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Mycobacterium tuberculosis is the etiological agent for tuberculosis in humans. The studies related to survival of this pathogen in the human host and development of drugs against reveal that the organism uses a complex physiology to adapt to the host environment. Many studies were targeted to key enzymes that allow this pathogen to either survive or remain latent within the host. Most of the models, which address the survival of pathogen, have evaluated limited dissolved oxygen and prevailing stress conditions. Hence, the truncated citric acid cycle, with the glyoxylate shunt was suggested as an option for survival of the pathogen and pathogenesis. We propose that the precursors to support this pathway could also be generated via enzymatic conversion involving poly-beta-hydroxybutyrate (PHB). We have used available genome sequence data and analyzed for the possible enzymatic conversions that can generate glyoxylate, acetyl CoA, and other enolases that can also be useful for various fatty acid transformations. The enzymes for accumulation and further hydrolysis of PHB were examined in sequence data analysis. The target enzymes were searched for in the genome using identified conserved domains. Using M. tuberculosis H37Rv as a model bacterium a supportive pathway has been envisaged and integrated with glyoxylate cycle to provide a complete option to pathogen for sustainable consumption of available carbon source(s). The study proposes that the enzymes of PHB synthesis and hydrolysis are possible targets for drug design, and that this should be considered when evaluating isocitrate lyase and malate synthase as targets.

In view of the realization that fossil fuels reserves are limited, various options of generating energy are being explored. Biological methods for producing fuels such as ethanol, diesel, hydrogen (H2), methane, etc. have the potential to provide a sustainable energy system for the society. Biological H2 production appears to be the most promising as it is non-polluting and can be produced from water and biological wastes. The major limiting factors are low yields, lack of industrially robust organisms, and high cost of feed. Actually, H2 yields are lower than theoretically possible yields of 4 mol/mol of glucose because of the associated fermentation products such as lactic acid, propionic acid and ethanol. The efficiency of energy production can be improved by screening microbial diversity and easily fermentable feed materials. Biowastes can serve as feed for H2 production through a set of microbial consortia: (1) hydrolytic bacteria, (2) H2 producers (dark fermentative and photosynthetic). The efficiency of the bioconversion process may be enhanced further by the production of value added chemicals such as polydroxyalkanoate and anaerobic digestion. Discovery of enormous microbial diversity and sequencing of a wide range of organisms may enable us to realize genetic variability, identify organisms with natural ability to acquire and transmit genes. Such organisms can be exploited through genome shuffling for transgenic expression and efficient generation of clean fuel and other diverse biotechnological applications.

Diagnosing tuberculous meningitis (TBM) remains problematic despite many new advanced diagnostic methods. Adenosine deaminase (ADA) assays have emerged as novel alternatives to other costly and time-consuming methods for TBM diagnosis. In the present study the usefulness of the ADA method was assessed for the diagnosis of TBM and compared with an in-house developed ELISA method for detecting the antigen 85 (Ag 85) complex of M. tuberculosis in cerebrospinal fluid (CSF) samples of suspected and culture-confirmed TBM patients.ADA activity in CSF was determined at 37 degrees C according to the method of Guisti and Galanti. ELISA, employing monoclonal antibodies against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in CSF from TBM patients. CSF samples were obtained from 153 patients in three different groups: confirmed TBM (n=27), clinically suspected TBM (n=39), and non-TBM (n=87).The ADA method yielded sensitivity and specificity of 83% and 86%, which are similar to those of the ELISA method (89% and 90%). The correlation between the Ag 85 complex activity in absorbance by ELISA and the ADA activity obtained in units per liter per minute (U/l/min) was also positive and significant. (Pearson's correlation coefficient r=0.3234, 95%CI: 0.1621-0.4679, p0.001).This study suggests that the ADA method can be performed for TBM diagnosis in low-income TB-affected regions where more sophisticated facilities are generally not available.

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H(2)) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H(2) constituted 63-69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34-38 mm) -Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56-62 mm) -Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) -B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H(2) producing abilities in the range of 0.26-0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) -B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H(2) - 0.63 mol/mol of glucose added and PHB - 420-435 mg/l medium.

