Antibacterial activity of plasma from crocodile (<it>Crocodylus siamensis</it>) against pathogenic bacteria

Abstract 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.</p

Идентификация На Щамове, Изолирани В Тайланд И Отнесени Към Родовете Kozakia И Swaminathania

Four isolates, isolated from fruit of sapodilla collected at Chantaburi and designated as CT8-1 and CT8-2, and isolated from seeds of ixora („khem” in Thai, Ixora species) collected at Rayong and designated as SI15-1 and SI15-2, were examined taxonomically. The four isolates were selected from a total of 181 isolated acetic acid bacteria. Isolates CT8-1 and CT8-2 were non motile and produced a levan-like mucous polysaccharide from sucrose or D-fructose, but did not produce a water-soluble brown pigment from D-glucose on CaCO3-containing agar slants. The isolates produced acetic acid from ethanol and oxidized acetate and lactate to carbon dioxide and water, but the intensity of the acetate and lactate oxidation was weak. Their growth was not inhibited by 0.35 % acetic acid (v/v) at pH 3.5. The isolates did not grow on 30 % D-glucose (w/v), and utilization of methanol was not found. Isolates SI15-1 and SI15-2 had peritrichous flagella and grew in the presence of either 0.35 % acetic acid (v/v) at pH 3.5, 3 % NaCl (w/v), or 1 % KNO3 (w/v). Acetate and lactate were oxidized to carbon dioxide and water, but the intensity was weak. The isolates grew on mannitol agar and glutamate agar as well as on 30 % Dglucose (w/v), but did not utilize methanol. The 16S rRNA gene sequence analysis and DNA-DNA hybridization indicated that isolates CT8-1 and CT8-2 and isolates SI15-1 and SI15-2 were unequivocally identified respectively as Kozakia baliensis and Swaminathania salitolerans .Изследвани са таксономично четири щама, изолирани съответно от плод на саподила в Чантабури, означени като CT8-1 и CT8-2, и от семена на иксора („кем” на тайвански) в Районг и означени като SI15-1 and SI15-2. Че- тирите щама са подбрани от общо 181 изолата на оцетнокисели бактерии. Щамове CT8-1 и CT8-2 са неподвижни и продуцират леваноподобен муко- полизахарид от захароза или D-фруктоза, но не отделят водноразтворим кафяв пигмент от D-глюкоза върху скосен агар, съдържащ CaCO3. Изола- тите произвеждат оцетна киселина от етанол и окисляват ацетат и лактат до въглероден двуокис и вода, но интензивността на окислението е слаба. Техният растеж не се инхибира от 0.35 % оцетна киселина (v/v) при pH 3.5. Изолатите не растат върху 30 % D-глюкоза (w/v), а усвояване на метанол не се установява. Щамове SI15-1 и SI15-2 имат перитрихално разположени ресни и растат в присъствие както на 0.35 % оцетна киселина (v/v) при pH 3.5, така и на 3 % NaCl (w/v) или 1 % KNO3 (w/v). Ацетатът и лактатът се окисляват до въглероден двуокис и вода, но интензивността е слаба. Изолатите растат върху агар с манитол или глутамат, както и върху 30 % D-глюкоза (w/v), но не усвояват метанол. Проведените секвен- ционен анализ на 16S рРНК и ДНК-ДНК хибридизация показват, че щамове CT8-1 and CT8-2 без съмнение се идентифицират като Kozakia baliensis , а изолати SI15-1 и SI15-2 като Swaminathania salitolerans

\u418\u434\u435\u43d\u442\u438\u444\u438\u43a\u430\u446\u438\u44f \u41d\u430 \u429\u430\u43c\u43e\u432\u435, \u418\u437\u43e\u43b\u438\u440\u430\u43d\u438 \u412 \u422\u430\u439\u43b\u430\u43d\u434 \u418 \u41e\u442\u43d\u435\u441\u435\u43d\u438 \u41a\u44a\u43c \u420\u43e\u434\u43e\u432\u435\u442\u435 Kozakia \u418 Swaminathania

