Genetic characterization of multidrug-resistant Escherichia coli harboring colistin-resistant gene isolated from food animals in food supply chain

Colistin is widely used for the prophylaxis and treatment of infectious disease in humans and livestock. However, the global food chain may actively promote the dissemination of colistin-resistant bacteria in the world. Mobile colistin-resistant (mcr) genes have spread globally, in both communities and hospitals. This study sought to genomically characterize mcr-mediated colistin resistance in 16 Escherichia coli strains isolated from retail meat samples using whole genome sequencing with short-read and long-read platforms. To assess colistin resistance and the transferability of mcr genes, antimicrobial susceptibility testing and conjugation experiments were conducted. Among the 16 isolates, 11 contained mcr-1, whereas three carried mcr-3 and two contained mcr-1 and mcr-3. All isolates had minimum inhibitory concentration (MIC) for colistin in the range 1–64 μg/mL. Notably, 15 out of the 16 isolates demonstrated successful transfer of mcr genes via conjugation, indicative of their presence on plasmids. In contrast, the KK3 strain did not exhibit such transferability. Replicon types of mcr-1-containing plasmids included IncI2 and IncX4, while IncFIB, IncFII, and IncP1 contained mcr-3. Another single strain carried mcr-1.1 on IncX4 and mcr-3.5 on IncP1. Notably, one isolate contained mcr-1.1 located on a chromosome and carrying mcr-3.1 on the IncFIB plasmid. The chromosomal location of the mcr gene may ensure a steady spread of resistance in the absence of selective pressure. Retail meat products may act as critical reservoirs of plasmid-mediated colistin resistance that has been transmitted to humans.


Introduction
Antimicrobial resistance (AMR) is a serious global health issue that affects both humans and animals.Misuse and overuse of antibiotics in clinical treatment and with livestock contribute to the growth and spread of antimicrobial resistance bacteria (Liu et al., 2021).Colistin is a common antibiotic that has been utilized in the veterinary field for decades, not only for prophylaxis and treatment, but also for growth promotion.It is now regarded as a last resort against antimicrobial agents in therapy infections caused by multidrug-resistant, Gram-negative bacteria in humans (Zajac et al., 2019).Inevitably, the increasing use of colistin in humans and animals has led to the emergence of colistin resistance in Gramnegative bacteria, with the rates of resistance continuing to increase (Kempf et al., 2016).
Normally, colistin resistance is thought to be chromosomally mediated (Meletis and Skoura, 2018).In 2016, a mobile colistin resistance (mcr) gene was identified in the IncI2-type plasmid of E. coli isolated from food animals and raw meat in China (Liu et al., 2016).The mcr gene encodes a membrane of phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine into lipid A to modify lipopolysaccharide (LPS), leading to a reduced negative charge on the outer membrane of Gram-negative bacteria (Vu Thi Ngoc et al., 2022).The mcr genes (mcr-1 to mcr-10) have been successively reported globally; the mcr-1 gene has been widely detected in animals, food products, humans and the general environment (Martiny et al., 2022).To date, the predominate plasmid carrying mcr has been reported in different plasmids based on their replicon types including IncX3, IncX4, IncI2, IncHI1, InHI2, IncK, IncP, IncF, IncFII, IncFIB, IncP, IncY, ColE10, and ColE10-like (Sun et al., 2018;Lu et al., 2020;Tang et al., 2022).Epidemiological studies have indicated that the spread of colistin-resistant mcr-carrying bacteria is not only a problem in hospitals, but also a major concern for environmental and food safety.In this regard, environmental sources, food-producing animals, foreign travel, and food trading have expedited the worldwide spread of mcr genes at the human-animalenvironment interface (Liu and Song, 2019;Liu et al., 2021;Lopes et al., 2021;Nakano et al., 2021).
Escherichia coli (E.coli) is a commensal pathogen that causes gastrointestinal tract disease in humans and animals.This bacterium has been isolated at various sites in the food chain, typically usually due to fecal contamination (Macori et al., 2021).Often, it is used as an indicator organism to monitor AMR in livestock, food products, and humans (Singh et al., 2018;Pormohammad et al., 2019;Puangseree et al., 2022) and represents a major reservoir of antimicrobial resistance genes, mostly acquired through horizontal gene transfer (Puangseree et al., 2022).The spreading of mcr-carrying E. coli along the food chain is the most common transmission mode of colistin resistance, the mcr-1 gene could have been disseminated to human populations through two primary pathways: the food supply chain and direct contact between animals and humans.Additionally, contamination of both freshwater and seawater systems likely played a role in the dissemination, subsequently leading to the contamination of vegetables and seafood (Chen et al., 2017;Lu et al., 2020).In Thailand, colistin-resistant mcr-carrying E. coli strains have been identified in humans ( (Khine et al., 2020;Khanawapee et al., 2021;Paveenkittiporn et al., 2021;Trongjit and Chuanchuen, 2021;Nguyet et al., 2022;Phuadraksa et al., 2022).In our previous study, mcr genes were detected in 7.8% of slaughtered pork in Thailand (Khanawapee et al., 2021).Consequently, to comprehend the genomic characteristics in mcr-harboring E. coli strains in food animals in Thailand, we characterized the genetic features of mcrharboring plasmids in E. coli that had been isolated from food animal products using whole-genome sequencing.

