Short communication: Characterization of Shiga toxin 2-carrying bacteriophages induced from Shiga-toxigenic Escherichia coli isolated from Italian dairy products

Forty samples of raw milk cheese and 25 samples of raw milk itself were subjected to enrichment culture for Shiga-toxigenic Escherichia coli (STEC), and a single Shiga toxin 2- (Stx₂) positive strain was obtained from one of the cheese samples. Thus, aged cheeses in which the curd is subsequently heat treated (48°C) cannot be presumed to be STEC free. Infective Stx₂ bacteriophages were induced from 3 STEC strains isolated elsewhere from raw milk and 1 STEC strain from aged cheese sourced in Italy. Data on E. coli host range, morphology, genome size, and genetic variation determined by restriction fragment length polymorphism and multi-locus genotyping are presented. Although all 4 bacteriophages were found to be short-tailed Podoviridae, they exhibited considerable variation in both genome size and content. This extended to the Stx₂ genes themselves, whose sequences contained several point mutations, but these did not translate to amino acid substitutions.     

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd

The use of raw milk in the processing of buffalo Mozzarella cheese is permitted, but the heat treatment used for stretching the curd must ensure that the final product does not contain pathogens such as Shiga toxin-producing Escherichia coli (STEC) that may be present on buffalo dairy farms. This study carried out challenge tests at temperatures between 68°C and 80°C for 2 to 10 min to simulate curd temperatures during the stretching phase. Curd samples were inoculated with 2 STEC strains (serotypes O157 and O26), and their inactivation rates were assessed in the different challenge tests. The curd samples were digested with papain to ensure a homogeneous dispersion of bacteria. The STEC cells were counted after inoculation (range 7.1–8.7 log cfu/g) and after heat treatments using the most probable number (MPN) technique. A plot of log MPN/g versus time was created for each separate experiment. The log linear model with tail was used to provide a reasonable fit to observed data. Maximum inactivation rate (k_max, min⁻¹), residual population (log MPN/g), decimal reduction time (min), and time for a 4D (4-log₁₀) reduction (min) were estimated at each temperature tested. A 4D reduction of the O26 STEC strain was achieved when curd was heated at 68°C for 2.6 to 6.3 min or at 80°C for 2.1 to 2.3 min. Greater resistance was observed for the O157 strain at 68°C because k_max was 1.48 min⁻¹. The model estimates can support cheesemakers in defining appropriate process criteria needed to control possible STEC contamination in raw milk intended for the production of Mozzarella.     

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.     

Occurrence of Campylobacter spp., Salmonella spp. and shiga toxin-producing Escherichia coli in inline milk filters from Swedish dairy farms

This study investigated the occurrence of shiga toxin-producing Escherichia coli (STEC), thermotolerant Campylobacter spp. and Salmonella spp. in Swedish dairy milk. A total of 302 inline milk filters were analyzed. Salmonella was not isolated from any filters. Polymerase chain reaction screening detected thermotolerant Campylobacter in 30.5% of the milk filters analyzed and it was isolated from 12.6% of filters. The stx genes (stx ₁, stx ₂, or both) were screened from 71% of the filters and STEC was isolated from 14% of these. Of the STEC isolates, 21 contained the stx ₁ gene, 19 the stx ₂ gene, and five a combination of both stx ₁ and stx ₂ genes. Whole genome sequence typing on 34 of the 45 STEC showed that they belonged to 21 different serotypes, of which STEC O145:H28 was the most common (2%). STEC O157:H7 was only found from one (0.3%) of the filters. A combination of stx ₂ and eae genes was found from 0.7% of the total number of inline milk filters analyzed, while stx _2a was found in 24% of the whole genome-sequenced isolates. There was a significant positive correlations between number of animals per farm and presence of pathogens on milk filters.     

Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources

Shiga toxin-producing strains of Escherichia coli (STEC) cause diarrhoea and haemorrhagic colitis in humans. Most human infections are attributed to consumption of STEC contaminated foodstuff. Food producing animals constitute important reservoirs of STEC and serve as source of food contamination. In this study, we have analyzed 593 foodborne STEC strains for their serotypes and for nine virulence genes (stx1, stx1c, stx1d, stx2, stx2b, stx2e, stx2g, E-hly and eae). The 593 STEC strains grouped into 215 serotypes, and 123 serotypes (57.2%) were represented each by only one STEC isolate. Fifteen serotypes (7.0%) were attributed to 198 (33.3%) of the 593 STEC strains. The foodborne STEC were grouped into different categories in relation to the species of the food producing animal (cattle, pigs, sheep, goats, red deer, wild-boar and hare). Univariate and multivariate statistical analyses revealed significant similarities between the animal origin of the food and the virulence markers of foodborne STEC. Significant associations (p < 0.001) were found for stx1 and for stx2 with bovine meat and milk products. The stx2e gene was significantly (p < 0.001) associated with STEC from pork and wild boar meat. Stx1c and stx2b genes were significantly (p < 0.001) more frequent in STEC from deer meat, as well as from meat and milk products derived from sheep and goats. Using logistic regression models we detected significant (p < 0.01) combinations between stx1, stx2 and E-hly genes and STEC from bovine meat. The combination of stx1c and stx2b genes was significant (p < 0.001) for STEC derived from red deer, sheep and goat products. The properties of foodborne STEC were compared with published data on faecal STEC from food producing animals. Virulence profiles and serotypes of STEC from food showed remarkable similarities to those of faecal STEC that were from the same animal species. The findings from our study clearly indicate that the food producing animals represent the most important source for the entry of STEC in the food chain. Sound hygiene measures implemented at critical stages of food production (milking, slaughtering, and evisceration) should be most effective in reducing the frequency of STEC contamination of food derived from domestic and wildlife animals.     

Micro-array for the identification of Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic Syndrome in humans

A micro-array has been developed, based on the GeneDisc® array, for the genetic identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia coli (STEC) including the most clinically relevant enterohemorrhagic E. coli (EHEC) serotypes. The genes selected for determination of the O antigens (rfbE_O157, wzx_O26, wzx_O103, wbd1_O111, ihp1_O145, wzx_O121, wzy_O113, wzy_O91, wzx_O104, wzy_O118, wzx_O45, and wbgN_O55) and H-types (fliC_H2, fliC_H7, fliC_H8, fliC_H11, fliC_H19, fliC_H21, and fliC_H28) showed a high specificity and concordance with serology. The micro-array also had a high specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2 (stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system (etpD), subtilase cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains, and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely associated with certain serotypes of typical EHEC and emerging EHEC strains. Virulence plasmid associated genes such as katP, espP, and etpD were more common in EHEC than in STEC strains; this supports their association with virulence. This array constitutes a valuable approach for the identification of STEC strains with a high potential for human virulence.     

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.     

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style)

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log₁₀ cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log₁₀ cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log₁₀ cfu/g after whey drainage and during ripening and storage decreased by 2.9 log₁₀ cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.     

Shiga toxin–producing Escherichia coli isolated from pasteurized dairy products from Bahia,Brazil

The presence of pathogenic Shiga toxin–producing Escherichia coli (STEC) in dairy products represents a public health concern because of its ability to produce the toxins Stx1 and Stx2, which cause intestinal diseases. Monitoring the stages of milk production and checking dairy products for contamination are crucial steps to ensure dairy safety. This study aimed to report the occurrence of thermotolerant coliforms, E. coli, and STEC strains in pasteurized dairy products and to evaluate the antibiotic resistance profiles, serotypes, and characterizations of the STEC isolates by pulsed-field gel electrophoresis. We obtained a total of 138 pasteurized dairy products from 15 processing plants in Bahia, Brazil, to examine coliforms, E. coli, and STEC strains. We found that 43% of samples (59/138) contained thermotolerant coliforms, and 30% (42/138) did not comply with Brazilian regulations. Overall, 6% (9/138) were positive for E. coli and 4% (5/138) were positive for STEC. We recovered 9 STEC isolates from pasteurized cream (2/9), Minas Padrão cheese (2/9), Minas Frescal cheese (4/9), and ricotta (1/9). All isolates were stx2-positive, and 2 were eae-positive. All isolates were negative for the “big 6” STEC serogroups, belonging instead to serotypes ONT:HNT, ONT:H12, O148:H?, OR:H40, OR:HNT, and O148:HNT. Pulsed-field gel electrophoresis revealed 100% genetic similarity among 3 isolates from 2 different samples produced in the same production facility, which may suggest cross-contamination. As well, we found isolates that were 98% similar but in samples produced in different production facilities, suggesting a mutual source of contamination or a circulating strain. Two STEC strains exhibited resistance to streptomycin. Although the isolates presented a low resistance profile and no strain belonged to the “big 6” pathogenic group, the circulation of stx2-positive STEC strains in ready-to-eat products highlights the importance of epidemiological surveillance inside the Brazilian dairy chain.     

