Phylogenetic and Phenotypic Analyses of a Collection of Food and Clinical Listeria monocytogenes Isolates Reveal Loss of Function of Sigma B from Several Clonal Complexes.

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.

Listeria monocytogenes environmental sampling program in ready-to-eat processing facilities: A practical approach.

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.

Microbiological quality of milk from farms to milk powder manufacture: an industrial case study.

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.

The effect of different precooling rates and cold storage on milk microbiological quality and composition.

The objective of this study was to measure the effect of different milk cooling rates, before entering the bulk tank, on the microbiological load and composition of the milk, as well as on energy usage. Three milk precooling treatments were applied before milk entered 3 identical bulk milk tanks: no plate cooler (NP), single-stage plate cooler (SP), and double-stage plate cooler (DP). These precooling treatments cooled the milk to 32.0 ± 1.4°C, 17.0 ± 2.8°C, and 6.0 ± 1.1°C, respectively. Milk was added to the bulk tank twice daily for 72 h, and the tank refrigeration temperature was set at 3°C. The blend temperature within each bulk tank was reduced after each milking event as the volume of milk at 3°C increased simultaneously. The bacterial counts of the milk volumes precooled at different rates did not differ significantly at 0 h of storage or at 24-h intervals thereafter. After 72 h of storage, the total bacterial count of the NP milk was 3.90 ± 0.09 log10 cfu/mL, whereas that of the precooled milk volumes were 3.77 ± 0.09 (SP) and 3.71 ± 0.09 (DP) log10 cfu/mL. The constant storage temperature (3°C) over 72 h helped to reduce bacterial growth rates in milk; consequently, milk composition was not affected and minimal, if any, proteolysis occurred. The DP treatment had the highest energy consumption (17.6 ± 0.5 Wh/L), followed by the NP (16.8 ± 2.7 Wh/L) and SP (10.6 ± 1.3 Wh/L) treatments. This study suggests that bacterial count and composition of milk are minimally affected when milk is stored at 3°C for 72 h, regardless of whether the milk is precooled; however, milk entering the tank should have good initial microbiological quality. Considering the numerical differences between bacterial counts, however, the use of the SP or DP precooling systems is recommended to maintain low levels of bacterial counts and reduce energy consumption.

Farm to Fork Quantitative Risk Assessment of Listeria monocytogenes Contamination in Raw and Pasteurized Milk Cheese in Ireland.

The objective of this study was to model and quantify the level of Listeria monocytogenes in raw milk cheese (RMc) and pasteurized milk cheese (PMc) from farm to fork using a Bayesian inference approach combined with a quantitative risk assessment. The modeling approach included a prediction of contamination arising from the farm environment as well from cross-contamination within the cheese-processing facility through storage and subsequent human exposure. The model predicted a high concentration of L. monocytogenes in contaminated RMc (mean 2.19 log10 CFU/g) compared to PMc (mean -1.73 log10 CFU/g). The mean probability of illness (P1 for low-risk population, LR) and (P2 for high-risk population, HR, e.g., immunocompromised) adult Irish consumers following exposure to contaminated cheese was 7 × 10(-8) (P1 ) and 9 × 10(-4) (P2 ) for RMc and 7 × 10(-10) (P1 ) and 8 × 10(-6) (P2 ) for PMc, respectively. In addition, the model was used to evaluate performance objectives at various stages, namely, the cheese making and ripening stages, and to set a food safety objective at the time of consumption. A scenario analysis predicted various probabilities of L. monocytogenes contamination along the cheese-processing chain for both RMc and PMc. The sensitivity analysis showed the critical factors for both cheeses were the serving size of the cheese, storage time, and temperature at the distribution stage. The developed model will allow food processors and policymakers to identify the possible routes of contamination along the cheese-processing chain and to reduce the risk posed to human health.

Absence of growth of Listeria monocytogenes in naturally contaminated Cheddar cheese.

