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Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00739-9
10.1016/j.psj.2024.104160
104160
MICROBIOLOGY AND FOOD SAFETY
Research note: characteristics of blaNDM and mcr-1 co-producing Escherichia coli from retail chicken meat
Zhao Wenbo *†1
Wan Shuigen *†1
Li Shihong *†
Li Wenjun *†
Kang Jin *†
Liu Yong *†
Huang Hexiang *†
Li Hao *†
Du Xiang-Dang *†
Xu Chunyan zmdchunyan@163.com
⁎†
Yao Hong yaoh0913@henau.edu.cn
⁎†2
⁎ College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
† Key Laboratory of Quality and Safety Control of Poultry Products, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Ministry of Agriculture and Rural Affairs, Zhengzhou 450046, China
2 Corresponding author. yaoh0913@henau.edu.cn
1 Equal contributors.

07 8 2024
11 2024
07 8 2024
103 11 10416019 6 2024
29 7 2024
© 2024 Published by Elsevier Inc. on behalf of Poultry Science Association Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Carbapenems and colistin are vital antimicrobials used to treat Enterobacteriaceae-caused infections. The present study aimed to characterize the coexistence mechanism of carbapenem and colistin resistance in an Escherichia coli isolated from retail chicken meat. A total of 4 E. coli isolates co-harboring carbapenem resistance gene blaNDM (2 E. coli isolates with blaNDM-5 and 2 with blaNDM-9) and colistin resistance gene mcr-1. Antimicrobial susceptibility testing exhibited that all the 4 E. coli strains had multidrug resistance profile and consistent with the resistance genes they carried. MLST showed that 3 E. coli isolates belonged to a pathogenic E. coli lineage ST354, which is closely associated with human infections and pose a serious threat to public health. Whole genome sequencing (WGS) showed that 4 mcr-1-positive plasmids with sizes of 60.4 kb to 67.4 kb all belonged to the IncI2 type. A total of 5 blaNDM-harboring plasmids ranged from 99.0 kb to 138.3 kb, among which 4 plasmids belonged to unknow type and only pCS5L-NDM belonged to IncFIA/IncFIB group of hybrid plasmids, a novel carrier for blaNDM. Comparative analysis exhibited that the mcr-1 or blaNDM-carrying plasmids of E. coli strains from chicken meat showed high identity with that from Enterobacteriaceae of human origin, which indicated the risk of mcr-1 or blaNDM dissemination from retail meat to human. The simultaneous occurrence of mcr-1 and blaNDM in E. coli emphasizes the significant of antimicrobial resistance surveillance in retail meat.

Key words

mcr-1
blaNDM
E. coli
retail meat
chicken
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pmcINTRODUCTION

The emergence and spread of antimicrobial resistance in Enterobacteriaceae pose a threat to public health and food safety worldwide. The increasingly prevalence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Enterobacteriaceae, especially carbapenem-resistant Enterobacteriaceae (CRE), has compromised the efficacy of antimicrobials used in clinic (Levy and Marshall, 2004). Colistin is classified as critically important antimicrobials by the World Health Organization (WHO) and is resurrected as last-resort antibiotics to treat CRE (https://www.who.int/foodsafety/ publications/antimicrobials-fifth/en/).

Carbapenem resistance is mainly mediated by production of carbapenemases, and New Delhi metallo-β-lactamases (NDM) represent a vital class of carbapenemases to confer resistance to most β-lactams except aztreonam. To date, more than 20 NDM variants have been identified since the first identification of NDM-1 in India in 2008. The encoding gene blaNDM is often located in transferable mobile genetic elements and co-exists with other resistance factors. mcr-1 encodes a PmrC-like pEtN transferase, which conveys pEtN from the cytomembrane to lipid A, leading to colistin resistance. Since the discovery of the transmissible colistin resistance gene mcr-1 in China, mcr variants have been reported in numerous studies from different countries (Liu et al., 2024).

