Evolution of triclosan resistance modulates bacterial permissiveness to multidrug resistance plasmids and phages.

The horizontal transfer of plasmids has been recognized as one of the key drivers for the worldwide spread of antimicrobial resistance (AMR) across bacterial pathogens. However, knowledge remain limited about the contribution made by environmental stress on the evolution of bacterial AMR by modulating horizontal acquisition of AMR plasmids and other mobile genetic elements. Here we combined experimental evolution, whole genome sequencing, reverse genetic engineering, and transcriptomics to examine if the evolution of chromosomal AMR to triclosan (TCS) disinfectant has correlated effects on modulating bacterial pathogen (Klebsiella pneumoniae) permissiveness to AMR plasmids and phage susceptibility. Herein, we show that TCS exposure increases the evolvability of K. pneumoniae to evolve TCS-resistant mutants (TRMs) by acquiring mutations and altered expression of several genes previously associated with TCS and antibiotic resistance. Notably, nsrR deletion increases conjugation permissiveness of K. pneumoniae to four AMR plasmids, and enhances susceptibility to various Klebsiella-specific phages through the downregulation of several bacterial defense systems and changes in membrane potential with altered reactive oxygen species response. Our findings suggest that unrestricted use of TCS disinfectant imposes a dual impact on bacterial antibiotic resistance by augmenting both chromosomally and horizontally acquired AMR mechanisms.

Three Fe(III)-reducing and nitrogen-fixing bacteria, Anaeromyxobacter terrae sp. nov., Anaeromyxobacter oryzisoli sp. nov. and Anaeromyxobacter soli sp. nov., isolated from paddy soil.

Three microaerophilic bacterial strains, designated SG22T, SG63T and SG29T were isolated from paddy soils in PR China. Cells of these strains were Gram-staining-negative and long rod-shaped. SG22T, SG63T and SG29T showed the highest 16S rRNA gene sequence similarities with the members of the genus Anaeromyxobacter. The results of phylogenetic and phylogenomic analysis also indicated that these strains clustered with members of the genus Anaeromyxobacter. The main respiratory menaquinone of SG22T, SG63T and SG29T was MK-8 and the major fatty acids were iso-C15 : 0, iso-C17 : 0 and C16 : 0. SG22T, SG29T and SG63T not only possessed iron reduction ability but also harboured genes (nifHDK) encoding nitrogenase. The genomic DNA G+C contents of SG22T, SG63T and SG29T ranged from 73.3 to 73.5 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between SG22T, SG63T and SG29T and the closely related species of the genus Anaeromyxobacter were lower than the cut-off values (dDDH 70 % and ANI 95-96 %) for prokaryotic species delineation. On the basis of these results, strains SG22T, SG63T and SG29T represent three novel species within the genus Anaeromyxobacter, for which the names Anaeromyxobacter terrae sp. nov., Anaeromyxobacter oryzisoli sp. nov. and Anaeromyxobacter soli sp. nov., are proposed. The type strains are SG22T (= GDMCC 1.3185T = JCM 35581T), SG63T (= GDMCC 1.2914T = JCM 35124T) and SG29T (= GDMCC 1.2911T = JCM 35123T).

Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study.

