Resistance Genes In Enterobacteriaceae Research Articles

Characterization of plasmid mediated quinolone resistance genes in Enterobacteriaceae in a University Hospital in Tunisia

Characterization of plasmid mediated quinolone resistance genes in Enterobacteriaceae in a University Hospital in Tunisia

The impact of genetic and environmental regulation on the expression of antibiotic resistance genes in Enterobacteriaceae

Antibiotic resistance is a major global concern and challenge in the 21st century. Enterobacteriaceae are one of the important pathogens of hospital-acquired infections. With the increasing use of antibiotics in clinical practice, a variety of drug-resistant Enterobacteriaceae, especially multidrug-resistant Enterobacteriaceae have emerged, posing an increasingly serious threat to human health. Bacteria can acquire antibiotic resistance by mutation or horizontal transfer of antibiotic resistance genes, and it is often possible to predict the corresponding resistance phenotype from known mechanism. However, recent findings suggest that genetic background and environmental factors could alter the expression of specific resistance genes and that a given genotype does not always generate the expected resistance phenotype. The genotype-phenotype segregation greatly hampers our ability to predict antibiotic resistance phenotype from a genetic perspective. In this review, we explore the genetic and environmental regulation of the expression of antibiotic resistance genes in a variety of Enterobacteriaceae, with the aim to provide scientific evidence for genetic prediction of antibiotic resistance phenotype and clinical guidance on drug use.

Characterization of antimicrobial resistance genes in Enterobacteriaceae carried by suburban mesocarnivores and locally owned and stray dogs.

The role of wildlife in the dissemination of antimicrobial-resistant bacteria and antimicrobial resistance genes (ARGs) in the environment is of increasing concern. We investigated the occurrence, richness and transmissibility potential of ARGs detected in the faeces of three mesocarnivore species: the coyote (Canis latrans), raccoon (Procyon lotor) and Virginia opossum (Didelphis virginiana), and of stray and owned dogs in suburban Chicago, IL, USA. Rectal swabs were collected from live-captured coyotes (n=32), raccoons (n=31) and Virginia opossums (n=22). Fresh faecal samples were collected from locally owned (n=13) and stray dogs (n=18) and from the live-captured mesocarnivores, when available. Faecal samples and rectal swabs were enriched to select for Enterobacteriaceae and pooled by mesocarnivore species and dog type (owned or stray). Pooled enriched samples were then analysed for the presence of ARGs using shotgun sequencing. The three mesocarnivore and stray dog samples had twice as many unique ARGs compared to the owned dog sample, which was partly driven by a greater richness of beta-lactamase genes (genes conferring resistance to penicillins and cephalosporins). Raccoon and stray dog samples had the most ARGs in common, suggesting possible exposure to similar environmental sources of ARGs. In addition to identifying clinically relevant ARGs (e.g. blaCMY and qnrB), some ARGs were linked to the class 1 integrase gene, intI1, which may indicate anthropogenic origin. Findings from this pilot investigation suggest that the microbial communities of suburban mesocarnivores and stray dogs can host ARGs that can confer resistance to several antimicrobials used in human and veterinary medicine.

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

First Case of Qnr B6 and Qnr B7 Genes in Enterobacteriaceae Producing Extended-Spectrum Beta-Lactamases in Abidjan, Côte D'ivoire

