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Prevalence, molecular identification and antimicrobial resistanceprofile of Salmonella serovars isolated from retail beef products inMansoura, Egypt

Khalid Ibrahim Sallam a,*, Mahmoud Ahmed Mohammed a, Mohammed Ahmed Hassan b,Tomohiro Tamura c

aDepartment of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, Mansoura, EgyptbDepartment of Food Control, Faculty of Veterinary Medicine, Benha University, EgyptcBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku,Sapporo 062-8517, Japan

a r t i c l e i n f o

Article history:Received 21 March 2013Received in revised form11 October 2013Accepted 19 October 2013

Keywords:Salmonella serovarsgyrB geneinvA genestn geneAntimicrobial resistanceBeef

a b s t r a c t

The present study was undertaken to determine the prevalence of Salmonella in 270 raw meat samples(90 each of fresh beef, ground beef, and beef burger) purchased on nine occasions from various super-markets and butchers’ shops in Mansoura city, Egypt. Using conventional biochemical identification,Salmonella species were recovered from 23.3% (21/90), 20% (18/90), and 12.2% (11/90) of fresh beef,ground beef and beef burger samples, respectively with an overall prevalence of 18.5% (50/270) amongall the meat products examined. Higher prevalence were obtained based on molecular identification, bydetecting gyrB and invA genes, which verified the presence of Salmonella species in 30.0% (27/90), 26.7%(24/90), and 16.7% (15/90) of fresh beef, ground beef, and beef burger samples, respectively with anoverall prevalence of 24.4% (66/270) among all the meat products tested. Of the 2635 presumptivecolonies tested, 228 were biochemically identified as Salmonella, while 272 were molecularly identifiedas Salmonella, which were all positive for the enterotoxin (stn) virulent gene. Of the 272 serologicallytested strains, 266 were serologically identified into six Salmonella serovars, while 6 strains wereuntypable. Salmonella Typhimurium and Salmonella Enteritidis were the most prevalent serovars with anincidence of 38.2% (104/272) and 34.6% (94/272), respectively. The other four serovars identified wereSalmonella Haifa, Salmonella Muenster, Salmonella Virchow, and Salmonella Anatum were detected atlower prevalences of 11% (30/272), 7.4% (20/272), 4% (11/272) and 2.6% (7/272), respectively. Interestingly,the antimicrobial susceptibility testing indicated that all of the 100 Salmonella serovars tested weremultidrug resistant (resistant to three or more antibiotics). Our findings demonstrated that the retail beefproducts tested were widely contaminated with multidrug-resistant Salmonella and such contaminationmay constitute a major public health concern.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The genus Salmonella contains two species, Salmonella bongoriand Salmonella enterica. S. enterica is divided into six subspecies;enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae(IV), and indica (VI) (Grimont & Weill, 2007). Currently, over 2587serovars have been identified in this genus, and 99% of Salmonellaserovars responsible for diseases in warm-blooded animals aremembers of group I (S. enterica) (Chan et al., 2003). The major

pathogenic serovars of S. enterica infected human from food ofanimal origin include Salmonella Typhimurium, Salmonella Enter-itidis, S. Newport, and S. Heidelberg (Hur, Jawale, & Lee, 2012).

It has been estimated that there are 9.4 million cases of food-borne illness each year in the United States; of which 55,961 casesare hospitalized and 1351 deaths; over one million and twentyseven thousand cases of this foodborne illness caused by non-typhoidal Salmonella, which resulting in 19,336 hospitalizationsand 378 deaths (Scallan et al., 2011).

Food animals harbor a wide range of Salmonella serovars andtherefore act as a source of contamination in non-typhoid humansalmonellosis (Acha & Szyfres, 2001, pp. 233e246). Contaminationof meat by Salmonella may occur at abattoirs during the removal of

* Corresponding author. Tel.: þ20 1000479670.E-mail address: khsallam@hotmail.com (K.I. Sallam).

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the gastrointestinal tract, contaminated abattoir equipment, floorsand personnel, while the pathogen can gain access to meat at anystage during butchering. Cross-contamination of carcasses andmeat products could continue during subsequent handling, pro-cessing, preparation and distribution (Adesiyun & Oni, 1989;Hendriksen et al., 2009).

The gyrB gene encoding the b-subunit of DNA gyrase and inva-sion A (invA) gene have been used as the target for the detectionand identification of Salmonella (Bülte & Jakob, 1995; Ye, Wang, &Lin, 2011). Moreover, the enterotoxin (stn) gene, which encodes aprotein that mediates severe diarrhea, was demonstrated as asuitable PCR target for detection of Salmonella strains (Prager, Fruth,& Tschäpe, 1995).

