Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets

Accepted Manuscript Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets

Abatcha Mustapha Goni, Mohd Esah Effarizah, Gulam Rusul PII:

S0956-7135(18)30085-9

DOI:

10.1016/j.foodcont.2018.02.039

Reference:

JFCO 6002

To appear in:

Food Control

Received Date:

14 November 2017

Revised Date:

28 January 2018

Accepted Date:

23 February 2018

Please cite this article as: Abatcha Mustapha Goni, Mohd Esah Effarizah, Gulam Rusul, Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets, Food Control (2018), doi: 10.1016/j.foodcont.2018.02.039

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ACCEPTED MANUSCRIPT

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Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars

2

in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh

3

food markets

4 5

Abatcha Mustapha Goniª, Mohd Esah Effarizah*ª, Gulam Rusulª

6 7

ªFood Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800

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Minden, Penang, Malaysia

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* Corresponding author. Email address: [email protected]

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Abstract

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This study was carried out to determine the prevalence, antibiotic resistance, resistance genes and

23

class 1 integrons of Salmonella serovars in raw leafy vegetables, chicken carcasses and related

24

environments. From April 2015 to May 2016, a total of 642 samples collected from fresh food

25

markets in Peninsular Malaysia were examined. The overall occurrence of Salmonella species was

26

29.1% (187/642) with 37 different serovars detected. The most prevalent serovars were S. Corvallis

27

(49/187), S. Brancaster (28/187), S. Albany (17/187), S. Weltevredent (15/187), S. Hvittingfoss

28

(6/178), S. Paratyphi B (6/178) and S. Typhimurium (6/178). Among the Salmonella isolates, the

29

highest antibiotic resistance was to streptomycin (66.6%), followed by tetracycline (44.4%),

30

sulfonamides

31

sulfamethoxazole (16.6%). All isolates of Salmonella were 100% susceptible to cephalothin. Fifty-five

32

percent of the isolates (103/187) were multidrug resistant. The multiple antibiotic resistance (MAR)

33

index of Salmonella serovars ranged from 0.08 to 0.83, and the most prevalent resistance pattern

34

was STeS₃. Eleven out of 16 resistant genes (tetA, tetB, blaTEM-1, temB, strA, strB, aadA, sulI, sulII, floR

35

and cmlA) were detected among the resistant Salmonella isolates. None of the isolates was positive

36

for tetC, tetG, cat1 and cat2. Seventeen isolates harboured class 1 integrons, which were grouped

37

into 5 different integrons profiles (IPs). DNA sequencing analyses have identified dfrA1, dfrA12,

38

aadA2, blaPSE−1, dfrA12-orf-aadA2 arrays of cassettes in variable regions on class 1 integrons.

(44.4%),

ampicillin

(26.7%),

chloramphenicol

(29.1%)

and

trimethoprim-

39 40

Keywords: Salmonella spp., antibiotic resistance, resistance genes, class 1 integrons

41 42

1. Introduction

43

Salmonella is one of the most important foodborne pathogens worldwide (Kirk et al., 2015; Fei

44

et al., 2017), causing as many as million cases of typhoid fever, billion cases of gastroenteritis and

2

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thousands of death each year (Bhunia, 2008). The estimated economic burden due to Salmonella

46

infections from all sources costs about 3.7 billion USD per year in the United States (ERS-USDA,

47

2013). In Malaysia, although Salmonellosis is not a notifiable disease, the incidence of Salmonella

48

isolated from humans has doubled in the past decade (Thong et al., 2016). Although Salmonella has

49

been isolated from several sources, human Salmonellosis is most often linked to the consumption of

50

contaminated poultry and poultry products (Fearnley et al., 2011), pork (Prendergast et al., 2009),

51

beef (Zhao et al., 2008), fish (Kramarenko et al., 2014), vegetables (Sant’Ana et al., 2011), and non-

52

pasteurized dairy products (Langer et al., 2012). The increasing prevalence trend across the world

53

(Pui et al., 2011) and growing number of vegetables and poultry related outbreaks (Guran et al.,

54

2017; CDC, 2015), have warranted the need for periodic surveillance of foods and environment in

55

order to prevent human Salmonellosis. These pathogens pose an imminent risk to public safety not

56

only due to its occurrence but also because many strains are resistant to a number of antimicrobial

57

agents (Hur et al., 2012).

58

The extensive use of antimicrobials by humans and in livestock production has led to

59

antimicrobial resistance (AMR) among several bacterial strains. Multidrug-resistant (MDR)

60

Salmonella enterica in foods have been previously reported in some parts of the world (Miko et al.,

61

2005; Van et al., 2007). Salmonella isolates from Malaysia and other countries have shown an

62

increased proportion in the number of multidrug-resistance (Thong and Modarressi, 2011). The

63

isolation

64

resistant Salmonella was of particular concern because these antibiotics are widely used for

65

treatment in medicine (Hur et al., 2012; Health Canada, 2009).

of

fluoroquinolones,

quinolones

and

extended-spectrum

cephalosporins

66

Contamination of food with MDR Salmonella is a major public health problem, as resistance

67

traits located on mobile genetic elements can easily be conveyed to other bacteria of clinical

68

significance (Thong and Modarressi, 2011; Van et al., 2007). The mechanisms of antimicrobial

69

resistance can be due to several factors, including changes in bacterial cell wall permeability,

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modification of the site of drug action, energy-dependent removal of antimicrobials via membrane-

71

bound efflux pumps and destruction or inactivation of antimicrobials (Chen et al., 2004).

72

Antimicrobial resistance genes are mainly found on mobile genetic elements like transposons,

73

integrons, and plasmids (Canal et al., 2016). A strong relationship between the presence of integrons

74

and increased resistance to numerous antibiotics has been reported (Moura et al., 2007; Van et al.,

75

2007). On top of that, four distinct classes of integrons encoding different integrase gene sequences

76

have also been investigated (Mazel, 2006). Among them, Class 1 integrons found to be the most

77

important contributor to MDR in Gram negative enteric bacteria and play a major role in

78

disseminating antimicrobial resistance genes (Krauland, 2009). Likewise, Class 1 integrons are very

79

frequent in MDR Salmonella (Khemtong and Chuanchuen, 2008). Moreover, in Malaysia, due to high

80

consumption of green leafy vegetables, chicken meat and increasing antimicrobial resistance in non-

81

typhoidal Salmonella (Van et al., 2012), the prevalence and antibacterial resistance

82

of Salmonella spp. in vegetables and the broiler chicken sold at the retail point need to be

83

monitored.

84

The objective of our study was to determine the prevalence of Salmonella serovars from raw leafy

85

vegetables, chickens carcasses and their related processing environments in Malaysian fresh food

86

markets. Salmonella serovars isolated were also examined for antibiotic resistance, resistance genes,

87

and Class 1 integrons.