Aims: The aim of this study was to characterize fluoroquinolone resistance genes in enterobacteriaceae that produce extended-spectrum β-lactamases, isolated in Abidjan.
 Place and Duration of Study: Pasteur Institute of Côte d'Ivoire and research unit on emerging tropical infectious diseases of Aix-Marseille University from January 2017 at July 2017.
 Methodology: The study included 90 enterobacteriaceae producing extended-spectrum β-lactamases isolated from biological products from various hospital services in Abidjan. These strains have been pre-identified and stored at the Center for Biological Resources (CeReB) of Pasteur Institute of Côte d'Ivoire. The identification of the strains was confirmed using the mass spectrometry MALDI-TOF (MS) and the antibiotic sensitivity test was performed using Müeller Hinton's agar diffusion method. The fluoroquinolone resistant genes were detected by conventional PCR and then, sequenced.
 Results: The strains studied were Escherichia coli (44), Klebsiella pneumoniae (31) and Enterobacter cloacae (15). High resistance rates to ceftriaxone (96.7%), cefotaxime (95.6%), aztreonam (95.6%) and cefoxitin (72.2%) were observed in all strains producing broad spectrum β-lactamases. The resistance rate to fluororquinolones represented by ciprofloxacin was 86.7%. The fluoroquinolone resistance genes detected were qnr A (3.3%) and qnr B (42.2%). Sequencing identified the qnr A1 (3.3%), qnr B1 (31.1%), qnr B6 (2.2%) and qnr B7 (1.1%) genes.
 Conclusion: This study made it possible to identify fluoroquinolone resistance genes in enterobacteriaceae producing β-lactamases which have an extended spectrum in Abidjan.

Development and validation of a multiplex polymerase chain reaction assay for detection of the five families of plasmid-encoded colistin resistance

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.

Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators.

Plasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, from Citrobacter freundii, is classified in the IVB subfamily and in the MPFI group, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely, orf35 and orf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the orf35 and orf36 deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication in Agrobacterium tumefaciens, Ralstonia eutropha, and Pseudomonas putida, though its conjugation system is capable of introducing plasmids bearing oriTpCTX-M3 into these bacteria, which are representatives of Alpha-, Beta-, and Gammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.IMPORTANCE Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in Enterobacteriaceae We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.

The detection of fosfomycin resistance genes in Enterobacteriaceae from pets and their owners

The aim of this study was to investigate the prevalence of fosfomycin resistance and molecular characteristic of fosfomycin-resistant strains isolated from companion animals and their owners. A total of 171 samples collected from pets and pet owners in a Chinese veterinary teaching hospital were screened for the presence of phenotype and genotype of fosfomycin-resistance by selective media containing fosfomycin and PCR & sequencing. Among 171 samples tested, nineteen isolates were resistant to fosfomycin. Sixteen and three of these fosfomycin-resistant isolates were positive for fosA3 and fosA genes, respectively. The fosA3 gene was detected both in chromosomes and plasmids in bacteria. All of the fosA3 gene-positive isolates except one were CTX-M producers and nearly half (7/16) of them also harbored the rmtB gene. The fosA3 gene-carrying plasmids, which were readily transferrable to recipient E. coli J53 by conjugation, conferred resistance to multiple antimicrobial agents. Genetic structures were IS26-385bp-fosA3-1810bp-IS26 (n=11) and IS26-385bp-fosA3-588bp-IS26 (n=5). Molecular typing indicated that two fosA3-positive isolates from dogs were genetically identical to the isolates from the pet owners. Our results indicated that active transmission of fosA3-mediated fosfomycin resistance has occurred among Enterobacteriaceae isolated from pets and their owners by both horizontal transfer and clonal expansion.

Prevalence and diversity of IncX plasmids carrying fluoroquinolone and β-lactam resistance genes in Escherichia coli originating from diverse sources and geographical areas.

