Antibacterial activity of blue light against nosocomial wound pathogens growing planktonically and as mature biofilms

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/cm2 to 108 J/cm2). 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

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

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

Role of DNA Repair and Protective Components in Bacillus subtilis Spore Resistance to Inactivation by 400 nm 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 products industries; particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light at ∼ 400 nm 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/β-type small acid-soluble spore proteins (SASP); ii) have an elevated spore core water content; or iii) lack enzymes involved in DNA repair, including homologous recombination and non-homologous end joining (HR and NHEJ), apurinic/apyrimidinic endonucleases, nucleotide- and base excision repair (NER & 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/β-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

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

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 5x10⁷ 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

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

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

Expression of BaTdp increases lipidation of cellular LC3.

<p>(A) HEK293T cells were transfected with plasmid encoding either BaTdp (wild type) or BaTdp (G164A) protein. After 24 or 48 hours post-transfection, cells were lysed in PBS containing 1% SDS and lysates were resolved by SDS-PAGE and analysed by Western blot using an anti-LC3B primary antibody or anti-GAPDH antibody for loading control. Result shown is representative of three independent experiments. Figures show cropped lanes from non-adjacent lanes of the same blot. Images have been cropped to only show the regions of interest. (Uncropped versions of blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.s001" target="_blank">S1 Fig</a>) (B) Western blots were analysed by densitometry using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158575#pone.0158575.ref043" target="_blank">43</a>]. For each sample, the LC3B-II value was normalised against corresponding GAPDH value. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. *P < 0.05 (by two-tailed Student’s t-test).</p

<i>In vitro</i> signalling assays indicate that BaTdp does not affect TLR signalling.

<p>(A) HEK-TLR4 cells were transfected with increasing amounts of vector DNA containing the gene encoding BaTdp (wildtype) or the BaTdp (G164A). 24 hours post-transfection, cells were challenged with LPS (0.1 μg/mL) and the production of inflammatory cytokines, TNFα and IL-8, was assessed by ELISA 24 hours later. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. (B) HEK-TLR2 cells were transfected with plasmid encoding for BaTdp or BaTdp (G164A) and challenged with LTA (1 μg/mL) 24 hours post-transfection, followed by assessment of IL-8 content in the culture supernatant by ELISA after an additional 24 hours. Bars represent mean values of three independent experiments. <i>Error bars</i>, SD of triplicates. (C) HEK-TLR4 cells were co-transfected with plasmids encoding FLAG-tagged MyD88 and GFP-tagged BaTdp. 24 hours post transfection, after being stained with mitotracker dye to stain cellular mitochondria, cells were fixed, permeabilised and stained with an anti-FLAG antibody and visualised with a red fluorescent secondary antibody. All scale bars, 10 μm. Original magnification ×100.</p