Ethics statement
Ethical review and approval were not required because no human or animal specimens or data were used in the current study.

Bacterial isolates and identification
In this study, a total of 16 E. coli isolates carrying mcr genes were analyzed.Ten of these isolates were collected from slaughtered pigs during 2014-2015, as reported in a previous study (Khanawapee et al., 2021).Furthermore, we identified mcr genes in six out of 40 E. coli isolates obtained from 63 retail meat samples collected between 2020-2021, (unpublished data) by using a polymerase chain reaction (PCR) methods as described elsewhere (Khanawapee et al., 2021).The geographical locations of sample collection are illustrated on the map in Figure 1.

Conjugation experiment
The transferability of mcr was assessed through a conjugation assay, utilizing mcr-harboring E. coli strains as donors and streptomycin-resistant E. coli UB1637 as a recipient.Briefly, both donor and recipient strains were culture in Luria-Bertani (LB) broth with shaking at 200 rpm at 37°C, then a 1:25 ration of donor to recipient strain was mixed in 1 ml of LB broth and incubated overnight at 37°C with gentle shaking 50 rpm.Transconjugants were selected on MacConkey agar plates containing streptomycin (3200 µg/mL) and colistin (4 µg/mL).The presence of mcr genes in transconjugants was detected via PCR method, and colistin MIC were determined to confirm the transconjugants as previously described (Phetburom et al., 2021).Geographical location of mcr-harboring E. coli in this study.(Khon Kaen = 3 isolates, Roi Et = 9 isolates, Lampang = 2 isolates and Chon Buri = 2 isolates).

Whole genome sequencing and bioinformatics analyses
Genomic DNA samples of the 16 mcr-1 harboring E. coli strains were extracted using a MagMAX DNA Multi-Sample Ultra 2.0 Kit (Appliedbiosystems by Thermo Fisher Scientific; Vilnius, Lithuania).DNA concentrations were determined using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific; Waltham, MA, USA).Whole-genome sequencing was performed using the HiSeq 3000 (Illumina; CA, USA) and MinION (Oxford Nanopore Technologies; Oxford, UK) platforms.The DNA library was generated using the NEBNex Ultra II DNA Library Prep Kit for Illumina (New England Biolabs; MA, USA) according to the manufacturer.The Illumina HiSeq platform was prepared with 150-bp paired-end reads.We applied the Fastp v0.19.5 program (Chen et al., 2018) with default parameters for quality filtering of the Illumina reads and adapter trimming for the sequences using the Skewer v0.2.2 program (Jiang et al., 2014).The FastQC v0.118 program1 was used for quality checking of the Illumina reads.
Library preparation for ONT sequencing followed the rapid barcoding DNA sequencing protocol using the SQK-RBK004 kit (Oxford Nanopore Technologies; Oxford, UK), and the libraries were sequenced using a single R9 version/FLO-MIN106D flow cell on a MinION Mk1B sequencer (Oxford Nanopore Technologies; Oxford, UK).We base called and demultiplexed the raw data using the Guppy v5.0.11(ONT) program.The ONT adapters were trimmed using the Porechop v0.2.4 program2 (Wick et al., 2019).Quality control of the ONT reads was undertaken using the NanoPlot v1.28.1 program3 (De Coster and Rademakers, 2023).Hybrid assemblies with the ONT and Illumina data were performed using Unicycler v0.48 (Wick et al., 2017) and the genome sequences were checked for quality using the QUAST v5.0.2 program (Gurevich et al., 2013).The genome sequences were processed using the DDBJ Fast Annotation and Submission tool (Tanizawa et al., 2018) for annotation.Default parameters were used for all software programs unless otherwise specified.
Genomes were in silico typed using the following databases: sequence types (MLST 2.0) (Larsen et al., 2012), serotypes (SerotypeFinder 2.0) (Joensen et al., 2015), subtypes (CHtyper 1.0) (Camacho et al., 2009), while FimH types were identified using the FimTyper 1.0 program (Roer et al., 2017).Plasmid replicon types were identified using the PlasmidFinder 2.1 program (Carattoli et al., 2014) from the Center for Genomic Epidemiology (CGE).Clermont phylotyping4 was performed.Acquired resistance genes and chromosomal mutation-mediated antimicrobial resistance genes were identified using the ResFinder 4.1 program (Zankari et al., 2012) from CGE and the resistance gene identifier from the Comprehensive Antibiotic Resistance Database (Alcock et al., 2023).A SNP-based phylogenetic tree was constructed to compare twelve E. coli strains isolated from patients in Thailand (Paveenkittiporn et al., 2021;Boueroy et al., 2022) (Supplementary Table 1) with the sixteen mcr-1 harboring E. coli strains from food-animal products in this study.The construction of the tree was carried out using REALPHY online tool (Bertels et al., 2014).Phylogenetic analysis of mcr carrying plasmids was applied using Mashtree (Katz et al., 2019) and visualized using the Interactive Tree of Life (iTOL) tool (Letunic and Bork, 2021).The Easyfig software (version 2.2.5) was used for the comparative and visualize the region of plasmids carrying mcr genes.(Sullivan et al., 2011).The sequence similarity search was performed using BLAST against the NCBI nucleotide database.In order to examine the distribution and relationships of other plasmid carrying mcr, plasmid sequences were downloaded from NCBI and compared (Supplementary Table 2).

