The modulatory effect of Lactobacillus gasseri ATCC 33323 on autophagy induced by extracellular vesicles of Helicobacter pylori in gastric epithelial cells in vitro.

Helicobacter pylori has been recognized as a true pathogen, which is associated with various gastroduodenal diseases, and gastric adenocarcinoma. The crosstalk between H. pylori virulence factors and host autophagy remains challenging. H. pylori can produce extracellular vesicles (EVs) that contribute to gastric inflammation and malignancy. Some probiotic strains have been documented to modulate cell autophagy process. This study was aimed to investigate the modulatory effect of cell-free supernatant (CFS) obtained from Lactobacillus gasseri ATCC 33323 on autophagy induced by H. pylori-derived EVs. EVs were isolated from two clinical H. pylori strains (BY-1 and OC824), and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The viability of AGS cells was assessed after exposure to different concentrations of H. pylori EVs, and L. gasseri CFS. Based on MTT assay and Annexin V-FITC/PI staining, 50 µg/ml of H. pylori EVs and 10 % v/v of L. gasseri CFS were used for further cell treatment experiments. Autophagy was examined using acridin orange (AO) staining, RT-qPCR analysis for autophagy mediators (LC3B, ATG5, ATG12, ATG16L1, BECN1, MTOR, and NOD1), and western blotting for LC3B expression. H. pylori EVs were detected to range in size from 50 to 200 nm. EVs of both H. pylori strains and L. gasseri CFS showed no significant effect on cell viability as compared to untreated cells. H. pylori EVs promoted the development of acidic vesicular organelles and the expression of autophagy-related genes (LC3B, ATG5, ATG12, ATG16L1, BECN1, and NOD1), and decreased the expression of MTOR in AGS cells at 12 and 24 h time periods. In addition, the production of LC3B was increased following 12 h of treatment in AGS cells. In contrast, L. gasseri CFS effectively inhibited EVs-induced autophagy, as evidenced by reduced acidic vesicular organelle formation and modulation of autophagy markers. Our study indicated that L. gasseri CFS can effectively suppress H. pylori EV-induced autophagy in AGS cells. Further investigations are required to decipher the mechanism of action L. gasseri CFS and its metabolites on autophagy inhibition induced by H. pylori.

In silico and In vitro Assessment of Antibacterial Activity, LPS Binding Affinity, and Toxicity of the GKY25 Peptide.

INTRODUCTION: Extensively and multi-drug resistant isolates of bacteria (MDR, XDR) have caused significant health problems and are responsible for high morbidity and mortality as well. In this critical condition, the discovery, design, or development of new antibiotics is of great concern. According to this necessity, antimicrobial peptides (AMPs) suggested as promising agents. Accordingly, this study aims to evaluate the GKY25 peptide to develop its future antibacterial applications as well as confirmation of LPS neutralization. METHODS: Predictions of 3D structure and helical wheel projection analysis of the peptide were performed by ITASSER and Heliquest servers. Binding affinity and antibacterial activity were performed using molecular docking and CAMPR4, respectively, followed by experimental binding assay as well as in vitro antibacterial assay. RESULTS: GKY25 was predicted as an alpha-helical peptide, and its helicity showed probable projection of hydrophobic and positively-charged amino acid residues. Docking studies showed binding affinity of GKY25 peptide to gram-positive and outer and inner gram-negative bacterial membranes as -5.7, -6.8, and -4 kcal/mole, respectively. CAMPR4 analysis predicted the peptide as an AMP. Experimental binding assay showed that the peptide binds LPS immediately and their interaction was observed at 274 nm. CONCLUSION: Gathering all in silico and in vitro data together, GKY25 is a good drug lead that could be examined further using clinical isolates of gram-negative bacteria in vitro.

Evaluation of the antibacterial effect of hydroalcoholic extract of the galls of Quercus infectoria on Aggregatibacter actinomycetemcomitans.

