Ro Osawa

Isolation of Tannin-Degrading Lactobacilli from Humans and Fermented Foods

ABSTRACT Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.

Streptococcus gallolyticus sp. nov. ; gallate degrading organisms formerly assigned to Streptococcus bovis

Summary Phenotypic charcteristics including the abilites two hydrolyze tannin and to decarboxylate gallic acid in 31 strains of S. bovis and 3 strains of S. equinus were compared with their dexoyribonucleic acid (DNA) relatedness. It was found that the strains capable of decarboxylating gallic acid all belong to a single DNA homology group which does not include either the type strain of S. bovis or the type strain of S. equinus . Thus it was concluded that the gallate degrading strains of S. bovis should be re-assigned to a new species, for which the name Streptococcus gallolyticus sp. nov. is proposed.

Genotypic Analyses of Vibrio parahaemolyticus and Development of a Pandemic Group-Specific Multiplex PCR Assay

ABSTRACT A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the “pandemic group”) were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

Lonepinella koalarum gen. nov., sp. nov., a New Tannin-Protein Complex Degrading Bacterium

Summary The phylogeny of bacteria that degrade a tannin-protein complex (T-PC) isolated from the fecal biota of koala was investigated. All T-PC degrading bacteria were facultatively anaerobic Gram-negative rods, producing tannase, but negative for catalase and oxidase activities. The 16S rDNA sequence analyses of eight T-PC degrading strains representing biovars a, b, c, and d, revealed that the strains formed a phylogeneric cluster within the family Pasteurellaceae . Furthermore seven strains of biovar a, b, and c formed a 16S rDNA sub-cluster with similarity values in the range 97.6 to 99.8%. As the strains also exhibit a combination of chemotaxonomic and physiological properties not found in other members of the family Pasteurellaceae , it was concluded that the T-PC degrading isolates of biovar a, b, and c represent a new genus and species, for which the name Lonepinella koalarum gen. nov., sp. nov. is proposed.

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917T

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.

Genotypic Analyses of Lactobacilli with a Range of Tannase Activities Isolated from Human Feces and Fermented Foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus are subjective synonyms.

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus have been shown to be subjective synonyms on the basis of their levels of 16S rRNA sequence similarity (98.3%) and DNA-DNA homology (> 70%) and the phenotypes of their type strains. S. gallolyticus has nomenclatural priority according to Rule 24b(2) of the International Code of Nomenclature of Bacteria.

Occurrence of Tannin-Protein Complex Degrading Streptococcus sp. in Feces of Various Animals

Summary Occurrence of tannin-protein complex (T-PC) degrading Streptococcus bovis in feces of 14 species of animals was investigated using a selective medium (colistin-oxolinic acid tannin-treated brain heart infusion). Strains of T-PC degrading S. bovis, all of which fermented mannitol, occurred commonly in browsing animals (koala, ringtail possum, deer) and omnivorous animals (guinea pig, pig, brushtail possum), whereas T-PC degrading S. bovis strains occurred less frequently in grazing herbivores (cattle, sheep, horse) and carnivores (dog, cat). Furthermore koalas and ringtail possums, whose diet was almost exclusively of tannin rich eucalypt leaves, had fecal streptococcal flora dominated by T-PC degrading S. bovis (59.9% for koalas; 59.1% for ringtail possums). Such dominance was never observed in any other animals examined.

Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, 26, and 33 based on DNA–DNA homology and sodA and recN phylogenies

To date, Streptococcus suis was divided into thirty-three serotypes based on its polysaccharide capsular antigens. Although 16S rRNA sequence similarities of serotypes 20, 22, 26, and 33 reference strains to the type strain NCTC 10234(T) were below the threshold value of 98.5% to assign them to S. suis species, no strong evidence support to reclassification. Here, their taxonomic identities were determined by DNA-DNA hybridization assays and by partial sequencing of the sodA and recN genes. Our results confirmed that the serotype 20, 22, 26, and 33 reference strains were distantly related to the type strain NCTC 10234(T) and the whole sequence strain P1/7 of S. suis. Moreover, the reference strains of serotypes 20, 22, and 26 were closely related to each other but distinct from the serotype 33 reference strain. Sequencing analyses of sodA and recN of a total 33 serotype reference strains showed that the serotype 20, 22, and 26 reference strains and the serotype 33 reference strain did not fall with not only other serotypes of S. suis, but also other streptococcal species (63 strains of 56 species for sodA and 87 strains of 55 species for recN). The evidence further substantiates the view that the reference strains of serotypes 20, 22, 26 and 33 should be taxonomically removed from S. suis, although their taxonomic designations and determinative phenotypic characteristics are yet to be addressed.

Effects of Repeated Subculturing and Prolonged Storage at Room Temperature of Enterohemorrhagic Escherichia coli O157:H7 on Pulsed-Field Gel Electrophoresis Profiles

ABSTRACT Three clinical strains of enterohemorrhagic Escherichia coli O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns. The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains.