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
51 - 100 of 107 results
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Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages
The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.
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How myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases
More LessUp to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014–2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host’s innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.
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An outbreak of a rare Shiga-toxin-producing Escherichia coli serotype (O117:H7) among men who have sex with men
More LessSexually transmissible enteric infections (STEIs) are commonly associated with transmission among men who have sex with men (MSM). In the past decade, the UK has experienced multiple parallel STEI emergences in MSM caused by a range of bacterial species of the genus Shigella, and an outbreak of an uncommon serotype (O117 : H7) of Shiga-toxin-producing Escherichia coli (STEC). Here, we used microbial genomics on 6 outbreak and 30 sporadic STEC O117 : H7 isolates to explore the origins and pathogenic drivers of the STEC O117 : H7 emergence in MSM. Using genomic epidemiology, we found that the STEC O117 : H7 outbreak lineage was potentially imported from Latin America and likely continues to circulate both in the UK MSM population and in Latin America. We found genomic relationships consistent with existing symptomatic evidence for chronic infection with this STEC serotype. Comparative genomic analysis indicated the existence of a novel Shiga toxin 1-encoding prophage in the outbreak isolates, and evidence of horizontal gene exchange among the STEC O117 : H7 outbreak lineage and other enteric pathogens. There was no evidence of increased virulence in the outbreak strains relative to contextual isolates, but the outbreak lineage was associated with azithromycin resistance. Comparing these findings with similar genomic investigations of emerging MSM-associated Shigella in the UK highlighted many parallels, the most striking of which was the importance of the azithromycin phenotype for STEI emergence in this patient group.
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Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential
Raimunda Sâmia Nogueira Brilhante, Jamille Alencar Sales, Maria Lucilene Queiroz da Silva, Jonathas Sales de Oliveira, Lucas de Alencar Pereira, Waldemiro Aquino Pereira-Neto, Rossana de Aguiar Cordeiro, José Júlio Costa Sidrim, Débora de Souza Collares Maia Castelo-Branco and Marcos Fábio Gadelha RochaPurpose. Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex.
Methodology. Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.
Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were β-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure.
Conclusion. These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.
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Relevance of antifungal penetration in biofilm-associated resistance of Candida albicans and non-albicans Candida species
More LessThe role of penetration limitation in Candida biofilm-associated antifungal resistance remains unclear. Most of the previous work has been done on Candida albicans, although non-albicans (NAC) species are also implicated in invasive candidiasis and the biofilm matrix has been shown to vary amongst different species. Only a few studies have evaluated clinical isolates. This study aimed to determine the relevance of penetration limitation in the antifungal resistance of biofilms formed by C. albicans and NAC clinical isolates, using an agar disk diffusion assay. The penetration of posaconazole and amphotericin B through the biofilms was significantly reduced. Fluconazole, voriconazole and caspofungin showed a superior penetration capacity in C. albicans, Candida tropicalis and Candida parapsilosis biofilms, but exhibited inter-species and strain/isolate variation. Candida krusei biofilms were the most resilient to antifungal permeation. All of the antifungal drugs failed to kill the biofilm cells, independent of penetration, suggesting that the other factors contribute markedly to the recalcitrance of the biofilms.
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Contribution of efflux to colistin heteroresistance in a multidrug resistant Acinetobacter baumannii clinical isolate
Purpose. The mechanisms underlying colistin heteroresistance in Acinetobacter baumannii are not fully understood. Here, we investigated the role of efflux in colistin-heteroresistant populations of a multidrug-resistant (MDR) A. baumannii clinical isolate.
Methodology. Three colistin-resistant A. baumannii strain variants isolated from the same clinical sample were studied for the presence of heteroresistance to colistin by drug susceptibility testing, genotyping and drug resistance target mutation analysis. The existence of active efflux was studied by synergism assays with efflux inhibitors, real-time efflux activity measurements and analysis of the mRNA transcriptional levels of selected efflux pump genes in response to colistin.
Results. All of the strain variants belong to the ST218, clonal complex 92, international clonal lineage II. Different colistin susceptibility levels were observed among the three strain variants, indicating that colistin-heteroresistant subpopulations were being selected upon exposure to colistin. No mutations were found in the genes lpxACD and pmrAB, which are associated with colistin resistance. The results showed the existence of synergistic interactions between efflux inhibitors and colistin and ethidium bromide. Real-time efflux assays demonstrated that the three strain variants had increased efflux activity that could be inhibited in the presence of the inhibitors. The efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS and abeM were found to be overexpressed in the strain variants in response to colistin exposure.
Conclusion. This study shows that efflux activity contributes to colistin heteroresistance in an MDR A. baumannii clinical isolate. The use of efflux inhibitors as adjuvants of the therapy can resensitize A. baumannii to colistin and prevent the emergence of drug resistance.
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Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China
Purpose. The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China.
Methodology. The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing.
Results. A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3′)-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3′)-IIa]. The oqxAB–aph(3′)-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin.
Conclusion. This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB–aph(3′)-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.
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Surveillance of antimicrobial resistance in Neisseria meningitidis strains isolated from invasive cases in Brazil from 2009 to 2016
Purpose. To describe the antimicrobial resistance profile of Neisseria meningitidis isolates causing invasive disease in Brazil from 2009 to 2016.