BACKGROUND: The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-ß-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context. METHODS: For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain. Bacteria were extracted from a hospital wastewater treatment plant (Fujian Provincial Maternity and Children's Hospital, Fuzhou, China) as the bacterial recipient community. We mixed this recipient community with the E coli donor strain carrying the gfp-tagged plasmid, both with and without sodium hypochlorite (NaClO) as an environmental stressor, and conducted several culture-based and culture-independent conjugation assays. The conjugation events were observed microscopically and quantified by fluorescence-activated cell sorting. We analysed the taxonomic composition of the sorted transconjugal pool by 16S rRNA gene amplicon sequencing and assessed the stability of the plasmid in the isolated transconjugants and its ability to transfer back to E coli. FINDINGS: We show that the plasmid pX3_NDM-5 has a broad host range and can transfer across various bacterial phyla, including between Gram-negative and Gram-positive bacteria. Although environmental stress with NaClO did not affect the overall plasmid transfer frequency, it reduced the breadth of the transconjugant pool. The taxonomic composition of the transconjugal pool was distinct from that of the recipient communities, and environmental stress modulated the permissiveness of some operational taxonomic units towards the acquisition of pX3_NDM-5. Notably, pX3_NDM-5 transconjugants included the Gram-positive pathogen Enterococcus faecalis, and the plasmid could subsequently be reconjugated back to E coli. These findings suggest that E faecalis could act as a natural shuttle vector for the wide dissemination of pX3_NDM-5 plasmids. INTERPRETATION: Our culture-independent conjugation model simulates natural environmental conditions and challenges the established theory that Gram-negative and Gram-positive bacteria rarely exchange clinically important plasmids. The data show that plasmids disseminate more widely across genera and phyla than previously thought. These findings have substantial implications when considering the spread of antimicrobial resistance across One Health sectors. FUNDING: The Laboratory of Lingnan Modern Agriculture Project, the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province of China, and the Outstanding Young Research Talents Program of Fujian Agriculture and Forestry University.

Mesophilic and thermophilic viruses are associated with nutrient cycling during hyperthermophilic composting.

While decomposition of organic matter by bacteria plays a major role in nutrient cycling in terrestrial ecosystems, the significance of viruses remains poorly understood. Here we combined metagenomics and metatranscriptomics with temporal sampling to study the significance of mesophilic and thermophilic bacteria and their viruses on nutrient cycling during industrial-scale hyperthermophilic composting (HTC). Our results show that virus-bacteria density dynamics and activity are tightly coupled, where viruses specific to mesophilic and thermophilic bacteria track their host densities, triggering microbial community succession via top-down control during HTC. Moreover, viruses specific to mesophilic bacteria encoded and expressed several auxiliary metabolic genes (AMGs) linked to carbon cycling, impacting nutrient turnover alongside bacteria. Nutrient turnover correlated positively with virus-host ratio, indicative of a positive relationship between ecosystem functioning, viral abundances, and viral activity. These effects were predominantly driven by DNA viruses as most detected RNA viruses were associated with eukaryotes and not associated with nutrient cycling during the thermophilic phase of composting. Our findings suggest that DNA viruses could drive nutrient cycling during HTC by recycling bacterial biomass through cell lysis and by expressing key AMGs. Viruses could hence potentially be used as indicators of microbial ecosystem functioning to optimize productivity of biotechnological and agricultural systems.

Silent circulation of BKC-1-producing Klebsiella pneumoniae ST442: molecular and clinical characterization of an early and unreported outbreak.

OBJECTIVE: To describe the undetected circulation of an epidemic BKC-1-producing Klebsiella pneumoniae ST442 clone, occasioning the first reported outbreak of the infrequent carbapenemase BKC-1. METHODS: Six hundred and forty-seven K. pneumoniae isolates (2008-2017) with reduced susceptibility to carbapenems were screened for blaBKC-1. BKC-1-positive isolates were typed using pulsed-field gel electrophoresis and multi-locus sequence typing. Susceptibility profiles were determined by broth microdilution, and additional antimicrobial resistance genes (ARGs) were investigated by polymerase chain reaction. Some isolates were submitted to full genomic characterization by whole-genome sequencing (Illumina MiSeq and MinIon), and in-vivo virulence studies using the Galleria mellonella model. RESULTS: Sixteen (2.5%) K. pneumoniae, from 15 patients, carrying blaBKC-1 were found between 2010 and 2012. Among these patients, the all-cause mortality rate was 54.5%. A major clone - A1-ST442 (13/16) - was isolated during the study period. The BKC-1-producing isolates had a multi-drug-resistant phenotype, remaining susceptible to gentamicin (87.5%) and ceftazidime-avibactam (100%) alone. The presence of two carbapenemases - blaBKC-1 and blaKPC-2 - was detected in six isolates, increasing the ß-lactam minimum inhibitory concentration significantly. Additionally, other ARGs were identified on A1-ST442 and B1-ST11 clones. The B1-ST11 clone was more virulent than the A1-ST442 clone. CONCLUSION: An undetected outbreak caused predominantly by a BKC-1-positive A1-ST442 clone between 2010 and 2012 was identified 10 years later in a Brazilian hospital. The misidentification of BKC-1 may have worsened the spread of resistant clones; this reinforces the need for correct and rapid identification of antimicrobial resistance mechanisms in hospitals.

