Concentrations in Cerebrospinal Fluid against Penicillin- Resistant Streptococcus pneumoniae Isolated

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1994, p Vol. 38, No /94/$4.+ Copyright C) 1994, American Society for Microbiology In Vitro Killing Activities of Antibiotics at Clinically Achievable Concentrations in Cerebrospinal Fluid against Penicillin Resistant Streptococcus pneumoniae Isolated from Children with Meningitis CATHERINE P. DOIT,1 STEPHANE P. BONACORSI,1 ANNICK J. FREMAUX,2 GENEVIEVE SISSIA,2 ROBERT COHEN,2 PIERRE L. GESLIN,2 AND EDOUARD H. BINGEN"* Laboratoire de Microbiologie, H6pital Robert Debre, 7519 Paris,' and Centre National de Reference du Pneumocoque, Centre Hospitalier de Creteil, 941 Creteil,2 France Received 1 May 1994/Returned for modification 13 June 1994/Accepted 22 August 1994 We evaluated the in vitro killing activities of ceftriaxone, imipenem, vancomycin, gentamicin, fosfomycin, and rifampin, alone and in combination, against 26 Streptococcus pneumoniae strains (penicillin G MICs, >.125 to 2,ug/ml) isolated from the cerebrospinal fluid of children with meningitis. The antibiotics were tested at clinically achievable concentrations in cerebrospinal fluid. After 5 h of incubation, imipenem was the most effective drug. None of the combinations had synergistic activity. Killing by 1Ilactam antibiotics or vancomycin was enhanced by the addition of gentamicin, reduced by the addition of rifampin, and unafected by the addition of fosfomycin. Pneumococcal meningitis in children is associated with considerable morbidity and mortality. As in many other parts of the world, there has recently been an increase in the prevalence of Streptococcus pneumoniae strains with diminished susceptibility to penicillin G in France; these strains represented about 12% of all S. pneumoniae strains isolated from cerebrospinal fluid (CSF) in France in 1992 (12). Clinical failure of S. pneumoniae meningitis therapy with delayed sterilization of CSF has been reported with such strains (11, 12, 16, 33), leading to recommendations that standard Plactam antimicrobial agents be replaced by broadspectrum cephalosporins in this setting (16, 33). However, S. pneumoniae strains resistant to broadspectrum cephalosporins have recently been reported in the United States and Spain, together with clinical treatment failure in cases of infection caused by strains for which the ceftriaxone or cefotaxime MIC is..5 jxg/ml (4, 5, 17, 29). New strategies such as the use of antimicrobial combinations are now being evaluated as firstline empiric treatment for meningitis (8, 9, 32). The aim of the study described here was to determine the comparative in vitro bactericidal activities of candidate antibiotics for use in childhood meningitis caused by S. pneumoniae strains with diminished susceptibilities to penicillin G. We used the timekilling curve method with clinically achievable CSF antibiotic concentrations and experimental conditions mimicking clinical conditions, such as a large inoculum (3) in the stationary growth phase (7). We studied 26 S. pneumoniae strains (serotype 23, n = 18; serotype 14, n = 3; serotype 9, n = 2; serotype 19, n = 1; serotype 6, n = 1; serotype 15, n = 1) for which penicillin G MICs were increased (MICs, >.125 to 2 p.g/ml). These strains represented all the strains isolated from the CSF of children with meningitis and collected by the French reference laboratory between 1987 and The MICs of penicillin G (Diamant, Puteaux, France), vancomycin (Lilly, Saint Cloud, * Corresponding author. Mailing address: Laboratoire de Microbiologie, H6pital R. Debre, 48 Bd. Serurier, 7519 Paris, France. Phone: 33 (1) Fax: 33 (1) France), ceftriaxone (Roche, NeuillysurSeine, France), imipenem (Merck, Sharp and DohmeChibret, Paris, France), gentamicin (ScheringPlough, LevalloisPerret, France), fosfomycin (Sanofi, Gentilly, France), and rifampin (CibaGeigy, RueilMalmaison, France) were determined by the dilution method on MuellerHinton agar supplemented with 5% sheep blood. The replicator prong delivered approximately 14 CFU per spot onto a blood agar plate. Killing studies were performed in microtiter plates (CML, Nemours, France) during a 5h incubation period with a stationaryphase culture adjusted to approximately 17 CFU/ml in MuellerHinton broth supplemented with 5% lysed defibrinated sheep blood. To achieve 14 