Disagreements between disc diffusion, and MIC-based susceptibility categorizations of ertapenem versus ESBL producers

Julie R. Samuel1,*, Manjula Natarajan1, Essam Rizkalla1, David M. Livermore2, Marina Warner2 and Peter Jenkinson1

1 Kettering General Hospital, Rothwell Road, Kettering, Northamptonshire NN16 8UZ, UK; 2 Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5HT, UK


* Corresponding author. Tel: +44-1536-492670; Fax: +44-1536-492704; E-mail: drmicro76{at}hotmail.com

Keywords: ß-lactamases , carbapenems , Enterobacteriaceae

Sir,

Ertapenem is the newest carbapenem to be licensed, approved by the US Food and Drug Administration in November 2001 and subsequently introduced worldwide for the treatment of severe community-acquired infections. It has good activity against most pathogens except non-fermenting Gram-negative bacilli, enterococci and methicillin-resistant staphylococci, along with the convenience of a once-a-day regimen.1 Activity is retained against most strains with high-level AmpC ß-lactamases or ESBLs,2 though there are reports of resistance in a few Enterobacteriaceae isolates that not only produce these enzymes, or metallo-ß-lactamases, but also have outer membrane protein changes that putatively reduce permeability.35

From December 2004, all Enterobacteriaceae isolates from urine and other samples at the Kettering General Hospital were screened for ESBL production, following BSAC- and Health Protection Agency-recommended guidelines and methodology.6 Isolates found to be ESBL-positive were examined for ertapenem susceptibility by the BSAC disc diffusion method and any found to be ertapenem-resistant were saved on Columbia agar for further study.

Surprisingly, 20 (35%) of the 58 putative ESBL producers screened (23 Escherichia coli, 22 Klebsiella spp., nine Enterobacter spp. and four others) appeared to be ertapenem resistant, giving zones of ≤33 mm to 10 µg discs. These 20 organisms were obtained between December 2004 and May 2005 and comprised Klebsiella spp. (10), Enterobacter spp. (5), Escherichia coli (4) and Citrobacter spp. (1). Nineteen were from urine and one from blood. Each isolate appeared positive for ESBL production using both cefpodoxime/clavulanate and ceftazidime/clavulanate combination discs (Oxoid, Basingstoke, UK) and with both cefotaxime/clavulanate and ceftazidime/clavulanate Etest strips (Biostat, Stockport, UK), although later reference laboratory testing suggested that one Enterobacter spp. isolate was AmpC-derepressed, based on a relatively low cefepime MIC (4 mg/L) that was not further reduced by the presence of clavulanic acid at 4 mg/L. It is possible that this isolate had lost an ESBL that originally was present.

The inhibition zone diameters for ertapenem 10 µg discs were re-determined at the reference laboratory, which confirmed that all 20 isolates gave zones ≤33 mm. Actual zones varied between 11 and 33 mm at Kettering, and 14.3 and 32.6 mm at ARMRL, with good correlation (R2 = 0.94) between the two data sets and no evidence of systematic bias. However, MIC determinations with Etests on Iso-Sensitest agar at both Kettering and ARMRL found only three of the 20 isolates required ertapenem concentrations greater than or equal to 4 mg/L for inhibition, whilst many isolates, including all the E. coli, were highly susceptible, with MICs ≤0.5 mg/L. Of the three resistant isolates, two were Klebsiella spp. and one was Enterobacter spp. with MICs between 4 and 16 mg/L. These results were further supported by agar dilution MICs, determined by BSAC methodology at the reference laboratory.

These data suggest that the present BSAC zone breakpoint of 33–34 mm for ertapenem 10 µg discs is too large, and will lead to exaggerated estimates of resistance prevalence. A zone/MIC data plot (Figure 1) suggested that a 2 mg/L dilution breakpoint corresponded to a zone breakpoint of ~22–24 mm, although the sample was too small for definitive assessment. Another limitation when proposing such a breakpoint would be that only ESBL-producing strains were tested in this study. Such a breakpoint would more closely resemble the 22–23 mm values for imipenem and meropenem, although these are matched against MIC breakpoints of ≤4 mg/L rather than ≤2 mg/L. Nevertheless, it is notable that a few of the isolates were genuinely resistant to ertapenem, with MICs of 4–16 mg/L, whilst none required an imipenem or meropenem MIC above 1 mg/L, as determined by agar dilution. Although combined resistance mechanisms were not investigated in the present isolates, these results are in accord with the view that ertapenem is more vulnerable to combinations of ESBLs (or AmpC) and impermeability than other carbapenems3,5 and that such combined mechanisms largely arise in Klebsiella spp. or Enterobacter spp., not E. coli. They are also compatible with a recent international survey7 which found that the resistance rate to ertapenem among ESBL-producing Enterobacteriaceae was ~6%, compared with 2–2.5% for imipenem and meropenem.



View larger version (10K):
[in this window]
[in a new window]
 
Figure 1. Plot of ARMRL zone diameters for ertapenem 10 µg discs, as determined at ARMRL versus MICs determined by Etest.

 
Transparency declarations

No declarations were made by the authors of this paper.

References

1. Livermore DM, Sefton AM, Scott GM. Properties and potential of ertapenem. J Antimicrob Chemother 2003; 52: 331–44.[Abstract/Free Full Text]

2. Jacoby G, Han P, Tran J. Comparative in vitro activities of carbapenem L-749,345 and other antimicrobials against multiresistant gram-negative clinical pathogens. Antimicrob Agents Chemother 1997; 41: 1830–1.[Abstract]

3. Ward ME, Woodford N, Warner M et al. Carbapenem-resistant, CTX-M-producing Klebsiella pneumoniae in the UK. In: Programs and Abstracts of the Fifteenth European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2005. Abstract P1266. ESCMID, Basle, Switzerland.

4. Koh TH, Babini GS, Woodford N et al. Carbapenem-hydrolysing IMP-1 ß-lactamase in Klebsiella pneumoniae from Singapore. Lancet 1999; 353: 2162.[CrossRef][ISI][Medline]

5. Jacoby GA, Mills DM, Chow N. Role of ß-lactamases and porins in resistance to ertapenem and other ß-lactams in Klebsiella pneumoniae. Antimicrob Agents Chemother 2004; 48: 3203–6.[Abstract/Free Full Text]

6. Health Protection Agency. Laboratory detection and reporting of bacteria with extended-spectrum ß-lactamases QSOP51, National Standard Method Guidance Note. http://www.hpa-standardmethods.org.uk/documents/qsop/pdf/qsop51.pdf (11 July 2005, date last accessed).

7. Paterson DL, Rossi F, Baquero F et al. In vitro susceptibilities of aerobic and facultative Gram-negative bacilli isolated from patients with intra-abdominal infections worldwide: the 2003 Study for Monitoring Antimicrobial Resistance Trends (SMART). J Antimicrob Chemother 2005; 55: 965–73.[Abstract/Free Full Text]





This Article
Extract
Full Text (PDF)
All Versions of this Article:
56/5/984    most recent
dki352v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Samuel, J. R.
Articles by Jenkinson, P.
PubMed
PubMed Citation
Articles by Samuel, J. R.
Articles by Jenkinson, P.