1b Isolates Research Articles

ZnO, TiO2 and Ag nanoparticles impact against some species of pathogenic bacteria and yeast.

The economic approaches for manufacturing the nanoparticles with physical and chemical effects and limited resistance to antibiotics have been progressed recently due to the rise of microbial resistance to antibiotics. This research aimed to study the antimicrobial efficacy of silver nanoparticles Ag, ZnO, and Tio2 nanoparticles against Salmonella typhimurium and Brucella abortus and Candida albicans. Two isolates of Salmonella and two isolates of Brucella abortus were isolated from food spastically meat and blood specimens, respectively. Candida albicans were isolated from the patient's mouth with oral candidiasis (oral thrush) and confirmed diagnosis by API 20C test. The antimicrobial susceptibility of Salmonella typhimurium and B. abortus isolates were performed against nine different antibiotics. Silver nanoparticles consisting of AgNPs size (90) nm, ZnO NPs size (20, 50) nm as well as TiO2 NPs size (10, 50) nm, were used. UV-Visible spectrophotometer was used to characterize silver nanoparticles. The highest resistance of Candida albicans was seen for fluconazole, Clotrimazole and Itraconazole. The results of the Minimum Inhibitory Concentration (MIC) of nanoparticles against Salmonella typhimurium showed the average MIC of Tio2-10nm and Tio2-50nm were 5000 and 2500 μg\ml for S1 and S2 isolates, respectively. The isolated Brucella abortus (B1 and B2) showed sensitivity to NPs with different MIC. The average MIC for Ag-90nm was 5000 and 2500 µg/ml for B1 and B2 isolates, respectively. The findings suggest NP solution has fungicidal and bactericidal impacts on the tested microorganisms so they can be suitable for multiple applications of the biomedical field such as developing new antimicrobial agents.

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence, resistance to biocides, genomic landscape and biocontrol.

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.

Astaxanthin-Producing Microalgae Identification Using 18S rRNA : Isolates from Bangkalan Mangrove Waters and Sowan Tuban Northern Waters, East Java, Indonesia

Microalgae are a group of micro-sized photosynthetic organisms that range from prokaryotic cyanobacteria to eukaryotic algae. Microalgae are widely used as a source of natural food, cosmetic ingredients, food ingredients, and a source of pigments. This study aims to identify species of four microalgae isolates named B1, B2, B3, and S2 from Bangkalan Mangrove Waters and Sowan Tuban Northern Waters, and to determine their astaxanthin pigment concentration under 1 M NaCl. Species identification was carried out through a molecular approach by utilization of an 18S rRNA gene marker. A quantitative test of astaxanthin concentration was carried out by spectrophotometric analysis. Molecular identification results show that isolates B1 and B3 are closely related to Chlorella sp., while isolates B2 and S2 are closely related to Picochlorum maculatum. Moreover, under salinity stress condition of 1 M NaCl shown a significant decrement of astaxanthin production compared to the control treatment. At 1 M NaCl, the astaxanthin content of isolate B1 was 4x10-5 mgL-1, isolate B2 was 2x10-5 mgL-1, isolate B3 was 1x10-5 mgL-1, and isolate S2 was 6x10-6 mgL-1. All in all, isolate S2 has the highest astaxanthin among the other isolates at normal conditions, while under salt stress regime, isolate B1 shown to be the best source for astaxanthin.

Profiles of Bacillus spp. Isolated from the Rhizosphere of Suaeda glauca and Their Potential to Promote Plant Growth and Suppress Fungal Phytopathogens.

