A survey of ß-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China.

The prevalence of ß-lactamase, 16S rRNA methylase genes, and plasmid-mediated fluoroquinolone-resistance (PMQR) determinants (qnrC and qnrD) was determined by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli isolated from a chicken farm, a pig farm, and a hospital in Shandong, China in 2007. The bla(TEM) and bla(CTX-M) were the most prevalent ß-lactamase genes in isolates from chickens (88.4%, 175/198 and 81.3%, 161/198) and hospitalized patients (87.8%, 122/139 and 69.1%, 96/139). The bla(TEM) was the most prevalent ß-lactamase gene observed in isolates from pigs (98.5%, 135/137). The gene bla(CMY-2) was also predominant among isolates from chickens (20.2%, 40/198). The bla(LAP-1) gene was first detected in one strain from chickens and humans (pig farm workers) in China. Only one strain from hospitalized patients was found to possess bla(SHV). The rmtB was the most prevalent 16S rRNA methylase gene detected in isolates from chickens (19.7%, 39/198) and hospitalized patients (15.8%, 22/139). To our knowledge, this is the first report of the detection of the qnrD gene in E. coli from chickens and pigs in China. The qnrC and bla(KPC) genes were not detected in any of the isolates. Results of southern hybridization revealed that PMQR determinants, ß-lactamases, and 16S rRNA methylase genes were located on the same plasmid in E. coli strains derived from patients. Also, PMQR determinants and ß-lactamase genes were localized on the same plasmid in an E. coli strain of animal origin. Results of conjugation experiments revealed that all of these plasmid-based resistance genes can be transferred by conjugation through horizontal transmission.

Prevalence of antimicrobial resistance among Salmonella isolates from chicken in China.

We evaluated the antimicrobial resistance of Salmonella isolated in 2008 from a chicken hatchery, chicken farms, and chicken slaughterhouses in China. A total of 311 Salmonella isolates were collected from the three sources, and two serogroups of Salmonella were detected, of which 133 (42.8%) consisted of Salmonella indiana and 178 (57.2%) of Salmonella enteritidis. The lowest percentage of S. indiana isolates was found in the chicken hatchery (4.2%), followed by the chicken farms (54.9%) and the slaughterhouses (71.4%). More than 80% of the S. indiana isolates were highly resistant to ampicillin (97.7%), amoxicillin/clavulanic acid (87.9%), cephalothin (87.9%), ceftiofur (85.7%), chloramphenicol (84.9%), florfenicol (90.9%), tetracycline (97.7%), doxycycline (98.5%), kanamycin (90.2%), and gentamicin (92.5%). About 60% of the S. indiana isolates were resistant to enrofloxacin (65.4%), norfloxacin (78.9%), and ciprofloxacin (59.4%). Of the S. indiana isolates, 4.5% were susceptible to amikacin and 5.3% to colistin. Of the S. enteritidis isolates, 73% were resistant to ampicillin, 33.1% to amoxicillin/clavulanic acid, 66.3% to tetracycline, and 65.3% to doxycycline, whereas all of these isolates were susceptible to the other drugs used in the study. The S. indiana isolates showed resistance to 16 antimicrobial agents. Strains of Salmonella (n = 108) carrying the resistance genes floR, aac(6')-Ib-cr, and bla(TEM) were most prevalent among the 133 isolates of S. indiana, at a frequency of 81.2%. The use of pulsed-field gel electrophoresis to analyze the S. indiana isolates that showed similar antimicrobial resistance patterns and carried resistance genes revealed six genotypes of these organisms. Most of these isolates had the common pulsed-field gel electrophoresis patterns found in the chicken hatchery, chicken farms, and slaughterhouses, suggesting that many multidrug-resistant isolates of S. indiana prevailed in the three sources. Some of these isolates were not derived from a specific clone, but represented a variety of genotypes of S. indiana.

First report of the multidrug resistance gene cfr and the phenicol resistance gene fexA in a Bacillus strain from swine feces.

