Amplified Ribosomal DNA Restriction Analysis Research Articles

Molecular identification of protease producer ORSK-4 strain and determination of optimum enzyme production conditions

A microorganism resistant to high temperatures and producing alkaline proteases was isolated from soil samples from a protein-rich region (Kırıkkale/Yahşihan). This isolate, with high protein production, was identified as ORSK-4 by determining its morphological and biochemical properties using the 16s rRNA molecular approach and the Amplified ribosomal DNA restriction analysis (ARDRA) technique employed in strain differentiation. The optimum enzyme production conditions of the strain ORSK-4 were found to be the enzyme media, 3 days of incubation, 27.0 °C, and pH 7.0. Different components were utilized to determine the effect of changing the medium content on enzyme activity. Under the optimal production conditions determined in this way, the enzyme activity of ORSK-4 was found to be higher than that of some ATCC reference Bacillus species. To purify the extracellular protease of ORSK-4, precipitation with ammonium sulfate (30% and 80%), dialysis, and DEAE ion exchange chromatography were performed. SDS-PAGE analysis determined the molecular weight of the purified enzyme as approximately 30 kDa. Although the enzyme showed activity at various pH ranges, it showed its maximum activity when increased up to pH 9.0. In conclusion, the stability of the obtained alkaline protease enzyme under different conditions shows that it can be used in industrial and environmental applications.

Biomolecular Characterization of Lactic Acid Bacteria (Lactobacilli and Cocci) Isolated from Nonnonkoumou, a Artisanal Milk Curd in Daloa, Côte D’ivoire

Nonnonkoumou is an artisanal sour milk consumed in Côte d'Ivoire, particularly in the town of Daloa. Its production is carried out under uncontrolled conditions and uses a variety of lactic acid bacteria. The main objective of this study was to characterize the cocci and lactobacilli present in the nonnonkoumou. The cocci had the highest loads going from 8.31 ± 0.14 log CFU / mL to 6.76 ± 0.01 log CFU / mL compared to the lactobacilli which had loads between 6.12 ± 0.30 log CFU / mL and 4.35 ± 0.42 log CFU / ml. This preponderance of cocci in all the nonnonkoumou samples taken in comparison to bacilli have been shown through the cocci / bacilli ratio which varied from 1.9 to 1.22. PCR followed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) after enzymatic digestion with Hae III and Hinf I have revealed 4 Hinf I profiles and 3 Hae III for lactobacilli and 2 Hae III profiles and 4 Hinf I profiles for cocci. This technique has revealed 4 species of lactobacilli isolated from nonnonkoumou which are Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus plantarum and Fructobacillus durionis and 6 groups of cocci corresponding to 6 species. The species identified during this study made it possible to understand their technological role and their contribution to the health quality of nonnonkoumou.

Mangroves: A hotspot for novel bacterial and archaeal diversity

Mangrove ecosystems are highly productive ecosystems that harbor diverse microbial lineage playing multiple roles in nutrient cycling. Though several novel mangrove rhizospheres associated with bacterial genera and species have been reported from these unexplored wetland ecosystems, but yet to be studied for their richness and diversity including actinobacterial and archaeal communities. Therefore, this study was undertaken to decode the bacterial and archaeal communities associated with the rhizosphere regions of true mangrove species-Avicennia marina and Rhizophora mucronata and their intersecting regions; and associated halophytic species -Suaeda maritima, and Salicornia brachiata of Pichavaram mangroves, Tamil Nadu, India. Amplified ribosomal DNA restriction analysis (ARDRA) and PCR denaturing gradient gel electrophoresis (PCR-DGGE) analysis techniques were adopted. About 567 culturable bacterial isolates with distinct morphology were purified, and nearly 88 distinct Operational Taxonomic Units (OTUs) were identified by ARDRA analysis. Phylogenetic analysis of representative groups indicated that the majority of the isolates belonged to Firmicutes (10 genera), followed by Proteobacteria- Alphaproteobacteria (10 genera) and Gammaproteobacteria (8 genera), Actinobacteria (8 genera), Bacteroidetes (1genera), and Halomonas (9 genera) representing 38 genera with 105 species. In addition, a few phylogenetically distinct novel groups belonging to the genera Mangrovibacter, Vibrio, Demequina, and Catenococcus were also isolated. PCR-DGGE with the RDP-Classifier program identified the prominent DGGE bands up to genus level namely Bacillus, Pseudomonas, Vibrio, Halomonas, etc. In addition, this study for the first time describes the genera Bhargavaea, Exiguobacterium, Hoeflea, Catenococcus, Albirhodobacter, Dietzia, Brachybacterium, Aerococcus, and Myroides association with mangrove rhizospheres. Likewise, DGGE analysis of archaea represented 2 major groups such as Crenarchaeota and Euryarchaeota, which were further classified into 7 different genera.

