Significant Reduction In Bacterial Burden Research Articles

Weight loss-induced adipose macrophage memory improves local Staphylococcus aureus clearance in male mice.

Different stimuli can induce innate immune memory to improve pathogen defense or worsen cardiometabolic disease. However, it is less clear if the same stimuli can induce both the protective and detrimental effects of innate immune memory. We previously showed that weight loss induces innate immune memory in adipose macrophages that correlates with worsened diabetes risk after weight regain. In this study, we investigated the effect of weight loss on macrophage cytokine production and overall survival in a mouse model of infection. Male C57Bl/6J mice were put on high-fat or low-fat diets over 18 weeks to induce weight gain or weight loss. Lean mice served as controls. All mice were then infected IV with 2.5×10^6 CFU Staphylococcus aureus . Tissues were collected from 10 mice/group at day 3 and the remaining animals were followed for survival. Weight gain mice had the highest blood neutrophils and the highest bacterial burden in the kidney. However, there was no significant difference in survival. The weight loss group had the highest plasma TNF-α and a significant reduction in bacterial burden in the adipose tissue that correlated with increased adipose macrophage cytokine production. Thus, weight loss-induced adipose macrophage memory may both improve local S.aureus clearance and worsen diabetes risk upon weight regain. Collectively, these findings support the notion that innate immune memory is an evolutionarily protective mechanism that also contributes to the development of cardiometabolic diseases.

Targeting purinergic signaling in platelets ameliorates the bacterial burden and reduces the cytokine storm in the peritoneal cavity during intra-abdominal sepsis

Sepsis is a complex clinical syndrome of the host response to an infection, involving a pathogenic cytokine storm that drives inflammatory injury in multiple organ systems. The abdomen is a major site of sepsis, contributing to high mortality. Platelets express the purinergic receptors P2Y1 and P2Y12; once activated, these receptors mediate the secretion of pro-inflammatory mediators that contribute to organ damage in sepsis. Systemic blockade of P2Y1 or P2Y12 decreased organ damage and inflammatory cytokine levels in a sex-related manner during sepsis. Here, we hypothesize that blocking P2Y1 or P2Y12 activity enhances pathogen clearance in the peritoneal cavity during intra-abdominal sepsis in a sex-specific manner. Male and female C57BL6/6J mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. P2Y1 or P2Y12 antagonists, MRS2279 (10 mg/kg) and Ticagrelor (30 mg/kg) respectively, were administered intraperitoneally after surgery. The peritoneal cavity fluid (PCF) was collected 24 hours post-surgery by peritoneal lavage. To assess cell recruitment in the cavity, we measured leukocyte and platelet counts using Hemavet® Multispecies Hematology System. To assess inflammation, we measured PCF levels of the cytokines MIP-1α and Rantes as well as bacterial clearance. The ileum of the small intestinal tissue samples were collected 24 hours post-surgery and platelet infiltration was measured by platelet factor 4 (PF4) content in the tissue using an ELISA kit. In response to the P2Y1 or P2Y12 blocker, we noted a significant reduction in the bacteria content in the PCF of CLP mice. Blocking P2Y12 but not P2Y1 reduced leukocyte recruitment, while platelet counts were reduced only with P2Y1 blockade in septic males. The sepsis-induced increase in MIP-1α levels was not noted upon P2Y1 but not P2Y12 blockage in the PCF of septic males. However, Rantes levels in the PCF were reduced for either treatment in males compared to the untreated CLP. The platelet marker PF4 in the ileum was increased during sepsis but reduced after either treatment in sepsis males as compared to the untreated CLP. In female CLP mice, a significant reduction in bacterial burden in the PCF in response to the P2Y1 but not the P2Y12 blocker was shown. Blocking P2Y1 or P2Y12 reduced leukocyte recruitment and platelet counts in female CLP mice. As observed in males, PF4 was increased in the ileum during sepsis but reduced following exposure to either treatment in females. No change was noted for MIP-1α or Rantes levels in the females among all the septic groups. Our data indicate that blocking purinergic signaling and in particular P2Y12 in the peritoneal cavity in males prevents bacterial spreading and diminishes cytokine levels, indicating that it could be an effective treatment for intra-abdominal sepsis. However, in female mice blocking the receptor P2Y1, but not P2Y12 has shown to be optimal. Targeting purinergic signaling is a promising sex-specific therapeutic strategy for sepsis. This work was supported by the Research grant AI156627-01 (EL) from the National Institute of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 μg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 μg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Evaluation of Pharmacokinetic and Pharmacodynamic (PK/PD) of Novel Fluorenylmethoxycarbonyl- Phenylalanine Antimicrobial Agent.

