GNAT toxin may have a potential role in Pseudomonas aeruginosa persistence: an in vitro and in silico study.

Aims: Persistent cells are primarily responsible for developing antibiotic resistance and the recurrence of Pseudomonas aeruginosa. This study investigated the possible role of GNAT toxin in persistence. Materials & methods: P. aeruginosa was exposed to five MIC concentrations of ciprofloxacin. The expression levels of target genes were assessed. The GNAT/HTH system was bioinformatically studied, and an inhibitory peptide was designed to disrupt this system. Results: Ciprofloxacin can induce bacterial persistence. There was a significant increase in the expression of the GNAT toxin during the persistence state. A structural study of the GNAT/HTH system determined that an inhibitory peptide could be designed to block this system effectively. Conclusion: The GNAT/HTH system shows promise as a novel therapeutic target for combating P. aeruginosa infections.

Antibiotics are used to treat infections caused by bacteria. Over time, some of these infections have become more difficult to treat. This is because the bacteria can slow their growth and tolerate the antibiotic, known as persistence. It is important to find new ways to treat infections caused by persistent bacteria. This study researched a toxin­antitoxin system, called GNAT/HTH, that may play a role in bacterial persistence. This system could be a target for new antibiotics.

Novel toxin-based mRNA vaccine against Clostridium perfringens using in silico approaches.

Clostridium perfringens is a bacterium that causes gastrointestinal diseases in humans and animals. The several powerful toxins such as alpha toxin (CPA), beta toxin (CPB), enterotoxin (CPE), Epsilon toxin (ETX), and theta toxin, play a major role in its pathogenesis. Traditional vaccine development methods are time-consuming and costly. In silico approaches offer an alternative strategy for designing vaccines by analyzing biological data and predicting immunogenic peptides. In this study, computational tools were utilized to design a RNA vaccine targeting C. perfringens toxins. Toxin protein sequences were retrieved and their linear B-cell, MHCI, and MHCII binding epitopes were predicted. Allergenicity, toxigenicity, and IFN-γ induction were assessed to select non-allergenic, non-toxic, and IFN-γ-inducing epitopes. Molecular docking was performed to identify epitopes that fit within the binding cleft of MHC alleles. A final peptide vaccine construct was designed with selected epitopes separated by a linker sequence. The antigenicity and physicochemical properties of the vaccine were evaluated. Immune response simulation showed enhanced secondary and tertiary immune responses, increased levels of immunoglobulins, cytotoxic T lymphocytes, helper T lymphocytes, macrophage activity, and elevated levels IFN-γ and interleukin-2. Docking analysis was done to assess interactions between the vaccine structure and Toll-like receptors. Codon optimization was performed, and a final RNA vaccine construct was designed. The secondary structure of the RNA vaccine was predicted and validated. Overall, this study demonstrates the potential of in silico approaches for designing an RNA vaccine against C. perfringens toxins, contributing to improved prevention and control of associated diseases.

Inhibitory Effects of Carvacrol on Biofilm Formation in Colistin Heteroresistant Acinetobacter baumannii Clinical Isolates.

BACKGROUND: The ability of bacteria to form biofilm is an essential strategy for creating stable infections. This issue is more critical in Acinetobacter bauamannii as a hospital pathogen. Today, the control of biofilm formation and solutions to prevent or remove biofilm is being developed. Carvacrol has been considered an anti-biofilm compound in significant bacteria. This study investigated the anti-biofilm effect of Carvacrol on biofilm formation in clinical colistin heteroresistant isolates of A. baumannii. METHODS: 22 clinical strains of A. baumannii were collected from Motahari Hospital in Tehran, Iran, in 2019. Biochemical and genotypic methods confirmed these isolates. Colistin heteroresistance was determined by the Standard PAP method. Carvacrol's antibacterial and anti-biofilm activity was determined according to the standard protocol. RESULTS: About 12 isolates were considered strong biofilm producers and were used for analysis. Six isolates had hetero-resistance to colistin. Carvacrol at a 512 g/ml concentration showed the best antibacterial activity against all isolates. The sub-MIC of Carvacrol (256 g/ml) reduced the biofilm formation capacity, which was statistically significant (p < 0.05). CONCLUSION: The results of this study showed that sub-MIC of Carvacrol has anti-biofilm effects in clinical A.baumannii colistin hetero-resistance isolates.

