Background

Superbug infections are one of the most serious threats to public and animal health nowadays. The emergence and rapid spread of multi-drug-resistant (MDR), extensively drug-resistant (XDR) and pan-drug-resistant (PDR) bacteria has been a major public health problem worldwide [1]. MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories [2]. The transmissibility of antimicrobial resistance mediated by mobile plasmids was reported to be an important reason for the generation of XDR and PDR bacteria [3].

In China, colistin is the last line of defense against carbapenem-resistant Escherichia. coli (CREC) [4, 5]. However, colistin has been used in animal production in China for decades as a treatment and feed additive [6]. In 2015, the plasmid-mediated mcr-1 gene was first discovered in food animals in South China [7]. Subsequently, nine different mcr alleles, e.g. mcr-1 to mcr-9, were found in different bacteria from many countries and regions [7,8,9,10,11,12,13,14,15].

Due to the extensive use of β-Lactam antimicrobial in human and veterinary medicine, the number of extended-spectrum β-lactamase (ESBL)-producing E. coli is increasing rapidly worldwide [16, 17]. More related studies on CREC were reported recently, for CREC is multi-drug resistant which is difficult to treat and cause a high lethality after infection [18, 19]. The occurrence of colistin resistant CREC aggravate the situation [20, 21].

Thus, this study aimed at investigate the phenotype of antimicrobial resistance of MCRPEC and its plasmid-carrying resistance genes from pigs in Guangxi, South China.

Results

Identification of mcr-1 positive E. coli (MCRPEC) isolates

A total of 142 E. coli isolates were isolated from pigs with diarrhea/dyspnea in Guangxi in 2018. Seventy-two (50.7%, 72/142) E. coli isolates were tested with colistin (MIC 3.5 mg/L). To investigate the proportion of mcr genes in E. coli, PCR amplification was performed to test mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8. 33 mcr-1-positive E. coli strains detected. The percentage of MCRPEC strains accounted for 45.8% (33/72) of colistin resistant strains and 23.2% (33/142) of all isolated strains. The thirty-three MCRPEC isolates were used for subsequent study.

The full-length 16S rRNA gene sequences of the 33 MCRPEC strains were used to generate a phylogenetic tree by means of Neighbor Joining method in MEGA-X (Fig. 1). Thirty-three MCRPEC strains were classified into eight distinct STs, including ST10, ST224, ST361, ST410, ST641, ST1408, ST3345, and an unknown ST. ST10 and ST224 were the dominant STs, which accounted for 69.7% (23/33) (Fig. 1). More information about MLST was included in the supplementary materials (supplementary materials Table 2).

Fig. 1
figure 1

STs, incompatibility plasmid groups and phylogenetic tree of the 33 swine-origin MCRPEC isolates. Phylogenetic tree was generated by Neighbor joining method in MEGA-X, and toggled scaling of tree

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Multiple PCR was used to identify incompatibility plasmid groups in MCRPEC by using plasmid DNA of MCRPEC isolates. As is shown in Fig. 1, six incompatibility plasmid groups were detected, including IncFI (97.0%, 32/33), IncHI (12.1%, 4/33), IncY (48.5%, 16/33), IncN (15.2%, 5/33), IncI1 (3.0%, 1/33) and IncX1 (6.1%, 2/33). Ten (30.3%, 10/33) MCRPEC isolates were detected to carry one incompatibility plasmid group (IncFI). Sixteen (16/33, 48.5%) MCRPEC isolates were detected to carry two incompatibility plasmid groups, among which eleven (11/16, 68.8%) isolates were the combination of IncFI and IncY, four (4/16, 25%) isolates displayed the combination of IncHI and IncFI, and one (1/16, 6.2%) isolate exhibited the combination of IncFI and IncX1. Six (6/33, 18.2%) MCRPEC isolates were detected to carry three incompatibility plasmid groups, among which four (4/6, 66.6%) isolates were the combination of IncFI, IncN and IncY, one (1/6, 16.7%) isolate was the combination of IncFI, IncI1 and IncY, and one (1/16, 16.7) isolate was the combination of IncFI, IncN and IncX1. In addition, one (1/33, 3.0%) MCRPEC isolate was not detected to carry incompatibility plasmid group.

