Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women.

PURPOSE: Sexually Transmitted Diseases (STDs) can cause sterility and many other problems for women planning pregnancy. Currently, almost 340 million people worldwide suffer from Sexually Transmitted Infections (STIs). This study made attempts to quickly identify STDs' most critical infectious agents using dedicated primers and probes. METHODS: The present study was done on the cervical samples of 200 infertile women. After extracting the total DNA of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium, quantitative methods were employed to determine the rate of target bacteria using multiplex real-time PCR. RESULTS: The multiplex qPCR showed the rates of 47%, 16%, 46%, and 16.5% for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women, respectively. In some patients, there were co-infections with two or three bacteria. The diagnostic approach used in our research could be employed as an alternative detection tool to identify the four most common STD-associated bacterial agents while detecting mixed infections. CONCLUSIONS: Infertile women with no biological problems could have their genital tract checked using this newly designed identification technique and get proper treatment for their infections as quickly as possible.

Evaluation of the effect of Saccharomyces cerevisiae on the expression of enterotoxin genes in Escherichia coli O157: H7 (EHEC) and Escherichia coli H10407 (ETEC).

Enterotoxigenic (ETEC) and enterohemorrhagic Escherichia coli (EHEC) are the most important intestinal pathogens. Probiotics play an effective role in reducing the expression of virulence factor genes in intestinal pathogenic bacteria. The aim of the present study is to investigate the effect of probiotic Saccharomyces cerevisiae S3 on the expression of enterotoxin genes in both ETEC and EHEC. Supernatant and lysate of S. cerevisiae S3 are prepared. Subminimal inhibitory concentrations (sub-MIC) of supernatant and lysate are individually exerted to O157: H7 and H10407. The genes' expression of enterotoxins (elt, est, stx1, and stx2) are then determined using real-time PCR technique. The results showed, the yeast supernatant could decrease the expression of the elt gene in ETEC and that of stx1 in EHEC. Of note, in other cases, stx1 and est genes' expression increased. The lysate had no inhibitory effect on the expressions of elt, est, and stx2 genes, but it increased the expression of genes in both ETEC and EHEC. Lysate extract only decreased the expression of stx1 in O157: H7. Our study shows some interesting results regarding the effectiveness of the compounds produced by S. cerevisiae S3 in the expressions of toxin genes in both ETEC and EHEC. We recommend more similar studies be performed in this regard.

Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater.

Background and Objectives: In the third world and developing countries, hospital sewage is mixed with municipal wastewater. The treated effluent contains dangerous bacteria released into the environment and used in the irrigation of agricultural products, and eventually these bacteria may endanger the human health through foods. Antibiotic-resistant bacteria are mostly found in hospital wastewater. In water and wastewater treatment plants, large amounts of toxic and polluting substances are removed and destroyed, but this process does not eliminate bacteria. Materials and Methods: Wastewater samples from 22 hospitals in Iran were collected and in the meantime specific phages (against drug-resistant pathogenic bacteria) extracted using the bilayer agar technique. Phage amplification was performed by employing a fermenter after phage identification. Amplified phages were added to the primary sedimentation pond using New-Brunwick biofermenter BioFlo/Celligen®115 and the bacterial count was evaluated for the desired bacteria. Results: Our phage cocktail was able to reduce 99.8%, 99.4%, 99.5%, 99.8%, 99.7%, 99.8%, 99.6% and 99.9% of E. coli, E. faecium, E. faecalis, K. pneumoniae, A. baumannii, P. aeruginosa, S. maltophilia and S. aureus counts respectively. Conclusion: The application of phage cocktails can remarkably help improve personal hygiene, the environment, and the optimization of surface water.

Phenotypic Identification and Genotypic Characterization of Plasmid-Mediated AmpC ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates in Iran.

One of the mechanisms of Klebsiella pneumoniae and Escherichia coli resistance to ß-lactam antibiotics is the production of ß-lactamase enzymes. Among these are the AmpC ß-lactamases, which confer resistance to a class of antibiotics. However, little is known about the AmpC ß-lactamases of K. pneumoniae and E. coli clinical isolates in Qazvin, Iran. This study was designed to assess the AmpC ß­lactamases-producing strains and also identify the prevalence of AmpC ß­lactamases genes. Antimicrobial susceptibility tests were performed on 435 K. pneumoniae and E. coli isolates using disk diffusion technique. Plasmid-mediated AmpC genes were studied using a multiplex PCR assay. The AmpC ß-lactamase-producer isolates were studied by employing cefoxitin disk diffusion test, AmpC induction test, AmpC cefoxitin-EDTA test, and boronic acid disk test. Our results showed that of 46 (18.4%) cefoxitin-insensitive E. coli isolates, 10 (21.7%) were positive for AmpC ß-lactamase genes, among them 4 (8.69%) isolates were positive for blaDHA genes and 6 (13%) for blaCIT genes. Of 57 (30.4%) cefoxitin-insensitive K. pneumoniae isolates, 10 (17.5%) were positive for AmpC gene with 4 (6.34%) and 6 (9.5%) isolates positive for blaDHA and blaCIT genes, respectively. However, no MOX, ACC, FOX, or EBC genes were detected in the isolates. Considering the results of different confirmatory phenotypic tests, the AmpC cefoxitin-EDTA test showed a higher discriminatory power for detecting AmpC ß-lactamase-producing strains. The specificity and sensitivity of AmpC cefoxitin-EDTA were 77%, 100% for K. pneumonia and 70%, 90% for E. coli higher than the other two tests, respectively. Also, the authors demonstrated high prevalence rate for resistance to certain antibiotics, such as cefuroxime, trimethoprim-sulfamethoxazole, ampicillin, and cefotaxime. In conclusion, our study provided valuable information regarding the plasmid-mediated AmpC ß-lactamase gene content, antibiotic resistance, and confirmatory phenotypic tests for AmpC ß-lactamases in E. coli and K. pneumoniae isolates from clinical sources.