Methodology. Among 3548 N. meningitidis isolates received, 2888 (81.4 %) were analysed for antimicrobial resistance using the broth microdilution technique, as recommended by the Clinical and Laboratory Standards Institute. Isolates were tested for ciprofloxacin, chloramphenicol, ceftriaxone, penicillin G, ampicillin and rifampin.
Results. All the isolates tested were susceptible to ceftriaxone, while 953 (33.0 %), 1307 (45.3 %) and 2 (0.07 %) isolates were penicillin G-, ampicillin- and rifampin-intermediate, respectively. Resistance to rifampin, ciprofloxacin and chloramphenicol was shown by three isolates (0.1 %), two isolates (0.07 %) and one (0.03 %) isolate, respectively. Although no isolates were resistant to penicillin G in the period of 2009–2016, our results show an upward trend in minimum inhibitory concentrations (MICs) for this drug as of 2010 (P<0.001). There was no significant difference between different gender and age groups of patients for reduced susceptibility to penicillin G. There was a higher frequency of isolates with reduced susceptibility to penicillin G in the South and Southeast regions (P<0.001). This reduced susceptibility was also associated with serotype 19 inside serogroup B (P<0.001).
Conclusion. Despite the decrease in susceptibility to penicillin G and ampicillin observed from 2010, the overall resistance of N. meningitidis isolates to the antimicrobials tested remained uncommon and sporadic, confirming their efficacy for chemoprophylaxis or treatment of invasive meningococcal disease (IMD) in Brazil. Continued surveillance of N. meningitidis antimicrobial susceptibility profiles is important in order to monitor variations in resistance either geographically, over time or in association with emergent clones.
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Prevalence, antimicrobial resistance and serotype distribution of group B streptococcus isolated among pregnant women and newborns in Rabat, Morocco
Purpose. Group B streptococcus (GBS) is an important cause of neonatal sepsis worldwide. Data on the prevalence of maternal GBS colonization, risk factors for carriage, antibiotic susceptibility and circulating serotypes are necessary to tailor adequate locally relevant public health policies.
Methodology. A prospective study including pregnant women and their newborns was conducted between March and July 2013 in Morocco. We collected clinical data and vagino-rectal and urine samples from the recruited pregnant women, together with the clinical characteristics of, and body surface samples from, their newborns. Additionally, the first three newborns admitted every day with suspected invasive infection were recruited for a thorough screening for neonatal sepsis. Serotypes were characterized by molecular testing.
Results. A total of 350 pregnant women and 139 of their newborns were recruited. The prevalence of pregnant women colonized by GBS was 24 %. In 5/160 additional sick newborns recruited with suspected sepsis, the blood cultures were positive for GBS. Gestational hypertension and vaginal pruritus were significantly associated with a vagino-rectal GBS colonization in univariate analyses. All of the strains were susceptible to penicillin, while 7 % were resistant to clindamycin and 12 % were resistant to erythromycin. The most common GBS serotypes detected included V, II and III.
Conclusion. In Morocco, maternal GBS colonization is high. Penicillin can continue to be the cornerstone of intrapartum antibiotic prophylaxis. A pentavalent GBS vaccine (Ia, Ib, II, III and V) would have been effective against the majority of the colonizing cases in this setting, but a trivalent one (Ia, Ib and III) would only prevent 28 % of the cases.
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Are commercial providers a viable option for clinical bacterial sequencing?
More LessBacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice.
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Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil
Patricia Fernanda Herkert, Jacques F. Meis, Gabriel Lucca de Oliveira Salvador, Renata Rodrigues Gomes, Vania Aparecida Vicente, Marisol Dominguez Muro, Rosangela Lameira Pinheiro, Arnaldo Lopes Colombo, Alexandre Vargas Schwarzbold, Carla Sakuma de Oliveira, Marcelo Simão Ferreira, Flávio Queiroz-Telles and Ferry HagenPurpose. Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil.
Methodology. We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.
Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson’s diversity index of this population was D=0.9856.
Conclusion. The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.
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Three novel proteins co-localise with polyhydroxybutyrate (PHB) granules in Rhodospirillum rubrum S1
More LessPolyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules’ associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.
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Antimicrobial activity against Mycobacterium tuberculosis under in vitro lipid-rich dormancy conditions
Although tuberculosis treatment is dependent on drug-susceptibility testing (DST) and molecular drug-resistance detection, treatment failure and relapse remain a challenge. This could be partially due to the emergence of antibiotic-tolerant dormant mycobacteria, where host lipids have been shown to play an important role. This study evaluated the susceptibility of Mycobacterium tuberculosis to two antibiotic combinations – rifampicin, moxifloxacin, amikacin and metronidazole (RIF-MXF-AMK-MTZ), and rifampicin, moxifloxacin, amikacin and pretomanid (RIF-MXF-AMK-PA) – in a lipid-rich dormancy model. Although their effectiveness in in vitro cultures with dextrose as a carbon source has been proved, we observed that none of the antibiotic mixtures were bactericidal in the presence of lipids. The presence of lipids may confer tolerance to M. tuberculosis against the mixture of antibiotics tested and such tolerance could be even higher during the dormant stages. The implementation of lipids in DST on clinical isolates could potentially lead to a better treatment strategy.
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Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway
Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d -xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway.
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Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages
There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.