CFU per spot and the inoculum for the killing studies, bacteria from an overnight agar culture were suspended in broth. A 5h incubation period was chosen because of the spontaneous in vitro autolysis of most of our strains (2 of 26). The antimicrobial agents were used at clinically achievable levels in CSF following the administration of the dosages currently used in the treatment of meningitis, as follows: ceftriaxone, 5 j±g/ml (19); imipenemcilastatin, 2,ug/ml each (25); gentamicin,.5,g/ml (23); vancomycin, 3,ug/ml (13); fosfomycin, 4,ug/ml (3); and rifampin, 1.5 jig/ml (27). Each 2655 antimicrobial agent was tested alone and in double or triple combinations. Ceftriaxone, imipenem, and vancomycin were each combined with fosfomycin, rifampin, or gentamicin. Ceftriaxone plus vancomycin, imipenem plus vancomycin, fosfomycin plus rifampin, and ceftriaxone plus vancomycin plus fosfomycin combinations were also tested. Colony counts were performed after 1:1 dilution (except for the control and gentamicin, 1:1,) by plating 5 RI of each dilution onto blood agar plates with a spiral plater (Spiral Systems Inc., Cincinnati, Ohio). Dilution and colony counts were performed in duplicate. Colonies were counted after 18 h of incubation at 37 C with 5% CO2 by the quadrant counting method. The CFU detection limit was 4,/ml. With the dilution and the spiral system used, antimicrobial agent carryover does not interfere with bacterial counts (35). The microdilution method used was initially compared with the macrodilution method with 13 strains from our strain collection for which MICs were Downloaded from on January 27, 219 by guest

2 2656 NOTES ANTiMICROB. AGENICS CHEMOTHER. TABLE 1. MICs of six antibiotics studied for 26 S. pneumoniae isolates from CSF placed in four classes according to the penicillin G MICs Class MIC range (pg/ml) Penicillin G Ceftriaxone Imipenem Gentamicin Vancomycin Fosfomycin Rifampin I (n = 3) II (n = 3) III (n = 5) IV (n = 15) representative. The mean difference in the bactericidal activity with mean killing that did not exceed 1.6 log1o CFU/ml for of the antibiotic tested against each strain between the two class III and IV strains. None of the two or threedrug methods was.2 log CFU/ml, with a range of.1 to +.5 combinations had synergistic or antagonistic activity. However, log1o CFU/ml. Student's paired t test showed that there was not some twodrug combinations were significantly more active significant difference between the two methods. Bactericidal than the most active agent used alone (Fig. 1). Gentamicin activity was defined as a reduction in CFU of more than 3 log1o significantly increased the activity of all of its partners against CFU/ml after 5 h of incubation. Bactericidal synergy was class IV strains (P.1) and that of ceftriaxone against class defined as an increase in killing of at least 2 log1o CFU/ml III strains (P =.5). In contrast, rifampin frequently reduced relative to the killing of the most active agent of the combina the killing activity of its partner. For example, against class IV tion used alone. Antagonism was defined as a similar decrease strains, ceftriaxone, imipenem, and vancomycin were signifiin killing (18). Student's paired t test was used to test for cantly more effective alone than when combined with rifampin statistical significance, and P values of less than.5 were (P.1). Surprisingly, while vancomycin did not modify the considered significant. activity of ceftriaxone, it induced a significant decrease in the The MICs of the antimicrobial agents tested are given in killing of class III and IV strains by imipenem (P.1). The Table 1. The strains were grouped into four classes (classes I, addition of fosfomycin did not affect the killing by its partners. II, III, and IV) according to the penicillin G MICs for the Among the combinations, imipenem plus gentamicin and strains (.25,.5, 1, and 2,ug/ml, respectively). Ceftriaxone ceftriaxone plus gentamicin displayed the best bactericidal MICs and imipenem MICs were less than the penicillin G activities (Table 2). MICs for all of the strains, which were also susceptible to the The increasing prevalence of S. pneumoniae strains with non,blactam antibiotics; MICs were in keeping with those reduced susceptibilities to penicillin G has led to a change in published previously (21, 22). The killing activities of the the firstline