Protective effect of microbisporicin (NAI-107) against vancomycin resistant Enterococcus faecium infection in a Galleria mellonella model

Increasing antimicrobial resistance in Enterococcus faecium necessitates the search for novel treatment agents, such as bacteriocins. In this study, we conducted an in vivo assessment of five bacteriocins, namely Lacticin Z, Lacticin Q, Garvicin KS (ABC), Aureocin A53 and Microbisporicin (NAI-107), against vanB-resistant Enterococcus faecium using a Galleria mellonella model. Our in vitro experiments demonstrated the efficacy of all five bacteriocins against vanB-resistant E. faecium with only NAI-107 demonstrating in vivo efficacy. Notably, NAI-107 exhibited efficacy across a range of tested doses, with the highest efficacy observed at a concentration of 16 µg/mL. Mortality rates in the group treated with 16 µg/mL NAI-107 were lower than those observed in the linezolid-treated group. These findings strongly suggest that NAI-107 holds promise as a potential alternative therapeutic agent for treating infections caused by resistant E. faecium and warrants further investigation.

G. mellonella has been shown to be an effective model host for investigating the efficacy of antimicrobial agents against various pathogens 21,22 .This model serves as a viable alternative to traditional mammalian in vivo systems.The immune response in Galleria mellonella encompasses both cellular mechanisms-such as phagocytosisand humoral responses, which include melanization, hemolymph clotting, and the synthesis of antimicrobial peptides [23][24][25] .Crucially, studies have found that the efficacies of antimicrobial agents on infected G. mellonella larvae closely correlate with the known drug susceptibilities of the pathogens in vitro and in mammalian models [26][27][28][29] .In addition, the use of G. mellonella enables the application of the 3Rs principles (replacement, reduction and refinement) in animal experimentation 29 .

E. faecium strains and minimum inhibitory concentrations (MIC) of bacteriocins and antimicrobial drugs
VanB-type E. faecium (ID 553) was used for all the experiments.Sixty-six bacteriocins from Syngulon PARAGEN collection and NAI-107 from Naicons, SRL were screened using the resazurin microtiter assay (REMA) 30,31 .The MIC for the antimicrobials listed in Table 1 were determined according to the clinical and laboratory standard institute (CLSI) guidelines and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference method for MIC determination in E. faecium 32,33 .

In vivo studies
(a) Determining the appropriate dose of E. faecium G. mellonella larvae were treated with a range of doses of vanB-type E. faecium (ID 553) (1 × 10 6 -1 × 10 9 CFU/ mL) and observed for death alongside phosphate buffer saline (PBS) controls.The dose that resulted in approximately 80% mortality was 2.7 × 10 7 CFU in 30 µl PBS, which was further used in the G. mellonella infection model.

(b) Galleria mellonella infection model
Injection of G. mellonella was carried out as described in Dijokaite et al. 21.The last larval stage of G. mellonella (Terramania, Arnhem, NL) were used for the experiments.Only non-discoloured, healthy larvae were selected.The larvae were injected in the last left proleg using 0.3 mL U-100 insulin syringes (BD Micro-Fine).
Table 1.The MICs of the antibiotics (n = 8) and five most promising bacteriocins were determined using the micro broth dilution method for van-B type E. faecium (ID 553).www.nature.com/scientificreports/Each test group consisted of two groups of 10 larvae per concentration of each bacteriocin or linezolid (10 µg/ mL).30 µl of PBS containing 2.7 × 10 7 CFU E. faecium was used to infect the G. mellonella larva.The larvae were treated with various concentrations of bacteriocins.One positive and two negative control groups were used.The positive control group received only the E. faecium inoculum in PBS.Out of the two negative control groups, one group underwent no manipulation, while the other group was injected with PBS only.Each antimicrobial agent was tested in a single experiment using larvae from the same batch and tested at the same time under identical conditions.Each group were incubated in sterile Petri dishes at 37 °C with a 5% (v/v) CO 2 atmosphere for the length of the experiments.The larvae were observed for 120 h for any indications of illness, necrosis, or paralysis, which enabled an evaluation of the bacteriocin's toxicity.Larvae were scored dead if they did not respond to touch stimuli by blunt sterile forceps and scored for 5 consecutive days (120 h).

Antibiotics
An overview of the in vivo assay in Galleria mellonella model is shown in Fig. 1.

Statistical analysis
Data were analyzed using GraphPad Prism v9.Survival plots were created using Kaplan-Meier survival curves.Statistical analysis was carried out using the Mantel-Cox test to compare survival curves between the PBS controls and each treatment arm.A P value of < 0.05 was considered statistically significant.

In vivo assay for efficacy of bacteriocins in Galleria mellonella E. faecium infection model
The in vivo efficacy of A53, GarKS (ABC), lacticin Q, lacticin Z and NAI-107 were determined against vanB-type E. faecium in comparison to linezolid.Despite the in vitro activity of all five of these bacteriocins, four of the bacteriocins [A53, GarKS (ABC), lacticin Q and lacticin Z) did not demonstrate any detectable efficacy in vivo (Fig. 2).NAI-107 was effective in vivo at all doses tested-2 µg/mL, 4 µg/mL, 8 µg/mL 16 µg/mL (P values 0.01 to < 0.0001; Fig. 3).Mortality was lowest in the 8 µg/mL and 16 µg/mL groups.The mortality in the 16 µg/mL was significantly lower than the linezolid treated group (P = 0.0221).