Members of the genus Bacillus are known to play an important role in promoting plant growth and protecting plants against phytopathogenic microorganisms. In this study, 21 isolates of Bacillus spp. were obtained from the root micro-ecosystem of Suaeda glauca. Analysis of the 16S rRNA genes indicated that the isolates belong to the species Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus aryabhattai and Brevibacterium frigoritolerans. One of the interesting findings of this study is that the four strains B1, B5, B16 and B21 are dominant in rhizosphere soil. Based on gyrA, gyrB, and rpoB gene analyses, B1, B5, and B21 were identified as B. amyloliquefaciens and B16 was identified as B. velezensis. Estimation of antifungal activity showed that the isolate B1 had a significant inhibitory effect on Fusarium verticillioides, B5 and B16 on Colletotrichum capsici (syd.) Butl, and B21 on Rhizoctonia cerealis van der Hoeven. The four strains grew well in medium with 1-10% NaCl, a pH value of 5-8, and promoted the growth of Arabidopsis thaliana. Our results indicate that these strains may be promising agents for the biocontrol and promotion of plant growth and further study of the relevant bacteria will provide a useful reference for the development of microbial resources.

Phylogenetic tree of NS5A gene of hepatitis C virus from infected Iraqi patients

Hepatitis C is a liver illness caused by the hepatitis C virus (HCV). Acute and chronic hepatitis can be caused by the virus. Hepatitis C infection affects an estimated 71 million people worldwide. Two hundred blood samples were collected (128 males and 72 females), ranged in age from 4 to 80 years and diagnosed through a rapid test and the real time-PCR from the teaching medical city hospital in Baghdad from October 2018 until April 2019. The results showed that 89 samples were positive for HCV by serological tests, and other samples are not. Also, the most frequent age of infection of HCV was between 51 and 80 years. In all samples, the partial region of the NS5A gene was successfully amplified and sequenced. PCR-sequencing revealed three subtyping were; subtype 1a, 1b, and 4a. Phylogenetic analysis revealed subtype concordance of 99 % with a bootstrap hepatitis C virus subtype 1b isolate AASN1 Iraq (ID:MT554101.1). The clusters including HCV subtype 1a isolate AASN20 Iraq (ID:MT554100.1) the identical 96 %, and the clusters including HCV subtype 4a isolate AASN35 Iraq (ID:MT554099.1) the identical 99%.

Biofilm Formation Capability of Clinical Helicobacter pylori Isolates on MKN-45 Cells

Background: In vitro biofilm formation of H. pylori is demonstrated; however, its potential role in the persistent infection of the human stomach has not yet been addressed. Objectives: The aim of this study was to assess the biofilm formation of clinical H. pylori isolates on an epithelial cell line, a line that produces mucin. Methods: H. pylori isolates consisting of an efficient (19B) and a weak (4B) biofilm formation ability, were selected from screening of the clinical isolates. Their adhesion index was determined after 2h incubation with the semi-confluent monolayers of MKN-45 cells. Their biofilm formation was evaluated after 24 and 72 h incubation with MKN-45 cells using a modified adherence assay developed in this work. Production of biofilm was quantitatively assessed by CFU enumeration and qualitatively by the immunofluorescence, and scanning-electron-microscopic (SEM) methods. Due to the importance of mucin in the binding of H. pylori and biofilm formation, the binding strength of the mucin binding protein, MUC5AC, and MUC1 with docking was investigated using cluspro webserver. Results: Using MKN-45 epithelial cell line as a model, significant differences were observed between the adhesion index of 19B and 4B isolates. After 24h, both isolates were able to form biofilms with significantly higher numbers of CFU for the 19B isolate. These results were confirmed by immunofluorescence and SEM such that after 24h, a cluster of coccoid bacteria on the MKN-45 cells in the form of microcolonies was observed. The docking results showed that MUC5AC demonstrated the most favorable interaction with H. pylori urease and BabA with docking energy scores of -931.1 and -906.3 kcal.mol-1, respectively. Conclusions: By developing an appropriate in situ biofilm assay, we investigated biofilm formation by clinical H. pylori isolates on the MKN-45 epithelial cell line. The establishment of such an in-situ model for studying the biofilm formation ability of clinical isolates can also be used to study cell-bacteria interactions in the context of a complex biofilm and also as a model for drug screening applications.

