Analysis of antibiotic resistance phenotypes and genes of Escherichia coli from healthy swine in Guizhou, China

This study was carried out to investigate the resistance phenotypes and resistance genes of Escherichia coli from swine in Guizhou, China. A total of 47 E. coli strains isolated between 2013 and 2018 were tested using the Kirby–Bauer (K–B) method to verify their resistance to 19 common clinical antimicrobials. Five classes consisting of 29 resistance genes were detected using polymerase chain reaction. The status regarding extended-spectrum β-lactamase (ESBL) and the relationship between ESBL CTX-M-type β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) genes were analysed. A total of 46 strains (97.9%) were found to be multidrug resistant. Amongst them, 27 strains (57.4%) were resistant to more than eight antimicrobials, and the maximum number of resistant antimicrobial agents was 16. Twenty antibiotic resistance genes were detected, including six β-lactamase genes blaTEM (74.5%), blaCTX-M-9G (29.8%), blaDHA (17.0%), blaCTX-M-1G (10.6%), blaSHV (8.5%), blaOXA (2.1%), five aminoglycoside-modifying enzyme genes aac(3′)-IV (93.6%), aadA1 (78.7%), aadA2 (76.6%), aac(3′)-II c (55.3%), aac(6′)-Ib (2.1%) and five amphenicol resistance genes floR (70.2%), cmlA (53.2%), cat2 (10.6%), cat1 (6.4%), cmlB (2.1%), three PMQR genes qnrS (55.3%), oqxA (53.2%), qepA (27.7%) and polypeptide resistance gene mcr-1 (40.4%). The detection rate of ESBL-positive strains was 80.9% (38/47) and ESBL TEM-type was the most abundant ESBLs. The percentage of the PMQR gene in blaCTX-M-positive strains was high, and the detection rate of blaCTX-M-9G was the highest in CTX-M type. It is clear that multiple drug resistant E. coli is common in healthy swine in this study. Extended-spectrum β-lactamase is very abundant in the E. coli strains isolated from swine and most of them are multiple compound genotypes.


Escherichia coli strains
A total of 112 faecal swab samples were collected from 47 large-scale pig farms in 13 counties and cities, including Guiyang, Anshun, Bijie, Qianxinan, Qiandongnan and Qiannan in Guizhou Province between 2013 and 2018. The samples were taken from diseased piglets with yellow and white scour symptoms, streaked out on Mackay medium and cultured at 37 °C for 24 h. Pink colonies were picked and purified on Luria-Bertani (LB) agar plate at 37 °C for 24 h, and the plates were stored at 4 °C for subsequent use. Isolated bacterial strains were cultured in LB medium at 37 °C for 24 h and tested according to Bergey's Manual of Systematic Bacteriology. The strains were further classified using 16s ribosomal ribonucleic acid (rRNA) sequences, which were sequenced using a pair of universal primers (16S-F, 5′-TGT GGG AAC GGC GAG TCG GAA TAC-3′; 16S-R, 5′-GGG CGC AGG GGA TGA AAC TCA AC-3′) (Shahi, Singh & Kumar 2013).

Drug susceptibility test
The Kirby-Bauer method (K-B method) was used to test the susceptibility to antimicrobials. Escherichia coli ATCC ® 25922 was used as the quality control strain in antimicrobial susceptibility testing. The breakpoints for tested drugs were used as recommended by the standards and guidelines described by the CLSI (2018b: M100) and veterinary CLSI (VET01-A4/VET01-S2) (CLSI 2018a(CLSI , 2018b. Based on the test, E. coli was classified as susceptible (S), intermediate (I) and resistant (R).

Extraction of bacterial deoxyribonucleic acid
Strains stored at −80 °C were inoculated in LB broth for recovery, streaked on eosin-methylene blue agar medium and incubated at 37 °C overnight. Fresh single colonies on the eosin-methylene blue plate were inoculated in 3 mL LB broth and cultured at 37 °C on a shaker operated at 200 revolutions per minute (r/min) for 12 h. The culture was then centrifuged at 12 000 r/min for 5 min. Bacterial pellet was suspended in 500 μL 1 × TE, vortexed and boiled for 10 min. Bacterial DNA was extracted using TIANamp Bacterial DNA Extraction Kit (TIANGEN, China) according to manufacturer's instruction.

Ethical consideration
No animal-related tests were involved in this study, and the faecal swab samples were provided by the farm owners who volunteered to participate in the study.

Isolated Escherichia coli strains were multidrug resistant
Drug susceptibility tests showed that 47 E. coli strains isolated from swine had the highest rates of resistance to tetracycline and doxycycline, which were both 95.7% (n = 45), followed (n = 15), respectively. The strains had relatively lower rates of resistance to ciprofloxacin, levofloxacin, neomycin and streptomycin, which were 29.8% (n = 14), 29.8% (n = 14), 19.1% (n = 9) and 17.0% (n = 8), respectively. In addition, the tests also showed that the strain had a rate of resistance to cefoxitin at only 8.5% (n = 4), and none of them was resistant to amikacin (Table 2).
When analysed based on isolation time (

Antibiotic resistance genes were detected in all isolates
In this study, 20 ARGs were detected (

