Extended Spectrum -Lactamases: Challenges in Laboratory Detection and Implications on Therapy

Dr. Iman M. Fawzy Clinical Pathology MD, PhD Mansoura, Egypt

ESBL
Extended spectrum -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.

-lactam antibiotics
Penicillin Cephalosporin Monobactam Carbapenem

 lactamases
Beta lactamases are enzymes produced by some bacteria that hydrolyze beta lactam antibiotics Penicillinases, Cephalosporinases Extended spectrum -lactamases (ESBL) Metallo 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 -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 intraabdominal 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-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

Functional Major Molecular group subgroup class Functional group Inhibition by clavulanate

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)
Penicillinases, confer resistance to all penicillins, primarily from Staphylococcus and enterococci Broad-spectrum -lactamases (penicillinases/cephalosporinases) , primarily from GNB. ESBLs, confer resistance to oxyimino-cephalosporins and monobactams. Inhibitor-resistant TEM (IRT) -lactamases Carbenicillin-hydrolyzing enzymes

2a 2b 2be 2br 2c

A A A A A

+ + + - (+ for tazobactam) +

Major groups of -lactamases

Functional Major Molecular group subgroup class Functional group Inhibition by clavulanate

2d
2e

D
A

Cloxacillin- (oxacillin)- hydrolyzing enzymes

Cephalosporinases, confer resistance to monobactams

+/+

2f
3 3a, 3b, 3c

A
B

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

Metallo--lactamases (zinc based carbapenemases), confer resistance to carbapenems and all -lactam classes, except monobactams. Miscellaneous unsequenced enzymes that do not fit into other groups

+
-

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

Selected -lactamases of gram-negative bacteria

-lactamase Broadspectrum Examples TEM-1, TEM-2, SHV-1 Substrates Penicillin G, aminopenicillins, carboxypenicillins, piperacillin, narrow-spectrum cephalosporins Broad-spectrum group plus cloxacillin, methicillin, and oxacillin Inhibition by Amblers class / clavulanate* Bushs class +++ A / 2b

OXA family

D / 2d

Extendedspectrum

TEM family, SHV family

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

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

++++

A / 2be

CTX-M family OXA family

++++ + ++++

A D / 2d A

Others (PER-1, PER-2, Same as for TEM family and SHV family BES-1, GES/IBC family, SFO-1, TLA-1, VEB-1, VEB2) *+, +++ , 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 Amblers clavulanate* class/ Bushs class 0 C/ 1

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

Carbapenemase IMP family, VIM family, Expanded-spectrum group plus GIM-1, SPM-1 (metallo-enzymes) cephamycins and carbapenems KPC-1, KPC-2, KPC-3 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 Same as for IMP family, VIM family, GIM-1, and SPM-1

0 +++ +

B/3 A / 2f D / 2d

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

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

Common ESBL producers

Klebsiella pneumoniae Escherichia coli Proteus mirabilis Enterobacter cloacae Non-typhoidal Salmonella (in some countries)

15

Common ESBL producers

Type
TEM, SHV

Major sources
E. coli, K. pneumoniae

Cefotaxime hydrolyzing S. Typhimurium, E. coli, K. pneumoniae (CTX-M)

Oxacillin hydrolyzing (OXA)

PER-1 PER-2 VEB-1

P. aeruginosa
P. aeruginosa, A. baumanii, S. Typhimurium S. Typhimurium E. coli, P. aeruginosa

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 -lactams co-presence of other resistance mechanisms (other -lactamases, efflux, altered permeability)

Survival of the fittest

Resistant bacteria survive, susceptible ones die

Mutant emerges slowly

Sensitive cells killed by antibiotic

Mutants progeny overrun

The Fight
PG

cell

LYSIS

The Fight
PG

-lactamase N cell

The Fight
PG

-lactamase N Inhibitor cell

The Fight
PG

-lactamase Inhibitor

cell

LYSIS

Sites of infection
Intra abdominal infections 6% Pneumonia 11% Skin and soft tissues 12% Others 5%