Data availability statement
The complete genomes were deposited in GenBank under Bioproject Accession No. PRJNA525849.

Antimicrobial susceptibility of mcrcarrying E. coli strains
In total, of the 16 E. coli carrying mcr isolates, 11 contained mcr-1, 3 isolates harbored mcr-3, and 2 isolates contained mcr-1 and mcr-3.The antimicrobial susceptibility results are shown in Table 1.All strains were defined as multidrug resistance (MDR) based on resistance to at least three classes of antibiotics (Magiorakos et al., 2012).Of the 16 isolates, 15 (93.75%) were resistant to ampicillin, 12 (75%) were resistant to trimethoprim and tetracycline, 11 (68.75%) were resistant to chloramphenicol, and 10 (62.5%) to ciprofloxacin, whereas there was no resistance to fosfomycin and carbapenem was detected.The colistin MIC of these isolates was in the range 1-16 µg/mL, as shown in Table 1.We detected only one isolate (RE46) did not resist to colistin (MIC = 1 ug/ml), whereas the rest isolates were resistant.We detected only one isolate (RE46) did not show resistance to colistin (MIC = 1 ug/ml), whereas the remaining isolates were resistant.

Conjugation
Of the 16 mcr harboring E. coli isolates, 15 successfully transferred the colistin resistant gene (mcr) by conjugation to the recipient strain (E. coli UB1637), with the KK3 strain being the exception.All transconjugants had harboring mcr-1 and mcr-3 depend on donor and colistin MIC values in the range 8-16 µg/mL, suggesting the transferred mcr genes were located on conjugative plasmids.3) including Clermont phylogroup, sequence types (ST), serotype, CHType, FimH type, plasmids replicon types, antibiotic resistance and virulence genes was shown in Table 2.The Clermont phylotyping identified 7 isolates (43.75%, 7/16) belonging to group A, 3 to group D, and 2 for each of groups C, B1, and B2 (Table 2).FimH398 was detected in 4 isolates (25%, 4/16).Two isolates had FimH54 and FimH23, while the remaining isolates belonged to individual FimH types.For the CHType, 27-398 was found in 2 isolates, with different CHType represented by the remaining isolates.The MLST revealed 13 STs among these isolates (Table 2).ST10 was found in 3 isolates, with 2 isolates belong to ST1114 and 11 STs.
To analyze the genetic relationship of the mcr-carrying E. coli isolated in this study, we conducted a phylogenomic analysis using available genomes of mcr-carrying E. coli (n = 12) isolated from patients in Thailand in another study (Boueroy et al., 2022).The phylogenetic tree had two main distinct clades.The mcr-carrying E. coli isolates from our study had a related genetic background in a cluster, except for C14.2 that was closely related to Thai human stains 54881, 54715, and 60000 (Figure 2).The strains KK76, RE40, RE46, and LP72 were located on the same cluster as the Thai human strains 53037, 2117, 62122, 58967, 56511, 53360, and V417 (Figure 2).
The phylogenetic relationships of the plasmid replicon type IncX4 and IncI2 carrying mcr-1 in E. coli in our study were closely related with the plasmids IncX4 and IncI2 harboring mcr-1 human E. coli.(Figure 4).The comparative analysis of the IncI2 and IncX4 types carrying mcr-1 in E. coli showed that both the human and meat strains were similar and contained mcr-1.1-pap2.(Figure 5).