Fabrication and characterization of Persian gum-based hydrogel loaded with gentamicin-loaded natural zeolite: An in vitro and in silico study.

The main purpose of this study is to synthesize and characterize Persian gum-based hydrogel composited with gentamicin (Gen)-loaded natural zeolite (Clinoptilolite) and to evaluate its biological properties. Clinoptilolite (CLN) was decorated with Gen, and the conjugation was confirmed using computational and experimental assessments. The Monte Carlo adsorption locator module was used to reveal the physicochemical nature of the adsorption processes of Gen on CLN and ALG and gum on Gen@ CLN in Materials Studio 2017 software. Based on the high negative results, the adsorption process was found to be endothermic in all studied cases, and the interaction energies were in the range of physisorption for Gen on CLN and ALG and gum on Gen@CLN. Dynamic light scattering (DLS) and zeta potential analysis showed that the size of pristine CLN was around 2959 nm and the conjugation decreased the size significantly to approximately 932 nm. The hydrogel characterizations showed that the Gen-decorated CLNs are homogenously dispersed into the hydrogel matrix, and the resultant hydrogels have a porous structure with interconnected pores. The release kinetics evaluation showed that around 80 % of Gen was released from the nanocomposite drug during the first 10 h. In vitro studies revealed hemocompatibility and cytocompatibility of the nanocomposite. Microbial assessments indicated dose-dependent antibacterial activity of the hydrogel against gram (+) and gram (-) bacteria. The results showed that the fabricated hydrogel nanocomposite exhibits favorable physicochemical and biological properties.

A pilot study on the relationship between Lactobacillus, Bifidibactrium counts and inflammatory factors following exercise training.

BACKGROUND: The current pilot study was carried out to examine the effect of aerobic exercise on Lactobacillus and Bifidobacterium as a function of weight loss and cytokine changes in overweight women. MATERIAL AND METHODS: Eighteen women with excessive weight (age = 19-30 years) were randomly assigned into exercise (10 weeks, aerobic exercise training, 3 sessions/week) and control groups. Lactobacillus and Bifidobacterium in stool and inflammatory factors in blood were evaluated before and after the intervention. RESULTS: The intervention induced significant improvements in body weight and in VO2 peak. There were significant time effects on Lactobacillus (p = .016) and significant time*exercise interaction effects on Bifidobacterium (p = .025). Lactobacillus and Bifidobacterium changes were negatively associated with body weight and IL-6 levels, respectively. CONCLUSIONS: The current results indicate that exercise training associated with weight loss can increase specific bacteria in people with excessive weight. Changes in Lactobacillus and Bifidobacterium were not significantly associated with cytokines.

In silico Study of the Proteins Involved in the Persistence of Brucella spp.

BACKGROUND: One of the major problems with Brucella infections is its tendency to become chronic and recurrent, providing a hindrance to the management of this infection. It has been proposed that chronicity is greatly affected by a phenomenon called persistence in bacteria. Several mechanisms are involved in bacterial persistence, including the type II toxin-antitoxin system, the SOS and oxidative and stringent responses. METHODS: In this in silico study, these persistence mechanisms in Brucella spp. were investigated. RESULTS: The structure and the interactions between modules involved in these systems were designed, and novel peptides that can interfere with some of these important mechanisms were developed. CONCLUSION: Since peptide-based therapeutics are a new and evolving field due to their ease of production, we hope that peptides developed in this study, as well as the information about the structure and interactions of modules of persistence mechanisms, can further be used to design drugs against Brucella persister cells in the hope of restraining the chronic nature of Brucellosis.

Development of innovative multi-epitope mRNA vaccine against Pseudomonas aeruginosa using in silico approaches.

The rising issue of antibiotic resistance has made treating Pseudomonas aeruginosa infections increasingly challenging. Therefore, vaccines have emerged as a viable alternative to antibiotics for preventing P. aeruginosa infections in susceptible individuals. With its superior accuracy, high efficiency in stimulating cellular and humoral immune responses, and low cost, mRNA vaccine technology is quickly replacing traditional methods. This study aimed to design a novel mRNA vaccine by using in silico approaches against P. aeruginosa. The research team identified five surface and antigenic proteins and selected their appropriate epitopes with immunoinformatic tools. These epitopes were then examined for toxicity, allergenicity and homology. The researchers also checked their presentation and identification by major histocompatibility complex cells and other immune cells through valuable tools like molecular docking. They subsequently modeled a multi-epitope protein and optimized it. The mRNA was analyzed in terms of structure and stability, after which the immune system's response against the new vaccine was simulated. The results indicated that the designed mRNA construct could be an effective and promising vaccine that requires laboratory and clinical trials.