Antimicrobial resistance in MCRPEC

The antimicrobial resistance proportion of thirty-three MCRPEC isolates were as follows: gentamicin (72.7%, 24/33), amikacin (48.5%, 16/33), ceftaroline (69.7%, 23/33), piperacillin-tazobactam (24.2%, 8/33), imipenem (36.4%, 12/33), meropenem (24.2%, 8/33), cefalexin (69.7%, 23/33), cefuroxime (57.6%, 19/33), cefotaxime (57.6%, 19/33), ceftriaxone (57.6%, 19/33), cefepime (39.4%, 13/33), cefoxitin (0%, 0/33), ciprofloxacin (75.8%, 25/33), sulfadiazine (24.2%, 8/33), trimethoprim-sulphamethoxazole (0%, 0/33), aztreonam (24.2%, 8/33), ampicillin (97.0%, 32/33), amoxicillin-clavulanic acid (0%, 0/33), ampicillin-sulbactam (24.2%, 8/33), chloramphenicol (84.8%, 28/33), fosfomycin (78.8%, 26/33), tetracycline (100%, 33/33), doxycycline (72.7%, 24/33), azithromycin (57.6%, 19/33), polymyxin B (100%, 33/33) and colistin (100%, 33/33) (Fig. 2a). In addition, each MCRPEC isolate showed significant antimicrobial resistance (Fig. 2b). According to the results of cephalosporin susceptibility test, nineteen strains (57.6%, 19/33) were resistant to the 3rd and 4th generation cephalosporins and twelve strains (36.4%, 12/33) were resistant to carbapenem (Fig. 2a). According to the definition of MDR, XDR, and PDR bacteria, all 33 MCRPEC isolates were identified as MDR (Fig. 2b) [2]. Among them, three of the MDR MCRPEC isolates were identified as XDR (Fig. 2c).

Fig. 2
figure 2

Antimicrobial resistance characteristics of the 33 swine-origin MCRPEC isolates from swine E. coli. a Antimicrobial resistance proportion, the bar chart showed the percentages of the 33 MCRPEC isolates that were sensitive (green), intermediate (yellow), or resistant (red) to 27 commonly used antimicrobials. b Statistics of the multi-antimicrobial category. c The first panel consists of 27 columns representing the sensitivity (green), intermediates (yellow), or resistance (red) of 27 antimicrobial agents. The next panel contains 18 columns indicating the antimicrobial category of the 33 MCRPEC isolates, including A: aminoglycosides, B: anti-MRSA cephalosporins, C: antipseudomonal penicillins + β-lactamase inhibitors, D: carbapenems, E: 1st and 2nd generation cephalosporins, F: 3rd and 4th generation cephalosporins, G: cephamycins, H: fluoroquinolones, I: folate pathway inhibitors, J: glycylcyclines, K: monobactams, L: penicillins, M: penicillins + β-lactamase inhibitors, N: phenicols, O: phosphonic acids, P: polymyxins, Q: tetracyclines, and R: Macrolides. The colors indicate resisitance to all kinds agents of one antimicrobial category (black), resistant to some of the agents of one antimicrobial category (gray), no agent (white). The rightmost section are judgments for MDR, XDR, or PDR. Multidrug resistant (MDR) is the acquired (but not natural) insensitivity (mediated or resistant) to three or more antimicrobial agents (at least one in each category). Extensive resistant (XDR) means that it is insensitive to all antimicrobial species (at least one in each category) except for those in the 1–2 category. PDR is defined as non-susceptibility to all agents in all antimicrobial categories. The MICs of antimicrobial resistance test were putted in supplementary material Table 4

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Coexistence of mcr-1 gene in plasmids with β-lactamase antimicrobial resistance genes and non-β-lactamase antimicrobial resistance genes in the MCRPEC isolates

There were 22 MCRPEC isolates harbored ESBL genes, including two blaOXA-1 and thirty-two blaCTX-M. The dominate blaCTX-M gene was blaCTX-M-14 (59.4%, 19/32), followed by blaCTX-M-123 (37.5%, 12/32) and blaCTX-M-24 (3.1%, 1/32) (Fig. 3a, b). Additionally, there were two and eight MCRPEC isolates with blaCMY-2 and blaNDM-5 respectively. (Fig. 3a, c).

