Quinolone resistance in Escherichia coli and Salmonella spp . isolates from diseased chickens during 1993-2008 in China

A total of 363 Escherichia coli and 224 Salmonella spp. were isolated from diseased chickens during 1993-2008 in China. The susceptibility to eight quinolones and prevalence of plasmid-mediated quinolone resistance (PMQR) determinants was investigated in these isolates. Among the E. coli isolates obtained during 1993-1999, 65.2% were resistant to nalidixic acid, while more than 50% of the E. coli isolates collected during 2000-2008 were resistant to 7 quinolones. All 101 Salmonella spp. isolates obtained during 1993-1999 were susceptible to quinolones, while more than 50% of the Salmonella spp. isolates collected during 2000-2008 were resistant to only nalidixic acid (82.9%). Among the 363 E. coli isolates, 4 (1.1%) were positive for aac(6 )-Ib-cr, 3 (0.8%) for qepA and 1 (0.3%) for qnrB10. No PMQR gene was identified in 224 Salmonella spp. isolates. The resistance of E. coli and Salmonella spp. to quinolones has been increasing in the past twenty years and the resistance of Salmonella spp. was much lower than that of E. coli, although they were separated in the same period. There is a rising trend of avian isolates harboring PMQR genes in China.


INTRODUCTION
E. coli and Salmonella are important pathogens that cause gastrointestinal infections and septicemia in human and animals as well as a range of secondary conditions, including respiratory tract infection in animals.Quinolones/ fluoroquinolones are broad-spectrum antibacterial agents used in human and veterinary medicine.The development of resistance to quinolone by Gram-negative pathogens is an important step in bacterial evolution (Hopkins et al., 2005).Quinolones were introduced into clinical use in 1962 in the form of nalidixic acid, which is a completely synthetic agent that in clinical concentrations has bactericidal effects on most members of Enterobacteriaceae.The extensive use of quinolones has been associated with an increasing level of quinolone resistance (Winokur et al., 2000;Jiang et al., 2008).*Corresponding author.E-mail: jiao@yzu.edu.cn.Tel: +86 514 8797 1136.Fax: +86 514 8799 1747.
All quinolones have the same mechanism of action, namely, the inhibition of DNA replication, regardless of whether they are used in clinical or veterinary medicine.Until 1998, it was believed that quinolone resistance could be acquired through chromosomal mutation.Later, it became clear that quinolone resistance may be associated with plasmid-mediated resistance genes (Martinez-Martinez et al., 1998;Robicsek et al., 2006a).Thus far, the following mechanisms have been reported to be involved in plasmid-encoded quinolone resistance: a quinolone-protective mechanism encoded by the qnr genes (Martinez-Martinez et al., 1998), a double class antibiotic-modifying enzyme encoded by aac(6 )-Ib-cr which involves in the acetylation of ciprofloxacin and norfloxacin (Robicsek et al., 2006b), and the qepA gene, which encodes an efflux pump belonging to the major facilitator subfamily (Yamane et al., 2007).In China, quinolones are commonly used for disease prevention and control in poultry production, and there were some surveys on the prevalence of antimicrobial resistance among E. coli and Salmonella spp.from food-producing animals (Yang et al., 2004;Dai et al., 2008;Yue et al., 2008;Huang et al., 2009;Pan et al., 2009;Wu et al., 2009).However, a longitudinal survey is still lacking on quinolone resistance determinants in E. coli and Salmonella spp.isolated from diseased chickens.
This study was conducted in order to compare the quinolone resistance and prevalence of plasmid-mediated quinolone resistance (PMQR) genes among E. coli and Salmonella spp.strains, which were isolated from ill chickens during 1993-2008 from 14 provinces of China.