The gyrB gene sequencing might be more useful for bacterialidentification to the species level. The gyrB is a single-copy gene,distributed universally among bacteria, which encodes the ATPasedomain of DNA gyrase, an enzyme essential for DNA replication.The amino acid sequences of gyrB are conservative enough to allowthe comparison of taxa which are not closely related (Yamamoto &Harayama, 1996). The chromosomally located invA gene containssequences unique to Salmonella is found in all known serovars andhas been established as an international standard suitable PCR forrapid, less expensive, and sensitive detection of this genus (Rahnet al., 1992). This virulent gene encodes a protein in the innermembrane of bacteria which is responsible for invasion of Salmo-nella into the host epithelial cells (Torpdahl, Skov, Sandvang, &Baggesen, 2005).

The widespread overuse and misuse of antimicrobial agents infood animal production has contributed to the development ofantimicrobial resistant pathogens such as Salmonella that hasemerged as a major health problem worldwide.

The goal of this study was to determine the incidence of Sal-monella spp. in fresh beef, ground beef, and beef burger samplessold in Mansoura city, Egypt using both conventional method,through biochemical and serological means, and a specific PCRdetection technique through detecting and sequencing of gyrB,invA, and stn genes among the isolated strains of Salmonella; also, todetermine the antimicrobial sensitivities of the isolated Salmonella.

2. Materials and methods

2.1. Collection of samples

Two hundred seventy meat product samples (90 each of freshbeef, ground beef, and beef burger) were purchased from differentsupermarkets and butchers’ shops distributed at Mansoura city,Egypt. Fresh beef samples were obtained from cattle carcassesslaughtered and dressed at Mansoura central province traditionalabattoir, while ground beef and beef burger samples were obtainedin a frozen packaged form (400e500 g) from various supermarkets.Samples were individually packed into a polyethylene bag thenmarked and transferred in ice box to the laboratory of Food Hygieneand Control Department, Faculty of Veterinary Medicine, MansouraUniversity, wherein the conventional bacteriological analyses weredone.

2.2. Isolation of Salmonella

The preparation of meat samples and the detection of Salmo-nella were done according to techniques recommended by theInternational Organization for Standardization (ISO 6579: 2002).Briefly, 25 g from each of beef samples were weighed, and ho-mogenized with 225 ml of sterile buffered peptone water (BPW;Oxoid CM0509) in a stomacher for 1 min. The pre-enrichmentbroth was then incubated at 37 �C for 18 h. An inoculum from

pre-enrichment broth was inoculated into each of Rappaport Vas-silliadis broth (RV; Oxoid, CM0669) and Muller-Kauffmann tetra-thionate/novobiocin broth (MKTTn; Oxoid, CM1048). The RV brothis incubated at 41.5 �C for 24 h, while the MKTTn broth at 37 �C for24 h. From these two cultures, 0.1 ml was streaked onto two se-lective solid media; Xylose-Lysine-Desoxycholate (XLD) agar(Oxoid, CM0469) and Brilliant Green Agar w/Sulfadiazine (BGA;Neogen, 7310). XLD plates were incubated at 37 �C for 24 h, whileBGA plates were incubated at 35 �C for 24 h. Presumptive colonieson XLD and BGA were picked up from each positive sample. A totalof 2635 presumptive Salmonella colonies were collected andcultured onto non-selective media for further identification by bothconventional and molecular methods.

2.3. Identification of suspect Salmonella isolates by conventionalmethod

Conventional identification of the 2635 presumptive Salmonellacolonies was performed using classical biochemical tests. Themolecularly verified Salmonella isolates (272 strains) were sero-logically typed by slide agglutination test according to Kauffmannwhite scheme (Kauffmann, 1974) with the use of rapid diagnosticSalmonella antisera sets (Wellcome Diagnostic, a Division of theWellcome Foundation Limited, Dartford DA15 AH, UK), whichtypically utilize polyvalent antisera for somatic (O) and flagellar (H)antigens.

Isolates were subcultured on nutrient slope for 24 h at 37 �C forapplication of slide agglutination technique. Two homogenoussuspensions were made on a slide by suspending a piece of sus-pected colony in a drop of sterile physiological saline. A drop of eachof separate O and H Salmonella factors were added separately toeach of the suspensions with standard loop thoroughly mixed tobring the microorganisms in close contact with antisera. Positiveagglutination occurred within a minute and could be easily seenwith the naked eye. A delayed or partial agglutination wasconsidered as negative or false result. Isolates with a typicalbiochemical profile, which agglutinate with both H and O antisera,were identified as Salmonella spp.

