Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches / Los plásmidos del grupo de incompatibilidad FIBK se encuentran en Klebsiella variicola en diversos nichos ecológicos

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.(AU)

IncHI1 plasmids mediated the tet(X4) gene spread in Enterobacteriaceae in porcine.

The tigecycline resistance gene tet(X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet(X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet(X4) gene spread among bacteria in a pig farm. The tet(X4) positive Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 (n = 11) and ST3830 (n = 1), E. cloacae and E. hormaeche belonged to ST524 (n = 1) and ST1862 (n = 1). All tet(X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by ISCR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet(X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet(X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet(X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches.

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.

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.

Mega-plasmid found worldwide confers multiple antimicrobial resistance in Salmonella Infantis of broiler origin in Russia.

Plasmids which are the mobile part of the bacterial genome can acquire and carry over genes conferring antimicrobial resistance, thus contributing to rapid adaptation of bacterial community to human-defined environment. In 2014, Israeli scientists have reported a large conjugative mega-plasmid pESI (plasmid for emerging S. Infantis) that provides multiple drug resistance (MDR) of Salmonella Infantis isolated from broilers. Later, very similar pESI-like plasmids have been found in Salmonella isolated from poultry in the United States, Italy, Switzerland, Hungary, and Japan. Here we report detection of pESI-like plasmids in Salmonella Infantis isolated from chicken food products in Russia. Whole genome sequencing of three MDR isolates revealed pESI-like plasmids in all three cases. These plasmids have such typical pESI features as a locus for siderophore yersiniabactin, a cluster of IncI1 conjugative genes, a cluster of type IV pilus genes, and three toxin-antitoxin modules. The pESI-like plasmids carry from two to five resistance genes in each isolate. In total, we observed six antimicrobial resistance genes associated with pESI-like plasmids (aadA1, blaCTX-M-14, dfrA14, sul1, tetA/tetR, tetM). Besides plasmid genes of antimicrobial resistance, all three MDR isolates of S. Infantis harbor a mutation in chromosomal gene gyrA (p.S83Y or p.D87Y) that is associated with resistance to fluoroquinolones. In addition, we performed a comparative bioinformatics meta-analysis of 25 pESI-like plasmids hosted by S. Infantis from the USA, Europe, Latin America, Israel, and Japan. This analysis identified a 173 kB sequence that is common for all pESI-like plasmids and carries virulence operons and toxin-antitoxin modules.

Plasmid Typing and Classification.

Plasmids identification and classification is an essential parameter in current bacterial typing. The most widely used PCR-based methods are the PCR-based replicon typing (PBRT) and the degenerate primer MOB typing (DPMT). PBRT targets the replicons on the plasmids and DPMT targets the relaxase genes. A finer resolution of phylogenetic relatedness can be obtained by plasmid multiLocus sequence typing available for the major plasmid types occurring in Enterobacteriaceae.

ORF-based binarized structure network analysis of plasmids (OSNAp), a novel approach to core gene-independent plasmid phylogeny.

OBJECTIVES: Systematic comparison of multiple plasmids remains challenging. We aimed to develop a new method for phylogenetic analysis of plasmids, open reading frame (ORF)-based binarized structure network analysis of plasmids (OSNAp). METHODS: With the OSNAp, the genetic structures of plasmids in a given plasmid group are expressed as binary sequences based on the presence or absence of ORFs regardless of their positions or directions. As a proof-of-concept, ORFs were collected from 101 complete I1 plasmid sequences, and their corresponding binary sequences were generated. A tree was generated using the neighbor-net, an algorithm for constructing phylogenetic networks based on distance between taxa, to visualize the plasmid phylogeny drawn from binary sequences. The results were compared with those of plasmid sequence types (pSTs) defined by plasmid multilocus sequence typing (pMLST). RESULTS: All I1 plasmids were placed on the phylogenetic tree constructed from the binary sequences. Most plasmids belonging to the same pSTs had Dice indices of ≥0.95 and were placed in the same OSNAp split. On the other hand, pST12 plasmids were distributed on separate splits due to differences in ORFs not used in pMLST, suggesting improved differentiation of the plasmids with OSNAp compared with pMLST. CONCLUSION: OSNAp is a novel holistic approach to assess relatedness of a population of plasmids in a given plasmid group based on nucleotide sequence data. It provides higher discrimination than pMLST, which may prove useful in tracing bacteria that harbor plasmids of shared origins.

