MULTIDRUG-RESISTANT SALMONELLA

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1 OCCURRENCE OF MULTIDRUG-RESISTANT SALMONELLA
2 ENTERICA IN RETAIL CHICKEN MEAT AND DEVELOPMENT
3 OF A SIX GENES-BASED MULTIPLEX PCR AS AN
4 ALTERNATIVE DIAGNOSTIC METHOD
5 H.S. AMINAQ5
1
, A.A. ABDELRAHMAN1
and G.S. ABDELLRAZEQ2,3
6 1
Department of Bacteriology, Animal Health Research Institute, Alexandria Provincial Laboratory, Dokky, Giza, Egypt
2
Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Rosetta-line, PO Box 22758, Egypt
3
Corresponding author.
TEL: 002 0452960306;
FAX: 0020452960450;
EMAIL: gaber.abdellatif@alexu.edu.eg
Received for Publication July 28, 2015
Accepted for Publication November 24, 2015
doi: 10.1111/jfs.12260
7 ABSTRACT
8 This study was aimed to develop a rapid discriminatory PCR-based assay for
9 detection and differentiation the most common Salmonella serovars in chicken
10 meat and their products. Definite six target genes were selected for their ability to
11 give unique results in a simple multiplex PCR assay to identify Salmonella
12 typhimurium, Salmonella kentucky and Salmonella enteritidis with multidrug
13 resistance properties. Three hundred chicken meat samples were analyzed using
14 conventional methods. The isolates were identified, serotyped and tested against 16
15 antibiotics. Isolated Salmonella serovars were further analyzed for the presence of
16 six target genes (invA, avrA, stn, fliC, stm and sefA) using two multiplex PCR
17 assays. 2.5, 0.5 and 0.5% of raw chicken meat were positive for S. typhimurium,
18 S. kentucky and S. enteritidis, respectively. All isolates were resistant, at least, to
19 three antibiotics. Multiplex PCR gave 284 bp (invA), 422 bp (avrA) and 617 bp
20 (stn) fragments belonging to the genus Salmonella, and additional three fragments
21 of 559 bp (fliC), 915 bp (stm) and 310 bp (sefA) which allowed distinct
22 identification of S. typhimurium, S. kentucky and S. enteritidis. The developed six
23 genes-based multiplex PCR in this study is a potential alternative method for
24 discrimination of the most common serotypes of Salmonella.
25 PRACTICAL APPLICATIONS
We developed a six genes-based multiplex PCR for effective discrimination of the
26 most common foodborne Salmonella serovars with multidrug resistance
27 prosperities. Although there are few studies about the incidence of Salmonella in
28 retail meat in Egypt, our study is the first one in Alexandria (on the Mediterranean
29 Sea in the north central part) about multidrug-resistant (MDR) serovars of
30 Salmonella in retail chicken meat and their products. If we analyze all these
31 findings to see the potential risk assessments in this region, our study could be a
32 leading report in near future.
33
34
35 INTRODUCTION
36 Salmonella has been recognized as an important foodborne
37 pathogen for humans over more than a century, causing
38 human foodborne illness as well as high medical and eco-
39 nomical costs (Lee et al. 2015). Approximately 93.8 million
40 human cases of gastroenteritis and 155,000 deaths world-
41wide are caused by Salmonella infection each year (Majowicz
42et al. 2010). Despite well-established instructions and meas-
43ures for preventing salmonellosis (Salmonella food poison-
44ing) in the developed countries, these considerations are
45lacking in the developing countries (Lee et al. 2015). Over
462,500 serovars of Salmonella have been reported (Fierer and
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47 Guiney 2001) based on the Kauffmann–White scheme, for
48 subtyping and strain differentiation (Kauffmann 1972). In
49 particular, Salmonella serotypes Enteritidis and Typhimurium
50 are of primary importance in human health (Hendriksen
51 et al. 2011). Salmonellosis caused by Salmonella enteritidis is
52 usually related to the consumption of eggs or poultry meat,
53 while foodstuffs of bovine, poultry or porcine origin are the
54 main sources of illness caused by Salmonella typhimurium
55 (Anonymous 2010).AQ2 Conversely, the inappropriate use of
56 antimicrobials both in human and in veterinary medicine
57 and the complex interactions between human health, animal
58 husbandry and veterinary medicine, have contributed to the
59 propagation and spread of antibiotic resistant Salmonella
60 (World Health Organization 2014).
