Isolation and characterization of Salmonella Enteritidis and Salmonella Typhimurium from chicken meat in Egypt

Introduction: Salmonella enterica serovars Enteritidis and Typhimurium represent the major serovars associated with human salmonellosis. Contamination of meat products with these serovars is considered the main source of infection. Methodology: In this study, 100 raw chicken meat samples were investigated for the presence of Salmonella spp., which were subsequently identified based on biochemical and serological tests as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) profile. Furthermore, the isolated serovars were examined using multiplex polymerase chain reaction (PCR) for the presence of virulence genes suspected to have a role in infection. Results: S. Enteritidis was isolated from two samples (2%), while S. Typhimurium was isolated from three samples (3%) of chicken meat. Of the 17 examined virulence genes using multiplex PCR, the sitC, sopB, sifA, lpfC, spaN, sipB, invA, spiA, and msgA genes were detected in S. Enteritidis. However, the sitC, iroN, sopB, sifA, lpfC, spaN, sipB, invA, and tolC genes were successfully amplified in S. Typhimurium. Conclusions: The detection of S. Enteritidis and S. Typhimurium in meat, even at low incidence, has important implications. In addition, the data presented here is the first attempt to identify a wide range of virulence genes in Egyptian Salmonella isolates recovered from meat products. A strict public health and food safety regime is urgently needed in order to decrease the human health hazard risk associated with salmonellosis.


Salmonella enterica represent the major cause of bacterial foodborne infection in United
States [1].Salmonella enterica serovars Enteritidis is considered the major cause of human salmonellosis out breaks in United states and Europe [2][3], linked in the main to consumption of contaminated poultry products, including eggs [4][5].Living poultry considered the main reservoir of salmonellae, microorganism present in the intestinal tract, skin, and feather of living birds.Bacterial contamination of poultry carcasses and their cuts are a main result of improper hygienic measures, improper cooking and abuse of temperature, this results in dissemination of infection throughout the plants during processing including evisceration, cooling, packaging and transport stages [6].According the food standards the presence of salmonellae in the foodstuff makes the food unsafe for the human consumption [7][8].Typhoid is still a major problem in the developing countries, mainly due to the lack of sanitation and hygiene standards [9][10].The disease is transmitted mainly via the contamination of foodstuff and water with the pathogen, the disease affecting more than 90 million people worldwide yearly with variable morbidity and mortality rates [11].The severity of the infection depends on many factors including, the strain of Salmonella, the standard of hygiene, age of the bird, route of infection, and immune status of the birds [12].Generally, the growth and multiplication of salmonellae within the host is governed by a central regulatory gene function.The virulence determinant genes of Salmonella spp. is associated either with a combination of chromosomal or plasmid factors [13].These genes have a role in adhesion, invasion, and enterotoxin production [13][14][15].So far, there is scarcity of data regarding the incidence of these genes in meat products in Egypt.
Therefore, present study was undertaken to isolate Salmonella enterica from raw and chicken meat as well as to screen their virulence genes profile within the isolates using multiplex PCR.

Isolation and identification of Salmonella
A total of hundred samples (100 g each) of raw and chicken meat (50 of each), were collected from different supermarkets in Minoufiya and Cairo Governorates.Samples were collected aseptically and transferred for further bacteriological examination.Samples were pre-enriched in buffered peptone water for 16-20 h at 35-37°C.One milliliter of each preenrichment culture was used to inoculate 10 ml of Muller-Kauffman tetrathionate (Oxoid Ltd, Hampshire, England), which were incubated at 37 °C, for 24 h.After thoroughly mixing each enrichment broth, a 3 mm (10 µl) loopful was removed and streaked onto xylose lysine desoxycholate (XLD; Oxoid Ltd, Hampshire, England).Following incubation at 37°C for agar medium for purification.Identification of isolated microbial strains was based on biochemical tests [16][17][18][19].Colonies were inoculated into triple sugar iron (TSI) agar and lysine iron agar and incubated at 37 °C for 24 h.Diagnosis was confirmed based on Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) profile according to previously developed protocol [20].Briefly, about 10 mg of cell material of the cultured strains were suspended in 300 μl of sterile water.After addition of 900 μl of absolute ethanol, the mixture was centrifuged at 10,000 rpm for 2 min.The supernatant was discarded and the pellet was suspended in 50 µl formic acid (70% v/v).After adding 50 µl acetonitrile (AN), the mixture was centrifuged at 10,000 rpm for 2 min and 1 μl of the clear supernatant was transferred to the MALDI target and allowed to dry followed by addition of 1 µl αcyano-hydroxy-cinnaminic acid (Bruker Daltonik GmbH) in a standard organic solvent mixture (2.5% trifluoroacetic acid, 50% AN in water).All chemicals used were of the highest quality (Merck, designated to be especially suitable for HPLC or MALDI-based techniques).Before each MALDI run, E. coli 1917 strain Nissle was analysed to serve as the positive control and calibration standard.The MALDI-TOF MS analysis was performed using a Bruker microflex LT mass spectrometer (Bruker Daltonik GmbH) and the spectra were automatically identified using the BrukerBioTyper™ 1.1 software.Serotyping was performed according to the Kauffmann-White typing scheme using slide agglutination with different "O" and "H" antisera (Difco Laboratories, Detroit, MI, USA).

