Milind S. Patole

Extracellular Synthesis of Crystalline Silver Nanoparticles and Molecular Evidence of Silver Resistance from Morganella sp : Towards Understanding Biochemical Synthesis Mechanism

There has been significant progress in the biological synthesis of nanomaterials. However, the molecular mechanism of synthesis of such bio‐nanomaterials remains largely unknown. Here, we report the extracellular synthesis of crystalline silver nanoparticles (AgNPs) by using Morganella sp., and show molecular evidence of silver resistance by elucidating the synthesis mechanism. The AgNPs were 20±5 nm in diameter and were highly stable at room temperature. The kinetics of AgNPs formation was investigated. Detectable particles were formed after an hour of reaction, and their production remained exponential up to 18 h, and saturated at 24 h. Morganella sp. was found to be highly resistant to silver cations and was able to grow in the presence of more than 0.5 mM AgNO3. Three gene homologues viz. silE, silP and silS were identified in silver‐resistant Morganella sp. The homologue of silE from Morganella sp. showed 99 % nucleotide sequence similarity with the previously reported gene, silE, which encodes a periplasmic silver‐binding protein. The homologues of silP and silS were also highly similar to previously reported sequences. Similar activity was totally absent in closely related Escherichia coli; this suggests that a unique mechanism of extracellular AgNPs synthesis is associated with silver‐resistant Morganella sp. The molecular mechanism of silver resistance and its gene products might have a key role to play in the overall synthesis process of AgNPs by Morganella sp. An understanding of such biochemical mechanisms at the molecular level might help in developing an ecologically friendly and cost‐effective protocol for microbial AgNPs synthesis.

Aeromonas culicicola sp. nov., from the midgut of Culex quinquefasciatus

The taxonomic position was examined of three isolates, MTCC 3249T, SH and SLH, from the midgut of female Culex quinquefasciatus and Aedes aegyptii mosquitoes. Numbers of cells of these isolates increased 2000-fold after a blood meal of the mosquitoes. 16S rRNA gene sequence analysis of the novel strains showed that they were highly homologous to strains of Aeromonas. DNA-DNA hybridization studies showed that DNA of strain MTCC 3249T was 96 and 88% similar to that of strains SH and SLH, respectively, and showed 54% relatedness to Aeromonas jandaei and 61% relatedness to Aeromonas sobria, which is below the cut-off value for species differentiation. The biochemical profiles of all three novel strains were identical. On the basis of a polyphasic approach using phenotypic analysis, 16S rRNA gene sequencing and DNA-DNA hybridization studies, a novel species is proposed for these isolates, Aeromonas culicicola sp. nov., with the type strain MTCC 3249T (= NCIM 5147T). Isolation of A. culicicola from the midgut of mosquitoes might help to explain the origin of Aeromonas infections caused without exposure to contaminated water, soil or food.

Phylogenetic characterization of bacteria in the gut of house flies (Musca domestica L.)

House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic insects that serve as mechanical or biological vectors for various microorganisms. To fully assess the role of house flies in the epidemiology of human diseases, it is essential to understand the diversity of microbiota harbored by natural fly populations. This study aimed to identify the diversity of house fly gut bacteria by both culture-dependent and culture-independent approaches. A total of 102 bacterial strains were isolated from the gut of 65 house flies collected from various public places including a garden, public park, garbage/dump area, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. Molecular phylogenetic analyses placed these isolates into 22 different genera. The majority of bacteria identified were known potential pathogens of the genera Klebsiella, Aeromonas, Shigella, Morganella, Providencia, and Staphylococcus. Culture-independent methods involved the construction of a 16S rRNA gene clone library, and sequence analyses supported culture recovery results. However, additional bacterial taxa not determined via culture recovery were revealed using this methodology and included members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phylum Bacteroidetes. Here, we show that the house fly gut is an environmental reservoir for a vast number of bacterial species, which may have impacts on vector potential and pathogen transmission.

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.

Decolorization and biodegradation of azo dye, reactive blue 59 by aerobic granules.

The present study deals with development of aerobic granules from textile wastewater sludge and challenged with different concentration of reactive blue 59 (RB59) to test their dye degradation potential. The granules efficiently degraded reactive blue 59 and also sustained higher dye loading of up to 5.0 g l(-1). The significant induction of enzymes azoreductase and cytochrome P-450 indicated their prominent role in the dye degradation while genotoxicity studies demonstrated that the biotransformed product of the dye as non-toxic. The microbial community of the textile dyes degrading aerobic sludge granules analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), revealed significantly diverse dye degrading microbial community belonging to alpha-, beta-, and gamma-proteobacteria.

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani.

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22322 unique peptides from a homology‐based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N‐terminally and one C‐terminally extended proteins based on the proteomic data search against the six‐frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.

Genus-Wide Physicochemical Evidence of Extracellular Crystalline Silver Nanoparticles Biosynthesis by Morganella spp

This study was performed to determine whether extracellular silver nanoparticles (AgNPs) production is a genus-wide phenotype associated with all the members of genus Morganella, or only Morganella morganii RP-42 isolate is able to synthesize extracellular Ag nanoparticles. To undertake this study, all the available Morganella isolates were exposed to Ag+ ions, and the obtained nanoproducts were thoroughly analyzed using physico-chemical characterization tools such as transmission electron microscopy (TEM), UV-visible spectrophotometry (UV-vis), and X-ray diffraction (XRD) analysis. It was identified that extracellular biosynthesis of crystalline silver nanoparticles is a unique biochemical character of all the members of genus Morganella, which was found independent of environmental changes. Significantly, the inability of other closely related members of the family Enterobacteriaceae towards AgNPs synthesis strongly suggests that AgNPs synthesis in the presence of Ag+ ions is a phenotypic character that is uniquely associated with genus Morganella.

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 micro-eukaryotes 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.

Phylogenetic Affiliation of Aeromonas culicicola MTCC 3249T Based on gyrB Gene Sequence and PCR-Amplicon Sequence Analysis of Cytolytic Enterotoxin Gene

We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.

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.