A note on challenge trials to determine the growth of Listeria monocytogenes on mushrooms (Agaricus bisporus)

peer-reviewedIn the EU, food is considered safe with regard to Listeria monocytogenes if the number of micro-organisms does not exceed 100 colony forming units (cfu)/g throughout its shelf-life. Therefore, it is important to determine if a food supports growth of L. monocytogenes. Guidelines for conducting challenge tests for growth assessment of L. monocytogenes on foods were published by the European Union Reference Laboratory (EURL) in 2014. The aim of this study was to use these guidelines to determine if refrigerated, fresh, whole, closed-cap, prepackaged mushrooms (Agaricus bisporus) support the growth of L. monocytogenes. Three batches of mushrooms were artificially inoculated at approximately 100 cfu/g with a three-strain mix of L. monocytogenes and incubated for 2 days at 8°C followed by 4 days at 12°C. L. monocytogenes numbers were determined (in triplicate for each batch) on days 0, 2 and 6. Water activity, pH and total bacterial counts were also determined. There was no increase in the number of L. monocytogenes above the threshold of 0.5 log cfu/g in any of the replicates. In 8 of 9 replicates, the numbers decreased indicating that A. bisporus do not support the growth of L. monocytogenes. As the EU regulations allow < 100 cfu/g if the food cannot support growth of L. monocytogenes, the significance of this study is that mushrooms with < 100 cfu/g may be within the regulations and therefore, quantitative rather than qualitative determination may be required.Safefoo

Complete Genome Sequences of vB_LmoS_188 and vB_LmoS_293, Two Bacteriophages with Specificity for Listeria monocytogenes Strains of Serotypes 4b and 4e

peer-reviewedListeria monocytogenes is responsible for the rare disease listeriosis, which is associated with the consumption of contaminated food products. We report here the complete genome sequences of vB_LmoS_188 and vB_LmoS_293, phages isolated from environmental sources and that have host specificity for L. monocytogenes strains of the 4b and 4e serotypes.This work was supported by the EU 7th Framework projects PROMISE (project no. 265877) and FOODSEG (project no. 266061) and by a safefood mini-project

Determination of Listeria monocytogenes numbers at less than 10 cfu/g

peer-reviewedListeria monocytogenes is a foodborne pathogen that causes a relatively rare foodborne disease called listeriosis, with a high mortality rate of 20%-30% and an undefined dose response. Current European Union regulations permit up to 100 colony-forming units (cfu)/g in food at the end of its shelf life, where the food has been shown not to support the growth of this pathogenic bacterium. Therefore, enumeration of L. monocytogenes at low numbers in food is important. The objective of this study was to reduce the detection limit of L. monocytogenes in food by a factor of 10. The International Organisation for Standardisation (ISO) 11290-2 method for enumeration of L. monocytogenes in food recommends spreading 0.1 mL of a 1:10 dilution of the food on the surface of an agar plate (detection limit 100 cfu/g), or 1.0 mL spread in equal parts on the surface of three agar plates (detection limit: 10 cfu/g). The pour-plate method (using 1 or 10 mL of an appropriate dilution) was compared to the spread-plate method using the ISO-approved chromogenic medium Agar Listeria according to Ottaviani and Agosti (ALOA). Using the pour-plate method, the colony morphology and halo formation were similar to the spread-plate method from pure cultures and inoculated foods. Using the pour-plate method in a 140 mm Petri dish, 10 mL of a 1:10 dilution of food allowed determination of numbers as low as 1 cfu/g. Applying this method, L. monocytogenes in naturally contaminated food samples were enumerated at numbers as low as 1-9 cfu/g.This work was supported by The Irish Department of Agriculture, Food and the Marine under the Food Institutional Research Measure, project 11/F/008, and by the Dairy Processing Technology Centre, supported by Enterprise Ireland and the Irish Dairy Industry, project number TC 2014 0016

Cronobacter Sakazakii ISO 22964:2017 Testing of Milk Powders Using Commercially Available PCR

