Development of a sequence-based molecular subtyping method for Bacillus cereus dairy isolatse

Open Access
Miller, Donna Marie
Graduate Program:
Food Science
Doctor of Philosophy
Document Type:
Date of Defense:
May 09, 2008
Committee Members:
  • Robert F Roberts, Dissertation Advisor
  • Stephanie Doores, Committee Chair
  • Robert F Roberts, Committee Chair
  • Edward G Dudley, Committee Member
  • Chitrita Debroy, Committee Member
  • Stephen John Knabel, Committee Member
  • Bacillus cereus
  • milk
  • multilocus sequence typing
  • subtyping
  • dairy
Recent research has suggested Gram-positive spore-forming microorganisms including Bacillus cereus are the predominant microorganisms in pasteurized milk during refrigerated storage. The presence of B. cereus in pasteurized milk is of concern to the dairy industry because this microorganism can influence the quality and safety of the product. During growth in milk, B. cereus can produce proteinases and lipases which cause quality defects in milk and limit shelf life. In addition, the presence of B. cereus in pasteurized milk may pose a health hazard to the consumer because B. cereus is a human pathogen and has been shown to cause emetic and diarrheal illnesses. Numerous international studies have been conducted to determine the incidence and level of B. cereus in pasteurized milk, but recent research in the U.S. is limited. The objectives of the first portion of this work were to evaluate the microbiological quality of commercial milk from four manufacturers and to determine the level and incidence of B. cereus. Microbiological analysis was conducted on whole, 2% milk fat, 1% milk fat, and skim milks stored at 4 and 7¡ÆC and included aerobic plate counts, mesophilic and psychrotrophic spore counts, and B. cereus counts. Analysis was conducted on the sell-by date (day 0), 12 days before the sell-by date (-12), 7 days before the sell-by date (-7), and 7 days after the sell-by date (+7). On the sell-by-date, milks stored at 4 and 7¢ªC, yielded aerobic plate counts ranging from ¡Â1.0 to 8.5 log10 CFU/mL and ¡Â1.0 to 8.6 log10 CFU/mL, respectively. Seven (25%) milk samples stored at 4¢ªC had aerobic plate counts ¡Ã4.3 log10 CFU/mL (20,000 CFU/mL), the limit outlined in the Pasteurized Milk Ordinance, while 23 (82%) milk samples stored at 7¢ªC exceeded the limit. Mesophilic and psychrotrophic spore counts of milks stored at 4 and 7¢ªC were low and ranged from ¡Â1.0 to 2.2 log10 CFU/mL and ¡Â1.0 to 1.7 log10 CFU/mL, respectively. The growth of B. cereus during refrigerated storage appeared to be influenced by storage temperature. On the sell-by date, B. cereus was detected in 2 of 28 (7%) cartons stored at 4¢ªC, and counts of milks ranged from ¡Â1.0 to 4.2 log10 CFU/mL. However, B. cereus was detected in 16 (57%) milk samples at 7¡ÆC, and counts ranged from ¡Â1.0 to 8.9 log10 CFU/mL. On the sell-by date, 9 (32%) of the milk samples stored at 7¡ÆC yielded B. cereus counts exceeding the minimum infectious dose for the diarrheal illness. The data from the first portion of the research suggested further work needed to be conducted to improve the microbiological quality of pasteurized milk. Such work would include tracking studies to identify contamination sites in milk production and processing. The second objective of this work was to develop a sequence-based subtyping method for B. cereus dairy isolates. Currently, the RAPD-PCR method developed by Nilsson et al. (1998) is the subtyping method used most extensively for tracking B. cereus in milk production and in the processing environment. This method is well-suited for large-scale typing studies because it is highly discriminatory and is relatively simple and inexpensive compared to other subtyping methods; however, this method has several disadvantages associated with fragment-based methods including difficulties in standardization and interpretation of the results and low portability of the data. The focus of the second portion of this work was to develop a two- or three-gene MLST scheme for tracking B. cereus dairy isolates using housekeeping and virulence gene sequences. Bacillus cereus possesses a number of virulence genes that could potentially be included in a MLST scheme, but in order for these genes to be included in a MLST scheme, the genes would need to be widely distributed among B. cereus isolates. The incidence of nine virulence genes was evaluated among 13 B. cereus reference strains and milk isolates, and two virulence genes, entFM and nheC were detected in all of the strains. The next part of the work involved comparison of the number of allelic types obtained with housekeeping gene sequences in the MLST scheme of Helgason et al. (2004) with sequences of virulence genes, entFM and nheC. It was expected that the virulence gene sequences would have a greater number of polymorphic sites and yield a greater number of allelic types than the housekeeping genes. The number of allelic types obtained with sequences of housekeeping genes in the MLST scheme of Helgason et al. (2004) ranged from four (adk, recF, and sucC) to seven (ftsA and glpT). The percentage of polymorphic sites among these genes ranged from 1.7% (ftsA) to 7.3% (glpT). Virulence genes entFM and nheC yielded seven and six allelic types, respectively, and these genes exhibited a higher percentage of polymorphic sites (10.2 and 12.1%) than the housekeeping genes. Finally, various combinations of housekeeping and virulence gene (entFM and nheC) sequences were evaluated in the development of a two- or three-gene MLST scheme for B. cereus. The number of sequence types obtained with the two-gene MLST schemes ranged from five (ccpA and pyrE, adk or recF and sucC) to eight (any housekeeping gene and nheC, or entFM and nheC). The addition of a third gene to the MLST scheme did not increase the number of sequence types. The seven-gene MLST scheme described by Helgason et al. (2004) yielded seven sequence types, and the combination of the seven housekeeping genes and two virulence genes yielded eight sequence types. The two-gene MLST schemes could be used for large-scale tracking studies to identify sources of B. cereus contamination. Such tracking studies could lead to a reduction in the level and incidence of B. cereus in pasteurized milk and result in improvements in the quality and shelf life of the product.