INTRODUCTION

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Methicillin-resistant Staphylococcus aureus (MRSA) strains represent a worldwide threat because of their virulence and their broad distribution in the hospital setting. Moreover, the MRSA strains are often resistant not only to -lactam agents but also to fluoroquinolones, chloramphenicol, clindamycin, tetracyclines, and aminoglycosides (20). Vancomycin is almost universally accepted as the drug of choice for the treatment of MRSA infections (13, 20). However, vancomycin used alone kills staphylococci slowly (1), resulting in delayed recovery of patients with life-threatening infections (19, 33). In addition, clinicians now have to face the emergence of strains with reduced susceptibility to vancomycin, i.e., so-called glycopeptide-intermediate S. aureus (GISA) (4, 5, 15, 16). Therefore, there is clearly a need for new antibiotic regimens with strong early bactericidal activity against MRSA. In this field, an alternative to the development of new classes of agents could be the use of combinations of well-known compounds. Some investigators have reported synergistic bacteriostatic and bactericidal (where tested) effects of different -lactams or different aminoglycosides combined with vancomycin against MRSA strains (3, 9, 11, 24, 28, 29, 30, 36-38), including some GISA isolates (8, 32). However, such findings have usually been observed either for very few of the antibiotic combinations (sometimes only one) tested or for a few strains and thus deserve to be confirmed in larger studies.

The purposes of this investigation were (i) to determine among 26 commonly available -lactams and among 8 aminoglycosides those that display the strongest beneficial bacteriostatic effect in combination with vancomycin against MRSA strains; (ii) to evaluate the bactericidal effects displayed by the most effective agents used in different -lactam-vancomycin, vancomycin-aminoglycoside and -lactam-vancomycin-aminoglycoside combinations; and (iii) to examine whether the effects of the different antibiotic combinations are dependent upon the aminoglycoside susceptibility pattern of the strains.

(Part of this work was presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy [abstr. E58, p. 185], San Diego, Calif., September 1998.)

	MATERIALS AND METHODS

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Bacterial strains. A total of 32 clinical MRSA strains selected from 32 individual patients attending the Rouen University Hospital between 1995 and 1997 were studied. Twenty-eight isolates were obtained from blood, two were from pleural fluid, one was from pericardial fluid, and one was from joint fluid. They were identified to the species level by conventional methods (colony morphology, Gram stain characteristics, coagulase reactions). All strains were methicillin resistant, as determined by a disk diffusion method with a 5-µg oxacillin disk (10) and by PCR amplification of the mecA gene (2). Strains were considered heterogeneously resistant to methicillin when partial growth within the inhibition zone or the presence of microcolonies around the oxacillin disk was observed. None of the 32 strains had reduced susceptibility to glycopeptides. The study population was chosen according to the strains' patterns of susceptibility to aminoglycosides as determined by a disk diffusion technique described by the Comité de l'Antibiogramme de la Société Française de Microbiologie (10). The aminoglycoside resistance mechanism was determined after applying the aminoglycoside resistance pattern to 12 aminoglycosides by a disk susceptibility test described by Miller et al. (21). Seventeen strains seemed to produce a bifunctional [AAC(6') + APH(2")] enzyme associated with ANT(4') (4 strains) or with APH(3') (13 strains), and 10 seemed to produce the ANT(4') enzyme alone. The production of the two enzyme combinations cited above notably confers resistance to kanamycin, tobramycin, and gentamicin (i.e., a Km^r Tm^r Gm^r phenotype), whereas production of the ANT(4') enzyme alone determines resistance to kanamycin and tobramycin (i.e., a Km^r Tm^r phenotype) (31). The five remaining strains were susceptible to aminoglycosides.

Media and antibiotics. Mueller-Hinton broth and agar (Becton-Dickinson, Le Pont-de-Chaix, France) and tryptic soy (TS) agar supplemented or not supplemented with blood (Bio-Rad, Marnes-la-Coquette, France) were used. All incubations were at 37°C for 24 h. The following antibiotics were kindly provided by the manufacturers: imipenem (Merck Sharp & Dohme, Paris, France), cefazolin (Panpharma S.A., Fougères, France), vancomycin (Eli Lilly & Co., Saint-Cloud, France), and netilmicin (Schering Plough, Levallois-Perret, France).