88 89

2. Materials and methods

90

2.1. Sample collection

91

A total of 642 no-repeat samples of raw leafy vegetables (405/642), chicken carcasses (35/642) and

92

their related processing environments (202/642) were obtained from fresh food markets in selected

93

states of Peninsular Malaysia (Penang, Kedah, Perlis and Selangor) during the period of April 2015 to 4

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May 2016. The vegetables selected were bean sprout (Vigna radiate), amaranth red (Amaranthus

95

tricolor), Chinese flowering cabbage (Brassica rapa var. Parachinensis), coriander (Coriandrum

96

sativum), lettuce salad (Lactuca sativa), amaranth green (Amaranthus tricolor), spring onion (Allium

97

fistulosum), winged bean (Psophocarpus tetragonolobus), laksa leaves (Poligonum minus), Indian

98

pennywort (Centenella asiatica), iceberg lettuce (Lactuca sativa), mint (Mentha arvensis), Japanese

99

parsley (Oenanther stolonifera), wild parsley (Cosmos caudatus), water spinach (Ipomoea aquatic)

100

and sweet basil (Ocimum basilicum) and were collected in sterile plastic bags. For chicken cuts and

101

whole chickens carcasses, the swab-sampling method described by Gill et al., (2005) was adopted by

102

which the inner-outer surfaces of the chicken were swabbed using 3M™ Dry-Sponge-Sticks (USA). On

103

the other hand, the environmental samples including transport crates, knifes, display tables, drums,

104

defeathering machines, drain crevices, floors, butcher aprons, chopping board and cages surface

105

area of 10 – 30 cm2 were swabbed using the 3M™ Dry-Sponge-Sticks as per manufacturer’s

106

instruction (https://multimedia.3m.com/mws/media/871382O/3m-sponge-stick.pdf). Sterile Schott

107

Duran® bottles were used in collecting all the water samples (wash water, scalding tank water,

108

bench water and drain water). Samples were delivered to the laboratory on ice in Polystyrene box

109

and processed immediately upon arrival to the laboratory.

110 111

2.2. Salmonella isolation and identification

112

Detection and isolation of Salmonella from samples were carried out according to ISO 6579:2002

113

Horizontal Method (ISO, 2002). Vegetable samples (25g), swab samples (3M™ Dry-Sponge-Sticks)

114

and 25 ml of water samples were pre-enriched in 225 ml of buffered peptone broth and were

115

incubated for 24 ± 2 hrs at 37 ± 1 °C. Then, pre-enriched 0.1 ml and 1ml cultures were incubated in

116

9.9 mL of Rappaport Vassiliadis Soy Broth (RVS) at 42 ± 1 °C and 9 ml of Muller-Kauffmann

117

Tetrathionate-Novobiocin (MKTTn) broth at 37 ± 1 °C for 24 ± 2 hrs, respectively. Loopfuls of RVS

118

cultures were streaked onto selective agar plates; Xylose-lysine-tergitol 4 (XLT4), Xylose Lysine

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Deoxycholate Agar (XLD) and Rambach Agar (RAM), then incubated for about 24 hrs at 37 °C.

120

Suspected Salmonella colonies were picked from each plate, purified and subjected to biochemical

121

tests (triple sugar iron, lysine iron agar). All media used were purchased from Merck, Germany.

122

Salmonella isolates were also serologically confirmed by using polyvalent O and H antisera (Remel

123

Europe, UK). The serotyping of Salmonella isolates were done at Salmonella reference centre at

124

Public Health Laboratory, of Ipoh district, Perak, Malaysia according to Kauffmann and White

125

Scheme (Grimont and Weill, 2007).

126 127

2.3. Antimicrobial susceptibility test of Salmonella serovars

128

Kirby–Bauer agar disk diffusion method was used to evaluate antimicrobial susceptibility against a

129

panel of 12 antimicrobial agents as suggested by Clinical and Laboratory Standard Institute (CLSI,

130

2012). These antimicrobials were ampicillin (10 μg), amoxicillin-clavulanic acid (20/10 μg),

131

chloramphenicol

132

trimethoprim-sulfamethoxazole (25 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), cephalothin

133

(30 μg), kanamycin (30 μg) and sulfonamides (300 μg) (Oxoid, UK). Escherichia coli (ATCC 25922) was

134

used as a control. The interpretation for the zones of inhibition was in accordance with CLSI

135

guidelines (CLSI, 2012). The Multiple Antibiotic Resistance (MAR) index was determined according to

136

the method stated by Krumperman (1983).

(30 μg),

gentamicin

(10 μg),

streptomycin

(10 μg),

tetracycline

(30 μg),

137 138

2.4. Polymerase chain reaction for detection of resistance genes and Class 1 integrons

139

Crude DNA was prepared by direct boiling of a suspension of the cell lysates, as previously described

140

by Ahmad et al. (2009). Sixteen pairs of oligonucleotides primers were used to target 16

141

antimicrobial resistance genes that confer resistance to five antimicrobial agents, including

142

ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline. The primer sequences and

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PCR conditions are presented in Table 1. All PCR amplifications contain 1× Taq buffer, 1.5 mM MgCl2, 6

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200 μM each dNTPs, 1 Unit of Taq Polymerase (Promega, Madison, USA), 0.5μM each primer (First

145

Base, Malaysia) and 50 ng DNA template. The isolates were screened for the presence of Class 1

146

integrons

147

AAGCAGACTTGACTGAT-3′) as previously described (Levesque et al., 1995), flanking the integrated

148

gene cassettes. Selected amplified PCR products were verified by DNA sequencing. The amplicons

149

were purified using a DNA purification kit (Qiagen, Germany) and sent to a commercial facility for

150

sequencing (First Base Laboratories, Malaysia). The resulted sequences were aligned and confirmed

151

using the GenBank database and BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

using

specific

primers

5′CS

(5′-GGCATCCAAGCACAAGC-3′)

and

3′CS

(5′-

152 153

2.5. Statistical analysis

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All statistical analyses were done using SPSS 18.0 (SPSS Inc., Chicago, IL), and the chi-squared test

155

was applied to assess any statistically significant (p<0.05) differences in the Salmonella prevalence

156

data.

157 158

3. Results and Discussion

159

3.1. Prevalence of Salmonella species

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The results showed that all 32 varieties of samples examined were contaminated with

161

Salmonella species. The overall prevalence of Salmonella serovars on leafy vegetables, on chicken

162

carcasses and from related processing environments is shown in Table 2. A total of 187/642 (29.1%)

163

samples were positive for Salmonella, of which 87/405 (21.5%) were vegetable samples, 17/35

164

(48.0%) were chicken samples and 83/202 (41.0%) were environmental samples. The prevalence of

165

Salmonella were significantly different between samples (P<0.05).

