Pharmacology Consult

Mechanisms of carbapenem resistance: CP or not CP? Is that the question?

Carbapenem-resistant Enterobacteriaceae, or CRE, pose an important threat to public health and a treatment challenge to clinicians. Further complicating the management of infections caused by these pathogens is uncertainty over how a pathogen developed carbapenem resistance: expression of carbapenemase enzymes (CP-CRE) or other mechanisms (non-CP-CRE).

Leah Molloy, PharmD
Leah Molloy

Understanding the mechanism of carbapenem resistance can help providers determine if broader spectrum agents are necessary. Newer beta-lactam/beta-lactamase inhibitor (BL/BLI) combination antibiotics like ceftazidime/avibactam and meropenem/vaborbactam are active against the most common carbapenemase enzyme — Klebsiella pneumoniae carbapenemase (KPC) — making them important treatment options for infections caused by CP-CRE. However, in non-CP-CRE infections in which other resistance mechanisms like outer cell membrane alterations are present, the added benefit of these agents could be minimal. Understanding resistance mechanisms can also help providers predict potential preserved utility of carbapenems in selected scenarios. A clinician would not hesitate to use meropenem to treat a pathogen susceptible to all carbapenems and would similarly avoid meropenem for a pathogen resistant to all carbapenems. However, discordant carbapenem susceptibility, such as a pathogen susceptible to meropenem but nonsusceptible to ertapenem and therefore classified as CRE, could create confusion. There may be less comfort using meropenem against a CP-CRE than a non-CP-CRE when resistance mechanisms could be unique to ertapenem, leaving meropenem unaffected. Additionally, infections caused by CP-CRE have been associated with worse outcomes than infections caused by non-CP-CRE.

Reduced susceptibility breakpoints vs. carbapenem detection tests

To improve the reliability of susceptibility testing as a means of identifying carbapenem resistance and predict successful treatment with carbapenems reported as active, interpretive breakpoints were reduced in 2010 (see Table) by the Clinical and Laboratory Standards Institute (CLSI). Subsequently, CLSI recommended that carbapenem susceptibility can be reliably interpreted from the current breakpoints, which negate the need for additional tests for carbapenemase production for patient care purposes, although such testing is necessary if a laboratory is still using old interpretive breakpoints. Otherwise, tests to detect carbapenemase production are reserved for research and epidemiology purposes, which may be of interest because the mobility of carbapenemase enzymes poses a bigger risk for transmission of CP-CRE between patients than non-CP-CRE.

For the previous example of determining the value of ceftazidime/avibactam or meropenem/vaborbactam, standard susceptibility testing is sufficient to guide their use. However, waiting until review and discussion of a finalized susceptibility panel indicating carbapenem resistance may further delay-identification of optimal treatment alternatives. To mitigate this, reflexive susceptibility testing of the newer BL/BLI antibiotics upon identification of CRE may be of value at the institutional level.

Table. Past and current Enterobacteriaceae susceptibility MIC breakpoints

Infection control interventions are recommended for patients infected with CRE. Resistance to any carbapenem, regardless of mechanism or discordant susceptibility, meets the current CDC definition of CRE. As such, the number of pathogens designated as CRE has increased to include non-CP-CRE. Pathogens intrinsically resistant to imipenem such as Proteus species, Morganella species and Providencia species are managed differently so that imipenem resistance alone, in the absence of resistance to any other carbapenems, does not meet the CRE definition. Although carbapenemase testing is not required for CRE identification or intervention, it could be used to tailor interventions differently between CP-CRE and non-CP-CRE. Examples provided by the CDC include prioritizing rigorous interventions like surveillance cultures of household contacts for patients infected with CP-CRE isolates while using less intense measures like standard contact precautions for patients infected with non-CP-CRE isolates.

Carbapenemase detection: Phenotypic vs. genotypic testing

Phenotypic tests, like the modified Hodge test (MHT), provide a “yes” or “no” answer to the question, “Does this species produce a carbapenemase?” without distinguishing the specific enzyme, which is often sufficient for clinical decision-making and infection control strategies. In contrast, genotypic tests identify the exact gene, which can be helpful for tracing related enzymes in an outbreak situation. Also, new antibiotics vary in their activity against different carbapenemase enzymes. For example, ceftazidime/avibactam, but not meropenem/vaborbactam, has demonstrated in vitro activity in the presence of certain oxacillinases. Both agents are active against KPC carbapenemases, but neither is active against organisms producing metallo-beta-lactamases (MBL). Only genotypic testing can determine which enzyme is causing carbapenem resistance, although again current susceptibility breakpoints should be sufficient to determine activity of these antibiotics against a given pathogen. Finally, resistance genes must be actively expressed to be detected by phenotypic testing. As such, an isolate harboring but not actively expressing a carbapenemase gene would yield a negative phenotypic test but a positive genotypic test. Genotypic tests come with their own limitations, namely they can identify enzymes only from a predetermined list. A carbapenemase not encoded by a gene being specifically looked for by a genotypic test will not be detected, but its activity will be seen using phenotypic testing. Examples of both types of tests are briefly reviewed later but do not constitute an exhaustive list of all available tests.

