Nisin treatments to control Listeria monocytogenes post-processing contamination on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages

Post-processing contamination and growth of Listeria monocytogenes in whey cheeses stored under refrigeration is an important safety concern. This study evaluated commercially available nisin (Nisaplin^®) as a biopreservative to control L. monocytogenes introduced post-processing on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages for up to 45 days. The whey used (pH 6.5–6.7) was from Feta cheese manufacture, and it was subjected either to natural acidification (pH 5.3, readjusted to 6.2 with 10% NaOH) prior to heating, or to direct acidification (pH 6.0–6.2) at 80°C with 10% citric acid. Nisin was added either to the whey (100 or 500 IU g⁻¹) prior to heating, or to the cheese (500 IU g⁻¹) prior to packaging, also inoculated with ca. 10⁴ cfu g⁻¹ of L. monocytogenes strain Scott A. In cheese samples without nisin, L. monocytogenes (PALCAM agar) exceeded 7 log cfu g⁻¹ after the first 10 days of storage, irrespective of the whey acidification method. All nisin treatments had an immediate lethal effect (0.7–2.2 log reduction) on L. monocytogenes populations at inoculation (day 0), which was more pronounced with 500 IU g⁻¹ added to the whey. This treatment also suppressed L. monocytogenes growth below the inoculation level for 30 and 45 days in naturally and directly acidified samples, respectively. All other treatments had weak antilisterial effects. Nisin reversed the natural spoilage flora of Anthotyros cheese from Gram-positive to Gram-negative, and this ecological alteration was far more pronounced in the most effective antilisterial treatments.     

Incidence of Listeria ssp. in Retail Meat Roasts

Interior muscle cores from 50 beef, 50 pork, and 10 lamb roasts, purchased at retail, were assayed for Listeria ssp., which was isolated from three beef roasts and three pork roasts, but not from lamb roasts. Five isolates of serotype la Listeria monocytogenes were obtained from the roasts, together with one strain each of Listeria innocua and Listeria welshimeri. Twice, L. monocytogenes was present at 10 CFU/ g; all other isolations of Listeria spp. required enrichment (<10 CFU/ g). The presence of listeriae in cores of whole-muscle meat roasts is probably not a result of environmental contamination, and may be due to antemortem exposure of the animal to Listeria.     

Development of a rapid, simple paddle-style dipstick dye immunoassay specific for Listeria monocytogenes

We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×10⁷ CFU ml⁻¹ for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0 CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.     

Development of a membrane filtration method for enumeration of Listeria monocytogenes from soft cheese

A new and simple analytical method has been developed to quantify low levels (≤50 cfu g⁻¹) of Listeria monocytogenes in soft cheese. The technique allows the analysis of 1 g of cheese instead of 0·1 g or 0·01 g using the ISO 11290-2 standard method. The analysis protocol combines filtration of the decimally diluted cheese suspension through a 0·45-μm pore size cellulose ester membrane, and a culture of the filter on a Palcam agar. A tween 80–trypsin treatment used to increase filterability of cheese did not reduce L. monocytogenes counts. The tested method provided more precise results (nearer to the true value) compared to the ISO 11290-2 standard method in the enumeration of L. monocytogenes from artificially contaminated cheese. However, it improves neither repeatability nor reproducibility since the selected medium, Palcam, does not allow distinction between the different Listeria species.     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

A study of the occurrence of Listeria species in raw sheep milk

The occurrence of bacteria from the genus Listeria in raw sheep milk and traditional local cheese was studied in three regions of the Karak district of Jordan. Conventional plating methods for the detection of Listeria species were followed to determine the average and the percentage of the contaminated samples. The result shows that there were significant differences between the regions in the study concerning the average and the percentage of positive occurrences of Listeria species in raw sheep milk. The results also showed that mainly L. monocytogenes and, to a lesser degree, L. ivanovii and L. innocua were found in the milk samples, while the occurrence of L. monocytogenes in cheese samples was very low.     

