Mahesh S. Dharne

Ignatzschineria indica sp. nov. and Ignatzschineria ureiclastica sp. nov., isolated from adult flesh flies (Diptera: Sarcophagidae)

Two Gram-negative-staining, aerobic, non-motile, rod-shaped bacteria, designated strains FFA1(T) and FFA3(T), and belonging to the class Gammaproteobacteria were isolated from the gastrointestinal tract of adult flesh flies (Diptera: Sarcophagidae). Phylogenetic analysis of 16S rRNA gene sequence data placed these two strains within the genus Ignatzschineria with similarities of 98.6 % (FFA1(T)) and 99.35 % (FFA3(T)) to Ignatzschineria larvae L1/68(T). The level of gene sequence similarity between strains FFA1(T) and FFA3(T) was 99 %, 97.15 % and 78.1 % based on the 16S rRNA, 23S rRNA and gyrB gene sequences, respectively. Strains FFA1(T) and FFA3(T) shared 24 % DNA-DNA relatedness. DNA-DNA hybridization revealed a very low level of relatedness between the novel strains (22 % for strain FFA1(T) and 44 % for strain FFA3(T)) and I. larvae L1/68(T) genomic DNA. The respiratory quinone was Q-8 in both novel strains. The DNA G+C contents were 41.1 mol% and 40.1 mol% for strains FFA1(T) and FFA3(T), respectively. The cell membrane of both strains consisted of phosphatidylglycerol, phosphatidylethanolamine, phospholipids and aminophospholipid. The major fatty acids for both strains were C(16 : 0), summed feature 8 (C(18 : 1)ω7c and/or C(18 : 1)ω6c), CyC(19 : 0)ω8c and C(14 : 0). The results of DNA-DNA hybridization between the two new strains and I. larvae L1/68(T), in combination with phylogenetic, chemotaxonomic, biochemical and electron microscopic data, demonstrated that strains FFA1(T) and FFA3(T) represented two novel species of the genus Ignatzschineria for which the names Ignatzschineria indica sp. nov. (type strain FFA1(T) = DSM 22309(T) = KCTC 22643(T) = NCIM 5325(T)) and Ignatzschineria ureiclastica sp. nov. (type strain FFA3(T) = DSM 22310(T) = KCTC 22644(T) = NCIM 5326(T)) are proposed.

Oxidative and membrane stress-mediated antibacterial activity of WS2 and rGO-WS2 nanosheets

Graphene-based materials have strong cytotoxic attributes against bacteria due to their unique physicochemical properties. We examined the antibacterial activities of nanosheets of the graphene analogue tungsten disulphide (WS2) and a composite of reduced graphene oxide-tungsten disulphide (rGO-WS2), comparing them with reduced graphene oxide (rGO) by a time and concentration dependent viability assay and growth curve studies against four bacterial strains: Gram negative Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium), and Gram positive Bacillus subtilis (B. subtilis) and Staphylococcus epidermidis (S. epidermidis). The nanosheets of the rGO-WS2 composite caused a more significant retardation in bacterial growth and inhibitory effect on the tested bacterial strains than WS2, followed by rGO. The tested E. coli and B. subtilis strains were more susceptible than the other strains. A mechanistic study revealed that rGO and WS2 did not produce the superoxide anion (O2˙−) or reactive oxygen species (ROS), but the nanocomposite of rGO-WS2 did produce both. However, all these materials did oxidize glutathione, which serves as a redox state mediator in bacteria. We conclude that the antimicrobial mechanism is due to the combined effect of initial cell deposition on the rGO-WS2 materials, the membrane stress due to direct contact with the nanosheets, and the produced superoxide anion-independent oxidation mechanisms. The beneficial aspects of the physicochemical properties of rGO-WS2, such as its size and conductivity, can be precisely customized to reduce its health and environmental risk factors.

Helicobacter pylori in areas of gastric metaplasia in the gallbladder and isolation of H. pylori DNA from gallstones

