to induce resistance to chemotherapeutic antibiotics using a new predictive protocol.
Results. The use of this predictive protocol with Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa showed that TRI and BZC significantly affected bacterial growth, MICs and minimum bactericidal concentrations (MBCs). There was no change in antibiotic susceptibility profile following exposure to BZC, but E. coli became intermediate resistant to tobramycin following treatment with TRI (0.00002 % w/v). HEPB did not change the antimicrobial susceptibility profile in P. aeruginosa and S. aureus but E. coli became susceptible to gentamicin. TRI exposure resulted in bacterial susceptibility profile alteration consistent with the literature and confirmed the use of TRI as a positive control in such a test.
Conclusion. Data produced on the propensity of a molecule to induce bacterial resistance is useful and appropriate when launching a new preservative.
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Antimicrobial resistance prevalence in commensal Escherichia coli from broilers, fattening turkeys, fattening pigs and veal calves in European countries and association with antimicrobial usage at country level
The aim of this article is to report on antimicrobial resistance (AMR) in commensal Escherichia coli from livestock from several European countries. The relationships with antimicrobial usage (AMU) at country level and harmonized indicators to cover the most relevant AMR aspects for human health in animal production were also investigated. E. coli were isolated in faeces from broilers and fattening pigs (from nine countries), and fattening turkeys and veal calves (from three countries) and screened against a fixed antimicrobial panel. AMU data were collected at farm and average treatment incidences stratified by antimicrobial class, country and livestock species were calculated. Associations between AMR and AMU at country level were analysed. Independent of animal species, the highest resistance was observed for ampicillin, sulphamethoxazole, tetracycline and trimethoprim. E. coli from broilers showed the highest resistance level for (fluoro)quinolones, and multidrug resistance peaked in broilers and fattening turkeys. Colistin resistance was observed at very low levels with the exception of fattening turkeys. High resistance to third- and fourth-generation cephalosporins was detected in broilers and fattening turkeys. The lowest levels of resistance were for meropenem, azithromycin and tigecycline (<1 %). Significant correlations between resistance and usage at country level were detected in broilers for polymyxins and aminoglycosides, and in fattening pigs for cephalosporins, amphenicols, fluoroquinolones and polymyxins. None of the correlations observed between AMR and AMU were statistically significant for fattening turkey and veal calves. The strength of the analysis performed here is the correlation of aggregated data from the same farms at country level for both AMU and AMR within antimicrobial classes.
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Origin, maintenance and spread of antibiotic resistance genes within plasmids and chromosomes of bloodstream isolates of Escherichia coli
Blood stream invasion by Escherichia coli is the commonest cause of bacteremia in the UK and elsewhere with an attributable mortality of about 15–20 %; antibiotic resistance to multiple agents is common in this microbe and is associated with worse outcomes. Genes conferring antimicrobial resistance, and their frequent location on horizontally transferred genetic elements is well-recognised, but the origin of these determinants, and their ability to be maintained and spread within clinically-relevant bacterial populations is unclear. Here, we set out to examine the distribution of antimicrobial resistance genes in chromosomes and plasmids of 16 bloodstream isolates of E. coli from patients within Scotland, and how these genes are maintained and spread. Using a combination of short and long-read whole genome sequencing methods, we were able to assemble complete sequences of 44 plasmids, with 16 Inc group F and 20 col plasmids; antibiotic resistance genes located almost exclusively within the F group. bla CTX-M15 genes had re-arranged in some strains into the chromosome alone (five strains), while others contained plasmid copies alone (two strains). Integrons containing multiple antibiotic genes were widespread in plasmids, notably many with a dfrA7 gene encoding resistance to trimethoprim, thus linking trimethoprim resistance to the other antibiotic resistance genes within the plasmids. This will allow even narrow spectrum antibiotics such as trimethoprim to act as a selective agent for plasmids containing antibiotic resistance genes mediating much broader resistance, including blaCTX-M15. To our knowledge, this is the first analysis to provide complete sequence data of chromosomes and plasmids in a collection of pathogenic human bloodstream isolates of E. coli . Our findings reveal the interplay between plasmids and integrative and conjugative elements in the maintenance and spread of antibiotic resistance genes within pathogenic E. coli .
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Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia
Introduction. Streptococcus pneumoniae is responsible for many community infections, with the main ones being pneumonia and meningitis. Pneumococcus has developed increased resistance to multiple classes of antibiotics. The evolution of antibiotic resistance in pneumococcus was influenced by changes in serotype distribution under vaccine selection pressure.
Aim. The aim of this study was to determine the genes involved in macrolide resistance, the antimicrobial susceptibility, the serotype distribution and the spread of international antibiotic-resistant clones among clinical isolates of S. pneumoniae .
Methodology. We investigated 86 erythromycin-resistant S. pneumoniae strains isolated from respiratory (n=74) or non-respiratory (n=12) samples in Tunisia. Antimicrobial susceptibility was tested using the disk diffusion method. Macrolide-resistant strains were analysed by polymerase chain reaction (PCR) for ermA, ermB, mefA and msrD. We also investigated the macrolide resistance mechanisms in eight isolates (9.3%) by sequencing the L4 and L22 riboprotein-coding genes, plus relevant segments of the three 23S rRNA genes. Capsular serotypes were detected by multiplex PCR. Sequence types (STs) were explored using multilocus sequence typing (MLST).
Results. Among the 86 studied strains, 70 (81.4 %) were resistant to penicillin G. The prevalent serotypes were 19F, 14, 19A and 23F. We observed that the cMLSB phenotype (66/86, 76.7%) was the most common in these pneumococci. In addition, ermB was the most frequent resistance gene. No mutation in ribosomal protein L22 or L4 or 23S rRNA was detected. Overall, 44 STs were identified in this study, including 16 that were described for the first time. Resistance to lincomycin, tetracycline and trimethoprim/sulfamethoxazole was observed in 55 (64 %), 34 (39.5 %) and 31 (36 %) isolates, respectively. Furthermore, an increase in fluoroquinolone use in particular may lead to the emergence of levofloxacin-resistant strains. Multidrug resistance was observed in 83 isolates (96.5%). Three global antibiotic-resistant clones were identified: Denmark14 ST230, Portugal19F ST177 and Spain9V ST156.
Conclusion. This study shows that macrolide resistance among S. pneumoniae isolated in Tunisia is mainly related to target site modification. Our observations demonstrate a high degree of genetic diversity and capsular types among strains resistant to macrolides.
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Antimicrobial resistance in Shiga toxin-producing Escherichia coli other than serotype O157 : H7 in England, 2014–2016
More LessIntroduction. Despite many ongoing surveillance projects and the recent focus on the veterinary and clinical ‘One Health’ aspects of antimicrobial resistance (AMR), evidence of the extent of any public health risk posed by animal reservoirs with respect to the transmission of resistant strains of Escherichia coli to humans remains varied and contentious. In the UK, the main zoonotic reservoir for the foodborne pathogen Shiga toxin-producing E. coli (STEC) is cattle and sheep. In this study, we adopt an alternative approach to the risk assessment of transmission of AMR E. coli from animals to humans, involving monitoring AMR in isolates of STEC, an established zoonotic, foodborne pathogen, from human cases of gastrointestinal disease.
Aim. The aim of this study was to determine the genome-derived AMR profiles for STEC from human cases to assess the risk of transmission of multidrug-resistant STEC from ruminants to humans.
Methodology. STEC belonging to 10 different clonal complexes (CCs) (n=457) isolated from human faecal specimens were sequenced and genome-derived AMR profiles were determined. Phenotypic susceptibility testing was undertaken on all isolates (n=100) predicted to be resistant to at least one class of antimicrobial.
Results. Of the 457 isolates, 332 (72.7 %) lacked identifiable resistance genes and were predicted to be fully susceptible to 11 classes of antimicrobials; 125/332 (27.3 %) carried 1 or more resistance genes, of which 83/125 (66.4 %) were resistant to 3 or more classes of antibiotic. The percentage of isolates harbouring AMR determinants varied between CCs, from 4% in CC25 to 100% in CC504. Forty-six different AMR genes were detected, which conferred resistance to eight different antibiotic classes. Resistance to ampicillin, streptomycin, tetracyclines and sulphonamides was most commonly detected. Four isolates were identified as extended-spectrum β-lactamase producers. An overall concordance of 97.7 % (n=1075/1100) was demonstrated between the phenotypic and genotypic methods.
Conclusion. This analysis provided an indirect assessment of the risk of transmission of AMR gastrointestinal pathogens from animals to humans, and revealed a subset of human isolates of the zoonotic pathogen STEC were resistant to the antimicrobials used in animal husbandry. However, this proportion has not increased over the last three decades, and thismay provide evidence that guidancepromoting responsible practice has been effective.
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Changes in epidemiologic characteristics and antimicrobial resistance of Streptococcus pyogenes isolated over 10 years from Japanese children with pharyngotonsillitis
Introduction. Pharyngotonsillitis caused by Streptococcus pyogenes (group A streptococci, or GAS) is among the most common infections treated with antibiotics in pediatric patients.
Aim. This study aimed to analyse changes in molecular epidemiology and antibiotic susceptibility among GAS isolates in three study periods spanning 10 years.
Methodology. GAS isolated from paediatric patients with pharyngotonsillitis during Period I (mid-2007 to 2008, n=235), Period II (2012, n=210), and Period III (2018, n=189) were analysed for emm type, multilocus sequence type (MLST), antibiotic susceptibility, and macrolide (ML)- and quinolone (QL)-resistance genes.
Results. Over 20 % of isolates represented emm1 and emm12 types, remaining common in all three periods. Among other emm types, emm4 was common in Period I, emm28 and emm89 in Period II, and emm3 and emm89 in Period III. All isolates remained highly susceptible to penicillins and cephalosporins. Isolates possessing mefA, ermA, or ermB genes mediating ML resistance increased from 34.9 % in Period I to 60.9 % in Period II, but fell to 27.5 % in Period III. QL-resistant isolates with amino acid substitutions affecting ParC and/or GyrA gradually increased from 11.5 to 14.3 %. Specific sequence types identified by MLST and emm typing were associated closely with ML or QL resistance.
Conclusion. Our findings indicate that even in ambulatory care, antibiotic choice for these infections should be based on rapid identification and characterization of causative pathogens.
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Haitian-like genetic traits with creeping MIC of Azithromycin in Vibrio cholerae O1 isolates from Puducherry, India
More LessIntroduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.
Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.
Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.
Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml−1 to 2 µg ml−1.
Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.
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Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: an inter-laboratory study
Ronan M. Doyle, Denise M. O'Sullivan, Sean D. Aller, Sebastian Bruchmann, Taane Clark, Andreu Coello Pelegrin, Martin Cormican, Ernest Diez Benavente, Matthew J. Ellington, Elaine McGrath, Yair Motro, Thi Phuong Thuy Nguyen, Jody Phelan, Liam P. Shaw, Richard A. Stabler, Alex van Belkum, Lucy van Dorp, Neil Woodford, Jacob Moran-Gilad, Jim F. Huggett and Kathryn A. HarrisAntimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.
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First outbreak of Haemophilus influenzae clone ST422 with low susceptibility to quinolones in paediatric patients in Japan
Introduction. Recently, a Haemophilus influenzae clone with low susceptibility to quinolones emerged in paediatric patients in Japan. Isolates of this clone survived for a long time when exposed to the therapeutic concentration of quinolones, despite being classified as ‘susceptible’ under the criteria of the Clinical and Laboratory Standards Institute. In the present study, we report the first outbreak of this clone in paediatric patients in 2018.
Aim. Our aim was to characterise the first outbreak of an H. influenzae clone with low susceptibility to quinolones.
Methodology. All H. influenzae isolates (n=62), collected at a Japanese teaching hospital in 2018, were characterized by both antimicrobial susceptibility tests and multilocus sequence typing. In addition, the similarity in genetic backgrounds was analysed by PFGE.
Results. Among all the isolates (n=62), quinolone low-susceptible isolates accounted for 19.4 % (n=12). Seven out of 12 isolates were identified as sequence type 422 (ST422) and showed more than 90 % similarity to each other by PFGE analysis. All ST422 isolates exhibited identical amino acid substitutions in both quinolone resistance-determining regions in GyrA and ParC. In addition, all these isolates were from paediatric patients who had been referred by different primary care clinics and had no relationship to each other.
Conclusion. In this study, we describe an outbreak of a quinolone low-susceptible ST422 clone in paediatric patients in Japan. Because ST422 isolates have already been reported in at least five other countries, it has the potential to spread worldwide.
