The document discusses extended spectrum beta-lactamases (ESBL), emphasizing their evolution, classification, detection methods, and clinical implications in treating infections caused by resistant bacteria. It highlights the importance of laboratory detection and appropriate antibiotic use, particularly avoiding third and fourth-generation cephalosporins for ESBL-producing organisms. Additionally, it outlines various therapeutic options and the significance of effective infection control measures in healthcare settings.

1. Extended Spectrum Beta Lactamases (ESBL): From Petri Dish to Patient Dr. Ashok Rattan, Super Religare Laboratories, India

2. Sir Alexander Fleming Ernest Boris Chain Sir Howard Florey

3. Beta lactams & β lactamases Oxyimino aminothiazolyl caphalosporin Cefuroxime, cefotaxime, ceftriaxone, Ceftazidime, cefepime, cefpirome

4. TEM & SHV β lactamases

8. ESBL An ESBL is any β lactamase, ordinarily acquired and not inherent to a species, that can rapidly hydrolyse or confer resistance to oxyimino cephalosporins David Liverpool 2008

9. Plasmid-mediated TEM and SHV  -lactamases Ampicillin 1965 TEM-1 E.coli S.paratyphi 1970s TEM-1 Reported in 28 Gm(-) sp 1983 ESBL in Europe 1988 ESBL in USA 2000 > 130 ESBLs Worldwide Extended-spectrum Cephalosporins 1963 Evolution of  -Lactamases Look and you will find ESBL

10. Classification of β lactamases Richards and Sykes (1971) substrate Ambler (1969) structure Bush, Jacoby, Medeiros (1995) Substrate; correlation with molecular structure 150 TEM; 88 SHV; 88 OXA, 53 CTX-M; 22 IMP; 12 VIM + smaller number of other enzymes (http://www.lahey.org)

11. β lactamases Penicillinases, Cephalosporinases ESBL Metallo β lactamases Amp C Carbapenemase

12. Redefining ESBL Balancing science and clinical need ESBL A ESBL M ESBL M-C ESBL M-D ESBL CARBA

14. Laboratory Detection of ESBL Phenotypic Methods Screening methods Confirmatory Methods Genotypic Methods

15. Double disk synergy test Jarlier V et al: Rev Infect Dis 1988;10: 867 - 878

16. E test Ceftaz/CA Ceftaz > 8 fold reduction in MIC in presence of CA= ESBL Cefotaxime/CA Cefepime/CA Cefotaxime Cefepime

17. Combination disk method Carter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232 Difference > 5 mm

22. Vitek ESBL confirmatory test Vitek 2 Advanced Expert Simultaneous assessment of antibacterial activity of cefepime, cefotaxime & ceftazidime either alone or + CA  interpretation through advanced expert system

23. Phoenix ESBL test (BD) Uses growth response in 5 wells containing to cefpodoxime, ceftazidime, Ceftazidime + CA, cefotaxime + CA ceftriaxone + CA Results are interpreted through a computer system

24. Microscan ESBL Panel Panels contain ceftazidime alone and in combination with CA And cefotaxime alone and in combination with CA

25. Routine “research” laboratory in a teaching hospital ?

26. Amp C

30. Cefoxitin Broader spectrum of activity than most first generation cephalosporins Greater resistance to  -lactamase enzymes The 7-methoxy group may act as a steric shield The urethane group is stable to metabolism compared to the ester Introducing a methoxy group to the equivalent position of penicillins (position 6) eliminates activity. Cephamycins Second Generation Cephalosporins

31. Aminothiazole ring enhances penetration of cephalosporins across the outer membrane of Gram -ve bacteria May also increase affinity for the transpeptidase enzyme Good activity against Gram -ve bacteria Variable activity against Gram +ve cocci Lack activity vs MRSA Generally reserved for troublesome infections Third Generation Cephalosporins Oximinocephalosporins R Aminothiazole ring

35. Labor intensive, can be technically challenging, can be difficult to interpret manual methods The gold standard, can detect all variants Nucleotide sequencing Requires a large number of oligonucleotide primers Can distinguish between a number of SHV variants LCR Requires special electrophoresis conditions Can distinguish between a number of SHV variants PCR-SSCP Nucleotide changes must result in altered restriction site for detection Easy to perform, can detect specific nucleotide changes PCR-RFLP Requires specific oligonucleotide probes, labor intensive, cannot detect new variants Detects specific TEM variants Oligotyping Cannot distinguish between ESBLs and non-ESBLs, cannot distinguish between variants of TEM or SHV Easy to perform, specific for gene family (e.g., TEM or SHV) PCR Labor intensive, cannot distinguish between ESBLs and non-ESBLs, cannot distinguish between variants of TEM or SHV Specific for gene family (e.g., TEM or SHV) DNA probes Disadvantages Advantages Test