Aims: To assess if the areas of gastric metaplasia in the gallbladder are colonised by Helicobacter pylori and to conduct a molecular study of gallstones for presence of H. pylori DNA. Methods: Sections from 111 gallbladders with evidence of gastric metaplasia on H&E and Alcian blue–periodic acid‐Schiff (pH 2.5) stain were stained with Loefflers methylene blue and Warthin Starry stain for demonstration of H. pylori. Presence of H. pylori was confirmed by immunohistochemistry. Formalin fixed mucosal tissues and gallstones from 11 cases showing heavy colonisation were subjected to molecular analysis. Results: Helicobacter pylori was present in 50 of 111 (45%) sections with gastric metaplasia. Areas adjacent to gastric metaplasia in gallbladder showed acute inflammation (6%) and lymphoid follicle formation in 58% of cases with H. pylori that were significantly higher than those seen in sections without H. pylori. In molecular study, 8 of 11 gallstones showed 16S rDNA. Amplification of material from one stone showed positivity for atpA, efp, mutY, ppa, trpC, UreI and vacA genes. Phylogenetic affiliation study of the isolates indicated that H. pylori sequence from the gallstones clustered with Indian strains of H. pylori. No considerable difference was observed in phylogenetic affiliations of eight stones studied. Conclusion: H. pylori colonises areas of gastric metaplasia in gallbladder producing histological changes similar to those seen in gastric mucosa. Isolation of H. pylori DNA from gallstones further support its presence in the gallbladder.

Complete metagenome sequencing based bacterial diversity and functional insights from basaltic hot spring of Unkeshwar, Maharashtra, India.

Unkeshwar hot springs are located at geographical South East Deccan Continental basalt of India. Here, we report the microbial community analysis of this hot spring using whole metagenome shotgun sequencing approach. The analysis revealed a total of 848,096 reads with 212.87 Mbps with 50.87% G + C content. Metagenomic sequences were deposited in SRA database with accession number (SUB1242219). Community analysis revealed 99.98% sequences belonging to bacteria and 0.01% to archaea and 0.01% to Viruses. The data obtained revealed 41 phyla including bacteria and Archaea and including 719 different species. In taxonomic analysis, the dominant phyla were found as, Actinobacteria (56%), Verrucomicrobia (24%), Bacteriodes (13%), Deinococcus-Thermus (3%) and firmicutes (2%) and Viruses (2%). Furthermore, functional annotation using pathway information revealed dynamic potential of hot spring community in terms of metabolism, environmental information processing, cellular processes and other important aspects. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of each contig sequence by assigning KEGG Orthology (KO) numbers revealed contig sequences that were assigned to metabolism, organismal system, Environmental Information Processing, cellular processes and human diseases with some unclassified sequences. The Unkeshwar hot springs offer rich phylogenetic diversity and metabolic potential for biotechnological applications.

Microbiology of the insect gut:tales from mosquitoes and bees.

The class Insecta consists of a large group of organisms with rich species diversity. There are an estimated 750,000 species of insects, but the actual number could be far more than this as some regions have been poorly studied and many ecosystems, especially in the tropics, have not been explored at all. Some estimates put the number as high as 10 million (Novotny et al 2002). All insect species are known to harbour a rich and complex community of microorganisms in their guts and other body regions. This microbiota participates in many types of interactions ranging from pathogenesis to obligate mutualism (Dillon and Dillon 2004). One reason for the microbial diversity is that different groups of insects have different feeding habits; this results in different gut structures and functions and promotes the establishment of different phylotypes. In recent years there has been renewed interest in the understanding of insect gut microorganisms for two reasons. First, this diverse microbiota is a potential source of novel bioactive compounds such as antimalarial, antiviral and antitumour peptides (Chernysh et al 2002), enzymes (Zhang and Brune 2004) and novel metabolites (Wilkinson 2001). Second, manipulating these microbial symbionts is thought to be an effective strategy for controlling the spread of pathogens that use insects as hosts (Mickes and Ferguson 1961; Lehane et al 1997; Beard et al 2002; Dillon et al 2005). Approaches for studying microbial community structure have changed phenomenally over in the last decade. Methods like 16S rDNA cloning and sequencing, and genetic profi ling using various methods – denaturant gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP) or terminal restriction fragment length polymorphism (T-RFLP) and fl uorescent in situ hybridization (FISH) among them – are being used to explore the bacterial diversity of various ecosystems without resorting to actually culturing the microorganisms in the laboratory (Reeson et al 2003; Mohr and Tebbe 2006; Thimm and Tebbe 2003). Before a few years, the microbiota of insect gut systems were only rarely examined using these techniques, an example being work on the microbial community structure of termite guts (Brauman et al 2001). Two recent studies (Lindh et al 2005; Mohr and Tebbe et al 2006) extend this knowledge to other insect groups and provide new insights into the interplay between gut microbiota and insect physiology, development and social behavior. These studies also provide background information on para-transgenic approaches to the control of vector-borne diseases (Durvasula et al 1997. The ‘paratransgenic’ method involves removing a microbial symbiont from an insect, genetically modifying it and re-introducing it into another insect that has previously been cured of its normally resident symbionts. Thus the transgene is carried by the insect but not in its own genome.). Pidiyar et al (2002, 2004) examined the mid-gut fl ora of Culex quinquifasciatus, responsible for the transmission of fi laria and probably Japanese encephalitis. They used both the conventional, culture-based approach and culture-independent, 16S rRNA gene-based PCR–clone–sequencing. Many cosmopolitan bacteria were found in the mosquito mid-gut, as also a new species of the genus Aeromonas, namely Aeromonas culicicola (Pidiyar et al 2002). This bacterial species was 2000-fold more abundant in mosquitoes that were blood fed as compared to those which were not. Subsequently, the same species was also found to inhabit the mid-guts of other mosquito species – Aedes aegyptii and Anopheles stephensi – found the same locality. In a later study, several species of bacteria were detected in the mid-guts of Anopheles gambiae and A. funestus by both methods (Lindh et al 2005). The isolates included at least two new species belonging to the genera Thorsellia and Janibacter (Kampfer et al 2006a,b). One isolate was identifi ed as Rhodococcus corynebacteroides which is a relative of R. rhodinii that is found in Rhodinus prolixus and has been successfully used in the para-transgenic approach (Durvasula et al 1997). The 16S rRNA approach revealed the presence of various subgroups of proteobacteria and genera representing intracellular bacteria such as Anaplasma and Spiroplasma. (The proteobacteria are a major group of Gram-negative bacteria; among them, the gamma-proteobacteria include many medicinally important and pathogenic forms, for example Enterobacteria, Vibrio, Salmonella, Yersinia and Pseudomonas.) Interestingly, both studies showed the presence of members of genera Bacillus, Stenotrophomonas and Pseudomonas in the mosquito mid-gut.

