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Genetic diversity of Salmonella enterica recovered from chickens farms and its potential transmission to human
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Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Genetic diversity of Salmonella enterica recovered from chickens farms and its potential transmission to human Omneya S. Magdy a , Ihab M. Moussa b,c,∗ , Hussein A. Hussein d , Mahmoud D. El-Hariri c , Ahmed Ghareeb e , Hassan A. Hemeg f , Khalid S. Al-Maary b , Aymen S. Mubarak b , Waleed K. Alwarhi b , Jakeen K. Eljakee c , Saleh A. Kabli g a
Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia c Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt d Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt e Medical Genetics Centre, Faculty of Medicine, Ain Shams University, Cairo, Egypt f Department of Medical Technology/Microbiology, College of Applied Medical Science, Taibah University, Madinah, Saudi Arabia g Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia b
a r t i c l e
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Article history: Received 27 March 2019 Received in revised form 27 August 2019 Accepted 17 September 2019 Keywords: Antibiotics Broiler S. Kentucky S. Enteritidis and S. Typhimurium sefA and fliC genes Sequencing
a b s t r a c t Background: Salmonella is a zoonotic bacterium transmitted through the food chain and is an important cause of disease in humans. The current study is aimed to characterize Salmonella isolates from broiler breeder chickens farms using, polymerase chain reaction (PCR) and sequencing analysis of representative isolates. Methods: S. Kentucky (n = 11), S. Enteritidis (n = 4), S. Typhimurium (n = 3), S. Breanderp (n = 1), and Sand S. Newport (n = 1), were identified from chicken farms. Antimicrobial sensitivity test among the strains were investigated using 13 antibacterial discs. The amplified fragments of fliC and sefA genes were used to characterize S. Kentucky, S. Enteritidis and S. Typhimurium strains. Sequence analysis of the amplified PCR products for Salmonella Kentucky, Enteritidis and Typhimurium were carried out. Results: Antimicrobial sensitivity testing revealed that 95% of the isolates were resistant to penicillin, 85% to norfloxacin and colistin sulfate (each), 75% to gentamicin, 70% to nalidixic acid and 60% to flumequine. The obtained sequences revealed the close identity of the isolated strains with other Salmonella reference strains in different countries. Conclusion: Analysis of the selected salmonellae confirm the report of Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Kentucky circulation among broiler breeder flocks and the need to determine antibacterial susceptibility pattern regularly to detect multidrug-resistant salmonellae. The present study reports the circulation of Salmonella Kentucky, Enteritidis and Typhimurium among broiler breeder farms in Egypt. Emergency control of salmonellae is a global public health concern. © 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction Salmonella is one of important economic problems food borne diseases in many counties [1,2]. It is a famous endemic zoonotic disease all over the world and difficult to be controlled [3,4]. Patient diagnosed with severe S. Enteritidis infections have an increased colon cancer risk [5].
∗ Corresponding author at: Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (I.M. Moussa).
In 1900s, vaccination can eradicate to some extend S. Pullorum and S. Gallinarum from poultry flocks, however, these eradication lead to propagation of S. Enteritidis, S. Typhimurium and other serovars, as S. Heidelberg and S. Kentucky, in the birds [6].Ubiquitous use of chemotherapeutic agents and growth promoters in our farms led to the appearance of multidrug-resistant microorganisms [7]. The correlation between the mortality rate in humans and infections with S. Typhimurium resistant to chloramphenicol, streptomycin, tetracycline, ampicillin and sulphonamide had been previously identified [8]. Following the use of antibacterial drugs in food animals, multidrug-resistant salmonellae are transmitted to humans, through the food [9]. In developing countries improper use of antimicrobial agents in human medicine
https://doi.org/10.1016/j.jiph.2019.09.007 1876-0341/© 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Magdy OS, et al. Genetic diversity of Salmonella enterica recovered from chickens farms and its potential transmission to human. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.09.007
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increase the occurrence of multidrug-resistant Salmonella strains [10]. PCR method is rapid, specific and sensitive method used for the detection of food borne pathogens [11]. The fliC gene, encoding for the flagellin protein, has been used as a target gene to determine the antigenic specificity and genetic diversity in Salmonella [12]. Nowadays, sequencing and phylogenetic analysis become an important molecular methods for characterization of pathogens. The objective of the present investigations was to characterize salmonellae isolated from broiler breeder chickens farms using antimicrobial sensitivity test, polymerase chain reaction and sequencing analysis of representative isolates. Materials and methods
Table 1 Number and sources of the positive Salmonella suspected samples. Source of the isolates
Number
Positive salmonellae
Fecal swabs Fecal swabs Fecal swabs Fecal swabs Dust swabs Drag swabs Drag swabs Litter swabs Drooping swab Hatched eggs Hatched eggs Hatched chicks Hatched chicks
5 1 3 1 1 2 1 1 1 1 1 1 1
S. Kentucky S. Enteritidis S. Typhimurium S. Breanderp S. Kentucky S. Kentucky S. Enteritidis S. Kentucky S. Kentucky S. Kentucky S. Enteritidis S. Newport S. Enteritidis
