Electrochemical and microbiological characterization of paper mill biofilms.

Biofilm samples collected from inside and outside the press and former sections of paper machines in a Northwestern Ontario paper mill for a period of 2 years were characterized microbiologically and electrochemically. Bacterial community profiling was done using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and selected bacterial isolates were identified using 16S rDNA analysis. The bacterial community showed the presence of Proteobacteria, Firmicutes, and Actinobacteria. Sphingomonas sp. was found to be the most common bacterial species, which showed the highest production of extracellular polymeric substances. Bacteria isolated from biofilms showed better adhesion properties than those from water samples. Cyclic voltammetry and electrochemical impedance spectroscopy studies showed that bacteria isolated from biofilms and feed water collected from inside the machine were more easily oxidized than those from outside, suggesting the need for a more rigorous biofilm abatement strategy for inside paper machines.

Enumeration of heterotrophs, fecal coliforms and Escherichia coli in water: comparison of 3M Petrifilm plates with standard plating procedures.

A total of 177 naturally contaminated water samples were analyzed by membrane filtration according to the Standard Methods for the Examination of Water and Wastewater published by the American Public Health Association. Filters were incubated in parallel on mHPC-agar and 3M Petrifilm Aerobic Count Plates (Petrifilm AC plates) for heterotrophic counts. Fecal coliforms and Escherichia coli were enumerated on mFC-agar and 3M Petrifilm E. coli/Coliform Count Plates (Petrifilm EC plates). Typical colonies on each media type were confirmed following standard procedures. Heterotrophic counts were between 10(3) and 10(4) CFU/mL and the average log10 counts obtained on Petrifilm AC plates were about two-fold lower than on mHPC-agar. Counts for fecal coliforms and E. coli were between 10(2) and 10(3) CFU/mL. Average log10 counts for confirmed fecal coliforms obtained on Petrifilm EC plates were slightly lower than on mFC agar with a correlation coefficient of 0.949. The average log10 counts for confirmed E. coli on Petrifilm EC plates and on mFC agar were statistically not different (P=0.126) with a correlation coefficient of 0.879. Specificity of Petrifilm EC plates and mFC agar was evaluated by comparing typical colony counts with confirmed counts. On mFC agar, counts for typical colonies were by 2 log10 CFU higher than the actual confirmed counts. In contrast, on Petrifilm EC plates typical colony counts were almost identical to confirmed colony counts for both fecal coliforms and E. coli. This comparison illustrates the high specificity of Petrifilm EC plates for enumeration of both fecal coliforms and E. coli in water.

Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains.

Thirteen Listeria monocytogenes strains were used to grow biofilms on glass surfaces in static conditions at 37 degrees C for up to 4 days. After the initial 3-h adhesion and in subsequent 1-day intervals, cell numbers were determined using standard plate count after swabbing the cells from the glass surface. The three-dimensional structure of in situ biofilms was determined by confocal scanning laser microscopy (CSLM). After 3 h incubation, bacterial cells for all 13 strains of L. monocytogenes were found attached to glass slides and all strains formed biofilms within 24 h. The strains varied significantly in their ability to adhere to the surface and significant differences for cell numbers after 24 h biofilm growth were found. Cell counts in biofilms formed by five L. monocytogenes strains were monitored over 4 days. The counts increased for the first 2 days reaching 10(5) cfu/cm2, except for L. monocytogenes 7148 (10(4) cfu/cm2). After 2 days, cell counts remained at 10(5) cfu/cm2 for four strains (tested on days 3 and 4), while L. monocytogenes 7148 continued to grow and reached 10(5) cfu/cm2 on day 4. This difference in biofilm growth was not related to variations in growth rates of planktonic cells suggesting that growth behaviour of Listeria in biofilms may be different from their planktonic growth. CSLM revealed that the biofilms grown under static conditions consisted of two distinct layers with 0.5 log10 higher cell numbers in the bottom layer as compared to the upper layer.

Identification of contamination sources of Bacillus cereus in pasteurized milk.

In order to determine the sources of Bacillus cereus in pasteurized milk, a total of 232 milk samples from various sampling points along milk processing lines and 122 environmental swabs were collected in two dairy plants between March and September, 1996. The incidence of B. cereus vegetative cells in raw milk from the plants was low (< or = 10%). However, the incidence and the average counts of B. cereus spores in the raw milk were very high and similar to those of B. cereus vegetative cells in pasteurized milk or final products after enrichment (> 80% and 1.1 x 10(5) cfu ml(-1), respectively). The incidence and average count of both vegetative cells and spores of B. cereus in environmental swabs was low. Using the microbial identification system (MIDI), a library of B. cereus fatty acid profiles comprising 229 B. cereus isolates from milk samples and environmental swabs was constructed using a critical Euclidian distance of 6.0 units as the cut-off value. Using this library, the relationship between 546 B. cereus isolates from the different sampling points along the milk processing lines and the environmental swabs was determined. Most B. cereus isolates obtained from the pasteurized milk and final products belonged to the same sub-groups as the B. cereus strains germinated from spores in raw milk. Furthermore, specific sub-groups were found in pasteurized milk, different dairy plants and at different sampling times. The results suggested that B. cereus spores in raw milk were the major source of B. cereus in pasteurized milk and that post-pasteurization contamination along the milk processing lines was possibly a minor source of B. cereus in pasteurized milk.

