Cross Streak Method Research Articles

A comprehensive review on in-vitro methods for anti- microbial activity

Microbes, also referred to as microscopic creatures because of their minuscule size and invisibility to human sight, are single-celled microorganisms. The first microorganism was discovered between 1665 and 1683 by Robert Hooke and Antoni van Leuwenhoek. There are two categories of techniques for examining microbial activity. methods used in vitro and in vivo. Experiments and tests carried out in a laboratory setting, typically without the presence of a living organism, are referred to as in vitro methods. By creating microbial cultures, cell lines, cell cultures, etc., we may do in vitro experiments. Scientists can study biological processes, assess the effects of medications, and understand systems without having to deal with the complexities of a whole organism by employing in vitro techniques. Growing cells outside of the body in a controlled setting for a range of studies is known as cell culture. Tissues are grown and kept in a lab setting for tissue culture. It is essential to research creating anti-infection therapies. To evaluate a substance's antibacterial activity, in vitro methods such as the brothdilution method and discdiffusion method are commonly used. There are different methods available for the testing of antimicrobial activity and the methods are the agar disk diffusion method, Agar well diffusion method, cross streak method, Bioautography, dilution method, Time kill test, ATP bioluminescence etc. Techniques for determining novel antimicrobial agents and testing for antibiotic susceptibility have been employed. Compared to in-vivo procedures, in vitro methods are less costly. To guarantee an accurate experimental approach, these techniques may be the answer.

Antagonistic activity of monocultures and consortia of lactobacilli against multidrug-resistant isolates of opportunistic bacteria as a screening of their probiotic potential

Background. In recent years, special attention has been paid to the studying the consortia of probiotic bacteria. In these associations, the properties of individual microorganisms can be enhanced, in particular, their antagonistic activity which is an effective indicator for screening of probiotic potential. The development of probiotics based on such consortia with antibacterial properties is critical in the light of the growing problem of drug resistance in microorganisms.The aim of the work. To study the antagonistic activity of monocultures and consortia of lactobacilli against multidrug-resistant isolates of opportunistic bacteria. Materials and methods. The antagonistic activity of lactobacilli monocultures and their consortia was assessed simultaneously by two methods: the cross streak method and the well diffusion method.Results. All strains of lactobacilli and their consortia, depending on the research method, had varying degrees of antagonistic activity. Five consortia had stronger antagonism to test cultures as compared to monocultures, while in one consortium, the effect of antagonistic activity was reduced compared to monocultures. The results of studying the antagonistic activity of two consortia (Limosilactobacillus fermentum 44/1 and Lacticaseibacillus rhamnosus 12L, Latilactobacillus curvatus LCR-111-1 and Lactiplantibacillus plantarum 8PAZ) contradict data on the biocompatibility of strains in these consortia. Differences in the degree of antagonistic effects of lactobacilli on gram-positive and gram-negative species of opportunistic bacteria were revealed.Conclusion. The study showed that both the biocompatibility of the probiotic strains and the antagonistic activity of the consortium are the important requirements for creating a probiotic consortium with effective probiotic potential. To study the antagonistic properties of lactobacilli, the number of isolates of target gram-positive and gram-negative bacteria and normobiota should be increased. This will allow us to determine effective strategies for using probiotics in conditions of the spread of drug resistance of microorganisms.

The Indiscriminate Chemical Makeup of Secondary Metabolites Derived from Endophytes Harvested from Aloe barbadensis Miller in South Africa's Limpopo Region.

The efficacy of 23 bacterial isolates obtained from surface-sterilized stems and leaves of three medicinal plants (Aloe barbadensis Miller, Artemisia afra, and Moringa oleifera) was investigated in an endeavour to prevent the growth of Mycobacterium bovis using the cross-streak method. Endophytes were isolated by incubating sterile plant materials on nutrient agar at 30 °C for 5 days. Two isolates showing activity were subsequently utilized to produce the extracts. Whole-genome sequencing (WGC) was used to identify the isolates. Secondary metabolites produced after 7 days of growth in nutrient broth were harvested through extraction with ethyl acetate. The extracts were chemically profiled using gas chromatography-high resolution time-of-flight mass spectrometry (GC-HRTOF-MS). NCBI BLAST search results revealed that the isolated endophytes belonged to the Pseudomonas and Enterobacter genera, based on WGC. Two endophytes, Aloe I4 and Aloe I3-I5 from Aloe barbadensis, exhibited potency based on the cross-streak method. The metabolite profiling of the selected endophytes identified 34 metabolites from Aloe I4, including ergotamine, octadecane, L-proline and 143 other metabolites including quinoline and valeramide, which inhibit microbial quorum sensing. These findings suggest that bacterial endophytes from medicinal plants, particularly Aloe barbadensis, hold promise as sources of antimycobacterial agents for human health applications.

