Prevalence of Foodborne Pathogens in Food Items in Quetta, Pakistan

This study was carried out in Quetta, Pakistan from February 2016 to December 2016. A total of 800 samples were collected randomly from different areas of Quetta city. Out of 800 samples, 200 chicken meat samples (100 raw fresh chicken + 100 processed), 200 beef meat samples (100 diced +100 minced), 200 raw milk samples (100 from shop + 100 dairy farm), 100 vegetables samples and 100 salad samples were collected and preserved in sterilized ice filled box and transported to the Bacteriology Lab of CASVAB.

ISO 16654 (2001) method was followed for detection of E. coli O157:H7. In brief 25 g of each sample was diluted in 225mL of modified tryptone soya broth (mTSB, Oxoid, UK), supplemented with novobiocin. The samples were then homogenized and incubated for 18–24 h at 41.5°C. After the enrichment the selective and differential isolation of enterohemorrhagic E. coli O157:H7 was carried out on Sorbitol MacConkey agar, supplemented with cefixime, and tellurite (CT-SMAC, Oxoid, UK) and incubated overnight at 37°C. From each sample, one Non Sorbitol fermenting (NSF) colony was transferred to tryptone soys agar (Oxoid) and incubated for 24 h at 37°C. Subsequently, one isolate from the subculture was further tested for agglutination with an E. coli O157:H7 latex agglutination test kit (Welcolex-Remel, UK) for serogroup O157:H7 confirmation.

ISO 6579 (2002) method was followed for detection of Salmonella spp. In brief each 25g food sample was diluted with 225mL of sterile Buffered Peptone Water (Oxoid - UK), homogenized and incubated for 18 h at 37°C. One milliliter of the culture was added to 10mL of Rappaport–Vassiliadis soy peptone and incubated at 42.5°C for 18 h. One loopful of the culture was then streaked onto xylose lysine deoxycholate agar (Oxoid, UK) and incubated at 37°C for 24h. The resulting presumptive Salmonella colonies were tested with biochemical screening on RapidID ONE test kits (Remel, UK)

For detection of L. monocytogenes, 25g of the food samples was mixed with 225mL of sterile Fraser Broth Listeria enrichment broth (Oxoid, UK), homogenized, followed by incubation for 48h at 30°C. One loopful of the culture broth was streaked onto Palcam agar (Oxoid, Hampshire, UK) and incubated at 37°C for 48 h. Presumptive colonies were streaked onto horse blood agar and TSA plates and incubated at 35°C for 48 h. The resulting presumptive Listeria colonies were submitted for biochemical screening (oxidase test, catalase test and Gram staining).

ISO 10272-1 (2006) method was followed for detection of Campylobacter. A 25 g of the food sample inoculated in 250mL of the enrichment medium Bolton Broth supplemented with 0.02g/L cefoperazone, 0.02g/L vancomycin, 0.02g/L trimethoprim lactate and 0.01g/L amphotericin-B without the addition of lysed defi brinated horse blood, which was incubated in a microaerobic atmosphere (5% O2, 10% CO2 and 85% N2) at 37°C for 4h to 6h, then at 41.5°C for 44 h (+/- 4h).

The enriched culture was inoculated with a sterile loop onto duplicate Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) supplemented with 0.032g/L cefoperazone and 0.01g/L amphotericin-B. Suspected colonies were taken from CCDA and streaked onto Blood Agar plates for confirmation. The plates were incubated in a microaerobic atmosphere at 41.5°C for 24h to 48h for examination of morphology, Gram staining, tests for catalase and oxidase, hippurate hydrolysis and hydrolysis identification of indoxyl acetate.

DNA was extracted through CTAB (Cetyletrimethyle ammonium bromide) method as described earlier by Minas et al. (2011).

PCR reactions mixture (30μl) consisted of 15µl of PCR Master mix (Gene All), 1µl of each primer (Macrogen), 10µl of Molecular grade water and 3µl of each extraction was used. Primers are enlisted in Table I. Amplification was performed in a gradient Thermocycler. Thermal cycler conditions were set as described by Shanmugasamy et al. (2011). An initial incubation at 94°C for 60 seconds, followed by 35 cycles of denaturation at 94°C for 60 seconds, annealing at 64°C for 30 seconds, elongation at 72°C for 30 seconds, and final extension period for 10 min at 72°C. A 50 bp DNA ladder (Gene One) was used. Deionized distilled water was used as a template for negative control.

