Journal Pre-proof

Prevalence of colistin-resistant Escherichia coli harbouring mcr-1 in raw beef and

ready-to-eat beef products in Egypt

Rana Fahmi Sabala, Masaru Usui, Yutaka Tamura, Samir Mohamed Abd-Elghany,
Khalid Ibrahim Sallam, Mohammed Mohammed Elgazzar

PII: S0956-7135(20)30352-2
DOI: https://doi.org/10.1016/j.foodcont.2020.107436
Reference: JFCO 107436

To appear in: Food Control

Received Date: 21 April 2020

Revised Date: 11 June 2020
Accepted Date: 16 June 2020

Please cite this article as: Sabala R.F., Usui M., Tamura Y., Abd-Elghany S.M., Sallam K.I. & Elgazzar
M.M., Prevalence of colistin-resistant Escherichia coli harbouring mcr-1 in raw beef and ready-to-eat
beef products in Egypt, Food Control (2020), doi: https://doi.org/10.1016/j.foodcont.2020.107436.

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 Author Statement

Manuscript title: Prevalence of colistin-resistant Escherichia coli harbouring mcr-1 in

raw beef and ready-to-eat beef products in Egypt

All persons who meet authorship criteria are listed as authors, and all authors certify
that they have participated sufficiently in the work to take public responsibility for the
content, including participation in the concept, design, analysis, writing, or revision of
the manuscript. Furthermore, each author certifies that this material or similar material
has not been and will not be submitted to or published in any other publication before
its appearance in the Food Control.

Authorship contributions

Rana Fahmi Sabala: Conceptualization, Data curation, Investigation, Resources, Writing

Masaru Usui: Conceptualization, Data curation, Investigation, Project administration,
Resources, Writing
Yutaka Tamura: Supervision
Samir Mohamed Abd-Elghany: Supervision
Khalid Ibrahim Sallam: Supervision
Mohammed Mohammed Elgazzar: Supervision
1 Prevalence of colistin-resistant Escherichia coli harbouring mcr-1 in raw beef

2 and ready-to-eat beef products in Egypt

4 Rana Fahmi Sabalaa,b, Masaru Usuib,*, Yutaka Tamurab, Samir Mohamed Abd-Elghanya, Khalid

5 Ibrahim Sallama, Mohammed Mohammed Elgazzara

a
6 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura

7 University, Mansoura, Egypt

b
8 Laboratory of Food Microbiology and Food Safety, School of Veterinary Medicine, Rakuno

9 Gakuen University, Ebetsu, Japan

10
*
11 Corresponding author:

12 Masaru Usui, D.V.M., Ph.D.

13 Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental

14 Sciences, School of Veterinary Medicine, Rakuno Gakuen University, 582 Midorimachi,

15 Bunkyodai, Ebetsu, Hokkaido 069-8501, Japan

16 Tel.: +81 11 388 4723; Fax: +81 11 388 4723

17 E-mail address: usuima@rakuno.ac.jp

18

1
19

20 ABSTRACT

21 The emergence of antibiotic-resistant bacteria in food, mainly contaminated meat and its

22 products, and their transfer to humans is of great concern. To evaluate the potential risk of such

23 contamination in Egypt, we isolated Escherichia coli from 78% (51/65) of raw beef and 53%

24 (24/45) of ready-to-eat beef products. Of the 210 E. coli isolates detected, 8 (3.8%) harboured

25 mcr-1 gene and were resistant to colistin, whereas 5 (2.4%) were positive for the blaCTX-M-28 gene

26 and were resistant to cefotaxime. Among the colistin-resistant isolates, three had both mcr-1 and

27 extended-spectrum beta-lactamase (ESBL) genes, constituting a great public health concern. The

28 sequence types of all these mcr and/or ESBL-positive isolates were variable, suggesting that

29 colistin and/or cephalosporin resistance spread through the mediation of plasmids harbouring

30 mcr-1 or ESBL genes in Egyptian beef. In addition, various extraintestinal virulence genes were

31 observed in some isolates. Colistin and cephalosporins are frequently used for livestock in Egypt;

32 hence the present results suggest colistin and cephalosporin-resistant pathogenic E. coli are

33 transferred from food animals to humans via meat and meat-derived products. Therefore, the

34 rational use of antimicrobials and the appropriate safety measures in food production are needed

35 in Egypt as well as in other developing countries.