To assess the pharmacokinetic profile, in-vivo toxicity, and efficacy of 9-Fluorenylmethoxycarbonyl-L-phenylalanine (Fmoc-F) as a potential antibacterial agent, with a focus on its suitability for clinical translation. An RP-HPLC-based bio-analytical method was developed and qualified to quantify Fmoc-F levels in mouse plasma for pharmacokinetic analysis. Oral bioavailability was determined, and in-vivo toxicity was evaluated following intra-peritoneal administration. Efficacy was assessed by measuring the reduction in Staphylococcus aureus burden and survival rates in BALB/c mice. The RP-HPLC method is highly sensitive, detecting as low as 0.8µgmL-1 (~ 2µM) of Fmoc-F in blood plasma. This study revealed that Fmoc-F has an oral bioavailability of 65 ± 18% and suitable pharmacokinetic profile. Further, we showed that intra-peritoneal administration of Fmoc-F is well tolerated by BALB/c mice and Fmoc-F treatment (100mg/kg, i.p.) significantly reduces Staphylococcus aureus burden from visceral organs in BALB/c mice but falls short in enhancing survival rates at higher bacterial loads. The study provides crucial insights into the pharmacokinetic and pharmacodynamic properties of Fmoc-F. The compound displayed favourable oral bioavailability and in-vivo tolerance. Its significant reduction of bacterial burden underscores its potential as a treatment for systemic infections. However, limited effectiveness for severe infections, short half-life, and inflammatory response at higher doses need to be addressed for its clinical application.

In vitro and in vivo antimicrobial potential of lithium complex against multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, accountable for lethal nosocomial infections, owing to high pathogenicity and multi-drug resistance (MDR). Hence, the World Health Organization has prioritized this bacterium against which new pharmacophores are required urgently. Therefore, this study evaluates the antimicrobial potential of lithium complex based on salicylic acid, and 1,10-phenanthroline {[Li(phen)2sal]} against MDR A. baumannii (MDRAB) through growth inhibition, growth curve assay, biofilm inhibition, and eradication alongside microscopy. The mechanism of action was proposed by flow cytometry and proteomic analysis. Moreover, in vivo efficacy was assessed using an A. baumannii-induced pneumonia model in mice. Results showed that lithium complex significantly inhibited the bacterial growth at 16-32 µg/mL. Growth kinetics revealed that it retarded the bacterial growth after 2-3 h of treatment. Lithium complex significantly inhibited and reduced >70% of biofilms at the MIC level which is a key problem of indwelling and prosthetic devices. Light and atomic force microscopy further indicated disarticulated biofilm and ruptured cellular membrane post-lithium complex treatment. Proteomic analysis revealed that the compound exerted oxidative stress on A. baumannii which may contribute to cell rupturing. Data are available via ProteomeXchange with identifier PXD044293. Flow cytometry confirmed its role in cytoplasmic membrane disintegration. The lithium complex ameliorated the pneumonia symptoms at non-toxic doses of 20 and 40 mg/kg in the pre-clinical A. baumannii-induced pneumonia model as evident by the significant reduction in bacterial burden and parenchymal inflammation. Taken together, these findings suggest that the lithium complex can be a potential drug candidate against MDRAB.IMPORTANCEMulti-drug resistance (MDR) by virtue of evolving resistance and virulence mechanisms among A. baumannii is a global concern which is responsible for lethal hospital-acquired infections. Therefore, it is crucial to develop new therapeutics against it. Metal complexes are compact structures with diverse mechanisms that the pathogens cannot evade easily which make them a strong drug candidate. In this study, we assessed the in vitro and in vivo efficacy of lithium complex {[Li(phen)2 sal]} against biofilm-forming MDR A. baumannii. The lithium complex displayed strong antimicrobial activity and reduced the pre-formed mature biofilm which is key barrier for antimicrobial action. Moreover, it employs oxidative stress as one of its mode of actions and causes cellular rupturing. Lithium complex was non-toxic and was significantly effective to overcome pneumonia in mice model. These results highlight the untapped potential of metal complexes that can be explored and utilized for combating notorious A. baumannii infections.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

The Effectiveness of Laser Applications and Photodynamic Therapy on Relevant Periodontal Pathogens (Aggregatibacter actinomycetemcomitans) Associated with Immunomodulating Anti-rheumatic Drugs.

