Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Diversity analysis of hilsa (Tenualosa ilisha) gut microbiota using culture-dependent and culture-independent approaches.

AIMS: The bacterial communities associated with the gastrointestinal (GI) tract are primarily involved in digestion, physiology, and the immune response against pathogenic bacteria for the overall development and health of the host. Hilsa shad (Tenualosa ilisha), a tropical anadromous fish, found predominantly in Bangladesh and India, has so far been poorly investigated for its gut bacterial communities. In this study, both culture-based and metagenomic approaches were used to detect intestinal isolates of hilsa, captured from both freshwater and seawater to investigate the community structure of intestinal microbiota. METHODS AND RESULTS: Culture-dependent approach allowed to isolate a total of 23 distinct bacterial species comprising 16 Gram-negative, and 7 Gram-positive isolates, where Proteobacteria and Firmicutes were identified as the two most dominant phyla. While metagenomic approach explored a wide range of important GI bacteria, primarily dominated by Proteobacteria, Firmicutes, and Bacteroidetes, with Proteobacteria and Firmicutes, being the most abundant in freshwater and seawater samples, respectively. CONCLUSIONS: A combination of these approaches provided the differential GI-associated bacterial diversity in freshwater and seawater hilsa with the prediction of overall functional potential. IMPACT STATEMENT: The study explored the diversity of gut microbiota in hilsa, one of the most preferred nutritious dietary fish, captured from freshwater and seawater habitats, which may encourage to comprehend the composition of the gut microbiome in relation to the migratory behavior and polyunsaturated fatty acid profile of anadromous fish in general.

Evaluation of distinct molecular architectures and coordinated regulation of the catabolic pathways of oestrogenic dioctyl phthalate isomers in Gordonia sp.

Bacterial strain GONU, belonging to the genus Gordonia, was isolated from a municipal waste-contaminated soil sample and was capable of utilizing an array of endocrine-disrupting phthalate diesters, including di-n-octyl phthalate (DnOP) and its isomer di(2-ethylhexyl) phthalate (DEHP), as the sole carbon and energy sources. The biochemical pathways of the degradation of DnOP and DEHP were evaluated in strain GONU by using a combination of various chromatographic, spectrometric and enzymatic analyses. Further, the upregulation of three different esterases (estG2, estG3 and estG5), a phthalic acid (PA)-metabolizing pht operon and a protocatechuic acid (PCA)-metabolizing pca operon were revealed based on de novo whole genome sequence information and substrate-induced protein profiling by LC-ESI-MS/MS analysis followed by differential gene expression by real-time PCR. Subsequently, functional characterization of the differentially upregulated esterases on the inducible hydrolytic metabolism of DnOP and DEHP revealed that EstG5 is involved in the hydrolysis of DnOP to PA, whereas EstG2 and EstG3 are involved in the metabolism of DEHP to PA. Finally, gene knockout experiments further validated the role of EstG2 and EstG5, and the present study deciphered the inducible regulation of the specific genes and operons in the assimilation of DOP isomers.

Biochemical and Multi-Omics Approaches To Obtain Molecular Insights into the Catabolism of the Plasticizer Benzyl Butyl Phthalate in Rhodococcus sp. Strain PAE-6.