Strains Twenty Salmonella isolates were identified biochemically [14] using API 20E (Biomeriux). Serological identification of the Salmonella isolates was carried out [15] using diagnostic poly and ¨ and ¨ antisera ¨ ¨ H (Mast). mono valent Salmonella O Antimicrobial sensitivity test Using ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, colistin, norfloxacin, flumequine, gentamicin, nalidixic acid, neomycin, oxytetracycline, penicillin-G and streptomycin discs (Oxoid) the sensitivity of the isolates was tested by disc diffusion method. The interpretation of the result was carried out as recorded by Refs. [16,17]. Characterization of the Salmonella isolates using polymerase chain reaction (PCR) DNA of Salmonella isolates were extracted using DNA Kit (Catalogue no.51304) according to QIAamp DNA mini kit instructions and Emerald Amp GT PCR master mix (Takara) Code No.RR310A. The Oligonucleotide primers sequences for detection of fliC gene (Fli15-F CGG TGT TGC CCA GGT TGG TAA T- Tym-R ACT CTT GCT GGC GGT GCG ACT T) [18] and for detection of sefA gene (SEFA2 5 -GCA GCG GTT ACT ATT GCA GC-3 and SEFA4 5 -TGT GAC AGG GAC ATT TAG CG-3 ) [19] were manufactured in Midland Certified Reagent Company-oilgos (USA). For detection of fliC gene during PCR, primary denaturation and activation of Taq DNA polymerase, 1 cycle at 94 ◦ C for 5 min. secondary denaturation 35 cycles at 94 ◦ C for 30 s annealing at 56 ◦ C for 30 s., extension at 72 ◦ C for 30 s and final extension at 1 cycle at 72 ◦ C for 10 min. For detection of sefA gene, primary denaturation and activation of Taq DNA polymerase 1 cycle at 94 ◦ C for 5 min, secondary denaturation 35 cycles at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, extension at 72 ◦ C for 30 s and final extension 1 cycle at 72 ◦ C for 10 min were carried out and the PCR products were analyzed [20]. Sequence analysis of the fliC gene of S. Typhimurium and S. Kentucky and sefA gene of S. Enteritidis PCR products were purified using DNA purification kit (Qiagen Inc. Valencia CA). Sequencing reaction was carried out at NLQP gene analysis unit and the obtained nucleotide sequence was analyzed using BioEdit7, mega 5softwares and the nucleotide sequence alignment was carried out using clustal W method [21]. A sequence distance showed the proportion of identical residues between all of the sequences in the alignment as they were currently aligned. The output was a 2-D matrix table which could either be able to eliminated or comma-delimited. The output depends completely upon the quality of the alignment. The sequences were not automatically aligned before the procedure was run.
Table 2 The percentage of resistance of the isolated among Salmonellae. Antibiotics
Resistance %
Ampicillin (A10 , 10 g) Chloramphenicol (C30 , 30 g) Cefotaxime (CTX30 , 30 g) Colistin Sulphate (CL10 , 10 g) Ciprofloxacin (CF5 , 5 g), Flumequine (UB30 , 30 g) Gentamicin (G10 , 10 g) Nalidixic acid (NA30 , 30 g) Norfloxacin (Nor10 , 10 g) Neomycin (N30 , 30 g) Penicillin-G (P10 , 10 I.U) Oxytetracycline (O30 , 30 g) Streptomycin (S10 , 10 g)
25 35 20 85 50 60 75 70 85 45 95 5 50
The Phylogenetic relationship was performed using (dnastar, lasergene, megaalign) software. Results Identification of the isolates The isolates were identified biochemically using API 20E (Biomeriux) and serologically using diagnostic poly and mono ¨ and ¨ antisera ¨ ¨ valent Salmonella O H (Mast). Twenty salmonellae (Salmonella Kentucky n = 11, Salmonella Enteritidis n = 4, Salmonella Typhimurium n = 3, Salmonella Breanderp n = 1 and Salmonella Newport n = 1 were identified from broiler breeder chickens farms as shown in Table 1. Results of antimicrobial sensitivity test for Salmonella isolates It is clear that 95% of isolates demonstrated resistance and intermediate resistance to penicillin, 85% to both norfloxacin and colistin sulphate, 75% to gentamicin, 70% to nalidixic acid and 60% for flumequine as shown in Table 2. Results of PCR for sefA gene amplification from Salmonella Enteritidis isolates The agarose gel electrophoresis with positive PCR amplification of 310 bp fragments of sefA gene of S. Enteritidis, whereas no amplification could be observed with the extracted DNA of the negative control Escherichia coli NCIMB 50034 as shown in Fig. 1. Results of PCR for fliC gene amplification from of Salmonella Typhimurium isolates Agarose gel electrophoresis with positive PCR amplification of 599 bp fragments of fliC gene of S. Typhimurium, whereas no ampli-
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Fig. 1. The amplified product of sefA gene of S. Enteritidis in the extracted DNA from the isolated SE. Lane M: 100–600 bp DNA ladder (Gel Pilot 100pb), Lanes 1–3: S. Enteritidis isolates positive for sefA gene, Lanes +: Positive control (S. Enteritidis ATCC 13076) and Lane −: Negative control (E. coli NCIMB 50034).
Fig. 2. The amplified product of fliC gene of S. Typhimurium i in the extracted DNA from the isolated ST. Lane M: 100–600 bp DNA ladder (Gel Pilot 100 bp), Lanes 2 and 3: S. Typhimurium isolates positive for fliC gene, Lane +: Positive control (S. Typhimurium ATCC 14028) and Lane −: Negative control (E. coli NCIMB 50034).
Fig. 3. DNA product of fliC gene of S. Kentucky isolates. Lane M: 100–600 bp DNA ladder (Gel Pilot 100 bp), Lanes 1–11 S. Kentucky isolates positive for fliC gene, Lane +: Positive control (S. Typhimurium ATCC 14028) and Lane −: S. Enteritidis ATCC 13076.
fication could be observed with the extracted DNA of the negative control E. coli NCIMB 50034 as shown in Fig. 2. Results of PCR for fliC gene from Salmonella Kentucky isolates Fig. 3 illustrated the agarose gel electrophoresis with positive PCR amplification of 599 bp fragments of fliC gene of S. Kentucky in 11 S. Kentucky isolates along with the positive and negative controls, whereas the positive control of the reaction was used Salmonella type ATCC 14028.
