دو ماهنامه میکروب شناسی پزشکی ایران
سال دوم شماره 3 (پاییز و زمستان 1387)

Bladder cancer is the second common genitourinary tract cancer in the world.Immunotherapy with intravesical Mycobacterium bovis BCG is the selective treatment for superficial bladder cancer. CD/5FC Enzyme-Prodrug system of gene therapy is an alternative technique for controlling bladder cancer. This research is aimed at the design and construction of mycobacterial shuttle vector containing hsp60 promoter, alpha antigen signal sequence and cytosine deaminase (CD) and consequent transformation to M.bovis BCG by electroporation. It is assumed that converting of 5-FC to 5-FU via CD will kill more tumor cells and increase the efficacy of this therapeutic method. After amplification of mycobacterial shuttle vector gene fragments containing hsp60 promoter, alpha antigen signal sequence and Saccharomyces cerevisiae CD gene by PCR and enzymatic digestion, they were cloned and subcloned in pBGGT and pVN2 respectively. The final plasmid was transformed to M. bovis BCG by electroporation method. Sequencing results confirmed the integrity and correctness of final vector which called pHARA. The cells which received pHARA, were cultured on Middlebrook 7H10 agar with 20μg/ml final concentration of kanamycin, and grown during 17 days. Mentioned recombinant BCG (rBCG) secreting CD enzyme in addition to adjuvant property and stimulation of local immunity responses against tumor cells, can also destroy more tumor cells through converting 5-FC to 5-FU via CD enzyme that intensifies BCG efficacy in treatment of superficial bladder cancer.

Nowadays, there is an emerging increase in drug resistance in medical centers due to extended use of third generation cephalosporins. This study was conducted to evaluate antibiotic sensitivity profiles of Esherichia coli and Klebsiella pneumoniae isolates collected from four medical centers (Imam Khomeini, Sina, Shohada and Pediatrics) in Tabriz and detection of presence of blaSHV gene among the isolates. In total 41 isolates of Escherichia coli and 47 isolates of Klebseilla pneumoniae were collected from medical centers. The antibiotic sensitivity pattern of the isolated were determined by using drug susceptibility test according to Kirby-Bauer method. Combined disk test was also used as confirmatory test, and the findings were adapted with CLSI standard.PCR technique was used for investigation of presence blaSHV gene in final step. Among the E. coli and K. pneumoniae isolates, 33 (80.49%) and 43 (91.48%) were resistant to ceftazidime and 32(78.05%) and 42 (89.36%) were resistant to cefotaxime respectively. Forthy out of 41 E. coli isolates (97.5%) were ESBL producers, which among them 7 isolates (17.0%) contained blaSHV gene. Among 47 K. pneumoniae isolates, 46 (97.8%) were ESBL producers which 12 isolates (25.5%) contained blaSHV gene. All of the E. coli and K. pneumoniae isolates were sensitive to imipenem. The high resistance profiles encountered in E. coli and K. pneumoniae isolates, underlines need for accurate sensitivity testing and also avoidance from inappropriate use of antibiotics in management of ESBL producing bacteria is recommended.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that cause acute and chronic infantile diarrhea. The stacked – brick appearance of EAEC strains to HeLa or Hep-2 cells is the gold standard assay for the detection of EAEC strains and is different from adherence pattern of EPEC and DAEC. In the present study, a multiplex PCR (mPCR) assay was used for detection of EAEC strains and its results was compared to HeLa cells adherence assay. In this study the E.coli strains isolated from diarrheal children from Tehran children medical center were used. The isolates were identified according to standard procedure. The 170 strains were subjected to mPCR assay. This assay is based on the presence of three plasmid borne genes i.e aaP, aggR, aatA.The mPCR positive and negative strains were further checked for their adherence patterns on HeLa cells. the results of mPCR of these strains revealed that 114 (67%) of these strains were identified as EAEC by this mPCR assay. The frequency of occurance of these genes was 100 %, 96.4 % and 70% for the aaP, aggR,and aatA genes respectively. The 86.9% of mPCR positive strains were HeLa cell adherent (stacked-brick) and only 13.1% of mPCR positive strains were not adherent. On the other hand 27.2% of st rains that on mPCR assay were negative, adhered to HeLa cells in a characteristic manner of EAEC isolates (stacked –brick).the overall results obtained in this study, showed that the mPCR assay used here is able to detect the strains with HeLa cells adherence characteristic of EAEC isolates. Moreover some strains non-adherent on HeLa cells was positive with the mPCR visa-versa. In conclusion it seems this mPCR assay is rapid and specific assay for detection of EAEC strains and especially is suitable for screening large number of isolates in epidemic situation.