Phthalate diesters are extensively used as plasticizers in manufacturing plastic materials; however, because of their estrogenic properties, these chemicals have emerged as a global threat to human health. The present study investigated the course of degradation of a widely used plasticizer, benzyl butyl phthalate (BBP), by the bacterium PAE-6, belonging to the genus Rhodococcus. The metabolism of BBP, possessing structurally dissimilar side chains, was evaluated biochemically using a combination of respirometric, chromatographic, enzymatic, and mass-spectrometric analyses, depicting pathways of degradation. Consequently, the biochemical observations were corroborated by identifying possible catabolic genes from whole-genome analysis, and the involvement of inducible specific esterases and other degradative enzymes was validated by transcriptomic, reverse transcription-quantitative PCR (RT-qPCR) and proteomic analyses. Nonetheless, phthalic acid (PA), an intermediate of BBP, could not be efficiently metabolized by strain PAE-6, although the genome contains a PA-degrading gene cluster. This deficiency of complete degradation of BBP by strain PAE-6 was effectively managed by using a coculture of strains PAE-6 and PAE-2. The latter was identified as a Paenarthrobacter strain which can efficiently utilize PA. Based on sequence analysis of the PA-degrading gene cluster in strain PAE-6, it appeared that the alpha subunit of the multicomponent phthalate 3,4-dioxygenase harbors a number of altered residues in the multiple sequence alignment of homologous subunits, which may play a role(s) in poor turnover of PA. IMPORTANCE Benzyl butyl phthalate (BBP), an estrogenic, high-molecular-weight phthalic acid diester, is an extensively used plasticizer throughout the world. Due to its structural rigidity and hydrophobic nature, BBP gets adsorbed on sediments and largely escapes the biotic and abiotic degradative processes of the ecosystem. In the present study, a potent BBP-degrading bacterial strain belonging to the genus Rhodococcus was isolated that can also assimilate a number of other phthalate diesters of environmental concern. Various biochemical and multi-omics analyses revealed that the strain harbors all the required catabolic machinery for the degradation of the plasticizer and elucidated the inducible regulation of the associated catabolic genes and gene clusters.

Molecular evaluation of the metabolism of estrogenic di(2-ethylhexyl) phthalate in Mycolicibacterium sp.

BACKGROUND: Di(2-ethylhexyl) phthalate (DEHP) is a widely detected plasticizer and a priority pollutant of utmost concern for its adverse impact on humans, wildlife and the environment. To eliminate such toxic burden, biological processes are the most promising ways to combat rampant environmental insults under eco-friendly conditions. The present study investigated the biochemical and molecular assessment of the catabolic potential of Mycolicibacterium sp. strain MBM in the assimilation of estrogenic DEHP. RESULTS: A detailed biochemical study revealed an initial hydrolytic pathway of degradation for DEHP followed by the assimilation of hydrolyzed phthalic acid and 2-ethylhexanol to TCA cycle intermediates. Besides the inducible nature of DEHP-catabolic enzymes, strain MBM can efficiently utilize various low- and high-molecular-weight phthalate diesters and can grow under moderately halotolerant conditions. Whole genome sequence analysis exhibited a genome size of 6.2 Mb with a GC content of 66.51% containing 6,878 coding sequences, including multiple genes, annotated as relevant to the catabolism of phthalic acid esters (PAEs). Substantiating the annotated genes through transcriptome assessment followed by RT-qPCR analysis, the possible roles of upregulated genes/gene clusters in the metabolism of DEHP were revealed, reinforcing the biochemical pathway of degradation at the molecular level. CONCLUSIONS: A detailed co-relation of biochemical, genomic, transcriptomic and RT-qPCR analyses highlights the PAE-degrading catabolic machineries in strain MBM. Further, due to functional attributes in the salinity range of both freshwater and seawater, strain MBM may find use as a suitable candidate in the bioremediation of PAEs.

Characterization of an antimicrobial pentapeptide produced by a drug-sensitive Pseudomonas aeruginosa strain PAST18.

The members of the genus Pseudomonas can secrete a wide range of ribosomally encoded antagonistic peptides and proteins, ranging from small microcins to large tailocins. In this study, a drug-sensitive Pseudomonas aeruginosa strain isolated from a high-altitude virgin soil sample showed a broad range of antibacterial activity against Gram-positive and Gram-negative bacteria. The antimicrobial compound, purified by affinity chromatography, ultrafiltration, and high-performance liquid chromatography, showed a molecular weight (M + H)+ of 494.7667 daltons, as revealed by ESI-MS analysis. The MS-MS analysis divulged the compound as an antimicrobial pentapeptide with the sequence NH2-Thr-Leu-Ser-Ala-Cys-COOH (TLSAC) and was further verified by evaluating the antimicrobial activity of the chemically synthesized pentapeptide. The extracellularly released pentapeptide, which is relatively hydrophobic in nature, is encoded in a symporter protein, as appraised from the whole genome sequence analysis of strain PAST18. The influence of different environmental factors was examined to determine the stability of the antimicrobial peptide (AMP), which was also assessed for several other biological functions, including antibiofilm activity. Further, the antibacterial mechanism of the AMP was evaluated by a permeability assay. Overall, the characterised pentapeptide, as revealed in this study, may find use as a potential biocontrol agent in various commercial applications.

