Extended Spectrum β-Lactamases:

Challenges in Laboratory Detection and Implications on

Therapy

Dr. Iman M. FawzyClinical Pathology MD, PhD

Mansoura, Egypt

ESBL

Extended spectrum β-lactamase (ESBL)-producing organisms pose unique challenges to

clinical microbiologists, clinicians, infection control professionals and antibacterial-discovery scientists.

Why we need esbl detection?

• ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world

• ESBL pose challenging infection control issues.

• ESBLs are clinically significant and indicate the appropriate antibacterial agents.

• Unfortunately, the laboratory detection of ESBLs can be complex and, at times, misleading.

– Penicillin– Cephalosporin– Monobactam– Carbapenem

β-lactam antibiotics

β lactamases

Beta lactamases are enzymes produced by some bacteria that hydrolyze beta lactam antibiotics

– Penicillinases, Cephalosporinases– Extended spectrum β-lactamases (ESBL)– Metallo β lactamases– Amp C– Carbapenemase

Definition of ESBL• ESBLs are enzymes

– hydrolyzing most penicillins and cephalosporins, and monobactam (aztreonam).

– but not cephamycins and carbapenems

– Susciptable to β-lactamase inhibitors (clavulanate, sulbactam and tazobactam)

Clinical significance

• ESBLs destroy cephalosporins, main hospital antibiotics, given as first-line agents to many severely-ill patients, including those with intra-abdominal infections, community-acquired pneumonias and bacteraemias.

• Delayed recognition inappropriate treatment of severe infections caused by ESBL producers with cephalosporins ↑↑mortality .

Clinical significance

• ESBL-mediated resistance is not always obvious to all cephalosporins in vitro.

• Many ESBL producers are multi-resistant to non-β-lactam antibiotics such as quinolones, aminoglycosides and trimethoprim, narrowing treatment options.

Spread

• direct and indirect contact

• with colonized/infected patients and

• contaminated environmental surfaces. • ESBLs are most commonly spread via unwashed

hands of health care providers.

Risk factors• Critically ill patients, Immunosuppression• Prolonged hospital or ICU unit stay • Invasive procedures: intubation, mechanical

ventillation, catheter • Long-term dialysis within 30 days • Family member with multidrug-resistant pathogens • Prior antibiotic use in last 3 months• High frequency of antibiotic resistance in the

community or in the specific hospital unit• Patient who previously had an antibiotic-resistant

organism (e.g., MRSA, VRE)

Major groups of -lactamases Functiona

l grou

p

Major

subgroup

Molecular class

Functional group

Inhibition by

clavulanate

1 C Cephalosporinases, often chromosomal enzymes in GNB but may be plasmid-encoded, confer resistance to all classes of -lactams, except carbapenems (unless combine with porin change)

-

2 2a A Penicillinases, confer resistance to all penicillins, primarily from Staphylococcus and enterococci

+

2b A Broad-spectrum -lactamases (penicillinases/cephalosporinases) , primarily from GNB.

+

2be A ESBLs, confer resistance to oxyimino-cephalosporins and monobactams.

+

2br A Inhibitor-resistant TEM (IRT) -lactamases

- (+ for

tazobactam

)

2c A Carbenicillin-hydrolyzing enzymes +

Functiona

l grou

p

Major

subgroup

Molecular class

Functional group

Inhibition by

clavulanate

2 2d D Cloxacillin- (oxacillin)- hydrolyzing enzymes

+/-

2e A Cephalosporinases, confer resistance to monobactams

+

2f A Carbapenem-hydrolyzing enzymes with active site serine (serine based carbapenemases)

+

3 3a, 3b, 3c

B Metallo--lactamases (zinc based carbapenemases), confer resistance to carbapenems and all -lactam classes, except monobactams.

-

4 Miscellaneous unsequenced enzymes that do not fit into other groups

-

Functional group classified by Bush-Jacoby-Medeiros.Molecular group classified by Ambler.

Major groups of -lactamases

Selected -lactamases of gram-negative bacteria-lactamase

Examples Substrates Inhibition by clavulanate*

Ambler’s class / Bush’s class

Broad-spectrum

TEM-1, TEM-2, SHV-1

Penicillin G, aminopenicillins, carboxypenicillins, piperacillin, narrow-spectrum cephalosporins

+++ A / 2b

OXA family Broad-spectrum group plus cloxacillin, methicillin, and oxacillin

+ D / 2d

Extended-spectrum

TEM family, SHV family

Broad-spectrum group plus oxyimino-cephalosporins, and monobactam (aztreonam)

++++ A / 2be

CTX-M family Expanded-spectrum group plus, for some enzymes, cefepime

++++ A

OXA family Same as for CTX-M family

+ D / 2d

Others (PER-1, PER-2, BES-1, GES/IBC family, SFO-1, TLA-1, VEB-1, VEB2)

Same as for TEM family and SHV family

++++ A

*+, +++ , and ++++ denote relative sensitivity to inhibition.

Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

Selected -lactamases of gram-negative bacteria-

lactamase

Examples Substrates Inhibition by clavulanate*

Ambler’s

class/ Bush’s class

AmpC ACC-1, ACT-1, CFE-1, CMY family, DHA-2, FOX family, LAT family, MIR-1, MOX-1, MOX-2

Expanded-spectrum group plus cephamycins

0 C / 1

Carbapenemase

IMP family, VIM family,GIM-1, SPM-1 (metallo-enzymes)

Expanded-spectrum group plus cephamycins and carbapenems

0 B / 3

KPC-1, KPC-2, KPC-3

Same as for IMP family, VIM family, GIM-1, and SPM-1

+++ A / 2f

OXA-23, OXA-24, OXA-25,OXA-26, OXA-27, OXA-40,OXA-48

Same as for IMP family, VIM family, GIM-1, and SPM-1

+ D / 2d

*+, +++ , and ++++ denote relative sensitivity to inhibition.

Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

15

• Klebsiella pneumoniae

• Escherichia coli• Proteus mirabilis• Enterobacter cloacae• Non-typhoidal

Salmonella (in some countries)

Common ESBL producers

Type Major sources

TEM, SHV E. coli, K. pneumoniae

Cefotaxime hydrolyzing (CTX-M)

S. Typhimurium, E. coli, K. pneumoniae

Oxacillin hydrolyzing (OXA)

P. aeruginosa

PER-1PER-2

P. aeruginosa, A. baumanii, S. TyphimuriumS. Typhimurium

VEB-1 E. coli, P. aeruginosa

Common ESBL producers

Mechanisms of resistance

• The majority of ESBLs are acquired enzymes, encoded by plasmids.

• Different resistance phenotypes to:– Different expression levels– Different biochemical characteristics such as

activity against specific β-lactams– co-presence of other resistance mechanisms

(other β-lactamases, efflux, altered permeability)

Survival of the fittest

Resistant bacteria survive, susceptible ones die

Mutant emergesslowly

Sensitive cellskilled by antibiotic

Mutant’s progenyoverrun

The Fight

cell

PG

NO LYSIS

The Fight

cell

PG

NO

β-lactamase

cell

PG

NO

β-lactamase

Inhibitor

The Fight

cell

PG

NO

β-lactamase

Inhibitor

LYSIS

The Fight

Sites of infection

UTI55%

Bactremia11%

Skin and soft tis-sue

s12%

Pneumonia

11%

In-tra ab-domi-nal in-fec-tion

s 6%

Others5%

Laboratory Detection of ESBL

Phenotypic MethodsScreeni

ng metho

ds

Genotypic Methods

CLSI 2013

CLSI 2013

Confirmatory methods

• 1- Combination disk– Uses 2 disks of 3rd cephalosporin alone and

combined with clavulanic acid– An increase of ≥5 mm in zone inhibition with use

of the combination diskDisc with cephalosporin

+ clavulanic acid

Disc with cephalosporin

alone

CLSI 2013

CLSI 2013

Ceftaz/CA

Cefotaxime/CA

Ceftaz

Cefotax

Ceftaz/CA

Ceftaz

Negative ESBL

Positive ESBL

Cefotax

Cefotaxime/CA

Difference > 5 mmCeftaz/CA

Cefotax/CA

Ceftaz Cefotax

Positive ESBL

Ciftazidim + Clav

Cefotaxim + Clav

CeftazidimCefotaxim

Difference > 5 mm

Difference > 5 mm

Difference > 5 mm

2- Double disk approximation or double disk synergy– Disk of 3rd cephalosporin placed 30 mm from amoxicillin-

clavulanic acid

– Result: Enhanced inhibition (A keyhole or ghost zone)

Phenotypic conformation

Amox-clavCefotaxime

Ceftriaxone

Ceftazidime

Azteonam

http://cmr.asm.org/content/vol14/issue4/images/large/cm0410042005.jpeg

CeftazidimAugmentin Cefotaxim

CeftazidimeAugmentin

Cefotaxime

AugmentinCefotaxime

Ceftriaxone

AMC AMC AMC

30 mm distance between discs (center to center)

20 mm distance between discs (center to center)

AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime; CPO, cefpirome.

