Characterization of antimicrobial resistance and related resistance genes in Escherichia coli strains isolated from chickens in China during 2007-2012

In the present study, the prevalence of antimicrobial-resistant chicken Escherichia coli strains and the resistance genes in E. coli was investigated. For this purpose, 1002 chicken E. coli strains isolated from layer and broiler flocks in Shaanxi, Henan and Gansu provinces in China during 2007-2012 were examined. Antimicrobial susceptibility of these E. coli strains against 18 antimicrobials was determined by the Kirby-Bauer disk diffusion method. Eight out of the twenty antimicrobial resistance genes were detected by polymerase chain reaction (PCR). The sequences of the resistance genes in chicken E. coli strains were compared with the previously published sequences. Our results revealed that the antimicrobial resistance prevalence of E. coli strains in western China to ampicillin, doxycycline, tetracycline and nalidixic acid were consistently kept at 62-100%. The E. coli resistance to nalidixic acid and ciprofloxacin had an increasing trend, as high as 100% for nalidixic acid while the resistance prevalence to gentamicin had a decreasing trend. The detection rates of the genes for tetA, tetB, blaTEM, and aac(3)-II in chicken E. coli strains were positively correlated with their antimicrobial resistance (P <0.01) during 2007-2012. Among 1002 chicken E. coli strains tested, all E. coli strains were resistant to more than three kinds of antimicrobials. Our results revealed that 499 of the 1002 (49.8%) chicken E. coli strains were resistant to more than eight kinds of antimicrobials. Considering all the 1002 isolates, the detection prevalence of the genes for tetA, tetB, blaTEM in chicken E. coli strains were constantly over 88.9%. The detection prevalence of the genes for floR, sul-I and cmlA in chicken E. coli strains increased, while aac(3)-II declined from 75.0 to 28.6%.


INTRODUCTION
Escherichia coli is one of the common pathogens in chicken production.For a long time, antibiotics have been widely used in the treatment and prevention of colibacillosis, even increasingly being used as animal growth promotion agents (Sarmah et al., 2006;Martinez, 2009).E. coli resistance rises with the selective pressure of the antibiotics, and then reduces the clinical efficacy of antibacterial drugs and increase the mortality of sick animals, *Corresponding author.E-mail: hjliu5257@nchu.edu.tw.Tel: 886-4-22840485.Ext: 243.Fax: 886-4-22874879.thereby causing economic losses (Dho-Mouline and Fairbrother, 1998; Barnes et al., 2008;Pan et al., 2009).
Avian pathogenic E. coli (APEC) cause aerosacculitis, polyserositis, septicemia and other mainly extraintestinal diseases in chickens, turkeys and other avain species (Dho-Moulin and Fairbrother, 1998).This disease results in significant morbidity and mortality, which gives rise to multimillion-dollar annual losses for all facets of the world's poultry industry.Plasmid-mediated antibiotic resistance genes are an important mechanism of resistance in E. coli.The resistance genes are not only vertical to offspring but also horizontally transmitted between different microbes, potentially affecting human health and causing economic loss in the breeding industry (Collignon and Angulo, 2006;James et al., 2007;Ben et al., 2010;Lu et al., 2010).In recent years, many reports have been published on the plasmid-mediated βlactams, aminoglycosides, chloramphenicol, sulfonamides, quinolones and tetracyclines in E. coli, but few reports have been published regarding chicken sources of E. coli resistance and resistance genes.Therefore, study of E. coli resistance and resistance genes in chickens is of great significance to public health (Collignon and Angulo, 2006).
In previous studies, it was found that quinolones and the first-generation of cephalosporins resistant strains occurred in the 1990s and drug-resistant strains to the third-generation of cephalosporins were found in 2003 (Li et al., 2010).The detection prevalence of resistance gene (blaCTX-M) was up to 75% (Li et al., 2010).Furthermore, previous study also suggested that the majority of E. coli strains from swine in China were resistant to streptomycin, chloramphenicol, norfloxacin and doxycycline, showing that the detection prevalence of the genes for cmlA and floR were 65 and 57%, respectively (Wang et al., 2011).The chicken E. coli strains in South Australian were found to be resistant to tetracycline, ampicillin, cotrimoxazole, streptomycin and neomycin, having the detection prevalence of the genes for tetA (19.1%) and blaTEM (17.1%) (Obeng et al., 2012).Karah et al. (2010) studied the plasmid-mediated quinolone resistance gene in E. coli strains isolated from human in Norway and Sweden.Among isolates that were ESBL producers and were resistant to nalidixic acid and/or had reduced susceptibility to ciprofloxacin, the detection prevalences of the genes for qnr and aac (6')-ib-cr genes were 9.1 and 52.3%, respectively (Karah et al., 2010).
The aim of this study was to investigate the occurrence of antimicrobial resistance of E. coli strains and the correlation between plasmid-mediated resistance genes and antimicrobial resistance of E. coli strains isolated from chickens in Shaanxi, Henan, and Gansu provinces in China during 2007-2012.

