High antimicrobial resistance and plasmid-carrying resistance genes in swine-origin mcr-1-positive Escherichia coli in Guangxi, South China

Background The discovery of mcr-1-positive Escherichia coli (MCRPEC), a notable superbug, attracted great attention worldwide. Swine-origin multi-drug resistance MCRPEC is a potential threat to public health and safety. To date, few detailed studies regarding swine-origin MCRPEC in Guangxi, South China, have been reported. Results In this study, thirty-three MCRPEC harbored mcr-1 genes were identified from 142 E. coli strains isolated from swine droppings and entrails in Guangxi in 2018. All MCRPEC isolates were assigned to 8 unique STs, including ST10, ST224 and ST410, which overlapped with the human-origin MCRPEC. Additionally, a total of six plasmid replicon types (IncFI, IncHI1, IncY, IncN, IncI1 and IncX1) were found. Moreover, the drug susceptibility of the MCRPEC isolates was tested with 27 antimicrobial agents belonging to 17 antimicrobial categories that are usually used in hospitals. There were 19 extended spectrum beta lactamase (ESBL) E. coli and 12 carbapenem resistant E. coli among the 33 MCRPEC strains. Importantly, the MCRPEC showed a high rate of resistance against two broad-spectrum carbapenem antibiotics, imipenem and meropenem, which are forbidden in livestock production use. Three MCRPEC strains were further identified to be extensively drug-resistant (XDR), and other isolates were recognized as multi-drug-resistant (MDR). Meanwhile, to detect whether plasmid-carrying antimicrobial resistance genes coexisted with the mcr-1 gene in the MCRPEC isolates, a total of 22 plasmid-carrying antimicrobial resistance genes were tested for. The results showed that four ESBL genes and one pAmpC gene were identified. Eight of the MCRPEC isolates also contained the carbapenem gene blaNDM-5, which could cause untreatable infections. Moreover, ten non-lactamase genes were also detected. Conclusion This study indicated that swine-origin MCRPEC isolated in Guangxi seemed to have a high rate of resistance to to reduce the chances of developing antimicrobial resistance. Some studies showed that the production and transfer of plasmid-carrying tigacycline resistance genes The detection of non-lactamase antimicrobial resistance genes in the E. coli isolates was performed by PCR amplification. The special primers included plasmid-encoded fluoroquinolone resistance genes (qnrA, qnrB, qnrS, aac(6’)-Ib-cr) [27], tetracycline resistance genes (tetA, tetB, tetX) [18], sulfonamide resistance genes (sul1, sul2), aminoglycoside resistance genes (aadA), and chloramphenicol resistance genes.


Abstract
Background The discovery of mcr-1-positive Escherichia coli (MCRPEC), a notable superbug, attracted great attention worldwide. Swine-origin multi-drug resistance MCRPEC is a potential threat to public health and safety. To date, few detailed studies regarding swine-origin MCRPEC in Guangxi, South China, have been reported. Results In this study, thirty-three MCRPEC harbored mcr-1 genes were identified from 142 E. coli strains isolated from swine droppings and entrails in Guangxi in 2018. All MCRPEC isolates were assigned to 8 unique STs, including ST10, ST224 and ST410, which overlapped with the humanorigin MCRPEC. Additionally, a total of six plasmid replicon types (IncFI, IncHI1, IncY, IncN, IncI1 and IncX1) were found. Moreover, the drug susceptibility of the MCRPEC isolates was tested with 27 antimicrobial agents belonging to 17 antimicrobial categories that are usually used in hospitals. There were 19 extended spectrum beta lactamase (ESBL) E. coli and 12 carbapenem resistant E. coli among the 33 MCRPEC strains. Importantly, the MCRPEC showed a high rate of resistance against two broad-spectrum carbapenem antibiotics, imipenem and meropenem, which are forbidden in livestock production use.
Three MCRPEC strains were further identified to be extensively drug-resistant (XDR), and other isolates were recognized as multi-drug-resistant (MDR). Meanwhile, to detect whether plasmid-carrying antimicrobial resistance genes coexisted with the mcr-1 gene in the MCRPEC isolates, a total of 22 plasmid-carrying antimicrobial resistance genes were tested for. The results showed that four ESBL genes and one pAmpC gene were identified.
Eight of the MCRPEC isolates also contained the carbapenem gene blaNDM-5, which could cause untreatable infections. Moreover, ten non-lactamase genes were also detected.
Conclusion This study indicated that swine-origin MCRPEC isolated in Guangxi seemed to have a high rate of resistance to both regular and final line of defense drugs as well as drug resistance genes, which pose a great threat to human public safety and health. 4 Background Superbug infection is one of the most serious threats to public and animal health. The emergence and rapid spread of multi-drug-resistant (MDR), extensively drug-resistant (XDR) and pan-drug-resistant (PDR) bacteria is a substantial public health concern around the world [1]. The transmissibility of antimicrobial resistance mediated by mobile plasmids is an important cause of XDR and PDR bacteria. For example, plasmids carrying the MCR-1 gene were found in E. coli, thus suggesting that polymyxin, a last line of defense against carbapenem resistant Enterobacteriaceae [2], was ineffective.
Colistin (polymyxin E) has been used as a veterinary medicine and a feed additive for decades in animal production [3]. In 2015, the plasmid-mediated mcr-1 gene was first discovered in food animals in South China [4]. At this time, a total of nine different mcr alleles, mcr-1 to mcr-9 [5][6][7][8][9][10][11][12], have been detected in different bacteria and spread to over 40 countries/regions. The full-length 16S rRNA nucleotide sequences from the 33 MCRPEC strains were used to generate a phylogenetic tree by MEGA-X (Table 1). Thirty-three MCRPEC strains were aligned to 8 unique STs, including ST10, ST224, ST361, ST410, ST641, ST1408, and ST3345, and an unknown ST. ST10 and ST224 were recognized as the dominant STs, which accounted for 69.7% (23/33) ( Table 1). All data about house-keeping genes were kept in one form (Supplementary material).