To describe the prevalence and diversity of IncX plasmids with antibiotic resistance genes in Enterobacteriaceae and to identify the most disseminated lineages of the plasmid family. IncX plasmids were screened in 1894 Enterobacteriaceae isolates resistant to cefotaxime (2 mg/L) or with reduced susceptibility to ciprofloxacin (0.05 mg/L) obtained from various sources in five continents using PCR. IncX plasmid-harbouring isolates were identified using MALDI-TOF or biochemical tests, and screened for antibiotic resistance genes using PCR and sequencing; their clonality was determined by PFGE. Horizontal transfer of plasmids was tested using transformation and conjugation. IncX plasmids were characterized by S1-nuclease and PFGE, RFLP and hybridization. A total of 164 Escherichia coli isolates (8.7%, n = 1894) carried at least one IncX subgroup. Seven isolates harboured two distinct subgroups. IncX1 subgroup was found in 93 isolates, followed by IncX2 (35 isolates), IncX4 (28) and IncX3 (15). IncX4 plasmids were not transferred horizontally as single plasmids and therefore excluded from further analysis. The most disseminated lineages of IncX plasmids included IncX1 harbouring qnrS1 and blaTEM-1,-135 found in 36 E. coli from different sources in Europe and Australia and IncX2 carrying qnrS1 and tet(A) detected in nine E. coli from wildlife in Europe. IncX3 plasmids harboured predominantly blaSHV-12 and qnrS1 or qnrB7. IncX plasmids were widely distributed in E. coli from wildlife in Europe and were predominantly associated with fluoroquinolone resistance genes. Plasmids showing indistinguishable restriction profiles were identified in E. coli from different sources and countries suggesting wide dissemination of certain plasmid lineages.

Partition locus-based classification of selected plasmids in Klebsiella pneumoniae, Escherichia coli and Salmonella enterica spp.: An additional tool

The dissemination of antimicrobial resistance genes in Enterobacteriaceae has been largely attributed to plasmids, circular DNA molecules capable of autonomous replication. Whereas high-copy-number plasmids primarily rely on passive diffusion for plasmid maintenance, low-copy-number plasmids utilize so-called partition (par) systems. Plasmid partition relies on three structures, i.e. a centromere like DNA site, a centromere-binding protein and an ATPase or a GTPase motor protein for plasmid positioning. Identification and classification of plasmids is essential for tracing plasmids conferring drug resistance. PCR-based replicon typing is currently the standard method for plasmid identification but there are new classification schemes, especially the relaxase gene typing (PRaseT). Here we developed a multiplex PCR set targeting par loci found on the plasmids most frequently encountered in Enterobacteriaceae. This method, called “plasmid partition gene typing” (PAR-T), was validated with 136 transconjugants or transformants harboring various replicon types. The method was tested with 30 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-4 replicons; all replicons were tested in parallel with PRaseT for comparison. Six multiplex PCRs and one simplex PCR including 18 pairs of primers recognized plasmids of groups IncA/C, FIA, FIB, FIC, FIIk, FII, HI1, HI2, I1, L/M, N, X. Our set of multiplex PCRs showed high specificity for the classification of resistance plasmids except for IncX replicons.

Plasmid-Mediated Quinolone Resistance Genes in Enterobacteriaceae from American Crows: High Prevalence of Bacteria with Variable qnrB Genes

Though wild birds are not normally exposed to use of antimicrobial agents, they can acquire antibiotic-resistant bacteria through the environment ([1][1]). It was also suggested that rooks may disseminate these bacteria over long distances and pose a risk of contaminating the environment ([2][2]). A

Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae

Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥2μg/mL were collected from seven major hospitals in Sydney (Australia) in 2008–2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC≥2μg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC≥8μg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5–10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.

Occurrence of antibiotic resistance and characterization of resistance genes and integrons in Enterobacteriaceae isolated from integrated fish farms in south China