Discussion
We investigated 47 E. coli strains isolated from swine in Guizhou from 2013 to 2018 for their resistance to 21 common antimicrobial agents. The results showed serious multidrug resistance in this region. The isolated strains have the highest resistance to the tetracycline antimicrobials, which are comparable to the rates of resistance to tetracycline (97.6%) and doxycycline (89.3%) in E. coli from swine in Guizhou in 2018 (Wang & Tan 2018). The rate of resistance to sulfaisoxazole is 89.4%, which is close to the rate of resistance to sulfonamide antimicrobials in 2018 (Kou, Liu & Tan 2018). The results may be related to the long-term irrational use of these three antibiotics in the prevention and treatment of piglet respiratory diseases and bacterial diarrhoea in pig breeding in Guizhou.   The strains are also resistant to β-lactam drugs, the rates of resistance to third-generation cephalosporin ceftazidime and cefotaxime were 70.2% and 31.9%, respectively, which were higher than that of second-generation cephalosporin drug cefoxitin (8.5%). This may be attributed to the irrational use of third-generation cephalosporins and the less use of secondgeneration cephalosporins in the pig farms in Guizhou in recent years.
For aminoglycoside resistance, the highest rates were resistant to kanamycin and gentamicin, followed by neomycin and streptomycin. However, none of the strain is resistant to amikacin. These results are consistent with earlier reports for Guangxi (Ge, Shi & Hu 2019), Chongqing (Chen & Liao 2019), Fujian (Yang, Luo & Xie 2018) and southeastern Shanxi (Shen, Kong & Guo 2019). This could be because of the less use of amikacin in the pig farms for disease treatment and prevention in Guizhou. The rates of resistance to aminoglycoside gentamicin, streptomycin, ciprofloxacin (a fluoroquinolone antimicrobial agent) and polymyxin (a polypeptide antimicrobial agent) are obviously lower than the rates of resistance (gentamicin 81.10%, streptomycin 53.66%, ciprofloxacin 53.05% and polymyxin 51.83%) observed in Guizhou in 2016 (Cao, Tan & Liu 2016), whilst the rate of resistance to florfenicol (an amphenicol agent) is obviously higher than previously reported rate (Cao et al. 2016). It is likely that the irrational use of fluorophenicol in the pig farms in recent years and the less use of gentamicin, streptomycin and polymyxins are responsible for the situation. At the same time, the reduction in ciprofloxacin resistance rate may be related to China's ban on the use of four antibiotics in aquaculture from 2016, as well as improved management of livestock form and changes of antimicrobials used and administration method of antimicrobials.
From 2 to 14 ARGs are detected in various stains and over a half of the strains are found to have more than eight resistance genes, indicating that the proportion and quantity of ARGs are high in these strains and the drug resistance problem is serious. In this study, five amphenicols resistant genes were detected, of which, floR (70.2%, 33/47) and cmlA (53.2%, 25/47) genes are frequent, which are believed to be the main cause of amphenicol resistance in the strains. The detection rates of four aminoglycoside-modifying enzyme genes (aac(3')-IV, aadA1, aadA2, aac(3')-IIc) are higher than 50%, and multiple aminoglycoside modifying enzyme genes are detected in the same strains, resulting in high-level resistance to aminoglycosides. In this study, the detection rates of aminoglycosides resistance genes are basically consistent with the resistance phenotypes. However, five aac(3′)-IV positive strains are found susceptible to aminoglycosides, probably because of infective expression of the gene in these strains. mcr-1i is also frequently found in the strains. mcr-1i is present in plasmid and chromosome, but mainly plasmids. Plasmids could carry multiple ARGs (Zhu 2019). The high detection rate (40.4%) of mcr-1 in this study likely results from overdose use of polymyxins in pig feed in Guizhou.
At present, the resistance of Gram-negative bacteria to β-lactam drugs is increasingly higher, and the most common resistance mechanism is that bacteria can produce three metalloenzymes: ESBL, cephalosporinase (AmpC enzyme) and carbapenems (Meini et al. 2019 Low-resistant PMQR genes conferring quinolone resistances are shown to be closely related to the prevalence of ESBLs (Ma et al. 2009;Wu et al. 2007;Yang, Zhuang & Hua 2018), especially qnr/oqxAB and bla CTX-M genes, which may be located on the same plasmid (Liu et al. 2014;Strahilevitz et al. 2009). In this study, PMQR genes were analysed in 17 E. coli strains and 14 strains were found to have bla CTX-M with a detection rate of 82.4%, of which, qnrS and oqxA are detected at 58.8% and 52.9% of the strains. The complex genotype bla CTX-M-9G +qnrS+oqxA is detected in four strains. It is, therefore, likely that the ESBLs and PMQR genes are located on the same plasmid in the E. coli stains, which would somewhat increase the risk of horizontal transmission of ARGs. Previously, it was shown that oqxA and oqxB frequently coexist ). However, this coexistence was not observed in our study, and further study is needed to clarify the situation.

Conclusion
Extended-spectrum β-lactamase is widespread in E. coli from pigs in Guizhou and TEM-type is the most prevalent ESBLs. Most of the E. coli strains have multiple ESBLs. In CTX-M strains, CTX-M-9G is the most frequent; PMQR carriers are often detected in bla CTX-M -positive strains, and the two genes are likely located on the same plasmids. Therefore, the co-transmission needs to be addressed in the future. Only oqxA was detected but not oqxB in this study, indicating a need to clarify their relationship. Co-existence of bla TEM +bla CTX-M-1G +bla CTX-M-9G in ECP4 and bla TEM +bla CTX-M-1G + bla CTX-M-9G +bla SHV in ECP27 may allow further investigation of the position and position effect. In addition, over 40% strains are found to contain mcr-1, and the location of the gene needs to be defined.