Immunogenicity of HspX/EsxS fusion protein of Mycobacterium tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants after subcutaneous administration in animal model.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the most common and dangerous infectious diseases in the world. Despite vaccination with BCG, it is still considered as a major health problem. Therefore, design and production of an effective novel vaccine against TB is necessary. Our aim was to evaluate immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX, PLUSCOM nano-adjuvants and MPLA through the subcutaneous route in mice model. METHODS: HspX/EsxS fused protein of M. tuberculosis was cloned, expressed and purified in the prokaryotic system. ISCOMATRIX and PLUSCOM nano-adjuvants were prepared by film hydration method. Subcutaneous immunization of BALB/c mice was performed by different formulations. IFN-γ, IL-4, IL-17 and TGF-ß cytokines levels as well as serum IgG1, IgG2a. RESULTS: Our results showed that subcutaneous administration of mice with HspX/EsxS along with three adjuvants, ISCOMATRIX, PLUSCOM and MPLA increased immunogenicity of multi-stage fusion protein of M. tuberculosis. Additionally, HspX/EsxS protein + ISCOMATRIX or + PLUSCOM nano-adjuvants induced stronger Th1, IgG2a and IgG1 immune responses compared to MPLA adjuvant. Totally, HspX/EsxS/ISCOMATRIX/MPLA, HspX/EsxS/PLUSCOM/MPLA and two BCG booster groups could significantly induce higher Th1 and IgG2a immune responses. CONCLUSION: With regard to ability of ISCOMATRIX, PLUSCOM and MPLA adjuvants to increase immunogenicity of HspX/EsxS protein through induction of IFN-γ and IgG2a immune responses, it seems that these adjuvants and especially ISCOMATRIX and PLUSCOM, could also improve BCG efficacy as a BCG booster.

A novel antigen of Mycobacterium tuberculosis and MPLA adjuvant co-entrapped into PLGA:DDA hybrid nanoparticles stimulates mucosal and systemic immunity.

BACKGROUND: Due to initiation of Mycobacterium tuberculosis infection via the mucosal tissue of the respiratory tract, intranasal administration of new tuberculosis vaccines is highly regarded to enhance mucosal immunity. Our outline was evaluation of mucosal and systemic immune responses in BALB/c mice after nasal delivery of HspX/EsxS fused antigen of Mycobacterium tuberculosis along with MPLA adjuvant entrapped in PLGA:DDA hybrid nanoparticles. METHODS: In this study, the double emulsion solvent evaporation method (w/o/w) was used to prepare different nanoparticle formulations containing HspX/EsxS protein and MPLA. Three weeks after the last nasal immunization of BALB/c mice, IgA antibody levels in nasal lavage and IFN-γ, IL-4, IL-17 and TGF-ß cytokines in supernatant of cultured splenocytes and also serum IgG1 and IgG2a titers were evaluated using ELISA method. RESULTS: Our results indicated that nasal vaccination with PLGA:DDA nanoparticles loaded with HspX/EsxS protein±MPLA, both with and without a prime dose of BCG could provide efficient Th1, Th17, IgA, IgG1 and IgG2a immune responses. CONCLUSION: These findings demonstrate that both PLGA:DDA hybrid nanoparticles as carrier/adjuvant and MPLA as adjuvant, could efficiently induce mucosal and systemic immune responses against HspX/EsxS antigen, alone or as a booster for BCG.

Are chitosan natural polymers suitable as adjuvant/delivery system for anti-tuberculosis vaccines?

Today, the effectiveness of the only approved tuberculosis (TB) vaccine, bacillus Calmette-Guerin (BCG), has encountered several serious problem in the control of TB infections including variable protection in adolescents and adults, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) as well as HIV/AIDS co-infection. Various studies have shown that chitosan, a natural polymer, can serve as a potent carrier for the delivery of various hydrophilic molecules such as peptide, protein and drug agents due to some of its excellent characteristics including low toxicity, biodegradable and biocompatible properties and stability. Currently, these polysaccharide polymers have gained more attention as candidates for the adjuvant/delivery of anti-TB vaccines due to better cellular uptake, muco-adhesive characteristics, prolonged control release, persistent stimulation of the immune system, more efficient uptake by antigen processing cells (APCs), adjuvant/immunopotentiator function, and preventing antigen degradation in-vivo. The present study showed that the new generation of TB vaccine candidates when used in combination with chitosan and its derivatives as adjuvant or delivery system, could potently induce both protective and cell-mediated (CD4 and CD8) immune responses in animal models. In addition, they could also enhance protection against Mtb infection in TB-challenged mice and act as booster-vaccines to improve BCG-primed immunity and excellent prime-vaccines. The results of this study showed that parenteral and non-parenteral immunization of chitosan-based TB vaccines can induce appropriate immune responses; however, we suggest that based on some advantages of chitosan polymers and mucosal delivery route, non-parenteral immunization may be a better administration route for chitosan-based TB vaccines.