61 Salmonella surveillance and monitoring which based on
62 the reliable and efficient detection methods, should help
63 improve the food safety (Rodriguez-Lazaro et al. 2007).
64 Conventional culture methods serve as the basis in food test-
65 ing laboratories despite rather laborious and time-
66 consuming protocols (Lee et al. 2015). Considerable progress
67 in rapid methods such as PCR yields faster answers and
68 higher throughput of samples (Lee et al. 2015). However, the
69 specificity of the target sequence becomes crucial for accu-
70 racy and precision of the PCR method (Liu et al. 2012).
71 Since there are various antigen genes in Salmonella serovars,
72 more than five primer sets would be designed in a multiplex
73 PCR to identify two serovars simultaneously.
74 The main objective of the study was to develop a rapid
75 discriminatory PCR-based assay for detection and differen-
76 tiation the most common Salmonella serovars in chicken
77 meat and their products. Definite six target genes were
78 selected for their ability to give unique results in a simple
79 multiplex PCR assay to identify S. typhimurium, Salmonella
80 kentucky and S. enteritidis with multidrug resistance prosper-
81 ities. To achieve this objective, chicken samples were col-
82 lected and processed for isolation of Salmonella enterica
83 using conventional methods then the isolates were tested for
84 their antimicrobial susceptibility against 16 antimicrobial
85 agents using the minimum inhibitory concentrations
86 (MICs) method. The isolated Salmonella serovars were fur-
87 ther investigated by multiplex PCR using six sets of primers
88 selected from different target gene sequences (invA, stn,
89 avrA, fliC, stm and sefA).
90 MATERIALS AND METHODS
91 Samples Collection
92 Three hundred chicken samples were purchased at random
93 over the period 2013–2014 from several retail stores and
94 supermarkets located in Alexandria city, Egypt. Each of the
95 samples purchased in a single retail store or supermarket.
96 The following samples were studied: (1) whole raw chickens
97(n 5 200), (2) ready to eat chicken meat products (n 5 100;
98250 g each) including chicken shawarma (traditional Egyp-
99tian fast food), and burger. After purchase, each sample was
100transported in its original container after individually packed
101into a polyethylene bag then marked and transferred in ice
102box to the laboratory of Bacteriology, Animal Health
103Research Institute, Alexandria, wherein the conventional
104bacteriological analysis were done.
105Isolation of Salmonella
106The preparation of samples for detection of Salmonella were
107performed according to the International Organization for
108Standardization (ISO 6579: 2002; International Organization
109for Standardization 2002). Briefly, 25 g from each of meat
110samples were minced into fine pieces using autoclaved
111equipment and placed in a separate stomacher bag contain-
112ing 225 mL of sterile buffered peptone water (Oxoid CM
113509) and homogenized using a stomacher. The pre-
114enrichment broth was then incubated at 37C for 18 h. An
115inoculum (1 mL) from pre-enrichment broth was inoculated
116into a tube of selective enrichment liquid media containing
11710 mL of Muller–Kauffmann tetrathionate/novobiocin broth
118(Oxoid CM1048) and incubated at 37C for 24 h. A loopful
119of inoculum from each of enrichment cultures were inocu-
120lated on the surface of two different plates containing
121Xylose-Lysine-Desoxycholate agar (XLD; Oxoid CM0469)
122and Brilliant Green Agar w/Sulfadiazine (BGA; Neogen-
1237310). XLD plates were incubated at 37C for 24 h, while
124BGA plates were incubated at 35C for 24 h.
125Presumptive Salmonella colonies isolated on plating
126media were used for Gram staining and biochemical confir-
127mation. Triple sugar iron agar (BD Difco-226540), lysine
128iron agar (BD Difco-284920), urea agar (BD BBL-221096)
129and Simmons citrate agar (Neogen-7156) were inoculated
130and incubated at 37C for 24 h. The isolates were further
131identified with API identification kits (bioMerieux, Marcy,
132France).
