Lipolytic and antimicrobial activities of Pseudomonas strains isolated from soils in Phetchaburi Province, Thailand

Purpose: To identify and determine lipolytic and antimicrobial activities, and  antibiotic susceptibility of bacterial isolates from soils in Phetchaburi Province, Thailand.Methods: Bacterial strains were isolated from surface soils by enrichment technique using lipolytic broth (LB) and then identified based on their phenotypic and genetic characteristics. The cell-free culture supernatant was determined for lipase activity by spectrophotometric assay. Disc diffusion assay was used to determine the crude ethyl acetate extract of the culture supernatant for antimicrobial activity and antibiotic susceptibility. The chemical profile of the crude ethyl acetate extract was analyzed by reverse-phase C-18 column high-performance liquid chromatography (HPLC).Results: On the basis of phenotypic properties and their 16S rRNA gene sequence analysis, five bacterial isolates, P1-2, P1-5, P1-6, P1-10 and P1-20 were identified as strains of Pseudomonas with sequence similarities (99.7 – 100 %). The extracellular lipase activity in LB supplemented with 1 % (v/v) of each of Tween 20, Tween 40, 60 or 80 as the substrate ranged from 11.61 ± 0.61 to 15.09 ± 0.42, 11.79 ± 0.28 to 15.75 ± 0.47, 12.65 ± 0.01 to 14.59 ± 0.87 and 12.71 ± 0.25 to 13.96 ± 0.21 unit/mL, respectively. The crude ethyl acetate extract of isolates P1-5, P1-6 and P1-20 contained quinoline alkaloid compounds and  exhibited antibacterial activity against Gram-positive Kocuria rhizophila ATCC 9341 and Staphylococcus aureus strains ATCC 25923, ATCC 6358 and ATCC 25913, but not against Gram-negative Escherichia coli ATCC 25922. All the  isolates were susceptible to cefepime, cefotaxime, ceftriaxone, ceftazidime,  amikacin, gentamicin, imipeneum, meropeneum and levofloxacin.Conclusion: The isolates demonstrate high lipolytic activity while the crude extracts exhibit antibacterial activity against Gram-positive bacteria. Thus, this lipase is a potential enzyme for pharmaceutical applications.Keywords: Antibiotic susceptibility, Antimicrobial activity, Lipolytic activity,  Pseudomonas, Thai soi

Diversity and antimicrobial activity of the tropical ant-derived actinomycetes isolated from Thailand

Antibiotic resistance is one of the most important global healthcare challenges and is responsible for the mortality of millions of people worldwide every year. It is a crisis attributed to misuse of antibiotics and a lack of new drug development. Actinomycetes constitute a group of Gram-positive bacteria known for their distinctive high guanine-cytosine (G+C) content in their genomic DNA. These microorganisms are widely recognized for their capability to generate a wide range of secondary metabolites with diverse biological activities. These versatile microorganisms are ubiquitous in diverse ecosystems, including soil, freshwater, marine sediments, and within the bodies of insects. A recent study has demonstrated that social insects, such as ants, host a diverse array of these bacteria. In this study, we involved the isolation and characterization of a total of 72 actinomycete strains obtained from 18 distinct ant species collected from various regions across Thailand. Utilizing 16S rRNA gene analysis, these isolated actinomycetes were classified into four distinct genera: Amycolatopsis (2 isolates), Micromonospora (1 isolate), Nocardia (8 isolates), and Streptomyces (61 isolates). Among the Streptomyces strains, 23 isolates exhibited antimicrobial activity against a panel of Gram-positive bacteria, including Bacillus subtilis ATCC 6633, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, Kocuria rhizophila ATCC 9341, and Methicillin-resistant Staphylococcus aureus (MRSA) DMST 20646. Additionally, two isolates displayed antifungal activity against Candida albicans TISTR 5554. Based on 16S rRNA gene sequence similarity studies, these two isolates, ODS25 and ODS28, were demonstrated to be closely related to Streptomyces lusitanus NBRC 13464T (98.07%) and Streptomyces haliclonae DSM 41970T (97.28%), respectively. The level of 16S rRNA gene sequence similarity below 98.65% cutoff indicates its potential as a novel actinomycete species. These findings underscore the potential of actinomycetes sourced from ants as a valuable reservoir of novel antimicrobials

Streptomyces krungchingensis Sripreechasak & Phongsopitanun & Tamura & Tanasupawat 2017, SP. NOV.