Each cheese producer is responsible by the legislation for the number of Listeria monocytogenes in cheese and is required to prove that numbers will not exceed 100 cfu/g throughout the shelf-life of the cheese. Even in the case of hard-cheese such as Cheddar cheese, the absence of growth of List. monocytogenes during ripening has to be demonstrated to comply with EU legislation. Studies dedicated to assessing List. monocytogenes growth throughout cheese shelf-life are generally based on artificially contaminated cheeses. Contrary to the majority of works, the current study focused on the growth of List. monocytogenes in naturally contaminated raw milk farmhouse Cheddar cheeses during a five-month ripening period. List. monocytogenes growth was assessed by direct count and its presence was detected by enrichment in two naturally contaminated cheese batches. In order to track routes of contamination, 199 processing environment samples from inside and outside the processing facility were taken, and their analysis for the presence of List. monocytogenes was performed on four occasions over a 9-month period. List. monocytogenes isolates were differentiated using PFGE and serotyping. List. monocytogenes never exceeded 20 cfu/g in the cheeses and could not be detected after five months of ripening. Eleven pulsotypes were identified. One pulsotype was found in the yard outside the processing facility, in a vat, on the processing area floor and in a cheese. This indicated that the outside environment constitutes a potential source of contamination of the processing environment and of the cheese. These results demonstrate that this farmhouse Cheddar cheese does not support List. monocytogenes growth and suggests that the efforts to reduce processing environment contamination are worthwhile.

Collaborative survey on the colonization of different types of cheese-processing facilities with Listeria monocytogenes.

Cross-contamination via equipment and the food-processing environment has been implicated as the main cause of Listeria monocytogenes transmission. The aim of this study, therefore, was to determine the occurrence and potential persistence of L. monocytogenes in 19 European cheese-processing facilities. A sampling approach in 2007-2008 included, respectively, 11 and two industrial cheese producers in Austria and the Czech Republic, as well as six Irish on-farm cheese producers. From some of the producers, isolates were available from sampling before 2007. All isolates from both periods were included in a strain collection consisting of 226 L. monocytogenes isolates, which were then typed by serotyping and pulsed-field gel electrophoresis (PFGE). In addition, metabolic fingerprints from a subset of isolates were obtained by means of Fourier-transform infrared (FTIR) spectroscopy. PFGE typing showed that six processing environments were colonized with seven persistent PFGE types of L. monocytogenes. Multilocus sequence typing undertaken on representatives of the seven persisting PFGE types grouped them into distinct clades on the basis of country and origin; however, two persistent strains from an Austrian and an Irish food processor were shown to be clonal. It was concluded that despite the fact that elaborate Hazard Analysis and Critical Control Point concepts and cleaning programs are applied, persistent occurrence of L. monocytogenes can take place during cheese making. L. monocytogenes sanitation programs could be strengthened by including rapid analytical tools, such as FTIR, which allow prescreening of potentially persistent L. monocytogenes contaminants.

Microbes versus microbes: control of pathogens in the food chain.

Foodborne illness continues as a considerable threat to public health. Despite improved hygiene management systems and increased regulation, pathogenic bacteria still contaminate food, causing sporadic cases of illness and disease outbreaks worldwide. For many centuries, microbial antagonism has been used in food processing to improve food safety. An understanding of the mode of action of this microbial antagonism has been gained in recent years and potential applications in food and feed safety are now being explored. This review focuses on the potential opportunities presented, and the limitations, of using microbial antagonism as a biocontrol mechanism to reduce contamination along the food chain; including animal feed as its first link. © 2014 Society of Chemical Industry.

Strategy for the reduction of Trichloromethane residue levels in farm bulk milk.

High fat dairy products, such as butter and margarine can be contaminated during the milk production process with a residue called Trichloromethane (TCM), which results from the use of chlorine based detergent solutions. Although, TCM concentrations in Irish products are not at levels that are a public health issue, such contamination can cause marketing difficulties in countries to which Irish products are being exported. In an attempt to reduce such milk residues, a template procedure was developed, tried and tested on 43 farms (from 3 processing companies). This involved identifying farms with high TCM milk, applying corrective action in the form of advice and recommendations to reduce TCM and re-measuring milks from these farms. Trichloromethane in milk was measured by head-space gas chromatography with electron capture detector. The TCM reduction strategy proved successful in significantly reducing the levels in milk in the farms tested, e.g. TCM was reduced from 0.006 to the target of 0.002 mg/kg (P < 0.05). The strategy was then applied to farms who supplied milk to six Irish dairy processors with the objective of reducing TCM in those milks to a level of ≤ 0.002 mg/kg. Initially, milk tankers containing milks from approximately 10-15 individual farms were sampled and analysed and tankers with high TCM (>0.002 mg/kg) identified. Individual herd milks contributing to these tankers were subsequently sampled and analysed and farms supplying high TCM identified. Guidance and advice was provided to the high TCM milk suppliers and levels of TCM of these milk supplies were monitored subsequently. A significant reduction (minimum P < 0.05) in milk TCM was observed in 5 of the 6 dairy processor milks, while a numerical reduction in TCM was observed in the remaining processor milk.

