A two-component, multimeric endolysin encoded by a single gene.

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis.

Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds.

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

Thioridazine reduces resistance of methicillin-resistant staphylococcus aureus by inhibiting a reserpine-sensitive efflux pump.

Previous studies suggested that the phenothiazine chlorpromazine (CPZ) could reverse or reduce the antibiotic resistance of bacteria. In some areas of the world, the majority of Staphylococcus aureus isolates are now resistant to methicillin, prompting this study to see whether such resistance can be altered by phenothiazine thioridazine (TZ), an agent with equal antibacterial activity, which is free of the severe side-effects associated with chronic administration of CPZ. The results indicated that, whereas methicillin-sensitive strains of Staphylococcus aureus (MSSA) were not rendered more susceptible to oxacillin, resistance to oxacillin by highly-resistant strains (MRSA) could be significantly reduced by sub-inhibitory concentrations of TZ. Reserpine, an inhibitor of efflux pumps, was also shown to reduce the resistance of MRSA strains to oxacillin in a concentration-dependent manner. The phenothiazines have been shown, by others, to inhibit the efflux pumps of bacteria and the mechanism by which MRSA are rendered more susceptible to oxacillin in the presence of TZ is believed to be due to a similar efflux pump.

Gamma delta T cell responses associated with the development of tuberculosis in health care workers.

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.

Inhibition of the Carpobrotus edulis methanol extract on the growth of phagocytosed multidrug-resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus.

The Carpobrotus edulis methanol extract, inactive against the methicillin-resistant Staphylococcus aureus or the multidrug-resistant Mycobacterium tuberculosis, does inhibit the growth of these two bacteria once they are phagocytosed by monocyte derived human macrophages.

Leishmaniasis: efflux pumps and chemoresistance.

Resistance of parasitic protozoa such as Leishmania to therapeutic drugs continues to escalate in developing countries. Treatment programs for human leishmaniasis are still based on pentavalent antimonials but resistance to these compounds has been a persistent problem. In many instances, resistance of the parasite is due to over-expressed ABC efflux pumps. In Leishmania different classes of ABC transporters extrude antimonials, azoles and folates resulting in drug-resistant phenotypes. Although some studies have focused on developing inhibitors against these resistant phenotypes, new efficient modulators that are able to inhibit drug efflux are needed.

Mycobacterial efflux pumps and chemotherapeutic implications.

The demonstration of the existence of active efflux pumps in mycobacteria raises the question of whether or not these can increase in number and activity rendering wild-type mycobacteria increasingly resistant to a given antibiotic. This could be a mechanism by which mutated resistant strains become better fit to the selective environment. Mycobacterium tuberculosis genome analysis reveals several genes encoding putative drug efflux pumps. During the course of tuberculosis chemotherapy many of these pumps might play a role in the survival of the mycobacterial populations. Compounds capable of inactivating these pumps could improve anti-tuberculous therapeutics.

Intracellular activity of clinical concentrations of phenothiazines including thioridiazine against phagocytosed Staphylococcus aureus.

The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3+ T cell production of IFN-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.

Phenothiazines alter resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) to oxacillin in vitro.

Mechanisms of antibiotic resistance of bacteria include efflux pumps which extrude the antibiotic prior to reaching its target. Phenothiazines inhibit the activity of some efflux pumps thereby altering the susceptibility of bacteria. This study demonstrated that chlorpromazine and thioridazine reduce the susceptibility of methicillin-resistant strains (MRSA) but not that of methicillin-susceptible Staphylococcus aureus (MSSA) strains to oxacillin (MIC of oxacillin reduced from >500 to 10 mg/l). Reserpine, an inhibitor of antibiotic efflux pumps also reduced the resistance of MRSA strains to oxacillin suggesting the presence of an efflux pump that contributes to antibiotic resistance of MRSA strains.

Cytokine expression during the outcome of canine experimental infection by Leishmania infantum.

In this study, the cytokines, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-12 p40, IL-6 and IL-10, expressed by peripheral blood mononuclear cells of 13 beagle dogs inoculated with Leishmania infantum amastigotes, were analysed during a period of up to 23 months. The course of infection was monitored through clinical and parasitological examinations, haematological alterations and serum antileishmania antibody levels. Dogs developed symptomatic infections with haematological alterations, humoral immune response and reduced specific lymphoproliferative response. Parasite presence was detected in bone marrow, popliteal lymph node and skin. Specifically stimulated cytokine transcripts were generally observed in a low proportion of dogs, except at months 9, 10 and 11 post-infection where there was a considerable increase in the proportion of dogs expressing IFN-gamma and IL-2 mRNA. IL-12 p40 and IL-10 transcripts were sporadically detected in few animals. In non-infected animals, IFN-gamma mRNA was the only detectable cytokine but only in cells cultured in the presence of concanavalin A (ConA). The low proportion of animals expressing specific cytokines, during the first 8 months of infection associated with evidences of parasite dispersion without clinical signs of disease, suggests the occurrence of a relatively "silent establishment" of the parasite avoiding adverse host-cell-mediated immunological reactions. The humoral immune response displayed in these animals, the cell-mediated immunosuppression, nor the disease severity could be related with the expression of IL-10. The predominance of a Th1 type response for a relatively short period indicates that these cytokines are required to control the infection delaying the appearance of progressive disease.