Discussion
In our study, we observed that among the bacteriocins investigated, only NAI-107 demonstrated effectiveness in protecting Galleria mellonella from infection caused by vanB-type Enterococcus faecium.We do not have a definitive explanation for this difference, but several pharmacodynamic and pharmacokinetic factors may play a role.One of these is that all the bacteriocins, except NAI-107 may be metabolized by the G. mellonella 29,34 .Another possible explanation is that the mechanisms of action of these bacteriocins may differ.For example, the leaderless peptides such as Lacticin Q, typically form stable pores in bacterial membranes.In contrast, NAI-107 which are ribosomally synthesized peptides that undergo posttranslational modifications causes depolarization of the membrane resulting in bacterial cell death without the formation of stable pores 35,36 .NAI-107 is a 23-amino acid lantibiotic (class I bacteriocin) produced by the actinomycete Microbispora sp.ATCC PTA-5024 12,35 .Like other lantibiotics, it contains a methyllanthionine and three lanthionine bridges 37 .Unlike the other lantibiotics, it also contains two unusually modified amino acids: 5-chlorotryptophan and 3,4-dihydroxyproline 37 .It has novel mechanisms of action, which include the inhibition of lipid II mediated synthesis of peptidoglycan as well as disrupting inner cell membrane protein interactions leading to slow membrane depolarization (Supplementary Fig. S1) 37 .Importantly, NAI-107 exhibits no known cross resistance with other antimicrobials 35 .Previous studies have revealed a low risk for the emergence of resistance during therapy 12 .One study found that after 20 subpassages at increasing concentrations of NAI-107, NAI-107 MICs of N. gonorrhoeae, E. faecalis (vanA), E. faecium (vanA) and S. aureus (MRSA and GISA) increased only occasionally and to a maximum of 2 to fourfold the initial MIC 12 .Thus far the predominant resistance mechanism to lantibiotics detected, has been the production of an immunity protein by the producer strain 12,35 .Its novel mechanism of  www.nature.com/scientificreports/action is a likely explanation for the fact that no cross resistance between NAI-107 and other antimicrobials has been found 35 .NAI-107 is not absorbed orally, but it does have good bioavailability after intramuscular, intravenous and subcutaneous administration 1,38 .In a mouse model, single subcutaneous dosing of 5, 20 and 80 mg/kg resulted in Cmax concentrations from 4 to 22 µg/ml, AUC values from 27 to 276 mg h/Liter, and elimination half-lives of 4.2-8.2h 39 .This characteristic implies that NAI-107 would require parenteral administration in multiple doses per day for therapeutic use.
There are a number of study limitations.We only evaluated the effects of the bacteriocins on a single strain of vanB-type E. faecium and did not include vancomycin-sensitive E. faecium (VSE) strains as a control.The hypothesis we were testing in this study was that NAI-107 would exhibit activity in vivo against VRE.This hypothesis was motivated by the growing problem of VRE infections.We acknowledge the limitations of this hypothesis.Inclusion of VSE strains in our hypothesis would have allowed us to assess if NAI-107 shows promise for both VSE and VRE infections.This would be of clinical use as the vancomycin susceptibility of clinical isolates is not typically known when they are first identified.Further studies will be required to assess if NAI-107 is active against VSE strains.Our findings need to be replicated in a broader range of strains of E. faecium.As already noted, we did not investigate the reasons why four of the five bacteriocins were active in vitro but not in vivo.We acknowledge that there is considerable variation in the therapeutic efficacy of linezolid between the different experiments testing each antimicrobial compound.This may be explained by differences in the larvae used in the different experiments.Each compound was assessed in a single experiment conducted with the same batch of larvae but each subsequent experiments was conducted with a new batch of larvae.This makes comparisons between experiments inappropriate.Our results may be reproduced using different infection vertebrate and invertebrate models, such as those involving mice, rats, rabbits, dogs, zebrafish, Caenorhabditis elegans and Drosophila melanogaster (reviewed in 40 ).However, Galleria mellonella has been successfully used to model various aspects of the colonization and infection of Enterococcus faecium 40 .Finally, we did not assess the toxicity of the bacteriocins as this has been done in previous studies 27 .NAI-107 exhibited no toxicity up to 64 µg/mL in a G. mellonella model 27 .Notably, NAI-107 has been found to be non-toxic and efficacious against methicillin-resistant Staphylococcus aureus in a rat model 38 .
If future studies confirm that NAI-107 is non-toxic and efficacious in human trials, it may emerge as a useful option for treating multi-resistant enterococcal infections, addressing a critical need in the field of antimicrobial therapy.

Figure 1 .
Figure 1.Overview of the in vivo assay in Galleria mellonella model.(Figure was generated using BioRender).