Date of Award

12-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Advisor

Jiang, Xiuping

Committee Member

Greene , Annel K

Committee Member

Dawson , Paul

Committee Member

Henson , J Michael

Abstract

In the United States, 1.3 billion tons of animal wastes are produced annually. Disposal of this huge amount of waste on agricultural land without proper treatment is a public health safety issue as animal waste is a potential source of several human pathogens. Therefore, composting of animal wastes is an economical solution to this problem. The high temperature reached during this process also brings about inactivation of pathogens in the waste. However, survival of pathogens has still been reported from different composting studies, indicating the complex nature of this process. The objectives of this study were to: 1) study thermal inactivation of heat-shocked Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in dairy compost under isothermal conditions, 2) evaluate thermal inactivation of E. coli O157:H7 and Salmonella spp. in different types of compost at elevated composting temperatures by simulating early stage of on-farm composting, 3) determine thermal inactivation of acid-adapted E. coli O157:H7 in fresh dairy compost by simulating early phase of composting process, 4) develop predictive mathematical model for thermal inactivation of stress-adapted E. coli O157:H7, Salmonella spp. and L. monocytogenes in compost, and 5) study stress and virulence gene expression of E. coli O157:H7 in fresh dairy compost using real-time PCR.
Thermal inactivation of E. coli O157, Salmonella, and L. monocytogenes was studied in different types of compost in an environmental chamber under either isothermal conditions or by simulating early phase of the composting process. A three strain mixture of pathogen (except gene expression study) was used in every study. Pathogens were inoculated at a final conc. of ca. 107 CFU/g for compost or liquid medium (107 CFU/ml) (broth or saline) study. The inoculated compost packed in Tyvek pouches were used for thermal inactivation study. An inactivation temperature of 50, 55, and 60°C was used for most of the study.

