ESBL and Classification of β-lactamases

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. coli transfer 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.

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.

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.

Sub group	Substrate	Defining character	Examples
2a	Penicillin	1. Predominant penicillinase in Staphylococci and enterococci 2. Preferentially hydrolyze benzylpenicillin and many penicillin derivatives, with poor hydrolysis of cephalosporins, carbapenems, or monobactams except nitrocefin hydrolysis 3. Are inhibited by clavulanic acid and tazobactam 4. Majority are chromosomal, although some staphylococcal penicillinases are plasmid-encoded	PC1
2b	Penicillins and early cephalosporins	1. Readily hydrolyze penicillins and early cephalosporins, such as cephaloridine and cephalothin 2. Strongly inhibited by clavulanic acid and tazobactam 3. Most common plasmid-mediated β lactamases	TEM-1, TEM-2, SHV-1
2be*	Extended-spectrum cephalosporins, monobactams	1. Increased hydrolysis of oxyimino-β-lactams (cefotaxime, ceftazidime, ceftriaxone, cefepime, aztreonam) 2. Are sensitive to inhibition by clavulanic acid, a feature used in their detection by clinical laboratories	TEM-3, SHV-2, CTX-M-15
2br	Penicillin	Have acquired resistance to clavulanic acid, sulbactam and tazobactam	TEM-30,SHV-10
2ber	Extended-spectrum cephalosporins, monobactams	1. Increased hydrolysis of oxyimino-β-lactams combined with resistance to clavulanic acid, sulbactam and tazobactam 2. Also known as CMT (complex mutant TEM) β-lactamases	TEM-50 (CMT-1)
2c	Carbenicillin	Ability to hydrolyze carbenicillin or ticarcillin Easily inhibited by clavulanic acid or tazobactam	PSE-1, CARB-3
2ce	Extended-spectrum cephalosporins	Increased hydrolysis of carbenicillin, cefepime, and cefpirome Inhibited by clavulanic acid or tazobactam	RTG-4 (CARB-10)
2d	Cloxacillin	Hydrolyze cloxacillin or oxacillin, also carbenicillin hence are termed OXA enzymes OXA-related enzymes now comprise the second largest family of β-lactamases	OXA-1 OXA-10
2df	Carbapenems	1. Hydrolyze cloxacillin or oxacillin and carbapenems 2. The enzymes, and their producing organisms, are typically unresponsive to inhibition by clavulanic acid	OXA-23 OXA-48
2e	Extended-spectrum cephalosporins	1. Hydrolyze cephalosporins. 2. Inhibited by clavulanic acid but not aztreonam	CepA
2f	Carbapenems	Increased hydrolysis of carbapenems, oxyimino-β lactams, cephamycins	KPC-2, IMI-1, SME-1

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.

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