Background Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients. Methods Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test. Results The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69). Conclusion The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.

Eight bacterial isolates closely related to Diaphorobacter sp. were isolated from activated biomass surviving on wastewater laden with dyes and nitro-substituted chemicals and were identified by 16S rDNA sequence analysis. The isolates showed sequence similarity of 99-100% to other Diaphorobacter strains such as ZY 2006b, F2, NA5, PCA039, D. nitroreducens KSP4, and KSP3 and 98-99% sequence homology to D. nitroreducens NA10B (type strain JCM 11421). Neighbor-joining tree revealed that all the eight strains formed tight cluster together and also showed close clustering with other Diaphorobacter strains. Isolates demonstrated the ability to perform simultaneous nitrification and denitrification under aerobic conditions. Strains HPC 805, 815, 821, and 856 gave highest chemical oxygen demand removal (85-93%) and ammonia removal (92-96%), which correlated well with higher growth rates of the cultures. Simultaneously, complete removal of nitrate supplied in the medium in presence of ammonium and acetate (electron donor) was observed in addition to aerobic nitrite release from ammonium. Thus, the above strains showed ability to perform partial nitrification followed by further aerobic removal of common intermediate nitrite, which indicated their potential application in treatment systems for treatment of high-nitrogen-containing wastewaters.

A common effluent treatment plant (CETP) is a biological wastewater treatment facility that receives wastewater from different industries. The activated biomass in the CETP survives on a wide range of chemicals with no fixed wastewater characteristics. We carried out a diversity analysis of this activated biomass using culture as well as culture-independent techniques. Using culture-based techniques, strains belonging to 26 different genera from the phyla Proteobacteria, Actinobacteria and Firmicutes were isolated. The gamma-proteobacteria was the best represented class, with 36.5% of the isolates. Bacterial diversity was also analyzed culture-independently by means of sequence determination of cloned 16S rRNA genes. Twenty-one different genera from the phyla Proteobacteria, Firmicutes, Planctomycetes and Bacteroidetes were identified. The total diversity of the activated biomass was composed of members of five known phyla, represented by 37 genera, with the Proteobacteria constituting the most abundant phylum detected. However, a very large fraction of the diversity represented a hitherto unidentified bacterial population. More than half (50.2%) of the 16S rDNA clones represented unidentified non-culturable bacteria, underlining the vast untapped diversity of CETP communities. Our results also indicate that both culture-based and culture-independent techniques should be combined to cover the microbial diversity of complex ecosystems.

Retroviral integration has recently been shown to be nonrandom, favoring transcriptionally active regions of chromatin. However, the mechanism for integration site selection by retroviruses is not clear. We show here the occurrence of Alu -like motifs in the sequences flanking the reported viral integration sites that are significantly different from those obtained from the randomly picked sequences from the human genome, suggesting that unique primary sequence features exist in the genomic regions targeted by human immunodeficiency virus type 1 (HIV-1). Additionally, these sequences were preferentially bound by SATB1, the T lineage-restricted chromatin organizer, in vitro and in vivo. Alu repeats make up nearly 10% of the human genome and have been implicated in the regulation of transcription. To specifically isolate sequences flanking the viral integration sites and also harboring both Alu -like repeats and SATB1-binding sites, we combined chromatin immunoprecipitation with sequential PCRs. The cloned sequences flanking HIV-1 integration sites were specifically immunoprecipitated and amplified from the pool of anti-SATB1-immunoprecipitated genomic DNA fragments isolated from HIV-1 NL4.3-infected Jurkat T-cell chromatin. Moreover, many of these sequences were preferentially partitioned in the DNA associated tightly with the nuclear matrix and not in the chromatin loops. Strikingly, many of these regions were disfavored for integration when SATB1 was silenced, providing unequivocal evidence for its role in HIV-1 integration site selection. We propose that definitive sequence features such as the Alu -like motifs and SATB1-binding sites provide a unique chromatin context in vivo which is preferentially targeted by the HIV-1 integration machinery.

