Plasmids In Enterobacteriaceae Research Articles

Antibiotic resistance plasmids in Enterobacteriaceae isolated from fresh produce in northern Germany.

This study showed that Enterobacteriaceae from raw vegetables carried plasmids ranging in size from 2,715 to 326,286 bp, of which about less than one-third carried antibiotic resistance genes encoding resistance toward antibiotics such as tetracyclines, aminoglycosides, fosfomycins, sulfonamides, quinolones, and β-lactam antibiotics. Some strains encoded multiple resistances, and some encoded extended-spectrum β-lactamases. The study highlights the potential of produce, which may be eaten raw, as a potential vehicle for the transfer of antibiotic-resistant bacteria.

Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Characterisation of ESBL/AmpC-Producing Enterobacteriaceae isolated from poultry farms in Peninsular Malaysia.

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp ( Ctenopharyngodon idella) and evidence of bla NDM-5-bearing IncHI2 plasmid transfer between ducks and fish.

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou, China. A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes. Characterization of blaNDM-5 positive isolates and plasmids was determined by antimicrobial susceptibility testing, conjugation experiments, Illumina HiSeq, and Nanopore sequencing. One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as blaNDM-5 carriers (3.57%, 7/196). TheblaNDM-5 genes were located on the IncX3 (n=5), IncHI2 (n=1), or IncHI2-IncF (n=1) plasmids. All blaNDM-5-bearing plasmids were transferred by conjugation at frequencies of ~10−4–10−6. Based on sequence analysis, the IncHI2 plasmid pHNBYF33-1 was similar to other blaNDM-5-carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, blaNDM-5 was embedded in a novel transposon, Tn7051 (IS3000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-tat-∆dct-IS26-∆umuD-∆ISKox3-IS3000), which was identical to the genetic structure surrounding blaNDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of theblaNDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ∆Tn1721. To the best of our knowledge, this is the first report on the characterization of blaNDM-5-bearing plasmids in fish in China. The IncHI2 plasmid pHNBYF33-1 may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong. Tn7051 is likely responsible for the transfer of blaNDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of blaNDM-5.

Molecular Characterization of Multidrug-Resistant Yersinia enterocolitica From Foodborne Outbreaks in Sweden.

The foodborne pathogen Yersinia enterocolitica causes gastrointestinal infections worldwide. In the spring of 2019, the Swedish Public Health Agency and Statens Serum Institut in Denmark independently identified an outbreak caused by Yersinia enterocolitica 4/O:3 that after sequence comparison turned out to be a cross-border outbreak. A trace-back investigation suggested shipments of fresh prewashed spinach from Italy as a common source for the outbreak. Here, we determined the genome sequences of five Y. enterocolitica clinical isolates during the Swedish outbreak using a combination of Illumina HiSeq short-read and Nanopore Technologies’ MinION long-read whole-genome sequencing. WGS results showed that all clinical strains have a fully assembled chromosome of approximately 4.6 Mbp in size and a 72-kbp virulence plasmid; one of the strains was carrying an additional 5.7-kbp plasmid, pYE-tet. All strains showed a high pathogen probability score (87.5%) with associated genes for virulence, all of which are closely related to an earlier clinical strain Y11 from Germany. In addition, we identified a chromosomally encoded multidrug-resistance cassette carrying resistance genes against chloramphenicol (catA1), streptomycin (aadA1), sulfonamides (sul1), and a mercury resistance module. This chromosomally encoded Tn2670 transposon has previously been reported associated with IncFII plasmids in Enterobacteriaceae: a Shigella flexneri clinical isolate from Japan in 1950s, a Klebsiella pneumoniae outbreak from Australia in 1997, and Salmonella enterica serovar Typhimurium. Interestingly, we identified an additional 5.7-kbp plasmid with tetB (encoding an ABC transporter), Rep, and its own ORI and ORIt sites, sharing high homology with small tetB-Rep plasmids from Pasteurellaceae. This is the first time that Tn2670 and Pasteurellaceae plasmids have been reported in Y. enterocolitica. Taken together, our study showed that the Swedish Y. enterocolitica outbreak strains acquired multi-antibiotic and metal-resistance genes through horizontal gene transfer, suggesting a potential reservoir of intraspecies dissemination of multidrug-resistance genes among foodborne pathogens. This study also highlights the concern of food-chain contamination of prewashed vegetables as a perpetual hazard against public health.

Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are diverse immune systems found in many prokaryotic genomes that target invading foreign DNA such as bacteriophages and plasmids. There are multiple types of CRISPR with arguably the most enigmatic being Type IV. During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a Type IV-A3 CRISPR-Cas system was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002–2003) and a single K. pneumoniae isolate from the United Kingdom (isolated in 2017). Sequence analysis of all other genomes available in GenBank revealed that this CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on a bacterial chromosome. This system is exclusively located on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Expression of the cas loci was confirmed in the available clinical isolates by RT-PCR. In all cases, the CRISPR-Cas system has a single CRISPR array (CRISPR1) upstream of the cas loci which has several, conserved, spacers which, amongst things, match regions within conjugal transfer genes of IncFIIK/IncFIB(K) plasmids. Our results reveal a Type IV-A3 CRISPR-Cas system exclusively located on IncHI1B/IncFIB plasmids in Enterobacteriaceae that is likely to be able to target IncFIIK/IncFIB(K) plasmids presumably facilitating intracellular, inter-plasmid competition.

Detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicons in Enterobacteriaceae using PlasmidSPAdes assembly of short-read sequence data.

Knowledge of the epidemiology of plasmids is essential for understanding the evolution and spread of antimicrobial resistance. PlasmidSPAdes attempts to reconstruct plasmids using short-read sequence data. Accurate detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicon genes is a prerequisite for the use of plasmid assembly tools to investigate the role of plasmids in the spread and evolution of ESBL production in Enterobacteriaceae . This study evaluated the performance of PlasmidSPAdes plasmid assembly for Enterobacteriaceae in terms of detection of ESBL-encoding genes, plasmid replicons and chromosomal wgMLST genes, and assessed the effect of k-mer size. Short-read sequence data for 59 ESBL-producing Enterobacteriaceae were assembled with PlasmidSPAdes using different k-mer sizes (21, 33, 55, 77, 99 and 127). For every k-mer size, the presence of ESBL genes, plasmid replicons and a selection of chromosomal wgMLST genes in the plasmid assembly was determined. Out of 241 plasmid replicons and 66 ESBL genes detected by whole-genome assembly, 213 plasmid replicons [88 %; 95 % confidence interval (CI): 83.9–91.9] and 43 ESBL genes (65 %; 95 % CI: 53.1–75.6) were detected in the plasmid assemblies obtained by PlasmidSPAdes. For most ESBL genes (83.3 %) and plasmid replicons (72.0 %), detection results did not differ between the k-mer sizes used in the plasmid assembly. No optimal k-mer size could be defined for the number of ESBL genes and plasmid replicons detected. For most isolates, the number of chromosomal wgMLST genes detected in the plasmid assemblies decreased with increasing k-mer size. Based on our findings, PlasmidSPAdes is not a suitable plasmid assembly tool for short-read sequence data for ESBL-encoding plasmids of Enterobacteriaceae .

Going around in circles: virulence plasmids in enteric pathogens.

Plasmids have a major role in the development of disease caused by enteric bacterial pathogens. Virulence plasmids are usually large (>40 kb) low copy elements and encode genes that promote host-pathogen interactions. Although virulence plasmids provide advantages to bacteria in specific conditions, they often impose fitness costs on their host. In this Review, we discuss virulence plasmids in Enterobacteriaceae that are important causes of diarrhoea in humans, Shigella spp., Salmonella spp., Yersinia spp and pathovars of Escherichia coli. We contrast these plasmids with those that are routinely used in the laboratory and outline the mechanisms by which virulence plasmids are maintained in bacterial populations. We highlight examples of virulence plasmids that encode multiple mechanisms for their maintenance (for example, toxin-antitoxin and partitioning systems) and speculate on how these might contribute to their propagation and success.

Worldwide dissemination of the NDM-type carbapenemases in Gram-negative bacteria.

