Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

Investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens as a vaccine candidate against S. dysenteri and S. flexneri.

BACKGROUNDS: Shigella spp. causes bloody diarrhea and leads to death, especially in children. Chimeric proteins containing virulence factors can prevent Shigella infection. The purpose of this study is to investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens against shiga toxin, S. dysenteri and S. flexneri in vitro and in vivo conditions. METHODS: Recombinant vector was transferred to E. coli BL21. The expression of the chimeric protein was confirmed by SDS PAGE and purified using the Ni-NTA column. Mice were immunized with recombinant protein and antibody titer was evaluated by ELISA. 10, 25 and 50 LD50 of Shiga toxin neutralization was evaluated in vitro (Vero cell line) and in vivo conditions. Also, the challenge of immunized mice with 10, 25 and 50 LD50 of S. dysentery and S. flexneri was done. RESULTS: The expression and purification of the recombinant protein with 60.6 kDa was done. ELISA showed increased antibody titer against the chimeric protein. MTT assay indicated that 1/8000 dilution of the sera had a 51% of cell viability against the toxin in Vero cell line. The challenge of mice immunized with toxin showed that the mice had complete protection against 10 and 25 LD50 of toxin and had 40% survival against 50 LD50. Mice receiving 10 and 25 LD50 of S. dysenteri and S. flexneri had 100% protection and in 50 LD50 the survival rate was 60 and 50%, respectively. Organ burden showed that the amount of bacterial colonization in immunized mice was 1 × 104 CFU/mL, which was significantly different from the control group. CONCLUSION: This study showed that chimeric proteins can create favorable immunogenicity in the host as vaccine candidates.

Quinolone resistance and biofilm formation capability of uropathogenic Escherichia coli isolates from an Iranian inpatients' population.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major pathogen of the urinary tract infection (UTI), and biofilm formation is crucial as it facilitates the colonization in the urinary tract. We aimed to investigate the antibiotic susceptibility pattern, biofilm formation capability, distribution of quinolone resistance genes, and phylogenetic groups among UPEC isolates from an Iranian inpatients' community. METHODS AND RESULTS: A collection of 126 UPEC obtained from hospitalized patients with symptomatic UTI at 3 teaching hospitals during 2016 were included. Antibiogram of all isolates against quinolone and fluoroquinolones was performed using the disk diffusion method. Phylogenetic groups and qnr A, B, and S genes were assessed by PCR. Susceptibility pattern showed that more than 50% and 81% of the isolates were resistant to fluoroquinolones and quinolones, correspondingly. The frequency of qnrS and qnrB genes was 22% and 13.5%, correspondingly. Our result indicated no significant association between the presence of fluoroquinolone genes and antibiotic resistance to them. The frequent common phylogroup was B2 (84.1%), followed by D (10.3%), A (3.2%) and B1 (2.4%) groups. Indeed, 80.2% of the isolates were biofilm producers, so that 42.1%, 16.7% and 21.4% of them were classified as weak, moderate and strong producers, respectively. CONCLUSIONS: Our results showed considerable fluoroquinolone and quinolone resistance among UPEC along with a remarkable rate of biofilm-producing isolates from symptomatic hospitalized patients, making them a serious health concern in the region. This survey highlights the need for awareness on quinolone resistance and careful prescription of them by physicians.

The highly conserved domain of RND multidrug efflux pumps in pathogenic Gram-negative bacteria.

RND (Resistance-Nodulation-Division) family transporters have a vital role in both intrinsic and acquired multi-drug resistance in Gram-negative bacteria. It is important to find a conserved domain in the RND family between different pathogenic bacteria for diagnostic and therapeutic purpose. Total sequences of three-component system RND efflux pumps were retrieved from NCBI nucleotide and protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. The phylogenetic tree for main transporters was drawn and the three-dimensional structure was also evaluated. From the sequence conservation analysis, highly conserved residues with 282 base pair (94 amino acid) long were identified. The location of the highly conserved domain is positioned in the domain 1 crystallographic structure of AcrB Escherichia coli and MexB Pseudomonas aeruginosa. The main transporter component phylogenetic tree shows the clusters of different genotypes and their evolutionary association.  Each of three components of RND proteins is crucial for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. Therefore, this highly conserved region can be used to disable the RND multidrug efflux pumps. In addition, this highly conserved can also be used for diagnostic aspects.

