The effect of type II toxin-antitoxin systems on methicillinresistant Staphylococcus aureus persister cell formation and antibiotic tolerance

Persister cells are defi ned as a subpopulation of bacteria in a dormant state with the ability to reduce bacterial metabolism and they are involved in antibiotic tolerance. Toxin-antitoxin (TA) systems have been previously suggested as important players in persistence. Therefore, this study aimed to study the involvement of TA systems in persister cell formation in methicillin-resistant Staphylococcus aureus following antibiotic exposure. Using TADB and RASTA database, two type II TA systems including MazF/MazE and RelE/RelB were predicted in S. aureus. The presence of these TA genes was determined in 5 methicillin-resistant S. aureus isolates and the standard strain S. aureus subsp. aureus N315 using PCR method. To induce persistence, isolates were exposed to lethal doses of ciprofl oxacin and the expression of the studied TA system genes was measured after 5 h using Real-Time PCR. According to our results, all the studied isolates harbored the TA system genes. S. aureus was highly capable of persister cell formation following exposure to sub-MIC of ciprofl oxacin and RT-qPCR showed a signifi cant increase in the expression of the MazEF and RelBE loci, indicating their potential role in antibiotic tolerance. Considering the importance of antibiotic tolerance, further studies on persister cell formation and TA systems involved in this phenomenon are required to effi ciently target these systems

Effect of fetal and adult bovine serum on pyocyanin production in Pseudomonas aeruginosa isolated from clinical and soil samples

Objective(s): Pyocyanin is a blue-greenish redox-active pigment, produced by Pseudomonas aeruginosa, with a wide range of biological and biotechnological applications. Pyocyanin biosynthesis is regulated by the quorum-sensing (QS) system in which the expression of QS genes and QS-controlled virulence genes may be affected by serum as a complex medium. In the current study, effects of adult bovine serum (ABS) and fetal bovine serum (FBS) on the production of pyocyanin were examined in order to develop it. Materials and Methods: The presence of pyocyanin-producing specific genes and proteins in clinical and soil isolates of P. aeruginosa was confirmed using PCR and SDS-PAGE. Isolates were inoculated to media containing different concentrations of complement-active/-inactivated ABS or FBS and pyocyanin concentration was measured by spectrophotometry. Extracted pigment was characterized by using UV-Visible spectrophotometry. Titration of ABS antibodies against studied isolates was performed by the tube agglutination test. Results: Adding ABS to P. aeruginosa culture medium decreased pyocyanin production compared to the control, while its production increased in FBS-containing media (113.21±2.581 vs. 55.26±0.827 μg.ml-1 and 126.80±2.036 vs. 30.56±0.382 μg.ml-1 of C11 and E8 pyocyanin concentration in the presence of 10% FBS vs. control, respectively). Conclusion: In this study, due to the presence of inhibitors such as complement proteins and antibodies in ABS samples, the use of FBS devoid of antibodies was effective to increase pyocyanin production in studied isolates

Pyocyanine Biosynthetic Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa and Detection of Pyocyanine’s Antimicrobial Effects with or without Colloidal Silver Nanoparticles

Objective: Pyocyanine plays an important role in the pathogenesis of Pseudomonas aeruginosa, (P. aeruginosa) and is known to have inhibitory and bactericidal effects. This study has aimed to detect the phenazine biosynthetic operon (phz ABCDEFG) and two phenazine modifying genes (phzM and phzS) by polymerase chain reaction (PCR) and detection of its possible protein bands by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The antimicrobial effects of pyocyanine alone and mixed with colloidal silver nanoparticles were studied.Materials and Methods: In this descriptive study, clinical and environmental species of P. aeruginosa were isolated by thioglycollate medium culture and cetrimide agar, respectively. The existence of a phenazine biosynthetic operon and two phenazine modifying genes as well as their protein products were confirmed by PCR and SDS-PAGE, respectively. Pyocyanine was extracted with chloroform and its antimicrobial effects against bacteria such as; Escherichia coli (E. coli), P. aeruginosaand Staphylococcus aureus (S. aureus) bacteria and yeast Candida albicans (C. albicans) were tested using well, spot and disk diffusion methods.Results: In this study, 3 out of 48 clinical strains were unable to produce pyocyanine on cetrimide and Mueller Hinton (MH) agar. Two strains did not have phenazine modifying gene bands. Another strain did not have the possible protein band of the phzM gene. Pyocyanine had antimicrobial effects against the microbial strains, which increased in the presence of silver nanoparticles.Conclusion: According to the results of the present study, some P. aeruginosa strains are unable to produce pyocyanine due to the absence of the phzM or phzS genes. Therefore, these genes have an important role in pyocyanine production in P. aeruginosa. Pyocyanine shows synergistic antimicrobial effects in the presence of silver nanoparticles against microbial strains

