Xiao-Ping Liao

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.

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.

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.

Co-transfer of blaNDM-5 and mcr-1 by an IncX3-X4 hybrid plasmid in Escherichia coli.

Carbapenem and colistin are the last-resort antibiotics used for treating multidrug-resistant Gram-negative pathogens. Here, we report, for the first time, co-transfer of resistance to both classes of antibiotics by a mobile IncX3–X4 hybrid plasmid in an Escherichia coli isolate. Spread of such a plasmid is of great concern for clinical therapy, and heightened efforts are needed to control its dissemination.

Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6′)-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.

Antimicrobial resistance, virulence genes, and phylogenetic background in Escherichia coli isolates from diseased pigs

Escherichia coli isolates from diseased pigs were examined for antimicrobial susceptibility to 12 antimicrobials and possession of virulence genes (VGs), and then grouped according to the phylogenetic background and genetic relatedness. Associations between antimicrobial resistance (AMR) and VGs and between AMR and phylogenetic group were subsequently assessed. The results showed that most isolates (91%) were epidemiologically unrelated. Multiple antimicrobial-resistant phenotypes (>or=5 antimicrobials) were observed in 89% of E. coli strains and the most frequent types of resistance were to sulfamethoxazole (95%), tetracycline (94%), chloramphenicol (89%), and streptomycin (84%). The majority of isolates belonged to phylogenetic group A (84%). The most prevalent VG was EAST1 (64%), followed by Stx2e (63%) and eae (47%). Resistance to ceftiofur was associated with the presence of certain VGs, whereas resistance to doxycycline and kanamycin was associated with the absence of certain VGs. These findings suggest that multidrug resistance phenotypes, a variety of VGs, and the clear associations between resistance and VGs are commonly present in E. coli strains from diseased pigs. These results indicate that there is a great need for surveillance programs in China to monitor AMR in pathogenic E. coli strains.

Prevalence of Serogroups, Virulence Genotypes, Antimicrobial Resistance, and Phylogenetic Background of Avian Pathogenic Escherichia coli in South of China

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. A variety of virulence-associated genes and serogroups are associated with avian colibacillosis caused by APEC strains. One hundred forty-eight E. coli isolates recovered from diagnosed cases of avian colibacillosis from Guangdong province between 2005 and 2008 were serotyped, and characterized for virulence-associated genes, phylogenetic backgrounds, antibiotic susceptibility, and genetic relatedness. Associations between virulence-associated genes and antimicrobial resistance were further analyzed. Although 148 APEC isolates belonged to 21 different serogroups, 81% were of one of eight serogroups: O65 (27%), O78 (10%), O8 (9%), O120 (9%), O2 (7%), O92 (6%), O108 (5%), and O26 (5%). Polymerase chain reaction analysis showed that the most prevalent gene was traT (90%), followed by iroN (63%), fimH (58%), hlyF (55%), cvaC (54%), and sitA (51%). The APEC strains mainly belonged to groups A (73%) and D (14%). Multiple antimicrobial-resistant phenotypes (greater than or equal to three antimicrobials) were detected in all E. coli isolates, with the majority of isolates displaying resistance to tetracycline (97%), sulfamethoxazole (93%) and fluoroquinolones (87% for ciprofloxacin and 84% for enrofloxacin), chloramphenicol (74%), and florfenicol (66%). All E. coli isolates were further genetically characterized by pulsed-field gel electrophoresis. A total of 125 different pulsed-field gel electrophoresis profiles were obtained, implying that the multiresistant E. coli isolates carrying virulence-associated genes and belonging to multiple serogroups were not derived from a specific clone, but represented a wide variety of chromosomal backgrounds. Statistical analysis showed that several virulence-associated genes were significantly present in APEC isolates susceptibility to multiple antimicrobials. The findings demonstrate that a wide variety of serogroups and potential virulence genes, multiple-resistances, and the clear association of susceptibility and virulence genes have commonly emerged in APEC strains, and these also suggest that antimicrobials should be prudently used to reduce the emergence and spread of resistant strains carrying virulence-associated genes.

Deciphering MCR-2 Colistin Resistance

ABSTRACT Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2. Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus. Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1. Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4′ position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition

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. ISApl1 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.