Antimicrobial and antibiofilm activity of silver, titanium dioxide and iron nano particles.

PURPOSE: To compare the antimicrobial effects of chlorhexidine, penicillin, erythromycin, clindamycin, tetracycline and vancomycin with silver, titanium dioxide and iron nanoparticles and also to consider the synergistic antibacterial and antibiofilm effects of nanoparticles in clinical and standard strains of Streptococcus mutans and Streptococcus sanguinis. METHODS: The specimens collected from 66 3-5 year-old children with detected S. mutans and S. sanguinis by PCR were then exposed to the antimicrobial activity of chlorhexidine, penicillin, erythromycin, clindamycin, tetracycline and vancomycin with silver, titanium dioxide and iron nanoparticles measured by microdilution and disc diffusion tests and the colony counted after 1 to 5 minutes. The antibiofilm activity was examined by microtiter test. RESULTS: Use of nanoparticles alone showed higher minimum inhibitory concentration (MIC) than using them synergistically. The most effective synergistic solution was the one containing TiO2, Ag and Fe3O4 showing 0.019 µg#47;ml in S. mutans and S. sanguinis. Furthermore, this solution had the lowest biofilm inhibitory concentration (BIC) and colony forming units than the other antibiotics and chlorhexidine. CLINICAL SIGNIFICANCE: The solution containing TiO2, Ag and Fe33O4 showed the lowest inhibitory and antibiofilm concentration against S. mutans and S. sanguinis compared to those of other nanoparticle containing solutions, antibiotics and chlorhexidine, thus it may be used for treating dental caries, dental plaque and oral infections.

An update on alternative therapy for Escherichia coli causing urinary tract infections; a narrative review.

BACKGROUND: Urinary tract infections (UTIs) are the most common type of nosocomial infection and severe health issues because of the difficulties and frequent recurrence. Today, alternative methods such as sonodynamic therapy (SDT), photodynamic therapy (PDT) and herbal materials use for treating infections like UTI in many countries. METHOD: We conducted searches of the biomedical databases (Google Scholar, Scopus, PubMed, and Web of sciences) to identify related studies from 2008 to 2023. RESULT: SDT aims to use ultrasound to activate a sonosensitizer, which causes a biological effect by raising reactive oxygen species (ROS). When bacteria are exposed to ROS, several important effects occur: oxidative damage, DNA damage, protein dysfunction etc. SDT with herbal medicine significantly reduced the number of colony-forming units and bactericidal activity for Klebsiella pneumonia and E. coli. PDT is a promising treatment for cancer and microbial infections, combining a photosensitiser, light and tissue molecular oxygen. It involves a photosensitizer, light source, and oxygen, with variations affecting microbial binding and bactericidal activity. Factors affecting antibacterial properties include plant type, growing conditions, harvesting, and processing. This review highlights the recent advancements in sonodynamic, photodynamic, herbal, and bio-material-based approaches in the treatment of E. coli infections. CONCLUSIONS: These alternative therapies offer exciting prospects for addressing UTIs, especially in cases where traditional antibiotic treatments may be less effective. Further research and clinical studies are warranted to fully explore the potential of these innovative treatment modalities in combating UTIs and improving patient outcomes.

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo.

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 µg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 µmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 µg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 µg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Ralstoniapickettii bloodstream infection in the patient with Guillain-Barre syndrome under plasmapheresis.

