دو ماهنامه میکروب شناسی پزشکی ایران
سال هشتم شماره 3 (پاییز 1393)

Type IV pilin molecule in Pseudomonas aeruginosa with wide functions, has important role in the diversity of the bacterial genome. According to the latest classification, Type IV pilin is divided to two main subtypes; IVa, IVb. The major subunit encoding Type IV pilin subtypes; IVa, IVb is pilA and pilS2 genes respectively which serve as a marker for the detection of the Type IV pilin subtypes. The purpose of the current study was to evaluate the frequency of pilA and pilS2 gene in cystic fibrosis, burns, and environmental isolates. The samples for P.aeruginosa were collected from cystic fibrosis, burns, and environment wastewater during the April 2013 to December 2013. Samples were cultured and identified using microbial and biochemical methods. DNA of isolates was extracted by commercial kit. PCR was performed using specific primers. Statistical analysis was done using SPSS 17.0 software. A total of 100 P.aeruginosa isolates were collected; 30 cystic fibrosis, 30 burn, 40 environmental. The prevalence of the pilA gene were 63.3%, 56.7% and 45/2% for cystic fibrosis, for burns and environmental strains. The PilS2 gene frequency was 57.5% of the environmental isolates, 67.5% of cystic fibrosis strains and 73.3 % of the isolates were burned. Our study showed that the frequency of P.aeruginosa T4P major pilin subunits in our P. aeruginosa population was high. The incidence of pilS2 gene was greater than pilA and was more frequent in Pseudomonas aeruginosa isolated from hospitalized burn patients.

Acinetobacter baumannii is opportunistic, gram negative, aerobic and nonfermentative coccobacilli. It is difficult to control and eradicate these bacteria, because of increased resistance to last-line antibiotic therapy and high peripheral resistance. The purpose of this study was to determine the prevalence of integron and its relationship with the antibiotic resistance in A. baumannii. 100 isolates of A. baumannii were gathered since 2012-2013 from ICU of three educational hospitals of Hamadan,Iran. After phenotypic identification of strains, they were confirmed with amplification of the blaOXA-51 gene by PCR. Antibiotic susceptibility pattern of isolates was determined by disk agar diffusion (DAD) method.. The presence of class 1, 2 and 3 integrons were analyzed using the PCR method. The most antibiotic resistance pattern was observed to ceftazidime (98%) and the highest sensitivity was seen to colistin sulfate (99%) and tigecycline (88%). Prevalence of integron class 1 and 2 were 97% and 31%, respectively. Class 3 integron was found in any of the isolates. In this study, a very high resistance to different classes of antibiotic and high prevalence of class 1 integron was detected in A. baumannii. Statistical analysis showed association of class 1 integron with drug resistant in this bacterium. Also no significant relationship between antibiotic resistance and the presence of class 2 integron was found in statistical analysis.

The increases in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) from outside healthcare settings are recently reported. This study was performed to determine the prevalence of community-acquired MRSA and antibiotic resistance of the isolates from nose of military training soldiers in Kerman city- 2012. Nasal samples were collected from 567 training soldiers. S. aureus was identified using standard Methods. MRSA phenotype was screened by oxacillin and cefoxitin disc. The MRSA was genetically confirmed by detection of mecA gene by PCR methods. Antibiotic susceptibility to six antibacterial agents was determined by standard agar disc diffusion method. Induction of resistance to clindamycin was performed using the D-test. samples were isolated from the nose of 39.8% of training soldiers. By the methods used 7.6% of the isolates were identified as MRSA all of them harbored mecA gene and were sensitive to vancomycin. The highest rate of resistance was detected against erythromycin (23.3%). Resistance to, clindamycin, gentamicin, trimethoprime sulfamethoxazole and ciprofloxacin were 14%, 16.3%, 14% and 9.3% respectively. Induced of Clindamycin resistance among MRSA isolates was not observed. The high frequency of isolation of S aureus from nose in military population studied is remarkable. The prevalence of MRSA in this group was %7.8.Although resistance to other antibacterial agents was not high in these isolates, but due to the increasing importance of community acquired MRSA frequent surveillance of this and similar type of population are required in intervals, and physicians should be informed about the presence of these bacteria to choose appropriate drug for therapy.

We carried out a descriptive study to determine the extent of nasal colonization and characteristics of Staphylococcus aureus and CA-MRSA isolates in 2-5 year old children of day care centers in Isfahan. The characteristics of isolates were determined using standard phenotypic profiles including colony morphology, Gram staining, catalase, hyaluronidase, coagulase and Dnase tests as well as mannitol fermentation. The MRSA detection was carried out according to CLSI guidelines with oxacillin agar screen test. Methicillin resistance was further confirmed by detection of a 310 bp fragment of mecA gene of MRSA by PCR. Drug susceptibility testing to antibiotics other than methicillin was conducted by disk diffusion. The Beta-lactamase production and inducible clindamycin resistance were also determined by performing the double-disc diffusion and D-test. Out of 323 children, 115 (35.6%) carried S. aureus and 11 (9.5%) carried MRSA. All MRSA strains were found to contain mecA gene. The susceptibility of strains to vancomycin, rifampicin and Linezolid were 100%. The susceptibility of strains to gentamicin, clindamycin, erythromycin, co-trimoxazole, amoxiclav, ciprofloxacin, tetracycline and penicillin were 99%, 97%, 94%, 94%, 93%, 88%, 44.4% and 1.8% respectively. Beta-lactamase production was seen in 19 (16.5%) of staphylococcal strains. Inducible clindamycin resistance was seen in 4 (3.5%) of the isolates. Our data indicates that the spread of CA-MRSA within Iranian population is worthy of consideration and merits further molecular investigation to determine the source and mode of transmission.

