Search

The aim of this study was to investigate the distribution of cefquinome in different dairy products during the processing of naturally contaminated milk or spiked milk. The analysis of cefquinome residues in milk, skimmed milk, buttermilk, whey, cream, butter, curd, and cheese samples was performed using a water:acetonitrile solvent extraction and C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) determination. The target concentration of cefquinome was achieved in the spiked milk (100 µg kg−1). During its processing, the antibiotic migrated primarily with the skimmed milk as opposed to cream (ratios of 3.6:1 and 2.8:1 for experiments A and B, respectively), and with the buttermilk during butter manufacture (ratios of 6.9:1 and 4.6:1), but was equal in the curd and whey during the manufacture of cheese. In the milk collected from treated animals, the measured concentration of cefquinome was considerably high (approx. 5000 µg kg−1). The results obtained from the dairy products were similar to those obtained in the spiked study (ratios of 8.2:1 and 3.1:1 for experiments A and B, respectively, during the separation of skimmed milk and cream, 6.0:1 and 5.0:1 for A and B, respectively, during the separation of buttermilk and butter). However, during cheesemaking, cefquinome migrated with the whey after cutting the curd, with ratios of 0.54:1 and 0.44:1 for experiments A and B, respectively. The difference in the migration of cefquinome between curd and whey in spiked and animal studies is probably due to the different concentration levels in the two different experiments. The results of this study showed that, in dairy products manufactured from milk containing cefquinome residues, the drug migrated primarily with the high-water-containing fractions.

Listeria monocytogenes is a foodborne pathogen that is frequently found in the environment. It can easily enter food processing environments and contaminate food, potentially causing public health issues. Food business operators (FBOs) are responsible for the control of L. monocytogenes in the food processing environment, particularly in facilities producing ready-to-eat food. The design and implementation of an effective environmental monitoring program (EMP) for L. monocytogenes is an integral part of controlling L. monocytogenes. An effective EMP, including all aspects from sampling, to analysis, to data interpretation, to implementation of corrective actions (including food disposition), is a tool that will help with identification and control of L. monocytogenes contamination. It should be used in conjunction with end product testing, not as a replacement for it. An EMP should be specifically designed for a particular facility on a case-by-case risk-based approach, by a food safety team within the facility. It should be reviewed regularly (at least every 6 months) and verified for its effectiveness. The control of L. monocytogenes in the food industry involves the full commitment of management and of all personnel involved with the safety of foods placed on the market, thus reducing the risk of listeriosis to consumers. Several regulatory and guidance documents provide recommendations for designing aspects of an effective L. monocytogenes EMP. However, a comprehensive review of the key components of an EMP in a single document is lacking. The objective of the present review is to provide FBOs with a practical guide to design, implementation, and verification of an EMP tailored by the food safety team for each food business.

To understand the molecular mechanisms that contribute to the stress responses of the important foodborne pathogen Listeria monocytogenes, we collected 139 strains (meat, n = 25; dairy, n = 10; vegetable, n = 8; seafood, n = 14; mixed food, n = 4; and food processing environments, n = 78), mostly isolated in Ireland, and subjected them to whole-genome sequencing. These strains were compared to 25 Irish clinical isolates and 4 well-studied reference strains. Core genome and pan-genome analysis confirmed a highly clonal and deeply branched population structure. Multilocus sequence typing showed that this collection contained a diverse range of strains from L. monocytogenes lineages I and II. Several groups of isolates with highly similar genome content were traced to single or multiple food business operators, providing evidence of strain persistence or prevalence, respectively. Phenotypic screening assays for tolerance to salt stress and resistance to acid stress revealed variants within several clonal complexes that were phenotypically distinct. Five of these phenotypic outliers were found to carry mutations in the sigB operon, which encodes the stress-inducible sigma factor sigma B. Transcriptional analysis confirmed that three of the strains that carried mutations in sigB, rsbV, or rsbU had reduced SigB activity, as predicted. These strains exhibited increased tolerance to salt stress and displayed decreased resistance to low pH stress. Overall, this study shows that loss-of-function mutations in the sigB operon are comparatively common in field isolates, probably reflecting the cost of the general stress response to reproductive fitness in this pathogen. IMPORTANCE The bacterial foodborne pathogen Listeria monocytogenes frequently contaminates various categories of food products and is able to cause life-threatening infections when ingested by humans. Thus, it is important to control the growth of this bacterium in food by understanding the mechanisms that allow its proliferation under suboptimal conditions. In this study, intraspecies heterogeneity in stress response was observed across a collection consisting of mainly Irish L. monocytogenes isolates. Through comparisons of genome sequence and phenotypes observed, we identified three strains with impairment of the general stress response regulator SigB. Two of these strains are used widely in food challenge studies for evaluating the growth potential of L. monocytogenes. Given that loss of SigB function is associated with atypical phenotypic properties, the use of these strains in food challenge studies should be re-evaluated.