DESCRIPTION OF STREPTOMYCES KRUNGCHINGENSIS SP. NOV. Streptomyces krungchingensis (krung.ching.en′ sis. N.L. masc. adj. krungchingensis pertaining to Krung Ching Waterfall National Park in Nakhon Si Thammarat Province of Thailand, where the type strain was isolated). Aerobic, mesophilic, filamentous, Gram-stain-positive actinomycete that produces extensively branched substrate and aerial mycelia. Flexuous spore chains are observed on aerial mycelia. The spore surface is smooth. White to light-greenish grey aerial mass is produced on ISP2, ISP3, ISP4 and nutrient agar but absent on ISP5, ISP6 and ISP7 media. The substrate mycelia appear greyish yellow on ISP2, ISP7 and nutrient agar, light olive brown on ISP3 and ISP4, brilliant yellow on ISP5 and greyish olive green on ISP6. Yellow to brown pigment is observed on most tested media except ISP5. Reduces nitrate to nitrite and hydrolyses starch but does not hydrolyse milk. Liquefaction of gelatin is weakly positive. Utilizes cellobiose, fructose, myo -inositol, D- mannitol, D- mannose, raffinose, ribose and sucrose as sole carbon sources, weakly utilizes salicin and D- xylose but does not utilize L- arabinose, cellulose, melezitose or D- sorbitol. Shows enzyme activities of acid phosphatase, alkaline phosphatase, esterase (C4), b-galactosidase, leucine arylamidase and N -acetyl-b- glucosaminidase, shows weak activities of esterase lipase (C8), b- glucosidase, naphthol-AS-BI-phosphohydrolase and valine arylamidase but does not show activities of a- chymotrypsin, cystine arylamidase, a- fucosidase, a- galactosidase, a- glucosidase, b- glucuronidase, lipase (C14), a- mannosidase or trypsin. Growth occurs at pH 5–9, at 15–37 Ǫ C and with 0–6 % (w/ v) NaCl. The cell-wall peptidoglycan contains LL- diaminopimelic acid. Ribose and glucose are observed as whole-cell sugars. The menaquinones are MK-9(H 8), MK-9(H 6) and MK-9 (H 4). The N -acyl type of muramic acid is acetyl. Phospholipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unknown lipids and an unidentified aminolipid. The major cellular fatty acids are anteiso-C 15: 0, C 16: 0, iso-C 16: 0, iso-C 15: 0 and iso-C 14: 0. The type strain, KC-035 T (= NBRC 110087 T = KCTC 29 503 T = TISTR 2402 T), was isolated from soil collected from Krung Ching Waterfall National Park, Nakhon Si Thammarat Province, Thailand. The DNA G+C content of the type strain is 72 mol%.Published as part of Sripreechasak, Paranee, Phongsopitanun, Wongsakorn, Tamura, Tomohiko & Tanasupawat, Somboon, 2017, Streptomyces krungchingensis sp. nov., isolated from soil, pp. 50-54 in International Journal of Systematic and Evolutionary Microbiology 67 (1) on page 53, DOI: 10.1099/ijsem.0.001570, http://zenodo.org/record/604838

Streptomyces krungchingensis sp. nov., isolated from soil

Sripreechasak, Paranee, Phongsopitanun, Wongsakorn, Tamura, Tomohiko, Tanasupawat, Somboon (2017): Streptomyces krungchingensis sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology 67 (1): 50-54, DOI: 10.1099/ijsem.0.001570, URL: http://dx.doi.org/10.1099/ijsem.0.00157

<i>Streptomyces rugosispiralis</i> sp. nov., a Novel Actinobacterium Isolated from Peat Swamp Forest Soil That Produces Ansamycin Derivatives and Nocardamines