Molecular typing and antibiotic resistance patterns among clinical isolates of Acinetobacter baumannii recovered from burn patients in Tehran, Iran.

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii.

Genotyping of Listeria monocytogenes isolates by high-resolution melting curve (HRM) analysis of tandem repeat locus.

Listeria monocytogenes is responsible for causing listeriosis, a type of food poisoning with high mortality. This bacterium is mainly transmitted to humans through the consumption of contaminated foods. Detection of L. monocytogenes through molecular methods is crucial for food safety and clinical diagnosis. Present techniques are characterized by low discrimination power and high cost, as well as being time-consuming and taking several days to give the final result. In our study, MLVA-HRM (Multiple-Locus Variable-number tandem repeats Analysis ‒ High-Resolution Melting) was investigated as an alternative method for a fast and precise method for the genotyping of L. monocytogenes isolates. Forty-eight isolates of L. monocytogenes obtained from the microbial bank of Department of Microbiology, Iran University of Medical Sciences, were typed by MLVA-HRM analysis using five Variable Numbers of Tandem Repeat (VNTR) loci. A total of 43 different types were obtained. This research demonstrated the usefulness of the MLVA-HRMA method and its ability to discriminate L. monocytogenes isolates. Since this method is easier and more efficient than existing methods, it can be widely used in food processing plants and diagnostic laboratories as a fast and accurate method.

In silico Investigation of Lon Protease as a Promising Therapeutic Target.

Considering the widespread occurrence of antibiotic resistance, the need for new therapeutic strategies is inevitable. Bacterial proteases are a broad set of enzymes that play a vital role in cell survival, stress response, and pathogenicity. This in silico study was aimed to focus on the crucial role of Lon protease in the regulation of toxin-antitoxin systems in E. coli and to design inhibitory peptides against the action of this protease. With the help of relevant servers and softwares, the communication network, the evolutionary history, and the interaction of Lon with the corresponding antitoxins were examined, following which the inhibitory peptides were designed against these interactions. The results showed that Lon protease plays a central role in the control of these systems and is a conserved protein, especially among the Enterobacteriaceae family. The docking results of the designed peptides with the Lon protease were significant. This study showed that Lon protease may have the characteristics of a new therapeutic target.

Microbiological and drug resistance patterns of bronchoalveolar lavage samples taken from hospitalized patients in Iran.

Introduction: Pulmonary diseases are amongst the most common causes of premature death and distressing disorders worldwide. This study aimed to detect the fastidious and routine infectious agents, and their drug resistance patterns in bronchoalveolar lavage (BAL) samples. Methods: A total of 44 BAL samples were collected by bronchoscopy from patients with respiratory disorders hospitalized at 2 teaching hospitals in Ilam, Iran. The samples were cultured on routine bacterial culture media to identify the bacterial agents and calculate the colony count. Antibiotic susceptibility was determined by disk diffusion method according to the CLSI protocol. PCR was used to detect the fastidious bacteria Mycoplasma pneumoniae and Chlamydia pneumoniae using the 16srRNA specific primers and Legionella pneumophila using the mip specific primers. Results: Overall, 100 bacterial isolates were isolated by culture from the 44 BAL samples including: Staphylococcus aureus (24, 31.2%), Streptococcus pyogenes (18, 23.4%), Enterococcus spp. (11, 14.3%), Acinetobacter baumannii (11, 14.3%), Pseudomonas aeruginosa (11, 14.3%), Enterococcus spp. (10, 13%), Micrococcus spp. (5, 6.5%), Staphylococcus epidermidis (5, 6.5%) and Klebsiella pneumoniae (5, 6.5%). PCR detected 4 positive samples (9.1%) for Chlamydia pneumoniae but no positive cases for Mycoplasma pneumoniae and Legionella pneumophila. Acinetobacter baumannii showed the highest resistance rate (81.8%) to aztreonam and ceftazidime. Seventy-five percent of the Staphylococcus aureus isolates were resistant to cefoxitin (MRSA) and 83.3% had the mecA gene. Vancomycin resistance was observed in 27.3% of the Enterococcus species (VRE). Resistance to piperacillin, cefotaxime, ciprofloxacin and imipenem was observed in 54.5%, 45.5%, and 36.4% of the Pseudomonas aeruginosa isolates, respectively. The frequency of organisms isolated from the ICU was higher (46%) than from other wards. Conclusions: The presence of MRSA, cephalosporins-resistant Enterobacteriaceae as well as Pseudomonas aeruginosa and Acinetobacter baumannii resistant against piperacillin, imipenem, cefotaxime, aztreonam and ciprofloxacin amongst different wards, especially the ICU ward of the surveyed hospitals, is a major healthcare concern and it is necessary to wisely scrutinize the preventive strategies for antibiotic resistant infections.