It has been observed that hydrocarbon treated wastewaters still contain high COD and a number of intermediates. This suggests that the required catabolic gene pool for further degradation might be absent in the system or, that its titer value is not significant enough. By providing the desired catabolic potential, the overall efficiency of the treatment system can be improved. This study aims to demonstrate this concept by bioaugmentation of a lab-scale reactor treating refinery wastewater with a consortium having the capacity to complement the alkB genotype to the available microbial population.Two reactors were set up using activated biomass collected from a refinery treatment plant and operated at a continuous mode for a period of 8 weeks. The feed to both reactors was kept constant. Crude oil was spiked regularly. One reactor was bioaugmented with a consortium previously described for crude oil spill remediation. The efficiency of the bioaugmented reactor was demonstrated by reduced COD. The changes in the microbial population over a period of time were analyzed by RAPD. Catabolic activity of the biomass in both reactors was monitored by PCR. The presence of the catabolic loci was confirmed by Southern Hybridization.52.2% removal of COD was observed in the bioaugmented reactor while only 15.1% reduction of COD was observed in the reactor without bioaugmentation. The change in microbial population can be seen from the 4th week, which also corresponds to improved catabolic activity. The presence of the bedA locus was seen in all samples, which indicates the presence of aromatic degraders, but the appearance of the alkB locus, from the 6th week onwards, which was observed only in the samples from the bioaugmented reactor. The results suggest that the gene pool of the bioaugmented reactor has catabolic loci that can degrade accumulated intermediates, thus improving the efficiency of the system.In this study, improvement of efficiency of bioremediation was demonstrated by addition of catabolic loci that are responsible for degradation. Bioaugmentation was carried out in biomass that was collected from an ETP (effluent treatment plant) treating hydrocarbon containing wastewater to study the strategies for improvement of the treatment system. Biostimulation, only marginally improved the efficiency, when compared to bioaugmentation. The improved efficiency was demonstrated by COD removal. The presence of the alkB locus suggests the importance of a catabolic gene pool that acts on accumulated intermediates. It is well documented that straight chain aliphatics and intermediates of aromatic compounds after ring cleavage, accumulate in refinery wastewater systems, thereby hindering further degradation of the wastewater. Supplementation of a catabolic gene pool that treats the lower pathway compounds and alkanes will improve the overall efficiency. In this study, results suggest that the alkB locus can also be used to monitor the degradative mode of the activated biomass.Pollution from petroleum and petroleum products around the globe are known to have grave consequences on the environment. Bioremediation, using activated sludge, is one option for the treatment of such wastes. Effluent treatment plants are usually unable to completely degrade the wastewater being treated in the biological unit (the aerator chambers). The efficiency of degradation can be improved by biostimulation and bioaugmentation. This study demonstrates the improved efficiency of a treatment system for wastewater containing hydrocarbons by bioaugmentation of a consortium that supports degradation. Further experiments on a pilot scale are recommended to assess the use of bioaugmentation on a large scale. The use of molecular tools, like DNA probes for alkB, to monitor the system also needs to be explored.

The diversity of Citrobacter isolated from effluent treatment plants (ETPs) was studied using three different parameters. Thirty Citrobacter strains were isolated from different ETPs treating wastewaters generated at various industries. All the isolates were characterized based on biochemical tests, antibiotic assay/functional analysis, and phylogenetic analysis. Results demonstrated that the pattern of grouping varied based on the selected criteria for analysis. Species that clustered together by biochemical analysis were found to vary by functional and 16S rDNA analysis and vice versa. This suggests that multiple methods approach needs to be carried out to understand the microbial diversity. Bacteria in effluent treatment plants are exposed to diverse categories of pollutants. Salicylate is a key intermediate formed during biodegradation of several aromatic compounds, a scenario expected in ETPs. Hence, the Citrobacter isolates were screened for their capability to utilize salicylate. In future studies, these isolates can be incorporated in a bioremediation program.

