Plasmid Content Research Articles

High conservation combined with high plasticity: genomics and evolution of Borrelia bavariensis

BackgroundBorrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge.ResultsWe present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia).ConclusionsWe present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents.

Borrelia miyamotoi strain LB-2001 retains plasmids and infectious phenotype throughout continuous culture passages as evaluated by multiplex PCR

Borrelia miyamotoi is a tick-borne spirochete of the relapsing fever borrelia group and an emerging pathogen of public health significance. The genomes of relapsing fever borreliae and Lyme disease borreliae consist of multiple linear and circular plasmids in addition to the chromosome. Previous work with B. burgdorferi sensu lato found diminished infectivity upon continuous in vitro culture passage that was attributable to plasmid loss. The effect of long-term culture passage on B. miyamotoi is not known. We generated a series of plasmid-specific primer sets and developed a multiplex PCR assay to detect the 14 known plasmids of B. miyamotoi North American strains LB-2001 and CT13−2396. We assessed the plasmid content of B. miyamotoi LB-2001 over 64 culture passages spanning 15 months and determined that strain LB-2001 retained all plasmids upon prolonged in vitro cultivation and remained infectious in mice. We also found that strain LB-2001 lacks plasmid lp20−1 which is present in strain CT13−2396. These results suggest that B. miyamotoi remains genetically stable when cultured and passaged in vitro.

Antibiotic Resistance and Mobile Genetic Elements in Extensively Drug-Resistant Klebsiella pneumoniae Sequence Type 147 Recovered from Germany.

Mobile genetic elements (MGEs), especially multidrug-resistance plasmids, are major vehicles for the dissemination of antimicrobial resistance determinants. Herein, we analyse the MGEs in three extensively drug-resistant (XDR) Klebsiella pneumoniae isolates from Germany. Whole genome sequencing (WGS) is performed using Illumina and MinION platforms followed by core-genome multi-locus sequence typing (MLST). The plasmid content is analysed by conjugation, S1-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot experiments. The K. pneumoniae isolates belong to the international high-risk clone ST147 and form a cluster of closely related isolates. They harbour the blaOXA-181 carbapenemase on a ColKP3 plasmid, and 12 antibiotic resistance determinants on an multidrug-resistant (MDR) IncR plasmid with a recombinogenic nature and encoding a large number of insertion elements. The IncR plasmids within the three isolates share a high degree of homology, but present also genetic variations, such as inversion or deletion of genetic regions in close proximity to MGEs. In addition, six plasmids not harbouring any antibiotic resistance determinants are present in each isolate. Our study indicates that genetic variations can be observed within a cluster of closely related isolates, due to the dynamic nature of MGEs. The mobilome of the K. pneumoniae isolates combined with the emergence of the XDR ST147 high-risk clone have the potential to become a major challenge for global healthcare.

Sediment Plasmidome of the Gulfs of Kathiawar Peninsula and Arabian Sea: Insights Gained from Metagenomics Data.

Plasmidomes have become the research area of interest for ecologists exploring bacteria rich ecosystems. Marine environments are among such niche that host a huge number of microbes and have a complex environment which pose the need to study these bacterial indicators of horizontal gene transfer events for survival and stability. The plasmid content of the metagenomics data from 8 sediment samples of the Gulfs of Kathiawar and an open Arabian Sea sample was screened. The reads corresponding to hits against the plasmid database were assembled and studied for diversity using Kraken and functional content using MG-RAST. The sequences were also checked for resistome and virulence factors. The replicon hosts were overall dominated by Proteobacteria, Firmicutes, and Actinobacteria while red algae specific to the Kutch samples. The genes encoded were dominant in the flagella motility and type VI secretion systems. Overall, results from the study confirmed that the plasmids encoded traits for metal, antibiotic, and phage resistance along with virulence systems, and these would be conferring benefit to the hosts. The study throws insights into the environmental role of the plasmidome in adaptation of the microbes in the studied sites to the environmental stresses.

Characterization of 16S rRNA methylase genes in Enterobacterales and Pseudomonas aeruginosa in Athens Metropolitan area, 2015-2016.

