Iranian Journal of Microbiology
Volume:1 Issue: 4, Dec 2009

PCR is a sensitive assay and could be used as an accurate diagnostic method for detecting various types of microorganisms'' genome in low concentration in biological specimens. The demand for sensitive, rapid, safe and easy detection of PCR products has led researchers to a combination of this method with ELISA Conserved sequences were selected for design of primers. Samples were tested by ELISA (for detection of specific HIV and HCV proteins) and real- time PCR for detection of specific nucleic acid and viral genome respectively. Viral genome was extracted and reverse transcription was performed with M-Mulv and the cDNA kept at -80º C. The PCR products were labeled by DIG-dUTP. Diluted PCR products were analyzed with both electrophoresis and ELISA methods. Thirty-five samples were tested with the PCR-ELISA method. False positive or negative reactions were not observed. ELISA results of diluted products were compared with results obtained by electrophoresis. In gel electrophoresis, dilution of 1/10 was positive, but in ELISA, optical density of 1/100 dilution was much more than the cut-off value. Detection limits for gel electrophoresis as well as ELISA have been evaluated. It was shown that the PCR-ELISA method is ten times more sensitive than conventional PCR.

A semi-quantitative Real-Time PCR strategy was developed to identify indicator organisms for an indication on anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method. The results showed that the semi-quantitative RT-PCR method has the clear potential to be useful as a powerful tool in early detection of anastomotic leakage.

Brucellosis is primarily a contagious disease of domestic animals causing abortion, so it is considered one of the most serious of the current public health problems, especially in developing countries. The main purpose of this study was finding the incidence of human and animal brucellosis and detection of any correlation between human and animal brucellosis in Khoy, one of the endemic regions in Iran. We carried out an ecological study in Khoy district in North West of Iran. We ascertained all new cases of human and animal brucellosis in the 2001-2004 period. Data were analyzed using Pearson correlation coefficient (r) and square of correlation coefficient (r2). Seasonal incidence was calculated for each species. The cumulative incidence rate of human brucellosis was detected to be 175/100,000, cattle brucellosis was 391/100,000, and sheep and goat brucellosis was 105/100,000. We detected direct and incomplete correlation between human and cattle (r=0.096, r2=0.009, p value 0.742), human and sheep (r=0.267, r2=0.071, p value=0.355), and cattle and sheep (r=0.797, r2=0.635, p value=0.001). The most effective routes to control the disease include pasteurization or boiling of milk for human consumption, cooking all food stuff derived from animal sources, vaccination of cattle against brucellosis, isolation and slaughtering of seropositive reactors for brucellosis and providing protective clothing for humans dealing with infected cattle.

Backgrounds and to obtain information about aflatoxigenicity of isolated Aspergillus flavus strains from shrimps. Forty - three isolates of Aspergillus flavus from cultured green tiger shrimps of Persian Gulf were examined for their ability to produce aflatoxins. Initially two media; Aflatoxin Producing Ability medium and Coconut Agar medium were used to detect fluorescence under UV light, later the presence of aflatoxin in culture extract was confirmed and quantified by high pressure liquid chromatography. Only 2 (4.6%) isolates fluoresced on Aflatoxin Producing Ability medium and Coconut Agar medium under UV light. In sum, 9 (20.93%) isolates (including the 2 above mentioned isolates) were confirmed to be aflatoxigenic by High Performance Liquid Chromatography. Eight (18.7%) of isolates produced aflatoxin B1 ranging from 0.32 to 12.18 ppb, while 1(2.3%) of isolates produced 18.88 ppb and 0.36 ppb of aflatoxin B1 and aflatoxin B2 respectively. Aspergillus oryzae did not produce any detectable aflatoxins. Although highest level of aflatoxin B1 (18.88 ppb) was detected in an isolate from a hepato-pancreatic sample, no histopathological change was observed in that tissue. Some Aspergillus flavus strains which were isolated from shrimps showed aflatoxin producing ability without any histopathological changes in tissues of contaminated shrimps.

