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Four strains of a hitherto unknown bacterium isolated from vacuum-packaged refrigerated beef were characterized by using phenotypic and phylogenetic methods. The novel strains were Gram-positive, catalase-negative, psychrophilic, rod-shaped bacteria with lactic acid-homofermentative mechanism. Comparative 16S rDNA gene sequencing demonstrated that the unknown strains represent a novel subline within the genus Lactobacillus, close to but distinct from Lactobacillus curvatus and Lactobacillus sakei. The unknown strains were readily distinguished from all currently described members of the genus Lactobacillus by biochemical properties and SDS-PAGE whole-cell protein profiles. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Lactobacillus fuchuensis sp. nov. The type strain is strain B5M10T (= JCM 11249T = DSM 14340T).

Five fresh beefcut samples were divided into 10 pieces, respectively, vacuum-packaged and stored at 2 degrees C for up to 6 weeks. The average pH values of five pieces from five beef samples were 5.62 +/- 0.04 at the start of storage and 5.12 +/- 0.07 after 6 weeks of storage. The pieces were homogenized, diluted and cultivated at weekly intervals on glucose-blood-liver (BL), de Man Rogosa and Sharpe (MRS) and trypticase soya (TS) agars at 7 degrees C for 10-14 days. From plates with 30-300 colonies, or the highest number if below 30, 15 colonies were randomly picked from each of the three media used for each beef sample. Based on morphologies, SDS-PAGE whole-cell protein profiles and physio-chemical characteristics, a total of 1493 strains isolated were identified as Brochothrix thermosphacta (64), Carnobacterium divergens (79), Carnobacterium piscicola (27), Lactobacillus algidus (637), Lactobacillus sp. (4), Lactococcus piscium (270), Leuconostoc gelidum (375), Acinetobacter (3), Aeromonas (1), Bacillus (10), Corynebacterium (3), Enterobacteriaceae (1), Pseudomonas (13) or Psychrobacter (6). A wider range of organisms was isolated from TS (13 organisms) and BL (7) agars than from MRS agar (3). Leuc. gelidum, Lc. piscium and L. algidas increased in numbers during the first 3 weeks of storage from about 5 x 10(3) cfu/g to the level of about 10(8) cfu/g and persisted at this level thereafter. C. divergens and C. piscicola were inconsistently detected, but seemed to persist at the relatively low level of about 5 x 10(7) cfu/g during the last 3 weeks of storage. B. thermosphacta increased to the level of about 7 x 10(5) cfu/g during the first 2 weeks of storage, but was not detected thereafter. Remaining organisms were detected sporadically at levels of <3.5 x 10(2) cfu/g during the first 2 weeks of storage.

The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans. The L. monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p < 0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses. The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%).

A total of 40 strains of Listeria monocytogenes which have been demonstrated to be serovar 1/2a, 1/2b, and 4b were genotyped by pulsed-field gel electrophoresis (PFGE) after separate digestion with Apa I, Asc I, Sma I, and Sse 8387 I. Twenty-seven unrelated strains including four representative strains showed distinctly different genotypes according to their PFGE profiles. Then nine strains isolated from shredded cheese of different lots and four strains isolated from the cheese-processing environment were shown to display the same genotype. Therefore, it is suggested that the Listeria was spread in cheese by cross-contamination from the cheese-processing environment. Thus, PFGE analysis has a good typeability and excellent discriminatory power, and has provided a useful tool for investigation of the source of Listeria contamination.

The mutagenicity of 15 household dog urine specimens were measured by the combination of blue rayon extraction and ultramicro forward-mutation method with Salmonella Typhimurium TM677 strain. A good dose-response relation was observed between the urine volume and mutation frequency. The minimum amount of urine required was 20 ml or less. The specific mutation frequency of urine greatly varied from one dog to another. The average specific mutation frequencies in the presence and absence of S9 mix were 28.7 +/- 51.5 (x 10(-4)) and 12.0 +/- 13.3 (x 10(-4)), respectively, and there was no significant difference between them. The mutation frequency markedly increased after the ingestion of broiled fish. Ten human urines specimens showed a similar level of specific mutation frequency to that of the dog urine specimens in both the presence and absence of S9 mix.

Among the 23 Salmonella strains isolated from 21 diseased chicken and 2 embryonated eggs in 6 poultry farms near Lusaka City in Zambia, serovars identified were S. Gallinarum (11 strains), S. Agona (7 strains), S. Alamo (1 strain), S. Infantis (1 strain), S. Virginia (1 strain), S. Haifa (1 strain), and S. Dublin (1 strain). S Gallinarum was detected at the highest incidence and from all the poultry farms. Fourteen serovars have been reported for the chickens in Zambia so far. Three serovars (S. Alamo, S. Haifa, S. Virginia) were newly identified in this study.

165 (18.1%) out of 910 rats captured at restaurants in 14 buildings in downtown Tokyo were positive for Staphylococcus aureus. The 165 S. aureus strains isolated were biotyped into A, B, C, D, G, and untypable groups (UT1 and UT2). The UT1 was the most frequent (72 strains, 43.6%), followed by biotype G (33 strains, 20.0%). The strains were classified into coagulase types I, II, III, IV, V, VI, VII, VIII, and an untypable group. Coagulase type V was the most frequent (92 strains, 55.8%), followed by coagulase type VII (25 strains, 15.2%). Enterotoxins A, B, C, or D were produced by 35 strains. Enterotoxins A and B were the most frequent (13 strains each). Toxic shock syndrome toxin-1 was produced by 3 strains. 65 strains were resistant to ampicillin, 2 to oxytetracycline, and 1 to erythromycin.