Molecular typing of multidrug resistant bacteria isolated from health care professionals’ mobile phone: A pilot study in Jashore, Bangladesh

Background: Nowadays, Health Care Professionals’ (HCPs) are increasingly using mobile phones which may act as reservoirs and vector for transmission of pathogens. The presence of multidrug resistant nosocomial microbes on the surface of mobile phones used by HCPs in hospitals can pose a great public health threat. So, this research was conducted to identify the concerned multi-drug resistant (MDR) bacteria and also to explore the recent status of bacterial contamination on mobile phones of HCPs in the Jashore region of Bangladesh and determine their antibiotic resistance pattern. Methods: Swab samples of mobile phones were collected between June and September 2019 from 24 different users (i.e., manager, worker, doctor and nurse) associated to four distinct hospitals of Jashore region, Bangladesh. After suitable morphological and biochemical identification, we determined their antimicrobial susceptibility by Kirby- Bauer disc diffusion method by using 18 antibiotics for Gram positive bacteria and 19 antibiotics for Gram negative bacteria. Later, the MDR isolates were grouped by amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA sequencing with phylogeny were performed to confirm the bacteria at species level. Results: A total of 38 bacterial isolates were obtained from the sample. Enterobacter spp. isolates showed maximum resistance against Amoxicillin, followed by Ampicillin and Aztreonam (80% each) and one isolate showed highest antibiotic resistance (15 out of 19) among all the isolates. In addition, Staphylococcus spp. and Exiguobacterium spp. isolates showed 100% resistance against Penicillin, Ampicillin, Oxacillin, Erythromycin, Lincomycin and Cefotaxime. On the contrary, all of the isolates of Escherichia spp., Bacillus spp., Proteus spp. were sensitive to all tested antibiotics. Surprisingly, 20 MDR isolates were showing resistance to at least 2 antibiotics. Subsequently, three distinct genera of these MDR isolates were identified by ARDRA; the strains Enterobacter cloacae (75%), Staphylococcus warneri (15%) and Exiguobacterium aurantiacum (10%) were confirmed by the 16S rRNA phylogenetic analysis. Conclusion: We found that cell phones can act as reservoirs of multidrug-resistant pathogens, causative agents for Hospital-acquired infections. An effective hygiene practice for health care personnel should be introduced to prevent the cross-contamination by their cell phone Bangladesh Journal of Medical Science Vol. 22 No. 03 July’23 Page : 643-656

Diversity analysis of endophytes with antimicrobial and antioxidant potential from Viola odorata: an endemic plant species of the Himalayas.

Viola odorata, also known as "Banafshah" in high altitudes of Himalayas, is well known for its pharmaceutical importance in Ayurvedic and Unani medicinal system. The plant is a source of various drugs for its anti-inflammatory, diaphoretic, diuretic, emollient, expectorant, antipyretic, and laxative properties. The endophytes of plants have been reported for their role in modulating various physiological and biological processes of the host plants. In the present study, a total of 244 endophytes were isolated in pure cultures from the roots of Viola odorata, and genetic diversity was evaluated using amplified ribosomal DNA restriction analysis (ARDRA) and enterobacterial repetitive intergenic consensus (ERIC). The molecular fingerprinting revealed variation among various rRNA types among morphologically different endophytes based on ARDRA and ERIC-PCR. The screening of endophytes showed antimicrobial activity of 11 bacterial isolates and one actinomycete SGA9 against various pathogens Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. The antioxidant activity revealed the majority of the bacterial isolates able to scavenge the free radical in the range of 10-50% and 8 bacterial isolates in the range of 50-85%. Principal component analysis separated eight isolates away from the central eclipse and form a separate group based on antimicrobial and antioxidant potential. The identification of these eight isolates showed affiliation with different species of the genus Enterobacter, Microbacterium, Pseudomonas, Rhizobium, and Streptomyces. This is the first report on the characterization of endophytic bacteria and actinomycetes from endemic Viola odorata. Results suggested that these endophytes could be explored for the production of antimicrobial and antioxidant products.

