Delafloxacin In Vitro Broth Microdilution and Disk Diffusion Antimicrobial Susceptibility Testing Guidelines: Susceptibility Breakpoint Criteria and Quality Control Ranges for an Expanded-Spectrum Anionic Fluoroquinolone

Delafloxacin, a recently approved anionic fluoroquinolone, was tested within an international resistance surveillance program. The in vitro susceptibilities of 7,914 indicated pathogens causing acute bacterial skin and skin structure infections (ABSSSI) were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution MIC testing methods.

Despite national and international efforts to combat antimicrobial resistance, it is notable that clinical microbiology laboratories struggle to generate accurate and actionable antimicrobial susceptibility test (AST) reports (17). Given that the majority of laboratories in the United States use automated AST systems (Vitek 2 [bioMérieux], MicroScan [Beckman Coulter], BD Phoenix [Becton Dickinson]) exclusively for routine testing, the ability to respond to the introduction of new antimicrobial agents and to adapt to emerging resistance mechanisms as well as to changes in interpretive criteria (breakpoints) are compromised by lag times between the approval of new agents or updates in clinical breakpoints and the clearance on commercial automated AST devices (17,18). In an effort to circumvent these barriers to providing AST data in a timely manner, laboratories may resort to using surrogate agents that may predict the susceptibility of the organism of interest to the newly introduced agent or to using manual AST methods, such as disk diffusion (DD) (17). The DD method advantages are the simplicity of the test, which does not require any special equipment, the provision of categorical results easily interpreted by clinicians, low cost, and flexibility in selecting drug disks for testing (18).
The recent regulatory approval of delafloxacin to treat ABSSSI (16) and the fact that there is no reliable surrogate among the FQs to predict the activity of delafloxacin against target pathogens mean that laboratories must adopt a nonautomated method, such as DD (delafloxacin disks are available in the United States from Hardy Diagnostics, Santa Maria, CA, USA), to provide AST information for this new broader-spectrum agent. The present study was designed to (i) assess the in vitro antibacterial activity of delafloxacin against contemporary (2014 to 2016) ABSSSI pathogens from the U.S. and European (EU) medical centers using FDA MIC interpretive criteria; (ii) determine the intermethod agreement between delafloxacin MIC and DD zone diameter values based on the FDA interpretive criteria; (iii) validate the FDA MIC quality control ranges for six aerobic quality control (QC) organisms.

Study designs.
International surveillance studies (involving 5,187 U.S. isolates and 2,727 EU isolates from 22 countries) monitored delafloxacin activity by reference BMD methods (19) during 2014 to 2016 via a central laboratory protocol (JMI Laboratories, North Liberty, IA). These 7,914 organisms from ABSSSI were collected as part of the SENTRY Antimicrobial Surveillance Program, and results were focused on the species listed in the delafloxacin product package insert (Table 1) (16). Key comparison agents were also tested (levofloxacin, oxacillin) to characterize drug-resistant subpopulations using breakpoint interpretive criteria of the CLSI and the European Committee on Antimicrobial Susceptibility Testing (20,21). QC was ensured by testing with ATCC reference strains and using performance guidelines found in published documents (16,20).
Delafloxacin BMD MIC and 5-g DD zone diameter comparison investigations utilized reference methods (19,22), FDA MIC and DD breakpoints (18; see also Table 2), and accepted intermethod analyses (23). Organisms of indicated species or groups were analyzed separately, attempting to achieve minimal intermethod categorical errors. The organisms studied were from clinical trial samples (n ϭ 2,214) and preclinical microbiology investigations (n ϭ 1,500). All isolates were identified by standard biochemical tests or matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker, Billerica, MA).
Determinations of QC limits for standardized tests (19,22) were guided by a multilaboratory study design of the CLSI (23). For the delafloxacin MIC and DD methods, three separate studies were completed (two for zone diameter limits and a single MIC trial).
In each trial, a minimum of seven laboratories contributed test results for the studied ATCC QC strains: S. aureus ATCC 25922 (DD only) and ATCC 29213 (MIC only), S. pneumoniae ATCC 49619, E. faecalis ATCC 29212 (MIC only), E. coli ATCC 25922, and P. aeruginosa ATCC 27853. These investigations used at least three medium lots from two or more manufacturers (BD, Remel, Tekova) and two disk lots (MAST). Ranges were calculated by methods found in CLSI M23 (23) or by the RangeFinder program (24).

