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

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


Extended-spectrum β -lactamase (ESBL) Production in Enterobacteriaceae. Daniel Garang Kuir . BBioMedSci , USQ M App Sci ( MedSci ), RMIT. What are Enterobacteriaceae?. Members of Enterobacteriaceae family are a heterogeneous group of gram negative bacteria. - PowerPoint PPT Presentation

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

Page 1: Extended-spectrum  β -lactamase (ESBL) Production in  Enterobacteriaceae

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

Daniel Garang Kuir.

BBioMedSci, USQM App Sci (MedSci), RMIT

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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 uropathogenic E. coli), Klebsiella spp,

Enterobacter spp, Citrobacter spp, Salmonella spp, Shigella spp, Yersinia pestis, Serratia marcescens, Proteus spp, Morganella spp, & Providencia spp.

Majority are often expediently termed as the “ESCPPM” organisms – which stands for Enterobacter spp, Serratia spp, Citrobacter freundii, Proteus vulgaris & penneri, Providencia spp, & Morganella morganii .

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.

What are Enterobacteriaceae?

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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.

What are Enterobacteriaceae? Contd..

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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 porin loss;◦ 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).

Mechanisms of antimicrobial resistance in Enterobacteriaceae

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ESBLs are novel β-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 AmpC β-lactamases – thus presence of ESBLs may be masked by AmpC.

What are ESBLs?

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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 classesNB : 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.

What are ESBLs? contd..

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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.

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Transmission of resistance genes between bacterial species.

Source: Partridge, S. (2014). Movement of resistance genes in hospitals. Microbiology Australia.

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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


Clinical significance of ESBL-producing Enterobacteriaceae (ESBL-PE)

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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. Increasing prevalence is reported in isolates recovered from

community-based patients. 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.

Clinical significance of ESBL-producing Enterobacteriaceae (ESBL-PE) contd…

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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

Risk factors for infections with ESBL-producing Enterobacteriaceae

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ESBLs were first reported in Germany in 1983. This followed introduction of broad spectrum 3G cephalosporins into

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.

Classification of β-lactamases

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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)

Classification of β-lactamases contd…

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Classification of β-lactamases contd…

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Snapshot of major ESBLs – SHV -, TEM- & CTX-M-types including rare and peculiar ESBLs

Classification of β-lactamases contd…

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Enzyme family

Functional group or subgroup

No. of enzymes Representative enzymes

       CMY 1, 1e 50 CMY-1 to CMY-50

TEM 2b, 2be, 2br, 2ber 172    2b 12 TEM-1, TEM-2, TEM-13  2be 79 TEM-3, TEM-10, TEM-26  2br 36 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM-

163  2ber 9 TEM-50 (CMT-1), TEM-158 (CMT-9)

SHV 2b, 2be, 2br 127    2b 30 SHV-1, SHV-11, SHV-89  2be 37 SHV-2, SHV-3, SHV-115  2br 5 SHV-10, SHV-72

CTX-M 2be 90 CTX-M-1, CTX-M-44 (Toho-1) to CTX-M-92

PER 2be 5 PER-1 to PER-5VEBGES



VEB-1 to VEB-7GES-2 to GES-7 (IBC-1) to GES-15

KPC 2f 9 KPC-2 to KPC-10SME 2f 3 SME-1, SME-2, SME-3

OXA 2d, 2de, 2df 158    2d 5 OXA-1, OXA-2, OXA-10  2de


48OXA-11, OXA-14, OXA-15OXA-23 (ARI-1), OXA-51, OXA-58

IMP 3a 26 IMP-1 to IMP-26VIM 3a 23 VIM-1 to VIM-23IND 3a 8 IND-1, IND-2, IND-2a, IND-3 to IND-7

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.

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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)

Epidemiology of ESBL-producing Enterobacteriaceae

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Country Study name or period

K. pneumoniae E. coli

    Number of isolates Percentage of ESBL positive

Number of isolates Percentage of ESBL positive

Canada SENTRY 1997-1999

386 4.9 1203 4.2

US and Canada

SENTRY 1998 192 4.2 - -

USA SENTRY 1997-1999

2017 7.9 4966 3.3

USA SENTRY 1997 409 44 771 4.7Latin America SENTRY 1997-

2000255 43.9 114 25.4

Latin America SENTRY 1997-2000

127 40 233 10.0

Latin America SENTRY 1997-2000

664 47.3 1239 6.7

Latin America SENTRY 1997-1999

897 45.4 2026 8.5

Europe SENTRY 1997-1999

946 22.6 3822 5.3

Italy 1999 946 20.0 

4604 1.2

Spain EARSS 2001 - - 1962 1.55France 1996-2000 6121 11.4 - -Germany PEG 2001 268 8.2 619 0.8Netherlands 1997 196 <1 571 <1Turkey 1997 43 48.8 530 1.1Western Pacific area

SENTRY 1997-1999

560 24.6 1104 7.9

Asian Pacific area

SENTRY 1998-1999

678 25.2 1337 10.1

China 1999 559 51 427 23.6Taiwan 2000 124 11.3 177 11.9Hong Kong 1998 472 13 702 11

Epidemiology of ESBL-producing Enterobacteriaceae contd…

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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 aztreonam discs. 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 (Vitek 2 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.

Detection of ESBLs in clinical isolates

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Detection of ESBLs in clinical isolates contd…

Can you tell a plate depicting ESBL positive in the Figure above?

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Setting ESBL positive ESBL negative Total

Hospital 30 259 289

community 75 402 477

Total 105 661 766

Frequencies at assigned age categories

0-20 years old 21-40 years old 41-60 years old ≥61 years old

11 26 15 53

Snapshots from ESBL studySummarized results of all isolates grouped by setting (hospital vs.

community), ESBL-producer status, and by age categories

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100%% Resistant_HP

% Resistant_CP

Major classes of antibiotics



ge r




Snapshots from ESBL study contd…

Comparison of percentage resistance of ESBL-producing isolates recovered from patients in hospital (HP) and community (CP) settings

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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???

Preventing growing threats of ESBL-mediated antimicrobial resistance

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Therapeutic options are very limited. Treatment usually involves a combination of drugs. These are usually the expensive, last line of antibiotics; Carbapenems (e.g meropenem, 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.

Treatment of infections caused by ESBL-producing Enterobacteriaceae.