Background Thrombolytic drugs are widely used for the management of cerebral venous sinus thrombosis patients. Several in vitro models have been developed to study clot lytic activity of thrombolytic drugs, but all of these have certain limitations. There is need of an appropriate model to check the clot lytic efficacy of thrombolytic drugs. In the present study, an attempt has been made to design and develop a new model system to study clot lysis in a simplified and easy way using a thrombolytic drug, streptokinase. Methods Whole blood from healthy individuals (n = 20) was allowed to form clots in a pre-weighed sterile microcentrifuge tubes; serum was removed and clot was weighed. After lysis by streptokinase fluid was removed and remnants of clot were again weighed along with the tube. Percentage of Clot lysis was calculated on the basis of the weight difference of microcentrifuge tubes obtained before and after clot lysis. Results There was a significant percentage of clot lysis observed when streptokinase was used. On the other hand with water (negative control), minimal (2.5%) clot lysis was observed. There was a significant difference between clot lysis done by streptokinase and water. Conclusion Our study could be a rapid and effective methodology to study clot-lytic effect of newly developed drugs as well as known drugs.

Background Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM. Methods A total of 160 CSF samples of different groups of patients (confirmed TBM {n = 18}, clinically suspected TBM {n = 62}, non TBM infectious meningitis {n = 35} and non-infectious neurological diseases {n = 45}) were analyzed by indirect ELISA method using mAb to 65 kD hsp antigen. The Kruskal Wallis test (Non-Parametric ANOVA) with the Dunnett post test was used for statistical analysis. Results The indirect ELISA method yielded 84% sensitivity and 90% specificity for the diagnosis of TBM using mAb to 65 kD hsp antigen. The mean absorbance value of 65 kD hsp antigen in TBM patients was [0.70 ± 0.23 (0.23–1.29)], significantly higher than the non-TBM infectious meningitis group [0.32 ± 0.14 (0.12–0.78), P < 0.001] and also higher than the non-infectious neurological disorders group [0.32 ± 0.13 (0.20–0.78), P < 0.001]. A significant difference in the mean absorbance of 65 kD hsp antigen was noted in the CSF of culture-positive TBM patients [0.94 ± 0.18 (0.54–1.29)] when compared with clinically suspected TBM patients [0.64 ± 0.20 (0.23–0.98), P < 0.05]. Conclusion The presence of 65 kD hsp antigen in the CSF of confirmed and suspected cases of TBM would indicate that the selected protein is specific to M. tuberculosis and could be considered as a diagnostic marker for TBM.

This study targeted the diversity of bacteria isolated from activated biomass of an ETP that treated wastewater from the dye industry. Phylogenetic analysis of 5′-partial 16S rDNA sequences revealed that the Proteobacteria (α, β, and γ) and Firmicutes were the major phyla in microbial community. Five unidentified culturable bacteria were also isolated. The activated biomass changed when acclimatized to wastewater with nitroaromatic residues. It was observed that the diversity of genera was reduced, but the degradative capacity of sludge was enhanced.

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.

Background Tuberculous meningitis (TBM) is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB). The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA) activity in cerebrospinal fluid (CSF) of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94), mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96) and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68), P < 0.0001 for both comparisons. A cut-off value of 11.39 U/L/min for the TBM patients was calculated from the mean + SD of the non-TBM patients. The ADA test gave a sensitivity of 82% and a specificity of 83% for infectious TBM when this cut-off value was used. Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.

Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% ( n = 5) of confirmed and 90% ( n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

We isolated a Staphylococcus aureus strain HPC-250 producing antibacterial agent against Paenibacillus strain HPC-251. Both strains were isolated from the same environmental niche. The bacteria were identified using the partial sequencing of their TA-cloned 16S rDNA. Spectrum of the antibacterial agent was also tested against routine observed bacteria with drinking water contamination such as Escherichia coli, Salmonella, Pseudomonas, and Vibrio and these were found to be sensitive. Bacteria like Acinetobacter and Burkholderia were found to be resistant. The differential antibacterial activity of the HPC-250 was observed for the genus Bacillus where B. subtilis remained resistant although B. sphaericus was sensitive.

