Influence of VanD Type Resistance on Activities of Glycopeptides In Vitro and in Experimental Endocarditis Due to Enterococcus faecium

The consequences of VanD type glycopeptide resistance on theactivity of vancomycin and teicoplanin were evaluated in vitroand in a rabbit model of aortic endocarditis with VanD typeclinical isolate Enterococcus faecium BM4339 (MICs: vancomycin,64 µg/ml; teicoplanin, 4 µg/ml) and its susceptiblederivative BM4459 (MICs: vancomycin, 1 µg/ml; teicoplanin,1 µg/ml). The two antibiotics were inactive against BM4339in vivo, in terms both of reduction of bacterial counts andof prevention of emergence of glycopeptide-resistant subpopulations,despite using teicoplanin at concentrations greater than theMIC for VanD strains. This could be due to the high inoculumeffect also observed in vitro with BM4339 and two other VanDtype isolates against both antibiotics. These results suggestthat detection of VanD type resistance is of major importancebecause it abolishes in vivo glycopeptide activity and allowsthe emergence of mutants highly resistant to glycopeptides.

INTRODUCTION

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Introduction

Acquired VanD type glycopeptide resistance in Enterococcus faeciumis characterized by moderate levels of resistance to vancomycin(MICs from 16 to 256 µg/ml) and to teicoplanin (MICs,2 to 64 µg/ml). Characterization of the first VanD typeE. faecium BM4339 clinical isolate (13) indicated that resistanceresulted from acquisition of the constitutively expressed vanDglycopeptide resistance operon and from a frameshift mutationin the chromosomal ddl gene for the D-alanine:D-alanine (D-Ala:D-Ala)ligase (12, 13). This combination of genetic events leads tosynthesis of precursors terminating in D-Ala-D-lactate (D-Ala-D-Lac)instead of the dipeptide D-Ala:D-Ala in susceptible bacteria(2, 12, 13).

The purpose of this work was to evaluate the impact of VanDtype resistance in E. faecium BM4339 on the activity of glycopeptidesin vitro and in rabbit aortic endocarditis. The efficiency ofvancomycin and teicoplanin was assessed by the reduction ofviable bacteria, both in vitro and in the valvular vegetations,and by their ability to select subpopulations with increasedglycopeptide resistance.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. E. faecium BM4339 is a VanD type glycopeptide-resistant clinicalisolate resistant to vancomycin (MIC, 64 µg/ml) and tolow levels of teicoplanin (MIC, 4 µg/ml). It is also resistantto the penicillins, tetracyclines, macrolide-lincosamide-streptograminB and high levels of aminoglycosides and streptomycin. E. faeciumBM4459 is a susceptible derivative of BM4339 obtained by insertionin the chromosome of a copy of the ddl gene for the D-Ala:D-Alaligase from E. faecium BM4147 (3). E. faecium BM4416 and 10/96Aare two unrelated VanD type clinical strains (4, 12). Enterococcusfaecalis JH2-2 is susceptible to glycopeptides and ß-lactams,intrinsically resistant to low levels of aminoglycosides, andresistant to rifampin and fusidic acid following mutations (7).Cultures and antibiotic susceptibility testing were performedin brain heart infusion (BHI) broth or agar (Difco Laboratories,Detroit, Mich.) at 37°C.

In vitro susceptibility testing and selection of mutants. The MICs of vancomycin (Eli Lilly France, Saint-Cloud, France)and teicoplanin (Aventis, Vitry-sur-Seine, France) were determinedby serial dilution in agar by the method of Steers et al. with10⁵ and 10⁸ CFU per spot (15) and in broth by a macrodilutionmethod (10) with an inoculum of 10⁵ or 10⁸ CFU per ml after24 h of incubation. For time-kill curves, exponentially growingE. faecium were diluted in broth to obtain ca. 10⁷ CFU/ml andincubated with vancomycin (16 and 40 µg/ml) or teicoplanin(4, 16, and 40 µg/ml). Aliquots (0.1 ml) were taken after0, 3, 6, and 24 h of incubation and plated on agar after serialdilutions to determine the surviving bacteria after 24 h ofincubation. Glycopeptide-resistant mutants were selected byplating 10⁹ CFU of overnight cultures of BM4339 and BM4459 onagar in the presence or absence of vancomycin or teicoplaninat fourfold the MICs. These were the lowest drug concentrationsthat resulted in the selection of resistant mutants (data notshown). The resistance of bacteria growing on antibiotic-containingagar was confirmed by determination of the MICs. Mutation frequencieswere determined by dividing the number of CFU obtained on selectivemedia by the number of those obtained on media devoid of antibioticafter 48 h of incubation. Each in vitro experiment was performedat least three times.