The aim of this study was to characterize the 16S rRNA methylase (RMT) genes in aminoglycoside-resistant Enterobacterales and Pseudomonas aeruginosa isolates in 2015-2016 in hospitals in Athens, Greece. Single-patient, Gram-negative clinical isolates resistant to both amikacin and gentamicin (n = 292) were consecutively collected during a two-year period (2015-2016) in five tertiary care hospitals in Athens. RMT genes were detected by PCR. In all RMT-producing isolates, ESBL and carbapenemase production was confirmed by PCR, and the clonal relatedness and the plasmid contents were also characterized. None of the 138 P. aeruginosa isolates harbored any of the RMT genes surveyed although some were highly resistant to aminoglycosides (MICs > = 512 mg/L). Among 154 Enterobacterales, 31 Providencia stuartii (93.9%), 42 Klebsiella pneumoniae (37.8%), six Proteus mirabilis (75%), and two Escherichia coli (100%) isolates were confirmed as highly resistant to amikacin, gentamicin, and tobramycin with MICs ≥ 512 mg/L, harboring mainly the rmtB (98.8%). All were carbapenemase producers. P. stuartii, P. mirabilis, and E. coli produced VIM-type carbapenemases. K. pneumoniae produced KPC- (n = 34, 81.0%), OXA-48 (n = 4, 9.5%), KPC- and VIM- (n = 3, 7.1%), or only VIM-type (n = 1, 2.4%) enzymes. Two groups of similar IncC plasmids were detected one harboring rmtB1, blaVEB-1, blaOXA-10, and blaTEM-1, and the other additionally blaVIM-1 and blaSHV-5. Among RMT-producing Enterobacterales, rmtB1 predominated and was associated with carbapenemase-encoding gene(s). Similar IncC plasmids carrying a multiresistant region, including ESBL genes, and in the case of VIM-producing isolates, the blaVIM-1, were responsible for this dissemination. The co-dissemination of these genes poses a public health threat.

Genotypic and phenotypic traits of blaCTX-M-carrying Escherichia coli strains from an UV-C-treated wastewater effluent

Wastewater treatment plants (WWTPs) are relevant sources of antibiotic resistance into aquatic environments. Disinfection of WWTPs’ effluents (e.g. by UV-C irradiation) may attenuate this problem, though some clinically relevant bacteria have been shown to survive disinfection. In this study we characterized 25 CTX-M-producing Escherichia coli strains isolated from a WWTP’s UV-C-irradiated effluent, aiming to identify putative human health hazards associated with such effluents. Molecular typing indicated that the strains belong to the phylogroups A, B2 and C and clustered into 9 multilocus sequence types (STs), namely B2:ST131 (n = 7), A:ST58 (n = 1), A:ST155 (n = 4), C:ST410 (n = 2), A:ST453 (n = 2), A:ST617 (n = 2), A:ST744 (n = 1), A:ST1284 (n = 3) and a putative novel ST (n = 3). PCR-screening identified 9 of the 20 antibiotic resistance genes investigated [i.e. sul1, sul2, sul3, tet(A), tet(B), blaOXA-1-like, aacA4, aacA4-cr and qnrS1]. The more prevalent were sul1, sul2 (n = 15 isolates) and tet(A) (n = 14 isolates). Plasmid restriction analysis indicated diverse plasmid content among strains (14 distinct profiles) and mating assays yielded cefotaxime-resistant transconjugants for 8 strains. Two of the transconjugants displayed a multi-drug resistance (MDR) phenotype. All strains were classified as cytotoxic to Vero cells (9 significantly more cytotoxic than the positive control) and 10 of 21 strains were invasive towards this cell line (including all B2:ST131 strains). The 10 strains tested against G. mellonella larvae exhibited a virulent behaviour. Twenty-four and 7 of the 25 strains produced siderophores and haemolysins, respectively. Approximately 66% of the strains formed biofilms. Genome analysis of 6 selected strains identified several virulence genes encoding toxins, siderophores, and colonizing, adhesion and invasion factors. Freshwater microcosms assays showed that after 28 days of incubation 3 out of 6 strains were still detected by cultivation and 4 strains by qPCR. Resistance phenotypes of these strains remained unaltered. Overall, we confirmed WWTP’s UV-C-treated outflow as a source of MDR and/or virulent E. coli strains, some probably capable of persisting in freshwater, and that carry conjugative antibiotic resistance plasmids. Hence, disinfected wastewater may still represent a risk for human health. More detailed evaluation of strains isolated from wastewater effluents is urgent, to design treatments that can mitigate the release of such bacteria.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link.

poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996-2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30-100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

Genetic Diversity of Xylella fastidiosa Plasmids Assessed by Comparative Genomics

