Processing environment monitoring in low moisture food production facilities: Are we looking for the right microorganisms?

Processing environment monitoring is gaining increasing importance in the context of food safety management plans/HACCP programs, since past outbreaks have shown the relevance of the environment as contamination pathway, therefore requiring to ensure the safety of products. However, there are still many open questions and a lack of clarity on how to set up a meaningful program, which would provide early warnings of potential product contamination. Therefore, the current paper aims to summarize and evaluate existing scientific information on outbreaks, relevant pathogens in low moisture foods, and knowledge on indicators, including their contribution to a "clean" environment capable of limiting the spread of pathogens in dry production environments. This paper also outlines the essential elements of a processing environment monitoring program thereby supporting the design and implementation of better programs focusing on the relevant microorganisms. This guidance document is intended to help industry and regulators focus and set up targeted processing environment monitoring programs depending on their purpose, and therefore provide the essential elements needed to improve food safety.

Guidance on validation of lethal control measures for foodborne pathogens in foods.

Food manufacturers are required to obtain scientific and technical evidence that a control measure or combination of control measures is capable of reducing a significant hazard to an acceptable level that does not pose a public health risk under normal conditions of distribution and storage. A validation study provides evidence that a control measure is capable of controlling the identified hazard under a worst-case scenario for process and product parameters tested. It also defines the critical parameters that must be controlled, monitored, and verified during processing. This review document is intended as guidance for the food industry to support appropriate validation studies, and aims to limit methodological discrepancies in validation studies that can occur among food safety professionals, consultants, and third-party laboratories. The document describes product and process factors that are essential when designing a validation study, and gives selection criteria for identifying an appropriate target pathogen or surrogate organism for a food product and process validation. Guidance is provided for approaches to evaluate available microbiological data for the target pathogen or surrogate organism in the product type of interest that can serve as part of the weight of evidence to support a validation study. The document intends to help food manufacturers, processors, and food safety professionals to better understand, plan, and perform validation studies by offering an overview of the choices and key technical elements of a validation plan, the necessary preparations including assembling the validation team and establishing prerequisite programs, and the elements of a validation report.

Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment.

Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments.

Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni.

The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.

Chicken juice enhances surface attachment and biofilm formation of Campylobacter jejuni.

The bacterial pathogen Campylobacter jejuni is primarily transmitted via the consumption of contaminated foodstuffs, especially poultry meat. In food processing environments, C. jejuni is required to survive a multitude of stresses and requires the use of specific survival mechanisms, such as biofilms. An initial step in biofilm formation is bacterial attachment to a surface. Here, we investigated the effects of a chicken meat exudate (chicken juice) on C. jejuni surface attachment and biofilm formation. Supplementation of brucella broth with ≥5% chicken juice resulted in increased biofilm formation on glass, polystyrene, and stainless steel surfaces with four C. jejuni isolates and one C. coli isolate in both microaerobic and aerobic conditions. When incubated with chicken juice, C. jejuni was both able to grow and form biofilms in static cultures in aerobic conditions. Electron microscopy showed that C. jejuni cells were associated with chicken juice particulates attached to the abiotic surface rather than the surface itself. This suggests that chicken juice contributes to C. jejuni biofilm formation by covering and conditioning the abiotic surface and is a source of nutrients. Chicken juice was able to complement the reduction in biofilm formation of an aflagellated mutant of C. jejuni, indicating that chicken juice may support food chain transmission of isolates with lowered motility. We provide here a useful model for studying the interaction of C. jejuni biofilms in food chain-relevant conditions and also show a possible mechanism for C. jejuni cell attachment and biofilm initiation on abiotic surfaces within the food chain.

Low-water activity foods: increased concern as vehicles of foodborne pathogens.

Foods and food ingredients with low water activity (a(w)) have been implicated with increased frequency in recent years as vehicles for pathogens that have caused outbreaks of illnesses. Some of these foodborne pathogens can survive for several months, even years, in low-a(w) foods and in dry food processing and preparation environments. Foodborne pathogens in low-a(w) foods often exhibit an increased tolerance to heat and other treatments that are lethal to cells in high-a(w) environments. It is virtually impossible to eliminate these pathogens in many dry foods or dry food ingredients without impairing organoleptic quality. Control measures should therefore focus on preventing contamination, which is often a much greater challenge than designing efficient control measures for high-a(w) foods. The most efficient approaches to prevent contamination are based on hygienic design, zoning, and implementation of efficient cleaning and sanitation procedures in the food processing environment. Methodologies to improve the sensitivity and speed of assays to resuscitate desiccated cells of foodborne pathogens and to detect them when present in dry foods in very low numbers should be developed. The goal should be to advance our knowledge of the behavior of foodborne pathogens in low-a(w) foods and food ingredients, with the ultimate aim of developing and implementing interventions that will reduce foodborne illness associated with this food category. Presented here are some observations on survival and persistence of foodborne pathogens in low-a(w) foods, selected outbreaks of illnesses associated with consumption of these foods, and approaches to minimize safety risks.

Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation.

Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.