Prevalence and Molecular Characterization of Antimicrobial-Resistant Escherichia coli in Pig Farms,Slaughterhouses,and Terminal Markets in Henan Province of China

Abstract

Escherichia coli is an important foodborne pathogen and also plays key roles in dissemination of antimicrobial resistance genes (ARGs). However, current data on the prevalence of antimicrobial-resistant E. coli at different nodes of the pork supplying chain are still limited. Herein, we investigated drug-resistant phenotypes and molecular characteristics of E. coli strains isolated from different pig farms, slaughterhouses, and terminal markets in the Henan Province of China. A total of 191 (70.74%), 140 (35.09%), and 77 (30.20%) E. coli strains were isolated from 270, 399, and 255 samples collected from pig farms, slaughterhouses, and retailing markets, respectively. Antimicrobial susceptibility testing revealed that these 408 strains showed severe antimicrobial resistance profiles. Approximately 93.19% (178/191), 66.43% (93/140), and 67.53% (52/77) of the isolates from farms, slaughterhouses, and terminal markets were resistant to three of the nine antibiotic classes tested, respectively. Multilocus sequence typing showed that sequence types (STs) 10 and ST101 were commonly identified among the isolates from farms, slaughterhouses, and terminal markets. Isolates belonging to these two STs carried multiple ARGs, conferring resistance to the antibiotics tested. Two important ARGs with great public health concerns (mcr-1 and bla _NDM-1) were found from these two STs. Isolates belonging to these two STs also carried several virulence factor-encoding genes, including astA, tsh, and traT, which might contribute to the pathogenesis of these isolates. The wide prevalence and distribution of these two STs in different nodes of pork supplying chain might represent a big public health threat and should receive more attention.

Introduction

The spread of drug-resistant bacteria is one of the most severe challenges to global public health (Low et al., 2019). It estimates that if no active actions are taken to slow it down, the rise of the infections by antibiotic-resistant bacteria will take 10 million lives per year and will result in a cumulative 100 trillion dollars of economic loss by the year 2050 (O'neill, 2015). The long-term use of antimicrobials in food-producing animals is regarded as an important source of drug-resistant bacteria, which might be transmitted to humans by animal production and other transmission routes (WHO, 2017).

Among the drug-resistant bacteria of great concern, Escherichia coli is a worldwide leading foodborne pathogen, and it is also an essential biomarker for monitoring antimicrobial resistance (AMR) (Brisola et al., 2019). This bacterial species is intrinsically sensitive to almost all clinically relevant antimicrobial agents, but it has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer (Nolivos et al., 2019). In recent years, E. coli has been found to play critical roles in the worldwide spread of plasmid-mediated colistin and tigecycline resistance in both medical and veterinary medicine, which represents a significant threat to global public health (Liu et al., 2016; He et al., 2019).

China is the largest pig-rearing and pork-consuming country in the world. However, pig production in China is very complex, with different sizes of pig farms and production models in various regions. From this point, the prevalence of drug-resistant bacteria in different nodes of the pork supplying chain in different regions in China remains to be addressed. In this study, we reported the prevalence and molecular characterization of drug-resistant E. coli in pig farms, slaughterhouses, and retailing meat markets in the Henan Province, one of the significant pork-producing and business regions in China.

Materials and Methods

Sample collection and bacterial isolation

In the second half year of 2018, we investigated five slaughterhouses in Xuchang City in Henan Province of China (Fig. 1A). In this investigation, we collected a total of 399 samples, including raw meat and swabs from the waiting lairage, carcass surface, slaughtering environment, tools, and vehicle surface (Table 1). In the first half year of 2019, we investigated six pig farms where pigs were sent to the above slaughterhouses for slaughtering as well as 28 different markets where pork products were supplied by the above slaughterhouses (Fig. 1A). From the six pig farms, we collected a total of 270 samples, including fecal samples of pigs and environmental swabs (pigsty's floor, carrier vehicles, feeding troughs, and drinking fountains) (Table 1). In different markets, we collected 255 raw pork samples. Each sample was stored in sterilized tubes (swabs) or bags (meat) and kept on ice for shipping.