Survey on phenotypic resistance in Enterococcus faecalis: comparison between the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells.

BACKGROUND: Phenotypic resistance is considered as a serious therapeutic challenge for which a definitive remedy has not been discovered yet. Biofilm and persister cell formation are two well-studied phenotypic resistance phenomena, leading to the recalcitrance and relapse of different types of chronic infections. The presence of persister cells in biofilm structures seems to be one of the main factors contributing to the relapse of infections and treatment failure. Given the dormant and inert nature of persister cells, they can be easy targets for the immune system factors. Biofilm formation can be a survival strategy for the defenseless persister cells. Thus, this study was aimed to evaluate the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells. METHODS: Vancomycin susceptibility and biofilm formation ability were investigated among 95 E. faecalis clinical isolates using microtiter broth dilution and microtiter plate assays, respectively. PCR was used to determine the presence of biofilm-related genes (gelE, esp, and agg) among the vancomycin-susceptible, biofilm producer E. faecalis isolates (91 isolates). Minimum bactericidal concentration for biofilms (MBCB) were determined for vancomycin using the MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biofilm-related genes was compared between the persister and non-persister isolates of E. faecalis using real-time qPCR. RESULTS: E. faecalis isolates showed a high level of susceptibility (95.8%) to vancomycin (MIC < 1 µg/mL). The gelE, esp, and agg genes were found in 91 (100%), 72 (79.12), and 74 (81.32) of the isolates, respectively. All the E. faecalis isolates were tolerant to vancomycin in the biofilm condition, showing a MBCB of > 2500 µg/mL. Based on the enzymatic lysis assay, only 3 isolates, out of the 91, had the ability to form persister cells. The expression of biofilm-associated genes was higher among the persister compared to non-persister E. faecalis isolates. CONCLUSIONS: Biofilm-associated persister cells indicated a high vancomycin tolerance compared to non-persister cells. Moreover, persister isolates showed a higher tendency for biofilm formation and a higher expression level of the biofilm-associated genes, compared to non-persister isolates.

Isolation of persister cells within the biofilm and relative gene expression analysis of type II toxin/antitoxin system in Pseudomonas aeruginosa isolates in exponential and stationary phases.

OBJECTIVES: Chronic infections and treatment failure are concerning issues in patients with Pseudomonas aeruginosa infections. Persister cell formation in biofilm is considered a key reason for antibiotic resistance and treatment failure. In this study, expression of type II toxin/antitoxin (TA) system genes (relBE, Xre-COG5654, vapBC and Xre-GNAT) in persister cells of biofilm was evaluated in the presence of the antibiotics ciprofloxacin and colistin during exponential and stationary phases. METHODS: The impact of antibiotics on biofilm persister cell formation during exponential and stationary phases of P. aeruginosa strains was examined by colony count at different time intervals in the presence of 5-fold minimum inhibitory concentration (MIC) of ciprofloxacin and colistin. Furthermore, expression of relBE, Xre-COG5654, vapBC and Xre-GNAT genes in P. aeruginosa strains undergoing antibiotic treatment for 3.5 h during exponential and stationary phases was examined by RT-qPCR. RESULTS: Formation of persister cells was observed in biofilms formed by P. aeruginosa strains in the presence of 5 × MIC of ciprofloxacin and colistin compared with the control group after 3.5 h of incubation both during exponential and stationary phases. The number of biofilm persister cells was higher in stationary phase than in exponential phase. According to the findings of RT-qPCR, ciprofloxacin and colistin may induce persister cell formation by enhancing the expression of type II TA systems during stationary and exponential phases. CONCLUSION: The result of this study indicate that ciprofloxacin and colistin have the potential to increase persister cells formation in biofilms by influencing the expression of type II TA systems.

