Partial rpoB Gene Sequencing Identification and Probiotic Potential of Floricoccus penangensis ML061-4 Isolated from Assam Tea (Camellia sinensis var. assamica).

Assam tea or Miang is a local name of Camellia sinensis var. assamica in northern Thailand. By the local wisdom, Assam tea leaves are used as the raw material in tea fermentation to produce "Fermented Miang" consumed by people in northern Thailand and the countries nearby. In this study, twenty-eight bacterial isolates were obtained from Assam tea leaf samples collected from Nan province, Thailand. Bacterial isolates were identified within 6 genera including Bacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus and Staphylococcus. Among these, the strain ML061-4 shared 100.0 and 99.4% similarity of 16S rRNA and rpoB gene sequence with F. penangensis JCM 31735T, respectively. This is the first discovery of F. penangensis in Thailand. F. penangensis ML061-4 exhibited probiotic characteristics including lactic acid production (9.19 ± 0.10 mg/ml), antibacterial activities (Escherichia coli ATCC 25922 and E. coli O157:H7 DMST 12743), acid and bile salt tolerance (71.1 and 54.9%, respectively), autoaggregation (97.0%), coaggregation (66.0% with E. coli O157:H7), cell surface hydrophobicity (90.0%), bacterial adhesion (82.9% with Lactobacillus plantarum FM03-1), competitive inhibition (17.8% with E. coli O157:H7) and competitive exclusion (34.9% with E. coli O157:H7). Overall, the data suggested that F. penangensis ML061-4 had a great potential to be a probiotic.

The visualization of biofilms in chronic diabetic foot wounds using routine diagnostic microscopy methods.

Diabetic foot wounds are commonly colonised by taxonomically diverse microbial communities and may additionally be infected with specific pathogens. Since biofilms are demonstrably less susceptible to antimicrobial agents than are planktonic bacteria, and may be present in chronic wounds, there is increasing interest in their aetiological role. In the current investigation, the presence of structured microbial assemblages in chronic diabetic foot wounds is demonstrated using several visualization methods. Debridement samples, collected from the foot wounds of diabetic patients, were histologically sectioned and examined using bright-field, fluorescence, and environmental scanning electron microscopy and assessed by quantitative differential viable counting. All samples (n = 26) harboured bioburdens in excess of 5 log10 CFU/g. Microcolonies were identified in 4/4 samples by all three microscopy methods, although bright-field and fluorescence microscopy were more effective at highlighting putative biofilm morphology than ESEM. Results in this pilot study indicate that bacterial microcolonies and putative biofilm matrix can be visualized in chronic wounds using fluorescence microscopy and ESEM, but also using the simple Gram stain.

Aerococcus urinae: polyphasic characterization of the species.

A polyphasic characterization of Aerococcus urinae is presented. In this study the intraspecies relationships between 26 strains of varying geographical origin were examined by phenotypic tests, ribotyping and multilocus enzyme electrophoresis. The results demonstrated two main phenotypic patterns that could be distinguished in tests for hydrolysis of aesculin, and acid production from amygdalin and salicin. Strains were either negative (n=19) or positive (n=6) in these tests. One strain had a deviating pattern. Heterogeneity within the 19 pattern I strains was demonstrated especially by phenotypic tests (acid production from ribose, mannitol, sorbitol, sucrose and D-arabitol) and by multilocus enzyme electrophoresis. However, DNA sequence analysis of the 16S rRNA (n=7) and gyrB genes (n=3) from strains representing the two main patterns showed no variation in sequences among strains. Comparison of A. urinae and representatives of related taxa by 16S rDNA sequence analysis showed that the taxon is related to, but distinct from, other Aerococcus spp.

Dynamics of the microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rRNA hybridization.

The microbial community developing during the spontaneous fermentation of sour cassava starch was investigated by cultivation-independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant organisms were all lactic acid bacteria (LAB), mainly close relatives of Bifidobacterium minimum, Lactococcus lactis, Streptococcus sp., Enterococcus saccharolyticus and Lactobacillus plantarum., Close relatives of Lb. panis, Leuconostoc mesenteroides and Ln. citreum were also found. A complementary analysis using hybridization of 16S rRNA with phylogenetic probes was necessary to detect the presence of the recently discovered species Lb. manihotivorans. Although it represented up to 13% of the total lactic acid bacteria of sour cassava starch, this species could not be detected by DGGE as the PCR product migrated to the same position as Lc. lactis. In addition, it was shown that a strong pH decrease in the time course of fermentation was most probably responsible for the competitive selection of acid-resistant LAB vs. both homo and heterofermentative acid-sensitive LAB.

