Prevalence of the enterotoxin gene and clonality of Clostridium perfringens strains associated with food-poisoning outbreaks.

The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR). The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases. The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination. Of the C. perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively. Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak. The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element.

Persistence in bovine mastitis of Staphylococcus aureus clones as assessed by random amplified polymorphic DNA analysis, ribotyping and biotyping.

Staphylococcus aureus isolates (N = 40) from bovine mastitis were characterized by random amplified polymorphic DNA-PCR (RAPD-PCR), ribotyping and biotyping. The isolates were collected in the veterinary surveillance area of the Ambulatory Clinic, Faculty of Veterinary Medicine, University of Helsinki from 20 quarters during the acute phase of infection and from the same quarters 3 weeks after cessation of therapy. The aim of the study was to compare the S. aureus isolates taken from the same quarter at different times to verify persistence of virulent strains in infected quarters and to compare the discriminatory power of the diagnostic methods. Using all methods (except for a commercial diagnostic test), the paired isolates of S. aureus were identical. Results suggest that the chronic nature of S. aureus infections was due to the persistence of the original infective strain. More laborious ribotyping and the more convenient RAPD-PCR method produced identical results. The molecular methods differentiated the 40 isolates into 6 distinct genotypes. Biotyping produced partially identical results to RAPD and ribotyping. A commercial diagnostic test system identified only 3 S. aureus biotypes.

Minimum growth temperatures of Hafnia alvei and other Enterobacteriaceae isolated from refrigerated meat determined with a temperature gradient incubator.

Minimum growth temperatures of Hafnia alvei (n = 156) and other Enterobacteriaceae isolates (n = 162) from refrigerated meat samples (n = 88) and control strains of H. alvei (n = 81) from clinical and environmental samples were determined with a plate-type continuous temperature gradient incubator on nutrient agar. The dominant species, Hafnia alvei and Serratia liquefaciens had mean minimum growth temperatures of 2.6 (range, 0.2-3.7 degrees C) and 1.7 (range, 0.2-2.6 degrees C), respectively. Values for other species included: Enterobacter agglomerans, 1.3 (0.7-1.7 degrees C); Escherichia coli, 8.7 (8.4-8.9); Escherichia vulneris, 1.6 (0.8-2.6 degrees C); and Serratia fonticola, 2.0 (1.1-3.0 degrees C). The H. alvei reference strains did not differ markedly from the meat isolates, with the exception of the diarrhoeagenic eae A positive strains (10.6, 10.2-11.5 degrees C). The representatives of H. alvei hybridization groups (HG) 1 and 2 did not differ in their minimum growth temperatures. The observed heterogeneity of the minimum growth temperatures of many Enterobacteriaceae species may be explained by limitations of the systems used for identification of enterobacteria, inadequacy of the Enterobacteriaceae taxonomy or true growth temperature heterogeneity within the various species.

Characterization of Lactobacillus sake strains associating with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns.

Randomly amplified polymorphic DNA (RAPD) patterns and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of ropy slime-producing Lactobacillus sake strains. The two most revealing commercially available primers (OPJ 12 and OPJ 16, Operon Inc. Alameda, USA) and two rare-cutting enzymes (AvrII and SmaI) were chosen from a pretested lot for the typing of 69 ropy slime-producing strains, 7 non-ropy isolates and 4 non-ropy reference strains. Both RAPD and PFGE patterns confirmed the group division established in previous studies and provided new information concerning ropy slime-producing strains. PFGE patterns were found to have the greatest discriminatory power, revealing the genetic variation of the main group of ropy slime-producing L. sake strains and distinguishing all non-ropy strains from slime-producers.

Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction.

A test protocol for the detection and enumeration of Clostridium botulinum in fish and sediment samples with specific identification of neurotoxin types A, B, E and F was developed. Specific amplification products generated by polymerase chain reaction (PCR) formed the basis of identification of the toxin-producing organism, whereas quantification of the results was achieved with the most probable number (MPN) method. Twenty-six C. botulinum strains studied with PCR assays after enrichment in trypticase-peptone-glucose-yeast extract (TPGY) broth gave identical results as with the mouse bioassay. The suitability of the detection method for food and environmental surveys was assessed by running it on 32 samples of rainbow trout inoculated with spore loads ranging from 10(2) to 10(6) C. botulinum type E spores per kg. The organism was detected in all samples, and MPN estimates corresponded well to inoculum levels. In order to assess possible natural contamination, 16 fish and 16 visceral samples of rainbow trout, as well as ten aquatic sediment samples were tested. Of these, eight (80%) of the sediment samples were positive, with estimated spore counts of C. botulinum type E ranging from 95-2710 per kg sample.

Characterization of Hafnia alvei by biochemical tests, random amplified polymorphic DNA PCR, and partial sequencing of 16S rRNA gene.