2.4. Molecular identification of presumptive Salmonella isolates byPCR

Molecular identification of the 2635 presumptive Salmonellaisolates was carried out at Bio-production Research Institute, Na-tional Institute of Advanced Industrial Science and Technology(AIST), Hokkaido, Japan. Genomic DNA of Salmonella isolates wasprepared by the method reported by Choo et al. (2007). S. Typhi-murium (RIMD 1985009) was used as a positive control strain,while Escherichia coli K12 DH5a was used as a negative controlstrain. Primer sets for PCR amplification have been previouslydescribed for gyrB (Ye et al., 2011), InvA (Chiu & Ou, 1996) and stn(Lee et al., 2009) genes that produce an amplified band size of 366,244, and 179 bp, respectively. The oligonucleotide primers weresynthesized by Hokkaido System Science Co. Ltd. (Hokkaido, Sap-poro, Japan). PCR was operated with the use of GeneAmp PCRsystem 2700 thermal cycler (Applied Biosystems, Foster City, CA,USA). PCR was carried out with a 12-ml reaction mixture consists of0.75 ml Salmonella DNA template, 1.25 ml each of forward andreverse primers (3 pmol each), 2.5 ml dNTPs (2 mM), 6 ml of 2 � PCRBuffer for KOD FX, and 0.25 ml KOD FX DNA Polymerase (Toyobo Co.,Ltd., Osaka, Japan). After an initial denaturation at 94 �C for 2 min,35 cycles (98 �C for 10 s, 58 �C for 30 s, and 68 �C for 45 s) wereperformed followed by a final extension at 68 �C for 5 min. PCRproducts of each reactionmixturewere separated by subjecting 3 mlaliquots to agarose (1.5%) gel electrophoresis for 30 min at 100 V

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followed by a 20-min staining in ethidium bromide solution. Theseparated PCR products were then visualized and photographedunder UV light.

For confirmation of the amplified genes, DNA sequencing wasperformed with the BigDye Terminator v3.1 Cycle Sequencing Kit(Applied BioSystems) according to the manufacturer’s instructionson an ABI Prism 3100 automated sequencer (Applied Biosystems).Primers used for sequencing were the same used for DNA ampli-fication. Nucleotide sequence data were then analyzed by theGENETYXMAC software, version 12 (GENETYX Corp., Tokyo, Japan).Homology searches of the obtained sequences against the alreadypublished genes in the GenBank were carried out using StandardNucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.5. Antimicrobial susceptibility testing

Antimicrobial susceptibility patterns for 100 selected Salmonellaisolates was determined according to the agar disk diffusion stan-dard method (CLSI, 2012) using MuellereHinton agar (Oxoid,CM0337) supplemented with 5% defibrinated horse blood. Theplates were incubated at 35 �C for 24 h. The twelve antibioticstested included penicillin (10 IU), amoxicillin (30 mg), erythromycin(15 mg), oxytetracycline (30 mg), ampicillin (10 mg), neomycin(10 mg), streptomycin (10 mg), chloramphenicol (30 mg), nalidixicacid (30 mg), ciprofloxacin (5 mg), kanamycin (30 mg), and genta-micin (10 mg). Susceptibility categorization was determined ac-cording to National Committee of Clinical Laboratory Standards(NCCLS) guidelines.

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was applied to comparethe prevalence values among the three meat products tested. Dataanalysis was performed using the VassarStats website for StatisticalComputation (http://vassarstats.net).

3. Results and discussion

3.1. Prevalence of Salmonella in fresh beef, ground beef and beefburger samples

The prevalence of Salmonella in meat product samples wasstudied by conventional and DNA based molecular techniques.Conventional method, based on biochemical tests, detected Sal-monella in 23.3% (21/90), 20% (18/90), and 12.2% (11/90) of freshbeef, ground beef and beef burger samples, respectively with anoverall mean of 18.5% (50/270) among all the meat productsexamined (Fig. 1). Molecular technique, however, verified thepresence of Salmonella at higher prevalences of 30.0% (27/90),26.7% (24/90), and 16.7% (15/90) in fresh beef, ground beef, and beefburger samples, respectively with an overall mean of 24.4% (66/270) among all the meat products tested (Fig. 1). The higher prev-alences of Salmonella positive samples that were obtained by PCR inthe present study in comparison with that of cultural conventionalmethod indicated that PCR is more sensitive and reliable than thephenotypic based culture method.

