A chimeric vaccine targeting Pseudomonas aeruginosa virulence factors protects mice against lethal infection.

Pseudomonas aeruginosa is an important and hazardous nosocomial pathogen in respiratory tract infections and rapidly achieves antibiotic resistance, so it is necessary to develop an effective vaccine to combat the infection. The Type III secretion system (T3SS) protein P. aeruginosa V-antigen (PcrV), outer membrane protein F (OprF), and two kinds of flagellins (FlaA and FlaB) all play important roles in the pathogenesis of P. aeruginosa lung infection and its spread into deeper tissues. In a mouse acute pneumonia model, the protective effects of a chimer vaccine including PcrV, FlaA, FlaB, and OprF (PABF) protein were investigated. PABF immunization prompted robust opsonophagocytic titer of IgG antibodies and decreased bacterial burden, and improved survival afterward intranasal challenge with ten times 50% lethal doses (LD50) of P. aeruginosa strains, indicating its broad-spectrum immunity. Moreover, these findings showed a promise chimeric vaccine candidate to treat and control P. aeruginosa infections.

Emerging role of microbiota derived outer membrane vesicles to preventive, therapeutic and diagnostic proposes.

The role of gut microbiota and its products in human health and disease is profoundly investigated. The communication between gut microbiota and the host involves a complicated network of signaling pathways via biologically active molecules generated by intestinal microbiota. Some of these molecules could be assembled within nanoparticles known as outer membrane vesicles (OMVs). Recent studies propose that OMVs play a critical role in shaping immune responses, including homeostasis and acute inflammatory responses. Moreover, these OMVs have an immense capacity to be applied in medical research, such as OMV-based vaccines and drug delivery. This review presents a comprehensive overview of emerging knowledge about biogenesis, the role, and application of these bacterial-derived OMVs, including OMV-based vaccines, OMV adjuvants characteristics, OMV vehicles (in conjugated vaccines), cancer immunotherapy, and drug carriers and delivery systems. Moreover, we also highlight the significance of the potential role of these OMVs in diagnosis and therapy.

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

Background: The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. Methods: In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). Results: The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 µg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 µg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 µg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 µg/mL, and MBEC values ranging from 40 to 10 µg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. Conclusion: In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Highly Synergistic Effects of Melittin With Vancomycin and Rifampin Against Vancomycin and Rifampin Resistant Staphylococcus epidermidis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) strains are increasingly emerging as serious pathogens because they can be resistant to many antibiotics called multidrug resistance (MDR) that limit the therapeutic options. In the case of vancomycin- and rifampin-resistant MDR-MRSE, the physicians are not allowed to increase the doses of antibiotics because of severe toxicity. Accordingly, we investigated the synergistic activity of melittin antimicrobial peptide with vancomycin and rifampin against vancomycin-resistant, and rifampin-resistant MDR-MRSE isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and fractional bactericidal concentration index (FBCi) of antimicrobial agents against isolates were determined. Coagulate activities and serum and salt stability as well as melittin cytotoxicity on the human embryonic kidney (HEK) 293 cells and human red blood cells (RBCs) at their synergistic concentrations. MIC and MBC values for melittin were in the range of 0.312-2.5 and 0.312-5, respectively. Results also showed that the interaction of melittin with drugs was highly synergistic in which the geometric means of FICi and FBCi were < 0.5. Induced synergism led to a decrease in melittin, rifampin, and vancomycin concentrations by 8-1,020, 2-16, and 4-16-folds, respectively. This phenomenon caused a reduction in melittin toxicity by which the synergistic concentration of melittin needed to kill bacteria did not show cytotoxicity and hemolytic activity. Besides, no coagulation activity was found for the synergistic and alone concentrations of melittin in both Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). Interestingly, the antibacterial activity of melittin in Mueller Hinton Broth (MHB) containing human serum did no significant differences between MIC and MBC values of melittin in MHB and MHB containing 10% human serum. The present findings showed that the therapeutic index of melittin was improved by 32.08- and 12.82-folds when combined with vancomycin and rifampin, respectively. Taken together, the obtained data show that melittin alone was effective against MDR-MRSE isolates and this antimicrobial peptide showed highly synergistic effects with vancomycin and rifampin without causing toxicity. Therefore, the combination of melittin and traditional antibiotics could be a promising strategy for the treatment of infections caused by MDR-MRSE.

