Isolation, phenotypic characterization and genotypic identification of Salmonella species isolated from food and water samples in Karachi, Pakistan.

Background: Foodborne illness is a global health problem, and Salmonella is one of the leading bacterial pathogens to cause salmonellosis and typhoid fever worldwide. These infections remain an obstinate risk to human and animal health despite substantial innovations and cleanliness practices. The high incidence of these infections is resulting not only from infected eggs and chicken but also from different food commodities. This research was designed in the direction of the certainties mentioned above and the high occurrence rate of extensively drug-resistant (XDR) infections in Pakistan. Methodology: The study focused on the isolation and identification of Salmonella species from food and water samples collected from different regions of Karachi, Pakistan. The standard protocol of enrichment, culturing, and biochemical confirmation from Biological Analytical Manual (BAM) was used to characterize Salmonella's phenotypic isolation and characterization. These isolates were then subjected to Polymerase chain reaction (PCR) and resolved on 1.5% agarose via electrophoresis. Results: From the total of 1010 samples, 10 genotypically confirmed Salmonella isolates were obtained by detecting the invA gene by using PCR. Food items of different kinds exhibited 80% of the total positive isolates, while 20% is from the water sample. Conclusion: The study exhibited the highest prevalence of Salmonella in chicken meat, which may indicate insufficient hygiene and sanitation practices during slaughtering and the supply chain. Incidence of Salmonella was also found in water and spices, signifying the improper sanitary systems and unhygienic handling of food items in many localities of the city, affecting human health.


Introduction
Foodborne illness is a global health problem, and Salmonella is one of the leading bacterial pathogens to cause foodborne disease worldwide 1 . Salmonella is the genus of gram negative rods belonging to the Enterobacteriaceae family, causing salmonellosis and typhoid fever after ingesting contaminated food. It is comprised of more than 3000 serovars which are categorized based on host specificity and invasiveness into three groups 2 . Group 1 is incredibly host adaptive and invasive such as Salmonella enterica subsp. enterica ser. Typhi; Group 2 is invasive but non-host-adaptive such as Salmonella enterica subsp. enterica ser. Enteritidis and Salmonella enterica ser. Typhimurium; while Group 3 consists of nonhost-adaptive but highly intrusive serotypes [3][4][5][6] .
Salmonellosis and other infections caused by Salmonella remains an obstinate risk to human and animal despite substantial innovations and cleanliness practices in plant and food manufacturing and production. High incidence of these infections resulting from infected eggs, meat, and various food commodities. These can be a source of illness as a consequence of insufficient cooking and crosscontamination of working areas 2,7 . Disease caused by Salmonella is generally self-limiting but may cause severe complications in a vulnerable group such as infants and older adults, depending on its serotype and host specificity 2,[8][9][10] .
According to World Health Organization (WHO), 3/4 of all broiler chicken meat was infected with Salmonella spp 11 . In recent years, outbreaks of salmonellosis involving beef, spicy sprout, black and red pepper, etc., have been reported 12 20 . Routes of transmission for non-typhoidal salmonellosis include eggs and poultry, milk and its products, and contact with pets. Person-to-person contact may also result in its transmission 21 . Consumption of contaminated tomatoes, sprouts, fruits, spinach, and peanuts is also the primary source of infection spread [22][23][24][25][26] . Drinking water is a major source of dissemination of Salmonella Typhi. Besides, food washed with contaminated water is also contributing in the spread of different Salmonella species 27,28 .
Antimicrobial resistance is a developing issue these days leading to the failure of present antibiotics, which may prolong nosocomial and community-acquired infections. Salmonella's Antimicrobial resistance genes are usually present on the mobile elements as plasmids, provides elasticity to host bacterium, and helps them distribute these genes across the diverse population 2, 29 .
Pakistan is viewed as an epidemic of XDR Salmonella infection ascending from Hyderabad and some other cities since November 2016 30 . A total of 5423 out of the 8630 laboratories investigated cases from ten different hospitals of Karachi were categorized as XDR Typhoid from Jan 2017 to Jun 2019 31 . The implementation of hygienic practices must be forced on the workers in slaughtering places, and the public needs to be educated for clean and safe food from the markets 32 . This study was designed due to the high occurrence rate of food poisoning and the XDR infections caused by Salmonella in Pakistan. In this research, the presence of Salmonella spp. was evaluated in water and different food products in Karachi city, Pakistan.

Sample Collection & Preparation
Different categories of export quality food products and samples of water and food items collected locally from different areas in Karachi in both raw and cooked form. A total of 1010 samples were collected, homogenized, and processed for Salmonella isolation and identification, based on the BAM with some modifications.