RESULTS
Antimicrobial activity of delafloxacin. The reference broth microdilution MIC distributions for the FDA-indicated organisms or organism groups (7,914 isolates) from global (U.S. and EU) surveillance in 2014 to 2016 are shown in Table 1. The MIC 90 values for U.S. and EU isolates of Gram-positive cocci (GPC) were within Ϯ 1 log 2 dilution step for each organism group except MSSA (MIC 90 , 0.06 and 0.008 g/ml for U.S. and EU isolates, respectively) (data not shown). Delafloxacin showed low MIC values (MIC 90 range, 0.015 to 2 g/ml) against Gram-positive pathogens (Table 1). Among S. aureus isolates, 98.5% of MSSA and 83.6% of MRSA were inhibited at the FDA susceptible (S) breakpoint of Յ0.25 g/ml. Levofloxacin resistance (R) was detected in 28.1% of S. aureus isolates (7.7% of MSSA and 61.3% of MRSA): 74.6% of levofloxacin-R isolates (80.8% of levofloxacin-R MSSA and 73.3% of levofloxacin-R MRSA) were S to delafloxacin (Table 1). Whereas delafloxacin showed decreased susceptibility rates among S. haemolyticus isolates (44.4% S at Յ0.25 g/ml; 17.8% S to levofloxacin), it was more active against S. lugdunensis (99.3% S at Յ0.25 g/ml; 98.6% S to levofloxacin).
Intermethod comparison. Based upon the FDA MIC breakpoints, DD method breakpoints have also been proposed for each group tested and the 5-g delafloxacin disk content under study (Table 2). Scattergrams depicting the intermethod accuracy of the proposed MIC and DD zone diameter breakpoints are presented in Fig. 1 and 2 and Fig. S1 to S5 in the supplemental material.
The 5-g disk content provided adequate separation of S and R strains of S. aureus and the Enterobacteriaceae, with rare very major error (VME; false susceptible at 0.0 to 0.4%) and no major error (ME; false resistant) for either organism group ( Fig. 1 and 2). The error rates for all organism groups were well within acceptable limits (Ͻ1.5% VME and Ͻ3.0% ME) (Fig. S1 to S5). There was only one VME observed with coagulatenegative staphylococci (CoNS) (0.4%) and E. faecalis (0.9%), respectively, and none with S. pyogenes and the S. anginosus group. The overall intermethod categorical agreement (CA) ranged from 83.7% (P. aeruginosa, only 1.0% ME) to 100.0% (S. pyogenes and S. anginosus group).
Quality control range studies. The broth microdilution (BMD) method QC study had nine participating laboratories and employed reference MIC panels produced by Trek Diagnostics (Cleveland, OH). All internal FQ (levofloxacin) control results were within published ranges, validating delafloxacin data. Two QC strains required MIC ranges of four log 2 dilution steps (S. aureus ATCC 29213 and E. faecalis ATCC 29212). A range of 0.001 to 0.008 g/ml was calculated for S. aureus ATCC 29213 (Fig. 3), which   (Table 3). QC study results for the 5-g delafloxacin DD method were produced by two investigations utilizing zone diameters (10 laboratories) for four QC organisms. In the initial study, data from eight laboratories determined the delafloxacin zone diameter QC ranges for E. coli ATCC 25922 and P. aeruginosa ATCC 27853 calculated from 478 to 480 zone diameter results per strain using Clinical and Laboratory Standards Institute (CLSI) M23 and/or statistical criteria (Table 3). For these two tested QC strains, 96.7 to 100.0% of reported zone diameter results were within the proposed 7-to 12-mm-wide ranges. The proposed zone diameter range for S. aureus ATCC 25923 was calculated from 414 results, producing a proposed QC range between 32 and 40 mm (9 mm wide), which included 98.8% of results and a modal zone diameter at 35 and 36 mm (91 results each). S. pneumoniae ATCC 49619 required an additional multilaboratory QC study to complete the calculated zone diameter range of 28 to 36 mm. Among 480 reported zones, 478 (99.6%) results were within the proposed range determined from 10 laboratories across two DD QC investigations. Although not a target species for ABSSSIs, S. pneumoniae is included here as an important strain for QC of delafloxacin testing of target streptococci (beta-hemolytic and viridans group streptococci).

