Verocytotoxin-producing Escherichia coli O157:H7 in the Swedish pig population.

Verocytotoxin-producing Escherichia coli O157:H7 (VTEC O157:H7) was detected in two of 2446 individual faecal samples collected from pigs slaughtered at five Swedish slaughterhouses, indicating a prevalence of 0.08 per cent, with a 95 per cent confidence interval from 0 to 0.16 per cent Four Swedish VTEC O157:H7-positive farms which kept ruminants and pigs were studied by repeated faecal sampling; VTEC O157:H7 was isolated from the ruminants and pigs on all the farms and the same strains were present in the pigs and the ruminants. On one of the farms, the organism persisted in the pig population for 11 months. On all four farms, management practices which might have influenced the isolation rate in pigs were identified. A group of young VTEC O157:H7-positive pigs was moved from one of the VTEC O157:H7-positive farms to a fattening herd where there were no ruminants. The number of VTEC O157:H7-positive faecal samples decreased gradually and after nine weeks the pigs were all negative; at slaughter none of the pigs was VTEC O157:H7-positive.

Bacteriological safety issues in red meat and ready-to-eat meat products, as well as control measures.

The importance of Eschericha coli O157, Listeria monocytogenes and Salmonella typhimurium DT104 as meat-borne pathogens is well established. Pathogenic bacteria such as Aeromonas spp., Arcobacter spp., psychrotrophic Bacillus cereus, Campylobacter spp., Clostridium botulinum and non-invasive Listeria monocytogenes can be regarded as rookies, but not yet firmly associated with today's production of red meat and meat products. The development of PCR and other DNA-based techniques will shed new light on so called emerging pathogens. Important safety issues in meat production, such as insufficient cleaning and disinfection (including the stable/lairage, processing environment), carcass decontamination and chilling, and cross contamination are discussed. Furthermore, probability modelling of survival and growth is identified as an important way to achieve a better understanding of how to deal with the complexity of further processing, including heat treatment and storage.

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs.

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

No sequence variation in part of the hexon and the fibre genes of adenovirus 8 isolated from patients with conjunctivitis or epidemic keratoconjunctivitis (EKC) in Norway during 1989 to 1996.

BACKGROUND/AIMS: Several local epidemics of keratoconjunctivitis/conjunctivitis caused by adenovirus type 8 (Ad8) occurred in Norway from August 1995 to May 1996. A smaller epidemic occurred in 1992. The Ad8 hexon forms the surface of the virion and contains the hypervariable regions loop I(1) and loop I(2). The fibre mediates the primary contact with cells. Sequence variation in hexon and fibre genes might play an important role in the pathogenicity of adenoviruses. The aim of this study was to investigate the genetic variability at the hexon and fibre genes in 26 strains of Ad8 isolated from 1989 to 1996. METHODS: The genetic variability of 26 strains of Ad8 isolated from 1989 to 1996 was studied by sequencing part of the hexon and fibre genes. The Ad8 sequences were compared with each other and with two Ad8 strains from the EMBL database. In addition, 14 of the 26 isolates were subjected to restriction endonuclease analysis. RESULTS: No significant sequence variation was seen during the six year period. CONCLUSION: The Ad8 strains causing epidemics of keratoconjunctivitis/conjunctivitis in Norway are genetically stable.

A model based on absorbance data on the growth rate of Listeria monocytogenes and including the effects of pH, NaCl, Na-lactate and Na-acetate.