Screening for the -lactam and for the aminoglycoside showing the best beneficial effect in combination with vancomycin. A one-disk diffusion technique previously described (27) was used to assess the effects of -lactam-vancomycin and vancomycin-aminoglycoside combinations against all of the 32 MRSA strains tested. Mueller-Hinton agar plates with or without vancomycin at one-fourth of the MIC were flooded with a bacterial suspension of 1.5 × 10⁶ CFU/ml. Twenty-six disks each impregnated with a -lactam (Bio-Rad) and 8 disks impregnated with an aminoglycoside were placed on top of the agar plates. For each strain, the inhibition zone around the disks in the absence and in the presence of vancomycin in agar plates was compared as described below. An initial score (S1) was established according to the inhibition zone on the plate without vancomycin to take into account the original susceptibility of the strain to the antibiotic tested. This inhibition zone was interpreted as described by the Comité de l'Antibiogramme de la Société Française de Microbiologie (10). S1 was 0 when the strain was resistant to the antibiotic tested, 1 when it was intermediate, and 2 when it was susceptible. A second score (S2) was determined by comparison of inhibition zones in the presence or absence of vancomycin; S2 was 0 in the absence of an increase in the diameter of the inhibition zone, 1 when the zone was increased but the strain remained in the same category, 2 when the strain changed by one category (i.e., moving from resistant to intermediate [RI] or from intermediate to susceptible [IS]), and 3 when the zone was increased so that the strain changed by two categories (RS). Lastly, depending upon the addition of these two scores (S3), the beneficial bacteriostatic effect was defined as absent (S3 = 0), weak (S3 = 1), moderate (S3 = 2), or strong (S3 = 3).

Susceptibility testing and checkerboard studies. The MICs of cefazolin, imipenem, netilmicin, and vancomycin were determined by the agar dilution method in accordance with standard guidelines (10, 23). The replicator prong delivered approximately 10⁴ CFU per spot (34). The MICs were interpreted in accordance with the recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie (10).

Killing studies. Killing experiments were performed to evaluate the bactericidal activities of two-drug and three-drug combinations of antibiotics, including a -lactam (imipenem or cefazolin) and/or netilmicin and vancomycin, against 10 of the 32 MRSA strains studied. The 10 strains tested were chosen according to their aminoglycoside susceptibility patterns and consisted of 4 strains with the Km^r Tm^r Gm^r phenotype, 4 strains with the Km^r Tm^r phenotype, and 2 strains susceptible to aminoglycosides.

	RESULTS

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Screening for the -lactam and the aminoglycoside showing the strongest beneficial bacteriostatic effect in combination with vancomycin. As shown in Table 1, the beneficial bacteriostatic effects of -lactam-vancomycin combinations were variable, depending on the -lactam tested. The -lactams which exhibited a strong beneficial effect against a high percentage of strains when combined with vancomycin were meropenem (66% of strains), ampicillin-sulbactam (62%), cefazolin (60%), and to a lesser extent imipenem, cefoxitin, and cefotiam (56% for each). Among these agents, ampicillin-sulbactam, cefazolin, and imipenem were those providing a moderate or strong beneficial effect against the most strains. Among these three drugs, cefazolin and imipenem, whose pharmacodynamic properties and therapeutic indications are markedly different, were both retained for the subsequent bacteriostatic and bactericidal studies.

View this table: [in this window] [in a new window]   TABLE 1.   Beneficial bacteriostatic effects of different -lactam-vancomycin combinations against 32 MRSA strains

MIC determinations and checkerboard studies. The characteristics of the 32 MRSA strains tested are shown in Table 3. The MICs of the antibiotics studied for 90% of the 32 MRSA strains tested were as follows: cefazolin, 256 µg/ml; imipenem, 64 µg/ml; netilmicin, 16 µg/ml; vancomycin, 4 µg/ml. On the basis of the MICs, 15 strains appeared susceptible to cefazolin and 17 appeared susceptible to imipenem. Seventeen of the 32 MRSA strains were susceptible to netilmicin, 9 were intermediate, and 6 were resistant at a low level (MIC, 16 or 32 mg/liter). All of the strains were susceptible to vancomycin.