A comprehensive assessment of the antimicrobial and immunomodulatory effects of frequently consumed fermented foods: insights in the management of COVID-19.

COVID-19, which is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), is the deadliest outbreak of this millennium. Despite adopting several precautionary strategies and guidelines, COVID-19 has spread rapidly, and the number of cases is still in escalation across the world. The various immune-boosting drugs with severe side effects and the vaccines approved after negotiated clinical trials have been struggling to cope with the emergence of new variants of the virus. Nevertheless, given a large number of asymptomatic cases, a high magnitude of recovery rate, and a relatively higher prevalence of morbidity and mortality among immunologically compromised individuals, those affected by an illness, and the elderly, it appears that a healthy microbiome and the associated immune responses are the key factors for survival. Incidentally, the consumption of traditionally popular and nutritious fermented foods, which are composed of biologically functional ingredients and several health-promoting probiotics, offers promising health benefits through the improvement of the immune system in general. Given the progress in functional food research, it has become crucial to understand the impact of a healthy microbiome and the immunomodulatory roles of fermented foods on the battles to combat infectious diseases. Based on the evidence of the impact of probiotics-based fermented foods, the beneficial roles of a few frequently consumed fermented foods in the management of various infections have been resolutely discussed in the present study, with a focus on their antagonistic and immune-modulating effects in the context of the current COVID-19 pandemic.

A systematic approach to understand hydrogeochemical dynamics in large river systems: Development and application to the River Ganges (Ganga) in India.

Large river systems, such as the River Ganges (Ganga), provide crucial water resources for the environment and society, yet often face significant challenges associated with cumulative impacts arising from upstream environmental and anthropogenic influences. Understanding the complex dynamics of such systems remains a major challenge, especially given accelerating environmental stressors including climate change and urbanization, and due to limitations in data and process understanding across scales. An integrated approach is required which robustly enables the hydrogeochemical dynamics and underpinning processes impacting water quality in large river systems to be explored. Here we develop a systematic approach for improving the understanding of hydrogeochemical dynamics and processes in large river systems, and apply this to a longitudinal survey (> 2500 km) of the River Ganges (Ganga) and key tributaries in the Indo-Gangetic basin. This framework enables us to succinctly interpret downstream water quality trends in response to the underpinning processes controlling major element hydrogeochemistry across the basin, based on conceptual water source signatures and dynamics. Informed by a 2019 post-monsoonal survey of 81 river bank-side sampling locations, the spatial distribution of a suite of selected physico-chemical and inorganic parameters, combined with segmented linear regression, reveals minor and major downstream hydrogeochemical transitions. We use this information to identify five major hydrogeochemical zones, characterized, in part, by the inputs of key tributaries, urban and agricultural areas, and estuarine inputs near the Bay of Bengal. Dominant trends are further explored by investigating geochemical relationships (e.g. Na:Cl, Ca:Na, Mg:Na, Sr:Ca and NO3:Cl), and how water source signatures and dynamics are modified by key processes, to assess the relative importance of controls such as dilution, evaporation, water-rock interactions (including carbonate and silicate weathering) and anthropogenic inputs. Mixing/dilution between sources and water-rock interactions explain most regional trends in major ion chemistry, although localized controls plausibly linked to anthropogenic activities are also evident in some locations. Temporal and spatial representativeness of river bank-side sampling are considered by supplementary sampling across the river at selected locations and via comparison to historical records. Limitations of such large-scale longitudinal sampling programs are discussed, as well as approaches to address some of these inherent challenges. This approach brings new, systematic insight into the basin-wide controls on the dominant geochemistry of the River Ganga, and provides a framework for characterising dominant hydrogeochemical zones, processes and controls, with utility to be transferable to other large river systems.