Ralstonia pickettii is a rare Gram-negative opportunistic bacterium that causes rare infections such as bacteremia, neonatal sepsis, endocarditis, and meningitis in hospitalized or immunocompromised patients. In this study, we identified and reported bloodstream infection caused by R. pickettii in a 15 -year-old boy patient with an autoimmune disease, Guillain-Barré syndrome, under plasmapheresis and intravenous immune globulin (IVIG) therapy. He was referred for admission to the neurology center of the teaching hospital of Shiraz, Iran for inability to walk, and lower extremity muscle weakness. After he was treated with plasmapheresis once during hospitalization, and after severe fever besides shivering blood cultures using BACT/ALERT®3D instrument were positive for R. pickettii.According to antibiotic susceptibility test reports, Ciprofloxacin (5 µg) was prescribed. Fortunately, after starting antibiotic treatment, blood culture results reported no growth after 5 days. Indeed, the patient was infected with nosocomial hepatitis A and URSOBIL (300 mg/BID/Po) was administered. Hence, after reporting the infection occurrence to the hospital infection control unit, initial and possible measures such as device infection control, replacement of potentially polluted plasmapheresis fluids, disinfecting the environment and replacing old sterile washing water with new sources were carried out in plasmapheresis unit. In conclusion, R. pickettii is a rare nosocomial infection that is responsible for the contamination of medical equipment, especially in hemodialysis, plasmapheresis devices and sterile solutions. Also, it is suggested that the role and importance of rare environmental bacteria as the causative agents of human infections should not be ignored in medical centers.

Molecular characterization of superbugs K. pneumoniae harboring extended-spectrum ß-lactamase (ESBL) and carbapenemase resistance genes among hospitalized patients in southwestern Iran, Western Asia.

Background: Detection of K. pneumoniae superbugs carrying Extended-spectrum ß-lactamase (ESBL) and Carbapenemase resistance genes among hospitalized patients is crucial for infection control and prevention. The aim of this molecular study was to investigate the spread of ESBL and Carbapenemase-producing K. pneumoniae in two hospitals located in Southwest Iran. Methods: One hundred clinical isolates of K. pneumoniae were randomly collected from two hospitals over a period of five months, from November 2023. The isolates were confirmed using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae isolates that exhibited resistance to at least one of the three indicator cephalosporins or carbapenems were selected for evaluation of ESBL and carbapenemase production. This was done using a combination disk confirmatory test and the modified carbapenem inactivation method (mCIM). Finally, the presence of ESBLs and carbapenemase resistance encoding genes was assessed using PCR and specific primers. Results: Out of the 100 isolates, the percentage of antibiotic resistance was cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Additionally, thirty isolated strains were found to be multidrug-resistant. Out of these, twenty-seven strains demonstrated a potential for ESBLs, twenty strains for Carbapenemase, and seventeen strains for both ESBLs and Carbapenemase production. Moreover, the occurrence of ESBLs and carbapenemase genes was as follows: bla SHV (25 %), bla TEM (23 %), bla CTX-M (20 %), bla OXA-48 (17 %), and bla VIM (13 %). It is important to mention that we did not detect the bla IMP and bla KPC. resistant genes among clinical isolates. Conclusion: Based on the results, the existence of this type of resistance in hospital centers needs to be reevaluated in terms of empirical antibiotic prescribing. Additionally, it is recommended that infection control measures should be taken for public health. Also, it's suggested that hospital-acquired infections caused by superbug K. pneumoniae resistant strains should be addressed.

Detection of Yersinia enterocolitica, Shigella spp. and Salmonella spp. in Rattus norvegicus captured from Tehran, Iran.

Background: The present study aims to determine the presence of Yersinia spp., Yersinia pestis, Yersinia enterocolitica pathogen, Listeria monocytogenes, Salmonella spp., Shigella spp., Francisella tularensis and Borrelia spp. in brown rats of Tehran, Iran. Methods: PCR was used to detect various bacteria in 100 brown rats, Also, ELISA was used to detect antibodies against the F. tularensis and Borrelia spp. Results: A total of 16% and 13% of fecal samples were positive for Yersinia spp. and Y. enterocolitica pathogen. ELISA results were negative for F. tularensis and Borrelia. No specific antibodies (IgG) were against these bacteria. Conclusion: According to the results of our analysis, rats are significant transmitters and carriers of a variety of illnesses that can spread to both people and other animals.

