IncI2-carrying multidrug-resistant Escherichia coli B1: ST101 isolated from meat and meat products in Egypt

Journal Pre-proof Draft Genome Sequence of a mcr-1/IncI2-carrying multidrug-resistant Escherichia coli B1: ST101 isolated from meat and meat products in Egypt Mustafa Sadek, Laurent Poirel, Patrice Nordmann, Tadashi Shimamoto

PII:

S2213-7165(19)30307-8

DOI:

https://doi.org/10.1016/j.jgar.2019.11.015

Reference:

JGAR 1102

To appear in:

Journal of Global Antimicrobial Resistance

Received Date:

11 November 2019

Accepted Date:

22 November 2019

Please cite this article as: Sadek M, Poirel L, Nordmann P, Shimamoto T, Draft Genome Sequence of a mcr-1/IncI2-carrying multidrug-resistant Escherichia coli B1: ST101 isolated from meat and meat products in Egypt, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.11.015

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Draft Genome Sequence of a mcr-1/ IncI2-carrying multidrug-resistant Escherichia 1 coli B1: ST101 isolated from meat and meat products in Egypt

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Mustafa Sadek 1,2, Laurent Poirel1,3,4, Patrice Nordmann1,3,4,5, and Tadashi

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Shimamoto6

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Medical and Molecular Microbiology, Faculty of Science and Medicine, University

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of Fribourg, Fribourg, Switzerland.

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Department of Food Hygiene and Control, Faculty of Veterinary Medicine, South

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Valley University, Qena, Egypt. INSERM European Unit (IAME), University of Fribourg, Fribourg;

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Swiss National Reference Center for Emerging Antibiotic Resistance (NARA),

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University of Fribourg, Fribourg

Institute for Microbiology, University of Lausanne and University Hospital Centre,

Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere

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Lausanne, Switzerland.

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Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan.

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Abstract

Objectives: The aim of this study was to investigate the occurrence of plasmid-encoded18 colistin resistance among gram-negative bacteria isolated from meat and meat products in19 Egypt and to report the draft genome sequence of an mcr-1/IncI2-carrying multidrug-20 21

resistant Escherichia coli B1:ST101.

Methods: A total of 128 colistin-resistant strains were isolated from different meat and meat22

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product samples in different cities in Egypt. Multiplex PCR screening for plasmid-mediated23 colistin-resistance genes was performed. WGS was performed using an Illumina NextSeq24 platform and was assembled using CLC Genomics Workbench 7.5.1.

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Results: A single mcr-1-positive multidrug-resistant E. coli strain was isolated from beef26

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sausages in Egypt. The genome size of E. coli strain was calculated at 5,044,715 bp,27

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comprising a total of 226 contigs, with a G+C content of 50.5%. The E. coli strain belonged28 to sequence type ST101 (phylogroup B1). The mcr-1 gene was located on an IncI2 type self-29

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conjugative plasmid of 64.6-kb in size. This strain showed a multidrug-resistant phenotype30 with an MIC of colistin at 4 mg/L. A large series of acquired antibiotic resistance genes was31

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identified, including those encoding resistance to colistin (mcr-1), beta-lactams (blaTEM-1),32 phenicol (floR), trimethoprim (dfrA12), aminoglycosides (aac(3)-IIa, aph(3'')-Ib and aadA2), 33

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macrolides (mphA and mdfA), tetracycline (tetA), sulfonamides (sul1 and sul2), and34 35

quinolones (qnrS1).

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Conclusion: Here we reported the first draft genome sequence of an mcr-1/IncI2-carrying36 multidrug-resistant E. coli B1:ST101 isolated from beef sausage in Egypt. This study 37 highlights the potential role played by food products in the spread of colistin resistance to38 humans.

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Keywords:

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mcr-1, IncI2, Escherichia coli, Whole-genome sequencing, Egypt

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The continuing dissemination of multidrug-resistant Gram-negative bacteria especially44 carbapenem-resistant bacteria is a global health concern and has led to the re-use of old45 antibiotics, namely colistin, for treating critical infections [1, 2]. Resistance to colistin had46 initially been linked to mutational changes identified within chromosomal genes that47 contribute to the biosynthesis of lipopolysaccharide. Recently, a plasmid-mediated colistin48

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resistance gene (mcr-1) was reported from humans, animals, food, and environment in China49 [3]. This gene encodes a phosphoethanolamine transferase responsible for acquired50 resistance to colistin that has now been reported worldwide.

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The aim of this work was to evaluate the occurrence of the plasmid-mediated colistin52

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resistance among gram negative bacteria isolated from meat and meat products in Egypt. A53

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total of 128 colistin-resistant strains (minimum inhibitory concentration [MIC] >2 μg/mL)54 were isolated from different meat and meat product samples (fresh beef, frozen beef, mutton,55

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minced meat, burger, sausage, luncheon, kofta, and pasterma) randomly collected from56 different supermarkets, slaughterhouses, and butchers’ shops in different cities in Egypt.57

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Those isolates were recovered by direct spreading on MacConkey agar supplemented with58 2 µg/ml of colistin. PCR screening for plasmid-mediated colistin-resistance genes (mcr-1 to59

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mcr-8) was performed but remained negative [4], except for a single E. coli isolate in which60 the mcr-1 gene was detected. This strain (named MC13) had been recovered from sausage61 62

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sample.