Source of strains
One thousand and two E. coli strains were isolated from liver samples of sick and dead layer and broiler flocks (Hy-Line Variety Brown) from Xi'an, Tongchuan City, Yangling Demonstration Zone in Shaanxi province; Sanmenxia and Luoyang Cities in Henan province; Tianshui, Dingxi Cities in Gansu province in China during 2007-2012.Samples were collected with visible enlargement of the liver, pericarditis, bladder inflammation and peritonitis during necropsy.The isolation information and E. coli source and distribution in three provinces are shown in Table 1.E. coli standard strain ATCC25922 was kindly provided by preventive veterinary medicine laboratory of Northwest Agriculture and Forestry University, China.

Antimicrobial susceptibility test
The E. coli strains were tested for susceptibility to 18 antimicrobial drugs by Kirby-Bauer disk diffusion method on ordinary agar plates.
The standard procedure of the clinical and laboratory standards institute guidelines were strictly followed throughout the testing procedure and the determination of results (CLSI, 2008a, b).The criteria for a drug to be classified as resistant or sensitive were judged as described previously (CLSI, 2008a, b).

Primers for amplification of resistance genes in chicken E. coli strains
The twenty sets of primer pairs (Table 2) used for polymerase chain reaction (PCR) amplification of β-lactams, aminoglycosides, chloramphenicol, sulfonamides, quinolones and tetracycline resistant genes, respectively were designed with Primer5.0 software based on the sequences deposited in GenBank.All E. coli strains and reference strains were grown on Luria-Bertani (LB) agar plates at 37°C overnight.E. coli colonies were suspended in 500 μL of deionized water and boiled for 10 min, followed by chilling on ice for 5 min and centrifugation at 10,000 xg for 5 min, the supernatant was used as the DNA templates for PCR amplification.The PCR mixture contained 10 μL of 2X PCR Master mix (including 2X Taq DNA polymerase, 2X PCR Buffer and 2X dNTP mixture) (TaKaRa), 1 μL of primer pairs, 4 μL of DNA template, and deionized water to a final volume of 25 μL.PCR was completed by an initial heat activation of 5 min at 95°C and then 30 cycles of 30 s at 94°C, 30 s at annealing temperatures and 45 s at 72°C; and an extension of 10 min at 72°C.PCR products were analyzed by 1% agarose gel electrophoresis and visualized after staining with ethidium bromide on a UV transilluminator.

Sequence analysis of PCR products
Resistance gene sequences were aligned and compared with related sequences in GenBank by DNAStar program.Longitudinal data on resistance and resistance genes in E. coli in the same farms at Shaanxi province, China was analyzed.The six farms of Tongchuan which raised about one million of layer at Shaanxi province in China were selected for study and the changes of resistance and resistance gene in E. coli strains were examined during 2007-2012.

Statistical analysis
Student's t-test was used to measure the correlations between resistance and resistance gene and to compare resistance prevalence between years.In all tests, p<0.05 was considered statistically significant.

