International Journal of Molecular and Clinical Microbiology
Volume:1 Issue: 2, Summer and Autumn 2011

In this study, Substitution at codon Ser315 of katG gene, a reliable marker for isoniazid (INH) resistance was analyzed and compared by three molecular methods such as DNA sequencing, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and PCR-single strand conformation polymorphism (PCR-SSCP) in 105 phenotypically resistant isolates obtained from various parts of India. Out of the 105 resistant isolates, 64 (61%) were found to be resistant by DNA sequencing, 54 (51%) by PCR-RFLP and 57 (54%) by PCR-SSCP methods. The results obtained using PCR-SSCP and PCR-RFLP methods were compared with those from DNA sequencing (gold standard). The sensitivity and specificity of PCR-RFLP were 84% and 100% respectively and corresponding values for PCR-SSCP method were 89% and 95% respectively. The study has shown the comparison of the simple, rapid and cost effective methods with DNA sequencing targeting codon Ser315 of katG gene and suggests that PCR-RFLP and PCRSSCP may be performed as alternative inexpensive methods in settings with a high prevalence of INH-resistant M. tuberculosis strains where sequencing cannot be afforded.

AIDS (Acquired Immune Deficiency Syndrome), a result of human HIV (Human Immunodeficinency Virus) infection, is one of the most troublesome world©s health problems. Extensive researches to find effective drugs for its treatment are running fast in huge capacities. IMOD (Immuno-Modulator Drug) is the name of an herbal drug that has modulatory effects of immune system. As a goal of this research, IMOD was tested to determine its effect on HIV-infected cells, and whether it can inhibit viral P24 gag protein production or not. Human PBMCs (Peripheral Blood Mononuclear Cell) isolated from peripheral blood by Ficoll-gradient centrifugation from two groups including 15 HIVinfected patients and 5 non-infected persons as the control group. Then the cells were cultured and the plates were incubated for 3 days in a humidified Co2 incubator at 37C.Then IMOD and AZT (Zidovodine) were added to the progeny of each flask. After 48 hours, the amount of P24 in supernatant was evaluated by Enzyme-linked immunosorbent assay (ELISA). It was demonstrated that the concentration of P24 in AZT treated flask was low but in IMOD treated flasks was variable. Our study reveals that IMOD couldn©t inhibit P24 production in HIV positive individuals, although this conclusion is only based in vitro study and specific research which must be tested in vivo. In conclusion, although AZT group has higher viral load but it can release P24 antigen more than IMOD treated groups.

Highly stabilized monodispersed silver nanoparticles (AgNPs) were synthesized by Rhizopus stolonifer and the antifungal efficacy of silver nanoparticles (AgNPs) against Candida sp. were studied. Characterization of biosynthesized nanosilver was made by TEM-EDS and AFM. Minimum Inhibitory Concentration (MIC) of biosynthesized AgNPs, Amphotericin B, and Fluconazole have been studied on pathogenic fungi and the changes on membrane reactions have been elucidated by Scanning Electron Microscopy (SEM). The present study indicates AgNPs has considerable antifungal activity comparison with other antifungal drugs Nanosilver showed potent activity against pathogenic fungi.The results showed nano-Ag exerted activity on the mycelia. Thus, the present study indicates nano-Ag may have effective antifungal activity, deserves further investigation for clinical applications.

Non-gonococcal infections causes are made up of Chlamydia trachomatis (one of the most common infection Non-Gonococcal), Ureaplasma urealyticum (a possible causal in acute pyelonephritis, pelvic inflammatory disease, curiuamniutis, bacterial vaginosis, preterm birth, Non-Gonococcal urethritis, miscarriage, infertility and spontaneous abortion), Gardnerella vaginalis and Streptococcus agalactiae (could be lead to colonization and disease in newborns). This study was achieved on 100 pregnant women that were conferred to the Alavi hospital of Ardabil city and done at the winter 1389 to late spring 1390. Three swab of vaginal discharge and 2 ml of blood were obtained from each woman. The samples were cultured on specific environments and analyzed by enzyme linked immunosorbent assay (ELISA). Finally, Ureaplasma urealyticum, Chlamydia trachomatis, Gardnerella vaginalis and Streptococcus agalactiae were isolated. In 26 cases Chlamydia trachomatis (%28.6), 18 cases Ureaplasma urealyticum (%19.8), 28 cases Gardnerella vaginalis (%30.8) and 18 cases Streptococcus agalactiae (%19.8), were isolated. Women who were spending their third trimester, had high outbreak chance rather than other women to these infections.

Substitution at codon Ser315 of katG gene، a reliable marker for isoniazid (INH) resistance and proliferate in spite of the loss of cell wall is called an L- form. Unstable L- forms can convert to normal cells but stble L-forms can not. cell wall - deffective state may be induced spontaneously or through the action of an appropriate agent such as antibiotic interfering with the synthesis of cell wall peptidoglycan. Vancomycin is one of these antibiotics which cause L-form formation in some strains of Staphylococcus aureus in presence of osmoprotective compounds. Horse serum and some of organic or mineral materials can be used for better isolation of L- forms in vitro. In this study، standard strain of Staphylococcus aureus (ATCC 25923) was inoculated to 2 media: i) L phase medium (LPM) agar containing horse serum & ii) LPM agar without horse serum. Sucrose was used in both above mentioned growth media as osmoprotective and vancomycin was used for inducing L-forms. Microscopic examination revealed presence of small and large colonies on LPM agar lacking horse serum and only large colonies on LPM agar containing horse serum after 3 - 4 days. induced by horse serum were unable to form colony in LPM agar containing horse serum. Results showed that unstable L-forms of Staphylococcus aureus in the presence of horse serum neither can survive nor convert to normal cells.

