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Graphical abstract, Highlights • Rhodomyrtone, a bioactive acylphloroglucinol compound isolated from the leaves of Rhodomyrtus tomentosa, has been scientifically evidenced as a potential antibacterial agent. • Rhodomyrtone did not cause any signs of toxicity in invertebrate (Galleria mellonella) and vertebrate (zebrafish, murine) models. • Rhodomyrtone at 256 μg/mL did not cause any observable human erythrocyte haemolysis. • As the minimal inhibitory concentration of rhodomyrtone against most Gram-positive pathogens is 0.5-1 μg/mL, the results suggest that it should produce no toxic effects at concentrations used in human., Background Rhodomyrtus tomentosa (Aiton) Hassk. has been traditionally used to relieve various diseases. Rhodomyrtone, a bioactive acylphloroglucinol compound isolated from the leaves of Rhodomyrtus tomentosa, has been scientifically evidenced as a potential antibacterial agent. This study aimed to assess safety of rhodomyrtone in both invertebrate and vertebrate models. Material and Methods Safety of rhodomyrtone was determined in an invertebrate model, Galleria mellonella as well as vertebrate models including zebrafish (Danio rerio) and murine. In addition, toxicity to human erythrocytes was also measured. Results Treatment of Galleria mellonella with rhodomyrtone at 100 mg/kg body weight up to four days showed no visible toxic effects (100 % survival). In zebrafish embryo model, at least 80 % survival of embryos was demonstrated when treated with rhodomyrtone at 0.5 μg/mL for three days. Prior to clinical trial, it is a prerequisite that rhodomyrtone has to be evaluated for its biocompatibility with human blood components. The results displayed that rhodomyrtone at 256 μg/mL did not cause any observable human erythrocyte haemolysis. Furthermore, preclinical assessment of rhodomyrtone formulation justified potential applications of rhodomyrtone in humans. Oral toxicity testing in a mouse model indicated the absence of systemic toxicity when the animals received up to 5000 mg/kg body weight of rhodomyrtone formulation for a period of fourteen days. Conclusions As the minimal inhibitory concentration of rhodomyrtone against most Gram-positive pathogens is 0.5−1 μg/mL, the results suggest that it should produce no toxic effects at concentrations used in human, thus support further development in pharmaceutical industries and public health applications.

The aim of the study was to determine the efficacy of dual β-lactam combination treatments derived from eight approved drugs against Galleria mellonella larvae infected with MDR strains of P. aeruginosa. Carbapenem-resistant P. aeruginosa NCTC 13437 and an unrelated clinical isolate were used to infect G. mellonella larvae and the efficacy of twenty-eight dual β-lactam combination therapies were compared to their constituent monotherapies. For the most potent combinations identified, penicillin-binding protein (PBP) inhibition profiles were measured and compared with each constituent antibiotic. Five of the dual β-lactam combinations resulted in greater than 70% survival of infected G. mellonella. Two combinations showed potent, enhanced efficacy versus both strains − ceftazidime + meropenem and aztreonam + meropenem. Comparison of PBP inhibition profiles revealed that the enhanced efficacy of these two dual β-lactam combinations could not be explained by more potent inhibition of PBPs or inhibition of a broader range of PBPs. A possible contribution to the enhanced efficacy of the combinations could be stimulation of innate immunity via increased haemocyte numbers compared to their constituent monotherapies. Combinations of β-lactam antibiotics show promise in overcoming MDR P. aeruginosa and are worthy of additional study and development.

Funding: University of St Andrews. Multi-drug resistant (MDR) Klebsiella pneumoniae represent a global threat to healthcare due to lack of effective treatments and high mortality rates. The aim of this research was to explore the potential of administering zidovudine (AZT) in combination with an existing antibiotic to treat resistant K. pneumoniae infections. Two MDR K. pneumoniae strains were employed, producing either the NDM-1 or KPC-3 carbapenemase. Efficacy of combinations of AZT with meropenem were compared with monotherapies against infections in Galleria mellonella larvae by measuring larval mortality and bacterial burden. The effect of the same combinations in vitro was determined via checkerboard and time-kill assays. In vitro, both K. pneumoniae strains were resistant to meropenem but were susceptible to AZT. In G. mellonella, treatment with either AZT or meropenem alone offered minimal therapeutic benefit against infections with either strain. In contrast, combination therapy of AZT with meropenem presented significantly enhanced efficacy compared to monotherapies. This was correlated with prevention of bacterial proliferation within the larvae but not elimination. Checkerboard assays showed that the interaction between AZT and meropenem was not synergistic but indifferent. In summary, combination therapy of AZT with meropenem represents a potential treatment for carbapenemase-producing MDR K. pneumoniae and merits further investigation. Postprint

Staphylococcus aureusis a major opportunistic pathogen that is exposed to antimicrobial innate immune effectors and antibiotics that can disrupt its cell membrane. An understanding ofS. aureuslipid composition and its role in defending the cell against membrane-disrupting agents is of fundamental importance. Common methods for characterising lipid profiles suffer shortcomings such as low sensitivity of detection and inferior resolution of the positional assignments of fatty acid chains in lipids. This present study developed a rapid and sensitive nano-electrospray ionisation tandem mass spectrometry (nESI-MS/MS) method to characterise the lipid composition of three commonly studiedS. aureusisolates: Newman, Mu50 and BB270. Confirming previous studies, nESI-MS/MS revealed that phosphatidylglycerols were most abundant inS. aureusmembranes, while diglucosyldiacylglycerols and lysyl-phosphatidylglycerols were also detected. Positional assignments for individual fatty acid chains within these lipids were also determined. Concomitantly, gas chromatography mass spectrometry of the fatty acids validated the molecular characterization and showed the principal species present in each strain were predominately anteiso- and iso-branched chain fatty acids. Though the fatty acid and lipid profiles were similar between theS. aureusstrains, this method was sufficiently sensitive to distinguish minor differences in lipid composition. In conclusion, this nESI-MS/MS methodology can characterise the role of lipids in antimicrobial resistance, and may even be applied to the rapid diagnosis of drug-resistant strains in the clinic.

