Demonstration of the Broad-Spectrum In Vitro Activity of Finafloxacin against Pathogens of Biodefense Interest

This study investigated the in vitro activity of finafloxacin against bacterial strain panels of the biodefense pathogens. Broth microdilution assays were performed at neutral and acidic pH to determine the effectiveness of the antibiotics under conditions typical of an intracellular environment. In all instances, finafloxacin demonstrated superior activity at low pH.

A ntimicrobial resistance is an evolving issue, and new therapeutics are needed to treat infections caused by the pathogens of biodefense interest and those that are considered to be of public health concern. It is important that new antimicrobials are evaluated under conditions that model those encountered within the environment of a host, including the low-pH environment of the cell (the phagolysosome) that is particularly relevant to intracellular pathogens and infected body sites. It has been shown previously that the activity of certain classes of antibiotics (including fluoroquinolones) can be affected by a reduction in pH (1)(2)(3)(4). Finafloxacin is a fluoroquinolone derivative with an 8-cyano substituent and 7-pyrrolo-oxazinyl moiety that is being developed for the treatment of urinary tract infections in hospitalized patients (5,6). This modification has conferred activity in low-pH environments, which has resulted in superior in vitro activity against a range of organisms, including Staphylococcus aureus and Acinetobacter baumannii (7,8).
The availability of formulations of finafloxacin that can be delivered orally and systemically makes finafloxacin a worthy alternative for the treatment of a range of infections. In addition to good safety and efficacy data obtained in patients suffering from complicated urinary tract infections and pyelonephritis, previous studies have also demonstrated efficacy against the biothreat agents Burkholderia pseudomallei and Francisella tularensis in vitro and in vivo (6,(9)(10)(11). The aim of this study was to further evaluate the in vitro activity of finafloxacin against larger strain panels of biodefense pathogens.
Antibiotic susceptibility was determined at pH 5 and pH 7 for B. pseudomallei (n ϭ 10), Burkholderia mallei (n ϭ 10), F. tularensis (n ϭ 10), Bacillus anthracis (n ϭ 10), and Yersinia pestis (n ϭ 10), held at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) ( Table 1). In addition, a B. pseudomallei strain panel (n ϭ 11) provided by the Biomedical Advanced Research and Development Authority (BARDA) was screened (Table 1) (12). These assays were performed under biosafety level 3 (BSL3) conditions. Antibiotic susceptibility was reported as the MIC 50 or MIC 90 , defined as the lowest concentration of the antibiotic at which the growth of 50% or 90% of the isolates, respectively, were inhibited. Finafloxacin was supplied by MerLion Pharmaceuticals GmbH, and all other antibiotics were sourced from the U.S. Pharmacopeia, Selleckchem, or Sigma. Broth microdilution assays were performed as detailed by the Clinical and Laboratory Standards Institute (CLSI) (13), with the exception of a medium supplement (2%), IsoVitaleX (Becton, Dickinson), used to support the growth of F. tularensis. The activity of finafloxacin was determined at pH 5 and pH 7 (if the bacterial species was able to be cultured) and the MICs determined.
At pH 5, the MICs for B. pseudomallei ranged from 0.12 to 2 g/ml, 16 to 64 g/ml, and 4 to 64 g/ml for finafloxacin, ciprofloxacin (CIP), and ceftazidime (CAZ), respectively, demonstrating the superior in vitro activity of finafloxacin at low pH. Although it is difficult to make comparisons between the efficacies of antibiotics simply by MIC, these values are lower than those determined for another fluoroquinolone, CIP, and a component of the treatment for B. pseudomallei infections in humans (CAZ) (Fig. 1A). At neutral pH, finafloxacin demonstrated a level of activity (0.5 to 8 g/ml) similar to those observed with CIP (1 to 4 g/ml) and CAZ (0.5 to 32 g/ml) (Fig. 1B).
A similar trend was observed with the other pathogens of biodefense interest. Finafloxacin had superior activity at pH 5 for B. anthracis, B. mallei, and Y. pestis compared to either CIP or azithromycin (AZM) (an antibiotic used for the treatment of B. mallei infection in humans and as a control in the in vitro assays) ( Table 2). Unfortunately, only two strains of F. tularensis could be cultured in this low-pH environment; therefore, the MIC 50 and MIC 90 at pH 5 could not be determined. The most striking difference was observed for B. mallei. Finafloxacin had 9-fold and 7-fold improved activity over that of AZM against a panel of these strains (MIC 50 , 0.12 g/ml compared to Ͼ64 g/ml; MIC 90 , 0.5 g/ml compared to Ͼ64 g/ml) when performed at pH 5 ( Table 2). At pH 7, finafloxacin demonstrated activity similar to those of the comparator antibiotics, with MIC 50 and MIC 90 of 0.5 g/ml (at both pHs) for B. mallei and 0.06 g/ml and 0.12 g/ml for B. anthracis, respectively ( Table 2).
The data set detailed in these studies demonstrates that finafloxacin has activity under both acidic and neutral conditions, with enhanced activity of finafloxacin in low-pH environments, where other antibiotics (including ciprofloxacin) have reduced activity. This has been demonstrated for all of the biodefense pathogens of interest and is in agreement with data generated by other groups (7,8,10,11). The improved activity of finafloxacin compared to that of ciprofloxacin (a typical treatment for infections caused by B. anthracis, Y. pestis, and F. tularensis) further highlights the importance of evaluating therapies under conditions considered to be more like those encountered within a host and identifies finafloxacin as a novel broad-spectrum fluoroquinolone that could be used for prophylaxis or treatment following exposure to a range of pathogens.
Of particular interest is the activity of finafloxacin against the Burkholderia species evaluated. It has been demonstrated previously that fluoroquinolones are not effective as treatment for melioidosis in humans mainly due to B. pseudomallei possessing resistance mechanisms, including efflux pumps (14)(15)(16)(17). The results detailed in this pseudomallei that confer resistance to other fluoroquinolones, possibly due to the effect of the modified chemical structure (7,10,18). The promising data generated for B. mallei suggest that finafloxacin is a potential alternative for the treatment of infection caused by this organism. Finafloxacin appears to have a wider spectrum of activity than the other fluoroquinolones and has the potential to be used to treat infections caused by all of the biothreat pathogens evaluated (19). It has also been shown to be safe and well tolerated in clinical trials (6). These encouraging in vitro findings warrant further investigation of finafloxacin which would determine whether this activity translates into comparable protection against all of these pathogens in vivo.

ACKNOWLEDGMENTS
This work was funded by the Defense Threat Reduction Agency (DTRA). We acknowledge the Biomedical Advanced Research and Development Authority (BARDA) for use of their B. pseudomallei strain panel. These strains were developed with federal funds from the Office of the Assistant Secretary for Preparedness and Response, BARDA, under contract number HHSO100201100008I, task order HHSO10033001T, contract number HHSO100201100005I, and task order HHSO10033001T.
The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.
We declare no conflicts of interest.