The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems' locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium

Cloning, expression and purification of pwo polymerase from pyrococcus woesei

Background and objectives: Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase (Pwo polymerase) that has proofreading activity. Materials and Methods: In this study, this microorganism was cultured, its DNA was extracted and the pwo gene polymerase was cloned, expressed and purified. The DNA sequence of the cloned gene was verified by sequencing. The pwo polymerase gene consists of 2,328 bps (775 amino acids with about 90 kD molecular weight). Cloning was done by GATEWAY TM Cloning System and for purification of recombinant protein; His6x-Tag was added to the C-terminus of the recombinant protein. Results and Conclusion: We could purify Pwo polymerase enzyme by Ni-NTA resin. PCR assay showed that Pwo polymerase activity is comparable to a commercial Pfu polymerase activity

The occurrence of extended-spectrum β-lactamase producing Shigella spp. in Tehran, Iran

Background and Objectives: The emergence of extended-spectrum β-lactamase (ESBL)-producing Shigella spp. is of increasing clinical concern specially in children worldwide. The aim of this study was to investigate the occurrence of extended-spectrum β-lactamase producing Shigella spp. in Tehran, Iran. Materials and Methods: The study included all Shigella isolates recovered from pediatric patients aged less than 12 years admitted to a major pediatric hospital in Tehran, Iran, from 2008 to 2010. Bacterial identification, antimicrobial susceptibility testing, extended spectrum β-lactamases (ESBLs) screening and confirmatory tests were performed according to the standard guidelines. Conjugal transfer experiments and plasmid analysis were also carried out. Polymerase chain reaction and sequencing were used to identify the genetic determinants responsible for ESBL production. Results: Four out of 55 Shigella isolates, including three S. sonnei and one S. flexneri, showed an ESBL-positive phenotype. Plasmid transfer of the ESBL phenotype was successful for the S. flexneri isolate only. By PCR and sequencing, one S. sonnei isolate tested positive for the CMY-59 gene, while the other two S. sonnei and the S. flexneri isolates tested positive for the blaTEM-1 and blaCTX-M-15 genes. Conclusion: We found the prevalence of ESBL producing Shigella isolates was higher than detection rates observed in many other countries. Our finding raise concerns about the dissemination of ESBL among the strains of endemic S. sonnei throughout the country, because this species is now the most frequently isolated Shigella species in Iran and shigellosis by such strains in the community can pose a significant threat to patients and presents a challenge for disease management

Epidemiological alteration in pathogens found in ground meat in Iran: unexpected predominance of vancomycin-resistant Enterococcus faecalis

Colonization of the human and animal intestinal tract with potential pathogenic bacteria is correlated with the risk of contamination of food products. The current study analyzed the prevalence of Enterococcus faecalis and Escherichia coli O157H7 in ground meat in Ilam, Iran. Both index organisms were identified following standard food microbiological methods. For E. faecalis, the susceptibility to vancomycin was tested, and PCR was used to check for the vanA gene. E. faecalis was present in all 24 ground meat samples, with no E. coli O157H7 detected in samples. The analysis showed the presence of the vanA gene in 5/24 vancomycin resistant enterococci. In conclusion, this study for the first time demonstrates the presence of vancomycin-resistant enterococci in ground meat in Iran. This observation warrants further epidemiologic investigation and should be followed up in the future

SURVEY IN IRAN OF CLARITHROMYCIN RESISTANCE IN HELICOBACTER PYLORI ISOLATES BY PCR-RFLP

The aims of this study were to assess primary resistance of H. pylori strains isolated from adult patients of Ilam, Iran to antibacterial agents (amoxicillin, clarithromycin, metronidazole and tetracycline) and detection of clarithromycin, azithromycin, clarithromycin, metronidazole and tetracycline resistance by disc diffusion. Fifty biopsies were taken from gastric mucosa of the antrum and body regions of adult patients by gastroscopy, and were cultured on Helicobacter pylori selective medium. The susceptibility of H. pylon strains showed that 44, 6, 6, 4 and 16 were resistance to metronidazole, amoxicillin, tetracycline, azithromycin, and clarithromycin, respectively. Polymerase chain reaction-restriction fragment length polymorphism analysis showed that all clarithromycin resistance isolates had A2143G mutation and PCR amplicons from these strains upon digestion by BsaI restriction enzyme resulted in 319 and 106 base pair fragments. Because most of physicians in Ilam do not use amoxicillin in triple therapy of H. pylon infection, isolates showed low rate of resistance to amoxicilli

The clinical and environmental spread and diversity of toxigenic Clostridium difficile diarrhea in the region of the Middle East.