Discussion
There have been reports of the prevalence of plasmid-mediated colistin resistance in E. coli in a wide range of Enterobacterales, especially in E. coli from various sources, environments (Lopes et al., 2021;Mohsin et al., 2021;Nakano et al., 2021) food-producing animals (Huang et al., 2017), livestock (Migura-Garcia et al., 2020), and humans (Li B. et al., 2018;Yamaguchi et al., 2020).Other studies have shown that the plasmid type is important in mcr dissemination due to horizontal gene transfer in various sources.In Thailand, the mcr gene has been identified in clinical samples (Paveenkittiporn et al., 2021;Boueroy et al., 2022) and animals (Khine et al., 2020;Khanawapee et al., 2021;Trongjit and Chuanchuen, 2021).Here, we characterized the genetic feature and accessory gene of 16 mcr-1 and mcr-3 carrying E. coli strains isolated from meat samples in the food supply chain.Almost all isolates exhibited resistance to colistin, however, only one isolate did not resist.This result is consistent with other studies that reported colistin MIC values below 2 µg/ml but present mcr gene ( (Khanawapee et al., 2021;Pungpian et al., 2021).
In recent decades, the incidence of extended-spectrum blactamases (ESBL)-producing E. coli, contributing to a growing incidence of community-acquired extra-intestinal infections in  humans, animals and wide range of food animals product, including beef, pork and chicken meat globally (Shafiq et al., 2019;Shafiq et al., 2021;Shafiq et al., 2022).Our study, E. coli harboring mcr-1 and mcr-3 were detected together with ESBL genes (bla TEM-1B , bla CTX-M-14 and bla CTX-M-55 ).Among them, bla TEM-1B was the most common found in thirteen mcr positive E. coli, these results correlated with previous findings in Thailand that demonstrated the concurrent carriage of mcr-1.1 with bla CTX-M-14 , bla CTX-M-55 , and bla TEM-1B (Leangapichart et al., 2023).Moreover, the coexistence of mcr with ESBL genes in E.coli was report from other studies such as China and Pakistan, E. coli co-harboring mcr with ESBL genes such as bla CTX-M-14 , bla CTX-M-55 , and bla TEM-1B (Shafiq et al., 2019;Shafiq et al., 2022).
Our study, identified 12 different STs, containing 3 ST10 strains and 1 ST34 strain, which could be considered as a broad range of hosts to carry mcr that are found in humans, animals, vegetables, and wastewater (Shen et al., 2020) that have been reported in humans and animals in Thailand (Khanawapee et al., 2021;FIGURE 4 Phylogenetic tree of mcr-harboring plasmid replicon types of E. coli, where blue color represents isolates from meat (pork and chicken) in this study and yellow represents isolates from human specimens.Paveenkittiporn et al., 2021;Boueroy et al., 2022).These STs were most common among the mcr-1 positive isolates (Li et al., 2022).The majority of phylogroups in our studies belonged to group A (43.75%), similar with other reports (Bosaḱ et al., 2019;Garcıá-Meniño et al., 2019).
Horizontal gene transfer (HGT) via plasmids plays a critical role in the dissemination of antibiotic resistance determinants among Gram-negative bacteria (Sun et al., 2017).Notably, the mcr genes, responsible for colistin resistance, are located on diverse plasmids, encompassing more than 20 incompatibility groups identified as carriers of mcr-1 (Liu et al., 2023).The IncX4, IncH2 and IncI2 replicon represent the majority, exceeding 90% of plasmids that have been identified globally.While IncX4 was the predominant plasmid group carrying the mcr-1 gene in Europe and IncI2 in Asia, recently indicates a global shift towards IncX4 prevalence (Sismova et al., 2023).Our analysis of mcr carrying plasmid sequences, revealing a significant predominance of mcr-1 on IncX4 replicons, with additional presence on IncI2, IncP1, IncFIB and IncFII plasmids.These finding resonate with our previously published data, demonstrating mcr-1 carriage by IncX4 and IncI2 replicon in E. coli in humans in Thailand (Boueroy et al., 2022).This supports the notion of mcr gene emergence within specific plasmid groups in Enterobacterales from various reservoirs, including animals, food, environment, and humans (Boonyasiri et al., 2023;Leangapichart et al., 2023).Additionally, our experiments revealed the conjugative transfer capacity of IncX4 plasmids harboring mcr-1.Although this observation is limited to our laboratory conditions, it may offer a partial explanation for the increasing dominance of IncX4 in mcr-1 dissemination.Its high self-transferable at high frequencies (Sun et al., 2017;Bai et al., 2018).The high efficiency of horizontal transfer via this plasmid family poses a significant risk, considering that colistin is considered a last-line antibiotic for the treating lifethreatening infections in humans (Sismova et al., 2023).
The pap2 superfamily protein was detected upstream but was not detected in the insertion sequence ISApl1 of any of the mcr-1bearing InX4 plasmids, which indicated that the mcr-1 located in IncX4 was stable.
The genetic context analysis surrounding the mcr genes in our studies was hampered by the inherent ambiguity of the conjugation assay for determining the origin of transferred genes.The conjugation assay inability to definitively differentiate between p l a s m i d a n d c h r o m o s o m a l t r a n s f e r c o u l d l e a d t o misinterpretations.This ambiguity stems from two scenarios.Firstly, mcr genes may reside on plasmids that integrated into the chromosome prior to conjugation.In such instances, the transferred genes would appear chromosomal despite their initial plasmid origin (Rafii and Crawford, 1988;Willett, 1988).
Furthermore, the ISAPl1 insertion sequence has been implicated as a crucial element facilitating the translocation of mcr-1 into diverse plasmid types.This sequence has been localized on the chromosome through the formation of circular intermediates (Trongjit and Chuanchuen, 2021).The close genetic association between ISApl1 and mcr-1 suggests that ISApl1 may play a pivotal role in the dissemination of mcr-1 (Li et al., 2017).The three isolates of mcr-1 located on the IncI2 plasmid contained ISAPl1-mcr-1-pap2, which is one pattern of the transposon Tn6330 (Snesrud et al., 2018).In our study, the mcr-3.1 gene was located on the IncFIB and IncFII plasmids, while mcr-3.5 was located on the IncP1 plasmid, to a study in China (Li J. et al., 2018).Both plasmids contained a diacylglycerol kinase (dgkA) upstream and this genomic context similar to another study (Phuadraksa et al., 2022).In addition, almost all the mcrcarrying plasmids could transfer to the recipient E. coli strain, which was in agreement with another study that concluded these plasmids were conjugative and dissemination could easily occur (Trongjit and Chuanchuen, 2021).
Other reports noted that the presence of the chromosomally encoded mcr gene is very rare (3.5-4%) (Li R. et al., 2018;Shen et al., 2020); however, a recent study reported E. coli strains harboring chromosomally encoded mcr-1 were found in 26-37% isolates from humans and animals in Vietnam (Vu Thi Ngoc et al., 2022).Recently, co-harboring of mcr-2 and mcr-3 on chromosomes in E. coli isolated from a healthy human was documented in Thailand (Phuadraksa et al., 2022).The current study revealed 2 mcr-1 harboring E. coli strains (KK3 and CB70) contained the segment of transposase ISApl1-mcr-1-pap2-ISApl1 (Tn6330) in the chromosome that would increase its stability and be highly mobilizable in spreading mcr genes due to vertical transfer.

