Universal Eubacterial Primers Research Articles

Evaluation of Antifungal Effect of Allium jesdianum Bacterial Endophytes on Rhizopus stolonifer, Penicillium, Cladosporium, and Trichophyton mentagrophytes

: Antimicrobial resistance (AMR) is the ability of microbes to survive and grow in the presence of antimicrobial drugs. Bacterial endophytes, which reside within plants, can promote plant growth and other essential activities. Endophytes have the potential to produce novel and unique products for use in modern medicine, agriculture, and industry. They may hold the key to combating drug-resistant strains by providing new antimicrobial compounds, potentially leading to the development of new medications. In this study, bacterial endophytes of Allium jesdianum were extracted and characterized. The antifungal effect of these endophytes on Rhizopus stolonifer, Penicillium, Cladosporium, and Trichophyton mentagrophytes was evaluated. Various parts of the plant were used to isolate ten bacterial endophytes to assess their antifungal properties. Universal eubacterial primers were used to amplify a 1500 bp region of the 16S rRNA gene. The resulting partial nucleotide sequences were compared with sequences available from the National Center for Biotechnology Information (NCBI) and EzBioCloud Database. Subsequent comparisons determined their similarity. Each isolated endophyte was cultured with equal amounts of R. stolonifer, Penicillium, Cladosporium, and T. mentagrophytes. This study measured the level of antagonism by assessing the extent of potential interactions. Ten bacterial endophytes, primarily cocci and bacilli, were extracted from different parts of A. jesdianum. The isolates were identified as Staphylococcus warneri AJB1, Staphylococcus succinus AJB2, Pseudomonas thivervalensis AJB3, Bacillus subtilis AJB4, Acinetobacter lwoffii AJB5, and Pantoea brenneri AJB6. AJB1 and AJB3 showed no antagonistic activity. AJB2, AJB5, and AJB6 displayed antagonistic action against R. stolonifer. Only AJB4 showed strong antagonistic activity against Penicillium. Further research is necessary to determine whether these endophytes possess antibiotic activity and can be used for food preservation.

Isolation and Identification of Actinomycetes-A Potential Bioprospective Candidates

Actinomycetes were isolated from the vegetable decomposing soil sample by serial dilution agar plating method. Five actinomycetes (A1 to A5) were obtained and the antimicrobial activity was carried out by Kirby Bauer agar diffusion method against selected eleven bacterial and eight fungal pathogens. Actinomycete strain A4 demonstrated the most activity. The most active actinomycete strain (A4) was identified phenotypically as Streptomyces spp. using macroscopic and microscopic examination. Following that, bioactive substances were purified using silica gel column chromatography and identified using Gas Chromatography-Mass Spectrometry. The major compounds identified were canadaline (35.79%), 2’4-diphromophenoxyphenol(31.07%). The ethyl acetate crude extract was assayed for anticancer activity by MTT method on HepG2cells with 10µl of extract showed 48.93% viability of HepG2 cells and significantly (p-value 0.000151 < 0.05 α-values) active among different concentration of crude A4 extract. The crude extract of A4 actinomyces exhibited the maximum inhibition of 47.55% at 10 10µl concentration and the activity were significant (p-value 0.01485) <0.05 α-values. The DNA was isolated from actinomycete strain (A4) and the 16SrRNA gene was amplified using universal eubacterial 16SrRNA gene primers and it was genotypically identified as Streptomyces coelicolor.

Isolation and identification of Ktedonobacteria using 16S rRNA gene sequences data