Effect of Filling Type and Heating Method on Prevalence of Listeria species and Listeria monocytogenes in Dumplings Produced in Poland

The count of Listeria monocytogenes was determined, before and after heat treatment, in 200 samples of dumplings of 9 brands and with different types of stuffing. Analyses were conducted according to ISO 11290–1 standard and with real‐time PCR method. The highest count of L. monocytogenes was found in meat dumplings (10² to 10⁴ CFU/g), whereas products with white cheese‐potato stuffing and vegetable‐mushroom stuffing contained significantly less Listeria, 20 to 80 and 5 to 32 CFU/g, respectively. In cooled meat dumplings the extent of contamination depended significantly on the producer. In addition, a significant (P < 0.05) correlation was determined between contamination level and meat content in the stuffing (rho = 0.418), especially in stuffing containing pork meat (0.464), contrary to beef‐containing stuffing (0.284). Heating dumplings in boiling water for 2 min completely eliminated L. monocytogenes in meat dumplings. In contrast, the microwave heating applied for 2 min at 600 W only reduced the count of L. monocytogenes by 1 to 2 logs. Hence, the microwave heating failed to reduce the risk of infection with this pathogen below the level permissible in the EU regulation, especially in the most contaminated samples. In this case, the efficacy of microwave heating was significantly (P < 0.05) affected by the initial count of L. monocytogenes (rho = 0.626), then by meat content in the stuffing (0.476), and to the lowest extent—by the type of meat (0.415 to 0.425). However, no Listeria sp. and L. monocytogenes were isolated from cooked dumplings with fruits (strawberries or blueberries).     

The probiotic,Leuconostoc mesenteroides,inhibits Listeria monocytogenes biofilm formation

Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the present study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 × 10⁶ colony forming units (CFU)/cm² L. monocytogenes ATCC19115 in single-species biofilms to 1.1 × 10⁵ CFU/cm² in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (>30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.     

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.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

High prevalence,low counts and uncommon serotypes of Listeria monocytogenes in linguiça, a Brazilian fresh pork sausage

Linguiça is a highly popular and appreciated pork product in Brazil, frequently consumed undercooked. Aiming at collection of data for a future risk assessment, this study evaluated the prevalence and counts of Listeria monocytogenes in linguiça samples collected at retail level in Sao Paulo, SP, Brazil. ISO methods were used for detection and enumeration of the pathogen (11290-1 and 11290-2, respectively). Isolates were submitted to Simplex-PCR for hlyA gene and those with biochemical features of L. monocytogenes and hlyA positive were serotyped using a Multiplex PCR. Ninety percent of the samples were positive for Listeria spp., and L. monocytogenes was detected in 42% of the samples, with counts below 10² CFU/g in all samples. A prevalence of uncommon serotypes 4a and 4c was observed.     

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).     

Evaluation of hygiene practices and microbiological quality of cooked meat products during slicing and handling at retail

Cooked meat ready-to-eat products are recognized to be contaminated during slicing which, in the last years, has been associated with several outbreaks. This work aimed to find out possible relation between the hygiene practice taking place at retail point during slicing of cooked meat products in small and medium-sized establishments (SMEs) and large-sized establishments (LEs) and the microbiological quality of sliced cooked meat products. For that, a checklist was drawn up and filled in based on scoring handling practice during slicing in different establishments in Cordoba (Southern Spain). In addition, sliced cooked meats were analyzed for different microbiological indicators and investigated for the presence of Listeria spp. and Listeria monocytogenes. Results indicated that SMEs showed a more deficient handling practices compared to LEs. In spite of these differences, microbiological counts indicated similar microbiological quality in cooked meat samples for both types of establishments. On the other hand, Listeria monocytogenes and Listeria inocua were isolated from 7.35% (5/68) and 8.82% (6/68) of analyzed samples, respectively. Positive samples for Listeria spp. were found in establishments which showed acceptable hygiene levels, though contamination could be associated to the lack of exclusiveness of slicers at retail points. Moreover, Listeria spp presence could not be statistically linked to any microbiological parameters; however, it was observed that seasonality influenced significantly (P < 0.05) L. monocytogenes presence, being all samples found during warm season (5/5). As a conclusion, results suggested that more effort should be made to adequately educate handlers in food hygiene practices, focused specially on SMEs.     

Evaluation of lactic acid and monolaurin to controlListeria monocytogeneson Stracchino cheese

The behaviour ofListeria monocytogeneswas evaluated during storage of Italian Stracchino cheese dipped in lactic acid (1.4%) or surface treated with (1)-monolauroyl-(rac)-glycerol (monolaurin, 200 μg cm⁻²). The cheese was surface inoculated with approximately 5×10²cfu cm⁻²ofL. monocytogenes, and stored under vacuum at 5°C for 12 days. The lactic acid treatment resulted in lower counts (P<0.05) ofL. monocytogenescompared with counts on untreated cheese washed with water. When lactic-treated cheese was stored at 5°C, levels ofL. monocytogenesdid not change appreciably. Treating cheese with monolaurin also significantly reduced the number ofL. monocytogenes. Furthermore, 12 day counts were less than the untreated control.     

Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese

The antilisteric activity of clove oil was examined in meat and cheese at both 30°C and 7°C. At concentrations of 0·5% and 1% clove oil restricted the growth of Listeria monocytogenes in the food items at both temperatures. The inhibitory activity of clove oil was more pronounced at a concentration of 1%.Listeria counts in treated samples were 1–3 log₁₀cfu g⁻¹less compared to controls at different intervals during storage. The results revealed the potential of clove oil as a natural preservative in meat and cheese.     

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR^® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.     

Behaviour of Listeria monocytogenes in the presence of Listeria innocua during storage of minced beef under vacuum or in air at 0°C and 10°C

Minced beef was inoculated with low levels (1·2–1·7 log₁₀cfu g⁻¹) of Listeria monocytogenes or Listeria innocua, or a combination of the two strains. Inoculated samples were stored at 0 or 10°C under two packaging atmospheres (aerobic and vacuum) for up to 28 days and surviving organisms recovered on Palcam Agar. The only significant increases in numbers of Listeria spp. occurred in samples held at 10°C under aerobic conditions. In vacuum packs, growth of both strains was inhibited. Under aerobic conditions meat pH increased from an initial value of pH 5·85 to c. 8·85 within 28 days. The pH of vacuum packaged meat declined to c. 4·95 during the same period. These differences in pH may be related to differences in the nature and effects of different background microflora that were observed to develop under each of these packaging conditions.Pseudomonas spp. predominated in aerobically stored beef, whereas in vacuum packed beef lactic acid bacteria predominated. No significant differences were observed between the growth rates of Listeria spp. inoculated into beef mince in pure and mixed culture. This suggests that the more frequent prevalence of Listeria innocua than Listeria monocytogenes in meat and meat products is not due to overgrowth or inhibition of the pathogen (Listeria monocytogenes) by the non-pathogen(Listeria innocua) during low-temperature storage.     

Inactivation of Listeria monocytogenes by Ultraviolet Energy

Short-wave UV-energy (100 μW/cm²) decreased the number of Listeria monocytogenes on Tryptose Agar (TA) ca. seven orders of magnitude in 4 min. Age of culture (48 vs 24 hr) did not alter the sensitivity of Listeria to this UV treatment. Increasing the intensity of irradiation (550 vs 100 μW/cm²) increased the rate at which L. monocytogenes was inactivated. Dry rather than moist cells of L. monocytogenes were most resistant to irradiation. In general, inactivation of Listeria with short-wave UV-energy yielded sigmoidal survivor curves. The population of Listeria on TA plates was not affected with long-wave UV-irradiation.     

Microbial analysis of commercially available US Queso Fresco

Queso Fresco (QF), a fresh Hispanic-style cheese, is often associated with Listeria monocytogenes outbreaks and recalls. Queso Fresco's susceptibility to bacterial contamination is partially due to its high pH and moisture content as well as Listeria's tolerance for the salt content typical for QF. Nine different brands of US QF, 2 packages from 4 different lots (to account for temporal variability), were sampled. The pH, salt content, and moisture content were analyzed in addition to microbial testing including yeasts and molds, coliforms, lactic acid bacteria enumeration, and L. monocytogenes counts. The cheeses were also inoculated with a cocktail of 5 food and human isolates of food-borne outbreak-associated Listeria monocytogenes strains to evaluate how the differences between brands influenced Listeria growth. Three of the cheeses underwent additional genus-level microbial analysis using extracted 16S rDNA, allowing for phylogenetic analysis between bacterial taxa including diversity and relative abundance. We found little variation between the sampled QF pH (range = 6.62–6.86), salt content (1.53–2.01%), and moisture content (43.90–54.50%). Yeasts and molds were below the detection limit of enumeration in all of the cheeses and coliforms were below the detection limit across the first 3 lots, but were detected at varying levels in the fourth lot (>3.0 most probable number/g) for 3 of the brands. Listeria monocytogenes was not isolated after enrichment in any of the samples. All cheeses tested positive for the presence of lactic acid bacteria, with only 1 of the cheeses being labeled as produced with added cultures having substantial counts. Fourteen days after inoculation with L. monocytogenes, at least 2.5 log10 cfu/g of growth was found for all QF brands stored at 4°C. Microbial genus analysis showed that, among the 3 brands, the microbial community was more similar within brand than when compared with the other 2 brands. Thermus, Anoxybacillus, and Streptococcus accounted for the dominant genera of brands A, B, and C, respectively. These variations within the microbial community may account for sensory differences and help manufacturers determine quality control consistency more readily than culture-based methods.