FIG. 1.

Sample collection and bacterial isolation in the present study. (A) A map showing the location of pig farms, slaughterhouses, and terminal markets for sample collection. The Chinese map was kindly provided by the Ministry of Natural Resources of the People's Republic of China. (B) Escherichia coli isolation rate from pig biological samples and environmental samples. Pig farm biological samples (88.07%, 96/109), environmental samples (59.00%, 95/161), slaughterhouse pig biological samples (41.72%, 68/163), and environmental samples (30.51%, 72/236). The results were analyzed using the chi-square test, and (*) means significant at p-value <0.05. Color images are available online.

Table 1.

Samples Collected and Escherichia coli Isolated in This Study

	Sample types	No. of samples	No. of E. coli isolates from samples	Isolation rate (%)
Pig farms (n = 6)	Diarrhea feces	60	55	91.67
	Rectal sponges	49	41	83.67
	Floor swabs	29	22	75.86
	Carrier vehicles	7	5	71.44
	Drinking fountains	34	24	70.59
	Trough swabs	35	21	60.00
	Feed	25	14	56.00
	Drinking water	31	9	29.03
	Subtotal	270	191	70.74
Slaughterhouses (n = 5)	Waiting lairage swabs	20	11	55.00
	Carcass swabs	163	68	41.72
	Slaughtering environment	114	40	35.09
	Slaughtering tools	37	12	32.43
	Vehicle swabs	17	5	29.41
	Raw meat	40	4	10.00
	Shower water	8	0	0.00
	Subtotal	399	140	35.08
Terminal markets (n = 28)	Pork	255	77	30.20
Terminal markets (n = 28)	Subtotal	255	77	30.20

All samples were treated immediately for bacterial isolation after collection. Briefly, 2 mL of Luria-Bertani broth was added into sterilized tubes containing swabs or homogenates of meats, and the tubes were shaken at 180 rpm at 37°C for 12–16 h. Afterward, sample cultures were streaked on MacConkey agar plates and then incubated at 37°C for 12–16 h. Presumptive colonies of E. coli were selected for polymerase chain reaction (PCR) amplification of the 16S rRNA gene as well as seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) (Wirth et al., 2006). PCR products were finally confirmed by nucleotide sequencing. Primer sequences used in this study are listed in Supplementary Table S1.

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) values of 28 types of antibiotics belonging to nine classes, including aminoglycosides (amikacin [AMK], gentamicin [GEN], tobramycin [TOB]), β-lactams (imipenem [IPM], meropenem [MRP], ertapenem [ETP], cefazolin [CFZ], cefuroxime [CFX], cefoxitin [FOX], ceftazidime [CAZ], ceftriaxone [CRO], cefepime [CPM], amoxicillin/clavulanate [AMC], ampicillin/sulbactam [AMS], piperacillin/tazobactam [PTZ], aztreonam [AZM]), phenicols (chloramphenicol [CHL]), tetracyclines (tetracycline [TET], minocycline [MIN], tigecycline [TGC]), fluoroquinolones (moxifloxacin [MXF], ciprofloxacin [CIP], levofloxacin [LVX], norfloxacin [NOR]), sulfonamides (trimethoprim/sulfamethoxazole [SXT]), fosfomycins (fosfomycin [FOS]), nitrofurantoins (nitrofurantoin [NIT]), and polymyxins (colistin [CL]) on E. coli strains isolated herein were determined by using the broth microdilution method following the protocol recommended by the U.S. Clinical & Laboratory Standards Institute.

Results were interpreted by using the breakpoints provided by the CLSI document (CLSI, 2018). If a CLSI breakpoint was not available, a EUCAST breakpoint was used (EUCAST, 2016). Each antibiotic was tested with three duplicates. E. coli ATCC 25922 was used as quality control. The concentration of antibiotics were illustrated at Supplementary Table S2.