Fresh beef samples in the present study showed the highestprevalence rate among the meat products tested, followed byground beef, while beef burgers were the lowest contaminatedsamples. Such difference in the prevalence rates, however, was notsignificant (P > 0.05) among the 3 products tested. Fresh beefsamples were obtained from animals slaughtered in Mansouraconventional abattoir in which floor dressing of the carcasses aswell as the poor slaughtering facilities and meat handling practicescould contribute greatly to the spread of this bacterium, and hence

the higher incidence of its presence in the beef samples tested.Ground beef and beef burger, however, were manufactured fromimported frozen beef that originated from developed countries, inwhich the cattle were slaughtered in modern abattoirs understandard levels of hygiene, with a minimal contamination, beforesubjecting to freezing and shipping to Egypt, where it is ground andprocessed into meat products in different meat plants in thecountry.

Our finding of Salmonella recovery is approximately similar tothat reported by other researchers especially in the developingcountries. For instance, Salmonella was recovered from 29.8% (43/144) of beef in Tunisia (Abbassi-Ghozzi et al., 2012), 20% (38/189) ofbeef in Iran (Dallal et al., 2010), 17% (13/78) of beef in China (Yanget al., 2010) and 20% (24/122) of minced beef samples examinedin Botswana (Mrema, Mpuchane, & Gashe, 2006). Much higherprevalence of 62% (31/50) was reported, however, in retail beef inVietnam (Van, Moutafis, Istivan, Tran, & Coloe, 2007). Conversely,much lower prevalences of Salmonella in meat were reported indeveloped countries. In Canada, none of the ground beef samples(n ¼ 134) tested were positive for Salmonella (Aslam et al., 2012),while only 4.2% (172/4136) of ground beef samples tested in USA(Bosilevac, Guerini, Kalchayanand, & Koohmaraie, 2009) and 1.1%(18/1514) of retail beef samples tested in the United Kingdom(Little, Richardson, Owen, de Pinna, & Threlfall, 2008) were positivefor Salmonella.

The prevalence of Salmonella in retail meat and meet productsvaries from one region to another. Many factors could be implicatedin the variations of Salmonella occurrence, for instance, the level ofslaughterhouse sanitation, the initial numbers of bacterial carriersentering the meat plant, the possible cross-contamination at retaillevel, improper handling, climate and temperature of meat storage,and the variations of isolation methods adopted for detection andisolation of the pathogen among different studies.

3.2. Distribution of Salmonella isolates among meat products

The distribution of identified Salmonella isolates among the beefproducts tested is shown in Fig. 2. It was evident that 228 isolates(96 isolates from fresh beef, 81 from ground beef, and 51 from beefburger) out of the 2635 presumptive colonies were biochemicallyidentified as Salmonella. Molecular identification based on detec-tion and sequencing of Salmonella specific invA and gyrB genes,however, verified that 272 isolates (117, 97, and 58 isolates from

Fig. 1. Prevalence of Salmonella in fresh beef, ground beef and beef burger samplesbased on both biochemical and molecular (genetic) identification.

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fresh beef, ground beef and beef burger, respectively) were Sal-monella (Fig. 2).

3.3. Salmonella serovars and their distribution among beef products

The distribution of Salmonella serovars among the tested beefproducts is shown in Table 1. Of the 272 serologically tested strains,266 were serologically identified into six Salmonella serovars, while6 strains were Salmonella untypable. S. Typhimurium and S.Enteritidis were the most prevalent serovars with an incidence of38.2% (104/272) and 34.6% (94/272), respectively. The other fourserovars identified were Salmonella Haifa, Salmonella Muenster,Salmonella Virchow, and Salmonella Anatum, which were detectedat lower prevalences of 11% (30/272), 7.4% (20/272), 4% (11/272) and2.6% (7/272), respectively. Most of S. Typhimurium serovars (46/104) originated from fresh beef, while most of S. Entritidis serovars(52/94) originated from beef burger. Only 3 serovars (S. Typhimu-rium, S. Entritidis, and S. Haifa) were detected in fresh beef, whileall of the six serovars identified were detected in both ground beefand beef burger samples (Table 2).

Salmonella serovars isolated from food vary greatly amonggeographic regions and type of food analyzed. Although all Sal-monella serovars considered as potential human pathogens, themajority of infections are caused by a very limited number ofserovars. Four serovars, Typhimurium, Enteritidis, Newport, andHeidelberg, are commonly identified serovars of Salmonella asso-ciated with human infections from food of animal origin (Hur et al.,2012). These four serovars are also reported to be themost commonserovars responsible for human hospitalizations in the UnitedStates. S. Typhimurium and S. Enteritidis together have beenimplicated in 60e90% of all human cases of salmonellosis world-wide (Nørrung, Andersen, & Buncic, 2009).

