Demetrios K. Papageorgiou

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 stx(2)-positive, five of which were also stx(1)-positive. The remaining isolate was positive only for the stx(1) gene. All stx-positive isolates (whether positive for stx(1), stx(2) or stx(1) and stx(2)) 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.

Fate of Listeria monocytogenes during the manufacture, ripening and storage of Feta cheese

The ability of Listeria monocytogenes to grow during the Feta cheese-making process, and to survive during ripening, and storage of the cheese was examined. Pasteurized whole cows milk was inoculated to contain ca. 5.0 × 103 L. monocytogenes [strain Scott A or California (CA)] cfu/ml and made into Feta cheese according to standard procedure. Lactobacillus bulgaricus , and Streptococcus thermophilus (1:1, v/v) were used as starter culture (1%, v/v). Fresh cheese was placed into sterile 12% salt brine and was held at 22°C for 24 h. Then it was placed into sterile 6% salt brine and held 4 d at 22°C after which it was stored in the same brine at 4°C. Milk, curd, whey, cheese, and brine were tested for numbers of L. monocytogenes and pH. Duplicate samples were used to enumerate L. monocytogenes by surface-plating on McBride Listeria Agar. Selected Listeria colonies were confirmed biochemically, L. monocytogenes was entrapped in curd during cheese-making with the population in curd being 0.92 Log10 cfu/g greater than in the inoculated milk; the whey contained an average of 3.2% of the initial inoculum. L. monocytogenes in cheese increased in numbers by ca. 1.5 Log10 cfu/g during the first 2 d of ripening, the population was 2.33 (S.D. ± 0.12) Log10 cfu/g greater in cheese than in the inoculated milk, with a maximum number of 1.5 × 106 cfu/g. The pH value of 2-d-old cheese decreased to 4.6 and then growth of L. monocytogenes ceased. Both strains of L. monocytogenes survived in Feta cheese for more than 90 d even at the low pH of 4.30 (S.D. ± 0.05) that Feta cheese had after ripening. Strain CA was significantly (P<0.006) less tolerant than strain Scott A, of conditions in the cheese during storage at 4°C.

Fate of Listeria monocytogenes During the Manufacture and Ripening of Blue Cheese

The ability of Listeria monocytogenes to grow during the manufacture of blue cheese and to survive during its ripening was examined. Pasteurized skim milk was standardized to a milk fat content of 3.7% by addition of pasteurized homogenized cream (35% milk fat), was inoculated to contain ca. 1.0-2.0 × 103 L. monocytogenes [strain Scott A or California (CA)] cfu/ml, and was made into blue cheese according to the modified Iowa method. Blue cheese was ripened at 9-12°C and a relative humidity of 90-98% for 84 d, and then cheese was stored at 4°C. Duplicate samples of milk, curd, whey, and cheese were tested for pH and for numbers of Listeria by surface plating of appropriate dilutions [made in Tryptose Broth (TB) with 2% sodium citrate] on McBride Listeria Agar (MLA). Initial TB dilutions were stored at 4°C and surface-plated on MLA after 2, 4, 6, and 8 weeks, if the pathogen was not quantitated in the original sample. Selected Listeria colonies were confirmed biochemically. L. monocytogenes was entrapped in curd during cheese-making with the population in curd before hooping being ca. 1.0 log10 cfu/g greater than in the inoculated milk; whey contained an average of 3.6% of the cells in the initial inoculum. L. monocytogenes in cheese increased in numbers by 0.58 to 1.22 log10 cfu/g during the first 24 h of the cheese-making process. Only modest growth (0.12 to 0.30 log10 cfu/g) was noted in two lots with rapid acid production. Growth of L. monocytogenes ceased when the pH of cheese dropped below 5.0. Populations of both strains of the pathogen decreased significantly (P≤ 0.005) during the first 50 d of ripening, by an average of 2.68 log10 cfu/g compared to populations of 1-d-old cheese. From days 50 to 120 the environment of blue cheese became more favorable (pH of cheese increased because of growth by Penicillium roqueforti ), and this resulted in improved survival but no growth of the pathogen. Strain Scott A survived without any more substantial decrease in numbers during days 50 to 120 of storage. Strain CA survived during days 50 to 80, and then populations of the pathogen decreased gradually so that direct plating at 110 d (one trial) and 120 d gave negative results, but the same samples gave positive results after cold enrichment.

Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH)

The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organisms fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

Enumeration and confirmation of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas sobria isolated from raw milk and other milk products in Northern Greece

A total of 138 raw cows and 57 raw ewes milk samples; 80 pasteurized cows milk samples; 39 Anthotyros cheese, 36 Manouri cheese, and 23 Feta cheese samples; and 15 rice pudding samples were examined for the presence and any countable population of Aeromonas species. Twenty-two (15.9%) of the 138 cows milk samples analyzed were contaminated with A. hydrophila. In 13 of these samples, populations of 3.0x10(2) to 5.0x10(3) CFU/ml were counted in starch ampicillin agar (SAA). Eighteen cows milk samples (13.0%) were contaminated with A. caviae, and in eight of these samples, populations of 2.0x10(2) to 3.0x10(3) CFU/ml were counted in SAA. Five cows milk samples (3.6%) were contaminated with A. sobria, and in two of these samples, populations of 2.5x10(3) and 5.0x10(3) CFU/ml were counted in SAA. Eleven cows milk samples (7.9%) were contaminated with other Aeromonas spp. not classified. Eight (14.0%) of the 57 ewes milk samples analyzed were contaminated with A. hydrophila. In these samples, populations of 5.0x10(2) to 5.0x10(3) CFU/ml were counted in SAA. Six ewes milk samples (10.5%) were contaminated with A. caviae, and populations of 1.5x10(2) to 1.0x10(3) CFU/ ml were counted in SAA. Two ewes milk samples (3.5%) were contaminated with A. sobria, and populations counted in SAA were 5.0x10(2) and 1.0x10(3) CFU/ml. Four samples (7.0%) were contaminated with other Aeromonas spp. not classified. A. hydrophila was recovered in 4 (10.2%) and 3 (8.3%) of the Anthotyros and Manouri cheese samples analyzed, respectively, but no countable populations were noted in SAA. None of the pasteurized milk, Feta cheese, and rice pudding samples yielded Aeromonas spp. The results of this work indicate that motile Aeromonas are common in raw milk in Greece. Also, the presence of A. hydrophila in the whey cheeses Anthotyros and Manouri indicates that postprocessing contaminations of these products with motile Aeromonas may occur during production.

Behavior of Escherichia coli O157:H7 during the Manufacture and Ripening of Feta and Telemes Cheeses

Pasteurized whole ewes and cows milk was used in the manufacture of Feta end Telemes cheeses, respectively, according to standard procedures. In both cases, the milk had been inoculated with Escherichia coli O157:H7 at a concentration of ca. 5.1 log CFU/ml and with thermophilic or mesophilic starter cultures at a concentration of ca. 5.3 to 5.6 log CFU/ml. In the first 10 h of cheesemaking, the pathogen increased by 1.18 and 0.82 log CFU/g in Feta cheese and by 1.56 and 1.35 log CFU/ g in Telemes cheese for the trials with thermophilic and mesophilic starters, respectively. After 24 h of fermentation, a decrease in E. coli O157:H7 was observed for all trials. At that time, the pH was reduced to 4.81 to 5.10 for all trials. Fresh cheeses were salted and held at 16 degrees C for ripening until the pH was reduced to 4.60. Cheeses were then moved into storage at 4 degrees C to complete ripening. During ripening, the E. coli O157:H7 population decreased significantly (P < or = 0.001) and finally was not detectable in Feta cheese after 44 and 36 days and in Telemes cheese after 40 and 30 days for the trials with thermophilic and mesophilic starters, respectively. The estimated times required for one decimal reduction of the population of E. coli O157:H7 after the first day of processing were 9.71 and 9.26 days for Feta cheese and 9.09 and 7.69 days for Telemes cheese for the trials with thermophilic and mesophilic starters, respectively.

