AmpC β-lactamases can interfere with extended-spectrum-β-lactamase (ESBL) confirmatory tests. Resulting failures to detect ESBLs can endanger patients because false susceptibility to cephalosporins may be reported (5). This problem occurs with CLSI and some other ESBL confirmatory tests, but there are tests that can be used to provide more accurate detection of ESBLs in AmpC-producing isolates (4, 5). While inaccurate susceptibility reports are never acceptable, it is of interest to know whether it is common for a lab to encounter ESBL-screen-positive, AmpC-producing isolates that yield negative CLSI confirmatory tests. That is, can it be common to encounter negative ESBL confirmatory test results of unknown accuracy? Apart from a small Escherichia coli study in which 20 of 26 cefpodoxime ESBL-screen-positive, cefoxitin-resistant E. coli isolates produced a plasmid-mediated AmpC β-lactamase (4), little is known about how often AmpC production creates uncertainty about the accuracy of CLSI ESBL confirmatory tests. Therefore, a study was conducted at a Louisville, KY, teaching hospital to determine how many ESBL-screen-positive isolates of E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis were AmpC positive and ESBL confirmatory test negative by CLSI methodology.
During the period of 1 May 2006 to 28 February 2007, 952 isolates from patients at the University of Louisville Hospital, Louisville, KY, were analyzed. They comprised 682 E. coli, 152 K. pneumoniae, 44 K. oxytoca, and 70 P. mirabilis isolates. ESBL screening was performed by using CLSI microdilution methodology using cefotaxime, ceftriaxone, ceftazidime, cefpodoxime, and aztreonam with positive isolates then tested by the CLSI ESBL disk confirmatory method (2). Screen-positive isolates with negative ESBL confirmatory tests were investigated for AmpC production using a Tris-EDTA-based disk test (1) to determine if they were AmpC producers. If this test was positive, it meant that the negative ESBL confirmatory result was of unknown accuracy. Representative AmpC-positive isolates of K. pneumoniae were investigated for gene identification by PCR using primers specific for blaDHA, blaFOX, blaCMY, blaENT, blaACT, and blaACC (3).
Only 13 of the 57 screen-positive isolates (22.8%) were ESBL producers by CLSI methodology, while 40 were AmpC producers (70.2%) (Table 1). Other resistance mechanisms were assumed to account for the other four positive screens. The screening-positive isolates comprised 37 E. coli, 16 K. pneumoniae, 3 Klebsiella oxytoca, and 1 P. mirabilis. Of these, 8 E. coli isolates (22% of screen-positive E. coli), 4 K. pneumoniae isolates (25% of screen-positive K. pneumoniae), and 1 P. mirabilis isolate were confirmed as ESBL producers. Of the isolates that gave an ESBL-negative result, 28 E. coli (76% of screen-positive isolates) and 12 K. pneumoniae (75% of screen-positive isolates) were AmpC positive. No K. oxytoca or P. mirabilis isolates were AmpC positive. The AmpC-positive K. pneumoniae isolates were assumed to produce a plasmid-mediated or imported AmpC β-lactamase because this organism's genome lacks an ampC gene. PCR testing of six representative K. pneumoniae isolates identified a FOX-like β-lactamase gene in each isolate. The AmpC-positive E. coli isolates were assumed to produce either a chromosomally mediated or plasmid-mediated AmpC β-lactamase.
TABLE 1.
ESBL-screen-positive isolates that produced extended-spectrum or AmpC β-lactamases
| Species | No. (%) of isolates with indicated test result |
||
|---|---|---|---|
| ESBL screening positive | ESBL positivea | AmpC positive | |
| E. coli | 37 | 8 (22) | 28 (76) |
| K. pneumoniae | 16 | 4 (25) | 12 (75) |
| K. oxytoca | 3 | 0 | 0 |
| P. mirabilis | 1 | 1 | 0 |
AmpC status was not investigated if ESBL-positive isolates were confirmed because such isolates were not those in which AmpC production interfered with ESBL detection.
In conclusion, the aim of the study was not to reconfirm that AmpC β-lactamases may interfere with ESBL detection or to define methodology to overcome this problem (5), but rather to determine if it was common for AmpC production to necessitate the use of a more reliable ESBL confirmatory test. In this study, E. coli and K. pneumoniae isolates yielded positive ESBL screening results three times more often for AmpC-producing isolates that yielded negative CLSI ESBL confirmatory test results than for isolates with positive CLSI ESBL confirmatory test results. That is, ESBL confirmatory test results of unknown accuracy occurred often enough to warrant the need for an alternative ESBL confirmatory test of greater accuracy for AmpC-producing isolates. The results also indicated that the CLSI ESBL screening recommendations are useful to screen for AmpC β-lactamases.
Acknowledgments
We acknowledge financial support for this work provided by Merck.
Footnotes
Published ahead of print on 2 December 2009.
REFERENCES
- 1.Black, J. A., E. S. Moland, and K. S. Thomson. 2005. AmpC disk test for detection of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking chromosomal AmpC β-lactamases. J. Clin. Microbiol. 43:3110-3113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement. CLSI M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
- 3.Pérez-Pérez, F. J., and N. D. Hanson. 2002. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153-2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Robberts, F. J., P. C. Kohner, and R. Patel. 2009. Unreliable extended-spectrum β-lactamase detection in the presence of plasmid-mediated AmpC in Escherichia coli clinical isolates. J. Clin. Microbiol. 47:358-361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Thomson, K. S. 2001. Controversies about extended-spectrum and AmpC beta-lactamases. Emerg. Infect. Dis. 7:333-336. [DOI] [PMC free article] [PubMed] [Google Scholar]