The detection of Cronobacter sakazakii in milk powder is important as a major health issue1 as it can survive for long periods in dry conditions2. Faster detection using PCR is possible and currently need to be compatible with traditionally standardised ISO methods. Viable cells are detected by PCR when dead cells and free DNA are diluted out, inhibited or destroyed. Biorad IQ–check DNA removal solution eliminates the detection of dead cells (based on endonuclease activity) or Biotecon Reagent D, (a light reactive aqueous reagent solution) is a dye designed to eliminate dead bacterial cell amplification, both avoid false-positive PCR results from dead cells. With ISO 20838: Real-Time PCR can be self-confirming and no further confirmation necessary with faster in house control and release of product quicker based on PCR results

A case of bovine raw milk contamination with Listeria monocytogenes

peer-reviewedDuring routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.Teagasc Walsh Fellowship; Irish Department of Agriculture, Fisheries and Food, Food Institutional Research Measure (Irish Microbial Risk Assessment Network project); European Union (EU), 6th Framework Programme (BIOTRACER project)

Editorial: Microbial Food Safety along the Dairy Chain

peer-reviewedMilk is susceptible to contamination with pathogenic and spoilage organisms and, therefore, Microbial food safety along the dairy chain is an important topic, from public health and industry perspectives. The dairy chain is an integral part of global food supply, with dairy food products a staple component of recommended healthy diets. The dairy food chain from production through to the consumer is complex, with various opportunities for microbial contamination of ingredients or food products, and as such interventions are key to preventing or controlling such contamination. Dairy foods often include a microbial control step in their production such as pasteurization, but in some cases may not, as with raw milk products. Microbial contamination may lead to a deterioration in food quality due to spoilage organisms, or may become a health risk to consumers should the contaminant be a pathogenic microorganism. As such food safety and food production are intrinsically linked

Evaluating the effect of storage conditions on milk microbiological quality and composition

peer-reviewedIn this study, the effect of storage temperature (2 or 4°C) on the composition of milk and microbiological load was investigated over 96 h. Milk samples were collected from farm bulk milk tanks after one complete milking and stored at 2 or 4°C over 96 h. Total bacterial count (TBC), psychrotrophic bacterial count (PBC) and proteolytic bacterial count (PROT) were affected by storage time and temperature and varied significantly between farms (P < 0.05). The levels of TBC, PBC and PROT bacterial count increased from 4.37 to 6.15 log cfu/mL, 4.34 to 6.44 log cfu/mL and 3.72 to 4.81 log cfu/mL, respectively, when the milk was stored for 96 h at 2°C. The milk samples stored at 4°C had higher increases in these bacterial counts after 72 h in comparison to milk samples stored at 2°C. The casein fraction content was lower in milk samples stored at 4°C, which could be due to high levels of PROT bacteria or enzyme activity in these samples. Milk stored for 96 h at 2°C has less impact on composition or processability parameters compared to milk stored at 4°C

A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

peer-reviewedThe development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods

Comparative Genomic Analysis of Two Serotype 1/2b Listeria monocytogenes Isolates from Analogous Environmental Niches Demonstrates the Influence of Hypervariable Hotspots in Defining Pathogenesis

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but differing infection abilities, were subjected to comparative genomic analysis. The results from this comparison highlight the importance of accessory genes (genes that are not part of the conserved core genome) in L. monocytogenes pathogenesis. In addition, a number of factors, which may account for the perceived inability of one of the strains to establish a systemic infection within its host, have been identified. These factors include the notable absence of the Listeria pathogenicity island 3 and the stress survival islet, of which the latter has been demonstrated to enhance the survival ability of L. monocytogenes during its passage through the host intestinal tract, leading to a higher infection rate. The findings from this research demonstrate the influence of hypervariable hotspots in defining the physiological characteristics of a L. monocytogenes strain and indicate that the emergence of a non-pathogenic isolate of L. monocytogenes may result from a cumulative loss of functionality rather than by a single isolated genetic event

Flow cytometric and 16S sequencing methodologies for monitoring the physiological status of the microbiome in powdered infant formula production

The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility