Biofilm Formation Of Meat-Borne Salmonella And Its Transferring

Salmonella sp. is one of the globally widespread food-borne pathogens, which has become the major cause of food poisoning and can result in numerous food recalls and outbreaks every year. The food contaminated by Salmonella can cause potentially huge economic losses to food industry and serious hazards to public healthy. Although there were many sources for contamination of food pathogens, the bacteria attached on food contact-surfaces have been demonstrated to be the principal source. In a suitable environments, the bacteria can form micro-colonies with 3-D structure on surfaces, commonly referred to as biofilm. The biofilm formation of Salmonella on food contact-surfaces can result in more serious cross-contamination, and many outbreaks have been demonstrated to result from the biofilm of Salmonella. Understanding the biofilm formation of Salmonella and its transferring were essential for food administrators to develop effective strategies to control the cross-contamination of Salmonella. Therefore, in my study, based on the isolation, identification and traceability of meat-borne Salmonella, two different growth media (a standard medium and a meat-based medium) were compared, then the attachment, the relationship with cells surfaces characteristics, extracellular polymeric substances, expression of functional genes and transferring of biofilm formed by meat-borne Salmonella on stainless steel surfaces were clarified, the context and results were shown as following:1. Tracing the contamination sources of Salmonella along the chicken slaughter-line and evaluating the attachment ability of isolatesA total of 104 samples collected from chicken carcass surfaces and processing contact-surfaces were obtained from three different processing points at a chicken slaughter line, and then Salmonella strains were isolated and confirmed by traditional and molecular methods. Based on the traceability of the isolates, the serotypes and attachment capacity of isolates were determined. The results showed that:a total of 188 strains were isolated by GB 4789.4-2010, and then 23 isolates were further confirmed by general PCR targeted with invA and hilA genes. The occurrence of Salmonella in the chicken processing line was 22.1%,19.2%(10/52) and 25.0%(13/52) were positive for Salmonella in samples from carcass surfaces and processing surfaces, respectively. Eight different serotypes were identified by serum and ERIC-PCR, including S. Indiana (9), S. Derby (3), S. Heidelberg (2), S. Agona (2), S. Infantis (4) and S. Typhimurium (1), and the other two isolates were unknown. There was significant variation in the occurrence of Salmonella at three different processing points, where incidence levels decreased with progress through the line. The ERIC-PCR fingerprints of isolates from post-chilling and post-grading points completely matched with the fingerprints of isolates from post-evisceration, indicating that the contact surfaces and carcass from evisceration point may serve as the principal sources of cross-contamination. Biofilm formation of Salmonella on polystyrene microplate surfaces were significantly influenced by growth medium types and incubation periods, S. Agona showed the greatest ability, whereas S. Heidelberg showing the weakest ability. These results supplied a solid foundation for the following study of biofilm formation.2. Biofilm formation of Salmonella and its relationship to cell characteristicsBiofilm formation of six different Salmonella on stainless steel surface and and its relationship to cell characteristics were evaluated, a meat-based medium (meat thawing losing broth, MTLB) and a standard medium (TSB) were used as cell growth medium, then they were compared. The results showed that:the ability of biofilm formation by Salmonella on stainless steel surfaces was significantly influenced by serotypes, incubation time and growth media, with S. Agona showing the greatest biofilm and the S. Derby and S. Heidelberg showing least biofilm. The biofilm formation significantly increased with the prolonging of incubation periods. Serotypes grown in TSB (> 7.5 Log CFU/cm2) showed greater biofilm formation than that in MTLB (< 6.5 Log CFU/cm2); There were significant differences in growth parameters, auto-aggregation, motility and cell surface hydrophobicity between each serotype; S. Typhimurium showed smaller μmax and greater L p and T than other serotypes regardless of growth medium types; S. Agona displayed greater auto-aggregation and motility, and S. Derby showing weaker. S. Agona and S. Indiana showed greater surface hydrophobicity, all the serotypes were electron-donor; A strong relationship was observed between the indices of cell surface characteristics, however, the relationships between biofilm formation and cell surface characteristics depend on growth medium types, showing a positive relationship with biofilm formation in TSB and no correlation with biofilm formation in MTLB. There was no relationship between biofilm formation and growth parameters. These results indicated that the biofilm formation of Salmonella were affected by environments factors and intrinsic factors, and there was no uniform tendency. There were great differences in biofilm formation of Salmonella grown in standard medium and meat-based medium.3. Micro-structure and extracellular polymeric substances of biofilm of meat-borne SalmonellaThe micro-structure of biofilm formed by multi-strains of meat-borne Salmonella, as well as the composition and distribution of extracellular polymeric substances (EPS), were determined in situ by CLSM, SEM, ATR-FTIR and Raman spectroscopy. The results showed that:biofilm formation of six-strain cocktail of Salmonella