166

In this study, the prevalence of Salmonella spp isolated from leafy vegetables was 21.5%. This

167

data was consistent with previous studies reported by other authors, who had described high 7

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prevalence rates for this pathogen in raw vegetables including 34% in Malaysia (Salleh et al., 2003),

169

29.1% in Brazil (Maistro et al., 2012) and 20.0% in Turkey (Aytac et al., 2010). Coriander had the

170

highest prevalence (52.0%) of Salmonella, followed by lettuce salad (32.0%), water spinach (31.0%),

171

amaranth red (28.0%), bean sprouts (28.0%) and amaranth green (27.0%). Salmonella spp. had

172

previously been isolated from leafy vegetables and other fresh produce such as lettuce, spinach,

173

bean sprouts, parsley, watercress, cucumber and potatoes (Maistro et al., 2012; Aytac et al., 2010).

174

Consumption of raw or minimally process leafy vegetables can be a potential source of human

175

Salmonellosis in Malaysia.

176

The use of untreated animal manure from livestock can be a major source of environmental

177

contamination with Salmonella spp. and some other foodborne pathogens may contaminate leafy

178

vegetables when applied during plant growing (Kotzekidou et al., 2016). Moreover, Salmonella is

179

shed into the soil directly by wildlife and livestock and can persist on the environment for months

180

(Liu et al., 2013). Likewise, Indian pennywort (Centella) is a topsoil creeper, and soil can be a source

181

of contamination if animal manure is used as fertilizer for the crop of this vegetable.

182

The water source for irrigation is one of the determining factors for the presence of pathogens

183

in vegetables (Cooley et al., 2014). The possibility of contamination via irrigation is increased, as

184

untreated wastewater is used for around 10% of crop irrigation (Anon, 2003). In the United States

185

and Senegal, 9% and 35% of the irrigation water samples analyzed were contaminated with

186

Salmonella, respectively (Pachepsky et al., 2011; Ndiaye et al., 2011). In addition, a number of

187

outbreaks related to contaminated irrigation water had been published. In 2005, iceberg lettuce

188

imported from Spain caused S. Typhimurium cases in Finland and UK after wastewater was used to

189

irrigate the crop (Takkinen et al., 2005).

190

Vegetables such as Japanese parsley (Oenanther stolonifera), laksa leaves (Poligonum minus)

191

and water spinach (Ipomoea aquatic) are often grown around swamps, river banks and irrigation

192

ditches, which are prone to contamination by refuse waste from industry, slaughter houses and 8

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processing plants (Sallah et al., 2003). According to Brackett, (1994) contamination of vegetables at

194

harvest and at post-harvest stages (transportation, processing, packaging, distribution and retail

195

levels) may be due to contaminated harvesting equipment, poor personal hygiene, inadequate

196

sanitation in the processing plant and retail handling. Hence, in this current study, the relatively high

197

rate of Salmonella contamination in leafy vegetables in the fresh food markets may have been

198

attributed to cross-contamination from the environment where the vegetables were cultivated,

199

prepared, handled by workers and consumers and stored.

200

In general, the prevalence of Salmonella was 48% for chicken carcasses and 41% for processing

201

environmental samples obtained from fresh food markets (Table 2). The results were incomparable

202

with the previous findings in Malaysia. Nidaullah et al. (2017) reported 88.46% of poultry carcasses

203

and related processing environmental samples from the small-scale plants and fresh markets were

204

contaminated with Salmonella. In addition, Modarressi and Thong (2010), reported a prevalence of

205

72.7% of Salmonella in chicken meat samples around Klang Valley, Malaysia from 2006 to 2009. In

206

another study, the prevalence of Salmonella in broiler carcasses obtained from wet markets and

207

processing plants was 35.5% and 50.0%, respectively (Rusul et al., 1996). The differences in the

208

results of these studies might be due to sampling approaches and geographical locations. As in this

209

study, samples were obtained from numerous processing stages of slaughtering to retailing at the

210

fresh food markets. The focus was to find contamination sites and to identify the area of Salmonella

211

dissemination during the processing. Among the studied samples (Table 2), drain water had the

212

highest prevalence (76.0%) of Salmonella, followed by floors (67.0%), chopping board (53.0%), wash

213

water (53.0%), display tables (50.0%), chicken cuts (50.0%), whole chicken (47.0%) and butcher

214

aprons (43.0%). Salmonella contamination was relatively high in our study, signifying that poultry

215

meat and processing environment may be a potential vector for transmitting Salmonella species.

216

This shows that Salmonella had established itself in poultry processing environments and on carcass

217

contact surfaces by colonizing the equipment surfaces forming biofilms which allows for longer

218

survival. Besides, the formation of biofilms by Salmonella on contact surfaces is of major food safety 9

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concern due to greater risk for cross-contamination (Carrasco et al., 2012). The existence of high

220

humidity in the fresh food markets environment may attribute to the presence of pathogen biofilm

221

on contact surfaces (Villa-Rojas et al., 2017). In most of the fresh food markets, chicken carcasses are

222

sold at ambient temperatures and exposed to the environment with the lack of cooling system and

223

ice is rarely used for chilling.

224

In live birds, Salmonella is carried asymptomatically in the gastrointestinal tract and can merely

225

transfer to carcasses in abattoir through faecal contamination. Further dissemination may perhaps

226

occur during processing if the carcasses become cross-contaminated (Trongjit et al., 2017). As in this

227

study, the live birds are mostly slaughtered, processed and retailed in the market without inspection

228

by the authority, and extensive human handling during each processing step almost certainly lead to

229

cross contamination.

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3.2. Salmonella isolate serotyping

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The 187 Salmonella isolates were divided into 37 different serovars (Table 3). The most

233

prevalent serovars were S. Corvallis (49/187), S. Brancaster (28/187), S. Albany (17/187), S.

234

Weltevredent (15/187), S. Hvittingfoss (6/178), S. Paratyphi B (6/178) and S. Typhimurium (6/178).

235

Other serovars isolated were S. Indiana (5/187), S. Aberdeen (4/187), S. Augustenborg (4/187), S.

236

Richmond (4/187), S. Mbandaka (4/187), S. Enteritidis (4/187), S. Braenderup (3/187), S. Give

237

(3/187), S. Redhill (3/187), S. Dusseldorf (2/187), S. Dumfries (2/187), S. Newport (2/187), S. Stanley

238

(2/187), and S. Planckendael (2/187). The frequencies of other serovars such as S. Bareilly, S. Cerrot,

239

S. Djugu, S. Gamira, S. Haifa, S. Kastrup, S. Kentucky, S. Lindenburg, S. Minnesota, S. Mkamba, S.

240

Molade, S. Obugu, S. Ohio, S. Salamae serovar II,19,12,Iv,z39, S. Tudu and S. Wandsworth were very

241

low (<2). Among the 37 different Salmonella serovars, 27 were isolated from leafy vegetables, 5

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different Salmonella serovars were isolated from chicken carcasses and 19 from processing

243

environments.

