دو ماهنامه میکروب شناسی پزشکی ایران
سال چهارم شماره 1 (بهار و تابستان 1389)

Streptococcus pneumoniae is one of the most important bacterial pathogens associated with pneumonia, meningitis, sinusitis, and otitis media. β-Lactam resistance in clinical S.pneumoniae strains is mediated by altered PBPs, specifically PBP2b. The purpose of this study was to determine the rates of penicillin and other antibiotics resistance in clinical strains of S. pneumoniae and to analyze possible mutations in pbp2b. Susceptibility testing was done by disc diffusion method for oxacillin, erythromycin, cefotaxime, cotrimoxazole, ampicillin, vancomycin and tetracycline. MIC was determined by broth micro dilution method for penicillin. Pbp2b gene was amplified by PCR and sequenced. From all 54 isolates, 44.4% (24 isolates) were penicillin intermediate, 25.9% (14 isolates) werepenicillin resistant, 51.9% (28 isolates) were cotrimoxazole resistant, 16.6% (9 isolates) were erythromycin resistant, 3.7% (2 isolates) were Cefotaxime resistant, and 18.51% (10 isolates) were tetracycline resistant. All penicillin resistant and the most penicillin intermediate resistant isolates had mutations in catalytic regions of PBP2b. Our results showed the increase of penicillin resistance in S. pneumoniae isolates, however prevalence of multiple resistant strains revealed a crisis in treatment of pneumococcal infections. Our investigation demonstrates that alterations in PBP2b tended to parallel with reduced susceptibility to penicillin. We have shown that susceptibility of S. pneumoniae to β-Lactams can generally be estimated by determining alterations in pbp2b gene.

The infection due to vancomycin resistant Enterococcus in immune deficient patient is a significant factor for increase in morbidity and mortality. The aim of this study was to determine VRE carriage rate and it’s molecular epidemiology and to identify risk factors for acquisition of VRE in children with ALL in Iran. In a cross-sectional study, stool samples of children with ALL in Aliasghar and Mahak hospital were randomly analyzed for detection of VRE. All isolates were sent to a reference centre for further investigation. Enterococci strains were identified by conventional methods and vancomycin resistance was studied by disk diffusion method and E-test. Van gene characterization was performed by a multiplex polymerase chain reaction (PCR). Of 130 children with ALL, 33 patients were colonized with vancomycin-resistant strains (VRE) and 62 patients were colonized with vancomycin-sensitive strains (VSE). Twenty- six isolates (78.8%) of VRE showed VanA phenotype and seven isolates (21.2%) exhibited VanB phenotype. In the univariate analysis, history of ICU admission (P=0.03) was significantly associated with Enterococcus colonization and had a trend with VRE colonization. Age (P=0,007) was significantly associated with VRE phenotype. There was no significant relationship between gender, duration of disease, number of admission, another disease, history of severe neutropenia among one month ago and history of antibiotic use in three month ago and VRE colonization. Our study demonstrated that VRE prevalence is high in children with ALL in Iran and indicated a prompt intervention strategy in the control of VRE particulary in PICU.

Increasing uses of antibiotics against bacterial infections have resulted in emergence of antibiotic resistance so extensive studies have been done on new antimicrobial drugs with higher efficacies particularly herbal drugs. The aim of this study was to evaluate the reciprocal pharmaceutical effects and antibacterial activity of Bunium persicum essential oil against some Gram positive and Gram negative bacteria.
Bunium persicum essential oil was purified from its fruit, and was subsequently analyzed chemically by gas chromatography and mass spectroscopy. Disk diffusion method and MIC (minimum inhibitory concentration) determination method were used for the evaluation of the essential oil effects on some standard bacterial strains. To determine synergistic and antagonistic effects, bacterial strains were cultured on a medium containing essential oil and then antibiotic disks were placed on the medium.
Chemical analysis revealed the extracted essential oil to contain 13 different chemicals. According to disk diffusion method, the largest growth inhibitory zone belonged to Bacillus cereus with 45 millimeter diameter. MIC and MBC results showed that the essential oil has the most inhibitory and bactericidal effect on Escherichia coli. The synergistic and antagonistic surveys showed synergistic effects of essential oil on gentamicin against Escherichia coli, but these results were variable against other bacterial strains.
Bunium persicum essential oil, singly or in combination with other antimicrobial agents, can be effective in the treatment of microbial infections. Also this essential oil can strengthen the effects of some antibiotics which particularly propose its usefulness in antimicrobial resistance cases.