Production of wara, a West African soft cheese using lemon juice as a coagulant

As an important protein source for West African consumers, wara cheese made from the leave extract of Calotropis procera has extremely short shelf life of only 2-3 days [Adegoke, G. O., Nse, E. N., & Akanni, A. O. (1992). Effects of heat, processing time, and pH on the microflora, aflatoxin content, and storability of wara, a soft white cheese. Die Nahrung, 36(3), 259-264; Umoh, V. J., & Solomon, O. (2001). Safety assessment and critical control point of milk product and some cereal beverages in Northern Nigeria. In: Proceedings of USDA/USAID/NIGERIA international conference on food safety and security, August 1-3 (pp. 122-127). Ibadan, Nigeria: IITA; Belewu, M. A., Belewu, K. Y., & Nkwunonwo, C.C. (2005). Effect of biological and chemical preservatives on the shelflife of West African soft cheese. African Journal of Biotechnology, 4, 1076-1079; Adetunji, A. O., Alonge, D. O., & Chen, J. (Unpublished). Microbial quality of wara, a southwestern Nigerian soft cheese]. Lemon juice was used in this study as a substitute coagulant during wara manufacture in order to improve the microbial quality of wara. The cheese was manufactured from pasteurized milk inoculated with 10¹ or 10² CFU ml⁻¹ of Listeria monocytogenes. Samples of the milk or cheese were taken along the manufacturing steps and during a 5 d storage period at 15 and 28 °C in order to determine the populations of L. monocytogenes, total aerobes, Enterobacteriaceae, and psychrotrophs, as well as mold and yeast. On the 4th day of storage, portions of the un-inoculated control cheese from 28 °C were deep fried in vegetable oil, mimicking the practice of West African local cheese processors. The results showed that L. monocytogenes, at both inoculation levels, did not survive the manufacture of wara. In samples initially inoculated with 10¹ CFU ml⁻¹ of L. monocytogenes, the Enterobacteriaceae counts decreased from the initial 1.78 to 1.00 Log₁₀ CFU g⁻¹ with the addition of lemon juice, and became undetectable (<1.00 Log₁₀ CFU g⁻¹) at the curdling point as well as during the 5 d storage period at both temperatures. The total aerobic counts increased from the undetectable level on the 1st day of storage to 7.65 and 3.39 Log₁₀ CFU g⁻¹, respectively at 28 or 15 °C on the 5th day of storage. The psychrotrophic, as well as the yeast and mold counts increased from the undetectable levels on the 1st day of storage to 7.11 and 5.03 Log₁₀ CFU g⁻¹, respectively at 28 °C. At 15 °C however, the population of pyschrotrophs remained undetectable throughout the 5 d storage period whereas, the yeast and molds count increased to 3.08 Log₁₀ CFU g⁻¹ on day 3 before quickly decreasing to the undetectable levels on the 5th day of storage. A similar trend was observed in cheese made from the milk with an initial Listeria inoculation level of 10² CFU ml⁻¹. The results of this study showed that lemon juice significantly reduced the populations of the sampled microorganisms, especially the populations of Enterobacteriaceae.     

A rapid procedure for the detection and isolation of enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables

Human infections with Enterohaemorrhagic Escherichia coli strains (EHEC) as agents of Haemorrhagic Colitis (HC) and Haemolytic Uraemic Syndrome (HUS) are frequently associated with the consumption of EHEC contaminated foodstuffs of different origins. EHEC O26, O103, O111, O118, O121, O145 and O157 strains are responsible for the majority of HC and HUS cases worldwide. In May 2011, the emerging aggregative EHEC O104:H4 strain caused a large outbreak with high HUS incidence in northern Germany. Contaminated sprouted seeds were suspected to be the vehicles of transmission. The examination of vegetables retailed for raw consumption revealed low numbers of E. coli (< 100 cfu/ g) together with high titres of Enterobacteriaceae and Pseudomonas (approx. 5.6 × 10⁷ cfu/ g). Specific methods of EHEC detection adapted to vegetables are not yet published. Therefore, we have developed a rapid and sensitive method for detecting low EHEC contamination in vegetables (1–10 cfu/25 g) with artificially EHEC contaminated ready-to-eat salads. A 6-hour enrichment period in BRILA-broth was sufficient to detect 1–10 EHEC from spiked samples after plating 0.1 ml portions of enrichment culture on selective TBX-agar and CHROMagar STEC plates that were incubated at 44 °C overnight. Unlike EHEC strains, the growth of bacteria of the plant flora was substantially inhibited at 44 °C. DNA for real-time PCR detection of EHEC characteristic genes (stx₁, stx₂, eae, ehxA, and O-antigen associated) was prepared with bacteria grown on TBX-agar plates. The storage of EHEC inoculated salad samples for 72 h at 6 °C resulted in a significant reduction (mean value 14.6%) of detectable EHEC, suggesting interference of EHEC with the resident plant microflora. CHROMagar STEC was evaluated as a selective medium for the detection of EHEC strains. Growth on CHROMagar STEC was closely associated with EHEC O26:[H11], O111:[H8], O118:H16, O121:[H19], O145:[H28], O157:[H7] and aggregative EHEC O104:H4 strains and with the presence of the terB gene (tellurite resistance). TerB sequences were found in 87.2% of 235 EHEC but only in only 12.5% of 567 non-EHEC strains. EHEC strains which did not grow on CHROMagar STEC were negative for terB as frequently observed with EHEC O103:H2 (52.9%) and sorbitol-fermenting O157:NM strains (100%). The enrichment and detection method was applied in the examination of sprouted seeds incriminated as vehicles in the EHEC O104:H4 outbreak in Germany. Aggregative EHEC O104:H4 could be detected and isolated from a sample of sprouted seeds which was suspected as vector of transmission of EHEC O104 to humans.     

