Characteristics of multiresistant Pseudomonas aeruginosa isolates from burn patients in Iran.

Infections caused by multidrug resistant (MDR) Pseudomonas aeruginosa isolates in burn patients restrict therapeutic strategies. The current study aimed to analyze antibiotic resistance genes and multilocus sequence typing (MLST) of P. aeruginosa strains isolated from burn patients in Shahid Motahari hospital in Tehran, Iran.Altogether 63 P. aeruginosa isolates were characterized in this study. Antibiotic susceptibility testing was performed by disc diffusion method. PCR was performed to determine the frequency of resistance genes. The expression rates of mexB, mexY genes were evaluated by Real-Time PCR. Genotyping of isolates was performed by MLST analysis. All isolates were MDR in this study. The highest resistance was detected against gentamicin, tobramycin, and cefoxitin (100%), while all isolates were susceptible to colistin. Altogether 14 resistance profiles were determined, and profile 1 included more than 50% of the isolates with the highest resistance. In this study blaampC, blaVIM-2, blaOXA-10, and aac(6')-Ib resistance genes were detected in all isolates. The expression levels of mexB and mexY genes were upregulated in 66.6 and 88.8% of MDR isolates, respectively. Overexpression of both genes was detected in 55.5% of the isolates.MLST analysis revealed five sequence types (STs), including ST235, ST664, ST532, ST2637, and ST230, which showed a significant relationship with antibiotic resistance profiles. The present study indicates an increase in antibiotic resistance against different antibiotic families among P. aeruginosa isolates. We describe the circulation of globally distributed STs among hospitalized patients, and we report ST235 as the most common MDR clone in our study.

NRAMP1 gene polymorphisms and cutaneous leishmaniasis: An evaluation on host susceptibility and treatment outcome.

BACKGROUND & OBJECTIVES: Association between polymorphisms in the natural resistance associated macrophage protein 1 (NRAMP1) gene and susceptibility to cutaneous leishmaniasis (CL) has been demonstrated worldwide; however, the reported results were inconsistent. This study aimed to determine the association of NRAMP1 variants with susceptibility to CL infection and patients' response to treatment in Isfahan province of Iran. METHODS: Peripheral blood samples were collected from 150 patients with CL and 136 healthy controls. The CL patients were treated with intralesional injection of meglumine antimoniate. The polymorphic variants at NRAMP1 (A318V and D543N) were analyzed using PCR-RFLP. The chi-square test and Fisher's exact test were used to compare frequencies of alleles and genotypes of polymorphisms between patient and healthy control populations. RESULTS: There was a statistically significant difference in the D543N (rs17235409) polymorphism between the CL patients and healthy controls (p=0.008). However, no significant association was detected for A318V (rs201565523) polymorphism between groups (p=0.26). In addition, there was a lack of association between D543N and A318V genotypes with response to treatment (p=0.54 and p=0.31, respectively). INTERPRETATION & CONCLUSION: The results indicated that genetic variations of D543N (rs17235409) might be associated with susceptibility to CL infection. These data may be used for detection of sensitive individuals and prevention of CL in endemic areas.

Approaches for the Treatment of SARS-CoV-2 Infection: A Pharmacologic View and Literature Review.

The emergence of a novel Coronavirus disease (COVID-19) inducing acute respiratory distress syndrome (ARDS) was identified in Hubei province of China in December 2019 and rapidly spread worldwide as pandemic and became a public health concern. COVID-19 disease is caused by a new virus known as SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), which has recently offered many challenges and efforts to identify effective drugs for its prevention and treatment. Currently, there is no proven effective approach and medication against this virus. Quickly expanding clinical trials and studies on Coronavirus disease 2019 increase our knowledge regarding SARS-CoV-2 virus and introduce several potential drugs targeting virus moiety or host cell elements. Overall, 3 stages were suggested for SARS-CoV-2 infection according to the disease severity, clinical manifestations, and treatment outcomes, including mild, moderate, and severe. This review aimed to classify and summarize several medications and potential therapies according to the disease 3 stages; however, it is worth noting that no medication and therapy has been effective so far.

Global Sequence Analysis and Expression of Azurin Gene in Different Clinical Specimens of Burn Patients with Pseudomonas aeruginosa Infection.

AIM: The purpose of this study was to analyze the sequence of azurin gene in relation to its expression in Pseudomanas aeruginosa strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of azurin gene using globally reported sequences was intended. MATERIALS AND METHODS: Fifty-nine multidrug-resistant P. aeruginosa isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the azurin gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The azurin gene sequences were compared with the sequence data from other countries. The expression level of azurin gene in P. aeruginosa isolates with different azurin sequences from different clinical specimens was evaluated by real-time PCR. RESULTS AND CONCLUSION: About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the azurin gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between azurin gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about azurin genetic variation and significance in P. aeruginosa pathogenesis.

Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from Pseudomonas aeruginosa against different bacterial species. MATERIALS AND METHODS: The azurin gene was cloned in the pET21a vector. The pET21a-azurin construct was transformed into Escherichia coli BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t-test and spearman correlation. RESULTS: The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 µg/mL in 24 h. Vibrio cholerae and Campilobacter jejuni displayed the most sensitivity to rAzurin (27.5 and 55 µg/mL, respectively) and the highest resistance (220 µg/mL) was displayed by P. aeruginosa and E. coli. The MIC for other species was 110 µg/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 µg/mL for Salmonella enterica and V. cholerae, 300 µg/mL for Shigella sonnei, Shigella flexneri and P. aeruginosa and 440 µg/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative. CONCLUSION: The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.

The immunohistochemistry and toluidine blue roles for Helicobacter pylori detection in patients with gastritis.

BACKGROUND: Helicobacter pylori, which is associated with many upper gastrointestinal diseases, is found in half of the population of the world. Several special stains and immunohistochemistry stain for H. pylori are available. The need for and usefulness of immunohistochemical (IHC) technique has been debated for years. Toluidine blue is a simple stain for microbiological studies and is easily available in laboratories. Therefore, this study was conducted to compare hematoxylin and eosin (H&E), Giemsa and toluidine blue staining with immunehistochemistry for detection of H. pylori in patients with gastritis and also to correlate the results of these staining methods with pathological grading. METHODS: We reviewed 54 consecutive gastric biopsy specimens stained by H&E and Giemsa as well as by toluidine blue and immunohistochemistry stains for H. pylori. RESULTS: H. pylori was positively identified by IHC in 43 (79.63%) patients, while positive samples were found in 18 (33.33%), 24 (44.44%) and 33 (61.11%) patients using H&E, Giemsa and toluidine blue staining methods. Our results showed that classical histological staining methods are not sensitive enough to identify low numbers or coccoid forms of organism, while toluidine blue and immunohistochemistry play an important role in detection of H. pylori infection. CONCLUSION: Toluidine blue has been proved to be much more reliable than H&E and Giemsa in detection of H. pylori. In addition, in post treatment biopsies and in biopsies with unexplained chronic active gastritis without histological evidence of H. pylori should have immunohistochemistry done to detect possible low density or coccoid form of organisms.