Extended – Spectrum beta – lactamase (ESBLs) are certain enzymes that are produced by Gram- negative bacilli such as K. pneumoniae and E .coli¡ which cause resistance of these organisms to penicillins¡ cephalosporins¡ and monobactams. The aim of this study was to determine prevalence of Extended-Spectrum β-lactamase (ESBL) producing E. coli and K. pneumoniea isolated from urine cultures in Milad hospital of Tehran in 2010.
Material and This study was performed on 735 Gram negative bacilli isolated from urine of patients admitted to Milad hospital of Tehran from March to June 2009 and which included 620 (84.3%) strains of E. coli and 115 (15.7%) strains of K. pneumoniea isolates. From antibacterial susceptibility testing as well as for beta-lactamase production disk diffusion method and combined disk diffusion technique were employed respectively¡ as recommended clinical laboratory standards institutes (CLSI).
ESBLs resistance was detected in 132 (21%) of the E.coli isolates and 18 (12%) of the K. pneumoniae strains. Of the 150 patients which had positive ESBLs isolates¡ 104 of them were outpatients and the other 46 were hospitalized. Resistance of ESBLs producing isolates of K. pneumoniae to carbenicillin¡ ampicillin and amikacin was 99.95%¡ 100% and 78 %¡ respectively. The least percentage of resistance among ESBLs positive isolates were observed against ofloxacin (28%). We observed high rate of resistance among isolates of E.coli to ampicillin (85%)¡ tetracycline (62%)¡ and trimethoprim /sulfamethoxazole (54%). However¡ the least rate of resistance was observed among 3 (0.5%) ESBLs positive isolates of E. coli against nitrofurantoin.
About one fifth of the E. coli and roughly one sixth of the K. pneumoniae isolates were producers of ESBLs. Resistance to other commonly used antibiotics among ESBLs producing isolateswere prevalent.

S-layer proteins form the outermost cell envelope component of a broad spectrum of bacteria and archaea which inhibits antibiotic entry into the bacterial cells. The aim of this research was to study the role of nanostructured surface layer and production of β– lactamase in penicillin resistant Bacillus cereus strains isolated from staff hands and hospital surfaces. This research was carried out on 274 samples during the years 2005-2007 in Azzahra hospital and Isfahan University. Bacterial strains were cultured in TSA for 16 h then surface proteins were separated using electrophoresis with 10X SDS-PAGE. Antibiogram was performed according to Kirby Bauer method. β–lactamase production was investigated using acidimetric method. Isolation frequency of B. cereus strains was %9.49. Eleven (84/6%) and one (7/7%) strains of B. cereus isolated from staff hands and hospital surfaces were producer of nanostructured surface layer respectively. Unlike to S-layer producer strains, non S-layer producer strains were more sensitive to antibiotics. All S-layer producer strains produced β–lactamase. A high prevalence of β–lactamase and S-layer producer B. cereus strains was observed in our setting that can contribute to an increase of the antibiotic resistance in nosocomial infection.

Control and treatment of Nosocomial infections due to methicillin resistant Staphylococcus aureus (MRSA) is a worldwide problem. The study aimed to detect the prevalence and risk factors of acquired methicillin resistant Staphylococcus aureus in educational and medical centers in Qazvin from 2005 to 2007. This cross sectional study was performed in three educational and medical centers in Qazvin (2005-2007). The nasal samples were obtained from patients on admission and after discharge. Isolation and detection of S. aureus isolates using Oxacillin screening plates were performed according to Clinical and laboratory standards institute (CLSI) procedure. Other personal informations were obtained by questionnaire, face to face conversation or from patient's documents. The data were analyzed by qui-square test. From the 1083 patients upon admission, 56 (5.2%) carried S. aureus, of which 51(4.7%) of them were methicillin sensitive S. aureus (MSSA) strains, and 5 (0.5%) were found to be MRSA. From the 793 remaining patients at the time of discharge, 79 (10%) and 15 (1.9%) were colonized with these strains, respectively. Risk factors of MRSA colonization were hospitalized duration, admission, prescribed antibiotics (especially Ciprofloxacin) and using Angiocat (P<0.05). There was no significant relation between other variations and MRSA colonization (P>0.05). These findings showed the existence of MRSA strains in Ghazvin population. The incidence of MSSA and MRSA colonization with hospitalizing increased 2.1 and 3.8 respectively in comparison with the carrier state on admission. This will be increased for MRSA and its isolation seems to have a direct relation with the length of hospitalization, especially in surgery and internal medicine words, ciprofloxacin use, and use of Angiocat.