Experimental endocarditis. Aortic endocarditis was induced in female New Zealand Whiterabbits (2.2 to 2.5 kg) by insertion of a polyethylene catheterthrough the right carotid artery into the left ventricle, aspreviously described (9). Twenty-four hours after catheter insertion,each rabbit was inoculated by the ear vein with 10⁸ CFU of E.faecium BM4339 or BM4459 in 1 ml of 0.9% NaCl. The catheterwas left in place throughout the experiment. Forty-eight hoursafter inoculation, animals received vancomycin intramuscularlyat 50 mg/kg thrice a day or teicoplanin 20 mg/kg twice a dayfor 5 days after a loading dose of 40 mg/kg. Control animalswere left untreated.

At sacrifice, the vegetations of each rabbit were excised, pooled,weighed, homogenized in 1 ml of sterile distilled water, andplated after serial dilutions onto agar to determine the survivingbacteria and onto agar containing vancomycin or teicoplaninat fourfold the MIC of the strain to screen for selection ofsubpopulations with increased resistance to glycopeptides. TheMICs of vancomycin and teicoplanin for the bacteria recoveredfrom the selective media were determined. The results were expressedas log₁₀ CFU per gram of vegetation.

Statistics. The results were expressed as means ± standard deviations.Comparison of bacterial counts in the vegetations of rabbitstreated with various regimens was performed by variance analysisfollowed, when significant, by the Scheffe test (14). A P valueof <0.05 was considered significant.

RESULTS AND DISCUSSION

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Results and Discussion

In vitro experiments. The vancomycin and teicoplanin MICs against VanD type E. faeciumBM4339 and its susceptible derivative BM4459 determined on agarwith the standard inoculum of 10⁵ CFU per spot are shown inTable 1. Insertion of a ddl gene for the D-Ala:D-Ala ligasein the chromosome of BM4339 restores vancomycin and teicoplaninsusceptibility. Subpopulations with increased resistance toglycopeptides were observed at mean frequencies of 7.5 x 10^-5and 6.0 x 10^-7 for BM4339 and BM4459, respectively, on agarcontaining fourfold the vancomycin MIC. These frequencies were4.2 x 10^-6 and 1.1 x 10^-7 for BM4339 and BM4459, respectively,when the bacteria were plated on agar containing fourfold theMIC of teicoplanin.

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TABLE 1. MICs of glycopeptides for standard (10⁵) and high (10⁸) inocula against enterococcal strains

The in vitro selection of teicoplanin-resistant mutants of VanDtype enterococci has been reported previously (11). Time-killcurves at 24 h showed that vancomycin or teicoplanin at 40 µg/mldid not diminish the bacterial counts of BM4339 and led to adecrease in bacterial counts of BM4459 (-1.7 and -2.3 log₁₀CFU/ml, respectively) (Fig. 1). Lack of vancomycin activityagainst VanD type BM4339 was expected at the concentration of40 µg/ml, which is lower than the MIC (64 µg/ml).By contrast, the lack of in vitro activity of teicoplanin atthe same concentration, which corresponds to 10-fold the MIC,was surprising.

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FIG. 1. Antibacterial activity of vancomycin (Vm) and teicoplanin (Tc) against VanD type E. faecium BM4339 (B) and susceptible derivative BM4459 (A).

Experimental endocarditis. Bacterial counts in the vegetations from animals treated for5 days and from controls sacrificed at the end of therapy areshown in Table 2. The glycopeptides displayed excellent bactericidalactivity against susceptible BM4459, leading to sterilizationof eight of nine (89%) and four of eight (50%) of the vegetationsfrom rabbits treated with vancomycin and teicoplanin, respectively.In addition, none of the animals infected with this strain andtreated with glycopeptides retained resistant subpopulations.By contrast, vancomycin at 50 mg/kg thrice a day had no activityagainst BM4339, as shown by bacterial counts in the vegetationssimilar to those of untreated animals at sacrifice (Table 2).This result could be anticipated from the peak and trough serumconcentrations obtained with this regimen (36.3 ± 2.1µg/ml and 15.0 ± 8.3 µg/ml, respectively)(8) that remained lower than the MIC.

