Host and Pathogen-Directed Therapies against Microbial Infections Using Exosome- and Antimicrobial Peptide-derived Stem Cells with a Special look at Pulmonary Infections and Sepsis.

Microbial diseases are a great threat to global health and cause considerable mortality and extensive economic losses each year. The medications for treating this group of diseases (antibiotics, antiviral, antifungal drugs, etc.) directly attack the pathogenic agents by recognizing the target molecules. However, it is necessary to note that excessive use of any of these drugs can lead to an increase in microbial resistance and infectious diseases. New therapeutic methods have been studied recently using emerging drugs such as mesenchymal stem cell-derived exosomes (MSC-Exos) and antimicrobial peptides (AMPs), which act based on two completely different strategies against pathogens including Host-Directed Therapy (HDT) and Pathogen-Directed Therapy (PDT), respectively. In the PDT approach, AMPs interact directly with pathogens to interrupt their intrusion, survival, and proliferation. These drugs interact directly with the cell membrane or intracellular components of pathogens and cause the death of pathogens or inhibit their replication. The mechanism of action of MSC-Exos in HDT is based on immunomodulation and regulation, promotion of tissue regeneration, and reduced host toxicity. This review studies the potential of mesenchymal stem cell-derived exosomes/ATPs therapeutic properties against microbial infectious diseases especially pulmonary infections and sepsis.

Immune response induced by recombinant pres2/S-protein and a pres2-S-protein fused with a core 18-27 antigen fragment of hepatitis B virus compared to conventional HBV vaccine.

Although comprehensive vaccination is the cornerstone of public health programs to control hepatitis B virus (HBV) infections, 5% of people who receive the existing vaccine do not develop proper immunity against HBV. To overcome this challenge, researchers have tried using various protein fragments encoded by the virus genome to achieve better immunization rates. An important antigenic component of HBsAg called the preS2/S or M protein has also received much attention in this area. The gene sequences of preS2/S and Core18-27 peptide were extracted from the GenBank (NCBI). Final gene synthesis was conducted with pET28. Groups of BALB/c mice were immunized with 10 µg/ml of recombinant proteins and 1 µg/ml CPG7909 adjuvant. Serum levels of IF-γ, TNF-α, IL-2, IL-4, and IL-10 were measured by ELISA assay method on spleen cell cultures on day 45, and IgG1, IgG2a, and total IgG titers obtained from mice serum were quantified on days 14 and 45. Statistical analysis did not show any significant difference between the groups regarding IF-γ level. There were, however, significant differences in terms of IL-2 and IL-4 levels between the groups receiving preS2/S-C18-27 with and without adjuvant and the groups receiving both preS2/S and preS2/S-C18-27 (Plus Recomb-Plus Recomb: the group of mice that received both preS2/S and preS2/S-C18-27 simultaneously). The strongest total antibody production was induced by immunization with both recombinant proteins without CPG adjuvant. The groups that received both preS2/S and preS2/S-C18-27, whether with or without adjuvant, were significantly different from those that received the conventional vaccine considering most abundant interleukins. This difference suggested that higher levels of efficacy can be achieved by the use of multiple virus antigen fragments rather than using a single fragment.

Interaction Between SARS-CoV-2 and Pathogenic Bacteria.

The novel human coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which results in the coronavirus disease 2019 (COVID-19), has caused a serious threat to global public health. Therefore, many studies are performed on the causes and prevalence of this disease and the possible co-occurrence of the infection with other viral and bacterial pathogens is investigated. Respiratory infections predispose patients to co-infections and these lead to increased disease severity and mortality. Numerous types of antibiotics have been employed for the prevention and treatment of bacterial co-infection and secondary bacterial infections in patients with a SARS-CoV-2 infection. Although antibiotics do not directly affect SARS-CoV-2, viral respiratory infections often result in bacterial pneumonia. It is possible that some patients die from bacterial co-infection rather than virus itself. Therefore, bacterial co-infection and secondary bacterial infection are considered critical risk factors for the severity and mortality rates of COVID-19. In this review, we will summarize the bacterial co-infection and secondary bacterial infection in some featured respiratory viral infections, especially COVID-19.

In silico and experimental methods for designing a potent anticancer arazyme-herceptin fusion protein in HER2-positive breast cancer.