Phthalate hydrolase: distribution, diversity and molecular evolution.

The alpha/beta-fold superfamily of hydrolases is rapidly becoming one of the largest groups of structurally related enzymes with diverse catalytic functions. In this superfamily of enzymes, esterase deserves special attention because of their wide distribution in biological systems and importance towards environmental and industrial applications. Among various esterases, phthalate hydrolases are the key alpha/beta enzymes involved in the metabolism of structurally diverse estrogenic phthalic acid esters, ubiquitously distributed synthetic chemicals, used as plasticizer in plastic manufacturing processes. Although they vary both at the sequence and functional levels, these hydrolases use a similar acid-base-nucleophile catalytic mechanism to catalyse reactions on structurally different substrates. The current review attempts to present insights on phthalate hydrolases, describing their sources, structural diversities, phylogenetic affiliations and catalytically different types or classes of enzymes, categorized as diesterase, monoesterase and diesterase-monoesterase, capable of hydrolysing phthalate diester, phthalate monoester and both respectively. Furthermore, available information on in silico analyses and site-directed mutagenesis studies revealing structure-function integrity and altered enzyme kinetics have been highlighted along with the possible scenario of their evolution at the molecular level.

Draft Whole-Genome Sequence of Sphingobium sp. Strain PNB, a Versatile Polycyclic Aromatic Hydrocarbon-Degrading Bacterium.

Sphingobium sp. strain PNB can completely degrade phenanthrene, naphthalene, and biphenyl as the sole carbon and energy source. The strain is also capable of cometabolizing benzo[a]pyrene, pyrene, acenaphthene, fluoranthene, etc. Here, we report the 5.69-Mb assembly and annotation of the genome sequence of strain PNB, obtained using Illumina sequencing.

Multidrug-Resistant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Pathotypes in North Eastern Region of India: Backyard Small Ruminants-Human-Water Interface.

A total of 648 diarrheagenic Escherichia coli (DEC) were isolated from calves (n = 219), lambs (n = 87), kids (n = 103), human (n = 193), and water (n = 46) samples. The presence of enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and shigatoxigenic E. coli (STEC) was confirmed by PCR-based detection of the Shiga toxin, intimin, hemolysin, and enterotoxin genes. All the isolates were tested for antimicrobial resistance (AMR) by disc diffusion assay. Extended-spectrum ß-lactamase (ESBL), carbapenemase, and metallo-beta-lactamase production were determined by double-disk synergy test, modified Hodge test, and combined disk test assays. AMR genes (blaTEM, blaSHV, blaCTX-M, blaCMY-2, blaNDM, blaKPC, blaVIM, and blaIMP) were detected by PCR using specific primers. Majority of the isolates from human and water exhibited resistance (>80%) against amoxicillin, ampicillin, aztreonam, cefotaxime, cefixime, gentamicin, ceftazidime, and cefalexin, and against imipenem (70.98%), doripenem (70.47%), and ertapenem (60.62%). Bovine isolates were sensitive to carbapenems. Many isolates (5.75-24.35%) from human, water, calves, kids, and lambs were multidrug resistant (MDR), with resistance against three or more classes of antimicrobials. A total of 170/648 (26.23%) isolates were classified as STEC (9.88%), EPEC (4.32%), and ETEC (12.04%). The AMR genes, including blaTEM, blaCMY2, blaCTX-M, and blaSHV were detected in the E. coli from all sources. but blaNDM and blaKPC were detected only in the isolates from human and water. Three STEC isolates from human origin possessed multiple ESBLs, carbapenemase and metallo-beta-lactamase genes reported for the first time. ESBLs producing EPEC and ETEC in lambs and kids are also reported under this study. Presence of MDR-DEC in domestic animals and common potable water poses public health concern in this region.

