1 MICROBIOLOGY · ANTIMICROBIAL RESISTANCE Extended-Spectrum β-Lactamases (ESBL) ◆ ◆ ◆ IN THIS CHAPTER 1 Background 2 ESBL and ESBL Producers 3 Classification and Evolution of ESBL 4 Mechanism of Resistance and Dissemination 5 Diagnostic Tools for Detection 6 Key Terms QUICK SUMMARY ◆ What ESBLs are: enzymes from Gram-negative Enterobacteriaceae that inactivate penicillins, 3rd/4th-generation cephalosporins and aztreonam, but not cephamycins or carbapenems. ◆ Blocked by: β-lactamase inhibitors (clavulanic acid, sulbactam, tazobactam), though not all β-lactamases are inhibited. ◆ Spread: genes sit on plasmids, transposons and insertion sequences and move by horizontal gene transfer (conjugation, transformation, transduction). ◆ Main families: TEM, SHV and CTX-M (now dominant). E. coli is the most common host, and ST131 is its most dominant clone. ◆ Classification: Ambler Classes A and D; Bush–Jacoby– Medeiros Group 2; modern groups ESBLA, ESBLM and ESBLCARBA. ◆ Detection: CDT (zone ≥ 5 mm larger), DDST (keyhole/augmented zone), gradient test (≥ 8-fold MIC drop).

EXTENDED-SPECTRUM Β-LACTAMASES 2 1 Background ◆ ESBLs are Gram-negative bacteria of the Enterobacteriaceae family that carry ESBL genes in their plasmids or chromosomes, produce β-lactam hydrolyzing enzymes, and are rightly considered to be among the most challenging pathogens by the World Health Organization (WHO). ◆ ESBL-producing Enterobacteriaceae (ESBL-E) confer resistance to penicillin—in addition to aztreonam and first, second, and third-generation cephalosporins—but are unable to hydrolyze cephamycin or carbapenems. Carbapenem has been the drug of first choice for treating ESBL-E-induced infection for a long time. This is changing, though, due to many factors including the recent emergence of carbapenemase-producing bacteria. Thus, there is an urgent need to develop alternative approaches. ◆ Misuse or overuse of antibiotics in both human and animal populations is responsible for the evolution of drug-resistant bacteria via gene mutations or horizontal transmission of resistance genes by plasmids. ◆ ESBL-E are commensal bacteria in both humans and animals and can be a major threat to food safety and food security. Commensal ESBL reservoirs in the environment have experienced recent dramatic increases due to the co- transmission of ESBL-E between the human and animal populations, which can occur through several direct and indirect routes of transmission. Pathogenic bacteria in the environment are able to acquire ESBL genes from commensal bacteria and can pose significant health risks to humans and animals. 2 ESBL and ESBL Producers ESBL ARE HYDROLYZING ENZYMES secreted by several Gram- negative bacteria of the family Enterobacteriaceae. They cause the

ESBL · ANTIMICROBIAL RESISTANCE 3 inactivation of broad-spectrum oxyimino-cephalosporin (third- and fourth-generation) and monobactam (aztreonam) but not cephamycin (cefoxitin) or carbapenems (meropenem, imipenem, ertapenem, and doripenem). Generally, these enzymes are neutralized by β-lactamase inhibitors (BLIs) such as clavulanic acid, sulbactam, and tazobactam. Genes that encode ESBL are mostly found on transposons or insertion sequences of plasmids in association with other resistance genes. As a result, they can spread rapidly, causing resistance to multiple antimicrobials such as aminoglycosides, trimethoprim, sulphonamides, tetracyclines, chloramphenicol, and fluoroquinolone. ESBL are produced by the nosocomial pathogens E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Among a wide range of Gram-negative bacterial species of different families harboring ESBL genes, E. coli is the most common host, followed by K. pneumoniae. Among the different variants of ESBL-producing E. coli, the ST131 clone is the most dominant. ◆ The ESBL-encoding genes are highly diverse in nature and can be classified into many families with unique characteristics such as blaTEM, blaSHV, and blaCTX-M. ◆ TEM-1, the first plasmid and transposon-mediated β-lactamase, was isolated from the blood culture of a patient named Temoniera in Greece in the early 1960s. It has spread worldwide and is now found in many species of the family Enterobacteriaceae, P. aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae. The SHV-1 type is common in Klebsiella spp. and E. coli. CTX-M-type ESBL are predominant in E. coli, K. pneumoniae, S. enterica serovar Typhimurium, and Shigella spp. The plasmid-mediated OXA and AmpC-type ESBL were discovered in P. aeruginosa and K. pneumoniae isolates, respectively. ◆ A series of Salmonella serovars, including S. enteritidis, S. newport, and S. paratyphi, have been characterized as ESBL