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The diversity of ice algal communities on the Greenland Ice Sheet as revealed by oligotyping
More LessThe Arctic is being disproportionally affected by climate change compared with other geographic locations, and is currently experiencing unprecedented melt rates. The Greenland Ice Sheet (GrIS) can be regarded as the largest supraglacial ecosystem on Earth, and ice algae are the dominant primary producers on bare ice surfaces throughout the course of a melt season. Ice-algal-derived pigments cause a darkening of the ice surface, which in turn decreases albedo and increases melt rates. The important role of ice algae in changing melt rates has only recently been recognized, and we currently know little about their community compositions and functions. Here, we present the first analysis of ice algal communities across a 100 km transect on the GrIS by high-throughput sequencing and subsequent oligotyping of the most abundant taxa. Our data reveal an extremely low algal diversity with Ancylonema nordenskiöldii and a Mesotaenium species being by far the dominant taxa at all sites. We employed an oligotyping approach and revealed a hidden diversity not detectable by conventional clustering of operational taxonomic units and taxonomic classification. Oligotypes of the dominant taxa exhibit a site-specific distribution, which may be linked to differences in temperatures and subsequently the extent of the melting. Our results help to better understand the distribution patterns of ice algal communities that play a crucial role in the GrIS ecosystem.
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Whole-genome sequencing revealed concurrent outbreaks of shigellosis in the English Orthodox Jewish Community caused by multiple importations of Shigella sonnei from Israel
In December 2013, Public Health England (PHE) observed an increase in the number of cases of Shigella sonnei linked to the Orthodox Jewish Community (OJC). Ultimately, 52 cases of S. sonnei phage type (PT) P and PT7 were notified between November 2013 and July 2014. Whole-genome sequencing (WGS) was performed on a HiSeq 2500 platform (Illumina) on isolates of S. sonnei submitted to PHE during the investigation. Quality trimmed sequence reads were mapped to a reference genome using BWA-MEM, and single-nucleotide polymorphisms (SNPs) were identified using GATK2. Analysis of the core genome SNP positions (>90 % consensus, minimum depth 10×, MQ≥30) revealed that isolates linked to the outbreak could be categorized as members of distinct monophyletic clusters (MPCs) representing concurrent regional outbreaks occurring in the OJCs across the United Kingdom. A dated phylogeny predicted the date of the most recent common ancestor of the MPCs to be approximately 3.1 years previously [95 % highest posterior density (HPD), 2.4–3.4]. Isolates of S. sonnei from cases from the OJCs in Israel included in the phylogeny, branched from nodes basal to the UK OJC outbreak clusters, indicating they were ancestral to the UK OJC isolates, and that the UK isolates represented multiple importations of S. sonnei into the UK population from Israel. The level of discrimination exhibited by WGS facilitated the identification of clusters of isolates within the closely related bacterial populations circulating in the OJC that may be linked to a unique point sources or transmission routes, thus enabling a more appropriate public health response and targeted interventions.
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Blad-containing oligomer: a novel fungicide used in crop protection as an alternative treatment for tinea pedis and tinea versicolor
Purpose. The lack of novel antifungal drugs and the increasing incidence and severity of fungal infections are major concerns worldwide. Herein, we tested the activity of the Blad-containing oligomer (BCO), a new antifungal molecule already in use for agriculture, on Malassezia spp. and dermatophytes, the causal agents of human tinea versicolor and tinea pedis. Given the lack of a standard method for Malassezia susceptibility testing and the plethora of published methods, we also developed an improved method for this genus.
Methodology. The efficacy of BCO was assessed in vitro and compared to that of the drugs currently utilized in the treatment of tinea versicolor (fluconazole and itraconazole) and tinea pedis (itraconazole and terbinafine). For dermatophytes, the standard microdilution broth-based method was used, with small adjustments, and several broth formulations and inocula sizes were tested to develop an improved susceptibility method for Malassezia spp.
Results. We successfully developed a microdilution broth-based method with considerable advantages over other available methods, and used it for all in vitro susceptibility tests of Malassezia spp. isolates. We report that, on a molar basis, BCO was more effective than fluconazole or itraconazole on most strains of Malassezia spp. isolated from clinical samples (n=29). By contrast, BCO was less effective than itraconazole or terbinafine on the common dermatophytes Trichophyton rubrum and Trichophyton interdigitale.
Conclusion. These data place BCO as a promising drug for the treatment of Malassezia-associated skin diseases. Further in vivo studies are now required to ascertain its applicability in the clinical setting.
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Efficient bio-production of citramalate using an engineered Escherichia coli strain
More LessCitramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l−1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l−1 in total) and 1.4 g l−1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l−1, with a productivity of 1.85 g l−1 h−1 and a conversion efficiency of 0.48 gcitramalate g−1 glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.
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Ram locus is a key regulator to trigger multidrug resistance in Enterobacter aerogenes
More LessPurpose. Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of E. aerogenes due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance.
Methodology. Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of E. aerogenes. Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed.
Results. In the majority of the MDR isolates tested, ramR and a region upstream of ramA were mutated but marR or marA were unchanged. Expression and cloning experiments highlighted the involvement of the ram locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects.
Conclusion. Mutations or deletions in ramR, leading to the overexpression of RamA predominated in clinical MDR E. aerogenes isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in ramR, and the self-regulating region proximal to ramA, probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing E. aerogenes towards the emergence of a MDR phenotype.