Behavior of Listeria monocytogenes at 4 and 22°C in Whey and Skim Milk Containing 6 or 12% Sodium Chloride

Autoclaved samples of skim milk and deproteinated whey were fortified with 6 or 12% NaCl, inoculated with Listeria monocytogenes strains Scott A or California (CA), to contain ca. 1.0 × 103 cfu/ml (in the products with 6% salt) or ca. 5.0 × 103 cfu/ml (in the products with 12% salt) and incubated at 4 and 22°C. The pH values of the 6% salted whey, 6% salted skim milk, 12% salted whey, and 12% salted skim milk were 5.65, 6.20, 5.50, and 6.00 respectively. These values remained relatively constant during the entire experiment. Listeria counts were obtained by surface-plating appropriate dilutions and/or undiluted samples on Trypticase Agar (TA). Samples in which L. monocytogenes was not detected, were re-examined after 2, 4, 6 and 8 weeks of cold-enrichment. Generation times of L. monocytogenes in 6% salted whey at 22°C (3.67 h and 3.56 h for strains Scott A and CA, respectively) were significantly shorter than those in 6% salted skim milk at 22°C (4.31 and 4.42 h for the two strains, respectively). Generation times in 6% salted products at 4°C ranged between 37.49 h and 49.43 h. Maximum populations reached at 22 and 4°C ranged from 7.58 to 8.10 Log10 cfu/ml, and were significantly higher in 6% salted whey than in 6% salted skim milk. In 12% salted whey and skim milk incubated at 22°C, L. monocytogenes gradually decreased in numbers. Strain CA was inactivated within 85 d in 12% salted skim milk or within 110 d in 12% salted whey, and was significantly less salt tolerant than strain Scott A which survived for more than 130 d under the same conditions. Loss of viability by both strains was similar in 12% salted whey and skim milk after 130 d of storage at 4°C, and the decreases in population were less than 0.7 order of magnitude.

Loss of viability of Listeria monocytogenes in contaminated processed cheese during storage at 4, 12 and 22 °C

The behaviour of Listeria monocytogenes in a processed cheese product was evaluated over time by inoculating the product with three different L. monocytogenes strains (Scott A, CA and a strain isolated from processed cheese) at three different inoculation levels (ca. 6x10(5), ca. 6x10(3) and 10(2)CFU/g of cheese or less) and after storage of the contaminated products at 4, 12 or 22 degrees C. Growth of L. monocytogenes was not observed in any of the experimental trials (experiments involving different combinations of strain, inoculum level and storage temperature) throughout the storage period. L. monocytogenes populations decreased over time with a rate that was strain- and storage temperature-dependent. Nonetheless, for cheeses that had been inoculated with the higher inoculum and stored at 4 degrees C viable populations of L. monocytogenes could be detected for up to nine months post-inoculation. The L. monocytogenes survival curves obtained from the different trials were characterised by a post-inoculation phase during which the populations remained essentially unchanged (lag phase) followed by a phase of logarithmic decline. The duration of the lag phase and the rate of inactivation of L. monocytogenes in the different trials were estimated based on data from the linear descending portions of the survival curves. In addition, a non-linear Weibull-type equation was fitted to the data from each survival curve with satisfactory results. The results of the present study emphasize that, according to the definition laid down in the European Union Regulation 1441/2007, the processed cheese product tested in this work should be considered and classified as one that does not support the growth of L. monocytogenes under reasonable foreseeable conditions of distribution and storage. However, post-processing contamination of the product should be austerely avoided as the pathogen can survive in the product for extended periods of time, particularly under refrigerated storage (4 degrees C).