EXTENDED-SPECTRUM Β-LACTAMASES 4 producers that have been linked to serious foodborne gastroenteritis in humans. 3 Classification and Evolution of ESBL β-lactamases can be defined and classified in two complementary ways: Ambler system Bush–Jacoby– Medeiros system Based on Molecular structure Function Divisions Four classes: A, B, C, D Groups 1 to 3, according to how they destroy substrates and react to inhibitors Where ESBLs sit Classes A and D, which use serine at their active center Group 2 (Ambler Classes A & D); an updated 2009 version still keeps them in Group 2 Modern Grouping of ESBLs Recently, ESBLs have been categorized into three main functional groups: ESBLA ESBLM ESBLCARBA Ambler Class A ESBL. The biggest group: most ESBLs in the world belong here. Miscellaneous ESBL. Other miscellaneous types. Carbapenem- degrading ESBL. Rare, dangerous types that can break down carbapenems, our strongest “last-resort” antibiotics.

ESBL · ANTIMICROBIAL RESISTANCE 5 KEY ESBLA TYPES ◆ TEM (Temoniera) ◆ SHV (Sulfhydryl reagent Variable) ◆ CTX-M (Cefotaxime-M), remember this is the one heavily used by E. coli ST131! ESBLA in Detail TEM-1 and SHV-1: the older β-lactamases ◆ What they originally did: – TEM-1 gave E. coli resistance to ampicillin, penicillin, and 1st-generation cephalosporins. – SHV-1 gave resistance to penicillin, tigecycline, and piperacillin. – Their limit: neither could break down oxyimino- cephalosporins. ◆ How they evolved in the 1980s: simple amino acid mutations allowed them to turn into ESBLs, giving them the new power to destroy newer drugs like 3rd-generation cephalosporins and aztreonam. CTX-M: the modern, dominant ESBL ◆ The new threat: CTX-M has largely replaced TEM and SHV as the primary cause of modern ESBL outbreaks. ◆ Origin: the resistance genes (bla genes) originally came from non-harmful soil/water bacteria called Kluyvera spp. ◆ How it spread: mobile genetic elements (like ISEcp1 and ISCR1) acted like cutting tools, moving these genes off Kluyvera chromosomes and onto plasmids inside E. coli. ◆ Classification: grouped into 6 major types (CTX-M-1, CTX-M- 2, CTX-M-8, CTX-M-9, CTX-M-25, and KLUC).

EXTENDED-SPECTRUM Β-LACTAMASES 6 Global spread and real-world impact ◆ Found everywhere: detected in over 80 variants across hospitals, communities, food animals, vegetables, and water. ◆ Key variants: – Humans: CTX-M-15 and CTX-M-14 are the most common worldwide. – Animals: CTX-M-1 is predominant. ◆ Regional preferences: CTX-M-2 and CTX-M-8 are common in South America; CTX-M-2 is common in Japan. Other ESBL Types ESBLM: miscellaneous ESBLs ◆ Sub-types: divided into ESBLM-C (Class C, AmpC) and ESBLM-D (Class D). ◆ AmpC features: resistance to penicillins, 3rd/4th generation cephalosporins, and sometimes carbapenems. – Inhibitors: blocked by cloxacillin and boronic acid. ◆ Includes some OXA-type enzymes as well. ESBLCARBA: carbapenem-resistant ESBLs ◆ Sub-types: divided into ESBLCARBA-A, ESBLCARBA-B, and ESBLCARBA-D. ◆ Super power: can degrade all β-lactam antibiotics (including carbapenems, our strongest drugs). ◆ Inhibitors: can be blocked by EDTA, DPA (for metallo-β- lactamases), boronic acid, or avibactam. OXA-type enzymes (Ambler Class D) ◆ Key features: different from TEM and SHV enzymes; they can break down oxacillin and cloxacillin. ◆ Resistant to standard blockers: they are not inhibited by clavulanic acid.

ESBL · ANTIMICROBIAL RESISTANCE 7 ◆ Where they are found: mostly in Pseudomonas aeruginosa, and rarely in E. coli (only 1–10%). Rare plasmid-transmitted ESBLs Other rare ESBL types passed around on plasmids include: ◆ PER (Pseudomonas extended-resistant) ◆ VEB (Vietnam ESBL) ◆ GES (Guiana extended-spectrum) ◆ IBC (Integron-borne cephalosporinase) 4 Mechanism of Resistance and Dissemination of Resistant Genes How bacteria destroy β-lactam antibiotics ◆ Enzyme action: Gram-negative bacteria release β-lactamase enzymes into their periplasm (the space between their inner and outer membranes). ◆ The trick: the antibiotic prefers to attach to the β-lactamase enzyme rather than its actual target, destroying the antibiotic before it can do harm. Where resistance genes live and how they evolve ◆ Location: genes encoding β-lactamases can live on: – The main chromosome (e.g., in Enterobacter species). – Mobile genetic elements (MGEs) like plasmids, transposons, or integrons. ◆ Evolution: bacteria get resistance by either: 1. Mutations in their own DNA. 2. Acquiring genes from other bacteria (same or different species).

