JOURNAL OF MEDICAL MICROBIOLOGY AND INFECTIOUS DISEASES
Year:2013 | Volume:1 | Issue:1|Papers Count:8

Target modification and reduced drug accumulation are the main resistance mechanisms against fluoroquinolone antibiotics in Pseudomonas aeruginosa. We performed a genotypic characterization of three major Mex multidrug efflux pumps (MexAB-OprM, MexXY-OprM and MexCD-OprJ) in ciprofloxacin resistant clinical isolates of P. aeruginosa, collected from Tehran, Iran; this was followed by sequencing and analyzing the type II topoisomerases encoding gyrA, gyrB, parC, and parE genes. Reverse transcription PCR (RT-PCR) and semi-quantitative RT-PCR methods were used to analyse the transcription of efflux pumps. Topoisomerase mutation analysis was carried out through PCR amplification and sequencing of the quinolone resistance determining region (QRDR) of the topoisomerases encoding genes. Some 11.1% of the strains actively expressed MexCD-OprJ and 15.5% hyperexpressed MexXY-OprM efflux pumps. No overexpression was detected for MexAB-OprM, whereas 4.4% of strains showed simultaneous expression of MexCD-OprJ and MexXY-OprM. In the sequencing results, a single point mutation in the QRDR of gyrA was detected in all tested strains, where Isoleucine was substituted by Threonine at position 83. No mutations were detected in QRDR of gyrB, parC and parE genes. We are the first to report the genotypic analysis of ciprofloxacin mediated efflux pump resistance from Iran. These findings emphasize the clinical significance of multidrug efflux pumps and topoisomerases mutations activity in conferring resistance to fluoroquinolone antibiotics, and support the use of genotypic methods for analysis of resistance elements in P.aeruginosa in developing countries such as Iran.

Distinction between brucellar epididymo-orchitis (BEO) and nonspecific epididymo-orchitis (EO) is an important medical issue. This study was conducted to compare demographic, clinical and laboratory features, treatment and outcome of patients with BEO and nonspecific EO in Arak city, Markazi Province, Iran. We compared the clinical and laboratory characteristics of 40 BEO and 40 non-specific EO patients. The diagnosis of brucellosis was based on the symptoms, compatible clinical findings and standard tube agglutination test. Epididymo-orchitis was diagnosed by swelling and tenderness of scrotal skin, testis and epididymis, which was confirmed by sonography. BEO can be distinguished from nonspecific EO based on having a history of living in rural areas, contact with domestic animals, and consumption of unpasteurized dairy products. Other criteria include seasonal pattern, gradual onset (P<0.05), sweating (P<0.001), arthralgia (P=0.02), associated lower urinary tract symptoms (P=0.004) and lower rate of leukocytosis and abnormal urine analysis (P=0.002). Our results showed that brucellosis should be considered as a cause of EO in endemic areas like Iran. Combination antibiotic therapy to manage BEO is usually effective and all patients in this study responded quite satisfactory to the treatment.

In Leishmania (L.) major-infected BALB/c mice Th2-type cells results in disease progression, whereas C57BL/6- infected mice mount a Th1-type response, which leads to control of the infection. Th2 response correlates with IgG1 whereas, Th1 response supports switching to IgG2a subclasses. Since IgG isotype-dominated response depends on different CD4+T cell subsets, we studied the antigenic profile of L. major promastigotes that induce IgG1, IgG2a and IgG2b isotypes in BALB/c and C57BL/6 mice to provide insight into Th1 and Th2 inducing antigens. Humoral immune responses were studied in sera from L. major-infected BALB/c and C57BL/6 mice by ELISA and immunoblot analysis. These techniques were used to detect IgG, IgG1, IgG2a, and IgG2b antibodies against crude promastigote antigens. BALB/c mice showed higher IgG, IgG1 and IgG2a antibody levels compared to C57BL/6 mice (p<0.01). The IgG2a/IgG1 ratio was higher in C57BL/6 mice. There was no significant difference in IgG2b level between susceptible (BALB/c) and resistant (C57BL/6) mice. Western blot analysis revealed that 27, 32, 117, 125 and 153 kDa bands reacted only with IgG1 from both mice strains. The 41, 45, 47, 52 and 55 kDa bands reacted only with IgG2a from C57BL/6. A very similar pattern of immunostaining was observed for IgG2a and IgG2b in each mouse strain. The results from both mouse strains indicate that some antigens react exclusively with IgG1, the antibody, which may be involved in generation of a predominant Th2 response. The antigens that reacted strongly with IgG2a in C57BL/6 are potential candidates for eliciting protective Th1 response.

Candida is an asexual, diploid, dimorphic fungus that is present on human body and his environment. Nowadays the number of patients, who are immunocompromised, aged, receiving prolonged antibacterial and aggressive cancer chemotherapy or undergoing invasive surgical procedures and organ transplantation, is on increase, and therefore candidiasis emerged itself as an alarming opportunistic disease. The aim of this study is to identify the most common Candida species in clinical samples, and their antifungal susceptibility patterns. During a cross-sectional study performed in the Department of Microbiology and Serology, Narayana Hrudayalaya Hospitals (India) from January to December 2012, some 213 fungal isolates from various samples were collected. All the isolates were identified to the species level, using Vitek 2 YST identification card (bioMerieux, France). Antifungal sensitivity was performed against amphotericin B (AMB), 5 flucytosine (5-FC), fluconazole (FLU) and voriconazole (VOR) using ASTYS06 (bioMerieux, France). The majority of the isolates were from urine (48%) followed by respiratory (17%) and blood samples (16%). The most common species among the 213 isolates were Candida tropicalis (56%) followed by Candida albicans (33%). Non-albicans Candida species are emerging as the major pathogens and mainly seen in patients on prolonged ventilation and central lines. Antifungal agents should be used cautiously due to increased resistance seen in these agents.

