Phenotypic Detection and Antibiogram of ß-lactamase-producing Proteus Species in a Tertiary Care Hospital, India.

BACKGROUND: Proteus species cause a variety of community- and hospital-acquired illnesses. Synthesis of ß-lactamases is the predominant mechanism for resistance to ß-lactam antibiotics. Among the ß-lactamases, extended spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases are the most common. AIM: The objective of this study was to determine the occurrence of ESBL and AmpC ß-lactamases in Proteus species among various clinical isolates at a tertiary care hospital, India. MATERIALS AND METHODS: This study was done to identify various species of Proteus from clinical samples (n = 3922). Antimicrobial susceptibility was performed by Kirby-Bauer disc diffusion method. ESBL production was detected by modified double-disc synergy test and indirect modified three-dimensional tests and AmpC ß-lactamase production by AmpC disc test and modified Hodge test. RESULTS: Proteus species were isolated in 5.4% (101/1876) specimens. Three Proteus species isolated were Proteus mirabilis 62.4% (63/101), Proteus vulgaris 29.7% (30/101), and Proteus penneri 7.9% (8/101). ESBL producers confirmed by both tests were of 88.1% (89/101). Only AmpC ß-lactamase was produced by four isolates. Coproduction of ESBL and AmpC ß-lactamase was observed in 58.4% (52/89) of isolates. Twelve isolates were non-ß-lactamase producers. Multidrug resistance (MDR) was found in 95.1% (96/101) of isolates, 50.5% (51/101) were possibly extensively drug resistant and none were pan drug resistant. None of the isolates were resistant to piperacillin-tazobactam. P. penneri isolates exhibited high resistance to most of the antibiotics. CONCLUSIONS: A high prevalence of ESBL and AmpC ß-lactamases was found that concurrently showed MDR. Phenotypic methods for the detection of ß-lactamases are easy and simple and can be implemented in routine diagnostic laboratories along with susceptibility testing. These data will assist the clinicians in the management and control of infections.

Possibility of scrub typhus in fever of unknown origin (FUO) cases: an experience from Rajasthan.

PURPOSE: Fever of unknown origin (FUO) has multiple causes. Scrub typhus is less known cause of FUO in India. The present study reports a recent epidemic of scrub typhus amongst cases of FUO from different areas of Rajasthan, India. There was high mortality in undiagnosed cases of FUO which lead to the diagnosis of scrub typhus. OBJECTIVE: To study the possibility of scrub typhus as a causative factor in FUO cases by qualitative detection of IgM antibodies with ELISA. MATERIALS AND METHODS: From September 2012 to December 2012, 271 serum samples of FUO cases were analysed for IgM antibodies to Orientia tsutsugamushi along with dengue, malaria, typhoid, tuberculosis and brucellosis. RESULTS: Scrub typhus IgM antibodies by ELISA were detected in 133 (49.1%) patients. Scrub typhus positivity was significantly higher among female in comparison to males (P<0.05). Maximum positivity of scrub typhus was found in females of 46-60 years age group. The laboratory parameters were abnormal in most of the patients as evident by thrombocytopenia (63%), deranged liver functions (56%) and renal functions (25%). CONCLUSION: The present study emphasises the importance of scrub typhus among cases of FUO especially after rainy season and during early cooler months. The study also highlights the significance of ELISA method for rapid and early reporting and ruling out scrub typhus in FUO cases.

Mixed hydrotropy: novel science of solubility enhancement.