Background and Aim: Teat disinfection is an important tool in reducing the incidence of bovine mastitis. Identifying the potential mastitis-causing bacterial species in milk can be the first step in choosing the correct teat disinfectant product. The objective of this study was to screen commercial teat disinfectants for inhibition against mastitis-associated bacteria isolated from various types of milk samples. Materials and Methods: Twelve commercially available teat disinfectant products were tested, against 12 mastitis-associated bacteria strains isolated from bulk tank milk samples and bacterial strains isolated from clinical (n=2) and subclinical (n=3) quarter foremilk samples using the disk diffusion method. Results: There was a significant variation (7-30 mm) in bacterial inhibition between teat disinfection products, with products containing a lactic acid combination (with chlorhexidine or salicylic acid) resulting in the greatest levels of bacterial inhibition against all tested bacteria (p

High-throughput biochemical screening techniques are an important tool in phenotypic analysis of bacteria. New methods, simultaneously measuring many phenotype responses, increase the output of such investigations and allow a more complete overview of the bacterial phenotype, facilitating large-scale correlation to related genotypes. This chapter describes the application of OmniLog phenotype microarray analysis, a high-throughput assay for the phenotypic characterization of bacterial strains across a variety of different traits such as nutrient utilization and antimicrobial sensitivity, to Listeria species.

PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.

In 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.

peer-reviewed Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the elderly. Under European Union legislation, EC 2073/2005, food business operators are encouraged to undertake sampling to ensure that the food processing environment, and required to ensure that food products, are free of Listeria monocytogenes. To determine the prevalence of L. monocytogenes in smaller food processing facilities in Northern Ireland, 24 companies submitted six processing environment swabs and two food samples every two months for eighteen months (July 2015 to November 2016) for L. monocytogenes examination. The prevalence of L. monocytogenes was 4.6% in food samples, and 6.3% in processing environment swabs. Over the duration of the study, 96 isolates of L. monocytogenes were obtained, one from each positive sample, except for two meat samples that had >100 cfu/g, where two isolates were obtained from each sample. No seasonality in occurrence of L. monocytogenes was seen for food isolates but significantly higher numbers of positive processing environment swabs were found in the warmer months of May, July and September (p = .007). Pulsed Field Gel Electrophoresis (PFGE) analysis revealed the presence of 27 pulsotypes; 9 pulsotypes were shared between different facilities and 9 were persistent. Based on a Combase predictive growth model, 77.5% (n = 130) of the foods tested were predicted to support the growth of L. monocytogenes. All of the isolates carried the pathogenicity genes inlA and actA and 71.4% carried qacH, which confers resistance to quaternary ammonium compounds which are frequently used in sanitizers. Whole genome sequencing of the isolates allowed multi-locus sequence typing to be undertaken. The data indicated that the sequence types identified included those with disease-causing ability, highlighting the disease-causing potential of the isolates.

