The effects of the level of ampicillin resistance and the inoculum size on the in-vitro bactericidal activity of the combination of ampicillin and ciprofloxacin against high-level gentamicin-resistant strains of Enterococcus faecium

J Antimicrob Chemother 1999; 44: 719–720

M. F. Tripodia,*, A. Rambaldia, G. Sarnataroa, E. Ragone a and R. Utilib

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 Mueller–Hinton 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 factors—the level of resistance to ampicillin and an inoculum effect—on 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 32–64 mg/L) and ciprofloxacin (MICs 2–4 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 time–kill 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 Mueller–Hinton II broth (MHB II; BBL Becton Dickinson, Cockeysville, MD, USA) containing increasing (4–8 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 time–kill 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 time–kill 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.


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Table. In-vitro activities of ampicillin and ciprofloxacin, alone or in combination, against E. faecium strain 662 and mutant derivatives of this isolate with varying MICs of ampicillin
 
In the experiment in which the effect of the inoculum size was investigated, neither ampicillin nor ciprofloxacin alone was bactericidal against the five strains, irrespective of the inoculum used (data not shown). The combination, however, was bactericidal against all five strains when the lower inoculum was used (–4.07 ± 0.32 change in log10 cfu/L after 24 h), but only bacteriostatic at the higher inoculum (–0.43 ± 0.19 change in log10 cfu/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 Back

References

1 . Tripodi, M. F., Utili, R., Rambaldi, A., Locatelli, A., Rosario, P., Florio, A. et al. (1996). Unorthodox antibiotic combinations including ciprofloxacin against high-level gentamicin resistant enterococci. Journal of Antimicrobial Chemotherapy 37,727 –36.[Abstract]

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]

3 . Tripodi, M. F., Locatelli, A., Rambaldi, A., Rosario, P. & Utili, R. (1998). In-vitro activity of combined ampicillin and ciprofloxacin against high-level gentamicin-resistant strains of Enterococcus faecium: influence of the medium on susceptibility test results. Journal of Antimicrobial Chemotherapy 41, 139–40.[Free Full Text]

4 . Amsterdam, D. (1992). The MIC: myth and reality. The Antimicrobic Newsletter2 , 9–16.

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, 734–6.[ISI][Medline]





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