Concurrence of blaNDM and mcr genes harbored by CRE, resulting in the emergence of MDR or XDR, is of particular concern. To date, co-occurrence of blaNDM and mcr in Enterobacteriaceae species originated from clinical settings, environment and animals has been described in various of studies from different countries (Zeng et al., 2023). Food of animal origin also pose a serious threat to public health as CRE co-existent blaNDM and mcr have been reported to be associated with it, including retail meat available in supermarket (Liu et al., 2019). However, the characteristics of E. coli of retail chicken meat origin co-harboring these 2 resistance genes have not been well explored. Herein, we characterized carriage of mcr-1 by E. coli strains harboring blaNDM from retail chicken meat origin.

MATERIALS AND METHODS

Bacterial Strains and Antimicrobial Susceptibility Testing

A total of 31 retail chicken meat samples were randomly collected from 3 supermarkets and 1 farmer's markets in Zhengzhou, Henan Province, China, in 2019. Randomly purchase nonduplicate chicken samples from randomly selected agricultural markets, then place the samples in separate sterile sealed bags and transport them back to the laboratory in a low-temperature environment. Cut a small amount of each sample and place it in a 2 mL centrifuge tube containing LB broth (Hopebio, China) for enrichment. The overnight cultures were streaked onto CHROMagar KPC plates (The Chromogenic Media Pioneer, France) and then incubated overnight at 37°C. Pink and blue colonies were selected and subcultured on brain heart infusion (BHI) (Hopebio, China) agar and incubated at 37°C for 24 h. Subsequently, the whole DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China), and amplicons of 16S rRNA gene were sequenced to identify the isolate species as described previously (Van Ba et al., 2018).

MICs for a range of antimicrobial agents, including aztreonam, cefazolin, cefoxitin, ceftazidime, ciprofloxacin, colistin, erythromycin, florfenicol, fosfomycin, gentamicin, meropenem and tigecycline were determined by using the broth dilution method and interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI M100-S30, 2020). The tigecycline MICs were interpreted based on the recommendations of EUCAST (https://www.eucast.org/ast_of_bacteria/). E. coli ATCC 25922 was used as the quality control strain.

Detection of the Carbapenem-Resistant and Colistin-Resistant Genes

Strains exhibiting resistance to both meropenem and colistin were identified by detecting the carbapenem resistance genes, including blaNDM, blaKPC, blaOXA, and blaIMP, and colistin resistance gene mcr with primers as previously described (Doyle et al., 2012). Then, PCR amplicons were sequenced by Sanger sequencing and further analyzed by using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Plasmid Conjugation

Conjugation by filter mating was performed as described in our previous study (Zhao et al., 2022). Briefly, the donor and recipient strains were cultured separately in LB broth for 6-8 h. Then, they mixed in a ratio of 1:4, and 0.1 mL was inoculated onto LB Nutrient Agar (LB agar) (Hopebio, China) plate for 14 h at 37°C. Colonies were subcultured on LB agar with 2 μg/mL meropenem or 1 μg/mL colistin and 100 μg/mL azide to select transconjugants.

Whole Genome Sequencing and Bioinformatic Analysis

Whole genome DNA of E. coli isolates co-producing blaNDM and mcr were extracted using a TianGen DNA Extraction kit (TianGen, Beijing, China) following the manufacturer's instructions. The 250 bp paired-end reads were obtained by using an Illumina MiSeq system (Illumina, San Diego, CA) and then draft genome were assembled using the SPAdes and BioNumerics v. 8.0 (Applied Maths, https://www.applied-maths.com). The genome sequence was annotated using RAST (https://rast.nmpdr.org/rast.cgi). Analysis of characteristics of the assembled genome was conducted by using bioinformatics tools (e.g., MLST v.2.0, ResFinder 4.1, and PlasmidFinder v.2.1), which were available on the Center for Genomic Epidemiology platform (CGE) (https://www.genomicepidemiology.org/). Easyfig 2.2.3 and BRIG v.0.95 were used to compare and analysis the plasmids.

