FOF, known for its broad-spectrum antimicrobial activity, particularly against S. aureus and methicillin-resistant staphylococci, is often explored in combination therapies to prevent the rapid emergence of resistant mutants and to sterilize the infection more rapidly [17, 18]. Notably, previous studies have shown promising synergistic effects with various beta-lactam and non-beta lactam antimicrobials, including cefpirome, cefazolin, cefotaxim, oxacillin, and imipenem [19,20,21,22].

The present study aimed to assess the synergistic effects of FLX and FOF against various S.s aureus strains, including methicillin-resistant isolates. Until now, only a limited number of studies have adopted a comprehensive multidimensional approach similar to ours, which integrates antimicrobial susceptibility testing, synergy testing, TKC’s, in vivo G. mellonella survival assays, and gene expression analyses of bacterial virulence factors.

The synergy testing revealed that the combination of FLX and FOF significantly lowered the MIC, particularly in methicillin-resistant strains ranging from 1/8 to 1/64 of their MIC. The susceptible breakpoint index (SBPI) values indicated that the combined MICs were equal to or lower than their respective breakpoints, as the greater the SBPI value (≥ 2), the more effective the antimicrobial combination (e.g., SBPI for MSSA 49–86 and for MRSA 2–139). Results of TKCs further supported this synergistic potential, demonstrating not only inhibition of bacterial growth but also prevention of regrowth, indicating sustained effectiveness. In both methicillin-susceptible and methicillin-resistant strains, the combination exhibited superior bacterial killing compared to individual antibiotic treatments. TKC data provided robust evidence for the relevance of the FOF and FLX combination, emphasizing its potential to enhance antibiotic efficacy. Although TKCs with MSSA strains ATCC 29213 and MSSA 231/20 exhibited a 1 log10 reduction in bacterial count with FOF alone, regrowth occurred after 24 h. FLX alone effectively inhibited growth, but when used in combination, it achieved a 2 log10 reduction in bacterial count for both strains without any subsequent regrowth. For MRSA strains 23622 DSMZ (FOF R) and ATCC 33592 (FOF S), only the combination of FOF and FLX achieved a 3 log10 reduction in bacterial count for both strains within the first 10 h. The FOF S strain demonstrated sustained growth inhibition up to 24 h, whereas the FOF R strain showed regrowth. Consequently, this data highlights significant potential against both MSSA and MRSA isolates. To further advance our research, we conducted in vivo testing to evaluate the potential combination in larvae. The decision to utilize the G. mellonella model for in vivo testing was informed by its established efficacy in simulating bacterial infections and assessing antimicrobial efficacy as discussed by Serrano et al. [23]. This model, first researched over 85 years ago, serves as a valuable tool for studying infections caused by various pathogens, screening antimicrobials, and exploring immune responses. Notably, its immune system exhibits similarities with mammals, and outcomes often align with mammalian and other invertebrate models [24]. Despite inherent limitations, G. mellonella effectively bridges the gap between in vitro and mammalian in vivo studies, reflecting the principles of the 3Rs in animal experimentation.

The G. mellonella survival assays offered insights into the in vivo efficacy of the FOF and FLX combination. The G. mellonella model provided a valuable platform for assessing the therapeutic advantage of the combination, with particularly promising results in MRSA infections. In MRSA infection, even the combination of FLX plus low-dose FOF significantly improved survival rates compared to monotherapies. These findings suggest that the FOF and FLX combination could be a viable option for treating S. aureus infections, including those caused by drug-resistant strains. For MSSA infections, however, this could not be proven at the beginning, as treatment with FLX or FLX plus low-dose FOF after infection with MSSA (ATCC 29213) surprisingly led to poorer survival than low-dose FOF alone and even placebo. In order to better understand this observation, further in vivo experiments were carried out with FLX alone, FOF alone, and the combination of both at different doses, in which the bacterial counts in the hemolymph were determined before administration of the therapy and after 24 h. In contrast to the results of the survival assays, however, there were no differences between the treatment groups. The complexity of regulatory circuits in S. aureus contributes to its adaptability and virulence, with genes encoding toxins (e.g., lukED, hla), cell surface proteins (e.g., protein A), and antimicrobial resistance (e.g., agrA) [25, 26]. Given the surprising results of the G. mellonella survival assay for infection with MSSA and FLX therapy, an expression analysis was added. The virulence factors of ATCC 29213 were determined using whole genome sequencing, and then agrA, a major global regulator of S. aureus virulence and two factors (hla, lukDE) that have already shown an effect on mortality in animal experiments after gene knockout or expression changes were examined. The gene expression analyses shed light on the impact of FOF and FLX alone and in combination at different concentrations on the of bacterial virulence factors. As suspected, monotherapy of FLX enhanced the gene expression of all virulence factors tested after 4 h, with lukED being the most pronounced [27,28,29]. In contrast, only the expression of lukED was significantly reduced after exposure to FOF alone, while the combination of FOF and FLX showed a lower reduction of lukED, but also of agrA and hla after exposure to 1/8 of the MIC. The lower influence on the expression of virulence factors under combination therapy with FOF plus FLX compared to FOF alone could therefore also explain the significantly reduced survival in the G. mellonella survival assay after infection with MSSA ATCC 29213.

However, MSSA ATCC 29213 is only one of many reference strains and it was unclear whether these results are transferable to other MSSA strains. For this reason, further in vivo experiments were carried out with another MSSA isolate (ATCC 6538). In a preliminary experiment, treatment with FLX at a dose of 100 mg/kg resulted in a survival rate of 91% compared to 0% in the control group, which was consistent with the previously proposed hypothesis (data not shown). In addition, survival rates of 5% in the control group, 55% in the group receiving low-dose FOF only, 35% in the group receiving FLX only, and 85% in the group receiving a combination of low-dose FOF and FLX were observed in the main experiment, similar to the studies with the MRSA isolate.

Based on the data obtained in this study and the current literature, the combination of FOF plus FLX could be prospectively investigated in clinical trials with specific areas of application. On the one hand, it might be investigated instead of imipenem plus FOF as a rescue therapy after treatment failure or necessary discontinuation due to pronounced side effects of classic MRSA agents, thereby ensuring that carbapenems are retained as last resort antibiotics for the treatment of critically ill patients. On the other hand, this combination therapy could be used especially in countries with low to moderate MRSA rates for the initial, calculated antimicrobial therapy of suspected S. aureus infections, such as delayed prosthetic joint infections or early prosthetic infective endocarditis, as well as in cases where S. aureus has been detected as the causative pathogen but the results of an antimicrobial susceptibility test are still pending or missing, in order to utilize the advantages of such combination therapy in the initial treatment phase.

In conclusion, this study provides substantial evidence supporting the synergistic effects of FLX and FOF against S. aureus, particularly in drug-resistant strains. The multidimensional approach, encompassing MIC assessments, TKC, in vivo assays, and gene expression analyses, provides a comprehensive understanding of the combination's potential. Nevertheless, it is important to acknowledge that findings from translational models, such as G. mellonella, may not always be directly extrapolatable to clinical settings. Differences in immune response, pharmacokinetics, and host–pathogen interactions highlight the limitations of preclinical models and underscore the need for further validation in human studies.