Rapid Identification of Vancomycin Resistant Enterococcus Faecalis Clinical Isolates using a Sugar Fermentation Method.

INTRODUCTION: Vancomycin Resistant Enterococci (VRE) can be found all over the world. Thus, rapid detection of the isolates could be of high importance in the treatment or prevention of the associated disease. AIM: To measure the turanose fermentation in Enterococcus faecalis clinical isolates for rapid differentiation of VRE and Vancomycin-Susceptible E. faecalis (VSE) isolates. MATERIALS AND METHODS: Forty E. faecalis samples were isolated from 200 clinical samples in Tehran Medical Center, Iran, from October 2012 to December 2012. These isolates were detected according to the standard microbial and biochemical tests. Detection of VRE isolates was originally performed by disk diffusion using 1 µg vancomycin disk, followed by Polymerase Chain Reaction (PCR) amplification of the vanA gene. Finally, the turanose consumption in 1%, 0.7% and 0.5% dilutions was detected by a phenotypic method. RESULTS: Among the 40 E. faecalis isolates, 20 vancomycin-susceptible and 20 vancomycin-resistant E. faecalis were isolated according to the disk diffusion and PCR of the vanA gene. There was a considerable difference between VRE and VSE isolates in 0.7% dilution of turanose. However, there was no significant difference between VRE and VSE in 1% and 0.5% dilutions of turanose. CONCLUSION: Since detection of VRE isolates is of high importance, especially in nosocomial infections, phenotypic methods may be highly useful for this purpose. In conclusion, our data indicate that VRE isolated from clinical samples could be distinguished from VSE isolates by turanose fermentation at dilution 0.7%.

Genotypic Diversity of Methicillin-Resistant Coagulase-Negative Staphylococci Isolated from Inpatients and Outpatients.

We investigated the prevalence of methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolated from hospitalized patients and outpatients (OP). Out of 350 staphylococcal isolates collected from three hospitals, 190 were coagulase-negative staphylococci (CoNS). These isolates were subjected to antimicrobial susceptibility tests, detection of mecA, and pulsed-field gel electrophoresis (PFGE) typing. Among the 190 isolated CoNS, Staphylococcus epidermidis (47.3%) and Staphylococcus haemolyticus (44.2%) were the most prevalent species. Other CoNS species that were isolated were Staphylococcus saprophyticus (2.1%), Staphylococcus warneri (2.1%), Staphylococcus simulans (1.6%), Staphylococcus capitis (1.1%), Staphylococcus schleiferi (1.1%), and Staphylococcus hominis (0.5%). The rate of resistance to methicillin was 60% with 58 (50%) S. epidermidis and 55 (49%) S. haemolyticus. The rate of resistance to 13 antibiotics tested with the lowest and highest to chloramphenicol and penicillin, respectively. High clonal diversity with different PFGE patterns was obtained for methicillin-resistant S. epidermidis and S. haemolyticus by 32 and 31 types, respectively. Our results indicated that the dissemination of MRCoNS is widespread in Tehran. The majority of these isolates showed distinct genotyping patterns. At the same time, the common patterns were found among the MRCoNS obtained from outpatient and inpatient isolates, suggestive of an epidemiological link.

Antibiotic Resistance and Biofilm Formation of Enterococcus faecalis in Patient and Environmental Samples.

BACKGROUND: Enterococci are opportunistic pathogens and are a major factor in nosocomial infections. They may contain ebp operon, which upon expression makes them highly prone to biofilm formation on biotic and abiotic surfaces. OBJECTIVES: The aim of the current study was to detect the polymorphism of ebp genes in Enterococcus faecalis. MATERIALS AND METHODS: Samples were isolated from patients (n = 58) and hospital environments (n = 32) of two hospitals in Tehran, Iran. All enterococcal species were identified by species-specific polymerase chain reaction (PCR); the antibiotic resistance pattern against nine antibiotics was determined. The ebp A, ebp B, ebp C and srt C genes were detected by PCR and the biofilm formation by the isolates was evaluated using the microtiter plate method. The genetic diversity of ebp genes was analyzed by restriction fragment length polymorphism (RFLP). RESULTS: The results indicated that, 86% of patient and 29% of environmental isolates carried ebp genes. The ability of the isolates to strongly attach was 62% and 71% for patient and environmental samples, respectively. The RFLP of the ebp showed no genetic variations amongst the isolates. CONCLUSIONS: The results of the antibiotic resistance and other data suggest that there is a possible common clone of E. faecalis, which could rapidly disseminate in patients and the environment.

