Vancomycin Acts Synergistically with Gentamicin against Penicillin-Resistant Pneumococci by Increasing the Intracellular Penetration of Gentamicin

Vancomycin and gentamicin act synergistically against penicillin-resistantpneumococci in vitro and in experimental rabbit meningitis.The aim of the present study was to investigate the underlyingmechanism of this synergism. The intracellular concentrationof gentamicin was measured by using the following experimentalsetting. Bacterial cultures were incubated with either gentamicinalone or gentamicin plus vancomycin for a short period (15 min).The gentamicin concentration was determined before and aftergrinding of the cultures by using the COBAS INTEGRA fluorescencepolarization system (Roche). The grinding efficacies rangedbetween 44 and 54%, as determined by viable cell counts. Inthe combination regimen the intracellular concentration of gentamicinincreased to 186% compared to that achieved with gentamicinmonotherapy. These data suggest that the synergy observed invivo and in vitro is based on an increased intracellular penetrationof the aminoglycoside, probably due to the effect of vancomycinon the permeability of the cell wall.

INTRODUCTION

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Introduction

Before the emergence of penicillin-resistant pneumococci, penicillinwas usually the first-line antibiotic used in the treatmentof pneumococcal infections. The increasing spread of resistantstrains has changed this situation. In meningitis, where thepenetration of antibiotics is limited, penicillin is ineffectiveeven against strains with intermediate resistance, and penicillinresistance is often associated with resistance to other ß-lactamantibiotics. Because treatment failures in meningitis have beenobserved with cephalosporin monotherapy, a combination of vancomycinand an extended broad-spectrum cephalosporin (ceftriaxone orcefotaxime) is usually recommended for the treatment of meningitiscaused by resistant strains (1, 7). Especially in the case ofß-lactam allergy, it is compelling for infectiousdisease specialists to elaborate new therapeutic strategies.We have recently investigated the role of gentamicin againstpenicillin-resistant pneumococci in the rabbit meningitis model(4). Although gentamicin monotherapy was only marginally effective,addition of vancomycin produced killing rates comparable tothose achieved by the standard treatment based on ceftriaxonein combination with vancomycin. The synergy between the twoantibiotic groups was also demonstrated in vitro in time-killingexperiments. The aim of this work was to study the underlyingmechanism of the synergism between gentamicin and vancomycinin vitro.

(This study was partially presented at the 41st InterscienceConference on Antimicrobial Agents and Chemotherapy, Chicago,Ill., December 2001.)

MATERIALS AND METHODS

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Materials and Methods

Pneumococcal strain. The serotype 6 penicillin-resistant strain (penicillin G MIC,4 mg/liter; ceftriaxone MIC, 0.5 mg/liter; cefepime MIC, 0.5mg/liter; vancomycin MIC, 0.25 mg/liter, gentamicin MIC, 4 mg/liter)used in this study was isolated from a patient with pneumoniaat the University Hospital of Bern. MICs were determined bythe broth macrodilution method.

In vitro assays. The penicillin-resistant strain was grown in C+Y medium (8)to an optical density at 590 nm of 0.3 and was then diluted40-fold to 10⁶ CFU/ml, which corresponds to the bacterial titerin the cerebrospinal fluid of rabbits before the initiationof therapy. Gentamicin was added at concentrations correspondingto one-fourth, one-half, one, and two times the MIC, alone orin combination with vancomycin at the MIC. Bacterial titerswere determined at hours 0, 2, 4, 6, and 8 by serial dilutionof samples; plated on agar plates containing 5% sheep blood;and incubated at 37°C for 24 h. The experiments were performedin triplicate, and results are expressed as means ± standarddeviations.

Measurement of intracellular gentamicin levels. The penicillin-resistant strain was grown in C+Y medium to anoptical density at 590 nm of 0.3. Then, antibiotics were addedat the following concentrations: gentamicin at the MIC, gentamicinat two times the MIC, vancomycin at the MIC, and the combinationof gentamicin (at the MIC) and vancomycin (at the MIC).