Background Tuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM. Methods We developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients. Results The cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM. Conclusion When induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond.

This study aims at development of an approach for selection of strain, which has capability for oxidation of broad-range of chloro-substitute phenols. A multiplex PCR was optimized targeting loci involved in phenol and chlorophenol degradation, which was used to select activated sludge samples and also to assess the degradative genotype of isolates. The isolated strains were screened on the basis of RAPD analysis. In parallel, physiological experiments were carried out with activated sludge samples and isolated bacteria by respirometric analysis. Based on cluster analysis of RAPD pattern and respirometric data, the isolate G20 was selected and identified by using 16S rDNA sequence analysis as Citrobacter freundii strain HPC255. The strain could oxidize different substituted chlorophenol molecules. Such strains could provide the pool of intermediates, which can further be degraded by the associated population, thus helping in maintaining the synergistic association of catabolic activity in activated sludge.

Tuberculous meningitis (TBM) is a serious central nervous system infection, which is seen in clinical practice fairly frequently in developing and underdeveloped countries. Our study was intended to develop a reliable and rapid diagnostic methodology for detecting mycobacterium tuberculous bacilli (MTB) in cerebrospinal fluid (CSF). Dot Enzyme Linked Immunosorbent assay (Dot ELISA) has been standardized to detect MTB antigens and antibodies against MTB in the CSF of TBM patients.CSF samples were collected from 156 registered patients suffering from various neurological disorders, including 56 cases of TBM. Polyclonal antibodies to Culture Filtrate Protein (CFP), H37Rv strain, an MTB antigen, obtained from Colorado State University (USA), were used to detect MTB antigen in the CSF of TBM patients using the Dot ELISA method.The methodology yielded a positive reaction to MTB antigens in 48 CSF samples (86%) obtained from all 56 cases of suspected TBM. The sensitivity achieved through the developed methodology could give reactivity with an antigen level 10ng/dl and above. Dot ELISA for MTB antigen was positive in only 5 out of 100 (5%) of the other neurological disorders, mainly pyogenic meningitis.This rapid methodology for the detection of MTB antigen in CSF is very simple, sensitive, specific, and cost effective. This technique can be easily and routinely employed in the pathology laboratory to support the clinical diagnosis.

Defining a microbial community and identifying bacteria, at least at the genus level, is a first step in predicting the behavior of a microbial community in bioremediation. In biological treatment systems, the most dominating groups observed are Pseudomonas, Moraxella, Acinetobactor, Burkholderia, and Alcaligenes. Our interest lies in identifying the distinguishing features of these bacterial groups based on their 16S rDNA sequence data, which could be used further for generating genus-specific probes. Accordingly, 20 sequences representing different species from each genus above were retrieved, which constituted a training set. A 16-dimensional feature vector comprised of transition probabilities of nucleotides was considered and each sampled sequence was expressed in terms of these features. A stepwise feature selection method was used to identify features that are distinct across the species of these five groups. Wilk's lambda selection criterion was used and resulted in a subset with six distinguishing features. The discriminating efficacy of this subset was tested through multiple group discriminant analysis. Two linear composites, as a function of these features, could discriminate the test set of forty-five sequences from these groups with 95% accuracy, thereby ascertaining the relevance of the identified features. The geometric representation of feature correlation in the reduced discriminant space demonstrated the dominance of identified features in specific groups. These features independently or in combination could be used to generate genus-specific patterns to design probes, so as to develop a tracking tool for the selected group of bacteria.

This study demonstrates the effect of various nitrogen levels in the medium along with different carbon sources in the media on the utilization of phenol by Pseudomonas CF600. Experiments were carried out using cultures derived from minimal media containing the carbon sources phenol and citrate, followed by varying nitrogen levels in the medium. Respirometric analysis under different conditions was carried out with cells using phenol and catechol as substrates. When nitrogen was limiting in the medium, phenol induced higher oxygen uptake rates, whereas catechol was independent of the nitrogen levels. These observations were also supported by the residual phenol levels in the medium with different levels of nitrogen as NH(4) ion. Results show that nitrogen-limiting conditions favor the phenol utilization by Pseudomonas CF600.

