Bacteriophages, known for their ability to kill bacteria, are hampered in their effectiveness because bacteria are able to rapidly develop resistance, thereby posing a significant challenge for the efficacy of phage therapy. The impact of evolutionary trajectories on the long-term success of phage therapy remains largely unclear. Herein, we conducted evolutionary experiments, genomic analysis, and CRISPR-mediated gene editing, to illustrate the evolutionary trajectory occurring between phages and their hosts. Our results illustrate the ongoing "arms race" between a lytic phage and its host, a carbapenem-resistant Klebsiella pneumoniae clinical strain Kp2092, suggesting their respective evolutionary adaptations that shape the efficacy of phage therapy. Specifically, Kp2092 rapidly developed resistance to phages through mutations in a key phage receptor (galU) and bacterial membrane defenses such as LPS synthesis, however, this evolution coincides with unexpected benefits. Evolved bacterial clones not only exhibited increased sensitivity to clinically important antibiotics but also displayed a loss of virulence in an in-vivo model. In contrast, phages evolved under the selection pressure against Kp2092 mutants and exhibited enhanced bacterial killing potency, targeting mutations in phage tail proteins gp12 and gp17. These parallel evolutionary trajectories suggest a common genetic mechanism driving adaptation, ultimately favoring the efficacy of phage therapy. Overall, our findings highlight the potential of phages not only as agents for combating bacterial resistance, but also a driver of evolution outcomes that could lead to more favorable clinical outcomes in the treatment of multidrug resistance pathogens.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae represents one of the leading pathogens for infectious diseases. With traditional antibiotics often being ineffective, phage therapy has emerged as a promising alternative. However, phage predation imposes a strong evolutionary pressure on the rapid evolution of bacteria, challenging treatment efficacy. Our findings illustrate how co-evolution enhances phage lytic capabilities through accumulated mutations in the tail proteins gp12 and gp17, while simultaneously reducing bacterial virulence and antibiotic resistance. These insights advance our understanding of phage-host interactions in clinical settings, potentially inspiring new approaches akin to an "arms race" model to combat multidrug-resistant crises effectively.

ABSTRACT The global rise of antibiotic resistance has renewed interest in phage therapy, as an alternative to antibiotics to eliminate multidrug-resistant (MDR) bacterial pathogens. However, optimizing the broad-spectrum efficacy of phage therapy remains a challenge. In this study, we addressed this issue by employing strategies to improve antimicrobial efficacy of phage therapy against MDR Klebsiella pneumoniae strains, which are notorious for their resistance to conventional antibiotics. This includes the selection of broad host range phages, optimization of phage formulation, and combinations with last-resort antibiotics. Our findings unveil that having a broad host range was a dominant trait of isolated phages, and increasing phage numbers in combination with antibiotics significantly enhanced the suppression of bacterial growth. The decreased incidence of bacterial infection was explained by a reduction in pathogen density and emergence of bacterial resistance. Furthermore, phage-antibiotic synergy (PAS) demonstrated considerable broad-spectrum antibacterial potential against different clades of clinical MDR K. pneumoniae pathogens. The improved treatment outcomes of optimized PAS were also evident in a murine model, where mice receiving optimized PAS therapy demonstrated a reduced bacterial burden in mouse tissues. Taken together, these findings offer an important development in optimizing PAS therapy and its efficacy in the elimination of MDR K. pneumoniae pathogens.IMPORTANCEThe worldwide spread of antimicrobial resistance (AMR) has posed a great challenge to global public health. Phage therapy has become a promising alternative against difficult-to-treat pathogens. One important goal of this study was to optimize the therapeutic efficiency of phage-antibiotic combinations, known as phage-antibiotic synergy (PAS). Through comprehensive analysis of the phenotypic and genotypic characteristics of a large number of CRKp-specific phages, we developed a systematic model for phage cocktail combinations. Crucially, our finding demonstrated that PAS treatments not only enhance the bactericidal effects of colistin and tigecycline against multidrug-resistant (MDR) K. pneumoniae strains in in vitro and in vivo context but also provide a robust response when antibiotics fail. Overall, the optimized PAS therapy demonstrates considerable potential in combating diverse K. pneumoniae pathogens, highlighting its relevance as a strategy to mitigate antibiotic resistance threats effectively.