Passive immunization with anti- chimeric protein PilQ/PilA -DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model.

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Antibacterial effect of cerium oxide nanoparticle against Pseudomonas aeruginosa.

BACKGROUND: Antibiotics have been widely used for the treatment of bacterial infections for decades. However, the rapid emergence of antibiotic-resistant bacteria has created many problems with a heavy burden for the medical community. Therefore, the use of nanoparticles as an alternative for antibacterial activity has been explored. In this context, metal nanoparticles have demonstrated broad-spectrum antimicrobial activity. This study investigated the antimicrobial activity of naked cerium oxide nanoparticles dispersed in aqueous solution (CNPs) and surface-stabilized using Pseudomonas aeruginosa as a bacterial model. METHODS: Gelatin-polycaprolactone nanofibers containing CNPs (Scaffold@CNPs) were synthesized, and their effect on P. aeruginosa was investigated. The minimum inhibitory and bactericidal concentrations of the nanoparticls were determined in an ATCC reference strain and a clinical isolate strain. To determine whether the exposure to the nanocomposites might change the expression of antibiotic resistance, the expression of the genes shv, kpc, and imp was also investigated. Moreover, the cytotoxicity of the CNPs was assessed on fibroblast using flow cytometry. RESULTS: Minimum bactericidal concentrations for the ATCC and the clinical isolate of 50 µg/mL and 200 µg/mL were measured, respectively, when the CNPs were used. In the case of the Scaffold@CNPs, the bactericidal effect was 50 µg/mL and 100 µg/mL for the ATCC and clinical isolate, respectively. Interestingly, the exposure to the Scaffold@CNPs significantly decreased the expression of the genes shv, kpc, and imp. CONCLUSIONS: A concentration of CNPs and scaffold@CNPs higher than 50 µg/mL can be used to inhibit the growth of P. aeruginosa. The fact that the scaffold@CNPs significantly reduced the expression of resistance genes, it has the potential to be used for medical applications such as wound dressings.

Transcriptome analysis of biofilm formation under aerobic and microaerobic conditions in clinical isolates of Campylobacter spp.

It has been well documented that Campylobacter is the leading cause of foodborne infections and bacterial enteritis in high-income countries. The gastrointestinal tract of most warm-blooded animals, such as mammals and poultry, is prone to this pathogen. Infections caused by this bacterium in humans have usually been associated with the consumption of contaminated poultry meat. The important point about Campylobacter is that this bacterium has adapted to harsh environmental conditions along the food chain (poultry digestive tract to the consumer's plate) and developed an adapted mechanism to those conditions. This study aimed to compare the ability of Campylobacter jejuni and Campylobacter coli strains to form biofilms under aerobic and microaerobic conditions. The presence and expression of flab, FliS, DnaK, luxs, CsrA, Cj0688, and cosR genes involved in biofilm formation were investigated. Finally, the correlation between the biofilm forming ability of Campylobacter isolates and the presence/expression of selected genes has been explored. A significant correlation was observed between the presence and expression of some genes and the degree of biofilm formation in C. jejuni and C. coli isolates. A strong biofilm production was detected in strains harboring all selected genes with greater expression levels. The ability of C. jejuni and C. coli strains in biofilm formation is associated with the coordinated function and convergent expression of the selected genes. Seemingly, stress response- and motility-related genes have the most involvement in biofilm formation of C. jejuni and C. coli strains, while other genes have an accessory role in this phenomenon.

Inhibitory Effects of Lactococcus lactis and its Supernatant on Listeria monocytogenes.