Background: Aggregatibacter actinomycetemcomitans (Aa) plays a vital role in some destructive forms of periodontitis. While mechanical and chemical plaque control is the first step in periodontitis treatment, side effects of adjunctive chemical agents such as chlorhexidine (CHX) mouthwash have led to the application of natural alternatives with minimal side effects. Therefore, this study evaluated the antibacterial effect of the hydroalcoholic extract of Quercus infectoria (Qi) galls on Aa in vitro. Methods: The hydroalcoholic extract of Qi was obtained by the maceration method, and Aa bacterial strain was cultured. The inhibition zone diameter was measured through the agar well diffusion method. Also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by the broth microdilution method. All the experiments were repeated three times. 0.2% CHX was used as a control. Results: The inhibition zone diameter of Aa increased with increasing concentration of Qi extract. While MIC and MBC values for CHX were 0.0039 and 0.0078 mg/mL, respectively, both MIC and MBC values of the Qi extract for this bacterium were similar, i.e., 2.5 mg/mL, which was significantly higherd. Conclusion: Since other in vivo studies have confirmed the other properties of this extract and its safety in terms of cytotoxicity and mutagenicity, hydroalcoholic extract of Qi may be used in mouthwashes or local delivery systems to affect periodontal biofilm.

Fast killing kinetics, significant therapeutic index, and high stability of melittin-derived antimicrobial peptide.

The emergence of multidrug-resistant (MDR) bacteria is a major challenge for antimicrobial chemotherapy. Concerning this issue, antimicrobial peptides (AMPs) have been presented as novel promising antibiotics. Our previous de novo designed melittin-derived peptides (MDP1 and MDP2) indicated their potential as peptide drug leads. Accordingly, this study was aimed to evaluate the kinetics of activity, toxicity, and stability of MDP1 and MDP2 as well as determination of their structures. The killing kinetics of MDP1 and MDP2 demonstrate that all bacterial strains were rapidly killed. MDP1 and MDP2 were ca. 100- and 26.6-fold less hemolytic than melittin and found to be respectively 72.9- and 41.6-fold less cytotoxic than melittin on the HEK293 cell line. MDP1 and MDP2 showed 252- and 132-fold improvement in their therapeutic index in comparison to melittin. MDP1 and MDP2 sustained their activities in the presence of human plasma and were found to be ca. four to eightfold more stable than melittin. Spectropolarimetry analysis of MDP1 and MDP2 indicates that the peptides adopt an alpha-helical structure predominantly. According to the fast killing kinetics, significant therapeutic index, and high stability of MDP1, it could be considered as a drug lead in a mouse model of septicemia infections.

Helicobacter pylori Infection Mediates Inflammation and Tumorigenesis-Associated Genes Through miR-155-5p: An Integrative Omics and Bioinformatics-Based Investigation.

Helicobacter pylori (H. pylori) is a major human pathogenic bacterium that survives in the gastric mucosa. The aim of this study is to evaluate the expression of the target gene network of miR-155-5p in H. pylori-related gastritis using a combination of public gene expression datasets and web-based platforms. To evaluate the expression of genes related to gastritis, we used two datasets from Gene Expression Omnibus (GEO) database. Then, we determined the overlaps between the predicted miR-155-5p target genes and gastritis-dysregulated GEO datasets genes; in the next step, we identified the possible miR-155-5p target-DEGs (Target-Differentially Expressed Genes). Also, we performed multiple bioinformatics analyses to identify the most important targets and downstream pathways associated with this miRNA. Using the UCSC cancer genomic browser analysis tool, we investigated the expression of hub genes in relation to gastric cancer and H. pylori infection, as well as the potential role of hub genes in gastritis, inflammation, and cancer. In this regard, 28 differentially expressed target genes of miR-155-5p were identified. Most of the captured target genes were correlated with the host immune response and inflammation. Based on the specific patterns of expression in gastritis and cancer, CD9, MST1R, and ADAM10 were candidates for the most probable targets of miR-155-5p. Although the focus of this study is primarily on bioinformatics, we think that our findings should be experimentally validated before they can be used as potential therapeutic and diagnostic tools.