UTI 55%

Bactremia 11%

Laboratory Detection of ESBL

Phenotypic Methods

Genotypic Methods

Screening methods

Confirmatory 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 disk
Disc with cephalosporin + clavulanic acid

Disc with cephalosporin alone

CLSI 2013

CLSI 2013

Positive ESBL

Cefotaxime/CA

Ceftaz
Cefotax

Ceftaz/CA

Ceftaz/CA

Difference > 5 mm Positive ESBL Cefotax/CA

Ceftaz

Cefotax Cefotaxime/CA

Negative ESBL

Ceftaz/CA Ceftaz

Cefotax

Difference > 5 mm

Cefotaxim

Ceftazidim

Cefotaxim + Clav

Ciftazidim + Clav

Difference > 5 mm
Difference > 5 mm

Phenotypic conformation
2- Double disk approximation or double disk synergy
Disk of 3rd cephalosporin placed 30 mm from amoxicillinclavulanic acid

Result: Enhanced inhibition (A keyhole or ghost zone)

Ceftriaxone

Amox-clav

Cefotaxime

Ceftazidime

Azteonam

Ceftazidim

Augmentin

Cefotaxim

Augmentin Ceftazidime Cefotaxime

Ceftriaxone

Cefotaxime

Augmentin

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 Microdilution
MIC 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 g/mL Ceftazidime + Clavulanate= 1 g/mL Or MIC ratio8

4- MIC broth dilution

MIC 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

MIC =16

Ceftaz

MIC= 0.25

Ceftaz/CA

Other confirmatory methods

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

No inhibitor Mercaptoacetic acid to inhibit MBL Clavulanate to inhibit ESBL

Boronic acid to inhibit AmpC

Other confirmatory methods

Brilliance ESBL agar identification of ESBLproducing 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

Other confirmatory methods

6- Automated instruments
Measure MICs and compare the growth of bacteria in presence of cephalosporin vs. cephalosporin clavulanic acid

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

Pitfalls in ESBL tests

AmpC -lactamases third-generation cephalosporins: resistance , cephamycins, e.g. a cefoxitin: resistance Cefepime: sensitive. Carbapenemases The presence of ESBLs may also be masked by carbapenemases

ESBLs vs AmpCs
ESBLs
Inhibitors (pip/tazo, amp/sulbactam, amox/clav)

AmpCs R R R

S S R

Cefoxitin, cefotetan Ceftazidime, ceftriaxone

Cefepime

S/R

Pitfalls in ESBL tests

ESBL+ AmpC -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.

Pitfalls in ESBL tests

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.

ESBL+ AmpC

Amox-Clav Cefepime

ESBL+ AmpC

ESBL+ AmpC
Cefotaxime Cefoxitin

Cefipime

Augmentin Cefpodoxime + Clavulanic

Ceftazidime

Cefpodoxime

ESBL+ AmpC
ESBL and AmpC
ESBL positive clavulanate enhancement present AmpC positive cefepime: S cefoxitin: R

ESBL+ AmpC
AmpC Fox: R Clav: R

ESBL Zone enhancement

AmpC
AmpC
cefepime : S cefoxitin : R no clavulanate enhancement=

ESBL negative

ESBL+ Carbapenemase
ESBL + carbapenemases
ESBL positive clavulanate enhancement present carbapenemase production resistance to carbapenem agents

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 highinoculum, 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 carbapenemresistant 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 multiresistance. Many ESBL producing organisms also express AmpC -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/-lactamase inhibitors can be predicted oral carbapenems are urgently needed

Recommendations
Empirical treatment strategies may need to be rethought 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):159177; . Drew RH. J Manag Care Pharm. 2009;15(2 Suppl):S18S23; Drew RH et al. Pharmacotherapy. 2009;29(5):593607.

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 ESBLproducing 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