IN VITRO MEROPENEM SUSCEPTIBILITY INDUCED BY PH ALTERATION IN METALLO-BETA-LACTAMASE POSITIVE PSEUDOMONAS AERUGINOSA

ABSTRACTObjective: The increasing emergence of metallo-beta-lactamase (MβL) producing Gram-negative bacteria such as Pseudomonas aeruginosa posea serious public health concern including in the treatment of urinary tract infection (UTI). This study was aimed to explore the combined effect ofin vitro pH alteration and antibiotic on the bacterial growth as a potential therapeutic approach against the drug resistant P. aeruginosa.Methods: Ten MβL producing P. aeruginosa isolates from the patients suffering from UTI were included. Bacteria were inoculated with or withoutmeropenem in media with varied pH range from 5 to 10. The variation of bacterial growth was determined by measuring the changes of opticaldensity at 620 nm and colony forming unit counts/ml.Result: The growth of bacteria was reduced both at very high and lower pH ranges. However, the growth was further reduced significantly with theaddition of meropenem at these extreme pH conditions.Conclusion: Alteration of pH especially at lower range of the medium might has changed the efficacy of MβL and thus helped the antibiotic meropenemto act on the bacteria, which was resistant toward the same at neutral pH. Combined effect of antibiotic and pH modulation of biological fluid like urineshould be explored for an effective alternative therapeutic approach against the drug-resistant bacteria.Keywords: Metallo-beta-lactamase, urinary tract infection, Pseudomonas aeruginosa, pH

ASSOCIATION OF GENERATION TIME WITH ANTI-TUBERCULAR DRUG(S) RESISTANCE PATTERN OF MYCOBACTERIUM TUBERCULOSIS ISOLATES AMONG TREATMENT FAILURE PULMONARY TUBERCULOSIS PATIENTS

ABSTRACTObjective: The emergence of drug resistance has complicated tuberculosis (TB) scenario and is associated to treatment failure. The causative agent,Mycobacterium tuberculosis is usually slow growing and has been implicated as a contributing factor for drug tolerance and development of resistantstrains. On the other hand, if rapidly growing bacilli, with shorter generation time emerge, mutations may lead to the development of drug resistance.From the hypothesis, this study was aimed to explore the whether there is any association between the generation time of Mycobacteria with theirdistinct drug resistant pattern.Methods: In-vitro generation time was determined from 77 mycobacterial isolates with varied drug resistance pattern, i.e. rifampicin resistant (RIFR),isoniazid resistant, multi-drug resistant (MDR), the sensitive clinical strains along with reference strains. The minimal inhibitory concentration wasalso determined for the respective resistant groups.Results: Among the individual group of clinical isolates, there was a significant negative association between generation time and drug resistancepattern of RIFR isolates.Conclusion: Keeping the current upsurge of the MDR-TB epidemic in India and the influence of generation time on dosing schedule and treatmentstrategy, necessary customization of dosing and therapeutic planning seemed urgent to minimize the operational and clinical potential for developmentof drug resistance among treatment failure pulmonary TB patients in this country.Keywords: Mycobacterium tuberculosis, Generation time, Multi-drug resistant, Treatment failure

PRIMARY CELL CULTURE OF AEDES ALBOPICTUS MIDGUT CELLS: A PROSPECTIVE MODEL FOR IN VITRO STUDY OF ARBOVIRUSES

  Objective: Midgut cells play a key role in the propagation of mosquito borne Arboviruses. The existing mosquito cell lines for studying viral pathogenesis are derived either from larvae or from eggs since there is no cell line available from the mosquito midgut. Therefore, to delineate the in situ viral interaction which naturally occurs within the mosquito midgut and represent cellular pathogenesis in human beings, the present work was aimed to develop a primary cell line from the midgut cells of Aedes albopictus.Methods: The midgut cells of A. albopictus were collected, cultured and incubated at 28°C to study the growth after every 24 hrs for 7 days.Result: The primary cell culture showed an increasing growth pattern of columnar cells up to 48 hrs followed by decrease in cell population afterward. However, the number of stem cells increased significantly throughout the study period, and their population outnumbered the columnar cells after 72 hrs. There was no significant change of goblet cells and regenerating cells which were scanty in number throughout the experiment.Conclusion: The present method will help to develop the individual cell lines from mosquito midgut and study the host pathogen interaction in arboviral diseases in future

ORAL ITRACONAZOLE FOR THE TREATMENT OF SEVERE SEBORRHOEIC DERMATITIS

Background : Seborrheic dermatitis (SD) is an inflammatory skin disorder in which colonies of Malassezia furfur have been found in affected areas. Aim: The aim of this study was to evaluate the efficacy of itraconazole in the treatment of severe SD. Materials and Methods: Itraconazole was given to 30 patients of SD in a dose of 100 mg twice daily for 1 week followed by 200 mg/day for first 2 days of the following 2 months. The response was noted on day 15, 30, 60, and 90. The clinical response was graded as markedly effective, effective, or ineffective. Results: Clinical improvement (evaluated as markedly effective or effective) was observed in 83.3% cases. Conclusion : The anti-inflammatory activity of oral itraconazole suggests that it should be the first-line therapy in severe SD

Bacteriological study of aerobic isolates from plantar ulcers of paucibacillary leprosy patients