Multilocus sequence typing identification

Multilocus sequence typing (MLST) was performed following the protocol provided on the EnteroBase E. coli MLST database website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). Sequence types (STs) were determined based on the schemes of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA).

PCR detection of AMR genes and virulence factor-encoding genes

The presence of 15 selected antimicrobial resistance genes (ARGs), including bla _NDM-1, bla _KPC, bla _OXA-48, bla _VIM, bla _IMP, bla _TEM, bla _SHV, bla _CTX-M, sul2, sul3, floR, qnrB, tetA, cfr, and mcr-1 (Saenz et al., 2004; Cavaco et al., 2008; González-Sanz et al., 2009; Poirel et al., 2011; Liu et al., 2016) and 13 virulence factor-encoding genes (VFGs), including astA (encoding enteroaggregative heat- stable enterotoxin), fimH (encoding the type I pili mannose-specific adhesin), fimC (encoding type 1 fimbriae), tsh (temperature-sensitive hemagglutinin), traT (encoding the complement resistance factor), ompT (encoding outer membrane protease), sitA (putative iron transport gene), iroN (encoding catecholate siderophore receptor), iutA (encoding ferric aerobactin receptor), hlyE (encoding hemolysin E), eae (encoding intimin), stx1 (encoding Shiga toxin I), and stx2 (encoding Shiga toxin II) (Rodriguez-Siek et al., 2005; Zhao et al., 2009; Peng et al., 2019b) among the isolates with predominate STs were detected by using PCR assays, with primers listed in Supplementary Table S1.

PCR was performed in a 20 μL mixture containing 1 μL bacterial DNA (10 ng) as template, 10 μL of 2 × Master Mix (TSINGKE, China), 1 μL of each primer (10 mM), and 7 μL ddH₂O. Thermo cycling conditions are 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, annealing at specific temperatures for 30 s (Supplementary Table S1), and 72°C for 2 min 30 s, with a final extension at 72°C for 5 min. PCR products were analyzed by electrophoresis on a 1% agarose gel.

Statistical analysis

The results were analyzed using the chi-square test embedded in GraphPad Prism 6.02 (Sabala et al., 2021). Value of p < 0.05 was considered significant.

Results

Antimicrobial-resistant phenotypes of E. coli strains isolated in pig farms, slaughterhouses, and terminal markets in Henan Province of China

A total of 408 E. coli strains were finally isolated from the 924 samples. In both pig farms and slaughterhouses, the isolation rates of E. coli from pig biological samples were higher than those of E. coli from environmental samples (Fig. 1B). Antimicrobial susceptibility testing (AST) on these isolates revealed that isolates from pig farms showed severe resistance profiles. A large proportion of E. coli isolates from pig farms, slaughterhouses, and terminal markets were resistant to TET (farms: 97.91% [187/191]; slaughterhouses: 87.86% [123/140]; markets: 87.01% [67/77]), CHL (farms: 90.58% [173/191]; slaughterhouses: 55.00% [77/140]; markets: 63.64% [49/77]), SXT (farms: 80.10% [153/191]; slaughterhouses: 64.29% [90/140]; markets: 72.73% [56/77]), and MXF (farms: 80.10% [153/191]; slaughterhouses: 52.86% [74/140]; markets: 41.56% [32/77]) (Fig. 2).

FIG. 2.

Percentages of Escherichia coli isolates showing resistance to the antimicrobials tested. TET: tetracycline; CHL: chloramphenicol; SXT: trimethoprim/sulfamethoxazole; MXF: moxifloxacin; CFZ: cefazolin; CFX: cefuroxime; CRO: ceftriaxone; AMS: ampicillin/sulbactam; GEN: gentamicin; AMC: amoxicillin/clavulanate; TOB: tobramycin; CPM: cefepime; CIP: ciprofloxacin; CAZ: ceftazidime; NOR: norfloxacin; AMK: amikacin; LVX: levofloxacin; FOX: cefoxitin; AZM: aztreonam; MIN: minocycline; ETP: ertapenem; IPM: imipenem; PTZ: piperacillin/tazobactam; MRP: meropenem; CL: colistin; FOS: fosfomycin; NIT: nitrofurantoin; TGC: tigecycline. The MIC breakpoints follow the CLSI, 2018, and EUCAST, 2016; “*” indicates a significant difference p-value <0.05. MIC, minimum inhibitory concentration. Color images are available online.

Resistance to cephalosporins was also a common phenotype of isolates from pig farms, as more than 50% of the farm isolates were resistant to CFZ 64.4% (123/191), CFX 60.2% (115/191), and CRO 58.1% (111/191), respectively (Fig. 2). However, less than 10% of the isolates from slaughterhouses and terminal markets displayed resistance to these cephalosporins (Fig. 2). In particular, ∼21.5% (41/191) and15.7% (30/191) of the farm isolates were resistant to carbapenems and CL, respectively. Only 0.71% (1/140) and 1.43% (2/77) of the isolates from slaughterhouses were resistant to carbapenems and CL, respectively. For the isolates from terminal markets, ∼6.49% of the isolates were resistant to CL, and none of them was resistant to carbapenems (Fig. 2). None of the isolates from farms and terminal markets showed resistance to TGC, while one isolate from slaughterhouses was resistant to this antibiotic (Fig. 2). Farms had significantly higher resistance rates when compared with slaughterhouses, however, there was almost no statistical significance between slaughterhouses and markets, except that markets had significantly higher resistance rates of CL (Fig. 2).

AST results also revealed that E. coli strains isolated from farms, slaughterhouses, and terminal markets displayed multidrug resistance (MDR) (Fig. 3). More than 65% of the isolates from farms, slaughterhouses, and terminal markets were resistant to three of the nine antibiotics tested classes. Particularly, 93.19% of the farm isolates were MDR strains (Fig. 3). AMR rates of E. coli from pig biological samples were overall higher than that from farm environments, but there was no statistical significance (Fig. 4). In slaughterhouses, the AMR rates of E. coli from the slaughterhouse environment were also higher than that from carcasses (Fig. 5).

FIG. 3.

Percentages of Escherichia coli isolates showing resistance to different antimicrobial classes. The nine classes of antimicrobials tested are aminoglycosides (amikacin, gentamicin, and tobramycin), β-lactams (imipenem, meropenem, ertapenem, cefazolin, cefuroxime, cefoxitin, ceftazidime, ceftriaxone, cefepime, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, and aztreonam), phenicols (chloramphenicol), tetracyclines (tetracycline, minocycline, and tigecycline), fluoroquinolones (moxifloxacin, ciprofloxacin, levofloxacin, and norfloxacin), sulfonamides (trimethoprim/sulfamethoxazole), fosfomycins (fosfomycin), nitrofurantoins (nitrofurantoin), and polymyxins (colistin). In the left Y axis, 0 means that these strains were sensitive to all classes of antimicrobials; 3 means that these strains were resistant to 3 classes of antimicrobials, which indicates the multidrug resistance; and 9 means these strains were resistant to 9 classes of antimicrobials. Color images are available online.

FIG. 4.

AMR rate comparison between Escherichia coli isolates from pig biological samples and environmental samples in farms. AMR rates of E. coli isolates from pig biological samples (n = 96) [diarrhea feces (n = 55), rectal sponges (n = 41)], and farm environmental isolates (n = 95) [floor swabs (n = 22), carrier vehicles (n = 5), drinking fountains (n = 24), trough swabs (n = 21), feed (n = 14), and drinking water (n = 9)]. AMR, antimicrobial resistance. Color images are available online.

FIG. 5.