244

The pattern of serovars distribution is quite different as S. Weltevredent was the dominant

245

serovar in leafy vegetables, followed by S. Corvallis, S. Brancaster, S. Paratyphi B and S. Hvittingfoss.

246

This is particularly similar to the findings by Salleh et al. (2003), in which they reported S.

247

Weltevredent as the predominant serovar isolated from raw leafy vegetables in the Central region of

248

Malaysia. In addition, between 1983–1992, Salmonella Weltevredent was third most prevalent

249

serovar isolated in the human clinical cases in Malaysia (Yasin et al., 1997). S. Weltevredent is an

250

emerging pathogen and increasingly being reported as a cause of invasive bacterial disease and

251

diarrhoea in the human population residing in the tropical regions of low income countries (Makendi

252

et al., 2016). This is of serious concern because S. Weltevredent had been reported to be the cause

253

of Salmonellosis in Scandinavia, Malaysia and Southeast Asia (Learn-Han et al., 2008;

254

Bangtrakulnonth et al. 2004; Padungtod and Kaneene, 2006).

255

On another note, the detection of S. Paratyphi B, S. Typhimurium and S. Enteritidis in

256

vegetables should have similarly raised concern to us because these serovars have been linked to

257

numerous outbreaks of foodborne Salmonellosis worldwide (Pui et al., 2011; Kirk et al., 2015).

258

Salmonella Paratyphi B has wide geographical distribution and has been isolated from different

259

sources causing enteric fever and self-limiting gastroenteritis in humans (Chart et al., 2005). In

260

Malaysia, the epidemiological knowledge of S. Paratyphi B is relatively scarce as compared to well-

261

known serovars like Typhimurium and Enteritidis. Only two studies reported S. Paratyphi B as the

262

second leading serovar isolated from children hospitalized with extra-intestinal non-typhoidal

263

Salmonellosis and those with non-typhoid gastroenteritis in Malaysia (Lee et at., 2000). However,

264

several reports of increasing incidence of S. Paratyphi B in Canada and Italy (Stratton et al., 2001;

265

Miko et al., 2002) show that this serovar will be significant in near future. The isolation of six S.

11

ACCEPTED MANUSCRIPT 266

Paratyphi B from different vegetables should not be underestimated, particularly in those lightly

267

cooked or eaten raw.

268

Some serovars may have epidemiological importance in certain geographical locations. For

269

instance, S. Mbandaka has been noted to emerge in the Poland, the UK and Australia (Hoszowski and

270

Wasyl, 2001; Reid et al., 1993; Scheil, Cameron et al., 1998). To our knowledge, this is the second

271

time S. Hvittingfoss had been isolated from vegetables obtained from the fresh food markets in

272

Malaysia as previously reported in 2003. An outbreak of S. Hvittingfoss associated with consumption

273

of rockmelon has been reported in Australia in 2016 (Food Safety News, 2016). A noteworthy finding

274

of several Salmonella serovars such as S. Augustenborg, S. Cerrot, S. Djugu, S. Dumfries, S. Kastrup, S.

275

Minnesota, S. Newport, S. Planckendael, S. Obugu, S. Ohio, S. Redhill, S. Richmond and S. Stanley to

276

mention a few is that these Salmonella serovars are among those isolated from vegetables but

277

infrequently reported in the Far East, particularly in Malaysia. The diversity of vegetables and study

278

locations have influenced the occurrence of many serovars obtained in this present study.

279

On chicken carcasses and the related processing environments, the majority of occurring

280

serovars were S. Corvallis, S. Brancaster and S. Albany. The Salmonella serovars identified in our

281

study are in conformity with that of reported by Nidaullah et al. (2017), in which S. Corvallis, S.

282

Brancaster and S. Albany were major serovars from wet markets and poultry processing plant. These

283

may be due to result of microbial adhesion and biofilm formation on equipment contact surfaces

284

such as cutting board, knife, scalding tank water, chilling tank, and defeathering machines which

285

allow these Salmonella serovars to be persistent for a longer period of time in biofilm and they tend

286

to protect the pathogens from sanitizers and detergents. Likewise, Strawn et al. (2014) stated that

287

certain Salmonella serovars may be more common in certain areas, probably due to persistence or

288

adaptation to specific hosts or abiotic environment found in a given geographical location. In

289

another study, 14 different Salmonella serovars were isolated from chicken carcasses obtained from

12

ACCEPTED MANUSCRIPT 290

retail outlets in Malaysia, with S. Muenchen (32.6%), S. Enteritidis (19.8%), S. Kentucky (17%), and S.

291

Blockley (12.8%) as the major serovars (Rusul et al., 1996).

292 293

3.3. Antimicrobial susceptibility of Salmonella isolates

294

The resistance of Salmonella isolates to 12 antimicrobial agents examined is shown in Table 4.

295

In total, 19 and 15 Salmonella isolates (10.2 and 8.0%) were resistant to one and two antibiotics,

296

respectively. In addition, 103 isolates of Salmonella (55.1%) were multi-drug resistant (MDR)

297

(resistance to 3 or more antimicrobials). MDR Salmonella isolates have been reported to be more

298

virulent than non-multiple drug-resistant (Foley et al., 2008). This level of MDR Salmonella isolates

299

was lower than that of previously reported by Thong and Moderassi (2011) in Malaysia (67%). In

300

another study, the level of MDR Salmonella isolates was reported in Vietnam (34%) by Van et al.

301

(2007) and in Morocco (44%) by Bouchrif et al. (2009). Notably, 16 different Salmonella serovars

302

were observed among the multidrug-resistant isolates.

303

In the present study, high prevalence of resistance was observed for streptomycin (66.6%),

304

tetracycline (44.4%), sulfonamides (44.4%), ampicillin (26.7%), chloramphenicol (29.1%),

305

trimethoprim-sulfamethoxazole (11.6%), nalidixic acid (12.8%) and kanamycin (11.2%). All isolates of

306

Salmonella were susceptible to cephalothin. In comparison, these findings are similar to earlier

307

reports showing that Salmonella isolates in vegetables, chicken and processing environment were

308

resistant to many antimicrobials, including tetracycline, streptomycin, sulfonamides and ampicillin

309

(Learn-Han et al., 2008). Much of the resistant isolates were from poultry and environmental

310

samples as compared to vegetables. In poultry, the emergence of antimicrobial resistant Salmonella

311

isolates may be a result of widespread use of antibiotics for growth promotion, therapeutic and

312

prophylactic uses in local poultry production in Malaysia. These isolates can then be transferred to

313

vegetables via the use of faeces from poultry houses as manure on vegetable farms. Even though

314

application of antibiotic in livestock's production is under strict veterinary supervision in most 13

ACCEPTED MANUSCRIPT 315

countries, farmers are still using antibiotics as prophylactic in intensive farming units, mainly poultry,

316

cattle and pigs without prescription (Usera et al., 2002).