Draft Genome Sequences of Bacillus licheniformis and Bacillus paralicheniformis Strains Isolated from Irish Skim Milk Powder.

Nineteen Bacillus licheniformis strains and four strains of the closely related species Bacillus paralicheniformis were isolated from a variety of Irish medium-heat skim milk powders. The draft genome sequences of these 23 isolates provide valuable genetic data for research work relevant to dairy products and process development. The isolates are available at Teagasc.

Physiological and transcriptional characterization of persistent and nonpersistent Listeria monocytogenes isolates.

This study aimed to characterize physiological differences between persistent and presumed nonpersistent Listeria monocytogenes strains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biolog's Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated "frequent persisters" because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons: pdu, cob-cbi, and eut. These genes may play a role in the persistence of L. monocytogenes outside the human host.

Molecular diversity of Listeria monocytogenes isolated from Irish dairy farms.

Many foods originate on the farm where cross-contamination with pathogens can occur, with implications for human health. This study characterized a bank of 51 Listeria monocytogenes isolates originating from 12 farms located in Ireland by pulsed-field gel electrophoresis (PFGE) to establish the molecular diversity of the isolate collection, and examine transmission patterns of L. monocytogenes across the farm environment, and also determined resistances against five different antibiotics (ampicillin, ciprofloxacin, erythromycin, penicillin G, and tetracycline). Analysis using a combination of AscI and ApaI digestion showed the 51 isolates comprised a total of 40 individual PFGE types, compared to individual restriction enzyme analysis, which was less discriminatory (36 types with ApaI analysis and 38 types with AscI analysis). Four of the PFGE types were common to multiple farms, and five farms had isolates with indistinguishable PFGE types in multiple locations on the farm. Indistinguishable PFGE types were common to multiple farms in different geographical locations up to ~200 km apart, and were found in a variety of different sample types, indicating multiple niches for the organism in the dairy farm environment. The presence of L. monocytogenes in samples related to animals other than cattle indicated that there are multiple possible vectors of contamination. The farm environment harbors a diverse collection of L. monocytogenes isolates that must be considered as possible agents of food contamination.

Heat adaptation and survival of Cronobacter spp. (formerly Enterobacter sakazakii).

Adaptation to sublethal stress can confer increased survival capability on foodborne pathogens. In this article, we report on the adaptive response of Cronobacter spp. to heat and compare the survival of heat adapted to unadapted Cronobacter spp. Five different isolates, representing at least three different Cronobacter spp., were adapted at 46°C for 30 min and subjected to a lethal stress at 52°C. All showed increased survival upon adaptation. Survival was greater in milk-grown cells, but broth-grown cells showed a higher degree of adaptation. The survival potential acquired following adaptation was not transferred to survival in a dry environment or to survival during reconstitution of artificially contaminated milk powder by conventional or microwave heat. The ratio of membrane unsaturated to saturated fatty acids decreased, possibly resulting in a more rigid membrane in adapted cells. Heat-adapted cells showed increased survival potential to lethal heat stress, but not to dry stress. Alterations in the ratio of fatty acids in the membrane may explain this adaptation.

Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Listeria monocytogenes.

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.

High-Throughput Characterization of Listeria monocytogenes Using the OmniLog Phenotypic Microarray.

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.

Effect of different cleaning procedures on water use and bacterial levels in weaner pig pens.

Pork is one of the most globally eaten meats and the pig production chain contributes significantly to the water footprint of livestock production. However, very little knowledge is available about the on-farm factors that influence freshwater use in the pig production chain. An experiment was conducted to quantify the effect of three different washing treatments on freshwater use, bacterial levels [(total bacterial counts; TBC), Enterobacteriaceae and Staphylococcus] and cleaning time in washing of pens for weaning pigs. Three weaner rooms were selected with each room having 10 pens and a capacity to hold up to 14 pigs each. Pigs were weaned and kept in the pens for 7 weeks. Finally, the pens were cleaned before the next batch of pigs moved in. The washing treatments used were power washing and disinfection (WASH); presoaking followed by power washing and disinfection (SOAK), and presoaking followed by detergent, power washing and disinfection (SOAK + DETER). A water meter was used to collect water use data and swab samples were taken to determine the bacterial levels. The results showed that there was no overall effect of washing treatments on water use. However, there was an effect of treatment on the washing time (p<0.01) with SOAK and SOAK+DETER reducing the washing time per pen by 2.3 minutes (14%) and 4.2 minutes (27%) compared to WASH. Nonetheless, there was an effect of sampling time (before or after washing) (p<0.001) on the levels of TBC and Staphylococcus, but no effect was seen on Enterobacteriaceae levels. Thus, the washing treatments used in this study had no effect on the water use of the pork production chain. Although there was no difference in both water use and bacterial load, from a producer perspective, presoaking and detergent use can save time and labour costs, so this would be the preferred option.