Molecular mechanisms and prevalence of colistin resistance of Klebsiella pneumoniae in the Middle East region: A review over the last 5 years.

The increasing prevalence of multidrug-resistant (MDR) Klebsiella pneumoniae is a serious clinical and public health problem, and colistin is the last-resort treatment option for MDR infections. However, resistance to colistin has been increasingly reported in the world, such as the Middle East region, where antibiotics are used more in the human and agriculture industry. In this paper, we review the available data on the molecular mechanisms and prevalence of colistin resistance of K. pneumoniae in the Middle East over the last 5 years. To the best of our knowledge, 590 colistin-resistant K. pneumoniae isolates were reported from six countries, including Turkey (438), Iran (86), Saudi Arabia (24), United Arab Emirates (31), Kuwait (5), Israel (3) and Lebanon (3), between 2013 and 2018. However, there has been no reports about colistin resistance among K. pneumoniae isolates in Iraq, Yemen, Syria, Jordan, Palestine, Oman, Qatar, Bahrain and Cyprus. Moreover, it seems that mutations and insertion sequence transpositions in the mgrB gene were the most common colistin resistance mechanisms among K. pneumoniae in the Middle East region, which is similar to other parts of the world.

Candida non albicans with a High amphotericin B resistance pattern causing candidemia among cancer patients.

BACKGROUND: Many scientists have reported Candida species to be of great concern because of the high frequency that they colonize and infect human hosts, particularly cancer patients. Moreover, in the last decades Candida species have developed resistance to many antifungal agents. Based on this, we aimed to identify and determine the prevalence of Candida spp from blood culture bottles among cancer patients and their antifungal resistance pattern. MATERIALS AND METHODS: From the blood culture bottles isolation and identification of the Candida spp were performed by conventional microbiological techniques. The in vitro antibiotic resistance pattern of the isolates was determined by CLSI guidelines. Genomic DNA was isolated and amplified. Each gene was separated by agar gel electrophoresis. RESULTS: Identification of Candida spp was based on the presence of yeast cells in direct examination, culture and DNA extraction. Of the 68 blood samples collected during the study period (April 2013 to October 2013), five (7.35%) were positive for the presence of Candida spp, 2 (40%) of which were identified as Candida albicans and 3 (60%) were Candida non-albicans. CONCLUSIONS: High resistance to amphotricin B was observed among all the Candida non-albicans isolates. Regular investigations into antifungal resistance will help us to get an updated knowledge about their antibiotic resistance pattern which may help the physician in selecting the antibiotics for empirical therapy.

Determination of ctxAB expression in Vibrio cholerae Classical and El Tor strains using Real-Time PCR.

Cholera is an infection of the small intestines caused by the bacterium V. cholerae. It is a major cause of health threat and also a major cause of death worldwide and especially in developing countries. The major virulence factor produced by V. cholerae during infection is the cholera toxin. Total mRNA extraction and reverse transcription was performed for making ctxAB cDNA. Relative Real-Time PCR analysis showed unequal enterotoxin production in V. cholerae strains. The results showed that, classical strain produces cholera toxin more than El Tor strain.

Quantitative expression of cholera toxin mRNA in Vibrio cholerae isolates with different CTX cassette arrangements.

Cholera toxin (CT) is the major virulence factor produced by Vibrio cholerae. Several genomic arrangements within the CTX cassette have been elucidated in V. cholerae. Previously, it was shown that three different CTX cassette arrangements, one complete CTX cassette (arrangement A), one complete and two incomplete CTX cassettes (arrangement B), and two complete CTX cassettes (arrangement C), exist within V. cholerae isolates. In the present study, the level of CT expression by V. cholerae isolates carrying different CTX cassette arrangements was evaluated. Real-time quantitative PCR analysis showed unequal production of CT mRNA in V. cholerae isolates with different CTX arrangements. V. cholerae with the CTX arrangement C expressed more CT mRNA than isolates with the other CTX arrangements. In addition, CT mRNA was expressed more in the isolates with CTX arrangement B than in those with arrangement A. Overall, these results suggest that the arrangement and number of regulatory elements (rstA) within the CTX cassette could affect the level of expression of CT.