Exploring virulence factors of Helicobacter pylori isolated from gastric biopsy.

BACKGROUND: Helicobacter pylori (H. pylori) colonizes human gastric mucosa and is classified as class one carcinogenic bacteria. In this regard, this study aimed to detect major virulence factors in H. pylori strains recovered from gastric biopsy in patients referred to Aras Clinique in Ardabil, northwest of Iran (2019-2021). MATERIALS AND METHODS: In this descriptive-cross sectional study, 287 dyspeptic patients were included. For bacterial isolation, gastric biopsy specimens (n=287) were taken from gastric antrum, then aseptically were cultured on the selective medium and incubated at 37C in microaerophilic conditions for 3-5 days. RESULTS: 25.18% of all (n = 70) patients were found to be infected with H. pylori upon endoscopy. Of them, 9 patients (12.857%) and 2 patients (2.875%) had peptic ulcer disease and gastric cancer respectively. According to the different patterns of virulence factors, 57 virutypes were identified in which oipA-vacAs1-vacAm2 (3, 4.28% n =) and oipA-vacAs1-vacAs2-vacAm2 (3, 4.28% n =) were the most common patterns. The simultaneous presence of vacAS2, vacAm2 and hopQ2 genes was observed in both patients with gastric cancer. OipA (n = 562.5%), VacAs1 (n = 6.75%), VacAs2 (n = 6.75%), and VacAm2 (n = 787.5%) were found to be the most prevalent virulence factor. CONCLUSION: According previous studies, it is confirmed that the cagPAI gene cluster and vacA gene alleles are strongly correlated with gastritis and gastrointestinal tract adenocarcinomas. Our study indicated that 50% of the indigenous strains of H. pylori harbor these oncogenic genes and they are hypervirulent.

Molecular detection and characterization of Shigella spp. harboring extended-spectrum ß-lactamase genes in children with diarrhea in northwest Iran.

Shigellosis is one of the acute bowel infections and remains a serious public health problem in resource-poor countries. The present study aimed to survey the distribution of extended-spectrum ß-lactamase (ESBL)-producing Shigella strains isolated from patients with diarrhea in northwest Iran. In the present cross-sectional study, from January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea in Ardabil, Iran. Multiplex PCR assay was applied for the presence of ipaH, invC, wbgZ, rfpB, and rfc genes to detect Shigella spp., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii, respectively. Phenotypic detection of ESBL-producing isolates was carried out using the Double Disc Test (DDT). The frequency of main ESBL encoding genes including blaCTX-M, blaSHV, and blaTEM was detected using multiplex PCR. The genetic similarity of S. sonnei isolates was determined using ERIC PCR. A total of 49 Shigella isolates (3.8%; 49/1280) including 42 (85.7%) S. sonnei, 5 (10.2%) S. flexneri, and 2 (4%) S. dysenteriae were identified. S. boydii was not detected in any fecal samples. ESBLs were produced by 10.2% of Shigella spp. including 3 S. sonnei, 1 S. flexneri, and 1 S. dysenteriae. The ESBL encoding genes include blaCTX-M and blaTEM found in 65.3% and 61.2% of isolates, respectively. blaSHV gene was not detected in any isolates. The ERIC-PCR profiles allowed the differentiation of 42 S. sonnei strains into 6 clusters. Our study revealed a high frequency of ESBL-encoding genes among Shigella spp. in northwest Iran. The high prevalence of S. sonnei harboring ESBL genes, in the present work, is the main challenge for dysentery treatment, and this concern justifies the need for effective and regular monitoring of antibiotic usage among patients.

Phenotypic and Molecular Characterization of Carbapenems Resistant Escherichia coli Isolated from Patients with Urinary Tract Infections in Ardabil Province, Iran.