Biofilm Formation in Mycobacterial Genus; Mechanism of Biofilm Formation and Anti-Mycobacterial Biofilm Agents.

Mycobacterium tuberculosis, Mycobacterium leprae, and non-tuberculous mycobacteria (NTM) are among the most significant human pathogens within the Mycobacterium genus. These pathogens can infect people who come into contact with biomaterials or have chronic illnesses. A characteristic pathogenic trait of mycobacteria is the development of biofilms, which involves several molecules, such as the GroEL1 chaperone, glycopeptidolipids, and shorter-chain mycolic acids. Bacterial behavior is influenced by nutrients, ions, and carbon sources, which also play a regulatory role in biofilm development. Compared to their planktonic phase, mycobacterial biofilms are more resilient to environmental stresses and disinfectants. Mycobacteria that produce biofilms have been found in several environmental studies, particularly in water systems. NTM can cause respiratory problems in individuals with underlying illnesses such as cystic fibrosis, bronchiectasis, and old tuberculosis scars. Mycobacteria that grow slowly, like those in the Mycobacterium avium complex (MAC), or rapidly, like Mycobacterium abscessus, can be pathogens. Infections related to biomaterials represent a significant category of biofilm-associated infections, with rapidly growing mycobacteria being the most frequently identified organisms. A biofilm produced by M. tuberculosis can contribute to caseous necrosis and cavity formation in lung tissue. Additionally, M. tuberculosis forms biofilms on clinical biomaterials. Biofilm formation is a major contributor to antimicrobial resistance, providing defense against drugs that would typically be effective against these bacteria in their planktonic state. The antibiotic resistance of biofilm-forming microbes may render therapy ineffective, necessitating the physical removal of biofilms to cure the infection. Recently, new approaches have been developed with potential anti-biofilm compounds to increase treatment effectiveness. Understanding biofilms is crucial for the appropriate treatment of various NTM diseases, and the recent discovery of M. tuberculosis biofilms has opened up a new field of study. This review focuses on the biofilm formation of the Mycobacterial genus, the mechanisms of biofilm formation, and anti-mycobacterial biofilm agents.

Intestinal Colonization by Campylobacter jejuni, Clostridium difficile, and Clostridium perfringens among Commensal Rattus norvegicus in the Urban Areas of Tehran, Iran.

Background: Rattus norvegicus (R. norvegicus) population plays a significant role in the spread of numerous diseases in urban environments. The present study is aimed at investigating the presence of Campylobacter jejuni (C. jejuni), C. coli, Clostridium difficile (C. difficile), C. difficile toxigenic, and C. perfringens in R. norvegicus captured from urban areas of Tehran, Iran. Methods: From October 2021 to October 2022, 100 urban rats were trapped in 5 different districts of Tehran, Iran. The genomic DNA was extracted from fecal samples, and the presence of C. jejuni, C. coli, C. perfringens, and C. difficile species was evaluated using PCR assay. Moreover, PCR was used to assess the toxicity of C. difficile isolates. Results: Overall, 30% (n = 30/100) of fecal samples were positive for zoonotic pathogens. Based on the PCR on hippuricase (hipO), glycine (gly), CIDIF, and phospholipase C (plc) genes, C. perfringens and C. difficile were isolated from 18.2% (n = 14/77) and 5.2% (n = 4/77) of male rats. The highest frequency of C. perfringens and C. jejuni was 25% (n = 5/20) related to the south of Tehran. Toxigenic C. difficile was not detected in all regions. Conclusion: According to the findings, rats are the main reservoirs for diseases. Therefore, rodent control coupled with the implementation of surveillance systems should be prioritized for urban health.

Superior Performance of Iron-Coated Silver Nanoparticles and Cefoxitin as an Antibiotic Composite Against Methicillin-Resistant Staphylococcus aureus (MRSA): A Population Study.

