Assessing in-house monitoring efficiency by tracing contamination rates in cheese lots recalled during an outbreak of listeriosis in Austria

A cluster of 34 cases of listeriosis was traced to consumption of quargel cheese, a sour milk specialty, in Austria, Germany and Czech Republic between 2009 and 2010. After recall from the retail market all soft cheese batches (n = 18) were sent for investigation and ISO 11290 based microbiological analysis revealed all red smear-ripened batches (16/18) to be positive for Listeria monocytogenes whereas mold ripened cheeses were negative. The 16 positive batches were grouped into three categories: those having exceeded shelf-life (G1), those around shelf-life (± 4 days, G2) and those within shelf-life (G3). Tracing the contamination levels as measured after recall (CL_R) to the theoretical contamination level after processing (CL₀) was considered to provide an estimate as to whether the in-house monitoring system would have been capable of unraveling the contamination scenario. Growth simulations starting from various hypothetical initial contamination levels of cheese at the plant and considering the potential variability in growth of L. monocytogenes due to model parameters and storage conditions suggested that a very low initial contamination level (e.g., < 1 CFU/g or 5 CFU/100 g) could justify the levels of L. monocytogenes enumerated in recalled samples of G1 and G2 lots. This in turn, may have resulted in low detection probability using ISO 11290:1996. In lots of G3 group, however, high initial contamination levels or temperature abuse at retail are inferred, based on simulated outputs.     

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.     

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.     

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.     

Short communication: Aflatoxin M1 in dairy products sold in Şanlıurfa,Turkey

The aim of this study was to detect the presence of aflatoxin M₁ (AFM1) in samples of raw milk (n = 38), UHT milk (n = 12), white pickled cheese (n = 50), and yogurt (n = 50) collected from the ?anl?urfa city markets and locally produced dairy products by ELISA. The mean contamination rates were 56.74 ± 40.32, 43.1 ± 23.19, 103.2 ± 29.13, and 55.28 ± 12.68 ng/kg, respectively, for raw milk, UHT milk, white pickled cheese, and yogurt. According to the data, 21 (55%) raw milk, 3 (25%) UHT milk, 10 (20%) white pickled cheese, and 10 (20%) yogurt samples were contaminated with AFM1 over the acceptable levels (≥50 ng/kg), ranging from 0.82 to 130.89 ng/kg. None of the white pickled cheese samples contained AFM1 levels above the Turkish legal limit (250 ng/kg). Consequently, the AFM1 contamination levels determined in this study in white pickled cheese were not considered to pose a serious public health hazard. However, the AFM1 levels in raw and UHT milk and yogurt samples indicate an increased human health risk in Turkey related to high aflatoxin levels. Therefore, milk and dairy products have to be monitored by the Turkish public health authorities continuously to detect AFM1 contamination.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzer-land represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows’, goats’, and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx₁ and stx₂ variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx₂, 6 strains for stx_2d2, 2 strains for stx_2-O118, 1 strain for stx_2-06, 1 strain for stx_2g, 1 strain for stx₂ and stx_2d2, and 1 strain for stx₂ and stx_2g. Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.     

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.     

Detection of milk powder and caseinates in Halloumi cheese

Halloumi cheese is traditionally manufactured from fresh milk. Nevertheless, dried dairy ingredients are sometimes illegally added to increase cheese yield. Lysinoalanine and furosine are newly formed molecules generated by heating and drying milk protein components. The levels of these molecular markers in the finished Halloumi have been investigated to verify their suitability to reveal the addition of skim milk powder and calcium caseinate to cheese milk. Because of the severe heating conditions applied in curd cooking, genuine Halloumi cheeses (n = 35), representative of the Cyprus production, were characterized by levels of lysinoalanine (mean value = 8.1 mg/100 g of protein) and furosine (mean value = 123 mg/100 g of protein) unusual for natural cheeses. Despite the variability of the values, a good correlation between the 2 parameters (R = 0.975) has been found in all cheeses, considering both the fresh and mature cheeses as well as those obtained from curd submitted to a prolonged cooking following a traditional practice adopted by a very small number of manufacturers. Experimental cheeses made by adding as low as 5% of skim milk powder, or calcium caseinate, or both, to cheese milk fell outside the prediction limits at ±2 standard deviation of the above-reported correlation regardless of curd cooking conditions or ripening length. This correlation may be adopted as a reliable index of Halloumi cheese genuineness.     

