The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

Detection and characterization of Listeria monocytogenes in Sao Jorge (Portugal) cheese production

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive disease in humans. Because human listeriosis cases have previously been linked to consumption of contaminated cheese, control of this pathogen throughout the cheese production chain is of particular concern. To understand the potential for L. monocytogenes transmission via São Jorge cheese, a Portuguese artisanal cheese variety that bears a Protected Denomination of Origin classification, 357 raw milk, curd, natural whey starter, and cheese samples representative of the production chain of this cheese were collected over one year and tested for the presence of L. monocytogenes and selected physicochemical parameters. Although neither L. monocytogenes nor other Listeria spp. were detected in whey, curd, or cheese samples, 2 of the 105 raw milk samples analyzed were positive for L. monocytogenes. These 2 raw milk isolates represented a ribotype that has previously been linked to multiple human listeriosis outbreaks and cases elsewhere, indicating the potential of these isolates to cause human listeriosis. On average, physicochemical parameters of São Jorge cheese ripened for 4 mo presented values that likely minimize the risk of L. monocytogenes outgrowth during ripening and storage (mean pH = 5.48; mean moisture = 37.79%; mean NaCl concentration = 4.73%). However, some cheese samples evaluated in this study were characterized by physicochemical parameters that may allow growth and survival of L. monocytogenes. Even though our results indicate that raw milk used for São Jorge cheese manufacture as well as finished products is rarely contaminated with L. monocytogenes, continued efforts to control the presence of this pathogen in the São Jorge cheese production chain are urged and are critical to ensure the safety of this product.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis,Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r² = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.     

Behaviour of Listeria monocytogenes in Lighvan cheese following artificial contamination during making,ripening and storage in different conditions

This study investigated the behaviour and fate of Listeria monocytogenes at different ripening temperatures and NaCl concentrations in traditional Lighvan cheese. L. monocytogenes was added to raw sheep's milk. After producing the cheese, they were stored in 8%, 12% and 15% NaCl at 4, 9 and 14 °C. Sampling was performed for 150 days. Different temperature and NaCl concentrations had a significant effect on the survival of L. monocytogenes (P < 0.001). The lowest growth and survival rates of L. monocytogenes were in 15% NaCl at 14 °C and 12% NaCl at 14 °C, respectively. Also, the highest growth and survival rates of the bacterium were in 8% NaCl at 4 °C.     

A survey of the microbiological quality of Sharri,a hard mountain cheese from Kosovo

The microbiological quality of a hard mountain unpasteurised sheep cheese from three randomly selected manufacturing locations in Kosovo was investigated. Forty‐eight samples of row milk, coagulum, 8–10 days ripening cheese and of ready to eat cheese (45‐days in brine) were tested. Seventy‐five per cent of raw milk samples failed to comply with EU regulation 853/2004. All of coagulum and ripened cheese failed to comply with EU regulation 2073/2005 on process hygiene criteria. Despite the high incidence of coagulase‐positive staphylococci even in the final product [>10⁵ colony‐forming units (cfu)/g], Staphylococcal enterotoxin was detected in none of the samples and no samples were positive for Listeria monocytogenes and Salmonella enteritidis.     

Foci of contamination of Listeria monocytogenes in different cheese processing plants

Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewe's milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L. monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI–ECIII) of L. monocytogenes was also investigated. Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewe's milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L. monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study.     

Microbiological quality of raw milk used for small-scale artisan cheese production in Vermont: Effect of farm characteristics and practices

This study 1) evaluated the overall milk quality and prevalence of 4 target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk used for small-scale artisan cheesemaking and 2) examined specific farm characteristics and practices and their effect on bacterial and somatic cell counts (SCC). Raw milk samples were collected weekly from 21 artisan cheese operations (6 organic) in the state of Vermont that manufactured raw-milk cheese from cow (12), goat (5), or sheep (4) milk during the summer of 2008. Individual samples were examined for standard plate counts (SPC), coliform counts (CC), and SCC. Samples were also screened for target pathogens both quantitatively and qualitatively by direct plating and PCR. Overall, 86% of samples had SPC <10,000 cfu/mL, with 42% <1,000 cfu/mL. Additionally, 68% of samples tested were within pasteurized milk standards for coliform bacteria under the United States’ Grade A Pasteurized Milk Ordinance at <10 cfu/mL. Log₁₀ SPC and CC did not differ significantly among species. Similarly, method of sample delivery (shipped or picked up), farm type (organic or conventional), and duration of milking (year-round or seasonal) did not have significant effects on farm aggregated mean log₁₀ SPC, CC, or SCC. Strong positive correlations were observed between herd size and mean log₁₀ SPC and between log₁₀ SPC and CC as well as SCC when data from all animal species were combined. Although SCC for cow milk were significantly lower than those for goat and sheep milk, 98, 71, and 92% of cow, sheep, and goat milk samples, respectively, were within the compliance limits of the United States’ Grade A Pasteurized Milk Ordinance for SCC. Fourteen of the 21 farms (67%) were positive for Staph. aureus, detected in 38% of samples at an average level of 20 cfu/mL. Neither L. monocytogenes, E. coli O157:H7, or Salmonella spp. were detected or recovered from any of the 101 samples tested. Our results indicate that the majority of raw milk produced for small-scale artisan cheesemaking was of high microbiological quality with no detectable target pathogens despite the repeat sampling of farms. These data will help to inform risk assessments that evaluate the microbiological safety of artisan and farmstead cheeses, particularly those manufactured from raw milk.     

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.     

Fate of Listeria monocytogenes in Gouda microcheese: No growth,and substantial inactivation after extended ripening times

This challenge study demonstrates that Listeria monocytogenes does not grow in Gouda cheese: during the first 8 weeks of ripening no growth was observed and between 8 and 52 weeks viable numbers declined significantly in a well-established Gouda microcheese system. Cheese milk was artificially contaminated just prior to addition of the starter culture. Three individual L. monocytogenes strains were used, including strains originating from cheese, a cheese plant environment and a reference strain. During curd formation, viable numbers of L. monocytogenes increased by 0.5 log cfu g⁻¹, resulting from entrapment in the curd. No growth was observed during the first 8 weeks of ripening. A significant decline in the viable numbers of L. monocytogenes was observed in Gouda cheese that was ripened for longer than 8 weeks. Two factors that could possibly control the fate of L. monocytogenes in Gouda cheese were lactic acid and water activity.     

Application of the SureTect Detection Methods for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria</Emphasis> spp. in Meat,Dairy, Fish,and Vegetable Products

The aim of this study was to evaluate the application of the Listeria monocytogenes and Listeria spp. SureTect detection methods for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, Spanish chorizo, pate, smoked salmon, raw sheep milk cured cheese, and ready-to-eat lettuce salad. The combination of a 24-h enrichment in the 24 Listeria Enrichment Broth (LEB) coupled to a rapid bacterial DNA extraction and real-time polymerase chain reaction (RTi-PCR) using the specific SureTect methods detected down to 2–6 L. monocytogenes CFU per sample in less than 27 h on the food categories tested. Furthermore, the applicability of L. monocytogenes and Listeria spp. SureTect detection methods in real samples was assessed using 303 food samples, obtaining at least the same analytical performance as the international reference method ISO 11290-1.     

A review of the microbiological hazards of dairy products made from raw milk

This review concentrates on information concerning microbiological hazards possibly present in raw milk dairy products, in particular cheese, butter, cream and buttermilk. The main microbiological hazards of raw milk cheeses (especially soft and fresh cheeses) are linked to Listeria monocytogenes, verocytotoxin-producing Escherichia coli (VTEC), Staphylococcus aureus, Salmonella and Campylobacter. L. monocytogenes, VTEC and S. aureus have been identified as microbiological hazards in raw milk butter and cream albeit to a lesser extent because of a reduced growth potential compared with cheese. In endemic areas, raw milk dairy products may also be contaminated with Brucella spp., Mycobacterium bovis and the tick-borne encephalitis virus (TBEV). Potential risks due to Coxiella burnetii and Mycobacterium avium subsp. paratuberculosis (MAP) are discussed. Pasteurisation ensures inactivation of vegetative pathogenic microorganisms, which increases the safety of products made thereof compared with dairy products made from raw milk. Several control measures from farm to fork are discussed.     

Incidence of Listeria in Egyptian meat and dairy samples

A total of 180 food samples including meat (raw lean beef, frozen lean beef, and frozen chicken) and dairy products (raw milk, Zabady and Kareesh cheese) were analysed for Listeria. Isolates were differentiated using morphological, cultural, and biochemical tests and an API-Listeria kit. Zabady cheese was completely free of Listeria. The highest incidence rate (13.33%) was in frozen lean beef. Raw lean beef and milk products showed an incidence rate of 6.67%. The lowest incidence rate (3.33%) was in Kareesh cheese and frozen chicken meat samples. L. monocytogenes showed the lowest incidence rate (0.55%), isolated from one frozen lean beef sample. L. ivanovii and L. grayi showed the highest incidence rate (2.22%), isolated from 4 samples. L. innocua and L. seeligeri were positive in 3 samples (1.67%), and L. welshimeri in 2 samples (1.11%). L. monocytogenes and L. ivanovii were positive for virulence factors (hemolytic properties, and extracellular enzyme activities).     

Survial of Listeria monocytogenes during the manufacture and ripening of Turkish White cheese

Listeria monocytogenes was enumerated during the manufacture and ripening of Turkish White cheese with particular reference to a) pasteurized milk, b) cheese milk after inoculation with L. monocytogenes (0 h( c) after curd formation (2 h( d) curd after pressing (6 h( e) curd after pH was reduced (17 h( f) curd after salting (32 h( and g) cheeses during ripening. Cheeses were also examined periodically for total solids, moisture and salt contents, pH values and aerobic plate count. An increase in the number ofL. monocytogenes was observed during manufacture. Following salting and throughout the storage period, numbers of L. monocytogenes decreased at a rate depending on the salt concentration, starter activity and storage time. The initial microbial number had a significant (P > 0.01) effect on the survival of L. monocytogenes during the storage period.     