CONTEXT: Breast cancer is the most prevalent type of malignancies among women worldwide and is associated with serious physical and mental consequences. Current chemotherapies may lack successful outcomes; thus, the development of targeted recombinant immunotoxins is plausible. The predicted B cell and T cell epitopes of arazyme of the fusion protein are able to elicit immune response. The results of codon adaptation tool of herceptin-arazyme have improved from 0.4 to 1. The in silico immune simulation results showed significant response for immune cells. In conclusion, our findings show that the known multi-epitope fusion protein may activate humoral and cellular immune responses and maybe a possible candidate for breast cancer treatment. METHODS: In this study, the selected monoclonal antibody constituting herceptin and the bacterial metalloprotease, arazyme, was used with different peptide linkers to design a novel fusion protein to predict different B cell and T cell epitopes by the means of the relevant databases. Modeler 10.1 and I-TASSER online server were used to predict and validate the 3D structure and then docked to HER2-receptor using HADDOCK2.4 web server. The molecular dynamics (MD) simulations of the arazyme-linker-herceptin-HER2 complex were performed by GROMACS 2019.6 software. The sequence of arazyme-herceptin was optimized for the expression in prokaryotic host using online servers and cloned into pET-28a plasmid. The recombinant pET28a was transferred into the Escherichia coli BL21DE3. Expression and binding affinity of arazyme-herceptin and arazyme to human breast cancer cell lines (SK-BR-3/HER2 + and MDA-MB-468/HER2 -) were validated by the SDS-PAGE and cell­ELISA, respectively.

The role of serum circulating microbial toxins in severity and cytokine storm of COVID positive patients.

The emergence of Coronavirus disease 2019 (Covid-19) is a global problem nowadays, causing health difficulty with increasing mortality rates, which doesn't have a verified treatment. SARS-CoV-2 infection has various pathological and epidemiological characteristics, one of them is increased amounts of cytokine production, which in order activate an abnormal unrestricted response called "cytokine storm". This event contributes to severe acute respiratory distress syndrome (ARDS), which results in respiratory failure and pneumonia and is the great cause of death associated with Covid-19. Endotoxemia and the release of bacterial lipopolysaccharides (endotoxins) from the lumen into the bloodstream enhance proinflammatory cytokines. SARS-CoV-2 can straightly interplay with endotoxins via its S protein, leading to the extremely elevating release of cytokines and consequently increase the harshness of Covid-19. In this review, we will discuss the possible role of viral-bacterial interaction that occurs through the transfer of bacterial products such as lipopolysaccharide (LPS) from the intestine into the bloodstream, exacerbating the severity of Covid-19 and cytokine storms.

Comparison of Immune Response in Mice Immunized with Recombinant PreS2/S-C18-27 Protein Derived from Hepatitis B Virus with Commercial Vaccine.

Background & Objective: The vaccine available to prevent Hepatitis B virus disease is ineffective in 5% of people due to the use of HBsAg as a weak immunogenic factor. In the present study, PreS2/S fused to C18-27 peptide fragment as an effective antigen and is proposed as a promising vaccine candidate compared with the conventional vaccine prescribed in the vaccination program. Methods: After the synthesis of PreS2/S genes and C18-27 peptide fragment in pET28a, the recombinant protein was confirmed by Western blotting. The efficacy of the PreS2/S-C18-27 protein was compared with the conventional vaccine injected into five groups of rats. Finally, the cytokine level of IF-r, IL-2, IL-4, IL-10, TNF-a, IgG1, and IgG2a were measured using the ELISA method. Results: This study showed no significant difference between the recombinant vaccine group and PBS control group in the IF-r test, but there was a significant difference between groups testing IL-2 and IL-10. In addition, the group receiving the recombinant vaccine with CPG adjuvant at a dilution of 1/10 in the IgG total test on days 14 and 45 after the first injection showed a significant difference in comparison with other groups. Conclusion: This study showed no statistically significant difference between the recombinant protein vaccine group and the conventional vaccine group. The Th1- mediated immune responses obtained from recombinant proteins with and without CPG performed better than conventional vaccines, possibly due to the functional deficiency of the available vaccines.

