Department of Food Science and Technology

Clostridium perfringens spores : inactivation, germination, and formation

Mon, 15 Apr 2013 00:00:00 GMT

Clostridium perfringens spores : inactivation, germination, and formation Udompijitkul, Pathima The enterotoxin-producing Clostridium perfringens type A isolates are responsible for the third most common foodborne illness in the United States and can also cause non-foodborne human gastrointestinal (GI) diseases such as antibiotic- associated and sporadic diarrheas. Three important factors contribute to the ability of C. perfringens to cause GI diseases, including its extremely rapid growth rate, its ubiquitous distribution in foods and environments, and its capability to form highly resistant endospores. In the first study, the antimicrobial peptide nisin was evaluated for its antimicrobial effect against enterotoxigenic C. perfringens food poisoning (FP) and non-foodborne (NFB) GI disease isolates. Nisin did not affect spore germination, whereas germinated spores were very susceptible to low concentration of nisin and thus spores outgrowth were arrested. Nisin also exerted its inhibitory effect against vegetative growth of C. perfringens FP and NFB isolates in rich medium; however, FP cells were less resistant to nisin than NFB cells. Nevertheless, nisin was not effective in controlling germination and outgrowth of C. perfringens spores in cooked meat products during storage at abusive temperature, even at ~ 4 times elevated concentration than the regulatory approved level. Strikingly, spores of NFB isolates also exhibited higher resistance to nisin than that of FP isolates in both laboratory medium as well as in meat systems. Collectively, despite its effectiveness in controlling spore outgrowth and vegetative cell growth in laboratory conditions, nisin showed no antimicrobial activity against C. perfringens spores inoculated into meat model systems. The main focus of the second study was to develop an effective spore inactivation strategy on food contact surfaces by inducing spore germination prior to inactivation of the more susceptible spores with commonly used surface disinfecting agents. The mixture of L-asparagine and KCl (AK) was the most effective germinant for spores of enterotoxigenic C. perfringens type A. Germination temperature had a significant influence on the germination extent and subsequent inactivation by variety of surface disinfectants. Implementation of germination step significantly increased the inhibitory effect of all tested disinfecting agents against spores of C. perfringens FP strain SM101 with lower efficacy against the spores of NFB strain NB16. Furthermore, spores of C. perfringens FP isolates could germinate with AK upon their adhesion onto stainless steel chips and were subsequently inactivated with disinfectant agents by i.e. 1.53 to 2.70 log reductions of colony forming units per chip. Overall, AK-induced germination followed by treatment with iodophore represents a promising strategy to inactivate spores of C. perfringens FP isolates on food contact surfaces. Spore germination is initiated upon sensing a variety of compounds, termed germinants, via the cognate germinant receptors. In the third study, we identified sodium ions and inorganic phosphate (NaPi) at pH ~ 6.0 as a novel germinant for spores of enterotoxin-producing C. perfringens FP isolates. The spores lacking germination proteins GerAA and GerKA-KC were severely impaired in their ability to germinate with NaPi, whereas GerKB-negative spores germinated to a similar extent as wild type spores with NaPi, but their initial rate of germination was lesser. Spores lacking GerO or GerO GerQ germinated to a lower extent and with a significantly slower rate than wild type spores. In contrast, gerQ spores exhibited only a slightly slower and lesser extent of germination with NaPi than its parent strains. Therefore, the germinant receptor proteins GerKA-KC, GerAA, and the putative antiporter GerO are essential for normal germination of C. perfringens spores with NaPi. In the fourth study, we demonstrated that polar, uncharged amino acids at pH 6.0 could efficiently trigger germination of spores of enterotoxigenic C. perfringens. While L-glutamine is a unique nutrient germinant for spores of C. perfringens FP isolates, L-asparagine, L-cysteine, L-serine, and L-threonine can induce germination of both FP and NFB spores. The germinant receptor GerKC is the major receptor involved in cysteine- and glutamine-induced germination and release of dipicolinic acid (DPA) from the spore’s core, whereas less pronounced germination defects were observed in gerAA and gerKB spores. GerKC also has a key role in L-asparagine germination. For serine and threonine (pH 6.0)-induced germination, GerKA is the dominant receptor and GerKC and GerKB are also required for efficient germination of FP spores. The objectives of the fifth study were to identify and characterize the putative sensor histidine kinases of C. perfringens. We identified six genes encoding putative sporulation-associated sensor histidine kinases in the genome of C. perfringens SM101. These putative kinase genes were highly expressed under sporulation- stimulating conditions. Two genes encoding putative orphan sensor histidine kinases, cpr1728 and cpr1055, were inactivated and roles of each putative kinase on various aspects in the life cycle of C. perfringens had been characterized. Inactivation of cpr1728 and cpr1055 significantly lowered C. perfringens sporulation capacity in two sporulation-inducing conditions. Moreover, sporulation delayed phenotype was also observed in strain lacking CPR1055. Inactivation of either cpr1728 or cpr1055 led to a marked defect in C. perfringens spore germination with all known germinants. Spores of two kinase mutants also exhibited slower outgrowth than their parental strain; however, no difference in colony forming efficiency was observed among tested strains. Additionally, mutations in cpr1728 and cpr1055 did not affect vegetative growth; however, both mutants grew at higher rate under sporulation-inducing conditions. In conclusion, this dissertation reports the experimental results that are relevant to various aspects of C. perfringens spores. These include the development of spore inactivation strategies in food products as well as on food contact surfaces, the identification of compounds triggering germination of spores of CPE-producing C. perfringens, and the insights into the roles of putative sensor histidine kinases in the process of spore formation and spore germination under a variety of conditions. Graduation date: 2013