Background & Objective: Carbapenem-resistant is Gram-negative bacteria representing a worldwide public health problem. The present study aims to survey the phenotypic and genotypic characteristics of carbapenem-resistant Escherichia coli isolates collected from hospitalized patients and outpatients in Ardabil province, Iran. Methods: Two hundred samples were collected from the patients who had already been referred to the hospitals in Ardabil, Iran, from January to June 2017. Each patient's social and demographic data were recorded in the first step. The resistance profile of all E. coli isolates against imipenem and meropenem antibiotics were determined using the Kirby-Bauer disk diffusion method. Moreover, the broth microdilution method determined the Minimum Inhibitory Concentration (MIC) of E. coli isolates to imipenem. The Carbapenem Inactivation Method (CIM) and Carba NP test were employed for screening carbapenem-resistant strains. The frequency of carbapenem-encoding genes was determined using Polymerase Chain Reaction (PCR) method. The Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR analysis was used to evaluate the genetic relatedness of E. coli isolates. Results: Out of 200 urine samples, 66% (n = 132) of the samples were collected from women. The patients' age varied from 1 month to 93 years. Results of the disk diffusion method revealed that 33% (n=66/200) of E. coli isolates were resistant to imipenem. However, imipenem resistance was detected in 37% (n = 74/200) of the E. coli isolates using broth microdilution method. All E. coli isolates were negative in CIM and Carba NP tests. Moreover, we could not detect any carbapenemase encoding genes among E. coli isolates. The ERIC-PCR method revealed the E. coli strains were classified into 39 clusters with 80% similarity. Conclusion: It appears that E. coli is the most common cause of urinary tract infection in Ardabil province.

Phenotypic and molecular characterization of extended-spectrum ß-lactamase/AmpC- and carbapenemase-producing Klebsiella pneumoniae in Iran.

BACKGROUND: The objective of the current study is to evaluate the phenotypic and molecular characterization of ESBL/AmpC- and carbapenemase-producing K. pneumoniae isolates in Iran. METHODS: From October 2018 until the end of April 2020, different clinical samples were collected and K. pneumoniae isolates were identified using conventional biochemical tests and PCR assay. Antibiotic susceptibility pattern was determined using the Kirby-Bauer disk diffusion method. Modified Hedge Test (MHT) was applied to the identification of carbapenemase-producing K. pneumoniae. ESBL and AmpC-producing K. pneumoniae were detected using Double Disc Test (DDT) and Disc Potentiation Test (DPT), respectively. The presence of carbapenemase, ESBL, and AmpC encoding genes was screened by Polymerase Chain Reaction (PCR) assay. RESULTS: A total of 100 K. pneumoniae isolates were collected. K. pneumoniae isolates had the highest resistance rate to cefazolin (66%) and cefotaxime (66%). Meropenem and amikacin with sensitivity rates of 76% and 69% were the most effective antimicrobial agents on K. pneumoniae isolates. It was found that 12 (12%), 27 (27%), and 9 (9%) K. pneumoniae isolates were positive in MHT, DDT, and DPT tests, respectively. Among the carbapenemase-encoding genes, blaOXA-48 (24%) and blaIMP (13%) genes had the highest frequency, while blaKPC and blaGIM genes were not detected among K. pneumoniae isolates. blaTEM (48%) and blaCMY (8%) genes had the highest frequency among ESBL and AmpC ß-lactamase-encoding genes, respectively. CONCLUSIONS: It is vital to adopt effective control strategies for K. pneumoniae infections and ensure rapid identification of antibiotic resistance profile.

