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Asset Tag: Bacterial genome

July 19, 2019

Comprehensive genome analysis of carbapenemase-producing Enterobacter spp.: new insights into phylogeny, population structure and resistance mechanisms.

Knowledge regarding the genomic structure of Enterobacter spp., the second most prevalent carbapenemase-producing Enterobacteriaceae, remains limited. Here we sequenced 97 clinical Enterobacter species isolates that were both carbapenem susceptible and resistant from various geographic regions to decipher the molecular origins of carbapenem resistance and to understand the changing phylogeny of these emerging and drug-resistant pathogens. Of the carbapenem-resistant isolates, 30 possessed blaKPC-2, 40 had blaKPC-3, 2 had blaKPC-4, and 2 had blaNDM-1 Twenty-three isolates were carbapenem susceptible. Six genomes were sequenced to completion, and their sizes ranged from 4.6 to 5.1 Mbp. Phylogenomic analysis placed 96 of these genomes, 351 additional Enterobacter genomes downloaded from NCBI GenBank, and six newly sequenced type strains into 19 phylogenomic groups-18 groups (A to R) in the Enterobacter cloacae complex and Enterobacter aerogenes Diverse mechanisms underlying the molecular evolutionary trajectory of these drug-resistant Enterobacter spp. were revealed, including the acquisition of an antibiotic resistance plasmid, followed by clonal spread, horizontal transfer of blaKPC-harboring plasmids between different phylogenomic groups, and repeated transposition of the blaKPC gene among different plasmid backbones. Group A, which comprises multilocus sequence type 171 (ST171), was the most commonly identified (23% of isolates). Genomic analysis showed that ST171 isolates evolved from a common ancestor and formed two different major clusters; each acquiring unique blaKPC-harboring plasmids, followed by clonal expansion. The data presented here represent the first comprehensive study of phylogenomic interrogation and the relationship between antibiotic resistance and plasmid discrimination among carbapenem-resistant Enterobacter spp., demonstrating the genetic diversity and complexity of the molecular mechanisms driving antibiotic resistance in this genus.Enterobacter spp., especially carbapenemase-producing Enterobacter spp., have emerged as a clinically significant cause of nosocomial infections. However, only limited information is available on the distribution of carbapenem resistance across this genus. Augmenting this problem is an erroneous identification of Enterobacter strains because of ambiguous typing methods and imprecise taxonomy. In this study, we used a whole-genome-based comparative phylogenetic approach to (i) revisit and redefine the genus Enterobacter and (ii) unravel the emergence and evolution of the Klebsiella pneumoniae carbapenemase-harboring Enterobacter spp. Using genomic analysis of 447 sequenced strains, we developed an improved understanding of the species designations within this complex genus and identified the diverse mechanisms driving the molecular evolution of carbapenem resistance. The findings in this study provide a solid genomic framework that will serve as an important resource in the future development of molecular diagnostics and in supporting drug discovery programs. Copyright © 2016 Chavda et al.

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July 19, 2019

Chromosomal integration of the Klebsiella pneumoniae carbapenemase gene, blaKPC, in Klebsiella species is elusive but not rare.

Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-?I-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated. Copyright © 2017 Mathers et al.

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July 19, 2019

The history of Bordetella pertussis genome evolution includes structural rearrangement.

Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology.IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen’s evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Comparative genomics reveals the diversity of restriction-modification systems and DNA methylation sites in Listeria monocytogenes.

