Deciphering Metallo-β-lactamases: Crucial Treatment Strategy

In the era of antibiotic resistance, clear understanding of β-lactamase-mediated resistance is more crucial than ever. Metallo-β-lactamases (MBLs), an unchallenged formidable subclass of β-lactamases, continue rising and becoming a key player, denying the use of some of the most potent antimicrobials available.

For pharmacists, microbiologists, and physicians, a thorough understanding of MBLs is not just academic but a necessary understanding for clinical practice. It should inform treatment decisions, impacts antibiotic stewardship policies, and shapes our approach to microbiological diagnostic. This deep dive into the molecular world of MBLs, particularly the blaNDM and blaVIM types, is intended to illuminate the complex interaction between these enzymes and various β-lactam antibiotics, such as aztreonam, cefiderocol, and other β-lactam agents. Understanding their molecular details and dynamic with β-lactams, should improve and even change our therapeutic and epidemiological perspective.

As a medical microbiologist, my clinical consulting role with providers is currently defined by the antimicrobial resistance challenge. My interaction and intervention have become more valuable when assisting in regards of antimicrobial therapy for resistant organisms such as those carrying MBLs. When there is no other therapeutic option, a full review of resistance mechanisms and rational implementation of synergistic agents becomes the next best option. It is here where having access to a clinical microbiologist can make a difference.

Molecular Structure and Function

Metallo-β-lactamases belong to the Ambler Class B of enzymes that confer resistance to a broad range of β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. But the two most technical and relevant characteristics of this group are its lack of activity against aztreonam and the lack of therapeutically available β-lactamase inhibitors. These enzymes are not inhibited by clavulanic acid, sulbactam, tazobactam, avibactam, vaborbactam, relebactam, or durlobactam.

Characterized by their requirement for zinc ions for catalytic activity, these enzymes have a unique ability to hydrolyze the β-lactam ring, which is crucial for the antimicrobial's bactericidal action. The molecular structure of MBLs is distinct than serine-based β-lactamases, featuring a characteristic αβ/βα sandwich fold, which houses the active site accommodating the zinc ions. The binding of zinc ions to the β-lactam is essential for the hydrolytic activity of these enzymes.

The structural configuration of zinc ions within MBLs is a key determinant of their activity. X-ray crystallography studies have revealed that in binuclear enzymes (two zinc ions) such as NDM and VIM, one zinc ion is often coordinated in a tetrahedral geometry, while the other adopts a more flexible, pentacoordinate arrangement. This configuration is critical for the hydrolysis of the β-lactam ring. The zinc ions create a unique electrostatic environment that activates water molecules, turning them into effective hydrolytic points capable of attacking the β-lactam ring.

While both MBLs and serine-β-lactamases share the same activity of β-lactam hydrolysis, their mechanisms of action differ significantly. Serine-β-lactamases utilize an active-site serine residue to initiate hydrolysis. This serine residue directly attacks the β-lactam ring, whereas MBLs rely on an additional zinc residue to stabilize the β-lactam in the active site. In MBLs one zinc hydrolyzes the β-lactam ring, while another one stabilizes the interaction by attaching it to the secondary ring. This difference in catalytic strategy not only defines their substrate affinity but also their molecular stability to inhibitors.

Due to the lack of a secondary ring or carboxyl group in current β-lactamase inhibitors, the secondary zinc ion is not able to stabilize the attachment, making MLBs immune to current inhibitors.

Enzymatic dynamics

NDM and VIM MBLs are pivotal in antimicrobial resistance, especially in the context of cephalosporins and carbapenems, two critical classes in the β-lactam arsenal. Despite similarities in their basic mechanism of action – hydrolyzing the β-lactam ring – these two MBLs exhibit distinct kinetic behaviors and substrate affinities influenced by their structural differences and zinc ion interactions.