Actinomycetes, especially the genus Streptomyces, are one of the most promising sources of bioactive natural products. In this study, a novel Streptomyces strain, RCU-064T, was isolated from a soil sample collected from a peat swamp forest in Thailand. Strain RCU-064T showed the highest 16S rRNA gene sequence similarity (99.06%) with Streptomyces malaysiensis NBRC 16446T. Based on a polyphasic approach, strain RCU-064T represents a novel species of the genus Streptomyces, for which the name Streptomyces rugosispiralis sp. nov. is proposed. The chemical isolation of the crude ethyl acetate extracts of the strain led to the isolation of six compounds: (1) geldanamycin, (2) 17-O-demethylgeldanamycin, (3) reblastatin, (4) 17-demethoxyreblastatin, (5) nocardamine, and (6) dehydroxynocardamine. These compounds were evaluated for their biological activities. All compounds showed no antimicrobial activity against tested microorganisms used in this study. Compounds (1)–(4) displayed cytotoxic activity against the NCI-H187 cell line, with IC50 values ranging from 0.045–4.250 µg/mL. Cytotoxicity against the MCF-7 cell line was found in compounds (1) and (3) with IC50 values of 3.51 and 1.27 µg/mL, respectively. Compounds (5) and (6) exhibited cytotoxicity only against Vero cells (IC50 of 16.57 µg/mL) and NCI-H187 cells (IC50 of 13.96 µg/mL), respectively. These results indicate that peat swamp forest soil remains a promising reservoir of novel actinomycetes capable of producing bioactive natural products

Streptomyces mimosae sp. nov., an endophytic actinomycete isolated from the root of Mimosa pudica in Thailand

An endophytic actinomycete, strain 3MP-10 T, isolated from the root of Mimosa pudica was taxonomically studied based upon polyphasic approaches. This strain formed spiral spore chains on aerial mycelia. ll -Diaminopimelic acid, glucose and ribose were found in the whole-cell hydrolysates. It belonged to the genus Streptomyces and was closely related to Streptomyces zhaozhouensis DSM 42101 T (98.9 %) and Streptomyces sedi JCM 16909 T (98.6 %) based on 16S rRNA gene sequence analysis results. The major menaquinones were MK-10(H 8 ), MK-10(H 6 ) and MK-9(H 8 ). The predominant cellular fatty acids were iso - C 16 : 0 , anteiso-C 15 : 0 and anteiso - C 17 : 0 . The detected phospholipids were diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol. Strain 3MP-10 T had a genome size of 7.2 Mb with a genome G+C content of 73.4 mol%. Results of in silico genome-based similarity analysis revealed ANIb values of 84.94 and 84.77 %, ANIm values of 88.01 and 87.92 %, and dDDH values of 29.9 and 29.6 % when compared with S. zhaozhouensis DSM 42101 T and S. sedi JCM 16909 T , respectively. Based on the polyphasic approach, digital DNA–DNA relatedness and average nucleotide identity, we propose that the novel actinomycete represents a novel species, Streptomyces mimosae , with type strain 3MP-10 T (=JCM 33328 T =TISTR 2646 T )

<i>Lactiplantibacillus argentoratensis</i> and <i>Candida tropicalis</i> Isolated from the Gastrointestinal Tract of Fish Exhibited Inhibitory Effects against Pathogenic Bacteria of Nile Tilapia