EXTENDED-SPECTRUM Β-LACTAMASES 8 How resistance genes spread (horizontal gene transfer) Bacteria pass these resistance genes around via transformation, transduction, or conjugation: ◆ Integrons (Classes 1–5): special DNA capture systems that act as “vehicles” to collect, store, and spread resistance genes across species. ◆ TEM types: mostly carried and spread by Tn1, Tn2, or Tn3- like transposons (“jumping genes”). ◆ SHV types: can be carried on both chromosomes and plasmids. ◆ CTX-M types: spread most commonly through conjugation (direct cell-to-cell plasmid transfer). Overcoming treatment and additional resistance tools ◆ Inhibitor failure: standard enzyme blockers (like clavulanic acid, sulbactam, and tazobactam) do not work against all β- lactamases. ◆ Other physical defenses: – Reduced permeability: DNA mutations close or shrink membrane pores, keeping antibiotics outside the cell. – Efflux pumps: active pumps flush antibiotics straight back out from the periplasm to the exterior. 5 Diagnostic Tools for Detection of ESBL A. Combination disk test (CDT) For each test, disks or tablets containing the cephalosporin alone (cefotaxime, ceftazidime, cefepime) and in combination with clavulanic acid are applied. The inhibition zone around the

ESBL · ANTIMICROBIAL RESISTANCE 9 cephalosporin disk or tablet combined with clavulanic acid is compared with the zone around the disk or tablet with the cephalosporin alone. POSITIVE RESULT Inhibition zone diameter is ≥ 5 mm larger with clavulanic acid than without. Figure 1 Combination disk test: cefotaxime alone vs. cefotaxime/clavulanate. B. Double-disk synergy test (DDST) Disks containing cephalosporins (cefotaxime, ceftazidime, cefepime) are applied to plates next to a disk with clavulanic acid (amoxicillin- clavulanic acid). The distance between the disks is critical and 20 mm centre-to-centre has been found to be optimal for cephalosporin 30 μg disks; however, it may be reduced (15 mm) or expanded (30 mm) for strains with very high or low levels of resistance, respectively.

EXTENDED-SPECTRUM Β-LACTAMASES 10 POSITIVE RESULT Inhibition zones around any of the cephalosporin disks are augmented, or there is a “keyhole” in the direction of the disk containing clavulanic acid. Figure 2 Double-disk synergy test (DDST). C. Gradient test method ESBL-specific gradient tests are set up, read and interpreted according to the manufacturer’s instructions. The test result is indeterminate if the strip cannot be read due to growth beyond the MIC range of the strip. In all other cases the test result is negative. According to the manufacturer, the ESBL gradient test should be used for confirmation of ESBL production only and is not reliable for determination of the MIC.

ESBL · ANTIMICROBIAL RESISTANCE 11 POSITIVE RESULT A ≥ 8-fold reduction in the MIC of the cephalosporin combined with clavulanic acid compared with the cephalosporin alone, or a phantom zone or deformed ellipse is present. Figure 3 ESBL gradient test strips. 6 Key Terms 1 Resistance gene — is the tool: a specific piece of DNA code that tells a bacterium how to block or destroy an antibiotic. 2 Horizontal transmission — is the delivery method: the process bacteria use to hand that resistance gene over to another bacterium. 3 Superbugs — are strains of bacteria, viruses, fungi, or parasites that have become resistant to most of the antibiotics and other medications commonly used to treat the infections they cause. 4 E. coli ST131 — (Sequence Type 131) is a globally spread, multidrug-resistant “superbug” clone. It is a major cause of extraintestinal infections, such as severe urinary tract infections (UTIs) and bloodstream infections (sepsis), both in hospitals and in the community.

EXTENDED-SPECTRUM Β-LACTAMASES 12 5 Transposons and insertion sequences — are essentially the “jumping genes” of the bacterial world. They play a huge role in how plasmids pick up antibiotic resistance in the first place. In simple terms: ◆ Insertion sequences: the simplest type of “jumping gene.” They are tiny pieces of DNA whose only job is to cut themselves out of one spot and paste themselves into another spot in the cell’s DNA. ◆ Transposons: a bit more advanced. They are larger jumping genes that carry extra cargo—such as an antibiotic resistance gene—along with them when they jump. 6 Nosocomial pathogens — (more commonly called hospital- acquired infections or HAIs) are bacteria, viruses, fungi, or parasites that patients catch while receiving medical care in a hospital, clinic, or nursing home. 7 Mobile genetic elements (MGEs) — are segments of DNA that can move around within a genome or transfer between different cells. Think of them as the “copy-paste tools” or “delivery trucks” of the genetic world. Instead of staying locked in one place, they can jump, copy themselves, and travel.

