Prevalence of Beta-Lactam Drug-Resistance Genes in Commensal

bioRxiv preprint doi: https://doi.org/10.1101/824516; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Prevalence of beta-lactam drug-resistance genes in commensal

2 Escherichia coli contaminating ready-to-eat lettuce

4 Ningbo Liao a,b, Julia Rubin a, Yuan Hu a, Hector A. Ramirez a, Clarissa Araújo 5 Borges a, Biao Zhoub, Yanjun Zhang b, Ronghua Zhang b, Jianmin Jiang b and 6 Lee W. Riley a† 7 8 9 a School of Public Health, Division of Infectious Diseases and Vaccinology, University of 10 California, Berkeley, California 94720, USA; 11 b Department of Nutrition and Food Safety, Zhejiang Provincial Center for Disease Control and 12 Prevention, Hangzhou 310006, China. 13 14 †Corresponding author 15 Phone: 510-642-9200 16 E-mail addresses: [email protected] 17 18 19 20 ABSTRACT 21 The objective of this study was to evaluate the prevalence of antibiotic resistance and 22 beta-lactam drug resistance genes in Escherichia coli isolated from ready-to-eat 23 lettuce, obtained from local supermarkets in Northern California. Bags of lettuce were 24 purchased from 4 chain supermarkets during three different periods—Oct 2018–Jan 25 2019, Feb 2019–Apr 2019 and May 2019–July 2019. From 91 packages of lettuce, we 26 recovered 34 E. coli isolates from 22 (24%) lettuce samples. All E. coli isolates were 27 genotyped by multilocus sequence typing (MLST), and we found 15 distinct sequence 28 types (STs). Five of these genotypes (ST2819, ST4600, ST2432, ST1198 and ST5143) 29 have been reported to cause infection in humans. Twenty (59%) E. coli isolates were 30 found resistant to at least one of the antibacterial drugs. They included resistance to 31 ampicillin (AMP, 85%) and ampicillin/sulbactam (SAM, 50%), cefoxitin (FOX, 40%) 32 and cefuroxime (CXM, 35%). We found 8 (40%) of 20 beta-lactam resistant E. coli

33 isolates to carry blaCTX-M; 5 (25%) tested positive for blaSHV, while only 4 (20%) 34 tested positive for blaTEM. Additionally, we identified a class A broad-spectrum 35 beta-lactamase SED-1 gene, blaSED, reported by others in Citrobacter sedlakii isolated 36 from bile of a patient. This study found that a large proportion of fresh lettuce carry 37 beta-lactam drug-resistant E. coli, which could serve as a reservoir for drug resistance 38 genes that could potentially enter pathogens to cause human infections. 39 40 41

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42 43 INTRODUCTION 44 Foodborne illnesses caused by various microorganisms including bacteria are major 45 concern to public health in both developed and developing countries (1). 46 Antimicrobial drugs have played an indispensable role in the treatment of foodborne 47 infectious diseases, but the frequent clinical use of antimicrobial agents has resulted in 48 the selection of antimicrobial drug-resistant (AMR) bacterial strains. However, factors 49 that contribute to the widespread dissemination of AMR bacterial strains in 50 community are not limited to just the human clinical use of antimicrobial agents (2). 51 Foods may contribute as an important source of antibiotic resistance genes found in 52 the human intestine. In particular, fresh produce carry saprophytic bacteria that harbor 53 drug-resistance genes, which can enter the human gut when it is eaten uncooked (3, 4). 54 Green leafy vegetables are frequently colonized with Gram-negative bacteria, 55 especially by members of Pseudomonadaceae and Enterobacteriaceae (5). Several 56 foodborne disease outbreaks caused by lettuce contaminated with recognized 57 pathogens (E. coli O157:H7, Salmonella) have been increasingly reported in the 58 United States in the last 10 years (6, 7, 8, 9, 10). 59 The primary reservoirs of these enteric pathogens are the food animal intestines, 60 and the green leafy vegetables are believed to become contaminated by food animal 61 manure. Thus, commensal E. coli in manure from such animals could also 62 contaminate green leafy vegetables. The objective of this study was, therefore, to 63 determine the prevalence of E. coli contamination of packaged, ready-to-eat lettuce 64 obtained from chain supermarkets in a Northern California community, and assess the 65 frequency of their antimicrobial drug resistance and beta-lactam drug resistance gene 66 carriage. 67 68 MATERIALS AND METHODS 69 In this study, ready-to-eat lettuce packages were purchased from chain 70 supermarkets located within a 7-mile vicinity of a university community in Northern 71 California during three different periods—Oct 2018–Jan 2019, Feb 2019–Apr 2019 72 and May 2019–July 2019. For each lettuce package, 25 g of lettuce were placed in a 73 UV-pretreated polyethylene bag containing 50 ml of sterile phosphate-buffered saline 74 (PBS; pH 7.4). The lettuce was incubated in PBS for 30 to 60 min at room 75 temperature after brief kneading. The PBS wash was then transferred to a 50-ml 76 conical tube and centrifuged at 12,000 g for 5 min at room temperature. The resulting 77 pellet was resuspended in 2 ml of PBS, and 1 ml of it was saved in a 10% glycerol 78 stock. The rest of the suspension (10μL) was cultured on a MacConkey agar plate to 79 isolate Gram-negative bacteria and presumptively identified by lactose fermentation 80 and indole test as E. coli. 81 Five colonies recovered on plate from each sample were randomly picked and 82 subtyped by the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay 83 according to standard procedures (11). Single colonies from tryptic soy agar plates 84 were selected and inoculated into 2 ml tryptic soy broth and incubated in a shaking 85 incubator for 15 h at 37°C. The 1-mL aliquots of grown cultures were centrifuged,