The aims of this study were to isolate and identify the members of Ktedonobacteria from soil samples in Cisolok geysers area, West Java, Indonesia. To isolate the bacteria, sample was spread on 10-fold-diluted Reasoner’s 2A medium and the plates were incubated at 30 °C until the Ktedonobacteria-like colonies appeared. Ktedonobacteria-like colonies showed the characteristics of a firm pale orange colony. The colonies were purified and colony PCR was performed using Ktedonobacteria specific and universal eubacterial primers for 16S rRNA gene. Almost full-length of 16S rRNA gene sequences were obtained in order to know the identity of the taxonomic affiliation of the isolates. Three Ktedonobacteria-like isolates, designated S3.2.1.5, S3.2.1.6 (isolated from soil under Bamboo tree) and S3.2.2.5 (isolated from soil in decayed Bamboo stems) were obtained. The detection using Ktedonobacteria specific primers showed positive results, which confirmed that all of three isolates belong to class Ktedonobacteria. The sequence homology search using EzBioCloud showed that these isolates (S3.2.1.5, S3.2.1.6, and S3.2.2.5) were belonged to the family Dictyobacteraceae and closely related to Bacterium SOSP1-79 (99.20%, 98.83%, 99.34%, respectively) and Dictyobacter aurantiacus S-27T (97.41%, 97.41%, 98.00%, respectively). The low percentage of sequence homology to type strains indicates that they could be a candidate of novel taxa of the genus Dictyobacter. Further characterizations such as morphology, physiology, and chemotaxonomy are needed in order to clarify the taxonomic position of these potential novel species of Dictyobacter.

Determination of bacterial species present in biofilm contaminating the channels of clinical endoscopes.

Outbreaks of endoscopy-related Carbapenem-resistant Enterobacteriaceae has highlighted failures in endoscope decontamination resulting in biofilm formation. Biofilms are tolerant to detergents and disinfectants. We evaluated decontaminated endoscope channels for residual bacterial contamination and biofilm presence. 64 channels were collected from 12 gastroscopes and 11 colonoscopes. Aerobic bacteria were isolated from inside the endoscope tubing by scrapping, sonication, and aerobic plate culture. Total number of contaminating bacteria was determined by quantitative real-time PCR with 16s rRNA eubacterial universal primers. Microbial diversity was assessed using next generation DNA sequencing. Biofilm presence was visually confirmed by confocal laser scanning and scanning electron microscopy. 47% of channels were culture positive, with α-haemolytic Streptococci from gastroscopes and coliforms from colonoscopes the most frequently isolated species. Sphingomonas spp., Staphylococcus spp., Streptococcus spp., and Pseudomonas aeruginosa were also isolated. An average of 1.2×103 bacteria/cm contaminated air-water channels, 2.8×102 and 6.6×102 bacteria/cm contaminated gastroscope and colonoscope working channels, respectively. Biofilm was on all 39 channels examined and was principally composed of environmental bacteria, although all samples contained potential pathogens. Biofilm is present on many endoscope channels obtained from Australian hospitals. Any soil including biofilm can compromise disinfectant action.

Aberrant crypt formation accompanies an increase of opportunistic pathogens/bacteria in the inflammatory gut of C57BL/6 mice fed a high‐fat diet

Obesity and high fat diet are risk factors for colon cancer, but the mechanism of this relationship remains to be determined. We tested the hypothesis that a high fat diet promotes the formation of aberrant crypt foci (ACF, preneoplastic lesions) in a manner associated with changes in hindgut bacterial taxa. We examined the susceptibility to azoxymethane (AOM)‐induced colonic ACF and microbiome composition in C57/BL6 mice fed a low fat (LF, 10% energy fat) diet or a high fat (HF, 45% energy fat) diet for 11 weeks. Mice receiving the HF diet exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the LF group. Consistent with a gut inflammatory phenotype, we observed an increase in colonic ACF, plasma interleukin 6 (IL6), tumor necrosis factor α (TNF α), monocyte chemoattractant protein 1 (MCP1), and inducible nitric oxide synthase protein in the ileum of the HF‐AOM group compared with those in the LF‐AOM group. Fecal DNA was extracted and 16S universal eubacterial primers were utilized to determine the microbiome composition using an amplicon pyrosequencing method. Although the HF and LF groups did not differ in bacterial alpha‐diversity, the HF and LF diets resulted in unique bacterial community structures in the hindgut. When treated with AOM, the abundance of certain short chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and the amount of fecal SCFA (e.g., acetic acid) were lower in the HF group compared with those in the LF group. Furthermore, we identified a high abundance of Anaeroplasma bacteria (opportunistic pathogens), accompanied with ACF formation in the HF group. Taken together, we demonstrate that HF feeding promotes ACF formation associated with an increase of opportunistic pathogens / bacteria in the hindgut of C57BL/6 mice.Support or Funding InformationThis work was funded by the US Department of Agriculture, Agricultural Research Service, Research Project 3062‐51000‐050‐00D.