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In vitro activity of imipenem-relebactam against resistant phenotypes of Enterobacteriaceae and Pseudomonas aeruginosa isolated from intraabdominal and urinary tract infection samples – SMART Surveillance Europe 2015–2017
Introduction. Infections attributable to carbapenem-resistant Gram-negative bacilli are increasing globally. New antimicrobial agents are urgently needed to treat patients with these infections.
Aim. To describe susceptibility to the novel carbapenem-β-lactamase inhibitor combination imipenem-relebactam and comparators of clinical isolates of non-Proteeae Enterobacteriaceae (NPE) and Pseudomonas aeruginosa from intraabdominal infections (IAIs) and urinary tract infections (UTIs).
Methods. Broth microdilution MICs were determined for isolates collected in 22 European countries in 2015–2017 and interpreted using EUCAST breakpoints; imipenem-relebactam MICs were interpreted using imipenem breakpoints.
Results. For NPE, 98.4 % of isolates from IAIs (n=10,465) and 98.5 % of UTI isolates (n=7,446) were susceptible to imipenem-relebactam, as were 42.4 % of imipenem-nonsusceptible (n=474), 98.6 % of Klebsiella pneumoniae carbapenemase (KPC)-positive (n=138), and 93.9 % of multidrug-resistant (MDR) isolates (n=4,424) from IAIs and UTIs combined. Molecular analysis demonstrated that two-thirds of imipenem-nonsusceptible isolates rendered susceptible by relebactam carried KPCs; 96 % (261/271) of imipenem-nonsusceptible isolates of NPE that remained nonsusceptible in the presence of relebactam carried metallo-β-lactamase (MBL)-type and/or OXA-48-like carbapenemases. Among P. aeruginosa , 94.4 % of IAI (n=1,245) and 93.0 % of UTI isolates (n=714) were susceptible to imipenem-relebactam, as were 74.4 % of imipenem-nonsusceptible (n=469) and 79.8 % of MDR isolates (n=595) from IAIs and UTIs combined. Among the 120 isolates of P. aeruginosa that remained nonsusceptible to imipenem upon addition of relebactam, 72 % carried MBLs. The distribution of NPE and P. aeruginosa carrying carbapenemases varied substantially across Europe, as did resistance to imipenem and imipenem-relebactam.
Conclusions. Continued surveillance of antimicrobial resistance and resistance mechanisms, including the study of imipenem-relebactam as it approaches regulatory approval, appears warranted.
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Raoultella spp. – reliable identification, susceptibility to antimicrobials and antibiotic resistance mechanisms
More LessIntroduction. Raoultella spp. representatives are Gram-negative rod-shaped bacteria of the Enterobacteriaceae family. These bacteria are commonly found in the natural environment.
Aim. The aim of the study was to indicate the reliable method for Raoultella spp. strains identification, evaluate the susceptibility of Raoultella spp. strains to selected antimicrobials and to detect their resistance mechanisms to beta-lactams.
Methodology. Susceptibility of the strains to chosen antimicrobials was determined using the automatic method. The presence of particular antimicrobial resistant mechanism and genes encoding ESBLs and MBLs was determined respectively with double-disc synergy test and commercially available kit – eazyplex SuperBug CRE test (Amplex Diagnostics) and standard PCR. For the selected strains, DNA sequencing was performed.
Results. Amongst 105 of the examined Raoultella spp. strains, majority were sensitive to: imipenem (99.0 %), meropenem (98.1 %), gentamicin (93.3 %) and ciprofloxacin (92.4 %). Of the tested Raoultella strains, thirteen (12.4 %) produced ESBLs and one strain simultaneously ESBLs and MBLs. The DNA sequencing results were as follows: for all the reference strains the correct species identification was achieved, for the analysed strains two were identified as R. planticola and one as R. ornithinolytica .
Conclusion. Although Raoultella spp. strains remain sensitive to antibiotics, there is a constant need to monitor the sensitivity of these bacteria to selected antimicrobials. Isolation of a multi-drug resistant R. ornithinolytica strain indicates that even the less frequently isolated species of Enterobacteriaceae family should be precisely identified because they might be of clinical importance and the particular strain can also produce enzymes that pose the greatest threat today.
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The characterization of mobile colistin resistance (mcr) genes among 33 000 Salmonella enterica genomes from routine public health surveillance in England
To establish the prevalence of mobile colistin resistance (mcr) genes amongst Salmonella enterica isolates obtained through public health surveillance in England (April 2014 to September 2017), 33 205 S . enterica genome sequences obtained from human, food, animal and environmental isolates were screened for the presence of mcr variants 1 to 8. The mcr-positive genomes were assembled, annotated and characterized according to plasmid type. Nanopore sequencing was performed on six selected isolates with putative novel plasmids, and phylogenetic analysis was used to provide an evolutionary context for the most commonly isolated clones. Fifty-two mcr-positive isolates were identified, of which 32 were positive for mcr-1, 19 for mcr-3 and 1 for mcr-5. The combination of Illumina and Nanopore sequencing identified three novel mcr-3 plasmids and one novel mcr-5 plasmid, as well as the presence of chromosomally integrated mcr-1 and mcr-3. Monophasic S. enterica serovar Typhimurium accounted for 27/52 (52 %) of the mcr-positive isolates, with the majority clustering in clades associated with travel to Southeast Asia. Isolates in these clades were associated with a specific plasmid range and an additional extended-spectrum beta-lactamase genotype. Routine whole-genome sequencing for public health surveillance provides an effective screen for novel and emerging antimicrobial determinants, including mcr. Complementary long-read technologies elucidated the genomic context of resistance determinants, offering insights into plasmid dissemination and linkage to other resistance genes.
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A rapid, point-of-care antibiotic susceptibility test for urinary tract infections
Introduction. The alarming rise in urinary tract infection (UTI) antimicrobial resistance has resulted from a combination of high prevalence, low specificity and the lack of a rapid, point-of-care (POC) antibiotic susceptibility test (AST), which has led to the overuse/inappropriate use of antibiotics.
Aim. This study aimed to evaluate the performance of a rapid POC phenotypic AST device in reporting susceptibility information within 2 h.
Methodology. Instrument calibration was performed with model bacteria and fluorescent microbeads to determine the dynamic range and limit of detection for quantifying concentrations of bacteria and demonstrate the ability to rapidly differentiate susceptible and resistant model bacteria. We then evaluated 30 presumptive UTI-positive patient urine samples in a clinical pilot study using a panel of 5 common UTI antibiotics plus a growth control and compared our results to the hospital standard of care AST.
Results. Our device was able to robustly detect and quantify bacteria concentrations from 50 to 105 colony-forming units (c.f.u.) ml−1. The high sensitivity of this measurement technique enabled the device to differentiate between susceptible and resistant model bacteria with 100 % specificity over a 2 h growth period. In the clinical pilot study, an overall categorical agreement (CA) of 90.7 % was observed (sensitivity=91.4 %, specificity=88.9 %, n=97) with performance for individual drugs ranging from 85 % CA (ceftazidime) to 100 % (nitrofurantoin).
Conclusions. By reducing the typical timeframe for susceptibility testing from 2–3 days to 2 h, our POC phenotypic AST can provide critical information to clinicians prior to the administration of antibiotic therapy.
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A simple phenotypic test for detecting the contribution of outer membrane permeability to carbapenem resistance
Introduction. The worldwide emergence of carbapenem resistance in Gram-negative bacteria makes the development of simple tests mandatory to identify antimicrobial resistance mechanisms. Enzymatic and membrane barriers are the prominent resistance mechanisms described in these bacteria. Several tests are currently used to detect carbapenemase activities.
Aim. However, a simple test for the identification of membrane-associated mechanisms of resistance is not yet available and this mechanism is often inferred after the exclusion of a carbapenemase in carbapenem-resistant Gram-negative bacteria.
Methodology. Different media (liquid and solid) containing a membrane permeabilizer were tested to identify the existence of a membrane barrier. Here, polymyxin B nonapeptide (PMBN) was selected to bypass the role of impermeability in clinical carbapenem-resistant Enterobacteriaceae , including Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae and Klebsiella aerogenes isolates. In parallel, the expression of porins (OmpC and OmpF types) was checked in the various bacterial strains in order to search for a correlation between the restoration of susceptibility and the expression of porin.
Results. Using a large number of clinical isolates, PMBN associated with a carbapenem allowed us to detect porin-deficient isolates with a sensitivity ranging from 89 to 93 % and a specificity ranging from 86 to 100 %.
Conclusion. This paves the way for a diagnostic assay allowing the detection of this membrane-associated mechanism of resistance in Enterobacteriaceae .
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In vivo acquisition and risk of inter-species spread of bla KPC-3-plasmid from Klebsiella pneumoniae to Serratia marcescens in the lower respiratory tract
In recent years, Serratia marcescens has emerged as an important agent of hospital-acquired infections, such as pneumonia, urinary tract infection, septicaemia and meningitis, particularly in vulnerable patients. Compared to Klebsiella pneumoniae and Escherichia coli , S. marcescens is less commonly associated with bla KPC genes, yet few cases of plasmid transmission at the gastrointestinal level from K. pneumoniae carbapenemase (KPC)-producing Enterobacterales to S. marcescens have been described. Here we report a case of in vivo acquisition, during a 3-month period of hospitalization in the intensive care unit, of a bla KPC-3 gene carried by a pKpQIL-IT plasmid, and its probable transmission at the bronchial level among different species of Enterobacterales , including K. pneumoniae and S. marcescens . By using whole genome sequence analyses we were able provide insight into the dynamics of carbapenem-resistance determinants acquisition in the lower respiratory tract, a novel anatomical region for such plasmid transmission events, that usually involve the gastrointestinal tract. The co-presence at the same time of both wild-type and resistant Enterobacterales could have been the critical factor leading to the spread of plasmids harbouring carbapenem-resistance genes, of particular importance during surveillance screenings. The possibility of such an event may have significant consequences in terms of antimicrobial treatment, with a potential limitation of therapeutic options, thereby further complicating the clinical management of high-risk critically ill patients.
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Population structure of KPC carbapenemase-producing Klebsiella pneumoniae in a long-term acute-care rehabilitation facility: identification of a new lineage of clonal group 101, associated with local hyperendemicity
In this work, we used a whole-genome sequencing (WGS) approach to study the features of KPC-producing Klebsiella pneumoniae (KPC-Kp) spreading in a large Italian long-term acute-care rehabilitation facility (LTACRF), and to track the dynamics of dissemination within this setting. Thirty-eight, non-replicated, KPC-Kp isolates from colonized patients (either already colonized at admission or colonized during admission), collected during 2016, were subjected to antimicrobial-susceptibility testing and WGS. All isolates were resistant to β-lactams, with the exception of ceftazidime/avibactam (97.4 % susceptible). The second most effective agent was fosfomycin, followed by colistin, trimethoprim/sulfamethoxazole, gentamicin and amikacin (92.1, 86.8, 60.5, 44.7 and 50 % of susceptibility, respectively). A large proportion of isolates (n=18/38, 47.4%) belonged to clonal group (CG) 101, and most of them (n=15) to a new sequence type (ST) designated as ST2502. All the CG101 isolates had a capsule locus type KL17. The ST2502 harboured the genes encoding for the yersiniabactin siderophore and the ArmA methylase, conferring high-level resistance to aminoglycosides. The second most represented lineage of isolates (16/38, 42.1%) belonged to ST512 of CG258. Analysing WGS data, we were able to ascertain the common origin of some isolates imported from other hospitals, and to track several clusters of in-LTACRF cross-transmissions. The results revealed that, in peculiar epidemiological settings such as LTACRF, new KPC-Kp clones different from those prevailing in acute-care hospitals and associated with uncommon resistance and virulence determinants can successfully emerge and disseminate.
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Two-component systems regulate ABC transporters in antimicrobial peptide production, immunity and resistance
More LessBacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central role in vital processes of self-immunity, antibiotic transport and resistance. The majority of ATP-binding cassette transporters are capable of detecting the presence of antibiotics in an external vicinity and are tightly regulated by two-component systems. The presence of an extracellular loop and an adjacent location of both the transporter and two-component system offers serious assistance to induce a quick and specific response against antibiotics. Both systems have demonstrated their ability of sensing such agents, however, the exact mechanism is not yet fully established. This review highlighted the three key functions of antibiotic resistance, transport and self-immunity of ATP-binding cassette transporters and an adjacent two-component regulatory system.
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Whole-genome analysis of extraintestinal Escherichia coli sequence type 73 from a single hospital over a 2 year period identified different circulating clonal groups
Sequence type (ST)73 has emerged as one of the most frequently isolated extraintestinal pathogenic Escherichia coli. To examine the localized diversity of ST73 clonal groups, including their mobile genetic element profile, we sequenced the genomes of 16 multiple-drug resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia, between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccurred, over a 2 year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 clone D i2 reference genome shows high bootstrap support and clusters into four major groups that correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes, but the presence of iss, pic and several iron-acquisition operons was notable.
