Daniel Garang Kuir.
BBioMedSci, USQ
M 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 AmpCproducers.
K. pneumoniae & E. coli are major producers of ESBLs in this
group of gram negative bacteria.
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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.
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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 thirdgeneration cephalosporins, most penicillins, and betalactam/beta-lactam-inhibitor (BL/BLI) combinations.
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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;
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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 βlactamases (e.g TEM, SHV or CTX-M).
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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;
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Plasmid-mediated AmpC β-lactamases;
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Carbapenem-hydrolysing β-lactamases (e.g. Klebsiella pneumoniae carbapenemases (KPC));
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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-, & thirdgeneration cephalosporins, and aztreonam.
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And are inhibited by β-lactamase inhibitors such as clavulanic acid,
sulbactam and tazobactam.
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ESBLs are plasmid-mediated enzymes that confer multi-drug resistance
to gram negative bacteria.
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ESBLs may be co-expressed &/or co-transmitted with chromosomallyencoded AmpC β-lactamases – thus presence of ESBLs may be masked
by AmpC.
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ESBLs hydrolyse all β-lactam antibiotics – penicillins and cephalosporins.
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β-lactamases possess either a serine moiety or a zinc atom in the active site,
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Either of which is vital for hydrolysis of the β-lactam ring of a β–lactam antibiotic.
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ESBLs are diverse, quickly evolving & therapeutically difficulty to eradicate.
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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.
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ESBL-mediated extensive antimicrobial resistance poses public health risks.
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ESBL-producing Enterobacteriaceae are essentially multidrug resistant bacteria.
Source: Rosário NA, Grumach AS. Allergy to beta-lactams in paediatrics: a practical
approach. J Pediatr (Rio J). 2006;82(5 Suppl):S181-8.
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, lifethreatening 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.
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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.
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ESBL-PE pose serious infection control challenges.
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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 communitybased 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.
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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
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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.
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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.
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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).
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The Bush-Jacoby-Medeiros functional system classifies βlactamases, on the basis of functional similarities/substrate and
profile inhibitor profile;
◦ 4 main groups (1, 2, 3 & 4).
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ESBLs are derived from group 2be β-lactamases;
◦ the `e’ of 2be denotes the extended-spectrum capability of the newly derived
enzyme.
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ESBLs are quite diverse.
Clinically important ESBLs are derived from 3 major types of
classic beta-lactamases; TEM-, SHV-, & CTX-M-type βlactamases.
◦ Temoniera – a Greek patient from whom this ESBL type was first isolated.
◦ SHV - Sulfhydryl Variable.
◦ CTX-M - Cefotaxime – Munich (first isolated in Munich)
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Snapshot of major ESBLs – SHV -, TEM- & CTX-M-types including rare
and peculiar ESBLs
Enzyme family
Functional group or
subgroup
CMY
1, 1e
TEM
2b, 2be, 2br, 2ber
SHV
No. of enzymes
50
CMY-1 to CMY-50
172
2b
2be
2br
12
79
36
2ber
9
2b, 2be, 2br
Representative enzymes
TEM-1, TEM-2, TEM-13
TEM-3, TEM-10, TEM-26
TEM-30 (IRT-2), TEM-31 (IRT-1), TEM163
TEM-50 (CMT-1), TEM-158 (CMT-9)
127
2b
2be
2br
30
37
5
SHV-1, SHV-11, SHV-89
SHV-2, SHV-3, SHV-115
SHV-10, SHV-72
CTX-M
2be
90
PER
VEB
GES
2be
2be
2f
5
7
15
CTX-M-1, CTX-M-44 (Toho-1) to CTXM-92
PER-1 to PER-5
VEB-1 to VEB-7
GES-2 to GES-7 (IBC-1) to GES-15
KPC
SME
2f
2f
OXA
2d, 2de, 2df
9
3
KPC-2 to KPC-10
SME-1, SME-2, SME-3
158
2d
2de
2df
5
9
48
OXA-1, OXA-2, OXA-10
OXA-11, OXA-14, OXA-15
OXA-23 (ARI-1), OXA-51, OXA-58
IMP
3a
26
IMP-1 to IMP-26
VIM
IND
3a
3a
23
8
VIM-1 to VIM-23
IND-1, IND-2, IND-2a, IND-3 to IND-7
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.
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Global epidemiology captures in major surveillance studies;
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AGAR (Australia)
SENTRY (US, Canada & Latin America)
SMART ( Global - US, SE Asia)
EARSS (European countries)
Country
Study name or
period
Italy
SENTRY 19971999
SENTRY 1998
SENTRY 19971999
SENTRY 1997
SENTRY 19972000
SENTRY 19972000
SENTRY 19972000
SENTRY 19971999
SENTRY 19971999
1999
Spain
France
Germany
Netherlands
Turkey
Western Pacific
area
Asian
Pacific
area
China
Taiwan
Hong Kong
EARSS 2001
1996-2000
PEG 2001
1997
1997
SENTRY 19971999
SENTRY 19981999
1999
2000
1998
Canada
US and Canada
USA
USA
Latin America
Latin America
Latin America
Latin America
Europe
K. pneumoniae
Number of isolates
386
E. coli
Percentage
positive
of
ESBL
Number of isolates
Percentage
positive
of
4.9
1203
4.2
192
2017
4.2
7.9
4966
3.3
409
255
44
43.9
771
114
4.7
25.4
127
40
233
10.0
664
47.3
1239
6.7
897
45.4
2026
8.5
946
22.6
3822
5.3
946
20.0
4604
1.2
6121
268
196
43
560
11.4
8.2
<1
48.8
24.6
1962
619
571
530
1104
1.55
0.8
<1
1.1
7.9
678
25.2
1337
10.1
559
124
472
51
11.3
13
427
177
702
23.6
11.9
11
ESBL
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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 .
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Screening using cefotaxime, cefpodoxime, ceftazidime, and aztreonam discs.
multiple 3GC agents reliably improves sensitivity by offering wider ESBL substrate base.
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A disc zone diameter difference of ≥5 mm between a cephalosporin and its respective cephalosporinclavulanate 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)
◦ 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).
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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).
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E-test, microScan panels and other discs-based methods are
also used.
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
105
661
766
Total
Frequencies at assigned age categories
0-20 years old
21-40 years old
41-60 years old
≥61 years old
11
26
15
53
100%
% Resistant_HP
90%
% Resistant_CP
80%
Percentage resistance
70%
60%
50%
40%
30%
20%
10%
0%
AMP
AMC
TIM
TZP FOX CRO MEM GM
Major classes of antibiotics
CIP
FT
SXT
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 ESBLmediated antibiotic resistance;
◦ Robust antibiotic stewardship – appropriate use of antibiotics
◦ Effective infection control measures in hospitals – effective preventive
measures to curb transmission;
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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???
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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;
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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.