Plasmids are a major source of horizontal gene transfer among bacteria contributing to their evolution and ecology. The known collection of plasmids carried by the plant pathogen Xylella fastidiosa increases as new strains from different origins and hosts are sampled and sequenced. Here we performed an extensive comparison of 61 publicly available sequences annotated as plasmids belonging to 38 X. fastidiosa strains isolated from different plant species and distinct geographical locations. Twenty-six strains exhibited at least one plasmid and up to four plasmids were found in a single strain. Plasmids sizes varied widely from 1.3 kbp to 64.3 kbp, ranging from 1 to 70 protein-coding sequences (CDS) encompassing 324 orthologs. Based on the presence of specific mobility proteins such as relaxases and type 4 secretion system-related genes, respectively 40 and 8 of the X. fastidiosa plasmids were classified as conjugative and mobilizable, while 13 were classified as non-mobilizable. X. fastidiosa plasmids did not carry known antibiotic resistance or virulence genes, and their stability seems to take advantage of toxin/antitoxin systems. The comparative analyses described here revealed similarity among plasmids of X. fastidiosa from different subspecies, geographical regions, and hosts, as well as with sequences found in plasmids from other bacterial species. Altogether, our results provide an in silico analysis of X. fastidiosa plasmid content and their main features, with applications in future studies of epidemiology, ecology, and evolution of this phytopathogen.

Plasmid Replicon Typing of Antibiotic-Resistant Escherichia coli From Clams and Marine Sediments.

Unlike human isolates, environmental Escherichia coli isolates have not been thoroughly investigated for the diversity and transferability of antibiotic-resistant plasmids. In this study, antibiotic-resistant strains from marine sediment (n = 50) and clams (n = 53) were analyzed (i) for their plasmid content using a PCR-based plasmid replicon typing (PBRT) kit and (ii) for the transferability of plasmid-associated antibiotic resistance (AR) traits by mating experiments. Fifteen of the thirty replicons targeted by the PBRT kit were detected in the isolates; 8/15 were identified in both sediment and clam isolates, although at different frequencies. The most frequent replicons in sediment (74%) and in clam strains (66%) alike, were FIA, FIB, or FII, which are associated with the IncF group, followed by the I1α replicon, which was more frequent in clam (24.5%) than in sediment (10%) strains. More than 50% of the strains contained multiple replicons; although 15 were untypable, S1-PFGE analysis demonstrated that 14/15 carried no plasmids. All cryptic strains were successfully typed and were positive for IncF or IncI replicons. Antibiotic-resistant strains accounted for 63% of all isolates and were significantly (p < 0.05) more frequent in phylogroup A. Most (35%) multidrug-resistant (MDR) strains belonged to phylogroup A, too. Although 25/26 MDR strains were positive for IncF plasmids (the exception being a clam strain), the FII-FIB rep combination was predominant (63%) among the sediment isolates, whereas most clam isolates (40%) carried the FII replicon alone. In mating experiments, selected MDR strains carrying FIB, FII, and I1α replicons, used as the donors, transferred multiple ARs together with the IncF or IncI plasmids at high frequency. Since IncI plasmids are common in E. coli and Salmonella enterica isolates from poultry, our findings suggest an animal origin to the E. coli clam strains carrying IncI plasmids. They also suggest a role for IncI plasmids in the spread of ARs among environmental Enterobacteriaceae and, through the food chain, to human isolates. In conclusion, the PBRT kit proved to be a useful tool to identify plasmids carrying antibiotic-resistant genes and to shed light on the factors underpinning their diffusion.

Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments.

Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38 clusters of attC sites lacking integron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.

A Retrospective Whole-Genome Sequencing Analysis of Carbapenem and Colistin-Resistant Klebsiella Pneumoniae Nosocomial Strains Isolated during an MDR Surveillance Program.

