Increasing prevalence of multidrug-resistant mcr-1-positive Escherichia coli isolates from fresh vegetables and healthy food animals in South Korea

Journal Pre-proof Increasing prevalence of multidrug-resistant mcr-1-positive Escherichia coli isolates from fresh vegetables and healthy food animals in South Korea Sung-Suck Oh, Jihyun Song, Junghee Kim, Jinwook Shin

PII:

S1201-9712(19)30498-9

DOI:

https://doi.org/10.1016/j.ijid.2019.12.025

Reference:

IJID 3878

To appear in:

International Journal of Infectious Diseases

Received Date:

25 November 2019

Revised Date:

17 December 2019

Accepted Date:

18 December 2019

Please cite this article as: Oh S-Suck, Song J, Kim J, Shin J, Increasing prevalence of multidrug-resistant mcr-1-positive Escherichia coli isolates from fresh vegetables and healthy food animals in South Korea, International Journal of Infectious Diseases (2019), doi: https://doi.org/10.1016/j.ijid.2019.12.025

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Increasing prevalence of multidrug-resistant mcr-1-positive Escherichia coli isolates from fresh vegetables and healthy food animals in South Korea

Authors: Sung-Suck Oh a,1, Jihyun Song b,1, Junghee Kim a,1 and Jinwook Shin b,*

Incheon Research Institute of Public Health and Environment, Incheon 22320, South Korea

b

Department of Microbiology, Inha University School of Medicine, Incheon 22212, South

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a

To whom correspondence should be addressed:

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*

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Korea

Jinwook Shin, Department of Microbiology, Inha University School of Medicine, 100 Inha-ro,

These authors contributed equally to this work.

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E-mail: [email protected]

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Nam-gu, Incheon 22212, Republic of Korea. Tel.: +82-32-860-9824, Fax: +82-32-881-8559,

Highlights

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 First detection of mcr-1-positive E. coli in vegetables from South Korea  Prevalence and characteristics of the isolates from vegetables and food animals  All of the isolates were multidrug-resistant  All of the isolates harbored both mcr-1 and -lactamase genes

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Abstract Colistin is a last-resort antimicrobial against multidrug-resistant gram-negative bacteria. The occurrence and spread of colistin resistance in humans and animals have been reported globally. In the present study, we investigated the prevalence and antimicrobial susceptibility of mcr-harboring colistin-resistant Enterobacteriaceae from retail vegetables and food animals in South Korea in 2018. The mcr-1 gene was detected in Escherichia coli isolates

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from 0.076% (1/1,324) of vegetables, 5.9% (2/34) of chickens, 6.8% (4/59) of pigs, and 0% (0/57) of cattle. Other mcr genes were not detected. All of the seven mcr-1-positive isolates showed multidrug resistance and coproduced -lactamases. Multilocus sequence typing

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analysis revealed five known E. coli sequence types (STs), including ST10 in the vegetable sample. Our findings demonstrated that the mcr-1 gene has emerged in vegetables and was

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increasingly detected in food animals in South Korea, highlighting the importance of continuous monitoring and control of colistin-resistant Enterobacteriaceae to prevent them

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from being transmitted to humans.

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Keywords: E. coli; colistin resistance; mcr-1; -lactamase; vegetable; food animal

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Since the plasmid-borne mobile colistin-resistant gene mcr-1 was first reported in China in 2015, eight additional homologs (mcr-2, -3, -4, -5, -6, -7, -8, and -9) have been identified (Carroll et al., 2019). The increasing spread of colistin resistance in particular in multidrug-resistant (MDR) bacteria has become a serious global threat. The mcr-1 gene has also been detected in patients and food animals in South Korea (Belaynehe et al., 2018, Lim et 2

al., 2016, Yoon et al., 2018). In the present study, we investigated the prevalence and antimicrobial susceptibility of mcr-harboring colistin-resistant Enterobacteriaceae from retail vegetables and food animals in South Korea during a national surveillance project in 2018. For this study, 1,324 fresh vegetables (879 leafy, 236 fruit, 170 stem, 37 root, and 2 other vegetables) and 150 fecal samples from healthy food animals (34 chickens, 59 pigs, and 57 cattle) from across the country were obtained from farmers’ markets and slaughterhouses

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and screened on MacConkey plates supplemented with 2 mg/L of colistin (SigmaAldrich). Seven colistin-resistant strains were isolated from one lettuce (0.076%), two chicken (5.9%) and four pig (6.8%) samples. No strain was recovered from cattle. All of the isolates were

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identified as Escherichia coli using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany). Colistin

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resistance was confirmed by the colistin minimum inhibitory concentration (MIC) values of 2