DISCUSSION
The in vitro activity of delafloxacin and support for S. aureus clinical breakpoints were presented by McCurdy et al. (10). Those data suggested a susceptible (S) breakpoint of Յ0.25 g/ml (16) with an intermediate (I) category at 0.5 g/ml ( Table 2).
Susceptible BMD clinical breakpoints were optimized by pharmacokinetic/pharmacodynamic (PK/PD) models, Monte Carlo simulations, and target attainment (TA) of the ratio of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC/MIC) for a cidal endpoint (1 log 10 CFU reduction) (25)(26)(27). Rubino and colleagues (27) observed a 96.8 to 98.5% TA for organisms with delafloxacin MIC values at 0.25 g/ml, and a stasis TA of 87.8 to 90.8% at 0.5 g/ml among the modeled ABSSSI clinical trial patients treated with the approved dosing regimens (16). The S breakpoints were further adjusted for the pathogen species MIC distribution and clinical success rates indexed by organism group (16,(23)(24)(25)(26)(27). As an example, the percentage of eradication rates for S. aureus in phase 3 ABSSSI clinical trials with delafloxacin MIC results at 0.25 and 0.5 g/ml were 97.2 and 100.0%, respectively (10). Furthermore, the applied fAUC/MIC target of 1 log 10 CFU cidality for delafloxacin with an associated Ն90% TA was consistent with the recent breakpoint recommendations of the U.S. Committee on Antimicrobial Susceptibility Testing organization fluoroquinolone breakpoint report (28).
Delafloxacin demonstrated potent activity against S. aureus, S. lugdunensis, and the indicated streptococci (MIC 90 results at 0.015 to 1 g/ml) ( Table 1). The levofloxacin-R strains of MRSA and S. haemolyticus tended to cluster in or near the I category for delafloxacin MIC testing results ( Table 1). As reported by McCurdy et al. (10), the overall microbiological response rate was 98.6% (81/82) for all S. aureus isolates with documented quinolone resistance-determining region (QRDR) mutations from ABSSSI patients treated with delafloxacin. Notably, the delafloxacin MIC values did not increase beyond 0.5 g/ml until at least double QRDR mutations in both gyrA and parC were observed, suggesting that delafloxacin may be useful in treating infections due to FQ-R staphylococci (10,16).
The decreased delafloxacin susceptibility for the E. faecalis and Gram-negative species was observed among the levofloxacin-R subpopulation. However, delafloxacin is known to display enhanced activity against Gram-negative isolates, such as E. coli and K. pneumoniae, when tested in an acidic environment, which suggests that the in vivo activity may be increased at an infection site (1,2,13,29).
The selection of correlate S DD zone diameters was made via scattergram intermethod error rate bounding analyses (23), which produced rare serious discords, with VME and ME rate ranges of 0.0 to 0.4% and 0.0 to 1.0%, respectively (Fig. 1, 2, and S1 to S5). Overall, the CA between BMD and DD method breakpoints was 83.7 to 100.0%. Only the P. aeruginosa breakpoints performed suboptimally, but the discords were  (23). Ranges approved by the FDA (16) and CLSI (20). BMD, broth microdilution; DD, disk diffusion; NA, not applicable. b DD range using a 5-g delafloxacin content.
In summary, these data extend the delafloxacin in vitro activity experience (11) into 2016 within a global surveillance program (Table 1) and also validate the susceptibility testing breakpoint criteria (16) for reference, standardized methods (19,22) used by clinical microbiology laboratories. These presented results appear to be robust, derived from testing thousands of ABSSSI clinical isolates among the clinically indicated species (16), and were supplemented with the determination of QC parameters for the in vitro test methods (16,20). The latter set of quality assurance guidance documents should enhance efforts to accurately assess the role of delafloxacin for treating FQ-R S. aureus and other pathogens, as well as for expanding clinical indications to respiratory tract infections (1,11,25,(30)(31)(32).

ACKNOWLEDGMENTS
We thank the following investigators who contributed study results/analyses: Sue Cammarata, Daniel Sahm, Clyde Thornsberry, and Leah Woosley.