Repurposing Mitomycin C in Combination with Pentamidine or Gentamicin to Treat Infections with Multi-Drug-Resistant (MDR) Pseudomonas aeruginosa.

The aims of this study were (i) to determine if the combination of mitomycin C with pentamidine or existing antibiotics resulted in enhanced efficacy versus infections with MDR P. aeruginosa in vivo; and (ii) to determine if the doses of mitomycin C and pentamidine in combination can be reduced to levels that are non-toxic in humans but still retain antibacterial activity. Resistant clinical isolates of P. aeruginosa, a mutant strain over-expressing the MexAB-OprM resistance nodulation division (RND) efflux pump and a strain with three RND pumps deleted, were used. MIC assays indicated that all strains were sensitive to mitomycin C, but deletion of three RND pumps resulted in hypersensitivity and over-expression of MexAB-OprM caused some resistance. These results imply that mitomycin C is a substrate of the RND efflux pumps. Mitomycin C monotherapy successfully treated infected Galleria mellonella larvae, albeit at doses too high for human administration. Checkerboard and time-kill assays showed that the combination of mitomycin C with pentamidine, or the antibiotic gentamicin, resulted in synergistic inhibition of most P. aeruginosa strains in vitro. In vivo, administration of a combination therapy of mitomycin C with pentamidine, or gentamicin, to G. mellonella larvae infected with P. aeruginosa resulted in enhanced efficacy compared with monotherapies for the majority of MDR clinical isolates. Notably, the therapeutic benefit conferred by the combination therapy occurred with doses of mitomycin C close to those used in human medicine. Thus, repurposing mitomycin C in combination therapies to target MDR P. aeruginosa infections merits further investigation.

Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.

The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against Galleria mellonella larvae infected with an MDR strain of P. aeruginosa and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected G. mellonella larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR P. aeruginosa strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.

Carbapenem-Only Combination Therapy against Multi-Drug Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and Mode of Action.

The aim of the study was to determine the efficacy of carbapenem-only combination treatments derived from four approved drugs (meropenem, doripenem, ertapenem and imipenem) against a MDR strain of P. aeruginosa in a Galleria mellonella larvae infection model. G. mellonella larvae were infected with P. aeruginosa NCTC 13437 (carrying the VIM 10 carbapenamase) and the efficacy of the six possible dual, four triple, and one quadruple carbapenem combination(s) were compared to their constituent monotherapies. Four of these combinations showed significantly enhanced survival compared to monotherapies and reduced the bacterial burden inside infected larvae but without complete elimination. Bacteria that survived combination therapy were slower growing, less virulent but with unchanged carbapenem MICs-observations that are consistent with a persister phenotype. In vitro time-kill assays confirmed that the combinations were bactericidal and confirmed that a low number of bacteria survived exposure. Mass spectrometry was used to quantify changes in the concentration of carbapenems in the presence of carbapenemase-carrying P. aeruginosa. The rate of degradation of individual carbapenems was altered, and often significantly reduced, when the drugs were in combinations compared with the drugs alone. These differences may account for the enhanced inhibitory effects of the combinations against carbapenem-resistant P. aeruginosa and are consistent with a 'shielding' hypothesis. In conclusion, carbapenem combinations show promise in combating MDR P. aeruginosa and are worthy of additional study and development.

Repurposing the anti-viral drug zidovudine (AZT) in combination with meropenem as an effective treatment for infections with multi-drug resistant, carbapenemase-producing strains of Klebsiella pneumoniae.

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.

Dual ß-lactam combination therapy for multi-drug resistant Pseudomonas aeruginosa infection: enhanced efficacy in vivo and comparison with monotherapies of penicillin-binding protein inhibition.

The aim of the study was to determine the efficacy of dual ß-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 ß-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 ß-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 ß-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 ß-lactam antibiotics show promise in overcoming MDR P. aeruginosa and are worthy of additional study and development.

Evaluation of greater wax moth larvae, Galleria mellonella, as a novel in vivo model for non-tuberculosis Mycobacteria infections and antibiotic treatments.

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 144 h. The effects of these anti-mycobacterial drugs on phagocytosis and circulating haemocyte 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 minimum inhibitory concentrations (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 haemocytes and reduced the phagocytic rate of haemocytes 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.

Effective immunosuppression with dexamethasone phosphate in the Galleria mellonella larva infection model resulting in enhanced virulence of Escherichia coli and Klebsiella pneumoniae.

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.

Enhanced efficacy of putative efflux pump inhibitor/antibiotic combination treatments versus MDR strains of Pseudomonas aeruginosa in a Galleria mellonella in vivo infection model.

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.

A Galleria mellonella infection model reveals double and triple antibiotic combination therapies with enhanced efficacy versus a multidrug-resistant strain of Pseudomonas aeruginosa.

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 ß-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.

Evaluation of Galleria mellonella larvae for measuring the efficacy and pharmacokinetics of antibiotic therapies against Pseudomonas aeruginosa infection.

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.

MRSA decolonization of cotton rat nares by a combination treatment comprising lysostaphin and the antimicrobial peptide ranalexin.

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.

A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus.

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.

Utility of Greater Wax Moth Larva (Galleria mellonella) for Evaluating the Toxicity and Efficacy of New Antimicrobial Agents.

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.

Wax moth larva (Galleria mellonella): an in vivo model for assessing the efficacy of antistaphylococcal agents.

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.

Global network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus.

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.

Survey of small antifungal peptides with chemotherapeutic potential.

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 Scottish Structural Proteomics Facility: targets, methods and outputs.

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.

In vivo efficacy of the antimicrobial peptide ranalexin in combination with the endopeptidase lysostaphin against wound and systemic meticillin-resistant Staphylococcus aureus (MRSA) infections.

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.

Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent synergistic killing of Candida albicans and Candida glabrata in vitro.

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.

Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface.

The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. 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, alpha-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.