Quantifying the effects of antibiotic treatment on the extracellular polymer network of antimicrobial resistant and sensitive biofilms using multiple particle tracking.

Novel therapeutics designed to target the polymeric matrix of biofilms requires innovative techniques to accurately assess their efficacy. Here, multiple particle tracking (MPT) was developed to characterize the physical and mechanical properties of antimicrobial resistant (AMR) bacterial biofilms and to quantify the effects of antibiotic treatment. Studies employed nanoparticles (NPs) of varying charge and size (40-500 nm) in Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA) biofilms and also in polymyxin B (PMB) treated Escherichia coli biofilms of PMB-sensitive (PMBSens) IR57 and PMB-resistant (PMBR) PN47 strains. NP size-dependent and strain-related differences in the diffusion coefficient values of biofilms were evident between PAO1 and MRSA. Dose-dependent treatment effects induced by PMB in PMBSens E. coli biofilms included increases in diffusion and creep compliance (P < 0.05), not evident in PMB treatment of PMBR E. coli biofilms. Our results highlight the ability of MPT to quantify the diffusion and mechanical effects of antibiotic therapies within the AMR biofilm matrix, offering a valuable tool for the pre-clinical screening of anti-biofilm therapies.

Compensatory mutations modulate the competitiveness and dynamics of plasmid-mediated colistin resistance in Escherichia coli clones.

The emergence of mobile colistin resistance (mcr) threatens to undermine the clinical efficacy of the last antibiotic that can be used to treat serious infections caused by Gram-negative pathogens. Here we measure the fitness cost of a newly discovered MCR-3 using in vitro growth and competition assays. mcr-3 expression confers a lower fitness cost than mcr-1, as determined by competitive ability and cell viability. Consistent with these findings, plasmids carrying mcr-3 have higher stability than mcr-1 plasmids across a range of Escherichia coli strains. Crucially, mcr-3 plasmids can stably persist, even in the absence of colistin. Recent compensatory evolution has helped to offset the cost of mcr-3 expression, as demonstrated by the high fitness of mcr-3.5 as opposed to mcr-3.1. Reconstructing all of the possible evolutionary trajectories from mcr-3.1 to mcr-3.5 reveals a complex fitness landscape shaped by negative epistasis between compensatory and neutral mutations. Our findings highlight the importance of fitness costs and compensatory evolution in driving the dynamics and stability of mobile colistin resistance in bacterial populations, and they highlight the need to understand how processes (other than colistin use) impact mcr dynamics.

Environmental dissemination of mcr-1 positive Enterobacteriaceae by Chrysomya spp. (common blowfly): An increasing public health risk.

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR.

Heavy Metal Resistance Genes Are Associated with blaNDM-1- and blaCTX-M-15-Carrying Enterobacteriaceae.

The occurrence of heavy metal resistance genes in multiresistant Enterobacteriaceae possessing blaNDM-1 or blaCTX-M-15 genes was examined by PCR and pulsed-field gel electrophoresis with S1 nuclease. Compared with clinical susceptible isolates (10.0% to 30.0%), the pcoA, merA, silC, and arsA genes occurred with higher frequencies in blaNDM-1-positive (48.8% to 71.8%) and blaCTX-M-15-positive (19.4% to 52.8%) isolates, and they were mostly located on plasmids. Given the high association of metal resistance genes with multidrug-resistant Enterobacteriaceae, increased vigilance needs to be taken with the use of heavy metals in hospitals and the environment.