Fig. 3
figure 3

Plasmid-carrying resistant genes of the 33 swine-origin MCRPEC isolates (a) The panel includes 22 columns indicating the presence or absence of plasmid-mediated resistance genes. Dark blue means presence, and light Blue means absence. b The distribution of the total ESBL/Carbapenemase/pAmpC genes in the MCRPEC isolates. c The distribution of blaCTX-M among thirty-two ESBL genes

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Furthermore, many non-β-lactamase antimicrobial resistance genes were also detected in MCPEC isolates, including fluoroquinolone resistance gene, tetracycline resistance genes, sulfanilamide resistance genes, aminoglycoside resistance genes and chloramphenicol resistance genes. As is shown in Fig. 3a, the rates of fluoroquinolone resistance related genes qnrA, qnrB, qnrS, and aac (6′)-Ib-cr were 36.4% (12/33), 36.4% (12/33), 33.3% (11/33), and 24.2%(8/33) individually. Tetracycline resistance related genes tetA, tetB, and tetX accounted for 100% (33/33), 18.2% (6/33), and 0% (0/33), respectively. Sulfanilamide resistance determinants sul1 and sul2 accounted for 90.0% (30/33) and 78.8% (26/33), respectively. Aminoglycoside resistance related gene aadA (100%, 33/33) and chloramphenicol resistance related gene floR (100%, 33/33) both have 100% detection rates.

Besides, virulence genes (Enterotoxigenic E. coli and Shigatoxin-pruducing E. coli) of thirty-three MCRPEC isolates from clinical diagnostic were detected. The results were shown in Table 3 of supplementary materials. Twenty-six (26/33, 78.8%) MCRPEC isolates were identified as pathogenic E. coli, of which twenty-two (22/26, 84.6%) isolates were identified as Enteroxigenic E. coli (ETEC), three (3/26, 11.5%) isolates were identified as Shigatoxin-pruducing E. coli (STEC), and one (1/26, 3.9%) isolate was both ETEC and STEC. Fifteen (15/22, 68.2%) ETEC isolates only carried STb gene, the rest of ETEC isolates (7/22, 31.8%) carried STb and LT genes. Three STEC isolates only carried stx2e gene. The ETEC/STEC isolate carried STb, LT and stx2e genes.

Discussion

China became the world’s lar raises and consumes about 500 million pigs a year, nearly half the world’s total, and is the world’s largest consumer of antibiotics [22]. The long term over antibiotics fast-growing of resistance [23]. In the challenge of antibiotic resistance, the Chinese government stipulated the veterinary drug prescription management measures in 2013 [24]. In recent years, plasmid-mediated colistin resistant genes mcr-1 to mcr-9 have been found worldwide [7,8,9,10,11,12,13,14,15]. Although the Chinese government began to tighten regulation of colistin in 2017, MCRPEC remains to be a chronic problem in pig farms [25,26,27]. Thus, we report extensive antimicrobial resistance and plasmid-carrying resistance genes in MCRPEC from pigs in Guangxi, China.

In this study, MCRPEC was found to be highly resistant to some of the β-lactam and non-β-lactam antibiotics commonly used in human medicine and veterinary medicine, such as penicillin, cephalosporins, fluoroquinolones, aminoglycosides, quinolones, sulfonamides and tetracycline. In addition, rare resistance to carbapenems in animal production such as imipenem and meropenem was also found in MCRPEC. According to veterinary prescription list, imipenem and meropenem are not permitted to use in livestock production. Recently, studies have found that MCRPEC contains a “new Delhi metallo-lactam resistance gene” that is resistant to almost all β-lactam antibiotics but monobactam [28, 29]. Thirty-three MCRPEC isolates were susceptible to tigecycline and cefoxitin. According to veterinary prescription list, tigecycline and cefoxitin are not permitted to use in livestock production [24, 25]. As the same time, Guangxi government published the notice explained tigecycline was special use level and cefoxitin was restricted use level (i.e. ordinary people is difficult to acquire) [30]. The presence of these multidrug-resistant phenotypes suggests that MCRPEC coexists with other drug-resistant genes.

Interestingly, there is evidence that ESBL E. coli has a higher level of mcr-1 than non-ESBL E. coli, and the rapid rise in ESBL also significantly increases the selective pressure for colistin resistance [17]. In this study, 63.6% (21/33) of MCRPEC strains were detected to contain different ESBL genes simultaneously. Among them, blaCTX-M (19/21, 90.5%) was dominant in MCRPEC plasmid mediated drug resistance genes. Among the 32 blaCTX-M sequences of 19 MCRPEC isolates, blaCTX-M-14 gene had the highest proportion blaCTX-M gene (59.4%, 19/32), followed by blaCTX-M-123 (37.5%, 12/32) and blaCTX-M-24 (3.1%, 1/32). Notably, all MCRPEC isolates carrying the blaCTX-M-14 genes were resistant to ciprofloxacin (Fig. 3a and c), consistent with another study [31]. Recently, several reports have shown that pig waste not only frequently carries mcr-1 and blaNDM but also transfers these genes by affecting the environment around farms and contaminating the food chain [22, 27, 32]. Therefore, the co-existence of mcr-1 gene with carbapenem-resistant gene blaNDM-5 (8/33, 24.2%) in E. coli has drawn our attention to the spread of such superbugs in Guangxi.