Heat stress is a common environmental factor in livestock breeding that has been shown to impact the development of antibiotic resistance within the gut microbiota of both human and animals. However, studies investigating the effect of temperature on antibiotic resistance in Enterococcus isolates remain limited. In this study, specific pathogen free (SPF) mice were divided into a control group maintained at normal temperature and an experimental group subjected to daily 1-h heat stress at 38 degrees C, respectively. Gene expression analysis was conducted to evaluate the activation of heat shock responsive genes in the liver of mice. Additionally, the antibiotic-resistant profile and antibiotic resistant genes (ARGs) in fecal samples from mice were analyzed. The results showed an upregulation of heat-inducible proteins HSP27, HSP70 and HSP90 following heat stress exposure, indicating successful induction of cellular stress within the mice. Furthermore, heat stress resulted in an increase in the proportion of erythromycin-resistant Enterococcus isolates, escalating from 0 % to 0.23 % over a 30-day duration of heat stress. The resistance of Enterococcus isolates to erythromycin also had a 128-fold increase in minimum inhibitory concentration (MIC) within the heated-stressed group compared to the control group. Additionally, a 2 similar to 8-fold rise in chloramphenicol MIC was observed among these erythromycin-resistant Enterococcus isolates. The acquisition of ermB genes was predominantly responsible for mediating the erythromycin resistance in these Enterococcus isolates. Moreover, the abundance of macrolide, lincosamide and streptogramin (MLS) resistant-related genes in the fecal samples from the heat-stressed group exhibited a significant elevation compared to the control group, primarily driven by changes in bacterial community composition, especially Enterococcaceae and Planococcaceae, and the transfer of mobile genetic elements (MGEs), particularly insertion elements. Collectively, these results highlight the role of environmental heat stress in promoting antibiotic resistance in Enterococcus isolates and partly explain the increasing prevalence of erythromycin-resistant Enterococcus isolates observed among animals in recent years.

Arthropods, such as houseflies, play a significant role in the dissemination of antimicrobial resistance (AMR); however, their impact has often been overlooked in comparison to other AMR vectors. Understanding the contribution of arthropods to the spread of AMR is critical for implementing robust policies to mitigate the spread of AMR across One Health sectors, affecting animals and environmental habitats as well as humans. In this study, we investigated the in situ transfer of a gfp-labelled AMR plasmid (IncA/C carrying an mcr-8 gene, pA/C_MCR-8) in the gut microbiota of houseflies (Musca domestica) by applying single-cell sorting, 16S rRNA gene amplicon sequencing and whole-genome sequencing. Our findings demonstrate that the pA/C_MCR-8-positive Escherichia coli donor strain is capable of colonizing the gut microbiome of houseflies and persists in the housefly intestine for 5 days; however, no transfer was detectable above the detection threshold of 10-5 per cell. The conjugative plasmid pA/C_MCR-8 demonstrated a high transfer frequency ranging from 4.1 x 10-3 to 5.0 x 10-3 per cell in vitro and exhibited transfer across various bacterial phyla, primarily encompassing Pseudomonadota and Bacillota. Phylogenic analysis has revealed that Providencia stuartii, a human opportunistic pathogen, is a notable recipient of pA/C_MCR-8. The conjugation assays further revealed that newly formed P. stuartii transconjugants readily transfer pA/C_MCR-8 to other clinically relevant pathogens (e.g. Klebsiella pneumoniae). Our findings indicate the potential transfer of AMR plasmids from houseflies to human opportunistic pathogens and further support the adoption of a One Health approach in developing infection control policies that address AMR across clinical settings.

IntroductionMatrine (MT) is a potential resistance reversal agent. However, it remains unclear whether MT can reverse the resistance of Haemophilus parasuis (H. parasuis) to β-lactams, and, if so, by what mechanism MT works.MethodsWe screened one cefaclor (CEC)-resistant strain (clinical strain C7) from eight clinical (H. parasuis) strains and determined the underlying resistance mechanism. Then, we investigated the reversal effect of MTon the resistance of this strain to CEC.Results and DiscussionThe production of β-lactamase, overexpression of AcrAB-TolC system, and formation of biofilm might not be responsible for the resistance of clinical strain C7 to CEC. Fourteen mutation sites were found in four PBP genes (ftsI, pbp1B, mrcA, and prcS) of clinical strain C7, among which the mutation sites located in ftsI (Y103D and L517R) and mrcA (A639V) genes triggered the resistance to CEC. The minimum inhibitory concentration (MIC) of CEC against clinical strain C7 was reduced by two to eight folds after MT treatment, accompanied by the significant down-regulated expression of mutated ftsI and mrcA genes. Based on such results, we believed that MT could reverse the resistance of H. parasuis to CEC by inhibiting the mutations in ftsI and mrcA genes. Our research would provide useful information for restoring the antimicrobial activity of β-lactams and improving the therapeutic efficacy of Glässer’s disease.

