Larvicidal Effects of Metabolites Extracted from Nocardia and Streptomyces Species against the Forth Larval Stage of Anopheles stephensi (Diptera: Culicidae).

Background: Larvicidal agents can be produced using microbial resources, which are environmentally friendly, biodegradable, and economical. The study's goal was to evaluate the larvicidal activity of metabolites isolated from Nocardia (N. fluminea, N. soli and N. pseudobrasiliensis) and Streptomyces (S. alboflavus) bacterial species against Anopheles stephensi. Methods: Four metabolites isolated from Nocardia and Streptomyces strains were exanimated for larvicidal activity. The experiments were performed for 24, 48, and 72 hours. 300, 350, 400, 450, 500, 550, and 600 µl of Actinobacteria metabolites were added to 100 cc of dechlorinated water. Fourth-stage larvae were placed in dechlorinated water as a control. LC50 and LC90 were calculated using toxicity data and analyzed. Results: All metabolites had a statistically significant influence on mosquito larvae (P< 0.05). At 24, 48, and 72 hours, the LC50 for N2 (N. fluminea) was 417, 386, and 370 ppm, respectively, and the LC90 was 650, 595, and 561 ppm. Moreover, LC50 for N4 (N. soli) was 389, 376, and 347 and LC90 were 591, 565, and 533 and LC50 for N5 (N. pseudobrasiliensis) was 390, 357, and 341 ppm and LC90 were 589, 532 ppm. In addition, LC50 for S921 (S. alboflavus) was 484, 416, and 382 ppm, and LC90 was 701, 612, and 574 ppm. Conclusion: The four bacterial metabolites tested in our study were found to have a notable effect on the mortality rate of Anopheles stephensi larvae, indicating their potential as natural larvicides. This is an effective technique for controlling Anopheles stephensi that has no detrimental environmental impact.

Transcriptional alteration of genes linked to gastritis concerning Helicobacter pylori infection status and its virulence factors.

BACKGROUND: Helicobacter pylori infection and heterogeneity in its pathogenesis could describe diversity in the expression of inflammatory genes in the gastric tissue. We aimed to investigate transcriptional alteration of genes linked to gastritis concerning the H. pylori infection status and its virulence factors. METHODS AND RESULTS: Biopsy samples of 12 infected and 12 non-infected patients with H. pylori that showed moderate chronic gastritis were selected for transcriptional analysis. Genotyping of H. pylori strains was done using PCR and relative expression of inflammatory genes was compared between the infected and non-infected patients using relative quantitative real-time PCR. Positive correlations between transcriptional changes of IL8 with TNF-α and Noxo1 in the infected and TNF-α with Noxo1, MMP7, and Atp4A in the non-infected patients were detected. Six distinct genotypes of H. pylori were detected that showed no correlation with gender, ethnicity, age, endoscopic findings, and transcriptional levels of host genes. Irrespective of the characterized genotypes, our results showed overexpression of TNF-α, MMP7, Noxo1, and ATP4A in the infected and IL-8, Noxo1, and ATP4A in the non-infected patients. CONCLUSIONS: A complexity in transcription of genes respective to the characterized H. pylori genotypes in the infected patients was detected in our study. The observed difference in co-regulation of genes linked to gastritis in the infected and non-infected patients proposed involvement of different regulatory pathways in the inflammation of the gastric tissue in the studied groups.

Evaluation of anti-biofilm potential of biosurfactant extracted from Nocardia species.

INTRODUCTION: Bacterial natural products such as biosurfactants and surface-active agents are important compounds which exhibit many applications in the fields of medicine. AIM: The aim of the present study was to isolate and identify Nocardia strains with high biosurfactant production and antibiofilm ability. MATERIALS AND METHODS: In the present study, a biosurfactant producing Nocardia species was isolated and identified by a laboratory method. Nocardia species were initially screened and then tested for their ability to produce biosurfactant. The oil spreading test and the surface tension measurements showed that one strain was a biosurfactant producer. The strain with the best surface activity results was selected for further studies and identified by 16S rRNA gene sequencing method. Fourier transform infrared spectroscopy (FTIR) and compositional analysis proved a biosurfactant structure. RESULTS: Oil spreading test and blue agar plate test confirmed biosurfactants and extracellular anionic glycolipids. E24% assay using olive oil revealed strong emulsifying characteristic of the extracted biosurfactant with 100% emulsifying strength. FTIR spectrum indicated the presence of aliphatic hydrocarbon chain (lipid) along with the polysaccharide portion, confirming the glycolipid nature of the biosurfactant. The stability of the biosurfactant produced in different conditions was significant. Increasing concentration of BS significantly inhibited Pseudomonas aeruginosa biofilm. CONCLUSIONS: N. coubleae can be a representative of the genus Nocardia for the production of biosurfactants with beneficial physicochemical properties.

Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

Nocardia isolation from clinical samples with the paraffin baiting technique.

BACKGROUND: The genus Nocardia is a cause of infection in the lungs, skin, brain, cerebrospinal fluid, eyes, joints and kidneys. Nocardia isolation from polymicrobial specimens is difficult due to its slow growth. Several methods have been reported for Nocardia isolation from clinical samples. In the current study, we used three methods: paraffin baiting technique, paraffin agar, and conventional media for Nocardia isolation from various clinical specimens from Iranian patients. METHODS: In this study, we examined 517 samples from various clinical specimens such as: sputum of patients with suspected tuberculosis, bronchoalveolar lavage, sputum of patients with cystic fibrosis, tracheal aspirate, cutaneous and subcutaneous abscesses, cerebrospinal fluid, dental abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. All collected specimens were cultured on carbon-free broth tubes (paraffin baiting technique), paraffin agar, Sabouraud dextrose agar, and Sabouraud dextrose agar with cycloheximide and were incubated at 35°C for one month. RESULTS: Seven Nocardia spp. were isolated with paraffin baiting technique, compared with 5 positive results with the paraffin agar technique and 3 positive results with Sabouraud dextrose agar with and without cycloheximide. The prevalence of nocardial infections in our specimens was 5.28%. CONCLUSION: In the present study, the use of the paraffin baiting technique appeared to be more effective than other methods for Nocardia isolation from various clinical specimens.

DNA extraction from nocardia species for special genes analysis using PCR.

BACKGROUND: Nocardia species have a complex cell wall structure similar to that of mycobacteria, and the extraction of DNA from this bacterium is extremely difficult. Currently, to identify Nocardia species particularly, it is essential to utilize molecular techniques. AIMS: In the present study, we investigated STET (sodium chloride-TRIS-EDTA-triton) buffer for the extraction of high-quality genomic DNA from 20 clinical and environmental isolates. MATERIALS AND METHODS: The extracted DNA was evaluated for portion of the 16S rRNA, 65-kDa heat-shock protein and 16S rRNA genes via polymerase chain reaction. RESULTS: The extracted DNA had high molecular mass, and its concentration and purity was suitable when tested in 1% agarose gel, and using UV spectrophotometry. Amplification of three different genes was successfully performed. CONCLUSION: This paper reveals an inexpensive, reproducible and efficient method of DNA extraction from Nocardia species, which is appropriate for accurate identification of this bacterium via polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism.

Diagnostic efficacy of lsa63 antigen for human leptospirosis.

BACKGROUND: Timely diagnosis of leptospirosis is essential for early and effective treatment, for there are many differential diagnoses for it.. Leptospiral researchers have an increasing interest in developing new serological methods with recombinant antigens to improve the Leptospirosis diagnosis. Several serological tests have been developed for the proper diagnosis of leptospirosis. OBJECTIVES: To improve the previous works we developed an enzyme linked immunosorbent assay (ELISA) with novel recombinant leptospiral surface adhesion (Lsa63) protein to offer a new test. MATERIALS AND METHODS: In an experimental study, Recombinant Lsa63 (rLsa63) was produced in Escherishia coli (E.coli) BL21 (DE3). By using rLsa63, we generated IgM and IgG ELISA. Performance of these tests was compared to microscopic agglutination golden test (MAT). Two hundred twenty human serum samples were obtained from individuals suspicious of leptospirosis who were referred to Guilan Province Central Leptospira Laboratory for definitive diagnosis. The sensitivity, specificity and other statistical indexes of Lsa63-ELISAs were also determined. RESULTS: Among 220 serum samples, 30% (n = 65) had positive MAT responses, and also 38% (n = 84) and 40.9% (n = 90) showed positive reaction to IgG and IgM rLsa63-ELISA, respectively. The sensitivity, specificity and accuracy were 93.8%, 81.29 % and 85.0 for IgM-Lsa63- ELISA and 83.07, 80, 64 and 81.36 for IgG-Lsa63- ELISA, respectively. CONCLUSIONS: Our results demonstrated that the sensitivity and specificity of Lsa63-ELISAs are promising for the detection of Leptospira serovars.