36

37 Keywords: Colistin, Food safety, mcr-1, Ready-to-eat beef products, Retail meat

38

39

2
40 Introduction

41 The emergence and spread of antimicrobial-resistant bacteria have reached disconcerting

42 proportions of global public health concern (WHO, 2015). Antimicrobials are largely used in the

43 animal sector, not only for the treatment of several infectious bacterial diseases, but also for

44 prophylactic purposes and growth promotion (Van Boeckel et al., 2015). The over- as well as

45 misuse of antimicrobials in the production of animals contributes to pervasion of multidrug-

46 resistant bacteria in food-producing animals (Economou & Gousia, 2015). Transmission of

47 antimicrobial-resistant bacteria from livestock to humans via food, especially meat and its

48 products, is one of the major public health concerns.

49 Food-borne diseases linked to pathogenic bacteria are a growing public health problem in

50 both developed and developing countries (El Sheikha, 2015; El Sheikha & Xu, 2017). Several

51 studies showed that antimicrobial-resistant Escherichia coli infections in humans were caused by

52 animal-derived strains (Johnson et al., 2009). It is not unreasonable to assume that the intestinal

53 carriage of antimicrobial-resistant E. coli in livestock for future consumption as food products

54 might increase the occurrence of infections with antimicrobial-resistant E. coli in humans

55 (Tadesse et al., 2018). Therefore, the establishment of food-traceability systems, especially for

56 meat and meat-derived products, is urgently required to improve the quality of food-processing

57 events and provide safe food for the final consumers (El Sheikha, 2017; El Sheikha, 2018).

58 Colistin, also known as polymyxin E, has historically been used for livestock across the

59 world and this has resulted in the emergence of colistin-resistant bacteria in livestock (Kempf,

60 Jouy, & Chauvin, 2016). Nonetheless, colistin is still used as the last option for treatment against

61 multidrug-resistant gram-negative bacteria in clinical situations (Lim et al., 2010). The usage of

3
62 this antibiotic for livestock was recently banned in some developed countries to avoid the

63 emergence of colistin-resistant bacteria (European Medicines Agency, 2016). The mechanisms

64 of resistance to colistin in gram-negative bacteria involve chromosomal mutations or acquiring

65 of mcr genes for resistance by the plasmid (Poirel, 2017). Various mcr genes (mcr-1 to mcr-9)

66 have been reported since the first identification of mcr-1 in China (Gharaibeh, & Shatnawi,

67 2019).

68 mcr genes are disseminated via horizontal transmission in different bacterial species (Sun,

69 Zhang, Liu, & Feng, 2018), and these genes (particularly mcr-1) are abundant in several sources

70 (food, food producing animals, and humans) worldwide (Poirel, 2017; Zurflu et al., 2017).

71 Recently, some reports showed that extended-spectrum beta-lactamase (ESBL)-producing

72 bacteria, which are resistant to cephalosporin and are sometimes treated with colistin, possess the

73 mcr genes. In these cases (both ESBL and mcr positive), therapeutic options are limited (Bitrus,

74 Chuanchuen, & Luangtongkum, 2018; Dandachi, Chabou, Daoud, & Rolain, 2018; Ghafur et al.,

75 2019). Therefore, the prevalence of mcr genes, especially in ESBL-producing gram-negative

76 bacteria, is a serious problem for public health.

77 In developing countries, including Egypt, colistin is still used for food-producing animals,

78 and cephalosporins are also frequently used (Dandachi, Chaddad, Hanna, Matta, & Daoud, 2019).

79 Bacteria harbouring mcr and ESBL genes were detected in food-producing animals in several

80 developing countries (Bitrus et al., 2018; Dandachi et al., 2018). In these countries, unlike in the

81 developed ones, bacteria derived from food-producing animals are more frequently transmitted

82 to humans via food owing to the poor facilities of hygiene and sanitation in food processing

83 sectors (Gwida, Hotzel, Geue, & Tomaso, 2014; Martin, & Beutin, 2011). In Egypt, ESBL-

84 producing E. coli has frequently been isolated from food-producing animals and meat (Braun et

4
 85 al., 2016; Moawad et al., 2017), whereas mcr-1 has been detected only in one subclinical mastitic

86 cow and one ready-to-eat meat product in previous studies (Khalifa et al., 2016; Sadek et al.,

87 2019), although mcr genes (mcr-1 and mcr-2) were detected from several sources, including the

88 environment and humans (Ahmed, Elshafiee, Khalefa, Kadry, & Hamza, 2019; Elnahriry et al.,

89 2016; Zafer et al., 2019). The existence of bacterial strains harbouring both mcr and ESBL genes

90 in food is a critical issue, especially when humans eat such food, because these bacteria show

91 resistance to both colistin and cephalosporin and result in serious bacterial infections with no

92 treatment options. Therefore, more studies are required to determine the prevalence of colistin

93 resistance, conferred by mcr and ESBL genes, in Egyptian foods.