therapy for S. pneumoniae menigitis. Optimal antibiotics alone and in combination are given in Table 2. therapeutic results require rapid bactericidal activity within the Imipenem was significantly more active than the other drugs, CSF, since delayed sterilization of the CSF is associated with a with bactericidal activity against class I and II strains and a poor prognosis and a high risk of sequelae in children (2). The decrease of more than 2.5 log1 CFU/ml after 5 h with class III timekilling curve method may provide a better means of and IV strains. Ceftriaxone and vancomycin were less active, predicting in vivo antimicrobial activity in CSF (6, 1). In our Antibiotica TABLE 2. Killing by ceftriaxone, imipenem, vancomycin, fosfomycin, gentamicin, and rifampin alone and in combination against 26 S. pneumoniae isolates from CSF according to the penicillin G MICs Mean (±SD) change in loglo CFU/ml after 5 h of incubation for classb: I (n = 3) II (n = 3) III (n = 5) IV (n = 15) CRO 2.62 (±.33) 2.26 (±.66) 1.56 (±.4) 1.61 (±.29) IMP 3.3 (±.16) 3.17 (±.46) 2.54 (±.4) 2.54 (±.37) VAN 2.25 (±.5) 1.84 (±.27) 1.24 (±.45) 1.4 (±.38) FOS 2.31 (±.28) 2.23 (±.2) 1.7 (±.22) 1.86 (±.26) RIF 2.45 (±.21) 1.85 (±1.15) 1.3 (±.6) 1.16 (±.19) GEN 1.7 (±.25) 1.82 (±.33) 1.47 (±.42) 1.87 (±.26) CROVAN 2.64 (±.56) 2.13 (±.23) 1.58 (±.77) 1.5 (±.43) CROFOS 2.72 (±.37) 2.52 (±.74) 1.72 (±.41) 1.8 (±.36) CRORIF 2.52 (±.17) 1.9 (±.98) 1.1 (±.17) 1.6 (±.25) CROGEN NDc ND 1.97 (±.19) 2.14 (±.26) IMPVAN 3.26 (±.23) 2.54 (±.29) 1.59 (±.68) 1.73 (±.44) IMPFOS 3.36 (±.5) 2.93 (±.51) 2.55 (±.41) 2.43 (±.4) IMPRIF 2.37 (±.16) 1.84 (±.88) 1.3 (±.15) 1.14 (±.28) IMPGEN ND ND 2.78 (±.23) 2.85 (±.41) VANFOS 2.6 (±.61) 2.4 (±.24) 1.26 (±.42) 1.57 (±.48) VANRIF 2.97 (±.44) 1.99 (±.71) 1.3 (±.23) 1.1 (±.36) VANGEN ND ND 1.48 (±.46) 1.84 (±.41) FOSRIF 2.42 (±.37) 1.8 (±.81).88 (±.6) 1.3 (±.26) CROVANFOS 2.76 (±.56) 2.4 (±.38) 1.45 (±.36) 1.71 (±.48) Control growth 1.75 (±.3) 1.9 (±.25) 1.56 (±.41) 1.8 (±.36) a CRO, ceftriaxone; IMP, imipenem; VAN, vancomycin; FOS, fosfomycin; RIF, rifampin; GEN, gentamicin. b See Table 1 for class definitions. c ND, not determined. Downloaded from on January 27, 219 by guest

3 VOL. 38, 1994 NOTES 2657, 22 *ti :1 AloglO UFC/m b 2 1 AloglO UFC/ml Downloaded from AloglO CFIJ/ml,5,25 eqw4 " (C4 IAloglO UFC/ml FIG. 1. Comparison of killing activities of antimicrobial agent combinations with that of the most active agent in the combination. For each class of S. pneumoniae, which was grouped according to the penicillin G MIC for the strain, horizontal bars represent the mean difference in killing between the combination and the most active single agent used alone (Alog1 CFU per milliliter) after 5 h of incubation. Positive values indicate that a combination was more effective than the single agent. *, P c.1. **, P =.5. CRO, ceftriaxone; FOS, fosfomycin; IMP, imipenem; VAN, vancomycin; GEN, gentamicin; RIF, rifampin. on January 27, 219 by guest study, we used experimental conditions as close as possible to those of clinical meningitis and studied the killing rate of the test agents only during the first few hours of incubation. Because of autolysis of our strains, the incubation time was limited to a 5h period. Friedland et al. (9) showed that results obtained at 4 h correlate with those obtained after 8 h; antibiotics or combinations which are the most efficient at 8 h are also the most efficient at 4 h. Used alone, imipenem had the best overall killing activity. However, its value in patients with meningitis is limited because of its seizure activity, especially in children with bacterial meningitis (34). New carbapenems like meropenem, which does not appear to be epileptogenic, should be evaluated. Ceftriaxone killing exceeded 2 log1o CFU/ml only against strains for which the penicillin G MIC was less than.5,ug/ml. Some investigators have used high doses of ceftriaxone or cefotaxime in this setting (32), but the concentrations in CSF obtained in this way are not known. In the present study, we used the concentrations obtained in CSF after administration of the usual dosage in clinical practice, and results could be different with higher

