Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria.

BACKGROUND: The 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. METHODS: Crude 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. RESULTS: Crude 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. CONCLUSIONS: The 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.

Identification of five reptile egg whites protein using MALDI-TOF mass spectrometry and LC/MS-MS analysis.

Proteomics of egg white proteins of five reptile species, namely Siamese crocodile (Crocodylus siamensis), soft-shelled turtle (Trionyx sinensis taiwanese), red-eared slider turtle (Trachemys scripta elegans), hawksbill turtle (Eretmochelys imbricate) and green turtle (Chelonia mydas) were studied by 2D-PAGE using IPG strip pH 4-7 size 7 cm and IPG strip pH 3-10 size 24 cm. The protein spots in the egg white of the five reptile species were identified by MALDI-TOF mass spectrometry and LC/MS-MS analysis. Sequence comparison with the database revealed that reptile egg white contained at least seven protein groups, such as serpine, transferrin precursor/iron binding protein, lysozyme C, teneurin-2 (fragment), interferon-induced GTP-binding protein Mx, succinate dehydrogenase iron-sulfur subunit and olfactory receptor 46. This report confirms that transferrin precursor/iron binding protein is the major component in reptile egg white. In egg white of Siamese crocodile, twenty isoforms of transferrin precursor were found. Iron binding protein was found in four species of turtle. In egg white of soft-shelled turtle, ten isoforms of lysozyme were found. Apart from well-known reptile egg white constituents, this study identified some reptile egg white proteins, such as the teneurin-2 (fragment), the interferon-induced GTP-binding protein Mx, the olfactory receptor 46 and the succinate dehydrogenase iron-sulfur subunit.

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses.

BACKGROUND: Fermented rice flour (khao-khab, a non-glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao-khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty-five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S-23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product.

Gluconobacter nephelii sp. nov., an acetic acid bacterium in the class Alphaproteobacteria.

Three strains, RBY-1(T), PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains were Gram-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) ( = BCC 36733(T) = NBRC 106061(T) = PCU 318(T)), whose DNA G+C content is 57.2 mol%.

Identification of Acetobacter, Gluconobacter, and Asaia strains isolated in Thailand based on 16S-23S rRNA gene internal transcribed spacer restriction and 16S rRNA gene sequence analyses.

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.