This paper describes a method that facilitates the extraction of PCR-compatible DNA from different activated sludge samples. The approach involves a novel preprocessing step in DNA extraction, which removes potential PCR inhibitors. The sludge was washed with different ratios of acetone and petroleum ether after pretreatment with 0.01% Tween-20 at 50 °C. It was observed that an initial washing step with 50 mM Tris–HCl, pH 9.0, before the detergent–solvent step, improved the quality of the extracted DNA. The extraction protocol resulted in amplifiable amounts of DNA when 10 mg of a sludge sample was used, even in the presence of phenol as a sludge contaminant. The usefulness of the extracted template was demonstrated by carrying out different PCR reactions. The random amplified polymorphic DNA (RAPD) patterns demonstrated the diversity of sludge samples.

Real time PCR provides a quantitative option for amplified DNA that could be of use in microbial quality control of drinking water.

Tuberculous meningitis (TBM) is still a serious cause of morbidity and mortality in developing nations, and the timing of treatment is the most crucial factor affecting the ultimate outcome. To establish a rapid diagnosis, we used Single Radial Immuno-Diffusion (SRID) to detect circulating mycobacterium antigen in the CSF of patients with clinically suspected TBM.Single radial immunodiffusion is the simplest of all immunotechniques for the quantitative determination of antigen or antibody. CSF was collected by standard lumbar puncture. Antiserum was raised against CSF from clinically suspected TBM patients by standard Immunization procedures.The developed protocol was tested with 73 CSF samples collected over a period of one year. The assay gave 94% sensitivity for the detection of mycobacterium antigen in the CSF of patients with clinically suspected TBM.This study suggests that single radial immunodiffusion is useful for quantitative as well as qualitative determination of mycobacterium antigen. The developed technique is of potential value in the laboratory diagnosis of TBM.

A simple and rapid protocol has been developed for monitoring of the phenol degrading population in the environmental samples. PCR protocol uses the total DNA prepared from the samples as a template in the amplification reaction. Primers have been designed from the sequence data for Dmp and Phe operon encoding multi-component and single component phenol oxidizing system, respectively. The phenol degrading genotype in various samples have been demonstrated using the developed PCR protocol.

Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA(Gly), integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.

The widespread problem of contaminated water by enteric microorganisms necessitates the need to develop a rapid protocol to detect pathogens in water bodies. Usual methods like plating, biochemical tests and use of DNA probes are time consuming which is a limiting factor especially in epidemic situations. Moreover, some cells can exist in a virulent viable but non-culturable state making detection very difficult by plating method. This paper describes a rapid method to detect the enteropathogens,E.coli, Salmonella andVibrio and enteric viruses from water samples by Polymerase Chain Reaction.E.coli was specially chosen as it is an indicator of fecal contamination. The sensitivity ofE.coli detection was improved to 10 cells to keep in tune with the WHO guidelines. The presence ofE.coli would also indicate the probable presence of other pathogens.

The major problem associated with petroleum refineries is the generation of various types of petroleum-field emulsions and oily sludges during recovery of light and heavy crudes. The emulsions as well as oily sludges generated are diverse in chemical composition and their accumulation in the environment creates disposal problems. Hence, management of refinery wastes has become a necessity and recovery of oil from these wastes would be the only environmentally friendly approach. A secondary metabolite of glucose grown Bacillus subtilis has been found to be a potent de-emulsifier. Ninety-eight percent of oil from the oil-in-water emulsion could be recovered using partially purified 0.6 mg/l of de-emulsifier. The same de-emulsifier is also successfully used to recover oil from oily sludges (Indian patent pending). The two-step process involves pretreatment of oily sludge with kerosene and/or methanol followed by recovery of 90% hydrocarbon. The biotechnological process developed is the competitive solutionn for management of petrochemical wastes in view of cost effectiveness and environmental harmony, when compared with the available technology.