94 The present study aimed to clarify the prevalence and characteristics of E. coli, especially

95 strains harbouring both colistin-resistant mcr-1 and ESBL genes in retail raw beef and ready-to-

96 eat beef products (RTE-BPs) from Egypt, and to highlight the risk of such strains on the public

97 health.

98

99 2. Material and methods

100 2.1. Collection of samples

101 A total of 65 retail raw beef samples and 45 RTE-BPs (15 each of burgers, kofta (balls of

102 minced meat mixed with spices and onion), and sausage sandwiches) were purchased from

103 October 2018 to January 2019 from eight categorised butcher shops and six markets in Mansoura

104 city, Egypt. All the samples were transported in sterile containers to the laboratory of Food

105 Control Department, Faculty of Veterinary Medicine, Mansoura University, Egypt, and were

106 tested within 24 h of collection.

5
107

108 2.2. Enrichment culture and isolation of E. coli

109 Twenty-five grams of food sample was blended in 225 mL Tryptone Soy Broth (Oxoid,

110 Thermo Fisher Scientific Inc., US), and then incubated at 37 °C for 18 h. A loopful of the

111 enriched culture was then streaked on MacConkey agar (Oxoid, Thermo Fisher Scientific Inc.)

112 plates, which were incubated at 37 °C for 24 h.

113

114 2.3. Biochemical identification of the isolated E. coli strains

115 Typical colonies chosen from the inoculated plates were subjected to biochemical

116 identification (Collee, Miles, & Watt, 1996). In addition, bacterial identification was conducted

117 by matrix-assisted laser desorption/ionisation-time of light mass spectrometry using the Bruker

118 MALDI Biotyper system (Bruker Daltonics, Bremen, Germany) according to the manufacturer’s

119 instructions, and as outlined by Croxatto, Prod’hom, & Greub (2012). For each sample, a

120 maximum of three colonies were identified as those of E. coli according to MALDI Biotyper and

121 were analysed further. When two or three isolates from any sample exhibited the same

122 antimicrobial susceptibility profile, they were considered as a single isolate.

123

124 2.4. Antimicrobial susceptibility

125 The susceptibility of the isolates to several antimicrobials (ampicillin, cefazolin,

126 cefotaxime, streptomycin, gentamicin, kanamycin, tetracycline, nalidixic acid, and ciprofloxacin;

127 all obtained from Sigma-Aldrich, MO, US) was tested by determining the minimum inhibitory

6
128 concentration (MIC) using the micro-broth dilution method and consensus 96-well microtiter

129 plates (Eiken Chemical, Japan) for all the E. coli isolates, according to Clinical and Laboratory

130 Standards Institute (CLSI) guidelines (CLSI, 2017). MIC against imipenem, meropenem, and

131 colistin (Sigma-Aldrich) was also determined using the micro-broth dilution method according to

132 CLSI guidelines (CLSI, 2017). Antimicrobials tested, dilution ranges, and interpretation of MIC

133 values were in accordance with the breakpoints (CLSI, 2017), except for streptomycin, for which

134 the National Antimicrobials Resistance Monitoring System guideline (NARMS, 2011) was

135 followed.

136

137 2.5. PCR screening for mcr genes

138 E. coli isolates having an MIC ≥ 4 mg/L against colistin were defined as colistin resistant

139 (CLSI, 2017). These colistin-resistant isolates were screened for the presence of mcr genes (mcr

140 1 to 8) using multiplex PCR (Rebelo et al., 2018). DNA was extracted using the Cica Geneus

141 DNA extraction kit (Kanto Chemical, Japan). All the PCR products were purified using Fast

142 Gene Gel/PCR extraction kit (Nippon Genetics, Japan) and sequenced in both directions using

143 the same primers designed for the mcr genes amplification. Nucleotide sequences were

144 determined using the BigDye Terminator, v. 3.1, Cycle Sequencing Kit (Thermo Fisher

145 Scientific Inc.) with an automated DNA sequencer (3730xl DNA Analyzer, Thermo Fisher

146 Scientific Inc.)

147

148 2.6. Detection of ESBL strains

7
149 To determine the ESBL-producing strains, Double Disk Synergy test (DDST) was done

150 for strains resistant to cefazolin (≥ 8 mg/L) and/or cefotaxime (≥ 4 mg/L) (CLSI, 2017), and then

151 these strains were screened for the presence of ESBL genes (TEM, CTX-M, SHV, and OXA)

152 using PCR amplification (Kojima et al., 2005; Xu, Ensor, Gossain, Nye, & Hawkey, 2005). All

153 the PCR products were sequenced using the above-mentioned method.