Multidrug-resistant Klebsiella pneumoniae (MDR Kp), in particular carbapenem-resistant Kp (CR-Kp), has become endemic in Italy, where alarming data have been reported on the spread of colistin-resistant CR-Kp (CRCR-Kp). During the period 2013–2014, 27 CRCR-Kp nosocomial strains were isolated within the Modena University Hospital Policlinico (MUHP) multidrug resistance surveillance program. We retrospectively investigated these isolates by whole-genome sequencing (WGS) analysis of the resistome, virulome, plasmid content, and core single nucleotide polymorphisms (cSNPs) in order to gain insights into their molecular epidemiology. The in silico WGS analysis of the resistome revealed the presence of genes, such as blaKPC, related to the phenotypically detected resistances to carbapenems. Concerning colistin resistance, the plasmidic genes mcr 1–9 were not detected, while known and new genetic variations in mgrB, phoQ, and pmrB were found. The virulome profile revealed the presence of type-3 fimbriae, capsular polysaccharide, and iron acquisition system genes. The detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX3, and IncFII(K) types. The cSNPs genotyping was consistent with the multi locus sequence typing (MLST) and with the distribution of mutations related to colistin resistance genes. In a nosocomial drug resistance surveillance program, WGS proved to be a useful tool for elucidating the spread dynamics of CRCR-Kp nosocomial strains and could help to limit their diffusion.

Metal-induced bacterial interactions promote diversity in river-sediment microbiomes.

Anthropogenic metal contamination results in long-term environmental selective pressure with unclear impacts on bacterial communities, which comprise key players in ecosystem functioning. Since metal contamination poses serious toxicity and bioaccumulation issues, assessing their impact on environmental microbiomes is important to respond to current environmental and health issues. Despite elevated metal concentrations, the river sedimentary microbiome near the MetalEurop foundry (France) shows unexpected higher diversity compared with the upstream control site. In this work, a follow-up of the microbial community assembly during a metal contamination event was performed in microcosms with periodic renewal of the supernatant river water. Sediments of the control site were gradually exposed to a mixture of metals (Cd, Cu, Pb and Zn) in order to reach similar concentrations to MetalEurop sediments. Illumina sequencing of 16S rRNA gene amplicons was performed. Metal-resistant genes, czcA and pbrA, as well as IncP plasmid content, were assessed by quantitative PCR. The outcomes of this study support previous in situ observations showing that metals act as community assembly managers, increasing diversity. This work revealed progressive adaptation of the sediment microbiome through the selection of different metal-resistant mechanisms and cross-species interactions involving public good-providing bacteria co-occurring with the rest of the community.

Comparison of Commensal and Clinical Isolates for Diversity of Plasmids in Escherichia coli and Klebsiella pneumoniae.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

Whole genome sequencing characterization of Slovenian carbapenem-resistant Klebsiella pneumoniae, including OXA-48 and NDM-1 producing outbreak isolates.

The first hospital outbreak of carbapenemase-producing Enterobacteriaceae in Slovenia occurred in 2014-2016. Whole genome sequencing was used to analyse the population of carbapenem-resistant Klebsiella pneumoniae collected in Slovenia in 2014-2017, including OXA-48 and/or NDM-1 producing strains from the outbreak. A total of 32 K. pneumoniae isolates were analysed using short-read sequencing. Multi-locus sequence typing and core genome multi-locus sequence typing were used to infer genetic relatedness. Antimicrobial resistance markers, virulence factors, plasmid content and wzi types were determined. Long-read sequencing was used for six isolates for detailed analysis of plasmids and their possible transmission. Overall, we detected 10 different sequence types (STs), the most common being ST437 (40.6%). Isolates from the initial outbreak belonged to ST437 (12/16) and ST147 (4/16). A second outbreak of four ST15 isolates was discovered. A new ST (ST3390) and two new wzi types (wzi-556, wzi-559) were identified. blaOXA-48 was found in 17 (53.1%) isolates, blaNDM-1 in five (15.6%), and a combination of blaOXA-48/NDM-1 in seven (21.9%) isolates. Identical plasmids carrying blaOXA-48 were found in outbreak isolates sequenced with long-read sequencing technology. Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbapenem producing K. pneumoniae in Slovenia. Transmission of the plasmid carrying blaOXA-48 between two STs was likely to have occurred. A previously unidentified second outbreak was also discovered, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones.

Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:- variants from clinical isolates.

To investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:-. A total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014-19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms. We identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles. Our findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates.

Characterization of the diverse plasmid pool harbored by the blaNDM-1-containing Acinetobacter bereziniae HPC229 clinical strain.

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Genomic Characterization of Prevalent mcr-1, mcr-4, and mcr-5 Escherichia coli Within Swine Enteric Colibacillosis in Spain.