Conventional furosemide tablets are practically insoluble in water, have slow onset of action (45-60 min) and poor bioavailability (39-53%), and therefore cannot be given in emergency clinical situations like hypertension or pulmonary edema. So purpose of research was to provide a fast dissolving oral dosage form of furosemide, which can provide quick onset of action by using concept of mixed hydrotropy. Initially solubility of furosemide was determined individually in 4 hydrotropic agents namely urea, sodium acetate, sodium benzoate, sodium citrate at concentration of 10, 20, 30 and 40% w/v solutions using purified water as solvent. Highest solubility was obtained in 40% sodium benzoate solution. Then different combinations of 2, 3 and 4 hydrotropic agents in different ratios were used to determine solubility, so that total concentration of hydrotropic agents was always 40%. Highest solubility was obtained in solution of urea+sodium benzoate+sodium citrate at optimum ratio of 15:20:5. This optimized combination was utilized in preparing solid dispersions by common solvent technique using distilled water as solvent. Solid dispersions were evaluated for flow properties, XRD, DSC, SEM and were also compressed to form tablets. Dissolution studies of conventional and prepared tablets were done using USP Type II apparatus. It was concluded that the concept of mixed hydrotropic solid dispersion is novel, safe and cost-effective technique for enhancing bioavailability of poorly water-soluble drugs by dissolving drug in nonionized form. The magical enhancement in solubility of furosemide is clear indication of its potential to be used in future for other poorly water-soluble drugs in which low bioavailability is major concern.

New spectrophotometric estimation of indomethacin capsules with niacinamide as hydrotropic solubilizing agent.

BACKGROUND: Hydrotropic solubilization process involves cooperative intermolecular interaction with several balancing molecular forces, rather than either a specific complexation event or a process dominated by a medium effect, such as co-solvency or salting-in. MATERIALS AND METHODS: In the present investigation, hydrotropic solution of 2 M niacinamide was employed as the solubilizing agent to solubilize the poorly water-soluble drug, indomethacin, from the capsule dosage form for spectrophotometric determination in ultraviolet region. RESULTS: Hydrotropic agent used did not interfere in the spectrophotometric analysis. In preliminary solubility studies, it was found that there was more than fivefold enhancement in the aqueous solubility of indomethacin (poorly water-soluble drug) in 2 M niacinamide solution as compared to its aqueous solubility at 28 ± 1°C. CONCLUSION: The proposed method is new, simple, safe, environmentally friendly, economic, accurate and cost-effective and can be successfully employed in routine analysis.

New spectrophotometric estimation of ornidazole tablets employing urea as a hydrotropic solubilizing additive.

Quantitative spectrophotometric analysis of poorly water-soluble drugs involves use of various organic solvents. Major drawbacks of organic solvents include high cost, volatility and toxicity. Safety of analyzer is affected by toxicity of the solvent used. In the present investigation the use of organic solvent has been avoided, making the method environmentally friendly. Urea has demonstrated enhancement in aqueous solubilities of a large number of poorly water-soluble drugs, thereby widely used as a hydrotropic agent. There was more than 10-fold enhancement in the solubility of ornidazole in 10 M urea solution as compared to its solubility in distilled water. In the present investigation, hydrotropic solution of urea (10 M) was employed as solubilizing agent to solubilize the poorly water-soluble drug, ornidazole, from fine powder of its tablet dosage form for spectrophotometric determination in ultraviolet region at 319 nm. Beer's law was obeyed in the concentration range of 5-25 µg/ml in presence of urea. Presence of urea did not interfere in the analysis. Proposed method is new, rapid, simple, accurate, and reproducible. Statistical data proved the accuracy, reproducibility and the precision of the proposed method.

Novel application of hydrotropic solubilizing additives in the estimation of aspirin in tablets.

Highly concentrated aqueous solutions of various hydrotropic agents like sodium benzoate, sodium salicylate, sodium acetate, sodium citrate, nicotinamide and sodium ascorbate have been observed to enhance aqueous solubilities of a large number of poorly water-soluble drugs. In the present investigation hydrotropic solubilization technique has been employed to solubilize poorly water-soluble aspirin (analgesic and antipyretic drug) by 0.5 M ibuprofen sodium solution to carry out titrimetric analysis of aspirin in tablet dosage form. Results of analysis by proposed method and Phamacopoeial method are very comparable. Proposed method is new, rapid, simple, accurate, and reproducible. Statistical data proved the accuracy, reproducibility and the precision of proposed method.

A novel method for quantitative determination of aceclofenac in bulk drug and tablets using sodium salicylate as a hydrotropic solubilizing agent.