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Wide genetic heterogeneity and low antimicrobial resistance of enteroaggregative Escherichia coli isolates from several rural communities
Enteroaggregative Escherichia coli (EAEC), a highly heterogeneous pathotype of E. coli classified as typical and atypical, are an emerging cause of acute and persistent diarrhea. We aimed to investigate whether population living in rural geographic areas, impacts in the heterogeneity, dissemination and antimicrobial susceptibility of EAEC strains. EAEC isolates (n=73) were analysed for the presence of 23 putative virulence factors, plasmid and antimicrobial resistance profiles, biofilm formation, pulsedfield gel electrophoresis (PFGE) and by multilocus sequence typing (MLST). The agg3A, agg4A, agn43, aap, shf, astA, pet, pic/set1A and sat genes, biofilm forming and antimicrobial resistance were statistically associated with typical EAEC. A low frequency of all isolates was resistant or showed a multidrug-resistance profile. No isolate showed the same plasmid profile. In total, 58 different pulsotypes were observed. Sixteen isolates analysed by MLST belonged to 15 different sequence types (ST) and showed a different PFGE pattern and virulence-gene profile. The fact that the communities are semi-isolated did not impact on the peculiar heterogeneity of EAEC, being characterized as epidemiologically independent strains.
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Shotgun proteomic analysis of nanoparticle-synthesizing Desulfovibrio alaskensis in response to platinum and palladium
More LessPlatinum and palladium are much sought-after metals of critical global importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them and synthesize elemental nanoparticles. Despite this ability, very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also seven proteins that were specific to either platinum or palladium. Overexpression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and is informative regarding how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as a way to capture contaminant metals from the environment.
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Outer membrane protein I is associated with poly-β-hydroxybutyrate granules and is necessary for optimal polymer accumulation in Azotobacter vinelandii on solid medium
Azotobacter vinelandii is a soil bacterium that is able to synthesize poly-β-hydroxybutyrate (PHB), a polymer used to produce biodegradable plastic. PHB is stored in the cytoplasm as granules surrounded by several proteins such as the major phasin PhbP, PHB synthase and PHB depolymerase, among others. Many studies have reported the presence of membrane proteins on PHB granules due to contamination during the polymer extraction procedures. Previously, the outer membrane protein I (OprI) was detected on the polymer granules in A. vinelandii . In this study, by using random transposon mutagenesis, we identified that a mutation in the oprI gene diminished PHB accumulation in A. vinelandii on solid medium. Electron microscopy confirmed the low polymer production by the oprI mutant. Analysis of PHB granules by Tricine-SDS-PAGE revealed that the absence of OprI affected the protein profile of the granules, suggesting that OprI could have a structural role in A. vinelandii . Thus, some membrane proteins on PHB granules may not be artefacts as previously described.
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Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa
Introduction. Pseudomonas aeruginosa is an important Gram-negative pathogen that is intrinsically multidrug-resistant (MDR) and frequently associated with healthcare-associated outbreaks. With increasing resistance to antibiotics and with very few novel drugs under development, clinicians often use combinations to treat critically ill patients.
Aim. The aim of this study was to evaluate the ability of epigallocatechin (EGCG) to restore the activity of aztreonam against clinical MDR strains of P. aeruginosa .
Methodology. Checkerboard and time–kill kinetic assays were performed to assess synergy in vitro and the Galleria mellonella model of infection was used to test the efficacy of the combination in vivo. Accumulation assays were performed to gain insight into the mechanism of action.
Results. The results demonstrate that synergy between aztreonam and EGCG exists [fractional inhibitory concentration indices (FICIs) 0.02-0.5], with the combination affording significantly (P=<0.05) enhanced bacterial killing, with a >3 log10 reduction in colony-forming units ml−1 at 24 h. EGCG was able to restore susceptibility to aztreonam to a level equal to or below the breakpoint set by the European Committee for Antimicrobial Susceptibility Testing. In G. mellonella, the combination was superior to monotherapy, with increased larval survival observed (94 % vs ≤63 %). We also demonstrated the relatively low toxicity of EGCG to human keratinocytes and G. mellonella larvae. Accumulation assay data suggest that the mechanism of synergy may be due to EGCG increasing the uptake of aztreonam.
Conclusion. EGCG was able to restore the activity of aztreonam against MDR P. aeruginosa . The data presented support further evaluation of the aztreonam–EGCG combination and highlight its potential for use in clinical medicine.
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Isolation and characterization of novel soil- and plant-associated bacteria with multiple phytohormone-degrading activities using a targeted methodology
More LessEthylene (ET), salicylic acid (SA) and indole-3-acetic acid (IAA) are important phytohormones regulating plant growth and development, as well as plant-microbe interactions. Plant growth-promoting bacteria (PGPB) naturally associate with plants and facilitate plant growth through a variety of mechanisms, including the ability to modulate the concentrations of these phytohormones in planta. Importantly, the wide presence of phytohormone degradation mechanisms amongst symbiotic and other soil- and plant-associated bacteria indicates that the ability to modulate phytohormone concentrations plays an important role in bacterial colonization and plant-growth promotion abilities. Obtaining phytohormone-degrading bacteria is therefore key for the development of novel solutions aiming to increase plant growth and protection. In this paper, we report an optimized targeted methodology and the consequent isolation of novel soil- and plant-associated bacteria, including rhizospheric, endophytic and phyllospheric strains, with the ability to degrade the phytohormones, SA and IAA, as well as the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). By using an optimized targeted methodology, we rapidly isolated diverse soil- and plant-associated bacteria presenting phytohormone-degrading abilities from several plants, plant tissues and environments, without the need for prior extensive and laborious isolation and maintenance of large numbers of isolates. The developed methodology facilitates PGPB research, especially in developing countries. Here, we also report, for the first time, the isolation of bacterial strains able to concomitantly catabolize three phytohormones (SA, IAA and ACC). Ultimately, the described targeted methodology and the novel phytohormone-degrading bacteria obtained in this work may be useful tools for future plant-microbe interaction studies, and in the development of new inoculant formulations for agriculture and biotechnology.
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Real-time PCR detection of a 16S rRNA single mutation of Helicobacter pylori isolates associated with reduced susceptibility and resistance to tetracycline in the gastroesophageal mucosa of individual hosts
The molecular mechanism of Helicobacter pylori resistance to tetracycline involves mutations in the primary binding site of the ribosome. A resistance or reduced susceptibility to tetracycline could be the result of single, double or triple mutations in the 16S rRNA gene of H. pylori . We investigated if the genotype was correlated to tetracycline resistance as determined phenotypically in vitro for 96 H . pylori isolates in the gastroesophageal mucosa of Venezuelan individual hosts. E-test for antimicrobial susceptibility test and real-time PCR for the detection of 16S rRNA gene mutations were performed in 96 H . pylori isolates (48 obtained from antrum, and 48 from oesophagus) from eight dyspeptic patients. In the gastric mucosa, 38 isolates were identified sensitive and 10 resistant to tetracycline by E-test, whereas 44 sensitive and 4 resistant isolates were found in the oesophagus. Real-time PCR detection of the 16S rRNA gene exhibited mutants with a single base-pair substitution (AGA926 GGA) in six antrum isolates and seven oesophagus isolates, whereas only three harboured a low level of tetracycline resistance in vitro. Our results indicate that real-time PCR detection of 16S rRNA is a reliable method to classify among tetracycline-resistant genotypes and useful in patients who have experienced a first-line treatment failure with triple therapy.
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In vitro activities of six antifungal agents and their combinations against Chaetomium spp.
Lingyue Sun, Zhe Wan, Ruoyu Li and Jin YuPurpose. To assess in vitro activities of six antifungal agents (amphotericin B, itraconazole, voriconazole, posaconazole, caspofungin and terbinafine) and the combined effects of eight pairs of them (caspofungin or terbinafine with amphotericin B, itraconazole, voriconazole or posaconazole) against 22 isolates of Chaetomium spp.
Methodology. The broth microdilution method drafted by the Clinical and Laboratory Standards Institute and the checkerboard method were used in this study to evaluate in vitro activities of antifungal drugs both alone and in combination against Chaetomium spp.
Results. Amphotericin B and triazoles exhibited lower geometric mean, MIC50 and MIC90 than caspofungin and terbinafine. Besides, all the paired drugs displayed varying degrees of synergism, with the interactions between caspofungin and itraconazole ranking first (86.36 %).
Conclusion. Our study illustrated varying degrees of synergism between caspofungin or terbinafine and itraconazole, voriconazole, posaconazole or amphotericin B towards Chaetomium spp., which could be a reference for the clinical treatment of Chaetomium spp. infections.
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In vitro activity of mecillinam and nitroxoline against Neisseria gonorrhoeae – re-purposing old antibiotics in the multi-drug resistance era
In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l−1 and MICs of cefotaxime ranged from < 0.016 to 1 mg l−1 . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l−1 whereas MICs of NIT ranged from 0.125 to 2 mg l−1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.
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In vitro efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent Clostridium difficile
Purpose. This study investigated the efficacy of the essential mineral, selenium (sodium selenite), in reducing the toxin production, spore outgrowth and antibiotic resistance of Clostridium difficile in vitro.
Methodology. Two hypervirulent C. difficile isolates were cultured in brain heart infusion broth with and without a sub-minimum inhibitory concentration (sub-MIC) of sodium selenite, and the supernatant and bacterial pellet were harvested for total toxin quantitation and RT-qPCR analysis of toxin-encoding genes, respectively. Additionally, C. difficile isolates were cultured in brain heart infusion broth containing 0.5 or 1× the minimum inhibitory concentration (MIC) of either ciprofloxacin or vancomycin with or without sub-MICs of sodium selenite. Further, the effect of sodium selenite on C. difficile germination and spore outgrowth was also determined by exposing C. difficile spores to a sub-MIC of sodium selenite in a germination medium and measuring the germination and outgrowth by measuring the optical density at 600 nm.
Results. Sodium selenite significantly reduced C. difficile toxin synthesis, cytotoxicity and spore outgrowth. Further, the expression of the toxin production genes, tcdA and tcdB, was downregulated in the presence of sodium selenite, while sodium selenite significantly increased the sensitivity of C. difficile to ciprofloxacin , but not vancomycin, as revealed by decreased bacterial growth in samples containing ciprofloxacin+selenium compared to the antibiotic control. Although the sub-MIC of sodium selenite did not inhibit spore germination, it was capable of completely inhibiting spore outgrowth.
Conclusion. Our results suggest that sodium selenite could potentially be used to control C. difficile and indicate that future in vivo studies are warranted.
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Microbiome digital signature of MCR genes – an in silico approach to study the diversity of methanogenic population in laboratory-developed and pilot-scale anaerobic digesters
The production of biogas by anaerobic digestion (AD) of organic/biological wastes has a firm place in sustainable energy production. A simple and cost-effective anaerobic jar at a laboratory scale is a prerequisite to study the microbial community involved in biomass conversion and releasing of methane gas. In this study, a simulation was carried out using a laboratory-modified anaerobic-jar-converted digester (AD1) with that of a commercial/pilot-scale anaerobic digester (AD2). Taxonomic profiling of biogas-producing communities by means of high-throughput methyl coenzyme-M reductase α-subunit (mcrA) gene amplicon sequencing provided high-resolution insights into bacterial and archaeal structures of AD assemblages and their linkages to fed substrates and process parameters. Commonly, the bacterial phyla Euryarchaeota , Chordata, Firmicutes and Proteobacteria appeared to dominate biogas communities in varying abundances depending on the apparent process conditions. Key micro-organisms identified from AD were Methanocorpusculum labreanum and Methanobacterium formicicum . Specific biogas production was found to be significantly correlating to Methanosarcinaceae . It can be implied from this study that the metagenomic sequencing data was able to dissect the microbial community structure in the digesters. The data gathered indicates that the anaerobic-jar system could throw light on the population dynamics of the methanogens at laboratory scale and its effectiveness at large-scale production of bio-methane. The genome sequence information of non-cultivable biogas community members, metagenome sequencing including assembly and binning strategies will be highly valuable in determining the efficacy of an anaerobic digester.
