THE epidemiological value of different methods of distinguishing types within a bacterial species depends on their discriminating ability, reliability and ease of performance. Discriminating ability depends primarily on the number of types distinguished, but is poor if a large proportion of strains falls into a single type. Reliability depends on the reproducibility of each isolate's results in repeated tests and on the stability of the typing characters among different isolates from the same epidemic episode.Probably the best single method for differentiating types in Salmonella typhimurium is the phage-typing system of Callow (1943, 1951) , 1972). This method is less discriminating and more laborious than phage-typing, but it distinguishes different biotypes within individual phage-types, so that its combination with phage-typing gives a finer discrimination than either method alone (Kallings and Laurell, 1957;Rische and Kretzschmar, 1962;Lewis and Stocker, 1971).In a combined phage-typing and biotyping study of S. typhimurium we found some serious defects in the Kristensen scheme.(1) The fermentation tests with d-, Z-and rn-tartaric acids and citric acid proved unreliable and often gave different results on different occasions of testing the same isolate. Improved, more reliable methods were developed for the tartaric acids by Alfredsson et al.(1972) and these methods are used in the new scheme described in the present paper.(2) In our series of strains, both the original and the modified tests identified several biotypes with patterns of reactions different from those of the twenty-one types already recognised, but the new types could not be accommodated in the Kristensen classification in such a way as to show their relations to the existing types. For example, a group of strains designated " type 19Xd " by Morgenroth and Duguid (1968) resembled biotype 6 except that it was rhamnose negative; if this new type had been added to the scheme as type 22, its close relation to typ6 6 would not have been apparent. (3) We found that discrimination in biotyping could be improved by the addition of tests for the fermentation of trehalose in peptone water, fermentation of rhamnose in Bitter's medium, fermentation of inositol at 25"C, gas production from glucose, growthfactor requirements and presence of flagella and fimbriae, but the results of these extra tests could not be shown in any convenient way within the Kristensen classification. Mutational fermentation. Alfredsson et al. (1972)found that a major cause of unreliability in tests with the tartaric acids was the power of many non-fermenting strains to produce ~ ~~

299 ~~~ ~ ~Thermop hi I ic, redd is h-coloured he tero t rop hic bacteria different from Thermus were isolated from submarine alkaline hot springs in Iceland. The bacteria were obligately aerobic, moderately halophilic, Gram-negative rods, about 0.5 pm in diameter and 2-2.5 pm long. Neither spores, flagella nor lipid granules were observed, but a slime capsule was formed on carbohydrate-rich medium. Optimum growth was at 65 "C, pH 7.0, and at about 2% (w/v) NaC1. The bacteria were oxidase negative, catalase positive and contained a carotenoid pigment with the main absorbance peak at 476 nm and shoulders at 456 and 502 nm. The GC content of the DNA was about 64mol %. Electron micrographs clearly showed an outer membrane, about 9 nm thick, and the cytoplasmic membrane together with the peptidoglycan layer was about 14 nm in thickness. The isolates were nutritionally different from Thermus. They utilized several common sugars but glutamate and aspartate were the only amino acids that most strains used. These bacteria are considered to represent a new genus which we name Rhodothermus, with the type species Rhodothermus marinus.

We describe the observed relationship of campylobacter in poultry operations to human cases in a closed environment. During 1999 in Iceland, domestic cases of campylobacteriosis reached peak levels at 116/100,000 and in 2000 dropped to 33/100,000. Approximately 62% of broiler carcass rinses were contaminated with Campylobacter spp. in 1999. During 2000, only 15% of the broiler flocks tested Campylobacter spp. positive. In 2000, carcasses from flocks which tested positive on the farms at 4 weeks of age were subsequently frozen prior to distribution. We suggest that public education, enhanced on-farm biological security measures, carcass freezing and other unidentified factors, such as variations in weather, contributed to the large reduction in poultry-borne campylobacteriosis. There is no immediate basis for assigning credit to any specific intervention. We continue to seek additional information to understand the decline in campylobacteriosis and to create a risk assessment model for Campylobacter spp. transmission through this well defined system.

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 8C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (k cat /K m ) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase . proteinase K . aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H 2 O 2 -oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.

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SUMMARYThe colour-change and lead acetate tests for fermentation of d-, 1-and m-tartaric acids and citric acid used in the Kristensen scheme for biotyping Salmonella typhimurium were found to be unreliable because, whatever the conditions of culture, they gave