The synergistic effects of antimicrobial nanostructures with antibiotics present a promising solution for overcoming resistance in methicillin-resistant Staphylococcus aureus (MRSA). Previous studies have introduced iron as a novel coating for silver nanoparticles (AgNPs) to enhance both economic efficiency and potency against S. aureus. However, there are currently no available data on the potential of these novel nanostructures to reverse MRSA resistance. To address this gap, a population study was conducted within the MRSA community, collecting a total of 48 S. aureus isolates from skin lesions. Among these, 21 isolates (43.75%) exhibited cefoxitin resistance as determined by agar disk diffusion assay. Subsequently, a PCR test confirmed the presence of the mecA gene in 20 isolates, verifying them as MRSA. These results highlight the cefoxitin disk diffusion susceptibility test as an accurate screening method for predicting mecA-mediated resistance in MRSA. Synergy tests were performed on cefoxitin, serving as a marker antibiotic, and iron-coated AgNPs (Fe@AgNPs) in a combination study using the checkerboard assay. The average minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of cefoxitin were calculated as 11.55 mg/mL and 3.61 mg/mL, respectively. The findings indicated a synergistic effect (FIC index < 0.5) between Fe@AgNPs and cefoxitin against 90% of MRSA infections, while an additive effect (0.5 ≤ FIC index ≤ 1) could be expected in 10% of infections. These results suggest that Fe@AgNPs could serve as an economically viable candidate for co-administration with antibiotics to reverse resistance in MRSA infections within skin lesions. Such findings may pave the way for the development of future treatment strategies against MRSA infections.

Green Synthesized Iron-Coated Silver Nanoparticles: Economic Bimetallic Nanoparticles Potential Against Methicillin-Resistance Staphylococcus aureus.

Iron coating was introduced as one of the novel techniques to improve physicochemical and biological properties of silver nanoparticles (AgNPs). In the current experiment, impact of iron coating on the antimicrobial potency of AgNPs was investigated against methicillin-resistance Staphylococcus aureus (MRSA). To obtain more accurate data about the antimicrobial potency of examined nanostructures, the experiment was done on the 10 isolates of MRSA which were isolated from skin lesions. AgNPs and iron-coated AgNPs (Fe@AgNPs) were fabricated based on a green one-pot reaction procedure. Minimal inhibitory concentration (MIC) of Fe@AgNPs was not significantly different with MIC of AgNPs against eight out of 10 examined MRSA isolates. Also, by iron coating a reduction in the minimal inhibitory concentration (MIC) of AgNPs was observed against two MRSA isolates. The average MIC of AgNPs against 10 MRSA isolates was calculated to be 2.16 ± 0.382 mg/mL and this value was reduced to 1.70 ± 0.638 mg/mL for Fe@AgNPs. However, this difference was not considered significant statistically (P-value > 0.05). From productivity point of view, it was found that iron coating would improve the productivity of the synthesis reaction more than fivefold. Productivity of AgNPs was calculated to be 1.02 ± 0.07 g/L, meanwhile this value was 5.25 ± 0.05 g/L for Fe@AgNPs. Iron coating may provide another economic benefit to reduce final price of AgNPs. It is obvious that the price of a particular nanostructure made of silver and iron is significantly lower than that of pure silver. These findings can be considered for the fabrication of economic and potent antimicrobial nanoparticles.

Bundle-forming pilus locus of Aeromonas veronii bv. Sobria.