Bacterial Co-infections in HIV/AIDS-positive Subjects: A Systematic Review and Meta-analysis.

BACKGROUND: Bacterial infections are the most common complications in people with HIV/AIDS. There has been no previous report on the prevalence of bacterial co-infections in Iranian HIV/AIDS-positive subjects. AIM: To evaluate the frequency of bacterial infections in hospitalized HIV/AIDS-infected patients in Iran. MATERIALS AND METHODS: Based on PRISMA guidelines, a computerized search in related data banks using relevant keywords was performed in both Persian and English languages for articles that were published until March 10, 2017. A total of 1118 original articles were systematically reviewed to identify eligible studies on the prevalence of bacterial co-infections in HIV/AIDS-infected patients from Iran. After screening for inclusion and exclusion criteria, we extracted data from 28 eligible articles for the meta-analysis. RESULTS: The overall bacterial infection rate among Iranian HIV/AIDS-positive individuals was estimated to be 48.6%. Gastrointestinal disorders (59.5%) were the most frequent bacterial infections in this group of patients followed by bacterial lymphadenopathy (38.9%), TB infection (38.2%), bacterial pneumonia (31.2%), brucellosis (26.3%), skin infections (13.3%) and sexually transmitted infections (9.7%). The prevalence of other bacterial infections including endocarditis, sepsis and Staphylococcus aureus (S. aureus) were 10%, 9.1%, and 6.9%, respectively. CONCLUSION: The prevalence of a wide spectrum of bacterial co-infections, especially endemic infections, in Iranian HIV/AIDS-infected patients, is alarming and calls for urgent need to improve the currently applied diagnostic and preventive methods. In addition, timely treatment of these infections is pivotal to decrease the morbidity and mortality rates in HIV/AIDS-infected patients.

High prevalence of multidrug-resistant Pseudomonas aeruginosa carrying integron and exoA, exoS, and exoU genes isolated from burn patients in Ahvaz, southwest Iran: A retrospective study.

Background: Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. This study evaluated the relative frequency of exoenzymes (exo) A, U and S genes and integron classes (I, II, and III) among multi-drug-resistant clinical P. aeruginosa isolates from burn patients in Ahvaz, southwest of Iran. Methods: In this cross-sectional study P. aeruginosa isolates were recovered from 355 wound samples. The antimicrobial susceptibility test was done by disk agar diffusion method on Muller-Hinton agar according to the Clinical and Laboratory Standards Institute. MDR isolates were defined if they showed simultaneous resistance to 3 antibiotics. Extensively drug-resistant was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories. The presence of class I, II, and III integrons and virulence genes was determined using a PCR assay on extracted DNA. Results: Overall, 145 clinical P. aeruginosa isolates were confirmed with biochemical and PCR tests. Overall, 35% (52/145) of the isolates were taken from males and 64% (93/145) from female hospitalized burn patients. The highest resistance rates of P. aeruginosa isolates to antibiotics were related to piperacillin 59% (n = 86/145) and piperacillin-tazobactam 57% (n = 83/145). A total of 100% of isolates were resistant to at least one antibiotic. MDR and XDR P. aeruginosa had a frequency of 60% and 29%, respectively. The prevalence of integron classes I, II, and III in P. aeruginosa was 60%, 7.58%, and 3.44%, respectively. IntI was more common in MDR and XDR P. aeruginosa isolates. In addition, 70(48%) of P. aeruginosa isolates did not harbor integron genes. Besides, exoA, exoS, and exoU in P. aeruginosa had a frequency of 55%, 55%, and 56%, respectively. Conclusion: It was found that P. aeruginosa as a potent pathogen with strong virulence factors and high antibiotic resistance in the health community can cause refractory diseases in burn patients.

Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Objectives: Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model. Materials and Methods: In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-ß concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined. Results: According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens. Conclusion: The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

Assessing the Potential Biological Activities of Postbiotics Derived from Saccharomyces cerevisiae: An In Vitro Study.