Pseudomonas aeruginosa is an opportunistic pathogen that can produce biofilm. Biofilm is a complex, three dimensional structure in which microorganisms are attached to a surface and embedded in a matrix made of extracellular polymers. Due to high resistance to antimicrobial agents, biofilms create difficulties in various situations in healthcare. In this study, antibiofilm activities of some biocides in P. aeruginosa were studied. The biofilm production ability of P. aeruginosa strain 214 (a clinical isolate) was determined in the presence of six biocides including of ethylene diamine tetra acetic acid (EDTA), silver nitrate (AgNO3), bismuth ethanedithiol (BisEDT), bismuth dimercaprol (BisBAL), bismuth-2-mercaptoethanol (BisMEO) and bismuth propanedithiol (BisPDT) using the modified microtiter plate method. Bactericidal activity of the biocides against biofilm and planktonic cells was investigated. In this study, permeation of biocides through alginate layer was evaluated with a sandwich cup method. The results demonstrated that in the presence of bismuth thiols, biofilm production in MIC and sub MIC concentrations was considerably inhibited. Bismuththiols had lower antibiofilm bactericidal activity than EDTA and silver nitrate. One possible mechanism of biofilm resistance is exopolysaccharide production which prevents the access of antimicrobial agents to cells inside the biofilm. Bismuth thiols could not penetrate, while EDTA and silver nitrate had high penetration rate. Due to the frequent use of silver nitrate and EDTA in various applications, low efficacy in the inhibition of biofilm production, unstudied toxicity of BTs for humans and high efficacy in the inhibition of biofilm production, it is suggested that combinatory effect of BTs with silver nitrate or EDTA on biofilms and biofilm production be investigated.

The development of molecular biological techniques provide a method for direct detection and identification of pathogens. This study attempted to compare bacterial culture and PCR to detect S. agalactiae in cattle milk samples. The PCR method was based on using primers V1 and V2 derived from the 16s rRNA gene. One hundred milk samples were collected from individual cattle herds of Urmia, Iran. Direct bacterial DNA extraction was attempted from100 milk samples. The PCR method was based on using primers V1and V2 derived from the 16s rRNA gene. In addition, two sets of positive control primer pairs (C1 and C2) were used to identify false negatives obtained with primers V1 and V2. In bacteriological culture, biochemical tests were carried out on suspected isolates. S. agalactiae was isolated from 8% of the 100 samples and 15% were positive in amplification of the 16S rRNA gene and all of the positive samples gave the expected size fragment of approximately 120bp. All isolates which were diagnosed as S. agalactiae in microbiological methods were also positive with PCR detection. Compared with culture, PCR is less time consuming. It takes less than 24h to complete, while identification of bacteria by conventional microbiological and biochemical methods requires more than 48h. The findings of our study revealed that PCR is more sensitive than culture for the detection of S. agalactiae in milk.

To determine the potential circulation of avian influenza viruses among different captive bird species, molecular surveillance was conducted at Tehran Zoo, Saiee Park and Pardisan Park of Tehran, Iran. These places are at risk for spread and transmission of influenza virus because of bird species diversity and close contact of birds with humans. During the influenza season in Tehran, in the cold weather (November 2008-February 2009), 76 cloacae samples were collected from 5 orders of Anseriformes, Galliformes, Columbiformes, Pelicaniformes and Phoenicopteriformes, including 13 bird species plus 5 hybrid species of ducks. Presence of avian influenza genome was monitored with RT-PCR as a sensitive and specific assay. The assay targeted a 132 bp fragment of the conserved M gene of influenza type A. Influenza type A virus was not detected in samples collected from November 2008 to February 2009. The sensitivity of RT-PCR based on M primers was 0.1ng total RNA. Interestingly, during the study period, there was no report of death or clinical signs of disease among the c aptive birds, whereas the birds did not have vaccinated history against influenza A virus. Although the results could be attributed at least partially to the presence of an undetectable amount of genomic RNA, based upon the sensitivity of the test our findings suggest that no RNA genome of influenza A viruses was present in the samples under study.

In marine actinomycetes, carotenoid production occurs in constitutive, light-dependent or cryptic manner. The present work deals with the fermentative production of carotenoids from marine actinomycetes. Marine actinomycetes namely Streptomyces strain AQBMM35 was isolated from the marine sponge Mycale mytilorum collected from South West coast of India using ISP media. The Streptomyces isolates were characterized for their colony characteristics, morphological properties, physiological and biochemical properties and were tentatively identified. Fermentation of the strain under fluorescent white light was carried out for the production of carotenoids. UV spectrum, TLC and HPLC analysis were done for the confirmation of carotenoids. The characteristics studied strongly suggest that the strain AQBMM35 belongs to the genus Streptomyces sp. It has been found that Streptomyces strain (AQBMM35) fermenting under fluorescent white light produced carotenoids. Spectrophotometric analysis of the carotenoid fraction revealed a peak at 280 nm. TLC analysis of the carotenoid extract showed the presence of phytoene (Rf of 0.81). HPLC confirmed the production of phytoene when compared with standards. The fermenting sponge-associated Streptomyces isolate (AQBMM35) produced carotenoids namely phytoene. If this symbiotic Streptomyces strain, from which secondary metabolite like carotenoids are derived, can be cultured under light, then it can be used for mass production of precursor pigment and it can be used as an antioxidant and also as a food additive.