4 2658 NOTES concentrations. Vancomycin has been shown to be effective in experimental meningitis caused by both penicillin intermediately resistant and resistant S. pneumoniae strains (24). By contrast, in our study vancomycin killing was less than 2 loglo CFU/ml against the strains for which the penicillin G MICs were >.25,ug/ml. Viladrich et al. (31) reported both successes and failures of vancomycin in the treatment of meningitis. CSF vancomycin levels varied over a wide range and were not clearly related to the simultaneous levels in serum, suggesting variability in bloodbrain barrier penetration. These results led the author to recommend the use of vancomycin in combination rather than alone (31). The use of antimicrobial agent combinations against S. pneumoniae meningitis has been reported in anecdotal cases. The most frequently proposed combinations involve broadspectrum cephalosporins or vancomycin with rifampin or fosfomycin (4, 8, 9, 15, 31). Most in vitro studies of drug combinations have used checkerboard titration methods (14), while the few investigators who have assessed drug interactions by means of timekilling techniques always used antimicrobial agent concentrations in the range of 1/4 to 4fold the MIC (1, 2, 9, 1). The use of subinhibitory concentrations optimizes the likelihood of detecting synergism, whereas clinically achievable concentrations can be used to predict the likely maximum killing rate. Contrary to our findings, Barakett et al. (2) reported that rifampin combined with vancomycin or cefotaxime usually had synergistic or indifferent activity. However, Barakett et al. used rifampin at one or twofold the MIC, whereas we used concentrations equivalent to 25 to 5 times the rifampin MICs. This, in addition to the fact that we used stationaryphase cultures, may explain this apparent discrepancy. There have been clinical reports of successful therapy of meningitis with antimicrobial agent combinations that include rifampin (4, 12, 15, 31). However, because the cell wallactive agent was used at a high dosage, it is difficult to conclude whether there is a real benefit from adding rifampin. The rifampininduced reduction in killing by the partner drug observed in our study supports the results of a recent report of vancomycinrifampin therapy in an animal model of meningitis, in which this combination reduced the activity of vancomycin during the first few hours (9). Fosfomycin has recently been used successfully in combination with other drugs (15). The combination of fosfomycin with cefotaxime has been described as synergistic in vitro (1), but this was not the case with ceftriaxone in our study. Moreover, fosfomycin gives concentrations in CSF close to the MICs, and this might facilitate the emergence of resistant mutants, especially when the partner drug diffuses slowly (e.g., vancomycin). The increase in killing observed in the present study with the addition of gentamicin is in keeping with the in vitro synergy observed between aminoglycosides and cell wallactive agents against streptococci (26). The beneficial effect obtained by adding an aminoglycoside to ampicillin, which is manifested by the faster clearance of bacteria from CSF, has been demonstrated for group B streptococci in experimental models of meningitis (28). However, the small increase in killing noted in the present study may be explained by the low concentration used (equivalent to 1/32nd the MIC) and the presence of C2, which was necessary for optimal S. pneumoniae growth. Friedland et al. (1), using gentamicin at onehalf the MIC (2 or 4,ug/ml), showed a synergistic effect with ceftriaxone at onehalf the MIC against three strains. Oncedaily aminoglycoside administration might give higher levels of drug in CSF. Pharmacokinetic and experimental studies are required to document the real benefit of this dosage regimen in patients with meningitis. Friedland et al. (9) tested the combination of vancomycin with ceftriaxone against two penicillinresistant S. ANTIMICROB. AGENTS CHEMOTHER. pneumoniae strains; a synergistic effect was found in vivo and in vitro by the timekilling curve method and by using fractional MICs, and these authors (9) recommended the use of that combination for the therapy of penicillinresistant S. pneumoniae meningitis. However, in our study this combination was found to be indifferent against 26 strains. Experimental and clinical studies are required to determine the relevance of our results. REFERENCES 1. Barakett, V., D. Lesage, F. Delisle, B. 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