Currently, antigenic determinants of Brucella cell wall such as outer membrane proteins (OMPs) and lipopolysaccharide (LPS) are considered as potential candidates to develop subunit vaccines. It has been shown that OMPs of Brucella abortus along with other bacterial components could stimulate synthesis of specific IgG antibody in rabbits. The aim of present study was evaluation of antibodyresponse to OMPs of Brucella abortus S99 by ELISA method. OMPs were extracted by deoxycholate extraction technique and further purification performed by sequential centrifugation and ultracentrifugation. Protein concentration determined by using the Nano drop ND – 10000. Sodium dodecyle sulfate polyacryl amid gel electrophoresis (SDS – PAGE) was used toproduce a profile of OMPs proteins which these were further differentiated on the basis of molecular weight.By using this method, the class II proteins belongs to porins, which comprised the major OMPs in B. abortus were purified. The groups consisted of 3 rabbits were immunized with three different compounds comprises of OMPs only, combined with LPS and combined with complete Freund's adjuvant (CFA). Sera from immunized animals were then evaluated for specific antibody response. the most immunogenic compounds were LPS+ porins and CFA+ porins and could promoted specific antibody response in experimental animals. The level of specific antibodies in rabbits immunized with above mentioned combinations was significantly higher compared to immunized rabbits with porins only (P<0.05). Based on the results, the combination of LPS+ porins can produce long term protective immunity against Brucella. So it could be probably considered for developing of subunit vaccine for prevention of human brucellosis.

Urinary tract infection (UTI) caused by E coli is one of the most common diseases in children. Presence of four virulence genes of pap, sfa, hly and cnf have important role in organism pathogenicity and adhesion to epithelial cell. The present study was aimed to evaluate the prevalence of these four important virulence genes in E. coli strains isolated from urine samples of children with UTI and theircorrelation with clinical data. E. coli strains were isolated from urine samples of children with UTI who presented during 2005-2006 in Motahary hospital, Jahrom, Iran. The isolates were then studied for presence of genes hly,cnf-1, pap and sfa by PCR terchnique. Totally 96 E. coli strains were isolated from urine samples of children with UTI aged from 1 month to 14 years (mean 21.8± 26.9 months). Cystitis was diagnosed in 49.2% and pyelonephritis in 50.8% of these patients. Prevalence of genes pap, sfa, hly and cnf-1 among the strains was 27.1%, 14.6%, 13.5% and 22.9 %,respectively. Overall 32 samples (33.3%) were positive for at least one of the genes and 6 samples (6.3%) were positive for all four genes. pap and sfa were more common in ages over 36 months but hly was more detected in age under 48 months (P<0.05). cnf-1 gene was significantly more common in samples of the patients with abnormal kidney sonography (P=0.049). This study showed that the prevalence of virulence genes hly, cnf-1, pap and sfa in E. coli isolates was similar to the results of other studies. Because of higher prevalence of pyelonephritis in presence of these genes, rapid detection of the genes in urine samples may help in more suspicious and rapid management of pyelonephritis.

Fluoroquinolones, in particular ciprofloxacin have recently become the drug of choice for antibiotic therapy of invasive Salmonella infections in many countries. however, several treatment failures due to Salmonella strains with decreased susceptibility to ciprofloxacin have been reported. These isolates are still considered to be susceptible to ciprofloxacin according to CLSI interpretive criteria (MIC≤1 μg/ml) while they can not be detected by disk diffusion test. CLSI has recommended nalidixic acid disk diffusion as an indicator test for screening of Salmonella isolates with decreased susceptibility to ciprofloxacin.In this study the value of nalidixic acid susceptibility testing for screening of clinical strains of Salmonella with decreased susceptibility to ciprofloxacin was evaluated. Salmonella spp. strains have been isolated from several provinces in Iran during 2007-2008. The strains were identified by standard biochemical tests and serology. The susceptibility of the isolates to nalidixic acid and ciprofloxacin was determined by disk diffusion method. MIC assay for ciprofloxacin was determined by E. test. 28 out of 53 Salmonella strains (52.8%) were resistant to nalidixic acid. Disk diffusion testing showed that all isolates were susceptible to ciprofloxacin however 8 (15%) resistant nalidixic acid isolates showed decreased susceptibility to ciprofloxacin (MIC= 0.125 μg/ml) when tested by E.test. The results showed that nalidixic acid disk diffusion test can be considered as a reliable and costeffective method for screening of Salmonella strains with low level resistance to ciprofloxacin.

