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Esbl
Extended spectrum β-lactamases (ESBLs) pose challenges for detection and treatment. ESBLs hydrolyze many penicillins and cephalosporins but are inhibited by β-lactamase inhibitors. Delayed or incorrect detection of ESBL producers can lead to inappropriate cephalosporin treatment and worse outcomes. Laboratory detection of ESBLs is complex, requiring screening methods like combination disks or double disk synergy tests followed by confirmatory tests. Genotypic methods can also detect ESBL genes. Accurate detection is important for infection control and antibiotic stewardship given ESBL producers' resistance and transmission risks.
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Esbl
- 1. Extended Spectrum β-Lactamases: Challenges inLaboratory Detection and Implications on Therapy Dr. Iman M. Fawzy Clinical Pathology MD, PhD Mansoura, Egypt
- 2. ESBL Extended spectrum β-lactamase(ESBL)-producing organisms pose unique challenges to clinical microbiologists, clinicians, infection control professionals and antibacterial-discovery scientists.
- 3. Why we needesbl 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.
- 4. β-lactam antibiotics – Penicillin –Cephalosporin – Monobactam – Carbapenem
- 5. β lactamases Beta lactamasesare enzymes produced by some bacteria that hydrolyze beta lactam antibiotics – Penicillinases, Cephalosporinases – Extended spectrum β-lactamases (ESBL) – Metallo β lactamases – Amp C – Carbapenemase
- 6. 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)
- 7. Clinical significance • ESBLsdestroy 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 .
- 8. Clinical significance • ESBL-mediatedresistance 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.
- 9. Spread • direct andindirect contact • with colonized/infected patients and • contaminated environmental surfaces. • ESBLs are most commonly spread via unwashed hands of health care providers.
- 10. Risk factors • Criticallyill 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)
- 11. 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- +
- 12. 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 -
- 13. Selected -lactamases ofgram-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
- 14. Selected -lactamases ofgram-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.
- 15. Common ESBL producers •Klebsiella pneumoniae • Escherichia coli • Proteus mirabilis • Enterobacter cloacae • Non-typhoidal Salmonella (in some countries) 15
- 16. 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
- 17. 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)
- 18. Survival of thefittest Resistant bacteria survive, susceptible ones die Mutant emerges slowly Sensitive cells killed by antibiotic Mutant’s progeny overrun
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- 23. Sites of infection Intraabdominal infections 6% Others 5% Pneumonia 11% Skin and soft tissues 12% Bactremia 11% UTI 55%
- 24. Laboratory Detection ofESBL Phenotypic Methods Screening methods Genotypic Methods Confirmatory Methods
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- 27. 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
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- 30. 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
- 32. Difference > 5mm Cefotaxim Ceftazidim Cefotaxim + Clav Ciftazidim + Clav
- 33. Difference > 5mm Difference > 5 mm
- 36. Phenotypic conformation 2- Doubledisk approximation or double disk synergy – Disk of 3rd cephalosporin placed 30 mm from amoxicillinclavulanic acid – Result: Enhanced inhibition (A keyhole or ghost zone)
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- 46. 30 mm distancebetween 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.
- 47. 30 mm distancebetween 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.
- 48. 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
- 49. 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)
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- 53. 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
- 54. Other confirmatory methods BrillianceESBL 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
- 55. Other confirmatory methods 6-Automated instruments • Measure MICs and compare the growth of bacteria in presence of cephalosporin vs. cephalosporin clavulanic acid
- 56. Vitek ESBL confirmatory test PhoenixESBL test (BD) Microscan ESBL Panel
- 57. Genotypic confirmation • Moleculardetection – PCR – RFLP – gene sequencing – DNA microarray-based method • Targets specific nucleotide sequences to detect different variants of TEM and SHV genes
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- 59. Pitfalls in ESBLtests 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
- 60. 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
- 62. Pitfalls in ESBLtests 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.
- 63. Pitfalls in ESBLtests • 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.
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- 67. ESBL+ AmpC ESBL andAmpC ESBL positive clavulanate enhancement present AmpC positive cefepime: S cefoxitin: R
- 68. ESBL+ AmpC AmpC Fox: R Clav:R ESBL Zone enhancement
- 69. AmpC AmpC cefepime : S cefoxitin: R no clavulanate enhancement= ESBL negative
- 70. ESBL+ Carbapenemase ESBL + carbapenemases ESBLpositive clavulanate enhancement present carbapenemase production resistance to carbapenem agents
- 71. ESBLs and theinoculum 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.
- 72. • Two antibiogramsof ESBL producing strain. Note the difference in zones and synergistic effect around the amoxicillin-clavulanate pills due to different inoculum concentration.
- 74. Reporting If ESBL: Resistant, forall penicillins, cephalosporins, and monobactams Report beta lactam inhibitor drugs as they test. If ESBL is not detected, report drugs as tested.
- 75. Treatment Carbapenems are thedrugs 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.
- 76. -lactam/-lactamase inhibitor on treatmentof 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.
- 77. Summary of cephamycinson 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)
- 78. ESBL are EmergingChallenges • • • • • • • multiple enzymes High-Risk clones globally disseminated hospital, community acquired High rates Challenge of intestinal carriage extra-human reservoirs
- 79. ESBL are morecomplex • 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
- 80. Prevention – ICU ishot spot – Hands of healthcare workers, family, visitors – thermometer – Ultrasound gel – Flag records – Education – Contact precautions – Transfer between wards & hospitals
- 81. Still the bestway to prevent spread of infections and drug resistance is ……
- 82. Prevention Individual patient level •Avoiduse 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
- 83. Recommendations • Older agentssuch 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
- 84. Recommendations • Empirical treatmentstrategies 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 .
- 85. Recommendations • Optimize appropriateuse 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.
- 86. 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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Editor's Notes
- #7 Intrinsic resistance = inherent or innate (not acquired) resistance, which is reflected in wildtype antimicrobial patterns of all or almost all representatives of a species. Intrinsic resistance is so common that susceptibility testing is unnecessary.• Acquired resistance = antimicrobial resistancein a bacterium that was previously susceptiblethat occurs as a result of:– Chance gene mutation– Acquisition of R genes from another bacterium