Date of Award

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Committee Member

Dr. Xiuping Jiang, Committee Chair

Committee Member

Dr. Annel K. Greene

Committee Member

Dr. Paul L. Dawson

Committee Member

Dr. T-R. Jeremy Tzeng

Abstract

Raw animal by-products destined for rendering process may contain high population of harmful microorganisms including hydrogen sulfide-producing bacteria (SPB) and Salmonella. SPB are the spoilage bacteria that can utilize sulfur-containing compounds of raw animal by-products to produce hazardous gas-hydrogen sulfide (H2S) which is toxic. Salmonella may contaminate the rendered animal meals resulting in an introduction of human pathogens into the food chain. Furthermore, both SPB and Salmonella are likely to form biofilms on the various surfaces in rendering processing environment, serving as the source of recontamination and causing persistent microbiological safety problems. Therefore, novel and practical strategies to control these harmful bacteria need to be explored. Bacteriophages are bacterial viruses that only infect specific species of bacteria without harming animals, plants and human, thus bacteriophage treatment has been explored as a novel biological method to control biofilms formed by persistent bacteria due to their high specificity and effectiveness. Therefore, the objectives of this study were: 1) to identify the sources of Salmonella contamination in rendering processing environment; 2) to optimize a scale-up production of Salmonella-specific bacteriophages; 3) to determine the effectiveness of bacteriophage treatment on reducing Salmonella and SPB attachment/biofilms on the surfaces under laboratory and greenhouse conditions; and 4) to apply bacteriophage treatment to reduce Salmonella and SPB attachment/biofilms on the surfaces in rendering processing environment. For the first objective, a microbiological analysis of Salmonella contamination was conducted in two rendering plants in order to investigate the potential cross-contamination of Salmonella in rendering processing environment. Sampling locations were pre-determined at the potential areas where Salmonella contamination may occur including raw materials receiving, crax grinding and the finished meal loading-out areas. Among 108 samples analyzed, 79 samples (73%) were Salmonella-positive after enrichment. Selected Salmonella isolates (n = 65) were identified to 31 unique PFGE patterns, and 16 Salmonella serotypes including Typhimurium and Mbandaka identified as predominant serotypes, and 10 Salmonella strains were determined as strong biofilm formers. Raw material receiving area was found as the primary source of Salmonella, whereas the surfaces surrounding crax grinding and the finished meal loading-out areas harbor Salmonella in biofilms. The same Salmonella serotypes found in both raw materials receiving and the finished meal loading-out areas also suggested a potential of cross-contamination between different areas in rendering processing environment. For the second objective, a mixed bacteriophage production in a single batch was developed. To scale up the production of Salmonella-specific bacteriophages with low cost for field study (fourth objective). Bacteriophage titer of mixed bacteriophage production yielded 10.3 log PFU/ml with optimized conditions of multiplicity of infection (MOI) of 0.01, agitation speed of 200 rpm, nalidixic acid at concentration of 0.06 μg/ml and incubation time of 8 h at 37°C. Additionally, final titer of bacteriophage production could reach up to 11.5 log PFU/ml with a PEG-6000 precipitation at concentration of 8% and sodium chloride at concentration of 3%. In the third objective, three SPB strains of Citrobacter freundii (n = 1) and Hafnia alvei (n = 2) were separately determined as strong biofilm formers using a 96-well microplate method. Application of 9 SPB-specific bacteriophages (107 PFU/mL) from families of Siphoviridae and Myoviridae resulted in 33-70% reduction of biofilm formation by each SPB strain. On stainless steel and plastic templates, bacteriophage treatment (108 PFU/mL) reduced the attached cells of a mixed SPB culture (no biofilm) by 2.3 and 2.7 log CFU/cm2 within 6 h at 30°C, respectively, as compared to 2 and 1.5 log CFU/cm2 reductions of SPB biofilms within 6 h at 30°C. To determine the efficacy of bacteriophage cocktail for reducing Salmonella attachment/biofilms on the surfaces, a mixture of 6 Salmonella-specific bacteriophage strains was selected among 