Nile tilapia is one of the most consumed farmed fish in the world. The outbreak of pathogenic bacterial diseases causes high mortality rates and economic losses in Nile tilapia farming. Antibiotic administrations are commonly utilized to inhibit and prevent bacterial infections. However, antibiotics are expensive and cause serious concerns for antibiotic resistance in fish that can be potentially transferred to humans. As an alternative solution, probiotics can be used to prevent infection of pathogenic bacteria in fish. In this work, both bacteria and yeast were isolated from fish gastrointestinal tracts and their inhibitory activity against Nile tilapia pathogenic bacteria was evaluated, as well as other probiotic properties. In this study, 66 bacteria and 176 acid tolerant yeasts were isolated from fish gastrointestinal tracts. Of all isolated microorganisms, 39 bacterial and 15 yeast isolates with inhibitory effect against pathogens were then examined for their probiotic properties (acidic and bile salt resistance, adhesion potential, and biofilm formation), formation of antibacterial factor survival rate under simulated gastrointestinal fluid, and safety evaluation. AT8/5 bacterial isolate demonstrated probiotic properties and the highest inhibition against all 54 tested pathogens while YON3/2 yeast isolate outperformed the inhibitory effect among all yeast isolates. These two probiotic isolates were further identified by 16S rDNA and the D1/D2 domain of 26S rDNA sequence analysis for bacterial and yeast identification, respectively. AT8/5 and YON3/2 showed the highest similarity to Lactiplantibacillus argentoratensis and Candida tropicalis, respectively. This is the first report on isolated L. argentoratensis and C. tropicalis with antipathogenic bacteria of Nile tilapia properties. Collectively, AT8/5 and YON3/2 could be potentially used as promising alternatives to existing antibiotic methods to prevent pathogenic bacteria infection in Nile tilapia farming

Alleviation of soil acidification and modification of soil bacterial community by biochar derived from water hyacinth Eichhornia crassipes

Abstract The highly acid sulfate Rangsit soil series of Rangsit, Pathum-Thani district, Thailand poses a major problem for agriculture in the area. Water hyacinth is a naturally occurring weed that can grow aggressively, causing eutrophication and leading to many severe environmental impacts. Here, through the pyrolysis process, we convert water hyacinth to biochar and use it for acid soil amendment. We found the ratio between biochar, soil, and sand suitable for the cultivation of water convolvulus to be 50 g of biochar, 400 g of soil, and 100 g of sand (1:8:2). This soil mixture improved the pH of the soil from 4.73 to 7.57. The plant height of the water convolvulus grown in the soil mixture was the greatest at 20.45 cm and the plant weight with and without roots was greatest at 2.23 g and 2.52 g, respectively. Moreover, we demonstrated the dominance and high abundance of Bacillus among the community in soil with biochar amendment. Here we provide the first assessment of the appropriate amount of water hyacinth-derived biochar for mitigation of soil acidity and promotion of optimal water convolvulus growth. Moreover, biochar can optimally modify soil bacterial communities that benefit plant development

Lactiplantibacillus argentoratensis and Candida tropicalis Isolated from the Gastrointestinal Tract of Fish Exhibited Inhibitory Effects against Pathogenic Bacteria of Nile Tilapia

Nile tilapia is one of the most consumed farmed fish in the world. The outbreak of pathogenic bacterial diseases causes high mortality rates and economic losses in Nile tilapia farming. Antibiotic administrations are commonly utilized to inhibit and prevent bacterial infections. However, antibiotics are expensive and cause serious concerns for antibiotic resistance in fish that can be potentially transferred to humans. As an alternative solution, probiotics can be used to prevent infection of pathogenic bacteria in fish. In this work, both bacteria and yeast were isolated from fish gastrointestinal tracts and their inhibitory activity against Nile tilapia pathogenic bacteria was evaluated, as well as other probiotic properties. In this study, 66 bacteria and 176 acid tolerant yeasts were isolated from fish gastrointestinal tracts. Of all isolated microorganisms, 39 bacterial and 15 yeast isolates with inhibitory effect against pathogens were then examined for their probiotic properties (acidic and bile salt resistance, adhesion potential, and biofilm formation), formation of antibacterial factor survival rate under simulated gastrointestinal fluid, and safety evaluation. AT8/5 bacterial isolate demonstrated probiotic properties and the highest inhibition against all 54 tested pathogens while YON3/2 yeast isolate outperformed the inhibitory effect among all yeast isolates. These two probiotic isolates were further identified by 16S rDNA and the D1/D2 domain of 26S rDNA sequence analysis for bacterial and yeast identification, respectively. AT8/5 and YON3/2 showed the highest similarity to Lactiplantibacillus argentoratensis and Candida tropicalis, respectively. This is the first report on isolated L. argentoratensis and C. tropicalis with antipathogenic bacteria of Nile tilapia properties. Collectively, AT8/5 and YON3/2 could be potentially used as promising alternatives to existing antibiotic methods to prevent pathogenic bacteria infection in Nile tilapia farming