Characterization of Actinobacteria strains in Lebanese soil with an emphasis on investigating their antibacterial activity.

With the alarming rise of drug resistant pathogens, the quest for new bioactive compounds from natural habitats has increased. Actinobacteria are Gram-positive bacteria, considered prominent natural antibiotic synthesizers. This study aimed at isolating Actinobacteria from agricultural soil samples of Tamnine El Tahta and Haddatha, with an emphasis on the physicochemical soil characteristics. It also aimed at screening and identifying the antibacterial-producing Actinobacteria, with a determination of the chemical composition of the extract. Forty-six Actinobacteria were isolated from six soil samples. Actinobacteria load exhibited a positive correlation with moisture content, and a negative correlation with pH, salinity, and organic matter content. Primary screening for antibacterial activity was performed against various Gram-positive and Gram-negative bacteria by cross-streak method. Fourteen Actinobacteria isolates were potent against the test microorganisms, and the most effective isolate (T25) was selected for identification, and extract preparation. The antibacterial activity of the extract was tested using secondary screening, in addition to minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) determination. T25 isolate exhibited a 92% similarity with Micrococcus luteus/lylae. MIC recorded was 12.5mg/ml and the MBC was higher than 100mg/ml against all test microorganisms. Total phenol content was estimated to be 18.5 ± 0.0015mg GAE/g dry weight using Folin-Ciocalteu method, and total flavonoid content recorded 2.3 ± 0.02mg RE/g dry weight using aluminum nitrate colorimetric method. This study revealed that the physicochemical parameters in soils impact the distribution of Actinobacteria. Moreover, it focuses on Micrococcus luteus/lylae strain, considered a promising antibacterial resource for further potential clinical investigations.

Evaluation of the Cytotoxicity of Secondary Bioactive Compounds Produced by Streptomyces in Soil against a Colon Cancer Cell Line.

Colorectal cancer is one of the most serious malignancies affecting humans. In this study, Streptomyces bioactivechemicals extracted from soil were analyzed for their anti-colorectal-cancer and antibacterial properties. A total of 100 soil samples were collected from Kerman-Iran, incubated in SCA media and the antimicrobial properties were tested using the cross-streak method. Three strains were cultured in ISP4 medium to obtain secondary bioactive compounds. After studying the effects of the bioactive compounds on the HT29 and human foreskin fibroblast (HFF) cell lines, the expression of the p53, p21, BAX, BCL2, Casp3 and Casp8 genes was analyzed by real-time PCR and flow cytometry to detect the presence of apoptosis.The isolates show high degree of identification with Streptomyces rochei, Streptomyces fungicidicus and Streptomyces maritimus due to 16SrDNA sequence homology. Compared to HT-29 cells, Streptomyces extracts had lower cytotoxicity against normal cells (SI=5.88), followed by HFF (SI=4.14). The cell lines demonstrated a dose-dependent significant increase in DNA fragmentation, an increase in the proportion of cells in sub-G1 phase and caused G2/M cell cycle arrest in HT-29 and HFF cells.The bacterial extracts obtained displayed strong antibacterial properties and inhibited the proliferation of HT-29 and HFF cell lines. The treated cells exhibited morphological changes caused by the activation of caspase and p53/p21 proteins. This confirms that Streptomyces-induced apoptosis is mediated by the activation of p21/p53. Anti-apoptotic Bcl-2 gene expression was downregulated by treatment with the extracts. Further studies are needed to understand the antimicrobial properties of Streptomyces.

