Peter McClure

Mechanisms of survival, responses and sources of Salmonella in low-moisture environments

Some Enterobacteriaceae possess the ability to survive in low-moisture environments for extended periods of time. Many of the reported food-borne outbreaks associated with low-moisture foods involve Salmonella contamination. The control of Salmonella in low-moisture foods and their production environments represents a significant challenge for all food manufacturers. This review summarizes the current state of knowledge with respect to Salmonella survival in intermediate- and low-moisture food matrices and their production environments. The mechanisms utilized by this bacterium to ensure their survival in these dry conditions remain to be fully elucidated, however, in depth transcriptomic data is now beginning to emerge regarding this observation. Earlier research work described the effect(s) that low-moisture can exert on the long-term persistence and heat tolerance of Salmonella, however, data are also now available highlighting the potential cross-tolerance to other stressors including commonly used microbicidal agents. Sources and potential control measures to reduce the risk of contamination will be explored. By extending our understanding of these geno- and phenotypes, we may be able to exploit them to improve food safety and protect public health.

Quantitative microbiological risk assessment : Principles applied to determining the comparative risk of salmonellosis from chicken products

Ensuring microbiological safety requires identification of realistic hazards and the means of controlling them. The risk assessment framework proposed by Codex Alimentarius allows the impact of raw materials and processes to be appreciated, and the output can be used for risk management and communication. Mathematical models allow numerical information to be processed by a computer and interpreted to give quantitative or comparative risk assessments. In this example, models have been put together according to the Codex. Alimentarius principles, providing a quantitative risk assessment (QRA) of salmonellosis from frozen poultry products. This model-based QRA takes into account three types of information: occurrence and distribution of the agent, sensitivity of populations to infection (e.g., normal or susceptible), and the effect of cooking (in the factory or home) on concentration of the agent and hence risks of infection after product consumption. It only demonstrates the impact of a single-process step (heating) and the effect of changes in population sensitivity, raw material quality, and cooking regime on the final risk. The effects of growth and recontamination are not considered. To aid risk communication, the models have been visualized by means of displays and slider controls on a computer screen because effective communication is essential to encourage manufacturers and their product designers to assess the effect of changes in processing or materials on risk.

The use of confocal scanning laser microscopy (CSLM) to study the germination of individual spores of Bacillus cereus

Abstract Confocal scanning laser microscopy (CSLM) has been used to study the germination of Bacillus cereus spores allowing generation of accurate quantitative data on loss of refractility of individual , germinating spores in a population. The technique also allowed the simultaneous study of changes in spore permeability to ethidium bromide by fluorescence microscopy. Statistical analyses were performed on the data obtained and the distributions of various germination parameters within the population studied. All the parameters studied were characterised by positively ‘skewed’ distributions indicating the high level of biovariability present in populations of bacterial spores. The lag time before a spore initiated germination was independent from the subsequent time to lose refractility, or germinate. The method described in this work, for the first time, allows changes in permeability during germination to be studied using the influx of a compound that is excluded from dormant spores (ethidium bromide). It is hoped that this will form the basis of a useful system for studying the kinetics and sequence of spore germination when combined with other permeability probes.

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores

ABSTRACT In this work, we have used spores of Bacillus subtilisthat specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0.5%), and a pasteurization step (90°C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the l-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate onl-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.

ProP Is Required for the Survival of Desiccated Salmonella enterica Serovar Typhimurium Cells on a Stainless Steel Surface

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods.

The impact of E. coli O157 on the food industry.

The relatively recent emergence of Escherichia coli O157 as a foodborne pathogen has had a significant impact on the food industry. This serovar possesses a number of undesirable characteristics that combine to make it one of the most serious threats to food safety in recent years. The widespread and sporadic occurrence in the gastrointestinal tracts of cattle, sheep, man and many other species, sometimes in the absence of any disease, and the complex nature of its ecology mean that it is not feasible to easily eradicate this serovar. In order to control and minimise foodborne disease by these organisms, it has been necessary to change primary production, processing, retailing and consumer-handling practices. Despite these new control measures, foodborne disease outbreaks caused by these microorganisms continue to occur and are accompanied by an increase in the number of cases of illness in many countries.

The effect of cationic microbicide exposure against Burkholderia cepacia complex (Bcc); the use of Burkholderia lata strain 383 as a model bacterium.

