Isolation and characterization of Lactococcus piscium strains from vacuum-packaged refrigerated beef.

AIMS: To characterize gram-positive, catalase-negative, psychrotrophic, lactic acid-homofermentative, non-motile cocci isolated from vacuum-packaged refrigerated beef using phenotypic and genotypic methods. METHODS AND RESULTS: A total of 89 strains was isolated at 2 and 6 weeks as one of the predominant microflora of five samples of vacuum-packaged beef stored at 2 degrees C. The strains were compared with reference strains of some gram-positive, catalase-negative cocci using SDS-PAGE whole-cell protein pattern analysis, biochemical characterization and 16S rDNA sequencing. The biochemical and physiological characteristics of the isolates resembled those of Lactococcus piscium GTC 552(T). Numerical analysis of the SDS-PAGE whole-cell protein patterns resulted in close clustering of the strains with L. piscium GTC 552(T) (r > 0.68). Other Lactococcus and Leuconostoc species could be distinguished from the isolates using SDS-PAGE whole-cell protein patterns (r < 0.58) and biochemical characteristics. The 16S rDNA sequencing of four randomly selected strains showed that the strains differed from L. piscium GTC 552(T) by two to three bases in the highly variable region of the sequence. This is the first report on the isolation of L. piscium from vacuum-packaged beef. CONCLUSIONS: The gram-positive catalase-negative cocci isolated from vacuum-packaged refrigerated beef have been identified as L. piscium. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of the microflora of vacuum-packaged refrigerated beef.

Lactobacillus algidus sp. nov., a psychrophilic lactic acid bacterium isolated from vacuum-packaged refrigerated beef.

Lactobacillus algidus sp. nov. is described on the basis of 40 strains isolated as one of the predominant bacteria from five specimens of vacuum-packaged beef collected from different meat shops and stored at 2 degrees C for 3 weeks. These strains were quite uniform in the overall characteristics examined. They are facultatively anaerobic, psychrophilic, Gram-positive, non-spore-forming, non-motile, lactic acid-homofermentative rods. The cells occurred singly and in pairs on agar media and in rather long chains in broth media. They differed in several cultural and biochemical characteristics from the authentic meso-diaminopimelic acid-positive or psychrophilic lactic acid bacteria in the genera Lactobacillus, Carnobacterium and Brochothrix. The SDS-PAGE whole-cell protein pattern was clearly distinctive. DNA-DNA hybridization and phylogenetic analysis of 16S rDNA also failed to associate these strains closely with any of the validly described organisms used. The phylogenetic analysis showed that these strains are rather remotely but most closely related to Lactobacillus mali (93% sequence similarity), which belongs to the Lactobacillus casei/Pediococcus group. Therefore, these strains should be included in the genus Lactobacillus and considered to represent a new species, Lactobacillus algidus sp. nov. The type strain is M6A9T (= JCM 10491T).

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Japan by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) patterns of 102 L. monocytogenes serovar 4b isolates from patients and foods examined in Japan were compared with 16 isolates from foodborne listeriosis episodes which occurred in North America or Europe. Using a combination of PFGE patterns with the restriction enzymes SmaI, ApaI, AscI and Sse8387I, 82 clinical isolates from Japan were categorized into 45 PFGE types: the largest group of 17 isolates (20.7%) were of the same PFGE type as cultures from the large foodborne outbreaks which occurred in California (1985) and Switzerland (1983-1987). Twenty cultures from foods on retail sale in Japan were classified into 12 PFGE types: four isolates were of three PFGE types also recognized among isolates of clinical origin from Japan, including the predominant clinical type.

Molecular typing of Listeria monocytogenes isolated in Japan by pulsed-field gel electrophoresis.

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.

Detection of Listeria monocytogenes in humans, animals and foods.

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%).

Mutagenicity of dog urine determined by blue rayon extraction and the ultramicro forward-mutation method.

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.

Recent characterization of Salmonella strains isolated from chickens in Zambia.

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.

Isolation and characterization of Staphylococcus aureus in rats trapped at restaurants in buildings in downtown Tokyo.

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.

Detection of genomic variability among isolates of Campylobacter jejuni from chickens by crossed-field gel electrophoresis.

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.

Macro-fingerprinting analysis at the chromosomal genomic DNA level of isolates of thermophilic Campylobacter coli and C. jejuni, by pulsed-field gel electrophoresis.

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.

Differentiation of thermophilic species of Campylobacter, in particular C. coli and C. jejuni, with atypical characteristics, by analysis of protein-banding profiles on non-denaturing polyacrylamide gels.

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.

Analysis of chromosome-sized DNA from the bacterial genome of thermophilic Campylobacter laridis by pulsed-field gel electrophoresis and physical mapping.

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.

Prevalence of Listeria monocytogenes in intestinal contents of healthy animals in Japan.

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.

Production of succinic Acid from citric Acid and related acids by lactobacillus strains.

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.