Ya-Hong Liu

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.

Isolation and identification of nitrogen‐fixing bacilli from plant rhizospheres in Beijing region

Aims:  To isolate and identify nitrogen‐fixing bacilli from the plant rhizospheres in Beijing region of China.

Dissemination and Characterization of Plasmids Carrying oqxAB-blaCTX-M Genes in Escherichia coli Isolates from Food-Producing Animals

Background The association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and bla CTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids. Methods The ESBL-encoding genes (bla CTX-M, bla TEM and bla SHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and bla CTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-bla CTX-M were also analyzed. The clonal relatedness was investigated by PFGE. Results Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried bla CTX-M, with bla CTX-M-14 the most common. We observed a significant higher prevalence of bla CTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and bla CTX-M was found in 18 of the 127 isolates carrying oqxAB-bla CTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination. Conclusion bla CTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-bla CTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, bla CTX-M, and floR on the same plasmids in E. coli.

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.

Emergence of NDM-5- and MCR-1-Producing Escherichia coli Clones ST648 and ST156 from a Single Muscovy Duck (Cairina moschata)

ABSTRACT Two Escherichia coli clones (sequence type 648 [ST648] and ST156) that coproduce NDM-5 and MCR-1 were detected from a single fowl in China. The blaNDM-5 gene was found on the two indistinguishable IncX3 plasmids from the two different E. coli isolates, whereas the mcr-1 gene was located on IncHI2 and IncI2 plasmids, respectively, suggesting that blaNDM-5 and mcr-1 have spread in avian intestinal flora. Also, the two strains harbor blaTEM-1, blaCTX-M-55, fosA3, and aac(6′)-Ib. The multiresistant E. coli strains (especially the epidemic clone ST648) might raise a potential threat to human health via food chain transmission.

Increasing prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in food animals and the diversity of CTX-M genotypes during 2003-2012

The aim of this study was to investigate the trends and the diversity of CTX-M types of extended-spectrum β-lactamase (ESBL) in Escherichia coli isolated from food animals in China over a ten-year period. From 2003 to 2012, 2815 E. coli isolates collected from diseased animals (chickens, pigs, and waterfowl) were screened for the prevalence of CTX-M genes. CTX-M-positive isolates were tested for their susceptibilities to 10 antimicrobial agents and the clonal relationship of CTX-M-producing E. coli isolates was also assessed. Overall, 677 (20.1%) of the 2815 E. coli isolates carried CTX-M genes. Eighteen different types of CTX-M ESBLs were identified, with CTX-M-14, CTX-M-55, and CTX-M-65 being the most dominant genotypes. The occurrence of CTX-M-producing E. coli increased significantly from 5.7% in 2003-2005 to 35.3% in 2009-2012 (p<0.0001). High genetic heterogeneities were observed in the CTX-M-producing E. coli isolates. Most CTX-M-producing strains were also resistant to other classes of antimicrobials. Compared to isolates carrying CTX-M-9 subgroup of ESBLs, isolates carrying CTX-M-1 subgroup ESBLs showed significantly higher resistance rates to ceftazidime, amikacin, and fosfomycin (p<0.01). The study reported the dramatic increase of CTX-M ESBLs in E. coli isolated from animals overtime in China. The increasing incidence of CTX-M-55 with high hydrolytic activity against ceftazidime and the widely spread co-resistance in CTX-M-producing isolates alarm the serious antimicrobial resistance situation in China and highlight the need for urgent control strategies to limit the dissemination of those resistant genes in China.

Prevalence and characterization of methicillin-resistant Staphylococcus pseudintermedius in pets from South China.

The aim of this study was to determine the presence of and characterize methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from pets in South China. From 2007 to 2009, 898 samples were collected from 785 pets in Guangdong Province. The identity of staphylococcal species and the presence of methicillin resistance were confirmed by phenotypic and genotypic assays. The genetic relationships of MRSP isolates were determined by multilocus sequence typing (MLST), PFGE and spa typing. SCCmec elements and antimicrobial resistance genes profiling were characterized by PCR amplification. A total of 144 S. pseudintermedius isolates were recovered from the dogs and cats tested, and 69 (47.9%) of these isolates were identified as MRSP. Most of the MRSP isolates exhibited simultaneous resistance to four or more different antimicrobial agents. However, valnemulin showed robust activity against MRSP (MIC(90)=1 μg/ml). Integron 1, 2 and 3 were not detected in MRSP isolates. Twenty-four different multilocus sequence types were found among the MRSP isolates, with ST4 (n=9), ST5 (n=8), and ST95 (n=7) being dominant sequence types. In addition, 8 new sequence types (ST134, 135, 136, 137, 138, 139, 140 and 148) were identified. Of the 69 MRSP isolates, SCCmecV was the most prevalent type (n=33), followed by SCCmecVII (n=13), SCCmecII-III (n=7), and SCCmecIII (n=4). This study demonstrates for the first time that the occurrence of MRSP in healthy pets in China and shows that MRSP in South China has high genetic diversity.