Sequence analysis of Salmonella isolates The nucleotide sequence of 310 and 599 bp PCR products of sefA and fliC genes of Salmonella Enteritidis, Salmonella Kentucky and Salmonella Typhimurium respectively had been sequenced using Applied Bio systems 3130 genetic analyzer (USA). The obtained sequences were blasted with the highly similar sequences which were downloaded and imported in BIOEDIT version 7.1.11. Multiple sequence alignments were conducted using Clustal W application embedded in the BIOEDIT.
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Fig. 4. Neighbor joining phylogenetic tree for Salmonella Enteritidis different strain. Partial (sefA) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
Fig. 5. Neighbor joining phylogenetic tree for Salmonella Typhimurium different strain. Partial (fliC) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
The Nucleotide and amino acid alignment of the obtained sequence in comparison with the imported reference strains from gene bank as shown in (Figs. 4–6). The obtained Nucleotide sequences were submitted in gene bank with access no. KF601584 for Salmonella Enteritidis strain Giza/VRLCU-144-5/2011(sefA) gene, KF601585 for Salmonella Typhimurium strain Giza/VRLCU144-6/2011(fliC) gene, KF601581, KF601582 and KF601583 for Salmonella Kentucky strain Giza/VRLCU-144-1/2011(fliC) gene, Salmonella Kentucky strain Giza/VRLCU-144-5/2011(fliC) gene and Salmonella Kentucky strain Giza/VRLCU-144-10/2011(fliC) gene respectively.
Discussion Food borne disease outbreaks due to Salmonella serovars is an important economic disease, mainly in developing countries [1]. It is one of the most problematic zoonotic diseases in addition to significant morbidity and mortality rates [2]. The Salmonella Enteritidis and Salmonella Typhimurium are the most important strains that infect human [22]. In the present study 20 isolates collected from broiler breeding farms (fecal swabs, dust swabs, drag swabs, litter swabs, drooping swabs, hatched eggs and hatched chicks) were identified as 11 S.
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Fig. 6. Neighbor joining phylogenetic tree for Salmonella Kentucky different strain. Partial (fliC) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
Kentucky, 4 S. Enteritidis, 3 S. Typhimurium, 1 S. Breanderp and 1 S. Newport. Salmonellae were recorded from environmental samples of poultry flocks [23,24]. Improper utilization of chemotherapeutic agents and growth promoters in our farms led to the appearance of multidrugresistant salmonellae. It is clear that 95% of isolates were resistant to penicillin, 85% to norfloxacin and colistin sulphate, 75% to gentamicin, 70% to nalidixic acid and 60% to flumequine. All Salmonella isolates were resistant to ampicillin, cefotaxime, erythromycin, kanamycin, nalidixic acid, neomycin, novobiocin, streptomycin, spectinomycin and tetracycline [25]. The prophylactic use of many antimicrobials in poultry feed can also lead to acquired antibiotic resistance [26]. There is an emergency need to control the use of growth promoters and antimicrobial agents in animals to minimize the emergence of resistant strains [27]. In the present study, two pairs of primers were used for detection of fliC and sefA genes among the isolates using PCR assay. Salmonella Typhimurium and Salmonella Enteritidis from hens’ eggs, using multiplex polymerase chain reaction had been detected before [28]. All S. Enteritidis isolates amplified sefA gene. The sefA is specific target gene for S. Enteritidis serovars and the fliC is specific target for serotypes Typhimurium and Kentucky [29]. fliC targets the gene encoding the flagellar antigens i expressed in S. Typhimurium and S. Kentucky and uncommon serovars: S. Aberdeen, S. Bergen and S. Kedougou [29]. The OmpR gene regions of Salmonella strains were previously amplified and sequenced [30]. The Salmonella fliC sequences had been applied by many authors [31,32], they found that these salmonellae were conserved at their terminal and variable in the central region between serotyped. The obtained nucleotide sequences were submitted in gene bank with access no. KF601584, KF601585, KF601581, KF601582 and KF601583 for S. Enteritidis, S. Typhimurium, and S. Kentucky strains. Conclusion The data demonstrated the high prevalence of multidrugresistant Salmonella serovars that indicated alarming in the veterinarian therapeutic treatment and there is a need to determine antibacterial susceptibility pattern regularly to detect multidrugresistant salmonellae. S. Enteritidis and S. Typhimurium were identified from the examined broiler breeder chickens farms, so
special biosecurity and control measures must be carried out for control of Salmonella and limit its potential transmission to human. Emergency control of salmonellae is a global public health concern. Sequence analysis of the selected five salmonellae in the present study confirm the report of Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Kentucky circulation among broiler breeder flocks under the present study. Funding No funding sources. Competing interests None declared. Ethical approval Not required. Acknowledgment The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for supporting the work through the research group project No: RG-162. References [1] Alves LMC, Costa NF, Silva MIS, Sales SS, Correia MR. Toxinfecc¸ãoalimentarp por Salmonella Enteritidis: relato de um surtoo corridor em São Luís-MA. Higiene Alimentar 2001;15:57–8. [2] Castiglioni Tessari EN, Iba Kanashiro AM, Stoppa GFZ, Luciano RL, De Castro AM, Cardoso AP. Important aspects of Salmonella in the poultry industry and in public health, Salmonella—a dangerous food borne ISBN 2012; (6): 978-953307-782-6. [3] Santo LR, Nascimento, VP, Flores, ML, Rosek H, D’Andrea A, Albuquerque MC, Rampanelli Y, Machado NP, Rios S, Fernandes SA. Salmonella Enteritidis isoladas de amostras clinicas de humanos e de alimentos envolvidos em episódios de toxinfecc¸ões alimentares, ocorridas entre 1995 e 1996, no Estado do Rio Grande do Sul Revista Higiene Alimentar, São Paulo. 2002; (6) (102/103), 93–99. [4] Antunes P, Reu C, Sousa JC, Peixe L, Pestana N. Incidence of Salmonella in poultry and their susceptibility to microbial agents. Int J Food Microbiol 2003;82:97–103. [5] Mughini-Gras L, Schaapveld M, Kramers J, Mooij S, Neefjes-Borst EA, Pelt WV, et al. Increased colon cancer risk after severe Salmonella infection. PLoS One 2018;13(1):e0189721, http://dx.doi.org/10.1371/journal.pone.0189721, eCollection 2018.
