Opening the Black Box of Phenotypic Carbapenemase Detection

May 6, 2019

The carbapenems are beta-lactam antimicrobials and one of the last lines of defense against multidrug resistant Enterobacteriaceae. The Clinical and Laboratory Standards Institute (CLSI) M100 document recommends use of molecular, carbapenem hydrolysis (Carba-NP), or phenotypic (mCIM/eCIM) assays to detect enzymes that degrade these drugs (carbapenemases) for epidemiologic purposes. In this article, I discuss beta-lactamases in general and how structural biology of these enzymes is important to our phenotypic carbapenemase detection assays.

What Are Beta-lactam Antimicrobials and How do They Work?

Beta-lactams are a class of antimicrobial compounds whose target is penicillin-binding protein (PBP), which, from a bacterial perspective, may be more aptly called “cell wall building protein.” PBPs mediate cross-linking of peptidoglycan and are irreversibly inhibited by beta-lactams. The eponymous beta-lactam ring, shown in red in Figure 1a, forms a covalently linked enzyme adduct with PBP. This adduct has a blocked active site, preventing cross-linking of peptidoglycan chains and weakening the cell wall, which leads to cell lysis from osmotic stress.
Figure 1. Mechanism of action of beta-lactams and beta-lactamases. (a) Beta-lactam antimicrobials bind irreversibly to penicillin binding protein (PBP), preventing bacteria from synthesizing the cell wall. (b) Beta-lactamase enzymes interact with beta-lactams but form only a temporary bond, leading to drug inactivation.
 

The Antimicrobial Arms Race

Unfortunately for us, beta-lactam antimicrobial resistance is frequently observed in clinical practice. In the Enterobacteriaceae, this is most commonly mediated by a beta-lactamase enzyme. These enzymes are structurally similar to PBPs but unlike PBPs, they do not form a stable adduct (Figure 1b). By binding to the beta-lactam antimicrobial, the beta-lactamase opens the beta-lactam ring, inactivating the drug, and it is then released from the antimicrobial by hydrolysis. This results in a nonfunctional drug and a beta-lactamase enzyme free to inactivate the next molecule.
 
Additional beta-lactam antibiotics resistant to hydrolysis have been developed to overcome this resistance, including penicillin derivatives, cephalosporins, monobactams, and carbapenems. Bacteria have kept pace with these developments and there are currently hundreds of different beta-lactamases with varying substrate specificity. To keep track of these, we can classify them by sequence similarity using a system developed by Ambler.

Beta-lactamase Classification

The Ambler classification broadly divides beta-lactamase enzymes into groups A through D. For the purpose of this article, the most important distinguishing characteristic between these groups is the active site. A, C, and D enzymes have a serine residue in the active site whereas class B enzymes have zinc ions.
 
Structural distinctions between enzymes that all perform a similar function may seem esoteric and outside the realm of clinical microbiology. However, structure is associated with substrate specificity and inhibitor profile, as codified by the Bush and Jacoby classification method. Nevertheless, the Clinical and Laboratory Standards Institute (CLSI) does not recommend testing or characterization of specific beta-lactamases since MICs are predictive of clinical outcome.
 
The one exception to this rule is testing for carbapenemases. What makes these enzymes special?

Carbapenemases: The Baddest Beta-lactamases 

Carbapenemases are a class of beta-lactamases active against carbapenems, drugs which are largely immune to hydrolysis by other enzymes. Bacteria with carpbaenemases have been reported in every state of the United States, and are (to date) most commonly associated with the so-called carbapenemase-producing Enterobacteriaceae (CPE). Unfortunately, relatively few treatment options remain for infections with these organisms so mortality may be as high as 50%.
 
Carbapenemases are typically encoded by genes on large transmissible plasmids. Given the potential for horizontal gene transfer it is not surprising that CPE outbreaks have been identified and transmission of carbapenemase-encoding plasmids between bacteria has been documented. To prevent outbreaks, CDC recommends that any patients with current or past CPE infection or colonization should be placed on contact precautions. Further, patient rooms and potential fomites should be thoroughly disinfected.

The ABD’s of Carbapenemases

There is not just one type of carbapenemase and each type has a unique resistance profile. The most common serine (Ambler class A) carbapenemase in the United States is currently the Klebsiella pneumoniae carbapenemase (KPC). Other serine carbapenemases such as the oxacillinases (OXA, Ambler class D), are found infrequently in the United States. Importantly, strains expressing KPC or OXA enzymes may be susceptible to newer beta-lactam/beta-lactamase inhibitor combinations such as ceftazidime/avibactam and meropenem/vaborbactam.

 
In contrast, strains expressing metallo-beta-lactamases, notably the New Delhi metallo-beta-lactamase (NDM, Ambler class B) are not inhibited by currently available beta-lactamase inhibitors. Although currently rare in the United States, NDM is increasing in frequency and at has been reported in the majority of states.

Carbapenem Resistant, Sans Carbapenemase

To complicate matters further, not all organisms that are resistant to carbapenems actually express carbapenemases. Some organisms acquire resistance through mutations in a porin protein (limiting drug penetration into the cell), which is often combined with expression of beta-lactamases that have slow carbapenem hydrolysis ability. These mechanisms are chromosomally based and are therefore not as much of an infection control concern as CPE.
 
So in the case of carbapenem resistance, we need to ask, “does the organism produce a carbapenemase?” and, if it does, “what carbapenemase is it?” (more details on this later). These are important questions to inform us which, if any, special infection control practices are required and to monitor which strains may be circulating in our facilities.
 
There are many ways to detect carbapenemases including enzyme activity assays, genotypic methods, and disk diffusion-based assays. For the purpose of this article however, I will focus on currently and previously CLSI recommended growth-based tests commonly performed in hospital-based laboratories.