Bacterial isolates and serogroup determination
A total of 363 E. coli and 224 Salmonella spp.field isolates were derived from clinically affected chickens in 14 provinces (Jiangsu, Shanghai, Shandong, Shanxi, Henan, Guangdong, Zhejiang, Hubei, Beijing, Anhui, Shanxi, Sichuan, Guizhou, and Guangxi) of China between 1993 and 2008.All E. coli and Salmonella spp.strains were isolated from diseased chickens for which sufficient amounts of bacteria could be obtained from systemic lesions (heart, liver, spleen, brain, and blood) for culturing.The samples were cultured directly on MacConkey agar (Difco) and were identified as E. coli and Salmonella spp.using biochemical procedures (Chen et al., 2004;Pan et al., 2009).Only one isolate from each diseased bird was examined.After isolation, organisms were stored at -70°C in Luria-Bertani (LB) broth after the addition of 15% glycerol.E. coli ATCC 25922 was used as a quality control strain for susceptibility testing.
E. coli serogroup typing was performed by using the standard methods followed at the China Institute of Veterinary Drug Control (Chen et al., 2004).Salmonella spp.strains were grown overnight in LB broth, and plate agglutination test was performed for monoclonal antibodies (Pan et al., 2009).

Antimicrobial susceptibility testing
The MICs of 363 E. coli and 224 Salmonella spp.isolates were determined using the broth microdilution method according to Clinical and Laboratory Standards Institute recommendations (CLSI, 2008;2009).The following antimicrobial agents were tested: nalidixic acid, enoxacin, fleroxacin, norfloxacin, lomefloxacin, ciprofloxacin, ofloxacin, and gatifloxacin (National Institute for the Control of Pharmaceutical and Biological Products, China).The method was controlled by parallel testing using quality control reference strain recommended by the CLSI.

Screening for PMQR genes in E. coli and Salmonella strains
The isolates were investigated for the presence of qnrB, qnrC, qnrD, qnrS, aac(6 )-Ib and qepA genes by PCR amplification with the primer sets described previously (Park et al., 2006;Cattoir et al., 2007;Yamane et al., 2008;Cavaco et al., 2009;Wang et al., 2009).The primers used for qnrA were 5´-AGAGGA TTTCTC ACGCCA GG and 5´-GCAGCA CTATKA CTCCCA AGG, giving a 619bp product.Both strands of the purified PCR products were sequenced and qnr alleles were assigned by referring to the qnr gene nomenclature (Jacoby et al., 2008).All positive PCR products of aac( 6)-Ib were further analyzed by digestion with Fokl(TaKaRa Biotechnology, Dalian, China) and/or direct sequencing to identify aac( 6)-Ib-cr, which lacks the Fokl restriction site present in the Chen et al. 3079 wild-type gene.

Conjugation assays
Conjugation experiments were attempted between the PMQR gene-positive E. coli donors and azide-resistant recipient strain E. coli J53AzR (Wang et al., 2003).Transconjugants were selected on tryptic soy agar plates containing sodium azide (100 mg/l) and tetracycline (20 mg/l), chloramphenicol (50 mg/l), gentamicin (8 mg/l) or amoxicillin (100 mg/l) to select for plasmid-encoded resistance.MICs for the donor, recipient, and transconjugant strains were measured by the broth microdilution method according to CLSI guidelines (CLSI, 2009).1.Among the 4 common serogroups, quinolone resistance in the O1 and O78 serogroup isolates (more than 50% of the strains were resistant to 6 and 5 quinolones, respectively) appeared to be higher than that of the O2 and O18 (more than 50% of the strains were resistant to 3 and 1 quinolones, respectively).

Comparison of the quinolone resistance of E. coli and Salmonella spp. isolates
In this study, 101 Salmonella spp.isolates obtained before the year 2000 were susceptible to all the 8 quinolone antimicrobial agents.While in the same period more than 50% of the E. coli isolates exhibited resistance to nalidixic acid.More than 50% of the Salmonella spp.isolates obtained during 2000-2008 were found to be resistant to nalidixic acid (82.9%), while more than 50% of the E. coli isolates collected during the period exhibited resistance to 7 quinolones (Table 2).

