Diagnostic Microbiology and Infectious Disease 95 (2019) 216–220 Contents lists available at ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio In vitro investigation of synergy among fosfomycin and parenteral antimicrobials against carbapenemase-producing Enterobacteriaceae Lindsay M. Avery a, Christina A. Sutherland a, David P. Nicolau a,b,⁎ a Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, CT, USA b Division of Infectious Diseases, Hartford Hospital, Hartford, CT, USA article info abstract Article history: Intravenous fosfomycin is undergoing clinical development in the United States for treatment of complicated Received 11 July 2018 urinary tract infections (cUTIs) and may be prescribed as a component of dual antibiotic regimens against Received in revised form 6 May 2019 carbapenemase-producing Enterobacteriaceae (CPE). Fosfomycin, aztreonam, cefepime, ceftazidime, ceftazi- Accepted 16 May 2019 dime/avibactam, ceftolozane/tazobactam, meropenem, piperacillin/tazobactam, and tobramycin minimum in- Available online 25 May 2019 hibitory concentrations (MICs) were determined by gradient diffusion strip (GDS) against CPE isolates (N = Keywords: 49). The GDS cross method was used to assess antibiotic interactions between fosfomycin and the aforemen- Enterobacteriaceae tioned parenteral antibiotics. The resultant fractional inhibitory concentration index was used to classify interac- Carbapenemase tions. Fosfomycin-containing combinations were evaluated only if nonsusceptible to the second agent. The Synergy fosfomycin MIC50 was ≥1024 mg/L by GDS. Synergy or additivity was detected in 80 (22%) fosfomycin- Fosfomycin containing combinations. Antagonism was not observed. Ceftolozane/tazobactam most frequently displayed synergy [8 (16.3%) isolates]. When CPE are isolated, clinical laboratories should consider performing GDS synergy tests to identify favorable antibiotic interactions. © 2019 Elsevier Inc. All rights reserved. 1. Introduction pharmacokinetic properties such as negligible protein binding and high tissue penetration allow for a potential utility in combination anti- Fosfomycin is a broad-spectrum antibiotic available as an intrave- biotic regimens for the treatment of MDR pathogens even in cases of nous (IV) formulation, fosfomycin disodium, for treatment of various deep-seated infection (Grabein et al., 2017). systemic infections in several European countries and Japan (Falagas Pharmacodynamic optimization of antimicrobial regimens, which et al., 2016; Grabein et al., 2017). In the United States, fosfomycin is ap- may include the use of combination therapy with 2 or more agents, is proved for treatment of uncomplicated urinary tract infection, but it is especially important in the treatment of CPE and other MDR Gram- available only as an oral formulation (Kaye et al., 2017, Monurol Pre- negative pathogens (Kollef et al., 2011; Monogue et al., 2016). The gra- scribing Information, 2007). However, IV fosfomycin is now being in- dient diffusion strip (GDS) cross method is an in vitro method used to vestigated in U.S. clinical trials to support its use in the treatment of investigate antibiotic interactions in terms of the fractional inhibitory complicated urinary tract infections (NCT02753946). concentration index (FICI), and it has been used to examine the activity Infections caused by multidrug-resistant (MDR) pathogens such as of dual antibiotic combinations against MDR organisms such as carbapenemase-producing Enterobacteriaceae (CPE) pose therapeutic Acinetobacter baumannii (Nageeb et al., 2015), Enterobacteriaceae challenges. Due primarily to acquisition of multiple broad-spectrum re- (Monogue et al., 2017; Samonis et al., 2012; White et al., 1996), Neisseria sistance mechanisms, a single agent (e.g., β-lactam) is unlikely to pro- gonorrhoeae (Wind et al., 2015), Pseudomonas aeruginosa (Samonis vide adequate empiric coverage of these pathogens in serious et al., 2012; White et al., 1996), and Stenotrophomonas maltophilia systemic infections (Kollef et al., 2011; Pogue et al., 2015). Fortunately, (Church et al., 2013). An anticipated role for IV fosfomycin in the U.S. worldwide clinical use of fosfomycin has remained infrequent since its will be as a component of dual antibiotic therapy when prescribed original discovery in 1969 (Grabein et al., 2017), and thus, the com- for CPE infections, especially those outside of the urinary tract. pound has retained activity against many MDR pathogens, including Thus, the purpose of the present study was to characterize antibiotic in- some CPE (Falagas et al., 2010,2016;. Furthermore, favorable teractions between fosfomycin and nonsusceptible antibiotics (aztreo- nam, cefepime, ceftazidime, ceftazidime/avibactam, ceftolozane/ tazobactam, meropenem, piperacillin/tazobactam, and tobramycin) ⁎ Corresponding author. Tel.: +860-972-3941; fax: +860-545-3992. among a genotypically diverse population of CPE isolates utilizing the E-mail address: [email protected] (D.P. Nicolau). GDS cross method. https://doi.org/10.1016/j.diagmicrobio.2019.05.014 0732-8893/© 2019 Elsevier Inc. All rights reserved. L.M. Avery et al. / Diagnostic Microbiology and Infectious Disease 95 (2019) 216–220 217 2. Materials and methods Table 1 Select resistance mechanisms of carbapenemase-producing Enterobacteriaceae isolates. 