A new biotherapeutic strategy involves the use of microbial bioactive substances (postbiotics) that exhibit optimum compatibility and intimate contact with the immune system of the host. This study was aimed at investigating the potential biological activities of postbiotics derived from Saccharomyces cerevisiae (PTCC 5269) (PSC) under in vitro circumstances. Based on the outcomes, the synthesized PSC possessing a high level of phenolic (102.46 ± 0.25 mg GAE/g) and flavonoid (19.87 ± 75.32 mg QE/g) content demonstrated significant radical scavenging activity (87.34 ± 0.56%); antibacterial action towards Listeria monocytogenes, Streptococcus mutans, Salmonella typhi, and Escherichia coli (in order of effectiveness) in both in vitro and food models (whole milk and ground meat); probiotics' growth-promoting activity in the fermentation medium; α-glucosidase enzyme-inhibiting and cholesterol-lowering properties in a concentration- and pH-dependent manner; reduction in the cell viability (with the significant IC50 values of 34.27 and 23.58 µg/mL after 24 and 48 h, respectively); suppressed the initial (G0/G1) phase of the cell's division; induced apoptosis; and increased the expression of PTEN gene, while the IkB, RelA, and Bcl-XL genes indicated diminished expression in treated SW480 cancer cells. These multiple health-promoting functions of PSC can be extended to medical, biomedical, and food scopes, as novel biotherapeutic approaches, in order to design efficient and optimized functional food formulations or/and supplementary medications to use as adjuvant agents for preventing or/and treating chronic/acute disorders.

Evaluating Inhibitory Effects of Paclitaxel and Vitamin D3 Loaded Poly Lactic Glycolic Acid Co-Delivery Nanoparticles on the Breast Cancer Cell Line.

Purpose: Paclitaxel (PTX) has transpired as a significant agent in the treatment of breast cancer. Meanwhile, polylactic glycolic acid (PLGA) nanoparticles (NPs) are able to increase the anticancer effect of the PTX in the blood. Methods: Nano-precipitation was used to prepare the PLGA-PTX-VitD3 co-delivery NPs. Drug loading, encapsulation efficiency, in vitro release profile, cell viability, migration, apoptosis, and bcl2 expression of NPs were evaluated. Results: The average size of co-delivery NPs was 231 ± 46 nm. Observed was a controlled release of the PTX and vitamin D3 from co-delivery NPs between 0.5 and 240 hours. MTT showed the ability of 8 µg.mL-1 of co-delivery NPs to kill 50 % of the MCF-7; likewise, the co-delivery NPs prevented MCF-7 migration. The co-delivery NPs led 46.35 % MCF-7 to enter primary apoptosis. 60.8% of MCF-7 in the control group were able to enter the G (1) phase of the cell cycle. The co-delivery NPs increased expression of bax. In addition to its higher toxicity against MCF-7 than that of PTX, co-delivery NPs were able to release drugs continuously for a long period, which indeed increased the efficiency of the drugs. Conclusion: The effect of co-delivery NPs on MCF-7 cell viability was different from that in other drugs. In fact, the co-deliver NPs were able to release drugs continuously for a long time, this could induce primary apoptosis in the MCF-7 and decrease the metastasis and toxicity of drugs.

Formulation and Optimization of a New Cationic Lipid-Modified PLGA Nanoparticle as Delivery System for Mycobacterium tuberculosis HspX/EsxS Fusion Protein: An Experimental Design.

Polymeric particles and liposomes are efficient tools to overcome the low immunogenicity of subunit vaccines. The aim of the present study was formulation and optimization of a new cationic lipid-modified PLGA nanoparticles (NPs) as a delivery system for Mycobacterium tuberculosis HspX/EsxS fusion protein. The cationic lipid-modified PLGA NPs containing HspX/EsxS fusion protein were prepared using a modified double emulsion solvent evaporation method. Scanning electron microscopy and dynamic light scattering (DLS) tools were used to determine physical properties of hybrid NPs. A multi-level full factorial design was used to evaluate the influence of two factors of PLGA:DDA weight ratio (w/w) and PVA concentration (%) on size, surface charge, polydispersity index, encapsulation efficiency and yield. Finally, the optimal formulation was achieved based on desired responses. Mathematical models were obtained to indicate the relation between the studied factors and responses. The DDA concentration showed an increasing effect on surface charge and also a decreasing effect on particle size, encapsulation efficiency and yield. Higher amounts of DDA increased surface charge of NPs; however, the size, encapsulation efficiency and yield were decreased. The influence of various concentrations of PVA on different physical characteristics of PLGA:DDA hybrid NPs was variable. The optimal formulation consisted of 0.91 (55:5, w/w) weight ratio of PLGA:DDA and 0.5% PVA. The hybrid NPs showed acceptable particle size distribution, strong positive surface charge, prolonged antigen release and good encapsulation efficiency in comparison to PLGA alone. However, further preclinical and clinical studies are needed.