Funding information: University of St Andrews. Purpose: To evaluate the suitability of Galleria mellonella larvae as an in vivo model and drug-screening tool for mycobacteria infections. Methodology: Larvae were infected using a range of inoculum sizes from a variety of rapid-growing mycobacteria, including strains of M. fortuitum, M. marinum and M. aurum. Larval survival, internal bacterial burden, and the effects of amikacin, ciprofloxacin, ethambutol, isoniazid and rifampicin treatment on larval survival were measured over 144h. The effects of these anti-mycobacterial drugs on phagocytosis and circulating hemocyte numbers were also examined using microscopy. Results: Larval survival decreased after infection with M. fortuitum and M. marinum in a dose-dependent manner, but remained unaffected by M. aurum. Heat-killed bacteria did not cause larval death. Where antibiotic monotherapy was efficacious, larval survival post-infection increased in a dose-dependent fashion. However, efficacy varied between different antibiotics and species of infecting mycobacteria and, apart from rifampicin, efficacy in vivo correlated poorly with the in vitro MICs. Combinations of antibiotics led to higher survival of infected larvae than antibiotic monotherapy. Selected antibiotic treatments that enhanced larval survival reduced the overall internal burden of infecting mycobacteria but did not eradicate the pathogens. Administration of amikacin or ethambutol to uninfected larvae induced an initial transient increase in the numbers of circulating hemocytes and reduced the phagocytic rate of hemocytes in larvae infected with M. marinum. Conclusions: This report demonstrates the potential of employing a wax moth larvae model for studying fast-growing mycobacteria infections, and as a cheap, effective system for initial screening of novel treatments. Postprint

The aim of this study was to determine whether Galleria mellonella larvae can be used (i) as an in vivo infection model for Pseudomonas aeruginosa and (ii) for evaluating the pharmacokinetics and efficacy of antipseudomonal antibiotics. Two strains of P. aeruginosa were employed, NCTC 10662 (antibiotic-susceptible) and NCTC 13437 (multidrug-resistant). Larvae were infected with increasing doses of either P. aeruginosa strain to investigate the effect of inoculum size on survival. Subsequently, infected larvae were treated with a range of antibiotics to examine whether these agents were effective against P. aeruginosa infection in vivo and whether the efficacy of these drugs matched the known susceptibilities of each bacterial strain. Larval burden of P. aeruginosa was also determined after infection and treatment with cefotaxime. Pharmacokinetic properties of the antibiotics tested were measured using a well diffusion assay to determine the concentration of antibiotics in larval haemolymph over time. Galleria mellonella larvae were sensitive to P. aeruginosa infection, and increasing inoculum doses of live cells resulted in greater larval mortality. Heat-killed bacteria had no detrimental effect on survival. Antibiotic efficacy against P. aeruginosa-infected G. mellonella correlated with the measured in vitro sensitivities of the two strains tested. The therapeutic benefit arising from administration of cefotaxime correlated with a reduced burden of bacteria present in the haemolymph. There was a clear correlation between measured antibiotic pharmacokinetics and the therapeutic effect. This study strongly supports future application of the G. mellonella infection model to initial studies of novel antipseudomonal treatments.

The objective of this study was to determine whether combinations of antimicrobial peptides (AMPs) with caspofungin display enhanced antifungal activity versus Candida albicans in vitro and in vivo. Three conventional AMPs that satisfied criteria favouring their potential development as novel antifungals were selected for investigation. Colistin sulphate was also included as a cyclic peptide antibiotic used in the clinic. Minimum inhibitory concentrations (MICs) were determined for each antifungal agent and checkerboard assays were used to determine fractional inhibitory concentration index (FICI) values for dual combinations of AMPs or colistin with caspofungin. Viability assays were performed for the same combinations in order to investigate fungicidal interactions. Synergistic antifungal combinations were then tested for efficacy in vivo and compared to monotherapies in wax moth larva and murine models of systemic C. albicans infection. In combination with caspofungin, each of the AMPs [hMUC7-12, DsS3(1-16), hLF(1-11)] and colistin were synergistic and candidacidal in vitro. The treatment of infected wax moth larvae with combinations of caspofungin with hMUC7-12, DsS3(1-16) or colistin resulted in significant enhancements in survival compared to treatment with monotherapies. Notably, the treatment of C. albicans-infected mice with a combination of caspofungin and DsS3(1-16) resulted in the enhancement of survival compared to groups treated with just the individual agents. This study demonstrates that combination therapies containing caspofungin and AMPs or colistin merit further development as potential novel treatments for C. albicans infections.