Amplified PCR products were then electrophoresed in 1.2% Agarose w/v gel stained with Ethidium bromide and was documented in gel documentation apparatus (Salehi et al., 2005).

Table II shows the prevalence of Salmonella spp., Listeria monocytogenes, Campylobacter jejuni and E. coli (O157:H7) in chicken meat, beef, milk and vegetables samples collected from market. Results revealed that out 0f 800 collected samples 48.37% were contaminated with above mentioned opportunistic bacteria. These results indicate alarming and poor food hygienic conditions prevalent in local market. Campylobacter was highest (28.99%) among the four investigated pathogens followed by Salmonella (19%), E. coli O157:H7 spp. (8%) and Listeria monocytogenes spp. (1.25%), respectively. It was also found that fresh chicken meat compared to processed chicken, minced beef compared to diced beef, milk purchased from retailer compared to one from dairy farms, and fresh salad compared to fresh vegetables had higher bacterial load for all types of bacteria tested.

C. j., Campylobacter jejuni; L. m., Listeria monocytogenes.

The identification of bacteria was also confirmed by PCR product of 284 bp for Salmonella, 589 bp for Campylobacter jejuni, 730 bp for Listeria monocytogenes and 625 bp for E. coli (O157:H7) fragment of rbfO157 and 259 bp for flicH7 gene (Supplementary Fig. S1).

Salmonellosis is considered as the major foodborne illness globally and especially a chief cause of death in non developed countries. In this study, the overall prevalence of Salmonella in food items was found to be 19% ranging from 4-24% in different food items. These results are in line with the previous reports of 32%-36% prevalence of Salmonella in processed frozen chicken in Egypt and Nigeria (Adeyanju and Shola, 2014). Lower prevalence of Salmonella has been reported in developed countries like New Zealand (1.3%) and Italy (9.9%) (Wong et al., 2007; Saraj-Uddin et al., 2015).

Raw cooked food items can be contaminated with Listeria. Under normal circumstances lesterial infections causes diarrhea and gastroinstinal problems, leading to death in 20% cases. In this study, contamination with Listeria was significantly low to other investigated pathogens. Similar findings have been reported previously (de Silva et al., 2014). However, several studies have reported higher prevalence rate in samples of cheese sprouts and other fresh food items.

Campylobacteriosis is cited as the most common bacterial infections of humans globally. It leads body cramps, fever and pain accompanied with bloody diarrhea or dysentery. In this study 28.99% of food samples were found contaminated with Campylobacter, which put it on top of the list of pathogens investigated. Surprisingly, 32% processed and 48% fresh chicken meat samples were found positive for Campylobacter. Previously several studies have reported very high (91%) prevalence in fresh poultry meat (Wong et al., 2007).

Escherichia coli are mostly harmless and play an important role in healthy human digestive system, but some are equipped with virulence and pathogenic factors. To date six different E. coli pathotypes have been reported and Shiga toxin-producing E. coli O157:H7 is the most prevalent strain. Our results revealed the 8% prevalence of E. coli O157:H7 in 800 analyzed samples. Beef, milk and salad samples showed the highest rate (12%) of this infectious agent which are in agreement to previous studies (Chang et al., 2013; El-Tawab et al., 2015; Rahimi et al., 2012; Sancak et al., 2015). On contrary, high prevalence (48%) of E. coli was reported in raw vegetables in Multan, Pakistan (Rabia et al., 2014). In Lahore, another city of Pakistan 41-50% incidence of E. coli O157:H7 were found in juices, fresh fruits and vegetables (Mehwish and Ayesha, 2014). Chang et al. (2013) reported 3.3% of the prevalence of E. coli O157:H7 were found n in Malaysia which is in range of our findings.

This study indicates high prevalence of foodborne infectious in local market of Quetta, Pakistan. Special measures are required to improve the hygiene and quality of food items.

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.sc17

Authors have declared no conflict of interest.