Susceptibility test of E. coli strains
The resistance information of chicken E. coli strains in three provinces in China to 18 common clinically used antibiotics is shown in Tables 3, 4 and 5.As shown in Table 3, the resistance prevalence of chicken E. coli strains to ampicillin in Shaanxi province was 100% during 2007-2012.Resistance prevalence to tetracycline and doxycycline was 70-100%.The increase in chicken E. coli strains resistance to nalidixic acid and ciprofloxacin was seen.The resistance rate to nalidixic acid has increased to 100% but a downward trend for neomycin and streptomycin was found.In 2007-2010, the resistance prevalence of chicken E. coli strains to ceftazidime increasingly was observed, but there was a slight decline in 2011-2012.A downward trend in resistance to gentamicin in  2007-2011 was seen, but there was a slight increase in 2012.
Table 4 shows that the resistance prevalence of chicken E. coli strains isolated from Henan province to ampicillin and doxycycline were 100% during 2007-2012 while tetracycline resistance prevalence was 60-100%.The resistance of chicken E. coli strains to tobramycin, florfenicol, nalidixic acid and cefaloxime showed an upward trend, and resistance rate of nalidixic acid has been up to 100% while resistance to neomycin and gentamicin showed a declining trend.
As seen in Table 5, the resistance prevalence of chicken E. coli strains isolated in Gansu province to ampicillin and doxycycline has remained at 100% from 2007-2012 while the resistance prevalence of tetracycline and nalidixic acid were 60-100%.The resistance prevalence to tobramycin showed an upward trend while gentamicin resistance prevalence showed a downward trend.Resistance rates to ceftazidime and ciprofloxacin increased in 2007-2010, but there was a slight decline in 2011-2012.
Overall, resistance prevalence of chicken E. coli strains to ampicillin in Shaanxi, Henan and Gansu provinces in China has been maintained at 100%.Doxycycline and tetracycline resistance prevalence were more than 80% and 60%, respectively.An upward trend to nalidixic acid and ciprofloxacin was seen while gentamicin resistance prevalence showed a downward trend.Resistance to kanamycin, tobramycin and trimethoprim sulfamethoxazole showed significant differences (P<0.05) and the rest of antibiotic resistance showed no significant difference (P>0.05).
Isolates showed multi-drug resistance (resistant to more than three kinds of antibiotics), and more than 3 were up to 100% of the drug-resistant strains, in which 8 resistant strains had the highest count for 19.2% (192/ 1002); isolates resistant to more than 8 antibiotics were up to 49.8% (499/1002), of which 14 chicken E. coli isolates resistant to 18 antibiotics, accounted for 1.4% (14/1002).

PCR detection of resistant genes in E. coli isolates
Among 1002 chicken E. coli strains, 8 of 20 resistance genes were detected by PCR.The electrophoretic patterns of these 8 resistance genes were indicated in Figure 1.The detection rates of the genes for tetA, tetB, blaTEM, aac(3)-II, sul-I, cmlA, floR and qnrB in three provinces in China during 2007-2012 were shown in Table 6.In 2007-2012, the tetB and tetA genes were detected with the highest prevalence in 86.6%-100% in chicken E. coli strains isolated from Shaanxi, Henan and Gansu provinces in China while the detection rates of the genes for floR, sul-I, cmlA and blaTEM increased gradually.It is interesting to note that a downward trend of detection prevalence of aac (3)-II gene and low detection rate of qnrB gene (0.4%) were seen (Table 6).

A continuous monitoring of E. coli resistance and resistance genes in the Tongchuan chicken farm
In 2007-2012, a continuous monitoring of E. coli resistance and resistance genes in the Tongchuan chicken farms was performed.The results are shown in Tables 7  and 8. Table 7 shows that E. coli strains had resistance to kanamycin since 2008 while that E. coli strains were resistant to florfenicol and ciprofloxacinb since 2009.E. coli strains were resistant to ceftazidime and norfloxacin since 2010 but were sensitive to gentamicin and neomycin.The number of antibiotics of E. coli resistance increased from 7 to 13 between 2007 and 2012.Table 8 indicates that, in 2007-2012, detection prevalence of the genes for blaTEM, tetA, and tetB in E. coli strains in this farm was more than 80%.The detection prevalence of the genes for sul-I and cmlA increased while the detection prevalence of the gene for aac(3)-II showed a declining trend.