133Serotyping
134Serological tests for biochemically confirmed isolates were
135performed according to Kauffmann–White Scheme (Kauff-
136mann 1972) by slide agglutination test with the use of rapid
137diagnostic Salmonella antisera sets (Wellcome Diagnostic),
138which typically utilize polyvalent antisera for somatic (O)
139and flagellar (H) antigens. A drop of each of separate O and
140H Salmonella factors were added separately to each of the
141cell suspensions then thoroughly mixed and the positive
142agglutination observed within a minute. A delayed or partial
143agglutination was considered as negative or false result. Iso-
144lates with a typical biochemical profile, which agglutinate
145with both H and O antisera, were identified as Salmonella
146serovars. All serovars were kept at 280C in brain–heart
OCCURRENCE OF MULTIDRUG-RESISTANT SALMONELLA ENTERICAAQ1 H.S. AMIN, A.A. ABDELRAHMAN and G.S. ABDELLRAZEQ
2 Journal of Food Safety 00 (2015) 00–00 VC 2015 Wiley Periodicals, Inc.

147 infusion broth (Oxoid CM1135) with the addition of 20%
148 (V/V) glycerol.
149 Antimicrobial Susceptibility Testing
150 The MICs of antibiotics were determined by an agar dilution
151 method with tested Salmonella isolates in Mueller–Hinton
152 agar (Oxoid CM0337) supplemented with 5% defibrinated
153 horse blood according to CLSI standards (Clinical and Labo-
154 ratory Standards Institute 2014). Aliquots of 5 3 104
cfu
155 were inoculated on agar plates that contained twofold serial
156 dilutions of the following 16 antibiotics (Britania): ticarcillin,
157 piperacillin, piperacillin 1 tazobactam, aztreonam, ceftazi-
158 dime, cefepime, imipenem, meropenem, gentamicin, tobra-
159 mycin, amikacin, ciprofloxacin, pefloxacin, minocycline,
160 colistin and trimethoprim/sulfamethoxazole. The MIC was
161 the lowest concentration of antibiotic that completely inhib-
162 ited visible growth after incubation for 18 h at 37C. Esche-
163 richia coli ATCC 25922 was used as a quality control strain
164 in accordance with CLSI guidelines (Clinical and Laboratory
165 Standards Institute 2014).
166 Multiplex PCR
167 Aliquots of the storage solutions were plated on trypticase
168 soy agar (BD BBL-211043) with overnight incubation at
169 37C. Bacterial genomic DNA was then extracted after growth
170 overnight in Luria–Bertani broth (BD Difco-244620) by
171 using the DNA extraction kit (Qiagen-69581) according to
172 the manufacturer’s protocol and stored at 4C (Lee et al.
173 2009).
174 For the identification of the isolated Salmonella serovars,
175 six target sets of primers from different genomic sequences
176 were chosen for their ability to give unique results to identify
177 three Salmonella serovars (S. typhimurium, S. kentucky and
178 S. enteritidis) in multiplex PCR assays (Table.T1 1). All primer
179 pairs were synthesized and supplied by Sigma-Aldrich. First
180three primer sets specific for the genus Salmonella amplified
181284 bp fragment within the invA gene (Oliveira et al. 2003),
182617 bp fragment within the stn gene (Murugkar et al. 2003)
183and 422 bp fragment within the avrA gene (Huehn et al.
1842010). The fourth primer set amplified a 559 bp fragment
185specific for S. typhimurium (Fli15) and S. kentucky (Tym)
186within the fliC gene (SOUMET et al. 1999a). The fifth
187primer set amplified a 310 bp fragment specific for S. enteri-
188tidis within the sefA gene (Rahn et al. 1992). The sixth
189primer set amplified a 915 bp fragment specific for S. typhi-
190murium within the STM gene (Liu et al. 2012).
191Two multiplex PCR assays were performed: The first assay
192was carried out to determine the positivity of each Salmo-
193nella serovar for the presence of the three common genes
194(invA, avrA, stn), in addition to the related discriminatory
195gene. In the first assay, three PCR tubes were used for each
196of S. typhimurium, S. kentucky and S. enteritidis, respectively.
197The first tube for positive control (C1), the second tube for
198tested serovar and the third tube for negative control (C2).
199The reaction was performed in PCR mixture (50 lL total
200volume) included 25 lL master mix (Qiagen-200403), 2.5
201lL each of upper and lower primers and 5 lL of template
202bacterial DNA.
