Isolation of Novel Afipia septicemium and Identification of Previously Unknown Bacteria Bradyrhizobium sp. OHSU_III from Blood of Patients with Poorly Defined Illnesses

Cultures previously set up for isolation of mycoplasmal agents from blood of patients with poorly-defined illnesses, although not yielding positive results, were cryopreserved because of suspicion of having low numbers of unknown microbes living in an inactive state in the broth. We re-initiated a set of 3 cultures for analysis of the "uncultivable" or poorly-grown microbes using NGS technology. Broth of cultures from 3 blood samples, submitted from OHSU between 2000 and 2004, were inoculated into culture flasks containing fresh modified SP4 medium and kept at room temperature (RT), 30°C and 35°C. The cultures showing evidence of microbial growth were expanded and subjected to DNA analysis by genomic sequencing using Illumina MiSeq. Two of the 3 re-initiated blood cultures kept at RT after 7–8 weeks showed evidence of microbial growth that gradually reached into a cell density with detectable turbidity. The microbes in the broth when streaked on SP4 agar plates produced microscopic colonies in ∼ 2 weeks. Genomic studies revealed that the microbes isolated from the 2 blood cultures were a novel Afipia species, tentatively named Afipia septicemium. Microbes in the 3^rd culture (OHSU_III) kept at RT had a limited level of growth and could not reach a plateau with high cell density. Genomic sequencing identified the microbe in the culture as a previously unknown species of Bradyrhizobium bacteria. This study reports on the isolation of novel Afipia and Bradyrhizobium species. Isolation of Bradyrhizobium species bacteria has never been reported in humans. The study also reveals a previously unrecognized nature of hematogenous infections by the 2 unique groups of Bradyrhizobiaceae. Our studies show that improvement of culture system plus effective use of NGS technology can facilitate findings of infections by unusual microbes in patients having poorly-defined, sometimes mysterious illnesses.     

Genome Sequence of Afipia birgiae,a Rare Bacterium Associated with Amoebae

Afipia birgiae is an alphaproteobacterium from the family Bradyrhizobiaceae, growing in amoebae, and a potential human pathogen. We sequenced the genome of type strain 34632^T. It is composed of 5,325,467 bp and contains 5,160 protein-coding genes and 53 RNA genes, including 3 rRNA genes.     

An investigation of conventional microbial culture for the Naja atra bite wound,and the comparison between culture-based 16S Sanger sequencing and 16S metagenomics of the snake oropharyngeal bacterial microbiota

Naja atra is a major venomous snake found in Taiwan. The bite of this snake causes extensive wound necrosis or necrotizing soft tissue infection. Conventional microbial culture-based techniques may fail to identify potential human pathogens and render antibiotics ineffective in the management of wound infection. Therefore, we evaluated 16S Sanger sequencing and next-generation sequencing (NGS) to identify bacterial species in the oropharynx of N. atra. Using conventional microbial culture methods and the VITEK 2 system, we isolated nine species from snakebite wounds. On the basis of the 16S Sanger sequencing of bacterial clones from agar plates, we identified 18 bacterial species in the oropharynx of N. atra, including Morganella morganii, Proteus vulgaris, and Proteus mirabilis, which were also present in the infected bite wound. Using NGS of 16S metagenomics, we uncovered more than 286 bacterial species in the oropharynx of N. atra. In addition, the bacterial species identified using 16S Sanger sequencing accounted for only 2% of those identified through NGS of 16S metagenomics. The bacterial microbiota of the oropharynx of N. atra were modeled better using NGS of 16S metagenomics compared to microbial culture-based techniques. Stenotrophomonas maltophilia, Acinetobacter baumannii, and Proteus penneri were also identified in the NGS of 16S metagenomics. Understanding the bacterial microbiota that are native to the oropharynx of N. atra, in addition to the bite wound, may have additional therapeutic implications regarding empiric antibiotic selection for managing N. atra bites.     

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn²⁺ and Co²⁺ resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595^T = BCC 40155^T).     

