The occurrence,growth, and biocontrol of Listeria monocytogenes in fresh and surface-ripened soft and semisoft cheeses

Listeria monocytogenes continues to pose a food safety risk in ready-to-eat foods, including fresh and soft/semisoft cheeses. Despite L. monocytogenes being detected regularly along the cheese production continuum, variations in cheese style and intrinsic/extrinsic factors throughout the production process (e.g., pH, water activity, and temperature) affect the potential for L. monocytogenes survival and growth. As novel preservation strategies against the growth of L. monocytogenes in susceptible cheeses, researchers have investigated the use of various biocontrol strategies, including bacteriocins and bacteriocin-producing cultures, bacteriophages, and competition with native microbiota. Bacteriocins produced by lactic acid bacteria (LAB) are of particular interest to the dairy industry since they are often effective against Gram-positive organisms such as L. monocytogenes, and because many LAB are granted Generally Regarded as Safe (GRAS) status by global food safety authorities. Similarly, bacteriophages are also considered a safe form of biocontrol since they have high specificity for their target bacterium. Both bacteriocins and bacteriophages have shown success in reducing L. monocytogenes populations in cheeses in the short term, but regrowth of surviving cells can commonly occur in the finished cheeses. Competition with native microbiota, not mediated by bacteriocin production, has also shown potential to inhibit the growth of L. monocytogenes in cheeses, but the mechanisms are still unclear. Here, we have reviewed the current knowledge on the growth of L. monocytogenes in fresh and surface-ripened soft and semisoft cheeses, as well as the various methods used for biocontrol of this common foodborne pathogen.     

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.     

A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheese

A cold enrichment method and a modified FDA procedure were compared for the isolation of Listeria monocytogenes from raw chickens and soft cheese. L. monocytogenes was isolated from a total of 23 of 222 cheese and 70 of 160 chicken samples by either one or both methods. Neither method alone yielded all isolates from the two food types. Only 12 cheese and 13 chicken samples were shown to be positive by both methods, although the serotypes isolated were not always identical. On some occasions one method yielded L. monocytogenes while the other produced a different Listeria sp. Reasons for differences in the performance of the two procedures and various points of technical interest are discussed.     

Listeria species in raw and ready-to-eat foods from restaurants

From September 1999 to March 2000, meat (pork, beef, and chicken), fish (salmon, hake, and sole), vegetable (lettuce and spinach), and Spanish potato omelette samples obtained at restaurants were collected and tested for the occurrence of Listeria spp. Listeria monocytogenes was isolated from 3 (2.9%) out of 103 studied samples. Other species isolated were Listeria grayi (13.6%), Listeria innocua (1.9%), Listeria ivanovii (5.8%), Listeria seeligeri (3.9%), and Listeria welshimeri (1.9%). Listeria was neither isolated from beef nor any type of fish.     

Effects of salts of organic acids on the survival of Listeria monocytogenes in soft cheeses

The purpose of this study was to determine the effect of sodium lactate and sodium propionate, both in combination with sodium acetate, on strains of Listeria monocytogenes in artificially inoculated soft cheeses. Minas Frescal and Coalho cheeses, inoculated with a mix of L. monocytogenes 1/2a and Scott A, underwent two treatments: 2% (w/v) sodium lactate in combination with 0.25% (w/v) sodium acetate and 2% (w/v) sodium propionate in combination with 0.25% (w/v) sodium acetate. The samples were analysed immediately and after 7 days at 10 °C. The growth of the pathogen was inhibited in cheeses containing the salts of organic acids, and the effects of treatment were statistically significant (P < 0.05). However, there was no difference between the types of treatment applied. Our data demonstrate that the effectiveness of the salts of organic acids depended on the initial concentration of L. monocytogenes and that a higher concentration of the salts is necessary to ensure sustained inactivation of target pathogens because they are weakly antilisterial when the soft cheeses are stored at 10 °C.     