Evaluation of Triple Fragment Vaccine HSPX (Rv2031c) + PPE44 (Rv2770c) + Mouse IgG1 (Fcγ2a) with Auxiliary Adjuncts IL-22 in Comparison with BCG Vaccine.

Background & Objective: Despite the vaccination with the BCG vaccine, tuberculosis (TB) remains one of the major health problems in the world. The aim of this study was to evaluate our newly designed vaccine using IL-22 as an adjuvant in comparison with the common BCG vaccine. Methods: The gene constructs were cloned into the expression vector of pET28a and then into the recombinant vector of PET28a - HSPX, and PPE44 was transformed into Escherichia coli BL21 (DE3). Finally, the immunogenicity of recombinant proteins with and without BCG and IL-22 in BALB/c mice was investigated. Results: The key cytokines INF-γ and TNF-α were elevated more greatly in BCG immunized group than in PHF immunized group. Immunization with PHF showed a significant increase in IL-4 levels versus the BCG group. Adding IL-22 to the vaccine formulations indicated a tiny increase in IL-4 levels compared to their related vaccine groups.Specific total IgG1 in the experimental groups showed an increase in comparison with control groups, but in the vaccinated groups, no significant differences were observed, and the presence of IL-22 in the vaccine formulations indicated a slight decrease compared with the related mere vaccine groups. Results of specific total IgG2a in the experimental groups revealed that only in the PHF group formulated with IL-22 a significant increase occurs compared with all other experimental groups. Conclusion: It seems that BCG, as the only licensed vaccine for TB infection, could be more potent than a recombinant vaccine in the induction of cellular and humoral immune responses.

Evaluating the Performance of PPE44, HSPX, ESAT-6 and CFP-10 Factors in Tuberculosis Subunit Vaccines.

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen causing long-term infection in humans that mainly attacks macrophages and can escape from the immune system with the various mechanisms. The only FDA-approved vaccine against M. tuberculosis (MTB) is Mycobacterium bovis bacillus Calmette-Guérin (BCG). The protection of this vaccine typically lasts 10-15 years. Due to the increasing number of people becoming ill with MTB each year worldwide, the need to develop a new effective treatment against the disease has been increased. During the past two decades, the research budget for TB vaccine has quadrupled to over half a billion dollars. Most of these research projects were based on amplifying and stimulating the response of T-cells and developing the subunit vaccines. Additionally, these studies have demonstrated that secretory and immunogenic proteins of MTB play a key role in the pathogenesis of the bacteria. Therefore, these proteins were used to develop the new subunit vaccines. In this review, based on the use of these proteins in the successful new subunit vaccines, the PPE44, HSPX, CFP-10 and ESAT-6 antigens were selected and the role of these antigens in designing and developing new subunit vaccines against TB and for the prevention of TB were investigated.

Targeting the EGFR in cancer cells by fusion protein consisting of arazyme and third loop of TGF-alpha: an in silico study.

The anticancer effects of arazyme, a bacterial metalloprotease, have been revealed in previous studies. Because of the overexpression of epidermal growth factor receptor (EGFR) in tumor cells, targeting this receptor is one of the approaches to cancer therapy. In the present study, we designed fusion protein by using a non-mitogenic binding sequence of TGFα, arazyme, and a suitable linker. The I-TASSER and Robetta web servers were employed to predict the territory structures of the Arazyme-linker-TGFαL3, and TGFαL3-linker-Arazyme. Then, models were validated by using PROCHECK, ERRAT, ProSA, and MolProbity web servers. After docking to EGFR, Arazyme-linker-TGFαL3 showed a higher binding affinity and was selected to be optimized through 100 ns Molecular dynamic (MD) simulation. Next, the stability of ligand-bound receptor was examined utilizing MD simulation for 100 ns. Furthermore, the binding free energy calculation and free energy decomposition were carried out employing MM-PBSA and MM-GBSA methods, respectively. The root mean square deviation and fluctuation (RMSD, RMSF), the radius of gyration, H-bond, and binding free energy analysis revealed the stability of the complex during simulation time. Finally, linear and conformational epitopes of B cells, as well as MHC class I and MHC class II were predicted by using different web servers. Meanwhile, the potential B cell and T cell epitopes were identified throughout arazyme protein sequence. Collectively, this study suggests a novel chimera protein candidate prevent cancer cells potentially by inducing an immune response and inhibiting cell proliferation. Communicated by Ramaswamy H. Sarma.