Clinical concentrations of thioridazine enhance the killing of intracellular methicillin-resistant Staphylococcus aureus: an in vivo, ex vivo and electron microscopy study.

Chlorpromazine (CPZ) is concentrated by human macrophages where it kills intracellular mycobacteria when the concentration outside the macrophage is sub-clinical. We have previously demonstrated that thioridazine (TZ), a much milder phenothiazine, has similar activity and kills intracellular methicillin-susceptible S. aureus at sub-clinical concentrations. We have extended this latter study to include methicillin-resistant S. aureus (MRSA) and show that TZ kills intracellular MRSA at clinically relevant concentrations. The ultrastructure of MRSA exposed to in vitro concentrations of TZ just below its MIC and that of MRSA phagocytosed by macrophages previously exposed to a clinically relevant concentration of TZ was also studied. TZ inhibits the replication of phagocytosed MRSA, affecting the structure of the cell envelope, resulting in lysis of the bacterium 6 hours post-phagocytosis. These ultrastructural changes are identical to those produced in vitro by a TZ concentration that is just below the MIC. Because macrophage intracellular MRSA is not killed by the macrophage and its intracellular location protects it from antibiotics that are unable to reach that site, recurrent infections which result may be successfully managed with the use of TZ.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapies.

Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the '3Rs' principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiology of infected diabetic wound healing and includes the current standard treatment (that is, debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for experiments with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.

Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes.

Expression of eight transporter genes of Escherichia coli K-12 and its DeltaacrAB mutant prior to and after induction of both strains to tetracycline resistance and after reversal of induced resistance were analyzed by quantitative reverse transcriptase PCR. All transporter genes were overexpressed after induced resistance with acrF being 80-fold more expressed in the DeltaacrAB tetracycline-induced strain.

Direct application of the INNO-LiPA Rif.TB line-probe assay for rapid identification of Mycobacterium tuberculosis complex strains and detection of rifampin resistance in 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis.

The INNO-LiPA Rif.TB assay for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampin (RIF) resistance has been evaluated with 360 smear-positive respiratory specimens from an area of high incidence of multidrug-resistant tuberculosis (MDR-TB). The sensitivity when compared to conventional identification/culture methods was 82.2%, and the specificity was 66.7%; the sensitivity and specificity were 100.0% and 96.9%, respectively, for the detection of RIF resistance. This assay has the potential to provide rapid information that is essential for the effective management of MDR-TB.

Chlorpromazine has intracellular killing activity against phagocytosed Staphylococcus aureus at clinical concentrations.

Chlorpromazine (CPZ) has in vitro antimicrobial activity against Staphylococcus aureus at concentrations that greatly exceed those achieved clinically. It is concentrated by tissues that are rich in macrophages and it is active against phagocytosed mycobacteria when the concentration in the medium is compatible with that achieved clinically. In this report we show that nontoxic concentrations of CPZ below clinical levels have killing activity against S. aureus phagocytosed by human monocyte-derived macrophages that have nominal killing activity against these bacteria. Little or no resistance to the antimicrobial activity of this compound is anticipated to result because of its large number of cellular targets. Therefore, CPZ may have a role in the management of intracellular staphylococcal infections that normally require the use of antibiotics whose potential toxicity exceeds that associated with short-term management with CPZ.

Isoniazid-induced transient high-level resistance in Mycobacterium tuberculosis.

An American Type Culture Collection reference strain and eight clinical strains of Mycobacterium tuberculosis, all of which were susceptible to isoniazid (INH) (mean MIC, 0.06 mg/liter) and negative for the Ser315Thr katG mutation, were left in their BACTEC 12B vials (for use with the BACTEC 460-TB method) containing 0.1 mg of INH per liter for periods of up to 28 days after the completion of the antibiotic susceptibility test. Each eventually grew to levels compatible with those of INH-resistant strains. Successive passages in INH-containing BACTEC 12B vials and onto solid media showed that the resistance noted above was maintained. Successive passages of these M. tuberculosis strains in which INH resistance had been induced into BACTEC 12B vials or solid media containing stepwise increases in INH concentrations eventually yielded organisms resistant to 20 mg of INH per liter. Transfer of cells in which INH resistance had been induced to drug-free medium followed by repeated passages in that medium eventually yielded organisms whose susceptibility to INH was identical to that of the original parent strains. The cycle of induced INH resistance could be repeated with these now INH-susceptible cells. The use of M. tuberculosis identification probes and IS6110-based restriction fragment length polymorphism analyses of cultures throughout the induction of INH resistance and the reversal of resistance in drug-free medium eliminated the possibility that the culture was contaminated or that the initial specimen had a mixed type of infection. Induced high-level resistance to INH (20 mg/liter) could be reduced 100-fold with a subinhibitory concentration of reserpine but not with verapamil. These results collectively suggest that high-level resistance to INH can be induced in INH-susceptible M. tuberculosis strains by the induction of a reserpine-sensitive efflux mechanism.