Antimicrobial Activity of Extremely Halophilic Archaea Isolated from Southern Thai Salt-Fermented Products and Solar Saltern of Pattani, Thailand

This research aimed to study the diversity and antimicrobial activity of culturable haloarchaea in soil samples of solar saltern in Pattani Province, Thailand and Southern Thai salt-fermented food. Seventy-seven extremely haloarchaea were isolated on Halophilic medium agar containing 25% NaCl at 37°C for 7-21 days. They were grouped by Amplified Ribosomal DNA Restriction Analysis (ARDRA) with two restriction enzymes, RsaI and HindIII. The ARDRA patterns illustrated 6 different Operation Taxonomic Units (OTUs). Partial 16S rRNA gene sequences (938 bp) of the representative of each OTUs were aligned with the GenBank database. The results showed that the representatives of OTU1, OTU2, OTU3, OTU4, OTU5, and OTU6 had 99-100% similarity to Halobacterium salinarum, 99-100% similarity to Halostagnicola larsenii, 99% similarity to Natronococcus sp., 99-100% similarity to Haloferax alexandrinus, 99-100% similarity to Natrialba sp. and 97% similarity to Halococcus sp., respectively. The antimicrobial activity testing of the 17 isolated haloarchaeal strains from solar saltern was performed against 5 tested strains of Hbt. salinarum and 1 strain of Natrialba sp. were isolated from fermented food. Seven (41.2%) were candidate halocin-producing strains. In addition, no phage activity was observed. The 14-day fermentation broth of Natronococcus sp. SS13 showed the highest antimicrobial activity against Hbt. salinarum PK08, BD07 and BD09 (>5,120 AU/ml).

COMPARATIVE IDENTIFICATION APPROACH FOR GUM-PRODUCING LACTOBACILLI IN PALM WINE

Introduction: Species of lactobacilli are responsible for the consistency and soluble white colouration of palm wine through the production of gum. Phenotypic and physiological characteristics as basis for identification of lactobacilli is often with low level of discrimination. Objection: This study aimed at comparative identification approach of gum-producing lactobacilli using amplified ribosomal DNA restriction analysis (ARDRA) and API kit 50 CHL. Methodology: Four hundred colonies of lactobacilli isolated from seventy-two fresh palm wine samples and two hundred were used for genomic DNA extraction, 16S rDNA analysis and digestion of amplicons with two endonucleases: Hae III and Bash 12361. Results: Twenty ARDRA pattern group were generated and representatives of each were randomly chosen for sequence and API kit analysis. The size of the amplified products (̴ 1500 bp), digested amplicons (100 - 1000 bp) and the ARDRA profiles; A-T. ARDRA screening revealed unique patterns among the two hundred isolates, with the same pattern for some of the isolates. The 16S rDNA gene sequence analysis showed that the isolates were divide into 20 groups of three genera: Lactobacillus (65%), Leuconostoc (30%), and Lactococcus (5%) whereas API kit identified two genera; Lactobacillus (75%) and Leuconostoc (25%). Gene sequence similarity of all the representative isolate for 16S rDNA (99 - 100%) while API kit (74.8 – 99.9%) with 55% overall similarity in bacterial identified. ARDRA showed higher similarities in identification and discrimination of bacterial species or between closely related groups isolated from palm wine compared to API kit. Conclusion: ARDRA is a good tool for lactobacilli identification based on the results from this study.