[The effect of Aerococcus viridans on the properties of salmonellae and staphylococci in vitro]. / Vplyv Aerococcus viridans na deiaki vlastyvosti sal'monel i stafilokokiv in vitro.

According to electron microscopy the repeated contacts of Aerococcus viridans 167 (industrial strain for the probiotic Aerobact production) with Salmonella typhimurium and Staphylococcus aureus caused the deep changes of cellular structure, down to bacteriolysis. Remaining viable pathogenic cells gave posterity with changed biochemical properties and virulence. S. typhimurium completely lost typical biochemical properties, its mobility and ability to be agglutinated by O-specific Salmonella serum reduced after the fifth joint passage. Sharp decrease of the leucotoxic and loss of the lethal and necrotic activity of the S. aureus toxin were observed. The latter was characterized by the smaller number of protein fractions in comparison with control series on electrophoregrams and densitograms. Pathogenic bacteria, playing a role in formation of the pathological intestinal microbiocenosis (S. typhimurium) and being hospital infections (S. aureus) acquired increased sensitivity to antibiotics. The carried out researches open some mechanisms of bactericidal and bacteriostatic action of A. viridans (group 12, Bergey, 1994) and their metabolic products, causing antagonistic effect during direct action on infectious microorganisms.

[The effect of Aerococcus viridans--the basis of the new therapeutic-prophylactic preparation M-bacterin--on the biological properties of Staphylococcus aureus]. / Vliianie Aerococcus viridans--osnovy novogo lechebno-profilakticheskogo preparata "M-bakterina"--na biologicheskie svoistva Staphylococcus aureus.

Representatives of the normal microflora from genus Aerococcus, in particular strain Aerococcus viridans 167 isolated from breast milk are studied for their effect on biological properties of Staphylococcus aureus in vitro and in vivo. It is established that the number of viable cells of the staphylococcus cultivated in the presence of antagonists in the beef-extract agar decreases progressively with each following passage, the population dying after the seventh-eight passage. Electronograms fix deep changes in the cell ultrastructure. A degree of changes in biological properties depends on the duration of the antagonist action. The results obtained reveal one of the mechanisms of the antagonistic action of aerococci-antagonists producing hydrogen peroxide.

Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls.

Membrane-walls from Gaffkya homari require a specific interrelationship between membrane and wall that functions in the incorporation of nascent peptidoglycan into the preexisting peptidoglycan of the wall. Two different methods were used to inhibit selectively this incorporation process: (i) sensitivity to sulfhydryl reagents and (ii) heat inactivation. Of the sulfhydryl reagents tested, 2.2 mM iodoacetamide inhibited the synthesis of wall peptidoglycan 50%, whereas greater than 100 mM was required to inhibit the synthesis of sodium dodecyl sulfate (SDS)-soluble peptidoglycan. Heat treatment at 37 degrees C (t 1/2 = 5.7 min) inhibited wall peptidoglycan synthesis without affecting SDS-soluble peptidoglycan synthesis. Inhibition of LD-carboxypeptidase by iodoacetamide and heat gave 50% inhibition and t 1/2 values similar to those observed for the incorporation process. Thus, it is suggested that the LD-carboxypeptidase may be one of the enzymes responsible for the sulfhydryl sensitivity and heat lability and that this enzyme may play a role in the relationship between membrane and wall in G. homari.

Ultrastructural surface changes associated with dextran synthesis by Leuconostoc mesenteroides.

When Leuconostoc mesenteroides NCDO 1875 was grown in MRS broth and fixed for electron microscopy in the presence of ruthenium red, the cell wall appeared as a triple-layered structure similar to other, gram-positive bacteria. When such logarithmic-phase cultures were exposed to sucrose, the appearance and growth of a uniform layer of electron-dense material was evident on the surface of the cell wall. After 2 h in the presence of sucrose, the formation of this surface coat (110 to 130 nm thick) was complete. For 85 to 90% of the cells, continued exposure to sucrose did not produce any further change in their appearance, but the rest of the population began to accumulate insoluble capsular dextran at the surface of their coat material. Within 18 h, these cells had produced a large capsule (maximum diameter, 6 micrometer) composed mainly of an extensive reticulum of fine filaments. Periodate-reactive carbohydrate was localized cytochemically in the capsular dextran and in the surface coat of all cells. It is suggested that the surface coat of sucrose-grown cells represents a cell-bound dextran-dextransucrase complex and that the acapsulate cells produce the relatively soluble S dextran reported by previous workers.