A mathematical model was developed for predicting the growth of L. monocytogenes at 9 degrees C in the presence of 70 ppm sodium nitrite, and at different levels of pH (5.5-6.5), sodium chloride (1.0-4.0%), sodium lactate (0-0.5%) and sodium acetate (0-0.6%). Collection of the growth data was done using absorbance measurements in broth cultures and the absorbance measurement was evaluated. The model was compared to the Food MicroModel, and against the growth of L. monocytogenes in a vacuum-packed meat product stored at 9 degrees C. A linear relationship was obtained, for the absorbance data on different dilutions of the inoculum, in the absorbance interval studied. There was also a linear relationship between the values of the maximum specific growth rates derived from the absorbance and the ones derived from viable count measurements; and corrections were made accordingly. The statistical evaluation showed that all the main factors, i.e. pH, sodium chloride, sodium lactate and sodium acetate were statistically significant for the growth rate of L. monocytogenes. Comparison to the Food MicroModel (FMM) showed a slight underprediction for the developed model (bias = 0.84). The predictions were, on average, within 20% of the FMM predictions (n = 10). Validation against the observed growth of L. monocytogenes inoculated into an emulsion type of sausage (n = 4) also showed a slight underprediction by the model. The predictions were, on average, 16% below the observed values in the sausage (Bias 0.84, Accuracy 1.26).

Using an electronic nose for determining the spoilage of vacuum-packaged beef.

The use of an electronic nose in the quantitative determination of the degree of spoilage of vacuum-packaged beef was evaluated. Beef from four different slaughterhouses was sliced, vacuum-packaged and stored at 4 degrees C for 8 weeks. Samples were withdrawn for bacterial (aerobic bacteria, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas and Enterobacteriaceae) and sensorial analyses and analysis of the volatile compounds during the storage period. A trained panel was used for the sensorial evaluations. The volatile compounds were analysed using an electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors, four Tagushi type sensors and one CO2-sensitive sensor. Four of the 15 sensors were excluded due to lack of response or overloading. Partial least-squares regression was used to define the mathematical relationships between the degree of spoilage of vacuum-packaged beef, as determined by the sensory panel, and the signal magnitudes of the sensors of the electronic nose. The mathematical models were validated after 6 months using a new set of samples. The stability of the sensors during this period was examined and it was shown that the sensitivity of five of the 11 sensors used had changed. Using the six remaining sensors, the signal patterns obtained from the meat from the different slaughterhouses did not change over a period of 6 months. It was shown that the degree of spoilage, as calculated using a model based on two Tagushi sensors, correlated well with the degree of spoilage determined by the sensory panel (r2 = 0.94).

Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components.

A multiplex PCR assay including sample preparation was developed to detect viable pathogenic strains of Yersinia enterocolitica in PCR-inhibitory samples, such as pork and enrichment media. The method developed was used to simultaneously detect the plasmid-borne virulence gene yadA and a Yersinia-specific region of the 16S rRNA gene. According to an auto-agglutination test for virulence-plasmid-bearing strains of Y. enterocolitica, all potential pathogenic strains tested were detected by the assay. A DNA extraction procedure, an aqueous two-phase system composed of polyethylene glycol 4000 and dextran 40 and a buoyant density centrifugation method, based on Percoll, were compared with regard to their efficiency in separating Yersinia enterocolitica from PCR inhibitors originating from enrichment media and pork. Using the density gradient centrifugation method resulted in a detection level of 4.0 x 10(2) CFU Y. enterocolitica per ml enrichment media. To ensure detection of viable bacteria a short enrichment step was included in the sample preparation together with the density gradient centrifugation. When this sample treatment method was evaluated with a selective enrichment medium together with a background flora inoculated with approximately 1.0 x 10(1) CFU per ml of Y. enterocolitica and incubated at 25 degrees C, a positive PCR result was obtained after 6 to 8 h. Our results indicate that selective enrichment followed by buoyant density gradient centrifugation provides a convenient and user-friendly sample preparation method prior to PCR.

Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4 degrees C.

A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments. A consumer acceptance test of products produced with and without the compounds was also performed. Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products. This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products. The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w/w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4-6 weeks. In addition, strict control of temperature during production and storage is very important.

Bacterial spoilage of meat and cured meat products.

