دو ماهنامه میکروب شناسی پزشکی ایران
سال دوم شماره 2 (تابستان 1387)

β-lactam antimicrobial agents represent the most common treatment for bacterial infections and continue to be the leading cause of resistance to β-lactam antibiotics among Gram negative bacteria world wide. Extended –spectrum β-lactamases(ESBLs) enzyme hydrolyze and inactivated β-lactam antibiotics. AmpC enzymes are in class C of ESBLs. Typical Ampc enzymes as clavulanate resistant cephalosporinases confer resistance to most oxyimino cephalosporins. AmpC enzymes are counted separately from ESBLs, but a taxonomic problem arises with AmpC mutants that have increased activity against cefepime and cefepirome, fourth generation of cephalosporins. Such mutants have arisen from inherent chromosomal AmpC types, but they could equally evolve from the plasmid AmpC types that are increasingly circulating in Klebsiella spp and E. coli. The aim of this study was to determine the Prevalenceof AmpC type extended spectrum beta lactamases genes in clinical isolates of Klebsiella pneumoniae. Phenotypic detection of ESBLs was used for screening of isolates by agar dilution method. The multiplex PCR assay was used for detection of AmpC genes in clinical isolates of Klebsiella pneumoniae. Of 168 clinical isolates, 119 isolates were positive for ESBL in initial screening tests and from them 99 isolates were positive in phenotypic confirmatory tests.10 isolates (5/95 %) were positive for AmpC genes in Klebsiella pneumoniae isolates. In this study, the existence of ESBLs and AmpC genes in clinical isolates of Klebsiella pneumoniae was shown.

Staphylococcus intermedius is a zoonotic bacteria which has been rarely found even among individuals with frequent exposures to animals. There were some reports on invasive infections in animals and humans. The objective of this study was identification of positive coagulase staphylococci other than S. aureus in health care workers. This descriptive cross sectional study was carried on 150 health care workers in three medical centers of Tehran University of Medical Sciences (TUMS) from July 2008 to December 2008. The samples were collected from anterior nasal region of cases using cotton swabs and sent to laboratory for identifying organisms by microbial and biochemical tests. Moreover, PCR was carried out with mecA specific primers to detect methicillin resistance. Out of 150 health care staff, 38 (25.3%) strains of coagulase positive staphylococci were isolated. Of these isolates, one strain was S. intermedius and the others were S. aureus. The presence of mecA gene in S. intermedius was confirmed by PCR. Transmission of S. intermedius from animals to human may result in expansion of uncommon infections and increasing of antibiotic resistance. Hence, identification of microbial colonization in health care staff has very important role for prevention of nosocomial infections.

Infections with Mycoplasma species can induce a variety of problems in living organisms and in in vitro cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. In order to circumvent those limitations, many nucleic acid technologypredicated procedures have been developed. PCR-based methods for detection of certain DNA regions of the Mycoplasma genome have proven both rapid and specific. Using SHAH-GPO-3, MGSO primers and standard Mycoplasma species PCR optimized and sensitivity and specificity evaluated by Known CFU samples and different strains. Cell culture DNA extracted and then tested by optimized PCR. Amplicon (272 bp) cloned by PCR-cloning and then sequenced by dideoxy chain termination. In this study, we describe our newly-developed sensitive PCR procedure for the detection of Mycoplasma genus contaminants. For amplification, the DNA regions of 16S rDNA were targeted using general Mycoplasma primers. The PCR, which generated DNA fragments of 272 bp, was found to be able to detect 10 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used.25 from 47 cell lines infected with Mycoplasmas. The improved 16S rRNA based PCR method for identification of Mycoplasma in cell culture and biological products, based on common and constant sequences is accurate and useful technique with high specificity and sensitivity. However, more researches should be developed in case of DNA extraction,samples concentration and target sequences and identification of PCR products.