Orange peel extract solution contains mucilage flavor and three types of glycosides. Limonene is one of the terpenoides in some vegetables, fruits and food; that plays as an antioxidant in juice. As it has anti-microorganism effects on some bacteria, we carried out the antifungal effects of limonene versus orange extract. In the present study the effects of orange extract and two types of limonene against Candida albicans, Aspergillus niger, Aspergillus sp. and Penicillium sp. has been studied using agar well diffusion and paper disc diffusion. (S)-(-)-Limonene had more inhibitory effect on examined fungi; in both well diffusion, and paper disc diffusion experiments. Aspergillus niger was more sensitive related other fungi. Orange peel extract didnt show any antifungal activity on tested fungi.The results showed that orange peel extract does not have any antifungal activity. However Limonene (S)-(-) type has been shown to have inhibitory effects on filamentous fungi.

Isoniazid، is the only antituberculous drug for which the relation between lack of virulence and acquisition of resistance was associated. INH-resistant mutants were shown to contain defective katG gene. Classical studies showed that INH-resistant south Indian isolates have lower virulence in guinea pigs and higher susceptibility to H2O2. It is of interest to assess the virulence in south Indian clinical mutants of KatG (catalase-peroxidase enzyme) associated with INH resistance. Five INH-resistant clinical isolates were selected on the basis of mutation in katG gene. Mutant isolates were used for infecting macrophage cell line (THP-1) and for the assessment of enzyme activity. In comparison to control H37Rv، Ser315Thr mutant exhibits similar virulence and reduction (4% and 17%) in catalase (C) and peroxidase (P) activity. For the mutants، Ser315Iso and Ser315Arg، the virulent nature was slightly reduced with 3% and 2%، and significant reduction was observed in the C (50%; 47%) and P (36%; 43%) activity respectively. Ser315Asn indicates 4% reduction in virulence with significant reduction in C (56%) and P (64%) activity. Asn138Ser mutant displayed low-level virulence، CP activity showed 47% and 56% reduction. The results indicate that similar level of virulence in all the mutants except Asn138Ser which showed relatively low-level whereas a significant decrease in C and P activity was observed in all mutants except Ser315Thr. The study suggests despite the fact that all mutants do not compromise survival or virulence yet provides INH resistance.

30 milled rice samples were collected from retailers in four states of Malaysia. These samples were evaluated for Eurotium spp. contaminations by direct plating on malt extract salt agar (MESA). All Eurotium were isolated and identified based on morphology and nucleotide sequences of internal transcribed spacer 1 (ITS1) and ITS2 of the rDNA. Four Eurotium species (E. rubrum, E. amstelodami, E. chevalieri and E. cristatum) dominated seed samples were identified. The main characteristics for morphological differentiation of Eurotium species were colony features on different culture media and ascospore surface ornamentations. The PCR-sequencing technique for sequences of ITS1 and ITS2 is a fast technique for identification of Eurotium species, but did not work perfectly for differentiating Eurotium species from each others. DNA sequence analysis showed a fixed sequence numbers in both ITS1 and ITS2 regions. These results suggest that sequencing of ITS regions could support morphological characteristics for identification of Eurotium species.

Clinical signs, para-clinical tests and gross lesion can be used for identification of parasite contamination. In most parasite contamination the clinical signs are not obvious. Thus, para-clinical tests and gross lesion play important roles in identifying parasite disease as all organs of animals such as skin, eyes, kidneys, liver and digestive tube can be affected by the parasites. Consequently, there is a possibility to use the feces of animals in order to observe parasite ovum and larva to identify the disease.This research was conducted from May to September 2011, and the number of 250 faecal samples of pigeon from mixed companion birds keeping in the cages. Faecal samples were examined by direct smear method whereas egg per gram (EPG) was counted by modified McMaster technique and centrifugal flotation method using Sheather’s saturated sugar solution. In this study, Raillietina spp, Tetramers, Sygnamus, Capillaria, Ascaridia colombae and oocyst protozoa, Phthiraptera, Ceratophyllus columbae contamination were studied and the frequency were found to be 24%, 8%, 9%, 14%, 4%, 7%, 8% and 6% respectively. The result of this study and the finding from other studies indicates that pigeons could be less susceptible to mixed infections as compared to chickens.

In many parts of the world there is a rich tradition in the use of herbal medicine for the treatment of many infectious diseases. Because of the side effects and the resistance that pathogenic microorganisms build against the antibiotics, much recent attention has been paid to extract the biologically active compounds from plant species used in herbal medicine. In this study different parts of Phoenix dactylifera from Kerman-Bam region were collected. Plant samples were dried in shade and extracted with methanol, chloroform and aqueous by maceration method for 10 days at room temperature. Microorganisms (five gram negative and three gram positive bacteria) were cultured on brain heart infusion agar and antibacterial activity tested by agar well diffusion assay. As a precaution for not missing any trace amounts of antimicrobials, a concentration of 40 mg/ml of each extract was prepared in dimethyl sulfoxide: methanol (1:1 v/v) solvent and administered in each well. Cultured plates were incubated at 35oC. After 48 hours the bioactivity was determined by the measurement of the diameter of inhibition zones (DIZ). Finally MIC and MBC were determined. Some extracts showed antibacterial activity against some bacteria and methalonic extract of palm seed had the most effects. In gram positive bacteria, Staphylococcus aureus (PTCC 1112) and in the gram negative bacteria Escherichia coli (PTCC 1330) were the most sensitive bacteria. MIC and MBC value were 1.25 and 2.5 mg/ml, respectively. According to the results from this study, it is suggested that different parts of Phoenix dactylifera may be used in the treatment of the infections including gram positive bacteria. Efforts should go on to screen more local flora in different regions, as many investigations have shown that environment is very effective in biological properties in plants.