317

A number of Salmonella isolates from vegetables in this study were also resistant to a few

318

antimicrobials agents, including streptomycin (36.8%), sulfonamides (25.3%), tetracycline (19.5%),

319

ampicillin (13.8%) and chloramphenicol (10.3%). Although a number of antibiotics used in vegetable

320

farming are modest (Schwaiger et al., 2011), primary sources for the spread of resistant bacteria into

321

the field were considered to be pesticides application, emission of residues from wastewater

322

treatment, antibiotic manufacturing and irrigation of crop with contaminated water (Brandl, 2006).

323

According to Segura et al. (1999), the formation of multidrug efflux systems from heavy metals and

324

plant metabolites can result in antibiotic resistance.

325

The antimicrobial resistance profile and MAR index of the Salmonella serovars are presented in

326

Table 5. One hundred and thirty-seven Salmonella isolates belonging to 25 different serovars

327

exhibited 51 different antibiogram patterns. S. Brancaster exhibited 20 different resistant patterns to

328

the antibiotics examined whereas various different resistant patterns were also shown by S. Albany,

329

S. Corvallis, S.Weltevreden, S. Give, S. Hvittingfoss, S. Indiana, S. Typhimurium, S. Enteritidis, S.

330

Mbandaka, S. Paratyphi, S. Stanley and S. Dumfries, respectively. The most prevalent resistance

331

pattern was STeS₃ and exhibited by S. Corvallis (28), S. Mbandaka (3), S. Typhimurium (1) and S.

332

Djugu (1). One S. Albany isolated from chopping board was resistant to 10 antibiotics and had the

333

highest MAR index of 0.83. One isolate of each S. Give and S. Weltevredent and 3 of S. Albany were

334

resistant to 8 antibiotics with a high MAR index of 0.66. Eight and 7 isolates were resistant to 7 and 6

335

antibiotics with MAR index of 0.58 and 0.50, respectively. The emergence of Salmonella serovars

336

having MAR Index of more than 0.2 originated from an environment where several antibiotics are

337

used more often as therapeutic or feed additive in animals (Krumperman, 1983).

338

14

ACCEPTED MANUSCRIPT 339

3.4. Antimicrobial resistance genes and class 1 integrons

340

Eleven out of the 16 resistance genes (tetA, tetB, blaTEM-1, temB, floR, cmlA, aadA, strA, strB, su1

341

and sul2) were detected in drug-resistant isolates by PCR (Table 6). A comparison of the DNA

342

sequences of these amplicons showed 95–100% similar identity with the available sequences in the

343

NCBI GenBank Database.

344

Out of the 117 streptomycin-resistant isolates, 38 harboured both strA and strB and only 32

345

harboured the aadA genes. Along with these, 14 isolates had all the three genes. The strA-strB genes

346

are widely disseminated in Salmonella and other Gram negative spp (Caratoliet al., 2008; Soudin,

347

2002). In Malaysia, strA-strB and aadA were detected in Salmonella serovars from raw beef and

348

chicken meat (Thong and Moderrasi, 2011). The use of streptomycin for treatment had been

349

reduced in human and veterinary medicine but the persistence might be caused by co-selection

350

(Peirano et al., 2006). Of the 83 tetracycline-resistant isolates, tetA alone was found in 76 isolates

351

and 3 were positive for both tetA/tetB, respectively. No isolates were positive for tetC and tetG.

352

Similar to tetA, tetB gene is widespread among Salmonella and has been located on transferable

353

plasmids (Lopes et al., 2016), and is easily transferred (Roberts, 2005).

354

Among the 83 sulphonamide-resistant isolates, 5 were positive for both sul1 and sul2, 7 were

355

positive for sul1 and 47 were positive for sul2 only. These genes most often found in integron

356

positive isolates that carried other genes (Mąka et al., 2015). In the United States and Canada, much

357

of the isolated MDR Salmonella enterica from humans, animals and retail meat harboured sul1

358

(26/56) and sul2 (23/56) (Glenn et al., 2013).

359

Twenty of the 41 chloramphenicol-resistant isolates harboured floR and 4 harboured cmlA only,

360

whereas 4 (1 Albany, 2 Brancaster, and 1 Corvallis) serovars had both floR and cmlA. None of the

361

chloramphenicol acetyltransferase genes, cat1 and cat2, were detected in the chloramphenicol-

362

resistant Salmonella. These two genes (cmlA and floR) are very much related to and encoded in

363

Chloramphenicol efflux pumps in Salmonella (Cabrera et al., 2004; White et al., 2001). In this study,

15

ACCEPTED MANUSCRIPT 364

floR genes appear to be very prevalent in Salmonella, whereas cmlA was less widely distributed. This

365

is in agreement with the report by Thong and Modarressi (2011), stating that the floR gene was

366

detected in 7 isolates (6 Typhimurium, 1 Newport) and cmlA was detected in 2 isolates (serovars

367

Istanbul and Wandsworth). Also in another study by Glenn et al. (2013), chloramphenicol resistance

368

genes including cat (36/56), floR (27/56) and cmlA (7/56) were detected in Salmonella enterica

369

serovars isolated from retail meat, animals and humans.

370

Out of 3 types of β-lactamase gene tested, 36 of 50 ampicillin-resistant isolates harboured both

371

blaTEM-1 and temB. At the same time, only 14 had temB. None of the temA was detected. In an earlier

372

study by Benacer et al. (2010), it was reported that all ampicillin-resistant Salmonella strains

373

harboured blaTEM-1, temA, and temB. In another similar study in Korea by Kim et al. (2013), temA

374

(1.6%) and blaTEM-1 (95%) genes were detected in resistant isolates while temB was not detected.

375

In this study, class 1 integrons were detected in 17 (12.4%) resistant Salmonella isolates (4

376

from vegetables, 6 from chicken carcasses and 7 from the environment). Five integron profiles (IP)

377

were identified. IP-1, IP-3 and IP-5 consist of one integron while IP-2 and IP-4 consist of two

378

integrons each as shown in Table 7. Five amplicons, which were 0.3 kb, 0.5 kb, 1.5 kb, 0.3+1.2 kb,

379

and 0.3+1.5 kb long were detected. In another study by Thong and Moderrasi (2011), they detected

380

12 class 1 integrons amongst 59 MDR Salmonella isolates from retail meat and street foods. Three IP

381

with variable amplicons were defined, which include IP-1 (0.7 kb) found in S. Typhimurium and S.

382

Weltevreden, IP-2 (1.2 kb) in S. Newport and S. Albany and IP-3 (1.5 kb) in S. Agona and S.

383

Typhimurium, respectively (Thong and Moderrasi, 2011).