2.1. Bacterial isolates and antimicrobial susceptibility testing N (%) Mechanism Citrobacter spp. E. coli E. cloacae Klebsiella spp. In total, 49 CPE isolates were studied, of which 37 were acquired (N =2) (N =8) (N =4) (N = 35)a from the FDA-CDC Antimicrobial Resistance Bank (Atlanta, GA) and 12 KPC 1 (50.0) 2 (25.0) 3 (75.0) 16 (45.7) were selected from the Center for Anti-Infective Research and Develop- NDM 1 (50.0) 6 (75.0) 1 (25.0) 14 (40.0) ment isolate library. Fosfomycin, aztreonam, cefepime, ceftazidime, cef- OXA-CP 0 (0.0) 0 (0.0) 0 (0.0) 5 (14.3) tazidime/avibactam, ceftolozane/tazobactam, meropenem, and VIM 0 (0.0) 0 (0.0) 0 (0.0) 3 (8.6) b tobramycin MICs were determined by Etest® (bioMérieux, Durham, ESBL 1 (50.0) 5 (62.5) 3 (75.0) 31 (88.6) fosA 0 (0.0) 0 (0.0)c 0 (0.0) 11 (44.0)d NC) in accordance with the manufacturer's instructions. Fosfomycin MICs were determined in duplicate, and in cases of discordance, the ESBL = extended spectrum β-lactamase; KPC = K. pneumoniae carbapenemase; NDM = New Delhi metallo-β-lactamase; OXA-CP = OXA-type carbapenemase. procedure was performed until a modal MIC was evident. MIC Test a fi Includes K. pneumoniae (N = 34) and K. ozaenae (N =1). Strip (Lio lchem®, Waltham, MA) was used to determine piperacillin/ b ESBL-positive isolate contained CTX-M-15 and/or SHV-12. tazobactam MICs due to lack of availability of the Etest product. For c Presence of fosA mutation not assessed in 2 E. coli isolates; fosfomycin GDS MICs were MIC and synergy assessments, P. aeruginosa ATCC 27853 was used as a 1 mg/L and 2 mg/L for these strains. d quality control organism, which is appropriate for extended spectrum Presence of fosA mutation not assessed in 10 K. pneumoniae isolates; among these 10, ≥ β – a total of 4 isolates displayed fosfomycin MICs 64 mg/L (high off-scale GDS MICs -lactamase (ESBL) producing Enterobacteriaceae according to the ≥1024 mg/L were observed in 3 of these 4 strains). manufacturer's instructions. 3.2. Antibiotic interaction assessments 2.2. Antibiotic interaction assessments Among 364 total fosfomycin–antibiotic combinations assessed, the For assessments of antibiotic interaction using GDS methodology, most frequent type of interaction observed was indifference (284/364, isolates were prepared in accordance to the manufacturer's instructions 77.8%, Table 3). This observation is reflected in the geometric mean for antimicrobial susceptibility testing of single agents. Each bacterial FICI for each fosfomycin–antibiotic combination as each value is N1.00. inoculum was prepared to a concentration of 1 to 5 × 108 CFU/mL Synergy with fosfomycin was observed most frequently with from a second subculture, which was confirmed by performing serial di- ceftolozane/tazobactam (C/T), and additivity was observed most fre- lutions and colony counts on agar media (BD™ Trypticase™ Soy Agar quently with aztreonam. with 5% Sheep Blood, Franklin Lakes, NJ). MICs of the agents in combina- An example of synergy between fosfomycin and aztreonam is shown tion were determined by the GDS cross method (Pankey et al., 2013) in Fig. 1. Antagonistic activity between fosfomycin and any of the 8 an- and used to calculate the FICI of each fosfomycin–antibiotic combina- tibiotics tested was not present in any CPE isolate assessed. tion. Briefly, the bacterial suspension was streaked onto Mueller Hinton When results were categorized by carbapenemase type, fosfomycin II agar (BD™ BBL™, Franklin Lakes, NJ) with a sterile swab to create a plus aztreonam was the only combination to display synergy (4/24, bacterial lawn; for each fosfomycin-antibiotic combination tested, anti- 16.7%) among isolates positive for Ambler class B enzymes (i.e., NDM biotic gradient strips were crossed at a 90° angle at the respective MICs and VIM), as shown in Fig. 2. Furthermore, additivity with aztreonam for each agent. To calculate the FICI, the MIC of each antibiotic in combi- was present in 8 (33.3%) isolates (Fig. 2). nation was divided by the MIC of the antibiotic alone, and the results Among KPC-positive isolates, synergy with C/T was observed in 7 were added together. In the