BACKGROUND: The application of biological compounds generated by lactic acid bacteria, especially Lactococcus lactis, is recently considered to be a natural preservative for improving quality and health of food. The purpose of this study is to investigate the inhibitory potential of L. lactis supernatant on the expression of inlA, plc, and hly genes related to L. monocytogenes virulence capacity. METHODS: L. lactis was cultured under anaerobic conditions for 16 - 18 hours. The supernatant and live bacteria were then separated by centrifuge. The antilisteria effects of L. lactis and supernatant were measured using the agar diffusion technique, and the effect on the expression of the virulence-related genes was calculated by real-time PCR. Also, the effects of live bacteria and its supernatant on the microbial count of milk and sausage infected by L. monocytogenes was evaluated by the colony count assay. RESULTS: After 24 hours, the highest non-growing hole diameter was obtained in the presence of acidic supernatant (pH = 3.5). The microbial count showed the inhibitory effect on the eighth day after incubation with L. lactis. qPCR data revealed a down-regulation of virulence-related genes of inlA (8 fold), hly (6 fold), and plc (1 fold) in L. monocytogenes after 24-hour incubation with the supernatant. CONCLUSIONS: Our findings showed that the supernatant of L. lactis has an effective inhibitory role in the growth of L. monocytogenes. In the presence of supernatant, among plc, inlA and hly genes, the expression of inlA and hly genes decreased after 2 hours, which could indicate the molecular inhibitory mechanism of L. lactis in L. monocytogenes.

Therapeutic effects, immunogenicity and cytotoxicity of a cell penetrating peptide-peptide nucleic acid conjugate against cagA of Helicobacter pylori in cell culture and animal model.

BACKGROUND AND OBJECTIVES: Helicobacter pylori causes several gastrointestinal diseases, including asymptomatic gastritis, chronic peptic ulcer, duodenal ulcer, lymphoma of the mucosa-associated lymphoid tissue (MALT), and gastric adenocarcinoma. In recent years, failure to eradicate H. pylori infections has become an alarming problem for physicians. It is now clear that the current treatment strategies may become ineffective, necessitating the development of innovative antimicrobial compounds as alternative treatments. MATERIALS AND METHODS: In this experimental study, a cell-penetrating peptide-conjugated peptide nucleic acid (CPP-PNA) was used to target the cagA expression. cagA expression was evaluated using RT-qPCR assay after treatment by the CPPPNA in cell culture and animal model. Additionally, immunogenicity and toxicity of the CPP-PNA were assessed in both cell culture and animal models. RESULTS: Our analysis showed that cagA mRNA levels reduced in H. pylori-infected HT29 cells after treatment with CPPPNA in a dose-dependent manner. Also, cagA expression in bacterial RNA extracted from stomach tissue of mice treated with PNA was reduced compared to that of untreated mice. The expression of inflammatory cytokines also decreased in cells and tissue of H. pylori-infected mice after PNA treatment. The tested CPP-PNA showed no significant adverse effects on cell proliferation of cultured cells and no detectable toxicity and immunogenicity were observed in mice. CONCLUSION: These results suggest the effectiveness of CPP-PNA in targeting CagA for various research and therapeutic purposes, offering a potential antisense therapy against H. pylori infections.

Monoclonal antibody directed to the PilQ -PilA DSL region in Pseudomonas aeruginosa improves survival of infected mice with antibiotic combination.

The infections caused by Pseudomonas aeruginosa are related to high mortality and morbidity in critically ill patients because of multidrug resistance. Thus, we performed the efficacy of the monoclonal antibody (mAb) against PilQ -PilA DSL region (QA) in combination with antibiotics in a model of P. aeruginosa infection. In the present study, three clinically applicable antibiotics (levofloxacin, ceftazidime and gentamicin) and the anti-QA mAb were utilized for treatment of P. aeruginosa sepsis in mice. Reliably, in comparison with other treatment groups (antibody or antibiotic administration), the combination of antibiotic and anti-QA mAb essentially enhanced the survival of mice infected with P. aeruginosa PAO1. This synergistic effect was due to improved bactericidal effect, which prevented bacterial dissemination to different organs. Consequently, the antibiotic and anti-QA mAb combination gives a new effective strategy for the treatment of P. aeruginosa sepsis, particularly when large numbers of exceptionally virulent strains are present.

