Mossambicus tilapia (Oreochromis mossambicus) collected from water bodies impacted by urban waste carries extended-spectrum beta-lactamases and integron-bearing gut bacteria.

Oreochromis mossambicus (Peters 1852) (Tilapia) is one of the most consumed fish globally. Tilapia thrives well in environments polluted by urban waste, which invariably contain antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Thus, Tilapia surviving in such polluted environments may serve as a potential source for dissemination of ARGs. To investigate this, we isolated bacterial strains from gut of Tilapia found in polluted rivers and lakes near Pune, India, and studied the prevalence of resistance genes bymolecularmethods. A total of 91 bacterial strains were obtained, which include fish pathogens and human pathogens such as Aeromonas hydrophila, Klebsiella pneumoniae, E. coli, Serratia marcescens, Enterobacter spp. and Shigella spp. Overall the prevalence of class 1 integrons, class 2 integrons, extended-spectrum betalactamases (ESBLs) blaCTX-M, blaSHV and aac(6')-Ib-cr gene was 38%, 24%, 38%, 31% and 31% respectively. Forty-two percent of the Enterobacteriaceae strains carried blaCTX-M gene, which is a common ESBL gene in clinics. The study demonstrates that tilapia found in the polluted waters can serve as reservoirs and an alternative route for human exposure to clinically important ARG-carrying bacteria. The consumption and handling of these fish may pose a potential health risk.

Transcriptome analysis of Anopheles stephensi embryo using expressed sequence tags.

Germ band retraction (GBR) stage is one of the important stages during insect development. It is associated with an extensive epithelial morphogenesis and may also be pivotal in generation of morphological diversity in insects. Despite its importance, only a handful of studies report the transcriptome repertoire of this stage in insects. Here, we report generation, annotation and analysis of ESTs from the embryonic stage (16–22 h post fertilization) of laboratoryreared Anopheles stephensi mosquitoes. A total of 1002 contigs were obtained upon clustering of 1140 high-quality ESTs, which demonstrates an astonishingly low transcript redundancy (12.1%). Putative functions were assigned only to 213 contigs (21%), comprising mainly of transcripts encoding protein synthesis machinery. Approximately 78% of the transcripts remain uncharacterized, illustrating a lack of sequence information about the genes expressed in the embryonic stages of mosquitoes. This study highlights several novel transcripts, which apart from insect development, may significantly contribute to the essential biological complexity underlying insect viability in adverse environments. Nonetheless, the generated sequence information from this work provides a comprehensive resource for genome annotation, microarray development, phylogenetic analysis and other molecular biology applications in entomology.

Comparative analysis of fecal microflora of healthy full-term Indian infants born with different methods of delivery (vaginal vs cesarean): Acinetobacter sp. prevalence in vaginally born infants.

In this study fecal microflora of human infants born through vaginal delivery (VB) and through cesarean section (CB) were investigated using culture-independent 16S rDNA cloning and sequencing approach. The results obtained clearly revealed that fecal microbiota of VB infants distinctly differ from those in their counterpart CB infants. The intestinal microbiota of infants delivered by cesarean section appears to be more diverse, in terms of bacteria species, than the microbiota of vaginally delivered infants. The most abundant bacterial species present in VB infants were Acinetobacter sp., Bifidobacterium sp. and Staphylococcus sp. However, CB infant’s fecal microbiota was dominated with Citrobacter sp., Escherichia coli and Clostridium difficile. The intestinal microbiota of cesarean section delivered infants in this study was also characterized by an absence of Bifidobacteria species. An interesting finding of our study was recovery of large number of Acinetobacter sp. consisting of Acinetobacter pittii (former Acinetobacter genomic species 3), Acinetobacter junii and Acinetobacter baumannii in the VB infants clone library. Among these, Acinetobacter baumannii is a known nosocomial pathogen and Acinetobacter pittii (genomic species 3) is recently recognized as clinically important taxa within the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex. Although none of the infants had shown any sign of clinical symptoms of disease, this observation warrants a closer look.

Molecular analysis of gut microbiota in obesity among Indian individuals.