MPT was the recipient of an ERASMUS training grant. FE is supported by the University of St Andrews. The aim was to evaluate whether immunosuppression with dexamethasone 21-phosphate could be applied to the Galleria mellonella in vivo infection model. Characterised clinical isolates of Escherichia coli or Klebsiella pneumoniae were employed, and G. mellonella larvae were infected with increasing doses of each strain to investigate virulence in vivo. Virulence was then compared with larvae exposed to increasing doses of dexamethasone 21-phosphate. The effect of dexamethasone 21-phosphate on larval haemocyte phagocytosis in vitro was determined via fluorescence microscopy and a burden assay measured the growth of infecting bacteria inside the larvae. Finally, the effect of dexamethasone 21-phosphate treatment on the efficacy of ceftazidime after infection was also noted. The pathogenicity of K. pneumoniae or E. coli in G. mellonella larvae was dependent on high inoculum numbers such that virulence could not be attributed specifically to infection by live bacteria but also to factors associated with dead cells. Thus, for these strains, G. mellonella larvae do not constitute an ideal infection model. Treatment of larvae with dexamethasone 21-phosphate enhanced the lethality induced by infection with E. coli or K. pneumoniae in a dose- and inoculum size-dependent manner. This correlated with proliferation of bacteria in the larvae that could be attributed to dexamethasone inhibiting haemocyte phagocytosis and acting as an immunosuppressant. Notably, prior exposure to dexamethasone 21-phosphate reduced the efficacy of ceftazidime in vivo. In conclusion, demonstration of an effective immunosuppressant regimen can improve the specificity and broaden the applications of the G. mellonella model to address key questions regarding infection. Publisher PDF

Many cationic peptides with antimicrobial properties have been isolated from bacteria, fungi, plants, and animals. These peptides vary in molecular size, potency and spectra of activities. This report surveyed the literature to highlight the peptides that have antifungal activity and greatest potential for development as new therapeutic agents. Thus, to be included in the evaluation, each peptide had to fulfil the following criteria: (i) potent antifungal activity, (ii) no, or minimal, mammalian cell toxicity, (iii) of ≤25 amino acids in length, which minimises the costs of synthesis, reduces immunogenicity and enhances bioavailability and stability in vivo, (iv) minimal post-translational modifications (also reduces the production costs). The ~80 peptides that satisfied these criteria are discussed with respect to their structures, mechanisms of antimicrobial action and in vitro and in vivo toxicities. Certainly, some of these small peptides warrant further study and have potential for future exploitation as new antifungal agents.

The SSPF was supported by grants from The Scottish Funding Council (references SSPF and SULSA), The Biotechnology and Biological Sciences Research Council (reference BB/S/B14450), European Union under framework 7 (reference Aeropath). The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology. Publisher PDF

Administering synergistic combinations of antifungals could be a route to overcome problems with toxicity and the development of resistance. Combination of the echinocandins caspofungin or anidulafungin with a range of structurally diverse antimicrobial peptides resulted in potent synergistic killing of Candida spp. in vitro. Fungicidal synergy was measured by calculating fractional inhibitory concentration indices from checkerboard assays as well as loss of viability. Inhibitory combinations of the antifungals did not induce cytotoxicity in vitro. However, in a murine model of systemic candidiasis, co-administration of caspofungin with one example of the cationic peptides tested, ranalexin, did not show enhanced efficacy compared with the single treatments alone. Further study using alternative peptides will identify whether this combination approach could represent a novel treatment for fungal pathogens.

In Listeria monocytogenes the alternative sigma factor σ B plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σ B regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic Δ sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σ B dependent; 17 of these proteins were found to require the presence of σ B for full expression, while 21 were expressed at a higher level in the Δ sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σ B in the general stress response and highlighted a probable role for σ B in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σ B in glycerol metabolism. Five newly identified members of the σ B regulon were shown to be under direct regulation of σ B using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σ B -dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σ B -dependent genes, as well as proteomic analyses, σ B was shown to play a major role in the stationary phase of growth in complex media.

Alzheimer patients have increased levels of both the 42 amyloid-beta-peptide (Abeta) and the amyloid binding alcohol dehydrogenase (ABAD), which is an intracellular binding site for Abeta. The overexpression of Abeta and ABAD in transgenic mice has shown that the binding of Abeta to ABAD results in amplified neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals, we have identified for the first time that the protein endophilin I increases in Alzheimer diseased brain. The increase in endophilin I levels in neurons is linked to an increase in the activation of the stress kinase c-Jun N-terminal kinase with the subsequent death of the neurons. We also demonstrate in living animals that the expression level of endophilin I is an indicator for the interaction of ABAD and Abeta as its expression levels return to normal if this interaction is perturbed. Therefore this identifies endophilin I as a new indicator of the progression of Alzheimer disease.

Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5'-phosphate-dependent aminotransferase with a molecular weight of 48,168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 A, alpha = beta = gamma = 90 degrees. Analysis of the systematic absences along the three principal axes indicated the space group to be P2(1)2(1)2(1). A complete data set was collected to 2.5 A resolution.

