In Vitro Activity of Finafloxacin against Panels of Respiratory Pathogens

This study determined the in vitro activity of finafloxacin against panels of bacterial strains, representative of those associated with infection in cystic fibrosis patients and predominately isolated from clinical cases of respiratory disease. Many of these isolates were resistant to various antimicrobials evaluated including the aminoglycosides, cephalosporins, carbapenems and fluoroquinolones. Broth microdilution assays were performed at neutral and acidic pH, to determine antimicrobial activity. Finafloxacin demonstrated superior activity at reduced pH for all of the bacterial species investigated, highlighting the requirement to determine the activity of antimicrobials in host-relevant conditions.


Introduction
Cystic fibrosis (CF) is an inherited disorder caused by a defective CF transmembrane conductance regulator (CFTR) gene, encoding the CFTR protein. This protein typically encodes a channel involved in the transport of ions, including chloride and bicarbonate, into and out of cells [1]. Mutations in this gene can result in the production of mucus which is thicker and more adherent in the lungs and other tissues including the gastrointestinal tract [1]. In addition, patients with CF suffer from bacterial colonization, chronic infections and inflammation of the respiratory tract [2].
Typically, these infections are caused by bacteria that can grow as a biofilm, making it difficult for antibiotic treatment to penetrate and completely clear the colonizing bacteria from the tissues. Common infectious agents include Staphylococcus aureus (in pediatric patients), Pseudomonas aeruginosa, Achromobacter spp., Stenotrophomonas maltophilia and members of the Burkholderia cepacia complex (Bcc), a group of closely related opportunistic Burkholderia species (in adult patients) [3,4]. P. aeruginosa and S. aureus are the most common causative agents of infections in CF lungs; however, infection etiology also includes the identification of species, including Achromobacter xylosoxidans and S. maltophilia, typically known to be environmental pathogens [5]. Although not part of the Bcc, Burkholderia gladioli is also commonly isolated from CF lungs [6].
Treatment of these respiratory infections includes inhaled tobramycin, which was approved by the US Food and Drug Administration (FDA) in 1997 for the treatment of P. aeruginosa infections in CF patients [7]; however, the use of suboptimal concentrations of tobramycin has been shown to drive resistance [8]. Other studies have also reported resistance and problems treating such pathogens with tobramycin in vitro and in patients [9,10].
The pH of the lung is typically 7.4, which can be reduced to a more acidic pH following the establishment of an infection and the subsequent inflammation that can follow [11,12]. A reduction in the airway surface liquid is one of the hypotheses as to how infections are established in CF lungs [13]. This alteration to a more acidic pH has also been observed in other tissues and human body fluids including the skin, peritoneal fluid, urine and the mucus of CF patients [14]. It has also been shown that the activity of many classes of antibiotics, e.g., the fluoroquinolones and the aminoglycosides (including tobramycin), is reduced in low pH conditions [15,16].
Finafloxacin is a fifth-generation fluoroquinolone that has been approved by the FDA and Health Canada for the topical treatment of ear infections and is currently being developed by MerLion Pharmaceuticals for the systemic treatment of complicated urinary tract infections and pyelonephiritis [17,18]. The antibacterial activity of finafloxacin increases as the pH of the media or environment becomes more acidic, a feature not observed with most of the other antibiotic classes (including previous generations of the fluoroquinolones (e.g., ciprofloxacin and levofloxacin), the folic acid synthesis inhibitors (e.g., co-trimoxazole) and the aminoglycosides (e.g., tobramcyin). This has been demonstrated against a variety of organisms including S. aureus, Escherichia coli, Klebsiella pneumoniae and P. aeruginosa [19].
Investigations to study the improved antibacterial activity of finafloxacin at low pH have suggested that it can rapidly accumulate in both eukaryotic and bacterial cells in acidic conditions. Due to this accumulation, higher intracellular concentrations of the drug can be achieved which may enable this superior antibiotic activity. At neutral pH, efflux proceeds at a rate similar to that of influx, suggesting that diffusion is the only process involved. In contrast, the efflux of finafloxacin out of the cell is much slower than influx at acidic pH, resulting in higher concentrations of the drug remaining within the cell for longer [20].
The activity of orally administered finafloxacin has also been investigated in a number of in vivo models of infection, including mice infected systemically with Moraxella catarrhalis. This organism (in mice) initially colonizes the lung and then disseminates to other tissues with a self-limiting nature; therefore, the bacterial load was quantified at 1 day postchallenge. Treatment with finafloxacin resulted in a reduction of the bacterial load in the lung that was 1-2 log10 greater than the reduction seen following equivalent doses of the other fluoroquinolones, moxifloxacin, ciprofloxacin and levofloxacin (unpublished data).
The in vivo efficacy of finafloxacin has also been evaluated in a rat respiratory tract infection model. Wistar rats were infected by the intratracheal route with a bacterial suspension of Streptococcus pneumoniae (1 × 10 5 CFU/animal). Finafloxacin treatment was administered orally at 1 h and 4 h post-challenge. At 24 h post-challenge, lungs were aseptically removed, homogenized and plated out for bacterial enumeration. Finafloxacin was very effective in this model, exhibiting an almost equivalent degree of activity to moxifloxacin. Both antibiotics reduced the bacterial load in the lungs by a factor greater than 4log 10 [21].
The aim of this study was to further evaluate the in vitro activity of finafloxacin against panels of bacteria typical of causing infections in CF patients, the majority isolated from clinical respiratory samples. Such bacterial strains can be difficult to treat; therefore, the ability of finafloxacin to retain activity in conditions of low pH (e.g., following the establishment of a bacterial infection) may identify an alternative therapeutic for the treatment of such infections.