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.

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-β-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.

Aminoacyl tRNA synthetases primarily function to attach specific amino acids to the corresponding tRNAs during protein translation. However, their roles in regulating plant growth and development still remain elusive. Here we reported a rice thermo-sensitive mutant yellow leaf chlorosis3 (ylc3) with reduced chlorophyll content, altered thylakoid structure, and substantially elevated levels of free aspartate, asparagine and glutamine in leaves under low temperature condition. Map-based cloning identified that YLC3 encodes an aspartyl-tRNA synthetase which is localized in cytosol and mitochondria. In addition, quantitative proteomics analysis revealed that both nuclear and chloroplast-encoded thylakoid proteins were significantly down-regulated in the mutant. On the other hand, proteins involved in amino acid metabolism and the process of protein synthesis were up-regulated in ylc3, particularly for key enzymes that convert aspartate to asparagine. Moreover, uncharged tRNA-Asp accumulation and phosphorylation of the translation initiation factor eIF2α was detected in the mutant, suggesting that YLC3 regulates the homeostasis of amino acid metabolism and chloroplast thylakoid development through modulation of processes during protein synthesis.

The chloramphenicol-florfenicol resistance gene cfr, also called as a multiresistance gene, not only confers phenicols and streptogramin A but also lincosamides, oxazolidinones and pleuromutilins antibiltics.1 The cfr gene has been detected in isolates of Gram-positive and -negative genera such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Jeotgalicoccus, Macrococcus, Mammaliicoccus, Escherichia, Proteus, Morganella, Pasteurella, Providencia, Vibrio and Leclercia.2–4 The cfr gene is not only found on the chromosomes, but also found on the plasmids. Studies have shown those plasmids can play a vital role in the dissemination of cfr in both Gram-negative and Gram-positive genera.5 Recently, an ∼40-kb conjugative plasmid pFJ6683 carrying cfr gene was identified by our group.6 Here, we report the complete nucleotide sequence of this plasmid. A unique plasmid pFJ6683 was extracted from the Pasteurella multocida FJ6683 with Qiagen plasmid extraction kits (Qiagen, Hilden, Germany). Sequencing was performed using the PacBio RSII sequencer, and data were assembled by Canu. Gene prediction and annotation were performed with the RAST tool.7 The sequence comparison and mapping were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and Easyfig v.2.1.8

Objectives: The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. Methods: A total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, 51 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. Results: Among 113 P. multocida isolates, strains F16671 and F16683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an similar to 40-kb conjugative plasmid in different genetic environments, including ISApl1(2)-cfr-IS26 and IS26-cfr-IS256. Conclusions: These results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted. (C) 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr-positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr-positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1-PFGE and Southern blotting. A 12 kb cfr-positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr-carrying ISEnfa5-composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr.

IncX4 plasmids are associated with the dissemination of the mcr-1 genes in Enterobacteriaceae. We screened IncX4 plasmids among 2,470 isolates of Enterobacteriaceae and determined the mcr-1 positive isolates. Forty-three isolates were observed to carry IncX4 type plasmid, among which 13 were identified to carry mcr-1 gene. Three representative mcr-1 -positive IncX4 plasmids were selected for high-throughput sequencing. Comparative genomics showed that the mcr-1 -carrying IncX4 plasmids exhibit remarkable similarity in the backbone, and the major distinction lies in the region containing mcr-1 . The major variable regions of all the IncX4 plasmids were fully characterized by PCR-RFLP. The results revealed that the mcr-1 was located on the Variable Region I of IncX4 plasmids in 11 E. coli isolates. Among them, nine E. coli strains possess an epidemic pCSZ4-like IncX4 plasmid containing mcr-1 . IS Apl1 was presumably involved in the transposition of the mcr-1 cassette and then was lost. Similar genetic contexts were found in different plasmids, even the E. coli chromosome, implying the acquisition of mcr-1 by a unique common mechanism.

ABSTRACT 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 [ bla CTX-M-55 , oqxA , oqxB , fosA3 , floR , tetA (A), tetA (R), strA , strB , sul2 , aph(3′)-II , and Δ bla TEM-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.

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.