Expression analysis of miRNA-155 level in Helicobacter pylori related inflammation and chronic gastritis.

Background and Objectives: Helicobacter pylori, is a major etiologic agent associated with gastritis. There is more evidence of noncoding microRNAs (miRs) dysregulation in gastrointestinal diseases, including inflammation caused by Helicobacter pylori. Also, the classification of gastrointestinal malignancies using the miRs profile is better than the protein profile. MiRNA-155(miRNA-155) among other miRs plays an important role in control of inflammation and gastric malignancy, so it can be remarkable prognosis marker of gastric cancer in the phase of chronic gastritis. The aim of this study was to compare the expression of miRNA-155 in gastric biopsy and serum samples of adult patients with chronic gastritis. Materials and Methods: Biopsy and blood samples were collected from endoscopy candidates at Taleghani hospital, Tehran, during 2019. H. pylori infection was detected using histology, culture and molecular PCR methods. Based on cagA and vacA genotyping, the toxicity of H. pylori isolates were determined. After RNA extraction, the expression rate of miRNA-155 was evaluated by real-time polymerase chain reaction (RT-PCR) in gastric tissue and serum of adults infected by H. pylori (n = 30) compared with control group without infection (n = 20). RNU6 housekeeping miRNA were used as endogenous control and statistical analyses were performed using SPSS, ANOVA and Student's t-test. Results: miRNA-155 expression in H. pylori infected adult patients increased significantly by 5.61 and 10.11 fold in serum and tissue respectively, compared to that observed in the control group. Evaluation of miRNA-155 expression pattern in relation to bacterial virulence factors showed that the increase in miRNA-155 expression is independent of CagA and VacA toxins. Conclusion: According to the differential expression patterns of miRNA-155 in serum samples of the infected adult patients, miRNA-155 has the potential to evaluate as chronic gastritis marker.

Prediction of Blood miRNA-mRNA Regulatory Network in Gastric Cancer.

BACKGROUND: The aim of the study was to suggest a high specific and sensitive blood biomarker for early GC diagnosis. METHODS: the expression data of miRNAs and mRNAs were collected from the blood samples of the GC patients based on literature mining. Bioinformatics tools and databases (PANTHER, TargetScan, miRTarBase, miRDB, STRING, and Cytoscape) were used to predict the regulatory relationship. Subsequently, expression level of the selected miRNA was evaluated in the blood samples of gastritis patients to recognize the common miRNA between the GC and gastritis patients. RESULTS: Analysis of 40 target genes by MCODE (installed in Cytoscape software) indicated 4 hub genes (WWP1, SKP2, KLHL42, and FBXO11) as a significant cluster in the PPI network related to miR-21, with Node Score Cutoff: 0.2, Degree Cutoff: 2 and K-Core: 2. In addition, the miRNA RT-qPCR results showed that, the expression level of miR-21 was significantly higher in gastritis group compared to the healthy group (p< 0.05). CONCLUSION: the present study clearly demonstrated the increasing level of blood miR-21 among the gastritis patients infected by H. pylori. Therefore, the altered miRNAs, especially overexpression of onco-miRs, may identify a potential link between miRNAs and pathogenesis of the H. pylori-related complications.

Cross-talk between immune system and microbiota in COVID-19.

INTRODUCTION: Human gut microbiota plays a crucial role in providing protective responses against pathogens, particularly by regulating immune system homeostasis. There is a reciprocal interaction between the gut and lung microbiota, called the gut-lung axis (GLA). Any alteration in the gut microbiota or their metabolites can cause immune dysregulation, which can impair the antiviral activity of the immune system against respiratory viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. AREAS COVERED: This narrative review mainly outlines emerging data on the mechanisms underlying the interactions between the immune system and intestinal microbial dysbiosis, which is caused by an imbalance in the levels of essential metabolites. The authors will also discuss the role of probiotics in restoring the balance of the gut microbiota and modulation of cytokine storm. EXPERT OPINION: Microbiota-derived signals regulate the immune system and protect different tissues during severe viral respiratory infections. The GLA's equilibration could help manage the mortality and morbidity rates associated with SARS-CoV-2 infection.