94 bacteriophages for bacteriophage treatment based on evaluating host ranges against the 10 selected Salmonella isolates obtained from rendering plants. The effectiveness of bacteriophage treatment with titers of 104-108 PFU/ml was evaluated against strong Salmonella biofilm formers using a colorimetric method in 96-well microplate. Furthermore, the bacteriophage treatment with a titer of 109 PFU/ml was applied for 7 days to reduce Salmonella attached to the stainless steel surfaces in laboratory and different seasons under greenhouse conditions. With bacteriophage treatment in 96-well microplate, the inhibition of biofilm formation and reduction of pre-formed biofilm of Salmonella reached up to 90 and 66%, respectively. Bacteriophage treatment reduced up to ca. 2.9 and 3.0 log CFU/cm2 of slightly formed biofilm/attachment of selected top 10 Salmonella biofilm former strains and strain 8243, respectively, under laboratory condition, as compared with 3.4, 1.4 and 3.0 log CFU/cm2 of Salmonella strain 8243 in summer, fall/winter and spring seasons under greenhouse condition, respectively. To test the efficacy of above Salmonella-specific bacteriophage cocktail for reducing Salmonella contamination on workers’ rubber boots, biofilms of Salmonella Typhimurium strain 8243 formed on rubber templates or boots were treated with the same Salmonella-specific bacteriophage cocktail of 6 strains (ca. 9 log PFU/ml) for 6 h under laboratory condition. Salmonella-specific bacteriophage treatments combined with sodium hypochlorite (400 ppm), 10-min pre-treatment with sodium hypochlorite (400 ppm) or brush scrubbing (30 sec) were also investigated for a synergistic effect on reducing Salmonella biofilms. SM buffer, sodium hypochlorite (400-ppm) or 10-min pre-treatment with sodium hypochlorite (400 ppm) were used as controls. Under laboratory condition, Salmonella biofilms formed on rubber templates and boots were reduced by 95.1-99.999% and 91.5-99.2%, respectively. For the fourth objective, our research on bacteriophage treatment of SPB and Salmonella was conducted in a rendering plant. For SPB application, indigenous SPB were allowed to form biofilms on the environmental surface, stainless steel, HDPE plastic, and rubber templates in a rendering plant for 7 days. A total of two trials were conducted for each season. With bacteriophage treatment (109 PFU/mL) for 6 h at room temperature, SPB biofilms were reduced by 0.7-1.4, 0.3-0.6 and 0.2-0.6 log CFU/cm2 in spring, summer and fall trials, respectively. To reduce contamination of indigenous Salmonella on workers’ boots, two trials of field study were conducted to apply Salmonella-specific bacteriophage treatments for 1 week at a rate of 3 times in rendering processing environment. In rendering processing environment (average temperature: 19.3ºC; average relative humidity: 48%), indigenous Salmonella populations on workers’ boots were reduced by 84.5, 92.9, and 93.2% after treated with bacteriophage cocktail alone, bacteriophages + sodium hypochlorite, and bacteriophage + scrubbing for 1 week, respectively. In summary, our study examined the current contamination rates of Salmonella in rendered animal meals and rendering processing environment, and indicated the high potential of finished meals being recontaminated with Salmonella biofilms during the post-rendering process. We also optimized a scale-up production of mixed bacteriophages in a single batch with reduced cost for field application. Moreover, our study demonstrated that bacteriophages could reduce the selected SPB and Salmonella attachment/biofilms formed on variou s surfaces effectively, suggesting that the use of bacteriophages on the hard surfaces in rendering processing environment could control H2S produced by SPB and Salmonella recontamination in rendered meals. Furthermore, the results of field study demonstrated the effectiveness of bacteriophage treatments in reducing indigenous SPB and Salmonella attachment/biofilms formed on the surfaces in rendering processing environment. Overall, our research findings validated bacteriophage treatment as an effective, non-corrosive and environmentally friendly biological control method to reduce SPB and Salmonella attachment/biofilms in rendering processing environment, thereby, helping the rendering industry to have a safe working environment for workers and produce high quality rendered animal meals free from Salmonella contamination.

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