Salmonella is recognized as a major food-borne pathogen in humans worldwide. Antimicrobial drug resistance is increasing among Salmonella spp. and causes significant therapeutic problems in the treatment of diseases caused by this organism. The aim of this study was to determine the aminoglycoside resistance pattern of Salmonella spp. isolated from clinical cases in Tehran. Salmonella spp. strains were isolated from several hospitals in Tehran during 2007-2008. The strains were identified by standard biochemical methods and serology. The susceptibility of the isolates to aminoglycoside antibiotics was determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The results showed that 44.1% of the strains were resistant to streptomycin, 22.8% to kanamycin,19.1% to neomycin, 0.7% to tobramycin and 0% to gentamycin. We found a diverse pattern of aminoglycosides resistance among Salmonella spp., the resistance to streptomycin and kanamycin was considerable, whereas to tobramycin and gentamycin was very low. While aminoglycoside resistance varied by Salmonella serogroups, continuous monitoring of resistance patterns and the use of antibiotic agents according to individual serogroup is recommended.

Staphylococcus epidermidis is the most important member of coagulase negative staphylococci. This organism has been reported as the third cause of nosocomial infections in the last decade and one of the most important causes of bacterimia. New generation β-lactam antibiotics are commonly used to cure these infections. Overproduction of β-lactamases not only results in bacterial resistance against β-lactams, but also causes false methicillin resistance in some strains lacking methicillin resistance gene. The aims of this study was to determine the susceptibility of clinical isolates of Staphylococcus epidermidis to some β-lactam antibiotics, to screen the isolates for β-lactamase production and detect the presence of the β-lactamase gene (blaZ). Sixty-nine clinical isolates of coagulase negative staphylococci were collected from three hospitals in Tehran. Susceptibility to 8 β –lactam antibiotics was determined using disc diffusion.β-lactamase production was screened by the iodometric colony test and presence of the blaZ gene was detected using specific primers and PCR. The antibiotic sensitivity results showed that 54 isolates (98.1%) were resistant to penicillin, 50 isolates (90.9%) to methicillin, 29 isolates (52.7%) to ceftriaxon, 27 isolates (49.09%) to ceftizoxime, 20 isolates (36.3%) to cefotaxim, 19 isolates (34.5%) to amoxicillin, 17 isolates (30.9%) to cefazolin and 13 isolates (23.6%) to cefalexin. The iodometric assay showed that all the isolates were β-lactamase producers and the PCR results confirmed the presence of blaZ gene in all isolates Overall, 24-53 % of the clinical isolates of S. epidermidis were resistant to the third generation β-lactam antibioticas by disc diffusion. On the other hand, iodometric colony tests showed that all isolates produced β-lactamase and the presence of the blaZ gene was confirmed in all by PCR.The results obtained in this study suggest that despite the presence of the β-lactamase gene, enzyme expression is variable in different isolates and disc susceptibility test alone is not often suitable for determining the course of antibiotic therapy.

Pseudomonas aeruginosa is important opportunistic pathogen and to produce widespread infection by numerous virulence factors. Drug delivery system that reduces the drugs toxicity while increasing their therapeutic index is a great interest and liposomes can provide the benefits. Liposomes are colloidal vesicules ranging from a few nanometers to several micrometers in diameter. The present invitro study was designated to evaluate the antimicrobial activity of free and liposomal amikacin against Pseudomonas aeruginosa (ATCC 27853). The minimal inhibitory concentrations (MICs) of free and liposomal amikacin for Pseudomonas aeruginosa (ATCC 27853) were determined by broth macro-dilution technique as recommended by CLSI (Clinical and laboratory standards institute). We therefore, encapsulated this drug in to liposome prepared by sonication. Change of number of bacteria in equal minimal inhibitory concentration of liposomal amikacin was compared with the change of number of bacteria in the present of variousconcentrations of free amikacin. The results showed that of liposomal amikacin had antimicrobial effect and MIC of liposomal amikacin were equal of 4μg/ml however minimal inhibitory concentration of free amikacin was 2μg/ml.Comparison of change of number of bacteria had shown that the effect of liposomal amikacin (4 μg/ml) after 8 h is equal of 6 μg/ml free amikacin after 4h. According to the results we can probably to use of liposomal amikacin for Pseudomonas infections, but we need more study and research.