Genotyping of Listeria monocytogenes isolates by high-resolution melting curve (HRM) analysis of tandem repeat locus

Abstract Listeria monocytogenes is responsible for causing listeriosis, a type of food poisoning with high mortality. This bacterium is mainly transmitted to humans through the consumption of contaminated foods. Detection of L. monocytogenes through molecular methods is crucial for food safety and clinical diagnosis. Present techniques are characterized by low discrimination power and high cost, as well as being time-consuming and taking several days to give the final result. In our study, MLVA-HRM (Multiple-Locus Variable-number tandem repeats Analysis ‒ High-Resolution Melting) was investigated as an alternative method for a fast and precise method for the genotyping of L. monocytogenes isolates. Forty-eight isolates of L. monocytogenes obtained from the microbial bank of Department of Microbiology, Iran University of Medical Sciences, were typed by MLVA-HRM analysis using five Variable Numbers of Tandem Repeat (VNTR) loci. A total of 43 different types were obtained. This research demonstrated the usefulness of the MLVA-HRMA method and its ability to discriminate L. monocytogenes isolates. Since this method is easier and more efficient than existing methods, it can be widely used in food processing plants and diagnostic laboratories as a fast and accurate method.

Transcriptome analysis of biofilm formation under aerobic and microaerobic conditions in clinical isolates of Campylobacter spp.

It has been well documented that Campylobacter is the leading cause of foodborne infections and bacterial enteritis in high-income countries. The gastrointestinal tract of most warm-blooded animals, such as mammals and poultry, is prone to this pathogen. Infections caused by this bacterium in humans have usually been associated with the consumption of contaminated poultry meat. The important point about Campylobacter is that this bacterium has adapted to harsh environmental conditions along the food chain (poultry digestive tract to the consumer's plate) and developed an adapted mechanism to those conditions. This study aimed to compare the ability of Campylobacter jejuni and Campylobacter coli strains to form biofilms under aerobic and microaerobic conditions. The presence and expression of flab, FliS, DnaK, luxs, CsrA, Cj0688, and cosR genes involved in biofilm formation were investigated. Finally, the correlation between the biofilm forming ability of Campylobacter isolates and the presence/expression of selected genes has been explored. A significant correlation was observed between the presence and expression of some genes and the degree of biofilm formation in C. jejuni and C. coli isolates. A strong biofilm production was detected in strains harboring all selected genes with greater expression levels. The ability of C. jejuni and C. coli strains in biofilm formation is associated with the coordinated function and convergent expression of the selected genes. Seemingly, stress response- and motility-related genes have the most involvement in biofilm formation of C. jejuni and C. coli strains, while other genes have an accessory role in this phenomenon.

Expression of type II toxin-antitoxin systems and ClpP protease of methicillin-resistant Staphylococcus aureus under thermal and oxidative stress conditions.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus is a main human pathogen that causes a variety of chronic to persistent infections. Across the diverse factors of pathogenesis in bacteria, Toxin-Antitoxin (TA) systems can be considered as an anti-bacterial target due to their involvement in cellular physiology counting stress responses. Here, the expression of TA system genes and ClpP protease was investigated under the thermal and oxidative conditions in S. aureus strains. MATERIALS AND METHODS: The colony-forming unit (CFU) was used to determine the effects of thermal and oxidative stresses on bacterial survival. Moreover, the expressions of TA system genes in S. aureus strains were evaluated 30 min and 1 h after thermal and oxidative stresses, respectively, by quantitative reverse transcriptase real-time PCR (qRT-PCR). RESULTS: The cell viability was constant across thermal stress while oxidative stress induction showed a significantly decrease in the growth of Methicillin-Resistant S. aureus (MRSA) strain. Based on the qRT-PCR results, the expression of mazF gene increased under both thermal and oxidative stresses in the MRSA strain. CONCLUSION: A putative TA system (namely immA/irrA) most likely has a role under the stress condition of S. aureus. The MRSA strain responds to stress by shifting the expression level of TA genes that has diverse effects on the survival of the pathogen due to the stress conditions. The TA systems may be introduced as potential targets for antibacterial treatment.