Considering the current context of the increasing resistance of bacterial species to antibiotics and other antimicrobial agents, a major objective is to develop other antimicrobial approaches, which would be able to inactivate pathogens with considerable effectiveness. Two such methods are photodynamic disinfection therapy and laser irradiation. In view of the immunocompromised status of some patients under immunosuppressive therapy and potential drug interactions that can be established between systemic antimicrobial agents, the research of local, minimally invasive methods of inactivating periodontal pathogens in the context of these systemic therapies with modifying drugs of the immune response is justified. This in vitro study evaluated the antimicrobial action of a diode laser, wavelength 940 nm, and photodisinfection therapy at 670 nm (photosensitizer, 3,7 dimethyl phenothiazine chloride) on a type strain of Aggregatibacter actinomycetemcomitans, a known periodontal pathogen, in the presence and absence of active substances used in autoimmune disease therapy (Etanercept, Infliximab, Metothrexate). The association of a conventional antirheumatic drug with anti-TNF-α therapy determined a significantly greater inhibition of the strain of A. actinomycetemcomitans compared to monotherapy, in vitro. Photodisinfection caused a significant reduction in bacterial burden after a 30 s exposure in vitro, regardless of the pharmaceutical associations of biological and conventional disease-modifying antirheumatic drugs (DMARDs). Irradiation with a diode laser for 30 s at a power of 5 W caused a greater reduction compared to irradiation with 1 W. The application of laser and photodisinfection induced a significant reduction in Aggregatibacter actinomycetemcomitans in vitro and could be considered important adjunctive measures for the eradication of this oral pathogen in the context of immunomodulating therapy.

Role of FruR transcriptional regulator in virulence of Listeria monocytogenes and identification of its regulon

Listeria monocytogenes is a ubiquitous opportunistic foodborne pathogen capable of survival in various adverse environmental conditions. Pathogenesis of L. monocytogenes is tightly controlled by a complex regulatory network of transcriptional regulators that are necessary for survival and adaptations to harsh environmental conditions both inside and outside host cells. Among these regulatory pathways are members of the DeoR-family transcriptional regulators that are known to play a regulatory role in sugar metabolism. In this study, we deciphered the role of FruR, a DeoR family protein, which is a fructose operon transcriptional repressor protein, in L. monocytogenes pathogenesis and growth. Following intravenous (IV) inoculation in mice, a mutant strain with deletion of fruR exhibited a significant reduction in bacterial burden in liver and spleen tissues compared to the parent strain. Further, the ΔfruR strain had a defect in cell-to-cell spread in L2 fibroblast monolayers. Constitutive activation of PrfA, a pleiotropic activator of L. monocytogenes virulence factors, did not restore virulence to the ΔfruR strain, suggesting that the attenuation was not a result of impaired PrfA activation. Transcriptome analysis revealed that FruR functions as a positive regulator for genes encoding enzymes involved in the pentose phosphate pathway (PPP) and as a repressor for genes encoding enzymes in the glycolysis pathway. These results suggested that FruR may function to facilitate NADPH regeneration, which is necessary for full protection from oxidative stress. Interestingly, deletion of fruR increased sensitivity of L. monocytogenes to H2O2, confirming a role for FruR in survival of L. monocytogenes during oxidative stress. Using anti-mouse neutrophil/monocyte monoclonal antibody RB6-8C5 (RB6) in an in vivo infection model, we found that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing. Overall, this work clarifies the role of FruR in controlling L. monocytogenes carbon flow between glycolysis and PPP for NADPH homeostasis, which provides a new mechanism allowing metabolic adaptation of L. monocytogenes to oxidative stress.

