Abstract
The global dissemination of colistin resistance has received a great deal of attention. Recently, the plasmid-mediated colistin resistance encoded by mcr-1 and mcr-2 genes in Escherichia coli (E.coli) strains from animals, food, and patients in China have been reported continuously. To make clear the colisin resistance and mcr gene spread in domestic animals in Jiangsu Province, we collected fecael swabs from pigs, chicken and cattle at different age distributed in intensive feeding farms. The selected chromogenic agar and mcr-PCR were used to screen the colisin resistance and mcr gene carriage. Colistin resistant E.coli colonies were identified from 54.25 % (440/811) pig faecal swabs, from 35.96 % (443/1232) chicken faecal swabs, and 26.92 % (42/156) from cattle faecal swabs. Of all the colisin resistant E.coli colonies, the positive amplifications of mcr-1 were significantly higher than mcr-2. The mcr-1 prevalence was 68.86 % (303/440) in pigs, 87.58 % (388/443) in chicken, and 71.43 % (30/42), compared with 46.82 % (206/440) in pigs, 14.90 % (66/443) in chicken, and 19.05 % (8/42) in cattle of prevalence of mcr-2. Co-occurrence of mcr-1 and mcr-2 was identified in 20 % (88/440) in pigs, 7.22 % (32/443) in chickens, and in 9.52 % (4/42) cattle. These data indicate that mcr was the most important colistin resistance mechanism. Interventions and alternative options are necessary to minimise further dissemination of mcr between food-producing animals and human.
IMPORTANCE Colistin is recognized one of the last defence lines for the treatment of highly resistant bacteria, but the emergence of resistance that conferred by a transferable plasmid-mediated mcr genes to this vital antibiotic is extremely disturbing. Here, we used E. coli as an index to monitor drug resistance in domestic animals (pigs, chicken and cattle). It was found that the colistin resistance widely occurred at all ages of domestic animals and the mcr-dependent mechanism dominated in E.coli. We also found that the elder and adult animals were a reservoir of resistant strains, suggesting a potential food safety issue and greater public health problems.
1 INTRODUCTUON
Colistin is recognized one of the last defence lines for the treatment of highly resistant bacteria, but the emergence of resistance that conferred by a transferable plasmid-mediated mcr-1 gene to this vital antibiotic is extremely disturbing. Actually, the mechanism of colistin resistance can be generally classified as mcr-independent or mcr-dependent. In a Morbidity and Mortality Weekly Report (MMWR) in September 2016, Vasquez and colleagues isolated a shiga-toxin-producing Escherichia coli (STEC) O157 with the mcr-1 gene in the whole genome sequence from stool [1]. In November 2016 in the Lancet Infectious Diseases, Liu et al. reported finding a transferable plasmid-mediated mcr-1 gene in E. coli isolates from animal food in China [2]. Compared with Klebsiella pneumoniae and Pseudomonas aeruginosa, in E. coli rare colistin resistance was mediated by chromosomal mutations and possibly imposed a fitness cost to the organism [3], which suggested that mcr-dependent colistin resistance perhaps was the major mechanism in E.coli, and would promote colistin resistance transmission among bacteria by plasmid transfer and chromosomal recombination. In China, [4]since the early 1980s colistin has been used in animals as a therapeutic drug and feed additive, which emphasizes that the use of colistin in animal feed has probably accelerated the dissemination of mcr gene in animals and subsequently humans.
2 MATERIALS AND METHODS
2.1 Sample collection
From March 2015 to December 2016, a surveillance of colistin resistant E.coli was conducted in Jiangsu Province, China. A total of 2199 faecal swab samples (Table 1) were collected from pigs, chicken and cattle. 811 faecal swab samples were collected from suckling piglets, weaned piglets, fattening pigs, and sows. 1232 faecal swab samples were collected from chicks, egg-laying growers and laying hens. 156 faecal samples were collected from calves, growing cows and milking cows.
2.2 Colistin resistance screening
E. coli has been identified as an index for monitoring drug resistance [5–6]. Here, we used E.coli selected chromogenic agar with 10 μg/mL of colistin sulphate [1] to test drug resistance to E.coli in domestic animal faeces. Each swab was dipped in 2 mL PBS for two hours at 4°C, and then homogenised by vortex. The homogenates were centrifuged at 500 rpm for 15 minutes. After the aspirated supernatants were centrifuged at 12,000 rpm for 5 min, the pellets were suspended with 1 mL PBS. Tenfold dilution series of 100 μL of the suspended pellets were plated onto E.coli selected chromogenic agar (HopeBio Biotech Corp., China) containing colistin sulphate. After overnight incubation at 37°C, the blue-green ones were counted as E.coli colonies (HopeBio Biotech Corp., China). If necessary, the faecal swabs were dropped into Tryptic Soy Broth (TSB) with antibiotics for enrichment and then bacterial culture was plated onto E.coli selected chromogenic agar (HopeBio Biotech Corp., China).