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TABLE 2. Results of treatment with vancomycin or teicoplanin for 5 days in rabbits with experimental endocarditis due to E. faecium BM4339 and BM4459

Surprisingly, teicoplanin at a dose producing peak and troughserum levels of 43.0 ± 4.0 µg/ml and 21.0 ±4.0 µg/ml (8), respectively, five times higher than theMIC during the entire dosing interval, was also ineffectiveand led to an increase of 1.5 log₁₀ CFU/g of vegetation in comparisonto animals sacrificed at the start of therapy (data not shown).Moreover, subpopulations with increased resistance to glycopeptideswere recovered from animals either untreated or treated withglycopeptides. The proportion of resistant subpopulations (10^-5to 10^-6 of the entire surviving population) was similar in controlsand in treated animals, which is consistent with lack of invivo activity of the two glycopeptides. The MICs () for thesesubpopulations were from 256 µg/ml to >1,024 µg/mlfor vancomycin and from 16 µg/ml to >1,024 µg/mlfor teicoplanin. Thus, the in vivo results confirm the in vitrodata indicating lack of glycopeptide activity against VanD typeBM4339. A possible explanation could be an inoculum effect ofVanD type strains for glycopeptides.

MICs of glycopeptides depending on bacterial inoculum. To screen for a putative inoculum effect as a cause for glycopeptideinactivity, the MICs of vancomycin and teicoplanin were determinedon agar and in broth at standard (10⁵) and high (10⁸) inocula,representative of the inocula observed in bacterial endocarditis.As shown in Table 1, there were wide variations in glycopeptideMICs against BM4339 and BM4459 depending on inoculum size thatwere more pronounced for teicoplanin than for vancomycin, asalready reported (5, 6). When the inoculum varied from 10⁵ to10⁸, the MICs of vancomycin and teicoplanin increased 8- and64-fold, respectively, by the agar dilution method, and 32-and 512-fold, respectively, by broth dilution. Similar resultswere obtained with the VanD type E. faecium BM4416 and 10/96Aisolates, which indicated that the inoculum effect was not straindependent (Table 1). The effect was greater with VanD type isolatesthan with susceptible E. faecium BM4459 and E. faecalis JH2-2(Table 1), suggesting a link with the VanD phenotype.

We have shown that the activity of vancomycin and teicoplaninagainst VanD type E. faecium BM4339 was abolished, both in vitroand in the rabbit model of aortic endocarditis. The lack ofin vivo activity of teicoplanin, despite apparent activity invitro, may be explained by an important inoculum effect whichwas observed in vitro for the three VanD type strains. The inoculumeffect may not be due to the presence of more glycopeptide-resistantsubpopulations in the larger inoculum. Indeed, the proportionof such subpopulations represented only 10^-5 to 10^-6 of theentire surviving subpopulation and was similar in controls andtreated animals. It is also not due to heteroresistance, sincethe phenotype of the resistant population appeared homogenouson agar plates. Rather, the inoculum effect observed for VanDtype isolates may be related to that previously observed forteicoplanin against E. faecalis (6) and for LY333328 againstVanA and VanC type enterococci (1).

The rabbit model of aortic endocarditis is characterized bya high inoculum, which varies between 10⁸ and 10⁹ CFU per gof vegetation at start of therapy. Although only a handful ofVanD type isolates have been described, none of which appearto transfer the resistance genes to other organisms (11, 13),our results emphasize the need to detect VanD type resistance,which abolishes the activity of both vancomycin and teicoplaninand favors the emergence of mutants highly resistant to glycopeptides.

ACKNOWLEDGMENTS

This work was supported in part by the "Programme de RechercheFondamentale en Microbiologie, Maladies infectieuses et Parasitaires"from the Ministère de l'Education Nationale de la Rechercheet de la Technologie.

FOOTNOTES

* Corresponding author. Mailing address: Service de Médecine Interne, Hôpital Beaujon, 100, boulevard du Général Leclerc, 92118 Clichy Cedex, France. Phone: (33) (1) 40 87 58 90. Fax: (33) (1) 40 87 10 81. E-mail: bruno.fantin{at}bjn.ap-hop-paris.fr.

REFERENCES

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