Phenotypic tests

The Rapidec Carba NP test is a rapid colorimetric test that, after up to 2 hours of incubation, shows a change from red to yellow to indicate the presence of carbapenemase. In the modified carbapenem inactivation method (mCIM), a meropenem disk is submerged and incubated in a broth containing the patient isolate. The disk is then retrieved and placed on an agar plate inoculated with a laboratory standard meropenem-susceptible strain of Escherichia coli. If growth of E. coli is not inhibited by the meropenem disk, the test is read as positive because the meropenem originally in the disk had been hydrolyzed by the patient isolate. An additional step reserved for Enterobacteriaceae with positive mCIM tests is the ethylenediaminetetraacetic acid (EDTA)-mCIM (eCIM), which differentiates MBLs such as IMP and VIM from serine carbapenemases (eg, KPC and New Delhi metallo-beta-lactamase, or NDM). In the eCIM, the same procedure is followed as in the mCIM test but with the addition of EDTA to the inoculated broth into which the meropenem disk is submerged. Because EDTA prevents meropenem hydrolysis by MBL, but not by serine carbapenemase, a positive eCIM test indicates the presence of serine carbapenemase and a negative result is reported as MBL. Importantly, concomitant production of both MBL and serine carbapenemase will yield a false-negative eCIM test because the two enzymes cannot be differentiated using this method. The MHT was once a frequently employed phenotypic test, but its use is no longer recommended by CLSI because of both false-positive and false-negative results.

Genotypic tests

Available genotypic tests are conducted on blood culture broths (Luminex’s Verigene and the BioFire FilmArray), rectal swabs (Cepheid’s Xpert Carba-R) or pure bacterial colonies (Xpert Carba-R). FilmArray can detect the presence of KPC, and Verigene and Xpert Carba-R can detect IMP, VIM, KPC, OXA-48 and NDM enzymes.

Conclusion

Reduced susceptibility interpretive breakpoints have simplified daily practice by improving the ability to identify carbapenem susceptibility. Phenotypic and genotypic tests are available for carbapenemase detection that may be of value for epidemiologic and infection control purposes. Clinicians should be aware of what can and cannot be ascertained from different testing methods and the utility of that information.

Disclosure: Molloy reports no relevant financial disclosures.

Carbapenem-resistant Enterobacteriaceae, or CRE, pose an important threat to public health and a treatment challenge to clinicians. Further complicating the management of infections caused by these pathogens is uncertainty over how a pathogen developed carbapenem resistance: expression of carbapenemase enzymes (CP-CRE) or other mechanisms (non-CP-CRE).

Leah Molloy, PharmD
Leah Molloy

Understanding the mechanism of carbapenem resistance can help providers determine if broader spectrum agents are necessary. Newer beta-lactam/beta-lactamase inhibitor (BL/BLI) combination antibiotics like ceftazidime/avibactam and meropenem/vaborbactam are active against the most common carbapenemase enzyme — Klebsiella pneumoniae carbapenemase (KPC) — making them important treatment options for infections caused by CP-CRE. However, in non-CP-CRE infections in which other resistance mechanisms like outer cell membrane alterations are present, the added benefit of these agents could be minimal. Understanding resistance mechanisms can also help providers predict potential preserved utility of carbapenems in selected scenarios. A clinician would not hesitate to use meropenem to treat a pathogen susceptible to all carbapenems and would similarly avoid meropenem for a pathogen resistant to all carbapenems. However, discordant carbapenem susceptibility, such as a pathogen susceptible to meropenem but nonsusceptible to ertapenem and therefore classified as CRE, could create confusion. There may be less comfort using meropenem against a CP-CRE than a non-CP-CRE when resistance mechanisms could be unique to ertapenem, leaving meropenem unaffected. Additionally, infections caused by CP-CRE have been associated with worse outcomes than infections caused by non-CP-CRE.

Reduced susceptibility breakpoints vs. carbapenem detection tests

To improve the reliability of susceptibility testing as a means of identifying carbapenem resistance and predict successful treatment with carbapenems reported as active, interpretive breakpoints were reduced in 2010 (see Table) by the Clinical and Laboratory Standards Institute (CLSI). Subsequently, CLSI recommended that carbapenem susceptibility can be reliably interpreted from the current breakpoints, which negate the need for additional tests for carbapenemase production for patient care purposes, although such testing is necessary if a laboratory is still using old interpretive breakpoints. Otherwise, tests to detect carbapenemase production are reserved for research and epidemiology purposes, which may be of interest because the mobility of carbapenemase enzymes poses a bigger risk for transmission of CP-CRE between patients than non-CP-CRE.