Gram –negative rods are the most common bacterial isolates in clinical laboratories around the word. Quick and accurate identification of these bacteria is a key to the effective therapeutic intervention and optimal clinical out come of infections caused by these bacteria. Multiple drug resistance gram-negative bacteria (MDR) are on increase and are reported frequently. Since in many clinical laboratories the standard antibiogram tests are not performed and very few tests are used for identification. The aim of this study was to evaluate the accuracy of identification and the susceptibility tests of gram negative bacilli performed by clinical laboratories compared to the standard procedures in Kerman. This study was performed on 948 clinical isolates reported to have the MDR phenotypes by disk diffusion method. The isolates were identified to species level by conventional biochemical tests. Sensitivity of the isolates to antibacterial agents and the Minimum Inhibitory concentration (MIC) of the isolates were determined using agar dilution method. For statistical analysis, x2 test was used. The rate of isolation of MDR bacteria by agar dilution methods was 76% of those reported by disk diffusion methods. For all antibiotics disk diffusion showed higher rate of resistance compared with agar dilution. The difference in case of tetracycline, gentamicin, ciprofloxacin, amoxicillin and cephalosporins (ceftizoxime, ceftazidime) and trimethoprime-sulfamethoxazole were significant (P=0.002).In the centers under study lower rate was reported for the isolation of Enterobacter spp and citrobacter spp, while Escherichia coli and Klebsiella spp were reported more than their actual presence. These results showed that the laboratory reports were relatively accurate. However since higher rate of bacterial resistance were reported by the disk diffusion methods, in case of serious and life threatening infections suitable drug for treatment should be confirmed by more sensitive tests. In the absence of suitable and low cost detection kits for the identification of gram negative bacilli, inaccurate identifications is an avoidable, therefore immediate action to design the accurate and low cost kits for the identification of these bacteria is recommended in the country.

Hepatitis viruses and Human Immunodeficiency Virus (HIV) are the most common blood-borne infections transmitted to healthcare workers. This study was performed to determine the prevalence of antibodies to hepatitis and AIDS viruses among dentists in Qazvin. All dentists in the city of Qazvin were asked to complete a questionnaire and donate a blood sample for analysis of hepatitis B surface antigen (HBsAg), anti-HBs titer, anti-HCV, and anti- HIV antibodies by ELISA. Positive samples for anti-HCV, and anti- HIV were assessed by RIBA and Western blot. Data analysis was carried out through SPSS software and Chi-square test. From 77 dentists who completed the questionnaire, 74 dentists (93.7%), including 49 general dentists (63.6%) and 24 specialists (36.4%) supplied blood samples. All blood samples were HBsAg,, anti-HCV and anti- HIV negative, and 40 general dentists (83.3%) and 24 specialists (92.3%) have used complete doses of vaccine. Among dentists, 34 general dentists (69.4%) and 12 specialists (24.9%) have visited patients suffering with hepatitis (1.5 times for general dentists). Antibody titer in 8 general dentists (10.8 %) was less than 10 mIU/ml, in 12 dentists (16.2%) was 10-100 mIU/ml in 23 dentists (31.1%) 100-500 mIU/ml and in 31 dentist (41.9%) was more than 500 mIU/ml. This level in 2 general dentists (2.7%) without prior vaccination was positive (10-500 mIU/ml). Between antibody level and vaccine dose (P= 0.04),as well as the education status (general and specialist) (P= 0.03) there was a significant correlation. The findings indicate HBsAg,, anti-HCV and anti- HIV are negative. Compliance of complete doses of vaccine and anti-HBs titers is satisfactory. But, antibody titer without prior vaccination indicates the continuous risk of HBV infection for dentists and in fact the risk of blood borne pathogens.There is a correlation between anti-HBs titer with vaccine doses and also with education status. Regarding with more hepatitic patients visited by general dentists, this group is more exposed to blood borne pathogens and emphasis the continuous education about control infection and surveillance of dentists by blood borne pathogens tests.