384

Based on the DNA sequence examination of variable regions of amplicons, the most

385

predominant gene cassette arrays carried by these integrons were dfrA1 and dfrA12 genes encoding

386

resistance to trimethoprim (GenBank accession no. KY965931), and aadA2 gene encoding for

387

aminoglycoside adenyltransferase AAD (3'') and confers resistance to streptomycin-spectinomycin

388

(accession no. KY965930). In addition, a 0.3 + 1.0-kb integron contains an aadA2 + blaPSE−1 gene

389

cassette (accession no. KY965929) encoding resistance to aminoglycosides and beta-lactamase. The 16

ACCEPTED MANUSCRIPT 390

1.5 kb dfrA12-orf-aadA2 gene cassette array (accession no. KY96592) found in 1 S. Corvallis and 1 S.

391

Kentucky confers resistance to sulphonamides, trimethoprim and aminoglycosides which was similar

392

to our finding (Thong and Moderrasi, 2011). Similar gene cassette of dfrA12-orf-aadA2 had been

393

found in 1.9 kb integrons in Thailand from serovars Rissen, Anatum,

394

Eppendorf, Stanley, Schwarzenrund and Typhimurium, (Khemtong and Chuanchuen, 2008), in Korea

395

from S. Gallinarum (Kwon et al., 2002), and in Taiwan from S. Choleraesuis (Hsu et al., 2006).

396

Identical gene cassette could be found from the same and different bacterial species (Hsu et al.,

397

2006) which indicates that integrons can be transferred between intra- and inter-species and play

398

significant roles in the spreading of antimicrobial resistance genes among bacteria.

Weltevreden, Kentucky,

399 400

4. Conclusions

401

In summary, our results had confirmed a relatively high rate of Salmonella contamination from

402

vegetables, chicken carcasses, and related processing environments in Malaysian fresh food

403

markets. These might act as the reservoirs for antimicrobial resistant Salmonella which harboured

404

mobile genetic elements. Class 1 integrons situated on transferable plasmids may contribute to the

405

dissemination of antibiotic resistance among Salmonella. Our findings highlight the need for

406

stringent sanitation and hygienic standards in fresh food markets to reduce the occurrence

407

of Salmonella as well as the cautious use of antibiotics in poultry production to limit the emergence

408

of antibiotic resistance in foodborne zoonotic bacterial pathogens.

409 410

Acknowledgments

411

This work was supported by the Universiti Sains Malaysia (grants number 1001/PTEKIND/811289).

412

The authors thank IPS-USM for awarding Mustapha Goni Abatcha with USM Global Fellowship to

413

undertake this study and Public Health Laboratory, Perak, Ministry of Health Malaysia for the

414

serotyping. 17

ACCEPTED MANUSCRIPT 415

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Van Belkum, A., Tassios, P., Dijkshoorn, L., Haeggman, S., Cookson, B., Fry, N., Fussing, V., Green, J., Feil, E., & Gerner-Smidt, P. (2007). Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clinical Microbiology and Infection, 13(s3), 1-46. Van, T. T. H., Moutafis, G., Istivan, T., Tran, L. T., & Coloe, P. J. (2007). Detection of Salmonella spp. in retail raw food samples from Vietnam and characterization of their antibiotic resistance. Applied and environmental microbiology, 73(21), 6885-6890. Van, T. T. H., Nguyen, H. N. K., Smooker, P. M., & Coloe, P.J (2012). The antibiotic resistance characteristics of non-typhoidal Salmonella enterica isolated from food-producing animals, retail meat and humans in South East Asia. International Journal of Food Microbiology, 154(3), 98-106. Villa-Rojas, R., Zhu, M.-J., Paul, N. C., Gray, P., Xu, J., Shah, D. H., & Tang, J. (2017). Biofilm forming Salmonella strains exhibit enhanced thermal resistance in wheat flour. Food Control, 73, 689-695. Wain, J., & Kidgell, C. (2004). The emergence of multidrug resistance to antimicrobial agents for the treatment of typhoid fever. Transactions of the Royal Society of Tropical Medicine and Hygiene, 98(7), 423-430. Weill, F.-X., Fabre, L., Grandry, B., Grimont, P. A., & Casin, I. (2005). Multiple-antibiotic resistance in Salmonella enterica serotype Paratyphi B isolates collected in France between 2000 and 2003 is due mainly to strains harboring Salmonella genomic islands 1, 1-B, and 1-C. Antimicrobial agents and chemotherapy, 49(7), 2793-2801. White, D. G., Zhao, S., Sudler, R., Ayers, S., Friedman, S., Chen, S., McDermott, P. F., McDermott, S., Wagner, D. D., & Meng, J. (2001). The isolation of antibiotic-resistant Salmonella from retail ground meats. New England journal of medicine, 345(16), 1147-1154. Yasin, R. M., Jegathesan, M., & Tiew, C. C. (1997). Salmonella serotypes isolated in Malaysia over the ten-year period 1983-1992. Asia-Pacific Journal of Public Health, 9(1), 1-5. Yoke-Kqueen, C., Learn-Han, L., Noorzaleha, A., Son, R., Sabrina, S., Jiun-Horng, S., & Chai-Hoon, K. (2008). Characterization of multiple-antimicrobial-resistant Salmonella enterica subsp. enterica isolated from indigenous vegetables and poultry in Malaysia. Letters in applied microbiology, 46(3), 318-324. Zhao, S., White, D., Friedman, S., Glenn, A., Blickenstaff, K., Ayers, S., Abbott, J., Hall-Robinson, E., & McDermott, P. (2008). Antimicrobial resistance in Salmonella enterica serovar Heidelberg 24

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isolates from retail meats, including poultry, from 2002 to 2006. Applied and environmental microbiology, 74(21), 6656-6662.

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ACCEPTED MANUSCRIPT Highlights 

The overall prevalence of Salmonella spp. was 29.1% (187/642) for all samples.



A total of 187 Salmonella isolates belonging to 37 different serovars were isolated.



Approximately 55% of the Salmonella spp. isolates were multidrug resistant.



Eleven different resistance genes were detected in antibiotic resistant isolates.



Class 1 integrons with variable gene cassettes were detected.