Antibacterial activity of Nocardia spp. and Streptomyces sp. on multidrug-resistant pathogens causing neonatal sepsis.

Neonatal sepsis leads to severe morbidity and occasionally death among neonates within the first week following birth, particularly in low- and middle-income countries. Empirical therapy includes antibiotics recommended by WHO. However, these have been ineffective against antimicrobial multidrug-resistant bacterial strains such as Klebsiella spp, Escherichia coli, and Staphylococcus aureus species. To counter this problem, new molecules and alternative sources of compounds with antibacterial activity are sought as options. Actinobacteria, particularly pathogenic strains, have revealed a biotechnological potential still underexplored. This study aimed to determine the presence of biosynthetic gene clusters and the antimicrobial activity of actinobacterial strains isolated from clinical cases against multidrug-resistant bacteria implicated in neonatal sepsis. In total, 15 strains isolated from clinical cases of actinomycetoma were used. PCR screening for the PKS-I, PKS-II, NRPS-I, and NRPS-II biosynthetic systems determined their secondary metabolite-producing potential. The strains were subsequently assayed for antimicrobial activity by the perpendicular cross streak method against Escherichia fergusonii Sec 23, Klebsiella pneumoniae subsp. pneumoniae H1064, Klebsiella variicola H776, Klebsiella oxytoca H793, and Klebsiella pneumoniae subsp. ozaenae H7595, previously classified as multidrug-resistant. Finally, the strains were identified by 16S rRNA gene sequence analysis. It was found that 100% of the actinobacteria had biosynthetic systems. The most frequent biosynthetic system was NRPS-I (100%), and the most frequent combination was NRPS-I and PKS-II (27%). All 15 strains showed antimicrobial activity. The strain with the highest antimicrobial activity was Streptomyces albus 94.1572, as it inhibited the growth of the five multidrug-resistant bacteria evaluated.

Investigation of Antimicrobial Activities and 16S rRNA Sequences of Actinomycetes Isolated from Karst Caves in the Eastern Black Sea Region of Türkiye

Considering that most antibiotics originate from actinomycete group bacteria, especially the Streptomyces genus, it is predicted that novel actinomycetes isolated from extreme environments such as caves may bring novel antibiotics to the medical world. The study aimed to screen the antimicrobial activity of actinomycetes isolated from the three karst caves in Türkiye and to identify selected isolates with antimicrobial activity by molecular methods. One hundred seventy-nine actinomycetes isolated from Akçakale, Kırklar (Altıntaş), and Köprübaşı Caves in Gümüşhane province in the Eastern Black Sea Region of Türkiye were included in the study. The antimicrobial activity of isolates was investigated using the modified cross-streak agar method against seven Gram-negative bacteria, three Gram-positive bacteria, and one yeast strain. Fifty-three isolates (29.6%) had antimicrobial activity against at least one of the tested microorganisms. The rate of isolates exhibiting antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Chromobacterium violaceum, Klebsiella pneumoniae, Salmonella Typhimurium, Escherichia coli, Acinetobacter haemolyticus, Pseudomonas aeruginosa, Enterobacter aerogenes, and Candida albicans was 21.2%, 20.0%, 16.8%, 12.8%, 3.4%, 2.8%, 2.2%, 1.1%, 0.6%, 0.6%, and 0.6%, respectively. An actinomycete isolate, TRMS 124, showed antimicrobial activity against ten test microorganisms. The 16S ribosomal RNA (16S rRNA) sequencing was performed for the identification and phylogenetic analysis of 26 isolates randomly selected among actinomycetes that exhibited antimicrobial activity against at least three test microorganisms. As a result, it was determined that 24 isolates showed homology with various Streptomyces species and two isolates with Embleya scabrispora and Couchioplanes caeruleus, respectively. These results showed that karst caves could be good sources for isolating actinomycetes with the potential to produce antimicrobial compounds.