Production of SO₂ Binding Compounds and SO₂ by Saccharomyces during Alcoholic Fermentation and the Impact on Malolactic Fermentation

Sat, 01 Jan 2011 00:00:00 GMT

Production of SO₂ Binding Compounds and SO₂ by Saccharomyces during Alcoholic Fermentation and the Impact on Malolactic Fermentation Wells, A.; Osborne, J. P. The objective of this study was to investigate the production of SO₂ and SO₂ binding compounds by wine yeast and the impact of the production of these compounds on the MLF at various time points during alcoholic fermentation. Fermentations were observed for a number of commercial wine yeasts in a synthetic grape juice and Pinot gris juice and SO₂, acetaldehyde, pyruvic acid and α-ketoglutarate. Measurements were taken at multiple time points during the fermentation. Samples were taken from the fermentations at weekly intervals, sterile filtered, and inoculated with 0. oeni strain VFO to induce MLF. Significant differences between the amount of SO₂, acetaldehyde and pyruvic acid produced by the various yeast strains were noted. Some yeast strains such as FX10, CK S102, F15 and M69, produced significantly higher SO₂ concentrations than other yeast strains and MLF was inhibited in these wines. Insignificant free SO₂ was measured, indicating that bound SO₂ rather than free SO₂ was responsible for inhibition. At almost all time points of the alcoholic fermentation, acetaldehyde bound SO₂ was determined to be the dominant species of bound SO₂ present, suggesting that MLF inhibition by bound SO₂ was due to acetaldehyde bound SO₂. This is the publisher’s final pdf. The published article is copyrighted by South African Society for Enology & Viticulture and can be found at: http://www.sasev.org/journal/?id=8.