1 2 3

Table 1 Primer sequences, PRC conditions and source of primers for amplification of antimicrobial resistance genes use for the study Genes tet A tet B tetC tet G str A str B aadA sul1 sul 2 blaTM1 temA temB cmlA cat1 ca2 floR

Primers sequence (5`to3`) F-GTAATTCTGAGCACTGTCG R-CTGCCTGGACAACATTGCTT F-CTCAGTATTCCAAGCCTTTG R-ACTCCCCTGAGCTTGAGGGG F-GGTTGAAGGCTCTCAAGGGC R-CCTCTTGCGGGAATCGTCC F-GCAGCGAAAGCGTATTTGCG R-TCCGAAAGCTGTCCAAGCAT F-CCAATCGCAGATAGAAGGC R-ATCGTCAAGGGATTGAAACC F-ATCGTCAAGGGATTGAAACC R-GGATCGTAGAACATATTGGC F-ATCCTTCGGCGCGATTTTG R-GCAGCGCAATGACATTCTTG TCA CCG AGG ACT CCT TCT TC CAG TCC GCC TCA GCA ATA TC F-GCGCTCAAGGCAGATGGCAT R-GCGTTTGATACCGGCACCCT F-ACCAATGCTTAATCAGTGAG R-ACCAATGCTTAATCAGTGAG F-ATGAGTATTCAACATTTCCG R-CTGACAGTTACCAATGCTTA F-TTTTCGTGTCGCCCTTATTCC R-CGTTCATCCATAGTTGCCTGACTC F-CGCCACGGTGTTGTTGTTAT R-GCGACCTGCGTAAATGTCAC F-CTTGTCGCCTTGCGTATAAT R-AACGGCATGATGAACCTGA F-AACGGCATGATGAACCTGAA R-ATCCCAATGGCTCGTAAAG F-CTGAGGGTGTCGTCATCTAC R-GCTCCGACAATGCTGACTAT

PCR conditions 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 53 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 53 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 53 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 53 °C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 35 cycles of 1 min at 94 C, 1 min. at 50 °C and 1 min at 72 °C; 10 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62°C and 1 min at 72 °C; 7 min at 72 °C 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62°C and 1 min at 72 °C; 7 min at 72 °C 10 min at 95 °C; 30 cycles of 30 s at 95 °C, 1 min at 55 °C and 1 min at 72 °C; 7 min 72 °C 10 min at 95 °C; 30 cycles of 30 s at 95 °C, 1 min at 55 °C and 1 min at 72 °C; 7 min 72 °C 10 min at 95 °C; 30 cycles of 30 s at 95 °C, 1 min at 55 °C and 1 min at 72 °C; 7 min 72 °C 10 min at 95 °C; 30 cycles of 30 s at 95 °C, 1 min at 55 °C and 1 min at 72 °C; 7 min 72 °C

1

Product Size 957bp

Reference Aarestrup et al., (2003)

414bp

Aarestrup et al., (2003)

505bp

Aarestrup et al., (2003)

662bp

Aarestrup et al., (2003)

548bp

Aarestrup et al., (2003)

507bp

Gebreyes and Altiers (2002)

282bp

Aarestrupet al., (2003)

435bp

Aarestrup et al., (2003)

293bp

Aarestrup et al., (2003)

857bp

Oslen et al., (2004)

867bp

Oliver et al., (2002)

798bp

Wain et al., (2003)

393bp

Chen et al., (2004)

508bp

Chen et al., (2004)

547 bp

Chen et al., (2004)

673 bp

Chen et al., (2004)

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Table 2 Prevalence of Salmonella species from vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets. Samples Chicken Chicken cuts Whole chicken Total Environment Display table Chopping board Wash water Drain water Butcher apron Knife Transport crate Floor Scalding tank Defeathering machine Cage Drain crevices Bench water Drum Total Vegetables Amaranth green Amaranth red Bean sprouts Coriander Water spinach Winged bean Laksa leaves Iceberg lettuce Mint Spring Onion Indian pennywort Wild parsley Lettuce salad Chinese f. Cabbage Sweet basil Japanese parsley Total Overall

No. of Samples tested

No. of positive samples

% Prevalence

18 17 35

9 8 17

50.0 47.0 48.0

20 17 15 17 14 15 13 15 15 13 16 10 9 13 202

10 9 8 13 6 6 1 10 3 3 4 4 1 5 83

50.0 53.0 53.0 76.0 43.0 40.0 8.00 67.0 20.0 15.0 25.0 40.0 11.0 38.0 41.0

26 25 25 25 26 25 26 25 25 25 25 25 25 26 26 25 405 642

7 6 7 13 8 5 4 2 3 1 6 6 8 4 2 5 87 187

27.0 28.0 28.0 52.0 31.0 20.0 15.4 8.0 12.0 4.0 24.0 24.0 32.0 11.5 8.0 16.0 21.5 29.1

9 10 11 12 13 14 2

ACCEPTED MANUSCRIPT 15 16 17 18

Table 3 Salmonella serovars isolated from vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets Serovar S. Aberdeen S. Albany S. Augustenborg S. Bareilly S. Braenderup S. Brancaster S. Cerrot S. Corvallis S. Djugu S. Duessedorlf S. Dumfries S. Enteritidis S. Gaminara S. Give S. Haifa S. Hvittingfoss S. Indiana S. Kastrup S. Kentucky S. Lindenburg S. Mbandaka S. Minnesota S. Mkamba S. Molade S. Newport S. Paratyphi B S. Planckendael S. Obugu S. Ohio S. Redhill S. Richmond S. Salamae serovar II,19,12,Iv,z39 S. Stanley S. Tudu S. Typhimurium S. Wandsworth S. Weltevredent

Vegetable 4 4 4 1 3 5 1 9 1 2 4 5 1 1 2 1 2 5 2 1 1 3 4 1

Chicken 3 5 7 1 1 -

-

Environment 8 18 33 2 1 2 1 1 4 1 2 1 1 1 -

Total (%) 4 (2.1) 15 (8.0) 4 (2.1) 1 (0.53) 3 (1.6) 28 (14.9) 1 (0.53) 49 (26.2) 1(0.53) 2 (1.1) 2 (1.1) 4 (2.2) 1(0.53) 3 (1.6) 1(0.53) 6 (3.2) 5 (2.7) 1(0.53) 1(0.53) 1(0.53) 4 (2.1) 1(0.53) 1(0.53) 1(0.53) 2 (1.0) 6 (3.2) 2 (1.1) 1(0.53) 1(0.53) 3 (1.6) 4 (2.1) 1(0.53)

1 5 14

-

1 1 1 1 3

2 (1.1) 1(0.53) 6 (3.2) 1(0.53) 17 (9.1)