First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Spicebush in Louisiana.

In the past two decades, laurel wilt disease has significantly affected members of the Lauraceae in the southeast United States, causing widespread mortality of native redbay (Persea borbonia (L.) Spreng), and incidence of infections in avocado (Persea americana Mill.), sassafras (Sassafras albidum L.) and swamp bay (Persea palustris [Raf.] Sarg.) (Fraedrich et al., 2008, 2015, Olatinwo, et al. 2019). Laurel wilt is a vascular disease caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva), a fungus vectored by a non-native ambrosia beetle Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). In August 2020, we investigated the mortality of a spicebush shrub (Lindera benzoin L.) (3.8 cm diameter at root collar, two m height) located ca. 17 mi northeast of Colfax, Grant Parish, Louisiana (31.750263° N, -92.643694° W). Evaluation of the dead shrub revealed brown, persistent foliage, and black vascular discoloration of the sapwood, typical symptoms of laurel wilt (Fig. S1). Although, beetle holes were observed on the sapwood, no beetle was found in galleries at the time. In the laboratory, a fungus consistently isolated from surface-sterilized sapwood tissues plated on potato dextrose agar (PDA) was identified as R. lauricola based on the morphological characteristics of the isolate (i.e., mucoid growth, conidiophores, and oblong/ovoid shape conidia [Harrington et al. 2008]). The fungal isolate was denoted as SB1. The identity of the fungus was confirmed by positive PCR amplification of the large subunit ribosomal RNA gene region using species-specific primers; rab-lsu-rl_F: CCCTCGCGGCGTATTATAG and rab-lsu-rl_R: GCGGGGCTCCTACTCAAA (Olatinwo, unpublished). The sequence of the isolate SB1 (GenBank Accession no. MW207371) showed 100% homology to the R. lauricola strain CBS 127349 sequence (GenBank Accession no. MH877933). The pathogenicity of SB1 on spicebush was evaluated on four healthy shrubs (average: 1 m height and 40 mm in diameter) at the location from which the original detection was made. Stems of two spicebush shrubs were inoculated with SB1 agar plugs from a 14-day old culture on PDA, while plain PDA plugs were used on the remaining two shrubs as non-inoculated controls. Agar plugs were placed in 5 mm (0.2 in) diameter hole punched on the bark with cork-borer as described by Mayfield et al (2008). After six weeks, the R. lauricola inoculated shrubs were wilted with noticeable blackened tissue discoloration in the sapwood, while the control trees remained healthy (Fig. S2). Raffaelea lauricola was re-isolated from tissue of the two inoculated, symptomatic shrubs, but not from the control trees. The sequence of the re-isolated R. lauricola isolate, denoted as SB3 (GenBank Accession no. MW207372), showed 100% homology to the R. lauricola strain CBS 127349 and isolate SB1. This first documentation of laurel wilt on spicebush in Louisiana is significant because, spicebush berries, leaves, and twigs are food sources for forest animals, birds, and insects including whitetail deer and spicebush swallowtail (Papilio troilus L.). Since its first report on sassafras in 2014 (Fraedrich et al. 2015), laurel wilt has spread across Louisiana on sassafras and swamp bay (Olatinwo et al. 2019) and has been confirmed in14 parishes. This report shows the relentless nature of the disease, as the pathogen moves from one vulnerable host to the next, expanding into new locations and threatening forest ecosystems across the southern United States.