Plasmid-mediated quinolone resistance determinants oqxAB and aac(6′)-Ib-cr and extended-spectrum β-lactamase gene blaCTX-M-24 co-located on the same plasmid in one Escherichia coli strain from China

Sir, With the common use of fluoroquinolones in both human and animal diseases, fluoroquinolone resistance in veterinary clinics has become an important public health problem since the discovery of horizontally transmissible elements, such as plasmids. To date, a number of plasmid-mediated quinolone resistance (PMQR) determinants have been described: the qnr genes (A, B, S, C and D); the aac(6′)-Ib-cr gene; and the qepA gene. However, OqxAB, a plasmid-encoded multidrug efflux pump that confers reduced susceptibility to multiple agents, including fluoroquinolones, was not recognized as a PMQR determinant until recently. The PMQR determinants can confer only low-level resistance to quinolones; however, they can facilitate the selection of resistant mutants upon exposure to ciprofloxacin. With the development of both quinolone resistance and b-lactam resistance, many studies on the association of PMQR genes and extended-spectrum b-lactamases (ESBLs) have been reported. There is a paucity of data with regard to the association of oqxAB and other resistance genes, although other PMQR genes were often found to be co-carried with genes encoding ESBLs or AmpCtype b-lactamases on the same plasmid. In this study, we investigated the dissemination mechanism of oqxAB and the coexistence of oqxAB and genes encoding ESBLs in an Escherichia coli strain. E. coli strain a6 was isolated from a liver sample of diseased chicken from a farm in Guangdong Province, China. Susceptibility to 15 antibiotics was measured by agar dilution methods and an ESBL production test was also performed according to the guidelines provided by the CLSI (2008). The genes blaCTX-M-9 group, blaTEM, qepA, qnrA, qnrB, qnrS, aac(6′)-Ib-cr, gyrA and parC were detected and sequenced by specific PCRs using previously described primers. – 7 The primers used for oqxA and oqxB were as follows: oqxA-F (5′-CTTGCACTTAGTTAAGCGCC-3′) and oqxA-R (5′-GAGGTTTTGATAGTGGAGGTAGG-3′) for oqxA; and oqxB-F (5′-GCGGTGCTGTCGATTTTA-3′) and oqxB-R (5′-TACCGGAA CCCATCTCGAT -3′) for oqxB. E. coli strain a6 showed a multidrug resistance phenotype and was positive for the ESBL production test. The high MICs of nalidixic acid, ciprofloxacin, olaquindox, chloramphenicol, ampicillin and cefotaxime were .512, 512, 128, 256, 512 and .256 mg/L, respectively. Amino acid changes were detected in both GyrA (Ser83Leu Asp87Tyr) and ParC (Ser80Ile) proteins. The genes qepA1, oqxAB and aac(6′)-Ib-cr were all detected in E. coli a6 harbouring the genes encoding CTX-M-24 and TEM-1b b-lactamases. A conjugation experiment was performed to investigate whether these genes were located on plasmids and whether the transfer of these genes contributed to the reduced susceptibility of the recipient E. coli towards antibiotics, by using E. coli C600 (streptomycin resistant) as the recipient. The transconjugants showed a multidrug resistance phenotype that included resistance to nalidixic acid, ampicillin, cefotaxime, trimethoprim/sulfamethoxazole, tetracycline and ceftiofur. Transconjugants showed 8-, 4and 8-fold increases in the MICs of nalidixic acid, ciprofloxacin and enrofloxacin, respectively, when compared with the recipient strain. With regard to the MICs of olaquindox and chloramphenicol, they both showed 8-fold increases. For the b-lactam antibiotics, the transconjugants showed 128-, 2048-, 32-, 256and 4-fold increases in the MICs of ampicillin, cefotaxime, ceftazidime, ceftiofur and cefoxitin, respectively, when compared with the recipient. The transconjugant strain a6T was detected to harbour oqxAB, blaCTX-M-24, blaTEM-1b and aac(6′)-Ib-cr simultaneously, while qepA and any amino acid changes in GyrA or ParC proteins were not detected. Southern blot hybridization was performed with digoxigenin-labelled probes specific for oqxB, blaCTX-M-24 and aac(6′)-Ib-cr. Notably, the results shown in Figure 1 revealed the coexistence of oqxAB, blaCTX-M-24 and aac(6′)-Ib-cr on the same plasmid of 54 kb in E. coli a6 and its transconjugant, a6T. Analysis of the genetic environment of the blaCTX-M-24 gene showed that the sequence upstream of it contained the ISEcp1 element; the orf477 sequence and the virulence gene iroN, a urovirulence factor in E. coli, were located downstream of the b-lactamase gene. According to their plasmid incompatibility group, plasmids were classified using the method described by Carattoli, and the conjugative plasmid harbouring blaCTX-M-24 in this study was found to

Complete Nucleotide Sequence of an IncI2 Plasmid Coharboring blaCTX-M-55 and mcr-1

ABSTRACT We report the complete nucleotide sequence of a plasmid, pA31-12, carrying blaCTX-M-55 and mcr-1 from a chicken Escherichia coli isolate. pA31-12 has an IncI2 replicon that displays extensive sequence similarity with pHN1122-1-borne blaCTX-M-55 and pHNSHP45-borne mcr-1. Insertion sequences ISEcp1 and ISApl1 are responsible for the mobilization of blaCTX-M-55 and mcr-1, respectively. The colocalization of mcr-1 with an extended-spectrum β-lactamase gene on a conjugative plasmid may accelerate the dissemination of both genes by coselection.