30 mm distance between discs (center to center)

20 mm distance between discs (center to center)

AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime; CPO, cefpirome.

Phenotypic conformation

• 3- Broth MicrodilutionMIC of 3rd cephalosporin alone and combined with clavulanic acid

>3-two fold serial dilution decrease in MIC of either cephalosporin in the presence of clavulanic acid compared to its MIC when tested alone.

Ceftazidim MIC =8 μg/mL

Ceftazidime + Clavulanate= 1 μg/mL

Or MIC ratio≥8

4- MIC broth dilutionMIC of 3rd cephalosporin alone and combined with clavulanic acid

MIC of 3rd cephalosporin alone and combined with clavulanic acid

A decrease in the MIC of the combination of > 3-two fold dilutions

Phenotypic conformation

• 5- E-test (MIC ESBL strips)• Two-sided strip containing cephalosporin on one side

and cephalosporin -clavulanic acid on the other• MIC ratio ≥8•>8 fold reduction in MIC in presence of CA= ESBL• or Phantom zone (deformed ellipse)

Cefotaxime

Cefotaxime+

clavulanate

Ceftaz/CA

CeftazMIC =16

MIC= 0.25

Other confirmatory methods

No inhibitor

Mercaptoacetic acid to inhibit MBL

Clavulanate to inhibit ESBL

Boronic acid to inhibit AmpC

Cica b-Testuses the chromogenic cephalosporin HMRZ-86,4,5 + inhibitors to determine rapidly whether an isolate has a metallo-β-lactamase (MBL), ESBL, or hyperproduced AmpC enzyme , a control strip with no inhibitor, to detect hydrolysis of extended-spectrum cephalosporins

Other confirmatory methodsBrilliance ESBL agar • identification of ESBL-

producing E. coli, Klebsiella, Enterobacter, Serratia and Citrobacter group, directly from clinical samples.

• two chromogens that specifically target enzymes green and blue colonies

• Negative pink. • Proteus, Morganella and

Providencia tan-coloured colonies with a brown halo

6- Automated instruments• Measure MICs and compare the growth of bacteria in

presence of cephalosporin vs. cephalosporin -clavulanic acid

Other confirmatory methods

Vitek ESBL confirmatory test Phoenix ESBL test (BD)

Microscan ESBL Panel

Genotypic confirmation

• Molecular detection – PCR– RFLP– gene sequencing– DNA microarray-based method

• Targets specific nucleotide sequences to detect different variants of TEM and SHV genes

Control strains

AmpC β-lactamases• third-generation cephalosporins: resistance , • cephamycins, e.g. a cefoxitin: resistance • Cefepime: sensitive.

CarbapenemasesThe presence of ESBLs may also be masked by

carbapenemases

Pitfalls in ESBL tests

ESBLs vs AmpCs

ESBLs AmpCs

Inhibitors (pip/tazo, amp/sulbactam, amox/clav) S R

Cefoxitin, cefotetan S R

Ceftazidime, ceftriaxone R R

Cefepime S/R S

Pitfalls in ESBL tests

ESBL+ AmpC β -lactamases: • Especially in Enterobacter spp., Citrobacter,

Morganella, Providencia and Serratia. • The AmpC enzymes may be induced by

clavulanate (which inhibits them poorly) and may then attack the cephalosporin, masking synergy arising from inhibition of the ESBL.

• Screening criterion for ESBL presence among AmpC producing Enterobacter, C. freundii and ‑Serratia is Cefepime MIC > 1 ug/ml (inhibition

zone< 26 mm).• Use of Cefepime is more reliable to detect

these strains because high AmpC production has little effect on cefepime activity.

Pitfalls in ESBL tests

Amox-Clav

Cefepime

ESBL+ AmpC

ESBL+ AmpC

CefoxitinCefotaxime

Cefipime

Ceftazidime Cefpodoxime

Cefpodoxime + Clavulanic

Augmentin

ESBL+ AmpC

ESBL and AmpC

ESBL positiveclavulanate enhancement present

AmpC positive cefepime: Scefoxitin: R

ESBL+ AmpC

ESBL+ AmpCAmpCFox: RClav: R

ESBLZone enhancement

AmpCcefepime : S cefoxitin : R

no clavulanate enhancement= ESBL negative

AmpC

ESBL + carbapenemases

ESBL positive clavulanate enhancement present carbapenemase production resistance to carbapenem agents

ESBL+ Carbapenemase

ESBLs and the inoculum effect

• In vitro: the MICs of cephalosporins rise as the inoculum of ESBL- producing organisms increases.