A multidrug resistance gene, cfr, and a phenicol resistance gene, fexA, were detected in a Bacillus strain, BS-01, isolated from swine feces. The cfr gene was carried on a novel 16.5-kb plasmid, designated pBS-01. A complete Tn917 structure, which harbors the macrolide-lincosamide-streptogramin B resistance gene erm(B), was located downstream of the cfr gene. The fexA gene was discovered in the chromosomal DNA of the BS-01 strain and identified in a Tn558 variant.

Characterization of antimicrobial resistance and molecular determinants of beta-lactamase in Escherichia coli isolated from chickens in China during 1970-2007.

The aim of this study is to investigate the trend of antimicrobial resistance of 696 Escherichia coli strains of chicken origin in China during 1970-2007, and to characterize the beta-lactamase determinants of these strains. Minimum inhibitory concentrations (MICs) of these strains against 11 antimicrobials were determined by the broth microdilution method. Quinolone-resistant, and first-generation cephalosporin-resistant strains have emerged since the 1990s; third-generation cephalosporin-resistant strains were not detected until 2003. Afterwards these resistant strains increased rapidly, and an obvious increase of isolates with decreased susceptibility to forfenicol was also observed. Three classes of beta-lactamase genes in E. coli strains were detected by PCR and confirmed by DNA sequencing. The detection rate of narrow-spectrum beta-lactamase genes decreased from 71.2% in the 1970s to 28.8% during 2004-2007. Extended-spectrum beta-lactamase (ESBL) genes did not emerge until 2004 and the detection rate was 18.5% between 2004 and 2007. The detection rate of bla(CMY-2), which emerged between 2000 and 2003, climbed to 6.6% during 2004-2007. bla(CTX-M) was the dominant genotype (75%) in ESBL gene-harboring E. coli strains. Fifty strains (17.6%), co-harboring multiple kinds of beta-lactamase genes, were detected in 284 beta-lactamase-producing strains. Southern hybridization revealed that two- or three kinds of ESBL and/or CMY-2 genes co-located on one plasmid in 9 strains. The rising trend of antimicrobial resistance and the high prevalence of beta-lactamase determinants in E. coli strains of chicken origin might be attributed to overuse of antimicrobials, especially beta-lactams, in poultry production.

Prevalence and antimicrobial resistance of Campylobacter isolates in broilers from China.

The prevalence and antimicrobial resistance of Campylobacter spp. in broiler chickens were determined in Shandong Province, China. In total, 275 Campylobacter isolates were obtained from 767 broiler cecal samples, including 208 Campylobacter jejuni, 53 Campylobacter coli, and 14 unidentified Campylobacter isolates. Minimal inhibitory concentrations of 11 antimicrobial agents were determined using the agar dilution method recommended by CLSI. More than 98% of the tested Campylobacter isolates were resistant to quinolones (nalidixic acid, ciprofloxacin and enrofloxacin) and tetracyclines (tetracycline and doxycycline). The C. jejuni isolates also exhibited a high rate of resistance to phenicol antibiotics and a moderate rate of resistance to macrolides and gentamicin. On the contrary, the C. coli isolates showed a high-level resistance to macrolides and gentamicin and little resistance to phenicol antibiotics. The vast majority of the Campylobacter isolates were classified as multidrug resistant. These findings reveal a broad extent of antimicrobial resistance in Campylobacter isolates from poultry in China and underline the need for prudent use of antibiotics in poultry production to minimize the spread of antibiotic resistant Campylobacter.

A survey of plasmid-mediated fluoroquinolone resistance genes from Escherichia coli isolates and their dissemination in Shandong, China.

