Occurrence of the mobile colistin resistance gene mcr-3 in Escherichia coli from household pigs in rural areas

Occurrence of the mobile colistin resistance gene mcr-3 in Escherichia coli from household pigs... Sir, Polymyxins are regarded as one of the few last-resort antibiotics for the treatment of serious infections caused by MDR Gram-negative bacteria, such as carbapenem-resistant Enterobacteriaceae. However, the discovery of the plasmid-mediated colistin resistance gene mcr-1 further reduced the treatment options.1 The gene mcr-1 has been identified in Enterobacteriaceae from different sources in more than 45 countries on five continents.2,3 The gene mcr-2 was identified in Escherichia coli from pigs and cattle in Belgium.4 This gene is rarely detected and seems to be limited to some European countries.4,5 Recently, the third plasmid-mediated colistin gene, mcr-3, has been reported in China.6 The mcr-4 and mcr-5 genes have been newly identified in Enterobacteriaceae from animals and humans in European countries.7,8 The gene mcr-3 shares 45% and 47% nucleotide sequence identity with mcr-1 and mcr-2, respectively, and the corresponding protein shares 33% and 32% amino acid identity with MCR-1 and MCR-2, respectively. To date, the knowledge about the occurrence of mcr-3 genes in Enterobacteriaceae from humans or animals is still limited. Here, for the first time (to the best of our knowledge), we report the occurrence of mcr-3 in backyard pigs from households in rural areas of China. We screened 417 pig faecal samples from 254 household backyard farms in 12 villages in the Shandong province in 2015, using ESwabs (Copan, Brescia, Italy). The faecal sample was first added to 1 mL of Nutrient broth (Hangzhou Binhe Microorganism Reagent Co., Ltd, Hangzhou, China) and was incubated overnight at 37°C. Total DNA from 500 μL of the enrichment culture of each sample was extracted using the TIANamp Bacteria DNA Kit (TIANGEN Biotech Co., Beijing, China). The total DNA was screened for mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 genes by PCR as previously described.1,4,6–8 A total of 71 (71/417, 17%) samples from 56 households were positive for mcr-1. Five (5/417, 1.2%) samples from five households were positive for mcr-3. All samples were negative for mcr-2, mcr-4 and mcr-5. A 1 μL sample of each mcr-3-positive culture was plated on Salmonella Shigella (SS) agar (Hangzhou Binhe Microorganism Reagent Co., Ltd) containing 2 mg/L colistin. Colonies with different morphology on the SS agar were picked and checked for the presence of the mcr-3 gene by PCR and sequencing. Five mcr-3-positive isolates, AZ20, BZ5, CZ11, LZ11 and IZ43, were detected from five different villages and all of them were identified as E. coli through 16S rDNA sequencing and MALDI-TOF MS analysis.2 Testing for susceptibility to eight antimicrobial agents by agar dilution and confirmation by broth dilution9,10 revealed that all isolates were resistant to colistin and gentamicin, and isolates AZ20 and BZ5 were also resistant to ciprofloxacin. All isolates were susceptible to meropenem and intermediate to ceftazidime (Table 1). Table 1 Antimicrobial susceptibility and resistome profiles of the five mcr-3-/mcr-3.5-carrying strains and their transconjugants Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – a Abbreviations and resistance breakpoints: CST, colistin (R >2 mg/L); PMB, polymyxin B; MEM, meropenem (R ≥4 mg/L); RIF, rifampicin; GEN, gentamicin (R ≥16 mg/L); CIP, ciprofloxacin (R ≥4 mg/L); FFC, florfenicol; CAZ, ceftazidime (R ≥16 mg/L). CST: EUCAST interpretation criteria. MEM, GEN, CIP and CAZ: CLSI interpretation