Occurrence of foodborne pathogens and characterization of Staphylococcus aureus in cheese produced on farm-dairies

The objective of this study was to address knowledge gaps identified in an earlier risk assessment of Staphylococcus aureus and raw milk cheese. A survey of fresh and short-time ripened cheeses produced on farm-dairies in Sweden was conducted to investigate the occurrence and levels of S. aureus, Listeria monocytogenes and Escherichia coli, to characterize S. aureus isolates with special emphasis on enterotoxin genes, antibiotic resistance, bio-typing and genetic variation, and to collect information related to production practices. In general, the hygienic quality of farm-dairy cheeses appeared to be of an acceptable microbiological quality, e.g. L. monocytogenes and staphylococcal enterotoxin were not detected in cheese samples. However, E. coli and enterotoxigenic S. aureus were frequently found in raw milk cheeses and sometimes at levels that are of concern, especially in fresh cheese. Interestingly, levels in raw milk fresh cheese were significantly lower when starter cultures were used. Up to five S. aureus colonies per cheese, if possible, were characterized and about 70% of isolates carried one or more enterotoxin genes, most common were sec and sea. The Ovine biotype (73%) was most common among isolates from goat milk cheese and the Human biotype (60%) from cow milk cheese. Of all isolates, 39% showed decreased susceptibility to penicillin, but the proportion of isolates from cows' cheese (66%) compared to isolates from goats' cheese (27%) was significantly higher. S. aureus isolates with different properties were detected in cheese from the same farm and, sometimes even the same cheese. Isolates with the same pulsed-field gel electrophoresis (PFGE)-pattern were detected on geographically distant dairies. This indicates that multiple sources and routes of contamination are important. To improve the safety of these products efforts to raise awareness of the importance of hygiene barriers and raw milk quality as well as improved process control can be suggested, e.g. use of starter cultures and monitoring of fermentation with a pH-meter. For future safety assessments, a better understanding of factors determining toxin production in these cheeses is needed.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

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.     

Utilization of microfiltration or lactoperoxidase system or both for manufacture of Cheddar cheese from raw milk

The objective of the present study was to determine if application of microfiltration (MF) or raw milk lactoperoxidase system (LP) could reduce the risk of foodborne illness from Escherichia coli in raw milk cheeses, without adversely affecting the overall sensory acceptability of the cheeses. Escherichia coli K12 was added to raw milk to study its survival as a non-pathogenic surrogate organism for pathogenic E. coli. Five replications of 6 treatments of Cheddar cheese were manufactured. The 6 treatments included cheeses made from pasteurized milk (PM), raw milk (RM), raw milk inoculated with E. coli K12 (RME), raw milk inoculated with E. coli K12 + LP activation (RMELP), raw milk inoculated with E. coli K12 + MF (MFE), and raw milk inoculated with E. coli K12 + MF + LP activation (MFELP). The population of E. coli K12 was enumerated in the cheese milks, in whey/curds during cheese manufacture, and in final Cheddar cheeses during ripening. Application of LP, MF, and a combination of MF and LP led to an average percentage reduction of E. coli K12 counts in cheese milk by 72, 88, and 96%, respectively. However, E. coli K12 populations significantly increased during the manufacture of Cheddar cheese for the reasons not related to contamination. The number of E. coli K12, however, decreased by 1.5 to 2 log cycles during 120 d of ripening, irrespective of the treatments. The results suggest that MF with or without LP significantly lowers E. coli count in raw milk. Hence, if reactivation of E. coli during cheese making could be prevented, MF with or without LP would be an effective technique for reducing the counts of E. coli in raw milk cheeses. The cheeses were also analyzed for proteolysis, starter and nonstarter lactic acid bacteria (NSLAB), and sensory characteristics during ripening. The concentration of pH 4.6 soluble nitrogen at 120 d was greater in PM cheese compared with the other treatments. The level of 12% trichloroacetic acid-soluble nitrogen at 120 d was greater in RM, RME, and RMELP cheeses compared with PM, MFE, and MFELP cheeses. This could be related to the fact that cheeses made from raw milk with or without LP (RM, RME, and RMELP) had greater levels of NSLAB compared with PM, MFE, and MFELP cheeses. Cheeses at 60 d, as evaluated by 8 trained panelists, did not differ in bitterness, pastiness, or curdiness attributes. Cheeses at 120 d showed no differences in acid-taste, bitterness, or curdiness attributes. Sensory analysis at 60 d showed that PM and MFELP cheeses had greater overall sensory acceptability than RM and RME cheeses. The overall sensory acceptability of the cheeses at 120 d showed that PM, MFE, and MFELP cheeses were more acceptable than RM and RME cheeses.     