Conjugation experiment and antimicrobial susceptibility testing
Quinolone resistance could be transferred by conjugation from three of the eight PMQR genes positive donors.Transfer from the other five donor strains were not successful despite conjugation experiments in broth and on filter surfaces and including separate selections with each of the antibiotics to which the donor was resistant (except quinolones).The MIC of ciprofloxacin against the transconjugants was 0.06 mg/l, representing an increase of 8-fold relative to that of the recipient E. coli J53AzR (Table 3).Various resistance to other antimicrobial agents were also transferred with the plasmids, all were resistance to ampicillin and chloramphenicol.All transconjugants had remarkable elevated MICs for amikacin, kanamycin and tetracycline.

DISCUSSION
Resistance to quinolones has increased markedly in some parts of the world since the introduction of these agents.Eight quinolones belonging to the first to fourth generations were selected for testing in this study.It was found that the isolates had high resistance to the first-generation quinolone like nalidixic acid and low resistance to the fourth-generation quinolone such as gatifloxacin.
E. coli isolates occur as non-pathogenic commensals in human or as animal pathogens, but the emergence of strains showing resistance to several quinolone antimicrobial agents is a public health concern.Before the early 1990s, clinical isolates of E. coli rarely showed resistance to quinolones.However, since then the frequency of resistance has significantly increased worldwide (Hopkins et al., 2005).In this study, E. coli was found to have high resistance to some quinolones after 1990.Resistance to 7 quinolone antimicrobial agents was observed in more than 50% of the isolates during 2000-2008.
High-level quinolone resistance is relatively uncommon in Salmonella spp.(Hopkins et al., 2005).In the year 2000, the results of antimicrobial susceptibility tests for isolates of human salmonellosis in 10 European countries revealed that clinical resistance to ciprofloxacin was rare, with only 0.5% of the isolates exhibiting CIP MIC greater than 1 mg/l (Threlfall et al., 2003).Quinolone resistance to Salmonella spp. was first observed in the year 2000 in this Plasmid-mediated quinolone resistance was first reported in 1998, the gene responsible for PMQR has been identified as qnr (Tran and Jacoby, 2002).The qnr gene has been identified in various bacterial species of the Enterobacteriaceae family in many countries.Only one qnrB10-positive isolate was detected among the 363 avian E. coli isolates in this study.A variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, aac(6 )-Ib-cr, seems to have emerged more recently, but might be more prevalent than qnr (Hopkins et al., 2005).Four (1.1%) E. coli isolates were positive for aac(6 )-Ib-cr.These 4 isolates were obtained from different henneries and belonged to 4 different serogroups.In this study, 3 (0.8%) E. coli isolates were positive for qepA, all of which were obtained from the same hennery and exhibited resistance to all 8 quinolones.Although these E. coli and Salmonella spp.strains were separated in the same period, the susceptibility to quinolones and the presence of the PMQR genes were different from each other.The exactly reasons for the differences are not known, but it possible because that E. coli strains are common both in intestinal tract and the environment.The pollution of natural ecosystems by antibiotics and resistance genes might have consequences for the evolution of the microbiosphere (Martinez, 2009).The opportunity for E. coli strains to acquire the resistance is much more than the Salmonella spp.strains, and the antimicrobial resistance of the commensal E. coli strains may also play an important role, transmission of resistance genes from normally nonpathogenic species to more virulent organisms is an important mechanism for acquiring antimicrobial-resistant organisms (Winokur et al., 2001).
In conclusion, the rate of quinolone resistance of E. coli and Salmonella isolates has increased considerably over the past twenty years.PMQR determinants appeared in avian E. coli and its prevalence is increasing in China.The prudent use of antimicrobial agents is thus necessary in veterinary medicine as well as in human medicine to minimize the spread of these resistance genes.

Table 1 .
Relationship between serogroups and quinolone resistance among 363 avian Escherichia coli isolates.
Salmonella spp.isolates All the Salmonella spp.isolates obtained during 1993-1999 were susceptible to quinolones, and a nalidixic acid-resistant Salmonella spp.isolate was first observed in the year 2000.Resistance to various quinolones in Salmonella spp.isolates during 2000-2008 was presented

Table 2 .
Results of antimicrobial resistance of 363 E. coli and 123 Salmonella spp.Isolates.

Table 3 .
Characteristics of donor strains and E. coli J53 transconjugants.