Diversity and characterization of antagonistic bacteria against Pseudomonas syringae pv. actinidiae isolated from kiwifruit rhizosphere.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is a severe global disease. However, effective biological control agents for controlling Psa are currently unavailable. This study aimed to screen potential biological control agents against Psa from the kiwifruit rhizosphere. In this study, a total of 722 isolates of bacteria were isolated from the rhizosphere of kiwifruit orchards in five regions of China. A total of 82 strains of rhizosphere bacteria showed antagonistic effects against Psa on plates. Based on amplified ribosomal DNA restriction analysis (ARDRA), these antagonistic rhizosphere bacteria were grouped into 17 clusters. BLAST analyses based on 16S rRNA gene sequence revealed 95.44%-100% sequence identity to recognized species. The isolated strains belonged to genus Acinetobacter, Bacillus, Chryseobacterium, Flavobacterium, Glutamicibacter, Lysinibacillus, Lysobacter, Pseudomonas, Pseudarthrobacter, and Streptomyces, respectively. A total of four representative strains were selected to determine their extracellular metabolites and cell-free supernatant activity against Psa in vitro. They all produce protease and none of them produce glucanase. One strain of Pseudomonas sp. produces siderophore. Strains of Bacillus spp. and Flavobacteria sp. produce cellulase, and Flavobacteria sp. also produce chitinase. Our results suggested that the kiwifruit rhizosphere soils contain a variety of antagonistic bacteria that effectively inhibit the growth of Psa.

Plant growth-promoting characteristics of halotolerant endophytic bacteria isolated from Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. of Ethiopian rift valley lakes.

Understanding plant microbes' intimate relationship and search for beneficial microbes is a sustainable alternative to improve plant growth and yield under a wide range of biotic and abiotic stress conditions. More than 20% of the total global agricultural land is affected by salinity. High salinity challenges crop plants by affecting several metabolic pathways and decreasing plant growth and yield. Unlike chemical fertilizers and pesticides, endophytic microbes offer an eco-friendly approach to increasing crop yield via various metabolites during salinity stress. The objective of this study was to isolate and characterize endophytic halotolerant bacterial isolates from haloalkaliphytes, investigate their plant growth-promoting (PGP) properties and tolerance for various stress conditions. Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. grass samples were collected from the shores of two Ethiopian soda lakes (Lakes Abijata, and Chitu, respectively). A total of 167 halotolerant endophytic bacterial isolates, that clustered into 21 ARDRA (Amplified ribosomal DNA restriction analysis) groups, affiliated to members of 11 bacterial genera, namely Halomonas, Agrobacterium, Exiguobacterium, Jonesia, Stenotrophomonas, Pseudomonas, Alishewanella, Kosakonia, Bacillus, Paracoccus and Pannonibacter, were identified based on 16S rRNA sequencing. Most of the strains were able to produce IAA (indole-3-acetic acid) and hydrogen cyanide, grow on a nitrogen-free medium and solubilize phosphate. In vitro tolerance tests reveal that isolates were tolerant to: 5.0-15% NaCl, up to 40% PEG 6000, temperatures up to 50°C, and pH 5-11. These characteristics of the isolates indicate their potential PGP application under various plant stress conditions.

Deep Subsurface Hypersaline Environment as a Source of Novel Species of Halophilic Sulfur-Oxidizing Bacteria.

The sulfur cycle participates significantly in life evolution. Some facultatively autotrophic microorganisms are able to thrive in extreme environments with limited nutrient availability where they specialize in obtaining energy by oxidation of reduced sulfur compounds. In our experiments focused on the characterization of halophilic bacteria from a former salt mine in Solivar (Presov, Slovakia), a high diversity of cultivable bacteria was observed. Based on ARDRA (Amplified Ribosomal DNA Restriction Analysis), at least six groups of strains were identified with four of them showing similarity levels of 16S rRNA gene sequences lower than 98.5% when compared against the GenBank rRNA/ITS database. Heterotrophic sulfur oxidizers represented ~34% of strains and were dominated by Halomonas and Marinobacter genera. Autotrophic sulfur oxidizers represented ~66% and were dominated by Guyparkeria and Hydrogenovibrio genera. Overall, our results indicate that the spatially isolated hypersaline deep subsurface habitat in Solivar harbors novel and diverse extremophilic sulfur-oxidizing bacteria.