203Another assay was carried out to investigate whether the
204regions designed for the primers of the discriminatory genes
205are not serovar unique region and there is another positivity
206found in the other Salmonella enterica.
207In this assay, the numbers of PCR tubes and reaction vol-
208umes were the same as the first assay for each of the three
209serovars. However, instead of using the first tube of each set
210as C1, we used a DNA mixture from the other two serovars
211(2.5 lL DNA from each of the two serovars). All PCR reac-
212tions were carried out under the following conditions: dena-
213turation at 95C for 10 min, followed by 35 cycles of
214amplification (denaturation at 95C for 1 min, annealing at
21558C for 1 min and extension at 72C for 1 min), ending with
TABLE 1. TARGET GENES USED IN THE MULTIPLEX PCR ASSAYS FOR DETECTION AND IDENTIFICATION OF SALMONELLA ISOLATES FROM
RETAIL CHICKEN MEAT
Salmonella
isolate Gene
Gene
function
Primer
sets
primer sequences
(50
! 30
)
Size
(bp) Reference
Salmonella spp. invA Invasion protein st 139-s
st141-as
GTGAAATTATCGCCACGTTCGGGCAA
TCATCGCACCGTCAAAGGAACC
284 Oliveira et al.
2003
Salmonella spp. stn Enterotoxin stn p1
stn m13
TTG TGT CGC TAT CAC TGG CAA CC
ATT CGT AAC CCG CTC TCG TCC
617 Murugkar et al.
2003
Salmonella spp. avrA Effector protein avra CCT GTA TTG TTG AGC GTC TGG
AGA AGA GCT TCG TTG AAT GTC C
422 Huehn et al.
2010
S. typhimutium
S. kentucky
fliC Flagellar protein fli15
tym
CGGTGTTGCCCAGGTTGGTAAT
ACTCTTGCTGGCGGTGCGACTT
559 SOUMET et al.
1999b
S. enteritidis sefA Fimbrial Protein sefa2
sefa4
GCAGCGGTTACTATTGCAGC
TGTGACAGGGACATTTAGCG
310 Rahn et al.
1992
S. Typhimurim stm4495 Inner membrane
protein
stm-4495 GGTGGCAAGGGAATGAA
CGCAGCGTAAAGCAACT
915 Liu et al.
2012
H.S. AMIN, A.A. ABDELRAHMAN and G.S. ABDELLRAZEQ OCCURRENCE OF MULTIDRUG-RESISTANT SALMONELLA ENTERICAAQ1

216 a final extension at 72C for 10 min. The samples were kept
217 at 14C and processed within 24 h.
218 The amplified products (10 lL) were separated by electro-
219 phoresis on a 1.5% agarose at 100 V gel run for 90 min in
220 TBE buffer (89 mmol/L Tris–Borate, 2 mmol/L EDTA, pH
221 8Á3) and a 100 bp DNA ladder was used as a size reference.
222 The gel was then stained with 0.5 lg/mL ethidium bromide
223 solution for 20 min and photographed under UV light. The
224 presence of a clear fragment with the correct amplification
225 size was assessed as a positive signal indicating the presence
226 of the gene.
227 RESULTS
228 Prevalence of Salmonella Serovars
229 A total of 300 samples (200 samples from fresh chicken
230 meat, 100 samples from ready to eat chicken meat products)
231 were processed by the conventional microbiological meth-
232ods. As presented in Table T22, the results of the bacteriological
233methods and serological serotyping revealed out of 200 fresh
234chicken meat samples, five (2.5%), one (0.5%) and one
235(0.5%) were positive for S. typhimurium, S. kentucky and S.
236enteritidis, respectively. No Salmonella enterica could be
237recovered in ready to eat products.
238Antimicrobial Susceptibility
239All of the three Salmonella serovars tested were resistant to
240gentamicin, topramycin and amikacin. Both S. kentucky and
241S. enteritidis were also resistant to ticarcillin, piperacillin,
242ciprofloxacin, pefloxacin, minocycline and trimethoprim/
243sulfamethoxazole (Table T33).