Antibiotics are still widely applied in animal husbandry to prevent diseases and used as feed additives to promote animal growth. This could result in antibiotic resistance to bacteria and antibiotic residues in animals. In this paper, Enterobacteriaceae isolated from four integrated fish farms in Zhongshan, South China were tested for antibiotic resistance, tetracycline resistance genes, sulfonamide resistance genes, and class 1 integrons. The Kirby-Bauer disk diffusion method and polymerase chain reaction (PCR) assays were carried out to test antibiotic susceptibility and resistance genes, respectively. Relatively high antibiotic resistance frequencies were found, especially for ampicillin (80%), tetracycline (52%), and trimethoprim (50%). Out of 203 Enterobacteriaceae isolates, 98.5% were resistant to one or more antibiotics tested. Multiple antibiotic resistance (MAR) was found highest in animal manures with a MAR index of 0.56. Tetracycline resistance genes (tet(A), tet(C)) and sulfonamide resistance genes (sul2) were detected in more than 50% of the isolates. The intI1 gene was found in 170 isolates (83.7%). Both classic and non-classic class 1 integrons were found. Four genes, aadA5, aadA22, dfr2, and dfrA17, were detected. To our knowledge, this is the first report for molecular characterization of antibiotic resistance genes in Enterobacteriaceae isolated from integrated fish farms in China and the first time that gene cassette array dfrA17-aadA5 has been detected in such fish farms. Results of this study indicated that fish farms may be a reservoir of highly diverse and abundant antibiotic resistant genes and gene cassettes. Integrons may play a key role in multiple antibiotic resistances posing potential health risks to the general public and aquaculture.

Complete nucleotide sequence of the IncN plasmid pKOX105 encoding VIM-1, QnrS1 and SHV-12 proteins in Enterobacteriaceae from Bolzano, Italy compared with IncN plasmids encoding KPC enzymes in the USA

We determined the complete nucleotide sequence of pKOX105, a 54 641 bp plasmid from a Klebsiella oxytoca strain that was isolated from a resident of a long-term-care facility in Bolzano, Italy. The plasmid was sequenced using a shotgun approach. Combinatorial PCRs, directed PCRs and walking reads were used to assemble the contigs and to fill in gaps. Gene sequences were compared with reference plasmids and aligned with GenBank data using BLAST and CLUSTAL W software. pKOX105 belonged to incompatibility group IncN, harboured bla(VIM-1), bla(SHV-12), qnrS1, aacA4 and dfrA14 and conferred resistance to carbapenems, oxyimino-cephalosporins, quinolones, aminoglycosides and trimethoprim. It was highly related to the p9 and p12 plasmids from Klebsiella pneumoniae and K. oxytoca strains isolated at a New York City hospital in 2005 carrying bla(KPC-2) and bla(KPC-3), respectively. IncN plasmids are broad host-range plasmids that have contributed significantly to the worldwide dissemination of many different resistance genes in Enterobacteriaceae from animal and human sources. This plasmid family is now playing a crucial role in the global spread of diverse carbapenemase genes in Klebsiella spp.

Detection of antibiotic resistance and tetracycline resistance genes in Enterobacteriaceae isolated from the Pearl rivers in South China

This study investigated antibiotic resistance profiles and tetracycline resistance genes in Enterobacteriaceae family isolates from the Pearl rivers. The Enterobacteriaceae isolates were tested for susceptibility to seven antibiotics ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, sulphamethoxazole/trimethoprim, tetracycline and trimethoprim. In Liuxi reservoir, with an exception to ampicillin resistant strains (11%) no other antibiotic resistance bacterial strains were detected. However, multiple drug resistance in bacterial isolates from the other sites of Pearl rivers was observed which is possibly due to sewage discharge and input from other anthropogenic sources along the rivers. Four tetracycline resistance genes tet A, tet B, tet C and tet D were detected in the isolates from the rivers. The genes tet A and tet B were widely detected with the detection frequencies of 43% and 40% respectively. Ciprofloxacin and levofloxacin resistant enteric bacteria were also isolated from the pig and duck manures which suggest a wider distribution of human specific drugs in the environment. This investigation provided a baseline data on antibiotic resistance profiles and tetracycline resistance genes in the Pearl rivers delta.

Emergence of plasmid-mediated quinolone resistance genes in Enterobacteriaceae isolated from chickens in China

Emergence of plasmid-mediated quinolone resistance genes in Enterobacteriaceae isolated from chickens in China