Yeast Biodiversity in Vineyard during Grape Ripening: Comparison between Culture Dependent and NGS Analysis

In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the “Nizza” Barbera d’Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching the harvest, a succession of oxidative or weakly fermentative copiotrophic species occurs, such as Saturnispora diversa, Issatchenkia terricola, Hanseniaspora opuntiae, Starmerella bacillaris and Hanseniaspora uvarum. The massive sequencing revealed a larger number of species, respect to the culture-dependent data. Comparing the two different approaches used in this work, it is possible to highlight some similarities since Aureobasidium, Rhodotorula and Sporobolomyces were detected by both methods. On the contrary, genera Hanseniaspora, Issatchenkia and Saturnispora were revealed by culture-dependent methods, but not by NGS, while Saccharomyces spp. were identified, with low frequency, only by NGS. The integrated application of NGS sequencing and culture-dependent techniques provides a comprehensive view of mycodiversity in the wine-growing environment, especially for yeasts with low abundance.

Gut associated culturable bacterial community in intertidal polychaete worms (Annelida: Polychaeta), their characterization and implications in captive shrimp aquaculture

Polychaetes supplements play a crucial role in the dietary requirements of shrimps reared under captive culture. Due to their high demand, wild captured worms are used extensively as supplementary feed for the broodstocks. However, the gut microbiota composition of the polychaetes is rarely investigated. In the present study, the culturable fraction of gut microbiota of two native polychaete species, Marphysa madrasi and Namalycastis sp., that are currently used as live supplemental feed by shrimp brooders, were analysed. The culture-dependent technique yielded a total of 39 bacterial isolates, which grouped into 23 unique phylotypic clusters based on amplified ribosomal DNA restriction analysis (ARDRA) profiling. The 16S rRNA gene sequencing of these 23 gut isolates revealed bacterial communities with probiotic potential and a few shrimp and human pathogens. The gut-associated bacterial community is dominated by three bacterial phyla, the Firmicutes, Actinobacteria, and Proteobacteria, composed of 10 different genera. Moreover, there were differences in the bacterial communities between the two polychaetes investigated in this study. The Shannon, Simpson, Chao1 and Abundance-based Coverage Estimator (ACE) index revealed that M. madrasi have significantly higher species diversity and richness than Namalycastis sp. in the gut. Screening for hydrolytic enzyme production revealed several bacterial species with probiotic applications, such as Bacillus subtilis, B. licheniformis, B. pumilus, B. megaterium, and B. halotolerans. On the other hand, a few opportunistic human pathogens, such as B. cereus, Gordonia terrae, Microbacterium paraoxydans, Staphylococcus epidermidis, and Staphylococcus gallinarum, were also identified. Overall, the results of this study revealed that the bacterial assemblages associated with the gut of intertidal polychaete worms can serve as excellent probiotic candidates.

Prevalence and Genetic Diversity of Escherichia coli Isolated from Sewage Samples of Hospitals in Different Regions of Bangladesh

Escherichia coli are a large and diverse group of bacteria found in the environment, foods and intestines of humans and animals. This study was carried out to observe the genotypic variations in E. coli found at different regions in Bangladesh using Amplified Ribosomal DNA Restriction Analysis (ARDRA) of the 16S rDNA and RAPD (Random Amplified Polymorphic DNA). Sewage samples were collected from different medical college hospitals and a total of 118 samples were observed in this study of which 26 were identified as E. coli based on their cultural, biochemical and molecular characteristics. Antibiotic susceptibility test was carried out where most isolates were resistant to Amoxicillin, Rifampicin and Erythromycin; and some were resistant to kanamycin, ceftriaxone, Chloramphenicol and Azithromycin; besides 73% (N=19 out of 26) of the test isolates were multi-drug resistant. Results of Polymerase chain reaction (PCR) showed that none of the isolates carried Stx1, Stx2 and LT gene. Two genotypic groups were found from Amplified Ribosomal DNA Restriction Analysis (ARDRA) by Alu1 restriction enzyme and seven different Random Amplification of Polymorphic DNA (RAPD) patterns were found. After analyzing, it was found that 24 out of 26 isolates showed similarity with Escherichia coli strain NBRC 102203 and the remaining two isolates showed similarity with Escherichia coli strain Mui T5 based on the restriction patterns of ARDRA and sequences of 16S rDNA. From the study, it was concluded that Escherichia coli strain NBRC 102203 was more prevalent than Escherichia coli strain Mui T5 isolated from sewage sample of hospitals from different regions of Bangladesh.
 Bangladesh J Microbiol, Volume 38, Number 2, December 2021, pp 39-44

