Modified Hodge test: A useful and the low-cost phenotypic method for detection of carbapenemase producers in Enterobacteriaceae members.

BACKGROUND: The global spread of antimicrobial resistance has acquired greater significance in the public health perspective. Drug resistance has posed a threat for the management of various hospital-acquired infections (HAI). For bacteria producing extended spectrum ß lactamase, carbapenems are the drug of choice. However, treatment failures are still a cause of concern due to carbapenemase producers. AIM: Various phenotypic and genotypic methods are available for the detection of carbapenemase producers. Studies thus far have mostly concentrated on comparing various methods for detection of carbapenemase producers. We used low-cost and the easily performed modified Hodge test (MHT) for detecting the carbapenemase producers in Enterobacteriaceae members isolated from various clinical specimens. MATERIAL AND METHODS: The study included 1072 clinical isolates of Enterobacteriaceae collected in India between April 2008 and February 2010. MHT was performed on all the isolates in accordance with CDC and CLSI guidelines. RESULTS: The carbapenemase activity was detected in 35.9% (385/1072) of the isolates. Klebsiella spp. 28.7% (80/278), Citrobacter spp. 20.4% (25/122), 11.3% (38/334) in E. coli, 20.3% (45/221) in Enterobacter spp., and 16.2% (9/117) in Proteus spp. revealed variable resistance activities against carbapenems. CONCLUSION: Enterobacteriaceae members are among the most common and easily transferable bacterial species responsible for severe HAI. This study revealed a high percentage of Enterobacteriaceae clinical isolates producing carbapenemases in India. Detection of such bacteria, formulating hospital antibiogram, and monitoring the usage of antimicrobial drugs is recommended.

A Study of Alternate Biomarkers in HIV Disease and Evaluating their Efficacy in Predicting T CD4+ Cell Counts and Disease Progression in Resource Poor Settings in Highly Active Antiretroviral Therapy (HAART) Era.

INTRODUCTION: Human Immunodeficiency Virus (HIV), the causative agent of AIDS, has been a challenge to medical fraternity since it was first discovered in 1983. About 40 million people are living with HIV infection globally and 99% of the infected people are in south East Asia (SEA). Traditionally, HIV disease and progression, initiation of HAART and response to therapy is monitored by assessing in regular intervals, the T CD4+ cell counts and plasma HIV/RNA viral load. Resource poor, low and low - middle income group countries still have no finances to acquire infrastructure and scientific technology for performing such tests. OBJECTIVES: Since very few studies are available, they have demonstrated the role of alternate biomarkers that can be used to predict CD4 cell counts and thereby, monitor HIV disease progression and HAART. We aimed to measure certain haematological parameters in HIV seropositive patients and to evaluate their efficacy in predicting TCD4+ cell counts. METHODS: The study group included 250 HIV seropositive patients with an age range of 18-65 years. 140(56%) males and 110(44%) females were included in the study. Absolute TCD4+cell counts and CD8+T cell counts were measured by using a flow cytometer. (MMWR Recommendations and Reports, 1992) TLC; HB%, AEC and ESR were estimated by using conventional haematological methods. CRP was evaluated by latex agglutination test (Immuno CRP Latex Agglutination Test). RESULTS: Among the tested haematological markers, a TLC of <1800 cells/mm(3) showed high specificity (100%) in predicting CD4 counts of < 200 cells/mm(3), with an accuracy of 61.46%. Haemoglobin and Absolute Eosinophilic counts showed high specificities of 84.09% and 94.32% respectively in predicting CD4 counts which were below 350 cells/mm(3). ESR with 98.98% sensitivity and AEC which had 83.67% sensitivity were able to predict CD4 counts of <200 cells/mm(3). CONCLUSION: Among the tested biomarkers, it was seen that Absolute Eosinophilic counts of more than 550 cells/mm(3), Blood Haemoglobin which was less than 10 g%, ESR which measured more than 20 mm, CRP values of >1.2 and TLC of <1800 cells/mm(3) could be helpful in predicting CD4 cell counts of < 350 and <200 cells/mm(3).

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.

Current prospective of aldose reductase inhibition in the therapy of allergic airway inflammation in asthma.

