Bao-Tao Liu

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.

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

Development of aminoglycoside and β-lactamase resistance among intestinal microbiota of swine treated with lincomycin, chlortetracycline, and amoxicillin

Lincomycin, chlortetracycline, and amoxicillin are commonly used antimicrobials for growth promotion and infectious disease prophylaxis in swine production. In this study, we investigated the shifts and resistance development among intestinal microbiota in pregnant sows before and after lincomycin, chlortetracycline, and amoxicillin treatment by using phylogenetic analysis, bacterial enumeration, and PCR. After the antimicrobial treatment, shifts in microbial community, an increased proportion of resistant bacteria, and genes related to antimicrobial resistance as compared to the day before antimicrobial administration (day 0) were observed. Importantly, a positive correlation between antimicrobial resistance gene expression in different categories, especially those encoding aminoglycoside and β-lactamase and antimicrobial resistance, was observed. These findings demonstrate an important role of antimicrobial usage in animals in the development of antimicrobial resistance, and support the notion that prudent use of antimicrobials in swine is needed to reduce the risk of the emergence of multi-drug resistant zoonotic pathogens.

Prevalence and Plasmid Characterization of the qnrD Determinant in Enterobacteriaceae Isolated from Animals, Retail Meat Products, and Humans

qnrD, unlike other qnr genes, is mainly located on small nonconjugative plasmids. We investigated the presence of qnrD among 1,373 Enterobacteriaceae isolates in China. Twelve qnrD-positive strains were detected, and all were nonsusceptible to fluoroquinolones. The complete sequence of plasmids showed that the qnrD determinants were located on two plasmids with a respective size of ~4.2 and 2.7 k-bp. Interestingly, the identification of qnrD in this study revealed the highest prevalence of Proteeae among Enterobacteriaceae identified.

Quantification of lincomycin resistance genes associated with lincomycin residues in waters and soils adjacent to representative swine farms in China.

Lincomycin is commonly used on swine farms for growth promotion as well as disease treatment and control. Consequently, lincomycin may accumulate in the environment adjacent to the swine farms in many ways, thereby influencing antibiotic resistance in the environment. Levels of lincomycin-resistance genes and lincomycin residues in water and soil samples collected from multiple sites near wastewater discharge areas were investigated in this study. Sixteen lincomycin-resistance and 16S rRNA genes were detected using real-time PCR. Three genes, lnu(F), erm(A), and erm(B), were detected in all water and soil samples except control samples. Lincomycin residues were determined by rapid resolution liquid chromatography-tandem mass spectrometry, with concentrations detected as high as 9.29 ng/mL in water and 0.97 ng/g in soil. A gradual reduction in the levels of lincomycin-resistance genes and lincomycin residues in the waters and soils were detected from multiple sites along the path of wastewater discharging to the surrounding environment from the swine farms. Significant correlations were found between levels of lincomycin-resistance genes in paired water and soil samples (r = 0.885, p = 0.019), and between lincomycin-resistance genes and lincomycin residues (r = 0.975, p < 0.01). This study emphasized the potential risk of dissemination of lincomycin-resistance genes such as lnu(F), erm(A), and erm(B), associated with lincomycin residues in surrounding environments adjacent to swine farms.

Co-location of the erm(T) gene and blaROB-1 gene on a small plasmid in Haemophilus parasuis of pig origin

Sir, Haemophilus parasuis is the causative agent of Glässer’s disease, characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs, which causes considerable economic losses in the swine industry. Macrolide antibiotics inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Although macrolides are widely used in the treatment of Gram-positive infections, they are also of clinical relevance in the treatment of infections caused by Gram-negative bacteria such as H. parasuis. The major mechanism of resistance to macrolides is the methylation of the adenine at position A2058 in domain V of the 23S rRNA and the methylases encoded by erm genes generally confer cross-resistance to macrolide, lincosamide and streptogramin B (MLSB) antibiotics (http://faculty.washington.edu/ marilynr/). In the present study, we report that the erm(T) gene is present in a blaROB-1-positive H. parasuis isolated in China. So far, the erm(T) gene has been described only in lactobacilli, streptococci, enterococci and staphylococci, – 6 all of which are Gram-positive bacteria. A total of 145 H. parasuis isolates were isolated from pigs suffering from polyserositis, pneumonia or meningitis in China during August 2010 to July 2011. Isolates were confirmed by biochemical tests and 16S diagnostic PCR. Due to the unavailability of a CLSI-approved method for H. parasuis, susceptibility tests were conducted using the broth microdilution method according to the CLSI recommendations for Actinobacillus pleuropneumoniae. The reference strain A. pleuropneumoniae ATCC 27090 served as a quality control strain. Among the 145 H. parasuis isolates, only strain FS39 exhibited high MICs of erythromycin (64 mg/L), lincomycin (64 mg/L), penicillin (32 mg/L), amoxicillin (256 mg/L) and cefaclor (32 mg/L), but showed low MIC values of ceftiofur (,0.064 mg/L) and cefotaxime (,0.064 mg/L). Subsequently, PCR screening for the genes erm(A), erm(B), erm(C), erm(F) and mef(A), which have been reported to occur in Research letters

