E. de Boer

Cross-contamination with Campylobacter jejuni and Salmonella spp. from raw chicken products during food preparation

Campylobacter jejuni was isolated from 170 (61%) of 279 samples of chicken products and Salmonella from 44 (54%) of 81 samples. Cross-contamination experiments showed that C. jejuni and to a lesser extent Salmonella were easily transferred from raw chicken products to cutting-boards, plates, and hands. These organisms were also isolated from raw vegetables and cooked chicken products, which were in contact with plates on which raw chicken products had been placed. Measures for the prevention of infection by cross-contamination through handling of raw chicken are mentioned.

Isolation and characterization of verocytotoxin-producing Escherichia coli O157 from slaughter pigs and poultry

Rectal contents and tonsils from Dutch slaughter pigs collected immediately after slaughter were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC). In addition, fresh fecal material from poultry layer flocks and turkey flocks collected on poultry farms was examined for the presence of O157 VTEC. E. coli O157 strains were isolated from two (1.4%) of 145 pigs. The strains were isolated from samples of rectal contents, all samples of tonsils being negative. While all 501 fecal samples from chicken flocks were found negative, E. coli O157 strains were isolated from six (1.3%) of 459 pooled fecal samples from turkey flocks. One of the porcine isolates and one of the turkey isolates contained the VT2 gene, the E. coli attaching-and-effacing gene, as well as the enterohemorrhagic E. coli hemolysin gene. Production of VT was confirmed by cytotoxicity tests on Vero cells. Based on these characteristics, the two stains were regarded as potentially pathogenic for humans. The porcine and the turkey isolate were further characterized as being of phage types 4 and 14, respectively. While biochemically typical of E. coli O157, the remaining six isolates were nonverocytotoxigenic and negative for both the E. coli attaching-and-effacing gene and the enterohemorrhagic E. coli hemolysin gene. All eight E. coli O157 isolates did not carry genes that encode E. coli heat-labile and heat-stable enterotoxins. It was concluded that pigs and poultry can be a source of O157 VTEC strains characteristic of those causing illness in man. The extent to which pigs and poultry play a role in the epidemiology of human O157 VTEC infection needs further research.

Survival of Campylobacter jejuni during poultry processing and pig slaughtering

Experiments were done to assess the survival of Campylobacter jejuni during different stages of poultry processing and pig slaughtering. Natural sources of Campylobacter contamination, i.e., spinchiller water, chicken intestinal contents and pig feces, were used for this purpose. C. jejuni in chicken intestinal contents had D-values ranging from 0.18 to 0.39 min at 60°C to 1.96 to 10.82 min at 52°C. Experiments with surfaces of pig carcasses contaminated with pig feces and held in the cooling room of a pig slaughterhouse showed an overnight reduction of Campylobacter until below the detection level. Further experiments in the laboratory showed that this reduction was due to drying of the skin surface. C. jejuni was very sensitive to drying. When contaminated spinchiller water was spread on tiles of different materials (aluminium, stainless steel, Formica and ceramic), the organism survived as long as a moistened surface could be observed. They could not be isolated once surfaces were visually dry. Freezing affected C. jejuni only during the first few hours; after an initial drop of number, Campylobacter could survive on chicken carcasses and chicken livers at -20°C for more than 64 and 84 d, respectively.

Occurrence and Survival of Verocytotoxin-Producing Escherichia coli O157 in Raw Cow's Milk in the Netherlands

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cows milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.

Occurrence of Escherichia coli O157 and Other Verocytotoxin-Producing E. coli in Retail Raw Meats in the Netherlands

Raw meats obtained from retail outlets in the Netherlands were examined for the presence of Escherichia coli of serogroup O157 and other verocytotoxin (VT)-producing E. coli (VTEC), in three different surveys. In the first survey O157 VTEC were detected and isolated by selective plating onto sorbitol MacConkey agar following selective enrichment in modified tryptone soy broth with acriflavin. The organisms were isolated from 2 (0.3%) of 770 samples of minced mixed beef and pork, but not detected in samples of raw minced beef (n = 1,000), minced pork (n = 260), or poultry products (n = 300). In the second survey an additional 360 raw meats were examined with the 3M Petrifilm™ Test Kit-HEC, after selective enrichment in modified E. coli broth containing novobiocin. VT-negative E. coli O157 strains were isolated from 22 (6.1%) samples. In the third survey 180 enrichment cultures of the first survey were screened for the presence of VT1 and VT2 genes with a polymerase chain reaction (PCR). Twenty-nine (16.1%) of the 180 enrichment cultures showed a positive PCR: one for the VT1 gene only, 17 for the VT2 gene only, and 11 for both the VT1 and VT2 gene. A total of 46 VTEC strains were isolated from 10 randomly selected PCR-positive samples. Serotyping revealed that 41 of the 46 VTEC isolates belonged to nine different O serogroups; the remaining five were unidentifiable. A number of the serogroups recovered have been associated with human disease.