The emergence of one of the most recently described carbapenemases, namely, the New Delhi metallo-lactamase (NDM-1), constitutes a critical and growingly important medical issue. This resistance trait compromises the efficacy of almost all lactams (except aztreonam), including the last resort carbapenems. Therapeutical options may remain limited mostly to colistin, tigecycline, and fosfomycin. The main known reservoir of NDM producers is the Indian subcontinent whereas a secondary reservoir seems to have established the Balkans regions and the Middle East. Although the spread of bla NDM-like genes (several variants) is derived mostly by conjugative plasmids in Enterobacteriaceae, this carbapenemase has also been identified in P. aeruginosa and Acinetobacter spp. Acinetobacter sp. may play a pivotal role for spreading bla NDM genes for its natural reservoir to Enterobacteriaceae. Rapid diagnostic techniques (Carba NP test) and screening of carriers are the cornerstone to try to contain this outbreak which threatens the efficacy of the modern medicine.

Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae

Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called “plasmid relaxase gene typing” (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1–7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.

Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India

Sir, The production of carbapenemases is the most common mechanism responsible for carbapenem resistance in Acinetobacter baumannii. They include metallo-b-lactamases (VIM, IMP and SIM types), which have been sporadically reported in some parts of the world, and acquired OXA-type carbapenemases which have been more frequently identified worldwide and are clustered in three major subfamilies (blaOXA-23, blaOXA-24 and blaOXA-58). 1 High levels of resistance to aminoglycosides due to the production of plasmid-encoded 16S rRNA methylase in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. have been documented since 2003. The present study was undertaken to determine the mechanism responsible for the carbapenem resistance. Three carbapenem-non-susceptible A. baumannii were isolated from patients in the intensive care unit of a tertiary care hospital in Chennai, India in April 2010. Species identification and antibiotic testing were carried out using an automated machine (VITEK-2). The MICs of carbapenems and aminoglycosides were determined using the CLSI agar dilution method, while tigecycline MICs were determined using the EUCAST broth microdilution method. All the isolates were resistant to all the b-lactams, aminoglycosides and quinolones, and were only susceptible to tigecycline and colistin. Double disc synergy test (DDST) and modified Hodge test (MHT) were used for detection of metallo-b-lactamases and other carbapenemases, respectively. PCR screening was performed for OXA-type carbapenemases (blaOXA-23, -24, -51 and -58-like) and the known metallo-b-lactamase genes. 3 – 5 All three isolates showed positivity for both DDST and MHT, and PCR yielded the products with expected sizes for blaOXA-51, 23-like and blaNDM-1 (Table 1). Sequencing of both blaOXA-23 and blaNDM-1 genes showed 100% identities with previously reported genes. In all three isolates, the blaOXA-23 gene was adjacent to insertion element ISAba1, which provides the promoter required for expression of linked resistance genes; blaOXA-51-like genes were also found in all of the isolates, but were not activated by ISAba1. Conjugation experiments using Escherichia coli J53 as the recipient were unsuccessful. Plasmids of Enterobacteriaceae were not detected among A. baumannii by PCR-based replicon typing. Susceptibility testing showed increased MICs of carbapenems and an unusual phenotype of broad-spectrum high-level resistance to aminoglycosides, including amikacin, gentamicin, netilmicin and tobramycin (MICs.256 mg/L), with susceptibility to tigecycline (MIC1⁄40.5–1 mg/L). PCR screening was performed for 16S rRNA methylase-encoding genes (armA, rmtA, rmtB, rmtC, rmtD and npmA). – 9 All three isolates showed positivity for the armA gene (Table 1). NDM-1 metallo-b-lactamase-mediated resistance to carbapenems in Enterobacteriaceae has been found in many parts of India. However, to our knowledge, this is the first

RepFIIA replicon of the natural Escherichia coli plasmid pRK100

The aim of the presented study was to identify the similarity of the plasmid pRK100 RepFIIA replicon (replication region) with similar replicons of other known plasmids of Enterobacteriaceae. For this purpose, within the determined nucleotide sequence of pRK100, theRepFIIA replicon rep genes/regions were identified. The nucleotide sequences of the pRK100 determined rep genes/regions were subsequently compared with the nucleotide sequences of other RepFIIA replicon rep genes/regions deposited in GenBank. Further, the nucleotide divergence between them was calculated. The obtained results clearly demonstrated, that the individual pRK100 rep regions are the same/most similar to rep regions from different plasmids. RepA2 of pRK100 is most similar to repA2 of pCP301, pINV_F6_M1382, pWR501 and R1, copA is the same as copA of plasmids pC15-1a and R100, repA6 of pRK100 is the same as repA6 in plasmids pC15-1a, pCP301, pINV_F6_M1382, pWR501, R1 and R100, repA1 is most similar to repA1 of the plasmid p1658/79, and repA4 of pRK100 is most similar to repA4 of pC15-1a. Hence, the composition of the pRK100 RepFIIA replicon is mosaic and unique among the plasmids.

