Microbiol. Res., Volume 2, Issue 1 (June 2011) – 10 articles

Resistance to β-lactam antibiotics by gramnegative bacteria, especially Escherichia coli (E. coli), is a major public health issue worldwide. The predominant resistance mechanism in gram negative bacteria particularly E. coli is via the production of extended spectrum beta lactamase (ESBLs) enzymes. In recent years, the prevalence of β-lactamase producing organisms is increased and identification of these isolates by using disk diffusion method and no-one else is not satisfactory. So, this investigation focused on evaluating the prevalence of ESBL enzymes by disk diffusion method and confirmatory test (Combined Disk). Five hundred clinical samples were collected and 200 E. coli isolates were detected by standard biochemical tests. To performing initial screening of ESBLs was used from Disk diffusion method on E. coli isolates. A confirmation test (Combined Disk method) was performed on isolates of resistant to cephalosporin's indicators. Up to 70% isolates exhibited the Multi Drug Resistance phenotype. In Disk diffusion method, 128(64%) E. coli isolates which resistant to ceftazidime and cefotaxime while in Combined Disk, among 128 screened isolates, 115 (89.8%) isolates were detected as ESBLs producers. This survey indicate beta lactamase enzymes are playing a significant role in antibiotic resistance and correct detection of them in phenotypic test by using disk diffusion and combined Disk is essential for accurate recognition of ESBLs. Full article

Japanese encephalitis virus (JEV) replicates in a variety of cells, the exact intracellular site of virus assembly is somewhat obscure. The aims of this study were to investigate the role Golgi apparatus in JEV maturation by utilizing two Golgi-disrupting agents- brefeldin A (BFA) and monensin (MN) that inhibit virus assembly at specific cellular sites. JEV-infected porcine kidney stable (PS) cells were treated with BFA (2 ug/ mL) or MN (10 uM/ mL) at different h post-infection (p. i.) and the virus contents were assayed after 48 h p. i. The treated cells were further subjected to immuno-fluorescence (IF) using antibodies directed against JEV envelope glycoprotein (gpE) for localization of intracellular viral antigen as well as the antigen expression on the cell surface. Addition of BFA or MN to cells immediately after virus adsorption or at 4 h and 12 h postinfection (p. i.), resulted in 4- or 8- fold reduction in infectious virus contents along with inhibition of its transport to the cell surface, indicating an essential role of the Golgi-associated membranes in JEV replication. Interestingly, the antigenicity of the virus, in contrast, remained unaffected as no difference in epitope presentation/ expression was observed in BFA/MN-treated and control (untreated) infected cells even though in the former cells a loss of hemagglutinating (HA) activity was observed. Further, BFA addition at 18 h or 24 h p. i. showed only a negligible effect on virus suggesting that once the viral-associated membranes are formed, these membranes appear to be stable. In contrast, the inhibition with MN persisted even after its addition to cells at 18 h and 24 h p. i., indicating its sustained effect on JEV. Although BFA inhibits protein transport from endoplasmic reticulum (ER) to the Golgi complex while MN inhibits transport from medial to trans cisternae of the Golgi complex, none of the two agents however affected the gpE synthesis and folding essentially required for the epitope presentation/expression within the cells. As flaviviruses are known to encode three glycoproteins (gps) within their genomes i. e., prM, E, and NS, it will be worthwhile in future to determine whether vesicular transport occurs within or between the virus-induced membranes and how the individual JEV-encoded proteins are transported to discrete compartments further remain to be seen. Full article

There are very few reports on agarases being produced by actinobacteria, Streptomyces coelicolor being the only one known since decades for its agar degrading property. Here we report an agar degrading diazotrophic actinobacterium other than Streptomyces coelicolor, isolated from the littoral soil of Lonar Lake situated in Buldhana district of Maharashtra, India, a lake characterised by high alkalinity, carbonates, bicarbonates, and algal blooms. The lake has a mean diameter of 1800 m. The Gram-positive filamentous rod grew in a simple medium of pH 10.5 containing agar as a sole source of carbon. The agar degrading property was detected by the appearance of depressions around each colony after 48 h of growth. The enzyme responsible for this degradation, agarase was also detected and estimated. The isolate also grew on Ashby’s Nitrogen free Mannitol Medium aerobically and fixed nitrogen. Morphological, physiological and biochemical characteristics of the isolate are presented in this paper. Full article