2.3 mcr-1 and mcr-2 screening
All blue-green colonies were picked into Luria-Bertani (LB) broth for 6 h enrichment, and bacterial culture were prepared DNA template by conventional boiling method. For the E.coli colonies identified, PCR was used to verify them by primer pairs of P1-F and P1-R [7] from the 16sRNA gene. For mcr-1 gene and mcr-2 screening, two primers pairs of P2-F/R [1] and P3-F/R [8] were used to amplify them. All the positive amplifications were sequenced by Genscript Corporation (Nanjing, China). Primers used in this study were listed in Table 2.
3. RESULT
3.1 Plate screening for colistin-resistant E.coli colonies
All blue-green colonies from E.coli selected chromogenic agar were recognized E.coli colonies after double identification using primer pairs of 16sRNA. In pigs, colistin resistant colonies were identified from 19.10 % (38/199) of sucking piglet, 40.76 % (86/211) of weaned piglet, 73.64 % (162/220) of fattening pig, and 85.08 % (154/181) of sow. In chicken, colistin resistant colonies were identified from 20 % (80/400) of chick, 37.44 % (152/406) of egg-laying grower, and 49.53 % (211/426) of laying hen. In cattle, colistin resistant colonies were identified from 14 % (7/50) of calve, 31.37 % (16/51) of growing cow, and 34.55% (19/55) of milking cow. Data on prevalence of colistin resistance in swab samples from all ages of domestic animals are presented in Fig 1.
3.2 Prevalence of mcr-1
The mcr-1 was identified in colistin-resistant E.coli colonies from all ages of pigs, chickens, and cattle. The mcr-1 prevalence was 68.86 % (303/440) in pigs, 87.58 % (388/443) in chicken, and 71.43 % (30/42) in cattle (Fig. 1). For pigs, the specific mcr-1 PCR identified the gene in 60.53 % (23/38) of suckling piglets, 60.47 % (52/86) of weaned piglets, 68.52 % (111/162) of fattening pigs, and 75.97 % (117/154) of sows. For chickens, the specific mcr-1 PCR identified the gene in 83.75 % (67/80) of chicks, 88.16 % (134/152) of egg-laying growers, and 88.63 % (187/211) of laying hens. For cattle, the specific mcr-1 PCR identified the gene in 57.14 % (4/7) of calves, 75.00 % (12/16) of growing cows, and 73.68 % (14/19) of milking cows.
3.2 Prevalence of mcr-2
The mcr-2 was identified in colistin-resistant E.coli colonies from all ages of pigs, chickens, and cattle. The mcr-2 prevalence was 46.82 % (206/440) in pigs, 14.90 % (66/443) in chicken, and 19.05 % (8/42) in cattle (Fig. 1). For pigs, the specific mcr-2 gene was amplified from 36.84 % (14/38) of suckling piglets, 39.53 % (34/86) of weaned piglets, 49.38 % (80/162) of fattening pigs, and 50.65 % (78/154) of sows. For chickens, the specific mcr-2 gene was amplified from 10 % (8/80) of chicks, 12.50 % (19/152) of egg-laying growers, and 18.48 % (39/211) of laying hens. For cattle, the specific mcr-2 gene was amplified from 28.57 % (2/7) of calves, 12.50 % (2/16) of growing cows, and 21.05 % (4/19) of milking cows.
3.3 Co-occurrence of mcr-1 and mcr-2
Both mcr-1 and mcr-2 positive amplifications were 20 % (88/440) in pigs, 7.22 % (32/443) in chickens, and 9.52 % (4/42) in cattle (Table 1). Dual positivity was identified in 7.89 % (3/38) of suckling piglets, 9.30 % (8/86) of weaned piglets, 20.37 % (33/162) of fattening pigs, 28.57 % (44/154) of sows, 5.00 % (4/80) of chicks, 4.61% (7/152) of egg-laying growers, 9.95 % (21/211) of laying hens, 6.25 % (1/16) of growing cows, and 15.79 % (3/19) of milking cows, but not in calves.
4. DISCUSSION
In the 1960s colistin was introduced into in food animal production in several countries for growth promotion, therapeutical and prophylactical purposes to control of Enterobacteriaceae infections, particularly for those caused by E.coli [5–6]. In 2016, Chinese scholars first reported that plasmid-mediated colistin resistance was encoded by the mcr-1 gene [1]. With this discovery, the higher prevalence of samples harboring mcr-1 gene in animal isolates compared to other origins raised alarm bell about the impact of colistin use on colistin resistance spread in animal production, livestock and poultry have been recognized as the major reservoir for colistin resistance transmission and amplification [9].