View this table: [in this window] [in a new window]   TABLE 4.   FIC indexes of -lactam-vancomycin and vancomycin-netilmicin combinations for 32 MRSA strains

Killing studies. The rates of killing of the 10 strains by antibiotics used alone or in double or triple combinations, determined at one-half of the MIC, are presented in Table 5. At this concentration, none of the monotherapies tested produced bactericidal effects after 24 h, except vancomycin against strain 4. It is noteworthy that vancomycin alone produced a >2-log₁₀ reduction in the counts of three additional strains. Regimens of netilmicin combined with either vancomycin or a -lactam (cefazolin or imipenem) were infrequently or never bactericidal. In contrast, regimens consisting of imipenem or cefazolin plus vancomycin were bactericidal after 24 h against 6 and 5 of the 10 strains studied, respectively, and were often synergistic. Both -lactam-vancomycin combinations were bactericidal against all of the strains harboring the Km^r Tm^r Gm^r phenotype, while only the imipenem-vancomycin combination produced a bactericidal effect against one strain with the Km^r Tm^r phenotype. It is noteworthy that all cases but one of the absence of a bactericidal effect of the -lactam-vancomycin regimen occured with strains for which FIC indexes of 0.5 was calculated. The addition of netilmicin markedly enhanced the killing activity of the combinations of cefazolin or imipenem plus vancomycin, but only for three of the four MRSA strains against which the -lactam-vancomycin combinations failed to produce a bactericidal effect. It is noteworthy that these strains (no. 21, 22, 27, and 30) were both susceptible to netilmicin and heterogeneously resistant to methicillin. As a result, the three-drug combinations each demonstrated a bactericidal effect against 8 of the 10 strains studied. The imipenem-vancomycin-netilmicin combination produced a greater bacterial count reduction and was more frequently synergistic than the cefazolin-vancomycin-netilmicin regimen.

	DISCUSSION

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In this study, we analyzed the activities of 8 aminoglycoside-vancomycin and 26 -lactam-vancomycin combinations against a set of 32 clinical MRSA strains. This represents 1,088 individual analyses, a work which would have been tremendously laborious with conventional methods for the study of antibiotic combinations such as checkerboard synergy testing. To circumvent such technical problems, we used a previously described disk diffusion method (27) that is easy to perform, reproducible, and easier to interpret than the double-disk potentiation methods (8). Compared with our original description (27), the calculation of the final score that analyzes the beneficial bacteriostatic effect observed was slightly modified to take into account the intrinsic -lactam activity against MRSA isolates.

Among the eight aminoglycosides tested, netilmicin was considered the best agent to combine with vancomycin, frequently showing a moderate or strong beneficial bacteriostatic effect, particularly against strains with a Km^r Tm^r phenotype and, interestingly, even against those with a Km^r Tm^r Gm^r phenotype (data not shown), despite the cross-resistance between gentamicin and netilmicin.

The screening test also revealed that most -lactam-vancomycin combinations exhibited a beneficial effect against MRSA strains and that the -lactams most often demonstrating a moderate or strong beneficial effect were ampicillin-sulbactam, imipenem, and cefazolin. This may be related to the binding affinities of these three -lactams for penicillin-binding protein 2a of MRSA, which are stronger than that of methicillin and allow the classification of these three agents among the -lactam antibiotics most active against MRSA in vitro (6, 7). Using a similar screening test, Climo et al. (8) recently reported a synergistic bacteriostatic effect of a combination of vancomycin and either oxacillin, ceftriaxone, ceftazidime, cefpodoxime, or amoxicillin-clavulanate against three clinical GISA isolates.

The favorable bacteriostatic effect of the imipenem- or cefazolin-vancomycin combinations was also demonstrated by the checkerboard studies. The concentrations at which synergism was observed were easily clinically achievable with both combinations. It is noteworthy that the imipenem-vancomycin regimen produced the best results, in terms of both FIC values and the antibiotic concentrations generating bacteriostatic synergy. By checkerboard studies, synergistic bacteriostatic effects of -lactam-vancomycin combinations against MRSA have been previously reported for imipenem and cephalosporins such as cefotiam, cefoperazone, and cefpirome (3, 9, 30, 35, 36), but not for oxacillin (8). However, unlike our results, the synergistic FIC values usually observed in such studies were frequently high, close to the 0.5 limit value for synergy (9, 30).

