DIAGN MICROBIOLINFECTDIS 1992;15:247-251

247

ANTIMICROBIAL SUSCEPTIBILITY STUDIES

In vitro Activity of Teicoplanin Compared with Vancomycin Against Methicillin-Resistant Staphylococcus aureus Derived from Cystic Fibrosis Sputum Antonio C. Arrieta, Harris R. Stutman, Joseph C. Akaniro, and Ofelia M. Vargas

We evaluated the in vitro activity of teicoplanin compared with vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) derived from cystic fibrosis (CF) sputum. Teicoplanin had a slightly lower median minimum inhibitory concentration (MIC) for these strains (0.25 p,g/ml) than did vancomycin (0.5 i~g/ml). Inoculum size increased the MICs similarly for both drugs, and pH variations did not sig-

nificantly affect their activity. The presence of serum and sputum in the growth media decreased the activity of both drugs, although this was more pronounced for teicoplanin which is highly protein bound. We conclude that teicoplanin has activity against this pathogen and might be evaluated in clinical protocols designed to address this emerging clinical problem.

INTRODUCTION

diphtheroids, Propionibacterium acnes, and Listeria monocytogenes (Kempf et al., 1989; Bibler et al., 1987). Because of their spectrum of activity, glycopeptide antibiotics may prove useful in the treatment of infections seen in patients with cystic fibrosis (CF), who very frequently have infections with S. aureus (Marks, 1990; Bauernfeind et al., 1988; Doring et al., 1987). Although semisynthetic penicillins have traditionally been associated with a low incidence of resistance, recent reports indicate the emergence of methicillin-resistant strains in CF patients (Marks, 1990; Bauernfeind et al., 1989; Boxerbaum and Jacobs, 1987). The treatment of bacterial pulmonary infections in CF is often complicated by the progressive development of resistance by causative organisms to available antibiotics. The special characteristics of the environment at the site of infection (for example, lower pH, larger inoculum, and increased protein) may interfere with antimicrobial action, thereby making eradication more difficult (Marks, 1990; Chartrand and Marks, 1988; Bauernfeind et al., 1987). Although resistance to vancomycin has not been

Teicoplanin is a novel compound that belongs to the glycopeptide antibiotic group. Like vancomycin, teicoplanin inhibits cell-wall peptidoglycan synthesis by interfering with transglycosylation, the last of a chain of events leading to the elongation of the cell wall (Reynolds, 1989). The antibacterial activity of teicoplanin is specific for Gram-positive bacteria, both aerobic and anaerobic. It is active against Staphylococcus aureus, including methicillin-resistant strains, Staphylococcus epidermidis, streptococci, enterococci, clostridia, From the Department of Pediatrics (A.C.A., H.R.S., J.C.A., O.M.V.), Division of Pediatric InfectiousDiseases, Memorial Miller Children's Hospital, Long Beach; and Universityof California-Irvine (A.C.A., H.R.S.), Irvine, California,USA. Address reprint requests to Dr. Antonio Arrieta, PediatricInfectious Diseases, MemorialMiller Children's Hospital, 2801 Atlantic Avenue, Long Beach, CA 90801-1428, USA. Received 21 February 1991; revised and accepted 1 July 1991. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00

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a problem, the potential side effects of nephro- and ototoxicity can be inconvenient, especially when vancomycin is used concomitantly with similarly toxic drugs, like the aminoglycosides. This is likely to be the case in CF, where S. aureus and Pseudomonas aeruginosa often coexist (Marks, 1990; Chartrand and Marks, 1988; Bauernfeind et al., 1987). Therefore, less-toxic alternatives to vancomycin should be sought. The present investigation was conducted to evaluate the in vitro activity of teicoplanin compared with that of vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) strains derived from CF sputum.

MATERIALS A N D METHODS Bacterial Strains We used bacterial isolates from CF sputum available at the Memorial Miller Children's Hospital Infectious Disease Research Laboratory, derived from CF centers in the USA. We also used 20 methicillin-sensitive S. aureus and 20 MRSA isolates obtained from the clinical microbiology laboratory at Long Beach Memorial Medical Center.

Antimicrobial Agents Teicoplanin powder was obtained from Merrell-Dow Laboratories. Vancomycin and all other agents (ciprofloxacin, rifampin, gentamicin, trimethoprim/ sulfamethoxazole, and oxacillin) were obtained from their respective manufacturers. All antimicrobials were dissolved in appropriate diluents as recommended by the manufacturer and then diluted to a stock concentration of 2560 mg/L. The drugs were stored at -70°C and used within 4 weeks of preparation. Antimicrobial Susceptibility Tests

