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Information about Esbl
Health & Medicine

Published on February 28, 2014

Author: monamustafa9212



Extended spectrum beta lactamases

Extended Spectrum β-Lactamases: Challenges in Laboratory Detection and Implications on Therapy Dr. Iman M. Fawzy Clinical 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.

β-lactam antibiotics – Penicillin – Cephalosporin – Monobactam – Carbapenem

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 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-βlactam 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 Functi Major Molec onal subgr ular group oup class 1 2 Functional group Inhibit ion by clavula nate 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) - 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- +

Major groups of -lactamases Functi Major Molec onal subgr ular group oup class 2 Functional group Inhibit ion by clavula nate Cloxacillin- (oxacillin)- hydrolyzing enzymes A Cephalosporinases, confer resistance to monobactams + 2f 4 D 2e 3 2d +/- A Carbapenem-hydrolyzing enzymes with active site serine (serine based carbapenemases) + 3a, 3b, 3c B 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 -

Selected -lactamases of gram-negative bacteria lactam ase Examples Broad- TEM- , TEM- , spectru SHVm OXA family Extend TEM family, SHV edfamily spectru m Substrates Penicillin G, aminopenicillins, carboxypenicillins, piperacillin, narrowspectrum cephalosporins Broad-spectrum group plus cloxacillin, methicillin, and oxacillin Inhibiti on by clavula nate* Ambler’s class / Bush’s class +++ A / 2b + D / 2d Broad-spectrum ++++ A / 2be group plus oxyiminocephalosporins, and Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8. monobactam

Selected -lactamases of gram-negative bacteria lactamas e Examples Substrates Inhibiti Ambler’ on by s class/ clavula Bush’s nate* class AmpC ACC- , ACT- , CFE- Expandedspectrum group CMY family, DHA- , plus FOX cephamycins family, LAT family, MIRMOX- , MOX- 0 C/ 1 Carbapen emase IMP family, VIM family, GIM- , SPM(metallo-enzymes) 0 B/3 +++ A / 2f Expandedspectrum group plus cephamycins and carbapenems KPC- , KPC- , KPC- Same as for IMP 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) P. aeruginosa PER-1 PER-2 P. aeruginosa, A. baumanii, S. Typhimurium S. Typhimurium VEB-1 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 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 emerges slowly Sensitive cells killed by antibiotic Mutant’s progeny overrun

The Fight PG O N cell LYSIS

The Fight PG β-lactamase O N cell

The Fight PG β-lactamase O N Inhibitor cell

The Fight PG β-lactamase Inhibitor O N cell LYSIS

Sites of infection Intra abdominal infections 6% Others 5% Pneumonia 11% Skin and soft tissues 12% Bactremia 11% UTI 55%

Laboratory Detection of ESBL Phenotypic Methods Screening methods Genotypic 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 Ceftaz Cefotax/CA 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 Ceftazidime Azteonam Cefotaxime

Ceftazidim Augmentin Cefotaxim

Augmentin Ceftazidime Cefotaxime

Ceftriaxone Cefotaxime Augmentin


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 μg/mL Ceftazidime + Clavulanate= 1 μg/mL Or MIC ratio≥8 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-β-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 β-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 AmpCs Inhibitors (pip/tazo, amp/sulbactam, amox/clav) S R Cefoxitin, cefotetan S R Ceftazidime, ceftriaxone R R S/R S Cefepime

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.

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 Cefotaxime Cefipime Cefoxitin 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 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 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):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 ESBLproducing bacterial infection. • Avoiding overuse or misuse of 3rd generation cephalosporins and implementing isolation and contact precaution to prevent and control the ESBL outbreak.


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