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86 and the pellets were collected to isolate DNA as described by Yamaji et al. (12). The 87 five colonies that had identical ERIC electrophoretic banding patterns by visual 88 inspection were considered to belong to the same clonal group, and one of these 89 colonies was selected for further analysis by multilocus sequence typing (MLST). If 90 samples contained more than one distinct electrophoretic banding pattern by 91 ERIC-PCR typing, all strains were included in the MLST analysis (12, 13). The allelic 92 number and the corresponding genotype number were designated by the curator of the 93 MLST based on the seven-gene scheme described at website 94 http://mlst.ucc.ie/mlst/dbs/Ecoli (13, 14). 95 All isolates from lettuce samples were assessed for susceptibility to ampicillin 96 (AMP), cefoxitin (FOX), cefotaxime (CTX), cefuroxime (CXM), nitrofurantoin (NIT), 97 ceftriaxone (CRO), ampicillin/sulbactam (SAM), ceftazidime (CAZ), fomycin 98 glucose (FOS), gentamicin (CN), trimethoprim-sulfamethozaxole (TMP-SMX), and 99 nalidixic acid (NA) by the standard disc diffusion assay (15). 100 The analysis of beta-lactamase genes was performed according to previously 101 published reports (16, 17). Primers (Table S1) used for PCR amplification of the

102 beta-lactams genes (blaTEM, blaSHV, blaCXT-M and blaOXA) were acquired from Sigma 103 Aldrich, USA. The PCR amplification was carried out with 5 μL of DNA template 104 (100-200 ng/μL) in 25 μL PCR mixture. Each reaction mixture contained 1 μL of each, 105 reverse and forward primer (10 μM), PCR grade water 1.5 μL, and Taq PCR master 106 mix (Bio-Rad, USA) 16.5 μL. Amplification reactions were accomplished with a 107 DNA thermo-cycler (Bio-Rad) as follows: initial denaturation for 30 seconds at 95°C, 108 followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing for 60

109 seconds (59°C, 60°C, 61°C and 65°C for blaTEM, blaSHV, blaCXT-M and blaOXA, 110 respectively). After final extension for 5 min at 72°C, amplified samples were 111 subjected to gel electrophoresis with QIAxcel advance system (QIAGEN, USA) (16).

112 DNA sequences of 400 bp or more for blaTEM, blaSHV and blaCXT-M were visually 113 inspected, aligned, and compared against sequences in GenBank with BLAST 114 (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Statistical analysis was performed by 115 Fisher’s exact test with significance set at p ≤0.05. 116 117 RESULTS AND DISCUSSION 118 Ninety-one lettuce packages were purchased from chain supermarkets located 119 within a 7-mile vicinity of a university community in Northern California. They 120 included organic (n=47) and non-organic (n=44) lettuce samples (Table S2). From 91 121 lettuce samples, we recovered 34 E. coli isolates from 22 (24%) samples. E. coli was 122 isolated from 18 (38%) organic and 9 (20%) non-organic lettuce packages (p=0.051, 123 Fisher’s exact test, 1-tailed). By MLST analysis, among 34 E. coli isolates, 24 were 124 assigned to 15 unique STs (Table 1). Two genotypes (ST8951 and ST5154) were 125 commonly detected in the lettuce samples. These genotypes have been reported in 126 deciduous forest soil and cattle, but not from human infections in the MLST database 127 (http://enterobase.warwick.ac.uk). We found strains ST2819, ST4600, ST2432, 128 ST5143 and ST1198, which have been reported in other regions of the world 129 including Japan (Fukuoka), USA (Nashville), China (Hangzhou and Shanghai), and