Bacterial profiling of healthy bone marrow using polymerase chain reaction technique

Background: The bone marrow of healthy individuals is conventionally considered sterile like other body fluids, but recently the scientists found genetic material from the bacteria inside their stem cells. The findings raise the possibility that other infectious agents may also reside in the bone marrow. Objectives: The study was planned to look for the presence of all type of bacteria by amplifying 16S rDNA sequences using eubacterial universal primers. Materials and Methods: A total of 18 bone marrow samples of apparently healthy individuals were collected from patients admitted having closed bone fractures. The DNA was isolated and was subjected to nested polymerase chain reaction (PCR) using Universal eubacterial 16S rDNA primers. The samples positive by universal PCR was further checked for the presence of Salmonella Typhi, Salmonella Paratyphi A, and/or Mycobacterium tuberculosis (Mtb) if any using a second nested PCR reaction. Results: A total of 16 (89%) samples could yield the desired amplicon through universal PCR. The secondary PCR of 16 samples, the desired amplicons were detected 3 (18.8%) for Mtb, 4 (25%) for S. Typhi, and 1 (6.2%) for S. Paratyphi A. Conclusions: Even in asymptomatic cases, other infectious agents such as Mtb, Salmonella spp., and many other microorganisms may be present as commensal.

Populations of Stored Product Mite Tyrophagus putrescentiae Differ in Their Bacterial Communities.

Background: Tyrophagus putrescentiae colonizes different human-related habitats and feeds on various post-harvest foods. The microbiota acquired by these mites can influence the nutritional plasticity in different populations. We compared the bacterial communities of five populations of T. putrescentiae and one mixed population of T. putrescentiae and T. fanetzhangorum collected from different habitats.Material: The bacterial communities of the six mite populations from different habitats and diets were compared by Sanger sequencing of cloned 16S rRNA obtained from amplification with universal eubacterial primers and using bacterial taxon-specific primers on the samples of adults/juveniles or eggs. Microscopic techniques were used to localize bacteria in food boli and mite bodies. The morphological determination of the mite populations was confirmed by analyses of CO1 and ITS fragment genes.Results: The following symbiotic bacteria were found in compared mite populations: Wolbachia (two populations), Cardinium (five populations), Bartonella-like (five populations), Blattabacterium-like symbiont (three populations), and Solitalea-like (six populations). From 35 identified OTUs97, only Solitalea was identified in all populations. The next most frequent and abundant sequences were Bacillus, Moraxella, Staphylococcus, Kocuria, and Microbacterium. We suggest that some bacterial species may occasionally be ingested with food. The bacteriocytes were observed in some individuals in all mite populations. Bacteria were not visualized in food boli by staining, but bacteria were found by histological means in ovaria of Wolbachia-infested populations.Conclusion: The presence of Blattabacterium-like, Cardinium, Wolbachia, and Solitalea-like in the eggs of T. putrescentiae indicates mother to offspring (vertical) transmission. Results of this study indicate that diet and habitats influence not only the ingested bacteria but also the symbiotic bacteria of T. putrescentiae.

Molecular identification and detection of Rickettsia endosymbiont in the green leafhopper: Cicadella viridis (Hemiptera: Cicadellidae)

The leafhopper Cicadella viridis (Hemiptera: Cicadellidae), a sap-sucking insect, is a notorious pest of fruit trees and seedlings. They harbor “Candidatus sulcia merii” which is an obligatory symbiotic bacterium housed in a special organ called the bacteriome. In this study universal eubacterial primers 27F and 1513R were used to amplify approximately 1462bp of symbiotic bacteria 16S rRNA gene from C. viridis. Cloning, sequencing, and phylogenetic analysis targeting the 16S rRNA gene revealed the presence of bacteria stably associated with C. viridis, and a Rickettsia endosymbiont was detected for the first time in this species. Rickettsia endosymbiont localization was studied using quantitative PCR which showed that it was found in all developmental stages. It was also co-localized in the bacteriomes, ovaries, testes, guts, and salivary glands. By an antibiotic treatment, the antibiotic-treated insects exhibited lower Rickettsia infection density than the untreated.