criteria. PMB, RIF and FFC: no interpretation criteria. b Bold formatting indicates resistance to the respective antimicrobial agents. c aadA1, aadA2, aacA4, strA, strB, aac(3)-IId, aph(3′)-Ia, aph(3′)-IIa and aph(3′)-Ic: aminoglycoside resistance genes. sul1, sul2 and sul3: sulphonamide resistance genes. dfrA12 and dfrA14: trimethoprim resistance genes. tet(A): tetracycline resistance gene. cmlA1, floR and catB3: phenicol resistance genes. blaTEM-1B and blaOXA-1: β-lactam resistance genes. fosA: fosfomycin resistance gene. arr-3: rifampicin resistance gene. oqxAB, qnrS1, qnrS2 and aac(6′)-Ib-cr: quinolone resistance genes. ND, not determined. Table 1 Antimicrobial susceptibility and resistome profiles of the five mcr-3-/mcr-3.5-carrying strains and their transconjugants Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – a Abbreviations and resistance breakpoints: CST, colistin (R >2 mg/L); PMB, polymyxin B; MEM, meropenem (R ≥4 mg/L); RIF, rifampicin; GEN, gentamicin (R ≥16 mg/L); CIP, ciprofloxacin (R ≥4 mg/L); FFC, florfenicol; CAZ, ceftazidime (R ≥16 mg/L). CST: EUCAST interpretation criteria. MEM, GEN, CIP and CAZ: CLSI interpretation criteria. PMB, RIF and FFC: no interpretation criteria. b Bold formatting indicates resistance to the respective antimicrobial agents. c aadA1, aadA2, aacA4, strA, strB, aac(3)-IId, aph(3′)-Ia, aph(3′)-IIa and aph(3′)-Ic: aminoglycoside resistance genes. sul1, sul2 and sul3: sulphonamide resistance genes. dfrA12 and dfrA14: trimethoprim resistance genes. tet(A): tetracycline resistance gene. cmlA1, floR and catB3: phenicol resistance genes. blaTEM-1B and blaOXA-1: β-lactam resistance genes. fosA: fosfomycin resistance gene. arr-3: rifampicin resistance gene. oqxAB, qnrS1, qnrS2 and aac(6′)-Ib-cr: quinolone resistance genes. ND, not determined. DNA from the five mcr-3-positive strains was extracted using the TIANamp Bacteria DNA Kit. Paired-end sequencing was conducted using an Illumina HiSeq 2500 (Berry Genomic Company, Beijing, China). Reads were assembled de novo using SPAdes 3.9.11 The MLST profiles, putative plasmids and antibiotic resistance genes (Table 1) were predicted by uploading the assembly to the bacterial analysis pipeline at the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org/). The mcr-3 gene was identical in the three isolates CZ11, LZ11 and IZ43, whereas the amino acid sequence encoded by the mcr-3 variant in isolates BZ5 and AZ20 differed from MCR-3 at three amino acid positions (M23V, A457E and T488I). This variant gene showed 100% nucleotide sequence identity to mcr-3.5 (GenBank accession no. NG_055782).12 S1-PFGE and Southern blot analysis revealed that mcr-3 and mcr-3.5 were located on different plasmids in the five isolates with their sizes ranging from ∼83 to ∼246 kb (Table 1). To test the transferability of the mcr-3 and mcr-3.5 genes, filter mating was performed with E. coli J53AzR as recipient.13 Transconjugants were selected on Brain Heart Infusion agar (Beijing Land Bridge Technology Ltd, Beijing, China) supplemented with 2 mg/L colistin and 300 mg/L sodium azide. Only isolates AZ20 and BZ5 carried conjugative plasmids, the IncP1-type plasmid pBZ5 and plasmid pAZ20 of an unknown Inc type. The two transconjugants exhibited 4- and 8-fold increased colistin and polymyxin B MICs compared with recipient E. coli J53, but were susceptible to the other six antimicrobial agents (Table 1). Electrotransformation was used for the remaining three mcr-3-carrying isolates. Two IncHI2 plasmids, pCZ11 and pLZ11, derived from isolates CZ11 and LZ11, respectively, were successfully transferred into E. coli DH5α (Takara Bio