The prevalence of asthma and costs of its care have been continuously increasing, but novel therapeutic options to treat this inflammatory disease have not been brought to the US market. Current therapies such as inhaled steroids, long-acting beta-agonist bronchodilators, antihistamines and immunomodulators may control the symptoms of allergic asthma but fail to modify the underlying disease. Excessive use of steroids and other immunosuppresents alter the patient's quality of life, produce undesirable toxicities, and increase the risk of other pathologies such as diabetes. Hence novel therapeutic options to manage asthma are desirable. In the present review, we have discussed the role of the polyol pathway enzyme aldose reductase (AR) in the amplification of allergic airway inflammation. Recent studies have indicated that AR inhibition prevents the NF-κB-dependent generation of pro-inflammatory cytokines and chemokines in mouse models of allergic airway inflammation indicating the potential use of AR inhibition as a novel tool to control allergic responses. Since orally available AR inhibitors have already undergone phase III clinical trials for diabetic neuropathy and appear to have a manageable side effects profile, they could be readily developed as potential new drugs for the treatment of asthma and related complications.

Emerging role of antioxidants in the protection of uveitis complications.

Current understanding of the role of oxidative stress in ocular inflammatory diseases indicates that antioxidant therapy may be important to optimize the treatment. Recently investigated antioxidant therapies for ocular inflammatory diseases include various vitamins, plant products and reactive oxygen species scavengers. Oxidative stress plays a causative role in both non-infectious and infectious uveitis complications, and novel strategies to diminish tissue damage and dysfunction with antioxidant therapy may ameliorate visual complications. Preclinical studies with experimental animals and cultured cells demonstrate significant anti-inflammatory effects of a number of promising antioxidant agents. Many of these antioxidants are under clinical trial for various inflammatory diseases other than uveitis such as cardiovascular, rheumatoid arthritis and cancer. Well planned interventional clinical studies in the field of ocular inflammation will be necessary to sufficiently investigate the potential medical benefits of antioxidant therapies for uveitis. This review summarizes the recent investigations of novel antioxidant agents for ocular inflammation, with selected studies focused on uveitis.

Bacterial cellulose nanocrystals exhibiting high thermal stability and their polymer nanocomposites.

Nanocrystals prepared from bacterial cellulose are considered as 'green nanomaterials' depending on their renewable nature and ease of production without the involvement of hazardous chemical treatments. In this investigation, a top down approach was followed for the preparation of bacterial cellulose nanocrystals (BCNC) using a commercially available cellulase enzyme so as to retain native properties of bacterial cellulose even in its nanodimensional form. The morphological and dimensional parameters of BCNC were studied using atomic force microscope (AFM) and transmission electron microscope (TEM). Thermal properties of BCNC produced using the novel enzyme treatment and conventional sulfuric acid hydrolysis were compared. The thermal stability of enzyme processed BCNC was almost two fold higher than sulfuric acid processed ones. Further, the activation energy required for decomposition of enzyme processed BCNC was much higher than the other. Using this enzyme processed BCNC, Polyvinylalcohol (PVA) nanocomposite films were prepared and characterized. Incorporation of these nanocrystals in polymer matrix resulted in a remarkable improvement in the thermal stability as well as mechanical properties of nanocomposite films. These nanocomposites exhibited higher melting temperature (Tm) and enthalpy of melting (ΔHm) than those of pure PVA, suggesting that the addition of nanocrystals modified the thermal properties of PVA. The effective load transfer from polymer chains to the BCNC resulted in an improved tensile strength from 62.5 MPa to 128 MPa, by the addition of just 4 wt% of BCNC. Furthermore, the elastic modulus was found to increase from 2 GPa to 3.4 GPa. The BCNC obtained through cellulose treatment under controlled conditions were associated with several desirable properties and appear to be superior over the conventional methods of nanocrystals production. The enzymatic method followed in this study is expected to contribute the fabrication of high performance polymer nanocomposites in a much greener and innovative manner.

Understanding the role of aldose reductase in ocular inflammation.

Aldose reductase, although identified initially as a glucose-reducing enzyme via polyol pathway, is believed to be an important component of antioxidant defense system as well as a key mediator of oxidative stress-induced molecular signaling. The dual role played by AR has made it a very important enzyme for the regulation of not only the cellular redox state by detoxifying the reactive lipid-aldehydes generated by lipid peroxidation which is crucial in the cellular homeostasis, but also in the regulation of molecular signaling cascade that may regulate oxidative stress-induced cytotoxic events. Search for the new molecular targets to restrain the oxidative stress-induced inflammation has resulted in the identification of AR as an unanticipated mediator of oxidative stress-induced signaling. Although, in last one decade or so AR has been implicated in various inflammation-related diseases conditions ranging from diabetes, sepsis, cancer, cardiovascular and ocular inflammation, however, a critical evaluation of the clinical efficacy of AR inhibitors awaits a better understanding of the role of AR in regulating inflammation, especially in ocular inflammation.