Characterization of an intracellular bacterium infecting the digestive gland of the South African abalone Haliotis midae

A Rickettsiales-like prokaryote was observed in digestive gland epithelium of farmed South African abalone Haliotis midae sampled for routine surveillance. Histological examination showed basophilic inclusions typical of rickettsial infections in shellfish (Mouton, 2000). This study aimed to identify the Rickettsiales-like prokaryote (RLP) colonising H. midae. Total genomic DNA was extracted from infected digestive gland sampled from cultured H. midae. PCR performed with “Candidatus Xenohaliotis californiensis” specific primers RA 5-1 and RA 3-6 did not yield positive results, indicating that the H. midae RLP is not the withering syndrome agent. The 16S rRNA gene sequence of the intracellular bacterium was determined following nested PCR using universal eubacterial primers fD1/Rp2 and the broad spectrum primers EHR16SD/EHR16SR which are specific for members of the Anaplasmataceae family within the Rickettsiales order. Phylogenetic and evolutionary distance analysis showed that the H. midae RLP is a unique member of the α-proteobacteria. The identity and location of the histologically observed intracellular bacterium was confirmed by in situ hybridization performed on sequential sections of infected H. midae digestive gland using a combination of three digoxigenin-labelled probes specific to various regions of the 16S rRNA gene of the RLP.Statement of relevanceThe manuscript shows that farmed abalone, showing no disease symptoms, can remain healthy despite the presence of intracellular bacteria colonising their digestive glands. The study shows that Rickettsiales-like prokaryotes detected in farmed abalone are not necessarily “Candidatus Xenohaliotis californiensis”, the causative agent of withering syndrome.

Enterococcus avium—a rare cause for limb loss in a type 2 diabetic patient

Enterococcus avium is a Gram-positive facultative anaerobe, found normally in the intestinal tract of human beings, pigs and chickens. E. avium has rarely been reported as a human pathogen. This bacterium has been reported previously along with other aerobes and obligate anaerobes .We report a case of diabetic foot infection with wet gangrene caused by E. avium that had led to morbidity in a 60-year-old woman due to her ignorance regarding health and self-care skills. Pus and exudates of the diabetic foot wound were processed for anaerobic cultivation, and the pure culture obtained was subjected to nested PCR using 16S rDNA universal eubacterial primers for rapid and precise identification. The clinical isolate obtained by anaerobic incubation was identified as E. avium. Aerobic culture detected scanty growth of Proteus mirabilis. This case report highlights the pathogenic potential of E. avium in anaerobiosis in the absence of obligate anaerobe accounting for serious health burden threatening patients’ quality of life.

Isolation and Identification of Rhizospheric Bacteria in Sugarcane (Saccharum spp. L.) Cultivated on Acrisols and Ferrasols of Dong Nai Province, the Southeast of Vietnam

Nitrogen-fixing and phosphate-solubilizing bacterial diversity and population dynamics in the Acrisol and Ferrasol rhizosphere of sugarcane grown in Dong Nai province, the eastern of South Vietnam was studied. Soil rhizosphere samples were taken in three districts (sites) of this region. Physical and chemical characteristics of soil samples and total nitrogen-fixing and phosphate-solubilizing bacteria counts were determined by drop plate count method together with 16S rRNA gene fragments amplified from DNA using eubacterial universal primers (27F and 1492R). A total of 31 isolates were isolated on two media (Burk’s N-free and NBRIP) and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis. Population of nitrogen-fixing and phosphate-solubilizing bacteria correlated with N total concentration and organic matter content in soil closely (P<0.05). The sequences from selected nitrogen-fixing and phosphate-solubilizing bacteria (12 isolates) showed high degrees of similarity to those of the GenBank references strains (between 98% and 99%). From 12 isolates, 7 belonged to Bacilli, while 2 were Beta-Proteobacteria, 1 was Acidobacteria and 2 were Bacteroides. Based on Pi value (nucleotide diversity), Bacilli group had the highest theta value and Theta values (persequence) from S of SNP for DNA polymorphism were calculated for each group and Bacilli group had the highest values in comparison to three groups. From these results showed that two strains (Bacillus megaterium B6 (Ferrasols) and Sphingomonas sp. P14 (Acrisols)) revealed promising candidates with multiple beneficial characteristics and they have the potential for application as inoculants adapted to poor soils and sugarcane tree because they are not only famous strains but also are safety strains for agricultural sustainable.

