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Asset Tag: Microbiology

April 21, 2020

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden.To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon.Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing.CPE populations increased during inoculation, plateauing at ~105?log10?cfu/mL in both models and persisting throughout the experiments (>65?days), with no evidence of CPE ‘washout’. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742?bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (~170?kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model).CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida.

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn2+ independent lignin depolymerization activity, while Mn2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 1012 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565?U/l) was achieved, providing novel insights for lignin bioconversion. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Clonal expansion and spread of the ceftriaxone-resistant Neisseria gonorrhoeae strain FC428, identified in Japan in 2015, and closely related isolates.

Ceftriaxone resistance in Neisseria gonorrhoeae is a major public health concern globally because a high-dose (1?g) injection of ceftriaxone is the only remaining option for empirical monotherapy of gonorrhoea. The ceftriaxone-resistant gonococcal strain FC428, cultured in Osaka in 2015, is suspected to have spread nationally and internationally. We describe the complete finished genomes of FC428 and two closely related isolates from Osaka in 2015, and examine the genomic epidemiology of these isolates plus three ceftriaxone-resistant gonococcal isolates from Osaka and Hyogo in 2016-17 and four ceftriaxone-resistant gonococcal isolates cultured in 2017 in Australia, Canada and Denmark.During 2015-17, we identified six ceftriaxone-resistant gonococcal isolates through our surveillance systems in Kyoto, Osaka and Hyogo. Antimicrobial susceptibility testing (six antimicrobials) was performed using Etest. Complete whole-genome sequences of the first three isolates (FC428, FC460 and FC498) from 2015 were obtained using PacBio RS II and Illumina MiSeq sequencing. The three complete genome sequences and draft genome sequences of the three additional Japanese (sequenced with Illumina MiSeq) and four international ceftriaxone-resistant isolates were compared.Detailed genomic analysis suggested that the Japanese isolates (FC428, FC460, FC498, KU16054, KM383 and KU17039) and the four international MLST ST1903 isolates from Australia, Canada and Denmark formed four linked subclades.Using detailed genomic analysis, we describe the clonal expansion of the ceftriaxone-resistant N. gonorrhoeae strain FC428, initially identified in 2015 in Japan, and closely related isolates. FC428 and its close relatives show some genomic diversity, suggesting multiple genetic subclades are already spreading internationally. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Evolution of Antibiotic Synthesis Gene Clusters in the Streptomyces globisporus TFH56, Isolated from Tomato Flower.

Streptomyces species are known to produce various bioactive metabolites that can prevent plant diseases. Previously, the Streptomyces strain TFH56 was found to inhibit the gray mold pathogen, Botrytis cinerea, in tomato flower. In this study, the genome sequence of strain TFH56 was acquired using the Pacific Biosciences RS II platform. Three linear sequences (7.67 Mbp in total) were obtained. Based on average nucleotide identity, strain TFH56 was classified as Streptomyces globisporus, which is consistent with the presence of a linear chromosome and linear plasmids. Moreover, as with other examples of S. globisporus, the genome of strain TFH56 included a caryolan-1-ol synthase gene, a conprimycin synthetic gene cluster, and a lidamycin synthetic gene cluster.Copyright © 2019 Cho, Kwak.

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April 21, 2020

Characterization of a blaIMP-4-carrying plasmid from Enterobacter cloacae of swine origin.

To characterize an MDR blaIMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China.Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media.Plasmid pIMP-4-EC62 is 314351?bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying blaIMP-4 and a group II intron. The blaIMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids.pIMP-4-EC62 represents the first fully sequenced IncHI2-type blaIMP-4-harbouring plasmid from E. cloacae in China. Co-location of blaIMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of blaIMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Complete genome sequence of Janthinobacterium sp. B9-8, a violacein-producing bacterium isolated from low-temperature sewage.

Janthinobacterium sp. B9-8, isolated from low temperature-sewage in Xinjiang, China, is capable of producing violacein, a promising antibiotic. Here we report the genome sequence of B9-8, which consist of 4,726,850 bp with a G + C content of 48.72%. The violacein biosynthesis gene cluster vioABCDE was identified and analyzed based on the genomic data, which revealed relatively low query coverage (3-44%) and identity (66-87%) with existing strains. Janthinobacterium sp. B9-8 grew fast and reached a high cell density and violacein content within 24?h?at 25?°C. The availability of this genome sequence will greatly benefit the industrial production of violacein and facilitate supplementary studies on the mechanism for violacein biosynthesis. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

WGS of 1058 Enterococcus faecium from Copenhagen, Denmark, reveals rapid clonal expansion of vancomycin-resistant clone ST80 combined with widespread dissemination of a vanA-containing plasmid and acquisition of a heterogeneous accessory genome.

From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized.WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems.Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in =99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ~29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes.Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Analysis of a poxtA- and optrA-co-carrying conjugative multiresistance plasmid from Enterococcus faecalis.

To investigate the presence and transferability of the poxtA gene and identify the genetic context of poxtA in two enterococcal plasmids from swine.MICs were determined by broth microdilution. A total of 114 porcine enterococci with florfenicol MICs of =16?mg/L were screened for the presence of the poxtA gene by PCR. Transferability of poxtA was investigated by conjugation and transformation. The poxtA-carrying plasmids were completely sequenced using the Illumina Miseq and PacBio RSII platform. The presence of circular intermediates was examined by inverse PCR.The poxtA gene was present in 57.9% (66/114) of the florfenicol-resistant porcine enterococci. Two poxtA-carrying plasmids, pE035 and pE076, were identified. The conjugative 121524?bp plasmid pE035 carried poxtA and optrA along with the resistance genes erm(A), erm(B), aac(A)-aph(D), lnu(G), fexB, dfrG and bcrABDR. Three mobile elements, comprising a mobile dfrG locus, a mobile bcrABDR locus and an unconventional circularizable structure containing aac(A)-aph(D), were located on this plasmid and all proved to be active by inverse PCR. The non-conjugative 19832?bp plasmid pE076 only carried poxtA and fexB. After transfer, both the transconjugant and the transformant displayed elevated MICs of the respective antimicrobial agents.To the best of our knowledge, this is the first report of the co-location of the oxazolidinone resistance genes poxtA and optrA on a conjugative multiresistance plasmid from a porcine enterococcal strain. In addition, the presence of three mobile elements in such a plasmid will aid in the persistence and dissemination of poxtA and optrA among enterococci. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Xylella fastidiosa in Olive in Apulia: Where We Stand.