Glycolysis is critical for granulocytic myeloid-derived suppressor cell (G-MDSC) activity during Staphylococcus aureus biofilm infection

Abstract Staphylococcus aureus (S. aureus) is a leading cause of prosthetic joint infection (PJI). These infections are often intractable due to biofilm formation, which are complex bacterial communities that adhere to biotic and abiotic surfaces. Within a biofilm, there is considerable heterogeneity in bacterial metabolism and gene expression that serves to create an anti-inflammatory milieu at the site of infection. This is due, in part, to the preferential recruitment of granulocytic myeloid-derived suppressor cells (G-MDSCs), a population of pathologically activated immature leukocytes with immunosuppressive features. Our prior work revealed that G-MDSCs are integral for promoting biofilm persistence via the production of interleukin-10 (IL-10) in response to biofilm-derived lactate. However, the mechanism responsible for G-MDSC anti-inflammatory activity in the context of biofilm infection is unknown. Using a mouse model of S. aureus PJI, time-course single-cell RNA sequencing (scRNA-seq) revealed a significant enrichment in glycolytic and hypoxic transcriptional signatures in infiltrating G-MDSCs. G-MDSCs significantly increased their glycolytic activity when co-cultured with S. aureus biofilm in vitro and inhibiting glycolysis during PJI in vivo with 2-DG nanoparticles significantly attenuated G-MDSC suppressive activity concomitant with a significant reduction in bacterial burden. This study demonstrates that glycolysis is required for G-MDSC-mediated immunosuppression and biofilm persistence during S. aureus PJI. Furthermore, our results demonstrate that G-MDSC metabolism can be targeted to improve infection outcomes. Supported by funding from NIH (2P01 AI083211) & UNMC (UNMC Graduate Fellowship)

Bi-directional elucidation of Lactiplantibacillus plantarum (RTA 8) intervention on the pathophysiology of gut-brain axis during Salmonella brain infection

BackgroundThere have been reports of patients suffering from typhoid fever, particularly those involving infants and immunocompromised patients, which at times present with Salmonella induced brain infection. Although rare, it has frequently been associated with adverse neurological complications and increased mortality. In this context, the gut-brain axis, involving two-way communication between the gut and the brain, holds immense significance as various gut ailments have been associated with psychiatric complications. In turn, several neurodegenerative diseases have been associated with an altered gut microbiota profile. Given the paucity of effective antimicrobials and increasing incidence of multi-drug resistance in pathogens, alternate treatment therapies such as probiotics have gained significant attention in the recent past.ResultsIn the current study, prophylactic effect of Lactiplantibacillus plantarum (RTA 8) in preventing neurological complications occurring due to Salmonella brain infection was evaluated in a murine model. Along with a significant reduction in bacterial burden and improved histoarchitecture, L. plantarum (RTA 8) administration resulted in amelioration in the level of neurotransmitters such as serotonin, norepinephrine and dopamine in the gut as well as in the brain tissue. Simultaneously, increased gene expression of physiologically essential molecules such as mucin (MUC1 and MUC3) and brain-derived neurotrophic factor (BDNF) was also observed in this group.ConclusionPresent study highlights the potential benefits of a probiotic supplemented diet in improving various aspects of host health due to their multi-targeted approach, thereby resulting in multi-faceted gains.

Multi‐Functional Small Molecules with Temporal Charge‐Switchability Tackle Infection and Inflammation