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130 Korea. They have been isolated from cases of human infection, as noted in the MLST 131 database. As shown in Table 1, E. coli strains ST4600 and ST1198 have been reported 132 as causes of diarrhea and urinary tract infection in USA and Korea, respectively. 133 Antibiotic susceptibility testing showed that most of the E. coli isolates were 134 resistant to at least one of the tested antibiotics (Table 2). Among 34 E. coli isolates 20 135 (59%) were resistant to one or more antibiotics; 17 (85%) were resistant to ampicillin 136 (AMP), 10 (50%) to ampicillin/sulbactam (SAM), 8 (40%) to cefoxitin (FOX), 7 137 (35%) to cefuroxime (CXM), and only 1 (5%) to nalidixic acid (NA). Nine (45%) of 138 these isolates were multidrug resistant (MDR), defined as resistance to three or more 139 classes of drugs (Table S3). Beta-lactam-resistant strains were examined for 140 beta-lactamase genes by PCR. Eight (40%) of 20 beta-lactam resistant E. coli strains

141 contained blaCTX-M, 5 (25%) tested positive for blaSHV, and 4 (20%) tested positive for 142 blaTEM. The high frequency of E. coli strains carrying blaCTX-M is surprising, since 143 most beta-lactam resistant clinical isolates of E. coli carry blaTEM and/or blaSHV (17, 144 18, 19). Of 9 MDR E. coli isolates that showed resistance to ampicillin,

145 ampicillin/sulbactam, cefoxitin and cefuroxime, 8 tested positive for blaTEM, blaCTX-M 146 or blaSHV genes. CTX-M beta-lactamases are the most common extended-spectrum 147 beta-lactamases (ESBL) expressed by clinical isolates of E. coli, which are 148 increasingly reported in extraintestinal infections from many regions of the world, 149 especially in healthcare settings (20, 21, 22). 150 To our surprise, in one strain (ST3222), we found a class A broad-spectrum

151 beta-lactamase SED-1 gene (Table 3), blaSED, reported by others to have been found 152 in Citrobacter sedlakii in the bile fluid of a patient (23, 24). This observation suggests 153 that some of these beta-lactamase genes are horizontally transferred among 154 saprophytic Gram-negative bacteria found in the environment, and that commensal E. 155 coli from mammalian intestine contaminating lettuce could acquire them. Such genes 156 can then enter the human intestine via ingestion of such contaminated food product 157 eaten uncooked. 158 The limitation of this study is that we analyzed the resistant E. coli strains for only 159 beta-lactamase genes. Since many of the strains were resistant to other classes of 160 drugs, they are likely to carry other drug-resistance genes. Hence, our study 161 underestimates the magnitude of these lettuce samples contributing to the spread of 162 these genes into the human intestine. 163 164 CONCLUSION 165 A large proportion of retail ready-to-eat lettuce samples contained E. coli, many of 166 which were resistant to commonly-used beta-lactam antibiotics. Although the 167 predominant STs (ST8951 and ST5154) were not pathogens, other genotypes ST2819, 168 ST4600, ST2432, ST1198 and ST5143, which were multidrug-resistant, have been 169 isolated from human infections in several regions of the world. A surprisingly large 170 proportion of the beta-lactam-resistant strains carried an ESBL beta-lactamase gene

171 blaCTX-M. While green-leafy vegetables could serve as sources of recognized enteric 172 pathogens, they may also serve as a common vector for drug-resistance genes that 173 ultimately enter human pathogens.