Background Malnutrition is a major risk factor for the development of tuberculosis (TB). In India, Melghat is among the tribal regions which consist of highest number of malnutrition cases. Because of the paucity of TB data from these malnourished areas there is an urgent need for the development and evaluation of improved TB diagnostic tests. In the present study, three in house developed diagnostic tests namely TB-Ag(antigen) ELISA, Adenosine deaminase (ADA) estimation and IS6110 polymerase chain reaction (PCR) assay were investigated for the detection of Mycobacterium tuberculosis (M. tb.) infection. Methods For investigation, blood samples were collected from 128 study subjects from six villages of Melghat tribal area and evaluated using three in house developed assays, namely TB-Ag ELISA, ADA estimation and IS6110 PCR. Results The TB-Ag ELISA method yielded 83% sensitivity and 94% specificity. The ADA and PCR assay gave a sensitivity of 61% and 49% and specificity of 62% and 98% respectively. A considerable good agreement of 82.81% (k=0.472) between TB-Ag ELISA and PCR was observed. The overall sensitivity of TB-Ag ELISA was significantly higher (p

Aim of the present study was to investigate serial changes in adenosine deaminase (ADA) and total homocysteine (tHcy) activities in male and female acute ischemic stroke (AIS) patients to determine their prognostic value. ADA and tHcy activities were measured in follow-up blood sample collected from AIS patients at admission, 24 hrs, 48 hrs, 72 hrs, and 144 hrs after admission and in control subjects. We did not observe any significant difference in ADA and tHcy activities between male and female of AIS and control subjects. ADA activity was significantly high in male AIS patients as compare to female throughout the follow-up. There were no significant difference in tHcy activity between male and female of AIS patients. Similarly there were no significant differences in ADA and tHcy activities on comparing follow-up samples of AIS patients with that of admission value. In addition, m-RS scale in female was better than male AIS patients. Findings of this preliminary study suggest that increase in ADA activity in male AIS patient may reflect poor outcome than female. The tHcy activity is not related with the severity and out come after AIS. Further prospective studies are needed to see if ADA is clinically useful in outcome prediction after AIS.Keywords: Acute ischemic stroke, adenosine deaminase, homocysteine, prognosisÖzetBu araştırmanın amacı erkek ve kadın akut iskemik inme hastalarında adenozin deaminaz (ADA) ve total homosistein (tHcy) aktivitelerindeki seri değişimin prognostik değerlerini belirlemektir. ADA ve tHcy aktiviteleri kontrol bireylerden ve kabulde, kabulden 24, 48, 72 ve 144 saat sonra akut istemik inme hastalarından ölçüldü. Kontrol ve akut iskemik inme olgularında kadın ve erkek bireylerin ADA ve tHcy aktiviteleri benzer bulundu. ADA aktivitesi erkek AIS hastalarında kadınlara göre belirgin şekilde yüksektir. Erkek ve kadın AIS hastalarında tHcy aktivitelerinde önemli bir fark yoktur. Benzer şekilde AIS hastalarının kabul ve takip ADA ve tHcy aktivitelerinde belirgin bir fark yoktur. Ayrıca kadın hastaların m-RS ölçüleri erkek AIS hastalarından daha iyidir. Bu öncül çalışmanın bulguları erkek AIS hastalarındaki ADA aktivitesinin artışı bayanlara göre daha önemsiz olmasıdır. tHcy aktivitesi AIS sonrası sonuç ve ölçüsü olamadığını gösterir. AIS sonrası tahminler için ADA'nın klinik olarak kullanılıp kullanılmayacağı için daha fazla prospektif çalışmaya ihtiyaç vardır. Anahtar sözcükler: Akut istemik inme, adenozin deaminaz, homosistein, prognoz, Bu araştırmanın amacı erkek ve kadın akut iskemik inme hastalarında adenozin deaminaz (ADA) ve total homosistein (tHcy) aktivitelerindeki seri değişimin prognostik değerlerini belirlemektir. ADA ve tHcy aktiviteleri kontrol bireylerden ve kabulde, kabulden 24, 48, 72 ve 144 saat sonra akut istemik inme hastalarından ölçüldü. Kontrol ve akut iskemik inme olgularında kadın ve erkek bireylerin ADA ve tHcy aktiviteleri benzer bulundu. ADA aktivitesi erkek AIS hastalarında kadınlara göre belirgin şekilde yüksektir. Erkek ve kadın AIS hastalarında tHcy aktivitelerinde önemli bir fark yoktur. Benzer şekilde AIS hastalarının kabul ve takip ADA ve tHcy aktivitelerinde belirgin bir fark yoktur. Ayrıca kadın hastaların m-RS ölçüleri erkek AIS hastalarından daha iyidir. Bu öncül çalışmanın bulguları erkek AIS hastalarındaki ADA aktivitesinin artışı bayanlara göre daha önemsiz olmasıdır. tHcy aktivitesi AIS sonrası sonuç ve ölçüsü olamadığını gösterir. AIS sonrası tahminler için ADA’nın klinik olarak kullanılıp kullanılmayacağı için daha fazla prospektif çalışmaya ihtiyaç vardır.