Inhibition of microbial pathogens and toxicity assessment of noformicin synthesized by Psychrobacter faecalis: An endophyte of Averrhoa carambola

Plants are known to harbor endophytic microbes which primarily reside in tissues beneath the epidermal cells. The current study was focused on exploring the endophytic bacteria of Averrhoa carambola for antimicrobial activity. The metabolites of endophytic isolate VITPMJ7 were found to be effective in cross streak method (antagonistic activity) as it inhibited the growth of Bacillus cereus and Escherichia coli. Furthermore, the effective isolate was fermented and chloroform extract showed antibacterial activity in the well diffusion method against E. coli, B. cereus and Staphylococcus aureus. Chloroform extract was fractionated and the lead molecules were obtained using solid phase extraction column (SPE) and Ultra performance liquid chromatography (UPLC). The purified lead molecule that showed inhibitory effects was identified to be noformicin by GC-MS. The presence of various functional groups such as amine, thiol, alcohol, alkene, sulfoxide identified by FT-IR supported and confirmed the lead molecule to be noformicin. The isolate VITPMJ7 capable of producing the lead compound was identified to be Psychrobacter faecalis by 16 S rRNA gene sequencing. Toxicity assessment of the metabolites in seed germination assay with Vigna radiata, Vigna mungo and Cajanus cajan revealed the non-toxic nature of the compounds.

Isolation of Probiotic Strains from Fishes Cultured in Sewage-fed Ponds

The research for the need of probiotics in aquaculture is increasing with the demand for environment friendly aquaculture. The present study aimed at to isolate probiotic strains from the gut of carps cultured in sewage-fed ponds. The fish gut was dissected longitudinally and the entire gut content was plated onto deMann Rogosa Sharp (MRS) Agar for allowing the Lactic Acid Bacteria (LAB) to manifest itself as white pinpoint colonies. The MRS Agar plates containing LAB were then over-layed with osft Tryptone Soya Agar (TSA) containing pathogenic Pseudomonas cells at 105 nos/ml. Those LAB which produced a clear inhibitory zone with a diameter more than 1 mm larger than that of the colony itself was judged to be a tentative probiotics strain. During the present study, four strains (SM1, SM2, ST1 and ST2) gave a clear inhibitory zone and were classified as tentative probiotic strains. The four tentative probiotic strains were further confirmed of its probiotic effect against three pathogenic strains of Pseudomonas, Aeromonas and Edwardsiella by antagonistic assay by cross streak method and among four only two strains SM2 and ST1 exhibited an inhibition of 2 mm and 1 mm, respectively against the three pathogenic test strains and confirmed their status as probiotic strains.

Endosymbiotic bacteria from donkeys milk in the inhibition of human pathogens

Donkey milk is known for its beneficial aspects such as low fat and cholesterol, total protein and casein content, high lactose, whey protein, calcium, selenium and vitamin D3 content. It also has nutraceutical and utilitarian properties that can boost invulnerability, regulate digestion and conveniently change gut microbiota. The current study was focused on the effect of probiotic bacterial metabolites from donkey milk against human bacterial pathogens. The effective isolate VITRAAJ14 was found to be non-hemolytic and was tolerant to acid, NaCl and bile salt. Antibacterial activity by cross streak method showed maximum zone of inhibition. Well diffusion method also exhibited effective inhibition against Escherichia coli (MTCC No. 9721), Staphylococcus aureus (MTCC No. 3160) and Salmonella enterica (MTCC No. 8587) with fractions obtained from chloroform: ethyl acetate. The metabolites capable of inhibiting pathogens were identified as oleyl alcohol by Gas chromatography- mass spectrometry (GCMS). Lead compounds detected in GCMS was studied for its functional group by Fourier transform infrared spectroscopy (FTIR), which revealed the presence of alcoholic stretch, carboxylic acid OH stretch, alkene and amide bond. The isolate VITRAAJ14 was identified to be the closest neighbour of Bacillus species (MH100985) by 16S rRNA gene sequencing. To our knowledge, current study is the first-time report on the presence of Bacillus sp. from donkey milk showing antibacterial activity due to its ability to produce oleyl alcohol. Scanning electron microscopic images revealed the presence of rod-shaped morphology of the effective bacterium VITRAAJ14.