Elimination of Listeria monocytogenes in soft and red smear cheeses by irradiation with low energy electrons

Soft and red smear cheeses are frequently contaminated by Listeria monocytogenes , sometimes at relatively high concentration (< 10⁵ CFUg^-1). This bacterium is radiosensitive (D₁₀ value of approximately 0.45 kGy) but irradiation of the whole cheese by X-rays induced off-flavours when the dose exceeded 1.0 kGy. Irradiation could be effective in eliminating L. monocytogenes only from lightly contaminated cheeses (> 10²CFU g^-1).
L. monocytogenes appears only in the rind (where the pH is greater than 6.3) and never grows in the core of the cheese. Under these conditions, a specific irradiation of the rind after ripening, with a low-energy electron beam at relatively high doses (up to 3.0 kGy), allows the total elimination of L. monocytogenes in heavily contaminated samples (10⁵-10⁶ CFU g^-1) without noticeable modifications of the organoleptic properties of the cheese.     

Control of Listeria monocytogenes in raw-milk cheeses

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.     

Occurrence and ribotypes of Listeria monocytogenes in Gorgonzola cheeses

In this study 1656 Gorgonzola cheeses, collected from October 2003 to April 2004 in the same industrial plant located in Lombardia (Italy), were analysed in order to evaluate their level of contamination with Listeria monocytogenes after packaging, as well as at the end of the shelf life. A subset of Gorgonzola isolates was submitted to automated EcoRI ribotyping to evaluate their DUP-IDs (DuPont identification library code) and their level of genetic diversity. The isolate ribotyping profiles were included in an on-line database named PathogenTracker database. The strain DUP-IDs and the similarities between Gorgonzola isolates and PathogenTracker human sporadic strains allowed to evaluate the potential virulence of Gorgonzola-associated strains. The L. monocytogenes detection rates observed in the cheese samples monitored after packaging and at the end of the shelf life were 2.1% and 4.8%, respectively. Seventy percent of the strains genotyped were classified in the same ribotype, labelled as 204 S5, indicating that L. monocytogenes population associated to Gorgonzola cheese shows a low level of genetic diversity. Ninety percent of the strains were classified in DUP-IDs belonging to the II pathogenicity lineage identified for L. monocytogenes. That lineage includes serotype 1/2a, 1/2c and 3c strains, associated to the 35% of the human sporadic isolates described in the literature as causing listeriosis. Moreover, 16.7% of Gorgonzola isolates showed a similarity >or=99% with PathogenTracker human sporadic strains. The results of this study showed that the incidence of L. monocytogenes in Gorgonzola cheeses commercialised by the plant tested was lower than that observed in other Italian blue-veined cheeses by other authors. However, it increased during cheese storage and it become double at the end of the cheese shelf life, ranging from 30 to 60 days after packaging.     

Occurrence of Listeria species in raw milk in farms on the outskirts of Mexico city

Listerosis may be transmitted by direct contact with infected animals or by consumption of contaminated vegetables or meat and milk products. In Mexico, raw milk is widely consumed and the incidence of milkborne disease is unknown. A total of 1300 raw milk samples were obtained from 20 l bulk tanks at four different dairy farms in southeast of Mexico City from June 1998 to June 1999. The samples were enriched for 48 h at 30°C and plated onto McBride's Modified Agar (MMA). Suspect colonies were biochemically tested to confirm identity. Overall, 23% of all raw milk samples examined tested positive forListeria species; 13% were positive for L. monocytogenes (45·6% were serotype-4b and 54·4% were serotype 1); 6% for L. ivanovii; 4% for L. seeligeri and 1% forL. innocua. L. monocytogenes contamination was more frequent during the spring and summer months as isolation rates were 12·2% from June to October 1998 and 17% from March to June 1999. Serotype-4b isolates were not pathogenic for the mouse, while for serotype-1, strains DL₅₀ranged from 1·8×10⁶to 4×10⁷CFU ml⁻¹. Additional studies are needed to assess the public health impact of contaminated milk in Mexico.     

