An Epidemiological Study of Drug Resistance and Resistance Genes in Bovine Escherichia coli Isolates in Heilongjiang Province of China

Background: To explore the epidemiology of bovine multidrug-resistant Escherichia coli isolates and resistance genes in Heilongjiang province of China. This study examined the prevalence of genes in bovine E. coli isolates, which confer resistance to antibiotics that are commonly used in the clinic, in regions of Baiquan, Shangzhi, and Songbei of Harbin. The purpose of the study was to investigate the epidemiology of the main resistance genes of bovine E. coli isolates in clinical veterinary medicine, and to provide a theoretical basis for preventing the spread of drug-resistant bacteria, as well as for rational drug use. Materials, Methods & Results: The sensitivity of 105 isolates to 22 antibiotics was determined using the KirbyBauer disk diffusion method, and the distribution of 19 kinds of common drug resistance genes was investigated using Polymerase Chain Reaction. The results showed that the resistance rate to nine antibiotics was over 50%, including rifampin (84.76%), ampicillin (73.58%), tetracycline (69.52%), and sulfisoxazole (59.05%). In total, 105 strains of bovine E. coli presented 21 spectra of drug resistance, including eight strains (7.62%, 8/105) that were resistant to one antibiotic and four strains (3.81%, 4/105) that were resistant to 21 antibiotics. The resistance gene detection results showed that the streptomycin-resistance gene strA was found in 73 isolates, accounting for 69.52% of the isolates, followed by the sulfanilamide-resistance genes sul3/sul2 and the aminoglycoside-resistance gene aphA, which accounted for 57.14%, 51.43%, and 50.48%, respectively, of the isolates. Discussion: This study revealed serious drug resistance of bovine E. coli isolates in some areas of Heilongjiang province. Of 105 E. coli isolates, more than 50% were resistant to the following antibacterial drugs: rifampicin, ampicillin, tetracycline, sulfisoxazole, and cephalothin. The isolates were the most sensitive to amikacin, with a sensitivity of 84.76%, followed by sensitivity to ofloxacin, ciprofloxacin, norfloxacin, cefoxitin, and tobramycin. Drug sensitivity tests showed that the drug resistance spectra of the bovine E. coli isolates was different in different regions, indicating that there were multidrug-resistant bovine E. coli isolates in different regions of Heilongjiang province, and that drug resistance differed among different regions. This may be due to prolonged use or overuse of antibiotics in a particular locality. Additionally, because of different management modes of livestock farms, the application of antimicrobial drugs in some farms may have imposed selective pressure on the intestinal flora including E. coli, resulting in the horizontal transmission of drug resistance among the bacteria. The study found that some strains had a resistance phenotype, but no resistance gene, while some had a resistance gene without expressing a resistance phenotype, which is consistent with relevant reports in the literature. This may be related to the same genotype corresponding to different resistance phenotypes, or different levels of gene expression, or different drug metabolic rates. In our study, some strains with certain drug resistance genes were sensitive to the corresponding drug, which may be due to mutations of drug-resistance genes, the loss of a strains resistance phenotype, or the loss of gene function. These issues require further study. This study revealed serious drug resistance of bovine E. coli isolates in some areas of Heilongjiang province. Of 105 E. coli isolates, more than 50% were resistant to the following antibacterial drugs: rifampicin, ampicillin, tetracycline, sulfisoxazole, and cephalothin. The isolates were the most sensitive to amikacin, with a sensitivity of 84.76%, followed by sensitivity to ofloxacin, ciprofloxacin, norfloxacin, cefoxitin, and tobramycin.


INTRODUCTION
Escherichia coli is a pathogen that causes anthropozoonoses, which result in serious hazards to the livestock industry, food production, and environmental water sources [4].E. coli is prone to drug resistance.Drugresistant strains are gradually increasing in the clinic, and the drug resistance spectrum is expanding.Meanwhile, the residual presence of antimicrobial drugs in animals, as well as the spread of drug-resistant strains among different animals, adversely affects the sustainable development of the livestock industry, as well as public health.
The selective pressure of antibiotics can lead to drug-resistant E. coli in the gut of animals [20].In vivo, E. coli not only obtains drug resistance through gene mutation and by capturing exogenous genes, but also participates in horizontal and vertical transmission of resistance genes, thus becoming a potential reservoir of drug-resistant genes in animals [18].E. coli has been identified as an indicator strain for drug resistance tests, which is important for monitoring drug resistance [1].Therefore, the detection of resistance genes is important to prevent bacterial resistance and to explore the mechanism of bacterial resistance.
This study examined the prevalence of genes in bovine E. coli isolates, which confer resistance to antibiotics that are commonly used in the clinic, in three regions Heilongjiang province of China (Baiquan, Shangzhi, and Songbei of Harbin).The purpose of the study was to investigate the epidemiology of the main resistance genes of bovine E. coli isolates in clinical veterinary medicine, and to provide a theoretical basis for preventing the spread of drug-resistant bacteria, as well as for rational drug use.