Stool samples of 1822 hospitalized patients with nosocomial diarrhea and 100 environmental samples were collected at three teaching hospitals and PCR amplification of rRNA intergenic spacer regions (ISR) was conducted. Bacterial cytotoxicity was assayed by conducting three assays namely toxigenic culture on vero cells, stool cytotoxin, and enzyme immunoassay. ISR was carried out using two universal primers complementary to conserved regions in the 16S and 23S rRNA genes. It was found that the toxigenic culture, stool cytotoxin and enzyme immunoassay showed close rates of detection of toxigenic C. difficile, 124, 121, and 122 /1822 (6.8, 6.64., and 6.7%) respectively. In addition, 32 different ribotypes for toxigenic C. difficile were detected, 28 in clinical and 6 in environmental isolates. The predominant ribotypes from the clinical isolates were 13-15, 35.6%, of isolates. Ribotypes were associated with age, location of isolation, and severity of symptoms of clostridial diarrhea (P<0.05). Ribotypes 6-9 affected children only. The most common ribotype of C. difficile , no. 13, as well as ribotypes 16, 20, and 4 covered almost the whole range of severity of symptoms. Ribotypes 21-27, 1, 3, 6, 7, 9, 11, 14, and 19 caused mild-moderate CDAD symptoms while ribotypes 5, 10 8, 12, 15, 17, and 28 were dominantly of severe symptoms (P<0.05). Environmental isolates showed 17% toxigenic strains composed of 4 different ribotypes while ribotypes 5 was shared with clinical isolates. These findings showed that C. difficile associated with diarrhea were genetically diverse and linked to environmental strains

Cloning of Vibrio cholerae outer membrane protein W in Pichia pastoris

BACKGROUND AND OBJECTIVE: The outer membrane protein W (ompW) of Vibrio cholerae is involved in stimulating the immune response via induction of protective immunity. It also plays an important role in bacterial pathogenesis by increasing the adaptability of pathogenic strains. In this study we aimed to clone V. cholerae ompW gene in the strain X-33 of Pichia pastoris. MATERIALS AND METHODS: A gene encoding ompW was cloned into the Ppicza vector downstream of alcohol oxidase promoter. Then recombinant vector was transformed into the genome of the strain X-33 of P. pastoris. After growth of zeocin-resistant transformants, clones were selected and subsequently confirmed for cloning by PCR enzymatic digestion and sequencing. RESULTS: PCR, enzymatic digestion and sequencing showed that the ompW gene was correctly cloned into P. pastoris genome. CONCLUSION: Results of our study showed that the methylotrophic yeast P. pastoris can be considered as an appropriate host instead of mammalian and prokaryotic systems for cloning of ompW. As far as data show, this is the first time that ompW of V. cholera is cloned into the methylotrophic P. pastoris

Phenotypic and Genotypic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli Isolates

Objectives: Drug resistance among gram-negative bacteria is a worldwide challenge. Due to the importance of drug-resistant Klebsiella pneumoniae and Escherichia coli strains in hospital-acquired infections, we aimed to determine the phenotypic and genotypic characteristics of ESBL-, AmpC-, and carbapenemase-producing isolates obtained from hospitalized patients in Tehran and Ilam (Iran). Materials and Methods: In total, 90 K. pneumoniae isolates and 65 E. coli isolates were collected from various infections. Phenotypic identification of bacterial isolates was performed using standard methods. Phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. Results: Phenotypic detection tests showed that 36 (40) K. pneumoniae and 23 (35.4) E. coli isolates were ESBL producers. Moreover, 18 (20) and 6 (9.2) K. pneumoniae and E. coli isolates were AmpC producers, respectively. Modified Hodge test results indicated that 39 (43.3) K. pneumoniae and 18 (27.7) E. coli isolates produced carbapenemase. Molecular tests showed that 40 of K. pneumoniae and 36.9 of E. coli isolates were ESBL positive. AmpC was detected in 24.4 and 13.8 of K. pneumoniae and E. coli isolates. Carbapenemase was detected in 34 (37.8) K. pneumoniae and 13 (20) E. coli isolates. -Conclusion: In this study, 3 K. pneumoniae isolates simultaneously carried ESBL, AmpC, and carbapenemase genes. Up-to-date strategies such as combination therapy or utilization of new antimicrobial agents might help to combat such drug-resistant organisms. © 2019 The Author(s) Published by S. Karger AG, Basel

The clinical and environmental spread and diversity of toxigenic Clostridium difficile diarrhea in the region of the Middle East

Abstract Stool samples of 1822 hospitalized patients with nosocomial diarrhea and 100 environmental samples were collected at three teaching hospitals and PCR amplification of rRNA intergenic spacer regions (ISR) was conducted. Bacterial cytotoxicity was assayed by conducting three assays namely toxigenic culture on vero cells, stool cytotoxin, and enzyme immunoassay. ISR was carried out using two universal primers complementary to conserved regions in the 16S and 23S rRNA genes. It was found that the toxigenic culture, stool cytotoxin and enzyme immunoassay showed close rates of detection of toxigenic C. difficile, 124, 121, and 122 /1822 (6.8, 6.64., and 6.7%) respectively. In addition, 32 different ribotypes for toxigenic C. difficile were detected, 28 in clinical and 6 in environmental isolates. The predominant ribotypes from the clinical isolates were 13-15, 35.6%, of isolates. Ribotypes were associated with age, location of isolation, and severity of symptoms of clostridial diarrhea (P&lt;0.05). Ribotypes 6-9 affected children only. The most common ribotype of C. difficile , no. 13, as well as ribotypes 16, 20, and 4 covered almost the whole range of severity of symptoms. Ribotypes 21-27, 1, 3, 6, 7, 9, 11, 14, and 19 caused mild-moderate CDAD symptoms while ribotypes 5, 10 8, 12, 15, 17, and 28 were dominantly of severe symptoms (P&lt;0.05). Environmental isolates showed 17% toxigenic strains composed of 4 different ribotypes while ribotypes 5 was shared with clinical isolates. These findings showed that C. difficile associated with diarrhea were genetically diverse and linked to environmental strains