Identification of gold nanoparticle-resistant mutants of Saccharomyces cerevisiae suggests a role for respiratory metabolism in mediating toxicity

Tue, 01 Jan 2013 00:00:00 GMT

Identification of gold nanoparticle-resistant mutants of Saccharomyces cerevisiae suggests a role for respiratory metabolism in mediating toxicity Smith, Mark R.; Boenzli, Matthew G.; Hindagolla, Vihangi; Ding, Jun; Miller, John M.; Hutchison, James E.; Greenwood, Jeffrey A.; Abeliovich, Hagai; Bakalinsky, Alan T. Positively-charged gold nanoparticles (0.8 nm core diameter) reduced yeast survival, but not growth, at a concentration of 10-100 μg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion. This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Society for Microbiology and can be found at: http://aem.asm.org/.

Survival of Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus in raw yellowfin tuna during refrigerated and frozen storage

Wed, 06 Mar 2013 00:00:00 GMT

Survival of Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus in raw yellowfin tuna during refrigerated and frozen storage Mou, Jing The consumption of seafood in the United States has increased rapidly in recent years due to high quality protein and health benefits of seafood. Seafood can be a carrier for bacteria normally distributed in the marine environment and, in some cases, can be contaminated by human pathogens. Therefore, there is a potential health risk if seafood is consumed raw or undercooked. However, information regarding prevalence of foodborne pathogens in retail seafood products and the ability of pathogens to survive in the products during refrigerated and frozen storage is limited. The objective of this study was to generate such information for a better understanding of distribution of foodborne pathogens in seafood products and provide data which might be used for risk assessment of foodborne infection associated with seafood consumption. A total of 45 seafood products were collected from local retail stores and analyzed for aerobic plate counts (APC) and psychrotrophic bacterial counts (PBC) as well as presence of foodborne pathogens, including Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus according to procedures described in the U.S. Food and Drug and Administration Bacteriological Analytical Manual (BAM). Presumptive isolates for each foodborne pathogen were further characterized by biochemical reactions using commercial identification kits and confirmed with polymerase chain reaction (PCR) assay. The samples had bacterial populations ranging from 1.90 to 6.11 CFU/g for APC and from 2.00 to 6.78 CFU/g for PBC. According to the microbiological criteria of International Commission on Microbiological Specifications for Foods (ICMSF), all 45 samples were considered acceptable quality (APC < 10⁷ CFU/g, E. coli < 3 MPN/g) with most samples (93.3%) being good quality (APC < 5 × 10⁵ CFU/g, E. coli < 3 MPN/g). No E. coli O157:H7, Salmonella, S. aureus, V. parahaemolyticus, and V. vulnificus was detected in any samples. Two previously frozen shrimp products (4.4%) were confirmed to carry L. monocytogenes. Studies of growth and survival of L. monocytogenes (3 strains), S. aureus (2 strains), and Salmonella (2 serovars) in raw yellowfin tuna meat stored at 5 - 7 °C for 14 days revealed that L. monocytogenes had the ability to multiply in the tuna meat during refrigerated storage while populations of S. aureus and Salmonella were reduced by 1 to 2 log CFU/g after 14 days at 5 - 7 °C. Studies of holding raw yellowfin tuna meat contaminated with L. monocytogenes, S. aureus, and Salmonella at -18 ± 2 °C for 12 weeks observed that all three pathogens, except Salmonella Newport, in tuna samples survived the frozen storage with less than 2- log of reductions in the populations over 12 weeks of storage. No viable cell of Salmonella Newport was detected in samples after 42 days storage at -18 °C. Raw seafood can be a carrier of foodborne pathogens, particularly L. monocytogenes, and many foodborne pathogens can survive in frozen products for several months. Consumption of raw or undercooked seafood products may lead to human infection if the products are contaminated with pathogens. Therefore, sanitation standard operating procedure (SSOP), good manufacturing practice (GMP) and hazards analysis and critical control points (HACCPs) programs shall all be implemented in the seafood industry to prevent seafood products from being contaminated with foodborne pathogens during handling and processing. Moreover, proper storage of raw seafood products and avoiding cross-contamination during handling at the retail levels also helps to minimize risk of human infection associated with ready-to-eat products. Graduation date: 2013