First Report of Goss's Bacterial Leaf Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis in North Dakota.

In August of 2011, the North Dakota State University Plant Diagnostic Lab received a hybrid corn (Zea mays) leaf sample from Burleigh County in south-central North Dakota (ND). The leaf had long, irregular, water-soaked lesions consistent with Goss's leaf blight of corn. Using a light microscope at 10× magnification, bacterial streaming was observed from the excised edge of leaf tissue. A bacterial suspension was created, streaked onto a semi-selective CNS medium (1), and incubated at 22°C. Dark yellow-orange colonies appeared on the medium after 5 days. Single colonies were subcultured onto additional CNS media. To verify the identity of the bacterial isolate, PCR amplification of the 16S ribosomal DNA from this isolate along with a known Clavibacter michiganensis spp. nebraskensis (Cmn) isolate collected in Indiana (4) was performed using the eubacterial universal primers 27f and 1525r (3). The 1,431-bp 16S rDNA region was obtained for each isolate and they were compared with each other and with those deposited in NCBI GenBank. Sequence alignment identified only one nucleotide difference between the ND isolate and the Indiana isolate. BLASTn search against the NCBI database showed the first 100 hits were described as C. michiganensis or unidentified Clavibacter sp. The ND isolate had a two-nucleotide difference with Cmn isolate NCPPB2581 (HE614873), and a three nucleotide difference was found with the C. michiganensis spp. michiganensis isolate NCPPB 382 (AM711867). To satisfy Koch's postulates, eight corn plants (Golden Cross Bantam) were grown in the greenhouse at 22 to 24°C. Four corn plants were inoculated at growth stage V4-V5 using a suspension of approximately 1 × 109 CFU/ml from cultures grown on CNS for 6 days. Wounds were created on the fifth leaf approximately 7 cm from the leaf tip using a tongue-seizing forceps outfitted with a rubber stopper composed of pins (2). Simultaneously, 1 ml of the bacterial suspension was delivered into the wounds through a hole on top of the rubber stopper. Four control plants were inoculated with sterile water in a similar fashion. No symptoms were observed on the control plants. After 6 days, long water-soaked symptoms were observed on leaves inoculated with the bacterial suspension. Using leaves with water-soaked lesions, the pathogen was re-isolated onto CNS media and subjected to PCR amplification, and the resulting amplicons were sequenced as before. The sequence of the amplicon from the re-isolation matched that of the original ND isolate. To our knowledge, this is the first account of Goss's leaf blight and wilt identified in ND. As the corn acreage and no-till production systems in the state have increased, the economic implications of this disease may become more significant. Recognition of symptoms and proper identification of this bacterial disease in the field should help reduce unnecessary foliar fungicide sprays.

Exploring the bacterial microbiota associated with native South American species of Aphis (Hemiptera: Aphididae).

Aphids harbor a variety of bacterial endosymbionts, including the obligate symbiont Buchnera aphidicola and diverse facultative symbionts. The former supplies its host with essential amino acids. The latter are not indispensable for insect survival, but often improve their host's fitness. To date, the study of such associations was restricted to aphids of Holarctic origin. The bacterial microbiota of seven Aphis species from Argentina was investigated. The presence of B. aphidicola was assessed by specific PCR. Additional symbionts were identified through PCR with eubacterial universal primers, cloning, and sequencing of nearly complete 16S rRNA gene, intergenic spacer region, and partial 23S rRNA gene and subjected to phylogenetic analysis. Infection with B. aphidicola was confirmed in every species analyzed. The facultative symbiont Serratia symbiotica was detected in Aphis malalhuina Mier Durante, Nieto Nafría & Ortego, 2003, Aphis senecionicoides Blanchard, 1944, and Aphis schinifoliae Blanchard, 1939, while Hamiltonella defensa was identified in Aphis mendocina Mier Durante, Ortego & Nieto Nafría, 2006. Arsenophonus sp. was found infecting Aphis melosae Mier Durante & Ortego, 1999, and a new, undescribed Aphis sp. In Aphis danielae Remaudière, 1994, no facultative symbionts could be recorded. When analyzing the highly conserved 16S rRNA gene, the phylogenetic tree grouped the S. symbiotica, H. defensa, and Arsenophonus isolates into three well-defined clusters showing little variability among clones corresponding to the same aphid host species. This article reports for the first time the endosymbionts associated with aphids native to South America. Despite their geographic origin, the qualitative composition of their microbiota revealed no evident differences from that described for aphids in the Northern Hemisphere.

