Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism.

Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man-Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high-throughput characterization of psychrotrophic bacteria prevailing in MAP meat.

Testing of amplified fragment length polymorphism (AFLP) technique as a tool for molecular epidemiology of Trichinella nativa.

A total of nine Trichinella nativa isolates were compared by amplified fragment length polymorphism (AFLP). Four hundred nanograms of genomic DNA from a pool of 10--20 larvae were digested using HindIII and MseI restriction endonucleases. Of the 16 primer combinations initially tested, Hind-C and Mse-C primers showed rich polymorphism with approximately 40--90 bands in the range of 30--270 bp. Genetic similarities were estimated visually. AFLP provided discriminatory banding patterns and may therefore be used as a method for detecting variation in T. nativa populations. However, the heterogeneous patterns obtained from pooled samples emphasize the need for further development of the sampling and numerical analysis of the patterns for epidemiological and taxonomical interpretation.

Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Finnish canine and feline flexispira strains.

The evolution and taxonomy of enterohepatic Helicobacter species with flexispira morphology were studied by a polyphasic approach including phenotypic characterization, analysis of 16S rRNA and ureB gene sequences and dot-blot DNA-DNA hybridization of whole genomic DNA. In addition, available phylogenetic data on the HSP60 gene were used in the analysis. The study included 14 Finnish canine and feline flexispira strains, the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Helicobacter bilis ATCC 51630(T). Phenotypically, all canine and feline strains were similar to H. bilis. Analysis of 16S rRNA gene sequences of these strains revealed a similarity of 97-99.5 %. Similarity of ureB nucleotide and amino acid sequences within the studied flexispira group was 97-100 % and 99-100 %, respectively, revealing close relatedness. ureB sequences of Helicobacter hepaticus had only 64-66 % similarity to the flexispira group. The similarity to Helicobacter trogontum was 81.5-82.1 %. High levels of DNA-DNA hybridization between the strains were found in dot-blot tests. Polyphasic analysis of the phenotypic and genotypic characteristics of the Finnish flexispira strains and the reference strains of taxa 2, 3 and 8 showed that they differed from other Helicobacter species and are members of the previously described species H. bilis. In addition, canine strain F56 differed in all phylogenetic analyses from the H. bilis group and probably represents a novel Helicobacter species.

Prevalence of Listeria monocytogenes in broilers at the abattoir, processing plant, and retail level.

The environment and products from two broiler abattoirs and processing plants and raw broiler pieces at the retail level were sampled for Listeria monocytogenes in order to evaluate the contamination level of the broiler carcasses and products. Sampling started in the slaughtering process and finished with raw broiler meat or ready-to-eat cooked product. Sampling sites positive for L. monocytogenes at the broiler abattoir were the air chiller, the skin-removing machine, and the conveyor belt leading to the packaging area. The L monocytogenes contamination rate varied from 1 to 19% between the two plants studied. Furthermore, 62% (38 of 61) of the raw broiler pieces, bought from retail stores, were positive for L. monocytogenes. Altogether, 136 L. monocytogenes isolates were obtained for serotyping and pulsed-field gel electrophoresis (PFGE) characterization performed with two rare-cutting enzymes (ApaI and AscI). Altogether three serotypes (1/2a, 1/2c, and 4b) and 14 different PFGE types were obtained using information provided from both ApaI and AscI patterns for discrimination basis. The two broiler abattoirs studied did not share the same PFGE types. However, the same PFGE types found in the raw broiler pieces at the retail level were also found in the broiler abattoirs where the broilers had been slaughtered.

Genotypes and enterotoxicity of Staphylococcus aureus isolated from the hands and nasal cavities of flight-catering employees.

Hand and nasal samples of flight-catering staff were collected from 1995 to 1997 to find employees carrying Staphylococcus aureus. Altogether 153 hand samples and 136 nose samples were taken. Nasal sampling showed a higher prevalence of S. aureus among food handlers (29%) than hand sampling (9%). A high proportion of the strains (46%) were enterotoxigenic, and a considerable amount of food handlers carried enterotoxigenic S. aureus, 6% and 12% according to hand and nasal sampling, respectively. Pulsed-field gel electrophoresis macrorestriction profiles revealed a total of 32 different types associated with the 35 employees carrying S. aureus. In most cases, the same type colonized both the hand and nose of a person. Despite the wide variety of types found, one strain colonized five persons and the second most common strain was associated with four food handlers. The predominant toxin produced was B, which was produced by the most common strain. The results showed that nasal sampling is a good way to detect S. aureus carriers, whereas hand sampling may fail to reveal carriers. The high proportion of enterotoxigenic strains show that a food handler harboring S. aureus must be considered a potential source of enterotoxigenic strains for airline meals.

Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions.

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.

Molecular epidemiology of an outbreak of febrile gastroenteritis caused by Listeria monocytogenes in cold-smoked rainbow trout.

Febrile gastroenteritis in five healthy persons was associated with the consumption of vacuum-packed cold-smoked rainbow trout containing Listeria monocytogenes. L. monocytogenes isolates from the incriminated fish product lot and the stool samples were all of serotype 1/2a and were indistinguishable by pulsed-field gel electrophoresis employing AscI and SmaI.

Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis.

One dominating strain of serotype 1/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant. Samples were taken from the production environment, equipment and ice cream during the years 1990-1997. Serotyping divided the isolates into two serovars, 1/2b and 4b. Three rare-cutting enzymes (ApaI, AscI and SmaI) were used in the creation of PFGE patterns. AscI resulted in the best restriction enzyme digestion patterns (REDPs) for visual comparison. Eight different AscI REDPs were obtained, whereas ApaI produced six and SmaI seven banding patterns. When one-band differences are taken into account, 12 different PFGE types were distinguished based on information obtained with all three enzymes. The dominant PFGE type was found to have persisted in the ice cream plant for seven years. Improved and precisely targeted cleaning and disinfection practices combined with structural changes making for easier cleaning of the packaging machine, resulted in eradication of L. monocytogenes from this plant.

Identification and characterization of Leuconostoc carnosum, associated with production and spoilage of vacuum-packaged, sliced, cooked ham.

Leuconostoc carnosum was shown to be the specific spoilage organism in vacuum-packaged, sliced, cooked ham showing spoilage during 3 weeks of shelf life. Identification of the specific spoilage organism was done by use of phenotypic data and ClaI, EcoRI, and HindIII reference strain ribopatterns. One hundred L. carnosum isolates associated with the production and spoilage of the ham were further characterized by pulsed-field gel electrophoresis (PFGE), together with some meat-associated Leuconostoc species: L. citreum, L. gelidum, L. mesenteroides subsp. dextranicum, and L. mesenteroides subsp. mesenteroides. ApaI and SmaI digests divided the industrial L. carnosum strains into 25 different PFGE types, ApaI and SmaI types being consistent. Only one specific PFGE type was associated with the spoiled packages. This type also was detected in air and raw-meat mass samples. The spoilage strain did not produce bacteriocins. Only seven isolates belonging to three different PFGE types produced bacteriocins. Similarity analysis of the industrial L. carnosum strains revealed a homogeneous cluster which could be divided into eight subclusters consisting of strains having at most three-fragment differences. The L. carnosum cluster was clearly distinguished from the other meat-associated leuconostoc clusters, with the exception of the L. carnosum type strain. Ribotyping can be very helpful in the identification of L. carnosum, but its discriminatory power is too weak for strain characterization. PFGE provides good discrimination for studies dealing with the properties of homogeneous L. carnosum strains.

Use of rRNA gene restriction patterns to evaluate lactic acid bacterium contamination of vacuum-packaged sliced cooked whole-meat product in a meat processing plant.

Molecular typing was applied to an in-plant lactic acid bacterium (LAB) contamination analysis of a vacuum-packaged sliced cooked whole-meat product. A total of 982 LAB isolates from the raw mass, product, and the environment at different production stages were screened by restriction endonuclease (EcoRI and HindIII) analysis. rRNA gene restriction patterns were further determined for different strains obtained from each source. These patterns were used for recognizing the spoilage-causing LAB strains from the product on the sell-by day and tracing the sources and sites of spoilage LAB contamination during the manufacture. LAB typing resulted in 71 different ribotypes, of which 27 were associated with contamination routes. Raw material was distinguished as the source of the major spoilage strains. Contamination of the product surfaces after cooking was shown to be airborne. The removal of the product from the cooking forms was localized as a major site of airborne LAB contamination. Food handlers and some surfaces in contact with the product during the manufacture were also contaminated with the spoilage strains. Some LAB strains were also able to resist cooking in the core of the product bar. These strains may have an effect on the product shelf life by contaminating the slicing machine. The air in the slicing department and adjacent cold room contained very few LAB. Surface-mediated contamination was detected during the slicing and packaging stages. Food handlers also carried strains later found in the packaged product. Molecular typing provided useful information revealing the LAB contamination sources and sites of this product. The production line will be reorganized in accordance with these results to reduce spoilage LAB contamination.