ESBL · ANTIMICROBIAL RESISTANCE 13 KEY TYPES OF MOBILE GENETIC ELEMENTS Plasmids: circular loops of DNA that can be shared directly between different bacteria (the main vehicle for horizontal transmission). Transposons and insertion sequences: “jumping genes” that can cut themselves out of one piece of DNA and paste themselves into another (like moving a resistance gene onto a plasmid). Bacteriophages (phages): viruses that infect bacteria and can accidentally carry resistance genes from one bacterium to another. References 1.Husna, A.; Rahman, M.M.; Badruzzaman, A.T.M.; Sikder, M.H.; Islam, M.R.; Rahman, M.T.; Alam, J.; Ashour, H.M. Extended-Spectrum β- Lactamases (ESBL): Challenges and Opportunities. Biomedicines 2023, 11, 2937. https://doi.org/10.3390/biomedicines11112937. 2.Das P, Mahapatra D, Mazumder SS. A Guide Towards the Phenotypic Detection of Extended-spectrum β-lactamases Production in Enterobacteriaceae: Alone or in Presence of Other Interfering Enzymes. J Pure Appl Microbiol. 2023;17(3):1410-1421. doi: 10.22207/JPAM.17.3.31

EXTENDED-SPECTRUM Β-LACTAMASES 14 Self-Check Quiz Multiple choice 1. Which antibiotics can ESBLs not hydrolyze? A. Penicillins B. Third-generation cephalosporins C. Carbapenems D. Aztreonam 2. Which agent generally neutralizes ESBLs? A. Clavulanic acid B. Cloxacillin C. EDTA D. Boronic acid 3. Which species is the most common ESBL host? A. Pseudomonas aeruginosa B. Klebsiella pneumoniae C. E. coli D. Acinetobacter baumannii 4. Which ESBL family has largely replaced TEM and SHV in modern outbreaks? A. OXA B. CTX-M C. PER D. GES 5. CTX-M resistance genes originally came from which bacteria? A. Kluyvera spp. B. Salmonella spp. C. Shigella spp. D. Neisseria spp.

ESBL · ANTIMICROBIAL RESISTANCE 15 6. ESBLs belong to which Ambler classes? A. A and B B. B and C C. C and D D. A and D 7. In the combination disk test, a positive result is a zone that is at least how much larger with clavulanic acid? A. 2 mm B. 5 mm C. 8 mm D. 10 mm 8. A positive gradient test shows at least what reduction in MIC with clavulanic acid? A. 2-fold B. 4-fold C. 8-fold D. 16-fold True or false 9. OXA-type enzymes are inhibited by clavulanic acid. 10. ESBLCARBA enzymes are common and can degrade all β- lactams except carbapenems. Short answer 11. Name the three mechanisms of horizontal gene transfer. 12. What is the difference between an insertion sequence and a transposon?

EXTENDED-SPECTRUM Β-LACTAMASES 16 Answer Key 1. C — ESBLs inactivate penicillins, oxyimino-cephalosporins and aztreonam, but not cephamycins or carbapenems. 2. A — Clavulanic acid, sulbactam and tazobactam are the β-lactamase inhibitors that normally block ESBLs. 3. C — E. coli is the most common host, followed by K. pneumoniae; its ST131 clone is the most dominant. 4. B — CTX-M is now the primary cause of modern ESBL outbreaks. 5. A — They were moved from Kluyvera chromosomes onto plasmids by elements such as ISEcp1 and ISCR1. 6. D — ESBLs are in Classes A and D, which use serine at the active center. 7. B — A zone ≥ 5 mm larger with clavulanic acid is positive. 8. C — A ≥ 8-fold MIC reduction (or a phantom zone / deformed ellipse) is positive. 9. False — OXA-type enzymes (Ambler Class D) are not inhibited by clavulanic acid. 10. False — They are rare and can degrade all β-lactams, including carbapenems. 11. Model answer — Transformation, transduction and conjugation. CTX-M types spread most commonly by conjugation. 12. Model answer — An insertion sequence is the simplest jumping gene: it only cuts and pastes itself. A transposon is larger and carries extra cargo, such as an antibiotic resistance gene.

ESBL · ANTIMICROBIAL RESISTANCE 17 Dedication These notes are dedicated to my lifelong friend, Dr. Mona Ahmed Issa, In appreciation of a friendship that has endured through the years. 12 October 2026 Regards, Ibtihal omer