Antimicrobial and antibiofilm potential of biosurfactants isolated from lactobacilli against multi-drug-resistant pathogens.

BACKGROUND: Biosurfactants (BS) are amphiphilic compounds produced by microbes, either on the cell surface or secreted extracellularly. BS exhibit strong antimicrobial and anti-adhesive properties, making them good candidates for applications used to combat infections. In this study, our goal was to assess the in vitro antimicrobial, anti-adhesive and anti-biofilm abilities of BS produced by Lactobacillus jensenii and Lactobacillus rhamnosus against clinical Multidrug Resistant (MDR) strains of Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus (MRSA). Cell-bound BS from both L. jensenii and L. rhamnosus were extracted and isolated. The surface activities of crude BS samples were evaluated using an oil spreading assay. The antimicrobial, anti-adhesive and anti-biofilm activities of both BS against the above mentioned MDR pathogens were determined. RESULTS: Surface activities for both BS ranged from 6.25 to 25 mg/ml with clear zones observed between 7 and 11 cm. BS of both L. jensenii and L. rhamnosus showed antimicrobial activities against A. baumannii, E. coli and S. aureus at 25-50 mg/ml. Anti-adhesive and anti-biofilm activities were also observed for the aforementioned pathogens between 25 and 50 mg/ml. Finally, analysis by electron microscope indicated that the BS caused membrane damage for A. baumannii and pronounced cell wall damage in S. aureus. CONCLUSION: Our results indicate that BS isolated from two Lactobacilli strains has antibacterial properties against MDR strains of A. baumannii, E. coli and MRSA. Both BS also displayed anti-adhesive and anti-biofilm abilities against A. baumannii, E. coli and S. aureus. Together, these capabilities may open up possibilities for BS as an alternative therapeutic approach for the prevention and/or treatment of hospital-acquired infections.

A Porcine Wound Model of Acinetobacter baumannii Infection.

Objective: To better understand Acinetobacter baumannii pathogenesis and to advance drug discovery against this pathogen, we developed a porcine, full-thickness, excisional, monospecies infection wound model. Approach: The research was facilitated with AB5075, a previously characterized, extensively drug-resistant A. baumannii isolate. The model requires cyclophosphamide-induced neutropenia to establish a skin and soft tissue infection (SSTI) that persists beyond 7 days. Multiple, 12-mm-diameter full-thickness wounds were created in the skin overlying the cervical and thoracic dorsum. Wound beds were inoculated with 5.0 × 104 colony-forming units (CFU) and covered with dressing. Results: A. baumannii was observed in the wound bed and on the dressing in what appeared to be biofilm. When bacterial burdens were measured, proliferation to at least 106 CFU/g (log106) wound tissue was observed. Infection was further characterized by scanning electron microscopy (SEM) and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) staining. To validate as a treatment model, polymyxin B was applied topically to a subset of infected wounds every 2 days. Then, the treated and untreated wounds were compared using multiple quantitative and qualitative techniques to include gross pathology, CFU burden, histopathology, PNA-FISH, and SEM. Innovation: This is the first study to use A. baumannii in a porcine model as the sole infectious agent. Conclusion: The porcine model allows for an additional preclinical assessment of antibacterial candidates that show promise against A. baumannii in rodent models, further evaluating safety and efficacy, and serve as a large animal in preclinical assessment for the treatment of SSTI.

Transcriptional profiling of Francisella tularensis infected peripheral blood mononuclear cells: a predictive tool for tularemia.

In this study, we analyzed temporal gene expression patterns in human peripheral blood mononuclear cells (PBMCs) infected with the Francisella tularensis live vaccine strain from 1 to 24 h utilizing a whole human Affymetrix gene chip. We found that a considerable number of induced genes had similar expression patterns and functions as reported previously for gene expression profiling in patients with ulceroglandular tularemia. Among the six uniquely regulated genes reported for tularemia patients as being part of the alarm signal gene cluster, five, namely caspase 1, PSME2, TAP-1, GBP1, and GCH1, were induced in vitro. We also detected four out of the seven potential biomarkers reported in tularemia patients, namely TNFAIP6 at 4 h and STAT1, TNFSF10, and SECTM1 at 16 and 24 h. These observations underscore the value of using microarray expression profiling as an in vitro tool to identify potential biomarkers for human infection and disease. Our results indicate the potential involvement of several host pathways/processes in Francisella infection, notably those involved in calcium, zinc ion binding, PPAR signaling, and lipid metabolism, which further refines the current knowledge of F. tularensis infection and its effects on the human host. Ultimately, this study provides support for utilizing in vitro microarray gene expression profiling in human PBMCs to identify biomarkers of infection and predict in vivo immune responses to infectious agents.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells.

BACKGROUND: Effective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs. METHODS: To detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays. RESULTS: We found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin. CONCLUSION: Host gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.

Temporal cytokine profiling of Francisella tularensis-infected human peripheral blood mononuclear cells.