Prevalence, serotyping and drug susceptibility patterns of Escherichia coli isolates from kidney transplanted patients with urinary tract infections.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) are among the main pathogens in urinary tract infections (UTIs) among kidney transplant patients (KTPs). AIM: To estimate the prevalence of ESBL-producing E. coli in KTPs and to evaluate the most prevalent serotypes and antibacterial susceptibility patterns of isolated bacteria in Tehran, Iran. METHODS: A total of 60 clinical isolates of uropathogenic E. coli were collected from 3 kidney transplant centers from April to May 2019. Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical Laboratory and Standards Institute. The serotyping of E. coli isolates was performed by the slide agglutination method. The presence of bla TEM, bla SHV, and bla CTX-M genes was evaluated by polymerase chain reaction. RESULTS: The frequency of ESBL-producing E. coli in KTPs was found to be 33.4%. All of the 60 E. coli isolates were found to be susceptible to doripenem (100%) and ertapenem (100%). High resistance rates to ampicillin (86%), cefotaxime (80%), and cefazolin (77%) were also documented. The most frequent serotypes were serotype I (50%), serotype II (15%), serotype III (25%), and serotype VI (10%). The gene most frequently found was bla TEM (55%), followed by bla CTX-M (51%) and bla SHV (41%). CONCLUSION: Molecular analysis showed that bla TEM was the most common ESBL-encoding gene. The high resistance to ß-lactams antibiotics (i.e., ampicillin, cefotaxime, and cefazolin) found in E. coli from KTPs with UTIs remains a serious clinical challenge. Further efforts to control ESBL-producing E. coli should include the careful use of all antibiotics as well as barrier precautions to reduce spread.

Anti-proliferative effects of cell wall, cytoplasmic extract of Lactococcus lactis and nisin through down-regulation of cyclin D1 on SW480 colorectal cancer cell line.

BACKGROUND AND OBJECTIVES: Colorectal cancer is one of the most types of cancer. Researchers have shown that lactic acid bacteria have antitumor activity. The cell wall of Lactococcus lactis, as the bacterial cytoplasmic extract and nisin can affect the proliferation of cancer cells. Since cyclin D1 plays an important role in the progression of the cell cycle, its regulation can also be a therapeutic approach. We investigated the antiproliferative effect of cell wall, cytoplasmic extract and nisin on SW480 cancer cell line and the expression level of cyclin D1 gene in treated cancer cells. MATERIALS AND METHODS: SW480 cell lines were treated with different concentrations of bacterial cell wall, cytoplasmic extract and nisin. MTT test was also performed. The expression level of cyclin D1 gene was determined using Real time PCR. Data were analyzed using Graph Pad Prism software. RESULTS: The growth rate of cancer cells treated with nisin has significantly decreased compared to the cancer cells treated by other two substances (p< 0.05). Survival rates of the cancer cells treated by nisin at a concentration of 2000 µg, cytoplasmic extract, and cell wall were 34%, 47% and 49%, respectively. Real-time PCR results showed that cyclin D1 mRNA expression has significantly decreased in nisin treated sw480 cells (P<0.05). CONCLUSION: The results of this study show that nisin, bacterial cytoplasmic extract, and bacterial cell wall have antiproliferative effects, which are associated with the decreased expression of cyclin D1 in SW480 cell line.