The efficiency of Soran M Medium in Selective Isolation of Antibiotic Producing Actinomycetes

To assess the role of enriched constituents forming Soran M medium in selectiveisolation of antibiotic producing actinomycetes, six soil samples were collected inNovember 2016 from different sites in Soran, Erbil, Iraq. To culture soil samples spreadplatetechnique was used on Soran M and standard medium such as Arginine GlycerolSalt agar. Preliminary screening was done using cross-streak method against fourbacterial pathogenic strains and Candida albicans. Nine isolates from total 40actinomycetes isolates showed antimicrobial activities against pathogens. Themorphological and cultural characterization of these isolates was performed and showedthat 3 isolates belonged to the genus Nocardia while the others belonged to Streptomyces.The potential strain MB3 with both broad spectrum antibacterial and antifungal activitywas isolated on both media but showed different abilities to produce antibiotics. It wasidentified as Streptomyces sp. Findings from this investigation revealed that nutrientmedia with different composition affect the isolation of antibiotic producingactinomycetes with different activities.

Inhibition of multi-species biofilm formation using chitosan-based film supplemented with essential oils

The biofilm development following microbial attachment on food-grade surfaces represents a main problem in the food sector, leading to significant economic losses. To overcome that, the present investigation aimed to develop biodegradable films that protect against heterogeneous biofilms using chitosan and essential oils (EOs). First, the cross-streak method revealed the absence of antagonism between Staphylococcus aureus, Enterococcus hirae, Escherichia coli, and Pseudomonas aeruginosa strains. The contact angle findings revealed a drop in the electron acceptor (γ+) and qualitative hydrophobicity (θw) associated with an increase in the Lifshitz-van Der Waals component (γLW) and electron donor (γ−) regarding the treated chitosan-based films (CH-films). The environmental scanning electron microscopy (ESEM) images showed that among all the treatments, the CH-film treated with the Pelargonium asperum EO exhibited the weakest antiadhesion activity (73.89 ± 1.42% of the covered surface) while the CH-film treated with the Pelargonium asperum, Thymus satureioides, Cinnamomum zeylanicum, and Eugenia caryophyllus EOs mixture had the strongest activity (9.23 ± 1.66% of the covered surface). From these results, the CH-film containing the EOs mixture presents a high potential for utilization in the food industry as an active coating and packaging that prevent the microorganisms' attachment and the eventual biofilm development.

Isolation, Identification and Screening of Saharan Actinomycete Strain Streptomyces fimbriatus AC31 Endowed with Antimicrobial Activity.

The increasing global public health concern of antimicrobial resistance (AMR) necessitates exploration of natural antimicrobial agents as potential alternatives. This study aimed to investigate antimicrobial activities of Saharan actinomycetes, with specific focus on the strain Streptomyces fimbriatus AC31, that holds promising potential as an alternative to combat AMR. In this context, 32 actinomycetes were isolated from El Atteuf (Ghardaïa), Algeria. Isolates obtained were characterized morphologically and biochemically. Screened isolate was identified by 16S rRNA gene sequencing. Classification of actinomycete isolates was carried out by UPGMA (Unweighted Pair Group Method with Arithmetic Mean). Then, they were screened for their antimicrobial activity by cross-streak method. Identification of 32 isolates revealed 5 genera: Streptomyces (65.63%), Nocardia (9.38%), Streptosporangium (9.38%), Nocardiopsis (9.38%) and Actinomadura (6.25%). According to the biochemical and physiological characteristics, UPGMA classified the isolates in 4 phenons. A number of 24 (75.00%) isolates were active against Gram-positive bacteria, 21 (65.63%) isolates were effective against Gram-negative bacteria, and 25 (78.13%) isolates inhibited Candida albicans. Screened strain Streptomyces fimbriatus AC31 showed highest antagonistic activity and revealed an inhibition zones of 41, 38, 41, 42, and 44 mm, against B. subtilis (ATCC 6633), E. coli (ATCC 8739), S. typhimurium (ATCC 13331), S. aureus (ATCC 6538) and C. albicans (ATCC 10231), respectively. Phylogenetic identification of the AC 31 isolate using 16S rRNA gene sequence showed similarity of 100% with Streptomyces fimbriatus NBRC 15411T. Actinomycete isolates characterized in this study were endowed with antimicrobial activity against various pathogenic microorganisms that could be used efficiently in developing new antimicrobial substances.