Simplification and optimization of deMan Rogosa Sharpe (MRS) medium for enhanced production of bacteriocin by Weissella paramesenteroides DFR-8.

Complex growth medium such as deMan Rogosa Sharpe (MRS) medium, commonly used for cultivation of fastidious lactic acid bacteria (LAB) interfere in bacteriocin purification. Sometimes all the ingredients of a defined medium are not required by all LAB strains for bacteriocin production. In the present study, composition of the MRS medium for the production of bacteriocin by Weissella paramesenteroides DFR-8, an isolate from cucumber (Cucumis sativus), was simplified and optimized with a step-wise strategy. In the first step, production profile, effect of incubation temperature, various C and N sources were investigated. In the second step, central composite rotatable design was employed to decide the optimal concentration of 3 key components (glucose, tryptone and pH) and the experimental results were fitted with a second order polynomial regression equation. According to the set criteria, the predicted bacteriocin titer from a medium containing 7.99% glucose, 9% tryptone, pH 7.5 (91.9% desirability) was 540 AU/ml and the observed bacteriocin titer was 538 AU/ml that indicated the validity of the developed model. Using optimized medium, bacteriocin titer of 674.5 AU/ml could be achieved after 72 h of fermentation that is nearly 2.5 fold higher than that obtained from unmodified MRS medium.

Staphylococcus aureus colonization of anterior nares of school going children.

OBJECTIVE: To determine the colonization rates of S. aureus in anterior nares of school going children, evaluate the antimicrobial resistance of such isolates against various antibiotics. METHODS: Nasal swabs taken for S. aureus in 392 healthy school going children aged between 5 and 15 yr belonging to three schools surrounding Kamineni Institute of Medical Sciences, Narketpally, Andhra Pradesh, India. Swabs were inoculated in to Mannitol Salt Agar (MSA) and incubation at 37 degrees C for 24hr, Staphylococcus aureus isolates were identified by standard microbiological methods such as Gram's stain, catalase and coagulase. MICs were determined by Agar dilution technique against Vancomycin, Ciprofloxacin, Sparfloxacin, Sparfloxacin beta Cyclodextrin. Antimicrobial resistance patterns of all the isolates against Oxacillin (1 microg) Penicillin (10 units), Ampicillin (10 microg), Co-trimoxazole (23.75 microg), Erythromycin (15 microg), Tetracycline (30 microg) and Gentamicin (10 microg) were tested using Kirby-Baeur disk diffusion method in accordance with CLSI standards. RESULTS: Of the 392 samples screened 63(16%) showed the growth of Staphylococcus aureus. 12(19%) isolates were found to be MRSA. Antimicrobial susceptibility testing using Agar dilution method against Vancomycin, ciprofloxacin, Sparfloxacin, and Sparfloxacin beta Cyclodextrin revealed MICs in the range of 0.52 microg/ml, 0.51 microg/ml, 0.5 microg/ml and <0.0312-0.250 microg/ml respectively. Disk diffusion method showed that all the isolates were resistant to Penicillin and Ampicillin. A resistance of 14.3%, 25% and 22.2% was observed against Co-trimoxazole, Erythromycin and Tetracycline respectively. Gentamiicin was the only antibiotic against which most of the isolates were sensitive. CONCLUSION: Our results suggests that healthy school going children under 16 yr of age are potential carriers of S. aureus and in particular MRSA and multi-drug resistant strains.

Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici.

AIM: To isolate and identify black pepper (Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease. METHODS AND RESULTS: Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici. Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in greenhouse trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa (Pseudomonas EF568931), IISRBP 25 as P. putida (Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium (B. megaterium EU071712) based on 16S rDNA sequencing. CONCLUSION: Black pepper associated P. aeruginosa, P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes.

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.

Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Chemoenzymatic synthesis of pyrrolo[2,1-b]quinazolinones: lipase-catalyzed resolution of vasicinone.

A facile synthesis of bronchodilatory pyrrolo[2,1-b]quinazoline alkaloids by azidoreductive cyclization strategy employing TMSCl-NaI and bakers' yeast is described. Both the chemical and enzymatic methods are mild and take place at room temperature in good yields. Further, synthesis and resolution of vasicinone has been carried out by employing different lipases. It has been observed that lipase PS provides acetate of (S)-vasicinone in 98% ee.

Characterization of the glutathione binding site of aldose reductase.

Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.