BACKGROUND AND PURPOSE: Francisella tularensis is an intracellular bacterium known to replicate in monocytes and macrophages and cause tularemia in humans. Because of its infectious nature, F. tularensis is considered a biowarfare agent. Early cytokine profiles of Francisella-infected human peripheral blood mononuclear cells were evaluated. METHODS: Populations of human peripheral blood mononuclear cells were infected in vitro with F. tularensis live vaccine strain at a very low multiplicity of infection of 1:10 (bacteria:cells). A multiplex bead kit which analyzes 30 cytokines, chemokines and growth factors was utilized to measure secreted cytokines in cell supernatants 1, 4, 8, 16, and 24 h post-infection. RESULTS: Compared with uninfected controls, infected cells showed no increase in cytokine secretion at 1 and 4 h, implying a threshold for activation of immune responses. Starting at 8 h post-infection and continuing through to 24 h, an array of cytokines and growth factors was secreted by the infected cells. Some cytokines not previously associated with Francisella infection in humans were detected at 8 h, including interleukin-17 and interleukin-1 receptor agonist and vascular endothelial growth factor. CONCLUSIONS: The cytokine profiles of F. tularensis-infected peripheral blood mononuclear cells indicate an intricate pattern of both pro- and anti-inflammatory responses, including early T-cell activation.

Transcriptional profiling of recall responses to Francisella live vaccine strain.

Global gene expression profile changes were monitored in human peripheral blood mononuclear cells (PBMCs) after challenge with the live vaccine strain (LVS) of Francisella tularensis. Because these PBMCs were from individuals previously immunized with LVS, stimulating these cells with LVS should activate memory responses. The Ingenuity Pathway Analysis tool identified pathways, functions, and networks associated with this in vitro recall response, including novel pathways triggered by the memory response. Dendritic cell (DC) maturation was the most significant among the more than 25 relevant pathways discovered. Interleukin 15, granulocyte-macrophage colony-stimulating factor, and triggering receptor expressed on myeloid cells 1 signaling pathways were also significant. Pathway analysis indicated that Class 1 antigen presentation may not be optimal with LVS vaccination. The top three biological functions were antigen presentation, cell-mediated and humoral immune responses. Network analysis revealed that the top network associated with these functions had IFNγ and TNFα in central interactive positions. Our results suggest that DC maturation is a key factor in the recall responses and that more effective antigen processing and presentation is needed for cytotoxic T lymphocyte responses. Taken together, these considerations are critical for future tularemia vaccine development studies.

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 µg/mL. The MIC of KS-30 fragment varied from 8.0 to 16 µg/mL and the KR-20 fragment MIC ranged from 16 to 64 µg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25-1 µg/mL. By 0.5h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 µg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 µg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 µg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64-128 µg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 µg/mL. At 24h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.

Th17 cytokines in recall responses against Francisella tularensis in humans.

To determine whether cytokines and T-cell subsets other than Th1 cells contribute to secondary immune responses against Francisella species, we investigated production of Th17-associated cytokines IL-17 and IL-22 in a recall response to Francisella tularensis. Peripheral blood mononuclear cells (PBMCs) from volunteers previously immunized with the F. tularensis live vaccine strain (LVS) were stimulated in vitro with bacterial lysates of LVS or a nonpathogenic type A B38 strain. Gene expression analysis by real-time PCR showed that IL-17 and IL-22 transcripts were induced in immune PBMCs at a significantly higher level than in cells from nonvaccinated volunteers stimulated with LVS or B38 antigens at 24 h. In addition, we detected both cell-associated and secreted IL-22 at 24 h after stimulation and IL-17 at 72 h post-stimulation. Intracellular IL-22 and IL-17 were observed in memory CD4+ cells and less in memory CD8+ cells. These findings suggest that Th17 responses in addition to the Th1 response may play an important role in adaptive immunity against Francisella.

Interferon-gamma associated cytokines and chemokines produced by spleen cells from Brucella-immune mice.

It is known that interferon (IFN)-gamma plays a critical role in protection against brucellosis. In this study we have investigated several cytokines and chemokines that are associated with IFN-gamma for potential in vitro correlates of protection. We cultured spleen cells in vitro from mice immunized orally with a live, attenuated Brucella melitensis vaccine candidate (WR201) and stimulated these cells with a lysate of B. melitensis. Differential gene expression of several cytokines and chemokines in stimulated spleen cells was analysed by real-time PCR, and secreted proteins were determined by ELISA. Immunized mice produced higher levels of both protein and gene transcripts for IFN-gamma, interleukin (IL)-2, IL-18 and MIP1-alpha. Immunized mice also had elevated gene expression levels for IL12-p40, IL23-p19, IP-10, MIG and MCP-1 when compared to normal mice. In this study we have identified new cytokines and chemokines as potential immune correlates in responses to protection in Brucella-vaccinated mice.

Identification of novel genes in the memory response to Brucella infection by cDNA arrays.

This study investigated memory responses in immune mice spleen cells to brucellosis by gene expression utilizing cDNA micro arrays. Out of a total of 1176 cDNA's 21 genes were differentially regulated in three independent experiments, and generally supported a Th1 type immune response. 10 genes were validated by real time PCR, and 3 genes (CD 86, CD 40 L and CD 132) were also analyzed by Flow Cytometry for surface protein expression. We extended these findings by studying the expression of five selected genes (IRF 1, SOCS 1, IL 2 R, IRF 7, and CXCR 4) in two independent groups of Brucella immunized mice. In this study we show the potential application of utilizing gene arrays to identify and establish new correlates of protection against a cell mediated immune response.

Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M.

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.