Jundishapur Journal of Microbiology
Year:2016 | Volume:9 | Issue:6|Papers Count:12

Background: Mycobacterium tuberculosis is the major pathogen of tuberculosis, which affects approximately one-third of the world’ s population. The 6 kDa early secreted antigenic target (ESAT6) and the 10 kDa culture filtrate protein (CFP10), which are secreted by the ESX-1 system in M. tuberculosis, can contribute to mycobacterial virulence. Objectives: The aim of this study was to research the effects of M. tuberculosis ESAT6-CFP10 protein on macrophages during a host’ s was first and second exposures to M. tuberculosis. Materials and Methods: In this study, the ESAT6 and CFP10 genes were amplified to create a fusion gene (ESAT6-CFP10) and cloned into the pET-32a(+) and pEGFP-N1 expression vectors, respectively. The recombinant pET-32a(+)-ESAT6-CFP10 plasmid was transformed into the Escherichia coli Origami strain, and the fusion protein was expressed and confirmed by SDS-PAGE andWestern blot analysis. The recombinant pEGFP-N1-ESAT6-CFP10 plasmid was transfected into rat alveolar macrophage cells (NR8383). The cell line expressing the ESAT6-CFP10 protein was selected with RT-PCR and designated as NR8383-EC. Finally, the effects of the ESAT6-CFP10 fusion protein on the NR8383 cell line, as well as on the newly constructed NR8383-EC cells, were further assessed. Results: The recombinant ESAT6-CFP10 protein wasexpressed in E. coliandin NR8383 rat alveolar macrophages. This protein affected the proliferation and nitric oxide (NO) generation of the NR8383 and NR8383-EC cells. AlthoughNOgeneration was inhibited in both cell lines, proliferation was inhibited in NR8383 while it was increased NR8383-EC. Conclusions: The data indicate that ESAT6-CFP10 could support the survival of M. tuberculosis in the host through altering the host immune response. It also indicates that the host may gain some level of protection from a second exposure to M. tuberculosis, as evidenced by increased proliferation of NR8383-EC.

Background: Biofilm formation is a primary cause of considerable bacterial destruction. Objectives: In an effort to combat these industrial and medical bacterial biofilm problems, our study aims to determine the antimicrobial effect of Euphorbia hebecarpa. Materials and Methods: The inhibition efficiency of alcoholic extracts on the planktonic form of six pathogenic bacteria was evaluated using a disk diffusion technique. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values were determined by means of a macrobroth dilution method. The effects of the extracts on biofilms were calculated using a microtiter plate method. Results: The results of the disk diffusion assay (MBC and MIC) confirmed that E. hebecarpa ethanolic extracts were more efficient than methanolic extracts in the inhibition of planktonic forms of bacteria. Also, the inhibitory effect of the extracts in a broth medium was greater than in a solid medium. Extracts of E. hebecarpa were found to inhibit biofilm formation better than demolish of biofilm and preventing metabolic activity of bacteria in biofilm structures. The greatest inhibitory effects of E. hebecarpa extracts were observed for the biofilm formation of B. cereus (92. 81%). In addition, the greatest demolition was observed for the S. aureus biofilm (74. 49%), and the metabolic activity decrement of this bacteria was highest (78. 21%) of all the tested bacteria. Conclusions: The results of this study suggest that E. hebecarpa extracts can be used to inhibit the planktonic and biofilm forms of these selected bacteria.