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[6] Foley SL, Nayak R, Hanning IB, Johnson TJ, Han J, Ricke SC. Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Appl Environ Microbiol 2011;77(13):4273–9. [7] WHO. World Health Organization. Drug-resistant Salmonella. Food safety Department Geneva, Fact sheet 2005; N 139. [8] Helms M, Vastrup P, Mølbak K. Antimicrobial drug-resistant Salmonella Typhimurium (reply to Dahl). Emerg Infect Dis 2003;9(10):1350–1. [9] Angulo FJ, Johnson KR, Tauxe RV, Cohen ML. Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb Drug Resist 2000;6:77–83. [10] Threlfall EJ, Ward LR, Rowe B. Increasing incidence of resistance to trimethoprim and ciprofloxacin in epidemic Salmonella Typhimurium DT104 in England and Wales. Euro Surveill 1997;2:81–4. [11] Olsen PE. Giant lava flows, mass extinctions, and mantle plumes. Science 1999;284:604–5. [12] Dauga C, Zabrovskaia A, Grimont PD. Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica. J Clin Microbiol 1998;36(10):2835–43. [14] Quinn PJ, Markey BK, Carter ME, Donnelly WJ, Leonard FC. Veterinary microbiology and microbial diseases. Oxford, London: Black Well Scientific Publications; 2002. [15] Popoff MY, Le Minor L. Antigenic formulas of the Salmonella serovars, 2001; 8th revision, World Health Organization Collaborating Centre for Reference and Research on Salmonella, Pasteur Institute, Paris, France. [16] NCCLS. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Wayne, PA: Clinical Laboratory Standard Institute; 2002. [17] Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 2010; Twenty-Fifth Informational Supplement (M100-S25). [18] Soumet C, Ermel G, Rose N, Rose V, Drouin P, Salvat G, et al. Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella sp., Salmonella Enteritidis and Salmonella Typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 1999;28(2):113–7. [19] Akbarmehr J, Zahraei Salehi T, Nikbakht GH. Identification of Salmonella isolated from poultry by MPCR technique and evaluation of their hsp groEL gene diversity based on the PCR-RFLP analysis. Afr J Microbiol Res 2010;4(15):1594–8.
[20] Sambrook J, Fritscgh EF, Meniates T. Molecular cloning. A laboratory manual, Vol. 1. New York: Cold Spring Harbor Laboratory Press; 1989. [21] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22(22):4673–80. [22] Afshari A, Baratpour A, Khanzade S, Jamshidi A. Salmonella Enteritidis and Salmonella Typhimurium identification in poultry carcasses. Iran J Microbiol 2018;10(1):45–50. [23] Davies R, Breslin M. Environmental contamination and detection of Salmonella enterica serovar Enteritidis in laying flocks. Vet Rec 2001;149(23):699–704. [24] Kinde H, Castellan DM, Kass PH, Ardans A, Cutler G, Breitmeyer RE, et al. The occurrence and distribution of Salmonella Enteritidis and other serovars on California egg laying premises: a comparison of two sampling methods and two culturing techniques. Avian Dis 2004;48:590–4. [25] Shah AH, Korejo NA. Antimicrobial resistance profile of Salmonella serovars isolated from chicken meat. J Vet Anim Sci 2012;2:40–6. [26] Rajagopal R, Mini M. Outbreaks of salmonellosis in three different poultry farms of Kerala, India. Asian Pac J Trop Biomed 2013;3(6):496–500. [27] El Jakee J, Ata NS, Abd El-Moez SI, Kandiel MM, Radwan NM. Assessment of the prevalence of Salmonellae in food. Int Curr Microbiol Appl Sci 2014;3(3):30–42. [28] El Jakee J, Khelfa DG, El-Safty MM, Seida AA, Marouf S, Hahne J, et al. Multiplex PCR-based detection of Salmonella Typhimurium and Salmonella Enteritidis in specific pathogen free (SPF) and commercial eggs. Clin Microbiol 2016;5(2):241. [29] O’Regan E, McCabe E, Burgess C, McGuinness S, Barry T, Duffy G, et al. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples. BMC Microbiol 2008;8:156. [30] Arunachalam R, Senthilkumar B, Senbagam D, Selvamaleeswaran P, Rajase karapandian M. Molecular phylogenetic approach for classification of, Salmonella Typhi. Res J Microbiol 2012;7(1):13–22. [31] Smith NH, Selander RK. Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella Typhimurium. J Bacteriol 1990;172(2):603–9. [32] Li J, Nelson K, McWhorter AC, Whittam TS, Selander RK. Recombinational basis of serovar diversity in Salmonella enterica. Proc Natl Acad Sci U S A 1994;91:2552–6.
