Extended-spectrum β -lactamase (ESBL) Production in Enterobacteriaceae

1. Extended-spectrum β-lactamase (ESBL) Production in Enterobacteriaceae Daniel Garang Kuir. BBioMedSci, USQ M App Sci (MedSci), RMIT

2. What are Enterobacteriaceae? • Members of Enterobacteriaceae family are a heterogeneous group of gram negative bacteria. • Are part of human’s normal enteric flora. • Are also abundantly distributed in nature. • Include some prominent, often opportunistic, human pathogens; • Such as E. coli (e.g uropathogenicE. coli), Klebsiella spp, Enterobacterspp, Citrobacterspp, Salmonella spp, Shigellaspp, Yersinia pestis, Serratiamarcescens, Proteus spp, Morganellaspp, & Providenciaspp. • Majority are often expediently termed as the “ESCPPM” organisms – which stands for Enterobacterspp, Serratiaspp, Citrobacterfreundii, Proteus vulgaris & penneri, Providenciaspp, & Morganellamorganii. • Several members of this group are ESBL - &/or AmpC- producers. • K. pneumoniae&E. coli are major producers of ESBLs in this group of gram negative bacteria.

3. What are Enterobacteriaceae? Contd.. • Production of β-lactamases in Enterobacteriaceae is a common mechanism of antimicrobial resistance. • These β-lactamases include the novel β-lactamases such as ESBLs, AmpC…etc, & others such as; • Penicillinase, cephalosporinase, broad-spectrum, extended-spectrum, carbapenemase. • AmpC β-lactamases are chromosomally encoded cephalosporinases (chromosomal bla genes). • AmpC are expressed in many Enterobacteriaceae and other organisms. • AmpC induce, by constitutive hyperproduction or mutation, wide-ranging resistance to first-, second-, and third-generation cephalosporins, most penicillins, and beta-lactam/beta-lactam-inhibitor (BL/BLI) combinations.

4. Mechanisms of antimicrobial resistance in Enterobacteriaceae • Production of novel β-lactamases e.g. ESBLs, AmpC; • In tandem with production of β-lactamases, Enterobacteriaceae employ other mechanisms of resistance such as; • enzymatic inactivation; • efflux pumps; • outer membrane porinloss; • target modifications; • transfer or acquisition of new genetic material, or • mutations – ESBLs are essentially derivative enzymes acquired through mutations - substitution or deletion of amino acids - in progenitor β-lactamases (e.g TEM, SHV or CTX-M).

5. What are ESBLs? • ESBLs are novelβ-lactamases - are newer β-lactamases of pathogenic gram negative bacteria (esp. Enterobacteriaceae family). • These novel β-lactamases also include; • Plasmid-mediated AmpC β-lactamases; • Carbapenem-hydrolysing β-lactamases (e.g. Klebsiella pneumoniae carbapenemases (KPC)); • Β-lactamases with reduced sensitivity to β-lactamases inhibitors • Definition: ESBLs are bacterial enzymes capable of hydrolysing and thus conferring resistance to all penicillins, first-, second-, & third-generation cephalosporins, and aztreonam. • And are inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. • ESBLs are plasmid-mediated enzymes that confer multi-drug resistance to gram negative bacteria. • ESBLs may be co-expressed &/or co-transmitted with chromosomally-encoded AmpCβ-lactamases – thus presence of ESBLs may be masked by AmpC.

6. What are ESBLs? contd.. • ESBLs hydrolyse all β-lactam antibiotics – penicillins and cephalosporins. • β-lactamases possess either a serine moiety or a zinc atom in the active site, • Either of which is vital for hydrolysis of the β-lactam ring of a β–lactam antibiotic. • ESBLs are diverse, quickly evolving & therapeutically difficulty to eradicate. • ESBL production in Enterobacteriaceae also render them resistant to other major classes of antibiotics such as; • Fluoroquinolones (e.g. ciprofloxacin, norfloxacin), • Aminoglycosides (e.g. gentamicin, tobramycin, amikacin) • Tetracyclines (e.g. tetracycline) • Trimethroprims-sulfamethoxazole (Cotrimoxazole) • Other antibiotic classes NB : β-lactamase production, co-expression of ESBL &/or AmpC, carriage of other resistance gene on the same plasmid account for multidrug resistance in this group of bacteria. • ESBL-mediated extensive antimicrobial resistance poses public health risks. • ESBL-producing Enterobacteriaceae are essentially multidrug resistant bacteria.