Inc., Beijing, China). The two transconjugants exhibited 16-fold increased colistin and polymyxin B MICs and exhibited 64-fold increased gentamicin MICs compared with E. coli DH5α (Table 1). WGS analysis of the five mcr-3- and mcr-3.5-carrying isolates revealed contigs ranging in size from 2.6 to 49.9 kb (Table 1). The core structure, mcr-3-dgkA, was identified in all five contigs, four of which exhibited >99% nucleotide sequence identity to the corresponding region of the original mcr-3-carrying plasmid pWJ1. The largest contig (49.9 kb) of plasmid pBZ5 showed 99% nucleotide sequence identity to that in plasmid pMCR3_WCHEC-LL123 (GenBank accession no. MF489760) of human E. coli from China.12 Plasmid pBZ5 contained toxin–antitoxin systems (higB and higA) as well as conjugative elements, which can maintain mcr-3.5 in its host and facilitate the horizontal transfer of this mcr-3 variant to other bacteria.14 Two of the E. coli isolates (CZ11 and LZ11) had identical resistome profiles, the same MLST type (ST3933) and similar MIC values (Table 1). The Parsnp tool in the Harvest suite was applied to compare the homology of the mcr-3-positive isolates.15 It revealed that the two isolates CZ11 and LZ11 were almost identical in their core-genome SNPs. The two E. coli isolates were from two different villages separated by a distance of ∼10 km, suggesting a clonal spread of mcr-3-carrying isolates among pigs from different villages, possibly by trade of their respective animals. In summary, this is—to the best of our knowledge—the first report of mcr-3 and mcr-3.5 in E. coli in backyard pig husbandry. This study revealed that mcr genes are not restricted to pigs from large-scale commercial farms, but also occur in pigs from small-scale backyard holdings. Therefore, adequate measures, such as raised awareness of rational usage of antimicrobial agents in both animals and humans, prudent usage of colistin in pigs for disease treatment and prevention, and good management/hygiene of backyard farming, should be taken into account to limit the spread of mcr genes, including mcr-3 and its variants. Funding This study is part of the Sino-Swedish IMPACT project funded by the National Natural Science Foundation of China (grant 81361138021) and the Swedish Research Council (grant D0879801). Transparency declarations None to declare. References 1 Liu YY , Wang Y , Walsh TR et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study . Lancet Infect Dis 2016 ; 16 : 161 – 8 . Google Scholar CrossRef Search ADS PubMed 2 Wang Y , Zhang R , Li J et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production . Nat Microbiol 2017 ; 2 : 16260. Google Scholar CrossRef Search ADS PubMed 3 Schwarz S , Johnson AP. Transferable resistance to colistin: a new but old threat . J Antimicrob Chemother 2016 ; 71 : 2066 – 70 . Google Scholar CrossRef Search ADS PubMed 4 Xavier BB , Lammens C , Ruhal R et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016 . Euro Surveill 2016 ; 21 : pii=30280. 5 AbuOun M , Stubberfield EJ , Duggett NA et al. mcr-1 and mcr-2 variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015 . J Antimicrob Chemother 2017 ; 72 : 2745 – 9 . Google Scholar CrossRef Search ADS PubMed 6 Yin W , Li H , Shen Y et al. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli . mBio 2017 ; 8 : e00543 - 17 . Google Scholar PubMed 7 Carattoli A , Villa L , Feudi C et al. Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016 . Euro Surveill 2017 ; 22 : pii=30589. 8 Borowiak M , Fischer J , Hammerl JA et al. Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B . J Antimicrob Chemother 2017 ; 72 : 3317 – 24 . Google Scholar CrossRef Search ADS PubMed 9 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 6.0, 2016. 