Therapeutic effects, immunogenicity and cytotoxicity of a cell penetrating peptide-peptide nucleic acid conjugate against cagA of Helicobacter pylori in cell culture and animal model.

BACKGROUND AND OBJECTIVES: Helicobacter pylori causes several gastrointestinal diseases, including asymptomatic gastritis, chronic peptic ulcer, duodenal ulcer, lymphoma of the mucosa-associated lymphoid tissue (MALT), and gastric adenocarcinoma. In recent years, failure to eradicate H. pylori infections has become an alarming problem for physicians. It is now clear that the current treatment strategies may become ineffective, necessitating the development of innovative antimicrobial compounds as alternative treatments. MATERIALS AND METHODS: In this experimental study, a cell-penetrating peptide-conjugated peptide nucleic acid (CPP-PNA) was used to target the cagA expression. cagA expression was evaluated using RT-qPCR assay after treatment by the CPPPNA in cell culture and animal model. Additionally, immunogenicity and toxicity of the CPP-PNA were assessed in both cell culture and animal models. RESULTS: Our analysis showed that cagA mRNA levels reduced in H. pylori-infected HT29 cells after treatment with CPPPNA in a dose-dependent manner. Also, cagA expression in bacterial RNA extracted from stomach tissue of mice treated with PNA was reduced compared to that of untreated mice. The expression of inflammatory cytokines also decreased in cells and tissue of H. pylori-infected mice after PNA treatment. The tested CPP-PNA showed no significant adverse effects on cell proliferation of cultured cells and no detectable toxicity and immunogenicity were observed in mice. CONCLUSION: These results suggest the effectiveness of CPP-PNA in targeting CagA for various research and therapeutic purposes, offering a potential antisense therapy against H. pylori infections.

Evaluation of Putative Toxin-antitoxins Systems in Clinical Brucella melitensis in Iran.

BACKGROUND: Toxin-antitoxin systems (TAs) are two-component elements, which are extensive in the bacterial genome and have a regulatory role in many cellular activities including, growth arrest, survival, biofilm formation, and bacterial persistence. OBJECTIVE: TAs have not well studied in Brucella spp. METHODS: We evaluated the presence of different toxin-antitoxin systems, including relE- rhhlike, Fic- Phd, Cog- Rhh, and cogT- cogAT in 40 clinical Brucella melitensis isolates using PCRbased sequencing assay. RESULTS: Our results showed the high presence of relE-rhh-like, Fic-Phd, Cog-rhh, and cogTcogAT s TAs genes in B. melitensis isolates that were 96.25%, 92.5%, 96.25%, and 95%, respectively. CONCLUSION: A high presence of TAs genes in clinical B. melitensis isolates revealed that the TA system could be an antibacterial target in B. melitensis but more investigation is necessitated to elucidate the exact roles of these genes.

Evaluation of gene expression and protein structural modeling involved in persister cell formation in Salmonella Typhimurium.

Persisters are phenotypic variants of the bacterial population that survive against lethal doses of bactericidal antibiotics.These phenotypes are created in numerous bacterial species, including those of clinical significance, such as Salmonella Typhimurium. Since persister cells are associated with the failure of antibiotic treatment and infection recurrence, it is crucial to identify the mechanisms that influence the formation of these cells. The aim of this study is to investigate the persister cell formation and expression analysis as well as to predict the 3D structure of the genes involved in the production of persister cells. The presence of persisters in S. Typhimurium was determined by time dependent killing of different types of bactericidal antibiotics and expression of genes associated with persister cell formation which was assessed five hours after the addition of antibiotics by the qRT-PCR. Indeed, the 3D structural model of the proteins studied was predicted by performing several computational methods of retrieved primary protein sequences. The results of the study showed that the S. Typhimurium produced high levels of persister cells in the exposure of bactericidal antibiotics. Furthermore, qRT-PCR resulted in the fact that the expression of related genes was different depending on the type of antibiotic. Overall, this study provides information on the creation of persister cells and the role of different genes in the formation of these cells and structure of proteins involved in the production of persister cells in S. Typhimurium.

Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori.

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 µM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 µM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.