244Efficacy of Developed PCR
245Six primer sets chosen from different genes were assessed to
246detect the genus Salmonella and their serovars. The first mul-
247tiplex PCR assay (Fig. F11. Lanes right to DNA ladder (L) lane)
TABLE 2. SALMONELLA SEROVARS FROM CHICKEN FRESH MEAT
Salmonella serovars
S. typhimurium S. kentucky S. enteritidis
Chicken samples No. of samples No. % No. % No. %
Fresh meat 200 5 2.5 1 0.5 1 0.5
Ready to eat products 100 0 0 0 0 0 0
TABLE 3. DISTRIBUTION OF RESISTANCE TO INDIVIDUAL ANTIMICROBIAL AGENTS AMONG SALMONELLA SEROVARS
Distribution of resistance to antimicrobials
S. typhimurium S. kentucky S. enteritidis
Antimicrobial MIC (mg/L) Interpretation MIC (mg/L) Interpretation MIC (mg/L) Interpretation
Penicillins Ticarcillin 8 S !128 R !128 R
Piperacillin 8 S !128 R !128 R
Piperacillin/
tazobactam
4 S 4 S 4 S
Other b-Lactams Aztreonam 1 S 1 S 1 S
Cephalosporins Ceftazidime 1 S 1 S 1 S
Cefepime 1 S 1 S 1 S
Carbapenems Imipenem 0.25 S 0.25 S 0.25 S
Meropenem 0.25 S 0.25 S 0.25 S
Aminoglycosides Gentamicin 1 R 1 R 1 R
Tobramycin 1 R 1 R 1 R
Amikacin 2 R 2 R 2 R
Quinolones Ciprofloxacin 0.25 S !4 R !4 R
Pefloxacin 0.25 S !16 R !16 R
Tetracyclines Minocycline 4 S !16 R !16 R
Miscellaneous
antibiotics
Colistin 0.5 S 0.5 S 0.5 S
Trimethoprim/
sulfamethoxazole
320 S !320 R !320 R
All Salmonella serovars tested were resistant to gentamicin, topramycin and amikacin. Both S. kentucky and S. enteritidis were also resistant to
ticarcillin, piperacillin, ciprofloxacin, pefloxacin, minocycline and trimethoprim/sulfamethoxazole.
S, sensitive; R, resistant.

248 gave positive results by all tested serovars that yielded three
249 fragments of 284 bp (invA), 422 bp (avrA) and 617 bp (stn)
250 belonging to the genus Salmonella (Fig. 1 a, c e bands). In
251 addition, S. typhimurium (T) was positive for stm4495 gene
252 (f band), S. kentucky (K) was positive for fliC gene (d band)
253 and S. enteritidis (E) was positive for sefA gene (b band).
254 In the other PCR assay (Fig. 1. Lanes left to DNA ladder
255 (L) lane), the Tym primer was used in combination with the
256 Fli15 primer to identify both S. typhimurium and S. ken-
257 tucky. However, the STM primer set was used to differentiate
258 between the two species. Under the conditions used, the fliC
259 gene (d bands) was positive by both S. typhimurium (T) and
260 S. kentucky (K). However, the stm4495 (f bands) and sefA (b
261 bands) genes were positive only by S. typhimurium (T) and
262 S. enteritidis (E), respectively.
263 DISCUSSION
264 Poultry is one of the most important reservoirs of Salmonella
265 that can be transmitted to humans through the dealing with
266 chicken meat and/or consumption of uncooked meat and
267 eggs (Wales and Davies 2011). Contamination of meat by
268 Salmonella may occur at abattoirs at any stage during butch-
269 ering and/or during subsequent handling, processing, prepa-
270 ration and distribution (Hendriksen et al. 2009).
271 Egypt is a developing country that lacks a routine Salmo-
272 nella surveillance (Barbour et al. 2015). The sporadic cases
273 of salmonellosis in Egypt were first reported by Ammar et al.
274 (2009), who documented the isolation of S. enteritidis from
275 chickens and other sources in Dakhlia governorate. Rabie
276 et al. (2012) documented a Salmonella food illness outbreak
277in humans, associated with Salmonella recovered from con-
278sumed poultry products and the common Salmonella sero-
279vars isolated were S. enteritidis and S. typhimurium. Adding
280to these documented reports, we report the incidence of Sal-
281monella serovars in retail chicken meat samples collected in
282Alexandria city. In this study, five (2.5%), one (0.5%) and
283one (0.5%) out of 200 fresh chicken meat samples, were pos-
284itive for S. typhimurium, S. kentucky and S. enteritidis,
285respectively. No Salmonella enterica could be recovered in
286ready to eat chicken meat products (n 5 100) in this study.