Bacterial resistance to fluoroquinolones result from mutations in the quinolone resistance-determining regions of the drug targets, overexpression of efflux pumps, and/or the more recently identified plasmid-mediated low-level resistance mechanisms. We investigated the prevalence of and characterized plasmid-mediated fluoroquinolone resistance genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli (n = 530) isolated from a chicken farm, a pig farm, and hospitalized patients in Shandong, China, in 2007. The aac(6')-Ib-cr gene was the most prevalent resistance gene that was detected in bacteria isolated from all sources. Next was the qnrS gene, which was predominantly present in isolates from the pig farm. Only eight (5.8%) isolates from hospital patients were found to possess the qepA gene, and these isolates were first reported in qepA-carrying E. coli from humans in China. The qnrA and qnrB genes were not detected in any of the isolates. Further, most of the isolates were also resistant to beta-lactams and aminoglycosides as determined by the broth microdilution method. Pulsed-field gel electrophoresis analysis of the E. coli isolates with similar resistance patterns that also carried resistance genes showed great genomic diversity among these bacteria, suggesting that the multiresistant E. coli isolates carrying the qnr, aac(6')-Ib-cr, or qepA genes were not derived from a specific clone, but represented a wide variety of different genotypes. The results of Southern hybridization revealed that qepA, qnrS, and parts of aac(6')-Ib-cr genes were localized on plasmids and/or chromosome. qepA and aac(6')-Ib-cr genes were colocalized with aac(6')-Ib-cr and qnrS genes, respectively, on the same plasmids. Our study demonstrated that two different genes (qepA and aac(6')-Ib-cr) were identified on the same plasmid in E. coli strains derived from patients and qnrS and aac(6')-lb-cr genes on the same plasmid in an E. coli strain of animal origin.

Increased prevalence of plasmid-mediated quinolone resistance determinants in chicken Escherichia coli isolates from 2001 to 2007.

To evaluate the temporal change in the plasmid-mediated quinolone resistance (PMQR) determinants from 2001 to 2007 in chicken, a total of 532 chicken Escherichia coli isolates were screened for PMQR determinants by polymerase chain reaction and sequencing. The prevalence of qnr genes, aa(6')-Ib-cr, and qepA were 9.8%, 11.7%, and 0.75%, respectively. Among the qnr determinants, qnrA-, qnrB-, and qnrS-type genes were detected in 4 (0.75%), 21 (3.9%), and 27 (5.1%) of the examined isolates, respectively. None of the isolates carried qnrC gene. Ciprofloxacin resistance increased over time (p < 0.01), and a clear trend of increase in the prevalence of qnr and aac(6')-Ib-cr genes among the isolates was shown from 2001 to 2007 (p < 0.01). Pulsed-field gel analysis showed that the PMQR-positive isolates were not clonally related and genetically diverse. Quinolone resistance was transferred by conjugation from qnrB-, qnrS-, and aac(6')-Ib-cr-positive isolates to recipient E. coli. The qnrB and aac(6')-Ib-cr alleles were located on the plasmids with the size of 49 and 50 kb, respectively. However, the qnrS alleles were located on different plasmids with sizes from 57.4 to 88.6 kb, indicating diverse genetic backgrounds. The increasing frequency of ciprofloxacin resistance in E. coli was associated with increasing prevalence of qnr genes and aac(6')-Ib-cr (r(s) = 0.964, p = 0.00045). This survey showed that PMQR determinants were highly prevalent in chicken E. coli isolates in China with a trend of increase from 2001 to 2007. Horizontal transfer and widespread use of quinolone antimicrobials may have contributed to the spread of PMQR determinants in the poultry production system. The widespread dissemination of PMQR could potentially fuel the rapid development of fluoroquinolones resistance.

Distribution of superantigenic toxin genes in Staphylococcus aureus isolates from milk samples of bovine subclinical mastitis cases in two major diary production regions of China.

To evaluate the distribution of most known staphylococcal superantigen (SAg) genes in Staphylococcus aureus isolated from bovine mastitis cases, a genetic analysis of 15 SAg genes and genotypes was performed in a total of 283 S. aureus isolates collected from milk samples of cows with subclinical mastitis in two major diary production regions of China. Almost 65% of the isolates possessed at least one toxin gene. The most frequently found genes were sea (36.0%) followed by sei (31.8%), seg (31.4%) and selm (26.9%). The genes see, selk, or selo were not found in any of the isolates tested. Overall, 28 SAg genotypes were observed, among which the genotypes sea-seg-sei-selm, seg-sei-selm-seln, and sea-sed-selj predominated at the rate of 8.8%, 7.4%, and 6.7%, respectively. Marked geographical variations were noticed in the distribution of individual SAg genes and genotypes among S. aureus isolates from the two different regions. The relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements (MGEs) was analyzed, revealing that majority of SAg genes were present in certain MGEs, which were in accordance with current knowledge about MGEs carrying those genes. However, some gene combinations suggest the possibility of the existence of variants or new types of MGEs.