Microbiological Quality of Commercial Tempeh in The Netherlands

A survey of the microbiological quality of commercial tempeh was done in The Netherlands. A total of 110 samples were examined. Most (98%) of the samples had an aerobic plate count above 107 CFU/g. Numbers of Enterobacteriaceae exceeded 105 CFU/g in 67% of the samples, whereas numbers of lactic acid bacteria exceeded 107 CFU/g in 81% of the samples. Staphylococcus aureus was found in 13%, Bacillus cereus in 11% and Escherichia coli in 3% of the samples at levels of 105 CFU/g. Yersinia enterocolitica was found in six samples, whereas Salmonella was absent in 25 g of all the samples examined. Many (69%) of the samples had a yeast count above 105 CFU/g. Trichosporon beigelii was the most frequent yeast species. Besides Rhizopus oryzae and Rizopus oligosporus , which obviously represent the mold species responsible for the fermentation, Mucor indicus was often associated with the mycoflora of the tempeh. The reasons for the poor microbiological quality are discussed and some recommendations are proposed.

Evaluation of media and lest kits for the detection and isolation of E coli 157 from minced beef

This study has evaluated the efficacy of selective enrichment and plating media used for the isolation of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157, by examining pure bacterial cultures. In addition, the performance of a variety of commercial test kits for the detection of E. coli O157 strains inoculated into minced beef was compared, using the Ampcor E. coli O157:H7 Kit, 3M Petrifilm™ Test Kit-HEC, Dynabeads anti- E coli O157, EHEC-TEK™, and the Tecra E. coli O157 visual immunoassay. The commercial Verotox F test for the determination of the VT type of VTEC isolates was compared with a polymerase chain reaction (PCR) assay for VT-coding genes. Modified E. coli broth containing novobiocin (mEC + n) and sorbitol MacConkey agar supplemented with cefixime and tellurite (CT-SMAC) were the most efficacious media for selective enrichment and isolation, respectively. After enrichment of the inoculated samples, all kits tested could detect less than one O157 VTEC cell per g of minced beef. While the results of the immunoassays need to be confirmed by isolating the organisms, the use of the immunomagnetic separation technique directly yields isolates. The results of the Verotox F test were consistent with PCR results. A sensitive and cost-effective method for the isolation of O157 VTEC from minced beef in food industry and epidemiological studies involving large numbers of samples is the following: enrichment in mEC + n at 37°C for 6 to 8 h with shaking at 100 rpm, followed by immunomagnetic separation using Dynabeads anti- E. coli O157 and spread plating of the concentrated target cells onto CT-SMAC. The Verotox F test can be used to determine whether the isolates produce VTl and/or VT2.

Comparison of Methods for Isolation and Confirmation of Clostridium perfringens from Spices and Herbs

Rapid Perfringens Medium (RPM), Perfringens Enrichment Medium (PEM) and Tryptose Sulfite Cycloserine agar medium (TSC) were compared for determination of Clostridium perfringens in spices and herbs. Of 147 samples, 62 (42%) contained C. perfringens when RPM was used; lower percentages of positive isolations were found with PEM (23%), TSC surface plate (19%) and TSC pour plates (26%). Heat-treatment of sample suspensions yielded additional isolates. C. perfringens was isolated by one or more of the techniques from 43 (80%) of 54 different kinds of spices and herbs and from 86 (59%) of 147 samples. Replacing glucose in RPM with raffinose and polymyxin and neomycin with cycloserine did not improve the efficacy of this medium. A good correlation was found between conventional confirmation tests for C. perfringens and tests for acid phosphatase, lecithinase, reverse CAMP test and an antiserum test.

Sensitivity to Natamycin1 (Pimaricin) of Fungi Isolated in Cheese Warehouses

The sensitivity to natamycin of molds and yeasts isolated in cheese warehouses where natamycin has been used for various periods was determined. After several years of continuous use of natamycin no natamycin-insensitive molds and yeasts were found. In 26 strains of molds isolated in cheese warehouses it was not possible under laboratory conditions to decrease the sensitivity for natamycin. Besides the sensitivity of fungi to natamycin, production of mycotoxins by the isolated molds was investigated. Strains of Penicillium viridicatum , Aspergillus versicolor , and Penicillium cyclopium isolated in the warehouses produced, under experimental conditions, ochratoxin A, sterigmatocystin, and penicillic acid, respectively.

General Purpose Enumeration and Isolation Media

This chapter contains results of studies done to evaluate media that may be useful for routine enumeration and isolation of fungi from foods. Prior to the workshop, questionnaires were sent to twenty-five laboratories to determine which media were used for enumeration of fungi in foods. A total of fifteen different media were reported to be routinely used. The media most often used were DRBC, RBC, PDA and OGY. Likewise, recommendations in texts as to the medium of choice for routine culture of foodborne fungi vary widely. Some recommended media are not optimal for enumeration or recovery of fungi from foods, e.g., acidified PDA (APDA). Thus, several scientists were asked to compare the media they routinely used with DRBC, DG18 or OGY. The following papers report the results of these studies and others describing media for general purpose enumeration and isolation of fungi from foods.