Objectives: Successful treatment of infections involving multiply drug-resistant methicillin-resistant Staphylococcus aureus (MRSA) is becoming increasingly difficult. In this work, we have investigated the potential of combining lysostaphin with cationic antimicrobial peptides to effectively inhibit Staphylococcus aureus. Methods: S. aureus strains were grown in 96-well plates in the presence of increasing concentrations of lysostaphin and the peptides ranalexin, dermaseptin S3(1-16) or magainin 2. Growth was determined visually after 48 h and the plates imaged, or by automated optical density readings in a plate reader. Susceptibility to the combination of lysostaphin and ranalexin was also determined by viable cell counts. The efficacy of combined lysostaphin and ranalexin on a solid surface was tested via disc diffusion assays. Results and conclusions: Combination of lysostaphin with ranalexin resulted in potent, synergistic inhibition of S. aureus MSSA476 and MRSA252. Synergistic inhibition was specific for lysostaphin-susceptible staphylococci and was not observed with clinical isolates of the Gram-negative Escherichia coli, or other Gram-positive organisms, such as Enterococcus faecalis. Lysostaphin was not specifically synergistic with ranalexin alone. Synergy was also observed with two other cationic antimicrobial peptides, magainin 2 and dermaseptin s3(1-16); although combination with ranalexin was most potent. Synergistic inhibition by ranalexin in combination with lysostaphin resulted in an enhanced bactericidal effect. Importantly, synergy between lysostaphin and ranalexin was also observed after impregnation and drying in filter paper discs that clearly inhibited growth of S. aureus on the surface of agar; a solid, porous matrix. Thus, the combination could represent a novel route to target wounds infected with drug-resistant MRSA via dressings impregnated with the two compounds.

Objectives The objectives of this study were to compare the antibiotic susceptibility of Pseudomonas aeruginosa strains with increased efflux pump expression in vitro and in vivo and to use these same strains to evaluate the efficacy of combinations of antibiotics with putative efflux pump inhibitors in vivo. Methods A collection of P. aeruginosa strains that overexpress three efflux pumps (MexAB-OprM, MexCD-OprJ and MexEF-OprN), in addition to a strain with all three Mex pumps deleted, were used. The virulence of these strains and their antibiotic susceptibility was measured in vivo using a Galleria mellonella larval infection model. The inhibitory effect of combinations of putative efflux pump inhibitors (trimethoprim and sertraline) with antibiotics on the strain overexpressing MexAB-OprM was also measured in vitro and compared with their efficacy in vivo in terms of larval survival and bacterial burden. Results Increased expression of the individual efflux pumps, or deletion of all three, had no significant effect on the virulence of P. aeruginosa in vivo. Expression levels of the efflux pumps clearly influenced antibiotic efficacy in vivo. The efficacy of levofloxacin, piperacillin and meropenem against larvae infected with the efflux pump mutants reflected susceptibility to the same drugs in vitro. Treatment of G. mellonella larvae infected with a strain that overexpressed MexAB-OprM with a combination of putative efflux pump inhibitors and levofloxacin resulted in enhanced therapeutic benefit compared with the constituent monotherapies. Conclusions This study has demonstrated the utility of using G. mellonella to screen for novel therapeutic options for MDR P. aeruginosa and has shown that antibiotic/efflux pump inhibitor combinations should be further investigated for clinical application.

Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca(2+) channel, CCH1, and a functional vacuolar membrane H(+)-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

In this mini-review, various aspects of homeostasis of microbial cells and its perturbation by antimicrobial agents will be discussed. First, outlining the position that the physiological studies on microbial behaviour using the modern molecular tools should have in food science sets the scene for the studies. Subsequently, the advent of functional genomics is discussed that allows full coverage of cellular reactions at unprecedented levels. Examples of weak organic acid resistance, the stress response against natural antimicrobial agents and responses against physicochemical factors show how we can now "open the black box" that microbes are, look inside and begin to understand how different cellular signalling cables are wired together. Using the analogy with machines, it will be indicated how the use of various signalling systems depends on the availability of substrates "fuel" to let the systems act in the context of the minimum energetic requirement cells have to let their housekeeping systems run. The outlook illustrates how new insights might be used to device knowledge-based rather than empirical combinations of preservation systems and how risk assessment models might be deviced that link the mechanistic insight to risk distributions of events in food manufacturing.

Yeast cells display an adaptive stress response when exposed to weak organic acids at low pH. This adaptation is important in the spoilage of preserved foods, as it allows growth in the presence of weak acid food preservatives. In Saccharomyces cerevisiae, this stress response leads to strong induction of the Pdr12 ATP-binding cassette (ABC) transporter, which catalyses the active efflux of weak acid anions from the cytosol of adapted cells. S. cerevisiae cells lacking the Cmk1 isoform of Ca2+–calmodulin-dependent protein kinase are intrinsically resistant to weak acid stress, in that they do not need to spend a long adaptive period in lag phase before resuming growth after exposure to this stress. This resistance of the cmk1 mutant is Pdr12 dependent and, unlike with wild-type S. cerevisiae, cmk1 cells are capable of performing Pdr12-specific functions such as energy-dependent cellular extrusion of fluorescein and benzoate. However, they have neither higher PDR12 gene promoter activity nor higher Pdr12 protein levels. The increased Pdr12 activity in cmk1 cells is therefore caused by Cmk1 exerting a negative post-transcriptional influence over the activity of the Pdr12 ABC transporter, a transporter protein that is constitutively expressed in low-pH yeast cultures. This is the first preliminary evidence that shows a protein kinase, either directly or indirectly, regulating the activity of a yeast ABC transporter.

