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 - 50 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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