Blood culture and antimicrobial susceptibility pattern of bacteria and fungi isolated from febrile neutropenic patients treated with chemotherapy at Taleghani hospital, Tehran.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the drug susceptibility pattern of the pathogens causing bacteraemia and fungemia in patients who have developed febrile neutropenia after chemotherapy. MATERIALS AND METHODS: A total of 95 patients with suspected or proven malignancy (50 patients) were admitted to the adult haematology ward at Taleghani Hospital in Tehran. Blood samples were inoculated into the bottles of Bact/Alert blood culture system and sent to Payvand's clinical and special laboratory immediately and then incubated at 35 ± 2°C. Culture from positive bottles were plated on appropriate media and incubated at 37°C and 30°C for bacterial and fungal isolation, respectively. A bacterial suspension with turbidity equal to 0.5 McFarland (1.5 × 108 CFU/mL) was prepared and used for the Vitec2 system (biomerioux). Statistical analysis using independent Fisher's exact test was conducted and a p-value of < 0.05 was considered as significant. RESULTS: Among 50 patients with approved malignancy, Acute Lymphoblastic Leukaemia (ALL) and Acute Myeloid Leukaemia (AML) were the most common underlying diseases. This study showed, 20% (n: 10) of febrile neutropenic episodes established positive blood culture. Of them, 3 were Gram-negative (30%) and 5 were-Gram-positive bacteria (50%) and 2 patients (20%) showed fungemia with Fusarium spp. CONCLUSION: It is crucial to know about the likely pathogens and their local antibiotic and antifungal sensitivity patterns. Such local findings will show if any modifications to treatment guidelines are necessary.

Rapid detection and molecular survey of blaVIM, blaIMP and blaNDM genes among clinical isolates of Acinetobacter baumannii using new multiplex real-time PCR and melting curve analysis.

BACKGROUND: Acinetobacter baumannii is a cosmopolitan bacterium that is frequently reported from hospitalized patients, especially those patients who admitted in the intensive care unit. Recently, multiplex real-time PCR has been introduced for rapid detection of the resistance genes in clinical isolates of bacteria. The current study aimed to develop and evaluate multiplex real-time PCR to detect common resistance genes among clinical isolates of A. baumannii. RESULTS: Multiplex real-time PCR based on melting curve analysis showed different Tm corresponding to the amplified fragment consisted of 83.5 °C, 93.3 °C and 89.3 °C for blaIMP, blaVIM and blaNDM, respectively. Results of multiplex real-time PCR showed that the prevalence of blaIMP, blaVIM and blaNDM among the clinical isolates of A. baumannii were 5/128(3.9%), 9/128(7.03%) and 0/128(0%), respectively. Multiplex real-time PCR was able to simultaneously identify the resistance genes, while showed 100% concordance with the results of conventional PCR. CONCLUSIONS: The current study showed that blaVIM, was the most prevalent MBL gene among the clinical isolates of A. baumannii while no amplification of blaNDM was seen. Multiplex real-time PCR can be sensitive and reliable technique for rapid detection of resistance genes in clinical isolates.

In vitro Antibacterial Effect of Hydroalcoholic Extract of Lawsonia inermis, Malva sylvestris, and Boswellia serrata on Aggregatibacter actinomycetemcomitans.

BACKGROUND: Considering the increased rate of microbial resistance to antibiotics and chemical side effects of antibiotics and antiseptics used for the treatment of periodontal disease, there is a need for an alternative antimicrobial agent with fewer complications. Medicinal herbs have recently become popular as novel antimicrobial agents. This study aimed to assess the antibacterial effects of hydroalcoholic extracts of Lawsonia inermis, Malva sylvestris, and Boswellia serrata on Aggregatibacter actinomycetemcomitans. MATERIALS AND METHODS: Hydroalcoholic extracts of the three medicinal plants were obtained by the maceration technique and A. actinomycetemcomitans was cultured. Antimicrobial efficacy of the three medicinal plants was compared with that of 0.2% chlorhexidine (CHX) according to the Clinical and Laboratory Standards Institute protocol using agar disc diffusion and broth microdilution techniques. All tests were repeated three times. RESULTS: Hydroalcoholic extracts of all three plants had antimicrobial activity against A. actinomycetemcomitans. The minimum inhibitory concentration (MIC) of L. inermis, M. sylvestris, and B. serrata was 78.1, 156.2, and 1666 µg/mL with no significant difference between them. The MIC of CHX was 3.33 µg/mL, which was significantly higher than that of B. serrata extract. CONCLUSION: Given that further in vivo studies confirm other properties of these extracts and their safety in terms of cytotoxicity and mutagenicity, hydroalcoholic extracts of L. inermis and M. sylvestris may be used in mouthwashes or local delivery systems to affect periodontal biofilm.