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Rapid antimicrobial susceptibility tests for sepsis; the road ahead
More LessCurrent methods for antimicrobial susceptibility testing (AST) are too slow to affect initial treatment decisions in the early stages of sepsis, when the prescriber is most concerned to select effective therapy immediately, rather than finding out what will not work 1 or 2 days later. There is a clear need for much faster differentiation between viral and bacterial infection, and AST, linked to earlier aetiological diagnosis, without sacrificing either the accuracy of quantitative AST or the low cost of qualitative AST. Truly rapid AST methods are eagerly awaited, and there are several candidate technologies that aim to improve the targeting of our limited stock of effective antimicrobial agents. However, none of these technologies are approaching the point of care and nor can they be described as truly culture-independent diagnostic tests. Rapid chemical and genomic methods of resistance detection are not yet reliable predictors of antimicrobial susceptibility and often rely on prior bacterial isolation. In order to resolve the trade-off between diagnostic confidence and therapeutic efficacy in increasingly antimicrobial-resistant sepsis, we propose a series of three linked decision milestones: initial clinical assessment (e.g. qSOFA score) within 10 min, initial laboratory tests and presumptive antimicrobial therapy within 1 h, and definitive AST with corresponding antimicrobial amendment within an 8 h window (i.e. the same working day). Truly rapid AST methods therefore must be integrated into the clinical laboratory workflow to ensure maximum impact on clinical outcomes of sepsis, and diagnostic and antimicrobial stewardship. The requisite series of development stages come with a substantial regulatory burden that hinders the translation of innovation into practice. The regulatory hurdles for the adoption of rapid AST technology emphasize technical accuracy, but progress will also rely on the effect rapid AST has on prescribing behaviour by physicians managing the care of patients with sepsis. Early adopters in well-equipped teaching centres in close proximity to large clinical laboratories are likely to be early beneficiaries of rapid AST, while simplified and lower-cost technology is needed to support poorly resourced hospitals in developing countries, with their higher burden of AMR. If we really want the clinical laboratory to deliver a specific, same-day diagnosis underpinned by definitive AST results, we are going to have to advocate more effectively for the clinical benefits of bacterial detection and susceptibility testing at critical decision points in the sepsis management pathway.
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Semisynthetic eugenol derivatives as antifungal agents against dermatophytes of the genus Trichophyton
Purpose. Eugenol, the main component of clove bud essential oil (Eugenia caryophyllus), has been linked to antimicrobial, anti-inflammatory, insecticidal and immunomodulatory properties. The purpose of this study was to evaluate the antifungal and cytotoxic activity of eugenol, the essential oil of Eugenia caryophyllus, and some semisynthetic derivatives of eugenol against dermatophytes of the genus Trichophyton.
Methodology. We evaluated the antifungal effect of the compounds, determining the minimum inhibitory concentrations (MICs) by the microdilution method and the minimum fungicidal concentrations by cultures from the inhibitions. Additionally, the inhibition of the radial growth of the mycelium of the dermatophyte fungi was tested by poisoned substrate. Cytotoxicity was measured by the colorimetric method on Vero cells.
Results. All of the eugenol compounds tested exhibited antifungal properties, showing MICs of 62.5–500 µg ml−1 , determined within three dermatophyte species: Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. Among these derivatives, methyl isoeugenol, at concentrations of 300 and 100 µg ml−1, was found to completely inhibit (100 %) radial growth of the mycelium of all three species after 20 days of treatment. Additionally, phenotypic variations related to the decrease in pigment production of T. rubrum were observed after treatment with O-ethyl and O-butyl isoeugenol derivatives. Meanwhile, all of the tested (iso)eugenol molecules exhibited moderate toxicity in Vero cells [50 % cytotoxic concentration (the concentration required for a 50 % reduction in cell viability; CC50): 54.06–265.18 µg ml−1 ).
Conclusion. The results suggest that the semisynthetic eugenol derivatives (SEDs) show promising antifungal activity and selectivity against dermatophyte fungi.
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Casing microbiome dynamics during button mushroom cultivation: implications for dry and wet bubble diseases
The casing material required in mushroom cultivation presents a very rich ecological niche, which is inhabited by a diverse population of bacteria and fungi. In this work three different casing materials, blonde peat, black peat and a 50 : 50 mixture of both, were compared for their capacity to show a natural suppressive response against dry bubble, Lecanicillium fungicola (Preuss) Zare and Gams, and wet bubble, Mycogone perniciosa (Magnus) Delacroix. The highest mushroom production was collected from crops cultivated using the mixed casing and black peat, which were not significantly different in yield. However, artificial infection with mycoparasites resulted in similar yield losses irrespective of the material used, indicating that the casing materials do not confer advantages in disease suppression. The composition of the microbiome of the 50 : 50 casing mixture along the crop cycle and the compost and basidiomes was evaluated through next-generation sequencing (NGS) of the V3–V4 region of the bacterial 16S rRNA gene and the fungal ITS2 region. Once colonized by Agaricus bisporus, the bacterial diversity of the casing microbiome increased and the fungal diversity drastically decreased. From then on, the composition of the casing microbiome remained relatively stable. Analysis of the composition of the bacterial microbiome in basidiomes indicated that it is highly influenced by the casing microbiota. Notably, L. fungicola was consistently detected in uninoculated control samples of compost and casing using NGS, even in asymptomatic crops. This suggests that the naturally established casing microbiota was able to help to suppress disease development when inoculum levels were low, but was not effective in suppressing high pressure from artificially introduced fungal inoculum. Determination of the composition of the casing microbiome paves the way for the development of synthetic casing communities that can be used to investigate the role of specific components of the casing microbiota in mushroom production and disease control.
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Alginate genes are required for optimal soil colonization and persistence by Pseudomonas fluorescens Pf0-1
More LessPseudomonas fluorescens strains are important candidates for use as biological control agents to reduce fungal diseases on crop plants. To understand the ecological success of these bacteria and for successful and stable biological control, determination of how these bacteria colonize and persist in soil environments is critical. Here we show that P. fluorescens Pf0-1 is negatively impacted by reduced water availability in soil, but adapts and persists. A pilot transcriptomic study of Pf0-1 colonizing moist and dehydrated soil was used to identify candidate genetic loci, which could play a role in the adaptation to dehydration. Genes predicted to specify alginate production were identified and chosen for functional evaluation. Using deletion mutants, predicted alginate biosynthesis genes were shown to be important for optimal colonization of moist soil, and necessary for adaptation to reduced water availability in dried soil. Our findings extend in vitro studies of water stress into a more natural system and suggest alginate may be an essential extracellular product for the lifestyle of P. fluorescens when growing in soil.
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Caribbean multi-centre study of Klebsiella pneumoniae: whole-genome sequencing, antimicrobial resistance and virulence factors
More LessThe surveillance of antimicrobial-resistant isolates has proven to be one of the most valuable tools to understand the global rise of multidrug-resistant bacterial pathogens. We report the first insights into the current situation in the Caribbean, where a pilot project to monitor antimicrobial resistance (AMR) through phenotypic resistance measurements combined with whole-genome sequencing was set up in collaboration with the Caribbean Public Health Agency (CARPHA). Our first study focused on Klebsiella pneumoniae , a highly relevant organism amongst the Gram-negative opportunistic pathogens worldwide causing hospital- and community-acquired infections. Our results show that not only carbapenem resistance, but also hypervirulent strains, are circulating in patients in the Caribbean. Our current data does not allow us to infer their prevalence in the population. We argue for the urgent need to further support AMR surveillance and stewardship in this almost uncharted territory, which can make a significant impact on the reduction of antimicrobial usage. This article contains data hosted by Microreact (https://microreact.org).
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Genomic surveillance of Escherichia coli in municipal wastewater treatment plants as an indicator of clinically relevant pathogens and their resistance genes
We examined whether genomic surveillance of Escherichia coli in wastewater could capture the dominant E. coli lineages associated with bloodstream infection and livestock in the East of England, together with the antibiotic-resistance genes circulating in the wider E. coli population. Treated and untreated wastewater was taken from 20 municipal treatment plants in the East of England, half in direct receipt of acute hospital waste. All samples were culture positive for E. coli , and all but one were positive for extended-spectrum β-lactamase (ESBL)-producing E. coli . The most stringent wastewater treatment (tertiary including UV light) did not eradicate ESBL- E. coli in 2/3 cases. We sequenced 388 E. coli (192 ESBL, 196 non-ESBL). Multilocus sequence type (ST) diversity was similar between plants in direct receipt of hospital waste versus the remainder (93 vs 95 STs, respectively). We compared the genomes of wastewater E. coli with isolates from bloodstream infection (n=437), and livestock farms and retail meat (n=431) in the East of England. A total of 19/20 wastewater plants contained one or more of the three most common STs associated with bloodstream infection (ST131, ST73, ST95), and 14/20 contained the most common livestock ST (ST10). In an analysis of 1254 genomes (2 cryptic E. coli were excluded), wastewater isolates were distributed across the phylogeny and intermixed with isolates from humans and livestock. Ten bla CTX-M elements were identified in E. coli isolated from wastewater, together with a further 47 genes encoding resistance to the major antibiotic drug groups. Genes encoding resistance to colistin and the carbapenems were not detected. Genomic surveillance of E. coli in wastewater could be used to monitor new and circulating lineages and resistance determinants of public-health importance.
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Molecular characterization and antifungal susceptibility testing of Candida nivariensis from blood samples – an Iranian multicentre study and a review of the literature
Purpose. Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates.
Methodology. Clinical C. glabrata species complex isolates from blood (n=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected.
Results. Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were susceptible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin.
Conclusion. In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients.
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Streptomyces fodineus sp. nov., an actinobacterium with antifungal activity isolated from mine area soil
More LessA novel actinobacterial strain producing an antifungal substance was isolated from a sample of acidic mine area soil, and its taxonomic position was evaluated. The novel strain, designated TW1S1T, formed white-grey aerial mycelium and yellow substrate mycelium on oatmeal agar. Growth occurred at 10–45 °C (optimum, 30 °C), pH 4–9 (pH 6–7) and in the presence of up to 8 % (w/v) NaCl. Melanin was produced on peptone–yeast extract–iron agar. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that the novel strain should be assigned to the genus Streptomyces , and the closest species was Streptomyces puniciscabiei S77T with 99.1 % sequence similarity, which was followed by Streptomyces durhamensis NRRL B-3309T (99.0 %), Streptomyces filipinensis NBRC 12860T (98.9 %) and Streptomyces yaanensis Z4T (98.7 %). The chemotaxonomic properties were consistent with those of Streptomyces . ll-Diaminopimelic acid was the diagnostic diamino acid, and alanine, glutamic acid and glycine were present in the peptidoglycan. The cell-wall hydrolysate also contained galactose, glucose, mannose and ribose. The predominant isoprenoid quinones were MK-9(H4) and MK-9(H6), the major polar lipids were phosphatidylglycerol and an unidentified phospholipid, and the main fatty acids were iso-C16 : 0 and anteiso-C15 : 0. However, strain TW1S1T could be distinguished from its neighbouring species by its phenotypic properties. In addition, the genome-based comparison with the closest species indicated that strain TW1S1T should be recognized as a separate species. The phylogenetic, phenotypic and chemotaxonomic as well as genomic evidence supported that TW1S1T represents a novel species of Streptomyces , for which the name Streptomyces fodineus sp. nov. is proposed (type strain, TW1S1T = KCTC 49013T = JCM 32404T).
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Use of whole genome sequencing in surveillance for antimicrobial-resistant Shigella sonnei infections acquired from domestic and international sources
More LessShigella species are a major cause of gastroenteritis worldwide, and Shigella sonnei is the most common species isolated within the United States. Previous surveillance work in Pennsylvania documented increased antimicrobial resistance (AMR) in S. sonnei associated with reported illnesses. The present study examined a subset of these isolates by whole genome sequencing (WGS) to determine the relationship between domestic and international isolates, to identify genes that may be useful for identifying specific Global Lineages of S. sonnei and to test the accuracy of WGS for predicting AMR phenotype. A collection of 22 antimicrobial-resistant isolates from patients infected within the United States or while travelling internationally between 2009 and 2014 was chosen for WGS. Phylogenetic analysis revealed both international and domestic isolates were one of two previously defined Global Lineages of S. sonnei , designated Lineage II and Lineage III. Twelve of 17 alleles tested distinguish these two lineages. Lastly, genome analysis was used to identify AMR determinants. Genotypic analysis was concordant with phenotypic resistance for six of eight antibiotic classes. For aminoglycosides and trimethoprim, resistance genes were identified in two and three phenotypically sensitive isolates, respectively. This article contains data hosted by Microreact.
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Antifungal susceptibilities, biofilms, phospholipase and proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals
Purpose. Non-albicans Candida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species.
Methodology. Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.
Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity.
Conclusion. Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.
