Extended-spectrum ß-lactamases in Enterobacter cloacae: underestimated but clinically significant!

B. Crowley* and G. Ratcliffe

Liverpool Public Health Laboratory, University Hospital Aintree, Lower Lane, Liverpool L9 7AL, UK

Keywords: ESBLs, Enterobacter cloacae

Sir,

Extended-spectrum ß-lactamase (ESBL)-producing organisms are a problem in hospitalized patients worldwide.1 ESBL-producing organisms characteristically have reduced susceptibility to oxyimino-aminothiazolylcephalosporins, such as cefotaxime, ceftazidime, cefepime and/or the monobactam, aztreonam.2 Detection of ESBL producers may be difficult because zone diameters or MICs of expanded-spectrum cephalosporins may not be interpreted clearly as resistant. The British Society for Antimicrobial Chemotherapy (BSAC) and the National Committee for Clinical Laboratory Standards (NCCLS) have described methods for detection of ESBL production in Klebsiella pneumoniae and Escherichia coli, based on antagonism by clavulanate of the action of ESBLs on cephalosporins, particularly ceftazidime.2,3 However, ESBLs are more difficult to detect in genera that have inducible AmpC chromosomal enzymes, such as Enterobacter spp. AmpC ß-lactamases are induced by clavulanate, and can then hydrolyse the indicator cephalosporin, thereby masking any synergy arising from inhibition of ESBLs by clavulanate.2 Cefepime, a fourth-generation cephalosporin, is a poor substrate for AmpC ß-lactamases.1 It has excellent in vitro activity against Enterobacter spp., including stably derepressed strains resistant to all third-generation cephalosporins. Therefore to differentiate between ESBL- and non-ESBL-producing strains of Enterobacter cloacae, a double disc potentiation test can be used to show synergy between a cefepime disc and an adjacent co-amoxiclav disc.4 The BSAC has claimed that ESBLs are rare outside klebsiellae, and the microbiologist is seldom called upon to confirm their presence.2 We undertook an 18 month retrospective study in our laboratory to determine the prevalence of patients with septicaemia caused by ESBL-producing E. cloacae.

A total of 15 non-replicate E. cloacae blood culture isolates were collected between January 2001 and June 2002 from patients in three different hospitals served by Liverpool Public Health Laboratory. There was no epidemiological relationship, either in time or place, between the patients. All strains were identified using the API 20E system (bioMerieux, Marcy l’Etoile, France). MICs of a range of antibiotics were determined by agar dilution, in accordance with recommendations of the NCCLS. Isolates with MICs >= 2 mg/L of cefotaxime and/or ceftazidime were suspected of ESBL production. This was confirmed by the double disc potentiation test using a cefepime disc (30 µg; Oxoid Ltd, Basingstoke, UK) placed 15 mm (edge to edge) from a co-amoxiclav disc (20 µg/10 µg). ESBL production was also confirmed by Etest (AB Biodisk, Solna, Sweden) with strips containing cefepime plus clavulanate versus cefepime alone (an MIC ratio >= 8 indicated ESBL production). Plasmid DNA was extracted with a DNA purification kit (Promega, Madison, WI, USA) and used as a template for amplification of blaSHV and blaTEM genes.5

ESBL production was suspected in five of 15 blood culture isolates. They had reduced susceptibility to ceftazidime (MICs 64–256 mg/L), but appeared susceptible to cefotaxime (MICs 2–4 mg/L); non-ESBL-producing isolates were susceptible to both ß-lactams. All isolates were resistant to cefoxitin (MIC > 128 mg/L), but no isolate was resistant to all third-generation cephalosporins, indicating the absence of stably derepressed strains of E. cloacae. ESBL production by the five isolates was confirmed by double disc potentiation (Figure 1) and by Etest, and genes encoding SHV-derived ESBLs were detected by PCR. Sequence analysis confirmed the presence of blaSHV-2 in one isolate and blaSHV-5 in the others. All five ESBL-producing isolates were susceptible to ciprofloxacin but resistant to gentamicin.



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Figure 1. Double disc potentiation test. There is a clear enhancement of the zone of inhibition around the cefepime disc (right) towards the clavulanate-containing disc (left).

 
Clinical failure due to ESBL production occurred in one patient who was treated with cefotaxime based on the organism being sensitive in vitro. Subsequent testing confirmed ESBL production. The isolate appeared susceptible to cefotaxime (MIC 2 mg/L) on MIC testing at the standard inoculum of 105 cfu/mL, but when the inoculum was increased to 107 cfu/mL the MIC rose dramatically (64 mg/L).

The proportion of E. cloacae blood culture isolates producing ESBLs was surprisingly high (33%) as production of ESBLs by Enterobacter spp. is considered rare.6 In the same period in our laboratory, ESBL production among E.coli and K. pneumoniae was only 2% and 18%, respectively. In our laboratory, there is a clinical need to test for ESBL production among blood culture isolates of E. cloacae with reduced susceptibility to cefotaxime and/or ceftazidime. Current guidelines do not address the issue of ESBL production among Enterobacteriaceae carrying inducible AmpC ß-lactamases.

Footnotes

* Correspondence address and present address. Central Pathology Laboratory, St James’ Hospital, James’ Street, Dublin 8, Republic of Ireland. E-mail: bcrowley{at}stjames.ie Back

References

1 . Bradford, P. A. (2001). Extended-spectrum ß-lactamases in the 21st century: characterization, epidemiology and detection of this important resistance threat. Clinical Microbiology Reviews 14, 933–51.[Abstract/Free Full Text]

2 . Livermore, D. M. & Brown, D. F. (2001). Detection of ß-lactamase-mediated resistance. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 59–64.[Abstract/Free Full Text]

3 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Susceptibility Testing: Tenth Informational Supplement, Document M100-S10. NCCLS, Wayne, PA, USA.

4 . Sirot, J. (1996). Detection of extended-spectrum plasmid-mediated ß-lactamases by disk diffusion. Clinical Microbiology and Infection 2, Suppl. 1, S35–9.[Medline]

5 . Vercauteren, E., Descheemaeker, P., Ieven, M., Sanders, C. & Goosens, H. (1997). Comparison of screening methods for detection of extended-spectrum ß-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital. Journal of Clinical Microbiology 35, 2191–7.[Abstract]

6 . Rice, L. B. & Bonomo R. A. (2000). ß-Lactamases: which ones are clinically important? Drug Resistance Updates 3, 178–89.[CrossRef][ISI][Medline]





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