In Vitro Activity of Eravacycline against Gram-Positive Bacteria Isolated in Clinical Laboratories Worldwide from 2013 to 2017

Eravacycline is a novel, fully synthetic fluorocycline antibiotic being developed for the treatment of serious infections, including those caused by resistant Gram-positive pathogens. Here, we evaluated the in vitro activities of eravacycline and comparator antimicrobial agents against a recent global collection of frequently encountered clinical isolates of Gram-positive bacteria. The CLSI broth microdilution method was used to determine in vitro MIC data for isolates of Enterococcus spp.

U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of complicated intra-abdominal infections (cIAI), including those caused by MDR pathogens (3, 4; https://clinicaltrials.gov/ct2/show/NCT01844856). Additionally, eravacycline has been demonstrated to have in vivo efficacy as a treatment in murine models of systemic, thigh, and lung infection and pyelonephritis (4,6,7).
Eravacycline is comprised of a tetracycline core with two novel modifications: a fluorine atom at the C-7 position and a pyrrolidinoacetamido group at the C-9 position, both of which are on the D ring (4,8). These novel modifications confer enhanced in vitro activity compared to that of other tetracyclines against resistant Gram-negative and Gram-positive bacteria, and the pyrrolidinoacetamido group allows for increased ribosomal binding and steric hindrance to avoid ribosome protection-based tetracycline resistance.
The objective of the current study was to determine the in vitro activity of eravacycline relative to that of other antimicrobial agents using a representative global collection of clinical isolates of Gram-positive bacteria.

RESULTS AND DISCUSSION
A total of 10,511 Gram-positive aerobic isolates collected between 2013 and 2017 were included in this study. The MIC distributions and the cumulative percentage of    Tables 2, 3, and 4 provide details on the in vitro activities of eravacycline and the comparator agents against staphylococci, enterococci, and streptococci, respectively, including percent susceptibility according to the CLSI and EUCAST breakpoints. The highest rates of nonsusceptibility in MRSA were reported for azithromycin, clindamycin, and levofloxacin (75.9%, 38.3%, and 65.9%, respectively, by CLSI criteria), while resistance rates were Ͻ1% for linezolid, daptomycin, and vancomycin ( Table 2). For compounds of the tetracycline class, tigecycline and minocycline, resistance rates were approximately 2 to 12% across FDA/CLSI and EUCAST breakpoints. Comparatively, due to overall lower breakpoints for eravacycline, the nonsusceptible rate was nearly 20% by the FDA criteria and 4.5% by the EUCAST criteria, but the MIC 90 value of eravacycline was 2-fold lower than that of tigecycline. Similarly, for E. faecalis the nonsusceptibility rates to linezolid and daptomycin were Ͻ1% and 5.6%, respectively, while the rates were 2% and 53%, respectively, for E. faecium (Table 3). Vancomycin retained activity against E. faecalis, with a resistance rate of 4.9%, but it was generally ineffective against E. faecium, in which the rate of resistance exceeded 40%. Both species of enterococci were resistant to minocycline, with nonsusceptibility rates ranging from 49 to 72%. While eravacycline and tigecycline nonsusceptibility rates were about 1 to 5%, the MIC 90 of tigecycline was 2 doubling dilutions higher than that of eravacycline. Notably, the rates of resistance for the comparators in this study were similar to those seen in other global surveillance studies (16,17).
When isolates were allocated to their respective geographic regions, eravacycline MIC 90 s were within 1 doubling dilution for all Gram-positive genera/species (see Table  S3 in the supplemental material). Similarly, there were no significant differences (a    (Table S4) or stratified by specimen source (Table S5) (4,6,7,15). Susceptibility rates, due to a difference in breakpoints, were similar between these two antibiotics. As tigecycline EUCAST breakpoints have recently been lowered for Gram-negative organisms, perhaps a review of the breakpoints for Grampositive organisms is also warranted for this agent.
This global surveillance investigation highlights the broad-spectrum potency of eravacycline against Gram-positive bacteria, including resistant isolates. As cIAIs are well-known to be polymicrobial, involving synergistic Gram-positive, Gram-negative, and anaerobic organism interactions, this study underscores the potential benefit of eravacycline for the empirical treatment of cIAIs. Furthermore, eravacycline may have a role in the treatment of other infections caused predominantly by Gram-positive pathogens, but the clinical utility in such disease states should be investigated.  Table S1 in the supplemental material summarizes the numbers of isolates collected in each of the four study periods (2013 to 2014, 2015, 2016, and 2017) by geographic region. Overall, approximately 54% of the isolates came from Europe, 35% of the isolates came from North America, and 10% came from the Asia-Pacific region. In total, there were 3,180, 2,082, 3,176, 956, and 1,117 isolates, respectively, from respiratory, intra-abdominal, urinary, skin, and other specimen sources (Table S2).