ABSTRACT 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 IS 26 and a truncated Tn 1331 was inserted.

The association of ESBLs (extended-spectrum beta-lactamases)/pAmpCs (plasmid-mediated AmpC β-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.

During a regular monitoring of antimicrobial resistance in a farrowing farm in Southern China, 117 Escherichia coli isolates were obtained from sows and piglets. Compared with the isolates from piglets, the isolates from sows exhibited higher resistance rates to the tested cephalosporins. Correspondingly, the total detection rate of the bla CMY-2/bla CTX-M genes in the sow isolates (34.2%) was also significantly higher than that of the piglet isolates (13.6%; p < 0.05). The bla CMY-2 gene had a relatively high prevalence (11.1%) in the E. coli isolates. MLST and PFGE analysis revealed the clonal spread of ST1121 E. coli in most (7/13) of the bla CMY-2-positive isolates. An indistinguishable IncHI2 plasmid harboring bla CMY-2 was also identified in each of the seven ST1121 E. coli isolates. Complete sequence analysis of this IncHI2 plasmid (pEC5207) revealed that pEC5207 may have originated through recombination of an IncHI2 plasmid with a bla CMY-2-carrying IncA/C plasmid like pCFSAN007427_01. In addition to bla CMY-2, pEC5207 also carried other resistance determinants for aminoglycosides (aacA7), sulfonamides (sul1), as well as heavy metals ions, such as Cu and Ag. The susceptibility testing showed that the pEC5207 can mediate both antibiotic and heavy metal resistance. This highlights the role of pEC5207 in co-selection of bla CMY-2-positive isolates under the selective pressure of heavy metals, cephalosporins, and other antimicrobials. In conclusion, clonal spread of an ST1121 type E. coli strain harboring an IncHI2 plasmid contributed to the dissemination of bla CMY-2 in a farrowing farm in Southern China. We also have determined the first complete sequence analysis of a bla CMY-2-carrying IncHI2 plasmid.

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.

A minoglycoside antibiotics such as amikacin and gentamicin, often along with (cid:2) -lactams, have been used for the treatment of serious infections caused by Gram-negative and Gram-positive pathogenic bacteria in clinical settings (1). However, bacteria have been furnished with various resistance mechanisms to become resistant to aminoglycosides (2). Recently, the spread of 16S rRNA methyltransferase (16S-RMTase), responsible for very-high-level resistance to various aminoglycosides (MIC (cid:3) 256 (cid:4) g/ml), has become apparent in both human and veterinary medicine (1). Since the first report in 2003, 10 16S-RMTase-encoding genes, named rmtA , rmtB , rmtC , rmtD , rmtE , rmtF , rmtG , rmtH , armA , and npmA , have been identified (3). Of the known 16S RMTases, rmtE has been reported in only two Escherichia coli strains, which were identified from a calf and a patient in the United States, respectively (4, 5). Here, we reported the first rmtE -producing E. coli strain isolated from swine in China. We the by random extension-based two-step PCR (RETS-PCR) (14), and the result indicated that Vs1 -like transposase was downstream of rmtE2

Previous study indicated that the multi-resistance gene cfr was mainly found in gram-positive bacteria, such as Staphylococcus and Enterococcus, and was sporadically detected in Escherichia coli. Little is known about the prevalence and transmission mechanism of cfr in E. coli. In this study, the presence of cfr in E. coli isolates collected during 2010–2012 from food-producing animals in Guangdong Province of China was investigated, and the cfr-positive E. coli isolates were characterized by PFGE, plasmid profiling, and genetic environment analysis. Of the 839 E. coli isolates, 10 isolates from pig were cfr positive. All the cfr-positive isolates presented a multi-resistance phenotype and were genetically divergent as determined by PFGE. In 8 out of the 10 strains, the cfr gene was located on plasmids of ∼30 kb. Restriction digestion of the plasmids with EcoRI and sequence hybridization with a cfr-specific probe revealed that the cfr-harboring fragments ranged from 6 to 23 kb and a ∼18 kb cfr-carrying fragment was common for the plasmids that were ∼30 kb. Four different genetic environments of cfr were detected, in which cfr is flanked by two identical copies of IS26, which may loop out the intervening sequence through homologous recombination. Among the 8 plasmids of ∼30 kb, 7 plasmids shared the same genetic environment. These results demonstrate plasmid-carried cfr in E. coli and suggest that transposition and homologous recombination mediated by IS26 might have played a rule in the transfer of the cfr gene in E. coli.