peer-reviewed As the new technology of whole genome sequencing (WGS) has been shown to have greater discriminatory power in differentiating strains than the much-used pulsed-field gel electrophoresis (PFGE), there is currently a transition from using PFGE to WGS for disease outbreak investigation. Therefore, there is a need for comparison of bacterial isolates using both PFGE and WGS. In this study, two pairs of L. monocytogenes strains with geographically diverse sources of isolation but which had indistinguishable or closely related PFGE profiles, were subjected to WGS analysis. Comparative analysis of their genomes showed that one pair of strains which had closely related PFGE profiles in fact differed significantly from one another in terms of their antibiotic and heavy metal stress resistance determinants, and mobile genetic elements. Therefore, this research demonstrated the ability of WGS analysis to differentiate very closely related strains and that WGS analysis represents the most effective tool available for subtyping L. monocytogenes isolates.

peer-reviewed Listeria 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.

Background The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called “substrate”; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. Results L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290‐2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. Conclusion These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross‐contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry., During the mushroom production, the current hygiene practices are not completely efficient in Listeria monocytogenes removal. Here, we investigate the effectiveness of hygiene practices in critical points throughout the mushroom production chain.

Background The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called “substrate”; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. Results L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290‐2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. Conclusion These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross‐contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry.

© The Author(s)., Listeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli (E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment., This research was funded by the Food Institutional Research Measure under project 14/F/881, grant BFU2014-53425-P from the Spanish Ministry of Science, Innovation and Universities and the Teagasc Walsh Fellowship Short-Term Overseas Training Awards.

The experiments reported in this research paper aimed to track the microbiological load of milk throughout a low-heat skim milk powder (SMP) manufacturing process, from farm bulk tanks to final powder, during mid- and late-lactation (spring and winter, respectively). In the milk powder processing plant studied, low-heat SMP was produced using only the milk supplied by the farms involved in this study. Samples of milk were collected from farm bulk tanks (mid-lactation: 67 farms; late-lactation: 150 farms), collection tankers (CTs), whole milk silo (WMS), skim milk silo (SMS), cream silo (CS) and final SMP. During mid-lactation, the raw milk produced on-farm and transported by the CTs had better microbiological quality than the late-lactation raw milk (e.g., total bacterial count (TBC): 3.60 ± 0.55 and 4.37 ± 0.62 log 10 cfu/ml, respectively). After pasteurisation, reductions in TBC, psychrotrophic (PBC) and proteolytic (PROT) bacterial counts were of lower magnitude in late-lactation than in mid-lactation milk, while thermoduric (LPC—laboratory pasteurisation count) and thermophilic (THERM) bacterial counts were not reduced in both periods. The microbiological quality of the SMP produced was better when using mid-lactation than late-lactation milk (e.g., TBC: 2.36 ± 0.09 and 3.55 ± 0.13 cfu/g, respectively), as mid-lactation raw milk had better quality than late-lactation milk. The bacterial counts of some CTs and of the WMS samples were higher than the upper confidence limit predicted using the bacterial counts measured in the farm milk samples, indicating that the transport conditions or cleaning protocols could have influenced the microbiological load. Therefore, during the different production seasons, appropriate cow management and hygiene practices (on-farm and within the factory) are necessary to control the numbers of different bacterial groups in milk, as those can influence the effectiveness of thermal treatments and consequently affect final product quality.

peer-reviewed The different customer and regulatory specifications for mesophilic and thermophilic aerobic and anaerobic spore numbers in skim-milk powder, in addition to some specifications on specific spore-forming bacteria, such as Bacillus cereus, can be challenging for the industry to meet. Twenty-two samples of medium-heat skim-milk spray-dried powder from eight sources were analysed in triplicate with 16 bacterial and spore enumeration tests to understand the variety of spore-forming bacteria population. Using 16S rDNA sequencing, the species were identified for 269 isolates that were representative of the various tests. Of the isolates identified, 68% were Bacillus licheniformis, a facultative anaerobe that can survive and grow at mesophilic and thermophilic temperatures, making it difficult to eliminate in manufacturing environments. Using whole genome sequencing, 16 of 23 isolates identified as B. licheniformis by 16S sequencing were confirmed as B. licheniformis, four were identified as Bacillus paralicheniformis and three were identified as Bacillus sp. H15-1.