E. coli is by nature sensitive to almost all clinically relevant antimicrobial agents, but this bacterium has a great capacity to accumulate resistance genes, mainly through horizontal gene transfer [33]. In this study, 11 plasmids carrying non-lactam genes were found in MCRPEC, including fluoroquinolone (qnrA, qnrB, qnrS, aac (6′)-Ib-cr), tetracycline (tetA, tetB), sulfonamide (sul1, sul2), aminoglycoside (aadA), and chloramphenicol (floR), suggesting that the effect of MCRPEC on spreading non-β-lactam genes should not be underestimated.

It has previously been reported that mcr-1 gene was found in the conjugative plasmids, IncI2, IncFII, IncX4, IncHI1, IncHI2, IncP, IncF, and IncY [34, 35]. We also detected these incompatibility types by PCR typing. The 33 MCRPEC isolates showed six different Inc. plasmid groups including IncHI, IncI1, IncN, IncFI, IncY, IncX1. Meanwhile, we found that IncFIA and IncFrepB were prevalent in 33 MCRPEC in this study. MLST results reflected that ST10 (13/33, 39.3%) was the most common ST among the 33 MCRPEC isolates, ST224 (10/33, 30.3%) and another ST (10/33, 30.3%) followed (Fig. 1). Recently, a study analyzed 616 whole genomes of mcr-1-positive E. coli isolates from NCBI online database, among them ST10 was the most abundant MCRPEC strains [36].

Conclusions

The study showed that thirty-three mcr-1-positive E. coli isolates in Guangxi had a wide range of antimicrobial resistance. Plasmid-carrying resistance genes might be the main cause of MCRPEC multidrug resistance. The results indicated that many ESBL genes (blaCTX-M, blaOXA-1) coexisted with mcr-1. The carbapenemase gene blaNDM-5 was detected in 8 MCRPEC strains. Furthermore, a number of non-β-lactam genes also coexisted with mcr-1 gene. Food animals and their feces are important sources of bacterial drug resistance transfer, our study highlights the necessity for long-term surveillance of mcr-1-positive E. coli in pigs.

Methods

Sample collection and detection of MCRPEC isolates

A total of 116 samples were collected from 44 pig farms that include 37 family farms and 7 swine breeding farms distributed in different towns of Guangxi, China in 2018. In which, 51 samples were collected from family farms and 65 samples were collected from swine breeding farms. These samples were collected from June 2018 to December 2018 and taken from rectal swabs or lung, intestinal tract, or lymph gland tissue collected from dead or unhealthy pigs with diarrhea or dyspnea. Before this study, these samples were sent to clinical veterinary laboratory of College of Animal Science and Technology of Guangxi University for molecular diagnosis. These farms managed about 27.8 thousand fattening pigs and 30.8 thousand breeding pigs during this study period. All fattening pigs belonged to family farms which were companies plus farmer model and small in size (number of pigs were between 400 to 1000 in one farm). Swine breeding farms adopted closed management and bigger in size (number of pigs were between 1000 to 5000 in one farm). In supplementary material Table 5, we provided description of 116 samples origin.

First, the collected samples were inoculated with MacConkey agar for 24 h at 37 °C. Then all single colonies of different forms on MacConkey agar were inoculated with eosin-methylene blue agar to screen suspected E. coli isolates. Colonies with a purplish black color or a metallic dark green color on the eosin-methylene blue agar were considered as suspected E. coli and further inoculated into LB Broth (Luria-Bertani Broth) for 8-10 h at 37 °C. Colistin resistance isolates were isolated by self-made SuperPolymyxin medium (i.e. a mixture of 10 ml Eosin-methylene blue agar and 35 μg colistin) as previous reported [37]. The genome DNA of Colistin resistance isolates was extracted with TIANamp Bacteria DNA Kit. Colistin resistance E. coli strains were determined by 16S rRNA gene sequencing and BLAST analysis (i.e. First, DNA and primers were used to amplify target fragment largely. PCR amplicons were purified by agarose gel electrophoresis and gel extraction from TIAN Gel Extraction Kit. Purified PCR products were sequenced by ABI 3730xl DNA Analyzer. SeqMan was used for sequence analysis. Finally, sequences were BLAST in NCBI.) [38]. Phylogenetic tree was generated by Neighbor joining statistical method in MEGA-X software. Thirty-three MCRPEC isolates would be tested in follow-up experiments. These isolates were from 116 pigs (un-weaned piglets, nursery piglets and sows) of 25 pig farms of different sizes distributed in 18 different towns of Guangxi, China in 2018 (Table 1). All E. coli isolates were stored in glycerol medium at − 80 °C.