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174 175 ACKNOWLEDGMENT 176 This work was funded by the U.S. Centers for Disease Control and Prevention 177 program to combat antibiotic resistance under BAA number 200-2016-91939, and the 178 National Natural Science Foundation of China (#31301715), the Program of Health 179 and Medicine Science in Zhejiang Province, China (#2017KY292), and the National 180 Health Commission Foundation of the People’s Republic of China (WKJ-ZJ-1917). 181 182 183 REFERENCES 184 1. Fletcher SM, McLaws ML, Ellis JT. 2013. Prevalence of gastrointestinal pathogens in 185 developed and developing countries: systematic review and meta-analysis. J Public 186 Health Res 2:42-53. https://doi.org/ 10.4081/jphr.2013.e9. 187 2. Butcher CR, Rubin J, Mussio K, Riley LW. 2019. Risk Factors Associated with 188 Community-Acquired Urinary Tract Infections Caused by Extended-Spectrum 189 β-Lactamase-Producing Escherichia coli: a Systematic Review. Curr Epidemiol Rep 190 6:300-309. https://doi.org/10.1007/s40471-019-00206-4. 191 3. Rolain JM. 2013. Food and human gut as reservoirs of transferable antibiotic resistance 192 encoding genes. Front Microbiol 4:173. https://doi.org/10.3389/fmicb.2013.00173. 193 4. Berman HF, Riley LW. 2013. Identification of novel antimicrobial resistance genes from 194 microbiota on retail spinach. BMC Microbiol 13:272. https://doi.org/10.1186/1471-2180 195 -13-272 196 5. Raphael E, Wong LK, Riley LW. 2011. Extended-spectrum beta-lactamase gene sequences 197 in gram-negative saprophytes on retail organic and nonorganic spinach. Appl Environ 198 Microbiol 77: 1601-1607. https://doi.org/10.1128/AEM.02506-10. 199 6. Heiman KE, Mody RK, Johnson SD, Griffin PM, Gould LH. 2015. Escherichia coli O157 200 outbreaks in the United States, 2003–2012. Emerg Infect Dis 21: 1293–1301. https://doi. 201 org/10.3201/eid2108.141364. 202 7. Harvey RR, Zakhour CM, Gould LH. 2016. Foodborne disease outbreaks associated with 203 organic foods in the United States. J Food Prot 79:1953-1958. https://doi.org/10.4315/ 204 0362-028X.JFP-16-204. 205 8. Gelting RJ, Baloch MA, Zarate-Bermudez M, Hajmeer MN, Yee JC, Brown T, Yee BJ. 206 2015. A systems analysis of irrigation water quality in an environmental assessment of an 207 E. coli O157: H7 outbreak in the United States linked to iceberg lettuce. Agric Water 208 Manag 150:111-118. https://doi.org/10.1016/j.agwat.2014.12.002. 209 9. Wadamori Y, Gooneratne R, Hussain M A. 2017. Outbreaks and factors influencing 210 microbiological contamination of fresh produce. J Sci Food Agric 97:1396-1403. 211 https://doi.org/10.1002/jsfa.8125. 212 10. Marder EP, Garman KN, Ingram LA, Dunn JR. 2014. Multistate outbreak of Escherichia 213 coli O157: H7 associated with bagged salad. Foodborne Pathog Dis 11:593-595. 214 https://doi.org/10.1089/fpd.2013.1726. 215 11. Wilson LA, Sharp PM. 2006. Enterobacterial repetitive intergenic consensus (ERIC) 216 sequences in Escherichia coli: evolution and implications for ERIC-PCR. Mol Biol Evol 217 23:1156 –1168. https://doi.org/10.1093/molbev/msj125.

5 bioRxiv preprint doi: https://doi.org/10.1101/824516; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