Clinical isolates of Escherichia coli strains
The strains were isolated from rectal swabs and stools from calves with diarrhea and healthy cattle on 5 farms in three regions of Heilongjiang province of China (Baiquan, Shangzhi, and Songbei of Harbin).The samples were streak-inoculated onto MacConkey agar and eosin methylene blue agar and cultured at 37°C for 24 h.The resulting colonies were picked and identified as E. coli by biochemistry and Polymerase Chain Reaction.The quality control E. coli strain ATCC 25922 was purchased from the China Industrial Microorganism Culture Collection Center.

Drug sensitivity tests
Drug sensitivity tests were performed with the Kirby-Bauer disk diffusion method using Mueller-Hinton agar.Bacteria were inoculated into BHI 2 medium and shaking-cultured at 37°C for 10 h.Then, normal saline was added to adjust the cultures to 0.5 McFarland units, the same as that of the standard tube, for comparative tests.Within 15 min, a sterile cotton swab was dipped into the culture broth and spread on a Mueller-Hinton agar plate 2 .Under aerobic conditions, the plate was cultured at 37°C for 24 h.E. coli ATCC 25922 served as a quality control strain, the diameter of the bacteriostatic zone was measured by a Vernier caliper, and the sensitivity of the isolates to antibacterial drugs was assessed according to American Committee for Clinical Laboratory Standards.

Resistance gene testing of isolates
Based on the drug resistance mechanisms of E. coli against β-lactams, aminoglycosides, tetracyclines, streptomycin, sulfonamides, quinolones, and chloramphenicol, 19 resistance genes, including SHV genes, floR, and aacA4, were selected to analyze the genotypes of the resistance genes from the bovine E. coli isolates.Amplification products were processed via 1.0% agarose gel electrophoresis (1 × TAE 3 , with ethidium bromide (EB) 3 staining, for 20 min at 120 V) and photographed with a gel-documenting system 4 .PCR primers and annealing temperatures are shown in Table 1.

Drug resistance spectra of bovine Escherichia coli isolates in different regions
In three areas of Heilongjiang province, a total of 105 bovine E. coli strains showed 21 drug resistance spectra, in which 8 strains (7.62%, 8/105) were resistant to 1 drug, and 4 strains (3.81%, 4/105) were resistant to 21 drugs.In the Shangzhi area, strains resistant to 13 or 15 drugs accounted for 15.91% of the strains, while strains resistant to 17, 19, or 1 drug accounted for 11.36% of the strains.In the Baiquan area, 34.88% of the strains were resistant to one drug, followed by strains resistant to 5, 3, or 13 drugs, accounting for 25.58%, 23.26%, and 9.30% of the strains, respectively.In the Songbei area, strains resistant to 7 or 11 drugs accounted for 22.22% of the strains, followed by strains resistant to 9 (16.67%), 15 (11.11%), and 19 drugs (11.11%) [Figure 1].

Resistance phenotypes of bovine Escherichia coli
Of the 105 bovine E. coli isolates, there were mainly 15 resistance phenotypes to nine antibiotics.AMP + S + TC + C + SIZ and AMP + SIZ resistant phenotypes accounted for 10.48% of the isolates (the maximal proportion), followed by resistance to AMP + CF + SIZ + S + TC, AMP + CF + SIZ + TC + S + GM, or AMP + CF + TC + S + GM + C, accounting for 8.57% of the total isolates (Table 3).

Resistance genes of bovine Escherichia coli isolates
Of the 105 bovine E. coli isolates, the test results for 19 resistance genes in 7 categories showed that the sulfonamide-resistant gene sul3 was the most widely distributed, as it was found in 30 isolates (28.57%), followed by the chloramphenicol-resistant gene floR, which was found in 28 strains (26.67%), the β-lactam-resistant gene blaTEM (25 strains, 23.18%), and the streptomycin-resistant gene strA (20 strains, 19.05%).Five strains carried the quinolone-resistant gene qepA, accounting for 4.76% of the strains (the lowest frequency), followed by the sulfonamideresistant gene sul1 (6.67%) and the chloramphenicolresistant gene clmA (7.62%) [Table 4].Of the 105 bovine E. coli isolates from the 3 areas, there were 7 kinds of resistant genotypes, including resistance to β-lactams, aminoglycosides, sulfonamides, tetracycline, chloramphenicol, quinolones, and streptomycin.Despite the presence of the same genotypes in all three areas, there were some differences between the different areas regarding the number of isolates carrying drug resistance genes (Table 5).

Correlation between resistance phenotype and resistance genes
Consistency analysis of the resistance phenotypes and the resistance genes to nine antibiotics showed that sulfamethoxazole had the highest consistency (83.87%), followed by chloramphenicol (68.29%), while norfloxacin had the lowest consistency (22.72%).It was observed that some isolates presented drug resistance without carrying resistance genes, whereas some isolates carried resistance genes without manifesting a resistance phenotype (Table 6).: number of E. coli isolates expressing phenotypic resistance to the indicated antimicrobial agent; N G * : number of E. coli isolates carrying the indicated resistance gene; P+/G-* : number of phenotypically resistant E. coli isolates (P+) with no resistance gene (G-) for the drug identified; P-/G+ * : number of phenotypically susceptible E. coli isolates (P-) with a resistance gene (G+) for the drug identified.