In titrimetric analysis costlier organic solvents are more often employed to solubilize the poorly water-soluble drugs. Volatility and pollution are drawbacks of such solvents. Various techniques are employed to enhance the aqueous solubility of poorly water-soluble drugs. Hydrotropic solubilization phenomenon has been widely used to enhance the aqueous solubility of large number of poorly water-soluble drugs. Aqueous solubility of aceclofenac bulk drug [a poorly water-soluble NSAID] was enhanced to a great extent i.e., 400 folds with 2.5 M sodium salicylate. The primary objective of the present investigation was to employ this hydrotropic solution to extract the drug from its dosage forms, precluding the use of costlier organic solvents. The proposed method of analysis is new, simple, accurate, environmentally friendly and reproducible. Statistical data proved the accuracy, reproducibility and the precision of the proposed method. The results of titrimetric analysis by use of hydrotropy compared very well with the results of Pharmacopoeial method.

Quantitative estimation of naproxen in tablets using Ibuprofen sodium as hydrotropic agent.

In the present investigation 0.5 M ibuprofen sodium solution has been used as hydrotropic solubilizing agent for naproxen, a poorly water-soluble drug and in it there was more than 350 fold enhancement in the solubility of naproxen as compared to the solubility in distilled water. Therefore, this hydrotropic solution was employed to extract out the drug from its tablet dosage form for quantitative estimation by titrimetry. The naproxen has been successfully analyzed in tablets. The results of analysis obtained by proposed method compared well with those by corresponding British pharmacopoeial method involving the use of methanol. The proposed method was also validated by recovery studies. Presence of ibuprofen sodium and common excipients did not interfere in analysis. Proposed method is new, simple, economic, safe, rapid, accurate, reproducible and environment-friendly.

Analysis of bulk sample of salicylic Acid by application of hydrotropic solubilization method.

In the present investigation, the poorly water-soluble drug, salicylic acid has been solubilized using 0.5 M ibuprofen sodium and 2.0 M sodium salicylate solution as hydrotropic agents for the titrimetric analysis precluding the use of organic solvents. Both hydrotropes are economic and pollution-free. The mean percent estimation of salicylic acid estimated in bulk sample by Indian Pharmacopoeial method is 98.78%. The mean percent estimation by ibuprofen sodium method and sodium salicylate method are 99.25% and 98.82%, respectively. The results of analysis by the proposed method are very close to the results of analysis by the standard method. This confirms the accuracy of the proposed method. The proposed method was validated statistically by low values of statistical parameters viz. standard deviation, percent coefficient of variation and standard error. The proposed method is new, accurate, simple and economic.

Tunicamycin enhances neuroinvasion and encephalitis in mice infected with Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis (VEE) viruses cause natural outbreaks in humans and horses and represent a significant biothreat agent. The effect of tunicamycin on the course of the disease in mice with VEE was investigated, and the combined effects of these agents was characterized. CD-1 mice given 2.5 microg of tunicamycin had >1,000-fold more virus in the brain 48 hours after infection with the virulent VEE strain V3000 and > or =100-fold of the attenuated strain V3034 at all tested times than did untreated mice, indicating enhanced neuroinvasion. Tunicamycin did not alter the viremia profiles of these viruses nor the replication of V3000 in the brain itself. Tunicamycin alone caused ultrastructural blood-brain barrier damage, yet neuroinvasion by V3000 in treated mice appeared to occur via the olfactory system rather than the blood-brain barrier. Tunicamycin-treated, V3000-infected mice also exhibited earlier and more severe weight loss, neurological signs, neuronal infection, neuronal necrosis and apoptosis, and inflammation than untreated, V3000-infected mice. The mean survival time of tunicamycin-treated, V3000-infected mice was 7.3 days versus 9.9 days for untreated, V3000-infected mice. These studies imply that animals that ingest toxins similar to tunicamycin, including the agent of annual ryegrass toxicity in livestock, are conceivably at greater risk from infections by encephalitis viruses and that humans and horses exposed to agents acting similar to tunicamycin may be more susceptible to encephalitis caused by VEE viruses. The exact mechanism of tunicamycin-enhanced neuroinvasion by VEE viruses requires further study.

Co-infection by Semliki forest virus and malarial parasite modulates viral multipucation, pathogenesis and cytokines in mice.