RESULTS AND DISCUSSION

Strain Identification, Antimicrobial Susceptibility Profile of E. coli Isolates Co-Harboring blaNDM and mcr-1

Clones with a pink color were picked from the KPC plates and were further identified as E. coli by 16S DNA sequencing. PCR detection results revealed that a total of 4 E. coli isolates harbored mcr-1 gene, 2 of them harbored blaNDM-5 and 2 isolates harbored blaNDM-9. To assess the transferability of the blaNDM and mcr-1-carrying plasmids, conjugation experiments were performed between these 4 E. coli isolates and the recipient isolate J53, respectively. The plasmids carrying blaNDM in CS5L, CS6L, and CS9F were transferred to E. coli J53 at frequencies of 6.8×10−7, 1.1×10−4, and 4.7×10−7, respectively. The plasmids carrying mcr-1 in CS5L, CS6L, and CS9F were also successful transferred at frequencies of 1.8×10−6, 5.4×10−5, and 3.5×10−7, respectively. Interestingly, when screening with meropenem, colistin, and sodium azide, co-transconjugants were also obtained with frequencies of 1.58×10−6, 2.2×10−5, and 1.7×10−7. However, the transconjugant of CM2 was not obtained.

Antimicrobial susceptibility testing showed that all the E. coli isolates co-harboring blaNDM and mcr-1 were resistant to multiple antimicrobial agents. In addition to meropenem and colistin, these isolates exhibited resistance or not susceptible to antimicrobials, such as ceftazidime (100%), cefoxitin (100%), cefazolin (100%), aztreonam (50%), gentamicin (100%), ciprofloxacin (100%) fosfomycin (100%) and florfenicol (100 %). However, all isolates were susceptible to tigecycline. These findings indicate that the E. coli isolates in our study showed resistance to multiple antibiotics and highlight that the surveillance of antimicrobial resistance in retail meat is important to ensure public health security.

Analysis of STs

The whole genome sequencing was performed to analyze the genetic characteristics of blaNDM and mcr-1 co-harboring E. coli isolates. Multilocus sequence typing (MLST) analysis showed that 3 E. coli isolates (CM2, CS5L and CS9F) were attributed to ST354 and CS6L belonged to unknow STs, indicating the potential epidemic clone ST354 in chicken meat samples.

ST354 is recognized as a pathogenic E. coli lineage, which could cause bloodstream infections, urinary tract infections and other human infections (Mottaghizadeh et al., 2020). E. coli ST354 has been reported in various countries, including Brazil, Australia, Spain, Siberia, South Korea, Thailand and China, and was originated from soil environment, food-producing animal, pet, raw vegetables and turkey meat in addition to clinical samples. These findings combined with the results in the current study suggested the wide distribution and high risk of ST354 clone. Therefore, the E. coli ST354 strain, particularly carrying multiple resistance genes and isolated from retail chicken meat, pose a serious threat to public health due to its possible transmission to human via the food chain, then leading to clinical treatment failures.

Characterization of Resistance Genes Carried by 4 E. coli Isolates

The 4 carbapenem-resistant and colistin-resistant E. coli isolates contained a chromosome of ∼5.0 Mb in size and several plasmids in accordance with the result indicated by WGS. CM2 and CS5L both contain blaNDM-5 and mcr-1, with CS5L carrying 2 different plasmids containing blaNDM-5, while the other 2 strains CS6L and CS9F contain blaNDM-9 and mcr-1. In addition to blaNDM and mcr-1, resistome analysis identified the presence of blaCTX-M (n = 3), blaOXA (n = 3) and blaTEM (n = 3), accounting for the resistance to carbapenem and other β-lactam antimicrobials. These strains also carried tetracyclines resistance gene tet(A) (n = 4); aminoglycosides resistance genes aac(3)-IId (n = 1), aac(3)-IV (n = 4), aadA (n = 4), ant(3′’)-Ia (n = 2), aph(3′’)-Ib (n = 3), aph(3′)-IIa (n = 1), aph(4)-Ia (n = 3), aph(6)-Id (n = 3) and aph(6′)-Ib-cr (n = 2); fluoroquinolones resistance genes qnrS1 (n = 2), oqxA (n=1) and oqxB (n = 1); macrolides resistance genes lnuF (n = 2), mdf(A) (n = 4) and mph (E) (n = 1); sulfonamides resistance genes sul1(n = 4) and sul2 (n = 4); phenicols resistance genes floR (n = 4) and cmlA (n=1); trimethoprims resistance genes dfrA12 (n = 3), dfrA14 (n = 2) and dfrA17 (n = 2); and fosA3 (n = 1) which accounts for fosfomycin resistance. This finding is in accordance with the antimicrobial resistance phenotype in our study. In consideration of the isolates with multi-drug resistance caused by various genes, it is significant to highlight that the future antimicrobial resistance surveillance should be carried out seriously.