Little is known about the colonization mechanisms of Aeromonas spp. Previous work has suggested that the type IV bundle-forming pilus (Bfp) is an aeromonad intestinal colonization factor. This study provides the first genetic characterization of this structure. To define the role of Bfp in Aeromonas veronii bv. Sobria adherence, a 22-kb locus encoding the bundle-forming pilus was isolated; this contained 17 pilus-related genes similar to the mannose-sensitive hemagglutinin (MSHA) of Vibrio cholerae. Reverse transcriptase PCR (RT-PCR) demonstrated that the locus had two major transcriptional units, mshI to mshF and mshB to mshQ. Transcriptional fusion experiments demonstrated the presence of two strong promoters upstream of mshI and mshB. The locus encoded four putative prepilin proteins, one of which (MshA) corresponded to the N-terminal sequence of the previously isolated major pilin protein. All the pilin genes were inactivated, mutation of each minor or major pilin gene greatly reduced the bacterium's ability to adhere and form biofilms, and complementation of each mutant in trans rescued this phenotype. Mutation of the major pilin MshA and MshB, a minor pilin, resulted in their loss. The position of the mshH gene is conserved within a number of bacteria, and we have shown it is not transcriptionally linked to the other msh genes; moreover, its mutation did not have a dramatic effect on either adhesion or biofilm formation. We conclude that the bundle-forming pilus is required for A. veronii bv. Sobria adherence and biofilm formation; furthermore, both the major and minor pilin proteins are essential for this process.

An in silico Design, Expression and Purification of a Chimeric Protein as an Immunogen Candidate Consisting of IpaD, StxB, and TolC Proteins from Shigella spp.

Background: Shigella spp. is the cause of dysentery and is widespread worldwide. On the other hand, antibiotic resistance is increasing in this bacterium. Bioinformatics is a new approach to vaccine and drug design involving the selection of appropriate antigens. This study aimed to design a chimeric protein consisting of IpaD, StxB, and TolC proteins from Shigella through a bioinformatics approach as an immunogen candidate. Methods: The sequences of ipaD, stxB, and tolC genes were obtained. Additionally, the immunogenic regions of the associated protein, physicochemical characteristics, protein structures, B and T cells epitopes, and molecular docking were determined using in silico servers. Besides, the chimeric gene was synthesized following sequence optimization by utilizing the codon usage of Escherichia coli (E. coli). The expression of the recombinant protein was confirmed via SDS-PAGE and Western blot technique. Results: The residues 41-160 of IpaD, 21-89 of StxB, and 40-335 of TolC were selected. According to half-life, instability, and buried indices, IpaD-StxB-TolC was selected as the best arrangement. The Ramachandran plot showed that 97.077% of the amino acids were in the favored area. Linear and conformational epitopes were also present throughout the chimeric protein sequence. Moreover, the C-ImmSim server indicated that IgG and IgM titers could reach desirable values by the third injection. Furthermore, the stability of the mRNA-optimized gene was enhanced, increasing the Codon Adaptive Index (CAI) to 0.9. Finally, the chimeric gene was transferred to E. coli BL21, and the expression of the 60.6 kDa recombinant protein was confirmed. Conclusion: The results indicated that the recombinant protein could act as a proper immunogen candidate against Shigella spp.

A global overview of the most important zoonotic bacteria pathogens transmitted from Rattus norvegicus to humans in urban environments.

Zoonotic pathogens, comprising over 61% of all pathogenic microorganisms, can be transmitted from different animals to individuals in numerous ways either in the presence or the absence of a vector. Causing new emerging human infectious diseases, these pathogens could be categorized into 4 groups, bacteria, viruses, parasites, and fungi. Among the wide range of reservoirs for zoonotic pathogens, tremendous attention has been attracted to wild rats, due to their global distribution not only in urban environments but also in the sylvatic and agricultural surroundings. For the nonce, zoonotic bacteria transmitted via wild rats have turned into a global public health problem probably due to their ability to induce re-emerging diseases even after eradication and controlling management. Despite the importance of wild rats in spreading pathogens, little data are available about the bacterial diversity present in urban wild rat populations. In this review, we present a complete list of zoonotic bacterial pathogens isolated from wild rats in urban environments.

Synthesis of mesoporous antimicrobial herbal nanomaterial-carrier for silver nanoparticles and antimicrobial sensing.