New Delhi Metallo-β-lactamase (NDM) and Verona Integron-encoded Metallo-β-lactamase (VIM) are among the most clinically significant MBLs. NDM, first identified in India, has a broad substrate profile, and can hydrolyze almost all β-lactams, including carbapenems. VIM, initially detected in Italy, also shows a wide range of activity against β-lactams, but highly dependable of the strength of the zinc attachment for its efficiency.

Enzymatic kinetics, which include parameters like turnover number (k_cat) that indicate the rate of hydrolysis and substrate affinity (K_m), vary significantly between NDM and VIM. NDM typically exhibits higher turnover numbers for a broader range of β-lactam antibiotics compared to VIM. This implies that NDM can hydrolyze certain β-lactam substrates more rapidly than VIM, making it a more potent contributor to antibiotic resistance. However, VIM enzymes can compensate for their lower turnover numbers with higher substrate affinity in certain cases, depending on the specific antibiotic in question. In basic terms, NDM is more efficient, where VIM can interact with a broader range of β-lactams.

In my own experience, NDM has been typically seen in Central Florida carried only by Enterobacterales, showing a typical pan-β-lactam resistant pattern, if we exclude aztreonam and consider the β-lactams in our formulary. In the case of VIM, exclusively seen on P. aeruginosa, certain β-lactams such as ceftazidime and cefepime, can show a susceptible rate up to 40%. These differences can suggest the lower affinity of VIM for certain β-lactams when compared to NDM, and it is caused by differences in the zinc ion binding strength.

Although MBLs exhibit varying degrees of efficiency in hydrolyzing different β-lactam antimicrobials, the molecular structure of the different classes directly influences their activity. For instance, cephalosporins with bulkier side chains are often less susceptible to hydrolysis by certain MBLs, whereas carbapenems are generally more vulnerable due to their specific ring structures. In clinical practice, this level of details or differences may be less significant, since NDM and VIM, as seen in clinical isolates, commonly affect all commonly tested agents.

Aztreonam

Aztreonam, a monobactam antimicrobial, stands out in its stability and resistance to hydrolysis by most MBLs, including NDM and VIM. This stability is attributed to its unique molecular structure. Monobactams (aztreonam) lack the usual secondary ring adjacent to the β-lactam ring found in penicillins, cephalosporins, and carbapenems. This singular structure not only renders it less recognizable to MBLs but also less susceptible to their catalytic mechanism that relies on a secondary zinc ion for its stabilization.

The lack of a secondary core ring in monobactams, that is needed for anchoring the β-lactams to the MBLs, is the molecular principle for the intrinsic stability and hydrolysis resistance by aztreonam. This characteristic that is important in the clinical practice, especially when treating patients with MBLs infections. An aztreonam resistant NDM-carrying Enterobacterales, is typically due to the presence of ESBLs and/or AmpCs, and the therapeutic strategy is not difficult to design when these characteristics are understood.

In NDM-carrying Enterobacterales, we haven’t seen the first resistant isolate against aztreonam, and although in VIM-carrying P. aeruginosa aztreonam resistance is common, the mechanism is based on porin restriction and expression of efflux pumps, two significant mechanisms to consider.

Cefiderocol

One of the most recent introduced β-lactams that is intrinsically stable and resistant to MBLs, especially NDM and VIM, is cefiderocol. A siderophore cephalosporin with a unique mechanism of cell entry. As a siderophore, when cefiderocol forms a complex with ferric ion, it is recognized by iron transport receptor/channels, providing cefiderocol with a backdoor into the periplasmic space. Cefiderocol molecule is a merge of ceftazidime/ceftolozane R1 structure in C7 and cefepime R3 in C3, creating an interesting hybrid. But it is the catechol functional group attached to R3 that provides more than its siderophore capability.

The entire molecular structure of cefiderocol plus its catechol group, makes this agent large, complex and three dimensionally bulky. Its structure makes difficult for most β-lactamases, including MBLs, to accommodate the molecule in the active site and stabilize it for hydrolysis. Although some β-lactams can hydrolase cefiderocol, including some NDM genotypes, this resistance rate is currently low.