AMR rate comparison between Escherichia coli isolates from slaughtered carcass samples and environmental samples in slaughterhouses. AMR rates of E. coli isolates from slaughtered carcasses (n = 72) [carcass swab isolates (n = 68) and raw meat isolates (n = 4)] and slaughterhouse environmental isolates (n = 68) [waiting lairage (n = 11), slaughtering environment (n = 40), slaughtering tools isolates (n = 12), vehicle swabs (n = 5)]. (*) means significant at p-value <0.05. (**) means significant at p-value <0.01. Color images are available online.

MLST of isolates from farms, slaughterhouses, and terminal markets

MLST analyses determined 73 catalogs of STs for the 191 isolates from pig farms, 56 catalogs of STs for the 140 isolates from slaughterhouses, and 40 catalogs of STs for the 77 isolates from terminal markets, respectively. Among these STs, ST10 and ST101 were the commonly predominant STs among isolates from pig farms, slaughterhouses, and terminal markets (Table 2). The resistance patterns of both ST10 and ST 101 strains from these nodes are exhibited in Figure 6. Besides, ST48 and ST542 were also the commonly determined STs among isolates from pig farms and slaughterhouses, while ST58, ST641, and ST1434 were determined among isolates from slaughterhouses and terminal markets (Table 2).

FIG. 6.

The AMR patterns of both ST10 and ST101 strains from different nodes (n = 75). (A) Resistance patterns for ST10 and ST101 Escherichia coli strains isolated from pig farms (n = 29). (B) Resistance patterns for ST10 and ST101 E. coli strains isolated from slaughterhouses (n = 31). (C) Resistance patterns for ST10 and ST101 E. coli strains isolated from terminal markets (n = 15). Square filled in red color means resistant to that antibiotic and in blank means sensitive to that antibiotic. Color images are available online.

Table 2.

Predominant Sequence-Type Isolates from Pig Farms, Slaughterhouses, and Terminal Markets

Pig farms n = 191			Slaughterhouses n = 140			Terminal markets n = 77
STs	No. of isolates	%	STs	No. of isolates	%	STs	No. of isolates	%
ST10	21	28.3	ST10	22	32.1	ST10	9	19.5
ST101	8		ST101	9		ST101	6	19.5
ST48	17		ST48	7		ST58	4	14.3
ST542	8		ST542	7		ST641	4
ST5229	8	4.2	ST58	9	15.7	ST1434	3
ST617	7	3.7	ST641	5		ST88	4	5.2
ST156	6	3.1	ST1434	8		ST2144	4	5.2
ST2930	6	3.1	ST744	5	3.6	ST542	2	2.6
ST746	6	3.1				ST156	2	2.6
ST165	4	2.1				ST1607	2	2.6
ST29	4	2.1
ST744	4	2.1

Bold indicates the predominant ST10 and ST101 presenting in all three links, the italic indicates both ST48 and ST542 which might spread from pig farms to slaughterhouses, and the bold italic of ST58, ST641, and ST1434 suggest that these three STs might spread from slaughterhouses to terminal markets.

STs, sequence types.

Determination of ARGs and VFGs among E. coli ST10 and ST101 isolates

Because ST10 and ST101 were the most commonly predominant STs shared among the isolates from pig farms, slaughterhouses, and markets (Table 2), we next detected the ARGs among these ST10 and ST101 isolates from pig farms, slaughterhouses, and terminal markets. Our results revealed the common presence of several ARGs, including sul2, sul3, floR, tetA, and bla_TEM , in the isolates from different nodes of the pork supplying chain (Fig. 7A). In addition, bla _NDM-1 and mcr-1 were also detected in E. coli strains from pig farms. Two bla _NDM-1-positive strains harbored sul2, sul3, floR, tetA, bla _TEM, bla _CTX-M simultaneously. Meanwhile, the mcr-1 positive isolate additionally carried sul3, floR, bla _TEM, and cfr ARGs (Fig. 7B).

FIG. 7.

Presence of selected ARGs in Escherichia coli ST10 and ST101 strains. (A) The detection rates (%) of different ARGs in E. coli ST10 and ST101 strains among those isolated from pig farms, slaughterhouses, terminal markets, and total isolates. (B) Presence of different ARG groups in E. coli ST10 and ST101 strains isolated from pig farms, slaughterhouses, and terminal markets. ARGs, antimicrobial resistance genes. Color images are available online.