Isolation, identification and characterization of Lactobacillus species diversity from Meekiri: traditional fermented buffalo milk gels in Sri Lanka

Traditional fermented buffalo milk gel; Meekiri, is a popular buffalo milk-derived product in Sri Lanka. Predominantly, it is produced using the back-slopping (adding a small amount of the previous fermentate) technique, following the life-long traditions available at the cottage level. Hence, diverse and unclassified starter cultures are likely to be established across the varying geographical regions of Meekiri production. In the present study, we aimed to elucidate the diversity of lactic acid bacteria (LAB) and their characteristics including probiotic properties from major Meekeri production areas (n = 22) in Sri Lanka. Lactic acid bacteria was isolated from locally produced Meekiri samples (n = 23) and characterized based on morphological, biochemical, physiological profiles and potential of probiotic properties. The isolates revealed five different colony and cell morphologies and were classified as heterofermenters, homofermenters and facultative heterofermenters based on CO2 production using glucose. None of the isolates showed the ability to grow either at 5 °C or 0 °C, while 71 % and 100 % survival of the isolates were observed at 15 °C and 45 °C, respectively. Amplified ribosomal DNA restriction analysis (ARDRA) primarily grouped the isolates into three distinct clusters based on their DNA banding patterns. Subsequently, 16S rRNA sequencing of isolates revealed the presence of four species namely, Limosilactobacillus fermentum (n = 18), Latilactobacillus curvatus (n = 2), Lactobacillus acidophilus (n = 2) and Lactiplantibacillus plantarum (n = 1) and in the phylogenetic analysis, it was represented by four distinctive clades. All the isolated species demonstrated promising probiotic potential with antibiotic sensitivity, antimicrobial properties, bile acid tolerance and acid tolerance. In conclusion, traditional back-slopping Meekiri in Sri Lanka contains diverse LAB, with a negligible geographical variation at species-level. Our work provides a strong foundation and insights into future applications in starter culture development for the fermentation of buffalo's milk.

Bioprospecting of endophytic bacteria from nodules and roots of Vigna radiata, Vigna unguiculata and Cajanus cajan for their potential use as bioinoculants

The haphazard and unwarranted use of chemical fertilizers has disturbed the soil microflora and groundwater by rendering them highly contaminated and reduced its agricultural productivity considerably. To revive the soil, groundwater and to restore the fertility of soil, a novel approach of using bioinoculants to restore both soil microflora and groundwater-detox with an added bonus of organic agriculture, is the major requirement at this moment. This study is dedicated towards isolation and characterization of the plant growth promoting endophytic bacteria (PGPEB) from the leguminous crops viz. Vigna radiata, V. unguiculata and Cajanus cajan. A total of 367 endophytic bacteria were isolated and characterized for plant growth promoting (PGP) attributes. Out of total, 97 endophytic bacteria were selected based upon morphological distinctness and plant growth promoting attributes. On the basis of amplified ribosomal DNA restriction analysis (ARDRA), these isolates were grouped into 45 genotypes. These genotypes were further evaluated for their PGP attributes and 28 representative strains selected for 16S rDNA sequence identification. The NCBI database analysis revealed the maximum identity of these genotypes towards different genera of three major phyla i.e. Firmicutes, Gamma (γ)-Proteobacteria and Actinobacteria. Firmicutes exclusively represented by 80% of isolates, encompassed three different genera Bacillus (10 species), Brevibacillus (2 species) and Lysinibacillus (1 species). The two genera included in γ-Proteobacteria were assigned to Enterobacter and Pseudomonas and two genera viz. Microbacterium (2 species) and Kocuria (1 species) to phylum Actinobacteria. B. panacihumi NMP3, B. australimaris NMP6, B. zhangzhouensis NMP7, M. barkeri NMP12, L. pakistanensis NAP3 and K. marina NAP9 are documented for the first time as PGPEB from these crops to the best of our knowledge. The analysis of in vivo pot experiment revealed that isolates E. cloacae NMP10, M. barkeri NMP12, B. licheniformis MHN12, B. paralicheniformis NMP13, M. arborescens NMP8, B. australimaris NMP6, P. stutzeri NAP4, B. megaterium NAP1, B. aerius NAP6 and B. megaterium NAP10 from V. radiata and C. cajan significantly increased all the growth parameters of their respective crops. Thus the study underlines the potential of these isolates to be harnessed as commercial bio-inoculants for integrated crop yield improvement.