In my own experience, cefiderocol has a 100% susceptible rate in VIM-carrying P. aeruginosa, what makes this agent an excellent option for this organism/resistance combination. On NDM-carrying Enterobacterales, although our experience is limited, the few cefiderocol tested have been susceptible.

Cephalosporins and Carbapenems

Cephalosporins, characterized by their β-lactam ring and a dihydrothiazine ring, are key targets of NDM and VIM. The kinetic efficiency of these enzymes against cephalosporins depends significantly on the side-chain substitutions of the antimicrobial. NDM and VIM show varying levels of affinity and catalytic rates for different cephalosporins. For instance, cephalosporins with bulkier side chains may hinder the access of the antibiotic to the active site of the enzyme, thereby reducing the hydrolysis rate. Two examples are ceftazidime and cefepime when interacting with VIM enzymes.

Carbapenems efficacy is severely compromised by NDM and VIM. These enzymes hydrolyze carbapenems efficiently, often leading to high MICs. The kinetic interaction between NDM/VIM and carbapenems is a significant concern, as it limits the therapeutic options available for treating infections caused by carbapenem-resistant organisms.

As therapeutic strategy and based on the affinity and kinetic of MBLs, the use of high doses of carbapenems is not a better option than cephalosporins. Although there is not justification for a patient suffering an infection by a MBLs-carrying organism to be treated with high doses of carbapenems or cephalosporins when appropriate agents are available, the old approaches for “carbapenem = better” may not have any rational in this case.

Based on our data, no carbapenem has tested susceptible in presence of a NDM or VIM carrying organism. Although not a unique pattern to MBLs, carbapenems in Enterobacterales are the best indicator for presence of carbapenemases, due to its sensitivity and specificity. In the case of P. aeruginosa, in addition to carbapenems, ceftolozane/tazobactam has better specificity for differentiating the presence of a carbapenemase from porin restriction.

Rapid Laboratory Detection

Although detection of carbapenemases by PCR or EIA should be the gold standard for laboratory testing, the presence of MBLs can be suspected based on the resistance pattern in Enterobacterales.

Every carbapenem resistant Enterobacterales (CRE) isolate should be routinely tested for the detection of a carbapenemase. PCR or EIA testing should not be held or delayed upon provider request, neither physician, pharmacist nor infection preventionist, should make the call for testing. A laboratory designed, validated and criteria-driven protocol, should be standardized and implemented routinely for all suspected organisms.

Including agents such as ceftazidime/avibactam, meropenem/vaborbactam or imipenem/relebactam, during the first line of antimicrobial testing against Enterobacterales, can provide a rapid preliminary assessment. A CRE susceptible or not to aztreonam that test also resistant to any of the BL/BLI mentioned above, offer a high level of suspicion for the presence of a MBL.

Therapeutic Relevance

The stability of aztreonam and cefiderocol to hydrolysis by MBLs has significant clinical implications. It remains the only two β-lactam antibiotics effective against MBLs-carrying organisms, highlighting their importance in the treatment of MBL-mediated infections. Their role is especially crucial given the increasing prevalence of MBL-producing bacteria in clinical settings.

As a microbiological and therapeutic rule:

? In MBLs-carrying Enterobacterales, especially NDM, aztreonam plus ceftazidime/avibactam is an ideal β-lactam treatment choice.

? NEVER prescribe aztreonam against a MBLs-carrying Enterobacterales without ceftazidime/avibactam, even when aztreonam tests susceptible in-vitro. Presence of secondary or regulated β-lactamases can be missed.

? Cefiderocol, with its siderophore structure, is an ideal agent against VIM-carrying P. aeruginosa, overcoming non-β-lactamase mediate resistance.

MBLs are becoming more and more prevalent. I believe that due to the necessary use of current anti-KPC agents such as ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam, we are selecting for MBLs to arise within Enterobacterales. The lack of defined protocols and testing for carbapenemase producing CRE is keeping the extent and magnitude of the problem under the radar. I will not be surprise if MBLs prevalence reaches and rivals the KPC prevalence in the coming years.

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