Environmental, technological and societal factors continue to have a dramatic effect on infectious diseases worldwide and are considered to be facilitating the emergence of several infectious diseases at a time. Co-infection with different species of viral and malaria infections are currently emerging problems of dual infection in the developing as well as developed countries. Understanding of interactions between the host, malaria and virus infection is of current concern and we have initiated studies to delineate the mechanisms involved during the progression of Semliki forest virus (SFV) and Plasmodium yoelii (P. yoelii) infection in mice. Enhanced virus multiplication and up-regulation of cytokine mRNA level in P. yoelii and SFV co-infected mice were observed on day 4 post-infection compared to respective controls. Collectively, our observations indicate that malaria infection may influence virus multiplication, pathogenesis and up-regulation of cytokine mRNA during co-infection in mice.

Enhancement of wound healing by shikonin analogue 93/637 in normal and impaired healing.

Wound healing is a complicated biological process, which involves interactions of multiple cell types, various growth factors, their mediators and the extracellular matrix proteins. In this study, we evaluated the effects of shikonin analogue 93/637 (SA), derived from the plant Arnebia nobilis, on normal and hydrocortisone-induced impaired healing in full thickness cutaneous punch wounds in rats. SA (0.1%) was applied topically daily as an ointment in polyethylene glycol base on wounds. SA treatment significantly accelerated healing of wounds, as measured by wound contraction compared to controls in hydrocortisone-impaired animals. SA treatment promoted formation of granulation tissue including cell migration and neovascularization, collagenization and reepithelialization. The expression of basic fibroblast growth factor (bFGF) was higher as revealed by immunohistochemistry in treated wounds compared to controls. However, the expression of transforming growth factor-beta(1) was not affected by SA treatment. Since bFGF is known to accelerate wound healing, the increased expression of bFGF by SA may be partly responsible for the enhancement of wound healing. These studies suggest that SA could be further studied for clinical use to enhance wound healing.

Exacerbation of soft tissue lesions in lead exposed virus infected mice.

OBJECTIVE: To investigate the effect of Lead (Pb) acetate exposure on Semliki forest virus (SFV) pathogenesis in mice. METHODS: Different doses (62.5, 125, 250 and 500 mg/Kg body weight) of Pb dissolved in normal saline were given to mice by oral intubation in a sub-acute (28 days) and sub-chronic (90 days) regimen followed by SFV infection. Morbidity, mortality, clinical symptoms, mean survival time (MST), changes in body and organ weight, accumulation of lead in soft tissues, virus titre in brain and histopathological alterations were compared between lead exposed and infected groups. RESULTS: Early appearance of virus symptoms, increased mortality, decreased MST, enhanced SFV titre and greater tissue damage were observed in lead exposed-SFV-infected mice. CONCLUSION: Pre-exposure to lead increases the susceptibility of mice towards SFV infection. Further studies are suggested in view of the persistence of lead in the environment and the possibility of infection by microbial pathogens.

Lead exposure enhances virus multiplication and pathogenesis in mice.

Suppression of the immune system by environmental xenobiotics may cause increased susceptibility of the host towards a variety of microbial pathogens an result in a life-threatening state. We investigated whether lead exposure would enhance susceptibility to Semliki Forest Virus (SFV). Mice orally exposed to lead acetate (62.5, 125, 250 or 500 mg/kg bw) exhibited increased mortality and decreased mean survival time compared to untreated animals on challenge with SFV. The mortality was associated with enhancement of high virus titer and earlier appearance of virus in lead-exposed mice. Histopathological studies observed enhancement of viral pathogenesis in a dose dependent pattern in the lead-dosed group challenged with SFV. The results indicate that exposure to lead enhanced susceptibility to viral infection. Environmental metal contamination and subsequent infection by pathogenic microbes points necessitate studies on the interaction of environmental pollutants on the immune system.

Effect of poly ICLC treatment on hepatic oxidative stress and antioxidant defense indices in Plasmodium yoelii nigeriensis infected mice.