Results
This additional screening against panels of respiratory pathogens demonstrated that, as previously described for other bacterial species, finafloxacin was more potent in conditions of low pH. Finafloxacin was more active at pH 5.8 compared to pH 7.2 for all bacterial species evaluated ( Table 1). The MIC 50 or MIC 90 for finafloxacin for each bacterial species was generally equivalent or lower than tobramycin at pH 7.2, with the exception of P. aeruginosa, where tobramycin was more active. At pH 5.8, the MIC 90 s for finafloxacin were lower than for tobramycin, with the exception of B. cepacia where the MIC 90 for both antibiotics against this pathogen was high at both pHs (64 or >64 µg/mL) while the MIC 50 for finafloxacin was 1.5 µg/mL in an acidic environment compared to >64 µg/mL for tobramycin. This is interesting considering the low MICs previously reported for the closely related CL3 Burkholderia species, B. pseudomallei and B. mallei. The MIC 90 s of finafloxacin were reported as 1 µg/mL and 2 µg/mL (for pH 5 and 7) and 0.5 µg/mL (at both pHs) for B. pseudomallei and B. mallei, respectively [24]. This huge difference could be due to some of the B. cepacia strains isolated and utilized in this study being less susceptible to antibiotic treatment and, once within a host, establishing a niche in the lung which is difficult for antibiotic treatment to penetrate. It is possible that the dose of antibiotics administered to treat these infections was not optimal which may have led to the development of resistance. (-too few strains to determine a MIC 50 or MIC 90 ).

Materials and Methods
The susceptibility of 150 clinical strains to finafloxacin at pH 5.8 and pH 7.2 was determined by IHMA Europe (Monthey, Switzerland). These were Achromobacter spp. . These MICs were compared to those generated for tobramycin, a standard of care antibiotic for the treatment of such infections. The resistant breakpoints of tobramycin for P. aeruginosa and Acinetobacter species were >16 µg/mL (the others were not determined). Breakpoints have not yet been determined for finafloxacin. As a group of 150 organisms and where a clinical breakpoint was available, the susceptibilities of comparator antibiotics varied widely. Susceptibility to amikacin, aztreonam and tobramycin was 52.9%, 34% and 30.8%, respectively. Concerning cephalosporins, cefepime susceptibility was 50%, and ceftazidime was 64%. Some 58.7% of isolates were susceptible to levofloxacin. Resistance to carbapenems ranged from 27.8% (meropenem) to 37.1% (imipenem), and 27.1% of isolates were resistant to piperacillin/tazobactam. Finafloxacin was supplied by MerLion Pharmaceuticals GmbH, Berlin, Germany and tobramycin was procured from the US Pharmacopeia (North Bethesda, MD, USA). Broth microdilution minimum inhibitory concentration (MIC) assays were performed as detailed by the Clinical and Laboratory Sciences Institute [27,28] at both neutral (7.2) and acidic (5.8) pHs. Antibiotic susceptibility was reported as MIC 50 (MIC 50 ) or 90 (MIC 90 ) (defined as the lowest concentration of the antibiotic at which the growth of 50% or 90% of the strain panel were inhibited), and the range of MICs was determined for each individual bacterial strain.