Analysis of virulence genes and molecular typing of Listeria monocytogenes isolates from human, food, and livestock from 2008 to 2016 in Iran.

The frequency of Listeria monocytogenes isolates collected from a total of 1150 samples including food (n = 300), livestock (n = 50), and human clinical (n = 800) was evaluated during 2008-2016. Antimicrobial resistance patterns, virulence factors, and molecular characteristics of these isolates were analyzed using disk diffusion method, sequencing, serotyping, and pulsed-field gel electrophoresis (PFGE). The analysis of 44 L. monocytogenes isolates showed that 72.7% (32 of 44) of all the isolates belonged to Serotype 1/2c, and 15.9% (7 of 44) belonged to Serotype 3c. All 44 isolates were resistant to one or more antimicrobial agents with the most frequent resistance to penicillin (75%) and tetracycline (47.7%). Of the 44 L. monocytogenes strains, 100, 69.2, and 62.5% of livestock, human, and food strains were resistant to penicillin, respectively. Using pulsed-field gel electrophoresis (PFGE) technique, the isolates' genetic diversity was determined, and 28 PFGE patterns with 8 common (CT) and 20 single types (ST) were identified. This study highlights the high prevalence of Serotype 1/2c in clinical and livestock samples, while different serotypes were observed in food samples. The presence of rare serotypes such as 4c, belonging to the Lineage III, as well as 4e and 1/2c which are infrequent in Iran indicates that paying attention to uncommon serotypes, especially 1/2c, during the listeriosis outbreaks is necessary.

Characterization of Antimicrobial Resistance Patterns of Klebsiella pneumoniae Isolates Obtained from Wound Infections.

CDATA[Background: Multidrug resistance among ESBL producing isolates has limited the administration of proper antibiotics. It is, therefore, important to monitor the resistance patterns of Klebsiella pneumoniae isolates and provide infection control strategies to prevent nosocomial outbreaks. This study was aimed to determine antimicrobial resistance patterns of K. pneumoniae isolates obtained from wound infections of patients in Tehran, Iran. METHODS: A total of 102 K. pneumoniae isolates were obtained from wound infections of patients in Tehran, Iran. The production of phenotypic ESBL and carbapenemase was assessed using the double-disc synergy test (DDST) and modified Hodge test (MHT), respectively. PCR was performed for the detection of ESBL, carbapenemase, quinolone and aminoglycoside resistance genes. RESULTS: Forty-six (45.1%) and 23 (22.5%) isolates, out of the 102 isolates, were phenotypically detected as ESBL and carbapenemase producers, respectively. The PCR results showed that 80/102 (78.4%) and 51/102 (50%) isolates possessed at least one of the assessed ESBL and carbapenemase genes, respectively. Quinolone resistance determinants (QRDs) and aac(6')-Ib genes were found amongst 50 (49%) and 67 (65.7%) isolates, respectively. Four isolates carried blaTEM, blaSHV, blaCTX-M, qnrB, qnrS and aac(6')-Ib genes, simultaneously. CONCLUSION: Due to the presence of multiple resistance genes among some K. pneumoniae strains, antibiotic agents should be used with caution to preserve their efficacy in case of life-threatening infections.

A genomic concept in cellular interaction of clinical Campylobacter spp. with human epithelial colorectal adenocarcinoma cells.

