Effect of antibiotic concentration on the killing of Staphylococcus aureus and Enterococcus faecalis: comparison of the novel penem, Men 10700, with other ß-lactam antibiotics

J Antimicrob Chemother 1999; 44: 418–420

J. M. T. Hamilton-Miller* and Saroj Shah

Department of Medical Microbiology, Royal Free and University College Medical School, Pond Street, London NW3 2QG, UK

Sir,

The so-called `paradoxical effect`, whereby antibiotics exhibit reduced bactericidal activities at high drug concentrations, has been observed mainly, although not exclusively, with ß-lactams and Gram-positive bacteria.1 The phenomenon was first described by Eagle & Musselman2 who found that Staphylococcus aureus, ß-haemolytic streptococci and, most strikingly, Enterococcus faecalis were killed much more slowly by high concentrations of benzylpenicillin than by lower (but still supra-MIC) concentrations.

The data sheets for many antibiotics recommend the administration of higher dosages when an infection is deemed `serious` and it is apparent that for ß-lactam antibiotics in particular, there is a theoretical risk that this practice may not always be beneficial to the patient. For this reason, we have investigated the `Eagle effect` as part of the microbiological evaluation of the novel penem, Men 10700.

The bacteria used in the study—S. aureus strains 957, 17090 and 17285 (all methicillin-susceptible ß-lactamase producers) and E. faecalis strains Lyons and FTS8—were clinical isolates used in previous studies.3 Men 10700 was obtained from Menarini Ricerche SpA (Florence, Italy), and imipenem, cefotaxime and amoxycillin were obtained from Merck Sharp & Dohme (Hoddesdon, UK), Roussel Laboratories (Uxbridge, UK) and SmithKline Beecham (Welwyn Garden City, UK), respectively. MICs were determined by a standard broth dilution method; the medium used was IsoSensitest broth (ISB; Unipath, Basingstoke, UK) and the inocula were between 5 x 109 and 5 x 1010 cfu/L. The bactericidal activities of Men 10700, imipenem and cefotaxime against the S. aureus strains, and Men 10700, amoxycillin and imipenem against the E. faecalis strains were investigated by time–kill studies. Overnight cultures of each bacterium (containing 7–11 x 1011 cfu/L) were diluted 1:10 in ISB, and 10 mL volumes were added to groups of five 25 mL conical flasks. For each antibiotic/bacterium combination, antibiotic was added to four flasks, giving final concentrations of 3 x, 10 x, 30 x or 100 x MIC; the fifth flask was a growth control. The flasks were shaken at 100 rpm at 37°C, and aliquots were withdrawn at 0, 4 and 24 h and inoculated on to nutrient agar plates (Unipath). Colonies were counted after incubation for 24 h and again after 48 h. From plots of log viable count versus time, the times taken to bring about 90% and 99.9% kill (t90 and t99.9, respectively) and the percentages of the original inoculum surviving at 4 and 24 h were calculated.

For the three strains of S. aureus incubated in the presence of Men 10700, cefotaxime or imipenem, the time–kill curves were virtually linear between 0 and 24 h, although strain 17285 was killed up to three-fold more slowly depending on the antibiotic and the concentration, than the other two strains (data not shown). There was no reduction in the rates of killing at higher concentrations of cefotaxime, but as the concentration of Men 10700 increased from 3 x to 100 x MIC, there was a 38% decrease in t99.9 for strain 957 only. On the other hand, imipenem at higher concentrations became less rapidly bactericidal against all three strains (t99.9 being reduced by 30–52%), particularly 17285. Analysis of the survival rates (the original method used to define the Eagle effect2) revealed no paradoxical effects at higher concentrations after 4 h, whereas after 24 h (Table), the pattern was similar to that observed with the time–kill plots, i.e. no effect with cefotaxime, a modest effect (three-fold increased survival) with Men 10700 against strain 957, and a marked effect (13-fold increased survival) with imipenem against strain 17285.