10 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement M100-S25 . CLSI , Wayne, PA, USA , 2015 . 11 Bankevich A , Nurk S , Antipov D et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing . J Comput Biol 2012 ; 19 : 455 – 77 . Google Scholar CrossRef Search ADS PubMed 12 Liu L , Feng Y , Zhang X et al. New variant of mcr-3 in an extensively drug-resistant Escherichia coli clinical isolate carrying mcr-1 and blaNDM-5 . Antimicrob Agents Chemother 2017 ; 61 : e01757-17 . Google Scholar CrossRef Search ADS PubMed 13 Pham Thanh D , Thanh Tuyen H , Nguyen Thi Nguyen T et al. Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate . J Antimicrob Chemother 2016 ; 71 : 2314 – 7 . Google Scholar CrossRef Search ADS PubMed 14 Yang QE , Walsh TR. Toxin-antitoxin systems and their role in disseminating and maintaining antimicrobial resistance . FEMS Microbiol Rev 2017 ; 41 : 343 – 53 . Google Scholar CrossRef Search ADS PubMed 15 Treangen TJ , Ondov BD , Koren S et al. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes . Genome Biol 2014 ; 15 : 524. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Occurrence of the mobile colistin resistance gene mcr-3 in Escherichia coli from household pigs in rural areas

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Abstract

Sir, Polymyxins are regarded as one of the few last-resort antibiotics for the treatment of serious infections caused by MDR Gram-negative bacteria, such as carbapenem-resistant Enterobacteriaceae. However, the discovery of the plasmid-mediated colistin resistance gene mcr-1 further reduced the treatment options.1 The gene mcr-1 has been identified in Enterobacteriaceae from different sources in more than 45 countries on five continents.2,3 The gene mcr-2 was identified in Escherichia coli from pigs and cattle in Belgium.4 This gene is rarely detected and seems to be limited to some European countries.4,5 Recently, the third plasmid-mediated colistin gene, mcr-3, has been reported in China.6 The mcr-4 and mcr-5 genes have been newly identified in Enterobacteriaceae from animals and humans in European countries.7,8 The gene mcr-3 shares 45% and 47% nucleotide sequence identity with mcr-1 and mcr-2, respectively, and the corresponding protein shares 33% and 32% amino acid identity with MCR-1 and MCR-2, respectively. To date, the knowledge about the occurrence of mcr-3 genes in Enterobacteriaceae from humans or animals is still limited. Here, for the first time (to the best of our knowledge), we report the occurrence of mcr-3 in backyard pigs from households in rural areas of China. We screened 417 pig faecal samples from 254 household backyard farms in 12 villages in the Shandong province in 2015, using ESwabs (Copan, Brescia, Italy). The faecal sample was first added to 1 mL of Nutrient broth (Hangzhou Binhe Microorganism Reagent Co., Ltd, Hangzhou, China) and was incubated overnight at 37°C. Total DNA from 500 μL of the enrichment culture of each sample was extracted using the TIANamp Bacteria DNA Kit (TIANGEN Biotech Co., Beijing, China). The total DNA was screened for mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 genes by PCR as previously described.1,4,6–8 A total of 71 (71/417, 17%) samples from 56 households were positive for mcr-1. Five (5/417, 1.2%) samples from five households were positive for mcr-3. All samples were negative for mcr-2, mcr-4 and mcr-5. A 1 μL sample of each mcr-3-positive culture was plated on Salmonella Shigella (SS) agar (Hangzhou Binhe Microorganism Reagent Co., Ltd) containing 2 mg/L