This study aimed to realize the genomic concept of cellular interaction of clinical Campylobacter spp. with human epithelial colorectal adenocarcinoma cells. It was indicated that the mean adherence and invasion rate of C.jejuni isolates was significantly higher than C.coli and the highest adhesion rate among the C.jejuni and C.coli belonged to strains harboring 4 (flaA, cadF, peb1A, and flpA) and 3 (flaA, cadF, and peb1A) adherence genes, respectively, which indicates that the adhesion potential of C.coli and C.jejuni strains is associated with the coordinate function and cumulative effect of selected virulence-associated genes. The highest invasion rate in C.jejuni (10.3%) and C.coli (8.4%) isolates belonged to strains which concomitantly contained 3 (ciaB, iamA, and tlp1) and 2 (ciaB and iamA) invasion-associated genes which emphasizes on the cooperative roles of these genes in C.jejuni and C.coli invasion to Caco-2 cells. The toxicity of C.jejuni for Caco-2 cells was proved higher than that of C.coli. There was a positive correlation between adherence, invasion and toxicity of both C.jejuni and C.coli isolates. Moreover, the expression levels of CDT-producing genes in C.jejuni strains was significantly higher than that of C.coli. The average cytotoxicity of the strains with all three CDT-encoding genes (cdtA, cdtB and cdtC) was statistically higher than those lacking one or more CDT subunits. A crucial contribution of CdtB to the cytotoxicity of Campylobacter strains was detected. Following the treatment of epithelial cells with C.jejuni or C.coli, IL-8 and TNF-α were significantly increased compared to untreated Caco-2 cells, and the highest IL-8 expression was observed in both C.jejuni and C.coli expressing all CDTs (cdtA, cdtB, and cdtC). We, for the first time, indicated the major contribution of TLR2 and TLR4 in campylobacter initiation of pathogenesis, while increased invasiveness and cytotoxicity was significantly associated with the increased expression of TLR4 in C.jejuni isolates.

Efficacy of low-dose local clindamycin in different times for microbial decontamination of autogenous particulate bone graft.

BACKGROUND: Clindamycin in low concentration (20 µg/mL) is safe for vitality and osteogenic potential of bone cells. The aim of this study was to evaluate the efficacy of local clindamycin (20 µg/mL) in two different exposure times, for microbial decontamination of particulate bone graft, collected during implant site preparation. This non-randomized parallel-group study was conducted on samples from 17 patients. The particulate bone collected during implant site preparation was divided into three portions by weight: in group S1, the particulate bone was immersed in thioglycolate broth without any antibiotic treatment; in group S2, the collected particulate bone was irrigated with 100 mL clindamycin solution (20 µg/mL); and in group S3, the collected particulate bone was soaked in one ml clindamycin solution (20 µg/mL) for 3 min. Samples in the three groups were cultured in aerobic and anaerobic media and species and CFU count of isolated bacteria were determined. RESULTS: Analysis of the data demonstrated a significant difference among the three groups in the mean count of total microorganisms (P = 0.001). The difference in the mean count of anaerobic and aerobic microorganisms in the three groups was statistically significant as well (P = 0.001). Pseudomonas aeruginosa was the only microorganism that was not affected with the mentioned antibiotic. CONCLUSIONS: Local use of low-dose clindamycin (20 µg/mL)-irrigation or 3 min immersing-is effective for the decontamination of particulate bone grafts.

Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori.

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 µM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 µM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.

A trivalent vaccine consisting of "flagellin A+B and pilin" protects against Pseudomonas aeruginosa infection in a murine burn model.

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly achieves antibiotic resistance, and thus, developing an effective vaccine is critically important for combating P. aeruginosa infection. Flagella and pili play important roles in colonization of P. aeruginosa at the burn wound site and its subsequent dissemination to deeper tissue and organs. In the present study, we evaluated protective efficacy of a trivalent vaccine containing flagellins A and B (FlaA + FlaB) + pilin (PilA) in a murine burn model of infection. "FlaA + FlaB + PilA" induced greater protection in P. aeruginosa murine burn model than the single components alone, and it showed broad immune protection against P. aeruginosa strains. Immunization with "FlaA + FlaB + PilA" induced strong opsonophagocytic antibodies and resulted in reduced bacterial loads, systemic IL-12/IL-10 cytokine expression, and increased survival after challenge with three times lethal dose fifty (LD50) of P. eruginosa strains. Moreover, the protective efficacy of "FlaA + FlaB + PilA" vaccination was largely attributed to specific antibodies. Taken together, these data further confirm that the protective effects of "FlaA + FlaB + PilA" vaccine significantly enhance efficacy compared with antibodies against either mono or divalent antigen, and that the former broadens the coverage against P. eruginosa strains that express two of the three antigens.

Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule.