PubMed: 31478167, Listeria monocytogenes is one of the most important foodborne pathogens and is a causal agent of listeriosis in humans and animals. The aim of this study was to determine the prevalence, serogroups, antibiotic susceptibility, virulence factor genes, and genetic relatedness of L. monocytogenes strains isolated from 500 poultry samples in Turkey. The isolation sources of 103 L. monocytogenes strains were retail markets (n = 100) and slaughterhouses (n = 3). L. monocytogenes strains were identified as serogroups 1/2a-3a (75.7%, lineage I), 1/2c-3c (14.56%, lineage I), 1/2b-3b-7 (5.82%, lineage II), 4a-4c (2.91%, lineage III), and 4b-4d-4e (0.97%, lineage III). Most of the L. monocytogenes strains (93.2%) were susceptible to the antibiotics tested. PCR analysis indicated that the majority of the strains (95% to 100%) contained most of the virulence genes (hylA, plcA, plcB, prfA, mpl, actA, dltA, fri, flaA inlA, inlC, and inlJ). Pulsed-field gel electrophoresis (PFGE) demonstrated that there were 18 pulsotypes grouped at a similarity of >90% among the strains. These results indicate that it is necessary to prevent the presence of L. monocytogenes in the poultry-processing environments to help prevent outbreaks of listeriosis and protect public health.

peer-reviewed The use of pre- and post-milking teat disinfectants can reduce teat bacterial load and aid in the collection of high-quality milk. The objective of this study was to compare the reduction in bacteria populations on teat skin after the application of different commercial teat disinfectant products. Ten teat disinfectant products were applied to the teats of 10 Holstein–Friesian cows. One cow received one teat disinfectant product at each sampling point before cluster application for milking. A composite swab sample was taken of the 4 teats of each cow before and after teat disinfectant application. Swab samples were placed on three different selective agars to enumerate bacterial counts of staphylococcal, streptococcal and coliforms isolates on teat skin. Staphylococcal isolates were the most prominent bacterial group recovered on teat swabs (49%), followed by streptococcal (36%) and coliform (15%) isolates before the application of disinfectant. The average bacterial reductions on teat skin were shown to be 76%, 73% and 60% for staphylococcal, streptococcal and coliform isolates, respectively. All of the teat disinfectant products tested reduced teat bacterial load for all three bacterial groups. Product 4 containing 0.6% w/w diamine was the most effective against bacterial populations of staphylococcal and streptococcal isolates on teat skin with a reduction of 90% and 94%, respectively. Whereas product 10, which contained 0.5% w/w iodine, resulted in the highest reduction in coliforms on teat skin with a reduction of 91%. Results from this study suggest that specific bacterial population loads on teats can be reduced using different teat disinfectant formulations.

Listeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli (E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment.

Listeria monocytogenes is a virulent pathogen that is the causative agent of listeriosis, a rare and potentially fatal foodborne disease. The aim of this study was to assess the behaviour of L. monocytogenes in milk obtained from a cow with a sub-clinical infection, representing naturally contaminated milk. Within four hours of milking, the inhibitors Nisin V and pediocin from Pediococcus acidilactici , were added (at a concentration previously found to be inhibitory) to this naturally contaminated milk. The naturally contaminated milk was also pre-chilled at 4 °C for 24 h and then the inhibitors added. All the milk samples were incubated at 30 °C for 10 h. Samples were taken at hourly intervals for pH measurement and L. monocytogenes enumeration. The naturally contaminated milk was used to manufacture a laboratory-scale semi-soft cheese, in triplicate, using fresh and frozen starter cultures, and ripened at 15 °C for 3 weeks. Using an isolated strain from the naturally contaminated milk, growth in milk and during cheesemaking and ripening was also studied. Increasing numbers of L. monocytogenes were measured in the naturally contaminated milk, even in the presence of the bacteriocins. The chilled naturally contaminated milk had a higher initial number of L. monocytogenes than the freshly analysed milk. This implies that in the naturally contaminated milk used in this study, ‘increasing numbers’ of L. monocytogenes was uncoupled from ‘growth’; the increasing numbers may be due to breaking of clumps of cells in the naturally contaminated milk directly from the cow into individual cells, implying growth. Liquid starter culture of direct vat starter culture had little impact on the behaviour of L. monocytogenes during cheesemaking or ripening, the final numbers being the same in the ripened cheese as in the starting milk. The study contributes to risk assessment of naturally contaminated milk with respect to L. monocytogenes .