• In vivo: Intra-abdominal abscesses and pneumonia are some of the clinical settings where organisms are present in high-inoculum, physicians should avoid cephalosporins if risk of ESBL-producing organism is suspected.

• Two antibiograms of ESBL producing strain. Note the difference in zones and synergistic effect around the amoxicillin-clavulanate pills due to different inoculum concentration.

Reporting

If ESBL: Resistant, for all penicillins, cephalosporins, and monobactams

Report beta lactam inhibitor drugs as they test.

If ESBL is not detected, report drugs as tested.

Treatment

Carbapenems are the drugs of choice.

Unfortunately, use of carbapenems has been associated with the emergence of carbapenem-resistant bacterial species

It may be advisable to use non carbapenem antimicrobials as the first line treatment in the less severe infections with ESBL producing strains.

-lactam/-lactamase inhibitor on treatment of ESBL-producing organisms

• Most ESBLs are susceptible to clavulanate and tazobactam in vitro,

• nevertheless some ESBL producers are resistant to -lactamase inhibitor due to– Hyperproduction of the ESBLs → overwhelm inhibitor– Co-production of inhibitor-resistant penicillinases or

AmpC enzyme– Relative impermeability of the host strain

• -lactam/-lactamase inhibitor should not be used to treat serious infections with ESBL-producing organisms.

Summary of cephamycins on treatment of ESBL-producing organisms

• Limited clinical data• Generally effective against Enterobacteriaceae

producing TEM-, SHV-, and CTX-M-derived ESBLs• Reports of cephamycins resistance development

during prolonged therapy– Loss of outer membrane porin (porin deficient

mutant)– Acquisition of plasmid-mediated AmpC -

lactamase (ACT-1)

ESBL are Emerging Challenges

• multiple enzymes • High-Risk clones• globally disseminated• hospital, community acquired• High rates• Challenge of intestinal carriage • extra-human reservoirs

ESBL are more complex

• Antibacterial choice is often complicated by multi-resistance. – Many ESBL producing organisms also express

AmpC β-lactamases – may be co-transferred with plasmids mediating

aminoglycoside resistance. – there is an increasing association between ESBL

production and fluoroquinolone resistance

Prevention

– ICU is hot spot– Hands of healthcare workers, family, visitors– thermometer– Ultrasound gel– Flag records– Education – Contact precautions– Transfer between wards & hospitals

Still the best way to prevent spread of infections and drug resistance is ……

Prevention

Individual patient level

•Avoid use of cephalosporins, aztreonam•Avoid unnecessary use of invasive devices •Ensure good hand hygiene before and after patient-care activities

Institutional level•Restrict use of 3rd-generation cephalosporins•Isolation of patient•Investigate environmental contamination

Recommendations• Older agents such as aminoglycosides need

reappraisal to spare the selective pressures of a carbapenem.

• new trials of cephalosporin/β-lactamase inhibitors can be predicted

• oral carbapenems are urgently needed

Recommendations

• Empirical treatment strategies may need to be re-thought where there is a significant risk.

• Use a carbapenem until the infection has been proved NOT to involve an ESBL producer, then to step down to a narrower- spectrum ab .

Recommendations• Optimize appropriate use of antimicrobials

– The right agent, dose, timing, duration, route

• Help reduce antimicrobial resistance– The combination of effective antimicrobial

supervision and infection control has been shown to limit the emergence and transmission of antimicrobial-resistant bacteria

Dellit TH et al. Clin Infect Dis. 2007;44(2):159–177; . Drew RH. J Manag Care Pharm. 2009;15(2 Suppl):S18–S23; Drew RH et al. Pharmacotherapy. 2009;29(5):593–607.

Take Home Messages• ESBL-producing bacterial infection is an emerging

problem worldwide.• These organisms are associated with multi-drug

resistance causing high rate of mortality and treatment failure.

• The significant risk factors for ESBL-producing bacterial infection are prior use of antibiotics, especially 3rd generation cephalosporins, and critically ill or debilitated patients.

• Need the ESBL-laboratory testing for establish the problem.

• Carbapenems is the drug of choice for serious ESBL-producing bacterial infection.

• Avoiding overuse or misuse of 3rd generation cephalosporins and implementing isolation and contact precaution to prevent and control the ESBL outbreak.

THANK YOU