Herbal nanoparticles (HNPs) were introduced as a novel generation of antimicrobial nanoparticles. But in the battle against superbugs we need nanostructures with boosted antimicrobial potency. So in the current experiment, for the first time a green approach was developed for the silver functionalization of HNPs which were fabricated from an antimicrobial herb Thymus vulgaris. Silver functionalized HNPs (AgHNPs) were found to be mesoporous and were further fortified with antimicrobial compounds. The resulted structures were re-tested against MRSA and P. aeruginosa as superbugs. It was found that silver functionalization can provide eight-fold increase in the antimicrobial potency of HNPs. Moreover, MIC was reduced from 20 mg/mL to 2.5 mg/mL. Another eight-fold reduction in the MIC (0.3 mg/mL) was achieved by fortification with antimicrobial compounds. So, the antimicrobial potency of HNPs was successfully increase approximately up to 64-folds. Obtained results illustrated that silver functionalization and fortification with antimicrobial compounds can be considered as effective approaches to achieve HNPs with boosted antimicrobial potency. These nanostructures have the potency to be loaded with other antimicrobial compound such as antibiotics toward synergistic effects of AgNPs and antibiotics. Resulted nanostructures can be employed in the formulation of powerful topical and surface disinfectants against superbugs. Also, these particles can be considered as a next generation of boosted antimicrobial nanostructures.

Determining spa-type of methicillin-resistant Staphylococcus aureus (MRSA) via high-resolution melting (HRM) analysis, Shiraz, Iran.

OBJECTIVES: Molecular typing methods are useful for rapid detection and control of a disease. Recently, the use of high-resolution melting (HRM) for spa typing of MRSA isolates were reported. This technique is rapid, inexpensive and simple for genotyping and mutation screening in DNA sequence. The aim of this study was to evaluate the ability of HRM-PCR to analysis spa genes amongst MRSA isolates. RESULTS: A total of 50 MRSA isolates were collected from two teaching hospitals in Shiraz, Iran. The isolates were confirmed as MRSA by susceptibility to cefoxitin and detection of mecA gene using PCR. We used HRM analysis and PCR-sequencing method for spa typing of MRSA isolates. In total, 15 different spa types were discriminate by HRM and sequencing method. The melting temperature of the 15 spa types, using HRM genotyping were between 82.16 and 85.66 °C. The rate of GC % content was 39.4-46.3. According to the results, spa typing of 50 clinical isolates via PCR-sequencing and HRM methods were 100% similar. Consequently, HRM method can easily identify and rapidly differentiate alleles of spa genes. This method is faster, less laborious and more suitable for high sample at lower cost and risk of contamination.

Prevalent bacterial infections in intensive care units of Shiraz University of medical sciences teaching hospitals, Shiraz, Iran.

Intensive care unit (ICU)-acquired infections can bring some degree of morbidity and mortality to patients staying in the ICU. In this study, over a 6-month period, the prevalence of infections and mortality rates among ICU patients, the predominant organisms and their resistance patterns were determined in a cross-sectional study. Samples of all the patients hospitalized longer than 48 h in two Shiraz university teaching hospitals in Shiraz, Iran, were microbiologically cultured, and antimicrobial susceptibility was determined using the disk diffusion method. We studied 123 specimens from 89 patients aged 1 month to 80 years (38.3-/+13.4), and among them 46 patients (51.7%; 95% CI, 41.3-62.1%) showed infection based on culture and clinical findings. Of these, 37 patients (41.6%) had more than one ICU-related nosocomial infection and 9 patients (10.1%) had only one ICU-related nosocomial infection. The overall mortality rate for ICU-acquired infections was 10.9% (5 patients). Gram-negative bacteria were significantly more involved in infections than were Gram-positive bacteria (P<0.05). The most frequently reported infections were urinary tract infections. The most frequently isolated bacteria were Pseudomonas (39.1%), which was mainly sensitive to amikacin and ceftazidime. Our study results underscore the importance of specific measures for infection control in critically ill patients in ICUs in Shiraz, Iran, especially when using urinary catheters.