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Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Genetic diversity of Salmonella enterica recovered from chickens farms and its potential transmission to human Omneya S. Magdy a , Ihab M. Moussa b,c,∗ , Hussein A. Hussein d , Mahmoud D. El-Hariri c , Ahmed Ghareeb e , Hassan A. Hemeg f , Khalid S. Al-Maary b , Aymen S. Mubarak b , Waleed K. Alwarhi b , Jakeen K. Eljakee c , Saleh A. Kabli g a
Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia c Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt d Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt e Medical Genetics Centre, Faculty of Medicine, Ain Shams University, Cairo, Egypt f Department of Medical Technology/Microbiology, College of Applied Medical Science, Taibah University, Madinah, Saudi Arabia g Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia b
a r t i c l e
i n f o
Article history: Received 27 March 2019 Received in revised form 27 August 2019 Accepted 17 September 2019 Keywords: Antibiotics Broiler S. Kentucky S. Enteritidis and S. Typhimurium sefA and fliC genes Sequencing
a b s t r a c t Background: Salmonella is a zoonotic bacterium transmitted through the food chain and is an important cause of disease in humans. The current study is aimed to characterize Salmonella isolates from broiler breeder chickens farms using, polymerase chain reaction (PCR) and sequencing analysis of representative isolates. Methods: S. Kentucky (n = 11), S. Enteritidis (n = 4), S. Typhimurium (n = 3), S. Breanderp (n = 1), and Sand S. Newport (n = 1), were identified from chicken farms. Antimicrobial sensitivity test among the strains were investigated using 13 antibacterial discs. The amplified fragments of fliC and sefA genes were used to characterize S. Kentucky, S. Enteritidis and S. Typhimurium strains. Sequence analysis of the amplified PCR products for Salmonella Kentucky, Enteritidis and Typhimurium were carried out. Results: Antimicrobial sensitivity testing revealed that 95% of the isolates were resistant to penicillin, 85% to norfloxacin and colistin sulfate (each), 75% to gentamicin, 70% to nalidixic acid and 60% to flumequine. The obtained sequences revealed the close identity of the isolated strains with other Salmonella reference strains in different countries. Conclusion: Analysis of the selected salmonellae confirm the report of Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Kentucky circulation among broiler breeder flocks and the need to determine antibacterial susceptibility pattern regularly to detect multidrug-resistant salmonellae. The present study reports the circulation of Salmonella Kentucky, Enteritidis and Typhimurium among broiler breeder farms in Egypt. Emergency control of salmonellae is a global public health concern. © 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction Salmonella is one of important economic problems food borne diseases in many counties [1,2]. It is a famous endemic zoonotic disease all over the world and difficult to be controlled [3,4]. Patient diagnosed with severe S. Enteritidis infections have an increased colon cancer risk [5].
∗ Corresponding author at: Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (I.M. Moussa).
In 1900s, vaccination can eradicate to some extend S. Pullorum and S. Gallinarum from poultry flocks, however, these eradication lead to propagation of S. Enteritidis, S. Typhimurium and other serovars, as S. Heidelberg and S. Kentucky, in the birds [6].Ubiquitous use of chemotherapeutic agents and growth promoters in our farms led to the appearance of multidrug-resistant microorganisms [7]. The correlation between the mortality rate in humans and infections with S. Typhimurium resistant to chloramphenicol, streptomycin, tetracycline, ampicillin and sulphonamide had been previously identified [8]. Following the use of antibacterial drugs in food animals, multidrug-resistant salmonellae are transmitted to humans, through the food [9]. In developing countries improper use of antimicrobial agents in human medicine
https://doi.org/10.1016/j.jiph.2019.09.007 1876-0341/© 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Magdy OS, et al. Genetic diversity of Salmonella enterica recovered from chickens farms and its potential transmission to human. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.09.007
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increase the occurrence of multidrug-resistant Salmonella strains [10]. PCR method is rapid, specific and sensitive method used for the detection of food borne pathogens [11]. The fliC gene, encoding for the flagellin protein, has been used as a target gene to determine the antigenic specificity and genetic diversity in Salmonella [12]. Nowadays, sequencing and phylogenetic analysis become an important molecular methods for characterization of pathogens. The objective of the present investigations was to characterize salmonellae isolated from broiler breeder chickens farms using antimicrobial sensitivity test, polymerase chain reaction and sequencing analysis of representative isolates. Materials and methods
Table 1 Number and sources of the positive Salmonella suspected samples. Source of the isolates
Number
Positive salmonellae
Fecal swabs Fecal swabs Fecal swabs Fecal swabs Dust swabs Drag swabs Drag swabs Litter swabs Drooping swab Hatched eggs Hatched eggs Hatched chicks Hatched chicks
5 1 3 1 1 2 1 1 1 1 1 1 1
S. Kentucky S. Enteritidis S. Typhimurium S. Breanderp S. Kentucky S. Kentucky S. Enteritidis S. Kentucky S. Kentucky S. Kentucky S. Enteritidis S. Newport S. Enteritidis