Pulmonary nocardiosis associated with cerebral abscess successfully treated by co-trimoxazole: a case report.

Nocardiosis is an acute or chronic infectious disease caused by the soil-borne filamentous bacteria belonging to the genus Nocardia. The organisms opportunistically infect both immunocompromised and immunocompetent individuals. The lungs are the primary site of infection and brain abscess is, by far, the most common complication following nocardial metastasis from pulmonary lesions. Although surgical intervention must always be considered in the treatment of nocardial brain abscess, it can obviously be cured by antibiotic therapy alone. This report describes a case infected by Nocardia cyriacigeorgica. Identification of the infectious agent was achieved by conventional and semi-nested PCR techniques. A 55-year-old woman with fever was referred to the infect disclinic of Imam Khomeini hospital in Tehran and was hospitalized after clinical assessment. She was a kidney transplant recipient for 4 years and was taking immunosuppressive treatment including azathioprine and methylprednisolone. Follow-up of the patient by CT scan revealed pulmonary infection and cerebral lesions. Specimens of the brain lesions contained filamentous bacteria. The patient received a combination of co-trimoxazole and ceftriaxone and brain abscesses as well as lung inflammation disappeared gradually during the course of antibiotic therapy within 3 months. The patient was discharged from the hospital after 2 months of therapy.

Evaluation of New ELISA based on rLsa63 - rLipL32 antigens for serodiagnosis of Human Leptospirosis.

BACKGROUND AND OBJECTIVES: Timely diagnosis of leptospirosis is essential for an effective treatment. Large diversity of clinical symptoms has led leptospirosis diagnosis difficult. Researchers have conducted many tests with wide-range of sensitivity and specificity to achieve novel diagnostic procedures which have higher sensitivity and specificity compared with previous tests and which are more reliable and available to public laboratories. This study aimed to introduce Lsa63 and LipL32 proteins-based ELISA tests with more sensitivity, specificity, accuracy and convenience for public laboratories. MATERIALS AND METHODS: Recombinant forms of Lsa63 and LipL32 proteins were first generated. After coating these proteins, IgM and IgG ELISA tests were performed. 220 patients with suspicion of leptospirosis infection were selected for serum collection. The sera tests were carried out using MAT, IgM and IgG ELISA tests. In order to assess the performance of ELISA, the results of this test were compared with MAT. RESULTS: 30% of serum samples (n=65) in MAT were positive for leptospirosis infection, while ELISA tests including rLipL32- rLsa63-IgM and rLipL32-rLsa63-IgG showed 40.45% (n=89) and 38.63% (n=80) positive reaction, respectively. CONCLUSION: Our results demonstrated that new ELISA tests based on mixing LipL32 and Lsa63 proteins, a novel mixture of recombinant antigens, are valuable to detect specific antibodies against pathogenic Leptospira in human serum and could be considered as helpful techniques in leptospirosis diagnosis.

Molecular analysis of vanHAX element in vancomycin resistant enterococci isolated from hospitalized patients in Tehran.

BACKGROUND: Vancomycin (glycopeptide)-resistant enterococci (VRE or GRE) can cause serious problems for hospitalized patients due to the limited options for treatment of VRE infections. As infection with VRE increases in hospitals, further knowledge about vancomycin resistant genes is needed. METHODS: Isolates of Enterococcus spp. were collected from hospitalized patients in Tehran (Iran) during 2006. Detailed molecular analysis was performed for vancomycin resistance genotype and vanHAX using conventional PCR and PCR- RFLP (restriction fragment length polymorphism), respectively. RESULTS: out of 830 enterococci spp., 48 VRE isolates (5.8 percent) were obtained. All of VRE isolates carried vanA gene. DdeI digestion of vanHAX element showed the presence of point mutation at 8234 position. CONCLUSION: This study indicates that vanA is a predominant genotype in Iranian isolates. In addition, PCR-RFLP analysis revealed the presence of two types of vanHAX element in vanA harboring transposons.