We present single-contig assemblies for Escherichia coli strain KV7 (serotype O27, phylogenetic group D) and its six plasmids, isolated from a healthy pig, as determined by PacBio RS II and Illumina MiSeq sequencing. The chromosome of 4,997,475 bp and G+C content of 50.75% harbored 4,540 protein-encoding genes. Copyright © 2017 Bateman et al.
Shigella flexneri is the primary cause of bacillary dysentery in the developing countries. S. flexneri serotype 1c is a novel serotype, which is found to be endemic in many developing countries, but little is known about its genomic architecture and virulence signatures. We have sequenced for the first time, the complete genome of S. flexneri serotype 1c strain Y394, to provide insights into its diversity and evolution.We generated a high-quality reference genome of S. flexneri serotype 1c using the hybrid methods of long-read single-molecule real-time (SMRT) sequencing technology and short-read MiSeq (Illumina) sequencing technology. The Y394 chromosome is 4.58 Mb in size and shares the basic genomic features with other S. flexneri complete genomes. However, it possesses unique and highly modified O-antigen structure comprising of three distinct O-antigen modifying gene clusters that potentially came from three different bacteriophages. It also possesses a large number of hypothetical unique genes compared to other S. flexneri genomes.Despite a high level of structural and functional similarities of Y394 genome with other S. flexneri genomes, there are marked differences in the pathogenic islands. The diversity in the pathogenic islands suggests that these bacterial pathogens are well adapted to respond to the selection pressures during their evolution, which might contribute to the differences in their virulence potential.
Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype. Copyright © 2017 American Society for Microbiology.
Genetic information about Enterococcus hirae is limited, a feature that has compromised our understanding of these clinically challenging bacteria. In this study, comparative analysis was performed of E. hirae R17, a daptomycin-resistant strain isolated from pork purchased from a retail market in Beijing, China, and three other enterococcal genomes (Enterococcus faecium DO, Enterococcus faecalis V583, and E. hirae ATCC™ 9790). Some 1,412 genes were identified that represented the core genome together with an additional 139 genes that were specific to E. hirae R17. The functions of these R17 strain-specific coding sequences relate to the COGs categories of carbohydrate transport and metabolism and transcription, a finding that suggests the carbohydrate utilization capacity of E. hirae R17 may be more extensive when compared with the other three bacterial species (spp.). Analysis of genomic islands and virulence genes highlighted the potential that horizontal gene transfer played as a contributor of variations in pathogenicity in this isolate. Drug-resistance gene prediction and antibiotic susceptibility testing indicated E. hirae R17 was resistant to several antimicrobial compounds, including bacitracin, ciprofloxacin, daptomycin, erythromycin, and tetracycline, thereby limiting chemotherapeutic treatment options. Further, tolerance to biocides and metals may confer a phenotype that facilitates the survival and adaptation of this isolate against food preservatives, disinfectants, and antibacterial coatings. The genomic plasticity, mediated by IS elements, transposases, and tandem repeats, identified in the E. hirae R17 genome may support adaptation to new environmental niches, such as those that are found in hospitalized patients. A predicted transmissible plasmid, pRZ1, was found to carry several antimicrobial determinants, along with some predicted pathogenic genes. These data supported the previously determined phenotype confirming that the foodborne E. hirae R17 is a multidrug-resistant pathogenic bacterium with evident genome plasticity and environmental adaptability.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins.In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5a and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared.MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones.There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some “epidemic” plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl, and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.© 2017 The Authors.
Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.
In this study, we present the complete genome sequence of a blaOXA-58-producing Acinetobacter baumannii strain, sampled from a Mexican hospital and not related to the international clones. Copyright © 2017 Pérez-Oseguera et al.
Histophilus somni is a fastidious Gram-negative opportunistic pathogenic Pasteurellaceae that affects multiple organ systems and is one of the principal bacterial species contributing to bovine respiratory disease complex (BRDC) in feed yard cattle. Here, we present seven closed genome sequences isolated from three beef calves showing sign of BRDC.
The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) ? (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine ß-naphthylamide (PAßN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
The type III arginine catabolic mobile element (ACME) was detected in three Staphylococcus epidermidis oral isolates recovered from separate patients (one healthy, one healthy with dental implants, and one with periodontal disease) based on ACME-arc-operon- and ACME-opp3-operon-directed PCR. These isolates were subjected to whole-genome sequencing to characterize the precise structural organization of ACME III for the first time, which also revealed that all three isolates were the same sequence type, ST329. Copyright © 2017 McManus et al.
Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Actinobacillus pleuropneumoniae is a bacterial pathogen causing highly contagious porcine pleuropneumonia. Due to limited information on this species, it is difficult to study the biology of A. pleuropneumoniae at the genome level. Here, we report the fully annotated genome sequence of A. pleuropneumoniae strain KL 16. Copyright © 2017 Park et al.
In 2015, plasmid-mediated colistin resistance was reported to be caused by a mobilized phosphoethanolamine transferase gene (mcr-1) in Enterobacteriaceae Here, we announce the complete genome sequence of the earliest d-tartrate-fermenting Salmonella enterica subsp. enterica serovar Paratyphi B isolate harboring mcr-1 from the collection of the German National Reference Laboratory for Salmonella. Copyright © 2017 Borowiak et al.
Carbapenem antibiotics are among the mainstay for treating infections caused by Acinetobacter baumannii, especially in the Northwest United States where carbapenem resistant A. baumannii remain relatively rare. However, between June 2012 and October 2014, an outbreak of carbapenem-resistant A. baumannii occurred in 16 patients from 5 healthcare facilities in the state of Oregon. All isolates were defined as extensively-drug resistant (XDR). MLST revealed that the isolates belonged to sequence type 2 (international clone 2, IC2), and were greater than 95% similar by rep-PCR analysis. Multiplex PCR revealed the presence of a blaOXA carbapenemase gene, later identified as blaOXA-237 Whole genome sequencing of all isolates revealed a well-supported separate branch within a global A. baumannii phylogeny. Pacific Biosciences (PacBio) SMRT sequencing was also performed on one isolate to gain insight into the genetic location of the carbapenem resistance gene. We discovered that blaOXA-237, flanked on either side by ISAba1 elements in opposite orientations, was carried by a 15,198 bp plasmid designated pORAB01-3, and was present in all 16 isolates. The plasmid also contained genes encoding for: a TonB-dependent receptor, septicolysin, a type IV secretory system conjugative DNA transfer family protein, an integrase, a RepB family plasmid DNA replication initiator protein, an a/ß hydrolase, and a BrnT/BrnA type II toxin-antitoxin system. This is the first reported outbreak associated with this specific carbapenemase. Particularly worrisome is that blaOXA-237 was plasmid encoded and found in the most prominent worldwide clonal group IC2, potentially giving pORAB01-3 great capacity for future widespread dissemination. Copyright © 2017 American Society for Microbiology.
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