Restriction endonuclease characterization of resistant plasmids in Enterobacteriaceae isolated from children in the Sudan

The investigation of plasmid similarity is an important component in the surveillance of antimicrobial resistance and in the detection of epidemic plasmids. The use of restriction endonucleases in the classification of transferable, multiply-resistant plasmids from faecal Enterobacteriaceae isolated at the Children's Emergency Hospital, Khartoum was investigated. Twenty-four transconjugant plasmids, coding for 11 different resistance patterns, each of molecular weight 62 MDa, were studied using four restriction enzymes; Pst I, EcoR I, Hind III and Ava II. Fifteen different digest profiles were obtained. Restriction profiles discriminated between plasmids with differing resistance patterns and demonstrated homology of plasmids with common resistance patterns. Restriction endonuclease digest patterns provide a potentially rapid and reproducible method of plasmid classification, that could contribute towards surveillance systems in tropical countries with a high prevalence of antimicrobial resistance.

Occurrence of multiple antibiotic resistance and R plasmids in Enterobacteriaceae isolated from children in the Sudan.

The prevalence of resistance to six commonly-used antimicrobial agents in faecal coliforms from children in Khartoum, Sudan was studied. A relatively high prevalence of resistance was found, ranging from 96% of children with isolates resistant to ampicillin to 70% of children with isolates resistant to chloramphenicol. Seventy-seven percent of children had isolates with high-level resistance to trimethoprim (MIC greater than 1000 micrograms/ml). Twenty-nine different resistance patterns were found. Thirty-nine percent of the children had isolates resistant to all six antibiotics studied, and 80% of children had isolates resistant to at least four. Transfer of resistance to each of the antimicrobials, in varying combinations, was demonstrated, but did not occur for all resistance patterns. Plasmid analysis showed plasmids ranging from 160 MDa to 2.8 MDa and isolates contained from one to five plasmids of different sizes. There were no consistent relationships between resistance pattern and plasmid profile, but multiple resistance transfer was mediated commonly by plasmids with a molecular weight of 62 MDa. The high prevalence of potentially transferable antibiotic resistance in gut commensals of children in the Sudan may be of importance in the management of enteric and other infections requiring antimicrobial treatment.

Multiple antibiotic resistance plasmids in Enterobacteriaceae isolated from diarrhoeal specimens of hospitalized children in Indonesia.

We studied the plasmid and antibiotic resistance characteristics of 35 strains of Enterobacteriaceae recovered from faecal specimens of children with diarrhoea in Central General Hospital, Bandung, Indonesia. Twenty three Escherichia coli, three Providencia, three Proteus, three Klebsiella, two Enterobacter and one Citrobacter were examined. All strains were multiply resistant, many carrying six to nine antibiotic resistances. Most of these resistances were transferable to a laboratory E. coli strain and were carried on large-sized plasmids. All recently-described tetracycline resistance determinants (Classes A----D) were represented; the most common was the Class B, or TN10 type. The TEM-1 beta-lactamase was detected in 17 out of 21 ampicillin-resistant strains examined. The OXA-1, PSE-1, and SHV-1 enzymes were also found. Of 23 plasmids tested, all could be classified into one of eight different incompatibility groups: IncFII, IncN, IncB, IncF1, IncI1, IncI2, IncH2 and IncT. These studies demonstrate the existence of large multiresistant transferable plasmids representing common incompatibility groups and bearing common tetracycline and ampicillin resistance determinants in enteric strains isolated from children hospitalized in Indonesia.

A rapid single colony lysate method for the selective visualization of plasmids in Enterobacteriaceae, including Serratia marcescens.

A rapid single colony lysate method for the selective visualization of plasmids in Enterobacteriaceae, including Serratia marcescens.

Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae.

SUMMARY: High-level resistance to trimethoprim (minimum inhibitory concentration > 1000 μ/ml) was conferred by R factors of the W compatibility group in Escherichia coli and Klebsiella spp. isolated from patients in three London hospitals. We suggest that we are observing the early stages in the spread of a new R factor.