Obesity is a consequence of a complex interplay between the host genome and the prevalent obesogenic factors among the modern communities. The role of gut microbiota in the pathogenesis of the disorder was recently discovered; however, 16S-rRNA-based surveys revealed compelling but community-specific data. Considering this, despite unique diets, dietary habits and an uprising trend in obesity, the Indian counterparts are poorly studied. Here, we report a comparative analysis and quantification of dominant gut microbiota of lean, normal, obese and surgically treated obese individuals of Indian origin. Representative gut microbial diversity was assessed by sequencing fecal 16S rRNA libraries for each group (n=5) with a total of over 3000 sequences. We detected no evident trend in the distribution of the predominant bacterial phyla, Bacteroidetes and Firmicutes. At the genus level, the bacteria of genus Bacteroides were prominent among the obese individuals, which was further confirmed by qPCR (P<0.05). In addition, a remarkably high archaeal density with elevated fecal SCFA levels was also noted in the obese group. On the contrary, the treated-obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced fecal SCFAs. In conclusion, the study successfully identified a representative microbial diversity in the Indian subjects and demonstrated the prominence of certain bacterial groups in obese individuals; nevertheless, further studies are essential to understand their role in obesity.

Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers.

The micro-eukaryotic diversity from the human gut was investigated using universal primers directed towards 18S rRNA gene, fecal samples being the source of DNA. The subjects in this study included two breast-fed and two formula-milk-fed infants and their mothers. The study revealed that the infants did not seem to harbour any microeukaryotes in their gut. In contrast, there were distinct eukaryotic microbiota present in the mothers. The investigation is the first of its kind in the comparative study of the human feces to reveal the presence of micro-eukaryotic diversity variance in infants and adults from the Indian subcontinent. The micro-eukaryotes encountered during the investigation include known gut colonizers like Blastocystis and some fungi species. Some of these micro-eukaryotes have been speculated to be involved in clinical manifestations of various diseases. The study is an attempt to highlight the importance of micro-eukaryotes in the human gut.

Life in (and on) the rocks.

Rocks, apart from being ancient records that enlighten us about the geological history of our planet, are dynamic repositories that support life forms central to the sustenance of our biosphere. It is the latter that our discussion will be largely focused on. Life associated with rocks has been documented as early as 1914 (Diels 1914), but it was in the 1960s that Friedmann and colleagues, with their extensive studies on rock-dwellers in hot and cold desert habitats, gave shape to this modern branch of geobiology. The presence of microscopic algae and bacteria was first demonstrated within exposed rocks from hot desert environments such as the Negev and the Sinai (Friedmann and Galun 1974) by electron microscopy and laboratory cultivation methods. Subsequent studies in ortho-quartzite rocks from the Dry Valley area of Antarctica also showed morphologically similar algae (related to the genus Gloeocapsa) to be colonizing areas ~1.5 mm wide parallel to and ~2 mm beneath the rock surface (Friedmann and Ocampo 1976). The results from the latter caught the attention of the scientific community as NASA had been testing their Voyager mission probes on the apparently lifeless cold deserts of Antarctica with the aim of studying a habitat analogous to Mars. In 2005, Walker and colleagues, through the use of culture-independent molecular methods, discovered the microbial colonization of rocks from the extremely acidic (pH~1) Yellowstone geothermal environment. By employing universal PCR primers that targeted 16S rRNA genes from all three domains of life, the authors were able to retrieve sequences phylogenetically related to extant red alga (Cyanidium sp.), bacteria (α-, β-, γ-Proteobacteria; Actinobacteria; Bacteroidetes and Firmicutes) and archaea (Euryarchaeota and Crenarchaeota). The astoundingly high diversity of microbial life forms present in such extreme habitats captured the imaginations of geo(micro)biologists, astrobiologists and microbial ecologists alike. Astrobiologists imagined that if rock interiors could support the major fraction of life in the harshest of environments on earth, then the same could be applicable to other planets such as Mars, and they sensed that there was a need to extend the scope of extraterrestrial life detection missions beyond the mere analysis of top soils. Microbial ecologists wondered if rock-associated life was ubiquitous in the biogeosphere, and geo(micro)biologists hypothesized that the rock micro-habitat offered life (a) protection from intense solar radiations, temperature and desiccation and (b) a supply of nutrients, moisture and growth surfaces. Today, with two dedicated international scientific journals – Geobiology and Geomicrobiology Journal and an ever growing number of papers dealing with rock-associated microbes in microbiological research journals, the concept of ‘life in/on the rocks’ has become as hardened as the rock itself. What is apparently lacking, however, is an understanding among the general public that a ‘dumblooking’ average rock in their gardens (on this planet and perhaps in the gardens of intelligent beings on other planets) could be home to a dynamic assortment of interesting yet diverse living organisms of inevitably microbial nature.