Effectiveness of current hygiene practices on minimization of Listeria monocytogenes in different mushroom production-related environments.

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 public health risk posed by Listeria monocytogenes in frozen fruit and vegetables including herbs, blanched during processing.

A multi-country outbreak of Listeria monocytogenes ST6 linked to blanched frozen vegetables (bfV) took place in the EU (2015-2018). Evidence of food-borne outbreaks shows that L. monocytogenes is the most relevant pathogen associated with bfV. The probability of illness per serving of uncooked bfV, for the elderly (65-74 years old) population, is up to 3,600 times greater than cooked bfV and very likely lower than any of the evaluated ready-to-eat food categories. The main factors affecting contamination and growth of L. monocytogenes in bfV during processing are the hygiene of the raw materials and process water; the hygienic conditions of the food processing environment (FPE); and the time/Temperature (t/T) combinations used for storage and processing (e.g. blanching, cooling). Relevant factors after processing are the intrinsic characteristics of the bfV, the t/T combinations used for thawing and storage and subsequent cooking conditions, unless eaten uncooked. Analysis of the possible control options suggests that application of a complete HACCP plan is either not possible or would not further enhance food safety. Instead, specific prerequisite programmes (PRP) and operational PRP activities should be applied such as cleaning and disinfection of the FPE, water control, t/T control and product information and consumer awareness. The occurrence of low levels of L. monocytogenes at the end of the production process (e.g. < 10 CFU/g) would be compatible with the limit of 100 CFU/g at the moment of consumption if any labelling recommendations are strictly followed (i.e. 24 h at 5°C). Under reasonably foreseeable conditions of use (i.e. 48 h at 12°C), L. monocytogenes levels need to be considerably lower (not detected in 25 g). Routine monitoring programmes for L. monocytogenes should be designed following a risk-based approach and regularly revised based on trend analysis, being FPE monitoring a key activity in the frozen vegetable industry.

Listeria monocytogenes in the Irish dairy farm environment.

Listeria monocytogenes is a potentially lethal foodborne pathogen commonly found in the environment. European Union hygiene legislation places responsibility for safety on primary production facilities, including farms, as part of a policy to introduce traceability throughout the food chain. This study aimed to determine the occurrence of L. monocytogenes in the Irish dairy farm environment and in particular the milking facility. Two hundred ninety-eight environmental samples were collected from 16 farms in the southern region of Ireland. A number of farms within the group supply raw milk to the unpasteurized milk cheese industry. The samples taken included cow feces, milk, silage, soil, water, etc. Samples were enriched in Listeria enrichment broth and incubated for 48 h, followed by plating on chromogenic agar Listeria Ottavani & Agosti and further incubation of the plates for 24 to 48 h. Presumptive L. monocytogenes isolates were purified and confirmed by PCR targeting the hly gene. Overall, 19% of the samples (57 of 298) were positive for L. monocytogenes. These were serotyped using conventional and PCR methods; serotypes 1/2a, 1/2b, and 4b made up 78% of the typeable isolates. A correlation was found between the level of hygiene standards on the farm and the occurrence of L. monocytogenes. There was little difference in the occurrence of L. monocytogenes between farms supplying milk to the unpasteurized milk cheese industry and those supplying milk for processing. This study demonstrates the prevalence of L. monocytogenes in the dairy farm environment and the need for good hygiene practices to prevent its entry into the food chain.

Occurrence of foodborne pathogens in Irish farmhouse cheese.

Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland. Three hundred and fifty one cheese samples, from 15 cheese producers, were analysed for microbiological quality on a monthly basis throughout the year. The analyses included enumeration of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (using the relevant agars) and enrichment for L. monocytogenes. The cheeses selected were produced from ovine, caprine and bovine milk. Both unpasteurised and pasteurised milk cheeses were sampled and these included hard, semi-hard and soft cheeses, internal/external mould-ripened and smear-ripened cheeses and the cheeses represented different geographic regions. Of the cheeses tested, 94% were free of L. monocytogenes, all were within the EU limits for E. coli and only one cheese variety had S. aureus levels above the recommended numbers for the first 6 months of the year. Due to a modified production process the numbers were within the guidelines for the second six months. The results indicate that Irish farmhouse cheeses are of a high microbiological quality.