Analysis for blaNDM and mcr-1-Carrying Plasmid

The mcr-1-positive plasmids with sizes of 60.4 kb to 67.4 kb all belonged to the IncI2 incompatibility (Inc) group and showed high nucleotide identity with each other (Figure 1). Furthermore, the complete sequence of a representative mcr-1-carrying plasmid in CS5L showed high similarity to some mcr-1-harboring plasmids isolated from animals and humans in the NCBI Genbank database, including p5CRE51-MCR-1 (accession number CP021176, E. coli plasmid from a human, China), pColR598_2 (MF175189, E. coli plasmid from a human, China), pD90-2 (CP022452, S. enterica plasmid from a chicken, China), pCREC-527-4 (KY657476, E. coli plasmid from human, South Korea) and pJIE3685-1 (KY795978, E. coli plasmid from a human, Australia) (Figure 1). None of these plasmids carried additional antimicrobial resistance genes in addition to mcr-1. The plasmids described above, together with the 4 mcr-1-harboring plasmids in this study, were aligned with reference plasmid pHNSHP45 (KP347127) which was first reported to carrying mcr-1 in E. coli from pigs in China. The result exhibited that the mcr-1-carrying plasmids of E. coli strains from chicken meat showed high identity with that from pig even human origin, which highlights the risk of mcr-1 IncI2 plasmid dissemination among various hosts. Genetic environment analysis showed that ISApl1 mobile element was not present upstream of the mcr-1 cassette in the plasmids analyzed in this study, while it was present in plasmid pHNSHP45. Among the genetic environments reported, ISApl1 is the most common IS element adjacent to mcr-1 at one or both sides. It has been demonstrated to play a vital role in mcr-1 gene dissemination, and the location of ISApl1 adjacent to mcr-1 may facilitate the translocation of mcr-1 onto the chromosome (Shi et al., 2020).Figure 1 Comparative analysis of 4 mcr-1 plasmids carrying this study and 5 plasmids with high similarity in GenBank database using BRIG. This figure presents a comparative visualization of plasmids generated using the BLAST Ring Image Generator (BRIG) software. The plasmids from the outside to the inside are pHNSHP45 (KP347127); pJIE3685-1 (KY795978); pCREC-527_4 (KY657476); pD90-2 (CP022452); pColR598_2 (MF175189); p5CRE51-MCR-1 (CP021176); pCS9F-mcr (CP158452); pCS6L-mcr (CP158449); pCS5L-mcr (CP158314); pCM2-mcr (CP158310). BLAST analysis results are represented by the intensity of the color, with darker shades indicating higher sequence similarity.

