Zhangliu Chen

High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants qnr, aac(6′)-Ib-cr, and qepA among Ceftiofur-Resistant Enterobacteriaceae Isolates from Companion and Food-Producing Animals

ABSTRACT Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants have been discovered and have been shown to be widely distributed among clinical isolates: qnr genes, aac(6′)-Ib-cr, and qepA. Few data on the prevalence of these determinants in strains from animals are available. The presence of PMQR genes in isolates from animals was determined by PCR amplification and DNA sequencing. The production of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in the strains was detected, and their genotypes were determined. The genetic environment of PMQR determinants in selected plasmids was analyzed. All samples of ceftiofur-resistant (MICs ≥ 8 μg/ml) isolates of the family Enterobacteriaceae were selected from 36 companion animals and 65 food-producing animals in Guangdong Province, China, between November 2003 and April 2007, including 89 Escherichia coli isolates, 9 Klebsiella pneumoniae isolates, and isolates of three other genera. A total of 68.3% (69/101) of the isolates produced ESBLs and/or AmpC β-lactamases, mainly those of the CTX-M and CMY types. Of the 101 strains, PMQR determinants were present in 35 (34.7%) isolates, with qnr, aac(6′)-Ib-cr, and qepA detected alone or in combination in 8 (7.9%), 19 (18.8%), and 16 (15.8%) strains, respectively. The qnr genes detected included one qnrB4 gene, four qnrB6 genes, and three qnrS1 genes. Five strains were positive for both aac(6′)-Ib-cr and qepA, while one strain was positive for qnrS1, aac(6′)-Ib-cr, and qepA. qnrB6 was flanked by two copies of ISCR1 with an intervening dfr gene downstream and sul1 and qacEΔ1 genes upstream. In another plasmid, aac(6′)-Ib-cr followed intI1 and arr-3 was downstream. PMQR determinants are highly prevalent in ceftiofur-resistant Enterobacteriaceae strains isolated from animals in China. This is the first report of the occurrence of PMQR determinants among isolates from companion animals.

Prevalence and Dissemination of oqxAB in Escherichia coli Isolates from Animals, Farmworkers, and the Environment

ABSTRACT OqxAB has recently been identified as one of the mechanisms of plasmid-mediated quinolone resistance (PMQR). Compared to what is observed for other PMQR determinants, there is a paucity of data with regard to the prevalence and epidemiology of OqxAB and its contribution to resistance to different antimicrobials. In this study, the prevalence and dissemination of oqxAB and other PMQR genes in Escherichia coli isolates from animals, farmworkers, and the environment in 2002 in China were investigated. Of the 172 E. coli isolates, 39.0% carried oqxA, while only 4.1%, 2.9%, and 0.6% carried qnr (1 qnrB6 isolate, 5 qnrS1 isolates, and 1 qnrD isolate), qepA, and aac(6′)-Ib-cr, respectively. Among the 33 isolates from farmworkers, 10 (30.3%) were positive for oqxA. oqxAB was associated with IS26 and was carried on the 43- to 115-kb IncF transferable plasmid. Transconjugants carrying oqxAB showed 4- to 16-fold increases in the MICs of quinolones, 16- to 64-fold increases in the MICs of quinoxalines, 8- to 32-fold increases in the MICs of chloramphenicol and trimethoprim-sulfamethoxazole, and 4- to 8-fold increases in the MICs of florfenicol compared to the levels for the recipient. The pulsed-field gel electrophoresis (PFGE) analysis showed that the high levels of prevalence and dissemination of oqxAB in E. coli in animal farms were primarily due to the transmission of plasmids carrying oqxAB, although clonal transmission between human and swine E. coli isolates was observed. It is concluded that oqxAB was widespread in animal farms in China, which may be due to the overuse of quinoxalines in animals. This study warrants the prudent use of quinoxalines in food animals.

Prevalence and characterisation of CTX-M β-lactamases amongst Escherichia coli isolates from healthy food animals in China

The impact of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration≥2 μg/mL). bla(CTX-M) genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n=40), CTX-M-55 (n=29) and CTX-M-65 (n=22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTX-M-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most bla(CTX-M) genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying bla(CTX-M), respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of bla(CTX-M) genes and may contribute to the dissemination and transfer of these β-lactamase genes throughout China.

Coprevalence of Plasmid-Mediated Quinolone Resistance Determinants QepA, Qnr, and AAC(6′)-Ib-cr among 16S rRNA Methylase RmtB-Producing Escherichia coli Isolates from Pigs

Plasmid-mediated quinolone resistance determinants, including Qnr peptides and AAC(6′)-Ib-cr, are increasingly identified worldwide among various clinical isolates of Enterobacteriaceae ([7][1], [9][2], [10][3]). Very recently, a novel plasmid-mediated fluoroquinolone-resistant determinant, QepA (

Dissemination of the Fosfomycin Resistance Gene fosA3 with CTX-M β-Lactamase Genes and rmtB Carried on IncFII Plasmids among Escherichia coli Isolates from Pets in China

ABSTRACT The presence and characterization of plasmid-mediated fosfomycin resistance determinants among Escherichia coli isolates collected from pets in China between 2006 and 2010 were investigated. Twenty-nine isolates (9.0%) were positive for fosA3, and all of them were CTX-M producers. The fosA3 genes were flanked by IS26 and were localized on F2:A−:B− plasmids or on very similar F33:A−:B− plasmids carrying both blaCTX-M-65 and rmtB. These findings indicate that the fosA3 gene may be coselected by antimicrobials other than fosfomycin.