colistin. Colonies with different morphology on the SS agar were picked and checked for the presence of the mcr-3 gene by PCR and sequencing. Five mcr-3-positive isolates, AZ20, BZ5, CZ11, LZ11 and IZ43, were detected from five different villages and all of them were identified as E. coli through 16S rDNA sequencing and MALDI-TOF MS analysis.2 Testing for susceptibility to eight antimicrobial agents by agar dilution and confirmation by broth dilution9,10 revealed that all isolates were resistant to colistin and gentamicin, and isolates AZ20 and BZ5 were also resistant to ciprofloxacin. All isolates were susceptible to meropenem and intermediate to ceftazidime (Table 1). Table 1 Antimicrobial susceptibility and resistome profiles of the five mcr-3-/mcr-3.5-carrying strains and their transconjugants Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – a Abbreviations and resistance breakpoints: CST, colistin (R >2 mg/L); PMB, polymyxin B; MEM, meropenem (R ≥4 mg/L); RIF, rifampicin; GEN, gentamicin (R ≥16 mg/L); CIP, ciprofloxacin (R ≥4 mg/L); FFC, florfenicol; CAZ, ceftazidime (R ≥16 mg/L). CST: EUCAST interpretation criteria. MEM, GEN, CIP and CAZ: CLSI interpretation criteria. PMB, RIF and FFC: no interpretation criteria. b Bold formatting indicates resistance to the respective antimicrobial agents. c aadA1, aadA2, aacA4, strA, strB, aac(3)-IId, aph(3′)-Ia, aph(3′)-IIa and aph(3′)-Ic: aminoglycoside resistance genes. sul1, sul2 and sul3: sulphonamide resistance genes. dfrA12 and dfrA14: trimethoprim resistance genes. tet(A): tetracycline resistance gene. cmlA1, floR and catB3: phenicol resistance genes. blaTEM-1B and blaOXA-1: β-lactam resistance genes. fosA: fosfomycin resistance gene. arr-3: rifampicin resistance gene. oqxAB, qnrS1, qnrS2 and aac(6′)-Ib-cr: quinolone resistance genes. ND, not determined. Table 1 Antimicrobial susceptibility and resistome profiles of the five mcr-3-/mcr-3.5-carrying strains and their transconjugants Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – Strain MLST Variant and size of mcr-3-carrying contig (kb) Plasmid size (kb) Plasmid type MIC (mg/L)a Resistome profilesc CST PMB MEM RIF GEN CIP FFC CAZ AZ20 ST155 mcr-3.5, 2.7 ∼83 unknown 8b 4 0.008 4 256 32 128 4 aac(3)-IId, aadA1, aadA2, aph(3′)-Ia, blaTEM-1B, cmlA1, floR, fosA, oqxAB, qnrS2, strA, strB, sul2, sul3, tet(A) BZ5 ST165 mcr-3.5, 49.9 ∼91 IncP1 8 4 0.016 >64 32 4 8 8 aac(6′)Ib-cr, aadA1, aadA2, arr-3, blaTEM-1B, cmlA1, dfrA12, oqxAB, qnrS2, sul3, tet(A) CZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 128 0.5 4 2 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) LZ11 ST3933 mcr-3, 2.6 ∼246 IncHI2 8 8 0.016 <2 256 1 4 8 aph(3′)-IIa, blaTEM-1B, oqxAB, strA, strB, sul1, sul2, tet(A) IZ43 ST10 mcr-3, 7.1 ∼180 unknown 4 4 0.016 16 >256 0.25 8 8 aac(6′)Ib-cr, aacA4, aph(3′)-Ic, arr-3, blaOXA-1, blaTEM-1B, catB3, dfrA14, qnrS1, strA, strB, sul1, sul3, tet(A) J53-AZ20 – – ∼83 unknown 8 4 0.008 4 1 0.008 4 0.5 ND J53-BZ5 – – ∼91 IncP1 4 4 0.016 4 1 0.032 4 1 ND J53 – – – – 1 0.125 0.016 4 1 0.004 8 1 – DH5α-CZ11 – – – IncHI2 4 4 0.008 2 16 0.032 4 2 ND DH5α-LZ11 – – – IncHI2 4 4 0.008 2 16 0.064 4 4 ND DH5α – – – – 0.25 0.25 0.008 2 0.25 0.032 4 0.25 – a Abbreviations and resistance breakpoints: CST, colistin (R >2 mg/L); PMB, polymyxin B; MEM, meropenem (R ≥4 mg/L); RIF, rifampicin; GEN, gentamicin (R ≥16 mg/L); CIP, ciprofloxacin (R ≥4 mg/L); FFC, florfenicol; CAZ, ceftazidime (R ≥16 mg/L). CST: EUCAST interpretation criteria. MEM, GEN, CIP and CAZ: CLSI interpretation criteria. PMB, RIF and FFC: no interpretation criteria. b Bold formatting indicates resistance to the respective