Population pharmacokinetics of cefquinome in pigs

This study was performed in 145 pigs to develop a population pharmacokinetics (PPK) model by i.m. administration of cefquinome (CEQ) at the dose of 2 mg/kg in the neck muscle. Serum physiological and biochemical parameters for each pig were determined before administration. After administration, 2-4 samples were collected at random, with the sampling point evenly distributed in the three periods (<1 h, 1-4 h and >4 h). The plasma concentration of CEQ was determined by high performance liquid chromatography with UV detector. The pharmacostatistical analyses of concentration-time data, weight, age, gender, serum physiological and biochemical parameters were performed with nonlinear mixed effect modeling (NONMEM). A one-compartmental model with first-order absorption and elimination adequately described the data from the study group. The optimal random effect model of pharmacokinetics parameters was of log-normal distribution and the residual errors assumed a mixed-type model (proportional and additive) to best explain intra-individual variability. Covariate analysis showed that body weight is positively correlated with apparent volume of distribution (V/F) and body clearance (CL/F). The typical PPK parameters of Ka , CL, and V were 0.564/h, 5.15 L/h, and 1.36 L, respectively.

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.

Pharmacokinetics and bioavailability of cefquinome in crossbred wild boars

The wild boar (Sus scrofa) is widely distributed in Europe, Asia, and Northern Africa and has been classified into at least 16 subspecies (Ruvinsky and Rothschild, 1998). With distinctive muscle characteristics (Oshima et al., 2009), crossbreeding domestic pigs with wild boars to produce wild boar crossbred pigs have been carried out as an industry (Ishiguro et al., 2002), especially in the Changbai Mountains of China. However, several clinically bacterial pathogens in wild boars, such as Salmonella, Actinobacillus, Escherichia coli, Haemophilus parasuis, and Streptococcus (Vengust et al., 2006; Baums et al., 2007; Wacheck et al., 2010), can cause seriously economic losses on crossbred wild boars farming and pose a threat to human health (Baums et al., 2007). Cefquinome (CEQ), which has been approved solely for veterinary use, is a broad-spectrum cephalosporin antibiotic. It is highly stable to beta-lactamases produced by the majority of clinically important bacteria and is approved for the treatment of respiratory tract diseases, acute mastitis and foot rot in cattle, calf septicemia, respiratory diseases in pigs and Metritis–Mastitis–Agalactia syndrome in sow (CVMP, 1995, 1999). The pharmacokinetic properties of CEQ in various species have been reported by Limbert et al. (1991), and later researches on pharmacokinetics of CEQ in fishes, bovine, piglets, cows, camels, sheep, and ducks have been published (San Martin et al., 1998; Ehinger et al., 2006; Li et al., 2008; Cagnardi et al., 2009; Al-Taher, 2010; Uney et al., 2011; Yuan et al., 2011). In those studies above, favorable pharmacokinetic features of CEQ, such as good absorption, high bioavailability, and primarily eliminated unchanged via the kidney are found. However, there is no information on pharmacokinetics of CEQ in crossbred wild boars up to now. So, the aim of this study was to investigate its pharmacokinetics and bioavailability in crossbred wild boars after single i.v. and i.m. administration, and to recommend a rational dosage schedule for CEQ. Six clinically healthy F2 cross-bred (Northeast wild boar of Changbai Mountain# · JiLin local black pig

Antimicrobial Resistance, Serotypes, and Virulence Factors of Streptococcus suis Isolates from Diseased Pigs

fi F1 wild boar crossbred pig