depended on the growth medium types and incubation times, after 5 days incubation, the biofilm in both media become mature; The percentage of live cells in the biofilm varied with the increasing of growth periods, at the same incubation time, there were significant differences in the percentage of live cells of biofilm grown in two media. Salmonella grown in both media were able to form biofilms on stainless steel surfaces via building a three-dimensional structure with multilayers of cells. Salmonella grown in MTLB showed "cloud-shaped" morphology in the mature biofilm, whereas when grown in TSB appeared "reticular-shaped". The ATR-FTIR and Raman analysis revealed that formation of EPS significantly depend on the incubation times, and great differences in ATR-FTIR and Raman spectra were observed between biofilms and corresponding planktonic cells. The composition of EPS contained carbohydrates, proteins, phospholipids, lipids and nucleic acids, polysaccharides and proteins were the main composition of EPS, which may serve as useful markers of biofilm formation, especial for the polysaccharides. Moreover, a completely different chemical composition of EPS were observed in biofilm grown in TSB and MTLB, the EPS components of biofilm in TSB were more abundant than that in MTLB. Taking into account the results in our present study, we speculated that the biofilm formation of Salmonella under our experimental conditions occurs through four different stages:initial cell attachment, a rudimentary biofilm formation, biofilm maturation and biofilm dispersal.4. Expression of functional genes of biofilm formed by meat-borne Salmonella Typhimurium and their relationshipsThe expression of functional genes of biofilm formed by meat-borne Salmonella Typhimurium were determined by RT-qPCR, and the relationships between genes expression were also evaluated. The results showed that:The genes expression of attachment, virulence and quorum sensing in biofilm were influenced by cells growth systems, the expression of each gene in different growth media showed great differences. The expression of genes in biofilm grown in TSB significant depend on the incubation time, after 7 days growth, each tested gene showed high expression. During a 7-day incubation period, the expression of genes in biofilm grown in MTLB increased and then decreased, or always decreased, the expression of virulence, auto-induce I and II greatly decreased after 7 days growth. Compared to the genes of biofilm grown in TSB, the genes expression of mature biofilm (5 and 7 days) grown in MTLB were lower, however, compared to the genes of planktonic cells grown in TSB, the genes expression of planktonic cells in MTLB were higher; Positive relationships between each gene expression in biofilm were observed, and a positive relationships between incubation time and genes expression in biofilm were also found. When the TSB media was used as a control group, positive relationships between each gene expression in biofilm and planktonic cells grown in MTLB were observed, negative relationships between incubation time and genes expression in biofilm and positive relationships between incubation time and genes expression in planktonic cells were also found. These results indicated that the gene regulatory networks of biofilm formation of Salmonella Typhimurium significantly depended on the growth environments, and the process of biofilm formation were coordinately regulated by several functional genes, the genes of CsgD, CsgB, CsrA, rpoS and PreA played important roles in the attachment and mature stages of biofilm formation.5. Transferring model of biofilm formed by meat-borne SalmonellaThe biofilm formation of meat-borne Salmonella (grown in TSB and MTLB) on stainless steel surfaces, which was the main surface type of meat processing equipments, were used as the contamination source, based on the analysis of detachment of biofilm from stainless steel surface to several meat products surfaces, the transferring models of biofilm from stainless steel surface to chicken meat surface were determined. The results showed that:the cells number of detachment and transfer rate significantly depend on the types of meat products, and there were significant differences in transfer rates between the biofilm grown in TSB and MTLB, bacon surface and emulsified sausage surface showed greater transfer rates, and pork chops transferred less cells. The results indicated that the meat products were possibly contaminated by the Salmonella biofilm formed on stainless steel surfaces during processing and pre-preparing procedures of meat. All the three different transferring model (typical logistic equation, exponential equation and multi roots function) could be used to describe the transferring of biofilm, which were proved by correlation coefficient (R2), the mean square error (MSE), accuracy factors (Af), bias factors (Bf) and F-test, and the relative errors were in acceptable range, indicating that each model was stable and reliable. There was no difference in attachment strength of biofilm grown in TSB and MTLB, which was supported by the parameters in three models (r in model I, D in model IT and B in model Ⅲ). Compared the fitting curves of three models, straight curves with small curvature were found in exponential equation, which could result in great errors in the prediction of cells number at the early and later transferring stage, a higher prediction of cells number in the first and the last three blots were observed in multi roots model, and logistic model could fit the transferring tendency better during the whole process. Therefore, the typical logistic equation can be used in risk assessment of Salmonella to accurately predict the cells number of biofilm.