antimicrobial agents. c aadA1, aadA2, aacA4, strA, strB, aac(3)-IId, aph(3′)-Ia, aph(3′)-IIa and aph(3′)-Ic: aminoglycoside resistance genes. sul1, sul2 and sul3: sulphonamide resistance genes. dfrA12 and dfrA14: trimethoprim resistance genes. tet(A): tetracycline resistance gene. cmlA1, floR and catB3: phenicol resistance genes. blaTEM-1B and blaOXA-1: β-lactam resistance genes. fosA: fosfomycin resistance gene. arr-3: rifampicin resistance gene. oqxAB, qnrS1, qnrS2 and aac(6′)-Ib-cr: quinolone resistance genes. ND, not determined. DNA from the five mcr-3-positive strains was extracted using the TIANamp Bacteria DNA Kit. Paired-end sequencing was conducted using an Illumina HiSeq 2500 (Berry Genomic Company, Beijing, China). Reads were assembled de novo using SPAdes 3.9.11 The MLST profiles, putative plasmids and antibiotic resistance genes (Table 1) were predicted by uploading the assembly to the bacterial analysis pipeline at the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org/). The mcr-3 gene was identical in the three isolates CZ11, LZ11 and IZ43, whereas the amino acid sequence encoded by the mcr-3 variant in isolates BZ5 and AZ20 differed from MCR-3 at three amino acid positions (M23V, A457E and T488I). This variant gene showed 100% nucleotide sequence identity to mcr-3.5 (GenBank accession no. NG_055782).12 S1-PFGE and Southern blot analysis revealed that mcr-3 and mcr-3.5 were located on different plasmids in the five isolates with their sizes ranging from ∼83 to ∼246 kb (Table 1). To test the transferability of the mcr-3 and mcr-3.5 genes, filter mating was performed with E. coli J53AzR as recipient.13 Transconjugants were selected on Brain Heart Infusion agar (Beijing Land Bridge Technology Ltd, Beijing, China) supplemented with 2 mg/L colistin and 300 mg/L sodium azide. Only isolates AZ20 and BZ5 carried conjugative plasmids, the IncP1-type plasmid pBZ5 and plasmid pAZ20 of an unknown Inc type. The two transconjugants exhibited 4- and 8-fold increased colistin and polymyxin B MICs compared with recipient E. coli J53, but were susceptible to the other six antimicrobial agents (Table 1). Electrotransformation was used for the remaining three mcr-3-carrying isolates. Two IncHI2 plasmids, pCZ11 and pLZ11, derived from isolates CZ11 and LZ11, respectively, were successfully transferred into E. coli DH5α (Takara Bio Inc., Beijing, China). The two transconjugants exhibited 16-fold increased colistin and polymyxin B MICs and exhibited 64-fold increased gentamicin MICs compared with E. coli DH5α (Table 1). WGS analysis of the five mcr-3- and mcr-3.5-carrying isolates revealed contigs ranging in size from 2.6 to 49.9 kb (Table 1). The core structure, mcr-3-dgkA, was identified in all five contigs, four of which exhibited >99% nucleotide sequence identity to the corresponding region of the original mcr-3-carrying plasmid pWJ1. The largest contig (49.9 kb) of plasmid pBZ5 showed 99% nucleotide sequence identity to that in plasmid pMCR3_WCHEC-LL123 (GenBank accession no. MF489760) of human E. coli from China.12 Plasmid pBZ5 contained toxin–antitoxin systems (higB and higA) as well as conjugative elements, which can maintain mcr-3.5 in its host and facilitate the horizontal transfer of this mcr-3 variant to other bacteria.14 Two of the E. coli isolates (CZ11 and LZ11) had identical resistome profiles, the same MLST type (ST3933) and similar MIC values (Table 1). The Parsnp tool in the Harvest suite was applied to compare the homology of the mcr-3-positive isolates.15 It revealed that the two isolates CZ11 and LZ11 were almost identical in their core-genome SNPs. The two E. coli isolates