Isolated Metallopeptidase from Lactobacillus casei: A Computational Study: Computational study of the metallopeptidase

One of the common studies on industrial proteins is to study the RNA Folding and stability, protein stability, physicochemical properties and conformational structure. In our previous proteomics study, Metallopeptidase (MP) was isolated from Lactobacillus casei, which in this study, it was evaluated with RNA fold, Protparam, I-TASSER and Phyre2 web servers. The results showed that it fairly has a suitable RNA folding in terms of minimum energy. In addition, Phyre2 could model the protein with 100% confidence and 68% coverage with human protease. Secondary structure of MP contains 45% alpha helix and 8% beta strand, 31% Disordered and 3% TM helix. The estimated TM-score for final predicted MP model was 0.38±0.13 and calculated RMSD was 15.9±3.2Aº. Phyre2 investigator showed that R217, G63, K64, F27 and G58 in active site participate in the reaction with the substrate. In conclusion, predicted structure of bacterial metallopeptidase in acidic conditions has 68% coverage with human protease. HIGHLIGHTS Metallopeptidase is a stable protein with suitable RNA folding. It has proper half-life for production in prokaryotic and eukaryotic systems. The amino acids R217, G63, K64, F27 and G58 in active site participate in the reaction with the substrate. Predicted structure of bacterial metallopeptidase has 68% coverage with human protease

RAPD PCR Profile, Antibiotic Resistance, Prevalence of armA Gene, and Detection of KPC Enzyme in Klebsiella pneumoniae Isolates

The increasing prevalence of multidrug-resistant Klebsiella pneumoniae strains isolated from hospitals shows the limitation of recent antibiotics used for bacterial eradication. In this study, 81 K. pneumoniae isolates were collected from three hospitals in Tehran. Antibiotic susceptibility test showed the highest rates of resistance to cefotaxim (85.5%) and ceftazidime (78.3%), and the lowest rates of resistance were detected for colistin (16.9%), streptomycin (16.8%), and chloroamphenicol (21.7%). Eleven different resistance patterns were observed. Sixty-six out of 81 isolates (81.5%) were found to be multidrug resistant (MDR), and 35.8% of them belonged to A3 resistance pattern. 7.4% and 66.7% were KPC enzyme and armA gene positive, respectively. RAPD PCR assay of these bacteria showed 5 clusters, 16 single types, and 14 common types, and there was not any correlation between genetic patterns of the isolates and presence of resistance agents. Simultaneous detection of resistance-creating agents could be an important challenge for combination therapy of MDR K. pneumoniae-caused infections

Prevalence of aac(3)-IIa gene among clinical isolates of uropathogenic Escherichia coli in Delfan, Lorestan

Backgrounds: Uropathogenic Escherichia coli strains are the predominant causative organisms of urinary tract infections (UTIs). Aminoglycosides are clinically useful antibiotics with bactericidal activity against this bacterium. The most common mechanism for resistance to these antibiotics are mediated through production of aminoglycoside modifying enzymes (AMEs). The most common of these enzymes are Aminoglycoside Acetyltransferases (AACs). The epidemiology of the dominant type of these enzymes, AAC(3)-II, varies from region to region. The aim of this study was to determine the antimicrobial susceptibility pattern with a focus on aminoglycosides and the prevalence of aac(3)-IIa gene among clinical isolates of uropathogenic Escherichia coli obtained from Delfan, Lorestan, Iran. Materials and Methods: In this descriptive study, a total of 100 uropathogenic Escherichia coli isolates were collected from BoAli hospital in Delfan city, Lorestan, from July to November 2010. Antibiotic susceptibility patterns of the isolates were determined using disk diffusion method according to Clinical and Laboratory Standards Institute )CLSI) guidelines. Prevalence of aac(3)-IIa gene was determined by PCR and the relationship between resistance phenotypes to aminoglycosides and presence of aac(3)-IIa gene was evaluated. Results: Among the 100 tested isolates, maximal resistance was seen to ampicillin (85%); whereas, no resistance to imipenem was found. Sixty percent of the isolates demonstrated resistance to at least one of the tested aminoglycosides. Resistance rate towards these agents were as followed: gentamicin 39%, kanamycin 26%, neomycin 31% and amikacin 1%. Forty–four isolates (44%) harbored the aac(3)-IIa gene. The maximal rate of gene presence (36 isolates, 92.3%) was detected in strains with gentamicin resistant phenotype (39 isolates, 39%). Conclusion: On the basis of our findings, use of antibiotics such as nitrofurantoin, amikacin or imipenem are recommended for empirical treatment of UTIs. In addition, locally widespread distribution of aac(3)-IIa gene will pose concerns about progressive resistance against potent aminoglycosides such as gentamicin, tobramycin and others in  the future