Strains Twenty Salmonella isolates were identified biochemically [14] using API 20E (Biomeriux). Serological identification of the Salmonella isolates was carried out [15] using diagnostic poly and ¨ and ¨ antisera ¨ ¨ H (Mast). mono valent Salmonella O Antimicrobial sensitivity test Using ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, colistin, norfloxacin, flumequine, gentamicin, nalidixic acid, neomycin, oxytetracycline, penicillin-G and streptomycin discs (Oxoid) the sensitivity of the isolates was tested by disc diffusion method. The interpretation of the result was carried out as recorded by Refs. [16,17]. Characterization of the Salmonella isolates using polymerase chain reaction (PCR) DNA of Salmonella isolates were extracted using DNA Kit (Catalogue no.51304) according to QIAamp DNA mini kit instructions and Emerald Amp GT PCR master mix (Takara) Code No.RR310A. The Oligonucleotide primers sequences for detection of fliC gene (Fli15-F CGG TGT TGC CCA GGT TGG TAA T- Tym-R ACT CTT GCT GGC GGT GCG ACT T) [18] and for detection of sefA gene (SEFA2 5 -GCA GCG GTT ACT ATT GCA GC-3 and SEFA4 5 -TGT GAC AGG GAC ATT TAG CG-3 ) [19] were manufactured in Midland Certified Reagent Company-oilgos (USA). For detection of fliC gene during PCR, primary denaturation and activation of Taq DNA polymerase, 1 cycle at 94 ◦ C for 5 min. secondary denaturation 35 cycles at 94 ◦ C for 30 s annealing at 56 ◦ C for 30 s., extension at 72 ◦ C for 30 s and final extension at 1 cycle at 72 ◦ C for 10 min. For detection of sefA gene, primary denaturation and activation of Taq DNA polymerase 1 cycle at 94 ◦ C for 5 min, secondary denaturation 35 cycles at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, extension at 72 ◦ C for 30 s and final extension 1 cycle at 72 ◦ C for 10 min were carried out and the PCR products were analyzed [20]. Sequence analysis of the fliC gene of S. Typhimurium and S. Kentucky and sefA gene of S. Enteritidis PCR products were purified using DNA purification kit (Qiagen Inc. Valencia CA). Sequencing reaction was carried out at NLQP gene analysis unit and the obtained nucleotide sequence was analyzed using BioEdit7, mega 5softwares and the nucleotide sequence alignment was carried out using clustal W method [21]. A sequence distance showed the proportion of identical residues between all of the sequences in the alignment as they were currently aligned. The output was a 2-D matrix table which could either be able to eliminated or comma-delimited. The output depends completely upon the quality of the alignment. The sequences were not automatically aligned before the procedure was run.
Table 2 The percentage of resistance of the isolated among Salmonellae. Antibiotics
Resistance %
Ampicillin (A10 , 10 g) Chloramphenicol (C30 , 30 g) Cefotaxime (CTX30 , 30 g) Colistin Sulphate (CL10 , 10 g) Ciprofloxacin (CF5 , 5 g), Flumequine (UB30 , 30 g) Gentamicin (G10 , 10 g) Nalidixic acid (NA30 , 30 g) Norfloxacin (Nor10 , 10 g) Neomycin (N30 , 30 g) Penicillin-G (P10 , 10 I.U) Oxytetracycline (O30 , 30 g) Streptomycin (S10 , 10 g)
25 35 20 85 50 60 75 70 85 45 95 5 50
The Phylogenetic relationship was performed using (dnastar, lasergene, megaalign) software. Results Identification of the isolates The isolates were identified biochemically using API 20E (Biomeriux) and serologically using diagnostic poly and mono ¨ and ¨ antisera ¨ ¨ valent Salmonella O H (Mast). Twenty salmonellae (Salmonella Kentucky n = 11, Salmonella Enteritidis n = 4, Salmonella Typhimurium n = 3, Salmonella Breanderp n = 1 and Salmonella Newport n = 1 were identified from broiler breeder chickens farms as shown in Table 1. Results of antimicrobial sensitivity test for Salmonella isolates It is clear that 95% of isolates demonstrated resistance and intermediate resistance to penicillin, 85% to both norfloxacin and colistin sulphate, 75% to gentamicin, 70% to nalidixic acid and 60% for flumequine as shown in Table 2. Results of PCR for sefA gene amplification from Salmonella Enteritidis isolates The agarose gel electrophoresis with positive PCR amplification of 310 bp fragments of sefA gene of S. Enteritidis, whereas no amplification could be observed with the extracted DNA of the negative control Escherichia coli NCIMB 50034 as shown in Fig. 1. Results of PCR for fliC gene amplification from of Salmonella Typhimurium isolates Agarose gel electrophoresis with positive PCR amplification of 599 bp fragments of fliC gene of S. Typhimurium, whereas no ampli-
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Fig. 1. The amplified product of sefA gene of S. Enteritidis in the extracted DNA from the isolated SE. Lane M: 100–600 bp DNA ladder (Gel Pilot 100pb), Lanes 1–3: S. Enteritidis isolates positive for sefA gene, Lanes +: Positive control (S. Enteritidis ATCC 13076) and Lane −: Negative control (E. coli NCIMB 50034).
Fig. 2. The amplified product of fliC gene of S. Typhimurium i in the extracted DNA from the isolated ST. Lane M: 100–600 bp DNA ladder (Gel Pilot 100 bp), Lanes 2 and 3: S. Typhimurium isolates positive for fliC gene, Lane +: Positive control (S. Typhimurium ATCC 14028) and Lane −: Negative control (E. coli NCIMB 50034).
Fig. 3. DNA product of fliC gene of S. Kentucky isolates. Lane M: 100–600 bp DNA ladder (Gel Pilot 100 bp), Lanes 1–11 S. Kentucky isolates positive for fliC gene, Lane +: Positive control (S. Typhimurium ATCC 14028) and Lane −: S. Enteritidis ATCC 13076.
fication could be observed with the extracted DNA of the negative control E. coli NCIMB 50034 as shown in Fig. 2. Results of PCR for fliC gene from Salmonella Kentucky isolates Fig. 3 illustrated the agarose gel electrophoresis with positive PCR amplification of 599 bp fragments of fliC gene of S. Kentucky in 11 S. Kentucky isolates along with the positive and negative controls, whereas the positive control of the reaction was used Salmonella type ATCC 14028.