AbstractThe alarming situation in public healthcare caused by ever‐increasing catastrophe of antimicrobial resistance, recurrent infections, and associated inflammation has accelerated the hunt for novel therapeutics which can address these diverse problems concomitantly. This article introduces a new class of multi‐functional amino acid conjugated small antibacterial molecules (ASAMs) which tackle complicated infections and associated inflammation. These molecules exhibit broad‐spectrum bactericidal activity against multi‐drug‐resistant bacteria. The phenylalanine‐bearing lead molecule (ASAM‐10) tackles bacterial dormant subpopulations, impenetrable biofilms, and intracellular pathogens simultaneously. Importantly, this molecule addresses the problem of toxicity associated with cationic lipopeptides like colistin through the temporal charge switching (cationic to zwitterionic) owing to the degradation of labile ester linkages. However, this does not affect its desired antibacterial action window. The substantial reduction in the overexpression of pro‐inflammatory cytokines (IL‐6, IL‐8, TNF‐α, and IL‐1β) upon treatment of infected macrophages with ASAM‐10 validates its anti‐inflammatory efficacy. Furthermore, bacteria exhibit diminished susceptibility toward resistance development against ASAM‐10 owing to its membrane‐active nature. ASAM‐10 displays significant reduction in bacterial burden (2 Log CFU/g) when administered intraperitoneally in mice for MRSA thigh infection. Overall, this new class of multi‐functional molecules is safe for anticipated advanced therapeutic applications to combat complex bacterial infections and inflammation.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202–347-SOD1–72-Ag85B173–330-74F1–148+669–786 as a fusion (‘MAP fusion protein particle’). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Recombinant Flagellin and Incomplete Freund’s Adjuvant Potentiate the Vaccine Efficacy of the Iron Acquisition Protein (HitA) of Pseudomonas aeruginosa

Objectives: Pseudomonas aeruginosa is a notorious bacterial pathogen that can cause a variety of infections with high morbidity and mortality. The increasing multi-drug resistance of this pathogen makes it urgent to develop an effective vaccine. This study aimed at evaluation of the immunoadjuvant effect of flagellin (FliC) of Salmonella enterica and incomplete Freund’s adjuvant (IFA) on the protective vaccine efficacy of P. aeruginosa recombinant iron acquisition protein (HitA). Methods: In this work, recombinant HitA, FliC and fused FliC-F-HitA proteins were expressed in Escherichia coli after cloning their respective genes into pQE31 plasmid vector. The proteins were purified using metal affinity chromatography. The immunoadjuvant effect of FliC was examined upon mixing as well as fusion with HitA antigen of P. aeruginosa in the presence of incomplete Freund’s adjuvant (IFA). This was tested by active immunization followed by challenge using P. aeruginosa murine infection model. Results: Two weeks after the last immunization dose, serum samples were tested for antibody response which showed significant HitA-specific IgG antibody response in all immunized groups compared to control groups. A significant reduction in bacterial burden of lungs from mice immunized with HitA/FliC/IFA mixture was observed after challenge. Opsonophagocytic assay and liver histopathological examination confirmed the previous results. Conclusion: Overall, HitA recombinant protein is considered a promising vaccine candidate against P. aeruginosa upon mixing with S. enterica flagellin protein FliC and IFA.

Use of wet-to-moist cleansing with different irrigation solutions to reduce bacterial bioburden in chronic wounds.

The influence of different irrigation solutions, in conjunction with wet-to-moist cleansing, on the reduction of sessile, non-planktonic bacteria which colonise wounds, has not been investigated. In this study, the antibacterial effect of different irrigation solutions, during a 20-minute wet-to-moist cleansing, has been evaluated in chronic wounds. This study was designed as a prospective cohort study with 12 study arms and was conducted between June 2011 and April 2016. Patients with chronic wounds present for more than three months, irrespective of previous treatments, were recruited into this study. Quantitative wound swabs were obtained before and after a 20-minute, wet-to-moist cleansing, using different wound irrigation solutions. Sterile 0.9% saline served as a control. We recruited 308 patients, of which 260 patients with 299 chronic wounds were eligible for analysis. Staphylococcus aureus was the most common recovered (25.5%) microorganism, of which 8% were meticillin-resistant Staphylococcus aureus (MRSA) strains. Although 0.9% saline supported cleansing of the wound bed, it did not significantly reduce the bacterial burden. The highest reduction of bacterial burden was achieved with an aqueous solution containing betaine, zinc and polyhexamethylene biguanide (polihexanide; ln RF=3.72), followed by a 3% saline solution containing 0.2% sodium hypochlorite (ln RF=3.40). The most statistically significant reduction of bacterial burden, although not the highest, was achieved with povidone-iodine (ln RF=2.98; p=0.001) and an irrigation solution containing sea salt 1.2% and NaOCl 0.4% (ln RF=2.51; p=0.002). If a reduction of bacterial burden is warranted, wound irrigation solutions containing a combination of hypochlorite/hypochlorous acid, or antiseptics such as polihexanide, octenidine or povidone-iodine, ought to be considered.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease-like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT-D). Immunization with rCPAF/CpG significantly reduced vaginal CT-D shedding and induced resolution of infection by day 24, compared to day 33 in CpG alone treated and challenged animals. Immunization induced robust anti-rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high level 4 weeks post challenge. Upregulation of antigen specific IFN-γ gene expression was observed in rCPAF/CpG vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG-immunized guinea pigs compared to CpG-immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

Natural Anti-Infective Pulmonary Proteins: In Vivo Cooperative Action of Surfactant Protein SP-A and the Lung Antimicrobial Peptide SP-BN.