Rapid Detection of Methicillin-Resistant Staphylococcus aureus Isolates by Turanose Fermentation Method.

BACKGROUND: Methicillin-Resistant Staphylococcus aureus (MRSA) is a major pathogen in the hospital and community settings. Rapid methods to diagnose S. aureus infections are sought by many researchers worldwide. The current study aimed to utilize a phenotypic method of turanose fermentation to identify methicillin-susceptible and resistant S. aureus. OBJECTIVES: The current study aimed to assay the turanose metabolism at different dilutions as a rapid phenotypic method to identify MRSA isolates. MATERIALS AND METHODS: A total of 150 Staphylococcus isolates were collected from Tehran health centers. Staphylococcus aureus isolates were identified based on cultural characteristics, biochemical reactions and positive tube coagulase test. Methicillin resistance was determined by the disk diffusion method. The Polymerase Chain Reaction amplification was used to detect the mecA gene in MRSA isolates. All the methicillin-resistant and susceptible isolates were evaluated for turanose metabolism with 1%, 0.7% and 0.5% dilutions using the microplate method. RESULTS: Out of the 150 staphylococcal isolates, 80 were identified as S. aureus. Among which 40 (50%) of the isolates were MRSA. The mecA gene was present in all S. aureus isolates resistant to methicillin. A considerable difference was also observed between susceptible and resistant isolates of S. aureus at a 0.7% dilution of turanose. CONCLUSIONS: Since it is highly important to rapidly detect MRSA isolates, especially in nosocomial infections, phenotypic methods may certainly be useful for this purpose. Resistance to methicillin in S. aureus shows a substantially increased ability in turanose metabolism. It is concluded that fermentation of turanose at 0.7% dilution could be a rapid detection method for primary screening of MRSA isolates.

Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis.

BACKGROUND: Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used. OBJECTIVES: During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method. MATERIALS AND METHODS: Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software. RESULTS: Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed. CONCLUSIONS: Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly important to consider in TB surveillance programs to prevent the spread of MDR-TB isolates in the population.

Comparison of an interferon-gamma release assay and the tuberculin skin test for diagnosis of latent tuberculosis in homeless people in Iran: a cross-sectional study.

BACKGROUND: The aim of this study was to evaluate of the concordance between the results of the tuberculin skin test (TST) and an interferon-γ release assay (QuantiFERON test, QFT-GIT) for diagnosis of latent tuberculosis infection (LTBI) in homeless people in Tehran city, Iran. METHODS: This cross-sectional study was conducted from June to August 2012. Homeless people were eligible to participate in the study if they were 18-60 years old. RESULTS: Among 569 homeless people, 46.22% (95% confidence interval (CI) = 42.16-50.33%) and 20.39% (95% CI = 17.28-23.9%) were QFT-GIT and TST positive, respectively. Among these participants, the prevalence of LTBI with positivity of at least one of the tests was 52.2%. The overall agreement between QFT-GIT and TST was 62.21% (kappa = 0.21, 95% CI = 0.13-0.29, p < 0.001). Factors associated with positive results in QFT-GIT and TST were older age, being male, having a longer history of homelessness and having a history of incarceration in the last 10 years. CONCLUSIONS: A high prevalence of LTBI was seen among homeless people in this study. There was a poor concordance between QFT-GIT and TST among this group. To better assess the utility of QFT-GIT in detection of LTBI further studies with a low prevalence of LTBI in this group are recommended.

Vulnerability of homeless people in Tehran, Iran, to HIV, tuberculosis and viral hepatitis.