154

155 2.7. Multilocus sequence typing

156 Multilocus sequence typing (MLST) for E. coli was performed as detailed on the MLST

157 website (https://pubmlst.org/escherichia/) for colistin- and/or cephalosporin-resistant isolates.

158

159 2.8. Plasmid extraction

160 Plasmid extraction from the mcr-1 positive colistin-resistant strains was conducted using

161 Plasmid Safe ATP-dependent DNase kit (Lucigen, U.S) according to the manufacture’s protocol.

162 The extracted plasmids were screened for mcr-1 gene by PCR to determine if this gene was

163 carried on the plasmid or on the chromosome.

164

165 2.9. Conjugation experiment

166 To determine whether colistin resistance gene is carried on a transferable plasmid, we

167 performed a conjugation experiment, the filter-mating assay, with slight modifications (Malwade,

168 Nguyen, Sadat-Mousavi, & Ingalls, 2017) using rifampicin-resistant E. coli MG 1655 as the

8
169 recipient strain. Briefly, the donor and recipient strains were grown in LB broth to an OD600 =

170 0.1 and then 2.5 mL of both the donor and recipient strains were mixed. The mixture was filtered

171 through 0.25-µm pore size membrane filters (Merck Millipore, Ireland), and these filters were

172 subsequently incubated overnight on LB agar. The filters were then suspended in 1.5 mL of

173 sterile saline and rigorously shaken to wash-off the bacteria adhering to their surface; 10 µL of

174 the suspension was plated on LB agar supplemented with 50 µg/mL rifampicin and 2 µg/mL

175 colistin and incubated overnight. Transconjugants (TCs) were selected from the incubated plates

176 and screened for mcr-1 by PCR; the susceptibility to several antibiotics tested for the donor was

177 examined for the TCs by MIC determination using the micro-broth dilution method.

178

179 2.10. Detection of virulence genes

180 All E. coli isolates were examined for 24 representative virulence genes present in

181 diarrhoeagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Among the DEC

182 and ExPEC virulence factors, screening for the following genes was done using multiplex PCR:

183 stx1, stx2, and eaeA for enterohaemorrhagic E. coli; astA for enteroaggregative E. coli; cdt, cnf,

184 hlyA, afa, aer, sfa/foc, pap, RPAi, ETTT, IroN, IHA, usp, kps, traT, FyuA, OmpT, ibeA, fimA1,

185 and fimH for ExPEC (Beutin et al., 2008; Khac et al., 2006; Moulin-Schouleur et al., 2006;

186 Takahashi et al., 2006; Tóth, Hérault, Beutin, & Oswald, 2003).

187

188 2.11. Statistical analysis

9
189 The variations in the resistance rates of E. coli strains isolated from raw beef and RTE-

190 BP samples against the tested antibiotics were assessed using the chi-square (χ2) test and the

191 SPSS software v. 23.0 (IBM, Armonk, NY, USA). Statistical significance was considered at P

192 values < 0.05 or < 0.01. In addition, the variations in positive strains for different types of

193 virulence genes were determined using the same methods.

194

195 3. Results

196 3.1. Prevalence of E. coli isolates in the examined samples

197 E. coli were isolated from 78% (51/65) of raw beef and 53% (24/45) of RTE-BP (4/15 of

198 burger, 10/15 of kofta, and 10/15 of sausage sandwich) samples (Fig. 1). In total, 210 strains

199 (150 from raw beef and 60 from ready-to-eat beef products) were isolated.

200

201 3.2. Antimicrobial susceptibility

202 Among the tested antimicrobials, the resistance rates of isolates from raw beef samples to

203 tetracycline, streptomycin, and ampicillin were higher (> 25%), whereas the isolates from RTE-

204 BPs showed relatively higher resistance rates against tetracycline and ampicillin (≥ 10%) (Fig. 2).

205 All the isolates were susceptible to ciprofloxacin and carbapenem antimicrobials (imipenem and

206 meropenem). The resistance rates of isolates derived from raw beef samples against the four

207 tested antimicrobials (ampicillin, streptomycin, tetracycline, and nalidixic acid) were

208 significantly higher than of those derived from RTE-BPs (P < 0.05 for ampicillin, and P < 0.01

209 for streptomycin, tetracycline and nalidixic acid) (Fig. 2).