Discussion
This is a very interesting dataset considering the typical susceptibility of the bacterial species investigated in this work to the fluoroquinolone class of antibiotics. It has been previously reported that wildtype strains of Achromobacter species are typically sensitive, whereas those strains isolated from the lungs of cystic fibrosis patients that are chronically colonized with Achromobacter are multi-drug resistant, including resistance to fluoroquinolones [29,30]. A similar pattern of fluoroquinolone resistance has emerged in isolates of A. baumannii [31]. A. baumannii is now non-susceptible to multiple antibiotic species including the fluoroquinolones [32].
A recent study determined the MICs for multiple antibiotics against 420 bacterial strains, the majority of which were respiratory samples collected from cystic fibrosis patients [33]. Of most relevance to the dataset described in this manuscript, this strain panel included A. xylosoxidans isolates (n = 50), S. maltophilia (n = 100), B. cenocepacia (n = 50), B. multivorans (n = 50), B. gladioli (n = 50) and B. cepacia (n = 20). The activity of the previous generations of fluoroquinolones, ciprofloxacin, levofloxacin and moxifloxacin against A. xylosoxidans (MIC 50 s of 4 µg/mL and MIC 90 s of >8 µg/mL for all) in this study was comparable to the activity of finafloxacin at pH 7.2 detailed in this short communication (MIC 50 s of 8 µg/mL and MIC 90 s of 32 µg/mL) and was reduced when finafloxacin was evaluated at pH 5.8 (MIC 50 s of 0.5 µg/mL and MIC 90 s of 1 µg/mL) [33].
The data generated in this study demonstrate that finafloxacin is active in both neutral and reduced pH conditions, with enhanced activity in acidic environments where other antibiotics (including the previous generations of the fluoroquinolones and the aminoglycosides) are less effective. Previous studies have demonstrated that this activity is antibacterial (due to greater cleavage activity with the bacterial rather than the eukaryotic Type II DNA topoisomerase enzymes) and not due to toxicity, demonstrated when finafloxacin was incubated with J774 mouse macrophages with no toxic effects observed (unpublished data).
The improved activity of finafloxacin when compared with tobramycin (a typical treatment for infections caused by P. aeruginosa particularly) demonstrates that determining activity in infection-relevant conditions is important and suggests that finafloxacin could be used to treat a wider spectrum of infections, particularly those caused by B. gladioli, A. xylosoxidans and P. aeruginosa. In addition, investigating the combined effectiveness of therapies may further benefit the treatment of infections caused by these pathogens. This is something we have previously demonstrated with finafloxacin and doxycycline against B. pseudomallei in vitro and in vivo [34].
In conclusion, we have demonstrated that finafloxacin has broad-spectrum activity against panels of clinical strains isolated predominantly from patients with respiratory infections, in addition to previously showing efficacy against ciprofloxacin-resistant A. baumannii, fluoroquinolone-resistant E. coli and the biothreat pathogens, most pertinent to this study, B. pseudomallei and B. mallei [22][23][24]35,36].
Furthermore, the current study showed the potent activity of finafloxacin against multiple isolates expressing resistance to various antibiotic classes. The need to combat antimicrobial resistance in multiple clinical settings has never been so important. If not addressed with novel agents or other therapeutic strategies as widely described, the impact of this phenomenon will become uncontrollable [36]. Importantly, the data from our current study support further investigation of finafloxacin against selected pathogens in vivo and may offer an alternative to tobramycin, colistin and levofloxacin in the treatment of bacterial respiratory infections in both CF and non-CF patients.