Sequence analysis of Salmonella isolates The nucleotide sequence of 310 and 599 bp PCR products of sefA and fliC genes of Salmonella Enteritidis, Salmonella Kentucky and Salmonella Typhimurium respectively had been sequenced using Applied Bio systems 3130 genetic analyzer (USA). The obtained sequences were blasted with the highly similar sequences which were downloaded and imported in BIOEDIT version 7.1.11. Multiple sequence alignments were conducted using Clustal W application embedded in the BIOEDIT.
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Fig. 4. Neighbor joining phylogenetic tree for Salmonella Enteritidis different strain. Partial (sefA) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
Fig. 5. Neighbor joining phylogenetic tree for Salmonella Typhimurium different strain. Partial (fliC) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
The Nucleotide and amino acid alignment of the obtained sequence in comparison with the imported reference strains from gene bank as shown in (Figs. 4–6). The obtained Nucleotide sequences were submitted in gene bank with access no. KF601584 for Salmonella Enteritidis strain Giza/VRLCU-144-5/2011(sefA) gene, KF601585 for Salmonella Typhimurium strain Giza/VRLCU144-6/2011(fliC) gene, KF601581, KF601582 and KF601583 for Salmonella Kentucky strain Giza/VRLCU-144-1/2011(fliC) gene, Salmonella Kentucky strain Giza/VRLCU-144-5/2011(fliC) gene and Salmonella Kentucky strain Giza/VRLCU-144-10/2011(fliC) gene respectively.
Discussion Food borne disease outbreaks due to Salmonella serovars is an important economic disease, mainly in developing countries [1]. It is one of the most problematic zoonotic diseases in addition to significant morbidity and mortality rates [2]. The Salmonella Enteritidis and Salmonella Typhimurium are the most important strains that infect human [22]. In the present study 20 isolates collected from broiler breeding farms (fecal swabs, dust swabs, drag swabs, litter swabs, drooping swabs, hatched eggs and hatched chicks) were identified as 11 S.
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Fig. 6. Neighbor joining phylogenetic tree for Salmonella Kentucky different strain. Partial (fliC) gene sequences showing the obtained sequence (defined by asterisk*) clustered with the sequence group of almost identical sequences that represent all tree sequences.
Kentucky, 4 S. Enteritidis, 3 S. Typhimurium, 1 S. Breanderp and 1 S. Newport. Salmonellae were recorded from environmental samples of poultry flocks [23,24]. Improper utilization of chemotherapeutic agents and growth promoters in our farms led to the appearance of multidrugresistant salmonellae. It is clear that 95% of isolates were resistant to penicillin, 85% to norfloxacin and colistin sulphate, 75% to gentamicin, 70% to nalidixic acid and 60% to flumequine. All Salmonella isolates were resistant to ampicillin, cefotaxime, erythromycin, kanamycin, nalidixic acid, neomycin, novobiocin, streptomycin, spectinomycin and tetracycline [25]. The prophylactic use of many antimicrobials in poultry feed can also lead to acquired antibiotic resistance [26]. There is an emergency need to control the use of growth promoters and antimicrobial agents in animals to minimize the emergence of resistant strains [27]. In the present study, two pairs of primers were used for detection of fliC and sefA genes among the isolates using PCR assay. Salmonella Typhimurium and Salmonella Enteritidis from hens’ eggs, using multiplex polymerase chain reaction had been detected before [28]. All S. Enteritidis isolates amplified sefA gene. The sefA is specific target gene for S. Enteritidis serovars and the fliC is specific target for serotypes Typhimurium and Kentucky [29]. fliC targets the gene encoding the flagellar antigens i expressed in S. Typhimurium and S. Kentucky and uncommon serovars: S. Aberdeen, S. Bergen and S. Kedougou [29]. The OmpR gene regions of Salmonella strains were previously amplified and sequenced [30]. The Salmonella fliC sequences had been applied by many authors [31,32], they found that these salmonellae were conserved at their terminal and variable in the central region between serotyped. The obtained nucleotide sequences were submitted in gene bank with access no. KF601584, KF601585, KF601581, KF601582 and KF601583 for S. Enteritidis, S. Typhimurium, and S. Kentucky strains. Conclusion The data demonstrated the high prevalence of multidrugresistant Salmonella serovars that indicated alarming in the veterinarian therapeutic treatment and there is a need to determine antibacterial susceptibility pattern regularly to detect multidrugresistant salmonellae. S. Enteritidis and S. Typhimurium were identified from the examined broiler breeder chickens farms, so
special biosecurity and control measures must be carried out for control of Salmonella and limit its potential transmission to human. Emergency control of salmonellae is a global public health concern. Sequence analysis of the selected five salmonellae in the present study confirm the report of Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Kentucky circulation among broiler breeder flocks under the present study. Funding No funding sources. Competing interests None declared. Ethical approval Not required. Acknowledgment The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for supporting the work through the research group project No: RG-162. References [1] Alves LMC, Costa NF, Silva MIS, Sales SS, Correia MR. Toxinfecc¸ãoalimentarp por Salmonella Enteritidis: relato de um surtoo corridor em São Luís-MA. Higiene Alimentar 2001;15:57–8. [2] Castiglioni Tessari EN, Iba Kanashiro AM, Stoppa GFZ, Luciano RL, De Castro AM, Cardoso AP. Important aspects of Salmonella in the poultry industry and in public health, Salmonella—a dangerous food borne ISBN 2012; (6): 978-953307-782-6. [3] Santo LR, Nascimento, VP, Flores, ML, Rosek H, D’Andrea A, Albuquerque MC, Rampanelli Y, Machado NP, Rios S, Fernandes SA. Salmonella Enteritidis isoladas de amostras clinicas de humanos e de alimentos envolvidos em episódios de toxinfecc¸ões alimentares, ocorridas entre 1995 e 1996, no Estado do Rio Grande do Sul Revista Higiene Alimentar, São Paulo. 2002; (6) (102/103), 93–99. [4] Antunes P, Reu C, Sousa JC, Peixe L, Pestana N. Incidence of Salmonella in poultry and their susceptibility to microbial agents. Int J Food Microbiol 2003;82:97–103. [5] Mughini-Gras L, Schaapveld M, Kramers J, Mooij S, Neefjes-Borst EA, Pelt WV, et al. Increased colon cancer risk after severe Salmonella infection. PLoS One 2018;13(1):e0189721, http://dx.doi.org/10.1371/journal.pone.0189721, eCollection 2018.