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Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence
Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral ‘pathogens’ cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.
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Fusion proteins towards fungi and bacteria in plant protection
More LessIn agriculture, although fungi are considered the foremost problem, infections by bacteria also cause significant economical losses. The presence of different diseases in crops often leads to a misuse of the proper therapeutic, or the combination of different diseases forces the use of more than one pesticide. This work concerns the development of a ‘super-Blad’: a chimeric protein consisting of Blad polypeptide, the active ingredient of a biological fungicide already on the market, and two selected peptides, SP10-5 and Sub5, proven to possess biological potential as antibacterial agents. The resulting chimeric protein obtained from the fusion of Blad with SP10-5 not only maintained strong antibacterial activity, especially against Xanthomonas spp. and Pseudomonas syringae, but was also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. However, the antibacterial activity of Sub5 was considerably diminished when fused with Blad, which seems to indicate that not all fusion proteins behave equally. These newly designed drugs can be considered promising compounds for use in plant protection. A deeper and focused development of an appropriate formulation may result in a potent biopesticide that can replace, per se, two conventional chemistries with less impact on the environment.
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An investigation of antifungal stewardship programmes in England
Purpose. We sought to explore the current status of antifungal stewardship (AFS) initiatives across National Health Service (NHS) Trusts within England, the challenges and barriers, as well as ways to improve current AFS programmes.
Methodology. An electronic survey was sent to all 155 acute NHS Trusts in England. A total of 47 Trusts, corresponding to 30 % of English acute Trusts, responded to the the survey; 46 Trusts (98 %) had an antimicrobial stewardship (AMS) programme but only 5 (11 %) had a dedicated AFS programme. Overall, 20 (43 %) Trusts said they included AFS as part of their AMS programmes. From those conducting AFS programmes, 7 (28 %) have an AFS/management team, 16 (64 %) monitor and report on antifungal usage, 5 (20 %) have dedicated AFS ward rounds and 12 (48 %) are directly involved in the management of invasive fungal infections.
Results/Key findings. Altogether, 13 acute Trusts (52 %) started their AFS programme to manage costs, whilst 12 (48 %) commenced the programme due to clinical need; 27 (73 %) declared that they would increase their AFS initiatives if they could. Of those without an AFS programme, 14 (67 %) responded that this was due to lack of resources/staff time. Overall, 12 Trusts (57 %) responded that the availability of rapid diagnostics and clinical support would enable them to conduct AFS activities.
Conclusion. Although a minority of Trusts conduct dedicated AFS programmes, nearly half include AFS as part of routine AMS activities. Cost issues are the main driver for AFS, followed by clinical need. The availability of rapid diagnostics and clinical support could help increase AFS initiatives.
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An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease
More Lessdoi: 10.1099/mgen.0.000121.001.
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children below 5 years of age in endemic areas, and is a primary cause of diarrhoea in travellers visiting developing countries. Epidemiological analysis of E. coli pathovars is traditionally carried out based on the results of serotyping. However, genomic analysis of a global ETEC collection of 362 isolates taken from patients revealed nine novel O-antigen biosynthesis gene clusters that were previously unrecognized, and have collectively been called unclassified. When put in the context of all isolates sequenced, one of the novel O-genotypes, OgN5, was found to be the second most common ETEC O-genotype causing disease, after O6, in a globally representative ETEC collection. It’s also clear that ETEC OgN5 isolates have spread globally. These novel O-genotypes have now been included in our comprehensive O-genotyping scheme, and can be detected using a PCR-based and an in silico typing method. This will assist in epidemiological studies, as well as in ETEC vaccine development.
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Population genetic structuring of methicillin-resistant Staphylococcus aureus clone EMRSA-15 within UK reflects patient referral patterns
Tjibbe Donker, Sandra Reuter, James Scriberras, Rosy Reynolds, Nicholas M. Brown, M. Estée Török, Richard James, East of England Microbiology Research Network, David M. Aanensen, Stephen D. Bentley, Matthew T. G. Holden, Julian Parkhill, Brian G. Spratt, Sharon J. Peacock, Edward J. Feil and Hajo GrundmannAntibiotic resistance forms a serious threat to the health of hospitalised patients, rendering otherwise treatable bacterial infections potentially life-threatening. A thorough understanding of the mechanisms by which resistance spreads between patients in different hospitals is required in order to design effective control strategies. We measured the differences between bacterial populations of 52 hospitals in the United Kingdom and Ireland, using whole-genome sequences from 1085 MRSA clonal complex 22 isolates collected between 1998 and 2012. The genetic differences between bacterial populations were compared with the number of patients transferred between hospitals and their regional structure. The MRSA populations within single hospitals, regions and countries were genetically distinct from the rest of the bacterial population at each of these levels. Hospitals from the same patient referral regions showed more similar MRSA populations, as did hospitals sharing many patients. Furthermore, the bacterial populations from different time-periods within the same hospital were generally more similar to each other than contemporaneous bacterial populations from different hospitals. We conclude that, while a large part of the dispersal and expansion of MRSA takes place among patients seeking care in single hospitals, inter-hospital spread of resistant bacteria is by no means a rare occurrence. Hospitals are exposed to constant introductions of MRSA on a number of levels: (1) most MRSA is received from hospitals that directly transfer large numbers of patients, while (2) fewer introductions happen between regions or (3) across national borders, reflecting lower numbers of transferred patients. A joint coordinated control effort between hospitals, is therefore paramount for the national control of MRSA, antibiotic-resistant bacteria and other hospital-associated pathogens.
