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

Daniel Garang Kuir.

BBioMedSci, USQM App Sci (MedSci), RMIT

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?

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..

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

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?

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..

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.

Transmission of resistance genes between bacterial species.

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

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.

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

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…

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

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

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…

Classification of β-lactamases contd…

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

Classification of β-lactamases contd…

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

2be2f

715

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

2df9

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.

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

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…

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

Detection of ESBLs in clinical isolates contd…

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

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

AMP AMC TIM TZP FOX CRO MEM GM CIP FT SXT0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%% Resistant_HP

% Resistant_CP

Major classes of antibiotics

Perc

enta

ge r

esis

tanc

e

Snapshots from ESBL study contd…

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

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

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.