Antimicrobial resistance in MCRPEC
To determine whether MCRPEC could threaten food safety and public health, rigorous drug resistance surveillance was vital and necessary. Twenty-seven antimicrobials that are widely used in human clinical treatment were tested ( Table 2).
On the basis of the definition standard for MDR, XDR, and PDR bacteria [13], all 33 MCRPEC isolates exhibited MDR (Fig. 1b). Moreover, three of the MDR MCRPEC isolates were identified as XDR (Fig. 1c). According to figure 1c, the swine original MCRPECs showed a great tendency to become XDR or PDR E. coli.
Coexistence of mcr-1 with plasmids carrying ESBL, pAmpC, carbapenem resistance genes and nonlactamase antimicrobial resistance genes among the

MCRPEC isolates
To detect whether the mcr-1 gene coexisted with ESBL, carbapenem and pAmpC genes and other β-lactamase genes in the MCRPEC strains, plasmids were isolated and purified without a chromosomal genome. Multiplex PCR was performed by using plasmids as a template. The results showed that β-lactamase genes existed in 23 (69.7%) of the MCRPEC isolates. The main β-lactamase gene types in this study were bla OXA-1 , bla CTX-M , bla NDM and bla CMY . Two MCRPEC isolates harbored bla OXA-1 genes. CTX-M group genes were found in 20 MCRPER isolates, among which, bla CTX-M-123 , bla CTX-M-14 and bla CTX-M-24 were found in 13,19 and 1 of the MCRPEC isolate(s) respectively . Two MCRPEC isolates were found to carry bla CMY-2 . Last, and most importantly, there were 8 MCRPEC isolates that contained the carbapenem gene bla NDM-5 (24.2%) (Fig. 2a, Fig. 2b), which could cause untreatable infections.
Monitoring plasmid-carrying non-lactamase antimicrobial resistance genes could effectively assess and prevent the threat of MCRPEC to public health safety. Twelve plasmid carrying non-lactamase antimicrobial resistance genes were detected by PCR. The qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes,, which are closely associated with fluoroquinolone resistance, were detected and account for 36.4%,36.4, 33.3% and 24.2%, respectively, of the isolates. The tetracycline resistance genes, tetA (100%), tetB (18.2%), and tetX (0%), were also present in the MCRPEC isolates. Moreover, sul1 (90.9%) and sul2 (78.8%) were prevalent in MCRPEC isolates, and the aadA (100%) genes were prevalent in the aminoglycoside-resistant MCRPEC isolates. The plasmid-encoded floR (100%) gene was the major resistance plasmid gene that encoded chloramphenicol resistance in the MCRPEC isolates (Fig. 2a). ST types (ST10, ST224, ST410) of swine-origin MCRPEC isolates were also found in humanorigin E. coli isolates [14]. This finding suggests that the three ST types of swine-origin MCRPEC are likely to infect humans under appropriate conditions. Notably, in previous works of bacterial antimicrobial resistance monitoring, ST224 E. coli strains from human and swine carried antimicrobial resistance genes that posed threats to public health [15].