287In line with our results, previous study showed no Salmo-
288nella in chicken wieners (Bohaychuk et al. 2006). However,
289other studies have shown Salmonella contamination in sev-
290eral ready to eat chicken meat products. Arumugaswamy
291et al. (1995) examined 112 ready-to-eat foods (cooked
292chicken, beef and prawns) obtained from street vendors and
293found 14% of samples were contaminated with Salmonella.
294Osaili et al. (2014) examined 478 ready to eat chicken prod-
295ucts (shawirma, roasted and burger) and the prevalence of
296Salmonella serovars was 0.8%. As previous reports have eval-
297uated contamination in raw and ready to eat chicken prod-
298ucts, these values are not comparable with those from this
299study. Several factors could explain it, such as differences in
300sampling procedures, origin, age of the animals at slaughter
301and level of sanitation (Antunes et al. 2003). Therefore, we
302are unable to compare them directly. Despite this, we believe
303that the frequency of contaminated retail chicken meat in
304this study cannot be neglected. However, further study with
305larger sample size is needed to evaluate Salmonella contami-
306nation in chicken meat and ready to eat chicken products in
307Alexandria, Egypt.
C
O
L
O
R
FIG. 1. AMPLIFICATION PROFILES OF THE MULTIPLEX PCR ASSAYS
The results of the two multiplex PCR assays for S. typhimurium (T), S. kentucky (K) and S. enteritidis (E) are indicated above each lane. Lane L: The
DNA ladder. The lanes right to lane L represent the results of the first PCR assay. The lanes left to lane L represent the results of another PCR assay.
The lanes for the first PCR assay show the three Salmonella serovars were positive for invA, avrA and stn genes (a, c e bands). S. typhimurium (T)
was positive for stm4495 gene (f band). S. kentucky (K) was positive for fliC gene (d band). S. enteritidis (E) was positive for sefA gene (b band).
The lanes for the other PCR assay show the specificity of fliC (d), stm4495 (f) and sefA (b) genes for S. typhimurium (T), S. kentucky (K) and S.
enteritidis (E), respectively. The fliC gene (d bands) was positive by both S. typhimurium (T) and S. kentucky (K). However, the stm4495 (f bands)
and sefA (b bands) genes were positive only by S. typhimurium (T) and S. enteritidis (E), respectively. C1: Positive control for each species. C2:
Nuclease free water. All of the primer sets produced the predicted sizes.

308 The emergence of MDR Salmonella serovars against the
309 current antibiotics, as the result of the widespread overuse
310 and misuse of antimicrobials for the disease treatment and
311 growth promotion in poultry and livestock, has become a
312 major concern worldwide (Siriken et al. 2015). The MDR
313 Salmonella serovars have been proposed to be more virulent
314 than non-MDR Salmonella (Foley and Lynne 2008). In this
315 study, the antimicrobial resistance of the isolated Salmonella
316 from retail chicken meat was determined using 16 antimi-
317 crobial agents (Table 3). The Salmonella isolates were MDR
318 (resistant to three or more antibiotics). Interestingly, all of
319 the three Salmonella serovars tested were resistant to genta-
320 micin, topramycin and amikacin. Both S. kentucky and S.
321 enteritidis were also resistant to ticarcillin, piperacillin, cipro-
322 floxacin, pefloxacin, minocycline and trimethoprim/sulfa-
323 methoxazole. These results are similar to those described in
324 previous studies showing that Salmonella isolates in retail
325 meat products are commonly resistant to multiple antimi-
326 crobials (Yang et al. 2010; Sallam et al. 2014). Presence of
327 MDR Salmonella isolates in retail chicken meat indicates the
328 need for the development of new antibiotics since these
329 strains were resistant to the antibiotics which are widely
330 used for treatment of salmonellosis. Further studies involving
331 larger and more randomly selected samples are necessary to
332 more precisely determine whether there are increases in the
333 antimicrobial resistance between Salmonella isolates from
334 different sources. In accordance with a previous report (Sal-
335 lam et al. 2014), the study emphasized the need to imple-
336 ment proactive measures of hygienic practices and the
337 application of hazard analysis and critical control point in
338 the preparation and processing of foods to reduce Salmonella
339 contamination and the risk of human infection. Moreover,
340 increased consumer awareness on proper cooking of meat
341 and meat products before consumption is necessary.