Quantitative detection of Listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid- and solid-based culture-dependent real time PCR approaches

A culture-independent and two culture-dependent real time (rt-) PCR approaches were developed to quantitatively identify Listeria monocytogenes in raw milk and soft cheeses. The optimised rt-PCR revealed 100% inclusivity and exclusivity. DNA- and cell-based standard curves showed a good linearity of response (R² ≥ 0.987 and R² ≥ 0.998, respectively) for five orders of magnitude (39 × 10⁵ – 3 × 10⁰ genome equivalents and 10⁶–10¹ CFU equivalents, respectively) with about 100% relative accuracy and inter-assay variability ≤0.90%. Up to 1 genome equivalent/and 10 CFU/reaction were quantified in the DNA and cell standard curves, respectively. The rt-PCR was then combined with a liquid- (MPN technique) or a solid- (ALOA and PALCAM) based enumeration. The diagnostic sensitivity of the different approaches was investigated in artificially contaminated raw milk and soft cheeses. The rt-PCR culture-independent approach performed well in raw milk and (with a lower sensitivity) in stracchino cheese-based standard curves. MPN/rt-PCR was the best approach to enumerate low levels of L. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification was more effective than the PALCAM-based. These performances were confirmed when 23 real samples of raw milk and soft cheeses by both the rt-PCR approaches were assayed.     

Important vectors for Listeria monocytogenes transmission at farm dairies manufacturing fresh sheep and goat cheese from raw milk

The aim of this study was to determine the transmission routs of Listeria spp. in dairy farms manufacturing fresh cheese made from ovine and caprine raw milk and to evaluate the impact of Listeria monocytogenes mastitis on raw milk contamination. Overall, 5,799 samples, including 835 environmental samples, 230 milk and milk product samples, and 4,734 aseptic half-udder foremilk samples were collected from 53 dairy farms in the dairy intensive area of Lower Austria. Farms were selected for the study because raw milk was processed to cheese that was sold directly to consumers. A total of 153 samples were positive for Listeria spp., yielding an overall prevalence of 2.6%; L. monocytogenes was found in 0.9% of the samples. Bulk tank milk, cheese, and half-udder samples were negative for Listeria spp. Because none of the sheep and goats tested positive from udder samples, L. monocytogenes mastitis was excluded as a significant source of raw milk contamination. L. monocytogenes was detected at 30.2% of all inspected farms. Swab samples from working boots and fecal samples had a significantly higher overall prevalence (P < 0.001) of L. monocytogenes (15.7 and 13.0%, respectively) than did swab samples from the milk processing environment (7.9%). A significant correlation was found between the prevalence of L. monocytogenes in the animal and in the milk processing environment and the silage feeding practices. Isolation of L. monocytogenes was three to seven times more likely from farms where silage was fed to animals throughout the year than from farms where silage was not fed to the animals.     

Molecular surveillance of Toxoplasma gondii in raw milk and Artisan cheese of sheep,goat, cow and water buffalo origin

This study is aimed at investigating the molecular prevalence of Toxoplasma gondii in raw milk and cheese of different animal species (sheep, goat, cow and water buffalo) in Kayseri Province, Türkiye, to provide a preliminary assessment for contamination risk. A total of 200 milk and cheese samples were analysed by real-time PCR. Toxoplasma gondii DNA was detected in two (8%) ewes and one (4%) goat raw milk sample, while none of the cheese samples were positive. These results indicated that the presence of T. gondii DNA in raw milk samples sold in Kayseri Province might be a risk factor for public health.     

Incidence of Listeria monocytogenes in different food products commercialized in Portugal

Several types of food products on sale in Portugal, were examined for the presence of Listeria monocytogenes. Secondary enrichments, in Fraser broth, were analysed by the mini-Vidas LMO, enzyme-linked fluorescent immunoassay technique. Positive samples were confirmed by isolation on Oxford and PALCAM selective agars followed by biochemical characterization. Of 1035 samples, 72 (7.0%) were positive for L. monocytogenes, the majority being from raw products (milk, meat, fish, flour) although some heat-processed or fermented foods (ready-to-eat) were also positive. In Portugal, a predilection for fresh cheese was indicated as a potential risk for consumers.     

Influence of temperature variations on growth,injury survival and inactivation of Listeria monocytogenes in goat milk samples at laboratory scale

Transmission of the thermo‐tolerant pathogen Listeria monocytogenes via contaminated milk and its products, can lead to serious food‐borne illness. In this study, the effects of selected temperatures on survival, percentage injury and inactivation of L. monocytogenes in goat milk samples collected from two different farms were evaluated. Low temperature ranges (0, 5, 10 °C) had a bacteriostatic effect; while at temperatures of 25 and 45 °C, this pathogen grew luxuriantly. However, growth was comparatively slow at 15 °C throughout a 12‐h stress period. Furthermore, a high temperature range (50, 55, 60 and 65 °C) resulted in the elimination of this pathogen within 4 h of stress. Results of Scanning Electron Microscopy showed morphological changes in the cells upon induction of stress temperatures.