Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

An Investigation into the Re-Emergence of Disease Following Cessation of Antibiotic Treatment in Balb/c Mice Infected with Inhalational Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a multifaceted disease. A proportion of the mortality and morbidity reported as a result of infection with this organism may be due to the premature cessation of antibiotic therapy typically lasting for several months. The progression of re-emergent disease was characterised in Balb/c mice following cessation of a 14 day treatment course of co-trimoxazole or finafloxacin, delivered at a human equivalent dose. Mice were culled weekly and the infection characterised in terms of bacterial load in tissues, weight loss, clinical signs of infection, cytokine levels and immunological cell counts. Following cessation of treatment, the infection re-established in some animals. Finafloxacin prevented the re-establishment of the infection for longer than co-trimoxazole, and it is apparent based on the protection offered, the development of clinical signs of disease, bodyweight loss and bacterial load, that finafloxacin was more effective at controlling infection when compared to co-trimoxazole.

Finafloxacin Is an Effective Treatment for Inhalational Tularemia and Plague in Mouse Models of Infection.

Infection with aerosolized Francisella tularensis or Yersinia pestis can lead to lethal disease in humans if treatment is not initiated promptly. Finafloxacin is a novel fluoroquinolone which has demonstrated broad-spectrum activity against a range of bacterial species in vitro, in vivo, and in humans, activity which is superior in acidic, infection-relevant conditions. Human-equivalent doses of finafloxacin or ciprofloxacin were delivered at 24 h (representing prophylaxis) or at 72 or 38 h (representing treatment) postchallenge with F. tularensis or Y. pestis, respectively, in BALB/c mouse models. In addition, a short course of therapy (3 days) was compared to a longer course (7 days). Both therapies provided a high level of protection against both infections when administered at 24 h postchallenge, irrespective of the length of the dosing regimen; however, differences were observed when therapy was delayed. A benefit was demonstrated with finafloxacin compared to ciprofloxacin in both models when therapy was delivered later in the infection. These studies suggest that finafloxacin is an effective alternative therapeutic for the prophylaxis and treatment of inhalational infections with F. tularensis or Y. pestis.

Role of DNA Repair and Protective Components in Bacillus subtilis Spore Resistance to Inactivation by 400-nm-Wavelength Blue Light.

The high intrinsic decontamination resistance of Firmicutes spores is important medically (disease) and commercially (food spoilage). Effective methods of spore eradication would be of considerable interest in the health care and medical product industries, particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light at a ∼400 nm wavelength is one such treatment that has drawn significant interest. This work has determined the resistance of spores to blue light in an extensive panel of Bacillus subtilis strains, including wild-type strains and mutants that (i) lack protective components such as the spore coat and its pigment(s) or the DNA protective α/ß-type small, acid-soluble spore proteins (SASP); (ii) have an elevated spore core water content; or (iii) lack enzymes involved in DNA repair, including those for homologous recombination and nonhomologous end joining (HR and NHEJ), apurinic/apyrimidinic endonucleases, nucleotide and base excision repair (NER and BER), translesion synthesis (TLS) by Y-family DNA polymerases, and spore photoproduct (SP) removal by SP lyase (SPL). The most important factors in spore blue light resistance were determined to be spore coats/pigmentation, α/ß-type SASP, NER, BER, TLS, and SP repair. A major conclusion from this work is that blue light kills spores by DNA damage, and the results in this work indicate at least some of the specific DNA damage. It appears that high-intensity blue light could be a significant addition to the agents used to kill bacterial spores in applied settings.IMPORTANCE Effective methods of spore inactivation would be of considerable interest in the health care and medical products industries, particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light radiation is one such treatment that has drawn significant interest. In this work, all known spore-protective features, as well as universal and spore-specific DNA repair mechanisms, were tested in a systematic fashion for their contribution to the resistance of spores to blue light radiation.

Species-specific antifungal activity of blue light.

Fungal pathogens represent a significant threat to immunocompromised patients or individuals with traumatic injury. Strategies to efficiently remove fungal spores from hospital surfaces and, ideally, patient skin thus offer the prospect of dramatically reducing infections in at-risk patients. Photodynamic inactivation of microbial cells using light holds considerable potential as a non-invasive, minimally destructive disinfection strategy. Recent data indicate that high-intensity blue light effectively removes bacteria from surfaces, but its efficacy against fungi has not been fully tested. Here we test a wide range of fungi that are pathogenic to humans and demonstrate that blue light is effective against some, but not all, fungal species. We additionally note that secondary heating effects are a previously unrecognized confounding factor in establishing the antimicrobial activity of blue light. Thus blue light holds promise for the sterilization of clinical surfaces, but requires further optimization prior to widespread use.

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms.

UNLABELLED: The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm(2) to 108 J/cm(2)). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE: Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further investigation.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 10(7) spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

Trimethoprim/sulfamethoxazole (co-trimoxazole) prophylaxis is effective against acute murine inhalational melioidosis and glanders.

Burkholderia pseudomallei is the causative agent of the disease melioidosis, which is prevalent in tropical countries and is intractable to a number of antibiotics. In this study, the antibiotic co-trimoxazole (trimethoprim/sulfamethoxazole) was assessed for the post-exposure prophylaxis of experimental infection in mice with B. pseudomallei and its close phylogenetic relative Burkholderia mallei, the causative agent of glanders. Co-trimoxazole was effective against an inhalational infection with B. pseudomallei or B. mallei. However, oral co-trimoxazole delivered twice daily did not eradicate infection when administered from 6h post exposure for 14 days or 21 days, since infected and antibiotic-treated mice succumbed to infection following relapse or immunosuppression. These data highlight the utility of co-trimoxazole for prophylaxis both of B. pseudomallei and B. mallei and the need for new approaches for the treatment of persistent bacterial infection.

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II.

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.

The cationic peptide magainin II is antimicrobial for Burkholderia cepacia-complex strains.

This study was undertaken to determine the antibacterial activity of eight cationic antimicrobial peptides towards strains of genomovars I-V of the Burkholderia cepacia complex (Bcc) in time-kill assays. All but one of the peptides failed to show activity against the panel of test strains. The exception was magainin II, a 23 aa peptide isolated from the epidermis of the African clawed frog, Xenopus laevis, which exhibited significant bactericidal activity for Bcc genomovars most frequently associated with lung infection of patients with cystic fibrosis. In vitro studies indicated that magainin II protected a human bronchial epithelial cell line (BEAS-2B) from killing by Bcc and suggest that this peptide may have therapeutic potential against these organisms.

Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis.

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 10(5) CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

Proteolytic degradation of human antimicrobial peptide LL-37 by Bacillus anthracis may contribute to virulence.

In this paper we report on the susceptibilities of a range of Bacillus species to the human antimicrobial peptide LL-37. B. subtilis showed a low level of resistance to killing by LL-37 (50% growth-inhibitory concentration [GI50], 1 microg/ml). B. cereus and B. thuringiensis showed intermediate levels of resistance to killing (GI50s, 33 microg/ml and 37 microg/ml, respectively). B. anthracis showed the highest level of resistance (GI50s, 40 to 66 microg/ml). The degradation of LL-37 by B. anthracis culture supernatant was blocked by the metalloprotease inhibitors EDTA and 1,10-phenanthroline, and the gene encoding the protease responsible for LL-37 degradation was not plasmid borne. Our findings suggest that alongside the classical plasmid-based virulence determinants, extracellular metalloproteases of B. anthracis may play a role in survival in the host.

The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria.

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA.

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.