Evaluation of Phytochemicals and Bioactive Properties in Mangrove Associate Suaeda monoica Forssk. ex J.F.Gmel. of Indian Sundarbans.

Suaeda monoica Forssk. ex J.F.Gmel. (Amaranthaceae), a mangrove associate and ethno-medicinal herb of Indian Sundarbans, was investigated as a promising source of bioactive compounds. Various polar and nonpolar solvent extracts of the leaf and root-shoot parts of the plant exhibited antioxidant, antibacterial, antifungal, allelopathic, mosquitocidal, antihaemolytic and antidiuretic potential. Moreover, to meet pharmacological requirements, the antioxidant ability of the plant was validated by both chemical and biological analyses. Extraction yield and presence of different phytochemicals like phenolics, flavonoids, tannins and saponins were compared in various solvent-extracted fractions. Principle component analysis revealed that the antioxidant property present in different extracts maintained a positive correlation with the occurrence of polyphenols (phenolics, tannins and flavonoids). Biochemical evaluation, HPLC examination and GC-MS analysis showed a differential level of the presence of various phytochemicals in different solvent extracts. In contrast to mosquitocidal, antioxidant, antihaemolytic and phytotoxic properties which were observed to be dominant in polar solvent extracts, maximum antibacterial potency was detected in nonpolar n-hexane fractions. Overall, the plant extract is nontoxic in nature and a dose amounting to 3,000 mg/kg was well tolerated by Swiss albino mice. A combination of HPLC and GC-MS analyses showed the presence of a large number of structurally diverse phytochemicals, many of which had already been reported as insecticidal, mosquitocidal, antibacterial, herbicidal, antidiuretic, antioxidant and anti-haemolytic compounds. All these findings support that the least explored traditional edible medicinal mangrove associate S.monoica is enriched with multiple bioactive molecules and may be considered as one of the richest sources of various lead molecules of pharmaceutical importance.

Characterization of Multidrug-Resistant Biofilm-Producing Escherichia coli and Klebsiella pneumoniae in Healthy Cattle and Cattle with Diarrhea.

This study describes comparative occurrence and characterization of multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae (KP) in healthy cattle (HC) and cattle with diarrhea (DC) in India. During 2018-2020, 72 MDR isolates, including 35 E. coli (DC: 27; HC 8) and 37 K. pneumoniae (DC: 34; HC: 3), from 251 rectal swabs (DC: 219; HC: 32) were investigated for extended-spectrum beta-lactamase (ESBL), AmpC type ß-lactamase and carbapenemase production, antimicrobial susceptibility profile, biofilm production, and efflux pump activity. Fifty-five MDR isolates were ESBL producers (ESBLPs) (DC: 50; HC: 5) and ESBLPs from DC were coresistant to multiple antibiotics. The blaCTX-M gene (50) was the most frequently detected ß-lactamases followed by blaAmpC (22), blaTEM1 (13), blaCMY-6 (6), blaOXA1 (5), blaPER (2), blaDHA, and blaFOX and blaSHV12 (1 each). Plasmid-mediated quinolone resistance determinants qnrB, qnrS, qnrA, and qepA were detected in 18, 16, 2, and 3 isolates, respectively. Twenty three isolates revealed mutation in gyrA and parC genes. Tetracycline-resistance markers tetA, tetB, tetC, and tetE were detected in 33, 10, 3, and 2 isolates, respectively. Only one of the 41 imipenem-resistant isolates harbored blaNDM-5 and two were colistin-resistant. Altogether, 20 MDR isolates were strong biofilm producers and 19 harbored different virulence factors. This is the first ever report from India on the presence of MDR Enterobacteriaceae with resistance to even last-resort antimicrobials in the bovine diarrhea.

Companion Animals Emerged as an Important Reservoir of Carbapenem-Resistant Enterobacteriaceae: A Report from India.