Listeria monocytogenes is a bacterial pathogen that is found in a wide variety of anthropogenic and natural environments. Genome sequencing technologies are rapidly becoming a powerful tool in facilitating our understanding of how genotype, classification phenotypes, and virulence phenotypes interact to predict the health risks of individual bacterial isolates. Currently, 57 closed L. monocytogenes genomes are publicly available, representing three of the four phylogenetic lineages, and they suggest that L. monocytogenes has high genomic synteny. This study contributes an additional 15 closed L. monocytogenes genomes that were used to determine the associations between the genome and methylome with host invasion magnitude. In contrast to previous findings, large chromosomal inversions and rearrangements were detected in five isolates at the chromosome terminus and within rRNA genes, including a previously undescribed inversion within rRNA-encoding regions. Each isolate’s epigenome contained highly diverse methyltransferase recognition sites, even within the same serotype and methylation pattern. Eleven strains contained a single chromosomally encoded methyltransferase, one strain contained two methylation systems (one system on a plasmid), and three strains exhibited no methylation, despite the occurrence of methyltransferase genes. In three isolates a new, unknown DNA modification was observed in addition to diverse methylation patterns, accompanied by a novel methylation system. Neither chromosome rearrangement nor strain-specific patterns of epigenome modification observed within virulence genes were correlated with serotype designation, clonal complex, or in vitro infectivity. These data suggest that genome diversity is larger than previously considered in L. monocytogenes and that as more genomes are sequenced, additional structure and methylation novelty will be observed in this organism.Listeria monocytogenes is the causative agent of listeriosis, a disease which manifests as gastroenteritis, meningoencephalitis, and abortion. Among Salmonella, Escherichia coli, Campylobacter, and Listeria-causing the most prevalent foodborne illnesses-infection by L. monocytogenes carries the highest mortality rate. The ability of L. monocytogenes to regulate its response to various harsh environments enables its persistence and transmission. Small-scale comparisons of L. monocytogenes focusing solely on genome contents reveal a highly syntenic genome yet fail to address the observed diversity in phenotypic regulation. This study provides a large-scale comparison of 302 L. monocytogenes isolates, revealing the importance of the epigenome and restriction-modification systems as major determinants of L. monocytogenes phylogenetic grouping and subsequent phenotypic expression. Further examination of virulence genes of select outbreak strains reveals an unprecedented diversity in methylation statuses despite high degrees of genome conservation. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Single-molecule sequencing (PacBio) of the Staphylococcus capitis NRCS-A clone reveals the basis of multidrug resistance and adaptation to the Neonatal Intensive Care Unit environment.

The multi-resistant Staphylococcus capitis clone NRCS-A has recently been described as a major pathogen causing nosocomial, late-onset sepsis (LOS) in preterm neonates worldwide. NRCS-A representatives exhibit an atypical antibiotic resistance profile. Here, the complete closed genome (chromosomal and plasmid sequences) of NRCS-A prototype strain CR01 and the draft genomes of three other clinical NRCS-A strains from Australia, Belgium and the United Kingdom are annotated and compared to available non-NRCS-A S. capitis genomes. Our goal was to delineate the uniqueness of the NRCS-A clone with respect to antibiotic resistance, virulence factors and mobile genetic elements. We identified 6 antimicrobial resistance genes, all carried by mobile genetic elements. Previously described virulence genes present in the NRCS-A genomes are shared with the six non-NRCS-A S. capitis genomes. Overall, 63 genes are specific to the NRCS-A lineage, including 28 genes located in the methicillin-resistance cassette SCCmec. Among the 35 remaining genes, 25 are of unknown function, and 9 correspond to an additional type I restriction modification system (n = 3), a cytosine methylation operon (n = 2), and a cluster of genes related to the biosynthesis of teichoic acids (n = 4). Interestingly, a tenth gene corresponds to a resistance determinant for nisin (nsr gene), a bacteriocin secreted by potential NRCS-A strain niche competitors in the gut microbiota. The genomic characteristics presented here emphasize the contribution of mobile genetic elements to the emergence of multidrug resistance in the S. capitis NRCS-A clone. No NRCS-A-specific known virulence determinant was detected, which does not support a role for virulence as a driving force of NRCS-A emergence in NICUs worldwide. However, the presence of a nisin resistance determinant on the NRCS-A chromosome, but not in other S. capitis strains and most coagulase-negative representatives, might confer a competitive advantage to NRCS-A strains during the early steps of gut colonization in neonates. This suggests that the striking adaptation of NRCS-A to the NICU environment might be related to its specific antimicrobial resistance and also to a possible enhanced ability to challenge competing bacteria in its ecological niche.

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July 19, 2019

Complete genome sequences of isolates of Enterococcus faecium sequence type 117, a globally disseminated multidrug-resistant clone.

The emergence of nosocomial infections by multidrug-resistant sequence type 117 (ST117) Enterococcus faecium has been reported in several European countries. ST117 has been detected in Spanish hospitals as one of the main causes of bloodstream infections. We analyzed genome variations of ST117 strains isolated in Madrid and describe the first ST117 closed genome sequences. Copyright © 2017 Tedim et al.

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July 19, 2019

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.