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.     

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA⁺, γ-tir⁺, stx2⁺, rfbE⁺, fliC⁺, hlyA⁺) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.     

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.     

Behaviour of Escherichia coli strains during semi-hard and hard raw milk cheese production

The behaviour of two Escherichia coli strains, one with a high and the other with a low thermotolerance phenotype, was investigated during production and ripening of Swiss semi-hard and hard raw milk cheese. In semi-hard cheese, counts of E. coli increased during production, before a log-linear decrease occurred during ripening, with a faster rate of reduction in core than in rind samples, and faster reduction of the more heat-sensitive strain in rind samples. Nevertheless, at the end of semi-hard cheese ripening, E. coli were present at least at 1.3 log₁₀ cfu g⁻¹ in rind samples and remained detectable by enrichment of core samples. During the first day of hard cheese production, both E. coli strains were almost completely inactivated. Detection by enrichment was possible in one of twelve spiked cheeses after 16 weeks, indicating the potential of a thermotolerant E. coli strain to survive until the end of ripening.     

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.     

Starter culture development for improving safety and quality of Domiati cheese

Eleven lactococci strains (sp. lactis and cremoris) were collected according to specific or selected characteristics for development of defined strain starter (DSS) to improve safety and nutritional quality of traditional and low salt Domiati cheese. Thirteen DSS; nisin-producing system or/and folate-producing strains were prepared. The behaviour of the strains in DSS was studied in milk and in two series of Domiati cheese; the first one made with 5% NaCl and salt tolerant strains, the second made with 3% NaCl and the control cheeses were made without starters. The population dynamics of strains and sensory evaluation of cheese corroborated the results in milk. All strains can grow well together and appeared to produce pleasant flavours, normal (typical) body and texture Domiati cheese. There was no apparent difference in cheese composition between cheeses in each series; the levels were within margins for composition of Domiati cheese. The levels of nisin (IU g⁻¹) ranged from 204 to 324 IU g⁻¹ in 3-months' cheeses. Folate concentration increased in cheeses made with DSS cultures than control and the level ranged from 5.5 to 11.1 μg 100 g⁻¹ in cheeses after 3 months. All results revealed that selected DSS can be used for improving Domiati cheese.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

The prevalence of Escherichia coli O157 and O157:H7 in ground beef and raw meatball by immunomagnetic separation and the detection of virulence genes using multiplex PCR

The present study was conducted to investigate the presence of Escherichia coli O157 and O157:H7 strains and to detect the presence of the stx1, stx2, and eaeA genes in isolates derived from 200 samples (100 samples from fresh ground beef and 100 samples from raw meatball). The samples were purchased from the Samsun Province in Turkey, over a period of 1 year. Enrichment-based immunomagnetic separation and multiplex polymerase chain reaction were applied for these analyses. E. coli O157 was detected in five of the 200 (2.5%) samples tested (one isolated from ground beef and four from meatball samples), whereas E. coli O157: H7 was not detected in any sample. During the analysis, eight strains of E. coli O157 were obtained. The genes stx1, stx2, and eaeA were detected in two E. coli O157 isolates obtained from two meatball samples, whereas only the eaeA and the stx2 genes were detected in four E. coli O157 strains that were isolated from one meatball sample. None of the stx1, stx2, and eaeA was detected in the E. coli O157 isolates obtained from the ground beef and the one meatball samples.