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Background and Aims: Many bacteria including Escherichia coli may enter into a viable but non-culturable (VBNC) state under unfavorable stresses, which are unable to be detected by culture-based methods. In this study, the use of Reverse Transcription PCR (RT-PCR) for detection of VBNC state of E. coli O157:H7 was investigated. Materials and Methods: Escherichia coli O157:H7 was inoculated in distilled water and 30 percent salt water at room temperature. The cultivability of bacteria was determined using routine culture and colony counting on MacConkey agar. The RT-PCR of 16S rRNA gene involved direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA was used to detect viable E. coli O157:H7 under studied treatments and was compared with the results of RT-PCR of 16S rRNA gene of heat- killed bacteria. Results: The cultivability of bacteria was maintained during the study period in distilled water; however, the use of 30% NaCl caused the bacteria to be non-cultivable on day 4. The RTPCR of 16S rRNA showed the positive expression of this gene in cultivable and non-cultivable bacteria during the study period, whereas heat-killed bacteria were negative for this gene, which indicated the efficacy of RT-PCR of 16S rRNA in differentiation of alive from dead bacteria. Conclusions: Escherichia coli O157:H7 entered into the VBNC under 30 percent NaCl which can be associated with serious human health problems. RT-PCR can be used to detect bacteria in the VBNC state. [ABSTRACT FROM AUTHOR]

Background and Aims: Brucellosis is an infectious zoonotic disease responsible for reproductive disorders among animals and significant economic losses to the meat and milk supplychain. The purpose of this study was therefore to evaluate and diagnose the species and strains of Brucella isolates among cattles via multiplex polymerase chain reaction. Materials and Methods: In this study, blood samples were taken from 22 cows diagnosed as having brucellosis by PCR test. Blood samples were cultured in BACTEC vials and incubated at 37 degrees Celsius for 5 days following which each sample was cultured on Brucella agar and kept for 3 days under Co2 condition.The 16SrRNA gene was used as the target for the detection of Brucella genus and the bcsp31 gene was used for the detection of Brucella biovars. The PCR products were electrophoresed on %1 agarose and compared with standard strains. Results: The PCR results showed a total of 22 positive samples for Brucella among cattles. Out of the 22 cattle positive samples, 19 were identified as B.abortus (biovars 1, 2, 3, 4, 5) and 3 as B. melitensis (biovars 1, 2, 3). Conclusions: Brucella abortus biovars 1 and 3 and B .melitensis biovar 1 are the most prevalent biovars among cattles in Lorestan province, Iran. Moreover, multiplex PCR method could be used to improve the qualitative level and the Brucella diagnosis speed in laboratories. [ABSTRACT FROM AUTHOR]

Background and Aim: The efflux pump in Pseudomonas aeruginosa inhibits the effect of ciprofloxacin by releasing quinolones out of the cell. It is important to find compounds to inactivate or inhibit its activity to continue using the antibiotic. The present study aims to investigate using sertraline as an efflux pump inhibitor in P. aeruginosa to reduce the antibiotic resistance. Methods: Pseudomonas aeruginosa strains were isolated from clinical sources and were identified by routine microbiological methods. Resistance of the isolates to ciprofloxacin was evaluated by Kirby–Bauer test. Resistance breakdown was investigated by adding sertraline to the Moller Hinton agar medium and determining the zone of inhibition of ciprofloxacin. Minimum inhibitory concentration (MIC) by microplate dilution method and Minimum bactericidal concentration (MBC) by culture and MTT method were determined for the isolates and ATCC 27853. The presence of the efflux pump was evaluated by phenotypic method using sertraline and serial dilution method of liquid medium in microplate, on ciprofloxacin resistant strains. The presence of the producing gene of this pump was determined by genotyping method in resistant strains by performing PCR. The standard PAO1 strain of Pseudomonas aeruginosa was used as a positive control. Ethical Considerations :This study was approved by the Ethics Committee of the Faculty of Medical Sciences of Islamic Azad University of Brojerd (Code: IR.IAU.B.REC.1401.011). Results: Based on Kirby Bauer's results, three strains were considered resistant to ciprofloxacin. MIC of drug-resistant strains was between 32 and 64 mg/ml and MBC was between 16 and 32 mg/ml. By performing electrophoresis on the PCR products, it was determined that the tested strains contain the mexA gene encoding the efflux pump. In the agar medium without sertraline, the zone of inhibition of around the ciprofloxacin disc was zero, but after adding sertraline, the diameter of the halo increased to 25 mm.The minimum inhibitory concentration of ciprofloxacin in the isolates before adding 25 micrograms of sertraline was 128 micrograms/ml and after adding sertraline, it was 4 micrograms/ml. Conclusion: It was concluded that sertraline inhibited the efficiency of the efflux pump in resistant P. aeruginosa isolates and reduced ciprofloxacin resistance. [ABSTRACT FROM AUTHOR]

Background: The genome of Enterococcus has a large number of repetitive sequences that are randomly distributed over DNA. In ERIC-PCR, a separate pattern is obtained for each strain and is considered a separate type. Prokaryotic and eukaryotic genomes include dispersed repeat sequences that are relatively short non-coding, and dispersed in the bacterial genome. BOX-PCR and ERIC-PCR primers are complementary to these repeat sequences and allow for dedicated binding and unique BOX-PCR fingerprint patterns and ERIC-PCR with reproducibility capability. Methods: In this cross-sectional descriptive study, based on previous studies and 95% confidence level using the formula n = z2P (1-P)/d2 and acceptable error 0.05, total of 60 Enterococcal Faecalis strains were cultured from sterile specimens on KF agar medium and incubated at 37°C for 24h and were identified by biochemical tests of suspected Enterococcal Faecalis. DNA samples were extracted and BOX-PCR and ERIC-PCR tests were performed. Results: All strains were distinguished in 25 distinct clusters at the level of similarity of 58%. Also, by analyzing the ERIC-PCR results, all strains were segregated into 15 separate clusters at a similar level of 58%. Conclusion: Molecular fingerprinting with BOX-PCR has a better subtraction and differentiation than ERICPCR for typing bacterial isolates and is widely used in epidemiological studies and trace source of infection and taxonomy. [ABSTRACT FROM AUTHOR]

Background and Aims: Mycoplasma pneumoniae is one of the species of mycoplasmas which could contaminate cell cultures. Identification of M. pneumoniae is significant because mycoplasmas could contaminate and have unsuitable effect on the cell cultures. Rapid detection of these contaminations is so important and it could be significant role in preventing and controlling of contamination in cell cultures. Materials and Methods: In this study, the isolation and detection of M. pneumoniae from 82 cell culture samples that were sent to the Mycoplasma Reference Laboratory using cell culture and PCR method were performed. M1F and M3R primers that have the ability to identify mycoplasma genus from the 16S rRNA gene were used. The P1 adhesin gene and MPF and MPR specific primers were used to initiate the PCR reaction to detect Mycoplasma pneumonia. Results: Of 82 samples, 31(37.8%) were positive and 51 (62.2%) were detected negative performing cell culture. Whereas, out of 82 samples, 48 (58.53%) were positive and 34 (41.47%) of the samples were negative using mycoplasma genus PCR as diagnostic method. M. pneumoniae was not detected from 82 samples in this study . Conclusion: The results of this study indicated that Mycoplasma pneumoniae is not a factor contributing to cell cultures in Iran. But PCR could be an alternative method instead of the culture because according to the results of this study, PCR has high accuracy, speed and it is cost-effective for detecting M. pneumoniae. [ABSTRACT FROM AUTHOR]