The impact of the gut microbiome on toxigenic bacteria.

Millions of symbiotic and pathogenic microorganisms known as microbiota colonize the host body. The microbiome plays an important role in human health and colonizes hundreds of different species of multicellular organisms so that they are introduced as the metaorganisms. Changes in the microbial population of the gut microbiome may cause resistance to pathogenic bacteria-induced infection. Understanding the principles of Host-Microbiota Interactions (HMIs) is important because it clarifies our insight towards the mechanisms of infections established in the host. Interactions between the host and the microbiota help answer the question of how a microorganism can contribute to the health or disease of the host. Microbiota can increase host resistance to colonization of pathogenic species. Studying the HMIs network can in several ways delineate the pathogenic mechanisms of pathogens and thereby help to increase useful and novel therapeutic pathways. For example, the potentially unique microbial effects that target the distinct host or interfere with the endogenous host interactions can be identified. In addition, the way mutations in essential proteins in the host and/or in the microbes can influence the interactions between them may be determined. Furthermore, HMIs help in identifying host cell regulatory modules.

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells.

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

The Association of Age, Sex, and RT-PCR Results with the Lymphocyte and Neutrophil Counts in SARS-CoV-2 Infection: A Cross-sectional Analysis of 1450 Iranian Patients with COVID-19.

Containment of pandemic infections mainly depends on prompt identification of carriers, achievable through strict surveillance and truthful diagnostic testing. Although molecular identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the gold standard method, its low sensitivity and long turnaround time are among major concerns. In this retrospective single-center study, we reviewed the results of the lymphocyte and neutrophil counts of 1450 Iranian patients with coronavirus disease 2019 (COVID-19) recruited at Baqiyatallah Hospital, Tehran, Iran. Of 1450 patients, 439 cases (30.3%) were polymerase chain reaction (PCR) negative; further emphasizing that getting negative molecular testing is not as reliable as a positive result. While the lymphocyte count in cases with less than 50 years old was 1.8×103/µL (1.2-2.5), it was 1.47×103/µL (0.84-2.16) in the older group (p<0.001). Also, men experienced lower lymphocytes as compared to women (1.53×103/µL vs 1.76×103/µL; p=0.002). Of particular interest, the lymphocyte count in the PCR-negative cases was 1.77×103/µL (0.98-2.45) which was significantly higher than its count in their positive counterparts (1.53×103/µL; p=0.004). Unlike lymphocytes, sex and PCR did not significantly affect the number of neutrophils. The odds ratio for neutrophilia in patients aged older than 50, either with a negative or a positive PCR, was 2.46 and 2.23, suggesting old age as the most significant associated factor. The number of lymphocytes along with increased neutrophil count may probably serve as simple, rapid, and economical biomarkers, and are seemingly appropriate items that should be taken into account in the identification of patients with COVID-19, especially those aged more than 50.

Effect of sub-lethal doses of nisin on Staphylococcus aureus toxin production and biofilm formation.

Staphylococcus aureus is one of the commonest food-borne pathogens that can cause gastroenteritis owing to having several enterotoxins. Also, biofilm formation can complicate infections caused by this microorganism. Nisin is a safe food bio preservative which is usually used as an agent to prevent pathogen growth; however, it is important to identify the exact impact of nisin on the growth of S. aureus and to determine the suitable concentration needed for elimination of this pathogen in food. In this study, after MIC determination of nisin against S. aureus ATCC 29213, this strain was treated with sub-MIC (1/2) of nisin (4 µg/ml) and transcript levels of toxin-encoding (hla, SEA, SEB, and SED) and biofilm-associated (fnb, ebpS, eno, and icaA) genes were determined using Quantitative Real-time PCR at 2, 8, and 24 h post exposure. All toxin genes were down-regulated following exposure to sub-MIC of nisin, whereas biofilm-associated genes were up-regulated. The expression levels of fnb and icaA in S. aureus were highest after 8 h (4.5-fold and 6.8-fold increase, respectively), while the expression levels of eno and ebpS genes were highest after 2 h (3.3 and 4.5-fold increase, respectively). According to these results, although transcriptional levels of toxin genes were reduced, sub-MIC concentrations of nisin could trigger the expression of biofilm-associated genes in S. aureus. This can further lead to bacteriocin tolerance such that even its higher concentrations cannot kill bacterial cells after exposure to sub-lethal doses. Therefore, it is pivotal to add appropriate concentrations of nisin to food products for preservation purposes.