To investigate the potential pathogenicity of these ST10 and ST101 strains, we detected several VFGs among these isolates. The results revealed that fimH, fimC, astA, and traT were commonly present in isolates from pig farms, slaughterhouses, and terminal markets (Fig. 8A). There were 23 different groups of VFGs among E. coli ST10 and ST101 STs (Fig. 8B). In particular, many isolates from pig farms, slaughterhouses, and terminal markets were also positive for astA, traT, and tsh simultaneously, which meant these strains had the potential ability to produce enteroaggregative E. coli heat-stable toxin and overcome serum bactericidal effect.

FIG. 8.

Presence of selected VFGs in Escherichia coli ST10 and ST101 strains. (A) The detection rates (%) of different VFGs in E. coli ST10 and ST101 strains among those isolated from pig farms, slaughterhouses, terminal markets, and total isolates. (B) Presence of different groups of VFGs in E. coli ST10 and ST101 strains isolated from pig farms, slaughterhouses, and terminal markets. VFGs, virulence factor-encoding genes. Color images are available online.

Discussion

In this study, we performed an isolation of E. coli and determined the AMR profiles of these isolates from both pig biological samples and environmental samples from pig farms, slaughterhouses, and terminal markets in the Henan Province, one of the major pig-rearing and pork-producing regions in China. Our AST results revealed that isolates from pig farms showed severe AMR as 93.19% (178/191) of the farm isolates exhibited MDR (Figs. 2 and 3). It has been widely accepted that E. coli is a common indicator bacterium for AMR (Brisola et al., 2019), and severe profiles of AMR displayed by E. coli from pig farms are suggestive that the AMR profile in these pig farms is still an anxious condition.

As the largest antimicrobial-producing and the largest pig-rearing country globally, antimicrobials are frequently used in livestock, particularly in the pig industry either for growth promotion or for health maintenance in China (Ying et al., 2017). The long-term use of antibiotics leads to a risky condition of AMR in farms (Van Boeckel et al., 2019). Apart from our present study, epidemiological studies on E. coli from pig farms in the other regions in China and on other bacterial species from pig farms also reported severe AMR phenotypes displayed by the bacterial isolates (Sun et al., 2019; Luo et al., 2020). These findings suggest that the current AMR condition in pig farms of Central China is still worrisome. Fortunately, the Chinese government is taking action to reduce the use of antimicrobials in livestock. The Ministry of Agriculture and Rural Affairs has issued a document to promote this process in 2018 (www.moa.gov.cn/nybgb/2018/201805/201806/t20180620_6152765.htm).

Our AST results also revealed that the AMR profiles of E. coli isolates from slaughterhouses and terminal markets are not as severe as those of the isolates from pig farms (Figs. 2 and 3). A main reason that might explain this finding is that antibiotics are not used in slaughterhouses and pork markets, which might not induce AMR in the environmental bacteria in these two places (Schwaiger et al., 2012). We should note that it is difficult for us to follow the same group of pigs from farms to slaughterhouses and markets during the investigation due to the outbreak of African swine fever in China since late 2018. This is also a main limitation of this work. However, the results from this study still provide knowledge of AMR profile of E. coli isolates from different nodes of the pork supplying chain.

It has been documented that antibiotics are commonly used for growth promotion and disease control; the extensive use of antibiotics for these purposes in pig farms may accelerate the development of drug-resistant E. coli in swine intestinal tracts (Van Boeckel et al., 2019). In daily production activities, drug-resistant E. coli from the intestinal tract of pigs may be discharged directly into the environment through dumping. In addition, the extensive use of antibiotics in pig farms may also lead to drug residue, which may aggravate the emergence of drug-resistant E. coli in the environment. These two reasons might explain the finding that the AMR profiles and resistance rates of E. coli from pig biological samples were similar to the isolates from environmental samples in pig farms (Fig. 4). Therefore, pig feces may be an important source of the contamination of drug-resistant E. coli in the environment.

