Population Biology Of Foodborne Pathogenic E. Coli And Salmonella Along The Production And Supply Chain Of Pork

E. coli and Salmonella are the most important food borne pathogens which are responsible for a variety of diseases in humans. These two pathogens can be transmitted through different routes including ingestion of food and water, person to person contact and animal to person contact. Consumption of food especially meat from food animals contaminated with E. coli and Salmonella act as a common cause of human infections. ExPEC and O157:H7 are the most important among E. coli strains which may cause life threatening diseases in humans. Salmonella enterica is responsible for 95% Salmonella infections in human. Antibiotic-resistant bacteria are becoming increasingly common which may cause global health crisis. Antibiotic resistance is encoded by several antibiotic resistant genes, many of which can transfer between bacteria.Little is known about the population biology of ExPEC, O157:H7 and Salmonella along the production and supply chain of pork (PSCP):the publish data about the whole-system from weaned piglets through to consumer-ready pig products is inadequate. China raises half population of pigs in the world, and has complicated PSCP:two types of pig farming (intensive farming with more than 1000 sows vs. backyard farming with less than 100 sows) and two types of retail pork markets (wet markets vs. super markets). The pork meat passes through several complex processes such as slaughtering, handling, transportation and cold chain before ready to consumer retail pork markets. These processes might contribute to the contamination of pork and pork products by bacteria including pathogenic ExPEC, E. coli O157:H7 and Salmonella. Therefore, we carried out this population biological study of E. coli and Salmonella along the PSCP. Whether ExPEC, O157:H7 and Salmonella are disseminating in the retail pork right from the production system or other processes may contribute in its contamination.Using MacConkey agar, E. coli were isolated from 285 samples including 125 tonsil swabs (4-6 weeks old healthy pigs from 5 farms),80 tissue samples from different slaughter houses (20 each intestine, liver, meat and kidney), and 80 samples from super-and wet-markets (each 20 meat and liver) collected both in summer and winter. Phylogenetic groupings (A, B1, B2 and D) and frequency distribution of 13 major virulence genes (fimH, pap A, papC, afa, focG, ire A, iutA, kpsmll, vat, fyuA and hlyD) were determined by polymerase chain reaction (PCR). Isolates were tested for 15 antibiotics (CRO, AMX, GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to the disc diffusion method and antibiotic resistant genes {tet(A), tet(B), tet(C), strA/strB, aadA, aac(3)IV), aadB, sull, sul2 and sul3, blaCMY-2, blaTEM and blaSHV} using mPCR. The most prevalent phylogenetic group was group B2 (59.3%), followed by groups B1 (28.1%), A (8.1%) and D (4.5%). The most prevalent virulence gene in group B2 isolates was kpsmⅡ(74%) followed by iutA (71%) and fimH (70%), papC (47%), cnf (40%) and hlyD (32%), while B1 isolates were higher in ireA (92%), fyu (77%) and vat (57%). Resistance for LIN was the highest in overall (96.3%, 77/80) isolates as well as from pig farms (100%,20/20) and different markets (100%, 20/20,85%,17/20). Resistance for other antibiotics such as AMX, TET, AMP and SXT was found 82.5%(66/80),63.7%(51/80),58.7%(47/80) and 50%(40/80), respectively. The most prevalent ARGs in the isolates recovered from pig farms was blaTEM’(100%, 20/20), followed by blaCMY-2 (80%,16/20), tetA & tetB (60%,12/20) and tetC (50%, 10/20). E. coli became more and more diverse along the PSCP with group B2 being the most prevalent. Besides multiple drug resistance, they share many traits with the human pathogenic isolates based on virulence gene contents that may pose a potential threat to public health.For detection and isolation of Salmonella enterica,325 meat and tissue samples were processed using Rappport-Vassiliadis Enrichment broth and Salmonella Chromogenic agar followed by confirmation through specific antisera and polymerase chain reaction (PCR) targeting their Specific Serovar Genomic Regions (SSGRS). Isolates were tested for 15 antibiotics (CRO, AMX, GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to the disc diffusion method and antimicrobial resistant genes (tet(A), tet(B), tet(C), strA/strB, aadA, aac(3)IV), aadB, sull, sul2 and sul3, blaCMY-2, blaTEM and blaSHV) using PCR. The overall prevalence of Salmonella enterica was 10.4%, being higher in wet markets (18.3%) followed by slaughter houses (15%), pig farms (7.2%) and super markets (3.3%). The MPN of all positive meat and tissue samples was found 3.6 MPN/gram (0.17-18). A total of 102 isolates were obtained, serovar Typimurium (75) and Dublin (18) were the most prevalent followed by Infantis (6) and Derby (3). Serovar dublin which is commonly adapted to cattle can be isolated along the PSCP. Antimicrobial resistance patterns were different in different serovars according to origin of Salmonella isolates. Overall isolates were highly resistant for LIN (89.2%, 91/102) followed by AMP (65.6%,67/102), AMX (63.7%,65), TET (52.9%,54/102) and STR (47.1%,48/102). Resistance for GAT and CRO was the lowest in all the isolates except the isolates of wet market which were 100% sensitive to these two antibiotics. The most common ARGs was blaTEM(73.5%,75/102), followed by blaSHV(53.9%,55/102) and aac(3)IV (52.9%,54/102).Shiga toxin Escherichia coli (STEC) O157:H7 is an important zoonotic food borne pathogen causing gastroenteritis that may lead to life threatening Hemorragic Colis (HC) and Hemorrhagic Uremic syndrome (HUS).325 meat and tissue samples were tested for enumeration of O157:H7 strains using MPN PCR targeting their specific genes (flicH7 and rfbO157) followed by isolation, sereotyping and pathogenicity testing. The overall prevalence of E. coli O157:H7 was 41.3%(134/325) along PSCP, being higher in supply chain (59%,118/200) as compared to pig farms (12.8%,16/125). Along the supply chain, the highest prevalence was found in slaughter houses (86.25%,69/80) followed by wet (53.3%,32/60) and super markets (28.3%,17/60). The MPN values ranged from 3-1100MPN/g in overall positive samples, being higher in slaughter houses followed by wet and super markets. Except from intestine and meat samples of slaughter house, the MPN was found higher in summer as compared to winter samples.8 STEC O157:H7 isolated from meat and liver samples were tested in Balb/C mice for pathogenicity. After development of clinical signs and symptoms,50-83.3% mortality were produced in the infected mice. Histopathological investigations revealed visible necrosis of intestinal epithelial cells, shedding of cellular debris in the intestine, while in the kidney, necrosis of renal cortical portion of tubular epithelial cells was observed. STEC O157:H7 is prevalent along PSCP around Hubei of China in different proportions being alarmingly higher in supply chain and markets which is a matter of concern for public health.