Esbl

Extended spectrum beta lactamases

1. Extended Spectrum β- Lactamases : Challenges in Laboratory 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 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 .

4. Penicillin Cephalosporin Monobactam Carbapenem β - lactam antibiotics

5. β lactam ase s 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

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 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 .

8. 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.

9. 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.

10. 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)

11. Major groups of - lactamases Functional group 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 +

12. Functional group 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

13. 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 B road-spectrum group plus cloxacillin, methicillin, and oxacillin + D / 2d Extended-spectrum TEM family, SHV family B road-spectrum group plus oxyimino-cephalosporins, and monobactam (aztreonam) ++++ A / 2be CTX-M family E xpanded-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.

14. 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 E xpanded-spectrum group plus cephamycins C / 1 Carbapenemase IMP family, VIM family, GIM-1, SPM-1 (metallo - enzymes) E xpanded-spectrum group plus cephamycins and carbapenems 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 15

16. 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-1 PER-2 P. aeruginosa, A. baumanii, S. Typhimurium S. Typhimurium VEB-1 E. coli, P. aeruginosa Common ESBL producers

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 the fittest Resistant bacteria survive, susceptible ones die Mutant emerges slowly Sensitive cells killed by antibiotic Mutant’s progeny overrun

19. The Fight cell PG N O LYSIS

20. The Fight cell PG N O β - lactamase

21. cell PG N O β - lactamase Inhibitor The Fight

22. cell PG N O β - lactamase Inhibitor LYSIS The Fight

23. Sites of infection

24. CLSI 2013

25. CLSI 2013

26. Confirmatory methods 1- Combination disk Uses 2 disks of 3 rd 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

27. CLSI 2013

28. CLSI 2013

29. Ceftaz/CA Cefotaxime/CA Ceftaz Cefotax Ceftaz/CA Ceftaz Negative ESBL Positive ESBL Cefotax Cefotaxime/CA Difference > 5 mm Ceftaz/CA Cefotax/CA Ceftaz Cefotax Positive ESBL

30. Ciftazidim + Clav Cefotaxim + Clav Ceftazidim Cefotaxim Difference > 5 mm

31. Difference > 5 mm Difference > 5 mm

32. 2- Double disk approximation or double disk synergy Disk of 3 rd cephalosporin placed 30 mm from amoxicillin- clavulanic acid Result: Enhanced inhibition (A keyhole or ghost zone) Phenotypic conformation

33. Amox-clav Cefotaxime Ceftriaxone Ceftazidime Azteonam

34. Ceftazidim Augmentin Cefotaxim

35. Ceftazidime Augmentin Cefotaxime

36. Augmentin Cefotaxime Ceftriaxone

37. AMC AMC AMC

38. 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.

39. 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.

40. 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 μ g/ mL Ceftazidime + Clavulanate = 1 μ g/ mL Or MIC ratio≥8 4- MIC broth dilution MIC of 3rd cephalosporin alone and combined with clavulanic acid MIC of 3 rd cephalosporin alone and combined with clavulanic acid A decrease in the MIC of the combination of > 3-two fold dilutions

41. 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)

42. Cefotaxime Cefotaxime + clavulanate

43. Ceftaz/CA Ceftaz MIC =16 MIC= 0.25

44. Other confirmatory methods No inhibitor Mercaptoacetic acid to inhibit MBL Clavulanate to inhibit ESBL Boronic acid to inhibit AmpC Cica b -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

45. Other confirmatory methods Brilliance 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

46. 6- Automated instruments Measure MICs and compare the growth of bacteria in presence of cephalosporin vs. cephalosporin - clavulanic acid Other confirmatory methods

47. Vitek ESBL confirmatory test Phoenix ESBL test (BD) Microscan ESBL Panel

48. 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

49. Control strains

50. 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 Pitfalls in ESBL tests

51. 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

52. 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.

53. 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

54. Amox-Clav Cefepime ESBL+ AmpC

55. ESBL+ AmpC

56. Cefoxitin Cefotaxime Cefipime Ceftazidime Cefpodoxime Cefpodoxime + Clavulanic Augmentin ESBL+ AmpC

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

58. ESBL+ AmpC AmpC Fox: R Clav : R ESBL Zone enhancement

59. AmpC cefepime : S cefoxitin : R no clavulanate enhancement= ESBL negative AmpC

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

61. 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.

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

63. 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.

64. 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.

65. - 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.

66. 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 )

67. ESBL are Emerging Challenges multiple enzymes High-Risk clones globally disseminated hospital , community acquired High rates Challenge of intestinal carriage extra-human reservoirs

68. 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

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

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

71. 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 3 rd -generation cephalosporins Isolation of patient Investigate environmental contamination

72. 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

73. 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 .

74. 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.

75. 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 3 rd 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 3 rd generation cephalosporins and implementing isolation and contact precaution to prevent and control the ESBL outbreak .

76. THANK YOU