Growth of Listeria monocytogenes in the Whey Cheeses Myzithra, Anthotyros, and Manouri during Storage at 5, 12, and 22°C

The fresh whey cheeses Myzithra, Anthotyros, and Manouri were inoculated with Listeria monocytogenes Scott A or California to contain ca. 5.0 × 102 CFU/g of cheese and incubated at 5, 12, and 22°C. The initial pH of the finished whey cheeses Myzithra, Anthotyros, and Manouri were 6.50, 6.41, and 6.30 respectively. Fat in dry matter was 16.3% in Myzithra, 65.9% in Anthotyros, and 71.7% in Manouri cheese; the moisture contents of the cheeses were 68.4, 66.9, and 52.2% respectively. The pH of the cheeses dropped gradually to between 5.30 to 4.97 during storage. Duplicate samples of whey cheeses were tested for numbers of L. monocytogenes and pH. Listeria counts were obtained by surface-plating on lithium chlorite-phenylethanol-moxalactam agar (LPM). Selected Listeria colonies were confirmed biochemically. The growth of L. monocytogenes in the whey cheeses was very rapid. Generation times of L. monocytogenes at 5°C ranged between 16.16 and 20.16 h and were significantly longer than those observed at 12°C, which ranged between 5.07 and 5.81 h. Generation times at 22°C ranged between 1.68 and 2.70 h. The generation time of L. monocytogenes Scott A at 5°C in Anthotyros cheese (20.16 h) was significantly longer than those of the same strain at 5°C in Myzithra cheese (16.16 h) and Manouri cheese (17.81 h). Also, the generation time of L. monocytogenes California at 22°C in Myzithra cheese (2.70 h) was significantly longer than the generation time of strain Scott A (1.93 h). Maximum populations of L. monocytogenes were reached after 24 to 30 days at 5°C, after 5 to 12 days at 12°C, and after only 56 to 72 h at 22°C and ranged from 6.92 to 8.81 log CPU/g. Maximum populations were significantly lower in Myzithra cheese at 5 and 12°C than maximum populations in Anthotyros and Manouri cheese at the same temperatures independent of the inoculated strain.

Chemical and bacteriological characteristics of Pichtogalo Chanion cheese and mesophilic starter cultures for its production

Sixty-two samples of Pichtogalo Chanion cheese traditionally produced in Crete, a few (3 to 6) days old, were analyzed for some chemical and bacteriological characteristics. The results of physicochemical analyses were as follows: (1) moisture content 61.63% (standard deviation 4.67); (2) fat in dry matter 54.03% (SD 7.73); (3) protein content 14.23% (SD 1.72); (4) salt content 1.02% (SD 0.38); (5) water activity (aw) 0.990 (SD 0.003); and (6) pH 4.36 (SD 0.25). None of the samples yielded Salmonella spp. Listeria monocytogenes and coagulase-positive staphylococci were present in 6.45% of the samples. Bacillus cereus and sulfite-reducing clostridia were isolated from 14.51% and 40.32% of the samples, respectively. High populations of coliforms were determined in the cheese samples. In 11.3% of the samples, Escherichia coli was not detected, while 88.7% of the samples yielded E. coli most probable number levels from 1.32 to 5.66 log10/g. The log10 CFU/g counts of enterococci were 6.89 (SD 0.84), of yeasts 6.79 (SD 0.61), of molds 4.68 (SD 0.69), and of psychrotrophic bacteria 7.63 (SD 0.62). The log10 CFU/g counts of lactic acid streptococci and lactococci were 7.91 (SD 0.68) and of lactobacilli 8.11 (SD 0.65). Lactic acid bacteria, mainly mesophilic, were isolated and confirmed using API 50 CH test trips. A pasteurized mixture of ewes and goats milk was made into Pichtogalo Chanion cheese according to standard procedure at 23 degrees C, after the addition of 4% commercial mesophilic starter culture or 2%, 3%, and 4% starter culture of the isolated and confirmed lactic acid bacteria and the addition of rennet. Results of this work indicated that high quality of Pichtogalo Chanion cheese can be produced using a pasteurized mixture of ewes and goats milk and 4% (vol/vol) of mesophilic starter culture.