Isolation and Characterization of Two Novel Bacteria Afipia cberi and Mesorhizobium hominis from Blood of a Patient Afflicted with Fatal Pulmonary Illness

We recently isolated and discovered new Bradyrhizobiaceae microbes from the cryopreserved culture broth of blood samples from 3 patients with poorly defined illnesses using modified SP4 media and culture conditions coupled with genomic sequencing. Using a similar protocol, we studied a previously cryopreserved culture broth of blood sample from a patient who had succumbed to an acute onset of fulminant pulmonary illness. We report that two phases of microbial growth were observed in the re-initiated culture. Biochemical and genomic characterization revealed microbes isolated from the first phase of growth were new Afipia species of Bradyrhizobiaceae, tentatively named A. cberi with a ~ 5 MB chromosome that was different from those of all previously known Afipia microbes including the newly discovered A. septicemium. The microbes isolated from the second phase of growth were prominent sugar assimilators, novel Phyllobacteriaceae, phylogenetically most closely related to Mesorhizobium and tentatively named M. hominis with a ~ 5.5 MB chromosome. All A. cberi isolates carry a circular ~ 140 KB plasmid. Some M. hominis isolates possess a circular ~ 412 KB plasmid that can be lost in prolonged culture or passage. No antibiotics resistant genes could be identified in both of the A. cberi and M. hominis plasmids. Antibiotic susceptibility studies using broth culture systems revealed isolates of A. cberi could be sensitive to some antibiotics, but all isolates of M. hominis were resistant to essentially all tested antibiotics. However, the cell-free antibiotics susceptibility test results may not be applicable to clinical treatment against the microbes that are known to be capable of intracellular growth. It remains to be determined if the 2 previously unknown Rhizobiales were indeed pathogenic and played a role in the pulmonary disease process in this patient. Specific probes and methods will be developed to re-examine the diseased lungs from patient''s autopsy.     

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage within the γ-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.     

A Novel Protein-Deamidating Enzyme from Chryseobacterium proteolyticum sp. nov., a Newly Isolated Bacterium from Soil

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670^T isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).     

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.     

Genomic characterization of the Yersinia genus

Background

New DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments.

Results

We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats.

Conclusions

Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.     

Surfactant-induced bacterial community changes correlated with increased polycyclic aromatic hydrocarbon degradation in contaminated soil

Bioremediation as a method for removing polycyclic aromatic hydrocarbons (PAHs) from contaminated environments has been criticized for poor removal of potentially carcinogenic but less bioavailable high molecular weight (HMW) compounds. As a partial remedy to this constraint, we studied surfactant addition at sub-micellar concentrations to contaminated soil to enhance the biodegradation of PAHs remaining after conventional aerobic bioremediation. We demonstrated increased removal of four- and five-ring PAHs using two nonionic surfactants, polyoxyethylene(4)lauryl ether (Brij 30) and polyoxyethylene sorbitol hexaoleate (POESH), and analyzed bacterial community shifts associated with those conditions. Eight groups of abundant bacteria were implicated as potentially being involved in increased HMW PAH removal. A group of unclassified Alphaproteobacteria and members of the Phenylobacterium genus in particular showed significantly increased relative abundance in the two conditions exhibiting increased PAH removal. Other implicated groups included members of the Sediminibacterium, Terrimonas, Acidovorax, and Luteimonas genera, as well as uncharacterized organisms within the families Chitinophagaceae and Bradyrhizobiaceae. Targeted isolation identified a subset of the community likely using the surfactants as a growth substrate, but few of the isolates exhibited PAH-degradation capability. Isolates recovered from the Acidovorax and uncharacterized Bradyrhizobiaceae groups suggest the abundance of those groups may have been attributable to growth on surfactants. Understanding the specific bacteria responsible for HMW PAH removal in natural and engineered systems and their response to stimuli such as surfactant amendment may improve bioremediation efficacy during treatment of contaminated environmental media.     

New Afipia and Bosea strains isolated from various water sources by amoebal co-culture

It has been suspected that some species belonging to the alphaproteobacteria might cause pneumonia in humans. It is thus of special interest to isolate new members of this phylum, and to further characterize their pathogenicity. The amoebal co-culture method allowed the isolation of various new bacterial species during the last few years, including fastidious alphaproteobacterial species that were isolated from complex environments. In this work, we isolated new bacterial strains from a drinking water network or from river water using amoebal co-culture with Acanthamoeba castellanii. One Afipia sp. strain and two Bosea sp. strains presented 16SrDNA and partial rpoB gene sequences suggesting that they could be representative of new species, and were thus further characterized using phenotypic tests.     