210

10
211 3.3. Characterisation of colistin-resistant isolates

212 Eight colistin-resistant strains (six strains from five raw beef samples and two strains from

213 two ready-to-eat sausage sandwiches) were isolated. All the eight colistin-resistant E. coli strains

214 had mcr-1. The sequence types (ST) of the eight colistin-resistant isolates were variable (Table

215 1). Of the eight colistin-resistant E. coli strains, three were phenotypically ESBL-producing; they

216 gave positive results in DDST, and co-harboured mcr-1 along with one of the ESBL genes (two

217 strains had blaCTX-M-28 and one had blaTEM-116) (Table 1).

218

219 3.4. Determination of ESBL genes

220 Six of the 210 (3%) cefazolin- and/or cefotaxime-resistant isolates gave positive result in

221 DDST. Among them, five strains were resistant to both the antibiotics and harboured blaCTX-M-28

222 (Table 1).

223

224 3.5. Plasmid extraction and conjugation experiment

225 The presence of mcr-1 on the extracted plasmids was confirmed in the eight colistin-

226 resistant isolates. TCs were observed from all the eight colistin-resistant isolates. The mcr-1 was

227 detected in all TCs. All the TCs showed the same resistance profiles for colistin and other tested

228 antibiotics, except for nalidixic acid and kanamycin, as of the donor; all the TCs showed

229 susceptibility to these antibiotics (Table S1).

230

231 3.6. Detection of virulence genes

11
232 The genes encoding type 1 fimbriae, fimH and fimA, were detected in 96% and 91% of the

233 E. coli isolates, respectively, whereas the type Ш secretion system gene (ETTT) was detected in

234 83% of the isolates (Fig. 3). In addition, the iron uptake system genes, fuyA, iroN, and aer, were

235 detected in 15%, 8%, and 6% of the isolates, respectively. Virulent factors, ompT, traT, and usp,

236 were determined in 16%, 15%, and 2%, of the isolates, respectively. Each of the genes encoding

237 cytotoxins, such as α-hlyA, cnf, cdt, were present in 3% of the isolates (Fig. 3). The astA gene

238 encoding enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) was found in 27% of the

239 isolates. All the genes were detected in raw beef and RTE-BP samples, except usp which was

240 detected only in RTE-BPs. In contrast, both cdt and cnf were detected only in raw beef samples

241 (Fig.3). The positive rates of isolates derived from raw beef samples of five virulence genes

242 (fimA, ETTT, fuyA, traT, and usp) were significantly different compared with those derived from

243 RTE-BPs (P < 0.05 for fimA, ETTT, traT, and P < 0.01 for fuyA, usp) (Fig. 3).

12
244

245 4. Discussion

246 This study shows that over 50% of the raw beef and RTE-BP samples collected from

247 Egypt were contaminated with E. coli. Several studies have also shown that the contamination

248 ratio in Egyptian raw beef is high (Gwida et al., 2014; Hussein, 2007). In developing countries,

249 including Egypt, meat is exposed to high levels of contamination with different types of bacteria

250 because of the use of conventional methods of slaughtering and evisceration, which is done

251 manually (Gwida et al., 2014; Shilenge, Shale, Matodzi, Machete, & Tshelane, 2017). The

252 contamination ratio of E. coli in RTE-BPs in Egypt (52% and 27%) has been reported in several

253 studies (Hussein, Eldaly, Seadawy, & El-Nagar, 2018; Salem, Gamel, Khalifa, AbdElHady &

254 Zeid, 2016). Incidence of bacterial contamination in ready-to-eat food is mostly due to the

255 unhygienic environment and improper handling (Hussein et al., 2018). The high contamination

256 ratio of these raw beef and RTE-BPs, not only by E. coli but also other pathogenic bacteria,

257 could be one of the causes of the high frequency of food-borne illness in these developing

258 countries (WHO, 2004; Gwida et al., 2014). Therefore, hygienic measures should be undertaken

259 to decrease the level of contamination of meat. Further, all food sectors should follow quality

260 control systems and must undergo periodic assessment.