Background: The endophytes of medicinal plants, such as Justicia adhatoda L., represent a promising and largely underexplored domain that is considered as a repository of biologically active compounds. Objectives: The aim of present study was isolation, identification, and biological evaluation of endophytic fungi associated with the J. adhatoda L. plant for the production of antimicrobial, antioxidant, and cytotoxic compounds Materials and Methods: Endophytic fungi associated with the J. adhatoda L. plant were isolated from healthy plant parts and taxonomically characterized through morphological, microscopic, and 18S rDNA sequencing methods. The screening for bioactive metabolite production was achieved using ethyl acetate extracts, followed by the optimization of different parameters for maximum production of bioactive metabolites. Crude and partially purified extracts were used to determine the antimicrobial, antioxidant, and cytotoxic potential Results: Out of six endophytic fungal isolates, Chaetomium sp. NF15 showed the most promising biological activity and was selected for detailed study. The crude ethyl acetate extract of NF15 isolate after cultivation under optimized culture conditions showed promising antimicrobial activity, with significant inhibition of the clinical isolates of Staphylococcus aureus (87%, n=42), Pseudomonas aeruginosa (> 85%, n = 41), and Candida albicans (62%, n = 24). Conclusions: The present study confirms the notion of selecting endophytic fungi of medicinal plant Justicia for the bioassayguided isolation of its bioactive compounds, and demonstrates that endophytic fungus Chaetomium sp. NF15 could be a potential source of bioactive metabolites.

Background: Spinosad (a mixture of spinosyns A and D) is a unique natural pesticide produced by Saccharopolyspora spinosa. With regard to attempts to improve S. spinosa by classical mutagenesis, we propose that the bottleneck of screening out high-spinosadproduction strains is probably caused by the fermentation media. Objectives: The current study aimed to identify a new medium to extensively investigate the potential of S. spinosa strains to produce spinosad. Materials and Methods: Statistical and regressive modeling methods were used to investigate the effects of the carbon source and to optimize the production media. Results: The spinosad production of S. spinosa Co121 increased 77. 13%, from 310. 44 21. 84 g/mL in the initial fermentation medium (with glucose as the main carbon source) to 549. 89  38. 59  g/mL in a new optimized fermentation medium (98. 0 g of mannitol, 43. 0 g of cottonseed flour, 12. 9 g of corn steep liquor, 0. 5 g of KH2PO4, and 3. 0 g of CaCO3 in 1 L of H2O; pH was adjusted to 7. 0 before autoclaving). After screening 4, 000 strains, an overall 3. 33-fold increase was observed in spinosad titers, starting from the parental strain Co121 in the original fermentation medium and ending with the mutant strain J78 (1035  34  g/mL) in the optimized medium. Conclusions: The optimized fermentation medium developed in this study can probably be used to improve spinosad production in screening industrial strains of S. spinosa.

Background: Enteroinvasive Escherichia coli (EIEC) isolates cause dysentery in humans. Several virulence factors associated with EIEC pathogenesis have been characterized. Multilocus variable-number tandem-repeat analysis (MLVA) is a PCR-based method that has been used for genotyping bacterial pathogens. Objectives: The aim of this study was to investigate the distribution of virulence factor genes in EIEC isolates from patients with diarrhea in Kerman, Iran, as well as the genetic relationships between these isolates. Patients and Methods: A total of 620 diarrheic stool samples were collected from patients attending two hospitals in Kerman from June 2013 to August 2014. All isolates were confirmed as EIEC by PCR for the ipaH gene. The EIEC isolates were evaluated by PCR for the presence of nine virulence genes (ial, set1A, sen, virF, invE, sat, sigA, pic, and sepA). MLVA was performed for all EIEC isolates. Results: A total of 11 EIEC isolates were identified, and all were positive for the ial gene. The invE and virF genes were observed in 81. 8% of the isolates, while sen, sigA, and pic were detected in 72. 7%, 63. 6%, and 27. 3% of the isolates, respectively. None of the isolates were positive for the sat, set, and sepA genes. Using MLVA, the 11 total isolates were divided into five types. Conclusions: By studying the profiles of virulence genes and MLVA, it can be concluded that EIEC isolates do not have high heterogeneity and are derived from a limited number of clones.