Toxin-antitoxin systems and their role in disseminating and maintaining antimicrobial resistance.

Toxin-antitoxin systems (TAs) are ubiquitous among bacteria and play a crucial role in the dissemination and evolution of antibiotic resistance, such as maintaining multi-resistant plasmids and inducing persistence formation. Generally, activities of the toxins are neutralised by their conjugate antitoxins. In contrast, antitoxins are more liable to degrade under specific conditions such as stress, and free active toxins interfere with essential cellular processes including replication, translation and cell-wall synthesis. TAs have also been shown to be responsible for plasmid maintenance, stress management, bacterial persistence and biofilm formation. We discuss here the recent findings of these multifaceted TAs (type I-VI) and in particular examine the role of TAs in augmenting the dissemination and maintenance of multi-drug resistance in bacteria.

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1.

The polymixin colistin is a "last line" antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.

Design, synthesis, and structure-activity relationship studies of novel pleuromutilin derivatives having a piperazine ring.

A series of novel pleuromutilin derivatives possessing piperazine moieties were synthesized under mild conditions. The in vitro antibacterial activities of these derivatives against Staphylococcus aureus and Escherichia coli were tested by the agar dilution method. Structure-activity relationship studies resulted in compounds 11b, 13b, and 14a with the most potent in vitro antibacterial activity among the series (minimal inhibitory concentration = 0.0625-0.125 µg/mL). The binding of compounds 11b, 13b, and 14a to the E. coli ribosome was investigated by molecular modeling, and it was found that there is a reasonable correlation between the binding free energy and the antibacterial activity.

Complete Sequence of the FII Plasmid p42-2, Carrying blaCTX-M-55, oqxAB, fosA3, and floR from Escherichia coli.

We sequenced a novel conjugative multidrug resistance IncF plasmid, p42-2, isolated from Escherichia coli strain 42-2, previously identified in China. p42-2 is 106,886 bp long, composed of a typical IncFII-type backbone (∼54 kb) and one distinct acquired DNA region spanning ∼53 kb, harboring 12 antibiotic resistance genes [blaCTX-M-55, oqxA, oqxB, fosA3, floR, tetA(A), tetA(R), strA, strB, sul2, aph(3')-II, and ΔblaTEM-1]. The spread of these multidrug resistance determinants on the same plasmid is of great concern and, because of coresistance to antibiotics from different classes, is therapeutically challenging.

IncF plasmid diversity in multi-drug resistant Escherichia coli strains from animals in China.

The purpose of this study was to characterize a collection of 103 multidrug resistance IncF plasmids recovered from Escherichia coli of food producing and companion animals between 2003 and 2012. A total of 103 incF plasmids were characterized using an established PCR-based IncF replicon sequence typing (RST) system to identify FII, FIA, and FIB (FAB) groups. Plasmids were also analyzed using-restriction fragment length polymorphism (RFLP). Antibiotic Resistance determinants bla CTX-M , plasmid-mediated quinolone resistance (PMQR) genes and rmtB and plasmid addiction systems (PAS) were identified by PCR screening. A total of 20 different RSTs from 103 IncF plasmids were identified. The groups F2 and F33 with the RST formulae A-: B- were the most frequently encountered types (63.1%). The antibiotic resistance genes (ARGs) bla CTX-M , rmtB, and oqxB were carried by 82, 37, and 34 IncF plasmids, respectively. Most of these plasmids carried more than one resistance gene (59.2%, 61/103). The IncF plasmids also had a high frequency of addiction systems (mean 2.54) and two antisense RNA-regulated systems (hok-sok and srnBC) and a protein antitoxin-regulated system (pemKI) were the most prevalent. Not surprisingly, RFLP profiles among the IncF plasmids were diverse even though some shared identical IncF-RSTs. This is the first extensive study of IncF plasmid-positive E. coli isolates from animals in China. Our results demonstrate that IncF is the most prevalent plasmid family in E. coli plasmids and they commonly carry multiple resistance determinants that render them resistant to different antibiotic classes simultaneously. IncF plasmids also harbor addiction systems, promoting their stability and maintenance in the bacterial host, under changing environmental conditions.

Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins.

The association of ESBLs (extended-spectrum beta-lactamases)/pAmpCs (plasmid-mediated AmpC ß-lactamases) with PMQR (plasmid mediated quinolone resistance) in gram-negative bacteria has been of great concern. The present study was performed to characterize the diversity, gene location, genetic context, and evolution of ampC and qnrB alleles in isolates of Citrobacter freundii. Fifteen isolates of C. freundii were identified from a total of 788 isolates of Enterobacteriaceae derived from humans, animals, animal food products, and the environment between 2010 and 2012. Co-existence of qnrB/ΔqnrB with ampC was detected in all C. freundii isolates. Both ampC and qnrB genes were found to be located on the chromosome, but were distantly separated on the chromosome. Seven and six novel alleles were discovered for the 10 ampC and qnrB variants detected in this study, respectively. Phylogenetic analysis showed that the new alleles differed a little from the variants of ampC/qnrB previously described in this genus. The genetic context surrounding ampC genes was AmpR-AmpC-Blc-SugE. However, five different genetic contexts surrounding qnrB/ΔqnrB genes were observed, but they occurred in all cases between the pspF and sapA genes. Additionally, cloning experiments showed that the regions containing different qnrB alleles, even with different genetic contexts, contributed to the reduction of quinolone susceptibility. Our results showed that the chromosomal ampC and qnrB alleles are closely related to C. freundii. However, unlike ampC, qnrB alleles seemed to be related to the genetic contexts surrounding them. The evolution of these two genes in C. freundii isolates might be through different pathways.

Dissemination of the chromosomally encoded CMY-2 cephalosporinase gene in Escherichia coli isolated from animals.

In this study, 619 individual Escherichia coli isolates from food-producing and companion animals were analysed to determine the prevalence of the cephalosporinase gene blaCMY-2. In total, 18 CMY-2-producers (2.9%) were detected and exhibited multidrug-resistant phenotypes. One of the CMY-2-producers was found to possess a novel blaCMY-2-like allele, blaCMY-130. The isolates belonged to distinct pulsotypes, suggesting that the blaCMY-2 gene was not disseminated by clonal expansion of blaCMY-2-positive strains. The blaCMY-2 genes were located on IncA/C-, IncHI2- or IncX-type plasmids in 9 (50%) of the 18 E. coli isolates. However, in the other nine isolates I-CeuI-PFGE and hybridisation analyses revealed that the blaCMY-2 gene was chromosomally located. A CMY gene-containing region composed of five open reading frames (ORFs) (ISEcp1-blaCMY-2-blc-sugE-ΔencR) was observed in plasmids from eight strains. A CMY gene-containing region composed of ten ORFs was observed in all of the nine chromosomally encoded blaCMY-2 genes, including a putative IS66-like element inserted in this conserved CMY genetic region in three strains. This conserved CMY genetic region was also found to be inserted into the oriVγ (putative gamma origin), part of the IncX plasmid backbone, by a complete transposition unit flanked by 5-bp DRs (direct repeat sequence) in pS62T. These results demonstrate the high prevalence of the chromosomally encoded blaCMY-2 gene in E. coli. This is the first study reporting a chromosomally encoded blaCMY-2 gene in E. coli. Chromosomally encoded blaCMY-2 might be a source of some plasmid-mediated blaCMY-2 genes and this probably facilitates the spread of cephalosporin-resistant strains.

Complete nucleotide sequence of cfr-carrying IncX4 plasmid pSD11 from Escherichia coli.