Isolation and Identification of Rhizospheric Bacteria in Acrisols of Maize (Zea Mays L.) in the Eastern of South Vietnam

Rhizobacterial diversity and population dynamics in the Acrisol rhizosphere of maize grown in the eastern of South Vietnam was studied. Soil rhizosphere samples were taken in three provinces of this region. Physical and chemical characteristics of soil samples and total nitrogen-fixing and phosphate-solubilizing bacteria counts were determined by drop plate count method together with 16S rRNA gene fragments amplified from DNA using eubacterial universal primers (8F and 1492R). A total of 149 isolates were isolated on two media (Burk’s N-free and NBRIP) and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis. Population of rhizobacteria correlated with soil pH and organic matter content in soil closely (P<0.05). The sequences from selected rhizobacteria (24 isolates) showed high degrees of similarity to those of the GenBank references strains (between 97% and 99%). From 24 isolates,13 belonged to Beta-Proteobacteria, while 11 were Firimicutes and Actinobacteria. Based on Pi value (nucleotide diversity), rhizobacteria (PGPR) group in Tay Ninh province had higher than rhizobacteria (PGPR) group in Baria-Vungtau province with the highest Theta value (per site). From these results showed that three strains (Burkholderia vietnamiensis VDN6a, VDB6a and VDN7c) revealed promising candidates with multiple beneficial characteristics and they have the potential for application as inoculants adapted to poor soils and local crops because they are not only famous strains but also are safety strains for agricultural sustainable

Isolation, Charcaterization and Phylogenetic Analysis of Endophytic Bacteria in Rice Plant Cultivated on Soil of Phu Yen Province, Vietnam

Endophytic bacterial diversity in Rice plant cultivated on soil of Phu Yen province, Vietnam was studied. Rice plant samples were taken in eight sites (districts) of Phu Yen provinces of this region. Endophytic bacteria were isolated in three kinds of medium (LGI, NFb, RMR) together with 16S rRNA gene fragments amplified from DNA using eubacterial universal primers (p515FPL and p13B). A total of 561 isolates were isolated on three media and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis but there were 73 isolates having the best characteristics and they were identified as rice endophytes and nifH gene. The sequences from selected endophytic bacteria (73 isolates) showed high degrees of similarity to those of the GenBank references strains (between 97% and 100%). From 73 isolates, 23 isolates belonged to Bacillus (31.54%), 44 isolates were Proteobacteria (60.24%), while 6 isolates were Bacteroides (8.22%). Based on Pi value (nucleotide diversity), Bacteroides group had the highest theta values in comparison of three groups and Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each group and Bacteroides group had the highest values in comparison of three group. From these results showed that thirteen strains (Bacillus megaterium TANa5, Bacillus methylotrophicus TAN17, Bacillus megaterium TALa14, Pseudomonas putida TAL1, Bacillus subtilis TAL4, Burkholderia kururiensis TAL22, Azospirillum amazonense SHL70, Bacillus subtilis DXL 136, Burkholderia kururiensis PHL87, Burkholderia vietnamiensis PHL103, Bacillus megaterium PHL105, Bacillus megaterium DHL154 and Bacillus subtilis SHIM60) revealed promising candidates with multiple beneficial characteristics and they have the potential for application as inoculants or bio-fertilizer adapted to poor soils and high-yielding rice because they are not only famous strains but also are safety strains for agricultural sustainable.