-

19 20 21 22 23 24 25 26 3

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Table 4 Antimicrobial resistance of Salmonella serovars from vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets Antimicrobials Vegetable Chicken Environment Total (n=187) (n=87) (n=17) (n=83) (%) resistant (%) resistant (%) resistant (%) Ampicillin 12 (13.8) 6 (35.3) 32 (38.5) 50 (26.7) Amoxicillin–clavulanate 0 (0) 0 (0) 4 (4.8) 4 (2.1) Cephalothin 2 (2.3) 1 (5.9) 6 (7.2) 9 (4.8) Chloramphenicol 9 (10.3) 6 (35.3) 26 (31.3) 41(21.9) Tetracycline 17 (19.5) 8 (47.0) 58 (69.8) 83 (44.3) Gentamycin 2 (2.3) 1 (5.9) 3 (3.6) 6 (3.2) Streptomycin 32 (36.8) 12 (70.6) 73 (84.9) 117 (62.6) Kanamycin 10 (11.5) 3 (17.6) 8 (9.6) 21 (11.2) Sulfonamides 22 (25.3) 9 (53.0) 52 (62.6) 83 (44.3) Trimethoprim–sulphamethoxazole 4 (4.6) 6 (35.3) 21 (25.3) 31 (16.6) Nalidixic acid 2 (2.3) 6 (35.3) 16 (19.3) 24 (12.8) Ciprofloxacin 0 (0) 0 (0) 0 (0) 0 (0) Resistance to 1 Antimicrobial 15 (17.2) 2 (17.8) 2 (2.4) 19 (10.1) Resistance to 2 Antimicrobial 6 (6.9) 0 (0) 9 (10.8) 15 (8.0 Resistance to ≥ 3 Antimicrobial 19 (21.8) 12 (70.6) 72 (86.7) 103 (55.1) 30 n= total number of isolates 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

4

54 55

Table 5 Antibiotic resistance profile, sources and multiple antibiotic resistance (MAR) indexes of individual Salmonella serovars obtained in this study Name of serovar Aberdeen

Antibiotic-resistant profile SS₃

No. of isolates 1

Source Vegetable

MAR index 0.16

Albany

Ste SS₃ AmpCNaSSxt AmpNaSSxtTeS₃ AmpCNaSSxtTeS₃ AmpCKNaSTeS₃ AmpKfCNaSSxtS₃ AmpCKNaSSxtTeS₃ AmpKfCNaSSxtTeAmc AmpKfCKNaSTeS₃ AmpKfCKNaSSxtTeS₃Amc K S

1 2 1 1 1 2 1 1 1 2 1 1 1

Vegetable Vegetable, Environment Chicken Chicken Environment Environment Chicken Environment Environment Vegetable Environment Vegetable Vegetable

0.16 0.16 0.41 0.5 0.58 0.58 0.58 0.66 0.66 0.66 0.83 0.08 0.08

AmpC Ste AmpKfS NaSTe AmpCS AmPCK CKSxt AmpSTe AmpCKTe AmpCSTe CKSSxtTe AmpCSSxtTe AmpCSTeS₃ AmpCCnSTeS₃ AmpCSSxtTeS₃

1 1 1 1 2 1 2 1 2 3 1 1 1 1 1

Environment Environment Environment Chicken Environment Vegetable Environments Environment Vegetable Environment Vegetable Environment Environment Chicken Environment

0.16 0.16 0.25 0.25 0.25 0,25 0.25 0.25 0.33 0.33 0.41 0.41 0.41 0.41 0.5

Augustenborg Brancaster Brancaster

5

Corvallis

Djugu Duessedorlf Dumfries Enteritidis Give Hvittingfoss Indiana Kentucky Lindenburg Mbandaka

AmpCSSxtTeS₃ AmpCKSSxtS₃ AmpCKSSxtTe AmpCKSSxtTeS₃ Na S SS₃ KTeS₃ STeS₃

1 1 2 3 1 1 2 1 28

KSTeS₃ AmpSTeS₃ NaSSxtS₃ STeS₃ AmpKfSSxtTeAmc AmpCNaSSxtS₃ AmpKSSxTS₃ S S₃ AmpCCnKNaSSxt AmpCNaSSxtTeS3 AmpCCnNaSSxtTeS3 Te Ste SS₃ S₃ S, Te, AmpSTe AmpCnSTeS₃ STe S₃ CnS Na STe S₃

1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 2

Environment Environment Vegetable Chicken, Environment (2) Chicken Vegetable Chicken Environment Vegetable (4), Environment (19), Chicken (5) Environment Vegetable Environment Vegetable Environment Environment Vegetable Vegetable Vegetable Environment Environment Environment Vegetable Vegetable Vegetable Environment Environment (2) Chicken Environment Vegetable Environment Vegetable Environment Environment, Vegetable 6

0.5 0.5 0.5 0.58 0.08 0.08 0.16 0.25 0.25 0.33 0.33 0.33 0,25 0.5 0.5 0.41 0.08 0.08 0.58 0.58 0.66 0.03 0.16 0.16 0.08 0.16 0.25 0.41 0.25 0.16 0.25

56 57 58

Molade Newport Paratyphi B

60

Richmond Salamae serovar II,19,12,Iv,z39 Stanley

61

Typhimurium

59

62 63 64 65 66 67 68

Tudu Weltevreden

AmpCSSxtTe S₃ Na AmpCSS₃ S S

1 1 1 1 2 1

Environment Vegetable Vegetable Vegetable Vegetable Vegetable

0.41 0.08 0.08 0.33 0.08 0.08

S AmpCSTeSxt Te STeS₃ AmpCSTe S₃ Te S NaS CSS₃ AmpCKNaSSxtTeS S₃

1 1 1 1 1 1 1 1 1 1

Vegetable Environment Vegetable Vegetable Environment Environment Vegetable Environment Vegetable Environment

0.08 0.41 0.08 0.25 0.41 0.08 0.16 0.25 0.25 0.66

Amp (Ampicillin), Kf (Cephalothin), Cip (Ciprofloxacin), C (Chloramphenicol), CN (Gentamycin), K (Kanamycin), Na (Nalidixic acid), S (Streptomycin), Sxt (Sulphamethoxazole/ trimethoprim 19:1), Te (Tetracyclin), S₃ (Compound sulfonamides), Amc (Amoxicillin/clavulanic acid)

7

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Table 6 Distribution of antibiotic resistance genes among Salmonella serovars from vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets Antimicrobial resistance Ampicillin

No. of resistant isolates 50

Genes detected blaTEM/ temB temB

No. of isolates 36 14

Chloramphenicol

41

floR cmlA floR/ cmlA

24 8 4

Sulphonamides

83

Sul1/Sul2 Sul1 Sul2

5 7 47

Streptomycin

117

strA/strB aadA strA/strB/aadA

38 32 14

Tetracycline

83

tetA tetA/tetB

76 3

74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 8

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107 108 109 110

Tables 7 Distribution of class 1 integron in antibiotic resistance Salmonella serovars from vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets Integron Profile (IP) Size (Kb) Genes cassettes IP-1 0.3 dfrA1, dfrA12 IP-2 0.3, 1.2 dfrA12-orf-aadA2 IP-3 1.5 dfrA12 IP-4 0.3, 1.0 aadA2-blaPSE−1 IP-5 0.5 dfrA12 n= total isolates that are carrying class 1 integron

9

No. of isolates (n=17)

Percentage profile

5 2 3 3 4

29.4 11.8 17.6 17.6 23.5