The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 μM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.

Comparative study of two antimicrobial dressings in infected leg ulcers: a pilot study

The aim of the study was to compare the efficacy of a microorganism-binding (MB) dressing with a silver-containing hydrofiber (SCH) dressing in controlling the bacterial loads of heavily colonised or locally infected chronic venous leg ulcers, before surgical management with homologous skin grafts. A randomised comparative single centre study recruited patients presenting with hard-to-heal critically colonised or locally infected leg ulcers, who could be treated with skin grafting. Inclusion criteria included; ulcers of vascular aetiology, over 18 years old, a wound duration ≥6 months and ankle brachial index (ABPI) >0.6. Patients were randomly assigned to treatment with SCH dressings (Aquacel Ag) or MB dressing (Cutimed Sorbact). Dressings were changed daily over a four-day observation period, after which they were taken for a skin grafting procedure. Swab samples from ulcer beds were taken in order to quantify the bacterial load at inclusion (D0) and at the end of the observation period day 4 (D4). No antibiotics were administered before or during the evaluation period. Both groups (n=20 SCH, n=20 MB) were similar in gender, age, pathophysiology (both had 15 patients with venous leg ulcers and 5 with arterial leg ulcers), ulcer surface, ulcer duration, treatment-related pain and initial bacterial load. Analysing bacterial load variation showed a significant reduction of bacterial burden at D4 in both groups. In the SCH group, we found an average bacterial load reduction of 41.6%, with an average reduction of 73.1% in the MB group (p< 0.00001). No serious adverse events were reported. Our evaluation confirmed that MB and SCH dressings are effective in reducing the bacterial burden in critically colonised or locally infected chronic leg ulcers, without inducing adverse events, with MB dressings significantly more effective. There were no external sources of funding for this study. The authors have no conflicts of interest to declare.

Correlates of Immune Protection following Cutaneous Immunization with an Attenuated Burkholderia pseudomallei Vaccine

Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.

Immunogenicity and protective efficacy of heterologous prime-boost regimens with mycobacterial vaccines and recombinant adenovirus- and poxvirus-vectored vaccines against murine tuberculosis

To evaluate regimens using bacillus Calmette-Guérin (BCG) or recombinant BCG (rBCG) overexpressing Ag85B for priming, followed by boosting with a modified vaccinia virus Ankara strain (MVA) and/or adenovirus vector (AD) expressing an Ag85B-ESAT6 fusion protein. Cellular and humoral immune responses were determined after subcutaneous vaccination, which was employed to trigger systemic immunity against intravenous infection in a mouse model of tuberculosis (TB). Bacterial loads and lung histology were evaluated. The relative IgG2a and IgG1 antibody levels indicated that the viral-vectored vaccines generated a T-helper type 1 (Th1)-biased response after two doses of viral boost vaccinations. Boosting BCG-primed mice with viral vaccines induced a Th1 immune response that included both CD4 and CD8 T-cells generating antigen-specific interferon-gamma (IFN-γ) and CD8 T cytotoxic activity. Only mice vaccinated with two different viral boosters after BCG priming exhibited a significant reduction in bacterial burden in the lung after challenge. Histology examinations confirmed the attenuation of lung damage and more compact granulomas. After mycobacteria priming, boosting with AD85B-E6 followed by MVA85B-E6 afforded better protection than the reverse order of administration of the viral vectors. This study demonstrates the potential of multiple heterologous viral booster vaccines, although the exact correlates of protection and optimal regimens should be further investigated for the rational design of future vaccine strategies.

Novel Structurally Designed Vaccine for S. aureus α-Hemolysin: Protection against Bacteremia and Pneumonia

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.