The aim of this study was to compare the inhibitory effect of antibiotic combinations in vitro with efficacy in Galleria mellonella larvae in vivo to identify efficacious combinations that target Pseudomonas aeruginosa. P. aeruginosa NCTC 13437, a multidrug-resistant strain resistant to β-lactams and aminoglycosides, was used. Susceptibility to cefotaxime, piperacillin, meropenem, amikacin, levofloxacin and colistin alone, or in dual or triple combinations, was measured in vitro via a 24 h time-kill assay. In vitro results were then compared with the efficacy of the same dual or triple antibiotic combinations versus G. mellonella larvae infected with P. aeruginosa. G. mellonella haemolymph burden of P. aeruginosa was determined over 96 h post-infection and treatment with the most potent combination therapies. Many dual and triple combinations of antibiotics displayed synergistic inhibition of multidrug-resistant P. aeruginosa in vitro. There was little correlation between combinations that were synergistic in vitro and those that showed enhanced efficacy in vivo versus infected G. mellonella larvae. The most potent dual and triple combinations in vivo were cefotaxime plus piperacillin, and meropenem plus piperacillin and amikacin, respectively. Fewer combinations were found to offer enhanced therapeutic benefit in vivo compared with in vitro. The therapeutic benefit arising from treatment with antibiotic combinations in vivo correlated with reduced larval burden of P. aeruginosa. This study has identified antibiotic combinations that merit further investigation for their clinical potential and has demonstrated the utility of using G. mellonella to screen for novel antibiotic treatments that demonstrate efficacy in vivo.

Aims: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. Methods and Results: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol‐thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. Conclusions: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. Significance and Impact of the Study: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol‐ thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.

A spore germination response requires the interaction of the germinant with a specific receptor on the spore (17). The gerA operon of Bacillus subtilis, required for germination in l-alanine (19, 33, 34), was the first characterized of a family of operons that is present in all spore formers so far examined; multiple family members are present in each genome. B. subtilis also contains the gerB (6) and gerK (10) operons required for germination in the alternative germinative combination of an amino acid, such as l-alanine or l-asparagine, in combination with sugars. Both gerA and gerB operons have been shown to be expressed in the developing forespore under the control of sigma G-associated RNA polymerase (5, 7). The gerA operon encodes three proteins: GerAA, which is predicted to comprise both hydrophobic and hydrophilic domains; GerAB, which is predicted to be an integral membrane protein and is a member of the single-component amino acid/polyamine/organocation transporter superfamily (11); and GerAC, a predicted lipoprotein. Spores of triple mutants in the gerA, gerB, and gerK operons will not germinate on nutrient media (22). Two other homologous operons in the B. subtilis genome (12) presumably encode components that respond to as-yet-unidentified germinants. In addition to the five operons in B. subtilis, there are homologues of demonstrated importance to inosine germination in B. cereus (4) and to germination in macrophages, encoded in the virulence cluster of plasmid pXO1 of B. anthracis (8). The levels of expression from gerA-lacZ fusions are very low and are only detectable by using fluorogenic substrates for β-galactosidase (7). If the Ger proteins are expressed only at a low level during sporulation, it is not likely to be practicable to detect the location of these proteins in spores by immunogold labeling. Fractionation of spores provides an alternative approach to defining their position(s) in the spore. The definition of the location of these proteins in the spore is of crucial importance to our formulation of models for the mechanism of spore germination.

Salmonella adaptation to low pH is a critical survival response and essential for virulence. Here, we show that another key virulence-associated process, flagellamediated cell motility, is co-regulated by low pH via the PhoPQ signal transduction system. Using a proteomic approach, we found that phase 1 and phase 2 flagellin were specifically down-regulated when acid-adapted (pH 5.0) Salmonella SL1344 cells were exposed to pH 3.0. Decreased flagellin expression and cell motility was dependent on activation of the PhoPQ pathway, which directly or indirectly negatively regulated transcription of the flagellin gene fliC. In contrast, the general stress sigma factor RpoS (S) positively regulated flagellar gene expression. Low external pH had no effect on the level of H-NS protein, a further regulator of flagellar gene expression. We suggest that flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol. These results highlight the power of proteomics to reveal unanticipated links between relatively well-characterised regulatory systems in bacteria.

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis , which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC , consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC -defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of l -carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting l -carnitine and which plays an important role in osmoregulation in this pathogen.

Preservative agents are required to ensure that manufactured foods remain safe and unspoiled. In this review, we will discuss the mode of action of both chemical and biological (nature-derived) preservatives and the stress response mechanisms induced by these compounds in microorganisms of concern to the food industry. We will discuss the challenges that food manufacturers face with respect to the assurance of food safety and the prevention of spoilage. Following this, chemical preservatives will be discussed, in particular, weak organic acids such as sorbic and benzoic acid which are widely used in preservation. Furthermore. the mechanisms of microbial inactivation with hydrogen peroxide mediated systems and chelators such as citric acid and EDTA and their potential use in preservation will be covered. We will then address the potential of naturally occurring "preservatives". Of the antimicrobial compounds present in nature, first to be discussed will be the nonproteinaceous compounds often present in herbs and spices and we will speculate on the stress response(s) that microorganisms may elicit to these natural compounds. Next to be addressed will be compounds that attack cell walls and membranes, for example, peptides, proteins and lytic enzymes. In discussing the resistance mechanisms against membrane and wall perturbation, the extensive knowledge of stress responses against osmotic stress and temperature stress will be refered to. Finally, in the concluding paragraphs, options for combination preservation systems are evaluated.

Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.