Investigations of virulence genes repertoire Extraction and purification of DNA
One milliliter of freshly enriched Salmonella was transferred to a micro-centrifuge tube with a capacity of 1.5 ml.The cell suspension was centrifuged for 10 min at 14,000 × g.
The pellet was resuspended in 300 µl of DNase-RNase-free distilled water and centrifuged at 14,000 × g for 5 min.The supernatant was carefully discarded and the pellet was resuspended in 200 µl of DNase-RNase-free distilled water, incubated for 15 min at 100 °C and immediately chilled on ice, then centrifuged for 5 min at 14,000 × g at 4 °C.An aliquot of 5 µl of the supernatant was used as the template DNA in the PCR [21].

Multiplex PCR procedures
The isolated Salmonella strains were examined by multiplex PCR for the presence of several genes thought to be involved in virulence.Targeted genes and their primer sequences used in the amplification studies are summarized in table (1).The primer sequence and PCR conditions was carried according to Skyberg and co-workers [22].Three reactions were used to amplify the seventeen genes; the first reaction (set 1) to amplify spvB, spiA, pagC, cdtB, and msgA; Set 2 to amplify invA, sipB, prgH, spaN, orgA, and tolC; Set 3 to amplify iroN, sitC, lpfC, sifA, sopB, and pefA genes.The cycling conditions and reaction mixtures were the same for each multiplex procedure used; only the primers differed among the three reactions.
The cycling conditions and reaction mixtures were the same for each multiplex procedure used; only the primers differed among the three reactions.The amplification was carried out in 50 µl reaction PCR tubes containing 5 µl master mix (10×), 5 µl of 20 mM dTNPs mix, 0.15 µl of Taq polymerase (5 U/1 µl), 1 µl of 0.1 mM forward and reverse primers and 1 µl of DNA template.The reaction was subjected to the following condition in the thermal cycler 5 min at 95 º C, 25 cycles of 30 sec at 94 º C, 30 sec at 66.5 º C, and 2 min at 72 º C, with a final cycle of 10 min at 72 º C, followed by a hold at 4 º C. PCR products obtained were subjected to horizontal gel electrophoresis in 1.5% agarose, and the size of the amplicon was determined by comparison with DNA marker.

Incidence of Salmonella spp in the examined meat samples
Suspected Salmonella colonies were confirmed using biochemical tests and MALDI-TOF MS profiles.Based on the serological tests, only Salmonella Enteritidis and Salmonella Typhimurium could be identified.The data presented in table (2) showed that, the recovered Salmonella spp.from the examined meat products.Salmonella Enteritidis was isolated from two samples (2%) of the examined raw meat and chicken meat.While in case of Salmonella Typhimurium, 2 samples (4%) and one sample (2%) of the examined 50 samples of raw meat and chicken meat, respectively, were positive.

Antibiotic sensitivity test
Salmonella Typhimurium isolates were resistant to ampicillin, amoxicillin, penicillin, neomycin, ofloxacin, doxycycline and chloramphenicol.However they were sensitive to tetracycline, streptomycin and erythromycin.
On the other hand, the isolated Salmonella Enteritidis showed complete resistance to all tested antibiotics.

Discussion
Salmonella induced food-borne illness has received more attention in comparison with other food-borne induced pathogens.In this study, only 3 Salmonella Enteritidis and 2 Salmonella Typhimurium isolates were recovered from 100 raw and chicken meat samples (Table 2).Salmonella enterica is highly diverse, containing over 2,500 different serovars.The Representative Serovars from this species are the most commonly isolated serovars during outbreaks of food-borne salmonellosis, including Salmonella Enteritidis, Salmonella Typhimurium, S. enterica serovar Virchow, and Salmonella Infantis [25].Salmonella Enteritidis and Typhimurium are the most predominant isolated organisms in most Salmonella cases associated with the consumption of contaminated poultry, pork and beef products [26].Contamination with Salmonella in poultry products can occur at multiple steps along the food chain, which includes production, processing, distribution, retail marketing, handling and preparation [27].
The manifest of salmonellosis pathogenic process and the severity of infection mainly ruled by an array of factors that work synergistically to maintain the growth of microorganism within the host and assist the microorganism in expressing its virulence, these factors briefly named as virulence genes [28].Herein, this study was mainly focused on 17 genes (spvB, spiA, pagC, cdtB, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, lpfC, sifA, sopB, and pefA) that known to have a role in salmonella infection in poultry.Out of 17 examined virulence genes, only 11 genes were successfully amplified using multiplex PCR table (3).
The Panel on Biological Hazards recommended assessing the public health risk posed by "Salmonella Typhimurium-like" strains, as Salmonella Typhimurium isolates are commonly found to be antimicrobial resistant [44].In the present study, the three Salmonella isolates were resistant to ampicillin, amoxicillin, penicillin, neomycin, ofloxacin, doxycycline, and chloramphenicol.They were sensitive to tetracycline, streptomycin and erythromycin.
Interestingly, the two Salmonella Enteritidis isolates were resistant to the all tested antibiotics.
It should be noted that these antimicrobials are frequently used on Egyptian poultry farms.
These results mainly confirmed by su et al., and Boukoucha et al., [45][46] who stated that the increasing antimicrobial resistance in non-typhoid Salmonella species expressed a serious problem for public health worldwide and declared that the high rate of resistance was hampering the use of conventional antibiotics.The growing resistance to newer antimicrobial agents is aggravating the situation as well.

Conclusion
From our study, some conclusions with potential implications for the isolation and identification of Salmonella from poultry meat could be drawn.First contamination of chicken meat with Salmonella indicates bad microbiological quality of chicken meat.
Secondly, the data presented is considered the first attempt conducted to identify wide range of virulence genes of the Egyptian salmonella isolates recovered from poultry meat products.
This study confirms the need for a strict public health and food safety regimes in order to decrease the human health hazard risk associated salmonellosis.

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Primer sequence and targeted amplicon size used for Salmonella typing