342 The purpose of this study was to develop a six genes-
343 based multiplex PCR to determine and differentiate the Sal-
344 monella serovars contaminated the retail chicken meat. The
345 multiplex PCR applied gave positive results by all tested
346 serovars that yielded three fragments of 284 bp (invA), 422
347 bp (avrA) and 617 bp (stn) belonging to the genus Salmo-
348 nella, and additional three distinct amplified fragments of
349 559 bp (fliC), 915 bp (stm) and 310 bp (sefA) which allowed
350 identification of S. typhimurium, S. kentucky and S. enteriti-
351 dis (Fig. 1).
352 Since the effector gene (AvrA) was found in 80%
353 (STRECKEL et al. 2004), the invasin gene (invA) was found
354 in 97.9% (Diarra et al. 2014) of Salmonella enterica serovars,
355 and the heat-labile Salmonella enterotoxin (stn) serve as
356 effector proteins, which are involved in the pathogenesis of
357 salmonellosis, so the primer sets designed in this study for
358 invA, avrA and stn genes allow a simultaneous identification
359 of all pathogenic Salmonella within the genus level. This
360 assay is suggested to be useful especially for rapid screening
361of large numbers of samples contaminated with different
362Salmonella species.
363Although, several studies have been focused on the molec-
364ular identification of S. typhimurium, and S. enteritidis iso-
365lated from retail chicken meat (Cortez et al. 2006; Abd-
366Elghany et al. 2015; Wang et al. 2015), few studies have been
367carried out on S. kentucky (Diarra et al. 2014). In this study,
368the Tym primer was used in combination with the Fli15
369primer to identify both S. typhimurium and S. kentucky.
370However, the STM primer set was used to differentiate
371between the two species. Under the conditions used, both S.
372typhimurium and S. kentucky gave a 559 bp amplified frag-
373ment with the Fli15-Tym primer set. However, only S. typhi-
374murium gave a 915 bp amplified fragment with the STM
375primer set (Fig. 1). These findings suggest the multiplex
376PCR assays described here are reliable for identification of
377Salmonella serovars (Fig. 1). This method could be an alter-
378native or a complementary method to the traditional identi-
379fication methods of Salmonella.
380In conclusion, our findings indicated that Salmonella con-
381tamination was common in retail chicken meats in Alexan-
382dria, Egypt. The Salmonella isolates were phenotypic and
383genetic diverse with the dominance of S. typhimurium, S.
384kentucky and S. enteritidis which harboring virulence deter-
385minants. Additionally, the isolates were resistant to multiple
386antimicrobials, a major concern for food safety and public
387health.
388The multiplex PCR described her can easily be imple-
389mented in diagnostic and food analysis laboratories for iden-
390tification and discrimination of the most common
391Salmonella species contaminate the retail chicken meat. Fur-
392ther studies on large scales are needed to establish the effi-
393cacy of the developed multiplex PCR assay and clarify its
394potential use as an ancillary method for identification and
395discrimination of Salmonella in foodstuffs.
396CONFLICT OF INTERESTS
397The authors declare that there is no conflict of interests.
398AUTHORS’ CONTRIBUTIONS
399GA and HA designed the experiment. HA, AA and GA con-
400ducted the experimental work. HA, AA, and GA were
401involved in scientific discussion and analysis of the data. GA
402drafted and revised the manuscript. All authors read and
403approved the final manuscript.
404ACKNOWLEDGMENTS
405This study was funded by Animal Health Research Institute,
406Alexandria Provincial Laboratory and by the Research
407Enhancement Program (ALEX REP), Alexandria University

408 Research Fund (AURF). The authors are thankful to Dr.
409 Samy Khaliel, Department of Microbiology, Faculty of Vet-
410 erinary Medicine, Alexandria University for providing facili-
411 ties for the current study.
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