Conclusion
Retail meat products (primarily pork and chicken), as a main food type and an important component of the food chain, were contaminated by mcr genes.Such meat products may serve as reservoirs of this antimicrobial-resistant gene that can affect humans.This finding supports the potential for the distribution and transmission of the colistin-resistant mcr gene is mediated by plasmids.Therefore, the mcr gene can spread via food chains, with the source of the strains suggesting that mcr-1 may be a public health risk by spreading to humans through farm to fork processes.Therefore, ongoing monitoring and investigations of mcr genes in agricultural sector are required to control and prevent their spread.

FIGURE 2
FIGURE 2Phylogenetic tree of mcr-harboring E. coli, where blue color represents isolates from meat (pork and chicken) in this study, while yellow represents isolates from human specimens.
FIGURE 5Schematic representation of sequence alignment analysis of IncI2 (A) and IncX4 (B) plasmids carrying mcr-1.1,comparing meat and human specimens.Boxed arrows represent position and transcriptional direction of ORFs, with mobile genetic elements (mcr-1.1) in red, pap2 protein in green, relaxase protein in pink, ISApl1 in orange, and other genes or hypothetical ones in blue.The ID strain in light blue box represents isolates from meat (pork and chicken) in this study and yellow box represents isolates from human specimens.

TABLE 1
Antimicrobial susceptibility profiles of mcr-harboring E. coli isolates.
Education, Science, Research and Innovation; and the Thailand Science Research and Innovation through the Kasetsart University Reinventing University Program 2021.