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Risk factors and molecular mechanisms associated with trimethoprim–sulfamethoxazole resistance in Stenotrophomonas maltophilia in Mexico
Purpose. Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen causing an increasing number of nosocomial infections. Our aim was to evaluate the risk factors and mechanisms associated with trimethoprim–sulfamethoxazole (SXT) resistance in S. maltophilia infections in Mexico.
Methodology. Clinical isolates and patients' demographic and clinical data were collected from February 2007 to August 2015 in two tertiary-care hospitals in Mexico. Antimicrobial susceptibility and analysis of sul and SmeABC and SmeDEF efflux pump overexpression were performed in all isolates.
Results/Key findings. In the 9-year period, 196 patients infected with S. maltophilia were identified. Most patients were male, and the mean age was 46.2 years. The mean Charlson score was 1.42, and the most frequent comorbidities were arterial hypertension (26.7 %), type 2 diabetes (21.2 %) and cerebral infarction (11.6 %). High drug resistance to meropenem (93.4 %), gentamicin (55.1 %), ceftazidime (52.3 %), cefotaxime (51.5 %), amikacin (42.3 %) and cefepime (32.1 %), and lower resistance to ciprofloxacin (26.0 %), SXT (25.0 %), chloramphenicol (14.3 %) and levofloxacin (2.6 %) were detected. SXT resistance was not associated with the sul genes. SmeABC overexpression was associated with gentamicin (P=0.001) and levofloxacin resistance (P=0.041), whereas SmeDEF overexpression was associated with ceftazidime resistance (P=0.003). Prolonged hospitalization (≥15 days) was an independent risk factor for SXT-resistant S. maltophilia infections (OR=3.05; 95 % CI=1.12–8.86; P=0.029).
Conclusion. Given the high SXT resistance rate, SXT is not an effective first-line therapy for our patients; instead, levofloxacin could be used as an appropriate therapeutic option against S. maltophilia infections.
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Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing
Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (bla OXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.
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Candidaemia in a tertiary care academic hospital in Italy. The impact of C. parapsilosis complex on the species distribution and antifungal susceptibility
More LessPurpose. To analyse the species distribution and the susceptibility profiles to the major antifungal agents of Candida isolated from bloodstream infections (BSIs) in both intensive care units (ICUs) and non-ICU wards in a tertiary care hospital in Italy from 2010 until 2015.
Methodology. Episodes of Candida BSI were recorded in a retrospective observational cohort study. Yeasts were isolated from both blood and intravascuIar devices (IVDs) and their susceptibility to antifungal drugs was tested using the microdilution method.
Results. 514 Candida BSIs were evidenced and 19 % of these episodes were associated with the presence of an IVD. The trend of the general incidence increased significantly throughout the study period, ranging from 1.42 to 3.63 (mean 2.52) episodes/1000 admissions. The incidence of Candida BSIs and IVD-associated candidaemia was significantly higher in ICUs relative to the other wards. The most frequently isolated species were C. albicans and C. parapsilosis complex, with the latter presenting a significant increased trend of isolation. C. parapsilosis complex was most frequently involved in IVD-related candidaemia, coinfections and late recurrent infections. Furthermore, the MIC50s of C. parapsilosis complex were significantly enhanced for echinocandins compared to the MIC50s for the same drugs and the other yeasts, while the MIC50s of C. albicans for amphotericin B showed a significant increase during the study period, ranging from 0.1 to 0.5 µg ml−1.
Conclusions. A progressively enhanced incidence of Candida BSIs, a relatively high impact of C. parapsilosis complex and changes in the susceptibility profiles of the isolated yeasts were evidenced during the observation period.
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Whole-genome sequencing to investigate a non-clonal melioidosis cluster on a remote Australian island
Melioidosis is a tropical disease caused by the bacterium Burkholderia pseudomallei. Outbreaks are uncommon and can generally be attributed to a single point source and strain. We used whole-genome sequencing to analyse B. pseudomallei isolates collected from an historical 2-year long case cluster that occurred in a remote northern Australian indigenous island community, where infections were previously linked to a contaminated communal water supply. We analysed the genome-wide relatedness of the two most common multilocus sequence types (STs) involved in the outbreak, STs 125 and 126. This analysis showed that although these STs were closely related on a whole-genome level, they demonstrated evidence of multiple recombination events that were unlikely to have occurred over the timeframe of the outbreak. Based on epidemiological and genetic data, we also identified two additional patients not previously associated with this outbreak. Our results confirm the previous hypothesis that a single unchlorinated water source harbouring multiple B. pseudomallei strains was linked to the outbreak, and that increased melioidosis risk in this community was associated with Piper methysticum root (kava) consumption.