Thermal resistance of heat-shocked (at 47.5°C for 1 h) E. coli O157:H7, Salmonella, and L. monocytogenes was compared to non-heat-shocked (control) cultures in finished dairy compost under isothermal conditions. The heat-shocked E. coli O157:H7, Salmonella and L. monocytogenes survived longer (P < 0.05) at 50°C with reductions of 2.7, 3.2 and 3.9 log CFU/g, respectively, within 4 h of heat exposure as compared with reductions of 3.6, 4.5, and 5.1 log CFU/g, respectively, in control cultures. The heat-shocked cultures of E. coli O157:H7, Salmonella, and L. monocytogenes had 1.2, 1.9 and 2.3 log reductions, respectively, within 1 h at 55°C, whereas the corresponding control cultures had 4, 5.6 and 4.8 log reductions, respectively. At 60°C, a rapid population reduction was observed during the come-up time of 14 min in control cultures of E. coli O157:H7, Salmonella, and L. monocytogenes with 4.9, 4.8 and 2.3 log reductions, respectively, as compared with 2.6, 2.4 and 1.7 log reduction, respectively, in heat-shocked cultures. L. monocytogenes survival curves for all three temperatures had extensive tailing. The mixed Weibulll distribution model was a good fit for the survival curves of pathogens, with difference in the shape parameter of heat-shocked and control cultures.
Thermal inactivation of E. coli O157:H7 was studied in fresh dairy compost by simulating the early phase of composting process (2 and 5 days of come-up time). Compost mixture with 40 or 50% initial moisture content was used. Optimal and suboptimal compost mixes, with carbon to nitrogen (C/N) ratio of 25:1 and 16:1, respectively, were also compared in this study. In the optimal compost mix E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the sub-optimal compost mix (C:N as 16:1), the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and 240, 72, and 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer with 5 days of come-up time compared with 2 days of come-up time.
In the study on thermal inactivation of Salmonella in fresh poultry compost, a compost mixture with 40 or 50% initial moisture was used. In poultry compost with optimal moisture content (50%), Salmonella spp. survived for 96, 72, and 24 h at 50, 55, and 60°C, respectively, as compared with 264, 144, and 72 h at 50, 55, and 60°C, respectively, in compost with suboptimal moisture (40%). Pathogen decline was faster during the come-up time probably due to higher ammonia volatilization.
In the investigation on cross-protection of acid-adaptation and non-adaptation (control) on thermal inactivation of E. coli O157 in compost, E. coli O157:H7 acid-adapted in tryptic soy broth without dextrose (TSB w/o D) (pH 5.0) was inoculated into fresh dairy compost. E. coli O157 grown in TSB w/o D with pH 7.0 served as control. In fresh dairy compost with 2 days of come-up time, acid-adapted and control E. coli O157:H7 survived for 19 and 17 days at 50°C, respectively, and 6 and 4 days for both types of culture at 55 and 60°C, respectively. Overall, pathogen survival was non-significant (P > 0.05) between control and acid-adapted cultures at all sampling intervals at all tested temperatures. In finished compost (Black Kow®), the survival of E. coli O157 was also non-significant (P > 0.05) at most of the sampling times between control and acid-adapted cultures at 55°C. However, the duration of survival for both cultures was short in comparison to that in fresh compost. In fresh compost with short come-up time (15 min) acid-adaptation provided E. coli O157 some cross-protection to heat at 55°C up to 30 min of exposure. In saline, acid-adapted E. coli O157 was inactivated at 55°C significantly slow as compared to control culture with short come-up time at 0.5 and 1 h of heat exposure.
To understand survival mechanisms for pathogens during composting, a two- step real-time PCR assay was used to evaluate expression of stress and virulent genes in E. coli O157:H7 heat-shocked in compost. E. coli O157:H7 (strain F07-020-1) was inoculated in autoclaved fresh dairy compost which was heat-shocked at 47.5°C for 10 min in water bath. Bacterial heat-shock was also done in tryptic soy broth (TSB) to serve as medium control. In compost heat-shock genes, clpB, dnaK, groEL, and alternative sigma factor (rpoH) were all up-regulated significantly (P < 0.05). There was no significant (P > 0.05) difference in the expression of trehalose synthesis genes. Virulent genes such as stx1 and fliC were up-regulated while rest of the virulent genes was down-regulated with no significant difference (P > 0.05). In toxin-antitoxin system, toxin genes mazF, hipA, and yafQ were up-regulated with no significant difference (P > 0.05), whereas antitoxin gene dinJ was up-regulated with level of expression significantly different (P < 0.05). Most of the other antitoxin genes were down-regulated. In broth as the heat-shock medium, all heat-shock genes were up-regulated with relative fold change significantly different (P < 0.05). There was no significant change (P > 0.05) in trehalose synthesis genes in broth medium either. Except for eaeA, rest of the virulent genes was down- regulated with no significant (P > 0.05) change. Majority of the toxin-antitoxin genes were down-regulated with relative fold change in toxin gene hipA and chpB only significantly different (P < 0.05).
These results suggest that the induction of heat-shock response in pathogens extended survival at temperatures typically considered lethal during composting process. Genetically, heat-shock genes play an important role in this process. Slow come-up time at the beginning of composting can be the reason for extended pathogen survival during composting due to better heat-adaptation. Additionally, both carbon to nitrogen (C/N) ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation. Thermal inactivation of pathogens during composting could be modeled by the mixed Weibulll model, with both moisture and come-up time identified as significant factors. Cross-protection against heat in E. coli O157:H7 due to acid adaptation was lost in fresh dairy compost during composting with 2 days of come-up time but was demonstrated in saline. In poultry compost, high nitrogen content is an additional factor contributing to Salmonella inactivation through ammonia volatilization during thermal exposure. Overall, physiological stage of pathogens, initial moisture level, composting come-up time, and controlled ammonia volatilization are some important parameters affecting the microbiological safety and quality of compost product.

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