Highly Synergistic Effects of Melittin with Conventional Antibiotics Against Multidrug-Resistant Isolates of Acinetobacter baumannii and Pseudomonas aeruginosa.

BACKGROUND: Morbidity and mortality due to multidrug-resistant (MDR) bacteria are of great concern in burn patients. In this critical condition, synergism between antimicrobial peptides and conventional antibiotics would be a promising strategy. Accordingly, this study aimed to determine the therapeutic value of melittin as a natural peptide by examining its synergistic effect with conventional antibiotics against MDR isolates of Acinetobacter baumannii and Pseudomonas aeruginosa. MATERIALS AND METHODS: Fifteen clinical isolates for each kind of bacteria were collected from burn patients. Antibiotic susceptibility of all isolates was evaluated by disk diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration for melittin, colistin, doripenem, doxycycline, and ceftazidime were also examined. Fractional inhibitory concentration (FIC) of melittin in combination with the antibiotics was determined for six MDR isolates. The cytotoxicity of melittin in combination with the antibiotics was examined on a normal human cell line. RESULTS: The geometric means of MIC (GMMIC) for melittin and doripenem after combination were reduced to 61.5- and 51.5-fold, respectively, against MDR A. baumannii isolates. These reductions for melittin-doripenem and melittin-ceftazidime against MDR P. aeruginosa isolates were (63.5 and 58)-fold and (16 and 11)-fold, respectively. FIC for melittin-doripenem against A. baumannii and FIC for melittin-doripenem and melittin-ceftazidime against P. aeruginosa strains were ≤0.5. This issue caused a decrease of up to 104-, 68-, and 17-fold, respectively, in the cytotoxicity of melittin. CONCLUSION: In conclusion, the synergism of melittin at its nontoxic dose with doripenem and ceftazidime could be of great therapeutic value as a topical drug against burn infections caused by MDR bacteria.

Phenotypic Investigation of the Antimicrobial Effect of Organic and Hydro-Alcoholic Extracts of Boswellia serrata on Oral Microbiota.

OBJECTIVES: Considering the emergence of resistant microbes and side effects of chemical drugs, in this study, the inhibitory effect of organic and hydro-alcoholic extracts of Boswellia serrata (B. serrata) on some oral microbiota was investigated. MATERIALS AND METHODS: In this experimental study, standard strains of Candida albicans (C. albicans; PTCC 5027), Candida glabrata (C. glabrata; PTCC 5295), Candida krusei (C. krusei; PTCC 5297), and Streptococcus mutans (S. mutans; PTCC 1688) were collected from the Iranian Research Organization for Science and Technology (IROST). Then, the minimum inhibitory concentration (MIC) of organic and hydro-alcoholic extracts of B. serrata was determined based on the CLSI protocol and using the micro-dilution method. The contents of each well were subcultured in Müller-Hinton agar (Candida species) and blood agar (S. mutans). The lowest concentration with no growth was considered as the minimum fungicidal concentration (MFC) or bactericidal concentration (MBC). Statistical analyses were performed using Mann-Whitney test. RESULTS: Hydro-alcoholic extract of B. serrata at the concentration of 50 mg/ml inhibited the growth of C. albicans and S. mutans. It also inhibited the growth of C. krusei and C. glabrata at the concentration of 100 mg/ml. Organic extract of B. serrata at the concentration of 200 mg/ml only inhibited the growth of C. glabrata. CONCLUSION: Hydro-alcoholic extract of B. serrata had a greater inhibitory effect on C. albicans and S. mutans compared to the organic extract.