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A chromosome 4 trisomy contributes to increased fluconazole resistance in a clinical isolate of Candida albicans
More LessCandida albicans is an important opportunistic fungal pathogen capable of causing both mucosal and disseminated disease. Infections are often treated with fluconazole, a front-line antifungal drug that targets the biosynthesis of ergosterol, a major component of the fungal cell membrane. Resistance to fluconazole can arise through a variety of mechanisms, including gain-of-function mutations, loss of heterozygosity events and aneuploidy. The clinical isolate P60002 was found to be highly resistant to azole-class drugs, yet lacked mutations or chromosomal rearrangements known to be associated with azole resistance. Transcription profiling suggested that increased expression of two putative drug efflux pumps, CDR11 and QDR1, might confer azole resistance. However, ectopic expression of the P60002 alleles of these genes in a drug-susceptible strain did not increase fluconazole resistance. We next examined whether the presence of three copies of chromosome 4 (Chr4) or chromosome 6 (Chr6) contributed to azole resistance in P60002. We established that Chr4 trisomy contributes significantly to fluconazole resistance, whereas Chr6 trisomy has no discernible effect on resistance. In contrast, a Chr4 trisomy did not increase fluconazole resistance when present in the standard SC5314 strain background. These results establish a link between Chr4 trisomy and elevated fluconazole resistance, and demonstrate the impact of genetic background on drug resistance phenotypes in C. albicans.
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Exposure to sub-inhibitory concentrations of gentamicin, ciprofloxacin and cefotaxime induces multidrug resistance and reactive oxygen species generation in meticillin-sensitive Staphylococcus aureus
More LessPurpose. The role of antibiotics below their MIC in the development of bacterial drug resistance is becoming increasingly important. We investigated the effect of sub-MICs of bactericidal antibiotics on the susceptibility pattern of Staphylococcus aureus and evaluated the role of free radicals.
Methodology. A total of 12 S. aureus strains were recovered from pus samples and their antibiograms determined. The test isolates were treated with sub-MIC levels of tetracycline, gentamicin, ciprofloxacin and cefotaxime. Alterations in their respective breakpoints were observed along with measurements of free radical generation by nitro blue tetrazolium test.
Results/Key findings. Gentamicin, ciprofloxacin and cefotaxime exposure significantly altered the breakpoints of exposed isolates against several tested antibiotics and higher levels of free radicals were generated after antibiotic exposure.
Conclusions. Our study demonstrates that sub-MIC levels of antimicrobials can lead to resistance and cross-resistance across several classes of antibiotics in wild strains of S. aureus, possibly by free radical production. The molecular mechanisms behind the acquisition of drug resistance at low antibiotic concentrations and the specific target genes of reactive oxygen speciesneed to be explored further.
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Physical contact and carbon transfer between a lichen-forming Trebouxia alga and a novel Alphaproteobacterium
More LessRecent progress in molecular techniques has begun to alter traditional recognition of lichens as symbiotic organisms comprised of a fungus and photosynthetic partners (green algae and/or cyanobacteria). Diverse organisms, especially various non-photosynthetic bacteria, are now indicated to be integral components of lichen symbiosis. Although lichen-associated bacteria are inferred to have functions that could support the symbiosis, little is known about their physical and nutritional interaction with fungi and algae. In the present study, we identified specific interaction between a lichen-forming alga and a novel bacterium. Trebouxia alga was isolated from a lichen, Usnea hakonensis, and kept as a strain for 8 years. Although no visible bacterial colonies were observed in this culture, high-throughput sequencing of DNA isolated from the culture revealed that the strain is composed of a Trebouxia alga and an Alphaproteobacterium species. In situ hybridization showed that bacterial cells were localized on the surface of the algal cells. Physiological assays revealed that the bacterium was able to use ribitol, glucose and mannitol, all of which are known to exist abundantly in lichens. It was resistant to three antibiotics. Bacteria closely related to this species were also identified in lichen specimens, indicating that U. hakonensis may commonly associate with this group of bacteria. These features of the novel bacterium suggest that it may be involved in carbon cycling of U. hakonensis as a member of lichen symbiosis and less likely to have become associated with the alga after isolation from a lichen.
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Systematics of haloarchaea and biotechnological potential of their hydrolytic enzymes
More LessHalophilic archaea, also referred to as haloarchaea, dominate hypersaline environments. To survive under such extreme conditions, haloarchaea and their enzymes have evolved to function optimally in environments with high salt concentrations and, sometimes, with extreme pH and temperatures. These features make haloarchaea attractive sources of a wide variety of biotechnological products, such as hydrolytic enzymes, with numerous potential applications in biotechnology. The unique trait of haloarchaeal enzymes, haloenzymes, to sustain activity under hypersaline conditions has extended the range of already-available biocatalysts and industrial processes in which high salt concentrations inhibit the activity of regular enzymes. In addition to their halostable properties, haloenzymes can also withstand other conditions such as extreme pH and temperature. In spite of these benefits, the industrial potential of these natural catalysts remains largely unexplored, with only a few characterized extracellular hydrolases. Because of the applied impact of haloarchaea and their specific ability to live in the presence of high salt concentrations, studies on their systematics have intensified in recent years, identifying many new genera and species. This review summarizes the current status of the haloarchaeal genera and species, and discusses the properties of haloenzymes and their potential industrial applications.
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Determination of antimicrobial resistance of Enterococcus strains isolated from pigs and their genotypic characterization by method of amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting)
More LessPurpose. In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms.
Methodology. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications.
Results. The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis.
Conclusion. Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.