Potent in vitro antitumor activity of B-subunit of Shiga toxin conjugated to the diphtheria toxin against breast cancer.

Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB). First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests. The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro. Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.

Design, Dimerization, and Recombinant Production of MCh-AMP1-Derived Peptide in Escherichia coli and Evaluation of Its Antifungal Activity and Cytotoxicity.

Fungal species resistant to current antifungal agents are considered as a serious threat to human health, the dilemma that has dragged attentions toward other sources of antifungals such as antimicrobial peptides (AMPs). In order to improve biological activity of a recently described antifungal peptide MCh-AMP1 from Matricaria chamomilla flowers, MCh-AMP1dimer (DiMCh-AMP1), containing 61 amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed and expressed in Escherichia coli, and its structure was analyzed using bioinformatics tools. DiMCh-AMP1 synthetic gene was cloned into pET-28a expression vector, which was then used to transform E. coli BL21 (DE3) strain. His-tag purification was achieved using metal-chelate affinity chromatography. Because there is no methionine residue in the DiMCh-AMP1 sequence, cyanogen bromide was successfully used to separate the target product from the tag. Reverse-phase high-performance liquid chromatography was used as the final step of purification. Results showed that recombinant peptide was produced in considerable amounts (0.9 mg/L) with improved antifungal activity toward both yeasts and molds compared to its monomeric counterpart. The minimum inhibition concentration and minimum fungicidal concentration values of DiMCh-AMP1 against Candida and Aspergillus species were reported in the range of 1.67-6.66 µM and 3.33-26.64 µM, respectively. Our results showed that while antifungal activity of dimerized peptide was improved considerably, its cytotoxicity was decreased, implying that DiMCh-AMP1 could be a potential candidate to design an effective antifungal agent against pathogenic yeasts and molds.

Genetic Diversity of ctxB Gene Among Classical O1 and El Tor Strains of Vibrio cholerae using High-Resolution Melting Curve Analysis.

BACKGROUND & OBJECTIVE: Vibrio cholerae is a natural inhabitant of the environment and causes severe diarrhea ailments (cholera) that affects thousands of people each year worldwide. The most important virulence factors of this pathogen are cholera toxin (cholera toxin CT) and Type IV pili (toxin co-regulated pili TCP), which are encoded within the genome of the filamentous bacteriophage CTXφ. In the present study, according to researchers' report on genotypic variations of cholera toxin, we evaluated the sequence of ctxB subunit obtained from 100 strains of patients infected with cholera in Iran. METHODS: The evaluation of genotype variations of cholera toxin was made by high-resolution melting curve analysis illustrating a single nucleotide change. Then, ctxB gene sequencing was performed. Through this analysis and the sequencing process, two standard samples were studied. RESULTS: Using serologic tests, all the strains analyzed in this study were identified to be in O1 serotype. However, there have been differences in sequences of ctxB as some were similar to V . cholerae O1 biovar El Tor str. N16961 while others were similar to the genotype of V . cholerae ATCC 14035. We did not observe any particular pattern within the process of mutation. CONCLUSION: The analysis of the new samples of ctxB showed that they were potentially different. It seems that these complicated species were affected by a new genetic exchange of El Tor and classic genotypes.

Clinical efficacy of convalescent plasma for treatment of COVID-19 infections: Results of a multicenter clinical study.

Since Dec. 2019 the new coronavirus (SARS-CoV-2) has infected millions and claimed life of several hundred thousand worldwide. However, so far no approved vaccine or drug therapy is available for treatment of virus infection. Convalescent plasma has been considered a potential modality for COVID-19 infection. One hundred eighty-nine COVID-19 positive patients including 115 patients in plasma therapy group and 74 patients in control group, registered in the hospitals with confirmed COVID-19 infection, entered this multi-center clinical study. Comparison of outcomes including all-cause mortality, total hospitalization days and patients' need for intubation between the two patient groups shows that total of 98 (98.2 %) of patients who received convalescent plasma were discharged from hospital which is substantially higher compared to 56 (78.7 %) patients in control group. Length of hospitalization days was significantly lower (9.54 days) in convalescent plasma group compared with that of control group (12.88 days). Only 8 patients (7%) in convalescent plasma group required intubation while that was 20 % in control group. This clinical study provides strong evidence to support the efficacy of convalescent plasma therapy in COVID-19 patients and recommends this treatment for management of these patients. Clinical efficacy, immediate availability and potential cost effectiveness could be considered as main advantages of convalescent plasma therapy.