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mg/L in Supplementary Table S1 using the broth microdilution method in accordance with the recommendations by the joint Clinical and Laboratory Standards Institute (CLSI)European Committee on Antimicrobial Susceptibility Testing (EUCAST) Polymyxin Breakpoints

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Working Group (EUCAST, 2016). Furthermore, the antimicrobial susceptibility testing of 21 agents from 14 antimicrobial classes was conducted using disk agar diffusion (Oxoid disk,

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Basingstoke, UK) (Table 1 and Supplementary Table S1): aminoglycoside (gentamicin,

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amikacin), carbapenem (ertapenem, imipenem, meropenem), non-extended spectrum cephalosporin (cefazolin), extended-spectrum cephalosporin (cefotaxime, ceftazidime, cefepime), cephamycin (cefoxitin), fluoroquinolone (ciprofloxacin), quinolone (nalidixic acid), folate pathway inhibitor (trimethoprim-sulfamethoxazole), glycylcycline (tigecycline), monobactam (aztreonam), penicillin (ampicillin, piperacillin), penicillin plus -lactamase 3

inhibitor (amoxicillin-clavulanic acid, ampicillin-sulbactam), phenicol (chloramphenicol), and tetracycline (tetracycline). All of the disk diffusion results except for those for tigecycline, which followed the EUCAST breakpoint version 7.1 (EUCAST, 2017), were interpreted according to the CLSI document M100-S27 (CLSI, 2017). All of the colistin-resistant isolates were non-susceptible (resistant or intermediate resistant) to cefazolin, ampicillin, and piperacillin, but susceptible to ertapenem, imipenem, meropenem, and tigecycline. All of the

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isolates showed MDR phenotypes ( 5 classes), as defined by resistance to at least one agent in three or more antimicrobial classes (Magiorakos et al., 2012).

The colistin-resistant E. coli isolates resolved into six different sequence types (STs)

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using multilocus sequence typing (MLST) based on the sequence profiles of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) according to EnteroBase

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(https://enterobase.warwick.ac.uk): ST10, ST898, ST2705, ST5229, ST6706, and unknown type (Table 1). Interestingly, two clones of ST5229 were identified in pigs from farms in

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different provinces. The mcr-1 gene was detected in all isolates by PCR and sequencing, but the mcr-2, -3, -4, -5, -6, -7, and -8 genes were absent (Table 1). Furthermore, PCR and

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sequencing analyses for blaTEM, blaSHV, and blaCTX-M groups 1, 2, 9, and 25 revealed that all isolates carried at least one -lactamase gene, including blaTEM-1, blaCTX-M-1, and blaCTX-M-55.

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None of the isolates investigated had blaSHV and blaCTX-M groups 2, 9, and 25. The primer sets

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used for the detection of mcr and bla genes are detailed in Supplementary Table S2. This is the first report of the mcr-1 gene in vegetable samples in South Korea. The

prevalence of mcr-1-harboring E. coli in chickens and pigs was about six times that of previous surveillance from samples collected between 2013 and 2017 (Belaynehe et al., 2018, Lim et al., 2016). A recent study by Wu et al. (Wu et al., 2018) demonstrated that mcr-1 occurred more 4

in extended-spectrum -lactamase (ESBL)-producing E. coli than in their non-ESBLproducing counterparts. Our study showed that 42.9% (3/7) of mcr-1-positive isolates were ESBL producers. Food animals have been considered major reservoirs for the dissemination of antimicrobial resistance. Given that colistin is widely used in food animals for infection control and that the manure of animals contaminated with mcr-1 can be reused as organic fertilizer on agricultural farms in South Korea, the mcr-1 gene in E. coli recovered from lettuce in this study

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may have been due to food animals contaminated with this gene, and there is a danger of direct transmission to humans through the food chain. Notably, E. coli ST10 of the lettuce isolate is common in human and animal sources (Manges et al., 2015). Thus, our study highlights the importance of surveillance strategies from One Health perspectives for controlling

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antimicrobial resistance in human together with animal, food, and environmental sources.

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Declarations Funding

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This research was supported by the Korea Centers for Disease Control and Prevention (2017ER540301), the Basic Science Research Program through the National Research

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Foundation (NRF) of Korea funded by the Ministry of Education (2018R1A6A1A03025523)

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and Inha University Research Grant (2018).

Conflict of interest statement: All authors have no conflict of interests to declare.

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Ethical approval: Not required.

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References Belaynehe KM, Shin SW, Park KY, Jang JY, Won HG, Yoon IJ, et al. Emergence of mcr-1 and mcr-3 variants coding for plasmid-mediated colistin resistance in Escherichia coli isolates from food-

producing

animals

in

South

Korea.