The extensive use of microbicides in a wide range of applications has been questioned with regard to their role in the development of bacterial resistance to antimicrobials. This study aims to measure the phenotypic and genotypic changes in Burkholderia lata strain 383 exposed to chlorhexidine gluconate (CHG) and benzalkonium chloride (BZC), two commonly used cationic microbicides.

Phenotypic characterization of Salmonella isolated from food production environments associated with low-water activity foods.

Salmonella can survive for extended periods of time in low-moisture environments posing a challenge for modern food production. This dangerous pathogen must be controlled throughout the production chain with a minimal risk of dissemination. Limited information is currently available describing the behavior and characteristics of this important zoonotic foodborne bacterium in low-moisture food production environments and in food. In our study, the phenotypes related to low-moisture survival of 46 Salmonella isolates were examined. Most of the isolates in the collection could form biofilms under defined laboratory conditions, with 57% being positive for curli fimbriae production and 75% of the collection positive for cellulose production, which are both linked with stronger biofilm formation. Biocides in the factory environment to manage hygiene were found to be most effective against planktonic cells but less so when the same bacteria were surface dried or present as a biofilm. Cellulose-producing isolates were better survivors when exposed to a biocide compared with cellulose-negative isolates. Examination of Salmonella growth of these 18 serotypes in NaCl, KCl, and glycerol found that glycerol was the least inhibitory of these three humectants. We identified a significant correlation between the ability to survive in glycerol and the ability to survive in KCl and biofilm formation, which may be important for food safety and the protection of public health.

Development of a Protocol for Predicting Bacterial Resistance to Microbicides

ABSTRACT Regulations dealing with microbicides in Europe and the United States are evolving and now require data on the risk of the development of resistance in organisms targeted by microbicidal products. There is no standard protocol to assess the risk of the development of resistance to microbicidal formulations. This study aimed to validate the use of changes in microbicide and antibiotic susceptibility as initial markers for predicting microbicide resistance and cross-resistance to antibiotics. Three industrial isolates (Pseudomonas aeruginosa, Burkholderia cepacia, and Klebsiella pneumoniae) and two Salmonella enterica serovar Typhimurium strains (SL1344 and 14028S) were exposed to a shampoo, a mouthwash, eye makeup remover, and the microbicides contained within these formulations (chlorhexidine digluconate [CHG] and benzalkonium chloride [BZC]) under realistic, in-use conditions. Baseline and postexposure data were compared. No significant increases in the MIC or the minimum bactericidal concentration (MBC) were observed for any strain after exposure to the three formulations. Increases as high as 100-fold in the MICs and MBCs of CHG and BZC for SL1344 and 14028S were observed but were unstable. Changes in antibiotic susceptibility were not clinically significant. The use of MICs and MBCs combined with antibiotic susceptibility profiling and stability testing generated reproducible data that allowed for an initial prediction of the development of resistance to microbicides. These approaches measure characteristics that are directly relevant to the concern over resistance and cross-resistance development following the use of microbicides. These are low-cost, high-throughput techniques, allowing manufacturers to provide to regulatory bodies, promptly and efficiently, data supporting an early assessment of the risk of resistance development.

Bacterial thermal death kinetics based on probability distributions: the heat destruction of Clostridium botulinum and Salmonella Bedford.

Despite the long history and excellent record of inactivation models used in thermal processing, there are relatively few approaches that attempt to describe the kinetics commonly observed. There are even fewer examples of models that allow the user to deal with the environmental conditions that influence these kinetics. We describe an approach that assumes a distribution of inactivation times within a population of bacterial cells. The concept allows for alternative interpretations of death kinetics and provides excellent descriptions of data generated with two important foodborne pathogens, Clostridium botulinum and Salmonella Bedford. The Salmonella Bedford data set used is unusual and perhaps unique in that it provides information where more than 50% of the population survival has been measured. These measurements are often overlooked or missed in experimental work but are essential when using a vitalistic approach, enabling calculation of a 50% lethal dose for destruction of bacteria. Use of the normal or Prentice distribution provided better fits to the data than other models commonly used to describe thermal death. There was no obvious bias in the fits even though significant tailing was evident. In addition, the procedure described allows data from all the conditions to be fitted rather than individual independent series. This enables a single equation to be derived that can be judged against the whole domain of the data. Approaches that provide accurate and unbiased descriptions of thermal death are likely to become increasingly important to ensure the safety of more marginal heat processes.