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Table. Bactericidal activities of Men 10700, imipenem and cefotaxime against three strains of S. aureus, and Men 10700, imipenem and amoxycillin against two strains of E. faecalis
 
In contrast to S. aureus, the time–kill curves for each of the E. faecalis strains were not linear, the rates of killing produced by the three antibiotics tested decreasing with time (data not shown). The survival data (Table) show marked paradoxical effects for all three drugs with both strains. When the effects of the lowest and highest concentrations are compared, the greatest (>=100-fold) increases in the percentages of survivors were observed when amoxycillin was tested against both strains and when Men 10700 was tested against strain FTS8.

As E. faecalis is killed only slowly by antibiotics, the MBC, as defined conventionally (i.e. >=99.9% killing), is often not achieved.4 Consequently, most of these drugs are regarded as having only bacteriostatic activities against strains belonging to this species. None the less, time–kill studies such as those described here reveal that some antibiotics have, albeit slow, bactericidal activities which are concentration dependent.

Not all of the paradoxical effects produced by ß-lactam antibiotics that have been reported in the literature5,6 are synonymous with the classic Eagle effect. The absence of autolytic enzymes, which is regarded as the basis of tolerance, may, in part, also account for this effect in enterococci,4 although tolerance appears to be a very different phenomenon.7

The Eagle effect is measured by comparing the percentages of survivors after varying periods of exposure to an antibiotic at high and low concentrations. Provided the time–kill plots are linear (as observed in this study with the S. aureus strains), the effect can be quantified from the t90s or t99.9s, but this does not apply if the time–kill plots are dog-legged (as occurred with the E. faecalis strains).

We have observed a marked Eagle effect in enterococci with the penam, penem and carbapenem antibiotics tested, but, at most, only a marginal effect in S. aureus. The common practice of increasing the dosages of antibiotics in seriously ill patients should not therefore compromise the bactericidal activities of these drugs against the latter pathogen, but may not be appropriate in patients with certain enterococcal infections, e.g. infective endocarditis.

Acknowledgments

We thank Dr M. De Luca (Menarini Ricerche), for helpful discussions and financial support.

Notes

* Corresponding author. Tel: +44-171-794-0500; Fax: +44-171-435-9694; E-mail: j.hamilton-miller{at}rfhsm.ac.uk Back

References

1 . Yourassowsky, E., VanderLinden, M. P., Lismont, M. J. & Schoutens, E. (1978). Qualitative study of the paradoxical zone phenomenon of penicillins against 17 bacterial species of clinical importance. Chemotherapy 24, 92–6.[ISI][Medline]

2 . Eagle, H. & Musselman, A. D. (1948). The rate of bactericidal action of penicillin in vitro as a function of its concentration, and its paradoxically reduced activity at high concentrations against certain organisms. Journal of Experimental Medicine 88, 99–131.

3 . Hamilton-Miller, J. M. T. & Shah, S. (1997). In-vitro microbiological assessment of a new penem, Men 10700. Journal of Antimicrobial Chemotherapy 39, 575–84.[Abstract]

4 . Fontana, R., Boaretti, M., Grossato, A., Tonin, E. A., Lleo, M. M. & Satta, G. (1990). Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin. Antimicrobial Agents and Chemotherapy 34, 314–20.[ISI][Medline]

5 . Kerry, D. W., Hamilton-Miller, J. M. T. & Brumfitt, W. (1976). Paradoxical effect of mecillinam on Providencia stuartii. Journal of Antimicrobial Chemotherapy 2, 386 –8.[Medline]

6 . Ikeda, Y. & Nishino, T. (1988). Paradoxical antibacterial activities of b-lactams against Proteus vulgaris: mechanism of the paradoxical effect. Antimicrobial Agents and Chemotherapy 32, 1073–7.[ISI][Medline]

7 . Puntorieri, M., Primavera, A., Privitera, O., {Delta}rquote Amico, G., Mezzatesta, M. L., Cowap, L. et al. (1994). Observations on the tolerance and the paradoxical effect in enterococci. Journal of Chemotherapy 6, 377–82.[ISI][Medline]





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