Aims This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by L. monocytogenes or to remove existing biofilms in mushroom production facilities. Methods and Results It is generally recognised that Listeria monocytogenes forms biofilms that can facilitate its survival in food processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterised based on heat and enzyme inactivation studies. Further characterisation was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of Nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. Conclusions Mushroom substrate can be source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. Significance and Impact of the Study The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment. This article is protected by copyright. All rights reserved.

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.

This aim of this study was to assess the microbiological and anthelmintic drug residue risks associated with raw milk used for cheesemaking and raw milk cheese, over an 18-month period. Samples of raw milk, milk filters, curd and cheese from nine raw milk artisan cheese producers in the south of Ireland were tested. Numbers of presumptive Bacillus cereus group, Escherichia coli, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes were determined. The determination of anthelmintic drug residues, including benzimidazoles, flukicides, macrocyclic lactone (avermectin and milbemycins), levamisole and morantel was also performed. Neither L. monocytogenes, nor Salmonella spp. were detected in any of the samples tested and no anthelmintic drug residues were detected. Only one of the samples did not conform with regulatory numbers for other bacteria. This survey has shown a good microbiological and residue quality (and low risk) of the raw milk cheese and raw milk used for raw milk cheese produced in Ireland. Moreover, it has shown the importance of frequent assessment of raw milk used for cheesemaking and for raw milk cheese, as it allows the identification of potential problems facilitating resolution of these issues before they cause any public health threat.

Sequential heating processes are commonly applied to milk by the dairy industry as part of their microbiological control strategy. Often pasteurisation at 72 °C is followed by a sequential high heat treatment step of up to 125 °C; however, such severe heat treatment can lead to reduced protein quality. Nine temperature combinations (80–90 °C) were evaluated to assess microbial reduction and whey protein nitrogen index values during pilot scale milk processing. A total of 110 bacterial isolates were identified to species level by 16S rDNA sequencing, with Bacillus licheniformis identified as the dominant species. While the experimental treatments did not achieve microbial reductions comparable with the control heating process, the results of this study provide a benchmark for milk processors relative to the effects of sequential heat treatments on milk and their impact on the survival of both thermally resistant microbial populations and thermally labile milk components during processing.

Listeria species are ubiquitous in nature and can adapt to survive in a variety of niches, including food processing environments. Listeria species that colonize these environments may also have the potential to persist. Food safety strategies designed to manage these niches include regular cleaning and disinfection with proven sanitizers containing biocide-active compounds. Typically, these sanitizers are effective against bacteria growing under planktonic conditions, but their efficacy may be compromised when bacteria are contained in biofilms. The susceptibility of persistent Listeria isolates, i.e., those capable of forming biofilms, to a selection of sanitizers was investigated. A quaternary ammonium compound-based sanitizer was the biocide most effective against planktonic bacteria, with a MIC of 0.0015 to 0.006%. In contrast, ethanol-based sanitizers were the least effective. Although, no triclosan tolerance was observed for planktonic Listeria isolates, triclosan was the only biocide that resulted in a significant biomass reduction. Differences between Listeria species were observed; L. monocytogenes and L. welshimeri biofilms were more tolerant to quaternary ammonium compound-based sanitizers than were L. innocua biofilms. These findings extend our understanding of the application of commonly used sanitizers in the food industry and the efficacy of these sanitizers against Listeria species and their associated biofilms.