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Kodoja: A workflow for virus detection in plants using k-mer analysis of RNA-sequencing data
More LessRNA-sequencing of plant material allows for hypothesis-free detection of multiple viruses simultaneously. This methodology relies on bioinformatics workflows for virus identification. Most workflows are designed for human clinical data, and few go beyond sequence mapping for virus identification. We present a new workflow (Kodoja) for the detection of plant virus sequences in RNA-sequence data. Kodoja uses k-mer profiling at the nucleotide level and sequence mapping at the protein level by integrating two existing tools Kraken and Kaiju. Kodoja was tested on three existing RNA-seq datasets from grapevine, and two new RNA-seq datasets from raspberry. For grapevine, Kodoja was shown to be more sensitive than a method based on contig building and blast alignments (27 viruses detected compared to 19). The application of Kodoja to raspberry, showed that field-grown raspberries were infected by multiple viruses, and that RNA-seq can identify lower amounts of virus material than reverse transcriptase PCR. This work enabled the design of new PCR-primers for detection of Raspberry yellow net virus and Beet ringspot virus. Kodoja is a sensitive method for plant virus discovery in field samples and enables the design of more accurate primers for detection. Kodoja is available to install through Bioconda and as a tool within Galaxy.
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Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii
Christine J. Boinett, Amy K. Cain, Jane Hawkey, Nhu Tran Do Hoang, Nhu Nguyen Thi Khanh, Duy Pham Thanh, Janina Dordel, James I. Campbell, Nguyen Phu Huong Lan, Matthew Mayho, Gemma C. Langridge, James Hadfield, Nguyen Van Vinh Chau, Guy E. Thwaites, Julian Parkhill, Nicholas R. Thomson, Kathryn E. Holt and Stephen BakerThe increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii . Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.
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LiSEQ – whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe
Anaïs Painset, Jonas T. Björkman, Kristoffer Kiil, Laurent Guillier, Jean-François Mariet, Benjamin Félix, Corinne Amar, Ovidiu Rotariu, Sophie Roussel, Francisco Perez-Reche, Sylvain Brisse, Alexandra Moura, Marc Lecuit, Ken Forbes, Norval Strachan, Kathie Grant, Eva Møller-Nielsen and Timothy J. DallmanWe present the LiSEQ ( Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.
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Plastic waste as a global challenge: are biodegradable plastics the answer to the plastic waste problem?
More LessThe strength, flexibility and light weight of traditional oil-derived plastics make them ideal materials for a large number of applications, including packaging, medical devices, building, transportation, etc. However, the majority of produced plastics are single-use plastics, which, coupled with a throw-away culture, leads to the accumulation of plastic waste and pollution, as well as the loss of a valuable resource. In this review we discuss the advances and possibilities in the biotransformation and biodegradation of oil-based plastics. We review bio-based and biodegradable polymers and highlight the importance of end-of-life management of biodegradables. Finally, we discuss the role of a circular economy in reducing plastic waste pollution.
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Transcriptomic analysis of Rhizobium leguminosarum bacteroids in determinate and indeterminate nodules
More LessTwo common classes of nitrogen-fixing legume root nodules are those that have determinate or indeterminate meristems, as in Phaseolus bean and pea, respectively. In indeterminate nodules, rhizobia terminally differentiate into bacteroids with endoreduplicated genomes, whereas bacteroids from determinate nodules are less differentiated and can regrow. We used RNA sequencing to compare bacteroid gene expression in determinate and indeterminate nodules using two Rhizobium leguminosarum strains whose genomes differ due to replacement of the symbiosis (Sym) plasmid pRP2 (strain Rlp4292) with pRL1 (strain RlvA34), thereby switching symbiosis hosts from Phaseolus bean (determinate nodules) to pea (indeterminate nodules). Both bacteroid types have gene expression patterns typical of a stringent response, a stressful environment and catabolism of dicarboxylates, formate, amino acids and quaternary amines. Gene expression patterns were indicative that bean bacteroids were more limited for phosphate, sulphate and iron than pea bacteroids. Bean bacteroids had higher levels of expression of genes whose products are predicted to be associated with metabolite detoxification or export. Pea bacteroids had increased expression of genes associated with DNA replication, membrane synthesis and the TCA (tricarboxylic acid) cycle. Analysis of bacteroid-specific transporter genes was indicative of distinct differences in sugars and other compounds in the two nodule environments. Cell division genes were down-regulated in pea but not bean bacteroids, while DNA synthesis was increased in pea bacteroids. This is consistent with endoreduplication of pea bacteroids and their failure to regrow once nodules senesce.
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Everybody hands-on to avoid ESKAPE: effect of sustained hand hygiene compliance on healthcare-associated infections and multidrug resistance in a paediatric hospital
Daniela De la Rosa-Zamboni, Sara A. Ochoa, Almudena Laris-González, Ariadnna Cruz-Córdova, Gerardo Escalona-Venegas, Georgina Pérez-Avendaño, Margarita Torres-García, Roselia Suaréz-Mora, Carmen Castellanos-Cruz, Yadhira V. Sánchrez-Flores, Adalberto Vázquez-Flores, Rosalinda Águila-Torres, Israel Parra-Ortega, Miguel Klünder-Klünder, José Arellano-Galindo, Rigoberto Hernández-Castro and Juan Xicohtencatl-CortesPurpose. Hand hygiene is the most important strategy for preventing healthcare-associated infections (HCAIs); however, the impact of hand hygiene in middle-income countries has been poorly described. In this work, we describe the impact of the programme ‘Let’s Go for 100’ on hand hygiene adherence, HCAIs rates and multidrug-resistant (MDR) bacteria, including the molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains.
Methodology. A multimodal, hospital-wide hand hygiene programme was implemented from 2013. ‘Let’s Go for 100’ involved all healthcare workers and encompassed education, awareness, visual reminders, feedback and innovative strategies. Monthly hand hygiene monitoring and active HCAI surveillance were performed in every ward. Molecular typing of MRSA was analysed by pulsed-field gel electrophoresis (PFGE).
Results/Key findings. Hand hygiene adherence increased from 34.9 % during the baseline period to 80.6 % in the last 3 months of this study. The HCAI rate decreased from 7.54 to 6.46/1000 patient-days (P=0.004). The central line-associated bloodstream infection (CLABSIs) rate fell from 4.84 to 3.66/1000 central line-days (P=0.05). Negative correlations between hand hygiene and HCAIs rates were identified. The attack rate of MDR-ESKAPE group bloodstream infections decreased from 0.54 to 0.20/100 discharges (P=0.024). MRSA pulsotypes that were prevalent during the baseline period were no longer detected after the 5th quarter, although new strains were identified.
Conclusions. A multimodal hand hygiene programme in a paediatric hospital in a middle-income country was effective in improving adherence and reducing HCAIs, CLABSIs and MDR-ESKAPE bloodstream infections. Sustaining hand hygiene adherence at a level of >60 % for one year limited MRSA clonal transmission.
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Changing the paradigm for hospital outbreak detection by leading with genomic surveillance of nosocomial pathogens
More LessThe current paradigm for hospital outbreak detection and investigation is based on methodology first developed over 150 years ago. Daily surveillance to detect patients positive for pathogens of particular importance for nosocomial infection is supported by epidemiological investigation to determine their relationship in time and place, and to identify any other factor that could link them. The antibiotic resistance pattern is commonly used as a surrogate for bacterial relatedness, although this lacks sensitivity and specificity. Typing may be used to define bacterial relatedness, although routine methods lack sufficient discriminatory power to distinguish relatedness beyond the level of bacterial clones. Ultimately, the identification of an outbreak remains a predominately subjective process reliant on the intuition of experienced infection control professionals. Here, we propose a redesign of hospital outbreak detection and investigation in which bacterial species associated with nosocomial transmission and infection undergo routine prospective whole-genome sequencing. Further investigation is based on the probability that isolates are associated with an outbreak, which is based on the degree of genetic relatedness between isolates. Evidence is provided that supports this model based on studies of MRSA (methicillin-resistant Staphylococcus aureus), together with the benefits of a ‘Sequence First’ approach. The feasibility of implementation is discussed, together with residual barriers that need to be overcome prior to implementation.
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Confirmation of Zika virus infection through hospital-based sentinel surveillance of acute febrile illness in Uganda, 2014–2017
Zika virus (ZIKV), transmitted by Aedes species mosquitoes, was first isolated in Uganda in 1947. From February 2014 to October 2017, the Uganda Virus Research Institute, in collaboration with the US Centers for Diseases Control and Prevention, conducted arbovirus surveillance in acute febrile illness (AFI) patients at St Francis hospital in Nkonkonjeru. Three hundred and eighty-four serum samples were collected and tested for IgM antibodies to yellow fever virus (YFV), West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and ZIKV. Of the 384 samples, 5 were positive for ZIKV IgM. Of these five, three were confirmed by plaque reduction neutralization test (PRNT) to be ZIKV infections. Of the remaining two, one was determined to be a non-specific flavivirus infection and one was confirmed to be alphavirus-positive by reverse transcriptase polymerase chain reaction (RT-PCR). This study provides the first evidence of laboratory-confirmed ZIKV infection in Uganda in five decades, and emphasizes the need to enhance sentinel surveillance.
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Naturally occurring polymorphisms in the virulence regulator Rsp modulate Staphylococcus aureus survival in blood and antibiotic susceptibility
Nasal colonization by the pathogen Staphylococcus aureus is a risk factor for subsequent infection. Loss of function mutations in the gene encoding the virulence regulator Rsp are associated with the transition of S. aureus from a colonizing isolate to one that causes bacteraemia. Here, we report the identification of several novel activity-altering mutations in rsp detected in clinical isolates, including for the first time, mutations that enhance agr operon activity. We assessed how these mutations affected infection-relevant phenotypes and found loss and enhancement of function mutations to have contrasting effects on S. aureus survival in blood and antibiotic susceptibility. These findings add to the growing body of evidence that suggests S. aureus ‘trades off’ virulence for the acquisition of traits that benefit survival in the host, and indicates that infection severity and treatment options can be significantly affected by mutations in the virulence regulator rsp.
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Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages
The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.
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How myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases
More LessUp to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014–2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host’s innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.
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An outbreak of a rare Shiga-toxin-producing Escherichia coli serotype (O117:H7) among men who have sex with men
More LessSexually transmissible enteric infections (STEIs) are commonly associated with transmission among men who have sex with men (MSM). In the past decade, the UK has experienced multiple parallel STEI emergences in MSM caused by a range of bacterial species of the genus Shigella, and an outbreak of an uncommon serotype (O117 : H7) of Shiga-toxin-producing Escherichia coli (STEC). Here, we used microbial genomics on 6 outbreak and 30 sporadic STEC O117 : H7 isolates to explore the origins and pathogenic drivers of the STEC O117 : H7 emergence in MSM. Using genomic epidemiology, we found that the STEC O117 : H7 outbreak lineage was potentially imported from Latin America and likely continues to circulate both in the UK MSM population and in Latin America. We found genomic relationships consistent with existing symptomatic evidence for chronic infection with this STEC serotype. Comparative genomic analysis indicated the existence of a novel Shiga toxin 1-encoding prophage in the outbreak isolates, and evidence of horizontal gene exchange among the STEC O117 : H7 outbreak lineage and other enteric pathogens. There was no evidence of increased virulence in the outbreak strains relative to contextual isolates, but the outbreak lineage was associated with azithromycin resistance. Comparing these findings with similar genomic investigations of emerging MSM-associated Shigella in the UK highlighted many parallels, the most striking of which was the importance of the azithromycin phenotype for STEI emergence in this patient group.
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Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential
Raimunda Sâmia Nogueira Brilhante, Jamille Alencar Sales, Maria Lucilene Queiroz da Silva, Jonathas Sales de Oliveira, Lucas de Alencar Pereira, Waldemiro Aquino Pereira-Neto, Rossana de Aguiar Cordeiro, José Júlio Costa Sidrim, Débora de Souza Collares Maia Castelo-Branco and Marcos Fábio Gadelha RochaPurpose. Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex.
Methodology. Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.
Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were β-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure.
Conclusion. These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.
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Relevance of antifungal penetration in biofilm-associated resistance of Candida albicans and non-albicans Candida species
More LessThe role of penetration limitation in Candida biofilm-associated antifungal resistance remains unclear. Most of the previous work has been done on Candida albicans, although non-albicans (NAC) species are also implicated in invasive candidiasis and the biofilm matrix has been shown to vary amongst different species. Only a few studies have evaluated clinical isolates. This study aimed to determine the relevance of penetration limitation in the antifungal resistance of biofilms formed by C. albicans and NAC clinical isolates, using an agar disk diffusion assay. The penetration of posaconazole and amphotericin B through the biofilms was significantly reduced. Fluconazole, voriconazole and caspofungin showed a superior penetration capacity in C. albicans, Candida tropicalis and Candida parapsilosis biofilms, but exhibited inter-species and strain/isolate variation. Candida krusei biofilms were the most resilient to antifungal permeation. All of the antifungal drugs failed to kill the biofilm cells, independent of penetration, suggesting that the other factors contribute markedly to the recalcitrance of the biofilms.