The growth rate of inulinolytic yeast isolates from yum (Pachyrhizus erosus L.) and their inulinase production capability

Inulinase (EC. 3.2.1.7) is a hydrolytic enzyme that hydrolyze inulin into fructose. This enzyme was very important in the high fructose syrup industry. Microbes producing inulinase can be isolated from various sources, among others from various types of tuber that contain inulin. The isolation of inulinolytic yeast from yum (Pachyrhizus erosus L.) obtained two potential isolates. Research on the growth rate of the two potential yeast isolates in producing inulinase is necessary to understand the pattern of the yeast growth and its ability to produce inulinase. The parameters observed in this study were specific growth rate (µ), generation time (g) and inulinase activity of two selected yeast isolates B2 and B3. The results showed that the specific growth rate (µ) of B2 isolate was 0.11 hours with generation time (g) 6.12 hours, whereas for B3 isolates the specific growth rate (µ ) was 0.21 hour and generation time 3.29 hours. The inulinase activity from yeast isolates B2 and B3 were 0.294 IU and 0.235 IU, respectively.

In vitro evaluation of the probiotic potential of Lactobacillus isolated from native swine manure.

Background and Aim:Using antimicrobials as a feed additive in swine production is prohibited because it is a major cause of the emergence of antimicrobial-resistant bacteria. Probiotics such as Lactobacillus spp. are an attractive alternative to reduce antimicrobial resistance and promote swine growth. This study aimed to evaluate the in vitro probiotic properties of Lactobacillus isolated from indigenous swine manure.Materials and Methods:A total of 30 fecal samples from healthy individual indigenous pigs were collected and isolated on de Man, Rogosa, and Sharpe agar. The preliminary screen identified candidates with antibacterial activity against six pathogens and >50% survival and tolerance to acid (pH 3.0) and 1% bile salt. Isolates that passed the initial screen will be tested for other probiotic properties.Results:Of the 314 isolates from 30 pig manure samples, 17 isolates satisfied all initial conditions for probiotic properties. Each isolate has unique, distinctive properties. Isolates B4, B5, B8, B17, B87, and B144 formed thick biofilms, whereas isolates B5, B8, and 27 adhered well to the intestinal wall and exhibited strong autoaggregation properties. Isolate B4 aggregated with Enterohemorrhagic Escherichia coli and Enteropathogenic E. coli. Tests in pH-adjusted cell-free medium indicated that the antibacterial activity resulted from bacterial acidification rather than bacteriocin formation. Sequence analysis (16S rRNA) revealed 16 of the isolates were Lactobacillus plantarum, and only one isolate was Lactobacillus salivarius.Conclusion:We isolated 17 Lactobacillus from swine manure and demonstrated that their probiotic properties might be useful as a probiotic cocktail for swine feed.

Comparative genome analyses of four rice-infecting Rhizoctonia solani isolates reveal extensive enrichment of homogalacturonan modification genes

BackgroundPlant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus.ResultsRice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes.ConclusionFour R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China.

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) μm long × (13-21) μm wide (n=30) in V8 juice agar, (22-38) μm long × (11-19) μm wide (n=30) in PCA, (21-41) μm long × (13-20) μm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 μm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

Isolation and characterization of lactic acid bacteria from Sabah (North Borneo) stingless bees for probiotic and food applications

ABSTRACT Stingless bees harbor lactic acid bacteria (LAB) which possesses multiple beneficial properties. However, there is no report on LAB in stingless bee from Sabah and their products. This study aimed to isolate LAB from several stingless bee species and their products as well as to perform characterization and safety assessments. A total of 104 strains were isolated and seven potential antimicrobial LAB isolates were identified from stingless bee and their products. Characterization, identification, and assessments were performed on seven (A2b, B3b, P1b, H4b, A6, B5, and B10) LAB that exert potential antimicrobial activity against Listeria monocytogenes ATCC 7644. The A6 isolate was closely related to the Lactobacillus pentosus species whereas isolates B5 and B10 were closely related to Weissella paramesenteroides species. Finally, isolates P1b, H4b, B3b, and A2b were closely related to the Enterococcus sp. These seven LAB isolates were able to survive in stimulated gastrointestinal tract conditions (acidic, salt, bile salt, and temperature). The carbon fermentation, proteolytic activities, acidification, milk coagulation, and wide antibiotic susceptibility testing of seven LAB isolates revealed their potential used as a probiotic and fermentation purposes. Therefore, more studies are warranted to investigate the potential of these LAB isolates towards applications in probiotic and fermentation.