DISCUSSION
The drug resistance of Escherichia coli is mainly acquired via mutation or the capture of exogenous resistance genes by plasmids, transposons, and other removable fragments, and exogenous genes play a major role in bacterial resistance [19].
β-lactamase plays a key role in the drug resistance of E. coli to broad-spectrum β-lactams [3,12].This study examined four kinds of β-lactam-resistance genes and found that more isolates carried blaTEM and blaCMY genes, while fewer isolates carried blaCTX and blaSHV genes.Of the 105 bovine E. coli isolates in our study, the overall detection rate of blaTEM was 15.24% (16/105), which was higher than that reported in China a few years ago [6,23], indicating that due to the increased use of cephalosporins, there was a rising trend of extended spectrum β lactamases (ESBLs) produced by bovine E. coli isolates.
The aminoglycoside-resistance gene aacA4 had the highest detection rate, and the resistance genes aacA4, aphA, and aadB were detected in all three regions, which was different from a previous report.This suggests that there are some differences in the distribution of aminoglycoside-resistance genes in different regions.
The streptomycin-resistance genes strA and strB co-mediate the streptomycin-resistance phenotype [17].In this study, the detection rates of strA and strB were 19.05% and 16.19%, respectively, indicating that almost all strains carrying strA also carried strB; thus, the consistency of the results was good.
It was reported that the most common tetracycline-resistance phenotype had more than 40 kinds of coding genes, but the two efflux pump genes, tetA and tetB, accounted for 90% of the resistance [16], which was also reported in gut commensal E. coli and pathogenic E. coli [10,11].In this study, the detection rates of tetA and tetB were 16.19% and 10.48%, respectively.However, only tetA was detected when testing tetracycline-resistance genes in the Sangzhi area, probably because the incompatibility between plasmids resulted in a negative correlation between tetB and tetA [2].
The test results for the chloramphenicol resistant genes floR and clmA showed that although chloramphenicol use is forbidden in livestock animals in China, the survey found a high degree of resistance to chloramphenicol, with a positive rate of 34.29%.
We suspected that the genes encoding chloramphenicol resistance were co-screened and preserved under selective pressure related to other antimicrobial agents.One study demonstrated that strains carrying floR also showed resistance to chloramphenicol, while strains carrying cmlA were not 100% resistant to florfenicol [22], which was consistent with the results of our study.Therefore, the mechanism of chloramphenicol resistance still needs to be further explored.
The results of sulfonamide-resistance gene tests showed that the detection rates of sul1, sul2, and sul3 were 6.67%, 14.29%, and 28.57%, respectively.In 2003, sul3 was first discovered by Perreten et al. [13].Afterwards, sul3 was found in E. coli strains in many countries.It was reported [7,8] that the main sulfonamide-resistance genes in bovine E. coli isolates were sul1 and sul2; the detection rate of sul2 was two to five times that of sul1, and only a few sul3 genes were observed.This was quite different from our study, in which the detection rate of sul3 was the highest, and the detection rate of sul2 was two times higher than that of sul1.
Since 1998, three plasmid-mediated quinolone resistance determinants including qnr genes (qnrA, qnrB, qnrS), aac(6')-Ib-cr, and qepA, have been reported in various Enterobacteriaceae genera worldwide [9].qnr encodes the QNR protein, which can protect DNA gyrase and topoisomerase IV from inhibition by quinolones; a qepA-encoded plasmid-mediated efflux pump can mediate low-level drug resistance by excreting hydrophilic quinolones [15].In this study, the detection rates of qnrS, qnrA, and qepA in 105 bovine E. coli isolates were 15.24%, 8.57%, and 4.76%, respectively, indicating that PMQR gene is prevalent in Heilongjiang province, and that qnrS is the dominant type.This is different from reports in recent years [14], which may be due to the different sources of strains and different medications.Studies have shown that qnr plasmids often carry integrons, transposons, and multi-drug resistance gene determinants, especially associated with ESBL-producing genes, which are helpful for the horizontal or vertical transmission of resistance genes among bacteria in the same or different genus under the selective pressure of different drugs [5].Therefore, how to promote the scientific and rational use of quinolones to reduce the production of quinolone-resistant strains becomes an important issue in future research.

Figure 1 .
Figure 1.Drug resistance spectrum of Escherichia coli from cattle in different areas of Heilongjiang province of China

Table 1 .
Primers used in Resistance gene detection of Escherichia coli isolated from cattle.

Table 2 .
The results of drug sensitivity test of 105 Escherichia coli isolated from cattle.

Table 3 .
Nine kinds of antimicrobial drug resistance phenotype of 105 Escherichia coli isolated from cattle.

Table 4 .
Carrier rate of different resistant genes of 105 Escherichia coli isolated from cattle.

Table 5 .
The distribution of resistant genes of Escherichia coli isolated from cattle fom regions of Baiquan, Shangzhi, and Songbei of Harbin -Heilongjiang province of China.

Table 6 .
Comparison of consistency in Escherichia coli isolates according to phenotypic and genotypic testing.