Diversity of culturable bacteria endowed with antifungal metabolites biosynthetic characteristics associated with tea rhizosphere soil of Assam, India

BackgroundRhizosphere soil is a crucial niche for the diverse beneficial microbial communities in plant-microbe interactions. This study explores the antagonistic potential and diversity of the rhizosphere soil bacteria from commercial tea estates of Assam, India which comes under the Indo-Burma mega-biodiversity hotspot. Rhizosphere soil samples were collected from six different tea estates to isolate the bacteria. The bacterial isolates were subjected to evaluate for the antagonistic activity against fungal pathogens. The potential isolates were investigated for chitinase production and the presence of chitinase gene. The bacterial genetic diversity was studied by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and BOX-PCR fingerprinting.ResultsA total of 217 rhizobacteria were isolated from tea rhizosphere soil, out of which 50 isolates exhibited the potential antagonistic activity against fungal pathogens. Among them, 12 isolates showed extracellular chitinase activity and the presence of chitinase genes. The chitinase genes were sequenced and the analysis of the sequences was performed by using PDB protein databank at the amino acid level. It showed the presence of ChiA and ChiA74 gene in the 6 most potent isolates which are involved in the hydrolysis of chitin. These isolates also exhibited antagonistic activity against all tested fungal pathogens. The diversity of 50 antagonistic bacterial isolates were analyzed through ARDRA and BOX-PCR fingerprinting. Diversity analysis and molecular identification of the rhizosphere isolates revealed that these antagonistic isolates predominantly belonged to the genus Bacillus followed by Enterobacter, Serratia, Lysinibacillus, Pseudomonas, and Burkholderia.ConclusionThe present study establishes that rhizobacteria isolated from the poorly explored tea rhizosphere soil could be a rich reservoir for the investigation of potential antagonistic bacterial candidates for sustainable agricultural and industrial applications.

Ensifer. meliloti sv. lancerottense nodulates Lotus creticus in alkaline soils of Northern Morocco

Lotus spp. are a plant of great importance as a forage to improve pastures, to restore degraded soils and the inoculation with best rhizobial strains is widely used in many countries. The aim of this work was to analyze the phylogeny of bacteria nodulating Lotus creticus collected from sandy alkaline saline soil in Tangier province Northern Morocco and determine their identity. Out of 44 bacterial isolates, 21 strains were selected for the phenotypic characterization and symbiotic efficiency evaluation. The enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) fingerprinting showed different patterns at 40% similarity, and the amplified ribosomal DNA restriction analysis (ARDRA) generated one ribotype. Based on both the ERIC-PCR and the ARDRA results, 2 isolates, LcT51 and LcA30, were then chosen for molecular analysis. The 16S rRNA gene sequences showed that the 2 strains are closely related to Ensifer meliloti LMG 6133T with similarity values of 99.69%% to 99.93%. The analysis of concatenated atpD, glnII, gyrB, rpoB and dnaK housekeeping genes sequences confirmed the affiliation of the two strains to E. meliloti LMG 6133T. The phylogenic analysis of the nodC and nifH symbiotic genes, confirmed that L. creticus is nodulated in Tangier alkaline soils by the symbiovar lancerottense of E. meliloti.

Novel Cyanobacterial Diversity Found in Costa Rican Thermal Springs Associated with Rincon de la Vieja and Miravalles Volcanoes: A Polyphasic Approach.