218 12. Yamaji R, Friedman CR, Rubin J, Suh J, Thys E, McDermott P, Hung-Fan M, Riley LW. 219 2018. A population-based surveillance study of shared genotypes of Escherichia coli 220 isolates from retail meat and suspected cases of urinary tract infections. mSphere 3: 221 e00179-18. https://doi.org/10.1128/mSphere.00179-18. 222 13. Riley LW. 2018. Laboratory Methods in Molecular Epidemiology: Bacterial Infections. 223 Microbiol Spectr 6: 1-23. https://doi.org/10.1128/microbiolspec.AME-0004-2018. 224 14. Tartof SY, Solberg OD, Manges AR, Riley LW. 2005. Analysis of a uropathogenic 225 Escherichia coli clonal group by multilocus sequence typing. J Clin Microbiol 43:5860 – 226 5864. https://doi.org/10.1128/JCM.43.12.5860-5864.2005. 227 15. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, 228 Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, 229 Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. 2012. Multidrug-resistant, 230 extensively drug-resistant and pandrugresistant bacteria: an international expert proposal 231 for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268 – 232 281. https://doi.org/10.1111/j.1469-0691.2011.03570.x. 233 16. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. 2010. Development of a set of 234 multiplex PCR assays for the detection of genes encoding important beta-lactamases in 235 Enterobacteriaceae. J Antimicrob Chemother 65:490–495. https://doi.org/10.1093/ 236 jac/dkp498. 237 17. Rubin J, Mussio K, Xu Y, Suh J, Riley LW. 2019. Prevalence of antimicrobial resistance 238 genes and integrons in commensal Gram-negative bacteria in a college community. 239 bioRxivorg 683524. https://doi.org/10.1101/683524. 240 18. Kim HS, Chon JW, Kim YJ, Kim DH, Kim MS, Seo KH. 2015. Prevalence and 241 characterization of extended-spectrum-β-lactamase-producing Escherichia coli and 242 Klebsiella pneumoniae in ready-to-eat vegetables. Int J Food Microbiol 207:83-86. 243 https://doi.org/10.1016/j.ijfoodmicro.2015.04.049. 244 19. Yamaji R, Rubin J, Thys E, Friedman CR, Riley LW. 2018. Persistent pandemic lineages of 245 uropathogenic Escherichia coli in a college community from 1999 to 2017. J Clin 246 Microbiol 56:01834-17. https://doi.org/10.1128/JCM.01834-17. 247 20. Lewis JS, Herrera M, Wickes B, Patterson JE, Jorgensen JH. 2007. First report of the 248 emergence of CTX-M-type extended-spectrum β-lactamases (ESBLs) as the predominant 249 ESBL isolated in a US health care system. Antimicrob Agents Chemother 51: 4015-4021. 250 https://doi.org/10.1128/AAC.00576-07. 251 21. Hayakawa K, Gattu S, Marchaim D, Bhargava A, Palla M, Alshabani K, Gudur UM, 252 Pulluru H, Bathina P, Sundaragiri PR, Sarkar M. 2013. Epidemiology and risk factors for 253 isolation of Escherichia coli producing CTX-M-type extended-spectrum β-lactamase in a 254 large US medical center. Antimicrob Agents Chemother 57:4010-4018. https://doi.org/ 255 10.1128/AAC.02516-12. 256 22. Tadesse DA, Li C, Mukherjee S, Hsu CH, Bodeis Jones S, Gaines SA, Kabera C, 257 Loneragan GH, Torrence M, Harhay DM, McDermott PF. 2018. Whole-Genome 258 sequence analysis of CTX-M containing Escherichia coli isolates from retail meats and 259 cattle in the United States. Microbial Drug Resistance 24: 939-948. https://doi.org/ 260 10.1089/mdr.2018.0206. 261 23. Petrella S, Clermont D, Casin I, Jarlier V, Sougakoff W. 2001. Novel Class A β-Lactamase

6 bioRxiv preprint doi: https://doi.org/10.1101/824516; this version posted October 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

262 Sed-1 from Citrobacter sedlakii: Genetic Diversity of β-Lactamases within the 263 Citrobacter Genus. Antimicrob Agents Chemother 45:2287-2298. https://doi.org/10.1128/ 264 AAC.45.8.2287-2298.2001. 265 24. Petrella S, Pernot L, Sougakoff W. 2004. Crystallization and preliminary X-ray diffraction 266 study of the class A β-lactamase SED-1 and its mutant SED-G238C from Citrobacter 267 sedlakii. Acta Cryst 60:125-128. https://doi.org/10.1107/S0907444903022765.

268 269 Table 1 E. coli isolates belonging to the 15 MLST genotypes found in lettuce samples 270 271 In MLST datas (http://enterobase.warwick.ac.uk/species/ecoli/search_strains?query=st_search) ST(total no. of ST Complex Location isolates typed ) Source Collection year Path/Nonpath (Country or City) ST8951(4) None Deciduous Forest; Deciduous Forest Soil; Soil 2015 None None ST2819(2) None human 2015 Japan(Fukuoka) Pathogen (Diarrhoea) ST5154(3) None Cattle 2017 Africa(Angola) None ST6374(2) None None None None None ST9285(1) None Poultry 2016 Africa(Gambia) None ST5143(2) None Faeces 2013 China(Shanghai) Pathogen ST2432(2) None human 2011 China(Hangzhou) Pathogen ST5351(1) ST568Cplx None None None None ST3567(1) None human 2012 Netherlands None ST2625(1) ST14Cplx human 2015 USA(Seattle) Pathogen ST4277(1) None Oryzomys sp 2015 Mexico None ST4600(1) None Human 2011 USA(Nashville) Pathogen (Diarrhoea) ST3222(1) ST648Cplx human 2008 France(Besancon) None ST1198(1) None human 2007 Korea Pathogen (UTI) ST6376(1) None None None None None unknown(10) None None None None 272 273 274 275 276