Rapid monitoring method to assess efficacy of sanitizers against Pseudomonas putida biofilms.

Biofilms of luminescent Pseudomonas putida were developed on rubber surfaces by incubation in brain heart infusion (BHI) broth. Scanning electron microscopy (SEM) and epifluorescence microscopy (EFM) were used to examine biofilm formation. To test the efficacy of two sanitizers commonly employed in dairy plants for CIP (cleaning in place) procedures, a novel bioluminescence method and aerobic plating were used to enumerate cells. Immediately after the sanitizer treatments an apparent 5-log reduction of biofilm-associated cells was determined. However, when the samples were resuscitated for 18 h in BHI broth, high numbers of cells were detected which reached levels close to those of nontreated controls. The results demonstrated that neither sanitizer could completely eliminate biofilm-associated P. putida. The microbial bioluminescence method proved to be the best way for assessing effectiveness of sanitizers against microbial biofilms.

Identification of Bacillus cereus by Fourier transform infrared spectroscopy (FTIR).

The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates. Ten B. cereus group isolates (comprising B. cereus, Bacillus mycoides, and Bacillus thuringiensis strains), five other Bacillus spp., and five non-Bacillus spp. were used. Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested. The results indicated that all B. cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm-1. These peaks were not affected by the growth medium. None of the other bacteria tested showed a similar peak after growth on BHI or TSA. Absorbance peaks between 1800 and 1500 cm-1 of members of the B. cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B. cereus group.

Epidemiological typing of Bacillus spp. isolated from food.

Biotypes, fatty acid profiles, and restriction fragment length polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB gene) were compared for 62 isolates of the Bacillus cereus group. Eleven isolates originated from various foods, and 51 isolates were obtained from pasteurized milk which had been processed by two different dairies. The isolates were clustered into 6 biotypes, 10 fatty acid groups, or 7 PCR-RFLP clusters. Isolates with mesophilic or psychrotrophic characteristics were preferentially distributed into specific fatty acid or PCR-RFLP groups (P = 0.004). Unique fatty acid clusters were predominantly found in milk samples of each dairy (P < 0.0001), suggesting that certain dairy plants may harbor plant-specific B. cereus which might constantly contribute to postpasteurization contamination.

Specific Oligonucleotide Primers for Detection of Lecithinase-Positive Bacillus spp. by PCR.

Volume 61, no. 1, p. 100, Table 3: in columns 7 and 8, row 3, "-" should read "+." [This corrects the article on p. 98 in vol. 61.].

Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR.

An assay based on the PCR has been developed to facilitate detection and identification of Bacillus cereus in foods. Three primers for the PCR have been designed within the sequence for cereolysin AB, a cytolytic determinant that encodes lecithin-hydrolyzing and hemolytic activities of B. cereus. With the PCR and hybridization, the specificity of the primers was tested with 39 isolates of the B. cereus group, with 17 other Bacillus spp., and with 21 non-Bacillus strains. Results demonstrate a high specificity of the three oligonucleotides for isolates of the B. cereus group. With a combined PCR-hybridization assay, the detection limit for B. cereus in artificially contaminated milk was 1 CFU/ml of milk.

Characterization of Bacillus licheniformis with the RAPD technique (randomly amplified polymorphic DNA).

RAPD technique (randomly amplified polymorphic DNA) was used for epidemiological subtyping of Bacillus licheniformis and other Bacillus spp. Within 46 isolates of B. licheniformis, up to 10 strain types could be determined when two 10-mer primers were used. RAPD patterns, which were found in eight further strains of Bacillus spp., clearly differed from those of B. licheniformis. Thus RAPD technique proved to be a promising tool for characterization of Bacillus spp.

[Quality assurance in food production: a challenge for veterinary food hygiene?]. / Qualitätssicherung in der Lebensmittel-produktion: Eine Herausforderung für die tierärztliche Lebensmittelhygiene?

Production liability, safety regulations as well as marketing strategies are important motivations for companies to establish in-house quality control measures which can be certified by an independent control body. Companies that advertise quality control measures place pressure on opposition companies to follow suite. It follows that certification in foodstuff production will lead to a chain reaction and spread quickly to other companies. In the establishment of a quality assurance system it is just as important to distinguish between quality control and quality assurance as it is to consider the guidelines laid down in the ISO-Standards 9000-9004. After certification according to the ISO-Standards, which dictates certain forms of quality assurance, there is no guarantee that all important risks, such as hygiene risks introduced with technology, can be excluded. In terms of public health monitoring an important responsibility awaits veterinary public health. Strategies and consequences for official monitoring of foodstuffs are presented and discussed.

Contamination of pig hindquarters with Staphylococcus aureus.

Swab specimens from 4357 pig hindquarters provided for production of cured raw ham were contaminated with Staphylococcus aureus in 22.7%. The bacterial counts for S. aureus on the rind surface of the raw, uncured ham were between 10(1) and 10(3) cfu/cm2 in 89% of the positive samples. In the remaining 11% of contaminated ham counts of 10(3) to 10(6) cfu/cm2 were determined. There were major differences in the rate of contamination between pork from different suppliers. Questioning the suppliers revealed that pork highly contaminated with S. aureus could be traced back to certain abattoirs. This suggests that the technique of slaughter, concomitant hygiene precautions and the subsequent refrigeration of the carcasses affect the contamination of the meat with staphylococci.