Potential Antimicrobe Producer of Endophytic Bacteria from Yellow Root Plant (Arcangelisia flava (L.)) Originated from Enggano Island.

Exploration studies of endophytic bacteria from Arcangelisia flava (L.) and their potential have not much been conducted. This research aims to explore and characterize the antimicrobial activity of endophytic bacteria in A. flava against pathogenic bacteria. This research consists of several steps including the isolation of bacteria, screening of the antimicrobial activity assay using the dual cross streak method, molecular identification through 16s rDNA analysis, and characterization of bioactive compound production through PKS-NRPS gene detection and GC-MS analysis. There are 29 endophytic bacteria that were successfully isolated from A. flava. The antimicrobial activity showed that there are four potential isolates AKEBG21, AKEBG23, AKEBG25, and AKEBG28 that can inhibit the growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The 16S rDNA sequence analysis showed that these isolates are identified as Bacillus cereus. These four isolates are identified as able to produce the bioactive compounds through the detection of polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS)-encoding genes. B. cereus AKEBG23 has the highest inhibition against pathogenic bacteria, and according to the GC-MS analysis, five major compounds are allegedly involved in its antimicrobial activity such as butylated hydroxytoluene (BHT), diisooctyl phthalate, E-15-heptadecenal, 1-heneicosanol, and E-14-hexadecenal. This result suggested that B. cereus AKEBG23 as the endophytic bacterium from A. flava has a beneficial role as well as the plant itself. The bacterium produces several bioactive compounds that are allegedly involved in its antimicrobial activity against pathogenic bacteria.

SCREENING OF ACTINOMYCETES FROM Cystoseira barbata (Stackhouse) C. Agardh COMPOST FOR THEIR ENZYME AND ANTIBACTERIAL ACTIVITIES

Bacterial secondary metabolites play an essential role in biotechnological and biomedical applications. Actinomycetes are important bacterial sources of antibiotics and enzymes. Most of the antimicrobials known today have been isolated from actinomycetes, especially from the genus Streptomyces. In this study, actinomycete isolation was performed from Cystoseira barbata (Stackhouse) C. Agardh compost, collected from the Black Sea coast, by serial dilution method. A total of 73 actinomycetes isolates (BSC) were obtained from the compost samples. The ability of the isolates to produce different extracellular enzymes was investigated qualitatively. It was determined that 68.5% of the isolates have amylase, 100% cellulase, 47.9% chitinase, 94.5% pectinase, 98.6% protease and 96.3% lipase/esterase activity. Antibacterial activities of the isolates were investigated primarily using cross-streak method. Isolates showed high antibacterial activities, with 98.6 and 84.9 % against Staphylococcus aureus Rosenbach and Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Bäl, respectively. Three out of six isolates (BSC-13, BSC-17, BSC-37, BSC-38, BSC-45, BSC-49) with high antibacterial activity, were screened secondarily for their antibacterial activities using double-layer method. At day 7, BSC-37 isolate showed a high inhibition (57 mm) against S. aureus. Furthermore, these six isolates were identified according to their morphological and physiological characteristics and 16S rDNA sequence analysis. 16S rDNA sequence analysis showed that the isolates with high antibacterial activity belong to Streptomyces genus. Results indicated that these isolates have great potential and may serve as a good source for the studies on bioactive natural products.

Lactic Acid Bacteria as Biocontrol Agents against Potato (Solanum tuberosum L.) Pathogens

Biological control offers an alternative to chemical pesticides, which are inconsistent with the global trend of “going green”. Biological control includes various approaches, from natural predators to biologically produced molecules. This article focuses on the selection of lactic acid bacteria (LAB) as biological control agents against potato pathogens. The scope included evaluating the antimicrobial activity of 100 LAB strains against ten phytopatogens (Pectobacterium carotovorum, Streptomyces scabiei, Fusarium oxysporum, Fusarium sambucinum, Alternaria solani, Alternaria, tenuissima, Alternaria alternata, Phoma exigua, Rhizoctonia solani, Colletotrichum coccodes) by cross-streak plate method. HPLC determined the metabolic profiles for the most active LAB strains, and lactic acid, acetic acid, propionic acid and ethanol were found in the largest quantities. The strain Lactiplantibacillus plantarum KB2 LAB 03 was finally selected and cultured on supplemented acid whey. After the selection in laboratory tests, the strain KB2 LAB 03 was assessed in situ on seed potatoes against phytopathogens. The test showed a 40–90% reduction of eight potato pathogens infestation; only F. sambucinum and F. oxysporum were not inhibited at all. L. plantarum KB2 LAB 03 was proposed as the potential biocontrol agent for the potato protection against phytopathogens.