Various adjuvants in combination with different antigens have been utilized as a vaccine candidate against leishmaniasis. However, the search for ideal adjuvants is still pursued due to the inefficiencies of current compounds. In the present study, the effect of imiquimod, as an adjuvant, is studied with soluble Leishmania antigens (SLA) in BALB/c mice. Four groups of mice were immunized with SLA, SLA plus imiquimod, SLA plus BCG, and PBS as control. Immunized mice received a boosting dose of SLA after 15 days. All groups were challenged with Leishmania major (L. major) promastigotes 2 weeks after the booster immunization. Our results showed that strong TH1 responses were induced in groups of SLA plus imiquimod as well as SLA plus BCGafter immunization. These responses included smaller footpad thickness, lower parasite load in lymph node, and higher proliferative response of lymph node cells to SLA, higher levels of interferon γ in culture supernatant of lymph node cells, and higher levels of IgG, and IgG2a in sera. The data supports the possibility of using imiquimod as a suitable adjuvant in leishmania vaccination.

Biofilm of Pseudomonas aeruginosa, an opportunistic pathogen, can cause serious health problems, such as chronic infections, especially in immunocompromised patients. Many studies have suggested administration of new generation of antibiotics, as P. aeruginosa biofilms have developed high resistance to antimicrobial drugs. This study reports the inhibitory effect of three medicinal plant extracts and an essential oil on biofilm formation by a clinical isolate of P. aeruginosa. In this study biofilm formation of P. aeruginosa strain 214 was determined in presence of three plant extracts, Cyclamen coum, Dianthus orieltalis and Origanum majorana, and Zataria multiflora Bio essential oil. Minimum Biofilm Inhibitory Concentrations (MBICs) were determined by microdilution techniques and XTT assay. The C. coam extract and Z. multiflora Bio essential oil inhibited biofilm formation completely at concentrations<0.062 mg/ml and 4 7l/ml, respectively. The D. orientalis and O. majorana extracts did not inhibit biofilm formation at the used concentrations (0.003–8 mg/ml). The results of this study indicate that some plant extracts at low concentrations may provide a complementary medication for biofilmassociated infections. Further evaluations are required to validate the antibiofilm effect of these medicinal plants.

Tuberculosis is a serious global public health problem and its high prevalence is strongly associated with the enhancement of drug resistance. In this study we demonstrate a multiplex allele-specific polymerase chain reaction (MAS) -PCR assay to simultaneously detect mutations in the first and third bases of the embB gene codon 306 ATG in ethambutol (EMB) resistant isolates of Mycobacterium tuberculosis. A total of 5029 patients were sampled between 2010 and 2012. All the specimens were examined microscopically for acid-fast bacilli and cultured in Lowenstein-Jensen medium. The susceptibility tests were performed for culture positive samples and the EMB resistant M. tuberculosis isolates were subjected to MAS-PCR targeting embB gene in the codon 306 ATG. Frothy eight of 176 isolates were EMB-resistant. None of the isolates showed mutation in the first base of the codon 306 ATG, but mutation in third base of the codon 306 ATG was detected in 14 isolates. We observed a correlation between culture-based phenotypic drug susceptibility and MAS-PCR method. The absence of mutation in resistant isolates can be attributed to possible involvement of other codon position at the same gene or other genes.

Pseudomonas aeruginosa is among the most important pathogens in the nosocomial infections. A genetic mobile element, the integron, is one of the major agents involved in dissemination of multi-drug resistance among gram negative bacteria. During a descriptive study from October 2009 to August 2010, some 130 P. aeruginosa clinical isolates were collected from different wards of three hospitals in Tehran. The Minimal inhibitory concentration (MIC) of 4 antibiotics conventionally used in clinical settings against the isolates was determined by E-test method. Also, the existence of integron classes and metallo-b-lactamases (blaVIM-1, blaIMP-1, and blaVIM-2) were investigated by PCR assay. Out of 130 isolates, 74 (56.9%) carried class 1 integron. None of the isolates harbored integrons class 2 and 3. Also, the blaVIM-1 gene was detected in 10 (13.3%) high level ceftazidime and imipenem- resistant isolates that carried class 1 integrons. The blaIMP-1 and blaVIM-2 genes were not detected in any isolates. In the present study, the antibiotic resistance rates in class 1 integron-positive isolates of P. aeruginosa were significantly higher than those lacking this integron, e.g., 82.6% resistance versus 17.3% sensitivity to ceftazidime. Also, 13.3% of ceftazidime and imipenem resistant isolates was metallo-b-lactamase producer. This indicates that all metallo-b-lactamase genes are correlated with class 1 integrons. These results imply that the blaVIM-1 gene has been presumably dispersed into P.aeruginosa isolates with the help of class 1 integron element.