دو ماهنامه میکروب شناسی پزشکی ایران
سال سوم شماره 4 (زمستان 1388)

Background and abjectives: Pseudomonas aeruginosa is the most common cause of burn wound infections in many centers. This bacterium shows high resistance to majority of antibiotics including β-lactams. The frequency of beta-lactam resistant P.aeruginosa is increasing in many countries due to production of Metallo beta lactamase enzymes. The aim of this study was to determine the antibiotic resistance patterns ofPseudomonas aeruginosa isolated from burn wound infections and to identify bla-VIM gene by application of phenotypic and genotypic methods. 79 isolates of Pseudomonas aeruginosa were recovered from 111 burn patients using microbiological methods. For all the isolates, antibiotic susceptibility tests were performed by Kirby–Bauer disc diffusion method. All imipenem resistant isolates were screened for MBLs production by IPMEDTA disk and in order to detect blaVIM gene, PCR analysis was performed. Resistance rate of the isolated strains were as fallows: imipenem 94.9% pipracilin 97.4% ciprofloxacin 98.7% tobramycin 95% ceftazidim and ticarcilin 100%. IPM-EDTA disk method showed that 41 out of 74 (55.4%) of imipenem resistant isolates of P. aeruginosa, were metallo-β-lactamase producers.The PCR method showed that 34 out of 79 P. aeruginosa isolates were positive for blaVIM. The results demonstrated that incidence of MBLs producing Pseudomonas aeruginosa in burn patients is higher than expected. Therefore detection of antibiotic resistance patterns of bacteria as well as detection of MBLs enzyme producing isolates are of great importance in prevention and control of these infections.

RNA extraction with efficient quality and quantity is very important for RT PCR,hybridization on northern blotting and gene expression analysis by real time PCR. Soluble and insoluble extracellular polysaccharides in Streptococcus mutans biofilm cells interfere with RNA extraction procedures. Therefore, finding efficient methods for polysaccharide removal, RNA extraction and purification is necessary for RNA based molecular research. The aim of present study was to evaluate the efficary of a few methods in RNA extraction from Streptococcus mutans. In this research we used Streptococcus mutans ATCC 35668 and one clinical isolate of S. mutans from dental plaque. RNA extraction was carried out from biofilms form in 24 well polystyrene microtiter plate using 3 methods. 1: Routine method for RNA extraction from planktonic cells with RNX-Plus solution (Cinagen), 2: Using HYBAID ribolyser Kit, instrument and tubes containing pearls. 3: Using HYBAID ribolyser instrument and RNX-Plus solution. Finally, determination of the isolated RNA purity and integrity was done. The results showed that RNA extraction from biofilm cells of S. mutans is challenging because of extracellular polysaccharides and the current RNA isolation protocols from planktonic cells (eg. protocol 1) are not suitable for biofilm cells. For this reason, we used HYBAID ribolyser instrument and tubes containing glass and plastic pearls with different size and shapes for maximum cell lysis without disturbing the RNA.The mean photo absorbtion from extracted RNA in latter methods showed statistical significantdifference compared to current in-use method (P< 0.05). RNA extraction from S.mutans biofilm cells needs techniques with maximum Polysaccharide removal and maximum cell lysis without disturbing the RNA. The 2nd and 3rd methods were more effective than 1st one.The 3rd protocol is recommended due to it`s lower cost.