Listeria monocytogenes causes listeriosis, a rare foodborne disease with a mortality rate of 20%-30%. The elderly and immunocompromised are particularly susceptible to listeriosis. L. monocytogenes is ubiquitous in nature and can contaminate food-processing environments, posing a threat to the food chain. This is particularly important for ready-to-eat foods as there is no heat treatment or other antimicrobial step between production and consumption. Thus, occurrence and control of L. monocytogenes are important for industry and public health. Advances in whole-genome sequence technology are facilitating the investigation of disease outbreaks, linking sporadic cases to outbreaks, and linking outbreaks internationally. Novel control methods, such as bacteriophage and bacteriocins, can contribute to a reduction in the occurrence of L. monocytogenes in the food-processing environment, thereby reducing the risk of food contamination and contributing to a reduction in public health issues.

peer-reviewed The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01103/full#supplementary-material The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups. This study was supported by the 7th Framework Programme projects PROMISE, contract number 265877.

In the EU, food is considered safe with regard toListeria monocytogenesif 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 ofL. monocytogenes. Guidelines for conducting challenge tests for growth assessment ofL. monocytogeneson 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 ofL. monocytogenes. Three batches of mushrooms were artificially inoculated at approximately 100 cfu/g with a three-strain mix ofL. monocytogenesand incubated for 2 days at 8°C followed by 4 days at 12°C.L. monocytogenesnumbers 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 ofL. monocytogenesabove the threshold of 0.5 log cfu/g in any of the replicates. In 8 of 9 replicates, the numbers decreased indicating thatA. bisporusdo not support the growth ofL. monocytogenes. As the EU regulations allow < 100 cfu/g if the food cannot support growth ofL. 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.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a relatively rare but life-threatening disease primarily affecting immunocompromised individuals. The aim of this study was to determine the prevalence of L. monocytogenes in the seafood processing industry in the Republic of Ireland. The occurrence of L. monocytogenes was determined by regular sampling of both food samples and processing environment swabs at eight seafood processing facilities over two calendar years. All samples were analyzed by the International Organization for Standardization 11290-1 standard method, and the isolates were characterized by PCR, pulsed-field gel electrophoresis, serotyping, and the occurrence of some genes related to survival under stress (SSI-1, Tn6188, and bcrABC). A prevalence of 2.5% in 508 samples (433 environmental swabs and 75 food samples) was found. From the isolates obtained, eight different pulsed-field gel electrophoresis profiles were identified, two occurring in more than one facility and one occurring in food and the environment. Five of the eight pulsotypes identified contained at least one of the three stress survival-related genes tested. The tolerance of the isolates to benzalkonium chloride, a representative quaternary ammonium compound, was also examined and ranged from 5.5 ± 0.5 to 8.5 ± 0.5 ppm of benzalkonium chloride. To evaluate the ability of smoked salmon to support the growth of L. monocytogenes, including the T4 widespread pulsotype that was isolated, a challenge test was performed on cold-smoked salmon obtained from two separate producers. The results showed clearly that both types of smoked salmon supported the growth of L. monocytogenes. Although occurrence of L. monocytogenes on seafood was low, this study showed that the smoked salmon used in this study can support the growth of L. monocytogenes; therefore, vigilance is required in the processing facilities to reduce the associated risk.

Staphylococcus aureus (S. aureus) can cause mastitis in cattle and, therefore, can be present in milk. This study was undertaken to determine the prevalence of coagulase positive S. aureus and its enterotoxin genes sea, seb, and sec in isolates recovered from raw milk, feta cheese, and human hand swabs of milk and cheese handlers in Beni-Suef province, Egypt. A total of 100 samples of raw milk and 50 samples of pasteurized-milk feta cheese were collected. In addition, 50 hand swabs from milk handlers and 25 hand swabs from cheese handlers were examined for the presence of coagulase positive S. aureus. The isolates were characterized by multiplex PCR for detection of sea, seb, and sec genes, and for resistance to 5 classes of commonly used antibiotics. Twelve (12/100), 12 (6/50), and 17% (13/75) of milk, cheese, and hand swab samples, respectively, were positive for coagulase positive S. aureus. One isolate was obtained from each positive sample (31 isolates), and none contained genes for SEA or SEC production. Twenty-five percent, 33%, and 31%, respectively, of the isolates contained the genes for SEB, resulting in 3%, 4%, and 5% of samples being positive for toxin producing coagulase positive S. aureus, respectively. At least one isolate was resistant to each of the antibiotics tested. Despite the low potential for SEB production shown, preventative measures, such as maintenance of the cold-chain and good hygienic practices should be implemented to further reduce the potential risk to public health from SEB, and to reduce the spread of antimicrobial resistance.