The correlation between resistance and resistance genes of the E. coli isolates
Resistance of 1002 chicken E. coli strains to different antibiotics and the related resistance genes are shown in Table 9.The ampicillin, streptomycin, trimethoprim sulfamethoxazole, florfenicol, ciprofloxacin and doxycycline resistance genes in 1002 chicken E. coli strains are shown in Table 9.Among the 1002 E. coli strains detected, detection prevalence of the genes for tetA, tetB, blaTEM, aac(3)-II, sul-I, cmlA, floR, and qnrB were 97. 4, 99.6, 88.9, 85.3, 41.7, 40.3, 26.5 and 0.5%, respectively.
The detection rates of the genes for tetA, tetB, blaTEM and aac(3)-II were positively correlated with the doxycycline, ampicillin and streptomycin-resistant coli strains, respectively (P < 0.01).Only 41.7% of the 223 trimethoprim sulfamethoxazole-resistant chicken E. coli strains carried the sul-I gene, showing no significant difference with the strains resistance to trimethoprim sulfamethoxazole (P > 0.05).In the 268 florfenicol-resistant chicken E. coli strains, the detection prevalence of cmlA and floR were 40.3 and 26.5%, respectively, showing no significant difference with the strains resistance to florfenicol (P > 0.05).Only 0.4% of the 742 ciprofloxacin-resistant strains carried the qnrB gene, showing no correlation with strains resistance to ciprofloxacin.

Sequence analysis of resistance genes in chicken E. coli strains
Among 1002 chicken E. coli strains tested, eight kinds of resistance genes in E. coli strains were detected by PCR (Figure 1).The sequences of these resistance genes have been sequenced and deposited with GenBank accession numbers JQ362472 (cmlA), JQ362473 (floR),