The influence of environmental factors (product composition and storage conditions) on the selection, growth rate and metabolic activity of the bacterial flora is presented for meat (pork and beef) and cooked, cured meat products. The predominant bacteria associated with spoilage of refrigerated beef and pork, are Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae, Lactobacillus spp., Leuconostoc spp., Pseudomonas spp. and Shewanella putrefaciens. The main defects in meat are off-odours and off-flavours, but discolouration and gas production also occur. Bacteria associated with the spoilage of refrigerated meat products, causing defects such as sour off-flavours, discolouration, gas production, slime production and decrease in pH, consist of B. thermosphacta, Carnobacterium spp. Luctobacillus spp. Leuconostoc spp. and Weissella spp. Analysis of spoilage as measured by bacterial and chemical indicators is discussed. It is concluded that a multivariate approach based on spectra of chemical compounds, may be helpful in order to analyse spoilage, at least for spoilage caused by lactic acid bacteria. The consequences of bacteria bacteria interactions should be evaluated more.

Hazard identification in swine slaughter with respect to foodborne bacteria.

Swine slaughter is an open process with many opportunities for the contamination of the pork carcass with potentially pathogenic bacteria; however, it does not contain any point where hazards are completely eliminated. Data on the prevalence of various pathogenic bacteria (Aeromonas hydrophila, Campylobacter coli/jejuni, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Yersinia enterocolitica) in pigs, their growth and survival characteristics and ability to become established on the slaughter line are presented. The presentation covers the processing steps from lairage to chilling and is based on swine slaughter practices in Denmark, Norway and Sweden. The major contamination points during swine slaughter are pig-related, such as faecal and pharyngeal, and environmental. HACCP (Hazard Analysis Critical Control Point) and GMP (Good Manufacturing Practice) in swine slaughter must be focused on limiting this spread. The pathogenic bacteria show differences in their general mechanism of distribution. The major contamination source of Campylobacter spp., Salmonella spp. and Y. enterocolitica is the pig, and the contamination of carcasses with these bacteria may be limited, provided that only strict slaughtering procedures are used. Other organisms such as Aeromonas spp., L. moncytogenes/Listeria spp. and S. aureus can be endemic in the processing environment. Since endemic bacteria can be controlled by proper cleaning and disinfection, these organisms are useful as indicators for the success of GMP rules. The following affiliation to CPs or CCPs made for specific steps during slaughter and dressing may serve as a guidance: (i) lairage (CP), (ii) killing (CP), (iii) scalding (CP), (iv) dehairing (CP), (v) singeing/flaming (CP), (vi) polishing (CP), (vii) circumanal incision and removal of the intestines (CCP), (viii) excision of the tongue, pharynx, and in particular the tonsils (CCP), (ix) splitting (CP), (x) post mortem inspection procedures (CCP) and (xi) deboning of the head (CCP).

Reduction of Yersinia enterocolitica and Listeria spp. on pig carcasses by enclosure of the rectum during slaughter.

By sealing off the rectum with a plastic bag immediately after it had been freed, the spread of Y. enterocolitica O:3/biovar 4 to pig carcasses could be considerably reduced. The organism was recovered from only 0.8% of carcasses when the plastic bag technique was employed. Y. enterocolitica O:3/biovar 4 was recovered from 10% of pig carcasses when eviscerating procedures did not include the use of the plastic bag technique. There was thus an obvious risk of the bacteria further contaminating meat cuts and other meat products. The plastic bag technique was effective both in connection with manual excision of the rectum/low throughput (90 per h), and mechanical freeing of the rectum/high slaughter rate (240 per h). L. monocytogenes was not detected in any of the samples taken from 120 pig carcasses in Norway or from 120 pig carcasses in Sweden. The plastic bag technique was used on half of these pigs. L. innocua was tested for in 120 pigs slaughtered in Sweden. The bacterium was recovered from 33% of the carcasses eviscerated without using a plastic bag, and from 10% of the carcasses in which this technique was employed. The results suggested that there were other, non-faecal, sources of contamination. Other measures in addition to the plastic bag technique are therefore required to limit the spread of Listeria spp. By incorporating the plastic bag technique into the slaughtering procedures, the meat industry would contribute to preventing the dissemination of Y. enterocolitica and other pathogens which spread via the faeces.

Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method.

A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase.

Predicting the aerobic growth of Y. enterocolitica O:3 at different pH-values, temperatures and L-lactate concentrations using conductance measurements.

The effect of the inoculum level and growth conditions on conductance response was studied for Yersinia enterocolitica O:3. The Gompertz equation, y = A+C exp (-exp(-B(time(-)-M))) was used in the fitting of conductance response curves. Inoculum levels between 3 and 7 log cfu/ml did not affect the B or C parameters. The M parameter was affected; the lower the inoculum level, the higher the M value. Conductance response was attained at 7.5 log cfu Y. enterocolitica/ml skim milk-SPYE medium used. Polynomial models for log B and log C were developed for Y. enterocolitica O:3 describing the effect of temperature (7-23 degrees C), pH (5.4-6.5) and L-lactate (0-1.2%), and combinations thereof, under aerobic conditions. Conductance response was attained in all combinations of L-lactate concentrations, pH levels and temperatures. The conductance rate (B.C/e) was of the same magnitude at 23 degrees C, pH 5.4 and 1.2% L-lactate as at 7 degrees C, pH 6.5 and 0% L-lactate. A high correlation was found between the conductance rates predicted from conductance polynomial models and rates predicted from an absorbance model taken from the literature. A large number of combinations of factors affecting the growth/activity of bacteria could be studied simultaneously, due to the large instrumental capacity of the Malthus 2000.

Lipolysis, proteolysis and formation of volatile components during ripening of a fermented sausage with Pediococcus pentosaceus and Staphylococcus xylosus as starter cultures.

Bacterial growth, formation of acids, lipolysis, proteolysis, fat oxidation, formation of volatile compounds and flavour characteristics were followed during ripening and storage of a fermented sausage. The starter culture used was composed of Pediococcus pentosaceus and Staphylococcus xylosus. The number of Pediococcus sp. increased by 1.5 log cfu/g during the first day of processing and remained constant at this level for 3 weeks. The corresponding initial increase in the numbers of Staphylococcus sp., 0.4 log cfu/g, was followed by a rapid decrease in the viable numbers. Lactic acid, mainly d-lactic acid, and acetic acid were formed during ripening. The triglycerides were hydrolysed to 1,2-diglycerides and free fatty acids at the beginning of ripening, followed by the formation of 1,3-diglycerides and monoglycerides, indicating lipolytic activity. Moreover, the nonprotein nitrogen increased during ripening as a result of the proteolytic activity. Most of the changes with respect to pH, formation of d-lactic acid, acetic acid, peroxides and flavour development occurred during the initial 3 days of ripening, when growth of Pediococcus sp. and Staphylococcus sp. occurred. Lipolysis as well as proteolysis continued after this initial period. The volatile compounds identified belonged to several chemical families, viz. aliphatic and aromatic hydrocarbons, aldehydes, ketones, alcohols, phenols, carboxylic acids, esters, nitrogen compounds, sulphur compounds, chloride compounds, terpenes and furans. Many of the volatile compounds probably originated from smoke and seasoning (onion/garlic and pepper), while others were a result of the activities of muscle enzymes and bacteria.

Contamination of beef carcasses by psychrotrophic Pseudomonas and Enterobacteriaceae at different stages along the processing line.

