Eija Hyytiä

A high prevalence of Clostridium botulinum type E in finnish freshwater and Baltic Sea sediment samples

The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined. A total of 110 samples were tested with a neurotoxin‐specific PCR assay. Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. Spore counts were quantified by the most probable number method, Cl. botulinum type E kg−1 averaging 940 in sea and 370 in freshwater samples. The overall prevalence and spore counts of Cl. botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature. These findings indicate the possibility of Cl. botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.

Prevalence of Clostridium botulinum type E in Finnish fish and fishery products.

The prevalence of Clostridium botulinum type E gene in fish and fishery products of commercial importance in Finland was determined using a quantitative PCR analysis. The contamination level in 438 raw fish samples from intestines, surface and whole fish and 208 fish roe samples varied from 10-40% and from 4-14% respectively, depending on the fish species studied. The presence of C. botulinum in European wild freshwater fish and roe was demonstrated for the first time by isolation of the organism from PCR-positive samples. Five percent of 214 vacuum-packed and 3% of 123 air-packed fishery product samples examined at retail level were positive for the botulinum neurotoxin type E gene. A contamination level of 10% in vacuum-packed hot-smoked whitefish was detected. The results demonstrate that C. botulinum type E poses a serious health risk for those consuming fishery products from the Baltic Sea area.

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.

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

The spoilage flora of vacuum-packaged, sodium nitrite or potassium nitrate treated, cold-smoked rainbow trout stored at 4°C or 8°C

The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4 degrees C and 8 degrees C, was studied. Of 620 isolates, lactic acid bacteria were the major fraction (76%), predominating in all samples of spoiled product. However, the phenotypical tests used were insufficient to identify the lactic acid bacteria to the species level. Gram-positive, catalase-positive cocci, gram-negative, oxidase-negative rods and gram-negative, oxidase-positive rods were found in 6%, 16% and 2% of the samples, respectively. Of 39 gram-positive, catalase-positive cocci, 29 were identified as staphylococci and 10 as micrococci. Eighty-five isolates were found to belong to the family Enterobacteriaceae, with 45 of those being Serratia plymuthica. Eleven isolates from the nitrate treated samples stored at 8 degrees C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8 degrees C, may cause problems with respect to the safety of the product. The types of lactic acid and other bacteria in the spoilage flora were generally reduced by the addition of nitrate or nitrite to fillets.

Type E botulism associated with vacuum-packaged hot-smoked whitefish

On January 16, 1997 two Germans got botulism after eating hot-smoked Canadian whitefish produced in Finland. The serum sample of one of the patients contained 6 MLD/ml of botulinum toxin. The type of toxin was identified as E by the toxin neutralization test and the botulinum neurotoxin type E (BoNT/E) gene was also amplified from the serum by polymerase chain reaction (PCR), but C. botulinum could not be isolated from the positive serum sample. The remains of the hot-smoked whitefish eaten by the patients contained botulinum toxin detected by the mouse bioassay and the BoNT/E gene as determined by PCR. C. botulinum was isolated from the fish sample and it was confirmed to be type E by the mouse bioassay and by PCR. Eleven other fish samples from the same lot did not contain botulinum toxin nor any BoNT gene. The incriminated food was processed on the 9th and 10th of January, 1997 from frozen whitefish imported to Finland from Canada. The pulsed-field gel electrophoretic pattern of the isolated C. botulinum strain resembled a reference strain of North American origin. It did not match any C. botulinum strains isolated from the Baltic sea-bottom or from the fish caught in the area indicating that the fish was contaminated by C. botulinum in Canada. The conditions resulting in toxin production could not be identified. The safety problems associated with vacuum-packaged hot-smoked fish seem to be of utmost concern and the product is one of the most important botulism food vehicles processed on an industrial scale. Temperature monitoring and the use of time-temperature indicators are to be recommended in order to ensure adequate storage temperature from processing through to consumption. Allowing the use of nitrate and nitrite together with sufficiently high NaC1 concentration in this particular product should also be considered.

Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism

Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism. Thirteen restriction enzymes were initially tested for suitability for ribotyping of C. botulinum, of which EcoRI and HindIII were selected. Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C. botulinum, and can be recommended for Group/species identification. Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C. botulinum groups was performed. EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret. All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates. The Clostridium strains diverged at 35+/-13% (mean+/-standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results. These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C. botulinum species identification.