7. βeta-lactam ring & the hydrolysing action of β-lactamases Source: Rosário NA, Grumach AS. Allergy to beta-lactams in paediatrics: a practical approach. J Pediatr (Rio J). 2006;82(5 Suppl):S181-8.

8. Transmission of resistance genes between bacterial species. Source: Partridge, S. (2014). Movement of resistance genes in hospitals. Microbiology Australia.

9. Clinical significance of ESBL-producing Enterobacteriaceae (ESBL-PE) • ESBL-producing Enterobacteriaceae (ESBL-PE) cause significant mortality and morbidity globally. • ESBL-PE cause a range of infections including uncomplicated UTIs, life-threatening bacteraemia, URTIs, gastroentritis, & colonising wound infections. • Mortality of patients with ESBL +ve sepsis is significantly higher than those with ESBL -ve sepsis – up to 30% of GNB-caused sepsis is fatal. • Are implicated in large scale outbreaks in hospital or community settings. • Cause localised or institutionalised outbreaks. • Infections caused by ESBL-PE are associated with rising healthcare cost. • Decreased productivity as a consequence of prolonged hospitalisation. • ESBL-PE are associated with increasing episodes of clinical treatment failure.

10. Clinical significance of ESBL-producing Enterobacteriaceae (ESBL-PE) contd… • ESBL producing organisms have important therapeutic and clinical ramifications for patients from whom they are isolated. • ESBL-PE pose significant public health risks. • ESBL-PE pose serious infection control challenges. • ESBL production in Enterobacteriaceae has been a consequence of widespread use of broad spectrum antibiotics in hospital settings. • Increasingprevalence is reported in isolates recovered from community-basedpatients. • ESBLs are transferrable via conjugative plasmids thus dissemination of resistance genes among bacterial populations can occur and spread in larger geographic regions. • Treatment of ESBL-PE involves a combination of antibiotics, some of which have undesirable side effects including nephrotoxicity.

11. Risk factors for infections with ESBL-producing Enterobacteriaceae • Risk factors for infections with ESBL-PE in healthcare- or community-acquired infections include; • Previous use of antibiotics including broad spectrum antibiotics e.g 3GC cephalosporins; • Recent or prolonged hospital admissions including admissions to ICU; • Recurrent UTIs; • Empiric antibiotic therapy • Increased age; female gender; institutionalised residential care e.g. nursing homes; • Intravenous therapy; • International travels to areas of established endemicity e.g India subcontinent, the Middle East and Africa; • Immunosuppressive chemotherapy; • Invasive procedures- indwelling urinary catheters; central venous catheter, and • Underlying comorbidities such as chronic renal insufficiencies, haemodialysis, liver disease, diabetes mellitus, malignancy, hypertension, heart disease, neutropenia, and HIV infection

12. Classification of β-lactamases • ESBLs were first reported in Germany in 1983. • This followed introduction of broad spectrum 3G cephalosporinsinto clinical use. • ESBLs have been reported in all parts of the world – except Antarctica. • ESBLs are derivatives of classic β-lactamases eg SHV-2 is derived from SHV-1. • ESBLs are occasioned by single mutations in progenitor (parent) enzymes • A mutation of few amino acids. • ESBLs exhibit fundamental changes in substrate spectra, substrate profile , reactions to inhibitors & isoelectric point – important distinguishing factors. • Over 200 ESBLs are characterised & classified – there is still no consensus on exact figure. • Β-lactamases have been variously classified over time. • Two commonly used classification schemes are; • Ambler molecular classification system • Bush-Jacoby-Medeiros functional classification system.

13. Classification ofβ-lactamases contd… • The Ambler molecular system classifies β-lactamases on the basis of protein homology (amino acid similarities); • 4 major classes (A, B, C & D). • The Bush-Jacoby-Medeiros functional system classifies β-lactamases, on the basis of functional similarities/substrate and profile inhibitor profile; • 4 main groups (1, 2, 3 & 4). • ESBLs are derived from group 2be β-lactamases; • the `e’ of 2be denotes the extended-spectrum capability of the newly derived enzyme. • ESBLs are quite diverse. • Clinically important ESBLs are derived from 3 major types of classic beta-lactamases; TEM-, SHV-, & CTX-M-type β-lactamases. • Temoniera– a Greek patient from whom this ESBL type was first isolated. • SHV - Sulfhydryl Variable. • CTX-M - Cefotaxime – Munich (first isolated in Munich)

14. Classification of β-lactamases contd…

15. Classification of β-lactamases contd… • Snapshot of major ESBLs – SHV -, TEM- & CTX-M-types including rare and peculiar ESBLs

16. Classification of β-lactamases contd…Major classes of β-lactamases of clinical significance [i] Enzyme families classified on the basis of amino acid structures (G. Jacoby and K. Bush, http://www.lahey.org/studies/). [ii]The sum of the subgroups in each family does not always equal to overall number of enzymes in each family due to withdrawn or non-classification of some enzymes.