Listeriosis is one of the most important food-borne diseases caused by Listeria species especially L. monocytogenes. The objective of this study was to determine the prevalence and antimicrobial resistance of Listeria species isolated from smoked and salted fish in Isfahan and Bandar anzali. From August 2009 to April 2011, a total of 120 samples of various smoked (n= 80) and salted Iranian fish (n= 40) were obtained from randomly selected retail stores in Isfahan and Bandaranzali cities and were evaluated for the presence of Listeria spp. using standard cultural and PCR methods. Then antibiogram tests were done for determination of antimicrobial resistance. 7 (8.8%) and 6 (15%) of smoked and salted fish samples were positive for Listeria spp. respectively. L. monocytogenes, L. innocua and L. seeligari were isolated from 2.5, 6.7 and 1.6% of fish samples. 9 of 13 Listeria isolates (69.2%) were resistant to one or more antimicrobial agents. Resistance to nalidixic acid (53.8%) and tetracycline (30.8%) were the most common finding. The results of this study indicate the potential risk of infection with Listeria in people consuming raw or under cooked smoked and salted fish. Also, the results obtained in this study indicate the need for an appropriate strategy of surveillance and epidemiological monitoring to control the development of resistance.

Colonization of gram – negative bacteria and fungi in oropharynx of hospitalized patients in intensive care units, is an important process in the development of aspiration pneumonia in these patients (ICU). The purpose of current study was to define effect of selective oropharyngeal decontaminatio (SOD) on colonization of the oral-pharynx in hospitalized patients in intensive care units. In this clinical trial study, 60 hospitalized patients in ICU have been randomly divided into two groups: control group and experimental group. Routine mouthwash with saline solution (QID 0.9%) was prescribed and after 24 hours, a cultural test has shown colonization rate. For experimental group, immediately after cultural test, SOD (paste containing polymyxin 2%, Tobramycin 2%, Amphotericin 2%), were used for a week. Routine mouthwash was continued for control group. After a week, the second culture was performed and the colonization rate in both groups was compared. SOD has decreased colonization rate in experimental group (positive 10 cases in the first culture to 4 cases in the second culture) and in control group, positive 8 cases in the first culture have increased to 18 cases in second culture. Statistical tests showed that the differences between these groups statistically were significantly meaningful (P= 0.002). In this study, SOD (paste containing polymyxin 2%, Tobramycin 2%, Amphotericin 2%) every 6 hours during a week, in hospitalized patients in ICU, significantly have led to decreasing colonization of oral–pharynx in comparison with control group. As a result, the usage of it for the patients in ICU is recommended.

Vulvovaginal candidiasis (VVC) is a common clinical problem that affects most adult women at least once during their life time. Many also have frequently recurring yeast infections (RVVC) caused by Candida albicans and non albicans Candida species. We studied use of CHROMagar Candida and PCR-RFLP to compare their differential ability and time consuming. Vaginal discharge samples of all women with clinical signs were cultured on CHROM agar Candida medium. After an overnight incubation at 37°C, growth coloration of Candida isolates compared with those of standard patterns. PCR-RFLP using restriction enzyme Msp I were used for the identification of more Candida isolates in the level of species. Totally 192 vaginal discharge samples were obtained from the women with vulvovaginitis clinical signs. 90 patients had Candida vulvovaginitis, 11 case were RVVC. The Candida species which were identified included, C.albicans (78.8%), C.glabrata (7.7 %), C. parapsilosis (6.6%), C. tropicalis (4.4 %) and C. krusei (2.2 %). We concluded that, use of PCR-RFLP is recommended for the identification of Candida species causing VVC and RVVC in accompany with the culture based methods, regarding to its differential power and speed.

Immunotoxin is a cytotoxic protein and have been proposed as a novel compound for cancer therapy. ONTAK is a recombinant immunotoxin composed diphtheria toxin fused to interleukin 2(IL2). In this study, optimization of defined culture medium for the expression of recombinant ONTAK immunotoxin has been investigated. In this study, the bacterial strain BL21 (DE3) was transformed with the recombinant plasmid PET-IDZ was used to express ONTAK. The medium composition such as carbon source (glucose, sucrose and glycerol), nitrogen source (ammonium chloride, urea and ammonium sulfate) and concentration of the inductor IPTG (0.1, 0.5, 1mM), induction time and concentration of various additives (different amino acids) were optimized based on Taguchi method for increasing expression on the shake flask cultivation. The cell concentration was measured by optical density at 600 nm and protein expression levels were analyzed by electrophoresis on SDS-PAGE gel. Modified defined culture medium containing (g/l): glucose,8.0; K2HPO4,15.0; KH2PO4,7.5; Citric acid,2; NH4Cl,3; MgSO4.7H2O,1; were determined as optimal culture medium. OD600nM=2.0 was determined as the best time for induction by IPTG at a concentration of 0.1mM. ONTAK expression was increased by adding Valine, 0.0502; Phenylalanine, 0.0132; Lysine, 0.0184; Aspartic acid, 0.0160 and Serine, 0.0251(g/l) amino acids to the medium. In present study, the Taguchi method analysis revealed that, nitrogen source type (NH4CL 3 g/l) significantly affects in the cell growth. The biomass production was on the optimized medium about over 3 times higher than that at M9 medium.