60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripened soft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared the growth potential of Listeria monocytogenes on cheeses manufactured from raw or pasteurized milk and held for > 60 days at 4 degrees C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate the holding period. Wheels were surface inoculated with a five-strain cocktail of L. monocytogenes at approximately 0.2 CFU/ cm2 (low level) or 2 CFU/cm2 (high level), ripened, wrapped, and held at 4 degrees C. Listeria populations began to increase by day 28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurized milk cheese reached maximums of 2.96 +/- 2.79 and 2.33 +/- 2.10 log CFU/g, respectively, after 60 days of holding. Similar growth was observed in cheese inoculated at high levels, where populations reached 4.55 +/- 4.33 and 5.29 +/- 5.11 log CFU/g for raw and pasteurized milk cheeses, respectively. No significant differences (P < 0.05) were observed in pH development, growth rate, or population levels between cheeses made from the different milk types. Independent of the milk type, cheeses held for 60 days supported growth from very low initial levels of L. monocytogenes introduced as a postprocess contaminant. The safety of cheeses of this type must be achieved through control strategies other than aging, and thus revision of current federal regulations is warranted.     

A survey of the occurrence of agaritine in U.K. cultivated mushrooms and processed mushroom products

The naturally occurring compound agaritine (beta-N-[gamma-L(+)glutamyl]-4-hydroxy-methylphenyl-hydrazine) has been determined in fresh, dried and processed mushrooms. A method was developed involving extraction of the toxin into methanol, clean-up where appropriate (for processed products) by high-performance size exclusion liquid chromatography and determination by reverse-phase HPLC with electrochemical detection. Diode array UV monitoring was used for confirmation. The method had a recovery of 90-98%, a relative standard deviation of 3-5% and a limit of detection of agaritine of 5 mg/kg on a dry-weight basis. Fresh cultivated mushrooms showed agaritine levels of 100-250 mg/kg and 80-190 mg/kg for two different commercial strains. There were slight differences in levels of agaritine between mushrooms of different sizes, and between those of the same size but harvested at different times (different breaks). Retail processed mushrooms products had low agaritine levels in the range 6-33 mg/kg, with the exception of one dried sliced mushroom product found to contain 6520 mg/kg.     

The occurrence of Listeria species in milk and dairy products: a national survey in England and Wales

A total of 4172 samples of milk, cheese and other dairy products were examined over a 1-year period for the presence of Listeria species. Strains of Listeria were found most frequently in soft, ripened cows milk cheese; 63 out of 769 (8.2%) samples contained Listeria monocytogenes, 25 samples contained species other than L. monocytogenes, and 18 samples contained both L. monocytogenes and other Listeria spp. Eleven samples of pasteurized cows milk (1.1%) from four dairies contained L. monocytogenes, and other Listeria spp. were isolated from a further five samples. Goats and ewes milk and their products, yogurt, cream and ice cream also occasionally contained Listeria spp. Levels of Listeria were usually low, but 20 samples of cheese contained more than 1000 cfu/g. Most strains of L. monocytogenes belonged to serotype 1/2 (58%) or serotype 4b (33%).     

The effect of raw milk microbial flora on the sensory characteristics of Salers-type cheeses

The sensory characteristics of Salers Protected Denomination of Origin raw-milk cheeses are linked to the biochemical composition of the raw material (milk) and to the resultant microbial community. To evaluate the influence of the microbial community on sensory characteristics, Salers-type cheeses were manufactured with the same pasteurized milk, reinoculated with 3 different microbial communities from 3 different filtrates from microfiltered milks. Each cheese was subjected to microbial counts (on selective media), biochemical tests, and volatile and sensory component analyses at different times of ripening. Adding different microbial communities to specimens of the same (biochemically identical) pasteurized milk lead to different sensory characteristics of the cheeses. Cheeses with fresh cream, hazelnut, and caramel attributes were opposed to those with fermented cream, chemical, and garlic flavors. The aromatic compounds identified (esters, acids, alcohols, and aldehydes) in these cheeses were quite similar. Nevertheless, one milk was distinguished by a higher content of acetoin, and lower 2-butanone and 3-methylpentanone concentrations. Over the production period of 1 mo, the different cheeses were characterized by the same balance of the microbial population assessed by microbial counts on different media. This was associated with the stability of some sensory attributes describing these cheeses. Nevertheless, there was no linear correlation between microbial flora data and sensory characteristics as measured in this study.     