Cellular resistance to tuberculosis (TB) infection depends on signals from mycobacterium tuberculosis-specific T lymphocytes activating mycobacterial killing mechanisms in infected macrophages. The network of soluble factors, or cytokines, responsible for these cellular communications has been progressively unraveled over the last 3 decades; however, new regulatory and effectors cytokines continue to be discovered. Infection of a host with a pathogen first result in activation of cells of the innate immune response including effectors molecules including cytokines such as TNF-α & IFN-γ. The aim of this study was to investigate the frequency of TNF-α, IFN-γ alleles, evaluating serum concentration of TNF-α and IFN-γ and relationship of between susceptibility to TB and TNF-α and IFN-γ gene variations. In this prospective case-control study, 93 patients with smear positive tuberculosis selected from Masih Daneshvari Hospital, Tehran, Iran. They were matched with 103 controls without any history TB. Genotype of 5 regions of TNF-α and 1 region of IFN-γ were distinguished by PCR-RFLP method, and level of serum concentration between case & control groups were evaluated by ELISA method.Data were analyzed with Mann-Whitney U & earning the cut off analyzed with ROC curve for ELISA method and the results of PCR-RFLP method were analyzed by SPSS, Fisher exact and X2. In PCR-RFLP method, the results showed a significant difference at TNF-308 and TNF-857 between two groups of control and patient (P < 0.05). In ELISA method, a significant difference in IFN-γ was observed between the groups of control and patient (P < 0.05). Also a cut off point as a serologic marker between the positive and negative states, for rapid TB examination about IFN-γ was found; the cutoff for IFN-γ was 0.19. Mutation in TNF-308 and TNF-857 regions were identified significantly, but it was not observed in other TNF-α and IFN-γ regions. In this study, ELISA method suggest that mycobacterial pathogens are frequently associated with production of cytokines such as IFN-γ and serologic tests considering the cut off patients, help us to detect TB in cases that we want to earn results rapidly.

Migraine is the most common headache in different communities and approximately 12-15% of individuals are suffering worldwide. Recent studies have revealed a relationship between Helicobacter pylori infection and migraine. The aim of presend study was to evaluate the antibody titer against H.pylori in patients with migraine and comparison to healthy subjects. This case – control randomized study was carried out in patients whose migraine has already been diagnosed referred to University neurology clinic and private medical sectors in Ilam, Iran.Seventy migraine patients as the case group and 70 healthy individuals as control group were participated in this study. The demographic criteria of both groups were identical. Blood samples were taken from all subjects and was used for evaluation of IgG antibody titer against H.pylori by using ELISA method. The mean antibody titer were compared in both groups. Household women had the highest prevalence of migraine (40%), which showed a correlation with menstruation in 21 women (45.7%). Fifty three patients with migraine (75.7%) had gastrointestinal disorders, which in 48 patients (68.6%), this was in correlation with certain nutritional habits. Stress was reported as the most important cause of migraine in 15 patients (21.4%) that in 91.4% of them, headache wasfollowed by anxiety. In 51 patients (72.9%), sleep disorder was seen. The mean OD value of antibody titre against H.pylori was 60.08 in case group and 21.82 in control group, which the difference was significant (P<0.05). The significant difference for the mean OD value to H.pylori among case and control groups in present study, shows the importance of investigation of H. pylori infection in patients with classic migraine,which needs more complementary tests. It is suggested that treatment of H.pylori infection may affect the disease and probably cure the headache partially.

Methicillin resistant Staphylococcus aureus (MRSA) is a main cause of nosocomial infections worldwide. Multidrug resistance property of strains causes difficulties in treatment of their infection. The aim of this study was to determine the frequency of MRSA and their resistance pattern to commonly used antibiotics in Health-educational centers of Gorgan. In this descriptive study, 121 clinical isolates of Staphylorcoccus aureus collected from different infections from September 2008 to August 2009. After confirmatory identification tests, the antibiotic susceptibility testing was performed by using disk diffusion method as per Clinical and Laboratory Standards Institute (CLSI) guideline. Data analyzed by Chi-square test and SPSS soft ware. Pvalue of <0.05 was determined as significant. Of 121 tested S.aureus, 104(85.9%)strains were MRSA. The highest resistance was demonstrated as:100% to Penicillin, 97.6% to CO- Amoxyclav, 71.4% to Cephotaxime and64.3% to Erythromycin. Our finding showed more MRSA isolates in urine (90.4%) and wound (89.2%) specimens. Prevalence of MRSA strains in our region was 85.9%. This is higher than other studies performed in major cities such as Tehtan,Mashhad and Shiraz. The treatment of infections caused by MRSA is difficult due to simultaneous multidrug resistance among the strains.