For the previous example of determining the value of ceftazidime/avibactam or meropenem/vaborbactam, standard susceptibility testing is sufficient to guide their use. However, waiting until review and discussion of a finalized susceptibility panel indicating carbapenem resistance may further delay-identification of optimal treatment alternatives. To mitigate this, reflexive susceptibility testing of the newer BL/BLI antibiotics upon identification of CRE may be of value at the institutional level.

Table. Past and current Enterobacteriaceae susceptibility MIC breakpoints

Infection control interventions are recommended for patients infected with CRE. Resistance to any carbapenem, regardless of mechanism or discordant susceptibility, meets the current CDC definition of CRE. As such, the number of pathogens designated as CRE has increased to include non-CP-CRE. Pathogens intrinsically resistant to imipenem such as Proteus species, Morganella species and Providencia species are managed differently so that imipenem resistance alone, in the absence of resistance to any other carbapenems, does not meet the CRE definition. Although carbapenemase testing is not required for CRE identification or intervention, it could be used to tailor interventions differently between CP-CRE and non-CP-CRE. Examples provided by the CDC include prioritizing rigorous interventions like surveillance cultures of household contacts for patients infected with CP-CRE isolates while using less intense measures like standard contact precautions for patients infected with non-CP-CRE isolates.

Carbapenemase detection: Phenotypic vs. genotypic testing

Phenotypic tests, like the modified Hodge test (MHT), provide a “yes” or “no” answer to the question, “Does this species produce a carbapenemase?” without distinguishing the specific enzyme, which is often sufficient for clinical decision-making and infection control strategies. In contrast, genotypic tests identify the exact gene, which can be helpful for tracing related enzymes in an outbreak situation. Also, new antibiotics vary in their activity against different carbapenemase enzymes. For example, ceftazidime/avibactam, but not meropenem/vaborbactam, has demonstrated in vitro activity in the presence of certain oxacillinases. Both agents are active against KPC carbapenemases, but neither is active against organisms producing metallo-beta-lactamases (MBL). Only genotypic testing can determine which enzyme is causing carbapenem resistance, although again current susceptibility breakpoints should be sufficient to determine activity of these antibiotics against a given pathogen. Finally, resistance genes must be actively expressed to be detected by phenotypic testing. As such, an isolate harboring but not actively expressing a carbapenemase gene would yield a negative phenotypic test but a positive genotypic test. Genotypic tests come with their own limitations, namely they can identify enzymes only from a predetermined list. A carbapenemase not encoded by a gene being specifically looked for by a genotypic test will not be detected, but its activity will be seen using phenotypic testing. Examples of both types of tests are briefly reviewed later but do not constitute an exhaustive list of all available tests.

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Phenotypic tests

The Rapidec Carba NP test is a rapid colorimetric test that, after up to 2 hours of incubation, shows a change from red to yellow to indicate the presence of carbapenemase. In the modified carbapenem inactivation method (mCIM), a meropenem disk is submerged and incubated in a broth containing the patient isolate. The disk is then retrieved and placed on an agar plate inoculated with a laboratory standard meropenem-susceptible strain of Escherichia coli. If growth of E. coli is not inhibited by the meropenem disk, the test is read as positive because the meropenem originally in the disk had been hydrolyzed by the patient isolate. An additional step reserved for Enterobacteriaceae with positive mCIM tests is the ethylenediaminetetraacetic acid (EDTA)-mCIM (eCIM), which differentiates MBLs such as IMP and VIM from serine carbapenemases (eg, KPC and New Delhi metallo-beta-lactamase, or NDM). In the eCIM, the same procedure is followed as in the mCIM test but with the addition of EDTA to the inoculated broth into which the meropenem disk is submerged. Because EDTA prevents meropenem hydrolysis by MBL, but not by serine carbapenemase, a positive eCIM test indicates the presence of serine carbapenemase and a negative result is reported as MBL. Importantly, concomitant production of both MBL and serine carbapenemase will yield a false-negative eCIM test because the two enzymes cannot be differentiated using this method. The MHT was once a frequently employed phenotypic test, but its use is no longer recommended by CLSI because of both false-positive and false-negative results.

Genotypic tests

Available genotypic tests are conducted on blood culture broths (Luminex’s Verigene and the BioFire FilmArray), rectal swabs (Cepheid’s Xpert Carba-R) or pure bacterial colonies (Xpert Carba-R). FilmArray can detect the presence of KPC, and Verigene and Xpert Carba-R can detect IMP, VIM, KPC, OXA-48 and NDM enzymes.

Conclusion

Reduced susceptibility interpretive breakpoints have simplified daily practice by improving the ability to identify carbapenem susceptibility. Phenotypic and genotypic tests are available for carbapenemase detection that may be of value for epidemiologic and infection control purposes. Clinicians should be aware of what can and cannot be ascertained from different testing methods and the utility of that information.

Disclosure: Molloy reports no relevant financial disclosures.