Action mechanism of melittin-derived antimicrobial peptides, MDP1 and MDP2, de novo designed against multidrug resistant bacteria.

The emergence and dissemination of multidrug resistant (MDR) bacteria are major challenges for antimicrobial chemotherapy of bacterial infections. In this critical condition, cationic antimicrobial peptides are 'novel' promising candidate antibiotics to overcome the issue. In this study, we investigated the antibacterial mechanism of new melittin-derived peptides (i.e., MDP1 and MDP2) against multidrug resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. MDP1 was designed with deletion of three amino acid residues, i.e., S18, W19, and I20, from the end of second hydrophobic motif of melittin. In the next step, VLTTG in MDP1 sequence was substituted with tryptophan residue. MDP1 and MDP2 had a high-antibacterial activity against MDR and reference strains of S. aureus, E. coli, and P. aeruginosa. DNA and calcein release and flow cytometry assays indicate a time-dependent antibacterial activity on the examined bacteria affected by both MDP1 and MDP2. Finally, SEM analyses highlighted dose- and time-dependent effects of MDP1 and MDP2 on S. aureus and E. coli bacteria by induction of vesicle or pore formation as well as cell lysis. In this study we successfully showed that rational truncation of large hydrophobic motifs can lead to significant reduction in toxicity against human RBCs and improving the antibacterial activity as well. Analyses of data from DNA release, fluorometry, flow cytometry, and morphological assays demonstrated that the MDP1 and MDP2 altered the integrity of both Gram-positive and Gram-negative bacterial membranes and killed the bacteria via membrane damages.

Evaluation of Clarithromycin Resistance Among Iranian Helicobacter pylori Isolates by E-Test and Real-Time Polymerase Chain Reaction Methods.

BACKGROUND: Helicobacter pylori is an important pathogen of human gastric mucosa. Antibiotic resistance, especially resistance to clarithromycin is a major factor for treatment failure of H. pylori infections. The main mechanism of clarithromycin resistance in these bacteria is related to point mutations in three different locations of 23S rRNA gene. OBJECTIVES: The aims of this study were to evaluate the resistance rate to clarithromycin among local H. pylori isolates by the E-test method and to determine the profile of point mutation in 23S rRNA by real-time polymerase chain reaction (PCR) method. PATIENTS AND METHODS: Eighty biopsy samples were collected from dyspeptic patients by endoscopy during 2011 - 2012. All samples were homogenized immediately and cultured on supplemented brucella blood agar and incubated under microaerophilic conditions. Further biochemical tests and ureC gene PCR was done for H. pylori confirmation. The H. pylori OC1096 strain was used as the control strain, simultaneously. Frequency of clarithromycin resistance was determined by the E-test method based on the clinical and laboratory standard institute (CLSI) standards. Point mutation profile was determined by real-time PCR and further analysis of melting curve, amplicon sequencing was done continuously. RESULTS: From 80 biopsy samples, 20 positive H. pylori isolates were detected and confirmed by biochemical tests and PCR method. Overall, 21.7% of the H. pylori isolates, showed clarithromycin resistance phenotype by use of the E-test. Also, the minimal inhibitory concentration of clarithromycin was determined as ≥ 0.5 mg/L by the E-test method. Only point mutation in the location of A2143G with melting temperature of 54.7°C was observed in all resistant isolates. CONCLUSIONS: This study showed that the frequency of H. pylori clarithromycin resistance in Iran is relatively high. Since clarithromycin is not commonly used in Iran for H. pylori eradication, the high rate of resistance could be related to cross-reactivity between other macrolides. Therefore, macrolide antibiotics must be prescribed with precaution in any case of treatment other than H. pylori infections. All resistant isolates showed A2143G mutation in 23S rRNA as the dominant pattern of point mutation at least in Tehran H. pylori isolates.