The roles of latency-associated antigens in tuberculosis vaccines.

Tuberculosis (TB) is a re-emerging disease and is caused by Mycobacterium tuberculosis (M. tuberculosis). TB is currently one of the leading causes of morbidity and mortality, worldwide. The only available vaccine against TB infection, Bacillus Calmette-Guérin (BCG), fails to adequately protect against reactivation of latent infections in adults. Furthermore, recently developed subunit vaccines, which are in various stages of clinical trials, are all prophylactic vaccines based on proteins expressed in replicating stage of M. tuberculosis and they are not preventive of reactivation of latent TB infection. Thus, an appropriate subunit post-exposure vaccine needs to be developed to control all forms of TB infection. To produce a multi-stage subunit vaccine, scientists should combine the early secreted M. tuberculosis antigens with latency antigens. For this purpose, some latency proteins are known which could be important antigens in the production of specific humoral and cellular immune responses in latent M. tuberculosis infected individuals. Several studies have evaluated the immunogenicity of these proteins in improving the TB vaccines. The present study is a comprehensive review of several studies on the role of the latency antigens in the development of TB vaccines. Overall, the studies indicate that the latency-associated antigens including the resuscitation-promoting factors, the Dormancy of survival regulon (DosR) proteins and the starvation stimulant proteins are potential candidates for the development of subunit vaccines against TB infection.

Prevalence of Antibiotic Resistance of Haemophilus Influenzae in Iran- A Meta-Analysis.

INTRODUCTION: Based on the World Health Organization reports, infections caused by ampicillin-resistant Haemophilus influenzae strains are a major threat to public health and need urgent therapy with new antibiotics. Given the lack of a previous comprehensive study on the prevalence of the antibiotic resistance of H. influenzae in Iran, this systematic review and meta-analysis was performed to increase the knowledge about antibiotic resistance status of this pathogenic agent. MATERIALS AND METHODS: For the purpose of the study, the articles related to the subject of interest and published up to August 2018 were searched in several English and Persian databases, including PubMed, Scopus, Web of Science, Scientific Information Database, and Magiran. The search process was accomplished using the following keywords: "Antibiotic resistance", "H. influenzae", and "Iran". The data were pooled from 13 eligible studies reporting the prevalence of antibiotic resistance of H. influenzae in Iran. RESULTS: The prevalence of H. influenzae resistance to various antibiotics in Iran, including ampicillin, amoxicillin, cephalexin, cefixime, ceftazidime, cefotaxime, and ceftizoxime, were obtained as 54.8%, 66.6%, 28.6%, 62%, 21.3%, 22.3%, 23.2%, respectively. These rates were reported as 27.7%, 46.7%, 53%, 82.6%, 40.3%, 30.8% for chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole, penicillin, erythromycin, and ciprofloxacin, respectively. Additionally, ceftriaxone, gentamicin, amikacin, kanamycin, rifampin, azithromycin, and clindamycin had the H. influenzae resistance rates of 33.1%, 40.2%, 45.8%, 44.4%, 18.5%, 17.4%, and 71.3%, respectively. CONCLUSION: The majority of the antibiotics tested in Iran showed a high rate of resistance to H. influenzae. This may cause serious problems in the treatment of infections in the future. Therefore, precautionary measures, such as monitoring antibiotic prescription and resistance and using the new classes of antibiotics, are necessary.

Potential of polymeric particles as future vaccine delivery systems/adjuvants for parenteral and non-parenteral immunization against tuberculosis: A systematic review.

OBJECTIVES: Production of effective tuberculosis (TB) vaccine is necessity. However, the development of new subunit vaccines is faced with concerns about their weak immunogenicity. To overcome such problems, polymers-based vaccine delivery systems have been proposed to be used via various routes. The purpose of this study was to determine the potential of polymeric particles as future vaccine delivery systems/adjuvants for parenteral and non-parenteral immunization against TB. MATERIALS AND METHODS: PubMed, Scopus, Science-Direct, and the ISI web of knowledge databases were searched for related keywords. A total of 420 articles, written up to June 25, 2016, were collected on the potential of polymeric particles as TB vaccine delivery systems after parenteral and non-parenteral immunization. Thirty-one relevant articles were selected by applying inclusion and exclusion criteria. RESULTS: It was shown that the immunogenicity of TB vaccines had been improved by using biodegradable and non-biodegradable synthetic polymers as well as natural polymers and they are better able to enhance the humoral and cellular immune responses, compared to TB vaccines alone. The present study revealed that various polymeric particles, after M. tuberculosis challenge in animal models, provide long-lasting protection against TB. PLGA (poly (lactide-co-glycolide)) and chitosan polymers were widely used as TB vaccine delivery systems/adjuvants. CONCLUSION: It seems that PLGA and chitosan polymers are well-suited particles for the parenteral and non-parenteral administration of TB vaccines, respectively. Non-biodegradable synthetic polymers in comparison with biodegradable synthetic and natural polymers have been used less frequently. Therefore, further study on this category of polymers is required.