17. Epidemiology of ESBL-producing Enterobacteriaceae • Stats of ESBL epidemiology are profoundly varied – all parts of the world have different rates of prevalence. • In general terms; • TEM-type ESBLs are predominantly reported in the United States, • SHV-type ESBLs are most frequently isolated in Western Europe. • CTX-M-type ESBLs have been detected in Australia, Latin America, Eastern Europe, and in specific countries such as Japan, Spain, & Kenya. • Global epidemiology captures in major surveillance studies; • AGAR (Australia) • SENTRY (US, Canada & Latin America) • SMART ( Global - US, SE Asia) • EARSS (European countries)

18. Epidemiology of ESBL-producing Enterobacteriaceae contd…

19. Detection of ESBLs in clinical isolates • Use of both genotypic and phenotypic techniques. • Phenotypic testing – a 2 steps process; • Screening; screening process aims to exclude potential ESBL-producing isolates by testing for resistance or reduced susceptibility to 3GC cephalosporins. • Screening using cefotaxime, cefpodoxime, ceftazidime, and aztreonamdiscs. • multiple 3GC agents reliably improves sensitivity by offering wider ESBL substrate base. • Confirmation; second step tests for synergy between 3GC cephalosporins &clavulanates (synergy between β-lactams and β-lactams-clavulanate combinations) – also known as DDST (double disc synergy test). • A disc zone diameter difference of ≥5 mm between a cephalosporin and its respective cephalosporin-clavulanate is taken as a phenotypic confirmation of ESBL production. • e.g an ESBL-producer tested against ceftazidime produces these resistance zones: ceftazidime zone = 16; ceftazidime-clavulanic acid zone = 21) • Automated (Vitek2 systems) MBD • Automated microbroth dilution - growth at or above screening concentrations (breakpoint) may indicate production of ESBL (that is, for E. coli and K. pneumoniae, MIC ≥ 2 μg/mL for ceftriaxone, ceftazidime, aztreonam, or cefpodoxime). • E-test, microScan panels and other discs-based methods are also used.

20. Detection of ESBLs in clinical isolates contd… Can you tell a plate depicting ESBL positive in the Figure above?

21. Snapshots from ESBL studySummarized results of all isolates grouped by setting (hospital vs. community), ESBL-producer status, and by age categories

22. Snapshots from ESBL study contd… Comparison of percentage resistance of ESBL-producing isolates recovered from patients in hospital (HP) and community (CP) settings

23. Preventing growing threats of ESBL-mediated antimicrobial resistance • What should be done to curb increasing threats pose by ESBL-mediated antibiotic resistance; • Robust antibiotic stewardship – appropriate use of antibiotics • Effective infection control measures in hospitals – effective preventive measures to curb transmission; • Contact precautions, • Hand hygiene, • Disinfections of inanimate objects, surfaces, medical devices in healthcare facilities • Public education – antibiotic resistance awareness campaign. • Controlling use of antibiotics in food chains – control & regulation of antibiotic use in agriculture. • Immunization – preventative & indirect • Development of newer, potent antibiotics against emerging multidrug resistant bacteria. • Timely detection, and reporting of ESBL producing bacteria by medical laboratories. • Instituting infection control measures in institutionalised care settings – eg nursing homes. • Active screening for multi-drug resistant Enterobacteriaceae. • Classifying ESBL-PE as notifiable infections???

24. Treatment of infections caused by ESBL-producing Enterobacteriaceae. • Therapeutic options are very limited. • Treatment usually involves a combination of drugs. • These are usually the expensive, last line of antibiotics; • Carbapenems (e.gmeropenem, ertapenem) • Fosfomycin. • β-lactam/β-lactam-inhibitor combination drugs (e.g Amoxicillin-clavulanate, piperacillin-tazobactam…etc) – supporting evidence from clinical studies is, however, controversial. • Limitation of therapeutic drugs is also compounded by other factors such as; • Site of infection, • Severity of infection, • Renal or liver functions of a patient, • Age, • Pregnancy or lactation status, • Other medications the patient may be taking.