Evaluation of Incompatibility Group I1 (IncI1) Plasmid-Containing Salmonella enterica and Assessment of the Plasmids in Bacteriocin Production and Biofilm Development.

Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.

Genomic Characterization of Extended-Spectrum Cephalosporin-Resistant Salmonella enterica in the Colombian Poultry Chain.

Salmonella enterica serovars have been isolated from Colombian broilers and broiler meat. The aim of this study was to investigate the diversity of ESBL/pAmpC genes in extended-spectrum cephalosporin resistant Salmonella enterica and the phylogeny of ESBL/pAmpC-carrying Salmonella using Whole Genome Sequencing (WGS). A total of 260 cefotaxime resistant Salmonella isolates, obtained between 2008 and 2013 from broiler farms, slaughterhouses and retail, were included. Isolates were screened by PCR for ESBL/pAmpC genes. Gene and plasmid subtyping and strain Multi Locus Sequence Typing was performed in silico for a selection of fully sequenced isolates. Core-genome-based analyses were performed per ST encountered. bla CMY-2-like was carried in 168 isolates, 52 carried bla CTX-M-2 group, 7 bla SHV, 5 a combination of bla CMY-2-like-bla SHV and 3 a combination of bla CMY-2-like-bla CTX-M-2 group. In 25 isolates no ESBL/pAmpC genes that were screened for were found. WGS characterization of 36 selected strains showed plasmid-encoded bla CMY-2 in 21, bla CTX-M-165 in 11 and bla SHV-12 in 7 strains. These genes were mostly carried on IncI1/ST12, IncQ1, and IncI1/ST231 plasmids, respectively. Finally, 17 strains belonged to S. Heidelberg ST15, 16 to S. Paratyphi B variant Java ST28, 1 to S. Enteritidis ST11, 1 to S. Kentucky ST152 and 1 to S. Albany ST292. Phylogenetic comparisons with publicly available genomes showed separate clustering of Colombian S. Heidelberg and S. Paratyphi B var. Java. In conclusion, resistance to extended-spectrum cephalosporins in Salmonella from Colombian poultry is mainly encoded by bla CMY-2 and bla CTX-M-165 genes. These genes are mostly associated with IncI1/ST12 and IncQ1 plasmids, respectively. Evolutionary divergence is observed between Colombian S. Heidelberg and S. Paratyphi B var. Java and those from other countries.

Complete Sequence of the IncA/C1 Plasmid pCf587 Carrying blaPER-2 from Citrobacter freundii.

The blaPER-2-harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C1 and is closely related to pRA1. It contains a large resistance island including the blaPER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.

Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages.

Evaluation and characterization of plasmids carrying CTX-M genes in a non-clonal population of multidrug-resistant Enterobacteriaceae isolated from poultry in Brazil.

The increasing presence of ESBL-producing bacteria in food-producing animals might impact on public health. In this study, ESBL-producing enterobacteria were investigated in the microbiota of chickens produced in Brazil. We detected blaCTX-M-2, blaCTX-M-8 and blaCTX-M-15 in 13 Escherichia coli isolates, within 9 different PFGE-types. Escherichia fergusonii and Klebsiella pneumoniae were found carrying blaCTX-M-2. Plasmid Inc groups found included repF, FIB, FIC, I1, Y, B/O, A/C, K and HI1. F plasmids were present in 87.5% of the isolates, however, no resistance gene was harbored in this replicon. The pMLST for IncI1 showed ST113 and the novel ST130, ST131 and ST132 harboring blaCTX-M-8. IncK plasmids carried blaCTX-M-2 in one E. coli isolate. Non-typeable plasmids carrying blaCTX-M-2 or blaCTX-M-15 had up to 260kb. blaCTX-M-2 was also associated with class I integron and ISCR1 and blaCTX-M-8 with IS10. Overall, similar resistance elements were disseminated among a diverse population of ESBL-producing enterobacteria.

Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates.

Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in blac-AmpC overexpression, blaac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. blaac-AmpC, alterations in the blac-AmpC promoter/attenuator, and PMQR genes [qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA] were characterised by PCR and sequencing. blac-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying blaac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21563 E. coli isolates, 240 (1.1%) overproduced AmpC ß-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the blac-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4, 8 aac(6')-Ib-cr, 6 qnrS1 and 3 qnrB19] and in 10% of c-AmpC isolates [5 aac(6')-Ib-cr and 1 qnrS1]. IncI1-ST12 and IncF were associated with blaCMY-2 and blaDHA-1, respectively. These results suggest that ac-AmpC ß-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity.

IncI1/ST113 and IncI1/ST114 conjugative plasmids carrying blaCTX-M-8 in Escherichia coli isolated from poultry in Brazil.

Extended-spectrum beta-lactamase-producing Escherichia coli isolated from poultry in Brazil showed blaCTX-M-8 gene. IS10 was found upstream of blaCTX-M-8, harbored on plasmids IncI1, ST113/ST114 subtypes. Genomic relationship revealed a heterogeneous E. coli population. The gene blaCTX-M-8 is established in South America in food-producing animals, which represent risk of dissemination for other countries.

IncI1/ST3 and IncN/ST1 plasmids drive the spread of blaTEM-52 and blaCTX-M-1/-32 in diverse Escherichia coli clones from different piggeries.

OBJECTIVES: The spread of ESBL-producing Enterobacteriaceae among food animals/products has raised concerns about their possible transmission through the food chain. We aimed to characterize piggeries (pigs, piggery environments) as reservoirs of TEM-52- and CTX-M-encoding plasmids and clones. METHODS: Forty-three samples from five Portuguese intensive production farms were studied (2006-07). Twenty-two ESBL-producing (13 TEM-52, 6 CTX-M-32, 3 CTX-M-1) Escherichia coli isolates from healthy pigs, feed and liquid manure were further characterized. Standard methods were used for clonal (PFGE, MLST) and plasmid (S1-PFGE, replicon typing, pMLST, RFLP) analysis. PCR and sequencing were used for analysis of blaCTX-M genetic context and plasmid-mediated quinolone resistance genes. RESULTS: TEM-52 (n = 13/22; 59%), CTX-M-32 (n = 6/22; 27%) and CTX-M-1 (n = 3/22; 14%) were identified in feed (36%), swine faeces (36%), swine hide (9%) and liquid manure (18%) at different farms. Diverse phylogenetic groups and clones were identified among TEM-52 (7 A, 3 B1, 2 B2, 1 D; 8 clones)-producing, CTX-M-1 (1 A, 1 B1, 1 D; 3 clones)-producing and CTX-M-32 (4 A, 2 B1; 4 clones)-producing isolates. However, the ST10 clonal complex was frequent among TEM-52 (n = 6) and CTX-M-32 (n = 3) producers. blaTEM-52 and blaCTX-M-1/-32 genes were identified within epidemic IncI1/ST3 and IncN/ST1 plasmid variants, respectively. CONCLUSIONS: We report for the first time a piggery reservoir for blaTEM-52. The spread of blaTEM-52 and blaCTX-M-1/-32 within and/or between different piggeries was mostly associated with epidemic plasmids and clones previously identified in humans and other animal hosts in different EU countries, highlighting possible distribution along the food chain.