261 Most of the E. coli strains isolated in this study have more than two virulence genes

262 identified in clinical extraintestinal pathogens. Based on previous molecular epidemiological

263 analyses, isolates are defined as ExPEC, if they are positive for two or more virulence genes

264 (Johnson, Kuskowski, Owens, Gajewski, & Winokur, 2003). In this study, 91% of ExPEC were

265 isolated from raw beef and RTE-BPs. There is an emerging body of evidence showing that

13
266 contaminated food of animal origin could be responsible for the spread of extraintestinal

267 pathogenic E. coli in the society (Ramchandani et al., 2005). Existence of such virulence genes

268 in food, especially in ready-to-eat products, could be a potential hazard for public health. In this

269 study, the prevalence of astA, encoding EAST1, in food was 27% in strains harbouring other

270 virulent genes. Multi-virulent EAEC was reported as a significant causative agent for paediatric

271 diarrhoea in Egypt (Ali et al., 2014). In addition, outbreaks in humans caused by E. coli

272 harbouring only EAST1 have been reported previously in the world (Ishiguro, Kyota, Mochizuki,

273 & Horikawa, 2005; Zhou et al., 2002). Therefore, the existence of EAST1 in food is a cause of

274 great concern in the spread of food-borne diarrhoeic infection caused by enteroaggregative E.

275 coli strains.

276 The rate of resistance of E. coli derived from raw meat and cooked meat products to

277 tetracycline, streptomycin, and ampicillin was determined to be high (Fig. 2), as in previous

278 studies (Moawad et al., 2017; Younis, Nasef, & Salem, 2019). Tetracyclines and beta-lactams,

279 including ampicillin, are still frequently used for livestock in Egypt (WHO, 2013). In addition,

280 streptomycin is frequently used for the treatment of mastitis (Younis, Elkenany, & Saleh, 2017).

281 A similar pattern of antimicrobial resistance of E. coli isolates from food of animal origin was

282 also observed in various developing countries, which resulted in human colonisation and

283 outbreak situations (Founou, Founou, & Essack, 2016). Although the actual situation of usage of

284 antimicrobials for livestock is not known in these countries (Founou et al., 2016), the frequent

285 use of antimicrobials in livestock would select the antibiotic-resistant bacteria, which enter the

286 food chain. Therefore, establishment of programs for monitoring the antibiotic usage and

287 antibiotic susceptibility of bacteria from livestock is needed.

14
288 The resistance rates of isolates derived from raw beef samples against the four tested

289 antimicrobials were significantly higher than of those derived from RTE-BPs. This may be

290 related to various factors, such as differences in the source of the meat or the source of

291 contamination, because it is known that in the processing of beef products (kofta, sausage, and

292 burger), the meat is mixed with several other ingredients, such as soybean, spices, and onions

293 (Gundogan, Citak, Yucel, & Devren, 2005). Other factors could be cross-contamination after

294 cooking with hands or raw food or the use of food additives (mayonnaise, salads, peppers, and

295 tahini (sesame seeds paste and oil) (Rane, 2011; Hussein et al., 2018). All these factors might

296 result in significant differences in the resistance profiles of E. coli isolated from raw beef and

297 RTE-BP samples against different antibiotics.

298 The resistance rate against colistin was 3.8% of the isolates in the present study, which is

299 consistent with the rates reported in previous studies on mcr-1 colistin-resistant E. coli from beef

300 (Mulvey et al., 2016; Kuo et al., 2016). In addition, mcr-1 was detected in all the colistin-

301 resistant E. coli in this study (Table 1). In contrast, Sadek et al. reported the detection of mcr-1 in

302 only one of the 128 colistin-resistant E. coli strains isolated from meat and meat product samples

303 (Sadek et al., 2019). Although the reason for differences in the mcr-1 positive ratio between the

304 present study and that of Sadek et al. (2019) is not clear, most of the colistin-resistant E. coli

305 strains (defined based on MIC determination using the micro-broth dilution method) were

306 reported to possess mcr-1 in recent studies (Elbediwi et al., 2019; Gharaibeh, & Shatnawi, 2019;

307 Carretto et al., 2018). These results suggest that the prevalence of mcr genes in different foods

308 contributes to the prevalence of colistin resistance in gram-negative bacteria.

309 Previous studies have reported the recovery of mcr-1 positive colistin-resistant E. coli

310 from clinical cases (5%), cloacal samples from poultry (8%), and ready-to-eat cheese (2%) in

15
311 Egypt (Zaki, ElKheir, & Mofreh, 2018; Moawada et al., 2018; Hammad et al., 2019). The

312 existence of mcr-1 gene, with a prevalence rate of 13%, in poultry samples dating back to 2010

313 (Barbieri et al., 2017), along with our results in the present study, provide the reason for the

314 increasing number of mcr-1 colistin-resistant cases in the clinics in Egypt.