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) are perceived as a serious public-health threat world-wide. Despite sporadic reports, no systemic study has been carried out on CRE in companion animals in Indian subcontinent. In total, 237 canine specimens collected from five veterinary polyclinics in and around Kolkata were analyzed for isolation, antimicrobial resistance profiling and molecular characterization of carbapenem-resistant (CR) E. coli. Of the 29 CR isolates, 19 were identified as metallo-ß-lactamase producers (MP-CRE) and 10 as metallo-ß-lactamase non-producers (MNP-CRE). Eleven of them were extended spectrum ß-lactamase and/or AmpC type ß-lactamase producers and harboured fluoroquinolone-, tetracycline-, sulfonamide- and aminoglycoside-resistant genes. Beside uropathogenic virulence determinants, they carried the adhesion factors mediating biofilm production which was remarkably higher in 6 MP-CRE and one MNP-CRE isolates. Although the CRE were of diverse origin including the healthy and the diseased dogs, these were more frequently isolated from canine pyometra. The MP-CRE harboured plasmids of IncF and IncA/C types. Phylo-type B1 was observed in 38% of the CR isolates, followed by A0 in 31% and rest were attributed to A1 and D1. The Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) revealed that these isolates were genetically diverse and constituted of a heterogenous population. Detection of CRE in pet dogs despite the fact that carbapenems are not used in animals in India emphasizes the need for active surveillance to identify the transmission and dynamics of such pathogens in companion animals.

Elucidating the network features and evolutionary attributes of intra- and interspecific protein-protein interactions between human and pathogenic bacteria.

Host-pathogen interaction is one of the most powerful determinants involved in coevolutionary processes covering a broad range of biological phenomena at molecular, cellular, organismal and/or population level. The present study explored host-pathogen interaction from the perspective of human-bacteria protein-protein interaction based on large-scale interspecific and intraspecific interactome data for human and three pathogenic bacterial species, Bacillus anthracis, Francisella tularensis and Yersinia pestis. The network features revealed a preferential enrichment of intraspecific hubs and bottlenecks for both human and bacterial pathogens in the interspecific human-bacteria interaction. Analyses unveiled that these bacterial pathogens interact mostly with human party-hubs that may enable them to affect desired functional modules, leading to pathogenesis. Structural features of pathogen-interacting human proteins indicated an abundance of protein domains, providing opportunities for interspecific domain-domain interactions. Moreover, these interactions do not always occur with high-affinity, as we observed that bacteria-interacting human proteins are rich in protein-disorder content, which correlates positively with the number of interacting pathogen proteins, facilitating low-affinity interspecific interactions. Furthermore, functional analyses of pathogen-interacting human proteins revealed an enrichment in regulation of processes like metabolism, immune system, cellular localization and transport apart from divulging functional competence to bind enzyme/protein, nucleic acids and cell adhesion molecules, necessary for host-microbial cross-talk.

Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates.

BACKGROUND: Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries. RESULTS: Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C ß-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C ß-lactamases but did not exhibit ß-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases. CONCLUSIONS: This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing ß-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Coinfection of diarrheagenic bacterial and viral pathogens in piglets of Northeast region of India.

