Investigation of Foodborne Salmonella spp. in Bangladesh and Development of Real-Time PCR Based Identification Method

Abstract

Background: Avian non-typhoidal salmonellosis is a major concern for to the development of poultry sector in Bangladesh. The conventional microbiological tests of Salmonella are time consuming, laborious and costly. Further confirmatory and rapid methods for detection of Salmonella spp. and its distribution are very crucial. Hypothesis: Salmonella spp., the etiological agent of salmonellosis is poultry derived zoonotic pathogen. Epidemiological studies based on the molecular genetics to identify clonal and strain distribution among particular locality or within the country are invaluable to track down the routes of transmission of Salmonella spp. and their distribution. Method: A total of 307 poultry samples were collected from fourteen poultry farms, live bird markets, hotel and household kitchens in the supply line from producers to consumers. Among these, 154 farm samples were found to have Salmonella spp. using selective culture and PCR amplification of invA gene. The isolates were further genotyped through Randomly Amplified Polymorphic DNA (RAPD), Amplified Ribosomal DNA Restriction Analysis (ARDRA), and Multi Locus Sequence Typing (MLST) methods. Antimicrobial profiles and genotypic variations were compared to address Multi Drug Resistance (MDR) among circulating genotypes. In addition to develop an economical and rapid method, all the 307 samples were analyzed using invA gene targeted SYBR green-qPCR to quantify the Salmonella spp. bacterial load and their characterization. Purified 284 amplicons of invA were cloned in the TOPO TA vector. Salmonella gDNA was used for the development of a standard template for SYBER green qPCR. The standard curve showed good linearity (R2 _ 0.97) and efficiency (99%). The bacterial load among farm samples were identified using the standard reference Ct value and the Ct values of the test samples. Results: Out of 687 isolates collected from farm samples, 200 (29.11%) were confirmed as Salmonella spp.. These 200 isolates were differentiated into 18 RAPD genotypes while MLST of these 18 groups assigned the isolates into 3 sequence types (STs) - ST198, ST11, and ST214. The prevalent MLST type, ST198 (50.5%) was represented as Salmonella enterica Kentucky, followed by ST214 (33%) representing S. enterica Litchfield and ST11 (16.5%) for S. enterica serovar Enteritidis. The present study revealed that farm-originated Salmonella spp. were multidrug-resistant, including the high level of resistance against doxycycline (96.49%) followed by ampicillin (88.30%), oxytetracycline (88.30%), and ciprofloxacin (66.08%). II We also developed a novel SYBR green-qPCR quantification method that detected the highest load of Salmonella spp. in the poultry dropping samples up to 1.3×107/ml followed by 6.8×106/ml in the cloacal swab, 3.8×105/ml count for poultry feed and 2.7 ×104/ml for poultry farm water samples respectively. Among the live bird market samples, water was analyzed and found to be highly contaminated with Salmonella (78%) through SYBR green-qPCR detection. Other market samples including cage of chicken (55%), processing board (60%), knives (40%) as well as transport van of chicken (from farm to bazar) (60%) were also found highly contaminated. Besides, a high percentage of Salmonella contamination among raw chicken (55%) and raw food (30%) processing areas of hotel kitchen indicates the possible means of transmission throughout the routes. Conclusion: The present investigation can be summarized as- (i) the collected Salmonella serovars from poultry farms are zoonotic in nature, indicating that poultry could be a major source of non-typhoidal zoonotic salmonellosis; (ii) the dominant MLST type, Salmonella ST198 with multidrug resistance traits in different farms confirm the probability of intra-farm transmission; (iii) the risk of Salmonella contamination is considerably high in different supply chain points; and (iv) SYBR Green Real-Time PCR can be a reliable and rapid method for Salmonella spp. detection.