Antagonistic Potential Marine Bacteria Against Luminescent Vibriosis Pathogen Vibrio Harveyi Obtained from Shrimp Farm, Marakkanam, Tamil Nadu, India

Aquaculture farming practices in India and other countries are facing a lot of economic loss due to luminescent vibriosis.Ecological and cost-effective practices may have been warranted to combat this tricky disease. A total of 57 morphologicallydifferent marine bacterial isolates were isolated from samples collected at different aquaculture ponds in Marakkanam, Tamil Nadu,India and screened for their antibacterial potential against luminescent vibriosis causing bacteria Vibrio harveyi. All marine bacterialisolates were screened for antibacterial activity by cross streak method against two different isolates V. harveyi GNC01 and V.harveyi GNC03. Secondary screening of antagonistic isolates by dual culture plate method and agar well diffusion method leads tothe identification of antagonistic isolates. The antagonistic isolates were characterized by molecular taxonomy (16S rRNA genesequence) and phylogeny and identified as Neobacillus niacini (GNC04), Bacillus halotolerans (GNC06), Bacillus amyloliquefaciens(GNC16), Lacticaseibacillus paracasei (GNC23) and Lactiplantibacillus plantarum (GNC24). The minimum inhibitory concentrationof all these selected bacterial crude secondary metabolites against both Vibrio harveyi isolates GNC01 and GNC03 ranged between0.062 to 0.50 mg/mL. The GC-MS profiling of the ethyl acetate extract revealed the presence of antibacterial compounds such asOxime-,methoxy-phenyl (26.63%), 2,3-Dihydroxybutane (9.52%), Phosphite, menthyldimethyl- (2.83%), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- (2.07%). The results of this study suggest that Lactiplantibacillus plantarum (GNC24) is apotential source for antagonistic secondary metabolites against luminescent vibriosis causing bacteria Vibrio harveyi. Further,biological control formulation with these isolates and their metabolites will be a reward for the integrated pest management inthe aquaculture sector.

Multidrug Resistant Strains Inhibition by Bacillus Species from the Gut of Oreochomis niloticus and Pomacea canaliculata

Antibiotic resistance is widespread in clinical settings, indicating a serious problem with infectious disease treatment. Novel strategies such as using natural products derived from microbes are being explored, generating increased research interest to address this issue. Here, the antimicrobial property of gut-associated Bacillus species against multidrug-resistant (MDR) strains; methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli producing extended-spectrum beta-lactamase (EsβL E. coli), and Pseudomonas aeruginosa producing metallo beta-lactamase (MβL P.aeruginosa) was evaluated using a cross-streak method and agar diffusion assay. The Bacillus isolates inhibited MRSA and ESβL E. coli with an average zone of inhibition of 9.57 ± 33.40 mm and 5.07 ± 32.69 mm, respectively, in the cross-streak method. The cell-free supernatant (CFS) of ten Bacillus species demonstrated anti-MRSA activity but was ineffective against ESβL E. coli and MβL P. aeruginosa. The relative enzyme activities of ten Bacillus isolates were determined in vitro, and amylase, caseinase, cellulase, lipase, and gelatinase production were confirmed. Isolates were identified as Bacillus siamensis, Bacillus velezensis, and Bacillus subtilis through biochemical tests and 16s rRNA sequence analysis. Minimum inhibitory concentrations (MICs) of the CFSs against MRSA range is between 12.5 and 25%. Bacillus species isolated from fish and snail guts exhibited antibacterial activity against MRSA. Therefore, it is imperative to confirm the presence of anti-MRSA active compounds in Bacillus CFS and characterize them further to determine their suitability for antimicrobial drug development.

