Iranian Journal of Microbiology
Volume:6 Issue: 3, Jun 2014

Pseudomonas aeruginosa is a Gram-negative bacterium that considered as important opportunistic human pathogen. One of the mechanisms that help bacteria to tolerate survival in adverse conditions and resistance to antibiotics is biofilm formation through quorum sensing (QS) signals and toxin-antitoxin (TA) systems. QS and TA are two systems that have important roles in biofilm formation. QS is a global regulatory mechanism that enable bacteria to communicate with each other by production of auto inducers (AI) molecules in population. Because of importance biofilm formation in P. aeruginosa infections, here, we studied frequency of QS and TA genes among clinical isolates of P. aeruginosa with ability of biofilm formation. One hundred and forty clinical isolates of P. aeruginosa were collected from Tehran and Ilam hospitals. The isolates were identified by biochemical tests. Biofilm formation was evaluated by microplate method. After DNA extraction by boiling method, the frequency of QS genes (lasIR, rhlIR), and TA genes (mazEF, relBE, hipBA, ccdAB and mqsR) were analyzed by PCR. Our results showed that maximum resistance is related to aztreonam (72.85%) antibiotic. Most of isolates were able to produce biofilm (87.15%) and the majority of them formed strong biofilm (56.42%). PCR results showed that frequency of mazEF, relBE, hipBA, ccdAB, mqsR, lasIR and rhlIR genes were 85.71, 100, 1.42, 100, 57.14, 93.57 and 83.57 percent, respectively. Clinical isolates of P. aeruginosa had high ability to form biofilm, and QS and TA system genes among these isolates were very high (except hipBA genes). There are significaut correlation between biofilm for mation and present of QS and TA system genes.

Rapid diagnosis of pertussis is important for the timely isolation of the infection source and early prevention measures among the contact persons, especially among non-vaccinated infants for whom pertussis is life-threatening. Targets IS481, IS1001, BP0026 and human GAPDH gene were used to develop a multiplex real-time PCR assay based on the TaqMan technology for detection and identification of Bordetella pertussis and Bordetella parapertussis in clinical samples. A total of 121 human clinical specimens obtained within 2012-2013 were used to evaluate the multiplex real-time PCR assay. Clinical specimens were also tested for culture and conventional PCR. Sensitivity and specificity for culture, conventional PCR, and multiplex real-time PCR were measured in comparison with a clinical standard for B. pertussis infection. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for IS481, IS1001 and 10 genomic equivalents per reaction for BP0026 target. When the B. pertussis assays were compared with a clinical standard for B. pertussis infection, sensitivity was 5, 59 and 89% the specificity was 100, 100 and 100% for culture, conventional PCR, and multiplex real-time PCR, respectively. Developed multiplex real-time PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections which is suitable for implementation in a routine laboratory diagnostics.

Essential oils are complex mixtures containing compounds of several different functional- group classes. Depending on the structure, we can distinguish monoterpenes, phenylpropanes, and other components. Here in this study two monoterpene compounds of essential oils, i.e. β-pinene and limonene were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1) in vitro. All antiviral assays were performed using RC-37 cells. Cytotoxicity was determined in a neutral red assay, antiviral assays were performed with HSV-1 strain KOS. The mode of antiviral action was evaluated at different periods during the viral replication cycle. Acyclovir was used as positive antiviral control. Beta-pinenene and limonenen reduced viral infectivity by 100%. The mode of antiviral action has been determined, only moderate antiviral effects were revealed by monoterpenes when these drugs were added to host cells prior infection or after entry of HSV into cells. However, both monoterpenes exhibited high anti-HSV-1 activity by direct interaction with free virus particles. Both tested drugs interacted with HSV-1 in a dose-dependent manner thereby inactivating viral infection. These results suggest that monoterpenes in essential oils exhibit antiherpetic activity in the early phase of viral multiplication and might be used as potential antiviral agents.