MATERIALS AND METHODS
Isolates were limited to one per patient, determined by the participating laboratory algorithms to be clinically significant, and collected irrespective of their antimicrobial susceptibility profile and independent of patient gender or age. The study was not designed to directly compare the prevalence of antimicrobial-resistant pathogens across specific geographic locations but, rather, was designed to   (21), as none currently exist. The FDA eravacycline susceptible breakpoint for the S. anginosus group was applied to beta-hemolytic streptococci. d The EUCAST eravacycline susceptible breakpoint for the S. anginosus group (Յ0.12 g/ml) was applied to beta-hemolytic streptococci. e The S. anginosus group (n ϭ 346) includes S. anginosus (n ϭ 302), S. constellatus (n ϭ 36), S. intermedius (n ϭ 7), and S. intermedius/S. milleri (n ϭ 1). f EUCAST tigecycline breakpoints for beta-hemolytic streptococci (Յ0.12 g/ml) were applied to the S. anginosus group. g NA, MIC breakpoint not available; -, not evaluable, as the tested MIC range did not extend high enough for the EUCAST susceptible breakpoint for S. pneumoniae. evaluate the in vitro activities of eravacycline and the comparator antimicrobial agents against a global collection of frequently encountered clinical isolates of Gram-positive bacteria collected from 2013 to 2017.
Antimicrobial susceptibility testing. The in vitro susceptibilities of the isolates were determined using the CLSI-defined broth microdilution method in 96-well broth microdilution panels (18,19). The antimicrobial agents used in panel production were acquired as laboratory-grade powders from their respective manufacturers or from a commercial source. The list of antimicrobial agents tested in each of the four study periods varied slightly, in that some agents, in addition to those tested in the 2013 to 2014 period, were included in the 2015, 2016, and 2017 testing periods. Of note, ampicillin, clindamycin, meropenem, and oxacillin were tested only in 2015, 2016, and 2017. The eravacycline MICs for Gram-positive bacteria were read following the current CLSI standard for dilution method testing; MIC endpoints were read following panel incubation at 35°C in ambient air for 16 to 20 h (Enterococcus and Staphylococcus spp.) or 35°C in ambient air for 20 to 24 h (Streptococcus spp.) (19). Quality control testing for eravacycline and the other antimicrobial agents was performed on each day of testing, as specified by the CLSI, using the CLSI-defined control strains E. faecalis ATCC 29212, S. aureus ATCC 29213, and S. pneumoniae ATCC 49619 (19).
MICs were interpreted using 2019 CLSI MIC breakpoints (19) and 2019 EUCAST MIC breakpoints (20), with the following exceptions. FDA MIC interpretative breakpoints were used for tigecycline (21) and eravacycline in place of CLSI MIC breakpoints, which are not currently published for these agents. Additionally, tigecycline breakpoints for vancomycin-susceptible Enterococcus faecalis were applied to vancomycin-resistant isolates and to Enterococcus faecium; EUCAST eravacycline breakpoints for the Streptococcus anginosus group were applied to beta-hemolytic streptococci; EUCAST tigecycline breakpoints for beta-hemolytic streptococci were applied to the S. anginosus group; and EUCAST eravacycline breakpoints for S. aureus were applied to coagulase-negative Staphylococcus species.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.3 MB.