were from two different villages separated by a distance of ∼10 km, suggesting a clonal spread of mcr-3-carrying isolates among pigs from different villages, possibly by trade of their respective animals. In summary, this is—to the best of our knowledge—the first report of mcr-3 and mcr-3.5 in E. coli in backyard pig husbandry. This study revealed that mcr genes are not restricted to pigs from large-scale commercial farms, but also occur in pigs from small-scale backyard holdings. Therefore, adequate measures, such as raised awareness of rational usage of antimicrobial agents in both animals and humans, prudent usage of colistin in pigs for disease treatment and prevention, and good management/hygiene of backyard farming, should be taken into account to limit the spread of mcr genes, including mcr-3 and its variants. Funding This study is part of the Sino-Swedish IMPACT project funded by the National Natural Science Foundation of China (grant 81361138021) and the Swedish Research Council (grant D0879801). Transparency declarations None to declare. References 1 Liu YY , Wang Y , Walsh TR et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study . Lancet Infect Dis 2016 ; 16 : 161 – 8 . Google Scholar CrossRef Search ADS PubMed 2 Wang Y , Zhang R , Li J et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production . Nat Microbiol 2017 ; 2 : 16260. Google Scholar CrossRef Search ADS PubMed 3 Schwarz S , Johnson AP. Transferable resistance to colistin: a new but old threat . J Antimicrob Chemother 2016 ; 71 : 2066 – 70 . Google Scholar CrossRef Search ADS PubMed 4 Xavier BB , Lammens C , Ruhal R et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016 . Euro Surveill 2016 ; 21 : pii=30280. 5 AbuOun M , Stubberfield EJ , Duggett NA et al. mcr-1 and mcr-2 variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015 . J Antimicrob Chemother 2017 ; 72 : 2745 – 9 . Google Scholar CrossRef Search ADS PubMed 6 Yin W , Li H , Shen Y et al. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli . mBio 2017 ; 8 : e00543 - 17 . Google Scholar PubMed 7 Carattoli A , Villa L , Feudi C et al. Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016 . Euro Surveill 2017 ; 22 : pii=30589. 8 Borowiak M , Fischer J , Hammerl JA et al. Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B . J Antimicrob Chemother 2017 ; 72 : 3317 – 24 . Google Scholar CrossRef Search ADS PubMed 9 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 6.0, 2016. 10 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement M100-S25 . CLSI , Wayne, PA, USA , 2015 . 11 Bankevich A , Nurk S , Antipov D et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing . J Comput Biol 2012 ; 19 : 455 – 77 . Google Scholar CrossRef Search ADS PubMed 12 Liu L , Feng Y , Zhang X et al. New variant of mcr-3 in an extensively drug-resistant Escherichia coli clinical isolate carrying mcr-1 and blaNDM-5 . Antimicrob Agents Chemother 2017 ; 61 : e01757-17 . Google Scholar CrossRef Search ADS PubMed 13 Pham Thanh D , Thanh Tuyen H , Nguyen Thi Nguyen T et al. Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate . J Antimicrob Chemother 2016 ; 71 : 2314 – 7 . Google Scholar CrossRef Search ADS PubMed 14 Yang QE , Walsh TR. Toxin-antitoxin systems and their role in disseminating and maintaining antimicrobial resistance . FEMS Microbiol Rev 2017 ; 41 : 343 – 53 . Google Scholar CrossRef Search ADS PubMed 15 Treangen TJ , Ondov BD , Koren S et al. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes . Genome Biol 2014 ; 15 : 524. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 16, 2018

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