A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecedented turmoil for the local economy and posed critical challenges for its management. With the new emerging threat to susceptible crops in the EU, efforts were devoted to gain basic knowledge on the pathogen biology, host, and environmental interactions (e.g., bacterial strain(s) and pathogenicity, hosts, vector(s), and fundamental drivers of its epidemics) in order to find means to control or mitigate the impacts of the infections. Field surveys, greenhouse tests, and laboratory analyses proved that a single bacterial introduction occurred in the area, with a single genotype, belonging to the subspecies pauca, associated with the epidemic. Infections caused by isolates of this genotype turned to be extremely aggressive on the local olive cultivars, causing a new disease termed olive quick decline syndrome. Due to the initial extension of the foci and the rapid spread of the infections, eradication measures (i.e., pathogen elimination from the area) were soon replaced by containment measures including intense border surveys of the contaminated area, removal of infected trees, and mandatory vector control. However, implementation of containment measures encountered serious difficulties, including public reluctance to accept control measures, poor stakeholder cooperation, misinformation from some media outlets, and lack of robust responses by some governmental authorities. This scenario delayed and limited containment efforts and allowed the bacterium to continue its rapid dissemination over more areas in the region, as shown by the continuous expansion of the official borders of the infected area. At the research level, the European Commission and regional authorities are now supporting several programs aimed to find effective methods to mitigate and contain the impact of X. fastidiosa on olives, the predominant host affected in this epidemic. Preliminary evidence of the presence of resistance in some olive cultivars represents a promising approach currently under investigation for long-term management strategies. The present review describes the current status of the epidemic and major research achievements since 2013.

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April 21, 2020

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

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April 21, 2020

Plastid genomes from diverse glaucophyte genera reveal a largely conserved gene content and limited architectural diversity.

Plastid genome (ptDNA) data of Glaucophyta have been limited for many years to the genus Cyanophora. Here, we sequenced the ptDNAs of Gloeochaete wittrockiana, Cyanoptyche gloeocystis, Glaucocystis incrassata, and Glaucocystis sp. BBH. The reported sequences are the first genome-scale plastid data available for these three poorly studied glaucophyte genera. Although the Glaucophyta plastids appear morphologically “ancestral,” they actually bear derived genomes not radically different from those of red algae or viridiplants. The glaucophyte plastid coding capacity is highly conserved (112 genes shared) and the architecture of the plastid chromosomes is relatively simple. Phylogenomic analyses recovered Glaucophyta as the earliest diverging Archaeplastida lineage, but the position of viridiplants as the first branching group was not rejected by the approximately unbiased test. Pairwise distances estimated from 19 different plastid genes revealed that the highest sequence divergence between glaucophyte genera is frequently higher than distances between species of different classes within red algae or viridiplants. Gene synteny and sequence similarity in the ptDNAs of the two Glaucocystis species analyzed is conserved. However, the ptDNA of Gla. incrassata contains a 7.9-kb insertion not detected in Glaucocystis sp. BBH. The insertion contains ten open reading frames that include four coding regions similar to bacterial serine recombinases (two open reading frames), DNA primases, and peptidoglycan aminohydrolases. These three enzymes, often encoded in bacterial plasmids and bacteriophage genomes, are known to participate in the mobilization and replication of DNA mobile elements. It is therefore plausible that the insertion in Gla. incrassata ptDNA is derived from a DNA mobile element.

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April 21, 2020

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone taxa was the highest under organic farming where agricultural intensification was the lowest. We also found a strong negative association (R2?=?0.366; P?

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April 21, 2020

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

Despite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.We have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.We found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10-7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.Our findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.

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April 21, 2020

Whole-Genome Analysis of Halomonas sp. Soap Lake #7 Reveals It Possesses Putative Mrp Antiporter Operon Groups 1 and 2.

The genus Halomonas possesses bacteria that are halophilic or halotolerant and exhibit a wide range of pH tolerance. The genome of Halomonas sp. Soap Lake #7 was sequenced to provide a better understanding of the mechanisms for salt and pH tolerance in this genus. The bacterium’s genome was found to possess two complete multiple resistance and pH antiporter systems, Group 1 and Group 2. This is the first report of both multiple resistance and pH antiporter Groups 1 and 2 in the genome of a haloalkaliphilic bacterium. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Microbial community and fermentation characteristic of Italian ryegrass silage prepared with corn stover and lactic acid bacteria.

The bacterial community determined via PacBio single molecule, real-time sequencing technology (SMRT) and the fermentation characteristics of Italian ryegrass (IR, 82% moisture) silage prepared with corn stover (CS) were investigated. A selected strain of Lactobacillus plantarum (L694) and a commercial inoculant stain of Lactobacillus plantarum (LP) were used as additives. Lactic acid bacteria (LAB) effectively improved silage quality. After fermentation, Lactobacillus plantarum was the dominant species in IR?+?LP and IR?+?L694 treatments, which led to higher (P?

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