Hafnia alvei strains which possess the attachment-effacement gene (eaeA) may have clinical importance as new diarrhea-causing pathogens and should therefore be differentiated from other H. alvei strains. We characterized diarrheal H. alvei strains, which were positive in the PCR test for the eaeA gene, using biochemical tests not routinely used for identification of members of the family Enterobacteriaceae, and compared them with eaeA-negative strains isolated from different clinical and nonclinical sources to find characteristics useful for identification. Random amplified polymorphic DNA (RAPD)-PCR and partial sequencing of the 16S rRNA gene were utilized to study the genetic diversity of the isolates. The eaeA-positive strains were found to have many characteristic biochemical properties. Negative reactions in the 2-ketogluconate and histidine assimilation tests and a positive reaction in the 3-hydroxybenzoate assimilation test may be useful in routine diagnostics. Nearly identical RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes indicated little genetic diversity among the eaeA-positive strains. The low level of homology (92%) in the partial 16S rRNA genes of eaeA-positive and -negative H. alvei strains raises questions about the taxonomic positioning of eaeA-positive H. alvei.

Phenotypic and molecular characteristics of Aeromonas salmonicida subsp. salmonicida isolated in southern and northern Finland.

Aeromonas salmonicida subsp. salmonicida causes outbreaks of furunculosis in salmonid fish. Furunculosis was first detected in Finland in 1986 on fish farms located on the Finnish coast of the Bothnian Bay. Molecular methods, SDS-PAGE, ribotyping, randomly amplified polymorphic DNA (RAPD) and plasmid profile analysis as well as phenotypic characteristics (biochemical characteristics, maximum growth temperature, pigment and elastase production) were used both for typing the strains and to study the possible routes of transmission of the organism to Finland and the spread of infection within Finland from 1986 to 1993. Ribopattern analysis of chromosomal DNA digested with SmaI, BglI, PstI and ClaI of 28 Finnish strains and eight foreign strains (Denmark, Sweden, Norway or Canada) showed that all Finnish strains and the Swedish strain originating from the Swedish coast of the Bothnian Bay had identical ribopatterns. All other foreign strains had distinct, unique ribotypes except for the second Swedish strain studied, the ribotype of which was identical with that of one Danish strain. RAPD typing, based on the results of two arbitrary primers, yielded 15 types for the Finnish strains. Except for both Danish strains, which had the RAPD type which was identical with that of one Finnish strain, the foreign strains had RAPD patterns differing from those of the Finnish strains. Plasmid profile typing and RAPD profile typing did not correlate. Ribotyping with four different enzymes proved to be the most sensitive method for studying genetically homogeneous Aer. salmonicida subsp. salmonicida. Ribopattern analysis showed that the infection which first started in 1984/1985 on the Swedish coast of the Bothnian Bay may have been transmitted to Finland where the first outbreaks occurred in 1986. The strains infecting Finnish fish farms were very homogeneous, with most differences seen, for example, in maximum growth temperature, plasmid profiles and the RAPD profiles of the strains.

Hafnia alvei in stool specimens from patients with diarrhea and healthy controls.

We found an epidemiological association of Hafnia alvei with diarrhea, because the organism was isolated from 12 of 77 (16%) adult Finnish tourists to Morocco who developed diarrhea and from 0 of 321 tourists without diarrhea (P < 0.001). From another group of 112 adult Finnish diarrheal patients, only 2 (2%) yielded H. alvei. In contrast to some Bangladeshi strains of H. alvei, the Finnish strains were negative for the attachment-effacement lesion by an in vitro fluorescent acting staining test and also did not show homology to the Escherichia coli attachment-effacement gene (eaeA) by PCR. These results suggest that a mechanism or mechanisms other than the attachment-effacement lesion may also be involved in the association of H. alvei with diarrhea.

Special treatment during slaughtering in Finland of cattle carrying an excessive load of dung; Meat hygienic aspects.

The aim of the study was to evaluate the microbiological consequences of the Finnish special hide regulations. These were agreed upon, by a committee including members representing meat production, industry and the state, to reduce the number of cattle carrying excessive loads of dung. The changes in the number of excessively dungy cattle in one beef abattoir have been recorded since the adoption of the special rules. The effect of excessive dunginess of the hide on microbial contamination of the carcasses was measured in the same abattoir using excision sampling technique. From 1983 to 1990 the proportion of excessively dungy cattle has decreased 85%, with the majority of them arriving from October to March. Excess dunginess resulted in statistically significantly increased microbial contamination at both sampling sites. Results indicate that the regulations are reasonable from the point of view of meat hygiene and that, for commercial-scale slaughtering, the drawbacks caused by the excess dunginess of the hide cannot be compensated for by greater care in the work procedures.

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.