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Contribution of efflux to colistin heteroresistance in a multidrug resistant Acinetobacter baumannii clinical isolate
Purpose. The mechanisms underlying colistin heteroresistance in Acinetobacter baumannii are not fully understood. Here, we investigated the role of efflux in colistin-heteroresistant populations of a multidrug-resistant (MDR) A. baumannii clinical isolate.
Methodology. Three colistin-resistant A. baumannii strain variants isolated from the same clinical sample were studied for the presence of heteroresistance to colistin by drug susceptibility testing, genotyping and drug resistance target mutation analysis. The existence of active efflux was studied by synergism assays with efflux inhibitors, real-time efflux activity measurements and analysis of the mRNA transcriptional levels of selected efflux pump genes in response to colistin.
Results. All of the strain variants belong to the ST218, clonal complex 92, international clonal lineage II. Different colistin susceptibility levels were observed among the three strain variants, indicating that colistin-heteroresistant subpopulations were being selected upon exposure to colistin. No mutations were found in the genes lpxACD and pmrAB, which are associated with colistin resistance. The results showed the existence of synergistic interactions between efflux inhibitors and colistin and ethidium bromide. Real-time efflux assays demonstrated that the three strain variants had increased efflux activity that could be inhibited in the presence of the inhibitors. The efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS and abeM were found to be overexpressed in the strain variants in response to colistin exposure.
Conclusion. This study shows that efflux activity contributes to colistin heteroresistance in an MDR A. baumannii clinical isolate. The use of efflux inhibitors as adjuvants of the therapy can resensitize A. baumannii to colistin and prevent the emergence of drug resistance.
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Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China
Purpose. The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China.
Methodology. The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing.
Results. A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3′)-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3′)-IIa]. The oqxAB–aph(3′)-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin.
Conclusion. This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB–aph(3′)-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.
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Surveillance of antimicrobial resistance in Neisseria meningitidis strains isolated from invasive cases in Brazil from 2009 to 2016
Purpose. To describe the antimicrobial resistance profile of Neisseria meningitidis isolates causing invasive disease in Brazil from 2009 to 2016.
Methodology. Among 3548 N. meningitidis isolates received, 2888 (81.4 %) were analysed for antimicrobial resistance using the broth microdilution technique, as recommended by the Clinical and Laboratory Standards Institute. Isolates were tested for ciprofloxacin, chloramphenicol, ceftriaxone, penicillin G, ampicillin and rifampin.
Results. All the isolates tested were susceptible to ceftriaxone, while 953 (33.0 %), 1307 (45.3 %) and 2 (0.07 %) isolates were penicillin G-, ampicillin- and rifampin-intermediate, respectively. Resistance to rifampin, ciprofloxacin and chloramphenicol was shown by three isolates (0.1 %), two isolates (0.07 %) and one (0.03 %) isolate, respectively. Although no isolates were resistant to penicillin G in the period of 2009–2016, our results show an upward trend in minimum inhibitory concentrations (MICs) for this drug as of 2010 (P<0.001). There was no significant difference between different gender and age groups of patients for reduced susceptibility to penicillin G. There was a higher frequency of isolates with reduced susceptibility to penicillin G in the South and Southeast regions (P<0.001). This reduced susceptibility was also associated with serotype 19 inside serogroup B (P<0.001).
Conclusion. Despite the decrease in susceptibility to penicillin G and ampicillin observed from 2010, the overall resistance of N. meningitidis isolates to the antimicrobials tested remained uncommon and sporadic, confirming their efficacy for chemoprophylaxis or treatment of invasive meningococcal disease (IMD) in Brazil. Continued surveillance of N. meningitidis antimicrobial susceptibility profiles is important in order to monitor variations in resistance either geographically, over time or in association with emergent clones.
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Prevalence, antimicrobial resistance and serotype distribution of group B streptococcus isolated among pregnant women and newborns in Rabat, Morocco
Purpose. Group B streptococcus (GBS) is an important cause of neonatal sepsis worldwide. Data on the prevalence of maternal GBS colonization, risk factors for carriage, antibiotic susceptibility and circulating serotypes are necessary to tailor adequate locally relevant public health policies.
Methodology. A prospective study including pregnant women and their newborns was conducted between March and July 2013 in Morocco. We collected clinical data and vagino-rectal and urine samples from the recruited pregnant women, together with the clinical characteristics of, and body surface samples from, their newborns. Additionally, the first three newborns admitted every day with suspected invasive infection were recruited for a thorough screening for neonatal sepsis. Serotypes were characterized by molecular testing.
Results. A total of 350 pregnant women and 139 of their newborns were recruited. The prevalence of pregnant women colonized by GBS was 24 %. In 5/160 additional sick newborns recruited with suspected sepsis, the blood cultures were positive for GBS. Gestational hypertension and vaginal pruritus were significantly associated with a vagino-rectal GBS colonization in univariate analyses. All of the strains were susceptible to penicillin, while 7 % were resistant to clindamycin and 12 % were resistant to erythromycin. The most common GBS serotypes detected included V, II and III.
Conclusion. In Morocco, maternal GBS colonization is high. Penicillin can continue to be the cornerstone of intrapartum antibiotic prophylaxis. A pentavalent GBS vaccine (Ia, Ib, II, III and V) would have been effective against the majority of the colonizing cases in this setting, but a trivalent one (Ia, Ib and III) would only prevent 28 % of the cases.
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Are commercial providers a viable option for clinical bacterial sequencing?
More LessBacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice.
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Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil
Patricia Fernanda Herkert, Jacques F. Meis, Gabriel Lucca de Oliveira Salvador, Renata Rodrigues Gomes, Vania Aparecida Vicente, Marisol Dominguez Muro, Rosangela Lameira Pinheiro, Arnaldo Lopes Colombo, Alexandre Vargas Schwarzbold, Carla Sakuma de Oliveira, Marcelo Simão Ferreira, Flávio Queiroz-Telles and Ferry HagenPurpose. Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil.
Methodology. We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.
Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson’s diversity index of this population was D=0.9856.
Conclusion. The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.
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Three novel proteins co-localise with polyhydroxybutyrate (PHB) granules in Rhodospirillum rubrum S1
More LessPolyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules’ associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.
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Antimicrobial activity against Mycobacterium tuberculosis under in vitro lipid-rich dormancy conditions
Although tuberculosis treatment is dependent on drug-susceptibility testing (DST) and molecular drug-resistance detection, treatment failure and relapse remain a challenge. This could be partially due to the emergence of antibiotic-tolerant dormant mycobacteria, where host lipids have been shown to play an important role. This study evaluated the susceptibility of Mycobacterium tuberculosis to two antibiotic combinations – rifampicin, moxifloxacin, amikacin and metronidazole (RIF-MXF-AMK-MTZ), and rifampicin, moxifloxacin, amikacin and pretomanid (RIF-MXF-AMK-PA) – in a lipid-rich dormancy model. Although their effectiveness in in vitro cultures with dextrose as a carbon source has been proved, we observed that none of the antibiotic mixtures were bactericidal in the presence of lipids. The presence of lipids may confer tolerance to M. tuberculosis against the mixture of antibiotics tested and such tolerance could be even higher during the dormant stages. The implementation of lipids in DST on clinical isolates could potentially lead to a better treatment strategy.
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Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway
Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d -xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway.
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Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages
There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.
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The diversity of ice algal communities on the Greenland Ice Sheet as revealed by oligotyping
More LessThe Arctic is being disproportionally affected by climate change compared with other geographic locations, and is currently experiencing unprecedented melt rates. The Greenland Ice Sheet (GrIS) can be regarded as the largest supraglacial ecosystem on Earth, and ice algae are the dominant primary producers on bare ice surfaces throughout the course of a melt season. Ice-algal-derived pigments cause a darkening of the ice surface, which in turn decreases albedo and increases melt rates. The important role of ice algae in changing melt rates has only recently been recognized, and we currently know little about their community compositions and functions. Here, we present the first analysis of ice algal communities across a 100 km transect on the GrIS by high-throughput sequencing and subsequent oligotyping of the most abundant taxa. Our data reveal an extremely low algal diversity with Ancylonema nordenskiöldii and a Mesotaenium species being by far the dominant taxa at all sites. We employed an oligotyping approach and revealed a hidden diversity not detectable by conventional clustering of operational taxonomic units and taxonomic classification. Oligotypes of the dominant taxa exhibit a site-specific distribution, which may be linked to differences in temperatures and subsequently the extent of the melting. Our results help to better understand the distribution patterns of ice algal communities that play a crucial role in the GrIS ecosystem.
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Whole-genome sequencing revealed concurrent outbreaks of shigellosis in the English Orthodox Jewish Community caused by multiple importations of Shigella sonnei from Israel
In December 2013, Public Health England (PHE) observed an increase in the number of cases of Shigella sonnei linked to the Orthodox Jewish Community (OJC). Ultimately, 52 cases of S. sonnei phage type (PT) P and PT7 were notified between November 2013 and July 2014. Whole-genome sequencing (WGS) was performed on a HiSeq 2500 platform (Illumina) on isolates of S. sonnei submitted to PHE during the investigation. Quality trimmed sequence reads were mapped to a reference genome using BWA-MEM, and single-nucleotide polymorphisms (SNPs) were identified using GATK2. Analysis of the core genome SNP positions (>90 % consensus, minimum depth 10×, MQ≥30) revealed that isolates linked to the outbreak could be categorized as members of distinct monophyletic clusters (MPCs) representing concurrent regional outbreaks occurring in the OJCs across the United Kingdom. A dated phylogeny predicted the date of the most recent common ancestor of the MPCs to be approximately 3.1 years previously [95 % highest posterior density (HPD), 2.4–3.4]. Isolates of S. sonnei from cases from the OJCs in Israel included in the phylogeny, branched from nodes basal to the UK OJC outbreak clusters, indicating they were ancestral to the UK OJC isolates, and that the UK isolates represented multiple importations of S. sonnei into the UK population from Israel. The level of discrimination exhibited by WGS facilitated the identification of clusters of isolates within the closely related bacterial populations circulating in the OJC that may be linked to a unique point sources or transmission routes, thus enabling a more appropriate public health response and targeted interventions.
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Blad-containing oligomer: a novel fungicide used in crop protection as an alternative treatment for tinea pedis and tinea versicolor
Purpose. The lack of novel antifungal drugs and the increasing incidence and severity of fungal infections are major concerns worldwide. Herein, we tested the activity of the Blad-containing oligomer (BCO), a new antifungal molecule already in use for agriculture, on Malassezia spp. and dermatophytes, the causal agents of human tinea versicolor and tinea pedis. Given the lack of a standard method for Malassezia susceptibility testing and the plethora of published methods, we also developed an improved method for this genus.
Methodology. The efficacy of BCO was assessed in vitro and compared to that of the drugs currently utilized in the treatment of tinea versicolor (fluconazole and itraconazole) and tinea pedis (itraconazole and terbinafine). For dermatophytes, the standard microdilution broth-based method was used, with small adjustments, and several broth formulations and inocula sizes were tested to develop an improved susceptibility method for Malassezia spp.
Results. We successfully developed a microdilution broth-based method with considerable advantages over other available methods, and used it for all in vitro susceptibility tests of Malassezia spp. isolates. We report that, on a molar basis, BCO was more effective than fluconazole or itraconazole on most strains of Malassezia spp. isolated from clinical samples (n=29). By contrast, BCO was less effective than itraconazole or terbinafine on the common dermatophytes Trichophyton rubrum and Trichophyton interdigitale.
Conclusion. These data place BCO as a promising drug for the treatment of Malassezia-associated skin diseases. Further in vivo studies are now required to ascertain its applicability in the clinical setting.
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Efficient bio-production of citramalate using an engineered Escherichia coli strain
More LessCitramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l−1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l−1 in total) and 1.4 g l−1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l−1, with a productivity of 1.85 g l−1 h−1 and a conversion efficiency of 0.48 gcitramalate g−1 glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.
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Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes
More LessPurpose. Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance.
Methodology. Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed.
Results. In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects.
Conclusion. Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.
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Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence
Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral ‘pathogens’ cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.
