ESBL and Beta-Lactamase Classification: Ambler vs. Bush-Jacoby, and Why an Enzyme Has Both

ESBL stands for extended-spectrum beta-lactamases, i.e., a group of enzymes produced by Gram-negative and Gram-positive bacteria that can hydrolyze all β- lactam agents except carbapenems and cephamycins. ESBLs belong to group 2be or group 2d (OXA-type) of the Bush-Jacoby-Medeiros classification scheme.

Transmission of ESBL

Being plasmid-mediated, genes encoding ESBL easily exchange among the same group and in different bacterial species. E.g., ESBL genes from E. colitransfer either to Klebsiella, Pseudomonas or even to gram-positive bacteria.

In a clinical or community setting, such ESBL-producing organisms transmit either by direct contact with an infected person’s bodily fluids (blood, drainage from a wound, urine, bowel movements, or phlegm) or contact with equipment or surfaces contaminated with such organisms (as in case of catheters or surgical instruments) or contaminated hands.

Why is ESBL-producing bacteria a problem?

ESBL-producing bacteria may cause a wide range of infections, from uncomplicated urinary tract infection (UTI), diarrhea, and skin infections to life-threatening bloodstream infections (such as septic shock), pneumonia, gastroenteritis, nosocomial infections, etc. Plasmids responsible for ESBL production also carry genes encoding resistance to other classes of antibiotics (for example, aminoglycosides, quinolones, etc). This limits the treatment options of ESBL-producing organisms and poses a threat to successful treatment.

This is why correctly identifying ESBL production at the susceptibility testing stage, through methods like the Kirby-Bauer disc diffusion test and accurate interpretation of S/I/R results, is critical to choosing an antibiotic that will actually work.

Classification of Beta lactamases

The Ambler molecular classification and the Bush-Jacoby-Medeiros functional classification are the two most commonly useful classification systems for β-lactamases.

Two Systems, the Same Enzymes

These aren't competing classifications, they're two different lenses on the same enzymes. Ambler groups beta-lactamases by molecular structure, what the enzyme's amino acid sequence looks like. Bush-Jacoby-Medeiros groups them by function, what substrate they hydrolyze and which inhibitors block them. A single enzyme carries a label in both systems at once.

Quick hook: A, C, and D all use serine for hydrolysis, think of them as the "standard" mechanism. B is the outlier, it needs a zinc ion instead, which is exactly why metal chelators like EDTA inhibit it while clavulanic acid, the inhibitor that works on the serine classes, does nothing against it.

Ambler classification

The most widely used classification of β-lactamases is the Ambler classification which divides β-lactamases into four classes (A, C, D, and B) based on their amino acid sequences. Ambler originally specified two classes (A and B) but later added C and D as new β-lactamases bore no resemblance to the existing class.

Class A, C, and D enzymes utilize serine for β-lactam hydrolysis, and class B metalloenzymes require divalent zinc ions for substrate hydrolysis.

Class A

contained the active-site serine β-lactamases

Class C

a member of serine β-lactamases, known as the ‘AmpC’ β-lactamases.

Class D

Another class of serine β-lactamases, commonly known as the OXA β-lactamases that had no resemblance to either class A or class C.

Class B

The metallo-β-lactamases that require a bivalent metal ion, usually zinc for activity.

Bush-Jacoby-Medeiros classification scheme

The Bush-Jacoby-Medeiros classification scheme groups β-lactamases according to functional similarities (substrate and inhibitor profile). There were four groups described by Jacob and bush but the fourth group has now been omitted as the enzyme properties were similar to that of the previous 3 groups, so currently, there are three main groups and multiple subgroups in this system.

Group I cephalosporinases

Ambler Class C beta-lactamases (also known as AmpC enzymes) fall in this group. Cephalosporin is the substrate. Distinctive characters are

• Beta-lactamase inhibitors like clavulanic acid or EDTA does not inhibit this group.
• Greater hydrolysis of cephalosporin than penicillinase hydrolyzes cephamycins
• Often chromosomal enzymes in gram-negatives, but some are plasmid-coded
• In this class, the enzyme is inducible. Thus any exposure of bacteria to beta-lactam antibiotics leads to an increase in enzyme production.
• The Group I producer beta-lactamases are resistant to beta-lactam/beta-lactamase inhibitor combinations, penicillins, cephamycins, and 1st, 2nd, and 3rd generation cephalosporins but sensitive to cefepime and carbapenems.
• The enzymes in group I are present in the Enterobacteriaceae family as well as Pseudomonas aeruginosa. Examples include E. coli AmpC, P99, ACT-1, CMY-2, FOX-1, MIR-1 enzymes.

Group I contains a subgroup 1e that can hydrolyze ceftazidime and often other oxyimino-β-Lactams. Examples include GC1, CMY-37.

Group II serine β-lactamases

Functional group 2 β-lactamases, including molecular classes A and D (ambler classification), represent the largest group of β-lactamases. There are various subgroups, each with a different property.

Group III MBLs

Group III β lactamases include Metallo-β -lactamases (MBLs) in class B of Amblers classification. They differ structurally from the other β-lactamases by their requirement for a zinc ion at the active site.

Distinguishing characters include

• Ability to hydrolyze carbapenems, but not monobactams.
• Clavulanic acid or tazobactam does not inhibit it. However, metal ion chelators such as EDTA, dipicolinic acid, etc inhibit it.
• Originally were identified as chromosomal enzymes in Gram-positive or occasional Gram-negative bacilli, such as Bacteroides fragilis or Stenotrophomonas maltophilia but now are plasmid-mediated hence can be detected on a wide variety of bacteria.

Only two functional subgroups are described.

1. Subgroup 3a includes the major plasmid-encoded MBL families, such as the IMP and VIM enzymes that have appeared globally, most frequently in non-fermentative bacteria but also in Enterobacteriaceae.
2. Subgroup 3b contains a smaller group of MBLs that preferentially hydrolyze carbapenems in contrast to penicillins and cephalosporins. Examples include L1, CAU-1, GOB-1, FEZ-1 enzymes.

Knowing an isolate produces an MBL or another carbapenemase only matters if the lab can detect it. Two phenotypic methods are commonly used: the older, still widely used Modified Hodge Test, and the faster, more specific Carba NP test.

Learning and Remembering

Clinical story: A urinary tract infection looks routine and gets treated empirically with a third-generation cephalosporin. It doesn't improve, the organism produces an ESBL, which hydrolyzes that entire drug class. The obvious fallback, a fluoroquinolone, often fails too, because the same plasmid that carries the ESBL gene frequently carries fluoroquinolone resistance genes alongside it. What started as a routine UTI ends up needing IV carbapenem therapy and hospital admission, all because one plasmid did the work of defeating two unrelated drug classes at once.

One sentence that captures it: ESBL resistance rarely travels alone, the same plasmid that defeats your first-choice antibiotic usually carries resistance to your backup too, which is exactly why ESBL-producing infections so often escalate from routine to hospitalization-requiring.

Exam facts

References and further readings

1. Hall B G. and Barlow M. Revised Ambler classification of β- lactamases. Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dki130 Advance Access publication 4 May 2005.
2. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother. 2010;54(3):969-976. doi:10.1128/AAC.01009-09
3. Rawat D, Nair D. Extended-spectrum β-lactamases in Gram Negative Bacteria. J Glob Infect Dis. 2010;2(3):263-274. doi:10.4103/0974-777X.68531