Putative routes of Listeria monocytogenes contamination, based on the workflow of the employees, were studied in a meat processing facility by investigating 226 samples collected from food contact surfaces, non-food contact surfaces, raw materials, and ready-to-eat meat products on four occasions over a 1-year period. In total, 19.7% of non-food contact surfaces, 22.9% of food contact surfaces, 45% of raw materials, and 20% of ready-to-eat meat products were positive for L. monocytogenes (analyzed by the International Organization for Standardization standard method ISO 11290). Pulsed-field gel electrophoresis (PFGE) profiles were determined for a representative subset of these isolates, and 11 distinct pulsotypes were identified, two of which were frequently isolated (T4 and T8) and considered persistent. Strains from the various pulsotypes were screened for the presence of bcrABC and qacH, the genes responsible for tolerance responses to quaternary ammonium compounds. Two strains harbored bcrABC, and these strains had a higher benzalkonium chloride tolerance; however, they were not considered persistent strains. The frequently isolated PFGE pulsotype T8 strains were highly adhesive to abiotic surfaces at 10 and 20°C; however, the pulsotype T6 strain, which was isolated only at the last sampling time, had the highest adhesion ability, and the pulsotype T4 strain (the second most persistent pulsotype) had only modest adhesion. Four putative cross-contamination routes were confirmed by mapping the persistent and other isolates. This information could allow a food safety manager to adjust the work flow to improve the hygienic conditions in a meat processing facility. This study revealed the prevalence and persistence of L. monocytogenes strains in a meat processing facility and established the importance of developing strategies to avoid cross-contamination, recalls, and outbreaks of listeriosis.

Persistence of Listeria monocytogenes strains in food processing environments remains relatively common but is difficult to control. Understanding the basis for such persistence represents an important step in the potential control or eradication of this pathogen from these environments. In this study, reverse transcription PCR was used to determine the relative and absolute expression of selected gene targets (pocR, eutJ, and qacH) among five persistent and four presumed nonpersistent L. monocytogenes strains. The quantification of these genes as markers for the persistent phenotype and the effect of benzethonium chloride (BZT) on their expression was investigated. Although no markers correlated with the ability of strains to persist in food processing facilities were found, expression of pocR was upregulated in three of the five persistent strains, in contrast to the four presumed nonpersistent strains, which showed down-regulation of this gene. These results provide further knowledge of the differential expression of genes of persistent and presumed nonpersistent strains of L. monocytogenes grown in the presence or absence of BZT and identifies upregulation of pocR as a potential response of persistent strains of L. monocytogenes to exposure to BZT.

Campylobacter jejuni CI 120 is a natural isolate obtained during poultry processing and has the ability to induce an acid tolerance response (ATR) to acid + aerobic conditions in early stationary phase. Other strains tested they did not induce an ATR or they induced it in exponential phase. Campylobacter spp. do not contain the genes that encode the global stationary phase stress response mechanism. Therefore, the aim of this study was to identify genes that are involved in the C. jejuni CI 120 early stationary phase ATR, as it seems to be expressing a novel mechanism of stress tolerance. Two-dimensional gel electrophoresis was used to examine the expression profile of cytosolic proteins during the C. jejuni CI 120 adaptation to acid + aerobic stress and microarrays to determine the genes that participate in the ATR. The results indicate induction of a global response that activated a number of stress responses, including several genes encoding surface components and genes involved with iron uptake. The findings of this study provide new insights into stress tolerance of C. jejuni, contribute to a better knowledge of the physiology of this bacterium and highlight the diversity among different strains.