Morphological and Molecular Identification of Colletotrichum spp. Associated with Chili Anthracnose Disease in Yogyakarta Region

Colletotrichum sp., the causal agent of anthracnose disease, is one of the important pathogenic fungi in chili which can cause considerable yield losses, especially during the rainy season. This study aimed to identify the species of Colletotrichum isolates obtained from chili cultivation area in The Special Region of Yogyakarta Province both morphologically and molecularly. As a comparison, a Colletotrichum isolate obtained from Magelang Regency, Central Java Province was used as comparison isolate. From the isolation result, it was obtained 14 isolates of Colletotrichum that generally had conidia that were fusiform to cylindrical with two pointed or slightly blunt ends, or crescent shapes with a various size range between 9.02-19.38 µm x 2.37–8.57 µm. Based on morphological observations using UPGMA analysis, these 14 isolates could be divided into 4 groups with 7 different types. Representative isolates of each type in different groups and a comparison isolate were identified molecularly by multi-gene analysis using the ITS1-4, gapdh and tub2 genes. The result showed that B1, G1, K2 and Mg isolates were closely related to Colletotrichum scovillei, J1 with C. truncatum; S1 and S2 with C. siamense; and J2 with C. makassarii. From the pathogenicity test on wounded chili, it showed that C. scovillei and C. siamense isolates had higher virulence than C. truncatum and C. makassarii isolates.

Antifungal Activities of Vernonia amygdalina Crude Extracts and Fractions against Strain1161, P37005 and RM1000

This research study was focused in determining antifungal activities of the crude extracts and fractions of Vernonia amygdalina against strain 1161, P37005 and RM1000. Vernonia amygdalina leaves were extracted by the reflux extraction protocol which was done in a successive method. This study also analysed quantitatively the phytochemical that were present in the crude leaf extracts of Vernonia amygdalina. The study identified the presence of tannin, flavonoid, alkaloids, saponin and phenols. The three strains used in this study were tested for their susceptibility, However, activity of the crude extract was assayed at a varying concentration of 40, 60, 80 and 100 mg/ml. The methanol leaf extract (MLE) at a concentration of 100mg/ml showed the highest zones of inhibition 15.33± 1.23 mm against strain 1161(isolate S5). The result of the minimum inhibitory concentration (MIC) and minimum fungicidal concentration showed for active crude extracts were 12.5 mg/ml and 50 mg/ml presented for the n- hexane crude extract against strain 1161(IsolateS5). The value for the methanol crude extract were 12.5 mg/ml and 50 mg/ml against strain 1161 (Isolate: S5). Furthermore, the value of 12.5 mg/ml and 100mg/ml were showed for n- hexane crude extract against P37005 (isolate: B4) However, The antifungal activity of the fractions against the strains were determined at a concentrations of 5mg/ml and 10mg/ml. The various fractions of Vernonia amygdalina showed inhibitory activity against all the strains.16.00± 0.0mm was the highest value that was presented for n-hexane fraction of Vernonia amygalina against strain 1161 and P37005. The result of the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) for active fractions (F1) are 12.5 mg/ml and 25 mg/ml showed for n- hexane fraction of Vernonia amygdalina against strain P37005(Isolate:B4). The value of 12.5 mg/ml and 25 mg/ml were showed for n- hexane fraction (F4) of Vernonia amygdalina against strain 1161 (isolate: S5). Fraction (F4) showed 3.125 mg/ml and 6.25 mg/ml against strain P37005 (isolate: B4). The methanol fraction (F1) showed values of 3.125 mg/ml and 25 mg/ml respectively against strain RM1000(isolate B2). Fraction F6 had values of 3.125 mg/ml and 6.25 mg/ml against strain P37005 (isolate: B4). It can be deduced from this study that the n- hexane and methanol crude extracts showed a significant result than the ethyl acetate crude extracts. Furthermore, there was inhibitory activity for the n-hexane, ethyl acetate and methanol fractions against all the tested strains.