Digestion with Sal I facilitated the subclassification of 41 strains of Campylobacter jejuni into seven types, and digestion with Sma I enabled subclassification into twelve types. Sma I was potentially more useful for the detection of variability among the 41 strains, but both restriction enzymes seemed to be potentially useful for detecting variability among crossed-field gel electrophoresis profiles of the strains. The results clearly demonstrated that C. jejuni strains from different sources and with different routes of transmission had invaded the three farms investigated. At farms Sa and Ai, approximately 70% (23 strains) of isolates of C. jejuni (33 strains) were subclassified into the two major genotypes (I and II) on the basis of cleavage profiles with both Sal I and Sma I. These two major genotypes appeared to have invaded, expanded in and occupied the two chicken farms. All nine strains from farm Ai belonged genotype I.

Chromosomal genomic DNA from 25 strains of thermophilic Campylobacter coli and C. jejuni were analysed after pulsed-field gel electrophoresis (PFGE) of restriction fragments generated by SalI and SmaI to evaluate the utility of macro-fingerprinting analysis and to assess the possible subtyping of the isolates. Two rare-cutting restriction enzymes, SalI and SmaI, allowed the subclassification of eight strains of C. coli into six types. SalI facilitated the subclassification of seventeen strains of C. jejuni into thirteen types and SmaI the subclassification of seventeen strains into twelve types. The diversity in the PFGE profiles was demonstrated and, therefore, the two enzymes were found to be potentially useful for detecting variability of PFGE profiles among strains of C. coli and C. jejuni. The two enzymes and PFGE may be useful in molecular epidemiology.

The protein-banding patterns after electrophoresis of 23 strains of thermophilic Campylobacter, including 17 strains of C. coli and C. jejuni which had atypical characteristics with respect to the hydrolysis of hippurate and susceptibility to nalidixic acid, were characterized. Of the atypical strains of C. coli and C. jejuni 16 out of 17 gave protein-banding patterns which were essentially identical to those of typical strains, and distinct patterns were obtained from strains of two other species. The banding patterns of soluble proteins on non-denaturing polyacrylamide gels after electrophoresis appear to be useful for the differentiation of atypical strains of C. coli and C. jejuni. Strain 11791 which was originally identified as C. coli was identified as C. lari from its protein-banding profile.

Three restriction enzymes ApaI, SalI and SmaI, among nine enzymes tested, were found to produce distributions of DNA fragments which were useful for analysis of chromosome-sized DNA from thermophilic Campylobacter laridis by pulsed-field gel electrophoresis. From experiments with C. laridis JCM2530T and four isolates of C. laridis, the size of the genome of C. laridis was calculated to range from 1,590 to 1,700 kb, with a mean of 1,640 kb. An SmaI restriction map was derived by the partial digestion of the DNA from C. laridis JCM2530T.

A total of 1,705 fecal specimens or ileo-cecal contents of cattle, pigs, dogs, cats, chicken and rats were submitted for the isolation of Listeria monocytogenes by the use of the combination of Oxford-LPM agar plates after the cold enrichment in PBS at 4 degrees C for 4-6 weeks. Prevalence of L. monocytogenes was found to be 1.9% in cattle, 0.6% in pigs, 0.9% in dogs and 6.5% in rats. However, none of L. monocytogenes was isolated from chicken or cats. Among 26 isolates of L. monocytogenes, 13 strains (50%) were classified into types 1/2a (3 strains), 1/2b (5 strains) and 4b (5 strains) and were often associated with human listeriosis. The majority of the Listeria spp. other than L. monocytogenes isolated from these animals was found to be L. innocua.

A colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf was characterized. The colicin type was identified as ColV by using reference ColV producers. The colicin plasmid was labeled with transposon Tn903 and subjected to conjugation. The transconjugants examined suggest that the colicin plasmid confers serum resistance. There was no difference in siderophore utilization ability between the transconjugants and host strain SF800. Bioassay for siderophore suggests that the colicin plasmid specifies the production of iron-chelating compounds available for the host strain.

A number of Lactobacillus strains produced succinic acid in de Man-Rogosa-Sharpe broth to various extents. Among 86 fresh isolates from fermented cane molasses in Thailand, 30 strains (35%) produced succinic acid; namely, 23 of 39 Lactobacillus reuteri strains, 6 of 18 L. cellobiosus strains, and 1 of 6 unidentified strains. All of 10 L. casei subsp. casei strains, 5 L. casei subsp. rhamnosus strains, 6 L. mali strains, and 2 L. buchneri strains did not produce succinic acid. Among 58 known strains including 48 type strains of different Lactobacillus species, the strains of L. acidophilus, L. crispatus, L. jensenii, and L. parvus produced succinic acid to the same extent as the most active fresh isolates, and those of L. alimentarius, L. collinoides, L. farciminis, L. fructivorans (1 of 2 strains tested), L. malefermentans, and L. reuteri were also positive, to lesser extents. Diammonium citrate in de Man-Rogosa-Sharpe broth was determined as a precursor of the succinic acid produced. Production rates were about 70% on a molar basis with two fresh strains tested. Succinic acid was also produced from fumaric and malic acids but not from dl-isocitric, alpha-ketoglutaric, and pyruvic acids. The present study is considered to provide the first evidence on the production of succinic acid, an important flavoring substance in dairy products and fermented beverages, from citrate by lactobacilli.