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In vitro antifungal susceptibilities of Candida species to liposomal amphotericin B, determined using CLSI broth microdilution, and amphotericin B deoxycholate, measured using the Etest
The antifungal susceptibilities of 598 isolates of Candida spp. (bloodstream and other sterile sites) to liposomal amphotericin B (L-AmB) versus amphotericin B (AmB) were determined. MICs were calculated using the Clinical and Laboratory Standards Institute broth microdilution (M27-A3) method for L-AmB and the Etest method for AmB. The MIC50/MIC90 (µg ml−1) values for L-AmB broth microdilution and AmB Etest were 0.25/1 and 0.19/0.5, respectively. The overall essential agreement (±2 dilutions) was 91.5 %, ranging from 37.5 % (Candida lusitaniae) to 100 % (Candida glabrata and Candida krusei). Categorical agreement between the two methods was categorized based on a previously published breakpoint (susceptible/resistant MIC cut-off of 1 µg ml−1). The overall categorical agreement at the 48 h reading was 97.3 %, ranging from 72.7 % (C. krusei) to 100 % (Candida albicans). Major and very major discrepancies occurred in 2.3 and 0.3 %, respectively. Spearman’s ρ was 0.48 (P<0.0001). These results demonstrate the utility of the AmB Etest as a surrogate marker to predict the sensibility and resistance of Candida spp. to L-AmB and thus to support its use in antifungal treatment.
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Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam
Multidrug resistance in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating multidrug-resistant (MDR) A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the bla OXA genes. The aim of this study was to determine the prevalence of antimicrobial-resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterize the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A . baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74 %) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32 µg ml−1) was determined for 109/117 (91.6 %) of the XDR imipenem-nonsusceptible organisms, of which the majority (86.7 %) harboured the bla OXA-51 and bla OXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis segregated the 160 A . baumannii into 107 different multiple-locus variable number tandem repeat analysis types, which described five major clusters. The biggest cluster was a clonal complex composed mainly of imipenem-resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the bla OXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and ongoing transmission within the Vietnamese healthcare system.
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Microbial nanowires: an electrifying tale
More LessElectromicrobiology has gained momentum in the last 10 years with advances in microbial fuel cells and the discovery of microbial nanowires (MNWs). The list of MNW-producing micro-organisms is growing and providing intriguing insights into the presence of such micro-organisms in diverse environments and the potential roles MNWs can perform. This review discusses the MNWs produced by different micro-organisms, including their structure, composition and mechanism of electron transfer through MNWs. Two hypotheses, metallic-like conductivity and an electron hopping model, have been proposed for electron transfer and we present a current understanding of both these hypotheses. MNWs not only are poised to change the way we see micro-organisms but also may impact the fields of bioenergy, biogeochemistry and bioremediation; hence, their potential applications in these fields are highlighted here.
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Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria
More LessCoagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to β-lactam (mecA) (15 %), aminoglycoside [aac(6′)/aph(2″)(11 %), aph (3′)-IIIa (15 %), ant(4′)-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062–0.5 µg ml−1 for tigecycline/rifampicin and 0.250–2 µg ml−1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.
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Pseudomonas putida as a platform for the synthesis of aromatic compounds
Aromatic compounds such as l -phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by ‘green’ alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l−1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheA fbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l−1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l−1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.
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The soil microbiome at the Gi-FACE experiment responds to a moisture gradient but not to CO2 enrichment
More LessThe soil bacterial community at the Giessen free-air CO2 enrichment (Gi-FACE) experiment was analysed by tag sequencing of the 16S rRNA gene. No substantial effects of CO2 levels on bacterial community composition were detected. However, the soil moisture gradient at Gi-FACE had a significant effect on bacterial community composition. Different groups within the Acidobacteria and Verrucomicrobia phyla were affected differently by soil moisture content. These results suggest that modest increases in atmospheric CO2 may cause only minor changes in soil bacterial community composition and indicate that the functional responses of the soil community to CO2 enrichment previously reported at Gi-FACE are due to factors other than changes in bacterial community composition. The effects of the moisture gradient revealed new information about the relationships between poorly known Acidobacteria and Verrucomicrobia and soil moisture content. This study contrasts with the relatively small number of other temperate grassland free-air CO2 enrichment microbiome studies in the use of moderate CO2 enrichment and the resulting minor changes in the soil microbiome. Thus, it will facilitate the development of further climate change mitigation studies. In addition, the moisture gradient found at Gi-FACE contributes new knowledge in soil microbial ecology, particularly regarding the abundance and moisture relationships of the soil Verrucomicrobia.
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Performance of the FilmArray® blood culture identification panel utilized by non-expert staff compared with conventional microbial identification and antimicrobial resistance gene detection from positive blood cultures
More LessUtilization of commercially available rapid platforms for microbial identification from positive blood cultures is useful during periods of, or in laboratories with, limited expert staffing. We compared the results of the FilmArray® BCID Panel performed by non-expert technologists to those of conventional methods for organism identification performed by skilled microbiologists. Within 8 h of signalling positive by a continuous monitoring blood culture system, positive bottles were analysed by the FilmArray BCID Panel. Data from these analyses were compared to standard-of-care testing, which included conventional and automated methods. To gauge the ease of use of the BCID Panel by non-expert staff, technologists unfamiliar with diagnostic bacteriology performed the testing without prior knowledge of the Gram stain results, or even whether organisms were detected. Identifications of 172/200 (86 %) positive blood cultures using the BCID Panel were consistent with identifications provided by standard-of-care methods. Standard-of-care testing identified organisms in 20 positive blood cultures, which were not represented on the BCID Panel. Seven (3.5 %) blood cultures demonstrated a discrepancy between the methods, which could not be attributed to either a lack of representation on the panel or unclear separate detection of organisms in a mixed blood culture of a shared genus or grouping of organisms, e.g. Staphylococcus or Enterobacteriaceae . One (0.5 %) blood culture yielded invalid results on two separate panels, so it was eliminated from the study. The easy-to-use FilmArray® technology shows good correlation with blood culture identification and antibiotic resistance detection performed by conventional methods. This technology may be particularly useful in laboratories with limited staffing or limited technical expertise.