Background: Plantar ulcers commonly occur in leprosy patients, which usually recur and cause morbidity in such cases. Aims: The aim of the study is to find out the bacteriological profile of these ulcers and to find out the antibiotic susceptibility of the isolates so that appropriate drugs may be chosen for treatment and for prevention of recurrence. Materials and Methods: Fifty-six samples from recurrent plantar ulcers of paucibacillary leprosy patients (attending the outpatient department of Calcutta School of Tropical Medicine) were studied for the purpose. Proper sample collection, gram staining, inoculation on culture media, and final identification by biochemical methods were undertaken. Antibiotic susceptibility testing was done for appropriate choice of drugs. Results: Mixed growth of bacteria was seen in 20 (36%) cases while single organism was isolated from the rest. Staphylococcus aureus is the predominant single isolate followed by E. coil, Proteus sp. and Pseudomonas sp. Chloramphenicol and gentamycin are the two drugs that have shown efficacy to the extent of 75 to 100% and 25 to 100% respectively in vitro studies. Conclusion: Bacteriological study of plantar ulcers of leprosy patients has revealed Staphylococcus aureus as the main pathogen. Treatment with chloramphenicol and gentamycin holds good prospect as per our study

Changes in viral load in different organs of Japanese Encephalitis virus-infected chick embryo under the influence of Belladonna 200C

Background: Japanese encephalitis(JE) is highly prevalent in many states of India. Belladonna 200C is widely used in the prevention and treatment of JE.The effect of Belladonna 200C in virus replication inside different tissues has not been studied. Objective: To study the effect of Belladonna 200C in virus replication inside different tissues utilising chick embryo model. Materials and Methods: Twelve-day-old fertilised eggs of Black Australorp were inoculated with JE via chorioallantoic membrane (CAM) route in different experimental sets: infection, Belladonna 200C treated and vehicle control, keeping matched blank sets. All experimental sets were incubated for 48 hours. After incubation, viable eggs were sacrificed humanly and different tissues were observed and collected for viral load determination by real-time-polymerase chain reaction (PCR). Results: The control group showed visible pocks over the CAM; brains were liquefied due to haemorrhagic liquefactive necrosis and white patches were found over the liver. However, the medicine-treated group was apparently normal; there were no visible changes in the brain and the liver was healthy like control. Real-time-PCR results showed high viral load in CAM and brain with absence of viral RNA in liver of the virus-infected group. Pre-treatment with Belladonna 200C significantly reduced the overall load (P < 0.05) in CAM and brain which correlated with the morbid pathological changes of the organs. Conclusion: Although Belladonna 200C did not completely inhibit JE viral replication in the brain, it reduced the severity of JE by diminishing the viral loads in this tissue

Immune Subversion by <i>Mycobacterium tuberculosis</i> through CCR5 Mediated Signaling: Involvement of IL-10

<div><p>Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During <i>Mycobacterium tuberculosis</i> infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that <i>Mycobacterium</i> infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, <i>in vivo</i> CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.</p></div

IL-10 augments the CCR5 expression in <i>H37Rv</i> infected macrophages via involving STAT3.

<p>Bone marrow derived macrophages (2×10⁶cells/ml) were pretreated with either anti IL-10 Ab (10 ug/ml) or with control siRNA and STAT3-specific siRNA and then infected with <i>Mycobacterium tuberculosis H37Rv</i> (MOI = 1∶10). Changes in messenger RNA (mRNA) expression of CCR5 and GAPDH were determined by semi quantitative RT-PCR (A). In a separate set, the pretreated and infected macrophages were lysed and subjected to Western blot with anti-CCR5 antibody as described in Materials and Methods (B). Infected macrophages were analyzed by flow cytometry for CCR5 (PE) expression as described in figure legend 1 (C). Data represented here are from one of three independent experiments, all of which yielded similar results. Murine macrophages (1×10⁶cells/ml) were treated with anti IL-10 Ab for 1 h and then subsequently followed by <i>Mycobacterium</i> tuberculosis infection for 45 min. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to acetylated H3 (IP acetyl-H3) (D) or STAT-3 (E), and conventional RT-PCR was performed using primers specific to the CCR5 promoter. Data represented here are from one of three independent experiments, all of which yielded similar results.</p

CCR5 silencing enhances pro inflammatory cytokine production and MHC-II expression in <i>H37Rv</i> infected C57BL/6 mice.

<p>C57BL/6 mice were transfected with CCR5 shRNA or control shRNA as described in materials and methods section prior to <i>Mycobacterium</i> infection. After 28 days of infection, the lung homogenates were assayed for the cytokine levels by ELISA as described in Methods (A–E). Data represented as means ± SD for 5 animals per group. ***<i>P</i><.001 and **<i>P</i><.05 for the comparison with infected mice. Total lung RNA was extracted and Changes in messenger RNA (mRNA) expression of IL-10, TGF-β, TNF-α, IL-12, IFN-γ and GAPDH were determined by semi quantitative RT-PCR (F). Data represented here are from one of three independent experiments, all of which yielded similar results. In separate experimental set, the lung homogenates were analyzed by flow cytometry for MHC-II (PE) expression as described in figure legend 1(G). Data represented here are from one of three independent experiments, all of which yielded similar results.</p