This randomized controlled study evaluated the association of Helicobacter pylori (H. pylori) with chronic periodontitis patients with and without type II Diabetes Mellitus. H. pylori is considered to be a pathogen responsible for gastritis, peptic ulcers and a risk factor for gastric cancer. The aim of the present study was to evaluate the association of H. pylori with chronic periodontitis patients with and without type II diabetes mellitus before and after treatment. The prevalence of H. pylori in periodontal pockets was determined by rapid urease test in a 36 patients, which were grouped as Group 1 (Healthy subjects), Group II (chronic periodontitis patients) and Group III (Chronic periodontitis patients with Type II Diabetes Mellitus), 12 in each group before treatment by collecting plaque samples. After treatment, 12 plaque samples were collected and prevalence H. pylori was detected. Group II and Group III had a significantly higher rate of positive results for H. pylori compared to healthy subjects before treatment. After treatment, H. pylori were not detected in Group II and in Group III Only one of 12 chronic periodontitis patients with Type II diabetes mellitus had H. pylori in the periodontal pocket. The prevalence of H. pylori did not differ significantly between the chronic periodontitis patients with and without type II diabetes mellitus. Meticulous scaling and root planning will reduce the prevalence of H. pylori in periodontal pockets. Full article

Opportunistic infections and invasive primary tumors represent major causes of morbidity and mortality in HIV-1-infected individuals. HIV-1 involvement of the central nervous system (CNS) affects nearly half of seropositive patients, being the primary CNS lymphoma (PCNSL) a hallmark neoplasia of this population. Interestingly, the incidence of other brain tumors (e.g. gliomas) is exceedingly rare in AIDS patients, and their co-morbidity has been limited to case reports. Here, we share our 20-year experience following brain tumors in HIV-1/AIDS patients from major referral hospitals in Mexico and Brazil. Additionally, we provide the most updated compilation of reported glioma cases in AIDS patients, with a thorough epidemiological analysis. Furthermore, we discuss HIV-1-driven mechanisms that would theoretically increase malignant transformation of glial cells; while offering newly reported explanations as to why protease inhibitors, key components of multi-drug anti-retroviral schemes, may be responsible for such a low co-incidence of gliomas in HIV-1 infected individuals.
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In Mycobacterium tuberculosis, genomic variation is generated mainly by insertions and deletions rather than by point mutations. RvD5 is one such deletion in M. tuberculosis H37Rv. Previous studies from our laboratory have shown the presence of moaA3 gene in the RvD5 region in a large number of clinical isolates, that is absent in M. tuberculosis H37Rv and H37Ra. The present study was aimed at investigating the RvD5 locus of the clinical isolates by a detailed PCR analysis. Here we report a new point of insertion of the mobile genetic element, IS6110 in the genome of one clinical isolate of M. tuberculosis. The insertion has disrupted the moaB3 gene, one of the ORFs in the RvD5 region, which is involved in the molybdopterin biosynthetic pathway. This insertion of IS6110 in the moaB3 of the clinical isolate is different when compared to the insertion in the moaB3 gene of M. tuberculosis H37Rv where 4kb RvD5 region has been lost by homologous recombination and only a truncated form of the gene is present. This finding is of relevance since IS6110 is a major element determining the genome plasticity of M. tuberculosis and its numerical and positional polymorphism has always been of special interest. Full article

Deletion in chemokine receptor CCR5 gene is reported to prevent development of tubal pathology among Dutch Caucasian C. trachomatis infected women. Hence, a pilot study was undertaken, to evaluate the involvement of CCR5 gene in tubal pathology among Indian women with or without Chlamydia infection. Three hundred women with or without Chlamydia infection and with different reproductive manifestations were screened for CCR5 gene using a standardized PCR. Only 6 C. trachomatis infected women without tubal block had heterozygous deletion for this gene. The rest had wild type CCR5 gene, that includes fertile women infected and non infected; infertile women infected and non infected with and without tubal block as well as infected women with history of recurrent spontaneous abortions. The study indicates no role of heterozygous CCR5 D32 gene deletion in prevention of tubal pathology in Chlamydia infected Indian women. Only 2% heterozygous deletion in CCR5 gene is observed in the studied population. Full article