Wolbachia and termite association: present status and future implications.

Inherited symbionts play an important role in the ecology and evolution of many species. One such inherited symbiont, Wolbachia, is known to have many interesting and diverse symbiotic associations with its arthropod and nematode hosts, ranging from parasitism to mutualism (Werren et al. 2008). The organism is notable for signifi cantly altering the reproductive capabilities of its arthropod hosts and manipulating their cell biology by inducing different phenotypes such as male killing, feminization, parthenogenesis, cytoplasmic incompatibility (sperm–egg incompatibility) and even speciation in certain species (Werren et al. 2008). These interactions become more interesting due to the spatial variation and phenotype of Wolbachia strains. Wolbachia strains that are genotypically very closely related can induce diverse phenotypic effects in different hosts, whereas different strains can induce similar phenotypic effects in the same hosts (Jiggins et al. 2002). Therefore, characterization of the Wolbachia genotype and its phenotypic effect in different hosts is important for understanding the ecology and evolution of different species. Since Wolbachia cannot be cultured outside host cells, traditional microbiological methods cannot be applied to study these bacteria. Currently, they are categorized into eleven different ‘supergroups’ (labelled alphabetically A–K) on the basis of clades formed in gene phylogenies (Lo et al. 2002; Bordenstein and Rosengaus 2005; Casiraghi et al. 2005; Ros et al. 2009). Termites are a group of social insects usually classifi ed in the taxonomic rank of order Isoptera, and described as ‘ecosystem engineers’ due to their important role in providing soil ecosystem services. They are major detrivores, particularly in the subtropical and tropical regions, and their recycling of wood and other plant matter is of considerable ecological importance (Harris 1971). Their ancient origin (Devonian period), great diversity and considerable ecological, biological and behavioural plasticity suggests that characterization of Wolbachia in this group is needed in order to understand the impact of the symbiont on termite reproduction, evolution and speciation (Roy and Harry 2007). The phenotypic effects of Wolbachia in Isoptera are still unknown but molecular data concerning these termite symbionts have recently become available. The available literature suggests the occurrence of Wolbachia in termite families Termopsidae, Kalotermitidae, Serritermitidae, Rhinotermitidae and Termitidae. Four phylogenetically different Wolbachia supergroups have been reported in termites. Twenty termite species have been reported to harbour Wolbachia. Out of these, thirteen species (Kalotermes fl avicollis, Coptotermes lacteus, Coptotermes acinaciformis, Cryptotermes secundus, Heterotermes sp., Nasutitermes takasagoensis, Nasutitermes sp., Nasutitermes nigriceps, Hospitalitermes mediofl avus, Microcerotermes sp., Apilitermes longiceps, Labiotermes labralis, Microtermes sp.) have supergroup F Wolbachia infection (Casiraghi et al. 2005; Lo et al. 2002; Lo and Evans 2007; Roy and Harry 2007). Two Zootermopsis species (Zootermopsis nevadensis and Zootermopsis angusticollis) carry supergroup H Wolbachia (Bordenstein and Rosengaus 2005). Cubitermes sp. affi nis subarquatus harbour diverse types of Wolbachia belonging to the supergroup A and B clade (Roy and Harry 2007), Incisitermes snyderi carry supergroup A Wolbachia (Baldo et al. 2006), while Wolbachia from Serritermes serrifer, Neotermes luykxi and Neotermes jouteli belong to a divergent sister clade within supergroup A (Lo and Evans 2007). Termites, like other groups of arthropods, can tolerate Wolbachia of more than one supergroup, although individual species can harbour only single infections. This provides some evidence for the horizontal transmission of Wolbachia. Infection with different Wolbachia supergroups in various termite species can be parsimoniously explained by independent acquisition of these lineages in termites, rather than a single ancient ancestral infection, with subsequent divergence and/or widespread loss (Bordenstein and Rosengaus 2005).

Characterization of Leptospira borgpetersenii isolates from field rats (Rattus norvegicus) by 16S rRNA and lipL32 gene sequencing

The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 percent, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.