ISOLASI DAN UJI ANTAGONIS BAKTERI ASAL TANDAN KOSONG SAWIT YANG DIAPLIKASIKAN PADA AREAL TANAMAN KARET TERHADAP PENYAKIT JAMUR AKAR PUTIH (R. microporus)

The concern of oil palm empty fruit bunches (OPEFB) utilization in the rubber field that potential to be a host of white root disease needs to be deeply investigated. The objective of this study was to assess the bacteria on the surface of OPEFB that able to inhibit the growth of R. microporus fungi. The antagonistic assay of eight isolates of bacteria against R. microporus fungi was conducted in the Plant Protection Laboratory of Sungei Putih Research Centre. The isolates were isolated from the surface of OPEFB applied in the rubber field. Nutrient agar (NA) was employed in the isolation and purification of bacteria. The antagonistic assay was carried out in vitro using the dual culture method. The observation was performed on 2, 4, 6, and 8 days after incubation (DAI). The result indicated that the eight isolates have varied in inhibition ability of R. Microporus fungi. The initial inhibition was started since 2 DAI particularly in the isolates B2, B3, B4, and B7. The percentage of inhibition were 30%, 60%, 65% dan 69% respectively. Until 8 HSI, the isolates B2, B3, B4, B6, B7, and B8 were still inhibiting the growth of R. microporus fungi > 80%. The isolates B3 and B4 showed the best performance in fungi inhibition with the percentage > 95%. Whereas, the ability of inhibition of the isolates B1 and B5 were very low as < 50%. The existence of such antagonistic bacteria perhaps led to OPEB was not role as the host of white root disease while applied in the rubber field

Isolation of Bacillus spp. from rhizosphere of garden soil: their potential role in amylase production and nitrogen cycle

Rhizobacteria influence plant growth by producing various substances like enzymes and play role in nitrogen cycle. Microbes are the most important sources of enzymes because of their stability and reduced price in production. In our present investigation, soil samples were collected from rhizosphere of garden soil and a total of eight isolates of Bacillus spp. were presumptively identified by morphological, cultural and biochemical analysis with the conventional technique. The isolates were named as A1, A3, A4, A5, B2, C1, D1 and D2. Amylase enzyme assay was performed by 3, 5-dinitrosalicylic acid method. The highest enzyme activity was observed with the isolate A4 (2.71 U/ml) followed by isolates D2 (2.54 U/ml) and D1 (2.52 U/ml). The highest ammonification was found to be positive with the isolates A4 and B2 followed by isolate D1. Denitrification potential was found to be highest with isolates A4 and A5. No isolates were found to fix atmospheric nitrogen in Jensen’s media after two weeks of incubation. The isolates have a great potential of amylase production and they can be used in different industries as well as in starch rich waste degradation. Involvement of these bacteria in nitrogen cycle may help promote growth of plants.
 Stamford Journal of Microbiology, Vol.10 (1) 2020: 12-15

Public health and clinical implications of multidrug-resistant shigellosis cases in a reception centre for refugees/migrants, Greece, October-December 2019.

From October to December 2019, 18 shigellosis cases (median age: 4.3 years, range: 0-21) were identified in the Reception Center for refugees/migrants on the Greek island of Samos. Fifteen cases (83.3%) were Afghani. Median time from arrival to symptoms onset was 53 days (40-101). Isolates from 14 cases, serotyped as Shigella flexneri 1b, and from three cases, serotyped as S. sonnei phase S (I), presented a multidrug-resistant phenotype. S. flexneri 1b isolates also produced extended-spectrum β-lactamases. Shigella flexneri 2a isolate from the remaining case was resistant to sulfomethoxazole, trimethoprim and pefloxacin. Improvement of hygiene and strengthening of laboratory investigation is needed.

