Background: The aim of this work was to induce the formation of antibiotic-tolerant and/or persister cells in vivo using antibiotic therapy on Galleria mellonella larvae infected with P. aeruginosa, isolate these surviving cells, and characterise their phenotype and genotype. Methods: Infected larvae were treated with effective doses of either ceftazidime or meropenem. Despite this, surviving P. aeruginosa colonies were isolated from living larvae, and antibiotic killing, fitness, virulence, antibiotic resistance and the whole genome sequence of a selection of these isolates were compared with their original parent strains. Results: The surviving isolates had an increased minimum duration to kill 99% of the population (MDK99) upon exposure to ceftazidime or meropenem and decreased growth rates in culture, but they showed no change to the MIC or virulence—consistent with an antibiotic-tolerant phenotype. Long-read genome sequencing of selected isolates revealed only one single nucleotide polymorphism (SNP) within bkdB, encoding the lipoamide acyltransferase component of the branched-chain α-keto acid dehydrogenase, present in two independent isolates. However, time-kill assays with ceftazidime of bkdB knockout strains showed no significant change in the MDK99. Concomitant with the antibiotic-tolerant phenotype, many of the isolates also had a reduced rate of killing when exposed to heat stress. Conclusions: P. aeruginosa cells that survived antibiotic therapy in vivo were found to be antibiotic-tolerant and thermotolerant but not antibiotic-resistant and had reduced growth rates under optimal conditions but unchanged virulence. In the absence of a convincing genetic explanation, the co-induction of enhanced thermotolerance with antibiotic tolerance indicated that both are conferred by a heritable phenotypic mechanism.

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.

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.

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.

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 mu g/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 mu g/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 mu g/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.

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.

Staphylococcus aureus is a major opportunistic pathogen that is exposed to antimicrobial innate immune effectors and antibiotics that can disrupt its cell membrane. An understanding of S. aureus lipid 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 studied S. aureus isolates: Newman, Mu50 and BB270. Confirming previous studies, nESI-MS/MS revealed that phosphatidylglycerols were most abundant in S. aureus membranes, 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 the S. aureus strains, 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.

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.

AbstractStaphylococcus 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.

Background This study aimed to evaluate the efficacy of combinations of steroidal alkaloids and conessine from the Thai medicinal plant Holarrhena antidysenterica with antibiotics against Pseudomonas aeruginosa strains possessing different efflux-pump-mediated multidrug-resistant (MDR) phenotypes in a Galleria mellonella infection model. Methods P. aeruginosa strains with defined mutations that result in the overexpression of the MexAB-OprM, MexCD-OprJ and MexEF-OprN efflux pumps, and a strain with all three of these pumps deleted, were used. In vitro, the effect of combinations of steroidal alkaloids and conessine with antibiotics was compared with antibiotic treatment alone via MIC determination and time-kill assays. Efficacy of combinations of the steroidal alkaloids and conessine with levofloxacin were compared with monotherapies against infections in G. mellonella larvae by measuring larval mortality and bacterial burden. Results Combination therapies of conessine or steroidal alkaloids with levofloxacin enhanced bacterial inhibition in vitro and restored antibiotic efficacy in vivo compared to the constituent monotherapies. Neither conessine nor the steroidal alkaloids induced any detectable toxicity in G. mellonella larvae. The enhanced efficacy of the combination treatments was most pronounced with conessine and correlated with reduced larval burden of infecting P. aeruginosa . Notably, the enhanced efficacy of conessine/levofloxacin combinations was only detected in the parent strain and strains that overexpressed the MexAB-OprM or MexEF-OprN efflux systems. Conclusions Steroidal alkaloids from Holarrhena antidysenterica , and particularly the principal active ingredient conessine, restored levofloxacin efficacy against resistant P. aeruginosa strains possessing efflux-mediated MDR phenotypes. The compounds should be investigated further as a potential novel therapy.

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.

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.

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.

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.

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.

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.

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 flares 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.

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.

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 A ˚ 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.