The therapeutic options for diseases caused by Escherichia coli are limited. In this study we investigated the presence of virulence factors among Enteropathogenic Escherichia coli (EPEC) strains and their antibiotic resistance patterns. The isolates were also checked for the presence of class1 integrons and gene cassettes. This study included 70 EPEC strains isolated from children. Antimicrobial resistance patterns were determined using diffusion methods. The broth microdilution methods was used to determine the minimum inhibitory concentration. PCR was used to detect eaeA, bfpA genes. The 5' and 3' conserved sequences (CSs) of class 1 integrons and intI gene were amplified to investigate the presence of integrons and gene cassettes. Antimicrobial susceptibility testing showed that 4 (5.7%), 3 (4.2%), and 2 (2.8 %) isolates were resistant to ampicillin, trimethoprim-sulfamethoxazole, and ceftazidime, respectively. Resistance rates to ciprofloxacin and aztreonam were 1.4%. Thirteen (18.5%) isolates showed resistance to tetracycline, and 4 (5.7%) were kanamycin resistant. Class I integron detected in 22 (31.4%) isolates. All the gene cassettes found in class I integrons corresponded to different variants of dfr and aadA genes. Prevalence of class I integrons in EPEC strains was high. Presence of aadA and dfr gene cassettes in integrons represents high distribution of resistance determinants in EPEC strains.

Reports on MRSA strains are increasing worldwide. The aim of this study was to find the prevalence of MRSA strains isolated from clinical specimens and to evaluate their resistance profile. Additionally we compared the phenotypic and genotypic methods for detection of methicillin resistance. In this cross-sectional study, a total of 41 isolates of S. aureus were collected from clinical specimens at two teaching hospitals in Ardabil, Iran. All isolates were identified at the species level by standard biochemical tests. The methicillin resistance were evaluated using three PCR for mecA gene, agar dilution for determination of oxacillin MIC and disk diffusion test to detect methicillin, oxacillin and cefoxitin resistance. Antimicrobial resistance patterns were determined by disk diffusion method. The results identified 19 (46.3 %) out of 41 isolates as MRSA. Most of the MRSA strains (68.4%) were isolated from patients hospitalized in ICU. All isolates were susceptible to vancomycin, mupirocin and linezolid. Among other antibiotics co-trimoxazole was more active against MRSA isolates. Using PCR as reference method all the phenotypic tests showed 100% specificity. The sensitivity for MIC test and cefoxitin was 100% and for methicillin and oxacillin disks was 77.7% and 89.5%, respectively. The prevalence of MRSA strains in our hospitals especially in ICU ward was high and disk diffusion testing using cefoxitin or oxacillin MIC test as an alternative to PCR for detection of MRSA is recommended.

Diarrheal disease is still a major health problem, especially in developing countries, where it is considered as one of the leading causes of morbidity and mortality especially in children. Studies showed that Diarrheagenic E.coli (DEC) such as STES and EPEC strains are among the most prevalent causative agents in acute diarrhea, particularly in children. Aim of the present study was to investigate the presence and the frequency of STEC and EPEC as etiologic agent of diarrhea in children less than 2 years of age with diarrhea in Shiraz. A total of 285 stool samples were collected from patients with diarrhea in Shiraz, in 2012. Diarrheagenic E.coli (DEC) strains were isolated by standard biochemical analysis. In this study, we used multiplex Real time PCR and single PCR to detect the presence of indicator genes stx1, stx2 and eaeA for STEC and EPEC strains, respectively. A total of 285 stool samples were tested in which 49 (17%) were identified as contaminated with E.coli by biochemical tests. Out of total samples, 15 STEC (31%) and 13 EPEC (27%) were identified using multiplex Real-Time PCR assay. Among STEC isolates, 2 strains were stx1+, 8 isolates stx2+, 3 isolates were stx1+, stx2+ and 2 isolates were stx1+, stx2+, eaeA+. In this study, we found rather high occurrence of STEC and EPEC virulence genes in children with diarrhea. The results of this study showed that, real time PCR can be used as a replacement for conventional PCR assay in the detecting virulence genes of STEC and EPEC strains. Real-time PCR offers the advantage of being a faster, more robust assay, because it does not require post-PCR procedures to detect amplification products.