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Fusion proteins towards fungi and bacteria in plant protection
More LessIn agriculture, although fungi are considered the foremost problem, infections by bacteria also cause significant economical losses. The presence of different diseases in crops often leads to a misuse of the proper therapeutic, or the combination of different diseases forces the use of more than one pesticide. This work concerns the development of a ‘super-Blad’: a chimeric protein consisting of Blad polypeptide, the active ingredient of a biological fungicide already on the market, and two selected peptides, SP10-5 and Sub5, proven to possess biological potential as antibacterial agents. The resulting chimeric protein obtained from the fusion of Blad with SP10-5 not only maintained strong antibacterial activity, especially against Xanthomonas spp. and Pseudomonas syringae, but was also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. However, the antibacterial activity of Sub5 was considerably diminished when fused with Blad, which seems to indicate that not all fusion proteins behave equally. These newly designed drugs can be considered promising compounds for use in plant protection. A deeper and focused development of an appropriate formulation may result in a potent biopesticide that can replace, per se, two conventional chemistries with less impact on the environment.
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An investigation of antifungal stewardship programmes in England
Purpose. We sought to explore the current status of antifungal stewardship (AFS) initiatives across National Health Service (NHS) Trusts within England, the challenges and barriers, as well as ways to improve current AFS programmes.
Methodology. An electronic survey was sent to all 155 acute NHS Trusts in England. A total of 47 Trusts, corresponding to 30 % of English acute Trusts, responded to the the survey; 46 Trusts (98 %) had an antimicrobial stewardship (AMS) programme but only 5 (11 %) had a dedicated AFS programme. Overall, 20 (43 %) Trusts said they included AFS as part of their AMS programmes. From those conducting AFS programmes, 7 (28 %) have an AFS/management team, 16 (64 %) monitor and report on antifungal usage, 5 (20 %) have dedicated AFS ward rounds and 12 (48 %) are directly involved in the management of invasive fungal infections.
Results/Key findings. Altogether, 13 acute Trusts (52 %) started their AFS programme to manage costs, whilst 12 (48 %) commenced the programme due to clinical need; 27 (73 %) declared that they would increase their AFS initiatives if they could. Of those without an AFS programme, 14 (67 %) responded that this was due to lack of resources/staff time. Overall, 12 Trusts (57 %) responded that the availability of rapid diagnostics and clinical support would enable them to conduct AFS activities.
Conclusion. Although a minority of Trusts conduct dedicated AFS programmes, nearly half include AFS as part of routine AMS activities. Cost issues are the main driver for AFS, followed by clinical need. The availability of rapid diagnostics and clinical support could help increase AFS initiatives.
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An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease
More Lessdoi: 10.1099/mgen.0.000121.001.
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children below 5 years of age in endemic areas, and is a primary cause of diarrhoea in travellers visiting developing countries. Epidemiological analysis of E. coli pathovars is traditionally carried out based on the results of serotyping. However, genomic analysis of a global ETEC collection of 362 isolates taken from patients revealed nine novel O-antigen biosynthesis gene clusters that were previously unrecognized, and have collectively been called unclassified. When put in the context of all isolates sequenced, one of the novel O-genotypes, OgN5, was found to be the second most common ETEC O-genotype causing disease, after O6, in a globally representative ETEC collection. It’s also clear that ETEC OgN5 isolates have spread globally. These novel O-genotypes have now been included in our comprehensive O-genotyping scheme, and can be detected using a PCR-based and an in silico typing method. This will assist in epidemiological studies, as well as in ETEC vaccine development.
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Population genetic structuring of methicillin-resistant Staphylococcus aureus clone EMRSA-15 within UK reflects patient referral patterns
Tjibbe Donker, Sandra Reuter, James Scriberras, Rosy Reynolds, Nicholas M. Brown, M. Estée Török, Richard James, East of England Microbiology Research Network, David M. Aanensen, Stephen D. Bentley, Matthew T. G. Holden, Julian Parkhill, Brian G. Spratt, Sharon J. Peacock, Edward J. Feil and Hajo GrundmannAntibiotic resistance forms a serious threat to the health of hospitalised patients, rendering otherwise treatable bacterial infections potentially life-threatening. A thorough understanding of the mechanisms by which resistance spreads between patients in different hospitals is required in order to design effective control strategies. We measured the differences between bacterial populations of 52 hospitals in the United Kingdom and Ireland, using whole-genome sequences from 1085 MRSA clonal complex 22 isolates collected between 1998 and 2012. The genetic differences between bacterial populations were compared with the number of patients transferred between hospitals and their regional structure. The MRSA populations within single hospitals, regions and countries were genetically distinct from the rest of the bacterial population at each of these levels. Hospitals from the same patient referral regions showed more similar MRSA populations, as did hospitals sharing many patients. Furthermore, the bacterial populations from different time-periods within the same hospital were generally more similar to each other than contemporaneous bacterial populations from different hospitals. We conclude that, while a large part of the dispersal and expansion of MRSA takes place among patients seeking care in single hospitals, inter-hospital spread of resistant bacteria is by no means a rare occurrence. Hospitals are exposed to constant introductions of MRSA on a number of levels: (1) most MRSA is received from hospitals that directly transfer large numbers of patients, while (2) fewer introductions happen between regions or (3) across national borders, reflecting lower numbers of transferred patients. A joint coordinated control effort between hospitals, is therefore paramount for the national control of MRSA, antibiotic-resistant bacteria and other hospital-associated pathogens.
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Risk factors and molecular mechanisms associated with trimethoprim–sulfamethoxazole resistance in Stenotrophomonas maltophilia in Mexico
Purpose. Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen causing an increasing number of nosocomial infections. Our aim was to evaluate the risk factors and mechanisms associated with trimethoprim–sulfamethoxazole (SXT) resistance in S. maltophilia infections in Mexico.
Methodology. Clinical isolates and patients' demographic and clinical data were collected from February 2007 to August 2015 in two tertiary-care hospitals in Mexico. Antimicrobial susceptibility and analysis of sul and SmeABC and SmeDEF efflux pump overexpression were performed in all isolates.
Results/Key findings. In the 9-year period, 196 patients infected with S. maltophilia were identified. Most patients were male, and the mean age was 46.2 years. The mean Charlson score was 1.42, and the most frequent comorbidities were arterial hypertension (26.7 %), type 2 diabetes (21.2 %) and cerebral infarction (11.6 %). High drug resistance to meropenem (93.4 %), gentamicin (55.1 %), ceftazidime (52.3 %), cefotaxime (51.5 %), amikacin (42.3 %) and cefepime (32.1 %), and lower resistance to ciprofloxacin (26.0 %), SXT (25.0 %), chloramphenicol (14.3 %) and levofloxacin (2.6 %) were detected. SXT resistance was not associated with the sul genes. SmeABC overexpression was associated with gentamicin (P=0.001) and levofloxacin resistance (P=0.041), whereas SmeDEF overexpression was associated with ceftazidime resistance (P=0.003). Prolonged hospitalization (≥15 days) was an independent risk factor for SXT-resistant S. maltophilia infections (OR=3.05; 95 % CI=1.12–8.86; P=0.029).
Conclusion. Given the high SXT resistance rate, SXT is not an effective first-line therapy for our patients; instead, levofloxacin could be used as an appropriate therapeutic option against S. maltophilia infections.
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Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing
Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (bla OXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.
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Candidaemia in a tertiary care academic hospital in Italy. The impact of C. parapsilosis complex on the species distribution and antifungal susceptibility
More LessPurpose. To analyse the species distribution and the susceptibility profiles to the major antifungal agents of Candida isolated from bloodstream infections (BSIs) in both intensive care units (ICUs) and non-ICU wards in a tertiary care hospital in Italy from 2010 until 2015.
Methodology. Episodes of Candida BSI were recorded in a retrospective observational cohort study. Yeasts were isolated from both blood and intravascuIar devices (IVDs) and their susceptibility to antifungal drugs was tested using the microdilution method.
Results. 514 Candida BSIs were evidenced and 19 % of these episodes were associated with the presence of an IVD. The trend of the general incidence increased significantly throughout the study period, ranging from 1.42 to 3.63 (mean 2.52) episodes/1000 admissions. The incidence of Candida BSIs and IVD-associated candidaemia was significantly higher in ICUs relative to the other wards. The most frequently isolated species were C. albicans and C. parapsilosis complex, with the latter presenting a significant increased trend of isolation. C. parapsilosis complex was most frequently involved in IVD-related candidaemia, coinfections and late recurrent infections. Furthermore, the MIC50s of C. parapsilosis complex were significantly enhanced for echinocandins compared to the MIC50s for the same drugs and the other yeasts, while the MIC50s of C. albicans for amphotericin B showed a significant increase during the study period, ranging from 0.1 to 0.5 µg ml−1.
Conclusions. A progressively enhanced incidence of Candida BSIs, a relatively high impact of C. parapsilosis complex and changes in the susceptibility profiles of the isolated yeasts were evidenced during the observation period.
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Whole-genome sequencing to investigate a non-clonal melioidosis cluster on a remote Australian island
Melioidosis is a tropical disease caused by the bacterium Burkholderia pseudomallei. Outbreaks are uncommon and can generally be attributed to a single point source and strain. We used whole-genome sequencing to analyse B. pseudomallei isolates collected from an historical 2-year long case cluster that occurred in a remote northern Australian indigenous island community, where infections were previously linked to a contaminated communal water supply. We analysed the genome-wide relatedness of the two most common multilocus sequence types (STs) involved in the outbreak, STs 125 and 126. This analysis showed that although these STs were closely related on a whole-genome level, they demonstrated evidence of multiple recombination events that were unlikely to have occurred over the timeframe of the outbreak. Based on epidemiological and genetic data, we also identified two additional patients not previously associated with this outbreak. Our results confirm the previous hypothesis that a single unchlorinated water source harbouring multiple B. pseudomallei strains was linked to the outbreak, and that increased melioidosis risk in this community was associated with Piper methysticum root (kava) consumption.
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A chromosome 4 trisomy contributes to increased fluconazole resistance in a clinical isolate of Candida albicans
More LessCandida albicans is an important opportunistic fungal pathogen capable of causing both mucosal and disseminated disease. Infections are often treated with fluconazole, a front-line antifungal drug that targets the biosynthesis of ergosterol, a major component of the fungal cell membrane. Resistance to fluconazole can arise through a variety of mechanisms, including gain-of-function mutations, loss of heterozygosity events and aneuploidy. The clinical isolate P60002 was found to be highly resistant to azole-class drugs, yet lacked mutations or chromosomal rearrangements known to be associated with azole resistance. Transcription profiling suggested that increased expression of two putative drug efflux pumps, CDR11 and QDR1, might confer azole resistance. However, ectopic expression of the P60002 alleles of these genes in a drug-susceptible strain did not increase fluconazole resistance. We next examined whether the presence of three copies of chromosome 4 (Chr4) or chromosome 6 (Chr6) contributed to azole resistance in P60002. We established that Chr4 trisomy contributes significantly to fluconazole resistance, whereas Chr6 trisomy has no discernible effect on resistance. In contrast, a Chr4 trisomy did not increase fluconazole resistance when present in the standard SC5314 strain background. These results establish a link between Chr4 trisomy and elevated fluconazole resistance, and demonstrate the impact of genetic background on drug resistance phenotypes in C. albicans.
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Exposure to sub-inhibitory concentrations of gentamicin, ciprofloxacin and cefotaxime induces multidrug resistance and reactive oxygen species generation in meticillin-sensitive Staphylococcus aureus
More LessPurpose. The role of antibiotics below their MIC in the development of bacterial drug resistance is becoming increasingly important. We investigated the effect of sub-MICs of bactericidal antibiotics on the susceptibility pattern of Staphylococcus aureus and evaluated the role of free radicals.
Methodology. A total of 12 S. aureus strains were recovered from pus samples and their antibiograms determined. The test isolates were treated with sub-MIC levels of tetracycline, gentamicin, ciprofloxacin and cefotaxime. Alterations in their respective breakpoints were observed along with measurements of free radical generation by nitro blue tetrazolium test.
Results/Key findings. Gentamicin, ciprofloxacin and cefotaxime exposure significantly altered the breakpoints of exposed isolates against several tested antibiotics and higher levels of free radicals were generated after antibiotic exposure.
Conclusions. Our study demonstrates that sub-MIC levels of antimicrobials can lead to resistance and cross-resistance across several classes of antibiotics in wild strains of S. aureus, possibly by free radical production. The molecular mechanisms behind the acquisition of drug resistance at low antibiotic concentrations and the specific target genes of reactive oxygen speciesneed to be explored further.