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277 278 279 Table 2 Beta-Lactamase gene types identified among antibiotic-resistant E. coli 280 isolates from lettuces. 281 Genotype No. of Drug resistance (no. of Beta-Lactamase gene type (total no. of susceptible isolates) a (no. of isolates) isolates typed) isolates

ST8951(4) 3 AMP+SAM(1) blaTEM type (1) c ST2819(2) 1 AMP+SAM+CXM(1) blaCTX-M type (1)

ST5145(3) 1 AMP(1) blaSHV type (1) AMP+SAM+FOX+CXM(1)c other (1)

ST6374(2) 1 AMP+CXM(1) blaCTX-M type (1)

ST9285(1) 0 AMP+SAM(1) blaTEM type (1)

ST5143(2) 0 AMP+FOX(1) blaSHV type+blaCTX-M type (1) AMP+FOX+SAM(1)c bla type (1) CTX-M ST2432(2) 0 AMP+FOX(1) blaCTX-M type (1) AMP+FOX+CXM+SAM(1)c bla type (1) CTX-M ST5351(1) 1 0 None ST3567(1) 1 0 None ST2625(1) 1 0 None ST4277(1) 0 AMP+SAM(1) other (1) c ST4600(1) 0 AMP+FOX+CXM+SAM(1) blaCTX-M type (1) c ST3222(1) 0 AMP+CXM+SAM(1) other type (1)

ST1198(1) 0 AMP+SAM(1) blaTEM type+ blaSHVtype (1)

ST6376(1) 0 AMP+SAM(1) blaSHV type (1)

unknown(10) 5 AMP+SAM(2) blaSHV type+blaTEM type (2) AMP+SAM+NA(1)c other (1)

AMP+FOX+SAM(1)c bla type+bla type (1) TEM CTX-M AMP+FOX+SAM+CXM(1)c other (1)

Total 14 20 282 a 283 AMP,ampicillin;FOX,cefoxitin;CTX,cefotaxime;CXM,cefuroxime;NIT,nitrofurantoin;CRO,ceftriaxon 284 e;SAM,ampicillin/sulbactam;CAZ,ceftazidime;FOS,fomycin glucose; CN, gentamicin; TMP, 285 Trimethoprim; NA,nalidixicacid b Other: Ampicillin-resistant isolates that did not have any bla -type, bla type, bla -type 286 TEM CTX-M OXA and bla -type 287 SHV c 288 Multidrug-resistant 289

290 291 Table 3 Genetic analysis of the beta-Lactamase -producing E.coli in lettuces 292 Size (bp) of Position (bp) of Sequence Lettuce Matched sequences in NCBI database (organism of origin,accession Strain sequence DNA sequence on matched type type no,sequence length [bp], % of Matches) fragment sequence

ST2819 E04270401 Organic 446 33-478 blaCTX-M gene, Citrobacter sp., KJ918511.1, 525, 99

ST4600 E06060204 Organic 425 53-478 blaCTX-M gene, Citrobacter sp., KJ918511.1, 525, 99

ST2432 E06200301 Organic 433 45-478 blaCTX-M gene, Citrobacter sp., KJ918511.1, 525, 99

ST3222 E06200202 Organic 455 270-725 blaSED gene, Citrobacter sedlakii, NG_049979.1,888, 98.68

ST1198 E50802 Organic 663 122-784 blaSHV-11 gene, Escherichia coli, MF402896.1, 861, 99.55

ST5143 E06270403 Non-organic 653 131-784 blaSHV-11 gene, Escherichia coli, MF402896.1, 861, 99.55

unknown E05180501 Non-organic 388 261-648 blaTEM gene, Acinetobacter baumannii, MK764360.1, 732, 100

unknown E06270303 Organic 433 212-768 blaSHV-11 gene, Citrobacter freundii, MF402898.1, 861, 89.59 293 294 295 296 297 298 299 300 301 302 303 304