Defining high-level gentamicin resistance in enterococci

M. J. Weinbrena, A. P. Johnsonb and N. Woodfordb,*

a Microbiology Department, Kingston Hospital, Kingston-upon-Thames KT2 7QD; b Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, London NW9 5HT, UK

Sir,

Enterococci cause c. 10–20% of cases of infective endocarditis (IE).1,2 The recommended treatment for patients with such infections is ampicillin/amoxycillin 12 g/day together with gentamicin 80 mg bd for 4 weeks.3 The rationale behind combination therapy is that penicillins alone have poor bactericidal activity and the addition of an aminoglycoside usually results in synergic killing. For enterococci that exhibit high-level gentamicin resistance, which is typically mediated by the bifunctional enzyme AAC(6')APH(2''),1 streptomycin can be substituted if the aetiological agent has been shown to be susceptible to it; otherwise, treatment with ampicillin/amoxycillin alone is recommended for a minimum of 6 weeks.3 We describe here a patient with IE caused by a strain of Enterococcus faecalis harbouring the aac(6')aph(2'') gene which is normally associated with high-level gentamicin resistance, although the isolate failed to fulfil the criteria for such resistance defined by the British Society for Antimicrobial Chemotherapy (BSAC)4 and the National Committee for Clinical Laboratory Standards (NCCLS).5

A 78 year-old man was admitted to hospital in December 1998 for elective total knee replacement made necessary by longstanding osteoarthrosis. His past medical history included insertion of a pacemaker in 1996 and replacement of an aortic valve in February 1998, the latter procedure having been complicated by haemarthrosis of the right knee joint and urinary retention. In January 1999 he underwent resection of the prostate for which he was given ampicillin and gentamicin as prophylaxis. On the seventh post-operative day he became pyrexial. Several blood cultures were obtained and a strain of E. faecalis susceptible to ampicillin, but apparently highly resistant to gentamicin (determined with a 100 µg disc), was isolated repeatedly. The patient received ampicillin 12 g/day and gentamicin for presumed IE. The isolate was referred to the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL), where susceptibility to ampicillin was confirmed, but high-level gentamicin resistance was not detected; although the MIC of gentamicin was >128 mg/L according to an agar dilution method, the isolate failed to grow on a plate containing gentamicin at a concentration of 2000 mg/L. One week after starting therapy the patient's renal function deteriorated. The gentamicin was therefore discontinued and the renal function improved. A cardiologist who subsequently reviewed the patient felt that IE was unlikely and the patient was discharged. However, in May he was readmitted complaining of pain in the right knee which had been present since the initial surgery. He was anaemic and the C-reactive protein concentration was elevated. The joint was aspirated and a strain of E. faecalis was isolated, but only after enrichment. Four sets of blood cultures, obtained before antibiotic therapy was initiated, yielded the same strain. A cardiology opinion was sought and on this occasion the impression was that the patient did indeed have IE, although it was unclear whether it was secondary to infection of the total knee replacement or vice versa.

The isolate was referred to ARMRL, where susceptibility testing yielded results identical to those for the primary isolate; the MIC of gentamicin, as determined by the Etest, was 512 mg/L. The two isolates were also indistinguishable by pulsed-field gel electrophoresis (PFGE). On disc testing,4 the zones of inhibition around 200 µg gentamicin discs after incubation for 24 h were 20–22 mm in diameter (thereby placing the isolates in the susceptible category), but a few tiny colonies were present within the zones. After incubation for 48 h the zones of inhibition were only 6 mm in diameter. Although the MICs were not consistent with high-level resistance according to BSAC and NCCLS criteria,4,5 concerns were expressed about the activity of gentamicin against a strain for which the MIC was eight-fold greater than the modal MIC for this species, i.e. 64 mg/L. Analysis of the isolates by PCR detected the aac(6')aph(2'') gene. Thus, the genotype of the isolates, together with the high MIC of gentamicin and the reduced inhibition zone diameter at 48 h, prompted us to conclude that gentamicin would not act synergically with penicillin against this bacterium. Gentamicin and ampicillin have previously been shown in time–kill studies not to act synergically against a strain of Enterococcus gallinarum for which the MIC of gentamicin was 256 mg/L and which expressed the APH(2')-Ic phenotype.6 Since the isolates were also resistant to streptomycin (MIC >2000 mg/L) and, notwithstanding the absence of vegetation on a transoesophageal echocardiogram, the patient was treated with ampicillin alone for 6 weeks, after which he was discharged much improved.

The most recent definition of high-level gentamicin resistance in enterococci, recommended by the BSAC Working Party,4 is either an MIC >=1024 mg/L or an inhibition zone diameter around a 200 µg gentamicin disc <=9 mm;4 similar criteria have been recommended by the NCCLS.5 However, if the endpoint of the disc diffusion test had not been read after an extended period of incubation, these criteria would have failed to detect resistance in the strains studied here, the patient from whom it was isolated having responded poorly to combination therapy and the bacteria harbouring the aac(6')aph(2'') gene. Several other isolates of E. faecalis and Enterococcus faecium for which the MICs of gentamicin were >128 mg/L but <2000 mg/L have been referred to ARMRL and all have harboured the aac(6')aph(2'') gene (N. Woodford, unpublished data). We therefore recommend caution in excluding high-level gentamicin resistance in enterococci with gentamicin MICs >128 mg/L but which fail to meet the BSAC or NCCLS criteria for phenotypic high-level resistance. Furthermore, we suggest that zones of inhibition produced by strains that are not clearly resistant at 24 h should be re-examined after 48 h, particularly if bactericidal combination therapy is required to treat patients infected with them, and that a genotypic method should be used to identify the presence of genes known to confer resistance to gentamicin, including aac(6')aph(2''), aph(2'')-1c6 and aph(2'')-1d.7

Acknowledgments

We are grateful to Marina Warner and Marie-France I. Palepou for technical assistance and to Polly Kaufmann for performing PFGE typing.

Notes

J Antimicrob Chemother 2000; 45: 404–405

* Corresponding author. Tel: +44-208-200-4400; Fax: +44-208-200-7449; E-mail: nwoodford{at}phls.nhs.uk Back

References

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