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Infect

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2018;72:22-4.

http://doi.org/10.1016/j.ijid.2018.05.011. Carroll LM, Gaballa A, Guldimann C, Sullivan G, Henderson LO, Wiedmann M. Identification

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of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, ColistinSusceptible Salmonella enterica Serotype Typhimurium Isolate. MBio 2019;10(3). http://doi.org/10.1128/mBio.00853-19.

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CLSI. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory

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Standards Institute. 2017. http://clsi.org.

EUCAST. European Committee on Antimicrobial Susceptibility Testing (EUCAST).

joint

CLSI-EUCAST

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Recommendations for MIC determination of colistin (polymyxin E) as recommended by the Polymyxin

Breakpoints

Working

Group.

2016.

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http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Recom mendations_for_MIC_determination_of_colistin_March_2016.pdf [accessed 1 August 2018].

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EUCAST. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint

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tables for interpretation of MICs and zone diameters. Version 7.1. 2017. http://www.eucast.org. Lim SK, Kang HY, Lee K, Moon DC, Lee HS, Jung SC. First Detection of the mcr-1 Gene in Escherichia coli Isolated from Livestock between 2013 and 2015 in South Korea. Antimicrob Agents Chemother 2016;60(11):6991-3. http://doi.org/10.1128/AAC.01472-16. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug7

resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18(3):268-81. http://doi.org/10.1111/j.1469-0691.2011.03570.x. Manges AR, Harel J, Masson L, Edens TJ, Portt A, Reid-Smith RJ, et al. Multilocus sequence typing and virulence gene profiles associated with Escherichia coli from human and animal sources. Foodborne Pathog Dis 2015;12(4):302-10. http://doi.org/ 10.1089/fpd.2014.1860.

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Wu CM, Wang YC, Shi XM, Wang S, Ren HW, Shen ZQ, et al. Rapid rise of the ESBL and mcr-1 genes in Escherichia coli of chicken origin in China, 2008-2014. Emerg Microbes Infect 2018;7(1):30. http://doi.org/10.1038/s41426-018-0033-1.

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Isolates

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http://doi.org/10.3343/alm.2018.38.6.555.

Korea.

Ann

Lab

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coli

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Enterobacteriaceae Isolates From Human Specimens: Comparison With Those in Escherichia

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Med

2018;38(6):555-62.

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Table 1 Characteristics of colistin-resistant E. coli isolates from vegetables and food animals Province

MLST

mcr

Antimicrobial agents and susceptibilitiesa

pr

Isolate Origin

bla

e-

and

COL GEN AMK ETP IPM MEM CFZ CTX CAZ FEP FOX CIP NAL SXT TGC ATM AMP PIP AMC SAM CHL TET

EC045 Pig

Gyeonggi ST5229 mcr- R

EC046 Pig

Chungnam ST898

S

1, R

S

Pr

genes

S

S

S

S

R

R

S

S

I

R

R

R

S

S

R

R R

I

R

R

S

S

S

S

I

S

S

S

S

R

R

R

S

S

R

R S

S

R

R

EC047 Pig

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TEM-

Chungnam unknown 1

S

S

S

S

S

I

S

S

S

S

S

S

S

S

S

R

R S

S

R

R

EC073 Chicken Chungbuk ST6706 mcr- R

S

S

S

S

S

R

R

S

I

S

R

R

R

S

I

R

R S

S

R

R

R

S

S

S

S

S

I

R

S

S

S

R

R

S

S

R

R

R S

S

S

R

R

R

R

S

S

S

R

R

I

I

S

R

R

R

S

R

R

R S

S

R

R

R

R

S

S

S

S

R

R

I

R

S

S

S

R

S

R

R

R I

I

R

S

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R

EC092 Chicken Incheon

ST2705 1,

TEM-

EC093 Pig

Chungnam ST5229

1

EC106 Lettuce Chungnam ST10

mcr-

9

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f

1, TEM-

pr

1 mcr-

e-

1, CTX-

mcr1,

Pr

M-1

na l

TEM1

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mcr1,

TEM1,

CTX-

10

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f

M-55 mcr-

pr

1, TEM-

e-

1, CTX-

The antimicrobial susceptibilities were indicated as resistant (R), intermediate resistant (I) and susceptible (S). Abbreviations: COL, colistin;

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a

Pr

M-55

GEN, gentamicin; AMK, amikacin; ETP, ertapenem; IPM, imipenem; MEM, meropenem; CFZ, cefazolin; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; CIP, ciprofloxacin; NAL, nalidixic acid; SXT, trimethoprim-sulfamethoxazole; TGC, tigecycline; ATM,

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aztreonam; AMP, ampicillin; PIP, piperacillin; AMC, amoxicillin-clavulanic acid; SAM, ampicillin-sulbactam; CHL, chloramphenicol; TET, tetracycline.

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