Multi-stage subunit vaccines against Mycobacterium tuberculosis: an alternative to the BCG vaccine or a BCG-prime boost?

INTRODUCTION: More than two billion people are latently infected with Mycobacterium tuberculosis. Most tuberculosis (TB)-subunit vaccines currently in various stages of clinical trials are designed for prevention of active TB, but not to prevent reactivation of latent TB-infection. Thus, there is an urgent need for an effective multi-stage vaccine based on early-expressed and latently-expressed antigens that prevents both acute and latent infections. AREAS COVERED: Here, we reviewed the published pre-clinical and clinical studies of multi-stage subunit vaccines against TB, and the protective capacities of the vaccines were compared with BCG, either alone or in combination with different vaccine delivery systems/adjuvants. The results revealed that multi-stage subunit vaccines induced a wide variety of immune-responses to all forms of TB, including CD8 + T-cell-mediated cytolytic and IFN-γ responses comparable to those induced by the BCG. They could potentially be used as a booster vaccine to improve the efficacy of the BCG. EXPERT COMMENTARY: Multi-stage TB-vaccines could boost BCG-primed immunity, decrease bacterial loads and provide efficient protection against progressive TB-infection, especially in the latent phase. These types of vaccines administered before and after TB-infection can act as pre-exposure, post-exposure and even therapeutic vaccines. In the near future, these vaccines could provide a new generation of prime-vaccines or BCG prime-boosters.

Antibiotic Resistance of Helicobacter pylori in Iranian Children: A Systematic Review and Meta-Analysis.

Antibiotic therapy for children infected with Helicobacter pylori is important. However, resistance to antibiotics is one of the main causes of treatment failure. This study was designed to evaluate the prevalence pattern of antibiotic resistance of H. pylori in Iranian children using a systematic review and meta-analysis of literature. A computerized search (until June 10, 2017) using related keywords in the national and international databases was performed. A total of 261 original articles on antibiotic resistance of H. pylori in Iranian children were collected. After screening for inclusion and exclusion criteria, six eligible articles were included in the meta-analysis. Resistance rates of H. pylori to different antibiotics were as follows: metronidazole: 71%, clarithromycin: 12.2%, amoxicillin: 20.4%, tetracycline: 8.4%, ampicillin: 21.4%, rifampin: 28.6%, furazolidone: 8.4%, ciprofloxacin: 16.2%, azithromycin: 19%, erythromycin: 15.3%, and nitrofurantoin: 0%. The prevalence of H. pylori resistance to metronidazole, amoxicillin, ampicillin, and rifampin among Iranian children was high. Therefore, a careful monitoring of antibiotic resistance to select the best treatment options and prevent treatment failure is required. Although resistance to some antibiotics such as clarithromycin, tetracycline, furazolidone, and ciprofloxacin was less prevalent, frequent consumption of these drugs in children should be controlled owing to their known adverse events.

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

BACKGROUND: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. METHODS: An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. RESULTS: The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. CONCLUSION: An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran.

Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. The most important of these factors are exotoxin A and type III secretion system (T3SS). The aim of this study was to determine the frequency of toxA, exoU and exoS genes among clinical isolates of P. aeruginosa. In this cross-sectional study from September 2011 to February 2012, 156 P. aeruginosa isolates were recovered from different clinical samples. Susceptibility testing against 10 antibiotics was performed on individual isolates by the disc diffusion method according to CLSI guidelines. Extracted DNA was subjected to PCR assay for determining the presence of toxA, exoU and exoS genes. Overall, the frequency of toxA, exoU and exoS genes were 90.4%, 66.7% and 65.4%, respectively. All of the abdominal and eye isolates were exoS (+). The frequency of exoS (+)/exoU (-) and exoS (-)/exoU (+) genotypes was estimated 19.2% and 16.2%, respectively. Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively. The highest and lowest antibiotic resistance rate was seen against azteroenam (94.2%) and amikacin (44.9%), respectively. Fluoroqinolone-resistant isolates were isolated with frequency of 45.8%. Multi-drug resistant (MDR) isolates were detected in 62.8% of isolates. The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates. The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.