Susceptibility testing was determined by disk diffusion according to the Clinical and63 Laboratory Standards Institute (CLSI) recommendations, except for colistin. Broth64 microdilution was performed for determination of MICs of colistin in cation-adjusted65 Mueller-Hinton broth (Bio-Rad, Marnes-La-Coquette, France) and results were interpreted66 according to the EUCAST/CLSI joint guidelines (www.eucast.org). E. coli MC13 was67

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resistant

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ampicillin,

chloramphenicol,

and

ciprofloxacin,

tetracycline

but

nalidixic

acid,

kanamycin,

remained

susceptible

to

gentamicin,68

broad-spectrum69

cephalosporins, carbapenems, and aztreonam. The MIC of colistin was 4 mg/L, being in the70 71

resistance range.

Whole genomic DNA was obtained from an over-night culture using the Purelink Genome72 DNA Mini kit (Invitrogen, Darmstadt, Germany), and a genomic library was prepared using73 an Illumina Nextera XT DNA Library with 2x300-bp paired-end reads on an Illumina MiSeq74

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instrument (Illumina, San Diego, CA, United States). De-novo genome assembly was75 performed using CLC Genomics Workbench 7.5.1. The assembled draft genome sequence76 of E. coli MC13 consisted of 226 contigs comprising 5,044,715 bp in size with of a 50.5%77

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G+C content. Multilocus sequence typing analysis (https://cge.cbs.dtu.dk/services/MLST/)78 showed that MC13 belonged to ST101, pathogenic phylogroup B1, a background which had79

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infection in South Africa [5].

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been already associated with the production of MCR-1 in a patient with a urinary tract80

Only a single mcr-1-positive E. coli human clinical isolate had been identified in Egypt so82 far, being from a patient with bacteremia [6]. With respect to animals, the mcr-1 gene was83

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identified in a E. coli isolate from a cow suffering from subclinical mastitis [4]. Furthermore,84 a single mcr-1-positive E. coli strain was isolated from raw milk cheese in 2019 [7].

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Serotype and FimH subtyping analyses were performed using SerotypeFinder 2.086 and

FimTyper

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(https://cge.cbs.dtu.dk/services/SerotypeFinder/)

(https://cge.cbs.dtu.dk/services/FimTyper/), respectively, and revealed the O15:H10-88 fimH86 profile. Virulence genes including cma (colicin M), ireA (siderophore receptor), gad89 (glutamate decarboxylase), ipfA (long polar fimbriae) and iss (increased serum survival)90 were detected by VirulenceFinder 2.0 (https://cge.cbs.dtu.dk/services/VirulenceFinder/).91 Various antibiotic resistance genes were predicted in the genome by ResFinder 3.292

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(https://cge.cbs.dtu.dk/services/ResFinder/), including blaTEM-1 (ampicillin resistance), floR93 (phenicol), dfrA12 (trimethoprim), aac(3)-IIa, aph(3'')-Ib and aadA2 (aminoglycosides),94 mph(A) and mdf(A) (macrolide), tetracycline (tetA), sul1 and sul2 (sulfonamides), and qnrS195 96

(quinolones).

The plasmid harboring mcr-1 (designated as pEGYMCR-1) was found to be 64,600-bp in-97 size, belonging to IncI2 (broad host range). Of note, the first reported mcr-1 gene being98 found in animal and human isolates in China had also been identified onto an IncI2-type99

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plasmid [3]. The sequence of this plasmid was identical to that of the mcr-1-positive plasmid100 pMCR-GN775 (accession no. KY471307) identified from an E. coli ST624 isolate recovered101 from a Canadian patient who previously received health care in Egypt [8].

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Generally, the mcr-1 gene is identified as being in association with the insertion sequence103 ISApl1 which may play a major role in its mobilization [2]. Noteworthy, the pEGYMCR-1104

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plasmid did not display an ISApl1 element in the vicinity of the mcr-1 gene. However, the105

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mcr-1-pap2 element was identified in a similar context, between the top (encoding a DNA106 topoisomerase III) and nikB (relaxase) genes, like in pMCR-GN775 and other IncI2 plasmids107 as previously reported [8].

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Transfer of the mcr-1 harbouring-IncI1 plasmid (pEGYMCR-1) was demonstrated by a filter109 mating assay using azide-resistant E. coli strain J53 as recipient. E. coli transconjugants were110

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selected on LB agar supplemented with sodium azide (150 μg/mL) and colistin (2 μg/mL).111

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The E. coli transconjugant was resistant only to colistin (MIC of 4 mg/L) and no other112 resistance determinant was co-transferred, indicating that only mcr-1 gene was located on 113 a 114

conjugative plasmid.

In conclusion, we report here the occurrence of a colistin-resistant mcr-1 gene in a multidrug-115 resistant E. coli strain recovered from meat products in Egypt. With the emergence of mcr-116 1-bearing bacteria in meat and meat products, it highlights the potential reservoir for colistin117

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resistance, thereby leading to a higher possibility of transmission of resistant bacteria from118 meat to humans. The strict monitoring and surveillance of resistant bacteria among foods of119 animal origin, particularly meat and meat products, are strongly needed to prevent their120 121

dissemination to humans.

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The draft genome sequencing project of E. coli strain MC13 had been deposited at GenBank 123 124

ACKNOWLEDGMENTS

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under accession no. PRJNA587479

Funding: This work has been funded by the Egyptian government, the University of Fribourg, 126

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Switzerland, and the Swiss National Science Foundation (project FNS-407240_177381). 127 Ethical approval: Not applicable.

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Competing interests: None to declare.

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