BACKGROUND: Homeless people are at risk of contracting communicable infectious diseases, as they indulge in risky behaviours and lifestyle. This study was conducted to determine the prevalence of the aforementioned infections and related risk behaviours among homeless people in Tehran. METHODS: In this study a convenience sample of 593 homeless individuals was studied. The ELISA method was used for the detection of HIV, HCV and HBV. Clinical symptoms, sputum cultures, acid fast bacilli smears, and chest X-rays were used to identify active pulmonary tuberculosis, and the Interferon Gamma Release Assay (IGRA) test was used to identify latent tuberculosis. RESULTS: The prevalence of HIV, HBV, HCV and latent tuberculosis was 3.4%, 2.6%, 23.3% and 46.7%, respectively. Active pulmonary tuberculosis was found in 7 persons (1.2%). Injection drug use was an independent risk factor for HIV, HCV and HBV infections. Older people had a higher proportion of Mycobacterium tuberculosis infection (OR: 2.6, 95%CI: 1.9, 3.7) and HCV positivity (OR: 1.7, 95% CI: 1.1, 2.5). CONCLUSION: Our findings highlighted that much more attention needs to be paid to the health of homeless people.

Status of pertussis in iran.

BACKGROUND: Pertussis is a respiratory and contagious disease which is mostly caused by Bordetella pertussis and B. parapertussis. It usually spreads from person to personduring the incubation or catarrhal phase of the disease. Despite of large-scale vaccination, whooping cough is still an endemic disease with several outbreaks. OBJECTIVES: The aim of this study was to determine the prevalence of pertussis and identify its causative agents, B. pertussis or B. parapertussis, from specimens collected from Iranian patients from 2004 to 2008. PATIENTS AND METHODS: Nasopharyngeal swab samples from 347 suspected pertussis cases were collected from 18 provinces of Iran. The patients were in different age groups and were either unvaccinated or vaccinated for pertussis with whole cell vaccine (WCV). Bacterial culture, agglutination tests and quantitative PCR (qPCR) targeting IS481 and IS1001 for B. pertussis and B. parapertussis were done for every specimen, respectively. RESULTS: The results showed that seven nasopharyngeal swab samples (2%) were positive for B. pertussis (1.7%) and B. parapertussis (0.3%) by culture and agglutination test and 30 patients had positive qPCR test results (9%). CONCLUSIONS: Despite the fact that bacterial culture is the golden standard for the detection of B. pertussis, direct detection of bacteria from nasopharyngeal specimens can be performed by a rapid qPCR assay. In this study, high percentage of positive qPCR cases may indicate that the patients might have recovered from pertussis following antibiotic treatment before samples were collected. Rapid detection by qPCR could be important for immediate diagnosis and treatment of patients with pertussis.

Clonal dissemination of methicillin-resistant Staphylococcus aureus in patients and the hospital environment.

BACKGROUND: Methicillin is the drug of choice to treat infections caused by resistant strains of Staphylococcus aureus. However, methicillin-resistant S. aureus (MRSA) is now becoming endemic in many hospitals worldwide and is the cause of nosocomial outbreaks. METHODS: To assess clonality and dissemination of MRSA strains in the hospitals of Tehran, a total of 60 MRSA strains were isolated from hospitalized patients (n=44) and hospital equipment and environment (n=16) of three metropolitan hospitals in Tehran between July 2009 and March 2010. These strains were subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and biochemical fingerprinting using the PhPlate system. RESULTS: Results showed the presence of between one and three dominant clonal groups within each hospital, with most equipment and environmental strains being identical to the dominant clones of hospitalized patient strains. The rate of resistance of these strains to the 13 antibiotics tested ranging from 2% to 100%, with resistance being highest for penicillin, ciprofloxacin, and tetracycline (>98% of the isolates). Comparison of the strains isolated from the three hospitals using a combination of PFGE and PhP types showed the presence of 11 clonal groups of MRSA among these hospitals; of these, three common clonal groups also had identical antibiotic resistance patterns and were found in more than one hospital. CONCLUSIONS: These data suggest dissemination of a few dominant clonal groups of MRSA strains in hospitals in Tehran, with high level resistance to other commonly used antibiotics.

