Activity of Daptomycin against Listeria monocytogenes Isolates from Cerebrospinal Fluid^▿

Listeria monocytogenes is the second or third most frequently isolated gram-positive microorganism from patients with meningitis (12, 18). L. monocytogenes is always penicillin sensitive, thus, ampicillin or amoxicillin is the agent of first choice to treat Listeria meningitis (18). For patients allergic to penicillin, trimethoprim-sulfamethoxazole and, possibly, meropenem are alternatives. The results of treatment with vancomycin are disappointing (18).

Daptomycin is a lipopeptide antimicrobial agent with bactericidal activity against gram-positive microorganisms (6, 9, 10, 11, 13, 16, 17). We tested the daptomycin susceptibility of 76 L. monocytogenes isolates from meningitis patients in The Netherlands to investigate whether daptomycin is an alternative treatment for Listeria meningitis.

The Netherlands Reference Laboratory for Bacterial Meningitis receives about 80% of all cerebrospinal fluid (CSF) isolates in The Netherlands (12, 15). All 76 L. monocytogenes CSF isolates, each representing one patient and received during 2001 to 2005, were used in this study (3).

Susceptibility tests were performed by broth dilution and Etest methods. Mueller-Hinton (MH) broth (Difco formulation; Becton Dickinson, Cockeysville, MD) with calcium (final concentration, 50 mg/liter) was used for broth dilution by following the procedure of the daptomycin manufacturer (Cubist Pharmaceuticals, Lexington, MA). The final inoculum in each well was 5 × 10⁵ CFU/ml. Colony counts of positive control wells were performed. The microdilution panels were incubated at 35°C in ambient air for 16 to 20 h prior to the visual determination of MICs. The control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were included with each test.

The Etest was performed with MH II agar BBL plates (Becton Dickinson) both without blood, as described by Jorgensen and Crawford (10), and with 5% sheep blood according to the Etest procedure of the manufacturer (AB Biodisk, Solna, Sweden). The addition of calcium was unnecessary, because calcium is incorporated in the daptomycin Etest strip. MH plates alone and MH plates with blood were inoculated using a 0.5 and 1.0 McFarland density suspension, respectively, and were incubated at 35°C in ambient air for 16 to 20 h.

From 2001 to 2005, the annual number of L. monocytogenes CSF isolates recovered varied between 10 and 19. Among the 76 strains collected, 49% were serotype 4b and 42% were type 1/2a. The daptomycin MICs at which 50 and 90% of the tested isolates were inhibited (MIC₅₀ and MIC₉₀, respectively), as determined by broth dilution, were both 4.0 mg/liter (Table 1). For the Etest on plates without blood, the MIC₅₀ and MIC₉₀ were 2.0 and 4.0 mg/liter, respectively. For 26 (34%) of the 76 strains, the MIC determined by Etest was identical to that determined by broth dilution. For 39 (51%) strains, the MIC derived by the Etest method was a twofold-step lower than that derived from the broth method, and for 11 (14%) strains the MIC derived by the Etest method was two steps lower than the MIC determined by the broth method. The MIC by Etest was never higher than the MIC by broth dilution. For a random sample of 25 strains (33%), the Etest was performed on MH plates with blood. The results did not differ significantly from those for the Etest performed on plates without blood (Table 1). No daptomycin interpretative breakpoints have been established for Listeria. Therefore, it is not possible to analyze these data with respect to interpretative errors between the Etest and broth dilution as the reference method.

In this study, 76 L. monocytogenes isolates from patients with meningitis in The Netherlands from 2001 to 2005 were tested for daptomycin susceptibility. We performed broth dilution on samples in MH without blood, whereas the Clinical and Laboratory Standards Institute recommends using MH with 2.5 to 5% blood (1). L. monocytogenes is not truly fastidious, and testing with MH without blood has been done satisfactorily (8). Therefore, and because MICs by Etest derived from MH agar with and without blood did not show a significant difference from each other, we do not think that broth dilution performed with MH with blood would give results deviating from our present data. Listeria strains have been included in a few other daptomycin susceptibility studies (9, 11, 13, 17). However, a collection of CSF isolates as large as ours has not been tested before. Only one of the previous studies provided data about the Etest (9). Our broth dilution results were similar to those of Huang et al. and Streit et al. (9, 17) but were higher than those of Piper et al. (13). However, Piper et al. used a fivefold smaller inoculum, 10⁵ CFU/ml. Our Etest results were similar to those of Huang et al. (9). However, we found that MICs determined by Etest were identical to or were a twofold step lower than those determined by broth dilution for 86% of the strains, and the MIC by Etest was never higher than the MIC by broth dilution. This is in contrast to the finding of Huang et al. that 35% of strains had a higher MIC by Etest than by broth dilution (9). Jorgensen and Crawford, who investigated enterococci, observed that daptomycin MICs determined by Etest tended to be lower than MICs determined by the dilution test (10). Because no breakpoints have been established for Listeria, it is not possible to analyze whether this would lead to interpretative errors with Etest.

The standard antimicrobial treatment of L. monocytogenes meningitis consists of ampicillin or amoxicillin. Only a few alternatives are available for patients allergic to penicillin (18). It is, therefore, essential that new antimicrobial agents be investigated with reference to their activity against meningitis pathogens. In animal studies of meningitis due to S. aureus (5, 7) and Streptococcus pneumoniae (2), daptomycin was more efficacious than vancomycin. However, daptomycin MICs for the S. aureus and S. pneumoniae strains used were 0.25 (7), 1.0 (5), and 0.06 (2), respectively, which are much lower than the daptomycin MIC₅₀ for L. monocytogenes. Furthermore, vancomycin is not the standard therapy for Listeria meningitis. The efficacy of an antibiotic in meningitis also depends on the concentration attained in CSF. The average daptomycin peak concentration in volunteer plasma is about 60 mg/liter with the presently recommended dosing regimen (4 mg/kg of body weight once daily) (4). In the rabbit meningitis model, the daptomycin CSF concentration does not exceed 6% of the plasma concentration (2, 5). In plasma, daptomycin is 92% protein bound. Hence, the concentration of free drug in CSF will be relatively higher due to the lack of protein in the CSF. For the successful treatment of bacterial meningitis, the antimicrobial agent should exert a bactericidal effect (18). In a mouse model, the daptomycin peak concentration needs to be at least seven times the MIC to produce a bactericidal effect (14). If this applies to humans, it can be calculated that the daptomycin CSF concentration should be at least 25 mg/liter to effectively treat 90% of patients with Listeria meningitis.

The conclusion of this study is that daptomycin cannot be recommended for the treatment of L. monocytogenes meningitis until more data are available on the penetration of daptomycin in human CSF and the amount of free drug present in CSF.