BACKGROUND: Plasmodium yoelii nigeriensis (P. y. nigeriensis) produces lethal malaria infection in Swiss albino mice. Reactive oxygen species (ROS) are important mediators of tissue injury during malaria infection. OBJECTIVE: To study the status of hepatic oxidative stress and antioxidant defense indices during Plasmodium yoelii nigeriensis (P. y. nigeriensis) infection and poly ICLC treatment of normal and P. y. nigeriensis infected Swiss albino mice. METHODS: Mice were divided into four groups viz., 1. Normal mice, 2. Normal mice treated with poly ICLC (5 mg/kg body weight, i.p.), 3. P. y. nigeriensis infected mice and 4. P. y. nigeriensis infected mice treated with poly ICLC (5 mg/kg body weight, i.p.). RESULTS: P. y. nigeriensis infection caused a significant increase in hepatic oxidative stress indices viz., xanthine oxidase and lipid peroxidation. This was accompanied by a significant increase in antioxidant defense indices viz., reduced glutathione (GSH), glutathione reductase while superoxide dismutase and catalase showed a significant decrease with respect to normal mice. Poly ICLC treatment of P. y. nigeriensis infected mice did not cure blood parasitemia. However, poly ICLC treatment of normal and P. y. nigeriensis resulted in an increased generation of hepatic oxidative stress and an associated increase in the antioxidant defense indices. CONCLUSION: poly ICLC therapy alone is not sufficient to treat the malaria infection caused by multiple drug resistant strain of P. y. nigeriensis. Therefore there is a need to develop newer antimalarias which can act alone or in combination with traditional antimalarials to be effective against drug resistant malarial parasite.

Inhibition of growth and regulation of IGFs and VEGF in human prostate cancer cell lines by shikonin analogue 93/637 (SA).

BACKGROUND: Insulin-like growth factors (IGFs) are important mitogens and are involved in normal and malignant cellular proliferation. IGFs and IGF binding proteins (IGFBPs) regulate the prostatic cell growth and reduction/blocking of IGFs has been suggested to be of therapeutic value in prostate cancer. beta,beta-dimethyl acryl shikonin, an extract from the roots of plant Arnebia nobilis has been shown to have anticancer properties but was found to be toxic. Subsequently, several analogoues of beta,beta-dimethyl acryloyl shikonin were synthesized and one of them shikonin analogue 93/637 (SA) was significantly less toxic compared to beta,beta-dimethyl acryloyl shikonin. MATERIALS AND METHODS: We have investigated the effect of SA on prostate cancer cell (DU 145, LNCaP and PC-3) growth and expression of IGFs (IGF-I, IGF-II and IGF-I receptor (IGF-IR)), IGFBP-3 and vascular endothelial growth factor (VEGF). RESULTS: SA had growth inhibitory effect on PC-3 cells in a dose dependent manner. It also showed slight inhibitory effect on the growth of DU 145 and LNCaP cells at low doses ranging from 250 nM to 1 microM and has moderate inhibitory effect at concentrations 2.5 microM and above. Lactate dehydrogenase (LDH) activity assays indicated cellular damage, only at higher concentrations of SA that are greater than 1 microM. Gene expression studies by RT-PCR have demonstrated a decrease in mRNAs of IGF-II in DU 145, IGF-I, and IGF-IR in LNCaP, and IGF-II and VEGF in PC-3 cells and an increase in IGFBP-3 in both DU 145 and PC-3 cells by treatment with SA. CONCLUSIONS: The results demonstrate the inhibitory effect of SA on cellular growth and IGFs specifically in PC-3 cells and suggest a potential therapeutic use in treatment of prostate cancer.

Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury.