We report the complete nucleotide sequence of a plasmid carrying the multiresistance gene cfr. This plasmid was isolated from an Escherichia coli strain of swine origin in 2011. This 37,672-bp plasmid, pSD11, had an IncX4 backbone similar to those of the IncX4 plasmids obtained from the United States and Australia, in which the cfr gene was flanked by two copies of IS26 and a truncated Tn1331 was inserted.

Characterization of plasmids carrying oqxAB in bla(CTX-M)-negative Escherichia coli isolates from food-producing animals.

To study the characteristics of plasmids harboring oqxAB among bla(CTX-M)-negative Escherichia coli isolates and search for oqxAB-harboring plasmids similar to plasmids carrying oqxAB-bla(CTX-M) reported previously, conjugation experiment was performed for 115 randomly selected oqxAB-positive but bla(CTX-M)-negative E. coli isolates from diseased animals in Guangdong, China. S1 nuclease pulsed-field gel electrophoresis (PFGE) and southern blotting experiments were performed to investigate the location of oqxAB and other resistance genes. The EcoRI digestion profiles of the plasmids with oqxAB were also analyzed. The clonal relatedness of donor isolates was investigated by PFGE. In this study, 32 oqxAB transconjugants were successfully obtained and most transconjugants showed multidrug resistances. Eleven replicon combination types were found in these transconjugants. floR and oqxAB were found on the same plasmids in all nine transconjugants resistant to florfenicol. The sequences between floR and oqxAB were identical in most transconjugants and the two genes were both linked with tnp in insertion sequences. Nine F18:A-:B1 plasmids with only oqxAB shared identical EcoRI digestion profiles and the profiles were also identical with that of a plasmid carrying oqxAB-bla(CTX-M) found previously. Co-transfer of plasmids carrying oqxAB and fosA3, respectively, was also observed in one isolate. This study demonstrates the dissemination of oqxAB among bla(CTX-M)-negative E. coli isolates was mainly mediated by identical F18:A-:B1 plasmids. A novel arrangement of regions between floR and oqxAB might play an important role in the dissemination of floR-oqxAB. This is the first description of the genetic environment of the relationship between oqxAB and floR in E. coli.

Comparison of plasmids coharboring 16s rrna methylase and extended-spectrum ß-lactamase genes among Escherichia coli isolates from pets and poultry.

A total of 247 Escherichia coli isolates (148 from diseased or dead poultry and 99 from diseased pets in the People's Republic of China) were screened for extended-spectrum ß-lactamase (ESBL) determinants by PCR and sequencing. Then, 16S rRNA methylase genes were detected among ESBL-producing isolates. Clonal relatedness of the E. coli isolates was examined by pulsed-field gel electrophoresis. Conjugation experiments were performed to investigate the association of 16S rRNA methylases and ESBLs, and plasmid contents were also characterized. Among 247 E. coli isolates, 74 (29.96%) isolates were positive for blaCTX-M genes, 42 from pets (12 from cats and 30 from dogs) and 32 from poultry (12 from chickens and 20 from ducks). The most common CTX-M type in isolates from pets was blaCTX-M-14, whereas blaCTX-M-27 was the most common for poultry. rmtB was dectected in 39 of the 74 blaCTX-M-positive isolates, 18 from pets and 21 from poultry. One strain from a pet was found to harbor blaCTX-M-14, blaCTX-M-15, and rmtB. blaCTX-M and rmtB were found to be colocated on the same transferable plasmid in 16 isolates. These genes were on the same or similar plasmids (eight F2:A-:B- and two IncN) in isolates from ducks, whereas they were colocated on the similar F2:A-:B- or similar F33:A-:B- plasmids in isolates of pets origin. In conclusion, similar F2:A-:B- plasmids and similar F33:A-:B- plasmids are responsible for the dissemination of both rmtB and blaCTX-Mgenes in E. coli isolates from poultry and pets, respectively.