Persistence at source of Listeria spp. in raw milk

From 36 of 315 bulk tank sources of raw milk found to harbour Listeria spp., 34 were available for resampling at intervals to determine persistence of the organisms. Listeriae were reisolated from 21 sources. In 16 Listeria spp. were isolated in one retest. From the other five listeriae were obtained in more than one retest. Listerial populations were not particularly persistent. In all but one instance listeriae were not reisolated more than 5 months after initial sampling. Intermittent variations in Listeria spp. isolated were observed. Some repeat samples yielded the same species as originally identified, but sometimes only one of originally two species was isolated. On occasions completely different or additional species were found. The aetiology of listeriosis in cattle and contamination of raw milk is discussed.     

A case of sporadic ovine mastitis caused by Listeria monocytogenes and its effect on contamination of raw milk and raw-milk cheeses produced in the on-farm dairy

We describe a case of listerial mastitis in a flock of 130 sheep. The animals were housed at a farm where the bulk raw ewe milk was processed to produce raw milk soft cheese. List. monocytogenes was shed from the right mammary complex. Shedding was observed over a period of 99 d. A mean level of 4-56 x 10(4) cfu (colony forming units) Listeria monocytogenes/ml was recovered from the raw milk originating from the infected udder. The numbers ranged from 9 x10(1) to 2.95 x 10(5). The bulk milk was contaminated by approx. 5.7 x 10(3) cfu/ml. In the cheese product, 2.0 x 10(2) cfu List. monocytogenes/g were constantly detectable for a period of 7 d post manufacture. The starter culture used for coagulation had a pivotal influence on the behaviour of List. monocytogenes during cheesemaking. Using the same mesophilic buttermilk culture as used by the farmer allowed numbers of Listeria to increase 60-fold within 12 h owing to a delayed acidification of the bulk milk. Addition of a thermophilic yogurt culture reduced the numbers of Listeria within 8 h of incubation.     

Comparison of a cold enrichment and the FDA method for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K

A cold enrichment and the modified FDA selective enrichment method were compared for their ability to detect Listeria monocytogenes and other Listeria species from various ready-to-eat foods on sale in the UK. Of 57 food samples examined using cold enrichment, five yielded L. monocytogenes, and two L. innocua. The FDA enrichment method yielded three samples positive for L. monocytogenes only. Foods examined included soft cheeses, fermented meat sausages, pates and salads.     

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.     

The incidence of Listeria species in retail foods in Japan.

Meat, fish and vegetable products obtained at retail shops in or around Tokyo were examined for Listeria contamination. Listeria spp. were isolated from 43 (56.6%) out of 76 samples of meat products. L. monocytogenes occurred in 26 (34%) of the samples, L. monocytogenes was isolated from 7 (6.1%) out of 114 samples of fish and fish products including 'ready-to-eat' foods. Listeria was not isolated from any of 21 samples of vegetable and vegetable product including 'ready-to-eat' foods investigated.     

Microbiota dynamics and lactic acid bacteria biodiversity in raw goat milk cheeses

Raw goat milk collected at 11 Andalusian dairy plants was used to individually manufacture cheeses without starter culture addition. Microbial groups were monitored during ripening for 60 days. A total of 1442 lactic acid bacteria (LAB) isolates randomly chosen were identified by molecular methods. Counts of mesophilic LAB exceeded 9 log cfu g⁻¹ on day 1 and decreased 0.5 log units during ripening whereas counts of lactobacilli and leuconostocs increased and those of enterococci remained constant. Thirty-three LAB species belonging to eight different genera were isolated, with higher biodiversity on day 1 (26 species) than on day 60 (20 species). Structural genes coding for nisin, lacticin 481, lactococcin B, six plantaricins and five enterocins were detected in LAB isolates. The information obtained on the technological properties of selected isolates may be useful in developing specific starters for goat milk cheeses, capable of maintaining their distinctive sensory characteristics.