315 Several STs were observed in mcr-1 harbouring isolates (Table 1). The diversity of STs

316 supported the spread of mcr genes via plasmids in accordance with several studies from across

317 the world, which confirmed the detection of mcr-1 in 112 unique STs (Matamoros et al., 2017).

318 The ST types of some isolates harbouring mcr-1 in this study include important human

319 pathogenic STs (ST10, ST58, ST155, and ST1431) (Mckinnon, Roy Chowdhury, & Djordjevic,

320 2018; Seiffert et al., 2017), suggesting that colistin-resistant E. coli harbouring mcr-1 are

321 transmitted through the food chain and are the cause of food-borne illness.

322 The ESBL gene, blaCTX-M-28, is being reported for the first time in Egypt in the present

323 study; blaCTX-M-1/15 and blaCTX-M-9 were the most frequently detected bla genes reported

324 previously (Braun et al., 2016). blaCTX-M-28 was reported to be highly prevalent in South Korea

325 and United Arabian Emirates marking a shift from the dominant blaCTX-M-1/15 gene (Alfaresi, Kim

326 Sing, & Senok, 2018). blaCTX-M-28 differs from blaCTX-M-15 in a single amino acid. Dramatic shifts

327 in the type and epidemiology of CTX-M-producing bacteria have been reported in many studies

328 from Japan, other Asian countries, and Europe (Bevan, Jones, & Hawkey, 2017). This might

329 explain the change in the genotype in the CTX-M type strains detected in this study from that

330 detected earlier in Egypt.

331 Both mcr-1 and ESBL genes in the same E. coli strain have been detected for the first

332 time in beef samples from Egypt in this study. Cases of mortality have been reported from

16
333 several developing countries where patients had infection with multidrug resistant bacteria that

334 were resistant to both cephalosporins and colistin (Dandachi et al., 2019). E. coli strains carrying

335 resistance and virulence genes are of great concern because such genes can aid the emergence of

336 pathogens that might be difficult to treat with antimicrobials (Ombarak et al., 2016). Co-

337 existence of both mcr-1 and ESBL genes in the same E. coli strains harbouring enterotoxin

338 (EAST1) and extraintestinal virulence genes in food is considered a serious threat for human

339 health. Therefore, strict hygienic measures are mandatory to control the spread of such resistant

340 and virulent bacteria through the food sector. Public should be made aware of such novel risks

341 and people in the livestock and agriculture sectors should be educated about the rational usage of

342 antibiotics.

343 5. Conclusion

344 This is the first study reporting the isolation and characterisation of E. coli strains

345 harbouring both the mcr-1 and ESBL genes from food products, particularly raw beef and RTE-

346 BP, in Egypt, indicating that these products could be a critical source of human infections in

347 Egypt since they harbour multi-virulent multi-drug resistant E. coli strains. Such infections

348 cannot be easily treated or might be untreatable, warranting the need to monitor and survey the

349 use of colistin and other types of antimicrobials for animals and in food industry, along with

350 implementation of strict measures to maintain hygiene during the production of food for human

351 consumption, especially RTE-BPs. In the future, further research using the one health approach

352 is needed to determine the significance of multi-drug resistant E. coli harbouring virulence genes

353 for food safety.

354

17
355 Acknowledgements

356 This work was supported by doctoral fellowship (channel system) from the Egypt-Japan

357 Education Partnership (EJEP).

358

359

360

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620

621 Figure Legends

622

623 Figure 1. The incidence (%) of occurrence of Escherichia coli in raw beef and ready-to-eat

624 beef products in Egypt.

625

626 Figure 2. Prevalence of antimicrobial resistance among Escherichia coli isolates from raw

627 beef and ready-to-eat beef products.

628 Antimicrobial resistance profiles of E. coli derived from raw beef (n = 150) and ready-to-eat

629 beef products (n = 61) in Egypt: colistin (CL/R ≥ 4 µg/mL, ampicillin (Amp/R ≥ 32 µg/mL),

630 cefazolin (CEZ/R ≥8 µg/mL), cefotaxime (CTX/R ≥ 4 µg/mL), streptomycin (SM/R ≥ 64

631 µg/mL), kanamycin (KAN/R ≥ 64 µg/mL), gentamicin (GM/R ≥16 µg/mL), tetracycline (TET/R

632 ≥16 µg/mL), nalidixic acid (NAL/R ≥ 32 µg/mL), ciprofloxacin (CIP/R ≥ 4 µg/mL), imipenem

633 (IMP/R ≥ 4 µg/mL), meropenem (MEPM/R ≥ 4 µg/mL); The minimum inhibitory concentration

31
634 (MIC) breakpoints follow the CLSI 2017 and NARMS 2011 guidelines); R: resistant; (*):

635 significant at P value < 0.05; (**): significant at P value < 0.01.