As an intracellular pathogen, Listeria monocytogenes can enter host cells where it can replicate and escape detection and eradication by the host immune response making the clearance of infection very challenging. Furthermore, with the advent of antimicrobial resistance, the need for alternative targets is inevitable. Internalin proteins are crucial to this bacterium as they contribute to bacterial entry to the systemic circulation. In this study, we targeted a highly conserved region of these proteins by an antisense sequence that was covalently conjugated to the cell penetrating peptides (CPP) to overcome the challenging delivery barriers. Then, we evaluated the efficiency of this construct in vitro. We also assessed the antigenicity, cytotoxicity, and probability of apoptosis induction by this construct. The studied CPP-PNA inhibited bacterial growth and suppressed the mRNA expression of internalins in a dose-dependent manner. In addition, at all studied concentrations, CPP-PNA significantly reduced the invasion rate of L. monocytogenes in the examined cell lines. Moreover, different concentrations of CPP-PNA did not have a significant antigenic, cytotoxic, and apoptotic properties compared to the control. These results suggest the effectiveness of CPP-antisense in targeting the mRNAs of internalins for various research, therapeutic and preventive purposes. However, additional research is required to evaluate the potency, safety, and pharmacokinetics of this compound for the prevention and treatment of listeriosis.

Molecular Analysis of PBP1A in Streptococcus pneumoniae Isolated from Clinical and Normal Flora Samples in Tehran, Iran: A Multicenter Study.

The emergence of high-level penicillin resistance in pneumococcal isolates has seriously complicated the treatment of pneumococcal infections in recent years. The purpose of this study was to determine the serotype, antimicrobial susceptibility, molecular typing, and genetic analysis of the penicillin-binding protein 1a (pbp1a) gene in pneumococcal isolates with high-level resistance to penicillin in Tehran, Iran. PCR amplification, sequencing, and data analysis of the pbp1a gene were carried out for isolates with high-level resistance to penicillin. Antibiotic susceptibility tests showed that the multiple drug resistance pattern "E-CD-OX-TS-T" was the most prevalent (18.0%). The most common serotypes were serotypes 14 (21%), 19F (17%), 23F (16%), and 3 (16%). The highest mutation rates were found in STMK conserved motifs, but no mutation was detected in the other two sequence motifs (SRN and KTG). High-level resistant isolates showed mutations at residues TSQF (574-577) NTGY. Pneumococcal isolates have experienced shifts toward higher penicillin minimal inhibitory concentration levels and other ß-lactams. The results of this study show that the presence of multiple substitutions in the pbp1a gene in pneumococcal isolates is highly associated with a reduced affinity to penicillin.

The diversity of class B and class D carbapenemases in clinical Acinetobacter baumannii isolates.

Wide distrubution of multidrug-resistant Acinetobacter baumannii strains has become a foremost concern in hospital environments. Treatment of infections caused by multidrug resistant strains has conventionally involved the use of ß-lactams such as carbapenems. In this study, we report the distribution of carbapenemase genes in A. baumannii isolated from hospitalized patients. The study was conducted on 110 non-repetitive A. baumannii isolates collected from hospitalized patients, over a nine-month period. Clinical isolates were examined by conventional susceptibility testing, using the disk-diffusion method and multiplex polymerase chain reaction to detect acquired carbapenemase genes. All of the isolates were completely resistant to TOB, SXT, IPM, MEM, CTX, CRO, FEP, CAZ, CIP, PTZ, PIP and were susceptible to colistin, but moderately susceptible TET (2.72%), AK (4.54%) and GEN (3.63%). The prevalence of bla-OXA-51like, bla-OXA-23like, bla-OXA-24like, bla-OXA-58like, blaSIM and blaSPM genes was 100%, 96.36%, 35.45%, 7.27%, 7.27% and 3.63%, respectively. bla-GIM and bla-VIM genes were not detected among the strains. Our results suggest that OXA-type carbapenemase genes plus class B ß-lactamases contribute to carbapenem resistance in the collected isolates. Therefore, quick identification of these resistant genes using molecular approaches is critical in limiting the spread of infections caused by A. baumannii. Drug administration correction of the physicians, based on antibiotic susceptibility testing and more knowledge on the nosocomial infection control policies as essential need.