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diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays
More LessMicrobial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of ‘tolerant’ cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.
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Enrichment of Desulfitobacterium spp. from forest and grassland soil using the O-demethylation of phenyl methyl ethers as a growth-selective process
More LessThe O-demethylation of phenyl methyl ethers under anaerobic conditions is a metabolic feature of acetogens and Desulfitobacterium spp. Desulfitobacteria as well as most acetogens are Gram-positive bacteria with a low GC content and belong to the phylum Firmicutes. The consumption of the phenyl methyl ether syringate was studied in enrichment cultures originating from five different topsoils. Desulfitobacterium spp. were detected in all topsoils via quantitative PCR. Desulfitobacteria could be enriched using the O-demethylation of syringate as a growth-selective process. The enrichment was significantly favoured by an external electron acceptor such as 3-chloro-4-hydroxyphenylacetate or thiosulfate. Upon cultivation in the presence of syringate and thiosulfate, which naturally occur in soil, a maximum number of 16S rRNA gene copies of Desulfitobacterium spp. was reached within the first three subcultivation steps and accounted for 3–10 % of the total microbial community depending on the soil type. Afterwards, a loss of Desulfitobacterium gene copies was observed. Community analyses revealed that Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the main phyla in the initial soil samples. Upon addition of syringate and thiosulfate as growth substrates, these phyla were rapidly outcompeted by Firmicutes, which were under-represented in soil. The main Firmicutes genera identified were Alkalibaculum, Clostridium, Sporobacterium, Sporomusa and Tissierella, which might be responsible for outcompeting the desulfitobacteria. Most of these organisms belong to the acetogens, which have previously been described to demethylate phenyl methyl ethers. The shift of the native community structure to almost exclusively Firmicutes supports the participation of members of this phylum in environmental demethylation processes.
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Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam
A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7 %). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8–EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6 %), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9 %). EV was most frequently detected in pigs ≤ 14 weeks old (∼95 %) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8–EV-G11, and five new types (EV-G12–EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6 %; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42 %) compared with domestic pigs (29 %), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.
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Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil
Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. ‘atroviride type B’ morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (l-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. ‘atroviride type B’ growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.
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Metabarcoding analysis of home composts reveals distinctive fungal communities with a high number of unassigned sequences
More LessHome composting has been strongly advocated in the UK, Europe and North America to divert organic waste away from conventional waste processing. Despite this, little attention has been given to microbial communities and their diversity in these systems. In this study, we examined the diversity of fungal species in 10 different domestic composts by 454 tag-encoded pyrosequencing. We report the recovery of 478 different molecular operational taxonomic units (MOTUs) from the 10 composts with a mean of 176.7 ± 19.6 MOTUs per compost and a mean of 12.9 ± 3.8 unique MOTUs per sample. Microascales (17.21 %), Hypocreales (16.76 %), Sordariales (14.89 %), Eurotiales (11.25 %) and Mortierellales (7.38 %) were the dominant orders in the community, with Pseudallescheria (9.52 %), Penicillium (8.43 %), Mortierella (3.60 %) and Fusarium (3.31 %) being the most abundant genera. Fungal communities in home composts were substantially different to large-scale commercial composts, with thermophilic and thermotolerant fungi present in much lower numbers. Significantly, 46.2 % of all sequences were identified as uncultured fungi or could not be assigned above the family level, suggesting there are a high number of new genera and species in these environments still to be described.
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Quantitative detection of syntrophic fatty acid-degrading bacterial communities in methanogenic environments
More LessIn methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H2, acetate and CO2 by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.
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Analysis of the Peltigera membranacea metagenome indicates that lichen-associated bacteria are involved in phosphate solubilization
More LessAlthough lichens are generally described as mutualistic symbioses of fungi and photosynthetic partners, they also harbour a diverse non-phototrophic microbiota, which is now regarded as a significant part of the symbiosis. However, the role of the non-phototrophic microbiota within the lichen is still poorly known, although possible functions have been suggested, including phosphate solubilization and various lytic activities. In the present study we focus on the bacterial biota associated with the foliose lichen Peltigera membranacea. To address our hypotheses on possible roles of the non-phototrophic microbiota, we used a metagenomic approach. A DNA library of bacterial sequence contigs was constructed from the lichen thallus material and the bacterial microbiota DNA sequence was analysed in terms of phylogenetic diversity and functional gene composition. Analysis of about 30 000 such bacterial contigs from the P. membranacea metagenome revealed significant representation of several genes involved in phosphate solubilization and biopolymer degradation.
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