Extended-spectrum ß-lactamases in blood culture isolates of Klebsiella pneumoniae: seek and you may find!

Brendan D. Crowley,*

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

Sir,

Extended-spectrum ß-lactamases (ESBLs) are plasmid-mediated enzymes that confer resistance to the monobactam antibiotic aztreonam, and the extended-spectrum cephalosporins, such as cefotaxime, ceftriaxone and ceftazidime. Such enzymes have been described predominantly in Klebsiella pneumoniae and Escherichia coli, although they may be found in other Enterobacteriaceae.1 However, ESBL-mediated resistance poses problems for in vitro susceptibility testing and reporting. MICs of third-generation cephalosporins for ESBL-producing organisms may not reach breakpoint values for resistance, yet treatment failures with these antibiotics can occur.1 ESBL activity can be detected by using a second test, based on susceptibility of ESBLs to inhibition by ß-lactamase inhibitors such as clavulanic acid, for example the ESBL disc detection test.2 However, no national consensus guidelines have yet been developed for laboratory testing of ESBLs in the UK. Lacking these guidelines, a 12 month retrospective study was performed at the Liverpool Public Health Laboratory to assess whether this laboratory should routinely test for ESBLs by determining the prevalence of ESBL production among blood culture isolates of K. pneumoniae.

Altogether, 20 non-replicate K. pneumoniae blood culture isolates were collected in 1999. All strains were identified using the API 20E system (bioMérieux, Marcy l'Étoile, France). MICs of the following antibiotics were determined by agar dilution, in accordance with recommendations of the National Committee for Clinical Laboratory Standards: amoxycillin, cefuroxime, cefotaxime, ceftazidime, cefoxitin, imipenem, co-amoxiclav (2:1), piperacillin/tazobactam (tazobactam at a fixed concentration of 4 mg/L), gentamicin and ciprofloxacin. Isolates with MIC values >=2 mg/L of cefotaxime and/or ceftazidime were considered as under suspicion of ESBL production. This was then confirmed by ESBL detection disc testing, using combination discs (produced by Oxoid Ltd, Basingstoke, UK) of cefotaxime/clavulanate (30 µg/10 µg) and ceftazidime/clavulanate (30 µg/10 µg). A >=5 mm increase in zone diameter for either cefotaxime or ceftazidime in combination with clavulanate, compared with the zone diameter when tested alone, indicated the presence of an ESBL, since this enzyme is inhibited by ß-lactamase inhibitors like clavulanate.2 Detection of genes coding for SHV ESBLs was performed by PCR amplification followed by restriction with NheI.3 Because of glycine (position 238) (SHV non-ESBL) to serine (position 238) (SHV-ESBL) substitution, only PCR-amplified fragments from genes encoding SHV-ESBLs are cleaved by NheI. For DNA analysis, plasmid DNA was extracted with a DNA purification kit (Promega, Madison, WI, USA) and used as a template for amplification of the SHV gene, using SHVspecific primers as used for PCR–NheI restriction.3

Five (25%) of the isolates were presumptive producers of ESBLs. They had reduced susceptibility to cefotaxime and ceftazidime, with MICs of 2–32 mg/L and >128 mg/L, respectively, while non-ESBL-producing isolates had MICs < 0.5 mg/L of these ß-lactams. The ESBL disc detection test confirmed ESBL production in these five isolates. Furthermore, genes coding for SHV-derived ESBLs were also detected by PCR–NheI restriction. Sequence analysis confirmed the presence of three SHV-2 and two SHV-5 ß-lactamase genes in these organisms. All five ESBLproducing organisms were sensitive to gentamicin and only one was resistant to ciprofloxacin.

None of the blood culture isolates had been categorized as an ESBL producer, and susceptibility results for extended-spectrum cephalosporins were not modified, with important clinical implications. Of the five patients from whom ESBL-producing bacteria were isolated, two were treated with third-generation cephalosporins. One patient with ventilator-associated pneumonia was treated with cefotaxime but, because of clinical deterioration, ciprofloxacin was added, to which he responded. The other patient, a diabetic with hospital-acquired pneumonia, responded to treatment after cefotaxime was changed to imipenem. The blood culture isolates in each of these two cases was reported as sensitive to cefotaxime but resistant to ceftazidime.

This investigation found that the proportion of K. pneumoniae blood culture isolates carrying ESBL ß-lactamase genes was high (25%). Furthermore, clinical failure with cephalosporins in two patients could be attributed to ESBL production, an important result that was overlooked during routine susceptibility testing. These results suggest that in this laboratory, it is now clinically necessary to institute additional testing on a routine basis to detect ESBL production, at least in blood culture isolates of K. pneumoniae. Clear guidelines on how clinical laboratories should approach the problem of ESBL-mediated resistance are needed from the British Society for Antimicrobial Chemotherapy and should include recommendations on when to test for ESBLs, what method to use and how the results should be reported.

Acknowledgments

I would like to thank John Corkhill, University of Liverpool, for scientific advice.

Notes

* Tel: +44-151-5294900; Fax: +44-151-5294918; E-mail: bcrowley{at}nw.phls.nhs.uk Back

References

1 . Jacoby, G. A. (1994). Genetics of extended-spectrum betalactamases. European Journal of Clinical Microbiology and Infectious Diseases 13, Suppl. 1, 2–11.[ISI]

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

3 . Nuesch-Inderbinen, M. T., Hachler, H. & Kayser F. H. (1996). Detection of genes coding for extended-spectrum SHV betalactamases in clinical isolates by a molecular genetic method, and comparison with the E-test. European Journal of Clinical Microbiology and Infectious Diseases 15, 398–402.[ISI][Medline]