Cell death following ischemia-reperfusion injury is a major concern in clinical issues such as organ transplantation and trauma. The need to identify agents with a potential for preventing such damage has assumed great importance. We have evaluated the efficacy of picroliv, a potent antioxidant derived from the plant Picrorhiza kurrooa, in protecting against hepatic ischemia-reperfusion injury in vivo. Picroliv was fed to male Sprague Dawley rats in a dose of 12 mg/kg once daily by oral gavage for 7 days prior to hepatic ischemia. Ischemia was induced by occluding the hepatic pedicel with a microaneurysm clip for 30 min and reperfusion was allowed thereafter for varying period (15-120 min) by releasing the microaneurysm clip. Picroliv pretreatment resulted in better hepatocyte glycogen preservation and reduced apoptosis. Reduction in apoptosis was associated with decreased mRNA expression of caspase-3 and Fas. Oxidant induced cellular damage as measured by tissue malondialdehyde (MDA) levels was significantly less following picroliv pretreatment. Both a reduction in neutrophil infiltration and an increased level of intracellular antioxidant enzyme superoxide dismutase possibly contributed to the reduction in tissue lipid peroxidation. Tissue inflammatory cytokines level of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) was also lower in picroliv group. Furthermore, picroliv pretreatment resulted in enhanced proliferating cell nuclear antigen (PCNA) immunoreactivity. These studies strongly suggest picroliv to be a promising agent for ameliorating injury following ischemia-reperfusion.

Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv.

Picroliv is a potent antioxidant extracted from the roots and rhizome of Picrorhiza kurrooa. It has been shown to impart significant hepatoprotective activities, partly by modulation of free radical-induced lipid peroxidation. Lipid peroxidation and reactive oxygen species are associated with tissue injury in post-ischemic acute renal failure. The efficacy of picroliv was assessed in an in vivo model of renal ischemia-reperfusion injury (IRI) in rats at a dose of 12 mg/kg orally for 7 days. The animals were killed at various times after reperfusion. Increased lipid peroxidation and apoptotic cell number reflected the oxidative damage following renal IRI. Picroliv-pretreated rats exhibited lower lipid peroxidation, improved antioxidant status, and reduced apoptosis, indicating better viability of renal cells. Immunohistochemical studies revealed that picroliv pretreatment attenuated the expression of intercellular adhesion molecule-1 in the glomerular region. These results suggested that picroliv pretreatment protects rat kidneys from IRI, perhaps by modulation of free radical damage and adhesion molecules.

Immunochemotherapy for Leishmania donovani infection in golden hamsters: combinatorial action of poly ICLC plus L-arginine and sodium stibogluconate (Stibanate).

We demonstrate that golden hamsters infected with Leishmania donovani amastigotes develop the capacity to eliminate intracellular pathogens on treatment with low-dose standard antileishmanial sodium stibogluconate (Stibanate) in combination with polyinosinic-polycytidilic acid stabilized with polylysine and carboxymethycellulose (poly ICLC), a potent inducer of interferon (IFN) and immune enhancer, plus L-arginine. Data suggest that low doses of both Stibanate and poly ICLC plus L-arginine provide marginal inhibition against L. donovani infection in golden hamsters. When given in combination, however, a significant inhibition was achieved without toxicity, as all the animals survived up to 45 or 60 days. These results suggest that combination therapy using Stibanate and poly ICLC plus L-arginine may be very effective in reducing the dose of Stibanate and, hence, its dose-dependent toxicity in clinical situations.

Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice.

Tissue repair and wound healing are complex processes that involve inflammation, granulation and tissue remodeling. Interactions of different cells, extracellular matrix proteins and their receptors are involved in wound healing, and are mediated by cytokines and growth factors. Previous studies from our laboratory have shown that curcumin (diferuloylmethane), a natural product obtained from the rhizomes of Curcuma longa, enhanced cutaneous wound healing in rats and guinea pigs. In this study, we have evaluated the efficacy of curcumin treatment by oral and topical applications on impaired wound healing in diabetic rats and genetically diabetic mice using a full thickness cutaneous punch wound model. Wounds of animals treated with curcumin showed earlier re-epithelialization, improved neovascularization, increased migration of various cells including dermal myofibroblasts, fibroblasts, and macrophages into the wound bed, and a higher collagen content. Immunohistochemical localization showed an increase in transforming growth factor-beta1 in curcumin-treated wounds compared to controls. Enhanced transforming growth factor-beta1 mRNA expression in treated wounds was confirmed by in situ hybridization, and laser scan cytometry. A delay in the apoptosis patterns was seen in diabetic wounds compared to curcumin treated wounds as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridyl triphosphate nick end labeling analysis. Curcumin was effective both orally and topically. These results show that curcumin enhanced wound repair in diabetic impaired healing, and could be developed as a pharmacological agent in such clinical settings.