Prevalence and characterization of Listeria monocytogenes isolated from soft cheese

A survey was made in 1995–1996 for Listeria spp. in 63 soft cheeses, made from raw ewe's milk using traditional methods, in the Province of Beira Baixa (Portugal). Listeria spp. were isolated from 47 (75%) of the cheeses, L.monocytogenes was isolated from 29 (46%), and L.innocua but not L.monocytogenes from 18 (29%). Of 24 isolates of L.monocytogenes that were serotyped, 20 were serotype 4b, three were serotype 1/2b and one was serotype 1/2a. Phage typing of isolates of L.monocytogenes and L.innocua showed that in some cases a particular phage type was associated with cheese from a particular source. Twenty four strains of L.monocytogenes tested were able to grow at 30°C in culture medium adjusted with HCl to a pH in the range from 4.4 to 6.0 within 3 days; in the pH range 4.4–6.8 a representative strain grew most rapidly at pH 6.8. The pH range in the cheeses during maturation was between about 5.2–6.4. Whether L.monocytogenes could multiply in the cheeses would depend on factors such as concentration of organic acids and of salt, and storage temperature.     

Short communication: Sensory profile of raw goat milk cheeses made with artisan kid rennet pastes from commercial-weight animals: Alternative to farmhouse goat cheeses

The loss of traditional kid rennet pastes in the Canary Islands (Spain), as in many other regions, is most likely due to the custom of using abomasa from very young animals killed below desirable commercial weight. In addition, the reasonable price of commercial rennets (CR) has resulted in the loss of typical sensory characteristics for most farmhouse raw goat milk cheeses, placing them at a disadvantage when local and international markets are full of different cheeses, often with aggressive marketing strategies. This paper analyzes the sensory characteristics of raw goat milk cheeses made with rennet pastes prepared from commercial kid abomasa in 2 ways: dried while full of ingested milk [full, commercial, artisan kid rennet (FCKR)], or dried after being emptied of ingested milk and refilled with raw goat milk [empty, commercial, artisan kid rennet (ECKR)]. This latter practice allows the use of empty abomasa, or abomasa with grass, soil, and so on. Sensory profiles of cheeses made with FCKR and ECKR rennets were compared with those made with CR by an expert panel (n = 7). The FCKR and ECKR cheeses had similar sensory profiles. Although scores for FCKR cheeses were somewhat higher than for ECKR cheeses, they were in the range found for traditional cheeses made with rennet prepared with abomasa from very young animals. The sensory profile of CR cheeses was very different. Almost 90% of consumer panelists (n = 90) preferred cheeses made with the experimental rennet pastes. These results demonstrate the possibility to prepare artisan rennet pastes from commercial-weight kids in an easy way for farmhouse cheese makers using local resources that would otherwise be destroyed in abattoirs.     

Lipolysis in Urfa cheese produced from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures during ripening

Lipolysis was evaluated in Urfa cheese made from raw and pasteurized goats’ and cows’ milk with mesophilic or thermophilic cultures. The acid degree values (ADVs) of the cows’ milk cheeses were significantly (P < 0.05) higher until 60 d of storage than that of cheese made from goats’ milk. Total free fatty acid (FFA) contents of goats’ milk cheese were significantly (P < 0.001) lower than that of cows’ milk cheese throughout ripening, whereas goats’ milk cheese flavour was higher (P < 0.05) than cows’ milk cheese. Pasteurization of milk prior to cheese-making has a negative influence, not only on the level of lipolysis throughout ripening, but also on the relative amounts of short chain FFAs and sensory properties of the cheeses (P < 0.001). Cheese produced without starter bacteria underwent significantly (P < 0.05) higher lipolysis than cheeses produced with mesophilic or thermophilic starter bacteria, while cheese made with thermophilic starter culture had similar flavour to cheese made without starter culture.     

Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells

The objective of this research was to evaluate the effects of 2 levels of raw milk somatic cell count (SCC) on the composition of Prato cheese and on the microbiological and sensory changes of Prato cheese throughout ripening. Two groups of dairy cows were selected to obtain low-SCC (<200,000 cells/mL) and high-SCC (>700,000 cells/mL) milks, which were used to manufacture 2 vats of cheese. The pasteurized milk was evaluated according to the pH, total solids, fat, total protein, lactose, standard plate count, coliforms at 45°C, and Salmonella spp. The cheese composition was evaluated 2 d after manufacture. Lactic acid bacteria, psychrotrophic bacteria, and yeast and mold counts were carried out after 3, 9, 16, 32, and 51 d of storage. Salmonella spp., Listeria monocytogenes, and coagulase-positive Staphylococcus counts were carried out after 3, 32, and 51 d of storage. A 2 × 5 factorial design with 4 replications was performed. Sensory evaluation of the cheeses from low- and high-SCC milks was carried out for overall acceptance by using a 9-point hedonic scale after 8, 22, 35, 50, and 63 d of storage. The somatic cell levels used did not affect the total protein and salt:moisture contents of the cheeses. The pH and moisture content were higher and the clotting time was longer for cheeses from high-SCC milk. Both cheeses presented the absence of Salmonella spp. and L. monocytogenes, and the coagulase-positive Staphylococcus count was below 1 × 10² cfu/g throughout the storage time. The lactic acid bacteria count decreased significantly during the storage time for the cheeses from both low- and high-SCC milks, but at a faster rate for the cheese from high-SCC milk. Cheeses from high-SCC milk presented lower psychrotrophic bacteria counts and higher yeast and mold counts than cheeses from low-SCC milk. Cheeses from low-SCC milk showed better overall acceptance by the consumers. The lower overall acceptance of the cheeses from high-SCC milk may be associated with texture and flavor defects, probably caused by the higher proteolysis of these cheeses.     

Genomic typing of enterococci isolated from bovine mammary glands and environmental sources

Enterococcal isolates (n = 102) from various sources of bovine origin on 1 farm were characterized using pulsed field gel electrophoresis analysis of SmaI restriction patterns. Isolates originated from feed samples (n = 6), bedding samples (n = 15), and bovine quarter-milk samples (n = 81). Isolates collected from milk samples included those from high-somatic cell count cows (n = 42), postpartum milk samples (n = 16), and clinical mastitis samples (n = 23). Species evaluated included Enterococcus faecium (n = 68), Enterococcus casseliflavus (n = 29), and Enterococcus faecalis (n = 5). A total of 20 clusters representing 44 isolates were detected when a similarity cut-off level of 75% was applied to interpret the pulsed field gel electrophoresis results. Fifteen of the clusters contained only isolates from milk samples. Four clusters contained isolates from bedding and milk samples. One cluster contained only isolates from feed samples. Clusters comprised of a single species represented 17 of the 20 total clusters. These results suggest enterococci from bovine origin were genetically diverse, whereas a limited number of isolates from various sources appeared to cluster together.     

Multiple-locus variable number of tandem repeat analysis (MLVA) of Listeria monocytogenes directly in food samples

Listeria monocytogenes is the etiologic agent of listeriosis responsible for severe and fatal infections in humans. Listeria contamination occurs quite often in a wide range of foods due to its ubiquitous nature. Isolates need to be characterized to a strain level for accurate diagnosis of Listeria infection, epidemiological studies, investigation of outbreaks and effective prevention and control of food-borne listeriosis. The purpose of this research was to evaluate the multiple-locus variable number of tandem repeat analysis (MLVA) for sub-typing L. monocytogenes isolates in pure cultures and in food matrices. Two multiplex PCR assays were formulated to amplify six specific loci using fluorescently-labeled primers; and the amplicons were analyzed by capillary electrophoresis. The MLVA method resulted in 34 unique DNA fingerprint patterns from 46 L. monocytogenes isolates of 10 serotypes which had 29 or 30 PFGE patterns with a single restriction enzyme and 34 AFLP patterns. The MLVA patterns of the 46 isolates remained unchanged in the presence of pre-enriched food matrices including sausage, ham, chicken, milk and lettuce. The MLVA method successfully typed L. monocytogenes strains spiked in cheese, roast beef, egg salad and vegetable samples after 48 h enrichment at the initial inoculation levels of 1-5 CFU per 25 g of food or higher. The limits of detection (typing) of the MLVA method were 10³-10⁴ CFU/mL of pre-enriched food broth when evaluated using post-spiked sausage, ham, chicken, milk and lettuce samples. The MLVA method was simple, highly discriminatory, and easy to perform with portable (numerical) results. To our knowledge, this is the first report that describes the application of the MLVA method directly to food samples and demonstrates the possibility to obtain rapid and accurate subtyping results before an isolate is obtained.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.