This report describes the use of the fluorescent probe, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), to determine pHi in growing cells of the spoilage yeast Saccharomyces cerevisiae. The technique is based on the pH-dependent intracellular fluorescence of the probe in the cytosol of the cell. The major advantage of this probe over other derivatives is the presence of a succinimidyl group which binds to aliphatic amines in cellular proteins. This allows for the determination of pHi under conditions that either permeabilise the cell membrane, or increase active extrusion, and would otherwise result in loss of the intracellular probe to the external medium. Optimal loading of the probe into cells only occurred after exposure to a mild stress regime of 37°C for 24 h in 100 mM citric/phosphate buffer, pH 4.0. This loading regime was selected because there was no appreciable effect on cell viability or subsequent growth rate in batch culture under optimal conditions. The pHi of yeast cells was measured after incubation in citric/phosphate buffer and gave values comparable to those measured by other techniques in the literature. Also, we were able to detect rapid changes in pHi induced by the addition of a known disruptor of pHi homeostasis in yeast, the weak-acid food preservative, sorbic acid. Finally, successful determination of pHi was made in growing cells of an isogenic parent (PMA1) and a mutant with reduced-expression of the membrane H+-ATPase, pma1-205. As might be expected, the pHi in the mutant strain was reduced compared to the parent. Comparison of growth and pHi in growing cells of either strain revealed no correlation between pHi and exit from lag phase. These results are discussed in relation to previous findings.

OBJECTIVES: To evaluate the in vivo effectiveness of a combination treatment containing ranalexin (a natural antimicrobial peptide) and lysostaphin (an antistaphylococcal endopeptidase) for reducing nasal burden of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The community-acquired MRSA strain S. aureus NRS384 (USA300-0114) was used in the present study because it is commonly isolated from human nares and it established consistent and reproducible colonization of cotton rat nares. This model was used to evaluate the efficacy of ranalexin/lysostaphin gels (0.1%-1% w/v; administered intranasally once or once per day for 3 consecutive days) for reducing nasal MRSA burden. Control animals were administered vehicle gel only (0.5% hydroxypropyl methylcellulose) or 2% mupirocin, which is used clinically for nasal decolonization of MRSA. Nasal MRSA burden was assessed at 192 h post-inoculation, which was at least 72 h after the final treatment had been administered. An additional study assessed the efficacy of 0.1% ranalexin/lysostaphin against a mupirocin-resistant MRSA strain (MUP20), which had been selected by serial passage of S. aureus NRS384 through subinhibitory concentrations of mupirocin. RESULTS: Gels containing 0.1% ranalexin/lysostaphin consistently reduced median nasal burden of MRSA to an extent similar to or greater than 2% mupirocin. Treatment with 0.1% ranalexin/lysostaphin was also effective against the MUP20 strain. There was evidence for only minimal irritancy in cotton rat nares administered three doses of 0.1% ranalexin/lysostaphin, suggesting that this agent is suitable for short-course therapy such as is employed currently for nasal decolonization with mupirocin. CONCLUSIONS: Ranalexin/lysostaphin could serve as an alternative to mupirocin for nasal decolonization of MRSA.

Listeria monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress, a response designated the acid tolerance response (ATR). Maximal acid resistance was seen when the organism was exposed to pH 5.0 for 1 h prior to challenge at pH 3.0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4.0 to 6.0. A 60 min adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of the ATR required de novo protein synthesis; chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Analysis of protein expression during the adaptive period by two-dimensional gel electrophoresis revealed a change in the expression of at least 23 proteins compared to the non-adapted culture. Eleven proteins showed induced expression while 12 were repressed, implying that the ATR is a complex response involving a modulation in the expression of a large number of genes. In addition to the exponential phase ATR, L. monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH-dependent ATR seen during exponential growth.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.

Confocal scanning laser microscopy (CSLM) has been used to study the germination of Bacillus cereus spores allowing generation of accurate quantitative data on loss of refractility of individual , germinating spores in a population. The technique also allowed the simultaneous study of changes in spore permeability to ethidium bromide by fluorescence microscopy. Statistical analyses were performed on the data obtained and the distributions of various germination parameters within the population studied. All the parameters studied were characterised by positively ‘skewed’ distributions indicating the high level of biovariability present in populations of bacterial spores. The lag time before a spore initiated germination was independent from the subsequent time to lose refractility, or germinate. The method described in this work, for the first time, allows changes in permeability during germination to be studied using the influx of a compound that is excluded from dormant spores (ethidium bromide). It is hoped that this will form the basis of a useful system for studying the kinetics and sequence of spore germination when combined with other permeability probes.

The structure of 3-methyladenine DNA glycosylase I in complex with 3-methyladenine is reported., The removal of chemically damaged DNA bases such as 3-methyladenine (3-­MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge–charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.

There is an urgent need for new antimicrobial agents to combat infections caused by drug-resistant pathogens. Once a compound is shown to be effective in vitro, it is necessary to evaluate its efficacy in an animal infection model. Typically, this is achieved using a mammalian model, but such experiments are costly, time consuming, and require full ethical consideration. Hence, cheaper and ethically more acceptable invertebrate models of infection have been introduced, including the larvae of the greater wax moth Galleria mellonella. Invertebrates have an immune system that is functionally similar to the innate immune system of mammals, and often identical virulence and pathogenicity factors are used by human pathogenic microbes to infect wax moth larvae and mammals. Moreover, the virulence of many human pathogens is comparable in wax moth larvae and mammals. Using key examples from the literature, this chapter highlights the benefits of using the wax moth larva model to provide a rapid, inexpensive, and reliable evaluation of the toxicity and efficacy of new antimicrobial agents in vivo and prior to the use of more expensive mammalian models. This simple insect model can bridge the gap between in vitro studies and mammalian experimentation by screening out compounds with a low likelihood of success, while providing greater justification for further studies in mammalian systems. Thus, broader implementation of the wax moth larva model into anti-infective drug discovery and development programs could reduce the use of mammals during preclinical assessments and the overall cost of drug development.