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ARTICLE IN PRESS O.S. Magdy et al. / Journal of Infection and Public Health xxx (2019) xxx–xxx
[6] Foley SL, Nayak R, Hanning IB, Johnson TJ, Han J, Ricke SC. Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Appl Environ Microbiol 2011;77(13):4273–9. [7] WHO. World Health Organization. Drug-resistant Salmonella. Food safety Department Geneva, Fact sheet 2005; N 139. [8] Helms M, Vastrup P, Mølbak K. Antimicrobial drug-resistant Salmonella Typhimurium (reply to Dahl). Emerg Infect Dis 2003;9(10):1350–1. [9] Angulo FJ, Johnson KR, Tauxe RV, Cohen ML. Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb Drug Resist 2000;6:77–83. [10] Threlfall EJ, Ward LR, Rowe B. Increasing incidence of resistance to trimethoprim and ciprofloxacin in epidemic Salmonella Typhimurium DT104 in England and Wales. Euro Surveill 1997;2:81–4. [11] Olsen PE. Giant lava flows, mass extinctions, and mantle plumes. Science 1999;284:604–5. [12] Dauga C, Zabrovskaia A, Grimont PD. Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica. J Clin Microbiol 1998;36(10):2835–43. [14] Quinn PJ, Markey BK, Carter ME, Donnelly WJ, Leonard FC. Veterinary microbiology and microbial diseases. Oxford, London: Black Well Scientific Publications; 2002. [15] Popoff MY, Le Minor L. Antigenic formulas of the Salmonella serovars, 2001; 8th revision, World Health Organization Collaborating Centre for Reference and Research on Salmonella, Pasteur Institute, Paris, France. [16] NCCLS. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Wayne, PA: Clinical Laboratory Standard Institute; 2002. [17] Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 2010; Twenty-Fifth Informational Supplement (M100-S25). [18] Soumet C, Ermel G, Rose N, Rose V, Drouin P, Salvat G, et al. Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella sp., Salmonella Enteritidis and Salmonella Typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 1999;28(2):113–7. [19] Akbarmehr J, Zahraei Salehi T, Nikbakht GH. Identification of Salmonella isolated from poultry by MPCR technique and evaluation of their hsp groEL gene diversity based on the PCR-RFLP analysis. Afr J Microbiol Res 2010;4(15):1594–8.
[20] Sambrook J, Fritscgh EF, Meniates T. Molecular cloning. A laboratory manual, Vol. 1. New York: Cold Spring Harbor Laboratory Press; 1989. [21] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22(22):4673–80. [22] Afshari A, Baratpour A, Khanzade S, Jamshidi A. Salmonella Enteritidis and Salmonella Typhimurium identification in poultry carcasses. Iran J Microbiol 2018;10(1):45–50. [23] Davies R, Breslin M. Environmental contamination and detection of Salmonella enterica serovar Enteritidis in laying flocks. Vet Rec 2001;149(23):699–704. [24] Kinde H, Castellan DM, Kass PH, Ardans A, Cutler G, Breitmeyer RE, et al. The occurrence and distribution of Salmonella Enteritidis and other serovars on California egg laying premises: a comparison of two sampling methods and two culturing techniques. Avian Dis 2004;48:590–4. [25] Shah AH, Korejo NA. Antimicrobial resistance profile of Salmonella serovars isolated from chicken meat. J Vet Anim Sci 2012;2:40–6. [26] Rajagopal R, Mini M. Outbreaks of salmonellosis in three different poultry farms of Kerala, India. Asian Pac J Trop Biomed 2013;3(6):496–500. [27] El Jakee J, Ata NS, Abd El-Moez SI, Kandiel MM, Radwan NM. Assessment of the prevalence of Salmonellae in food. Int Curr Microbiol Appl Sci 2014;3(3):30–42. [28] El Jakee J, Khelfa DG, El-Safty MM, Seida AA, Marouf S, Hahne J, et al. Multiplex PCR-based detection of Salmonella Typhimurium and Salmonella Enteritidis in specific pathogen free (SPF) and commercial eggs. Clin Microbiol 2016;5(2):241. [29] O’Regan E, McCabe E, Burgess C, McGuinness S, Barry T, Duffy G, et al. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples. BMC Microbiol 2008;8:156. [30] Arunachalam R, Senthilkumar B, Senbagam D, Selvamaleeswaran P, Rajase karapandian M. Molecular phylogenetic approach for classification of, Salmonella Typhi. Res J Microbiol 2012;7(1):13–22. [31] Smith NH, Selander RK. Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella Typhimurium. J Bacteriol 1990;172(2):603–9. [32] Li J, Nelson K, McWhorter AC, Whittam TS, Selander RK. Recombinational basis of serovar diversity in Salmonella enterica. Proc Natl Acad Sci U S A 1994;91:2552–6.
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