Genetic Differentiation of Verticillium dahliae Populations Recovered from Symptomatic and Asymptomatic Hosts.

Verticillium dahliae is a soilborne fungal pathogen affecting many economically important crops that can also infect weeds and rotational crops with no apparent disease symptoms. The main research goal was to test the hypothesis that V. dahliae populations recovered from asymptomatic rotational crops and weed species are evolutionarily and genetically distinct from symptomatic hosts. We collected V. dahliae isolates from symptomatic and asymptomatic hosts growing in fields with histories of Verticillium wilt of potato in Israel and Pennsylvania (United States), and used genotyping-by-sequencing to analyze the evolutionary history and genetic differentiation between populations of different hosts. A phylogeny inferred from 26,934 single-nucleotide polymorphisms (SNPs) in 126 V. dahliae isolates displayed a highly clonal structure correlated with vegetative compatibility groups, and isolates grouped in lineages 2A, 2B824, 4A, and 4B, with 77% of the isolates in lineage 4B. The lineages identified in this study were differentiated by host of origin; we found 2A, 2B824, and 4A only in symptomatic hosts but isolates from asymptomatic hosts (weeds, oat, and sorghum) grouped exclusively within lineage 4B, and were genetically indistinguishable from 4B isolates sampled from symptomatic hosts (potato, eggplant, and avocado). Using coalescent analysis of 158 SNPs of lineage 4B, we inferred a genealogy with clades that correlated with geographic origin. In contrast, isolates from asymptomatic and symptomatic hosts shared some of the same haplotypes and were not differentiated. We conclude that asymptomatic weeds and rotational hosts may be potential reservoirs for V. dahliae populations of lineage 4B, which are pathogenic to many cultivated hosts.

Methylotrophic bacteria from underarm of human: Isolation, molecular detection, antibiofilm activity, and toxicity property

Background: Methylotrophic bacteria are a group of bacteria that capable in utilize one-carbon and reduced carbon compounds as a source of carbon and energy for their growth. This ability was supported by the presence of mxaF gene that encodes methanol dehydrogenase (MDH) enzyme. Previous studies have reported the presence of these bacteria in the mouth and feet of human. However, research about the presence of methylotrophic bacteria in human underarm is still limited. Biofilm is known as one of the self-defense mechanisms against the antibacterial agents, possessed by several bacteria, resulting in resistance and many chronic infections. This research is aimed to isolate methylotrophic bacteria from underarm of human, detect the genes encode MDH, screen the antibiofilm activity of these bacteria, and continue to determine their toxicity property. Methods and materials: Methylotrophic bacteria were isolated from 80 participants. Subsequently, molecular detection of mxaF gene as marker of these bacteria was conducted by performing polymerase chain reaction (PCR) using specific primer, namely f1003 and r1561. Then, antibiofilm activity assay was performed in two different mechanisms (inhibition and destruction activity) at two different concetration treatment (5 and 10% of crude extract). Finally, toxicity assay was performed by using brine shrimp lethality assay (BSLA) to determine LC50 values of these isolates. Results: In total, 120 isolates were isolated from 80 participants, and mxaF genes of 54 isolates were successfully amplified. Most of the methylotrophic bacteria exhibited specific antibiofilm activity against specific pathogen at specific concentration. The highest antibiofilm activity was demonstrated by isolate B2.4, which was identified as Serratia marcescens through 16S rRNA gene sequencing method. This isolate showed the highest inhibition activity against ETEC and also the destruction activity against Staphylococcus aureus ATCC 25923 with the result up to 65.09% and 58.56%, respectively. Then, five selected isolates showed LC50 values greater than 1000 μg/mL, indicating that these isolates had no cytotoxic effect. Conclusion: Further study needs to be conducted to explore other molecular detection of methylotrophic bacteria, other than using mxaF gene as a molecular marker. Moreover, it is also important to characterize, purify, and do more advanced toxicity assay on the potential bioactive compound.

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).