The impact of Aggregatibacter actinomycetemcomitans biofilm-derived effectors following antimicrobial photodynamic therapy on cytokine production in human gingival fibroblasts.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is an effective adjunctive therapeutic modality for the treatment of local infections, including periodontitis and peri-implantitis. After receiving aPDT, microbial cells in the biofilm structure may produce and/ or release soluble biofilm-derived effectors (BDEs), which may affect the biology of the host cells in the community context of their surrounding microenvironment. Given the fact that no study has investigated the role of BDEs following aPDT in the pathogenesis of infectious diseases, the aim of the current study was to determine the effect of BDEs of Aggregatibacter actinomycetemcomitans following exposure to sub-lethal doses of indocyanine green (ICG)-aPDT on human gingival fibroblasts (HGFs) in terms of cytokines produced. MATERIALS AND METHODS: In this study, we evaluated the effect of biofilm-conditioned medium (BCM) resulting from the treatment of A. actinomycetemcomitans biofilm with a sub-lethal dose of aPDT on cytokines production, including IL-6, IL-8, CXCL10, TGF-ß, and bFGF of HGFs using enzyme-linked immunoassays (ELISA). The sensitivity of cytokines to BDEs was determined by micro-titer plates. RESULTS: The maximal sub-lethal dose of ICG-PDT was 20.15 µM/mL ICG at a fluence of 31.2 J/cm2. The BCM of ICG-PDT-treated viable A. actinomycetemcomitans significantly reduced IL-6, IL-8, and CXCL10 levels compared to the BCM of untreated viable A. actinomycetemcomitans (78-, 93-, and 61.6-fold reduction, respectively; all P < 0.01). TGF-ß and bFGF were strongly induced by BCM of ICG-PDT treated viable A. actinomycetemcomitans (by 57.7 and 36.1 folds, respectively; both P < 0.05). The BCM of untreated viable A. actinomycetemcomitans degraded most of the CxCL10, TGF-ß and bFGF (58.8, 61.5, and 71.6%, respectively) in 24 h, while it degraded 9.3% of IL-6 and 15.1% of IL-8 after 24 h. CONCLUSION: The results of the current study revealed that a sub-lethal dose of ICG-aPDT through the effect of BCM on HGFs could not only significantly reduce the production of pro-inflammatory cytokines but also promoted their role in periodontal regeneration due to increasing the bFGF level. Altogether, ICG-aPDT, with it's antimicrobial effects reduces inflammation and induces of tissue regeneration resulting from BCM, can be considered an efficient adjunctive therapeutic method for the treatment of local infections.

Current concepts on immunopathogenesis of hepatitis B virus infection.

Hepatitis B virus (HBV) infection is a leading cause of liver damage and hepatic inflammation. Upon infection, effective antiviral responses by CD8+ T cells, CD4+ T cells, Natural killer (NK) cells, and monocytes can lead to partial or complete eradication of the viral infection. To date, many studies have shown that the production of inhibitory cytokines such as Interleukin 10 (IL-10), Transforming growth factor beta (TGF-ß), along with dysfunction of the dendritic cells (DCs), and the absence of efficient innate immune responses could lead to T cell exhaustion, development of persistent infection, and inability to eradicate the viral infection from liver. Understanding the immunopathogenesis of the virus could be useful in providing further insights toward novel strategies in the eradication of HBV infection.

A study on the immune response induced by a DNA vaccine encoding Mtb32C-HBHA antigen of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) has still remained a global health issue. One third of the world's population is infected with tuberculosis and the current BCG vaccine has low efficiency; hence, it is necessary to develop a new vaccine against TB. The aim of the current study was to evaluate the efficiency of a novel DNA vaccine encoding Mtb32C-HBHA antigen in inducing specific immune responses against Mycobacterium tuberculosis. MATERIALS AND METHODS: A DNA plasmid vaccine expressing Mtb32C-HBHA fusion protein was constructed and its ability in protein expression was examined by RT-PCR and Western blot methods. Female BALB/c mice were vaccinated with 100 µg of purified recombinant vector in an attempt to assess its immunogenicity and protective efficacy. Further, the cytokines, IFN-γ, IL-12, IL-4, IL-10, and TGF-ß were assessed. RESULTS: The levels of all the studied cytokines were significantly increased (P<0.05) compared with the control group. IFN-γ production in the group receiving DNA vaccine plus BCG was increased compared with those receiving only DNA vaccine or BCG (P<0.001). CONCLUSION: The immunogenicity of the new chimeric DNA vaccine was confirmed alone and in combination with BCG. Based on the results of the current study, the constructed DNA vaccine induced the expression of Mtb32C-HBHA fusion protein efficiently in vitro. Furthermore, high levels of the specific cytokines were induced in mice. By using this DNA vaccine as a booster after BCG, higher amounts of IFN-γ will be produced.

Characterization of clarithromycin-resistant Helicobacter pylori strains in Iran: A systematic review and meta-analysis.