There is an urgent need for new antimicrobial agents to combat infections caused by drug-resistant pathogens. Once a compound is shown to be effective in vitro, it is necessary to evaluate its efficacy in an animal infection model. Typically, this is achieved using a mammalian model, but such experiments are costly, time consuming, and require full ethical consideration. Hence, cheaper and ethically more acceptable invertebrate models of infection have been introduced, including the larvae of the greater wax moth Galleria mellonella. Invertebrates have an immune system that is functionally similar to the innate immune system of mammals, and often identical virulence and pathogenicity factors are used by human pathogenic microbes to infect wax moth larvae and mammals. Moreover, the virulence of many human pathogens is comparable in wax moth larvae and mammals. Using key examples from the literature, this chapter highlights the benefits of using the wax moth larva model to provide a rapid, inexpensive, and reliable evaluation of the toxicity and efficacy of new antimicrobial agents in vivo and prior to the use of more expensive mammalian models. This simple insect model can bridge the gap between in vitro studies and mammalian experimentation by screening out compounds with a low likelihood of success, while providing greater justification for further studies in mammalian systems. Thus, broader implementation of the wax moth larva model into anti-infective drug discovery and development programs could reduce the use of mammals during preclinical assessments and the overall cost of drug development.

A method was developed to predict spoilage of minced meat at chill temperatures, based on the difference in proton efflux from and influx into bacterial cells. This difference depends on the number of organisms present, the available glucose in the meat sample and the ability of the organisms to metabolize amino acids. The proton efflux/influx of a meat filtrate containing bacteria was measured at 25 degrees C with a pH/ion meter in the presence of peptone with or without glucose. There was a noticeable rate of change of mV h-1 of the meat filtrate prior to the organoleptic detection of spoilage which may be used semi-predictively to determine the remaining shelf-life of meat at different storage temperatures. The method could be investigated further, encompassing type and relative numbers of organisms, incubation temperature, meat type and composition (i.e. available glucose) to produce a spoilage prediction model. The method does not require sophisticated equipment, only a standard pH/ion meter, is cheap, needing only peptone and glucose, is relatively simple, and takes less than 2 h to perform.

Objectives: To investigate whether the wax moth larva, Galleria mellonella, is a suitable host for assessing the in vivo efficacy of antistaphylococcal agents against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) infections. Methods: Wax moth larvae were infected with increasing doses of S. aureus to investigate the effect of inoculum size on larval survival. In addition, infected wax moth larvae were treated with daptomycin, penicillin or vancomycin to examine whether these agents were effective against S. aureus and MRSA infections in vivo. Results: Increasing inoculum doses of live S. aureus cells resulted in greater larval mortality, but heat-killed bacteria and cell-free culture filtrates had no detrimental effects on survival. Larval mortality rate also depended on the post-inoculation incubation temperature. After larvae were infected with S. aureus, larval survival was enhanced by administering the antistaphylococcal antibiotics daptomycin or vancomycin. Larval survival increased with increasing doses of the antibiotics. Moreover, penicillin improved survival of larvae infected with a penicillin-susceptible methicillin-susceptible S. aureus (MSSA) strain, but it was ineffective at similar doses in larvae infected with MRSA (penicillin resistant). Daptomycin and vancomycin were also effective when administered to the larvae prior to infection with bacteria. Conclusions: This is the first report to demonstrate that antibiotics are effective in the wax moth larva model for the treatment of infections caused by Gram-positive bacteria. The new wax moth larva model is a useful preliminary model for assessing the in vivo efficacy of candidate antistaphylococcal agents before proceeding to mammalian studies, which may reduce animal experimentation and expense.

Background Staphylococcus aureus is a major human pathogen and strains resistant to existing treatments continue to emerge. Development of novel treatments is therefore important. Antimicrobial peptides represent a source of potential novel antibiotics to combat resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA). A promising antimicrobial peptide is ranalexin, which has potent activity against Gram-positive bacteria, and particularly S. aureus. Understanding mode of action is a key component of drug discovery and network biology approaches enable a global, integrated view of microbial physiology, including mechanisms of antibiotic killing. We developed a systems-wide functional association network approach to integrate proteome and transcriptome profiles, enabling study of drug resistance and mode of action. Results The functional association network was constructed by Bayesian logistic regression, providing a framework for identification of antimicrobial peptide (ranalexin) response modules from S. aureus MRSA-252 transcriptome and proteome profiling. These signatures of ranalexin treatment revealed multiple killing mechanisms, including cell wall activity. Cell wall effects were supported by gene disruption and osmotic fragility experiments. Furthermore, twenty-two novel virulence factors were inferred, while the VraRS two-component system and PhoU-mediated persister formation were implicated in MRSA tolerance to cationic antimicrobial peptides. Conclusions This work demonstrates a powerful integrative approach to study drug resistance and mode of action. Our findings are informative to the development of novel therapeutic strategies against Staphylococcus aureus and particularly MRSA.

International audience; Drug-resistant staphylococci constitute a serious problem that urgently requires the discovery of new therapeutic agents. There has been a resurgence in interest in using lysostaphin (a specific anti-staphylococcal enzyme) as a treatment for infections caused by these important pathogens. However, bacterial resistance to lysostaphin is a problem, but the use of a combination treatment may surmount this issue. In this present study, using viable counts from suspension incubations, lysostaphin is shown to be synergistically bactericidal in combination with various conventional antimicrobial peptides, the antimicrobial protein bovine lactoferrin, a lantibiotic (nisin), and certain lipopeptides used clinically (colistin, daptomycin and polymyxin B). Combinations that act in synergy are of clinical importance as these reduce the doses of the compounds needed for effective treatments and decrease the chances of resistance being selected. The use of lysostaphin in combination with a peptide may represent a new avenue in tackling drug-resistant staphylococci.