Novel cyromazine imprinted polymer applied to the solid-phase extraction of melamine from feed and milk samples

A water compatible molecularly imprinted polymer (MIP) using cyromazine as a mimic template, methacrylic acid as the functional polymer and ethylene glycol dimethacrylate as the cross-linker was synthesized and used to extract melamine from feed and milk samples via a molecularly imprinted solid-phase extraction (MISPE) protocol. Optimum retention of melamine on the MISPE cartridge was achieved using methanol, and the interferences in the samples were effectively washed out. The binding capacity of the polymer toward melamine was found to be about 500 microg of melamine/g of polymer. The recoveries of 2 microg and 20 microg melamine standard spiked into water extract of blank feeds and milk samples were between 83.4% and 103%, with relative standard deviation <5.6%. The cyromazine-MIP demonstrated high cross-reactivity for melamine and low affinity to cyanuric acid. The ionic bond interaction was regarded as the main factors that dominated the retention of the melamine on the MISPE cartridge.

Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm

ABSTRACT In this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). bla CTX-M, bla SHV-12, bla CMY-2, and bla DHA-1 were identified in 108, 5, 9, and 1 isolates, respectively. The most common bla CTX-M genes were bla CTX-M-27 (n = 34), bla CTX-M-55 (n = 27), bla CTX-M-24e (n = 22), and bla CTX-M-105 (n = 20), followed by bla CTX-M-14a, bla CTX-M-14b, bla CTX-M-24a, and bla CTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. bla CTX-M genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene bla CTX-M-105 was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of bla CTX-M genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of bla CTX-M in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.

F33:A−:B− and F2:A−:B− Plasmids Mediate Dissemination of rmtB-blaCTX-M-9 Group Genes and rmtB-qepA in Enterobacteriaceae Isolates from Pets in China

ABSTRACT This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the blaCTX-M-9 group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A−:B−, carrying rmtB-qepA, and F33:A−:B−, carrying the rmtB-blaCTX-M-9 group genes (and especially blaCTX-M-65), shared restriction patterns within each incompatibility group.

Complete nucleotide sequence of pHN7A8, an F33:A-:B-type epidemic plasmid carrying blaCTX-M-65, fosA3 and rmtB from China

OBJECTIVES To characterize a representative self-transmissible multidrug resistance plasmid pHN7A8 isolated from an Escherichia coli from a dog in China, classified as F33:A-:B- by replicon sequence typing and carrying the bla(TEM-1b), bla(CTX-M-65), fosA3 and rmtB genes conferring resistance to penicillins, cephalosporins, fosfomycin and aminoglycosides, respectively. METHODS pHN7A8 was sequenced using a whole-genome shotgun approach and the sequence analysed by comparison with reference plasmids. RESULTS pHN7A8 is a circular molecule of 76 878 bp. bla(CTX-M-65), fosA3 and rmtB are found in known contexts, interspersed with different mobile elements including ISEcp1, IS1, Tn2, IS1294, IS903 and four copies of IS26. This multiresistance region has only a single nucleotide difference from that of pXZ, an F2:A-:B- plasmid isolated from poultry in China. The pHN7A8 backbone carries genes encoding addiction and partitioning systems that promote plasmid maintenance and has a similar organization to pXZ, as well as IncFII plasmids such as R100, pC15-1a/pEK516 and pHK23, isolated in Japan, Canada/the UK and China, respectively, but with varying levels of identity, suggesting recombination. CONCLUSIONS pHN7A8 is a chimera that may have resulted from the acquisition, by recombination in the plasmid backbone, of the multiresistance region found in pXZ. This region appears to have evolved from the resistance determinant R100 through the stepwise integration of multiple antimicrobial resistance determinants from different sources by the actions of mobile elements and recombination. The successful dissemination of this multidrug resistance plasmid presents further challenges for the prevention and treatment of Enterobacteriaceae infections.

Dissemination of IncFII plasmids carrying rmtB and qepA in Escherichia coli from pigs, farm workers and the environment

Fifty-one fluoroquinolone-resistant Escherichia coli isolates recovered from pigs, workers and environmental samples in one pig farm were screened for 16S rRNA methylase genes and qepA, a fluoroquinolone efflux pump gene, by PCR. Clonal relatedness of the E. coli isolates was examined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and phylogenetic analysis. Plasmids from the E. coli isolates were characterized by incompatibility group, restriction enzyme digestion and Southern hybridization analysis. The genetic environment of rmtB and qepA was also determined by PCR mapping. Eleven isolates that were highly resistant to amikacin and fluoroquinolones were positive for rmtB and qepA. All of these isolates belonged to phylogenetic group A, but most of them had different PFGE patterns or belonged to different sequence types (STs). Four isolates from different sources (two from pigs, one from a farm worker and one from an environmental sample) belonged to the same ST (ST160). Both rmtB and qepA were located on approximately 75-kb IncFII conjugative plasmids with nearly the same EcoRI digestion pattern. Tn3, IS26 and ISCR3 were found to be associated with rmtB and qepA. This study has found, for the first time, the transmission of rmtB and qepA among E. coli isolates from pigs, farm workers and the environment. Both horizontal transfer of IncFII plasmids and clonal dissemination have occurred and been seen to contribute to the dissemination of these resistance genes in a pig farm.