DISCUSSION
Antibiotics are widely used in the treatment and prevention of disease and can also promote the growth of animals.Under the pressure of antibiotic selectivity, drugresistant bacteria appear.To date, there are many reports regarding E. coli resistance in many countries and regions (Harada et al., 2012;Holzel et al., 2012;Johns et al., 2012;Ryu et al., 2012a,b).The E. coli resistance has become a global problem (Alan et al., 2007;Szmolka and Nagy, 2013).To date, the comprehensive studies on chicken E. coli resistance genes are relatively few.Soufi et al. (2011) studied resistance of 166 chicken E. coli strains in Tunisia and found that resistant rates of different strains to ampicillin, streptomycin, nalidixic acid, sulfonamide and tetracycline are 66-95%.To date, E. coli resistance problem is very serious in China.Dai et al. (2008) found that the resistant rates of chicken E. coli strains in China between 2001 and 2006 to ampicillin and doxycycline is more than 70%.The resistance of chicken E. coli strains to ampicillin and doxycycline are very serious.Our results revealed that resistant rates of chicken E. coli strains isolated from three provinces in China between 2007-2012 to ampicillin and doxycycline was 100 and 80%, respectively while the resistant prevalence of amikacin was below 30%.In the present study, we also found that quinolone resistance among E. coli from chicken in China is rising, which is consistent with a previous study (Zhang et al., 2010).The occurrence of antibiotic resistance of chicken E. coli strains isolated from three provinces in China during 2007-2012 is different, and this may be related to the use of different antibiotics in the farms in different provinces.In-feed or therapeutic antibiotics were used in these farms for all major classes of antibiotics except vancomycins.Ampicillin, tetracycline, doxycycline and nalidixic acid and ciprofloxacin were usually added into animal feed or drinking water in each sampling farm of these provinces, and the resistance of chicken E. coli strains isolated to the above antibiotics increased gradually.For example, a chicken farm in Gansu province in China used kanamycin to prevent and treat layer yolk peritonitis caused by E. coli for four years, the resistance rate of E. coli strains to kanamycin during 2009-2012 increased from 11.0 to 65.0%.
The resistance mechanism of E. coli is complicated.The resistance genes mediated by plasmid can make the resistance spread among different bacteria, which make bacteria obtain resistance genes more easily and thus produce multiple resistances (Li, 2005;Roberts, 2005;Zhang et al., 2009;Liu et al., 2012;Mosquito et al., 2012).This mechanism is that resistance genes can directly code enzymes which result in damage antibiotic effect (Skold, 2000;Yoo et al., 2003;Li et al., 2007;Ramirez and Tolmasky, 2010).Yu et (2009) found that the aminoglycoside resistance gene in human E. coli strain is main aac (3)-II.Previous studies on β-lactamase genes in the French E. coli strains indicated that the detection prevalence of the genes for blaTEM and blaCTX-M among 8 ceftiofur-resistant strains were 62.5 and 100%, respectively (Meunier et al., 2006).The results are similar to a previous study suggesting that blaTEM is the main β-lactamase resistance gene in the human E. coli (Yang et al., 2011).Tang et al. (2011) detected the E. coli drug-resistant gene in pigs in China during 2004-2007 and found that the β-lactamase resistance gene is mainly blaTEM and the detection rate is 87%.They also found that the resistance genes of aminoglycoside, tetracycline, and Sulfa are mainly aphA, tetB and sul-II, respectively.The detection prevalence for these resistance genes were 82.6, 49.8 and 55.4%, respectively (Tang et al., 2011).The detection rates for E. coli drug-resistant genes show differences which may be due to the strains from various countries and regions and the difference of serum type or antibiotic usage mode.During 2007-2012, we conducted the detection of resistance and resistance genes at chicken farms in Tongchuan City of Shaanxi province in China for 6 years, and found that the numbers of antibiotic resistance increased from 7 to 13.The detection prevalence of resistance genes for sul-I and cmlA increased gradually.The resistance genes of tetB, tetA, blaTEM and aac(3)-II in E. coli strains are positively correlated with the resistance of bacterial strain (P < 0.01).
In addition, only few quinolone resistance genes were detected from quinolone resistant strains in this study.Whether its resistance is associated with other types of resistance genes or other mechanisms of resistance remains to be further elucidated.Resistance genes were detected from several aminoglycosides and florfenicolsensitive strains, indicating the resistance genes in a silent state under the pressure of antibiotic.These strains are likely to develop into drug-resistant strains.Therefore, the detection of resistance and associated resistance genes in animal source of pathogenic isolates will be of great significance to the rational use of antibiotics in clinical and public health.
In summary, our results revealed that 1002 chicken E. coli strains isolated in three provinces in China during 2007-2012 showed multiple drug resistance.Of all isolated strains, 499 of 1002 E. coli strains (69%) were resistant to more than eight kinds of antibiotics, of which resistance gene tetB, tetA, blaTEM and aac(3)-II showed a positive correlation (P <0.01) with the E. coli strains resistance to antibiotics.The current results provide useful information on the drug prevention of chicken colibacillosis in China and resistance mechanisms of E. coli.

Table 1 .
E. coli strains isolated from chickens in three provinces in China during2007-2012.

Table 2 .
Primers used for PCR amplification of resistance genes in chicken E. coli. bReverse.

Table 3 .
Antimicrobial resistance of E. coli isolated from chickens of Shaanxi province during 2007-2012.

Table 4 .
Antimicrobial resistance of E. coli strains isolated from chickens of Henan province during 2007-2012.

Table 5 .
Antimicrobial resistance of E. coli strains isolated from chickens in Gansu province during2007-2012.

Table 7 .
Antimicrobial resistance of chicken E. coli strains isolated from the Tongchuan farms at Shaanxi province during 2007-2012.

Table 8 .
Resistance genes in chicken E. coli strains isolated from the Tongchuan farms at Shaanxi province during 2007-2012.

Table 9 .
Resistance and related resistance genes in 1002 chicken E. coli strains to different antibiotics.