Streptomyces filamentosus strain KS17 isolated from microbiologically unexplored marine ecosystems exhibited a broad spectrum of antimicrobial activity against human pathogens

The continuous development of antimicrobial resistance and appearance of new diseases necessitate use of novel and effective antimicrobial molecules. This research work was planned to determine the antimicrobial and metabolite profiling of Streptomyces filamentosus strain KS17 from unexplored regions of Devbag beach, India. The Streptomyces filamentosus strain KS17 was screened and was subjected to various characterization techniques. Antagonistic activity of ethyl acetate extract was carried out and finally spectral and Gas Chromatography-Mass Spectrometry (GC-MS) analysis was done. The Streptomyces filamentosus strain KS17 displayed a strong antimicrobial activity on cross streak method. The organism produced greyish and light yellow mycelia and optimal growth was observed at 30–35 ℃ and expressed an evolutionary relationship with Streptomyces filamentosus strain Act74 (KT996121). The UV spectrum exhibited active peak at 213 nm. The ethyl acetate extract exhibited significant antimicrobial activity against bacteria and yeasts, such as S. flexneri, E. coli, K. pneumoniae, B. cereus, S. aureus, E. faecalis, B. subtilis, C. albicans and C. glabrata. The gram negative bacteria P. aeruginosa was unsusceptible. The metabolite profiling through GC-MS acquainted the presence of 30 bioactive compounds having antibacterial, anticancer properties. Pentane, 2,4-dimethyl- (40.75%), Stigmast-5-en-3-ol, oleate (17.09%), 2-methylhexacosane (4.52%), 9-Octadecenoic acid, 1,2,3-propanetriyl ester, (E,E,E)- (3.85%) and Phthalic acid, di(2-propylpentyl) ester (2.82%) were the leading compounds available in extract. These compounds are known to have strong antibacterial and antifungal activity. In conclusion, the results clearly suggest the possibility of Streptomyces filamentosus strain KS17 as optimistic candidate for the development of potential antimicrobial drugs after structure elucidation and detail characterizations of bioactive metabolite.

Anti-bacteria and toxicity potential of a rare Actinobacterium Pseudonocardia sp. SM1A, isolated from Mangrove Park, West Kalimantan, Indonesia

Abstract. Nofiani R, Rizky, Briliantoro R, Ardiningsih P. 2021. Anti-bacteria and toxicity potential of a rare Actinobacterium Pseudonocardia sp. SM1A, isolated from Mangrove Park, West Kalimantan, Indonesia. Biodiversitas 23: 453-458. This study aimed to identify and evaluate antibacterial and toxicity activities of a rare Actinobacterium isolated from mangrove mud, Mempawah District, West Kalimantan. The mangrove mud sample was inoculated on ISP4 agar dissolved with seawater enriched meropenem (75µg/mL) and nystatin (100 µg/mL) using pour plate procedure and incubated at room temperature until the appearance powdery colony. One of the 2 powdery colonies successfully isolated was SM1A. SM1A was morphologically and biochemically identified to determine its genus and tested antibacterial activities using the cross streak method. SM1A also was cultivated and shaken at room temperature. After 10 days, the culture was extracted using ethyl acetate and then the extract was tested antibacterial activities using a well diffusion method and toxicity using brine shrimp lethality test (BSLT). SM1A was identified as Pseudonocardia sp., then was called Pseudonocardia sp. SM1A. Pseudonocardia sp. SM1A tested anti-bacteria using the cross streak method showed active against Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Vibrio cholera, and Streptococcus mutants for the ISP4 agar medium and S. typhi, S. mutants, and V. cholera for the ISP1. Pseudonocardia sp. SM1A extract prepared from ISP1 broth medium was active against S. typhi, E. coli, V. cholera, and Streptococcus mutants. In addition, Pseudonocardia sp. SM1A extract cultivated on ISP1 broth medium showed LC50=11.2570 µg/mL based on the BSLT and categorized as moderately toxic level. Pseudonocardia sp. SM1A contained secondary metabolites having high potential as lead compounds for drug discovery.