Enterococci are natural inhabitants of the gastrointestinal tract of humans and animals. Over the past decade, enterococci have become one of the leading causes of nosocomial infections,because they have acquired resistance to many antimicrobial agents such as vancomycin and gentamicin.Development of high-level gentamicin resistance among enterococci represents a serious therapeutic problem as it precludes synergy between aminoglycosides and cell-wall active agents. High-level gentamicin resistance (MIC ≥ 500 mg/ml) in enterococci is predominantly mediated by aac (6')-Ie-aph (2'')-Ia gene.. The aim of present study was determination of high level gentamicin resistance in Enterococcus species isolated fromhealthy human in Tehran. After enrichment of the fecal samples collected from healthy volunteers in BHI broth containing gentamicin, the isolates were subcultured on m-Enterococcus agar containing gentamicin. The isolates were identified to the genus and species levels and MIC was determined. Antibiotic susceptibility of the isolates was tested by agar diffusion method for 8 various antibiotics. The aac(6')-Ie-aph(2'')-Ia gene was identified by PCR technique. gentamicin resistant enterococci(GRE) were isolated from 76(15.2%) of 500 fecal samples. The isolated species were: E. faecalis 45(59.2%), E. faecium 29(38.1%) and E. gallinarum 2(2.63%). GRE isolates were highly resistant to gentamicin(MIC ≥ 500 μg/ml). Eleven different antimicrobial resistance patterns were observed in this study. All of isolates carried the aac(6')-Ie-aph(2'')-Ia resistance gene. These results represent a rather high prevalence of gentamicin resistance enterococci as a normal flora in the community. The aac(6')-Ie-aph(2'')-Ia gene was the most common gene among the gentamicinresistant isolates evaluated in this stud.

Enterococcus faecalis is a normal commensal in the human intestinal flora but can leads to nosocomial infections. Several virulence factors have been described in E. faecalis, including aggregation substance(Agg), entrococcal surface protein(Esp), cytolysin (Cyl) having both hemolytic and bactericidal activity, and gelatinase(Gel). These factors act synergistically to enhance virulence, resulting tissue damage and tissue invasion. The aims of this study was phenotypic determination of virulence factors of E.faecalis isolates recovered from patients with urinary tract infections. This descriptive study were carried out in 95 clinical E. faecalis isolates recovered from patients with urinary tract infections in Shahid Beheshti hospital in Kashan, Iran, between 2007 and 2008. The production of biofilm, hemolysin and gelatinase by these isolates was also examined and the associated medical records of patients were retrospectively reviewed. Data were analyzed using Chi Square and Exact Fischer tests. Gelatinase activity was detected in 19 of the 95 (20%) E. faecalis isolates, hemolysin was detected in 42 of 95 (44.2%) E. faecalis isolates as beta hemolysis 35.8% and alpha hemolysis 7.4%, and 79 out of 95(83.2%) isolates showed weak, 6 out of 95(6.3%) moderate and 10 out of 95 (10.5%) strong biofilm formation. There was no significant correlation between biofilm formation and age, sex, previous history of antibiotic therapy, catheterization, production of haemolysin and gelatinase activity. No single factor was detected as virulence prediction. It seems that the factors act synergistically.

Enteropathogenic E.coli (EPEC) strains have high prevalence in children and are of the most important diarrheagenic agents in developed and developing countries that may cause severe diarrhea even death. Prevalence and antibiotic resistance patterns differ in different geographic areas.Knowledge of the patterns is necessary for physicians in order to treatment of infections using appropriate antibiotics. This research aimed to detection of common EPEC serogroups in diarrheal under 5 year old children and study the patterns of antibiotic resistance of these strains. 278 samples collected from children with diarrhea visited in Ali Asghar hospital,Tehran. Bacterial isolates confirmed as EPEC serogroups on the basis of standard biochemical and serological tests. Antibiotic susceptibility testing was performed using 16 different antibiotic discs by disc diffusion agar (Kirby-Bauer) method. In this study 19 strains (6.8%) of EPEC isolated that prevalence of EPEC strains particularly serogroup poly IV was more common in children under one year old. All strains were sensitive to meropenem and imipenem, 63.2 % were resistant to nalidixic acid, tetracycline and amoxicillin and 89.5 % of strains were sensitive to ceftazidime and gentamycin. Results indicated that most strains completely or intermediately were resistant to nalidixic acid, tetracycline and amoxicillin. Antibiotic resistance of EPEC strains to nalidixic acid, tetracyclin and amoxiciline is increasing. Due to increasing resistance towards antibiotics, we should use new and effective antibiotics according to antibiotic susceptibility testing for treatment of infections.