In contrast, the combination of vancomycin with netilmicin resulted in an indifferent effect, even against MRSA strains susceptible to netilmicin. Such results are in accordance with recent studies performed with gentamicin (17, 18) or netilmicin; D. Ince and H. Eraksoy, 8th Int. Congr. Infect. Dis., abstr. 12.038, p. 20, 1998).

The potential bactericidal effects of double or triple combinations including a -lactam (imipenem or cefazolin) and/or vancomycin and/or netilmicin were further evaluated by killing experiments with 10 strains. The antibiotics were tested at one-half of the MICs to improve the detection of synergy (12) and also to reflect clinical conditions. As expected from the antibiotic concentrations used in these experiments, the monotherapies and the -lactam-netilmicin combinations generally failed to produce bactericidal effects after 24 h. The bacterial count reductions sometimes observed in broth with vancomycin alone were maybe related to a lack of accuracy of the vancomycin MICs, which were determined by agar and not by broth dilution.

As expected from the high FIC indexes, the vancomycin-netilmicin combination was found to be poorly active against the MRSA strains studied, whatever their aminoglycoside susceptibility pattern. Previous studies examining the bactericidal activity of vancomycin-aminoglycoside combinations against MRSA most of the time tested gentamicin instead of netilmicin, but their authors globally reported similar indifferent results (22; Ince and Eraksoy, 8th Int. Congr. Infect. Dis.). Synergism was shown not to be predictable from the aminoglycoside MIC (22). On the other hand, some studies have reported that gentamicin (14, 17) or netilmicin (26) can enhance the bactericidal activity of vancomycin but these results were observed at inhibitory antibiotic concentrations and against only one to three strains.

The most interesting results of our study are the frequent and strong bactericidal effects of the -lactam (cefazolin or imipenem)-vancomycin combination and, moreover, of the -lactam-vancomycin-netilmicin combination. The results of our bitherapies compared favorably with those of previous works evaluating the killing activity of combinations of vancomycin and either cefotiam (35) or cefpirome (29) against MRSA: for the cefotiam-vancomycin combination, only a weak synergistic bacteriostatic effect was reported; for the cefpirome-vancomycin regimen, the bactericidal effect at 24 h was observed only against the two strains homogeneously resistant to methicillin. In a previous work performed with a single MRSA strain (36), the bactericidal effect of the imipenem-vancomycin combination has been reported to be dependent on the imipenem concentration.

To our knowledge, this is the first work studying the efficacy of triple combinations of a -lactam, a glycopeptide, and an aminoglycoside against MRSA strains. For the strains both homogeneously resistant to -lactams and resistant to netilmicin, the triple combinations had no advantage over the -lactam-vancomycin combinations in terms of bactericidal effect. For four of the six strains both heterogeneously resistant to methicillin and susceptible to netilmicin, the -lactam-vancomycin combinations failed to provide a bactericidal effect, probably because of the heterogeneous methicillin resistance. As expected for these strains, the addition of netilmicin markedly increased the killing effects of the -lactam-vancomycin combinations, except for one strain. However, as all of the four Km^r Tm^r Gm^r strains tested were homogeneously resistant to methicillin and all of the four Km^r Tm^r strains were heterogeneously resistant, these results do not allow determination of what is significant in the phenotype of the strains to predict the bactericidal effects of the combinations.

In conclusion, this study shows that vancomycin combined with imipenem or cefazolin, and even with netilmicin in a triple combination (depending on the aminoglycoside susceptibility pattern of the strains), at subinhibitory concentrations can be bactericidal after 24 h at higher rates than vancomycin-netilmicin. Further studies are needed before generalizing the concept of the usefulness of adding a -lactam such as imipenem or cefazolin, and potentially an aminoglycoside such as netilmicin, to vancomycin for the treatment of MRSA infections. Interestingly, the choice between cefazolin and imipenem could be based on in vitro tests but also on the clinical context: cefazolin in cases of proven monomicrobial infection due to MRSA and imipenem in cases of suspected or proven mixed infections. Lastly, the recent description of synergistic activities of different -lactams and vancomycin against some GISA isolates (8, 32) indicates that further studies in this field are warranted.