Anti-microbial resistance of Enterococci isolated from urinary tract infections in Iran.

BACKGROUND: During the last decade, enterococci have become important nosocomial pathogens, representing the second leading cause of urinary tract infections. This increasing prevalence has been paralleled by the occurrence of multi-drug resistant (MDR) and high-level gentamicin resistant (HLGR) strains. METHODS: From September 2005 to 2006, a total of 638 enterococcal isolates were collected from urine samples among 9 medical centers in Tehran (Iran). Confirmation of species and detection of gentamicin resistance genes were done by PCR method. Anti-microbial susceptibility test was determined with disk diffusion and minimal inhibitory concentration of gentamicin among HLGR isolates assayed by microdilution methods. RESULTS: The isolates were found to consist of Enterococcus faecalis (77.8%) and Enterococcus faecium (22.2%). The results obtained from PCR showed a high rate of agreement with phenotypic assays for both species. MDR to most prevalent anti-microbials was present in 29% and 72% of the E. faecalis and E. faecium isolates, respectively. HLGR phenotype was detected in 64% of E. faecalis and 92% of E. faecium isolates. The aac(6')-Ie-aph(2'')-Ia gene were identified in 83% of E. faecalis and 100% of E. faecium HLGR isolates. E. faecalis and E. faecium isolates differed in their susceptibilities to different antibiotics. CONCLUSION: Emergence of multi-resistant enterococci and high level resistance to gentamicin shown by enterococcal strains is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS.

OBJECTIVE: To examine the clonal diversity of vancomycin-resistant enterococci (VRE). METHODS: A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE) typing. RESULTS: Forty-nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC >or= 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D(i) = 0.93) and belonged to 24 PhP-types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C-->A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. CONCLUSION: The isolated clinical VRE strains were highly diverse in Tehran.

Clonal diversity of Salmonella enterica serotype Typhi isolated from patients with typhoid fever in Tehran.

In this study, antimicrobial susceptibility test and genetic typing were used to characterize 15 Salmonella enterica serotype Typhi (S. Typhi) isolates recovered from sporadic cases of typhoid fever in Tehran, Iran during 2004. Antimicrobial susceptibility test showed that all isolates were susceptible to 20 antimicrobials examined in this study. Analysis of insertion elements showed that 2 IS200 types with 10 and 11 copies were present. 11 of the 15 isolates were found to possess 10 IS200 elements residing on fragments from 23 to 2.3 kb. Comparison of the RiboPrinter (automated ribotyping) patterns of S. Typhi showed that 60% (9/15) of the isolates belonged to a single ribotype. PCR based random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) and pulsed-field gel electrophresis (PFGE) were also performed. ERIC and RAPD-PCR method showed 2 and 3 genotyping patterns amongst the isolates, respectively. The PFGE typing was carried out by using XbaI restriction enzyme, and 7 restriction patterns were observed. Overall, the molecular typing methods applied in this study showed that the isolated S. Typhi populations were highly polyclonal as shown by PFGE.

Distribution of enterococcal species and detection of vancomycin resistance genes by multiplex PCR in Tehran sewage.

BACKGROUND: Enterococci are important because of their role as the leading cause of nosocomial infections which have a significant role in the dissemination and persistence of antimicrobial resistance genes. METHODS: In this study, we determined the distribution of enterococcal species in the sewage treatment plants in Iran. Furthermore, we improved a rapid and specific PCR method using primers (sodA and ddl genes) for identification of enterococci spp. RESULTS AND CONCLUSION: A total number of 712 enterococci spp. were isolated and the results showed that 56%, 24%, 12%, 4%, 2%, 1% and 1% isolates were E. faecium, E. hirae, E. faecalis, E. gallinarum, E. casseliflavus, E. mundtii and other enterococcal spp., respectively. The use of species-specific PCR was in agreement with the biochemical tests. Furthermore, multiplex PCR was developed to study the presence of vancomycin resistant genes in E. faecium or E. faecalis. The multiplex PCR appeared to be a useful, rapid and specific method for detecting and discriminating genotypes for vancomycin-resistant Enterococcus.