Four isolates, isolated from fruit of sapodilla collected at Chantaburi and designated as CT8-1 and CT8-2, and isolated from seeds of ixora (\u201ekhem\u201d in Thai, Ixora species) collected at Rayong and designated as SI15-1 and SI15-2, were examined taxonomically. The four isolates were selected from a total of 181 isolated acetic acid bacteria. Isolates CT8-1 and CT8-2 were non motile and produced a levan-like mucous polysaccharide from sucrose or D-fructose, but did not produce a water-soluble brown pigment from D-glucose on CaCO3-containing agar slants. The isolates produced acetic acid from ethanol and oxidized acetate and lactate to carbon dioxide and water, but the intensity of the acetate and lactate oxidation was weak. Their growth was not inhibited by 0.35 % acetic acid (v/v) at pH 3.5. The isolates did not grow on 30 % D-glucose (w/v), and utilization of methanol was not found. Isolates SI15-1 and SI15-2 had peritrichous flagella and grew in the presence of either 0.35 % acetic acid (v/v) at pH 3.5, 3 % NaCl (w/v), or 1 % KNO3 (w/v). Acetate and lactate were oxidized to carbon dioxide and water, but the intensity was weak. The isolates grew on mannitol agar and glutamate agar as well as on 30 % Dglucose (w/v), but did not utilize methanol. The 16S rRNA gene sequence analysis and DNA-DNA hybridization indicated that isolates CT8-1 and CT8-2 and isolates SI15-1 and SI15-2 were unequivocally identified respectively as Kozakia baliensis and Swaminathania salitolerans .\u418\u437\u441\u43b\u435\u434\u432\u430\u43d\u438 \u441\u430 \u442\u430\u43a\u441\u43e\u43d\u43e\u43c\u438\u447\u43d\u43e \u447\u435\u442\u438\u440\u438 \u449\u430\u43c\u430, \u438\u437\u43e\u43b\u438\u440\u430\u43d\u438 \u441\u44a\u43e\u442\u432\u435\u442\u43d\u43e \u43e\u442 \u43f\u43b\u43e\u434 \u43d\u430 \u441\u430\u43f\u43e\u434\u438\u43b\u430 \u432 \u427\u430\u43d\u442\u430\u431\u443\u440\u438, \u43e\u437\u43d\u430\u447\u435\u43d\u438 \u43a\u430\u442\u43e CT8-1 \u438 CT8-2, \u438 \u43e\u442 \u441\u435\u43c\u435\u43d\u430 \u43d\u430 \u438\u43a\u441\u43e\u440\u430 (\u201e\u43a\u435\u43c\u201d \u43d\u430 \u442\u430\u439\u432\u430\u43d\u441\u43a\u438) \u432 \u420\u430\u439\u43e\u43d\u433 \u438 \u43e\u437\u43d\u430\u447\u435\u43d\u438 \u43a\u430\u442\u43e SI15-1 and SI15-2. \u427\u435- \u442\u438\u440\u438\u442\u435 \u449\u430\u43c\u430 \u441\u430 \u43f\u43e\u434\u431\u440\u430\u43d\u438 \u43e\u442 \u43e\u431\u449\u43e 181 \u438\u437\u43e\u43b\u430\u442\u430 \u43d\u430 \u43e\u446\u435\u442\u43d\u43e\u43a\u438\u441\u435\u43b\u438 \u431\u430\u43a\u442\u435\u440\u438\u438. \u429\u430\u43c\u43e\u432\u435 CT8-1 \u438 CT8-2 \u441\u430 \u43d\u435\u43f\u43e\u434\u432\u438\u436\u43d\u438 \u438 \u43f\u440\u43e\u434\u443\u446\u438\u440\u430\u442 \u43b\u435\u432\u430\u43d\u43e\u43f\u43e\u434\u43e\u431\u435\u43d \u43c\u443\u43a\u43e- \u43f\u43e\u43b\u438\u437\u430\u445\u430\u440\u438\u434 \u43e\u442 \u437\u430\u445\u430\u440\u43e\u437\u430 \u438\u43b\u438 D-\u444\u440\u443\u43a\u442\u43e\u437\u430, \u43d\u43e \u43d\u435 \u43e\u442\u434\u435\u43b\u44f\u442 \u432\u43e\u434\u43d\u43e\u440\u430\u437\u442\u432\u43e\u440\u438\u43c \u43a\u430\u444\u44f\u432 \u43f\u438\u433\u43c\u435\u43d\u442 \u43e\u442 D-\u433\u43b\u44e\u43a\u43e\u437\u430 \u432\u44a\u440\u445\u443 \u441\u43a\u43e\u441\u435\u43d \u430\u433\u430\u440, \u441\u44a\u434\u44a\u440\u436\u430\u449 CaCO3. \u418\u437\u43e\u43b\u430- \u442\u438\u442\u435 \u43f\u440\u43e\u438\u437\u432\u435\u436\u434\u430\u442 \u43e\u446\u435\u442\u43d\u430 \u43a\u438\u441\u435\u43b\u438\u43d\u430 \u43e\u442 \u435\u442\u430\u43d\u43e\u43b \u438 \u43e\u43a\u438\u441\u43b\u44f\u432\u430\u442 \u430\u446\u435\u442\u430\u442 \u438 \u43b\u430\u43a\u442\u430\u442 \u434\u43e \u432\u44a\u433\u43b\u435\u440\u43e\u434\u435\u43d \u434\u432\u443\u43e\u43a\u438\u441 \u438 \u432\u43e\u434\u430, \u43d\u43e \u438\u43d\u442\u435\u43d\u437\u438\u432\u43d\u43e\u441\u442\u442\u430 \u43d\u430 \u43e\u43a\u438\u441\u43b\u435\u43d\u438\u435\u442\u43e \u435 \u441\u43b\u430\u431\u430. \u422\u435\u445\u43d\u438\u44f\u442 \u440\u430\u441\u442\u435\u436 \u43d\u435 \u441\u435 \u438\u43d\u445\u438\u431\u438\u440\u430 \u43e\u442 0.35 % \u43e\u446\u435\u442\u43d\u430 \u43a\u438\u441\u435\u43b\u438\u43d\u430 (v/v) \u43f\u440\u438 pH 3.5. \u418\u437\u43e\u43b\u430\u442\u438\u442\u435 \u43d\u435 \u440\u430\u441\u442\u430\u442 \u432\u44a\u440\u445\u443 30 % D-\u433\u43b\u44e\u43a\u43e\u437\u430 (w/v), \u430 \u443\u441\u432\u43e\u44f\u432\u430\u43d\u435 \u43d\u430 \u43c\u435\u442\u430\u43d\u43e\u43b \u43d\u435 \u441\u435 \u443\u441\u442\u430\u43d\u43e\u432\u44f\u432\u430. \u429\u430\u43c\u43e\u432\u435 SI15-1 \u438 SI15-2 \u438\u43c\u430\u442 \u43f\u435\u440\u438\u442\u440\u438\u445\u430\u43b\u43d\u43e \u440\u430\u437\u43f\u43e\u43b\u43e\u436\u435\u43d\u438 \u440\u435\u441\u43d\u438 \u438 \u440\u430\u441\u442\u430\u442 \u432 \u43f\u440\u438\u441\u44a\u441\u442\u432\u438\u435 \u43a\u430\u43a\u442\u43e \u43d\u430 0.35 % \u43e\u446\u435\u442\u43d\u430 \u43a\u438\u441\u435\u43b\u438\u43d\u430 (v/v) \u43f\u440\u438 pH 3.5, \u442\u430\u43a\u430 \u438 \u43d\u430 3 % NaCl (w/v) \u438\u43b\u438 1 % KNO3 (w/v). \u410\u446\u435\u442\u430\u442\u44a\u442 \u438 \u43b\u430\u43a\u442\u430\u442\u44a\u442 \u441\u435 \u43e\u43a\u438\u441\u43b\u44f\u432\u430\u442 \u434\u43e \u432\u44a\u433\u43b\u435\u440\u43e\u434\u435\u43d \u434\u432\u443\u43e\u43a\u438\u441 \u438 \u432\u43e\u434\u430, \u43d\u43e \u438\u43d\u442\u435\u43d\u437\u438\u432\u43d\u43e\u441\u442\u442\u430 \u435 \u441\u43b\u430\u431\u430. \u418\u437\u43e\u43b\u430\u442\u438\u442\u435 \u440\u430\u441\u442\u430\u442 \u432\u44a\u440\u445\u443 \u430\u433\u430\u440 \u441 \u43c\u430\u43d\u438\u442\u43e\u43b \u438\u43b\u438 \u433\u43b\u443\u442\u430\u43c\u430\u442, \u43a\u430\u43a\u442\u43e \u438 \u432\u44a\u440\u445\u443 30 % D-\u433\u43b\u44e\u43a\u43e\u437\u430 (w/v), \u43d\u43e \u43d\u435 \u443\u441\u432\u43e\u44f\u432\u430\u442 \u43c\u435\u442\u430\u43d\u43e\u43b. \u41f\u440\u43e\u432\u435\u434\u435\u43d\u438\u442\u435 \u441\u435\u43a\u432\u435\u43d- \u446\u438\u43e\u43d\u435\u43d \u430\u43d\u430\u43b\u438\u437 \u43d\u430 16S \u440\u420\u41d\u41a \u438 \u414\u41d\u41a-\u414\u41d\u41a \u445\u438\u431\u440\u438\u434\u438\u437\u430\u446\u438\u44f \u43f\u43e\u43a\u430\u437\u432\u430\u442, \u447\u435 \u449\u430\u43c\u43e\u432\u435 CT8-1 and CT8-2 \u431\u435\u437 \u441\u44a\u43c\u43d\u435\u43d\u438\u435 \u441\u435 \u438\u434\u435\u43d\u442\u438\u444\u438\u446\u438\u440\u430\u442 \u43a\u430\u442\u43e Kozakia baliensis , \u430 \u438\u437\u43e\u43b\u430\u442\u438 SI15-1 \u438 SI15-2 \u43a\u430\u442\u43e Swaminathania salitolerans