Central America is one of the most important biodiversity hot spots in the world, and Costa Rican microbial communities from thermal springs are the best characterized in the isthmus. Miravalles is an inactive quaternary stratovolcano, and the Rincón de la Vieja is a unique active volcano, in whose slopes diverse hydrothermal springs, such as Las Lilas, are located. These springs harbor extensive microbial mats, whose diversity has been studied. Based on their importance as primary producers, in this study we focused on cultured cyanobacterial diversity from two geothermal environments of northern Costa Rica. Several cultural, molecular and taxonomic techniques were employed to maximize the results of a polyphasic approach. Sample collection sites were physicochemically described, and strains were isolated and characterized by light and electron microscopy. Phylogenetic analysis was performed using 16S rRNA gene sequences and amplified ribosomal DNA restriction analysis (ARDRA). Fifty-six phylotypes were isolated and classified into 21 morphotypes and identified in 14 genera, some of them might be new species within these genera. Furthermore, according to phylogenetic analysis, there are three possible new genera in our collection. Miravalles and Las Lilas thermal springs are reservoirs of novel phylogeographic lineages of phototrophic microorganisms. This study is the first report of strains that belong to the genera Gloeocapsa, Stanieria, Microseira, Klisinema and Oculatella isolated from thermal springs and growing at temperatures above 50°C. We also obtained isolates assigned to Synechococcus, Leptolyngbya spp., and Fischerella, which are considered typical strains in these environments.

Phenetic and Molecular Diversity of Nitrogen Fixating Plant Growth Promoting Azotobacter Isolated from Semiarid Regions of India

In the present study, 24 Azotobacter strains were isolated from soils of different areas of southern Rajasthan and characterized at biochemical, functional, and molecular levels. The isolated Azotobacter strains were gram negative and cyst forming when viewed under the microscope. These strains were also screened for their plant growth promoting activities and the ability of these isolates to survive under abiotic stress conditions viz. salt, pH, temperature, and drought stress. All the isolates showed IAA, siderophore, HCN, and ammonia production, whereas seven Azotobacter strains showed phosphate solubilization. Amplified Ribosomal DNA Restriction Analysis (ARDRA) revealed significant diversity among Azotobacter strains and the dendrogram obtained differentiated twenty-four of the strains into two major clusters at a similarity coefficient of 0.64. Qualitative and quantitative N2 fixation abilities of these strains were also detrained, and the amounts of acetylene reduced by Azotobacter strains were in the range of 1.31 to 846.56 nmol C2H4 mg protein−1 h−1. The strains showing high nitrogen fixation ability with multiple PGP activities were selected for further pot studies, and these Azotobacter strains significantly increased the various plant growth parameters of maize plantlets. Furthermore, the best Azotobacter isolates were subjected to 16S rRNA sequencing and confirmed their identities as Azotobacter sp. The indigenous Azotobacter strains with multiple PGP activities could be further used for commercial production.

Evaluation of Vitek-MS™ and Microflex LT™ commercial systems for identification of Acinetobacter calcoaceticus–baumannii complex

IntroductionAcinetobacter is a genus that comprises a group of opportunistic pathogens responsible for a variety of nosocomial infections. The Acinetobacter calcoaceticus–Acinetobacter baumannii (Acb) complex includes some species of clinical importance, mainly A. baumannii, A. pittii and A. nosocomialis, which share phenotypic similarities that make it very difficult to distinguish between them using a phenotypic approach. The aim of this study was to evaluate two commercial matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems for the identification of different Acinetobacter species, with a special focus among those belonging to the Acb complex. MethodsOne hundred and fifty-six Acinetobacter spp. clinical strains, identified by amplified ribosomal DNA restriction analysis (ARDRA) and rpoB gene sequencing, were analysed by two different MALDI-TOF systems. ResultsConsidering only the 144 strains of the Acb complex evaluated in this study, the Vitek-MS™ and Microflex LT™ systems correctly identified 129 (89.6%) and 143 (99.3%) strains, respectively. ConclusionAfter analysing 156 strains belonging to Acinetobacter spp., both Vitek-MS™ and Microflex LT™ proved to be rapid and accurate systems for the identification of Acb complex species showing a good correlation. However, both manufacturers should improve their databases to include new species in them.