BackgroundThe Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains.MethodsCrude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity.ResultsCrude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates.ConclusionsThe crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.
Forty-five acetic acid bacteria, which were isolated from fruits, flowers and other materials collected in Thailand by an enrichment culture approach, were assigned to the genus Gluconobacter by phenotypic and chemotaxonomic characterisations. On the basis of 16S-23S rRNA gene ITS restriction and 16S rRNA gene sequence analyses, the forty-five isolates were grouped into five groups and identified at the specific level as follows: 1) seventeen isolates were grouped into Group A and identified as G. oxydans; 2) twelve isolates were grouped into Group B and identified as G. cerinus; 3) nine isolates were grouped into Group C and identified as G. frateurii; 4) six isolates were included into Group D and identified as G. thailandicus; 5) one isolate was grouped into Group E, characterised by a restriction pattern comprised of 667 and 48-bp fragments in AvaII digestion, differing from those of strains of Group C or G. frateurii and Group D or G. thailandicus, and unidentified.
Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thailand. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and considered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization.
Three strains, RBY-1 T , PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains wereGram-negative, aerobic rods with polar flagella, produced acetic acid from ethanol and did not oxidize acetate or lactate. In phylogenetic trees based on 16S rRNA gene sequences and 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, the strains formed a cluster separate from the type strains of recognized species of the genus Gluconobacter. The calculated 16S rRNA gene sequence and 16S-23S rRNA gene ITS sequence similarities were respectively 97.7-99.7 % and 77.3-98.1 %. DNA G+C contents ranged from 57.2 to 57.6 mol%. The strains showed high DNA-DNA relatedness of 100 % to one another, but low DNA-DNA relatedness of 11-34 % to the tested type strains of recognized Gluconobacter species. Q-10 was the major quinone. On the basis of the genotypic and phenotypic data obtained, the three strains clearly represent a novel species, for which the name Gluconobacter nephelii sp. nov. is proposed. The type strain is RBY-1 T (5BCC 36733 T 5NBRC 106061 T 5PCU 318 T ), whose DNA G+C content is 57.2 mol%.In the genus Gluconobacter, 12 species have been described at the time of writing: Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter frateurii, Gluconobacter asaii, Gluconobacter albidus, Gluconobacter thailandicus, Gluconobacter kondonii, Gluconobacter roseus, Gluconobacter japonicus, Gluconobacter sphaericus, Gluconobacter kanchanaburiensis and Gluconobacter wancherniae (De Ley, 1961;Skerman et al., 1980;Gosselé et al., 1983; Yamada & Akita, 1984a, b;Mason & Claus, 1989; Yukphan et al., 2004aYukphan et al., , 2005Yukphan et al., , 2010 Tanasupawat et al., 2004Tanasupawat et al., , 2005 Malimas et al., 2007 Malimas et al., , 2008a Malimas et al., , b, c, 2009a. However, G. asaii was shown to be a later heterotypic synonym of G. cerinus (Katsura et al., 2002;Tanaka et al., 1999;Yamada et al., 1999). This paper describes Gluconobacter nephelii sp. nov. for three strains isolated in Thailand.The three strains, RBY-1 T , PHD-1 and PHD-2, were isolated from rambutan and litchi by an enrichment-culture approach using the glucose/ethanol/acetic acid medium described previously (Yamada et al., 1976(Yamada et al., , 1999(Yamada et al., , 2000Yukphan et al., 2004c PHD-2 as described previously (Malimas et al., 2007; Yukphan et al., 2004a, b, c). Multiple sequence alignments were performed with the program CLUSTAL_X (version 1.81) (Thompson et al., 1997). Sequence gaps and ambiguous bases were excluded. Distance matrices were calculated by the two-parameter method of Kimura (1980). The neighbour-joining (Saitou & Nei, 1987), maximumparsimony and maximum-likelihood (Felsenstein, 1981(Felsenstein, , 1983) methods were used for constructing phylogenetic trees. The confidence values of individual branches in the phylogenetic trees were determined by using the bootstrap analysis of Felsenstein (1985) based on 1000 replications with the program MEGA (version 4.0;Tamura et al., 2007). Pairwise sequence similarities were...
Acetic acid bacteria (AAB) are commonly distributed in vinegar, fruits, fl owers, etc. AAB are nowadays classifi ed into 13 genera in the family Acetobacteraceae:
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