During 2015-2016, we collected 2199 faecal swabs from pigs, chicken and cattle to make clear prevalence of colisitn resistance in intensive breeding farms of Jiangsu Province. Our study using selected chromogenic agar with colistin showed that E.coli resistance to colistin occurred widely in pigs (54.25 %), poultry (35.96 %) and cattle (26.92 %), suggesting that colistin resistance was considerably serious, especially in pigs. From 2013 to 2014, it was reported that a high frequency of colistin resistance in E. coli from pigs (26.5%), from chickens (14.0%), and from cattle (0.9%) on farms in different geographic areas of China, including Jiangsu Province [10]. Increasing use of colistin in fodder in recent years may be the reason of the high prevalence of colistin resistance in these food animals. Here, in 811 pig samples, colistin resistant colonies were identified from 85.08 % (154/181) of sows and 73.64 % (162/220) of fattening pigs, significantly higher than 19.10 % (38/199) of sucking piglet, 40.76 % (86/211) of weaned piglets. The same patterns also were found in chicken 1232 samples and 156 cattle samples. The highest proportions of resistant E.coli colonies were identified from the adult animals, implying that the long-term selective pressure resulted in not only the highest prevalence of colistin resistance among E. coli isolates from adult animals found in this study, but also bacterial evolution and adaption from the piglet groups to adult groups [11]. Compared with the isolates from pigs and chickens recovered during 2013-2014, E. coli isolates collected during 2007-2008 (5.5%) and 2010-2011 (12.4%) showed significantly lower frequency of colistin resistance [12]. A high frequency of colistin resistance in E. coli from pigs on farm (24.1%) and from chickens on farm (14.0%) led to a high prevalence of colistin at pig slaughter (24.3%) and chicken slaughter (9.5%) in 2013-2014 [12]. The adult animals generally entered the slaughter house and the food chains, drug-resistant strains inevitably invaded our dining table for consumers to cause public health events. Sows are the reservoir of resistant strains, they give not only life to piglets, but also resistant strains to them, which promote drug resistance circulation among Chinese farms [13]. The link between animals and humans in terms of colistin resistant E. coli strain transfer following direct contact has recently been confirmed [14]. The overuse of antibiotics will promote the unrestricted expansion and circulation of drug-resistant strains among human-animals-environment.
While colistin is a last-line antibiotic used to treat multidrug resistant Gram-negative bacteria (GNB) isolated from food animals, raw meat, and humans in several countries [15], its efficacy is being compromised by the detected mobile colistin resistance genes, mcr-1 at the end of 2015 [2], and subsequently mcr-2, mcr-3, mcr-4, mcr-5[8, 16]. Of all the colisin resistant E.coli colonies in our study, the mcr-1 was the predominant gene for the colistin resistance of E.coli, higher than mcr-2. The mcr-1 prevalence was 68.86 % (303/440) in pigs, 87.58 % (388/443) in chicken, and 71.43 % (30/42) in cattle, compared with mcr-2 prevalence of 46.82 % (206/440) in pigs, 14.90 % (66/443) in chicken, and 19.05 % (8/42) in cattle. The mcr variant gene prevalence reported by [17] was considerably higher than ours and those previously reported in China which was based on the presence of the mcr in bacterial isolates. They directly detected the clinical samples instead of isolated E.coi strains and sequenced three variants of mcr-1, mcr-2, and mcr-3 [17]. The mcr-1 and mcr-2 occurred widely in pigs and poultry of Chinese farms [17–18]. Except harbouring the mcr genes, a mcr-independent mechanism behind the remaining colistin resistant E.coli colonies, for example, lipopolysaccharide modification [19], other (transferable) colistin-resistant mechanisms, and undefined mechanisms exist. The implication of the mcr gene wide spread will be enormous if plasmid-mediated colistin resistance was readily passed between E. coli strains, and also be passed to Klebsiella pneumoniae and Pseudomonas aeruginosa strains like descriptions in the Lancet Infectious Disease published by Liu Yi-Yun and colleagues [2]. Since 1 April 2017, the Chinese government has implemented the withdrawal of colistin as a food additive for growth promotion in food animal [20], this policy is in line with international policy of One Health.
5. CONCLUSION
The management of colistin resistance at the human-animal-environment interface requires the urgent use of the One Health approach for effective control and prevention. Our study will provide new data about colistin resistance prevalence worldwide. The colistin resistance widely occurred at all ages of domestic animals and the mcr-dependent mechanism dominated in E.coli. We also found that the older and adult animals were a reservoir of resistant strains, suggesting a potential food safety issue and greater public health problems.
AUTHOR CONTRIBUTIONS
Zhang XH and He KW conceived and designed the experiments. Zhang BC analyzed the data. Yu ZY, GuoYY, WangJ, Zhao PD, and Liu JJ performed the experiments; Zhang XH and Zhang BC wrote the paper.
Funding
This work was supported by the National Natural Science Foundation of China (31572503) and Jiangsu R&D plan (BE2017341-1).
Competing interests
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethical approval
Ethical approval was granted by the Ethics Committee of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences (Nanjing, Jiangsu, China) [20150212].