3.4. Molecular identification of Salmonella by detection andsequencing of gyrB and invA genes

The gyrB gene has been used as the target sequence for thedetection and identification of several bacterial species by PCR-based assays including the phylogenetic studies of Salmonella,Shigella, and E. coli (Fukushima, Kakinuma, & Kawaguchi, 2002). Allof the 2635 presumptive colonies that were obtained from theexamined beef samples were PCR screened for the detection andsequencing of gyrB gene (using gyrB-targeted Salmonella-specific

primers) as well as the Salmonella-specific invA gene. Two hundredand seventy two isolates analyzed were genetically confirmed asSalmonella positive by the appearance of an amplified PCR fragmentof 366 bp for gyrB gene (Fig. 3A) and 244 bp for invA gene (Fig. 3B).Sequence analysis using BLASTN Search verified that the amplifiedgyrB and invA amplicons shared 100% similarity with the corre-sponding genes of Salmonella deposited in the GenBank database.

Our results concerning the presence of the invA gene are inagreement with those previously reported by many other authors(Kumar, Surendran, & Thampuran, 2008; Nagappa, Tamuly,Brajmadhuri, Saxena, & Singh, 2007; Oliveira et al., 2002), whoconcluded that amplification of invA gene allowed detection of allSalmonella serovars and that this gene was a good target for Sal-monella detection.

3.5. Detection of enterotoxin (stn) gene

The exact mechanism by which Salmonella induces diarrhea isnot fully understood. Salmonella harbors an enterotoxin similar tothe enterotoxins in E. coli. This enterotoxin production is mediatedby the stn gene (Chopra, Houston, Peterson, Prasad, & Mekalanos,1987). The stn gene is one of the virulent genes that assist Salmo-nellae in expressing its virulence in the host cells and manifestationof pathogenic processes, mainly diarrhea (Chopra et al., 1987). Inthis study, PCR assay carried out for the detection of the stn geneconfirmed its presence in all of the 272 molecularly identifiedisolates. The amplified 179-bp fragment for stn gene is shown inFig. 3C. Sequence analysis using BLASTN search verified that theamplified amplicon shared 100% similarity with stn gene of Sal-monella deposited in the GenBank database. Similar to our findings,earlier studies (Murugkar, Rahman, & Dutta, 2003; Prager et al.,1995; Rahman, 1999) reported that all the examined Salmonellaisolates from food as well as clinical samples carried the stn gene.

3.6. Antimicrobial resistance of Salmonella isolates

Administration of antimicrobial agents in food animals for ther-apy, prophylaxis and growth promotion has led to the emergence ofantimicrobial resistant pathogens, including Salmonella that may beimplicated in foodborne antimicrobial resistant Salmonella infectionin humans (Barbosa & Levy, 2000). Of the 272 Salmonella isolatesrecovered from beef samples, 100 selected isolates represented thesix serovars identifiedwere tested for antibiotic resistance against 12antimicrobial agents (Table 2). Interestingly, all of the 100 Salmonellaserovars tested were multidrug resistant (resistant to three or moreantibiotics). These 100 Salmonella isolates were resistant to peni-cillin, amoxicillin and erythromycin. Resistance was also mostfrequently observed to oxytetracycline (96%), ampicillin (93%),neomycin (87%), streptomycin (83%), chloramphenicol (76%), fol-lowed by nalidixic acid (70%) and ciprofloxacin (63%), and to a lesser

Fig. 2. Distribution of Salmonella isolates among the meat products tested based onbiochemical, and genetic identifications.

Table 1Distribution of identified Salmonella serovars among the meat products tested.

Salmonella serotype Meat product Total

Fresh beef Ground beef Beef burger

Salmonella Typhimurium 46 28 30 104Salmonella Enteritidis 32 10 52 94Salmonella Haifa 19 7 4 30Salmonella Muenster 0 11 9 20Salmonella Virchow 0 7 4 11Salmonella Anatum 0 6 1 7Untypable 2 1 3 6Total 99 70 103 272

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extent kanamycin (39%), and gentamycin (23%). Multidrug resistantSalmonella serovars have been proposed to be more virulent thannon-multidrug resistant Salmonellae (Fluit, 2005; Foley & Lynne,2008). High percentages of antimicrobial resistant Salmonella fromretail meat products were also reported worldwide by several re-searchers (Chen et al., 2004; Dallal et al., 2010; Yanget al., 2010; Zhaoet al., 2008).