Streptococcus pneumoniae is a bacterial pathogen most commonly associated with acute otitis media and pneumonia and the second most important pathogen in cases of meningitis in children under 2 years old. Currently more than 90 pneumococcal serotypes have been identified based on their antigenic differences in the capsular polysaccharides, with distribution varying according to age group, clinical symptoms, and geographic area. The aim of this study was to determine the most prevalent serotypes of Streptococcus pneumoniae associated with different infectious diseases in Tehran medical centers. In total 50 clinical isolates of Streptococcus pneumoniae were collected from Tehran medical centers and re – identified using standard identification tests. Quelling test was used for serotyping of the isolates as per Statens Serum Institute(SSI) guideline. The data were analyzed using SSI guideline. In this study, the distribution of isolated Streptococcus pneumoniae in relation to site of infection were:respiratory tract, 18 isolates (36%); blood, 13 isolates (26%); eye, 10 isolates (20%) and other sites, 7 isolates (14%). The most common serotypes were 6A and 6B. The prevalent serotypes in adults and infants were serotype 2 and 19 (19A, 19B, 19C, 19F) respectively. The prevalent serotypes according to the site of infection were: respiratory tract, 7; blood, 4; and eye, 19. The finding of this study showed that serotype 6 in common serotype in both adults and infants.

Ice cream is made from milk, cream, sugar, food additive and may contain preservatives and edible colors as well. This product provides good condition for bacterial growth.This study was conducted to determine the frequency of E.coli and/or Staphylococcus aureus contaminated traditional non-pasteurized ice cream in Semnan. In this descriptive cross sectional study, during two years, 136 samples of nonpasteurized ice creams were investigated in Semnan food and cosmetics laboratory. The samples were investigated for contamination with E. coli and Staphylococcus aureus according to Iranian standard guideline.The isolated bacteria were further identified by conventional bacteriologic tests. Of the 136 samples, 96 (70.6%) were contaminated with E. coli, 53 (38.9%) contaminated with Staphylococcus aureus, 52 (38.2%) contaminated with both bacteria and 39 (28.7%) were non contaminated. Results of this study showed that frequency of traditional non-pasteurized ice creams which were contaminated with E.coli and Staphylococcus a ureus was significantly higher than other surveys.

The adaptive power of Staphylococcus aureus (S.aureus) to antibiotics leaded,in the early 1960s, to the emergence of methicillin-resistant S. aureus (MRSA). The early MRSA clones were hospital-associated (HA-MRSA). However, from the late 1990s, community-associated MRSA (CAMRSA) clones emerged worldwide. Patient’s study on the admission and the discharge provides an important information about the position of CA-MRSA, the rate of HA-MRSA colonization, and risk factors for colonization. This information could be used for prevention of organism transmission, control of hospital environment, and prevention of hospital organisms spread into community. The aim of this study was to determine the prevalence of and some risk factors of methicillin- resistant S.aureus nasal colonization in hospitalized children. Nasal swab specimens were collected on the admission and the discharge of patients admitted in the Qods educational and health pediatrics center of Qazvin. After isolation and identification the strains, the methicillin resistance was determined by Clinical and laboratory standards institute(CLSI) oxacillin screening plate method. Other information were collected by questionnaire. The data were analyzed by Chi-square test.. A total number of 200 patients (2 to 12 years old) were studied. The carrier state of methicillinsensitive S. aureus (MSSA), and MRSA strains were 6 (3%),and 1 (0.5%) respectively. From 193 remainder on discharging 14 (9%), and 3 (1.9%) patients were colonized with MSSA, and MRSA strains respectively.Ceftriaxone was administrated for all patients colonized with MRSA strains. There was no significant correlation between colonization with MRSA strains and studied variables. Our findings confirm the presence of MRSA strain in children population in Qazvin city. The incidence of colonization with MSSA, and MRSA, in comparison of the carrier state with the same strains,were 2/4, and 3.2 times. This rate is higher in the case of MRSA, which is correlated with administration of Ceftriaxon.