Isolation, Characterization and Identification of Endophytic Bacteria in Sugarcane (Saccharum spp. L.) Cultivated on Soils of the Dong Nai Province, Southeast of Vietnam

Endophytic bacterial diversity in sugarcane plant cultivated on Latosols and Acrisols of the Eastern of South Vietnam was studied. Sugarcane plant samples were taken in four sites (districts/towns) (Xuanloc, Dinhquan, Vinhc uu and Trangbom) of Dong Nai province. Endophytic bacteria were isolated in LGI medium together with 16S rRNA gene fragments amplified from DNA using eubacterial universal primers (p515FPL and p13B). A total of 27 isolates were isolated on LGI medium and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis but there were 10 isolates having the best characteristics. The sequences from selected endophytic bacteria (10 isolates) showed high degrees of similarity to those of the GenBank references strains (between 98% and 99%). All of them are Proteobacteria (gram-negative bacteria) and 3 isolates belonged to Alpha-proteobacteria (30%), 2 isolates were Beta-proteobacteria (20%), 5 isolates were Gamma-proteobacteria (50%). Based on Pi value (nucleotide diversity), Gamma-proteobacteria group had the highest theta values and Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each group and Gamma-proteobacteria group had the highest values in comparison of two groups. From these results showed that Enterobacter oryzae LT7, Achromobacter xylosoxidans T16, Achromobacter insolitus R15b and Pantoea agglomerans T12 revealed promising candidates with multiple beneficial characteristics and they have the potential for application as inoculants or bio-fertilizer adapted to poor latosols and acrisols because they are not only famous strains but also are safety strains for sustainable agriculture.

Isolation, Characterization and Identification of Endophytic Bacteria in Maize (Zea Mays L.) Cultivated on Acrisols of the Southeast of Vietnam

Endophytic bacterial diversity in Maize plant cultivated on Acrisols of the Eastern of South Vietnam was studied. Maize material was collected from five sites (provinces/cities) of this region. Endophytic bacteria were isolated in two kinds of medium (LGI, NFb) together with 16S rRNA gene fragments amplified from DNA using eubacterial universal primers (p515FPL and p13B). A total of 301 isolates were isolated and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis but there were 30 isolates having the best characteristics and they were identified as maize endophytes and nifH gene owners. The sequences from selected endophytic bacteria (30 isolates) showed high degrees of similarity to those of the GenBank references strains (between 97% and 100%). Among the selected isolates were 6 isolates belong to Bacillus (20.00%) and 24 isolates belong to Proteobacteria (80.00%) including 2 alpha-proteobacteria (7%), 6 beta-proteobacteria (20%), and 16 gamma-proteobacteria (53%). Based on Pi value (nucleotide diversity), Proteobacteria group had the highest Theta values and Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each group and Proteobacteria group had the highest values in comparison of two groups. From these results showed that 3 isolates including DTN1b (Azotobacter vinelandii), VTN2b (Bacillus subtilis) and VTN7 (Enterobacter cloacae) proposed as potential microbial inoculants or biofertilizers for sustainable corn production in poor Acrisols in Vietnam because of their benefit and biosafety.

Pseudomonas fluorescens-like bacteria from the stomach: A microbiological and molecular study

To characterize oxidase- and urease-producing bacterial isolates, grown aerobically, that originated from antral biopsies of patients suffering from acid peptic diseases. A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production, acid tolerance and aerobic growth. The selected isolates were further characterized by molecular techniques viz. amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers. The amplicons were subjected to restriction analysis and partial sequencing. A phylogenetic tree was generated using unweighted pair group method with arithmetic mean (UPGMA) from evolutionary distance computed with bootstrap test of phylogeny. Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10(-7) mol/L to 10(-1) mol/L. Of the 258 antral biopsy specimens collected from patients, 179 (69.4%) were positive for urease production by rapid urease test and 31% (80/258) yielded typical Helicobacter pylori (H. pylori) after 5-7 d of incubation under a microaerophilic environment. A total of 240 (93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation. The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species. A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens (P. fluorescens). On the basis of HSP60 sequences applied to the UPGMA phylogenetic tree, it was observed that isolated strains in an aerobic environment were likely to be P. fluorescens, and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences. Interestingly, this bacterium was acid tolerant for hours at low pH. Further, a total of 250 (96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P. fluorescens-specific conserved putative outer membrane protein gene sequences. This study indicates that bacterial isolates from antral biopsies grown aerobically were P. fluorescens, and thus acid-tolerant bacteria other than H. pylori can also colonize the stomach and may be implicated in pathogenesis/protection.

