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 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.

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 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).

5. What are ESBLs?
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.

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.
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.

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 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.

13. Classification of β-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. Functional group or subgroup Representative enzymes
Classification of β-lactamases contd… Major classes of β-lactamases of clinical significance
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 30
SHV-1, SHV-11, SHV-89 37
SHV-2, SHV-3, SHV-115 5
SHV-10, SHV-72
CTX-M 90
CTX-M-1, CTX-M-44 (Toho-1) to CTX- M-92
PER
PER-1 to PER-5
VEB
GES 2f 7 15
VEB-1 to VEB-7
GES-2 to GES-7 (IBC-1) to GES-15
KPC
KPC-2 to KPC-10
SME 3
SME-1, SME-2, SME-3
OXA
2d, 2de, 2df
158 2d
OXA-1, OXA-2, OXA-10
2de
2df 48
OXA-11, OXA-14, OXA-15
OXA-23 (ARI-1), OXA-51, OXA-58
IMP 3a 26
IMP-1 to IMP-26
VIM 23
VIM-1 to VIM-23
IND 8
IND-1, IND-2, IND-2a, IND-3 to IND-7
[i] Enzyme families classified on the basis of amino acid structures (G. Jacoby and K. Bush,
[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)
AGAR - Australian Group on Antimicrobial Resistance, SMART - Study for Monitor Antimicrobial Resistance Trends, EARSS - European Antibiotic Resistance Surveillance System (EARSS).

18. Epidemiology of ESBL-producing Enterobacteriaceae contd…
Country
Study name or period
K. pneumoniae
E. coli
Number of isolates
Percentage of ESBL positive
Canada
SENTRY
386
4.9
1203
4.2
US and Canada
SENTRY 1998
192 -
USA
2017
7.9
4966
3.3
SENTRY 1997
409 44
771
4.7
Latin America
SENTRY
255
43.9
114
25.4
127 40
233
10.0
664
47.3
1239
6.7
897
45.4
2026
8.5
Europe
946
22.6
3822
5.3
Italy
1999
20.0
4604
1.2
Spain
EARSS 2001
1962
1.55
France
6121
11.4
Germany
PEG 2001
268
8.2
619
0.8
Netherlands
1997
196
<1
571
Turkey 43
48.8
530
1.1
Western Pacific area
560
24.6
1104
Asian Pacific area
SENTRY
678
25.2
1337
10.1
China
559 51
427
23.6
Taiwan
2000
124
11.3
177
11.9
Hong Kong
1998
472 13
702 11

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 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.

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

21. Frequencies at assigned age categories
Snapshots from ESBL study Summarized results of all isolates grouped by setting (hospital vs. community), ESBL-producer status, and by age categories
Setting
ESBL positive
ESBL negative
Total
Hospital 30
259
289
community 75
402
477
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
ESBL positive: 10.4% (30/289) of isolates_HP, 15.7% (75/477) of isolates recovered from CP, > 1/2 (50.5% i.e. (53/105)) of all ESBL-positive isolates were isolated from patients aged 61 years and over. Within this sub-group, 75% of the patients were aged in their 70s.

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.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.