Table 1 MCRPEC samples collection information
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The MCRPEC isolates were preliminarily screened by PCR amplification using genome DNA and special primer pairs for the mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes (supplementary material Tables 1), [39, 40]. To identify MCRPEC strains, the mcr-1 gene sequences in the E. coli strains were determined by direct sequencing from the PCR products and BLAST analysis [38].

Detection of multilocus sequence typing (MLST) and incompatibility plasmid groups

MLST analysis was performed by PCR amplicons of seven housekeeping genes, namely adk, fumC, gyrB, icd, mdh, purA, and recA using genome DNA. PCR amplicons were sequenced after purified by agarose gel electrophoresis and gel extraction by TIAN Gel Extraction Kit. The gene sequences for seven housekeeping genes were uploaded to the EnteroBase database to obtain the sequence type (ST) of corresponding E. coli isolate [41].

Incompatibility plasmid groups were assigned by PCR-based replicon types (HI1, HI2, I1, L/M, N, FIA, FIB, FIC, Frep, W Y, P, A/C, T, FIIS, K/B, B/O) [42]. Additional PCRs were performed for the IncX (X1, X2, X3, X4) replicon types [43]. The primers of house genes and replicon, and PCR reaction conditions were included in supplementary material Table 1.

Antimicrobial susceptibility testing

According to the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) consensus, a total of 27 commonly used human antimicrobials from 18 antimicrobial categories were selected in this study, including gentamicin, amikacin, ceftaroline, piperacillin-tazobactam, imipenem, meropenem, cefalexin, cefuroxime, cefotaxime, ceftriaxone, cefepime, cefoxitin, ciprofloxacin, sulfadiazine, trimethoprim-sulphamethoxazole, aztreonam, ampicillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, chloramphenicol, fosfomycin, tetracycline, doxycycline, azithromycin, polymyxin B and colistin (Table 2), [2]. Minimum inhibitory concentrations (MICs) were determined by using the agar microdilution (Mueller-Hinton Agar) method according to the Clinical and Laboratory Standards Institute [44]. The MICs of each drug were measured and recorded. E. coli ATCC25922 was used as a quality control. Resistant breakpoints of other antimicrobial abided by the CLSI-M100 document [45]. CLSI breakpoints are not available for colistin and cefalexin. So, in this study, we adopted the European Committee on Antimicrobial Susceptibility Testing Resistant/Susceptible breakpoints for determine colistin and cefalexin MICs. MICs of ≤2 mg/L and ≤ 16 mg/L are considered as susceptible (S) for colistin and cephalexin, respectively, according to the EUCAST guidelines [46].

Table 2 The antimicrobial agents for 17 antimicrobial categories used to define the E. coli antimicrobial resistance
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Molecular identification of ESBL, pAmpC, and carbapenem resistance genes

The ESBL, plasmid-mediated AmpC (pAmpC), and carbapenem genes were detected by multiplex PCR in plasmid DNA of MCRPEC isolates. The ESBL genes (blaCTX-M, blaTEM, blaOXA-1, and blaSHV), plasmid-mediated AmpC (pAmpC) genes (blaCMY, blaFOX, blaDHA), and carbapenem resistance genes (blaNDM, blaKPC, blaOXA-48, and blaIMP) were amplified using specific primers, as previously reported [47]. The DNA sequences for the ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase genes were determined by using BLAST analysis [38]. The primers of β-lactamase resistance genes and PCR reaction conditions were included in supplementary material Table 1.

Detection of non-β-lactamase antimicrobial resistance genes

The non-β-lactamase antimicrobial resistance genes were detected by PCR in plasmid DNA of MCRPEC isolates. The special primers included plasmid-encoded fluoroquinolone resistance genes (qnrA, qnrB, qnrS, aac (6′)-Ib-cr) [48], tetracycline resistance genes (tetA, tetB, tetX) [49], sulfonamide resistance genes (sul1, sul2), aminoglycoside resistance genes (aadA), and chloramphenicol resistance genes (floR), respectively. The primers of non-β-lactamase resistance genes and PCR reaction conditions were included in supplementary material Table 1.