Determination of antimicrobial susceptibilities was performed by microdilution technique using Mueller-Hinton broth adjusted to pH 7.2-7.4, and supplemented with 50 mg of calcium and 25 mg of magnesium per liter of solution. Microdilution trays were prepared by adding 0.1 ml of medium-antimicrobial broth to each well, forming a concentration gradient ranging from 64 to 0.03 ~g/ml in doubling dilutions for each antibiotic. Bacterial inocula were grown in trypticase soy broth and incubated overnight at 37°C. By using a nephelometer, we adjusted the bacterial density to obtain a turbidity comparable to that of 0.5 McFarland standard. This was consistent with an inoculum size of 10s colony-forming units (CFU)/ml. Then microtiter

A.C. Arrieta et al.

wells were inoculated to a final concentration of 2.0 x 10 6 CFU/ml. This concentration was used to preserve a uniform inoculum size when performing comparative experiments. The m i n i m u m inhibitory concentration (MIC) was defined as the last well that showed no visible turbidity after an incubation period of 18-20 hr at 37°C in ambient air. Reference strain S. aureus 29213 was used to control for the stability of the incubation conditions and antimicrobial agents through the different experiments; the MIC for this reference change did not vary by more than + 1 dilution between different experiments.

Variation of Growth Conditions Pooled human sera obtained from healthy volunteers was added in a 1:1 dilution to appropriately supplemented Mueller-Hinton broth. By using the serum-Mueller-Hinton broth medium as diluent, we determined MICs. In addition, sputum obtained from CF patients was used as a diluent. The sputum was processed with dithiothreitol, sterilized, and mixed in a 1:1 ratio with Mueller-Hinton broth. Due to the turbidity of the medium (because of the tenacious sputum), MIC determinations for teicoplanin and vancomycin were impossible, so the m i n i m u m bactericidal concentration (MBC) was used and defined as the concentration of antibiotic needed to kill 99.9% of the initial bacterial inoculum. For this assay, 10~1 were aspirated from each well after mixing and subcultured onto blood agar plates. At a final inoculum density of 2.0 x 106 CFU/ml, isolates were exposed to teicoplanin and vancomycin at pH 4, 5, 6, and 7. Similarly, MICs were determined for teicoplanin and vancomycin at inoculum densities of 10 4, 10 5, 10 6, 10 7 and 108 CFU/ml.

RESULTS Results of the activity of teicoplanin and vancomycin against MRSA and methicillin-sensitive S. aureus are shown in Table 1. The activity of teicoplanin and vancomycin against MRSA derived from CF sputum is shown in Table 2. We also evaluated the activity of other antimicrobial agents frequently used against S. aureus and found that vancomycin and teicoplanin were the only ones with consistent activity against MRSA either from clinical isolates or CF sputum. This also confirms recent observations that ciprofloxacin is not active against S. aureus resistant to [3lactams. The activity of teicoplanin was stable at inoculum sizes up to 10 6 CFU/ml. At an inoculum size of ~ 1 0 7 CFU/ml, the activities of both vancomycin and teicoplanin were significantly reduced. Changes in pH

Teicoplanin for MRSA in Cystic Fibrosis Sputum

TABLE 1

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Antimicrobial Activity of Teicoplanin and Comparative Antibiotics Against Clinical Isolates of Staphylococcus aureus MIC (~g/ml) Range A.

50%

90%

1 0.5 0.5 0.5 0.004 1 0.25/4.75

2 1 1 1 0.008 2 1/19

Methicillin Sensitive (n = 20) 0.5-2 0.12-4 0.25-1 0.12-1 -128 0.12/2.5-16/304

did not seem to affect the activity of either drug except for an en h a nc e m ent of ~8-fold at a pH of 4. By adding serum to the growth media as previously described, we observed a decrease in the activity for both vancomycin and teicoplanin. There was a twofold diminution in the activity of vancomycin, whereas teicoplanin activity decreased by fourfold (Table 3). There was a striking increase in the MBC for both drugs wh en used in media containing CF sputum. This was again greater for teicoplanin which showed

TABLE 2

2 0.5 16 16 0.008 4 0.25/4.75

2 1 > 16 32 2 >128 16/304

an increase of ~256-fold as compared with vancomycin, which was increased by 16-fold (Table 3).

DISCUSSION Staphylococcus aureus appears to have an important role in the pathogenesis of infection in the lung of CF patients. Ongoing research protocols have focused on prophylactic regimens aimed at the prevention or delay of S. aureus colonization in these

Activity of Teicoplanin and Comparative Antibiotics Against Methicillin-Resistant Staphylococcus aureus Derived from Cystic Fibrosis Sputum MIC (~g/ml) Range

n=9 Vancomycin Teicoplanin Ciprofloxacin Oxacillin Rifampin Gentamicin Trimethoprim-sulfamethoxazole

0.12-2 0.06-1 0.25-16 0.5-32 --

In vitro activity of teicoplanin compared with vancomycin against methicillin-resistant Staphylococcus aureus derived from cystic fibrosis sputum.

We evaluated the in vitro activity of teicoplanin compared with vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) derived from cys...
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