4. Conclusion

The present study demonstrated widespread contaminationby multidrug-resistant Salmonella serovars in beef products

retailed in Mansoura, Egypt and highlighted the dominance ofS. Typhimurium and S. Enteritidis, which constitute a possiblerisk to humans from consumption of these products. Moreover,the detection level of Salmonella in meat samples by the PCR-based technique was higher than that obtained by the con-ventional method; and hence, it can be used for the rapid andaccurate detection of Salmonella. The study also emphasizedthe need to implement proactive measures of hygienic prac-tices and the application of Hazard Analysis and Critical ControlPoint (HACCP) in the preparation and processing of foods toreduce Salmonella contamination and the risk of humaninfection.

Table 2Number and percentages of antimicrobial-resistant Salmonella serovars isolated from meat products.

Antibiotics Salmonella serovar

S. Typhimurium(n ¼ 40)

S. Enteritidis(n ¼ 30)

S. Haifa(n ¼ 10)

S. Muenster(n ¼ 10)

S. Virchow(n ¼ 5)

S. Anatum(n ¼ 5)

Total(n ¼ 100)

Penicillin (10 IU) 40 (100%) 30 (100%) 10 (100%) 10 (100%) 5 (100%) 5 (100%) 100 (100%)Amoxicillin (30 mg) 40 (100%) 30 (100%) 10 (100%) 10 (100%) 5 (100%) 5 (100%) 100 (100%)Erythromycin (15 mg) 40 (100%) 30 (100%) 10 (100%) 10 (100%) 5 (100%) 5 (100%) 100 (100%)Oxytetracycline (30 mg) 38 (95%) 30 (100%) 9 (90%) 9 (90%) 5 (100%) 5 (100%) 96 (96%)Ampicillin (10 mg) 40 (100%) 26 (100%) 8 (90%) 9 (90%) 5 (100%) 5 (100%) 93 (93%)Neomycin (10 mg) 40 (100%) 27 (90%) 7 (70%) 8 (80%) 2 (40%) 3 (60%) 87 (87%)Streptomycin (10 mg) 38 (95%) 26 (86.7%) 6 (60%) 7 (70%) 3 (60%) 3 (60%) 83 (83%)Chloramphenicol (30 mg) 37 (92.5%) 24 (80%) 4 (40%) 6 (60%) 2 (40%) 3 (60%) 76 (76%)Nalidixic acid (30 mg) 33 (100%) 22(100%) 5 (100%) 6 (100%) 2 (100%) 2 (100%) 70 (70%)Ciprofloxacin (5 mg) 27 (67.5%) 22 (73.3%) 6 (60%) 4 (40%) 2 (40%) 2 (40%) 63 (63%)Kanamycin (30 mg) 18 (45%) 14 (46.7%) 3 (30%) 2 (20%) 1 (10%) 1 (10%) 39 (100%)Gentamycin (10 mg) 11 (27.5%) 8 (26.7%) 1 (10%) 2 (20%) 0 (0%) 1 (10%) 23 (100%)

Fig. 3. Representative agarose gel electrophoresis for PCR products showed the expected molecular size of 366 bp for gyrB gene (A), 244 bp for invA gene (B), and 179 bp for theenterotoxin (stn) gene (C) in Salmonella spp. isolated from different meat products. Chromosomal DNA from the presumptive Salmonella isolates was used as a template for PCRamplification using primer sets for Salmonella-specific gyrB, invA, and stn gene. Three microliters from the PCR product were separated by electrophoresis on 1.5% agarose gel andvisualized under UV light. M: DNA marker (Gene Ladder 100) used as a reference for fragment size; Lane C�: E. coli K12 DH5a as a negative control strain; Lane Cþ: SalmonellaTyphimurium (RIMD 1985009) as a positive control strain; Lanes with the key numbers from 8 to 228 are representative positive strains.

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References

Abbassi-Ghozzi, I., Jaouani, A., Hammami, S., Martinez-Urtaza, J., Boudabous, A., &Gtari, M. (2012). Molecular analysis and antimicrobial resistance of Salmonellaisolates recovered from raw meat marketed in the area of ‘‘Grand Tunis’’,Tunisia. Pathologie Biologie, 60, 49e54.

Acha, P. N., & Szyfres, B. (2001). Zoonoses and communicable diseases common to manand animals (3rd ed.). Washington DC: Pan American Health Organization.

Adesiyun, A. A., & Oni, O. O. (1989). Prevalence and antibiograms of Salmonellae inSlaughter cattle, slaughter areas and effluents in Zaria abattoir Nigeria. Journalof Food Protection, 52, 232e235.