Synergistic antimicrobial activity of melittin with clindamycin on the expression of encoding exfoliative toxin in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic human pathogens, with the ability to produce a series of virulence factors that contribute to the severity of infections. Exfoliative toxins (ETs) are one of the important virulence factors that participating in staphylococcal scalded skin syndrome. Melittin has different biological activities, comprising of antiviral, broad spectrum antibacterial, antiprotozoal, antifungal and anti-inflammatory effects. Twelve clinical isolates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were obtained from wound infection in the burn patients. The MIC plus three sub-inhibitory concentrations (I, II and III) of clindamycin and melittin were tested. Next, the synergistic effects of melittin and clindamycin were evaluated using the broth microdilution checkerboard assay. The detection of exfoliative toxin A and B genes were examined by PCR method. Then the effects of sub-MIC melittin on the expression levels of eta and etb were assessed by quantitative real-time PCR (qRT-PCR) assay. Melittin MIC values against MRSA and MSSA planktonic cells were 0.25-0.5 and 0.25-1 µg/ml, respectively. The clindamycin MIC values against MRSA and MSSA were between 0.5 and 8 µg/ml and 0.5-2 µg/ml, respectively. The results of the time-kill kinetics assay (3.5log10 and 3log10) against MSSA and MRSA planktonic cells were determined within 24 h using melittin. The mean expression of eta in MRSA and MSSA was significantly downregulated to approximately 3.5 and 4 fold, respectively. Moreover, the mean expression of etb in MRSA and MSSA were significantly downregulated to approximately 2.5 and 3 fold, respectively. Hemolytic assay showed that the extracted melittin indicates a strong hemolytic activity (HD50 = 2 µg/ml). Melittin at 0.5 µg/ml induced cell lysis and stimulated the formation of vesicles in S. aureus strains. Melittin could reduce the expression of eta and etb as encoding exfoliative toxin A and B genes. This component appears to be a good candidate for the treatment of MRSA and MSSA strains. So, melittin in combination with clindamycin can be classified as a complementary treatment of wound infections in burn patients.

Molecular characterization of Shigella species isolated from diarrheal patients in Tehran, Iran: phylogenetic typing and its association with virulence gene profiles and a novel description of Shigella invasion associated locus.

The present study aims to employ a multiplex PCR-based method for phylogenetic typing of Shigella and determine the frequency of several virulence genes among Shigella phylogenetic clades and species. Species identification, phylogenetic typing of 44 previously diagnosed Shigella isolates, and frequency of virulence genes and loci, virA, virB, virF, ipaBCD, ial, sen, and set1A were investigated through performing several PCR assays. Distribution of virulence genes among Shigella phylogenetic clades and species was determined by the statistical analysis. The identities of 40 isolates out of 44 were confirmed as Shigella, and these isolates were classified in four phylogenetic clades, S1 (7.5%), S2 (52.5%), S3 (20%), and S5 (20%) and 4 species, S. sonnei (52.5%), S. flexneri (22.5%), S. dysenteriae (20%), and S. boydii (5%). The prevalence of virA, virB, virF, ipaBCD, ial, sen, and set1A was determined as 67.5%, 72.5%, 72.5%, 65%, 75%, 40%, and 5%, respectively. The presence of sen, uidA, or set1A was found to be statistically correlated with either of Shigella phylogenetic clades or species. A significant statistically association was also determined between set1A and Shigella phylogenetic clades. Furthermore, the nucleotide sequence of invasion-associated locus (ial) was determined and mapped on Shigella genome through in silico analysis. The current study shows the distribution of Shigella isolates and its key virulence genes within the five recently described phylogenetic clades for the first time in the Asia. This is also the first description of ial nucleotide sequence and its exact location on Shigella genome after its initial identification.