In VitroActivity of Fosfomycin against a Collection of Clinical Pseudomonas aeruginosa Isolates from 16 Spanish Hospitals: Establishing the Validity of Standard Broth Microdilution as Susceptibility Testing Method

Abstract

The broth microdilution method for fosfomycin and Pseudomonas aeruginosa was assessed and compared with the approved agar dilution method in 206 genetically unrelated P. aeruginosa clinical isolates. Essential agreement between the two methods was 84%, and categorical agreement was 89.3%. Additionally, Etest and disk diffusion assays were performed. Results validate broth microdilution as a reliable susceptibility testing method for fosfomycin against P. aeruginosa. Conversely, unacceptable concordance was established between Etest and disk diffusion results with agar dilution results. Multi-drug resistance (MDR) in clinical Pseudomonas aeruginosa isolates has been widely reported and is of particularly concern in severe infections for which few therapeutic options remain available (1). Fosfomycin shows no cross-resistance with other antimicrobials and has demonstrated safety and efficacy over a broad range of infections and organisms (2). This antibiotic may act synergistically with many antimicrobials, and the intravenous form could be administered in combination for the treatment of systemic infections caused by MDR P. aeruginosa (3,4).The approved in vitro antimicrobial susceptibility testing method for fosfomycin is agar dilution (5); however, broth microdilution is the basis of automated systems currently used in clinical microbiology laboratories. Neither the Clinical and Laboratory Standards Institute (CLSI) (5) nor the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (6) includes fosfomycin breakpoints for P. aeruginosa, although EUCAST has defined an epidemiological cutoff (ECOFF) value (6).Our study was designed to evaluate the in vitro fosfomycin activity against a collection of P. aeruginosa isolates by standard broth microdilution and to compare that method's performance with the performance of agar dilution, considered in this study as the reference method. In addition, agar dilution and broth microdilution were also assessed without glucose-6-phosphate (G6P) against 25 strains exhibiting different fosfomycin MIC values. Etest and disk diffusion were also assessed. A total of 206 genetically unrelated clinical P. aeruginosa isolates, 148 carbapenem susceptible and 58 non-carbapenem susceptible, were included (7). P. aeruginosa ATCC 27853 was used as the control.(Part of this research was presented at the 22nd European Congress of Clinical Microbiology and Infectious Diseases, London, United Kingdom, 31 March to 3 April 2012.) Susceptibility to fosfomycin (Laboratorios Ern, S.A., Barcelona, Spain) was determined concomitantly by agar dilution and broth microdilution using BBL Mueller-Hinton II cation-adjusted agar and broth, respectively (Becton, Dickinson [BD], Sparks, MD), both supplemented with 25 g/ml of G6P (SigmaAldrich Chemical Co., St. Louis, MO). The MIC was defined as the lowest antibiotic concentration that inhibited visible growth of the organism. MIC interpretation was done using an ECOFF value of Յ128 g/ml (6). Fosfomycin disk diffusion using disks of two different ...

Susceptibility of Multiresistant Gram-Negative Bacteria to Fosfomycin and Performance of Different Susceptibility Testing Methods

“…For P. aeruginosa, the opposite occurred and results for A. baumannii were more uniform. Etest performed very poorly for P. aeruginosa, as already reported recently (24). For A. baumannii and Enterobacter, there were unacceptable proportions of minor errors, and for K. pneumoniae, there were unacceptable major errors.…”

Fosfomycin

“…This finding was consistent regarding K. pneumoniae and Enterobacter cloacae, whereas the prevalence of resistance of P. aeruginosa and S. maltophilia was affected by the choice of MIC (63). On the other hand, particularly for P. aeruginosa, a recent study suggested that broth microdilution is a reliable method, whereas no concordance was observed between agar dilution and disk diffusion/Etest (61). Finally, a recent study evaluating agar dilution, disk diffusion, and Etest for contemporary multidrug-resistant (MDR) Gram-negative pathogens suggested that disk diffusion had poor performance for Acinetobacter baumannii and Enterobacteriaceae and that Etest performed poorly for all tested pathogens (64).…”

“…MIC 50 and MIC 90 values were usually one dilution lower in ESBL-producing K. pneumoniae strains than in CR/KPC-producing K. pneumoniae strains. All CR A. baumannii strains were also resistant to fosfomycin (94), while 80.6% of CR P. aeruginosa strains were reported to be susceptible in one study (61).…”

“…In this regard, Mueller-Hinton agar or broth supplemented with 25 g/ml G-6-P is recommended, as it results in maximal enhancement of fosfomycin activity (60). A recent study suggested that regarding P. aeruginosa, which lacks a specific G-6-P transporter, the addition of G-6-P in agar or broth does not affect fosfomycin activity (61). According to the Clinical and Laboratory Standards Institute (CLSI) standard criteria, the approved susceptibility testing methods are disk diffusion and agar dilution for urinary E. coli and E. faecalis isolates, whereas broth microdilution should not be performed (48).…”

“…The above factors, as well as the limited options of novel antibiotic agents, necessitated the reevaluation of fosfomycin as a potential therapeutic option for infections caused by contemporary isolates with advanced antimicrobial resistance. Table 2 shows the susceptibility of contemporary bacteria to fosfomycin from the larger studies published from 2010 onwards (36,56,57,61,. Regarding Gram-positive bacteria, contemporary studies showed that fosfomycin is active against the majority of S. aureus strains (Ͼ90%), including MRSA, as well as coagulase-negative staphylococci (CoNS) (36,56,61,72,75,82,83,87,88,90).…”