Comparing pili producing gene (mtp-flp) in susceptible and resistant dormant Mycobacterium tuberculosis strains with active clinical isolates

Background: Pili producing genes in different life cycles of Mycobacterium tuberculosis (M. tuberculosis) were assessed. M. tuberculosis has two life cycles: dormant and active states. We aimed to assess the pili producing genes such as curli pili of M. tuberculosis (mtp) encoded by the mtp gene (Rv3312A) and fimbrial low-molecular-weight protein encoded by flp gene (Rv3656c) which were compared and analyzed. Methods: Two hundred M. tuberculosis isolates were investigated both at active and dormant states for production and expression of pili. The dormant M. tuberculosis was achieved by incubation in a sealed tube (modified Wayne method). The susceptibility of M. tuberculosis was evaluated on genes, rpob, inh, katg, and gyra by using multiplex polymerase chain reaction (PCR) and single-strand conformational polymorphism methods. The PCR–restriction fragment length polymorphism was used to express pili genes mtp and flp and then the PCR products was digested using restriction enzyme Fnu4HI, XmaI, and MspJI and AciI, TagII, and HaeII, respectively. The transmission electron microscopy was also used to detect pili in different isolates. The result was compared and analyzed using H37RV as a gold standard. Results: The mtp and flp PCR products were 263 and 122 bp in the studied strains irrespective of M. tuberculosis different life cycles, respectively. The PCR products were analyzed on 8% Polyacrylamide gel electrophoresis (PAGE), and in the 180/200 (20%), producing five fragments of 25,40,45,63,90 bp with the Fun4HI and two fragments of 126,138 bp with the XmaI and uncut with the MspJI for mtp gen were obtained at the dormant and active states of M. tuberculosis (P 0.05). Conclusion: Pili were shown by electron microscopy, although at the gene expression, the insignificant difference was observed at the dormant strains in comparison to active states. Therefore, we may conclude that other genes might be involved in pili production of M. tuberculosis that needs further investigation. Although, the resistance phenomena might influence the pili producing gene expression that showed in our results

Determination of Asymptomatic Chlamydia Trachomatis Infections by Omp1 Gene Based -PCR

Objective: The objective of this research was to determine the prevalenceof genital C. trachomatis infection in asymptomatic women by using highlysensitive nested-polymerase chain reaction (PCR) in urine sample.Materials and Methods: One hundred-forty asymptomatic women wererandomly selected from those who attended gynecology out patient departmentof Hazraat e Rasool Hospital in Tehran. First catch urine specimen were collectedfrom all the participants. DNA extraction was performed by means of HighPure PCR Template Preparation Kit (HPPTP) according to the manufacture’sinstructions. Extracted DNA was tested by omp1 gene based nested-PCR,using sets of primers to amplify C. trachomatis omp1 gene. Visualizationof a 1027 bp fragment from omp1 gene in agarose gel electrophoresis wasconsidered as a positive result.Results: In total, 140 urines were tested for determination of C. trachomatisinfection. C. trachomatis omp-1 was detected in 22.1% of cases (31/140). Theoverall prevalence rates of C. trachomatis in the urine sample as determined byomp1 based nested-PCR were 4.3% in group I (age, <25 years), 12.1% in groupII (age, 25-34 years), 5.0% in group III (age, 35-44 years) and 0.7% in group IV(>44 years). The highest prevalence of C. trachomatis infection (12.1%) wasseen in women aged 25-34 years. This finding was not statistically significant(p=0.710). Also, there was not relation between C. trachomatis infection andsome probable risk factors such as young age (<25 years), STD history andmissing use of barrier contraceptive in this study.Conclusion: The prevalence of C.trachomatis infection in the women notseeking health care warrants more comprehensive study using high sensitiveomp1 based nested- PCR to identify and treat a large number of infectedwomen in Iran