Notably, drug-resistant E. coli in pig farms may have a circulation between pigs and environments through specific medium agents such as pig feces, drinking water, and food in the farm. Therefore, pig farms should improve their biosecurity and management level to help control diseases and promote productivity rather than over reliance on the use of antibiotics. Although the isolation rate of drug-resistant E. coli from pig biological samples was higher than that of E. coli from environmental samples in slaughterhouses, the isolation rate of drug-resistant E. coli from environmental samples was still 30.51% (Fig. 1B). This isolation rate might be a bit high since disinfectants are frequently used in slaughterhouses. However, considering there are so many carcasses that need to be handled during the operation, it is inescapable for the staff in slaughterhouses to cut pig guts, which might lead to the release of drug-resistant bacteria (Loretz et al., 2011; Buncic et al., 2014). Therefore, pig guts are recognized as a main source for the contamination of drug-resistant bacteria in the environment of slaughterhouses (Loretz et al., 2011).

Our MLST results revealed that ST10 and ST101 were the common STs of E. coli from farms, slaughterhouses, and terminal markets (Table 2). It has been reported that both E. coli ST10 and ST101 were widely determined in different nodes of pork-producing chain and humans, and both represent significant public health concerns (Oteo et al., 2009; Ranjan et al., 2016; Zhang et al., 2017; Bai et al., 2019). Our investigation on the presence of ARGs revealed that both STs carried multiple ARGs, conferring resistance phenotypes to the antibiotics tested. Moreover, several important ARGs with a tremendous public health concern, including bla _NDM-1 and mcr-1, were observed from these two STs (Fig. 7), suggesting that these two STs might have the ability to spread resistance to colistin and broad-spectrum β-lactams, including carbapenems. These antibiotics were currently regarded as the last option for treating infection caused by MDR Gram-negative bacteria (Peng et al., 2019a).

In addition, our PCR amplification on VFGs revealed that both ST10 and ST101 STs carried several VFGs (Fig. 8). Of particular note were astA, tsh, and traT, which were frequently detected in pathogenic E. coli associated with different diseases in humans and animals (Johnson and Stell, 2000; Gutiérrez et al., 2015; Maluta et al., 2017). These findings suggested that these isolates might possess potential pathogenicity to humans. A wide determination of these two STs from pig farms, slaughterhouses, and terminal markets should receive more attention.

Conclusion

In summary, we characterized the E. coli isolates from farms, slaughterhouses, and terminal markets in one of the main pig-rearing and pork-producing regions in China in this study. Our results revealed that E. coli isolates from farms, slaughterhouses, and terminal markets showed severe AMR profiles. Importantly, some isolates from pig farms showed phenotypes of resistance to carbapenems, broad-spectrum cephalosporins, and colistin. Regarding these worrisome conditions of AMR, active measures should be taken to reduce the use of antibiotics particularly in pig farms. We found ST10 and ST101 to be the most prevalent STs shared among the total isolates, and these two predominant STs carried several important ARGs with a tremendous public health concern. Importantly, these two STs also harbored several important VFGs that would be beneficial for bacterial pathogenesis. Since it has been reported that E. coli ST10 and ST101 STs represent significant public health concerns, the wide prevalence and distribution of these two STs in farms, slaughterhouses, and terminal markets should receive more attention.

Footnotes

Authors' Contributions

All authors have read this article and would like to have it considered exclusively for publication.

Acknowledgment

We sincerely thank the Xuchang Animal Husbandry Bureau for helping collect the samples from the slaughterhouses.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported, in part, by the National Key Research & Development Program of China (2017YFC1600101 and 2017YFC1600103), the earmarked fund for the China Agriculture Research System (CARS-35), the Hubei Provincial Natural Science Foundation of China (2020CFB525), the Walmart Foundation (project # 61626817) and supported by Walmart Food Safety Collaboration center.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

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