Aslam, M., Checkley, S., Avery, B., Chalmers, G., Bohaychuk, V., Gensler, G., et al.(2012). Phenotypic and genetic characterization of antimicrobial resistance inSalmonella serovars isolated from retail meats in Alberta, Canada. Food Micro-biology, 32, 110e117.

Barbosa, T. M., & Levy, S. B. (2000). The impact of antibiotic use on resistancedevelopment and persistence. Drug Resistance Updates, 3, 303e311.

Bosilevac, J. M., Guerini, M. N., Kalchayanand, N., & Koohmaraie, M. (2009). Preva-lence and characterization of Salmonellae in commercial ground beef in theUnited States. Applied and Environmental Microbiology, 75, 1892e1900.

Bülte, M., & Jakob, P. (1995). The use of a PCR-generated invA probe for the detectionof Salmonella spp. in artificially and naturally contaminated foods. InternationalJournal of Food Microbiology, 26, 335e344.

Chan, K., Baker, S., Kim, C. C., Detweiler, C. S., Dougan, G., & Falkow, S. (2003).Genomic comparison of Salmonella enterica serovars and Salmonella bongori byuse of an S. enterica serovar typhimurium DNA microarray. Journal of Bacteri-ology, 185, 553e563.

Chen, S., Zhao, S., White, D. G., Schroeder, C. M., Ran, L., Yang, H., et al. (2004).Characterization of multiple-antimicrobial-resistant Salmonella serovars iso-lated from retail meats. Applied and Environmental Microbiology, 70, 1e7.

Chiu, C. H., & Ou, J. T. (1996). Rapid identification of Salmonella serovars in feces byspecific detection of virulence genes, invA and spvC, by an enrichment brothculture multiplex PCR combination assay. Journal of Clinical Microbiology, 34,2619e2622.

Choo, E., Jang, S. S., Kim, K., Lee, K. G., Heu, S., & Ryu, S. (2007). Prevalence andgenetic diversity of Bacillus cereus in dried red pepper in Korea. Journal of FoodProtection, 70, 917e922.

Chopra, A. K., Houston, C. W., Peterson, J. W., Prasad, R., & Mekalanos, J. J. (1987).Cloning and expression of the Salmonella enterotoxin gene. Journal of Bacteri-ology, 169, 5095e5100.

CLSI, Clinical and Laboratory Standards Institute. (2012). Performance standards forantimicrobial susceptibility testing; twenty-second informational supplement. InCLSI document (Vol. 32(3)). M100-MS22. Wayne, PA, USA.

Dallal, M. M. S., Doyle, M. P., Rezadehbashi, M., Dabiri, H., Sanaei, M., Modarresi, S.,et al. (2010). Prevalence and antimicrobial resistance profiles of Salmonellaserovars, Campylobacter and Yersinia spp. isolated from retail chicken and beef,Tehran, Iran. Food Control, 21, 388e392.

Fluit, A. D. (2005). Towards more virulent and antibiotic-resistant Salmonella? FEMSImmunology and Medical Microbiology, 43, 1e11.

Foley, S. L., & Lynne, A. M. (2008). Food animal-associated Salmonella challenges:pathogenicity and antimicrobial resistance. Journal of Animal Science, 86, E173eE187.

Fukushima, M., Kakinuma, K., & Kawaguchi, R. (2002). Phylogenetic analysis ofSalmonella, Shigella, and Escherichia coli strains on the basis of the gyrB genesequence. Journal of Clinical Microbiology, 40, 2779e2785.

Grimont, P. A. D., & Weill, F. X. (2007). Antigenic formulae of the Salmonella serovars(9th ed.). Paris, France: WHO Collaborating Centre for Reference and Researchon Salmonella Institut Pasteur http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-089.

Hendriksen, R. S., Seyfarth, A. M., Jensen, A. B., Whichard, J., Karlsmose, S., Joyce, K.,et al. (2009). Results of use of WHO Global Salm-Surv external quality assurancesystem for antimicrobial susceptibility testing of Salmonella isolates from 2000to 2007. Journal of Clinical Microbiology, 47, 79e85.

Hur, J., Jawale, C., & Lee, J. H. (2012). Antimicrobial resistance of Salmonella isolatedfrom food animals: a review. Food Research International, 45, 819e830.

International Organization for Standardization (ISO). (2002). Microbiology of foodand animal feeding stuffsdHorizontal method for the detection of Salmonella spp..ISO 6579:2002 (4th ed.). Geneva, Switzerland: The International Organizationfor Standardization.

Kauffmann, G. (1974). Kauffmann white scheme. WHO, BD 172, L. Rev. 1. ActaPathologica et Microbiologica Scandinavica, 61, 385.

