a Institute of Medical Therapy b Department of Tropical Diseases, Second University of Naples, Naples, Italy
Sir,
Optimal therapy of patients with
infections caused by high-level gentamicin-resistant (HLGR) enterococci remains to be
determined. A combination of ampicillin and ciprofloxacin has been shown to be bactericidal in vitro against HLGR strains exhibiting low-level resistance to ciprofloxacin
(MICs 4 mg/L),1 but, in an endocarditis animal
model, failed to sterilize vegetations infected with a HLGR strain of Enterococcus faecium which was also highly resistant to ampicillin.2 We
have recently reported that the in-vitro activity of this combination was influenced by the
composition of the medium in which the activity was evaluated, i.e. ampicillin together with
ciprofloxacin exhibited bactericidal activity in MuellerHinton broth (MHB), but only
bacteriostatic activity in the enriched media, Brain Heart Infusion (BHI) broth and human
serum-supplemented MHB.3 The reduced activity of the
combination in the latter media was attributed, at least in part, to the high cation content, which
is known to be inhibitory to ciprofloxacin. The present study was undertaken to assess the effects
of two additional factorsthe level of resistance to ampicillin and an inoculum
effecton the in-vitro activity of the combination against HLGR enterococci.
The strains included in the study, five HLGR E. faecium isolates exhibiting
low-level resistance to ampicillin (MICs 3264 mg/L) and ciprofloxacin (MICs
24 mg/L), were stored at 70°C in BHI broth (Difco, Detroit, MI, USA)
containing 10% glycerol. The effects of the variables were investigated by timekill
studies. The medium used was MHB (Difco) and the strains were incubated in the presence of
ampicillin alone, ciprofloxacin alone, both ampicillin and ciprofloxacin or no antibiotic (control),
as described previously.1 The concentrations of ampicillin
and ciprofloxacin were 20 and 2 mg/L, respectively, and the suspensions initially contained
5 x 108 cfu/L. Bactericidal activity was defined
as a reduction in the initial suspension of 3 log10 cfu/L after incubation at
37°C for 24 h. All experiments were carried out at least twice and the results expressed as
the means.
High-level resistance to ampicillin was induced in one of the five strains, No. 662 (initial MIC 32 mg/L), following daily passage in MuellerHinton II broth (MHB II; BBL Becton Dickinson, Cockeysville, MD, USA) containing increasing (48 mg at a time) concentrations of ampicillin and subsequent passage on agar that also contained ampicillin. Each resultant mutant for which the MIC had increased two-fold underwent at least 10 passages in antibiotic-free medium, after which the MIC of ampicillin was redetermined. This procedure yielded three mutants, identified as numbers 662.1, 662.2 and 662.3 and confirmed to be strains of E. faecium by conventional laboratory techniques, including the API Strep test (bioMérieux, Marcy l'Etoile, France). The MICs of ampicillin for the mutants were 64, 128 and 150 mg/L, respectively, these being the true MICs (defined as the actual inhibitory concentrations which lie anywhere between two two-fold dilutional steps);4 the MICs of ciprofloxacin and gentamicin for the mutants were the same as those for the parental strain. The effect of the level of ampicillin resistance on the activity of the combination was assessed by repeating the timekill studies with both the parental and mutant strains in MHB.
The influence of the size of the initial inoculum on the activity of the combination was also assessed. Overnight cultures of the five strains were diluted in MHB to give inocula of 108 cfu/L and 1010 cfu/L and the timekill studies were repeated, as described above, with each inoculum.
As shown in the Table, there was an inverse relationship between the
level of ampicillin
resistance and the activity of the ampicillin/ciprofloxacin combination (which produced changes
in log10 cfu/L after incubation for 24 h ranging from 3.92 to 0.45).
The combination exerted only bacteriostatic activity against strains for which the MICs of
ampicillin were 64 mg/L. This observation is at variance with a previous report in which
we described the combination as being bactericidal against strains with MICs of 64 mg/L.1 The discrepancy might be explained by differences in the
methods used to determine the MICs in the two studies. In the earlier study, the antibiotic
concentrations tested were based on the traditional two-fold dilutions; with this technique, the
true MIC tends to be lower than the observed endpoint and may actually have been any value
between 32 and 64 mg/L. In the present study, however, we determined the true MIC to be 64
mg/L, growth having been observed on MHB II plates containing ampicillin at a concentration of
60 mg/L, but not on those containing ampicillin at a concentration of 64 mg/L.
|
The present study demonstrates that the level of ampicillin resistance in the study strain and the size of the inoculum have profound effects on the in-vitro activity of the ampicillin/ciprofloxacin combination against a HLGR strain of E. faecium. The poor activity of this combination in the endocarditis animal model2 may therefore be accounted for by both the high level of ampicillin resistance exhibited by the strain used in the study and the high density of bacteria in the valvular vegetations.
The study has also defined the strengths and limitations of the ampicillin/ciprofloxacin
combination in terms of its activity against multidrug-resistant enterococci. Regardless of the
limitations, we believe that the combination might be effective therapy for some patients with
severe infections caused by HLGR strains for which the MICs of ampicillin and ciprofloxacin
are 32 and
4 mg/L, respectively. Two elderly patients with endocarditis caused
by HLGR enterococci that were susceptible to ampicillin and exhibited low-level resistance or
susceptibility to ciprofloxacin were recently treated successfully by us with combinations of
ampicillin and a fluoroquinolone; one of the strains was also resistant to glycopeptides (VanA
phenotype).5 Further studies to evaluate the efficacies of
combinations of newer quinolones and ß-lactams as treatment of patients with infections
caused by multidrug-resistant enterococci are warranted.
Acknowledgments
This study was supported by grant MURST 1998.
Notes
* Correspondence address: Istituto di Terapia Medica, II
Università di Napoli, Via
D. Cotugno 1 (c/o Ospedale Gesù e Maria), 80135 Napoli, Italy. Tel:+39-81-566-6229;
Fax:+39-81-566-6230; E-mail: Mtripodi{at}unina.it
References
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2 . Landman, D., Quale, J. M., Mobarakai, N.& Zaman, M. M. (1995). Ampicillin plus ciprofloxacin therapy of experimental endocarditis caused by multidrug-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 36,253 8.[Abstract]
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Tripodi, M. F., Locatelli, A., Rambaldi, A., Rosario, P. &
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5 . Tripodi, M. F., Locatelli, A., Adinolfi, L.E., Andreana, A. & Utili, R. (1998). Successful treatment with ampicillin and fluoroquinolones of human endocarditis due to high-level gentamincin-resistant enterococci. European Journal of Clinical Microbiology and Infectious Diseases 17, 7346.[ISI][Medline]