Introduction: The Streptococcus anginosus group of bacteria are low-virulence bacteria existing as commensals in the oral flora and gastrointestinal tracts of humans. S. anginosus may spread to the blood in individuals with poor oral hygiene in cases of oral infections, such as gingivitis and tooth abscesses, that develop following the loss of mucosal unity. This may lead to infections in the whole body, primarily as brain and liver abscesses. Case Presentation: A 32-year-old male patient presented with complaints of nausea, vomiting, and diffuse abdominal pain. Diffuse abdominal tenderness and rebound tenderness were detected particularly in the epigastrium and right upper quadrant. Laboratory assessment revealed a leukocyte count of 20, 500/mm3. Free fluid around the liver and heterogeneous areas of abscess formation in the right lateral gallbladder were revealed on abdominal computed tomography. Diffuse adhesions between the bowel and seropurulent free liquid in the abdomen were detected on surgical exploration, and a sample was taken for cultures. The patient was discharged without complications on the sixth postoperative day and his antibiotic course was completed with 4 weeks of oral treatment. We reviewed the literature for similar cases of disseminated pyogenic infections caused by the S. anginosus group. Conclusions: It should be kept in mind that the oral flora bacterium S. anginosus may cause transient bacteremia and deep-seated organ abscesses in immunodeficient patients with poor oral hygiene. Such patients with intra-abdominal abscesses should be treated with antibiotics and surgery.

Background: Haemophilus influenzae type b (Hib) is the leading cause of bacterial meningitis, otitis media, pneumonia, cellulitis, bacteremia, and septic arthritis in infants and young children. The Hib capsule contains the major virulence factor, and is composed of polyribosyl ribitol phosphate (PRP) that can induce immune system response. Vaccines consisting of Hib capsular polysaccharide (PRP) conjugated to a carrier protein are effective in the prevention of the infections. However, due to costly processes in PRP production, these vaccines are too expensive. Objectives: To enhance biomass, in this research we focused on optimizing Hib growth with respect to physical factors such as pH, temperature, and agitation by using a response surface methodology (RSM). Materials and Methods: We employed a central composite design (CCD) and a response surface methodology to determine the optimum cultivation conditions for growth and biomass production of H. influenzae type b. The treatment factors investigated were initial pH, agitation, and temperature, using shaking flasks. After Hib cultivation and determination of dry biomass, analysis of experimental data was performed by the RSM-CCD. Results: The model showed that temperature and pH had an interactive effect on Hib biomass production. The dry biomass produced in shaking flasks was about 5470 mg/L, which was under an initial pH of 8. 5, at 250 rpm and 35° C. Conclusions: We found CCD and RSM very effective in optimizing Hib culture conditions, and Hib biomass production was greatly influenced by pH and incubation temperature. Therefore, optimization of the growth factors to maximize Hib production can lead to 1) an increase in bacterial biomass and PRP productions, 2) lower vaccine prices, 3) vaccination of more susceptible populations, and 4) lower risk of Hib infections.

Background: Infections caused by Leishmania are becoming major public health problems on a global scale. Many species of Leishmania around the world are obtaining resistance levels of up to 15 folds, as estimated by theWorld Health Organization. Leishmania showing resistance is relatively difficult to observe and maintain in laboratory settings. Objectives: The current study deals with the generation of Leishmania tropica strains that are resistant to amphotericin B (amp B). Materials and Methods: The L. tropica strain was attenuated using continuous passaging 20 times. The infectivity of L. tropica was confirmed in BALB/c mice. The L. tropica resistant strain was produced in vitro using a continuous increase in drug pressure. The cross resistance of L. tropica to other drugs was also investigated. Results: After 20 continuous passages, the BALB/c mice tested negative in the development of leishmaniasis. At a concentration of 0. 1  g/mL, L. tropica showed resistance to amp B. The newly developed promastigotes were 16 times more resistant compared to the resistance of the wild type promastigotes. The resistant L. tropica strain showed cross resistance to itraconazole and had a resistance index that was greater than five. The resistant strain displayed maximum stability for more than three months in the drug-free medium. Conclusions: The resistant strain of L. tropica can be produced in laboratories using continuous drug pressure. The attenuated resistant strain has significant implications (both medically and academically) in the ability to overcome resistance.