The Modified Hodge Test Detects Carbapenemase Activity 

The first CLSI recommended growth-based carbapenemase detection test was the modified Hodge test (mHT). The original Hodge test was published in 1978 for detection of penicillinase production in Neisseria gonorrhoeae. It was subsequently modified to use meropenem in place of penicillin and Enterobacteriaceae in place of N. gonorrhoeae. Although currently not recommended by CLSI, >50% of laboratories in the United States and Europe still use this method for carbapenemase detection.
 
The mHT is based on the ability of carbapenemase enzymes to diffuse from cells and protect otherwise susceptible organisms. The mHT is agnostic to carbapenemase type so it can only answer the question “is there a carbapenemase present” but provides no information about its class.
 
The test is performed by spreading a susceptible lawn of Escherichia coli on an agar plate with a meropenem disk. Carbapenemase-producing controls and test strains are streaked across the plate and into the expected zone of inhibition. If present, the carbapenemase will diffuse away from the streaks and hydrolyze the meropenem in the surrounding media. This allows the susceptible E. coli strain to grow further into the zone, evidenced by an indentation (Figure 2, positive control).
 
Figure 2. The modified Hodge test (mHT). A susceptible lawn of E. coli is spread across the plate with a meropenem disk with. Positive controls, negative controls, and test strains are streaked across the expected zone of inhibition. Diffusible carbapenemases protect otherwise susceptible E. coli from killing and are seen as an indentation in the zone of inhibition. A limitation of the mHT is that these indentations are sometimes difficult to interpret (Unknown #2). Figure courtesy K.P. Smith.

The mHT has some limitations, notably insensitivity for detection of metallo-beta-lactamase enzymes. This is likely because they are anchored to the bacterial outer membrane and therefore cannot diffuse. As stated previously, mHT is not able to distinguish between metallo- and serine beta-lactamases. Further, results may be difficult to interpret if the indentation is not pronounced (Figure 2, Unknown #2). Finally, the mHT has poor specificity as it can be positive due to the presence of a potent non-carbapenemase beta-lactamase (for example, an extended spectrum beta-lactamase) in the presence of a porin mutation that reduces drug entry.

The Carbapenemase Inactivation Methods (mCIM/eCIM) are Currently Recommended by CLSI

In 2018, CLSI replaced the mHT with the modified carbapenem inactivation method (mCIM, the unmodified version used a shorter incubation and water instead of broth). Additionally, they introduced the EDTA-mCIM (eCIM). The mCIM may be set up alone, or the eCIM/mCIM assays may be set up simultaneously. Performed together, they can determine whether a carbapenemase is present and, if so, whether it is a metallo-beta-lactamase.
 
For the mCIM test, organisms are incubated with a meropenem disk in tryptic soy broth. For the eCIM, EDTA is added to the broth to chelate metal ions necessary for metallo-beta-lactamase function. After incubation, the disks are fished out and placed on a lawn of susceptible E. coli to determine whether the test organisms destroyed the meropenem. Zone diameters are measured and interpreted, making this test less subjective than the mHT.
 
The first assay to be interpreted is the mCIM, which asks the question “does the organism produce any type of carbapenemase?” (Figure 3a and b). If a carbapenemase is present (at this point it doesn’t matter which one), it will destroy the meropenem in the disk and there will be no significant zone of inhibition. If not, the meropenem remains untouched and a zone of inhibition will be evident.
 
Figure 3. The mCIM and eCIM test. In the mCIM assay, (a) Meropenem disks are incubated in tryptic soy broth with test organisms then (b) placed on a lawn of susceptible E. coli. Test organisms producing any type of carbapenemase destroy the meropenem and show no zone of inhibition. Those with no carbapenemase do not affect the meropenem and a zone of inhibition is evident. The eCIM assay is performed to differentiate carbapenemase producers after a positive mCIM assay. (c) Disks are incubated as with the mCIM but with addition of EDTA (purple color for illustration only). Metallo-beta-lactamases are inhibited in the presence of EDTA and show a zone of inhibition. Serine carbapenemases are unaffected by EDTA and disk retains activity. Figure courtesy K.P. Smith.

In the case of a positive mCIM, the eCIM asks the question “what type of carbapenemase is being produced?” The EDTA in this assay chelates the cations necessary for metallo-beta-lactamase activity. Therefore, if a metallo-beta-lactamase is present, meropenem is not destroyed and a zone of inhibition will be seen. Conversely, serine beta-lactamases are not dependent on cations and are not inhibited. A simplified diagram for mCIM/eCIM interpretation is shown in Figure 4.
 
Figure 4. The mCIM and eCIM flowchart. Simplified flowchart for eCIM and mCIM interpretations. Indeterminate results are omitted for simplicity. Figure K.P. Smith.

Conclusion

Carbapenem resistance in the Enterobacteriaceae is a significant challenge and carbapenemase detection is important for infection control and surveillance. CLSI currently recommends the combined mCIM/eCIM assay for this purpose. Together, these tests can not only identify carbapenemases but also differentiate between the Ambler class A/D (serine) and Ambler class B (metallo) enzymes. Interpretation of these tests is made simpler when we compare our results to the expected mechanisms of each enzyme class.

Do you have a commonly asked question for phenotypic susceptibility testing? Leave a comment below and yours could be the next issue addressed in our Opening the Black Box series!

The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

Author: Kenneth (K.P.) Smith

Kenneth (K.P.) Smith
Dr. Kenneth (K.P.) Smith is a postdoctoral fellow at Beth Israel Deaconess Medical Center. His research interest is in development of drugs and diagnostics for multidrug-resistant gram-negative bacteria.