Hospital surfaces can serve as reservoirs of potential pathogenic bacteria. Staff hands are the main source of bacterial transmission in hospital. The prevalence of bacteria harboring β–lactamase enzyme in staff hands and hospital surfaces, leads to spread of β–lactamase producing bacteria and ultimate increase of antibiotic resistance nosocomial infections. The aim of this study was to investigate the frequency of β-lactamase producing bacteria and susceptibility pattern of isolated bacteria from staff hands and high and low contact hospital surfaces of Alzahra hospital in Isfahan, Iran. This laboratory-based study was performed in Alzahra hospital in Isfahan during 2005-2007 years. Overally, 274 samples (194 strains from surfaces and 80 strains from staff hands) were screened during the study. Environmental samples were collected by using swabs in Nutrient Broth (NB) and samples from staff hands were collected with Finger Print method. Bacterial identification was performed by conventional biochemical identification tests. For determination of β–lactamase production,acidometric method was used, and antibiotic susceptibility testing was performed by Kirby Bauer method. the 194 isolated strains from hospital surfaces were: Staphylococcus spp. 105 (53.7%),Bacillus spp. 74 (24%) Enterobacteriaceae 21 (10.7%), Pseudomonas spp. 9 (4.6%), Streptococcus spp. 2 (1%), other gram negative bacilli 10 (5.15%), and the 80 strains isolated from staff hands were:Bacillus spp. 48 (60%), Staphylococcus spp. 28 (35%), Enterobacteriaceae 4 (0.5%). The isolated bacteria from both sources were highly resistant to tested antibiotics. According to the results from acidometric test, 125 (61.54%) strains isolated from hospital surfaces and 46 (61.85%) strains isolated from staff hands, were β-lactamase producers. The results show the high frequency of antibiotic resistant and β–lactamase producing bacterial strains on staff hands and hospital surfaces in present study.

Infections due to Herpes simlex virus type 2 (HSV-2) often causes genital herpes in men and women, infant herpes and nonsupporative meningitis. These diseases are caused in relation to HIV infection, and might be transfected from mother to baby.In this study the prevalence of HSV-2 was determined for the first time by PCR method in clinical samples collected from Isfahan and Chaharmahal-va-Bakhtiari provinces. One hundred HSV-2 infected sera with high titer of IgG and IgM were collected from suspected patients. In total 82 samples from Isfahan province and 18 samples from Chaharmahal-va-Bakhtiari province were collected. Viral DNA was extracted and PCR was performed by using specific primers for gD gene of HSV-2. The positive results of HSV-PCR and the 1013 bp segment of HSV-2 gD gene was detected in 8 of 100 (8%) of serum samples. In Isfahan province 6.09 % and in Chaharmahal-va-Bakhtiari province 16.66 % were positive for HSV-2. The results of this study demonstrated the prevalence of HSV-2 in studied region similar to other regions of the world. Besides, PCR method is presented as a useful procedure for detection of HSV-2 in infected sera.

The intracellular Wolbachia pipientis is a Rickettsia-like bacterium. The gene of W. pipientis is usually congenitally-inherited in insects and can cause cytoplasmic incompatibility (CI),which is potentially useful for driving genes through populations.In present study, W. pipientis has been detected in Phlebotomus papatasi, the vector of rural cutaneous leishmaniosis in Iran by using PCR to amplify fragments either of the 16S ribosomal RNA gene (16S rRNA) or of the major Wolbachia surface protein gene wsp. sandflies were screened for the presence of W. pipientis by PCR using the nonstrain specific primers wsp 81F with 691R and 16S 99F with 994R. The sequences obtained were edited and aligned using SequencherTM v. 3.1 to identify W. pipientis haplotypes, which were analysed phylogenetically using PAUP* software. from 165 Individual wild-caught P. papatasi from Iran, the gene fragment of wsp was found in 124 cases (75.2%), and 16s RNA gene fragment was identified in 123 cases (74.5%). Only one haplotype was obtained for each gene, from which it is inferred that only one A-group genetic strain of W. pipientis occurs in P. papatasi throughout much of this sandfly's range. based on the results of this study there is possibility of using just one genetically modified strain of W. pipientis to drive through wild sandfly populations transgenes for intervening in the transmission of L. major by P. papatasi. This genetically modified strain would have to show a CI phenotype in P.papatasi, in order to be spread quickly through wild sandfly populations. Due to natural infection of Wolbachia in sandflies and for their behavior of Cytoplasmic Incompatibility(CI), Wolbachia can be used as a transferring gene in populations of sandflies to control Leishmaniasis.