Discussion
Meanwhile, all ST224 MCRPEC isolates in this study showed extremely high levels of multidrug-resistance, and 3 strains of ST224 MCRPEC were identified as XDR. As the number of test samples was not large in this study, more meaningful work that separates zoonotic MCRPEC strains and analyzes ST types is needed in future studies.
Bacterial antimicrobial resistance is constantly evolving and horizontal gene transfer through plasmids plays a major role [16]. The identification of plasmid characteristics provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of antimicrobial resistance determinants. To date, the mcr-1 gene has been found in eight different plasmid incompatibility groups (IncI2, IncFII, IncX4, IncHI1, IncHI2, IncP, IncF, and IncY) [17]. In this study, we found that IncFIA and IncFrep were highly carried in our 33 MCRPEC isolates. Moreover, bacteria contained more antimicrobial resistance genes when carrying IncFIA, IncFIB, IncFIC, IncFrep, and IncY. This result suggests that a combination of plasmid replicon types made it easier to obtain antimicrobial resistance genes following plasmid transmission.
Previous studies regarding swine-origin MCRPEC mostly focused on genes and transfer.
However, the exploration of antimicrobial-resistance phenotypes has not often been pursued. In other words, antimicrobial selection was not comprehensively evaluated.
Consequently, a total of 27 antimicrobials against E. coli were selected from 18 antimicrobial categories in this study. Antimicrobial-resistance phenotypes usually indicate two problems: damage from the bacteria themselves and a hazard below the surface. As noted above, three MCRPEC ST types that have been isolated from humans were observed in this study. According to the antimicrobial resistance test results, this is a warning sign, as nine isolates were ESBL E. coli and eight isolates were CRE in ten of the ST224 MCRPEC isolates. Meanwhile, nine isolates were ESBL E. coli in thirteen of the ST10 MCRPEC isolates. In many past studies, ST10 and ST224 E. coli were usually observed to be MDR. This problem implies that ST10 and ST224 MCRPEC are typical zoonotic E. coli types and can pose a great threat to public health. Although it turns out that humans with superbug infections are increasingly limited, the development of new drugs could offer a glimmer of hope, such as tigecycline (0%). Of course, rational use of these antimicrobials is required to reduce the chances of developing antimicrobial resistance. Some studies showed that the production and transfer of plasmid-carrying tigacycline resistance genes were influenced by a high-pressure environment of the same antimicrobial type [18].
Plasmid-carrying resistance genes are a hidden threat, as they are the key to antimicrobial resistance transfer. The identification of plasmid-carrying resistance genes is essential for studying resistance genes transfer. In this study, we found seventeen ESBL E. coli that contained CTX-M group genes, which included bla CTX-M-123 , bla CTX-M-14 , and bla CTX-M-24 , that were also found in ESBL E. coli from humans or other animals [19,20].
Alarmingly, eight of the MCRPEC isolates carried bla NDM-5 and bla CTX-M-14 at the same time. Although some studies have foundMCRPEC that carried bla NDM-5 , most bacteria were isolated from hospitals, as carbapenems are strictly forbidden in animal use in China. A new study [21] showed that resistance gene transfer in E. coli occurs regardless of gender and age, but gene transfer has much to do with industrial and social attributes. The report also showed that women and children who farmed pigs and poultry were more likely to be vehicles for ESBL and carbapenem gene transfer. This finding implies that the earlier source of the bla NDM gene in MCRPEC was from humans, and bla NDM can be transferred between animal groups and humans with close contact.

Conclusions
This study indicated that swine-origin MCRPEC isolated in Guangxi seemed to have a high rate of resistance to both regular and final line of defense drugs as well as drug resistance genes. The results of molecular detection showed coexistence of bla NDM-5 and mcr-1, coexistence of ESBL genes (bla OXA-1 , bla CTX-M14 , bla CTX-M123 , and bla CTX-M24 ) and mcr-1 in E. coli isolates. The coexistence of bla NDM-5 , ESBL genes and mcr-1 in one E. coli isolate was worst. From a public health perspective, continued surveillance of MCRPEC is essential in swine, other food animals, and companion animals.

Sample collection and detection of MCRPEC isolates
A total of 142 nonrepetitive E.coli strains were collected retrospectively from 44 pig farms in Guangxi, China in 2018. The swine isolates were from anal swabs or lung, intestinal tract, heart, or lymph gland tissue collected from dead or unhealthy pigs. Samples from the collected tissues were inoculated onto MacConkey agar for 24 h at 37°C, followed by inoculation with eosin-methylene blue agar and Luria-Bertani (LB) agar. Then, 16S rRNA gene sequencing was used to confirm the E. coli isolates, and the full-length nucleotide sequence of the 16S rRNA genes among the E. coli strains were determined by direct sequencing from PCR products and BLAST analysis (http://www.ncbi.nlm.nih.gov/). Then, a phylogenetic tree was generated with MEGA-X software. The plasmid genome of MCRPEC was extracted with a TIANpure Midi Plasmid Kit. These strains were stocked at -80°C in glycerol medium.

Detection of multilocus sequence typing (MLST) and plasmid replicon types
The seven housekeeping genes, namely, adk, fumC, gyrB, icd, mdh, purA, and recA, were used to perform MLST analysis. PCR amplicons were purified, and DNA was sequenced. The gene sequences for seven housekeeping genes were uploaded to the EnteroBase database types, additional PCRs were conducted, as described previously [23,24].

Antimicrobial susceptibility testing
According to the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) consensus, a total of 27 commonly used human antimicrobials were selected from 18 antimicrobial categories (Table 2) in this study [13]. MICs≥4 mg/L as nonwild type (non-WT) and colistin MICs≤2 mg/L as wild type (WT). In this study, we adopted the EUCAST Resistant/Susceptible breakpoints.

Detection of non-lactamase antimicrobial resistance genes
The detection of non-lactamase antimicrobial resistance genes in the E. coli isolates was performed by PCR amplification. The special primers included plasmid-encoded fluoroquinolone resistance genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr) [27], tetracycline resistance genes (tetA, tetB, tetX) [18], sulfonamide resistance genes (sul1, sul2), aminoglycoside resistance genes (aadA), and chloramphenicol resistance genes. Availability of data and materials The data were presented in the main manuscript and available to readers.