Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks.

The antimicrobial susceptibility of 103 Riemerella anatipestifer isolates obtained from ducks during 2008 and 2010 in Southern China, to 23 antimicrobial agents was investigated using the agar dilution method. The MIC(50) and MIC(90) values of streptomycin, kanamycin, gentamicin, apramycin, amikacin, neomycin, nalidixic acid and sulfadimidine were high (32-≥ 128 µg/ml) among the 103 R. anatipestifer isolates. However, relatively low MIC(90) values (8 µg/ml) of ampicillin and florfenicol were observed among these isolates. The presence of resistance genes and integrons was determined using PCR. The genes bla(TEM-1), aph(3')-VII, aadA1, aadA2, aac(3')-IV, aac(3')-IIc, aac(6')-Ib, cat2, cmlA, floR, tet(A), tet(B), tet(C), sul1, and sul2 were detected in 1, 2, 24, 35, 11, 4, 67, 16, 26, 10, 6, 1, 9, 36 and 2 isolates, respectively. Twenty isolates contained one or two class 1 integrons carrying aadA2 or aac(6')-II-catB3-aadA1 gene cassette(s). Mutation analysis of the quinolone resistance-determining regions (QRDRs) of 43 R. anatipestifer isolates with nalidixic acid MICs ≥ 32 µg/ml, showed that the most prevalent mutations in gyrA were those resulting in the amino acid exchanges Ser83-Ile (n=37), followed by Asp87-His (n=7) and Ser83-Arg (n=5). Point mutations in parC (Arg120-Glu) were observed in 5 isolates with a ciprofloxacin MIC of >16 µg/ml. No plasmid-mediated quinolone resistance gene was detected. PFGE analysis showed that the clonal spread of multi-drug resistant R. anatipestifer isolates occurred in the same farm or between different farms. Our results reported, for the first time, the mechanism of quinolone resistance in R. anatipestifer.

High prevalence and widespread distribution of multi-resistant Escherichia coli isolates in pigs and poultry in China.

Escherichia coli play an important ecological role within resistant bacteria populations, and can be used as a bio-indicator of antimicrobial resistance. The aim of the present study was to use this feature of E. coli to investigate the prevalence of antimicrobial resistance and the degree of cross-species transmission of bacteria in pigs and poultry in China. A total of 592 E. coli strains, isolated from pigs and poultry (healthy and diseased animals), were tested for resistance to 22 antimicrobials representing eight antimicrobial drug types. E. coli isolates had high rates of resistance to ampicillin (99.5%), doxycycline (95.6%), tetracycline (93.4%), trimethoprim-sulfamethoxazole (74.3%), amoxicillin (65.1%), streptomycin (54.7%), and chloramphenicol (50.2%). Resistance to cephalosporins, quinolones, and aminoglycosides was also quite prevalent. The majority (81%) of isolates demonstrated multi-antimicrobial resistance, most commonly to 5-6 different antimicrobial types. One isolate was resistant to all 22 antimicrobials. Twenty-two cultures exhibiting multi-antimicrobial resistance were analysed by pulsed-field gel electrophoresis (PFGE) to assess their distribution between farms. Three distinct PFGE types were identified, indicating inter-farm transmission of multi-antimicrobial resistant bacteria. The study confirmed the presence and transmission of multi-antimicrobial-resistant E. coli strains amongst pigs and poultry in China and highlights the urgent need for appropriate monitoring programmes.

[Effects of enrofloxacin on DNA sequence diversity of soil cultural bacterial community].

In order to understand the effects of remained enrofloxacin (ENR) in environment on the diversity of soil microbial communities, amplified ribosomal DNA restriction analysis (ARDRA) approach and genomic fingerprinting technique enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR) were used to analyze the molecular diversity of 16S rDNA from soil bacteria after ENR addition. The results showed that after the ENR addition for 35 days, the total count of soil bacteria was less than that of CK, and decreased with increasing ENR concentration. The ARDRA divided the separated soil bacteria into different operational taxonomic units (OTU) groups, and the count of group I to group VI was 15, 13, 10, 8, 6, and 6, respectively. Genomic fingerprinting analysis indicated that the Shannon-Wiener index of group I to group VI was 2.78, 2.14, 1.78, 1.11, 0.69 and 0.31, respectively, and the Margalef index, Simpson index, and Pielou index of soil microbial community in CK were higher than that in the soils in which ENR was added.

Molecular variability of DR-1 and DR-2 within the pvpA gene in Mycoplasma gallisepticum isolates.

A total of 15 Mycoplasma gallisepticum (MG) isolates from Chinese poultry farms and three reference strains (S6, BG44T, and F36) were characterized by nested polymerase chain reaction and sequence analysis for two identical and directly repeated sequences, DR-1 and DR-2, within the putative cytadhesin pvpA gene. The molecular variation patterns of the pvpA genes among the 15 MG isolates were identical to the reference strains S6 and BG44T, that is, a 60 bp deletion in DR-1 and DR-2 and repetition of 1) a proline residue 33 times and 2) a tetrapeptide motif 10 times (Pro-Arg-Pro-X, where X is Met, Gly, Asn, or Gln for 6, 1, 1, or 2 times, respectively). However, the variation pattern is quite different from that of the vaccine strain F36, in which only the DR-1 region is retained, 24 of the 25 peptides comprising the linkage sequence between DR-1 and DR-2 are missing, and the entire DR-2 sequence is deleted. A comparison of the sequences within the DR-1 and DR-2 repeated regions among clinical isolates from different geographic sites suggested that > or = 30 proline residue repeats and 7-10 repeats of the tetrapeptide motif may exert an important role in the functionality of PvpA as an adhesin molecule. Size variation and differences in deletion patterns in the C-terminal coding region of the pvpA gene were observed among the field isolates and vaccine strain F, providing the basis for strain differentiation.

Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China.

OBJECTIVES: To investigate the occurrence of 16S rRNA methylases conferring high-level resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms in China. METHODS: Enterobacteriaceae isolated from 151 pig rectal swab samples and 9 environmental samples were screened for the presence of the rmtA, rmtB, armA and rmtC genes by PCR and sequencing. Conjugation experiments were carried out to study the transferability of the 16S rRNA methylase genes. All isolates and their transconjugants were tested for susceptibility to antimicrobial agents. The clonal relatedness of RmtB-producing Escherichia coli was assessed by PFGE with XbaI. RESULTS: Of 152 Enterobacteriaceae isolates recovered from pigs, 49 (32%) were positive for the rmtB gene, including 48 E. coli and a single isolate of Enterobacter cloacae. Of the nine Enterobacteriaceae isolates from environmental samples, no 16S rRNA methylase gene was identified. The 49 rmtB-positive isolates showed resistance to ampicillin, tetracycline and trimethoprim and also carried a bla(TEM) gene. Transfer of the rmtB and bla(TEM) genes by conjugation experiments of all 49 isolates was successful, suggesting that the rmtB-containing plasmids in the E. coli and E. cloacae isolates were self-transmissible. Conjugative transfer frequencies varied from 2.2 x 10(-10) to 1.3 x 10(-6) transconjugants per recipient. The transfer of non-aminoglycoside antimicrobial resistance traits was also observed in most cases. Forty-four rmtB-positive E. coli showed 30 different PFGE types. CONCLUSIONS: The rmtB gene was detected on conjugative plasmids of porcine E. coli and E. cloacae isolates. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of 16S rRNA methylases in Enterobacteriaceae isolates is described for the first time in China. This is also the first report of rmtB-positive Enterobacteriaceae among healthy food-producing animals.

Detection and characterisation of CTX-M and CMY-2 beta-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China.

The objective of this study was to characterise the beta-lactamase genes of cephalosporin-resistant Escherichia coli isolated from farm animals in Guangdong Province of China. Of 592 E. coli isolates recovered from farm animals from 2003-2005, 50 (8.4%) showed cephalosporin resistance. Polymerase chain reaction and sequencing analysis showed that 14 isolates (2.4%) from chickens, ducks, pigs and partridges were positive for the bla(CTX-M) gene (10 for bla(CTX-M-14) and 4 for bla(CTX-M-27)). CMY-2 was detected for the first time in mainland China in six E. coli isolates (1.0%) from chickens and goose. Except for one isolate, bla(CTX-M)- and bla(CMY-2)-containing isolates also harboured the bla(TEM-1b) gene. Conjugation experiments demonstrated that the bla(CTX-M) and bla(TEM) genes could be transferred to E. coli DH5alpha. Pulsed-field gel electrophoresis (PFGE) showed that the 14 CTX-M-producing isolates belonged to 12 different types. Two isolates (one from a chicken, the other from a pig) containing CTX-M-14 showed indistinguishable PFGE patterns, indicating clonal dissemination of this strain among animals from different farms. This study describes for the first time the emergence of CTX-M- and CMY-2-producing E. coli among farm animals in China, with the CTX-M-9 group being the predominant extended-spectrum beta-lactamase detected.

Tissue pharmacokinetics of florfenicol in pigs experimentally infected with Actinobacillus pleuropneumoniae.

UNLABELLED: The aim of this study has been to determine the tissue pharmacokinetic parameters of florfenicol in the pigs experimentally infected with Actinobacillus pleuropneumoniae. 21 crossed-bred (Duroc x Landrace x Yorkshire) local species of pigs were infected experimentally with Actinobacillus pleuropneumoniae serotype 1 and confirmed as typical sub-acute pleuropneumonia. A single dose of 20 mg/kg body weight of florfenicol, a novel animal-using antibiotic, was administrated intramuscularly in the pigs and then samples of blood, lung, trachea with bronchi, liver, kidney and muscle were taken at scheduled time points. Drug concentrations were determined by high performance liquid chromatography (HPLC) with an ultraviolet detector via extraction with ethyl acetate under nitrogen flow. The statistic moment theory (SMT) mathematic package was applied to calculate the tissue pharmacokinetic parameters of florfenicol in the infected model. AUC of lung, trachea with bronchi, liver, kidney and muscle were 121.69, 79.37, 81.05, 181.2, and 94.07 mg/l x h, respectively, MRT were from 34.66 to 90.17 h, and t1/2beta from 24.75 to 69.34 h, respectively. CONCLUSIONS: Florfenicol was widely distributed in these tissues and maintained the effective therapeutic concentrations especially in the respiratory tract tissues that are the target organs of Actinobacillus pneuropneumoniae. CLINICAL SIGNIFICANCE: Tissue pharmacokinetic data could be evidence for regime designing of florfenicol in treatment of porcine pleuropneumonia.