After a short incubation (15 min), the cultures were centrifugedat 3,600 rpm in a Hettich Universal 30RF washed with saline(three times), and ground with glass beads (diameter, 212 to300 µm) for 15 min. The numbers of CFU were determinedafter 15 min of incubation, after each washing step, and aftergrinding. Then, the cultures were resuspended in a small volumeof saline. The gentamicin concentration was determined beforeand after grinding by using the COBAS INTEGRA fluorescence polarizationsystem (Roche) (5). In brief, probes were incubated with a mousemonoclonal antibody, and then a tracer reagent was added. Thelight emission was measured at 515 nm. The light emission isproportional to the gentamicin concentration of the probe. Thegentamicin levels were expressed in micromoles per liter. Thesensitivity and specificity of the method are excellent, thedetection level for gentamicin is 0.08 µg/ml. No cross-reactivityhas been observed with vancomycin at concentrations up to 400µg/ml. The gentamicin concentrations obtained with theCOBAS INTEGRA system have been compared to those determinedby a commercially available fluorescence polarization immunoassaymethod with an excellent correlation (correlation coefficient,0.992; slope, 0.955; intercept, -0.015 µg/ml [Roche, personalcommunication]).

Grinding efficacy was determined by plating an aliquot of thesurviving microorganisms on blood agar plates overnight at 37°C.The grinding efficacies ranged between 44 and 55% for all groups.Data have been statistically analyzed by the nonparametric Mann-Whitneytest.

The gentamicin level measurements were kindly performed by U.Schilt, MCL Laboratories, Bern, Switzerland.

Checkerboard method. Checkerboards and fractional inhibitory concentration (FIC)indices were determined by the method of Eliopoulos and Moellering(6). Checkerboard runs were repeated three times, and the FICindex was calculated. Synergy was defined as an FIC index of $<=$ 0.5, indifference was defined as an FIC index of >0.5 and $<=$ 4, and antagonism was defined as an FIC index of >4 (11).

RESULTS AND DISCUSSION

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Results and Discussion

The permanent increase in the numbers of penicillin- and cephalosporin-resistantpneumococci has jeopardized the successful treatment of pneumococcalmeningitis, underlining the need for alternative therapy. Asa potential alternative, a combination of gentamicin and vancomycinshowed antibacterial activity similar to that of the standardregimen based on ceftriaxone and vancomycin in the rabbit meningitismodel (4). The antibacterial activities of the different regimensin time-killing assays over 8 h are shown in Fig. 1A and B.By using concentrations under the MIC (one-fourth and one-halfthe MIC), gentamicin monotherapy did not show antibacterialactivity. Gentamicin at the MIC, which corresponded to the levelachieved in the cerebrospinal fluid of rabbits during experimentalmeningitis (4), led to a negligible decrease in the viable cellcounts (0.3 log₁₀ CFU/ml over 8 h). On the other hand, a concentrationover the MIC (two times the MIC) produced activity that washighly bactericidal and that managed to sterilize the culturesafter 6 h (Fig. 1A).

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FIG. 1. (A) Killing rates (•) of monotherapy with gentamicin at different concentrations (one-fourth, one-half, one, and two times the MIC) over 8 h. ${circ}$ , untreated controls. The experiments were performed in triplicate, and the results are expressed as means ± standard deviations. (B) Killing rates ( ${blacksquare}$ ) of different concentrations of gentamicin in combination with vancomycin (Vanco; at the MIC) over 8 h. x, monotherapy with vancomycin (at the MIC); ${circ}$ , untreated controls. The experiments were performed in triplicate, and the results are expressed as means ± standard deviations.

The vancomycin concentration was chosen in order to producea minimal intrinsic activity, thus avoiding masking of a potentialsynergy between the two antibiotics. Although the bactericidalactivity was more pronounced than that of gentamicin, vancomycin(at the MIC) produced only modest bactericidal activity (1.8log₁₀ CFU/ml over 8 h). Surprisingly the combination therapies(Fig. 1B) were highly synergistic, even when each drug was usedat a concentration below the MIC (one-fourth and one-half theMIC), and managed to sterilize the majority of the culturesafter 8 h. The synergy was less pronounced for the combinationof gentamicin (at two times the MIC) and vancomycin becauseof the highly bactericidal activity of gentamicin monotherapy(Fig. 1A).