Role of Aders and OXA23 Genes among Imipenem Resistant Acinetobacter baumannii Isolates from Two Hospitals of Tehran, Iran.

BACKGROUND: This study aimed to evaluate the role of efflux pump regulator and OXA-23 genes in imipenem resistance Acinetobacter baumannii strains isolated from hospitalized patients in Tehran, Iran. METHODS: This study was conducted on 60 A. baumannii isolates collected from patients admitted to the Shahid Motahari and Taleghani Hospitals in Tehran during 2013-14. Antibiotic susceptibility tests (AST) and minimal inhibitory concentration (MIC) was determined by broth micro dilution methods according to CLSI 2014 guidelines. The frequency of efflux pump adeRS and OXA-23 genes were detected by PCR and further sequencing. RESULTS: The resistance of A. baumannii isolates to tested antibiotics was 100% to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole and piperacillin/tazobactam, 97% to imipenem, 94% to gentamicin, 83% to amikacin, 76% to tetracycline, and 0.0% to colistin. The MIC of 58 (96.6%) strains to imipenem was highly decreased in the presence of efflux pump inhibitor (PaßN), by 4 to 64 folds. The adeR and adeS genes were detected in 36 (60%) and 59 (98.3%), respectively and the frequency of OXA-23 gene was 57 (95%) of isolates. CONCLUSION: Existence of adeRS and OXA-23 genes in more than 50% of A. baumannii isolates in this study shows the presumptive role of efflux pump in simultaneous of carbapenemase production. Therefore, using new strategies are required in order to stop the vertical or horizontal exchanges mentioned genes from the resistant A. baumannii isolates to sensitive strains.

Efflux Pump Inhibitor Phenylalanine-Arginine Β-Naphthylamide Effect on the Minimum Inhibitory Concentration of Imipenem in Acinetobacter baumannii Strains Isolated From Hospitalized Patients in Shahid Motahari Burn Hospital, Tehran, Iran.

BACKGROUND: Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients. OBJECTIVES: The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients. MATERIALS AND METHODS: This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing. RESULTS: In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%). CONCLUSIONS: The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing nosocomial infections.

Occurrence of Potentially Pathogenic Bacterial-Endosymbionts in Acanthamoeba Spp.

BACKGROUND: Acanthamoeba- bacteria interactions enable pathogenic bacteria to tolerate harsh conditions and lead to transmission to the susceptible host. The present study was aimed to address the presence of bacterial endosymbionts of Acanthamoeba isolated from recreational water sources of Tehran, Iran. To the best of our knowledge this is the first study regarding occurrence of bacteria in environmental Acanthamoeba spp. in Iran. METHODS: A total of 75 samples of recreational water sources were collected. Samples were cultured on non- nutrient agar 1.5% plates. Positive Acanthamoeba spp. were axenically grown. DNA extraction and PCR reaction was performed using JDP1-2 primers. All positive samples of Acanthamoeba were examined for the presence of endosymbionts using staining and molecular methods. The PCR products were then sequenced in order to determine the genotypes of Acanthamoeba and bacteria genera. RESULTS: Out of 75 samples, 16 (21.3%) plates were positive for Acanthamoeba according to the morphological criteria. Molecular analysis revealed that Acanthamoeba belonged to T4 and T5 genotypes. Five isolates (35.7%) were positive for bacterial endosymbionts using staining method and PCR test. Sequencing of PCR products confirmed the presence of Pseudomonas aeruginosa and Agrobacterium tumefasiens. CONCLUSION: The presence of Acanthamoeba bearing pathogenic endosymbionts in water sources leads us to public health issues including improved sanitation and decontamination measures in recreational water sources in order to prevent amoebae-related infection. To the best of our knowledge this is the first report regarding the isolation of A. tumefasiens from Acanthamoeba in Iran and worldwide.