Background: Diarrhea is the most frequent health problem among children in developing countries. Defining the etiology of acute diarrhea is critical to disease therapy and prevention. Some anaerobic bacteria such as Enterotoxigenic Bacteroides fragilis (ETBF) strains cause diarrheal disease by production of enterotoxin in children less than 5 years old. Objectives: This study aimed to evaluate the prevalence of ETBF among common bacteria and viruses causing diarrhea in children aged less than five years. Materials and Methods: One hundred diarrheal stools were cultured for detection of aerobic and anaerobic pathogen bacteria by direct plating on selective media and antibiotic susceptibility tests were performed according to clinical and laboratory standards institute (CLSI) guidelines on isolates of ETBF. The enterotoxigenic gene among B. fragilis isolates was also investigated using the polymerase chain reaction (PCR) method. Detection of viral pathogens was carried out using the latex agglutination test. Results: Ten B. fragilis were isolated from 100 diarrheal fecal specimens. All isolates were susceptible to metronidazole, while 10% were susceptible to clindamycin. Four (40%) ETBF were isolated. Rotaviruses (57. 2%) and adenoviruses (18. 6%) were the most frequently detected etiological agents. Conclusions: ETBF is one of the etiological agents that may cause diarrhea in children but it is not the commonest of them. Metronidazole is still an effective antibiotic against B. fragilis. Viruses are the most important etiological agents of diarrhea in children less than 5 years of age.

Objectives: This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. Materials and Methods: A variety of nucleic acid detection methods that target bothDNAand RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. Results: Of the 24 isolates, 21 had 350 bp bands (87. 5%) and three had 450 bp bands (12. 5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. Conclusions: The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for the disease are appearing in parts of this region.

Background: Bovine leptospirosis is a widespread zoonotic disease, leading to serious economic losses in animal production and causing potential hazards to human health. Leptospiral lipopolysaccharide (L-LPS) plays an important role in leptospirosis pathogenicity. Objectives: With respect to L-LPS endotoxin-like activity, we examined bovine immune response to L-LPS and the inhibitory ability of bovine myeloid antimicrobial peptide-28 (BMAP-28) against L-LPS-induced immune activation in bovine cells. Materials and Methods: In this study, L-LPS-induced proinflammatory cytokine production in bovine cells was quantitatively measured with real-time PCR and ELISA, and we determined which cell membrane receptors (toll-like receptor [TLR]2 and TLR4) played a major role. In addition, the ability of BMAP-28 to inhibit L-LPS-induced endotoxin-like immune activation in bovine cells was determined by the decrease in cytokine secretion. Results: L-LPS showed the ability to induce cytokine production in bovine cells, and its induction was TLR2-dependent. BMAP-28 was used to inhibit L-LPS-induced endotoxin-like activity. The function of BMAP-28 was to inhibit LPS-induced TLR2 expression and cytokine production. Conclusions: In this study, the L-LPS immune response of bovine cells was significant, indicating that TLR2 is the predominant receptor for L-LPS. Due to L-LPS endotoxin-like activity, we found a strategy through using BMAP-28 to prevent L-LPS-induced TLR2-dependent immune activation in bovine cells.

Background: Acute gastroenteritisstemmingfrom viral causes is verycommonduring the childhood period. Rotavirusandenteric adenovirus are the most common factors of acute gastroenteritis encountered in infants and children. However, the epidemiology of rotavirus and enteric adenovirus gastroenteritis in the east Anatolia region is not well-known. Objectives: Weaimedto evaluate the relationship between the distribution of antigen positivity in rotavirusandenteric adenovirus antigen tests required cases and demographic data retrospectively in pediatric patients admitted to our hospital. Patients and Methods: The records of stool sample analyses for 1154 patients admitted to our hospital from June 2011 to December 2011 with complaints of diarrhea were retrospectively examined. The presence of rotavirus and enteric adenovirus antigens in stool specimens was investigated by means of an immunochromatographic test. Results: Viral antigens were detected in 327 (28. 3%) stool specimens out of 1154. Among the positive results, the frequency was 73. 7% for rotavirus and 26. 2% for adenovirus. While the detected rotavirus antigen rate was high for all age groups, it was highest for children under the age of 2, with a rate of 57. 1%. Moreover, the rotavirus infections were observed at a rate of 44. 3% in winter and of 24. 6% in autumn. Conclusions: The most important factor in childhood acute gastroenteritis in east Anatolia is the rotavirus. Rotavirus and adenovirus antigens should be routinely investigated as a factor in fresh stool samples for the accurate diagnosis and treatment of gastroenteritis in children in the winter and autumn months.