The decreased vancomycin susceptibility and subsequent emergence of vancomycin resistant Staphylococcus aureus (VRSA) strains already multi-resistant to antibiotics is a major public health problem. In 2009, the Clinical and Laboratory Standards Institute (CLSI) altered its guidelines for vancomycin susceptibility testing in S. aureus and recent data suggests the possibility that VRSA may emerge more frequently than previously expected. Against this background, we conducted a study to ascertain the susceptibility status of clinical S. aureus isolates to vancomycin in our environment using vancomycin agar screen, disk diffusion and broth dilution methods. Of the total 49 S. aureus invasive strains isolated, 25 (51.0%) had vancomycin MIC of ≤2µg/ml by the CLSI standard broth dilution method and are classed as vancomycin susceptible; 18 (36.7%) had MIC of 4-8µg/ml (vancomycin intermediate resistant) and 6 (12.2%) had MIC of >256µg/ml (high level vancomycin resistant). Vancomycin agar screen with Mueller-Hinton agar containing 3µg/ml vancomycin (MHA-V3) correctly identified 20 of 25 (80%) vancomycin susceptible isolates; detected all 6 vancomycin resistant isolates and 16 of 18 (88.9%) vancomycin intermediate strains. Similarly, Mueller-Hinton agar containing 6µg/ml vancomycin (MHA-V6) correctly identified 23 of 25 (92%) vancomycin susceptible isolates and all 6 vancomycin resistant isolates but detected 14 (77.8%) of 18 vancomycin intermediate strains. Vancomycin disk diffusion test correctly identified all the 25 vancomycin susceptible S. aureus isolates giving 100% specificity but detected only 1 of 18 (5.6%) vancomycin intermediate and none (0%) of vancomycin resistant isolates. This result shows the occurrence of VISA and high level VRSA isolates in our environment, which contrary to current belief, may indicate widespread dissemination of VRSA. MHA-V3 agar is a useful alternative screening medium for vancomycin non-susceptibility detection in clinical S. aureus isolates but vancomycin disk diffusion is not useful in this regard. Full article

ORF69 (Ac69) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in Spodoptera frugiperda Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an ac69 knockout AcMNPV bacmid mutant and analyses of the influence of ac69 deletion on the viral infectivity in Sf-9 cells and Trichoplusia ni larvae so as to investigate the role of ac69 in the viral life cycle. Results indicated that ac69 deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect AcMNPV infectivity for Trichoplusia ni larvae in LD₅₀ and LT₅₀ bioassay when administered orally. These results suggest that ac69 may be dispensable for viral infectivity both in vitro and in vivo. Full article

Linezolid is approved for complicated and uncomplicated skin and soft tissue infections. We have evaluated the efficacy of this drug in murine as well as in rat skin and soft tissue infection models using Staphylococcus aureus ATCC and clinical strains. In thigh infection model the dose of linezolid required for more than 1 log₁₀ kill from baseline inoculum in neutropenic mice and rats was 100 mg/kg and 50 mg/Kg, b.i.d/day, respectively, which was 5 and 4 folds more than that in immunocompetent animals, respectively. Dose required to achieve 1 log₁₀ killing was similar against different strains of S. aureus in immunocompetent mouse thigh infection model. However, in murine groin abscess infection model, a dose of 100 mg/kg, b.i.d/day of linezolid produce static effect in 2 days, but revealed to be superior in 4 days treatment and showed approximately 1 log₁₀ killing from base line inoculums. Based upon pharmacokinetic profile, a 24-h AUC/MIC required for linezolid efficacy in murine groin abscess model was 91.5 for the strain used in this study. As linezolid is taken as a gold standard drug in the evaluation of new chemical entity, this data could be useful for comparing the preclinical efficacy of new anti-MRSA agents. Full article