On the other hand, the results of the checkerboard techniquewere less evident. In this experimental setting the combinationwas repeatedly tested and was marginally synergistic (FIC index $<=$ 0.5) or indifferent (FIC index = 1), underlining the fact thatthe time-killing assays in vitro correspond more closely tothe situation in vivo.

In a further step the intracellular gentamicin concentrationswere calculated for all regimens.

Due to the limits of the method used in this experimental setting,we were not able to localize precisely the intrabacterial compartmentin which gentamicin accumulated. Therefore, we use the term"cell-associated gentamicin" to describe intrabacterial gentamicinaccumulation. In order to avoid technical bias due to the intrinsicbacteriolytic activity of vancomycin, the cultures were incubatedwith the different regimens only for a short period (15 min)and were then washed three times with saline and extensivelyground with glass beads. This procedure did not influence theviable cell count for any of the regimens except the combinationregimen (gentamicin with vancomycin; reduction, 0.5 log₁₀ CFU/ml).The grinding efficacies, estimated by counting the survivingmicroorganisms, were similar for all groups and ranged between44 and 55% of the starting inoculum. The results are presentedin Fig. 2. After washout and before grinding of the cultures,no gentamicin could be detected in any of the groups. Aftergrinding of the cultures, a relative concentration of 0.78 ±0.16 µmol of gentamicin per liter was measured when thecultures were incubated with gentamicin alone. The additionof vancomycin drastically increased the cell-associated levelof gentamicin to 186% (1.45 ± 0.21 µmol/liter),even higher than the levels obtained with higher doses of gentamicin(1.10 ± 0.14 µmol/liter). It is interesting thatthe cell-associated gentamicin level was probably underestimatedin the group that received the combination due to the reductionin the number of CFU after as soon as 15 min (0.5 log₁₀ CFU/ml);the reduction corresponded to an approximately threefold decreasein the cell population. This decrease in the numbers of viablecells was due to cell lysis and was confirmed by a decreasein the optical density for the group treated with the combination(optical density decrease, 0.330 to 0.280). Thus, the real concentrationin the group treated with the combination (vancomycin at theMIC plus gentamicin at the MIC) was probably three times higher,i.e., 4.35 µmol/liter. One-fourth of that value wouldapproximately correspond to the gentamicin concentration obtainedwith two times the MIC. This could explain why the activityof gentamicin at a lower concentration (one-fourth the MIC)in combination with vancomycin was still synergistic and whygentamicin showed pronounced bactericidal activity (Fig. 1B).

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FIG. 2. Relative gentamicin (G) concentration (in micromoles per liter) in the supernatant before and after grinding of the cultures, as measured by using the COBAS INTEGRA fluorescence polarization system (Roche). The experiments were performed in triplicate, and the results are expressed as means ± standard deviations. Due to the small number of probes used (three for each group), the differences were not significant (by the nonparametric Mann-Whitney test).

These findings may explain the mechanism of the synergy betweengentamicin and vancomycin in pneumococci. The underlying mechanismis not completely understood, but these data strongly suggestthat the inhibition of cell wall synthesis by vancomycin altersthe permeability of the cell wall, which probably acts as anatural barrier to aminoglycosides, and facilitates the penetrationof gentamicin into the cell.

These results are reminiscent of the synergism between streptomycinand penicillin against enterococci observed by Moellering andcolleagues (9, 10) in the 1970s. Although the experimental settingwas completely different, the synergy was explained by increasedlevels of uptake of radiolabeled streptomycin by enterococci(9, 10).

These data might provide a plausible explanation for the similarsynergism observed with either vancomycin (3, 13) or ß-lactamantibiotics and quinolones (2, 12) and might open the avenueto new therapeutic options for pneumococcal diseases.

FOOTNOTES

* Corresponding author. Mailing address: Department of Internal Medicine, Inselspital Bern, Bern, Switzerland. Phone: 41-31-632-34-72. Fax: 41-31-632-38-47. E-mail: pcottagn{at}insel.ch.

REFERENCES

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