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July 19, 2019

Complete genome sequence of Tessaracoccus sp. strain T2.5-30 isolated from 139.5 meters deep on the subsurface of the Iberian Pyritic Belt.

Here, we report the complete genome sequence of Tessaracoccus sp. strain T2.5-30, which consists of a chromosome with 3.2 Mbp, 70.4% G+C content, and 3,005 coding DNA sequences. The strain was isolated from a rock core retrieved at a depth of 139.5 m in the subsurface of the Iberian Pyritic Belt (Spain). Copyright © 2017 Leandro et al.

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July 19, 2019

Complete genome sequence of Vibrio campbellii strain 20130629003S01 isolated from shrimp with acute hepatopancreatic necrosis disease.

Vibrio campbellii is widely distributed in the marine environment and is an important pathogen of aquatic organisms such as shrimp, fish, and mollusks. An isolate of V. campbellii carrying the pirAB(vp) gene, causing acute hepatopancreatic necrosis disease (AHPND), has been reported. There are no previous reports about the complete genome of V. campbellii causing AHPND (VCAHPND). To extend our understanding of the pathogenesis of VCAHPND at the genomic level, the genome of V. campbellii 20130629003S01 isolated from a shrimp with AHPND was sequenced and analysed.The complete genome sequence of V. campbellii 20130629003S01 was generated using the PacBio RSII platform with single molecule, real-time sequencing. The 20130629003S01 strain consists of two circular chromosomes (3,621,712 bp in chromosome 1 and 2,245,751 bp in chromosome 2) and four plasmids of 70,066, 204,531, 143,140, and 86,121 bp. The genome contains a total of 5855 protein coding genes, 134 tRNA genes and 37 rRNA genes. The average nucleotide identity value of 20130629003S01 and other reference V. campbellii strains was 97.46%, suggesting that they are closely related.The genome sequence of V. campbellii 20130629003S01 and its comparative analysis with other V. campbellii strains that we present here are important for a better understanding of the genomic characteristics of VCAHPND.

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July 19, 2019

Complete genome sequence of undomesticated Bacillus subtilis strain NCIB 3610.

Bacillus subtilis is a Gram-positive bacterium that serves as an important experimental system. B. subtilis NCIB 3610 is an undomesticated strain that exhibits phenotypes lost from the more common domesticated laboratory strains. Here, we announce the complete genome sequence of DK1042, a genetically competent derivative of NCIB 3610. Copyright © 2017 Nye et al.

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July 19, 2019

Comparative analysis of extended-spectrum-ß-lactamase CTX-M-65-producing Salmonella enterica serovar Infantis isolates from humans, food animals, and retail chickens in the United States.

We sequenced the genomes of ten Salmonella enterica serovar Infantis containing blaCTX-M-65 isolated from chicken, cattle, and human sources collected between 2012 and 2015 in the United States through routine NARMS surveillance and product sampling programs. We also completely assembled the plasmids from four of the isolates. All isolates had a D87Y mutation in the gyrA gene and harbored between 7 and 10 resistance genes (aph (4)-Ia, aac (3)-IVa, aph(3′ )-Ic, blaCTX-M-65, fosA3, floR, dfrA14, sul1, tetA, aadA1) located in two distinct sites of a megaplasmid (~316-323kb) similar to that described in a blaCTX-M-65-positive S. Infantis isolated from a patient in Italy. High-quality single nucleotide polymorphism (hqSNP) analysis revealed that all U.S. isolates were closely related, separated by only 1 to 38 pairwise high quality SNPs, indicating a high likelihood that strains from humans, chicken, and cattle recently evolved from a common ancestor. The U.S. isolates were genetically similar to the blaCTX-M-65-positive S. Infantis isolate from Italy, with a separation of 34 to 47 SNPs. This is the first report of the blaCTX-M-65 gene and the pESI-like megaplasmid from S. Infantis in the United States, and illustrates the importance of applying a global One Health, human and animal perspective to combat antimicrobial resistance. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production.In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon.Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

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July 19, 2019

Characterization of a large antibiotic resistance plasmid found in enteropathogenic Escherichia coli strain B171 and its relatedness to plasmids of diverse E. coli and Shigella.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline (tetA), sulfonamides (sulI), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor (csi). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 (csi and traI) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10?kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n?=?21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (=9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

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