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Physical contact and carbon transfer between a lichen-forming Trebouxia alga and a novel Alphaproteobacterium
More LessRecent progress in molecular techniques has begun to alter traditional recognition of lichens as symbiotic organisms comprised of a fungus and photosynthetic partners (green algae and/or cyanobacteria). Diverse organisms, especially various non-photosynthetic bacteria, are now indicated to be integral components of lichen symbiosis. Although lichen-associated bacteria are inferred to have functions that could support the symbiosis, little is known about their physical and nutritional interaction with fungi and algae. In the present study, we identified specific interaction between a lichen-forming alga and a novel bacterium. Trebouxia alga was isolated from a lichen, Usnea hakonensis, and kept as a strain for 8 years. Although no visible bacterial colonies were observed in this culture, high-throughput sequencing of DNA isolated from the culture revealed that the strain is composed of a Trebouxia alga and an Alphaproteobacterium species. In situ hybridization showed that bacterial cells were localized on the surface of the algal cells. Physiological assays revealed that the bacterium was able to use ribitol, glucose and mannitol, all of which are known to exist abundantly in lichens. It was resistant to three antibiotics. Bacteria closely related to this species were also identified in lichen specimens, indicating that U. hakonensis may commonly associate with this group of bacteria. These features of the novel bacterium suggest that it may be involved in carbon cycling of U. hakonensis as a member of lichen symbiosis and less likely to have become associated with the alga after isolation from a lichen.
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Systematics of haloarchaea and biotechnological potential of their hydrolytic enzymes
More LessHalophilic archaea, also referred to as haloarchaea, dominate hypersaline environments. To survive under such extreme conditions, haloarchaea and their enzymes have evolved to function optimally in environments with high salt concentrations and, sometimes, with extreme pH and temperatures. These features make haloarchaea attractive sources of a wide variety of biotechnological products, such as hydrolytic enzymes, with numerous potential applications in biotechnology. The unique trait of haloarchaeal enzymes, haloenzymes, to sustain activity under hypersaline conditions has extended the range of already-available biocatalysts and industrial processes in which high salt concentrations inhibit the activity of regular enzymes. In addition to their halostable properties, haloenzymes can also withstand other conditions such as extreme pH and temperature. In spite of these benefits, the industrial potential of these natural catalysts remains largely unexplored, with only a few characterized extracellular hydrolases. Because of the applied impact of haloarchaea and their specific ability to live in the presence of high salt concentrations, studies on their systematics have intensified in recent years, identifying many new genera and species. This review summarizes the current status of the haloarchaeal genera and species, and discusses the properties of haloenzymes and their potential industrial applications.
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Determination of antimicrobial resistance of Enterococcus strains isolated from pigs and their genotypic characterization by method of amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting)
More LessPurpose. In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms.
Methodology. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications.
Results. The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis.
Conclusion. Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.
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In vitro antifungal susceptibilities of Candida species to liposomal amphotericin B, determined using CLSI broth microdilution, and amphotericin B deoxycholate, measured using the Etest
The antifungal susceptibilities of 598 isolates of Candida spp. (bloodstream and other sterile sites) to liposomal amphotericin B (L-AmB) versus amphotericin B (AmB) were determined. MICs were calculated using the Clinical and Laboratory Standards Institute broth microdilution (M27-A3) method for L-AmB and the Etest method for AmB. The MIC50/MIC90 (µg ml−1) values for L-AmB broth microdilution and AmB Etest were 0.25/1 and 0.19/0.5, respectively. The overall essential agreement (±2 dilutions) was 91.5 %, ranging from 37.5 % (Candida lusitaniae) to 100 % (Candida glabrata and Candida krusei). Categorical agreement between the two methods was categorized based on a previously published breakpoint (susceptible/resistant MIC cut-off of 1 µg ml−1). The overall categorical agreement at the 48 h reading was 97.3 %, ranging from 72.7 % (C. krusei) to 100 % (Candida albicans). Major and very major discrepancies occurred in 2.3 and 0.3 %, respectively. Spearman’s ρ was 0.48 (P<0.0001). These results demonstrate the utility of the AmB Etest as a surrogate marker to predict the sensibility and resistance of Candida spp. to L-AmB and thus to support its use in antifungal treatment.
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Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam
Multidrug resistance in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating multidrug-resistant (MDR) A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the bla OXA genes. The aim of this study was to determine the prevalence of antimicrobial-resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterize the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A . baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74 %) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32 µg ml−1) was determined for 109/117 (91.6 %) of the XDR imipenem-nonsusceptible organisms, of which the majority (86.7 %) harboured the bla OXA-51 and bla OXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis segregated the 160 A . baumannii into 107 different multiple-locus variable number tandem repeat analysis types, which described five major clusters. The biggest cluster was a clonal complex composed mainly of imipenem-resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the bla OXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and ongoing transmission within the Vietnamese healthcare system.
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Microbial nanowires: an electrifying tale
More LessElectromicrobiology has gained momentum in the last 10 years with advances in microbial fuel cells and the discovery of microbial nanowires (MNWs). The list of MNW-producing micro-organisms is growing and providing intriguing insights into the presence of such micro-organisms in diverse environments and the potential roles MNWs can perform. This review discusses the MNWs produced by different micro-organisms, including their structure, composition and mechanism of electron transfer through MNWs. Two hypotheses, metallic-like conductivity and an electron hopping model, have been proposed for electron transfer and we present a current understanding of both these hypotheses. MNWs not only are poised to change the way we see micro-organisms but also may impact the fields of bioenergy, biogeochemistry and bioremediation; hence, their potential applications in these fields are highlighted here.
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Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria
More LessCoagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to β-lactam (mecA) (15 %), aminoglycoside [aac(6′)/aph(2″)(11 %), aph (3′)-IIIa (15 %), ant(4′)-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062–0.5 µg ml−1 for tigecycline/rifampicin and 0.250–2 µg ml−1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.
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Pseudomonas putida as a platform for the synthesis of aromatic compounds
Aromatic compounds such as l -phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by ‘green’ alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l−1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheA fbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l−1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l−1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.
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The soil microbiome at the Gi-FACE experiment responds to a moisture gradient but not to CO2 enrichment
More LessThe soil bacterial community at the Giessen free-air CO2 enrichment (Gi-FACE) experiment was analysed by tag sequencing of the 16S rRNA gene. No substantial effects of CO2 levels on bacterial community composition were detected. However, the soil moisture gradient at Gi-FACE had a significant effect on bacterial community composition. Different groups within the Acidobacteria and Verrucomicrobia phyla were affected differently by soil moisture content. These results suggest that modest increases in atmospheric CO2 may cause only minor changes in soil bacterial community composition and indicate that the functional responses of the soil community to CO2 enrichment previously reported at Gi-FACE are due to factors other than changes in bacterial community composition. The effects of the moisture gradient revealed new information about the relationships between poorly known Acidobacteria and Verrucomicrobia and soil moisture content. This study contrasts with the relatively small number of other temperate grassland free-air CO2 enrichment microbiome studies in the use of moderate CO2 enrichment and the resulting minor changes in the soil microbiome. Thus, it will facilitate the development of further climate change mitigation studies. In addition, the moisture gradient found at Gi-FACE contributes new knowledge in soil microbial ecology, particularly regarding the abundance and moisture relationships of the soil Verrucomicrobia.
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Performance of the FilmArray® blood culture identification panel utilized by non-expert staff compared with conventional microbial identification and antimicrobial resistance gene detection from positive blood cultures
More LessUtilization of commercially available rapid platforms for microbial identification from positive blood cultures is useful during periods of, or in laboratories with, limited expert staffing. We compared the results of the FilmArray® BCID Panel performed by non-expert technologists to those of conventional methods for organism identification performed by skilled microbiologists. Within 8 h of signalling positive by a continuous monitoring blood culture system, positive bottles were analysed by the FilmArray BCID Panel. Data from these analyses were compared to standard-of-care testing, which included conventional and automated methods. To gauge the ease of use of the BCID Panel by non-expert staff, technologists unfamiliar with diagnostic bacteriology performed the testing without prior knowledge of the Gram stain results, or even whether organisms were detected. Identifications of 172/200 (86 %) positive blood cultures using the BCID Panel were consistent with identifications provided by standard-of-care methods. Standard-of-care testing identified organisms in 20 positive blood cultures, which were not represented on the BCID Panel. Seven (3.5 %) blood cultures demonstrated a discrepancy between the methods, which could not be attributed to either a lack of representation on the panel or unclear separate detection of organisms in a mixed blood culture of a shared genus or grouping of organisms, e.g. Staphylococcus or Enterobacteriaceae . One (0.5 %) blood culture yielded invalid results on two separate panels, so it was eliminated from the study. The easy-to-use FilmArray® technology shows good correlation with blood culture identification and antibiotic resistance detection performed by conventional methods. This technology may be particularly useful in laboratories with limited staffing or limited technical expertise.
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diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays
More LessMicrobial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of ‘tolerant’ cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.
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Enrichment of Desulfitobacterium spp. from forest and grassland soil using the O-demethylation of phenyl methyl ethers as a growth-selective process
More LessThe O-demethylation of phenyl methyl ethers under anaerobic conditions is a metabolic feature of acetogens and Desulfitobacterium spp. Desulfitobacteria as well as most acetogens are Gram-positive bacteria with a low GC content and belong to the phylum Firmicutes. The consumption of the phenyl methyl ether syringate was studied in enrichment cultures originating from five different topsoils. Desulfitobacterium spp. were detected in all topsoils via quantitative PCR. Desulfitobacteria could be enriched using the O-demethylation of syringate as a growth-selective process. The enrichment was significantly favoured by an external electron acceptor such as 3-chloro-4-hydroxyphenylacetate or thiosulfate. Upon cultivation in the presence of syringate and thiosulfate, which naturally occur in soil, a maximum number of 16S rRNA gene copies of Desulfitobacterium spp. was reached within the first three subcultivation steps and accounted for 3–10 % of the total microbial community depending on the soil type. Afterwards, a loss of Desulfitobacterium gene copies was observed. Community analyses revealed that Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the main phyla in the initial soil samples. Upon addition of syringate and thiosulfate as growth substrates, these phyla were rapidly outcompeted by Firmicutes, which were under-represented in soil. The main Firmicutes genera identified were Alkalibaculum, Clostridium, Sporobacterium, Sporomusa and Tissierella, which might be responsible for outcompeting the desulfitobacteria. Most of these organisms belong to the acetogens, which have previously been described to demethylate phenyl methyl ethers. The shift of the native community structure to almost exclusively Firmicutes supports the participation of members of this phylum in environmental demethylation processes.
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Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam
A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7 %). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8–EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6 %), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9 %). EV was most frequently detected in pigs ≤ 14 weeks old (∼95 %) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8–EV-G11, and five new types (EV-G12–EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6 %; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42 %) compared with domestic pigs (29 %), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.
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Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil
Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. ‘atroviride type B’ morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (l-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. ‘atroviride type B’ growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.
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Metabarcoding analysis of home composts reveals distinctive fungal communities with a high number of unassigned sequences
More LessHome composting has been strongly advocated in the UK, Europe and North America to divert organic waste away from conventional waste processing. Despite this, little attention has been given to microbial communities and their diversity in these systems. In this study, we examined the diversity of fungal species in 10 different domestic composts by 454 tag-encoded pyrosequencing. We report the recovery of 478 different molecular operational taxonomic units (MOTUs) from the 10 composts with a mean of 176.7 ± 19.6 MOTUs per compost and a mean of 12.9 ± 3.8 unique MOTUs per sample. Microascales (17.21 %), Hypocreales (16.76 %), Sordariales (14.89 %), Eurotiales (11.25 %) and Mortierellales (7.38 %) were the dominant orders in the community, with Pseudallescheria (9.52 %), Penicillium (8.43 %), Mortierella (3.60 %) and Fusarium (3.31 %) being the most abundant genera. Fungal communities in home composts were substantially different to large-scale commercial composts, with thermophilic and thermotolerant fungi present in much lower numbers. Significantly, 46.2 % of all sequences were identified as uncultured fungi or could not be assigned above the family level, suggesting there are a high number of new genera and species in these environments still to be described.
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Quantitative detection of syntrophic fatty acid-degrading bacterial communities in methanogenic environments
More LessIn methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H2, acetate and CO2 by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.
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Analysis of the Peltigera membranacea metagenome indicates that lichen-associated bacteria are involved in phosphate solubilization
More LessAlthough lichens are generally described as mutualistic symbioses of fungi and photosynthetic partners, they also harbour a diverse non-phototrophic microbiota, which is now regarded as a significant part of the symbiosis. However, the role of the non-phototrophic microbiota within the lichen is still poorly known, although possible functions have been suggested, including phosphate solubilization and various lytic activities. In the present study we focus on the bacterial biota associated with the foliose lichen Peltigera membranacea. To address our hypotheses on possible roles of the non-phototrophic microbiota, we used a metagenomic approach. A DNA library of bacterial sequence contigs was constructed from the lichen thallus material and the bacterial microbiota DNA sequence was analysed in terms of phylogenetic diversity and functional gene composition. Analysis of about 30 000 such bacterial contigs from the P. membranacea metagenome revealed significant representation of several genes involved in phosphate solubilization and biopolymer degradation.
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