Kumar, R., Surendran, P. K., & Thampuran, N. (2008). An eight-hour PCR-basedtechnique for detection of Salmonella serovars in seafood. World Journal ofMicrobiology and Biotechnology, 24, 627e631.

Lee, K., Iwata, T., Shimizu, M., Taniguchi, T., Nakadia, A., Hirota, Y., et al. (2009).A novel multiplex PCR assay for Salmonella subspecies identification. Journal ofApplied Microbiology, 107, 805e811.

Little, C. L., Richardson, J. F., Owen, R. J., de Pinna, E., & Threlfall, E. J. (2008).Campylobacter and Salmonella in raw red meats in the United Kingdom: prev-alence, characterization and antimicrobial resistance pattern, 2003e2005. FoodMicrobiology, 25, 538e543.

Mrema, N., Mpuchane, S., & Gashe, B. A. (2006). Prevalence of Salmonella in rawminced meat, raw fresh sausages and raw burger patties from retail outlets inGaborone, Botswana. Food Control, l7, 207e212.

Murugkar, H. V., Rahman, H., & Dutta, P. K. (2003). Distribution of virulence genes inSalmonella serovars isolated from man & animals. Indian Journal of MedicalResearch, 117, 66e70.

Nagappa, K., Tamuly, S., Brajmadhuri, Saxena, M. K., & Singh, S. P. (2007). Isolation ofSalmonella Typhimurium from poultry eggs and meat of Tarai region of Uttar-anchal. Indian Journal of Biotechnology, 6, 407e409.

Nørrung, B., Andersen, J. K., & Buncic, S. (2009). Main concerns of pathogenic mi-croorganisms in meat. In F. Toldrá (Ed.), Safety of meat and processed meat (pp.3e29). Springer-Verlag New York Inc.

Oliveira, S. D., Santos, L. R., Schuch, D. M. T., Silva, A. B., Salle, C. T. P., & Canal, C. W.(2002). Detection and identification of Salmonella from poultry-related samplesby PCR. Veterinary Microbiology, 87, 25e35.

Prager, R., Fruth, A., & Tschäpe, H. (1995). Salmonella enterotoxin (stn) gene isprevalent among strains of Salmonella enterica but not among Salmonellabongori and other Enterobacteriaceae. FEMS Immunology and Medical Microbi-ology, 12, 47e50.

Rahman, H. (1999). Prevalence of enterotoxin gene (stn) among different serovars ofSalmonella. Indian Journal of Medical Research, 110, 43e46.

Rahn, K., De Grandis, S. A., Clarke, R. C., McEwen, S. A., Galán, J. E., Ginocchio, C.,et al. (1992). Amplification of an invA gene sequence of Salmonella Typhimu-rium by polymerase chain reaction as a specific method of detection of Sal-monella. Molecular and Cellular Probes, 6, 271e279.

Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L.,et al. (2011). Foodborne illness acquired in the United States-major pathogens.Emerging Infectious Diseases, 17, 7e15.

Torpdahl, M., Skov, M. N., Sandvang, D., & Baggesen, D. L. (2005). Genotypic char-acterization of Salmonella by multilocus sequence typing, pulsed-field gelelectrophoresis and amplified fragment length polymorphism. Journal ofMicrobiological Methods, 63, 173e184.

Van, T. T., Moutafis, G., Istivan, T., Tran, L. T., & Coloe, P. J. (2007). Detection of Sal-monella spp. in retail raw food samples from Vietnam and characterization oftheir antibiotic resistance. Applied and Environmental Microbiology, 73, 6885e6890.

Yamamoto, S., & Harayama, S. (1996). Phylogenetic analysis of Acinetobacter strainsbased on the nucleotide sequences of gyrB genes and on the amino acid se-quences of their products. International Journal of Systematic Bacteriology, 46,506e511.

Yang, B., Qu, D., Zhang, X., Shen, J., Cui, S., Shi, Y., et al. (2010). Prevalence andcharacterization of Salmonella serovars in retail meats of marketplace inShaanxi, China. International Journal of Food Microbiology, 141, 63e72.

Ye, X., Wang, Y., & Lin, X. (2011). A gyrB-targeted PCR for rapid identification ofSalmonella. Current Microbiology, 63, 477e483.

Zhao, S., White, D. G., Friedman, S. L., Glenn, A., Blickenstaff, K., Ayers, S. L., et al.(2008). Antimicrobial resistance in Salmonella enterica serovar Heidelberg iso-lates from retail meats, including poultry, from 2002 to 2006. Applied andEnvironmental Microbiology, 74, 6656e6662.

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