636

637 Figure 3. Prevalence of different virulence factors and genes in Escherichia coli isolated

638 from raw beef and ready-to-eat beef products.

639 fimH, fimA; type 1 fimbriae adhesin, ETTT; type III secretion system, ompT; outer membrane

640 protease T, fyuA; yersiniabactin receptor for ferric yersiniabactin uptake, traT, serum resistance

641 associates, iroN; catecholate siderophore receptor, aer; aerobactin, usp; uropathogenic specific

642 protein, hyl; α-haemolysin, Cdt; cytolethal distending toxins, cnf; cytotoxic necrotizing factor,

643 EAST1; EAEC heat-stable enterotoxin 1. (*): significant at P value < 0.05; (**): significant at P

644 value < 0.01.

32
 Table 1. Molecular characterisation and antimicrobial resistance profile of mcr-1 and/or ESBL positive Escherichia coli isolates derived from food

Strain ST
Origin ESBL gene mcr gene Resistance phenotype Virulence genes
No. type
mcr-
19 Sausage 1720 blaTEM-116 mcr-1 CL-AMP-CEZ-TET usp, OmpT, fimA, fimH
1/ESBL
194 Raw beef 1431 blaCTX-M-28 mcr-1 CL-AMP-CEZ-CTX-SM-TET-NAL fimA, fimH, ETTT
201 Raw beef 10 blaCTX-M-28 mcr-1 CL-AMP-CEZ-CTX-SM-TET EAST1, cdt, fyuA, ETTT
mcr-1 20 Sausage 58 ND mcr-1 CL-CEZ-NAL fimA, fimH, ETTT
123 Raw beef 1172 ND mcr-1 CL-AMP-TET traT, OmpT, fimA, fimH, ETTT
157 Raw beef 58 ND mcr-1 CL-AMP-SM-KAN-TET-NAL EAST1, traT, fimA, fimH, ETTT
174 Raw beef 109 ND mcr-1 CL EAST1, fyuA, aer, IroN, fimA, fimH, ETTT
202 Raw beef 155 ND mcr-1 CL-AMP fyuA, fimA, fimH, ETTT
ESBL 121 Raw beef 3580 blaCTX-M-28 ND AMP-CEZ -CTX EAST1, fimA, fimH, ETTT
212 Raw beef 1125 blaCTX-M-28 ND AMP-CEZ-CTX-SM-KAN-GM-TET Trat, fimA, fimH, ETTT
216 Raw beef 1431 blaCTX-M-28 ND AMP-CEZ-CTX-SM-TET EAST1, FuyA, ETTT
ST, Sequence type.
ESBL, extended-spectrum beta lactamase
ND, not detected
CL, colistin (≥4 µg/mL); Amp, ampicillin (≥32 µg/mL); CEZ, cefazolin (≥8 µg/mL); CTX, cefotaxime (≥4 µg/mL); SM, streptomycin (≥64
µg/mL); TET, tetracycline (≥16 µg/mL); GM, gentamicin (≥16 µg/mL); KAN, kanamycin (≥64 µg/mL); NAL, nalidixic acid (≥32 µg/mL); the
minimum inhibitory concentration (MIC) breakpoints follow the CLSI 2017 and NARMS 2010 guidelines.
EAST1, EAEC heat-stable enterotoxin 1; Cdt, cytolethal distending toxins; traT, serum resistance associates; aer, aerobactin; ETTT, type III
secretion system; fimH, fimA, type 1 fimbriae adhesin; fyuA, yersiniabactin receptor for ferric yersiniabactin uptake; iroN, catecholate
siderophore receptor; ompT, outer membrane protease T; usp, uropathogenic-specific protein.
Highlight
• Spread of antibiotic-resistant bacteria through food is a global issue.
• Multidrug-resistant pathogenic E. coli were detected in meat products in Egypt.
• Contaminated food with pathogenic E. coli is a source of infection for human.
• Three colistin-resistant isolates had both the mcr-1 and ESBL genes.
• CTX-M-28 was detected, for the first time, in meat products in Egypt.