Carbapenem-resistant Klebsiella pneumoniae
Carbapenem-resistant Klebsiella pneumoniae
Abstract & Commentary
By Ellen Jo Baron, PhD, D(ABBM), Professor of Pathology and Medicine, Stanford University Medical School Director; Clinical Microbiology Laboratory, Stanford University Medical Center, is Associate Editor for Infectious Disease Alert.
Dr. Baron reports no financial relationships relevant to this field of study.
Most of us microbiologists were rudely awakened to the insufficiency of our susceptibility testing methods last year when the College of American Pathologists (CAP) sent out a carbapenemase-producing Klebsiella pneumoniae as an unknown proficiency testing sample. My laboratory, like most others in the United States, incorrectly reported the isolate as susceptible to imipenem and meropenem. Hooray for CAP for helping us to recognize our failings. We now retain that strain as our internal quality control (QC) organism to run along with the patient's isolate when we perform our newly implemented susceptibility test for KPC presence (Klebsiella pneumoniae carbapenemase). But the invitro phenotypic test for this group of enzymes is not uniformly recognized among microbiologists.
This lack of knowledge was recently addressed by a new "case study" on a widely used website devoted to antibacterial susceptibility testing.1 Although clinicians may not be aware of it, this very useful website, dealing with susceptibility testing methods for antibacterial resistance, has been available since 2001. Initially developed by Janet Hindler (UCLA) and Fred Tenover (CDC), the site continues to provide timely case studies, protocols, and educational resources for microbiologists worldwide. The most recent addition to the site is of special interest to infectious diseases practitioners, as it outlines a laboratory method for detecting Klebsiella pneumoniae carbapenemases (KPCs), a rather recently expanding class of resistance factors for which there are currently no detection methods given in the reference standard that most laboratories depend on for all susceptibility testing decisions: M100-S18, the Clinical and Laboratory Standards Institute (CLSI) annually updated document, due out again in January 2009.
Laboratories in the United States that are accredited by agencies such as the College of American Pathologists, or occasionally The Joint Commission, are assured of acceptable performance ratings on their susceptibility tests if they follow the CLSI standards to the letter. Never mind that European standards set by the European Committee for Antimicrobial Susceptibility Testing (EUCAST)2 are sometimes different, and possibly more predictive clinically, from those agreed upon by CLSI (with input from FDA), we are still effectively bound by CLSI rules. Given that CLSI standards do not include a method, many US laboratories are not testing isolates for the KPC group of enzymes, and may be erroneously reporting some organisms as susceptible to carbapenems. Case reports of treatment failures are beginning to be published.3,4
Carbapenemases were first characterized in the mid-1990s,5 but they have only been recognized in clinical infections since 2001. Recently, several outbreaks in specific locales have been reported.6 Some classes of carbapenemases (Class B) contain zinc at the active site and, are thus, termed "metallo-βeta-lactamases." In addition to Enterobacteriaceae, the carbapenemases are found in other organisms, including E. coli Citrobacter, Acinetobacter, Salmonella and, of particular worry, Pseudomonas aeruginosa.7 Unfortunately, standard laboratory testing methods such as MicroScan (Dade Behring), Vitek 2 (bioMerieux), and even disk diffusion (Bauer-Kirby) do not reliably detect this resistance mechanism.8
What's a laboratory to do? Experts such as Dr. Kenneth Thomson at Creighton and the authors of the recent MASTER site case study recommend the modified Hodge test, also called the cloverleaf test, because the pattern of the zone of inhibition exhibited on a petri dish showing a positive result resembles a 3- or 4-leaf clover.9 The test is performed by inoculating a fully susceptible quality control strain of E. coli on the standard susceptibility plate (Mueller-Hinton agar) and placing a carbapenem disk on the plate; our laboratory uses meropenem. Then freshly subcultured QC organisms, positive and negative for KPC production, are inoculated by drawing a thin line of inoculum from right next to the disk outward about 1 inch or more, usually at 90° angles to each other or on opposite sides of the disk. The test organism (unknown patient isolate) is also inoculated in a thin line outward from the disk, equidistant from the other lines. As many as four lines (two controls and two test organisms) can be drawn from one disk, hence the four-leaf clover appearance. A hint from Frans J. Robberts, a Mayo Clinic post-doctoral Fellow, is to use the sharp edge of a coverslip (not necessary that it be sterile) to draw the thinnest possible line, allowing better observation of the inward curve of the growth of E. coli that signifies a positive test. If the test organism produces a carbapenemase, the enzyme will diffuse into the medium around the line of growth and break down the antibiotic diffusing outward in a clean circle from the disk, allowing the E. coli to grow up along the line of the test organism, making a rounded corner to the zone of inhibition on either side of the line. The edge of the inhibition zone continues in a smooth circle past the line of an organism that does not produce a carbapenemase (see Figure 1).
Commentary
Although reduced susceptibility (MICs near the breakpoint, for example) of most any gram-negative rod to any carbapenem (imipenem, meropenem, ertapenem, doripenem), or resistance to a third-generation cephalosporin, are potential flags that could be used to trigger performance of the modified Hodge test, it is possible that a few strains harboring these enzymes may not exhibit either characteristic. It is the best clue we have right now, however. Clearly,
laboratories will be delaying, for one day, a number of results on gram-negative rods if they implement this protocol. Implicated isolates also may produce extended-spectrum βeta-lactamases, which are detected with a separate test method.
The plethora of new resistance mechanisms is leading to the necessity for expanded susceptibility testing methods and, in some cases, slower turnaround times. Clinicians will need to exhibit patience with their microbiologists, who are scrambling to remain up-to-date and one step ahead of those crafty microbes that adapt a lot faster than we do.
References
- http://wwwn.cdc.gov/dls/master/default.aspx
- http://www.escmid.org/research_projects/eucast/
- Elliott E, et al. In vivo development of ertapenem resistance in a patient with pneumonia caused by Klebsiella pneumoniae with an extended-spectrum βeta-lactamase. Clin Infect Dis. 2006;42:e95-e98.
- Walsh TR, et al. Metallo-βeta-lactamases: the quiet before the storm? Clin Microbiol Rev. 2005;18:306-325.
- Rasmussen BA, et al. Characterization of IMI-1 βeta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother. 1996;40:2080-2086.
- Bratu S, et al. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob Agents Chemother. 2005;49:3018-3020.
- Zavascki AP, et al. Risk factors for nosocomial infections due to Pseudomonas aeruginosa producing metallo-βeta-lactamase in two tertiary-care teaching hospitals. J Antimicrob Chemother. 2006;58:882-885.
- Tenover FC, et al. Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg Infect Dis. 2006;12:1209-1213.
- Anderson KF, et al. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J Clin Microbiol. 2007;45:2723-2725.
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