Figure 1

The blaNDM-positive plasmids, with size range from 99.0 kb to 138.3 kb, were more diverse than mcr-1-carrying plasmid. To be noted, 2 blaNDM-harboring plasmids were present in 1 E. coli isolate CS5L. A total of 5 blaNDM-harboring plasmids were categorized into different replicon types. One plasmid in CS5L belonged to IncFIA/IncFIB group of hybrid plasmids. The rest of the plasmids (pCM2-NDM, pCS5L-NDM-p2, pCS6L-NDM and pCS9F-NDM) belonged to unknown replicon types. The plasmids pCM2-NDM, pCS5L-NDM-p2, pCS6L-NDM and pCS9F-NDM in our study exhibited a high degree of homologies with some blaNDM-harboring plasmids isolated from chicken retail meat and human in the NCBI GenBank database, including pNDM-T2 (MN335919, chicken), p1106-NDM (MG825375, chicken), pC (CP069173.1, human) and pHNTH02-1 (MG196294, retail meat) (Figure 2A). Interestingly, plasmid pCS5L-NDM showed low identity with other 4 plasmids in our study. Then, using BLAST with the plasmid pCS5L-NDM sequence as the query, it shared 99.9% identity with plasmids pA98_p0 (CP142584.1, swine), pHB42-1 (CP104078.1, dog) and pTEM1-GZEC065 (CP048026.1, human), while with 78%, 78% and 74% query coverage, respectively (Figure 2B). However, the 3 similar plasmids in GenBank database didn't carry blaNDM gene. It is speculated that pCS5L-NDM may originate from pA98_p0 like plasmid and integrate blaNDM gene. These results suggested that the plasmids identified in this study was important carriers of blaNDM. Numerous documents indicated that IncX3 plasmid was the most common vehicle to carry blaNDM gene (Ariyoshi et al., 2022). However, IncFIA/IncFIB group of hybrid plasmids carrying blaNDM has never been report. Our findings suggested IncFIA/IncFIB plasmid was novel carrier to habor blaNDM and widened the range of carrier for this carbapenem resistance gene.Figure 2 Comparative analysis of plasmid structures carrying blaNDM using BRIG. This figure presents a comparative visualization of plasmids generated using the BLAST Ring Image Generator (BRIG) software. (A) The plasmids from the outside to the inside are pNDM-T2 (MN335919.1); pCS9F-NDM (CP158451); pCS6L-NDM (CP158448); pHNTH02-1 (MG196294.1); pC (CP069173.1); p1106-NDM (MG825375.1); pCS5L-NDM-p2 (CP158312); pCM2-NDM (CP158309). (B) The plasmids from the outside to the inside are pCS5L-NDM (CP158313), pHB42-1 (CP104078.1), pA98_p0 (CP142584.1), pTEM1-GZEC065 (CP048026.1). (A) Contains blaNDM-5 for CM2 and CS5L, blaNDM-9 for CS6L and CS9F. Although blaNDM are different, the genetic environment is similar. The BLAST analysis results are represented by the intensity of the color, with darker shades indicating higher sequence similarity.

Figure 2

In summary, our study reported the occurrence of colistin resistance gene mcr-1 and carbapenem resistance gene blaNDM in E. coli isolated from retail chicken meat, particularly including pathogenic E. coli lineage ST35 closely associated with human infections. mcr-1 was found located on IncI2 type plasmid while blaNDM-harboring plasmids belonged to IncFIA/IncFIB group of hybrid plasmids (a novel carrier for blaNDM) or unknow type. Both mcr-1 or blaNDM-carrying plasmids of E. coli strains from chicken meat showed high identity with that from Enterobacteriaceae of human origin, which indicated the risk of mcr-1 or blaNDM dissemination from retail meat to human and pose a serious threat to public health. The simultaneous occurrence of mcr-1 and blaNDM in E. coli emphasizes the important of antimicrobial resistance surveillance in retail meat.

DISCLOSURES

The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by grants from National Key Research and Development Program of China (2023YFD1801000 ), Natural Science Foundation of Henan Province (no. 232300421037 ), and Young Talent Lifting Project in Henan Province (2023HYTP001 ), and Henan Province University Young Backbone Teachers Training Program (2023GGJS031 ).

Data availability: The chromosomes and described plasmid assemblies of the 4 strains of Escherichia coli in this study are stored in GenBank, Bioproject number: PRJNA1122937.
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