The extent of the contamination of beef carcasses with psychrotrophic Pseudomonas spp. and Enterobacteriaceae during slaughter, chilling and cutting was estimated by introducing a new analytical procedure; the contamination index. Comparisons were made between the initial viable counts and the contamination index. The contamination index was calculated as the sum of the bacterial counts obtained during aerobic cold storage of excised meat samples. The presence and composition of spoilage bacteria in the slaughter environment and on the carcasses was also determined at one plant. Rapid chilling was identified as a critical processing step by the contamination index. In addition to this, the dehiding and the chilling in cold storage rooms were implicated as critical operations, with respect to aerosol contamination and surface cross-contamination. Comparison of the composition of spoilage bacteria in the slaughter environment and the bacteria proliferating on the carcass surface samples taken at the corresponding steps showed similar distributions of the identified Pseudomonas spp. In five surveys at two plants, the contamination of beef carcasses along the processing line was estimated. Statistically significant variations between different processing steps were more pronounced for the contamination index than for the conventional counts. It was concluded that the contamination index could be used for identifying critical processing steps, with respect to the extent of contamination of carcasses by psychrotrophic spoilage bacteria.

Evaluation of bacterial contamination at separate processing stages in emulsion sausage production.

The contamination with spoilage bacteria at separate production steps during the production of emulsion sausages was evaluated using a special sampling and evaluation method. Heat processed and chilled sausages were aseptically transferred directly to cold storage, cutting down or packing. Upon completion of the particular production step the sausages were vacuum-packed and stored at 8 degrees C. During storage, the microbial growth of the sausages was followed and the area under the plot of aerobic count versus storage time was calculated. No correlation was found between the total aerobic count of unstored samples and bacterial growth during storage, defined as area under growth curves. Furthermore, the count of lactic acid bacteria on unstored sausages was often below the detection limit. However, the area reflected the extent of contamination during processing with bacteria able to grow on cold-stored vacuum-packed sausages. Storage in a cold storage room was identified as a critical point with respect to bacterial recontamination and shelf-life.

Chemical, microbial and sensory changes during the anaerobic cold storage of beef inoculated with a homofermentative Lactobacillus sp. or a Leuconostoc sp.

Slices of beef were inoculated with about 3.5 log cfu/cm2 of Lactobacillus sp. 93 SMRICC 235 (homofermentative) or Leuconostoc sp. 89 SMRICC 189 and stored in 5% CO2 + 95% N2 at 4 degrees C. The microbial, chemical (glucose, L-lactate, D-lactate, acetate, formate, ethanol, H2S) and sensory changes of the beef slices were studied. For beef inoculated with Lactobacillus sp. 93 the flavour score started to decrease when the maximum bacterial count was reached. Leuconostoc sp. 89 caused a rapid decrease in the flavour score before reaching the maximum bacterial count. Concentrations of acetate and D-lactate increased while glucose and L-lactate decreased in beef slices inoculated with Lactobacillus sp. 93. In the presence of Leuconostoc sp. 89 ethanol and D-lactate increased while glucose decreased. Lactobacillus sp. 93 formed the highest level of H2S, and a sulphurous off-odour was noted only in the presence of this strain. D-Lactate and acetate indicated high numbers of Lactobacillus sp. 93 on the meat surface, while D-lactate and ethanol indicated high numbers of Leuconostoc sp. 89. More studies are needed in order to correlate levels of D-lactate, acetate and ethanol with sensory changes.

Identification of major contamination sources during processing of emulsion sausage.

The extent of contamination of an emulsion type of sausage with lactic acid bacteria was determined along the processing line. This was done by aseptically removing sausages after five different processing stages (heat processing, chilling, cold storage, cutting down and packing). Removed sausages were vacuum-packed and stored at 8 degrees C. The microbial growth was followed during storage and the microbiological shelf-life obtained at the different stages of the processing was determined. The spoilage flora of stored sausages was identified/grouped. Two major hygienic problems were identified: (1) a heat tolerant flora of Lactobacillus viridescens which survived the heat processing and was never outgrown by the recontaminating flora; (2) recontamination with a flora dominated by Lactobacillus sp. group 5, which occurred in the cold storage room; this flora dominated in the absence of L. viridescens. The heat tolerant L. viridescens SMRICC 193 survived at 68 degrees C for 40 min. Being exposed to a slowly increasing temperature, only a 10 cfu/ml decrease took place when the temperature increased from 60 degrees C to 70 degrees C over a period of 30 min.