Draft genome of Ochrobactrum intermedium strain M86 isolated from non-ulcer dyspeptic individual from India

BackgroundOchrobactrum intermedium is an emerging opportunistic pathogen of humans that is closely related to members of the genus Brucella. Earlier, we reported the case of an Indian subject with non-ulcer dyspeptic symptoms whose urease positive gastric biopsy revealed the presence of Helicobacter pylori along with non-Helicobacter like bacteria, eventually cultured and identified as O. intermedium strain M86.ResultsHere, we describe the unclosed draft genome of the strain M86 with a length of 5,188,688 bp and mean G+C content of 57.9%. We have also identified many putative gene clusters that might be responsible for its persistence in the gastric mucosa.Comparative analysis of genomic features of Ochrobactrum intermedium strain M86 and Ochrobactrum intermedium LMG 3301T was also done.ConclusionsThis paper attempts to gain whole-genome based insights into the putative gene determinants of O. intermedium for survival in the highly acidic stomach lumen environment .Identification of genes putatively involved in the various metabolic pathways may lead to a better understanding of the survival of O. intermdedium in acidic condition.

Antibiofilm activity of tert-BuOH functionalized ionic liquids with methylsulfonate counteranions

A series of varying alkyl chain length substituted tert-BuOH-functionalized-imidazolium mesylate salts [alkyl-tOHim][OMs] were synthesized and evaluated for antimicrobial activity and antibiofilm potential on selected pathogenic microorganisms including bacteria (Gram positive and Gram negative), yeast, and fungi. The dodecyl substituted ionic liquid [C12-tOHim][OMs] significantly prevented the biofilm formation of S. epidermidis at 100 µM concentration as well as showed noteworthy antimicrobial activity. We conclude that the ionic liquids (ILs) bearing chain lengths lower than the dodecyl length were found to be less effective against most of the tested pathogenic microorganisms.

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin

Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin.

Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

ABSTRACT Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche.

Genome sequencing analysis reveals virulence-related gene content of Ochrobactrum intermedium strain 229E, a urease-positive strain isolated from the human gastric niche.

We report draft genome sequence of Ochrobactrum intermedium strain 229E concurrent with Helicobacter pylori in urease positive gastric biopsy of non-ulcer dyspeptic individual from Southern part of India. Since the role of Ochrobactrum in human gastric environment is poorly understood, comprehensive pathological, microbiological, and genome level understanding are necessary to evaluate its association with H. pylori in the gastric niche. Comparative analysis of O. intermedium 299E strain revealed functional similarities with virulence related gene clusters present in H. pylori genomes, which probably might aid in its ability to persist in the human gastric mucosa. However, H.pylori specific vacuolating cytotoxin (vacA) involved in vacuolization, cytotoxicity, and T-cell inhibition was absent in the O. intermedium 229E genome. Taken together, O. intermedium 229E shared numerous features like secretion system, urease, and flagella with H.pylori genome sequence that might aid concurrence in the gastric niche.