Culture-Based Method
The samples were subjected to non-selective pre-enrichment by homogenizing 25 g of the sample (for liquids; 25 ml) with 225 ml of sterile Lactose broth (LB). This homogenized mixture was incubated for 24 ± 2 h at 35ºC after kept for 1 h at room temperature. For spices, Trypticase Soy Broth (TSB) was used with 0.5% K2SO3 instead of LB due to high microbial load. A total of 0.1 ml of the overnight incubated lactose broth was added to 10 ml Rappaport-Vassiliadis (RV) medium and 1 ml to Tetrathionate (TT) broth, respectively for selective enrichment of Salmonella species, and incubated for another 24±2h at their appropriate incubation temperatures in circulating, thermostaticallycontrolled water bath.
A loopful of the inoculum from both of the enrichment broths was streaked on hektoen enteric agar (OXIDE), bismuth sulfite agar (OXIDE), and xylose lysine deoxycholate agar (OXIDE) separately, and incubated for 24 ± 2 h at 35ºC. Gram's staining reaction confirmed the presumptive Salmonella isolates, as well as by biochemical identification on triple sugar iron slants and lysine iron agar slants and hydrogen sulphide production 33 .

b. Primers Set and PCR Amplification
The invA gene (389 bp) was amplified to detect Salmonella spp. by using specific primers.
The sequence of the primers used were Salm3 (5'-GCTGCGCGCGAACGGCGAAG-3') and Salm4 (5'-TCCCGGCAGAGTTCCCATT-3') 33 . The PCR mix was prepared by GoTag® Green Master Mix (Promega), Forward Primers, Reverse Primers, and Nuclease-Free Water. A total of 23 μl of the mixture was added to each PCR tube. Bacterial lysate (2.5 μl) was then added to make the volume up to 25.5 μl. S. Typhimurium ATCC 14028 was used as a reference strain. PCR was run by using Thermal Cycler (Bio-Rad) with the following condition: preliminary denaturation at 95ºC (5 min), followed by 35 cycles of denaturation at 95ºC (90 sec), annealing at 62ºC (60 sec) and elongation at 72ºC (90 sec). Termination of the cycle was done with the final extension at 72ºC (7 min) 34 .
c. Preparation of Agarose Gel and Resolution by Electrophoresis 1.5% agarose gel was prepared by dissolving 1.5g agarose powder in 100 ml Tris-Borate EDTA (TBE) buffer by heating. After reaching room temperature, 4 µl of Ethidium bromide was added and immediately poured into the gel cast. After placing the gel in an electrophoretic tank, TBE buffer was added. A total volume of 12 µl for each PCR-amplified product was loaded in each well, and 9 µl of the DNA ladder (100 bp) was loaded as a marker. The electrophoresis was done at 120 V for 1.5 to 2 hours. The gel was exposed to UV transillumination to visualize the DNA bands using a Bio-Rad Gel doc system.

Data Collection and Analysis
Data was collected by calculating the frequencies and percentages based on colony morphology on selective media, change in color during biochemical reactions, production of hydrogen sulphide gas, and observing the shape of cells and gram staining reaction in microscopy. Genotypically, the assessment was done on the basis of the presence of the invA gene.

Genotypic Identification of the Salmonella Isolates
Salmonella isolates were tested genotypically by Polymerase Chain Reaction. The invA gene was amplified by PCR for the detection of Salmonella spp. and was resolved on 1.5% agarose Tris-EDTA gel by electrophoresis. Bands that coincide with the positive control band of Salmonella Typhimurium ATCC 14028 were considered positive for Salmonella ( Figure 6). The total occurrence of Salmonella was 0.99% (10/1010), from which 80% were isolated from different food items (Green veges 25%, Processed food 25%, Raw chicken 25%, Ready to eat food 12.5% & Spices 12.5%), while only 20% were from water samples.

Discussion
The current study is focusing on the isolation and characterization of Salmonella from the water and food items collected from different areas in Karachi city, Pakistan. In the present research, 21 isolates were recognized as Salmonella from the total 1010 examined samples using a conventional culture-based identification method. Samples were of export as well as local quality, having raw and cooked both types. The detection of Salmonella was done on the basis of their morphological characteristics and biochemical reactions. Identification based on genotype was also performed. The invA gene was exponentially amplified using PCR and resolved on agarose gel by electrophoresis 34  Pullorum was showed to be the most prevalent in both sick and healthy chicken. From these three isolates, last one was found only in healthy chickens 36 . El-Sharkawy et al. (2017) also designed the prevalence of S. enterica in broiler chicken. Sixty-seven isolates of Salmonella enterica were obtained, which showed the presence of S. Typhimurium and S. Enteritidis 2 . The prevalence of Salmonella was also found high in different spices.
This study provides a good comparison between two different identification systems; i.e., phenotypic and genotypic methods, though it was limited till genotype level rather than to identify different serovars of Salmonella.

Conclusion
From this study, it is concluded that Salmonella is most prevalent in chicken meat, which may indicate the unhygienic slaughtering process or the insufficient general hygiene and cleaning. The study showed that the improvement in sanitation systems and the implementation of hygienic practices could lessen the contamination of Salmonella and eventually the incidence of human diseases.

Conflicts of Interest
The authors have declared that no competing interests exist.