Significance of bacteria in oviposition and larval development of the sand fly Lutzomyia longipalpis

BackgroundMicrobial ecology of phlebotomine sand flies is not well understood although bacteria likely play an important role in the sand fly biology and vector capacity for Leishmania parasites. In this study, we assessed the significance of the microbial community of rabbit feces in oviposition and larval development of Lutzomyia longipalpis as well as bacterial colonization of the gut of freshly emerged flies.MethodsSterile (by autoclaving) and non-sterile (control) rabbit feces were used in the two-choice assay to determine their oviposition attractiveness to sand fly females. Bacteria were identified by amplification and sequencing of the 16S rRNA gene with universal eubacterial primers. Sterile, control (non-sterile), and sterilized and inoculated rabbit feces were used to assess the significance of bacteria in L. longipalpis development. Newly emerged adult flies were surface-sterilized and screened for the bacterial population size and diversity by the culturing approach. The digestive tract of L4 sterile and control larvae was incubated with Phalloidin to visualize muscle tissues and DAPI to visualize nuclei.ResultsTwo-choice behavioural assays revealed a great preference of L. longipalpis to lay eggs on rabbit feces with an active complex bacterial community (control) (85.8 % of eggs) in comparison to that of sterile (autoclaved) rabbit feces (14.2 %). Bioassays demonstrated that L. longipalpis larvae can develop in sterile rabbit feces although development time to adult stage was greatly extended (47 days) and survival of larvae was significantly lower (77.8 %) compared to that of larvae developing in the control rabbit feces (32 days and 91.7 %). Larval survival on sterilized rabbit feces inoculated with the individual bacterial isolates originating from this substrate varied greatly depending on a bacterial strain. Rhizobium radiobacter supported larval development to adult stage into the greatest extent (39 days, 88.0 %) in contrast to that of Bacillus spp. (76 days, 36.0 %). From the complex natural bacterial community of rabbit feces, R. radiobacter survived pupation and colonized the newly emerged females most successfully (82.6 % of all bacteria cultured); however, only 25 % of females were positive for bacteria in the digestive tract upon emergence. Immunohistochemistry did not reveal any obvious differences in anatomy of the digestive tract between control and axenic larvae.ConclusionsThe bacterial community in the sand fly larval habitat affects oviposition and larval development although bacteria are not essential for successful development of L. longipalpis. Different bacteria contribute to larval development to various degrees and some, e.g. Rhizobium radiobacter, survive pupation and colonize the digestive tract of newly emerged females. With the establishment of the axenic rearing system, this study opens new venues to study the effect of bacteria on the gut epithelial immunity and vector competence of sand flies for Leishmania parasites with a goal to develop paratransgenic approaches for Leishmania control.

English

Arcanobacterium (Actinomyces) pyogenes, are animal pathogens, they produce hemolytic exotoxin, pyolysin (PLO) and are opportunistic pathogens of economically important livestock such as dairy, beef cattle and sheep. Samples were collected from pleural fluid of 10 diseased and 10 healthy sheep.&nbsp; Samples were from neighboring village to gazelle breeding center, Riyadh Saud Arabia. The objectives of this study were to investigate&nbsp;A. pyogenes&nbsp;in these sheep. The isolates were grown on brain heart infusion (BHI; Difco) agar plates, supplemented with 5% bovine blood at 37&deg;C and 5% CO2. Biochemical identification of 20 isolates revealed beta-hemolytic on blood agar, hydrolyzed gelatin, starch, were unable to hydrolyze esculin or reduce nitrate and did not produce urease. DNA was&nbsp;extracted from isolates using&nbsp;DNeasy Blood and Tissue Kits (Qiagen, NY, USA). Molecular tool using 16S r- deoxyribonucleic acid (DNA)&nbsp;universal eubacterial forward primer 16F27 (5&acute;-AGA GTT TGA TCC TGG CTC AG-3&acute;) and reverse primer 16SrDNA R1525 (5&acute;-AAG GAG GTG ATC CAG CCG CA-3&acute;), MWG Biotech AG (Ebersberg, Germany) identified the isolates&nbsp;successfully as&nbsp;A. pyogenes. &nbsp; Key words:&nbsp;Sheep, pathogen, esculin, urease, 16SrDNA,&nbsp;Antinomies pyogenes.