Antimicrobial agents are crucial for the treatment of many bacterial diseases in pigs, however, the massive use of critically important antibiotics such as colistin, fluoroquinolones and 3rd–4th-generation cephalosporins often selects for co-resistance. Based on a comprehensive characterization of 35 colistin-resistant Escherichia coli from swine enteric colibacillosis, belonging to prevalent Spanish lineages, the aims of the present study were to investigate the characteristics of E. coli clones successfully spread in swine and to assess the correlation of the in vitro results with in silico predictions from WGS data. The resistome analysis showed six different mcr variants: mcr-1.1; mcr-1.10; mcr-4.1; mcr-4.2; mcr-4.5; and mcr-5.1. Additionally, blaCTX–M–14, blaCTX–M–32 and blaSHV–12 genes were present in seven genomes. PlasmidFinder revealed that mcr-1.1 genes located mainly on IncHI2 and IncX4 types, and mcr-4 on ColE10-like plasmids. Twenty-eight genomes showed a gyrA S83L substitution, and 12 of those 28 harbored double-serine mutations gyrA S83L and parC S80I, correlating with in vitro quinolone-resistances. Notably, 16 of the 35 mcr-bearing genomes showed mutations in the PmrA (S39I) and PmrB (V161G) proteins. The summative presence of mechanisms, associated with high-level of resistance to quinolones/fluoroquinolones and colistin, could be conferring adaptive advantages to prevalent pig E. coli lineages, such as the ST10-A (CH11-24), as presumed for ST131. SerotypeFinder allowed the H-antigen identification of in vitro non-mobile (HNM) isolates, revealing that 15 of the 21 HNM E. coli analyzed were H39. Since the H39 is associated with the most prevalent O antigens worldwide within swine colibacillosis, such as O108 and O157, it would be probably playing a role in porcine colibacillosis to be considered as a valuable subunit antigen in the formulation of a broadly protective Enterotoxigenic E. coli (ETEC) vaccine. Our data show common features with other European countries in relation to a prevalent clonal group (CC10), serotypes (O108:H39, O138:H10, O139:H1, O141:H4), high plasmid content within the isolates and mcr location, which would support global alternatives to the use of antibiotics in pigs. Here, we report for first time a rare finding so far, which is the co-occurrence of double colistin-resistance mechanisms in a significant number of E. coli isolates.

246. Carbapenem-resistant Klebsiella (CRK) Bloodstream Infections (BSIs) Are Caused by Bacterial Populations That Are Genotypically and Phenotypically Diverse

BackgroundThe majority of bacterial BSIs are believed to stem from a single, clonal organism. We hypothesized that most CRK BSIs are caused by genetically diverse, clonal strains that exhibit different phenotypes.MethodsBlood cultures (BCs) that were positive for CRK from each of 10 patients (patients) were streaked onto agar plates, and 100 individual colonies were chosen for Illumina whole-genome sequencing. Strains from 3 patients were tested for in vitro phenotypes and virulence in mice.ResultsPatients had BSIs due to ST258 K. pneumoniae (Kp; 6), non-ST258 Kp (3), and K. michiganensis (Km; 1). 5 patients were infected with strains that differed by core genome single nucleotide polymorphism phylogeny (2–5 unique genotypes/patient) [figure]. 6 patients were infected with strains that differed by gene or plasmid content, and/or gene deletions [table]. Differences in individual patients encompassed antibiotic resistance and putative virulence genes (including mixtures of blaKPC+ and blaKPC– strains, and various capsular (CPS) and porin mutant strains). In total, BSIs in 8 of 10 patients were caused by a genotypically diverse population. In each of 3 patients, genotypically diverse ST258 Kp strains demonstrated significant differences in antibiotic susceptibility, CPS content, mucoviscosity, adherence, resistance to serum killing, and mortality rates and tissue burdens during BSIs of mice. ST258 strains from a pt with and without a KPC-bearing IncFIA plasmid differed in β-lactam susceptibility, but were equally virulent. Progressive loss of CPS in ST258 strains from another patient enhanced serum killing and adherence, and attenuated virulence. Using PCR markers to test 96 colonies per positive BC bottle, we demonstrated that strains selected by the clinical micro lab accounted for 2% to 98% of a population in different patients.ConclusionCRK causing BSIs in most patients demonstrated remarkable genotypic diversity, which impacted antibiotic susceptibility, virulence and other phenotypes. Differences were not recognized during hospitalization since clinical labs select single, morphologically distinct colonies for evaluation. Studies are needed to understand the clinical implications of our findings, diversity during other BSIs, and whether clinical lab practices need revision. Disclosures All authors: No reported disclosures.