Microbial Diversity of Some Sabkha and Desert Sites in Saudi Arabia

Several studies isolated fungal and bacterial species from extreme environments, such as Sabkha and hot deserts, as their natural habitat, some of which are of medicinal importance. Current research aimed investigating the microbial (fungi and bacteria) diversity and abundance in Sabkha and desert areas in Saudi Arabia. Soil samples from nine different geographical areas (Al-Aushazia lake, AlQasab, AlKasar, Tabuk, Al-Kharj, Al-Madina, Jubail, Taif and Abqaiq) were collected and cultured for microbial isolation. Isolated fungi and bacteria were identified by molecular techniques (PCR and sequencing). Based on 18S rDNA sequencing, 203 fungal species belonging to 33 genera were identified. The most common fungal genera were Fusarium, Alternaria, Chaetomium, Aspergillus Cochliobolus and Pencillium, while the most common species were Chaetomium globosum and Fusarium oxysporum. By 16S rDNA sequencing 22 bacterial species belonging to only two genera, Bacillus and Lactobacillus, were identified. The most commonly isolated bacterial species were Bacillus subtilis and Lactobacillus murinus. Some fungal species were confined to specific locations, such as Actinomyces elegans, Fusarium proliferatum, Gymnoascus reesii and Myzostoma spp. that were only isolated from Al-Aushazia soil. AlQasab soil had the highest microbial diversity among other areas with abundances of 23.5% and 4.4% of total fungi, and bacteria, respectively. Findings of this study show a higher degree of fungal diversity than that of bacteria in all studied areas. Further studies needed to investigate the connection between some isolated species and their habitat ecology, as well as to identify those of medicinal importance.     

A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex

The Mycobacterium abscessus complex can cause fatal pulmonary disease, especially in cystic fibrosis patients. Diagnosing M. abscessus complex pulmonary disease is challenging. Immunologic assays specific for M. abscessus are not available. In this study seven clinical M. abscessus complex strains and the M. abscessus reference strain ATCC19977 were used to find species-specific proteins for their use in immune assays. Six strains showed rough and smooth colony morphotypes simultaneously, two strains only showed rough mophotypes, resulting in 14 separate isolates. Clinical isolates were submitted to whole genome sequencing. Proteomic analysis was performed on bacterial lysates and culture supernatant of all 14 isolates. Species-specificity for M. abscessus complex was determined by a BLAST search for proteins present in all supernatants. Species-specific proteins underwent in silico B- and T-cell epitope prediction. All clinical strains were found to be M. abscessus ssp. abscessus. Mutations in MAB_4099c as a likely genetic basis of the rough morphotype were found in six out of seven clinical isolates. 79 proteins were present in every supernatant, of which 12 are exclusively encoded by all members of M. abscessus complex plus Mycobacterium immunogenum. In silico analyses predicted B- and T-cell epitopes in all of these 12 species-specific proteins.     

Microbial and pigment profile of the reddish patch occurring within Tuber magnatum ascomata

Tuber magnatum Pico, the delectable white truffle, is the most prized truffle species. In this study, we examined the reddish pigmentation that frequently occurs in T. magnatum ascomata for the presence of pigment-producing bacteria.The inner part of the reddish-pigmented region of three T. magnatum ascomata collected in North–Central Italy was analysed. This reddish part was used to establish a bacterial culture collection and to extract the total genomic DNA in order to obtain a library of 16S rRNA genes representative of the bacterial community. The molecular approach revealed limited microbial diversity within the reddish-pigmented regions compared to the wider range of bacterial species commonly found at the same maturation stage and season in T. magnatum ascomata. The pigmented regions showed a prevalence of specific bacterial species belonging to α-, β- and γ- Proteobacteria, Actinobacteria and Firmicutes. From the tandem mass spectrometry analysis of the extracted pigment, four compounds were identified: i) bixin, ii) β-carotene, iii) cis-1-glycosyl-apo-8′- lycopene and iv) the fucoxanthin. Carotenoid producing species such as Microbacterium and Chryseobacterium emerged as the most likely cause of the peculiar reddish pigment production. Indeed, our findings suggest that the peculiar reddish pigment might be produced by these bacterial species.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area.     

Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.     

Midgut Microbial Community of Culex quinquefasciatus Mosquito Populations from India

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.     

Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

Background

Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods.

Results

Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations.

Conclusions

This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality rates.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.