New treatments are urgently required for infections caused by meticillin-resistant Staphylococcus aureus (MRSA) as these strains are often resistant to multiple conventional antibiotics. Earlier studies showed that ranalexin, an antimicrobial peptide (AMP), in combination with lysostaphin, an antistaphylococcal endopeptidase, synergistically inhibits the growth of MRSA, meaning that it deserved consideration as a new anti-S. aureus therapy. Using haemolysis and Vero cell viability assays, ranalexin with lysostaphin is proven to be non-toxic at antibacterial concentrations. In human serum, ranalexin with lysostaphin is significantly more effective against MRSA than treatment with either component alone. In a rabbit model of wound infection, ranalexin with lysostaphin reduced MRSA in the wound by ca. 3.5log(10) colony-forming units (CFU) compared with the untreated control. The combination is significantly more effective than treatment with ranalexin or lysostaphin alone. In a mouse model of systemic infection, ranalexin with lysostaphin reduced MRSA kidney burden by ca. 1log(10)CFU/g compared with untreated controls or treatment with ranalexin or lysostaphin alone. Importantly, the combination is synergistically bactericidal against various S. aureus isolates in vitro, including those with reduced susceptibility to lysostaphin or vancomycin. Ranalexin and lysostaphin could be incorporated in wound dressings for the prevention and treatment of topical S. aureus infections. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci.

Aims: To characterize the antibacterial synergy of the antimicrobial peptide, ranalexin, used in combination with the anti-staphylococcal endopeptidase, lysostaphin, against methicillin-resistant Staphylococcus aureus (MRSA), and to assess the combination’s potential as a topical disinfectant or decolonizing agent for MRSA. MRSA causes potentially lethal infections, and pre-operative patients colonized with MRSA are often treated with chlorhexidine digluconate and mupirocin cream to eradicate carriage. However, chlorhexidine is unsuitable for some patients, and mupirocin resistance is increasingly encountered, indicating new agents are required. Methods and Results: Using an ex vivo assay, ranalexin and lysostaphin tested in combination reduced viable MRSA on human skin to a greater extent than either compound individually. The combination killed bacteria within 5 min and remained effective and synergistic even in high salt and low pH conditions. Conclusions: The combination is active against MRSA on human skin and under conditions that may be encountered in sweat. Significance and Impact of the Study: Although the exact mechanism of activity remains unresolved, considering its specific spectrum of activity, fast killing kinetics and low likelihood of resistance arising, the combination of ranalexin with lysostaphin warrants consideration as a new agent to eradicate nasal and skin carriage of Staph. aureus, including MRSA.

The outermost layer of theCandida albicanscell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants ofC. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic,α-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.

Conventional heating was used to expose cells of Listeria monocytogenes, either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10(6) upon reaching 70 degrees C. Simulated microwave cooking of L. monocytogenes in situ, on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85 degrees C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10(6). To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70 degrees C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10(6) and 10(8). These results show that when a temperature of 70 degrees C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes. The survival of this organism during microwave heating when temperatures of over 70 degrees C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.

In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an α-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae . Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.

The dermaseptins are a family of antimicrobial peptides from the tree-frog Phyllomedusa sauvagii. Yeast exposed to dermaseptin S3(1-16), a truncated derivative of dermaseptin S3 with full activity, showed diagnostic markers of yeast apoptosis: the appearance of reactive oxygen species and fragmentation of nuclear DNA. This process was independent of the yeast caspase, Yca1p. Screening of a non-essential gene deletion collection in yeast identified genes that conferred resistance to dermaseptin S3(1-16): izh2Delta, izh3Delta, stm1Delta and aif1Delta, all known to be involved in regulating yeast apoptosis. The appearance of apoptotic markers was reduced in these strains when exposed to the peptide. Dermaseptin S3(1-16) was shown to interact with DNA, and cause DNA damage in vivo, a process known to trigger apoptosis. Supporting this, a dermaseptin S3(1-16) affinity column specifically purified Stm1p, Mre11p and Htb2p; DNA-binding proteins implicated in yeast apoptosis and DNA repair. Thus, amphibians may have evolved a mechanism to induce cell suicide in invading fungal pathogens.

Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 A, alpha = 87.2, beta = 81.2, gamma = 68.4 degrees . A complete data set has been collected to 2.3 A resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model.

Alzheimer's patients have increased levels of both the 42 beta amyloid-beta-peptide (Abeta) and amyloid binding alcohol dehydrogenase (ABAD) which is an intracellular binding site for Abeta. The over-expression of Abeta and ABAD in transgenic mice has shown that the binding of Abeta to ABAD results in exaggerating neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals we identified that peroxiredoxin II levels increase in Alzheimer's diseased brain. This increase in peroxiredoxin II levels protects neurons against Abeta induced toxicity. We also demonstrate, for the first time in living animals, that the expression level of peroxiredoxin II is an indicator for the interaction of ABAD and Abeta as its expression levels return to normal if this interaction is perturbed. Therefore this indicates the possibility of reversing changes observed in Alzheimer's disease and that the Abeta-ABAD interaction is a suitable drug target.