In recent years, Enterococci have been increasingly witnessed for causing serious clinical and nosocomial infections. Resistance to multiple antimicrobial agents, especially vancomycin, ampicillin and high levels of aminoglycosides is typical in these isolates. Rapid and accurate identification of vancomycin-resistant enterococci (VRE) is crucial in the management and treatment of both colonized and infected patients, to allow selection of appropriate antimicrobial treatment and to prevent the spread of VRE. In this study 432 stool or rectal swab samples were studied for detection of VRE species. Minimum inhibitory concentration (MIC) of vancomycin for enterococcal species isolated from stool specimens were determined by agar dilution and/or E-test. PCR was carried out to detect the presence of vanA/vanB and also E. faecalis/E. faecium genes. Of 291 Enterococci species isolated by culture, (64.9%) were E. faecalis and (29.5%) were E. faecium. MICs determination confirmed resistance to vancomycin in 7.3% strains. PCR confirmed vanA and vanB genes with moderate (MICs, 8-16 μg/ml) and high level resistance to vancomycin (MICs, ≥256 μg/ml) in this species. According to findings of this study, prevalence of intestinal colonization by vancomycin resistant Enterococci among hospitalized patients is a medical concern proposing improvement in antibiotic policies.

Escherichia coli is the most common cause of nosocomial infection including urinary tract infections (UTIS). Due to the presence of various resistance mechanisms against antibacterial agents specially extended spectrum β -lactamases (ESBLs), the treatment of infections caused by these bacteria is problematic. Since detection of bacterial resistance and SHV and TEM type ESBLs which are the common type of β - lactamases is required for a better management of infection caused by these bacteria, the aim of this study was to determine the antibacterial resistance pattern and the prevalence of bla SHV and blaTEM lactamases genes in clinical isolates of E.colifrom urine of hospitalized patient in Kerman. This study was performed on 115 E.coli strains isolated from urine specimens of hospitalized patients in 3 hospitals in Kerman. Agar dilution method was used for determination of minimum inhibitory concentration to selected antibacterial agents. ESBLs production was detected by combined disc method. TEM and SHV type β -lactamases were detected by PCR method using plasmid and chromosomal DNA. Of the total isolated bacteria, 86(74.7%) showed resistance to 3 antibacterial agents from different classes and were considered as multiple drug resistance (MDR). Totally, 76 (66%) of the isolates produced ESBLs. The PCR assay performed on the plasmid and chromosomal DNA, totally 48 (4107%) of the isolates showed TEM, 39 (33.9%) showed SHV, and 20 (17.3%) of the isolates had both TEM and SHV genes.
The lowest level of resistance, only in 7 of the isolates (14.7%), was seen towards ceftizoxim. The highest level of resistance was detected to amoxicillin and trimethoprim /sulfamethoxazole which was in the order of 108 (94%) and 110 (95%) isolates, respectively. MDR trait in urine isolates is common in this area. Due to high resistance to trimethoprim /sulfamethoxazole and amoxicillin, these antibacterial agents are not recommended for treatment of urinary tract infections. In general, the prevalence of TEM gene was found to be much higher than the SHV.

Human Adenoviruses are one of the most common and serious etiologic agents of acute gastroenteritis in children younger of 5 years old in word wide¡ especially developing countries. The purpose study was a survey of molecular epidemiology of enteric Adenoviruses in acute gastroenteritis in hospitalized children in Bahrami hospital in Tehran¡ 2008-2008.
We obtained 100 stool specimens from hospitalized children under 5 years of age with clinical presentation of acute gastroenteritis in Tehran from June 2008 to June 2009. Viral DNA was extracted by extraction kit of DNA. Then extracted viral DNA by use of PCR method with genus specific and type 40¡ 41 primers¡ enteric Adenoviruses were diagnosed.
Adenoviruses Ad40¡ Ad41 have been detected in 8 (8%) of patients with diarrhea.
The highest prevalence of infection occurred in children aged 0 to 24 months (75%). The rates of infection were 60% for male and 40%for females. Our data indicated that the outbreaks of Adenoviruses infection were in winter(50%).The frequency of the Adenoviruses Ad40¡Ad41 among breast-fed and bottle-fed¡ was 12.5%and 62.5%respectively.

Molecular diagnostic methods should be with high features such as PCR and effective step in the recognition of these factors can be harvested as the appropriate strategy for treatment adopted.

