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

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

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.PostprintPeer reviewe

Repurposing mitomycin C in combination with pentamidine or gentamicin to treat infections with multi-drug resistant (MDR) 3 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.Peer reviewe

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

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.PostprintPeer reviewe

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

The structure of 3-methyladenine DNA glycosylase I in complex with 3-methyladenine is reported

Demographics of extra-articular calcaneal fractures: Including a review of the literature on treatment and outcome

Introduction: Extra-articular calcaneal fractures represent 25-40% of all calcaneal fractures and an even higher percentage of up to 60% is seen in children. A disproportionately small part of the literature on calcaneal fractures involves the extra-articular type. The aim of this study was to investigate the incidence of extra-articular calcaneal fractures in a Level 1 trauma centre, define the distribution of the various types of fractures and compare patient demographics between extra- and intra-articular calcaneal fractures. In addition the literature was reviewed for the most common types of extra-articular calcaneal fractures with regard to incidence, treatment and clinical outcome. Methods: The radiological records between 2003 and 2005 were reviewed for intra- and extra-articular calcaneal fractures. Patient gender-distribution and age were compared. A literature search was conducted for the treatment of extra-articular calcaneal fractures. Results: In this 3-year study period a total of 49 patients with 50 extra-articular calcaneal fractures and 91 patients with 101 intra-articular fractures were identified. The median age for the first group was 32.7 years, and for the second group 40.3 years; P = 0.04. Male predominance was significantly less pronounced for extra-articular (63%) compared with intra-articular fractures (79%; P = 0.04). Conclusion: One-third of all calcaneal fractures are extra-articular. Significant differences exist between the intra- and extra-articular groups, in terms of lower age and male-female ratio. The literature study shows inconsistencies in treatment options, but most extra-articular fractures are well manageable conservatively

Analysis combining correlated glaucoma traits identifies five new risk loci for open-angle glaucoma

Open-angle glaucoma (OAG) is a major cause of blindness worldwide. To identify new risk loci for OAG, we performed a genome-wide association study in 3,071 OAG cases and 6,750 unscreened controls, and meta-analysed the results with GWAS data for intraocular pressure (IOP) and optic disc parameters (the overall meta-analysis sample size varying between 32,000 to 48,000 participants), which are glaucoma-related traits. We identified and independently validated four novel genome-wide significant associations within or near MYOF and CYP26A1, LINC02052 and CRYGS, LMX1B, and LMO7 using single variant tests, one additional locus (C9) using gene-based tests, and two genetic pathways - “response to fluid shear stress” and “abnormal retina morphology” - in pathway-based tests. Interestingly, some of the new risk loci contribute to risk of other genetically-correlated eye diseases including myopia and age-related macular degeneration. To our knowledge, this study is the first integrative study to combine genetic data from OAG and its correlated traits to identify new risk variants and genetic pathways, highlighting the future potential of combining genetic data from genetically-correlated eye traits for the purpose of gene discovery and mapping