AIM: This study aimed to study the prevalence of the coinfection of enteric bacterial and viral pathogens, namely Escherichia coli, Salmonella, Rotavirus, and Picobirnavirus from fecal samples of pre-weaned piglets in Northeast region of India. MATERIALS AND METHODS: A total of 457 fresh fecal samples were collected from piglets under 9 weeks old during 2013-2015 from organized (n=225) and unorganized (n=232) farms of Manipur, Meghalaya, Mizoram, and Nagaland. Samples were collected from diarrheic (n =339) and non-diarrheic (n=118) piglets including local indigenous (n=130) and crossbreed (n=327) piglets in different seasons during the study period. The samples were processed for the isolation of E. coli and Salmonella and detection of their putative virulence genes by polymerase chain reaction (PCR). Samples were also processed for the detection of Rotavirus and Picobirnavirus by RNA-polyacrylamide agarose gel electrophoresis and reverse transcriptase-PCR (RT-PCR). RESULTS: A total of 11 (2.40%) samples were found positive for two or more coinfecting enteric bacterial and viral pathogens. All the 11 positive fecal samples were recovered from diarrheic piglets. Salmonella Typhimurium (enterotoxin, stn gene) and Picobirnavirus genogroup 1 were found to be more frequent as coinfecting agents. Coinfection was recorded higher in unorganized (3.87%) compared to organized farm (0.88%). Again, higher detection was recorded in crossbreed (2.75%) than local indigenous piglets (1.53%). The occurrence of coinfection was found to be more common during summer (4.68%) followed by winter (2.27%) season. CONCLUSION: The present study highlighted the significance of E. coli, Salmonella, Rotavirus, and Picobirnavirus as important diarrheagenic pathogens causing coinfection in piglets in Northeast region of India. Probably, this is the first systematic study of the coinfection of four important diarrheagenic bacterial and viral agents associated with piglet diarrhea in India.

Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster.

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and para-isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.

Genomic Identity of Fluoroquinolone-Resistant blaCTX-M-15-Type ESBL and pMAmpC ß-Lactamase Producing Klebsiella pneumoniae from Buffalo Milk, India.

We investigated the occurrence of extended-spectrum ß-lactamase (ESBL) and AmpC-type ß-lactamase (ACBL) producing quinolone-resistant Klebsiella pneumoniae (KP) in milk samples of apparently healthy buffaloes (n = 348) and buffaloes (n = 19) with evidence of subclinical mastitis from seven districts of West Bengal, India. In total, 12 ESBL producing KP were isolated with blaCTX-M-15 gene and 7 of them were ACBL producers, as well. The blaCTX-M-15 genes were carried by transposable element ISEcp1. The plasmid-mediated quinolone resistance genes-qnrS, qnrA, qnrB, qepA, and aac(6')-Ib-cr were detected in five, one, three, four, and one isolate (s), respectively. In addition, eight isolates carried mutation in gyrase (gyrA) and six in topoisomerase IV (parC). Resistance markers/genes for sulfonamide (sul1), tetracycline [tet(A) and tet(B)], and aminoglycoside (aacC2) were also detected in eight, four, and one isolate(s), respectively. The class I integrons identified in five isolates carried aad2/aad5 and dfrA12/dfrA17 gene cassettes. The enterobacterial repetitive intergenic consensus-PCR revealed that all the isolates were genetically diverse and comprised a heterogeneous population. Isolation of multidrug-resistant KP, a typical nosocomial pathogen from buffalo milk, reiterates the need to monitor farm animals for ESBL producing Enterobacteriaceae and emphasizes on judicious use of antibiotics in animal husbandry sector.

Characterization of a topologically unique oxygenase from Sphingobium sp. PNB capable of catalyzing a broad spectrum of aromatics.

A Rieske non-heme iron ring-hydroxylating oxygenase (RHO) from Sphingobium sp. PNB involved in the initial oxidation of a wide range of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs) was investigated. The RHO was shown to comprise of the gene products of distantly located ahdA1f-ahdA2f, ahdA3 and ahdA4 genes, which encoded the oxygenase α- and ß-subunits, ferredoxin and reductase, respectively. In silico structural analysis of AhdA1f revealed a very large substrate-binding pocket, satisfying the spatial requirements to accommodate high molecular weight substrates. In addition, an atypical substrate access channel was noticed from the topology analysis of the oxygenase. Guided by molecular docking studies, dioxygenation of several PAHs as well as alkyl- and aryl benzenes was examined with the recombinant AhdA1fA2f expressed in Escherichia coli. Chromatographic and mass spectrometric analyses confirmed that AhdA1fA2f displays broad substrate specificity towards a wide range of aromatic hydrocarbons including potential xenobiotics, demonstrating metabolic robustness of strain PNB.