Bovine respiratory syncytial virus (BRSV) are from the pneumovirus genus and belongs to Paramixoviridea family. The growth place of this virus is the respiratory tract. The infection mostly appears in calves and young animals which are often kept in close stables. For serological and identification antibody study of this virus¡ natural serum test¡ indirect heamoglutanation and ELISA assays can be used. The percentage of sensitivity of these tests for antibody presence against virus in serum is 92%¡ 95%¡ and 100%¡ respectively. The aim of this study was to analyze the rate of prevalence of BRSV antibody infection in dairy farms cattle population of Ilam province.
This study had been performed from autumn 2008 to the end of autumn 2009. Blood samples were collected from 400heads of dairy cattle with different ages. 356 (89%) female and 44 (11%) male cows were randomly selected from different regions in the province and in different seasons of the year. In overall¡ 85% of the samples were from industrial and the remaining 15% were from native dairy farms. %% Antibody against BRSV were measured using a specific ELISA assay. The data were analyzed using qui-square test.
Of the total number of cows under study¡ 298 (83.7%) heads of female and 21(47.7%) heads of male cows had antibody against BRSV. This was equivalent to 79.9% (319 cows) of the total animal population under study.
The rate of cattle infection is significantly highin Ilam province and it appears as though the infection rate has a direct correlation with features like sex¡ age¡ and season. Therefore¡ it is postulated that by proper management and effective handling of the respiratory ailments¡ we can observe a significant improvement on cattle health¡ as well as¡ in milk and beef production.

Giardiasis has multiple clinical manifestations and its prevalence is relatively high in Hamadan province. This study was conducted to determine the most frequent clinical sign and symptoms of giardiasis in Hamadan province in 2006. In a descriptive cross sectional study, sixty four patients infected with Giardia were recruited. Anti glidin anti-body and TTG tests were done to roll out celiac disease. The chi-square test was used for statistical analysis. Giardiasis was the most common in cases aged 16-20 years old (20.3%), in males (59.4%) and in patients with educational status of primary school (31.25%). The most frequent symptom was abdominal pain (42.1%). Bloating and diarrhea with 23.4% and 9.4% were the next ones. We found that the clinical manifestations of giardiasis are similar to other gastrointestinal diseases such as celiac, so giardiasis should be considered as probable diagnosis in patients with gastrointestinal problems.

In recent decades, the incidence of fungal infections has increased. Invasive Candida nfection is an increasing cause of morbidity and mortality in the immunocompromised patients, for example, neuropenic patient with hematological malignancies, recipients of allogeneic transplants and HIV-infected individuals. There is an increasing incidence of bloodstream infections caused by Candida species. In this study, we applied Seminested PCR for detection and identification of Candida species in blood specimens.
A total of 2516 blood culture samples in Baghiyetollah hospital were investigated for the presence of yeast cells duringthe year 2009. Fourteen blood samples which were found to be infected with Candidaspecies, as proved by culture, were analysed. All yeasts cells were identified by germ tube test, chlamydospor production on cornmeal agar and using CHROMagar Candida medium. DNA was extracted by DNG-plus kit from whole blood. The universal outer primers (CTSF & CTSR) amplified the Internal Transcribed Spacer2 (ITS2). The species-specific primers (CADET, CPDET, CTDET, CGDET and CDDET), complementary to unique sequences within the ITS2 of each test species, amplified speciesspecific DNA in the reamplification step of snPCR. PCR products were identified by electrophoresis. Among the 14 (0.5%) blood samples, 16 Candidaspecies were detected and identified by using snPCR. Using snPCR for Candidaspecies identification indicated that 6 isolates were C. albicans, 4 isolates of C. parapsilosis, 4 isolates of C. glabrata, and 2 isolates of C. tropicalis. Two of the patients had dual infection with C. tropicalisand either C. albicansor C. glabrata. C. dubliniensiswasn’t detected in blood samples. The Seminested PCR that was applied and evaluated in this study is a rapid, specific and sensitive method for the diagnosis of candidemia or hematogenous candidiasis caused by common pathogenic Candidaspecies.

Influenza viruses, especially the subtype H1N1, arecontinuously evolving viruses, which have caused significant morbidity and mortality in Eastern Asian countries as well as in the Middle East. The aim ofthis study was to analyze genetic variations in hemaglutinin (HA) gene of the H1N1 viruses circulating in the countries neighboring Iran during 2003 till 2006. In this study we used multiple sequence alignment software for analysis of sequences. Genetic analyses were performed on 64 hemagglutinin gene in influenza A(H5N1) viruses derived from GeneBank reported in Iran and neighboring countries between 2003 and 2006. HA phylogenetic analysis of these viruses showed multiple clades circulated around the world with regional prevalence patterns. Our analysis seems to indicate that the H1N1 viruses have initially entered Iranian borders from its northern bordering countries, such as Russia and the other CIS countries. They have subsequently spread inward towards the interior of mainland Iran. This study suggested that influenza A (H5N1) viruses might have migrated from neighboring countries of Iran to this country.