Hemant J. Purohit

Quenching the quorum sensing system: potential antibacterial drug targets

Emergence of antibiotic and multi-drug resistant pathogenic bacteria has created the need for new drugs and drug targets. During pathogenesis bacteria release signals which regulate virulence and pathogenicity related genes. Such bacteria co-ordinate their virulent behaviour in a cell density dependent phenomenon termed as quorum sensing (QS). In contrast, microbes interfere with QS system by quenching the signals, termed quorum quenching (QQ). As a consequence of disrupted QS, pathogens become susceptible to antibiotics and drugs. In this article, the biodiversity of organisms with potential to quench QS signals and the use of QQ molecules as antibacterial drugs have been reviewed.

Development of an in vitro model to study clot lysis activity of thrombolytic drugs

BackgroundThrombolytic 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.MethodsWhole 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.ResultsThere 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.ConclusionOur study could be a rapid and effective methodology to study clot-lytic effect of newly developed drugs as well as known drugs.

Simultaneous nitrification and denitrification by diverse Diaphorobacter sp.

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.

Neurological complications of Chikungunya virus infection.

BACKGROUND In May 2006, there was a large Chikungunya virus infection (CHIKV) outbreak in the Nagpur district of Maharashtra, a province in western India. Usually, CHIKV is a self-limiting febrile illness. However, neurological complications have been described infrequently. AIM To study the clinical characteristics of various neurological complications associated with CHIKV infections. MATERIALS AND METHODS Patients with neurological complications following CHIKV infection during the outbreak were the subjects of the study. On the basis of clinical features and investigative findings, patients were grouped into various neurological syndromes: Encephalitis, myelopathy, peripheral neuropathy, myeloneuropathy, and myopathy. Cerebrospinal fluid (CSF) samples were also collected for biochemical and serological studies. RESULTS Of the 300 patients with CHIKV infection seen during the study period, June-December 2006, 49 (16.3%) [M : F: 42:7] had neurological complications. The neurological complications included: Encephalitis (27, 55%), myelopathy (7, 14% ), peripheral neuropathy (7, 14%), myeloneuropathy (7, 14%), and myopathy (1, 2%). Reverse Transcriptase polymerase chain reaction (RT-PCR) and real-time PCR was positive in the CSF in 16% and 18%, respectively. CONCLUSION Recent CHIKV infection was associated with various neurological complications, suggesting neurotropic nature of the virus. The outcome of the neurological complications is likely to be good.

Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis

BackgroundAtherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This studys aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not.MethodsAn in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control.ResultsUsing an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%).ConclusionThrough our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.

Anaerobic and aerobic biodegradation of chlorophenols using UASB and ASG bioreactors

Chlorophenol degradation was studied by combined anaerobic–aerobic treatments as a single or multi-substrate system. 2,4-Dichlorophenol (2,4-DCP) was degraded to the extent of 52 and 78% in up-flow anaerobic sludge blanket (UASB) and aerobic suspended growth (ASG) reactors respectively, at organic loading rates of 0.18 kg/m3/day and hydraulic retention time of 26.4 h in the presence of glucose. The UASB represents the dominating facultative anaerobic microbial population. When the effluent from the anaerobic reactor (UASB) was subjected to aerobic treatment on the ASG reactor, 2,4-DCP and COD removals of 86 and 95% respectively were achieved. Aerobic degradation of chlorophenol by acclimated mixed bacterial isolates was found to be sequential: 2-Chlorophenol (2-CP) and 4-CP were degraded first, followed by 2,4-DCP and 2,4,6-Trichlorophenol (2,4,6-TCP) while the contrary was obtained in anaerobic degradation. In anaerobic degradation by acclimated mixed bacterial cells, 2,4-DCP and 2,4,6-TCP were degraded first followed by mono-chlorophenols. The anaerobic/aerobic bioreactors were most efficient when operated in sequence (series) rather than in parallel.

Genomic Analysis Reveals Versatile Organisms for Quorum Quenching Enzymes: Acyl-Homoserine Lactone-Acylase and -Lactonase

Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and –acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and –acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation.

Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383

The present study describes the production and characterization of a feather hydrolyzing enzyme by Serratia sp. HPC 1383 isolated from tannery sludge, which was identified by the ability to form clear zones around colonies on milk agar plates. The proteolytic activity was expressed in terms of the micromoles of tyrosine released from substrate casein per ml per min (U/mL min). Induction of the inoculum with protein was essential to stimulate higher activity of the enzyme, with 0.03% feathermeal in the inoculum resulting in increased enzyme activity (45U/mL) that further increased to 90U/mL when 3d old inoculum was used. The highest enzyme activity, 130U/mL, was observed in the presence of 0.2% yeast extract. The optimum assay temperature and pH for the enzyme were found to be 60 degrees C and 10.0, respectively. The enzyme had a half-life of 10min at 60 degrees C, which improved slightly to 18min in presence of 1mM Ca(2+). Inhibition of the enzyme by phenylmethyl sulfonyl fluoride (PMSF) indicated that the enzyme was a serine protease. The enzyme was also partially inhibited (39%) by the reducing agent beta-mercaptoethanol and by divalent metal ions such as Zn(2+) (41% inhibition). However, Ca(2+) and Fe(2+) resulted in increases in enzyme activity of 15% and 26%, respectively. The kinetic constants of the keratinase were found to be 3.84 microM (K(m)) and 108.7 microM/mLmin (V(max)). These results suggest that this extracellular keratinase may be a useful alternative and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial processes.

Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.

Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

BackgroundTuberculous 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.MethodADA 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.ResultsThe 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.ConclusionThis 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.