We report here a rapid protocol for the detection of Vibrio and Salmonella in drinking water using a duplex PCR reaction. The developed protocol can detect as few as 500 cells in a single reaction, which has been achieved by optimizing the temperature steps and magnesium chloride concentration for the reactions. The described PCR protocol could detect Vibrio and Salmonella spiked in drinking water.

Objective: To investigate the effect of Hemidesmus indicus extract on activities of human peripheral blood lymphocytes in vitro . Materials and Methods: The total extract of the raw herb was obtained by methanol: isopropyl alcohol: acetone extraction and used at different concentrations. Human peripheral blood lymphocytes (PBLs) were isolated, stimulated to proliferate using phytohaemagglutinin (PHA) or lipopolysaccharide (LPS), with and without different concentration of herbal extracts. Adenosine deaminase (ADA) activity and immunoglobulin (IgG) secretion from cultured PBLs were studied with the herbal extracts and appropriate controls. Results: Hemidesmus indicus extract stimulated the cell proliferation at 1 mg/ml concentration significantly, after 72 h in culture. Viability of extract-treated PBLs was also maintained after culture. The extract increased the IgG production from cultured PBLs, when used at 1 mg/ml concentration. It also increased the ADA activity of PBLs after 72 h in culture. Conclusion: An immunomodulatory activity of H.indicus, related to IgG secretion and ADA activity, is revealed during the study. The herbal extract has shown to promote the release of IgG by lymphocytes and also the ADA activity after 72 h of culture.

The requirements for treated wastewater are becoming increasingly more stringent, and therefore the improved efficiency of biological treatment processes is indispensable at industrial effluent treatment plants (ETPs). Microorganisms such as bacteria play an important role in the natural cycling of materials and particularly in the decomposition of organic wastes. The knowledge of the interactions among these microbial populations needs to be harnessed for optimum evaluation and functioning of effluent treatment plants. Modern molecular techniques have revolutionized the methods of assessing these microbial populations. The combination of the results of these microbial assessments along with the on-site parameters at ETPs would favor an efficient treatment. In this review, the various approaches and importance of correlating the microbial population dynamics and treatment of wastewater at industrial ETPs has been elaborated.

Tamarind seed powder (TSP) was used as raw material for microbial production of citric acid. The maximum citric acid production was observed when the TSP-basal medium (TSP with inorganic nitrogen and phosphorus source) was supplemented with Mg 2+ , Fe 2+ , Cu 2+ and Zn 2+ . The use of methanol and ferrocyanide as a modulator for citric acid production was studied. On methanol supplementation to the medium, ammonium nitrate was found to be better than ammonium carbonate as a source of inorganic nitrogen. Addition of ferrocyanide after sterilization to the medium containing methanol gave the maximum citrate production of 363 μg/ml.

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.