Carbapenemases (CPs) are enzymes produced by Gram-negative bacteria, such as Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, that chemically neutralize carbapenem antibiotics. Carbapenems, including meropenem and imipenem, are often considered the last line of defense for treating severe, multidrug-resistant infections. CPs dismantle these powerful drugs, creating organisms resistant to nearly all \(\beta\)-lactam antibiotics. The rapid global spread of carbapenemase-producing organisms (CPOs) represents a major public health crisis. CPO infections are associated with prolonged hospital stays, increased healthcare costs, and higher mortality rates due to limited effective treatment options.
Classification of Carbapenemases
Carbapenemases are grouped using the Ambler classification system, which categorizes \(\beta\)-lactamases into four molecular classes: A, B, C, and D. Carbapenem-hydrolyzing enzymes primarily fall into Classes A, B, and D. Structural differences among these classes determine their mechanism of action, which informs laboratory detection and clinical treatment choices.
Classes A and D are serine carbapenemases, distinguished by a conserved serine residue at their active site crucial for the enzymatic process. Class A is represented by the Klebsiella pneumoniae Carbapenemase (KPC) family, which is widely distributed globally. Class D carbapenemases, often called oxacillinases (OXA-types), include the clinically important OXA-48 enzyme family.
Class B carbapenemases are mechanically distinct metallo-\(\beta\)-lactamases (MBLs) because their enzymatic activity requires one or two zinc ions at the active site. Common MBL examples include the New Delhi Metallo-\(\beta\)-Lactamase (NDM), Verona Integron-encoded Metallo-\(\beta\)-Lactamase (VIM), and Imipenemase (IMP) families. MBLs are not inhibited by traditional \(\beta\)-lactamase inhibitors, posing a unique therapeutic challenge.
The Enzymatic Mechanism of Carbapenem Resistance
Carbapenems, like all \(\beta\)-lactam antibiotics, contain a \(\beta\)-lactam ring, the drug’s functional component. Normally, the drug acts by binding to and inactivating bacterial enzymes responsible for building the cell wall. Carbapenemases are hydrolytic enzymes that specifically target and break the amide bond within this \(\beta\)-lactam ring.
Hydrolysis opens the \(\beta\)-lactam ring, rendering the antibiotic inactive and preventing it from binding to its target enzymes. Serine carbapenemases (KPC, OXA-48) function through a two-step process: acylation and deacylation. The active site serine residue attacks the \(\beta\)-lactam ring, forming a temporary covalent acyl-enzyme intermediate, which a water molecule rapidly breaks down to regenerate the enzyme.
Metallo-\(\beta\)-lactamases (Class B) use a different pathway, relying on bound zinc ions to activate a water molecule. This activated water molecule directly attacks the \(\beta\)-lactam ring, causing hydrolysis without forming a stable covalent intermediate. This mechanistic distinction explains why MBLs are unaffected by serine-targeting inhibitors and require metal-chelating agents.
Laboratory Identification and Testing
Timely identification of carbapenemase-producing organisms is crucial for patient treatment and infection control. Clinical laboratories use phenotypic and molecular methods to detect these resistance mechanisms. Phenotypic tests observe the enzyme’s physical activity by detecting carbapenem substrate hydrolysis.
Phenotypic Assays
The Carba NP test is a rapid biochemical assay that detects carbapenem hydrolysis by monitoring a color change. When the carbapenem breaks down, acidic byproducts are released, causing a pH indicator to change color, providing results within two hours. The Carbapenem Inactivation Method (CIM) involves incubating a bacterial isolate with a carbapenem disk before placing the disk on a plate seeded with a susceptible indicator organism.
Molecular Detection
Molecular methods, such as Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS), directly identify the genes responsible for carbapenemase production. PCR assays rapidly detect epidemiologically significant genes, including \(bla_{\text{KPC}}\), \(bla_{\text{NDM}}\), \(bla_{\text{VIM}}\), and \(bla_{\text{OXA-48}}\). Although more expensive than phenotypic tests, molecular methods offer high sensitivity and specificity, providing definitive identification of the resistance gene. Newer Lateral Flow Immunoassays (LFIAs) use antibodies to detect the specific carbapenemase enzyme protein, offering quick results, often within 15 minutes, which aids in prompt isolation and treatment decisions.
Therapeutic Strategies for Inhibition
Therapeutic strategies focus on overcoming carbapenemase activity by combining a \(\beta\)-lactam antibiotic with a novel \(\beta\)-lactamase inhibitor (BLI). This protects the antibiotic from hydrolysis, allowing it to remain intact and exert its antibacterial effect. The choice of combination therapy depends heavily on the specific class of carbapenemase identified.
Ceftazidime-avibactam (CAZ-AVI) pairs the cephalosporin ceftazidime with avibactam, a non-\(\beta\)-lactam inhibitor. Avibactam is effective against Ambler Class A (KPC) and many Class D (OXA-48-like) carbapenemases by forming a stable, reversible bond with the active site serine residue. However, avibactam cannot inhibit zinc-dependent Class B metallo-\(\beta\)-lactamases (MBLs), making CAZ-AVI ineffective against NDM or VIM-producing organisms.
Meropenem-vaborbactam (MER-VAB) combines meropenem with vaborbactam, a boronic acid-based inhibitor. Vaborbactam is a potent inhibitor of Class A carbapenemases, particularly KPC, functioning by mimicking the \(\beta\)-lactam ring structure to competitively bind the enzyme’s active site. Like avibactam, vaborbactam has limited activity against MBLs. The development of these novel BLIs provides effective options against common serine carbapenemase threats.