BACKGROUND: The high prevalence of clarithromycin-resistant strains of Helicobacter pylori is a main challenge for the successful treatment of gastrointestinal infections. Point mutations in the 23S rRNA gene are one of the main mechanisms leading to the resistance to clarithromycin in Iran. The purpose of the present review was to evaluate the prevalence of clarithromycin-resistant H. pylori strains in Iran and to identify the major molecular mechanisms of resistance in the resistant isolates. METHODS: Using related keywords and computer search in English and Persian databases (up to November 21, 2016), available data about prevalence of clarithromycin-resistant H. pylori strains and molecular mechanisms of resistance in Iran were retrieved. Relevant articles were selected using pre-defined inclusion and exclusion criteria. RESULTS: The results of the meta-analysis showed that the overall prevalence of clarithromycin-resistant H. pylori strains in Iran is 14.7%. The highest and the lowest resistance to clarithromycin were reported from Kashan (33.7%) and Rasht (5.5%), respectively. The most prevalent point mutations in Iran were A2143G (59.1%), A2142G (17.8%), A2142C (8.8%), and A2144G (6.2%), respectively. CONCLUSION: The prevalence of clarithromycin-resistant H. pylori strains was in an acceptable level in our region. Therefore, clarithromycin can be used for the eradication of H. pylori infection in Iran. However, it seems that investigation about the role of other mechanisms involved in the induction of resistance to clarithromycin is needed.

Antimicrobial activity of photodynamic therapy in combination with colistin against a pan-drug resistant Acinetobacter baumannii isolated from burn patient.

Nosocomially-acquired multi-, extensively-, and pandrug resistant (MDR, XDR, and PDR) strains of microorganisms such as Acinetobacter baumannii remain a serious cause of infection and septic mortality in burn patients. Treatment of patients with nosocomial burn wound infections is often complicated by drug-resistant strains of A. baumannii. Today, many researchers are focusing on the investigation of novel non-antibiotic strategies such as photodynamic therapy (PDT). We report a new PDT strategy that suppresses colistin resistance in PDR A. baumannii by interfering with the expression of a pmrA/pmrB two-component system. In the current study, A. baumannii with a PDR feature isolated from a burn patient was used as a test strain. PDT was carried out using toluidine blue O (TBO) and light-emitting diode (LED) as a photosensitizer and radiation source, respectively. The antimicrobial susceptibility profiles were assessed for cells surviving PDT. The effects of sub-lethal PDT (sPDT) on the expression of the pmrA/pmrB two-component signal transduction system were evaluated by real-time quantitative reverse transcription PCR. Results of drug susceptibly testing (DST) in LED and TBO groups separately showed that the bacteria were resistant to all tested antibiotics, while the DST result of the LED+TBO group showed highly declining bacterial growth when compared with the control group. Reduction in the expression of pmrA and pmrB was observed in the treated strains after sPDT. This represents the first conclusive example of a direct role for the PDT in breaking antibiotic resistance by directly modulating two-component system activity.

Evaluation of ELISA and Brucellacapt tests for diagnosis of human Brucellosis.

BACKGROUND AND OBJECTIVES: Brucellosis is one of the most common zoonotic diseases in Iran and human brucellosis is endemic in all parts of the country. Because of the difficulty in the diagnosis of brucellosis, particularly in endemic areas, the use of new and feasible diagnostic tests seem to be of great importance for resolving the diagnostic obstacles. We evaluated the usefulness of a new serological test based on an immunocapture-agglutination technique in comparison with ELISA test for serological diagnosis of brucellosis. MATERIALS AND METHODS: A total of 11 patients with brucellosis, who had positive blood cultures for Brucella species, and 47 suspected patients were included in this study. Serum samples collected from these patients were tested by brucellacapt and ELISA and the results were, consequently, compared. RESULTS: In patients with positive blood culture, all the samples gave positive results with brucellacapt test while IgM ELISA, IgG ELISA and (IgG + IgM) ELISA tests were positive in 8, 9 and 11 patients, respectively. Out of the 46 suspected patients, (IgG + IgM) ELISA, Brucellacapt, IgG ELISA and IgM ELISA were positive in 37, 15, 34 and 37 patients, respectively.The best cut-off point of ELISA-IgG was 10.78 IU/ml which produced the maximal sensitivity and specificity for the diagnosis of human brucellosis. CONCLUSION: Both the (IgG + IgM) ELISA and Brucellacapt tests demonstrate a high specificity in this study. According to the results of the current study, it is found that both tests are valuable tools for diagnosis of brucellosis in Iran as an endemic area of brucellosis. It is strongly suggested that a combination of both tests to be used for the diagnosis of brucellosis.