Staphylococcus aureus (S. aureus) nasal carriage may be responsible for some serious infections in hemodialyzed patients. The main target of this study was to estimate the prevalence of S. aureus nasal carriage in hemodialysis outpatients and medical staff in hemodialysis centers specifically in Fez region. The second target is to identify the risks of colonization, resistance pattern of isolates and their virulence toxin genes.Patients and Nasal swab specimens were obtained from 143 hemodialyzed outpatients and 32 medical staff from January to June 2012. Each participant completed a short questionnaire. Nasal carriage of S. aureus was demographically related (age, gender, hemodialysis duration), comorbidity (diabetes, malignancy) and exposure to health care (dialysis staff, hospitalization). PCR were used on all the isolates in the research of twelve staphylococcal enterotoxins genes. Also, PCR was used to investigate on the three factors epidermal cell differentiation inhibitors; three exfoliatin toxins; two leukotoxins; the toxic shock syndrome toxin-1 and the hemolysin beta genes. Nasal screening revealed 38.16%, 50% and 18.75% S. aureus carries in chronic, acute hemodialysis patients and medical staff, respectively. Only young participants were likely to be S. aureus carries (p = 0.002). there were no gender differences between the isolate carriers and non-carriers or some comorbidity factors such as viral hepatitis B and C, HIV infections, diabetes, chronic smoking, recent hospitalization or antibiotic therapy. Out of all isolates, only one (1.61%) was methicillin-resistant and Twenty-one (33.87%) had at least two virulence toxin genes. Knowledge and monitoring of antibiotic resistance profile and virulence of S. aureus carriage are essential in the treatment of infections generated by this pathogen, as well as in the control of clonal dissemination and prevent the spread of S. aureus resistance.

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. 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. 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. 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.

Tetanus and diphtheria are vaccine-preventable, infectious diseases with significant morbidity and mortality. Immunization by the diphtheria and tetanus toxoid (DT) has been applied in Iran for almost 50 years. However, there are very few data about the rate of immunity to these diseases in the adult population. the humoral immunity to tetanus and diphtheria among blood donors in Arak city, central provice of Iran were investigated.Patients and A total of 530 consecutive blood donor samples were collected from Blood Transfusion Organization, Central province of Iran. All samples were tested for diphteria and tetanus IgG antibodies using enzyme-linked immunosorbent assay (ELISA). From 530 cases, 91.9% were male and 8.1% were female. 99.6% of cases had protective levels of diphtheria antibody. Protective levels of tetanus antibody were found in 96% of subjects. There was not any significant difference between diphtheria and tetanus antibodies levels and age and sex. The obtained data showed that high proportion of the adult population in Arak have sufficient protection against diphtheria and tetanus. The high protective level of immunity to diphtheria and tetanus in Iran can be due to widespread use of booster vaccines in Iranian high schools and during the military services or for pregnant women in their 3rd trimester.

Neisseria gonorrhoeae is the second most sexually transmitted diseases agents in developing countries. Antimicrobial resistance strains have created serious health concern. The aim of this study was to determine the frequency of endocervical gonococcal infection and antimicrobial susceptibility of N.gonorrhoeae in Kashan, Iran. In this study, 294 endocervical swabs were collected from married women referred to the obstetrics and gynecology clinics in Kashan from December 2012 to May 2013. The samples were cultured in modified Thayer Martin in 37°C with 5-10% CO2 for 72 hours. Gram staining, oxidase, catalase and carbohydrate utilization tests were used to identify the isolated species. All isolates were tested for their susceptibilities to antimicrobials using the Kirby Bauer-disk diffusion techniques. N.gonorrhoeae was detected in 2.38% of studied cases (95% confidence interval [CI] 1.5-3.26%). All isolates were resistance to ceftriaxone, penicillin G, ciprofloxacin, cefepime, and two isolate (28.5%) showed intermediate sensitivity to tetracycline. Continued monitoring of prevalence of N.gonorrhoeae is important for preventing the dissemination of this microorganism. The present study emphasizes the importance of surveillance of antimicrobial resistance of N.gonorrhoeae in order to manage the rate of resistant strains and to revise the treatment policies.