41. A. TEM, B. SHV

42. Clinical implications It is important to identify ESBL producing isolates ? No ! particularly if there is no indication of such organisms in the hospital in question K Bush: Eur J Clin Microbiol Infect Dis 1996, 15: 361 – 364

43. Emery CL & Weymouth LA JCM 1997; 35: 2061 – 2067 Detection & clinical significance of ESBL in a tertiary care medical center (Virginia, US) Prevalence of ESBL unknown, national guidelines for testing & reporting not developed 750 bedded hospital, 6M, in vitro , MIC (if > 1 ug/ml) -> ESBL by DDS & response from records 50 isolates from 23 pts (1.2%) Prevalence of ESBL is low Respond favorably to antibiotic therapy based on MIC No clinical failure May not be clinically necessary or cost effective to institute additional testing to detect ESBL production on a routine basis.

44. 12 hospitals in USA, Taiwan, Australia, South Africa, Turkey, Belgium & Argentina Jan ’96 – Dec ’97 Blood culture positive for K.pneumoniae Monitored for 1 M, 188 observational prospective study In vitro susceptibility of isolates (%) Antibiotics <1 2 4 8 16 32 >64  g / ml Cefotaxime 5.6 18.1 5.6 19.4 13.9 15.3 22.2 Ceftriaxone 4.2 5.6 15.3 11.1 16.7 15.3 31.9 Ceftazidime 4.2 4.2 5.6 5.6 8.3 5.6 66.5 Cefepime 23.6 22.2 23.6 9.7 4.2 9.7 6.9 Cefoxitin 0 2.8 59.7 18.1 9.7 4.2 5.6 Cefotetan 65.3 19.4 8.3 1.4 1.4 2.8 1.4 Paterson et.al. 2001. JCM.39:2206-12

45. Outcome of Serious ESBL Infections When Treated with 3 rd gen Cephalosporin Paterson et.al. 2001. JCM.39:2206-12 54% (15/28) Total 18% (2/11) 27% (3/11) ≤ 1 S 0 (0/3) 67% (2/3) 4 S 0 (0/3) 33% (1/3) 2 S 33% (2/6) 100% (6/6) 8 S Died within 14 d of bacteremia Failure MIC ( µ g/ml) 3 rd gen cef

47. Fatality rates for episodes of bloodstream infections Kim et. al. 2002. AAC.46:1481 – 91. 4 / 78 1 / 9 7 / 39 5 / 6 Subtotal 4 / 36 0 / 25 1 / 1 1 / 8 2 / 2 Competent 13 / 96 4 / 53 0 / 8 6 / 31 3 / 4 Compromised - + - + Shock -> Total Non ESBL ESBL group No. of fatal episodes (%) Immune status

48. Molecular correlation for the treatment outcomes in bloodstream infections caused by E. coli & K. pneumoniae with reduced susceptibility to Ceftazidime UCLA, 12 years retrospective study 23 E. coli , 13 K. pneumoniae ; CTZ MIC > 2  g/ml CTZ treatment was associated with failure of therapy in all patients Wong Beringer et. al. 2002. CID.34:135 - 46 .

49. Conclusion: Do not use 3 rd or 4 th generation cephalosporins if infection is caused by ESBL +ve bacteria, even if sensitive in vitro Message to the lab: Always test for ESBL in E.coli & K.pneumoniae (Screen & confirm)

50. Therapeutic options in infections caused by ESBL producing E.coli, K.pneumoniae Cephalosporins (3 rd or 4 th generation) Cephamycins  lac +  lac inhibitor combinations Aminoglycosides Fluoroquinolones Carbapenems

51. Available  lac +  lac inhibitors Amoxacillin + Clavulonic acid Ampicillin + Sulbactum Ticarcillin + Clavulonic Acid Cefoperazone + Sulbactum Piperacillin + Tazobactum Cefotaxime + Sulbactum (India only)

52. MIC (  g/ml) of Ceftazidime alone and in combination (4 +1) with Pot. Clavulonic Acid MIC E.coli 16 E.coli 435 E.coli 387 E.coli 324 kleb. pneum.700603 Kleb. pneum.WHO1 K.aeruginosa KL 139 Klebseilla spp. 53 Proteus spp. Proteus spp. P23 Proteus spp. P18 Proteus spp. P10 Pseudomonas 210 Pseudomonas 204 Pseudomonas 35 Serratia 196 Serratia 63 H S.aureus 240 S.aureus 29213 Ceftazidime Cefta.+ Pot. Clav 32 8 16 0 64 128 256

53. In vivo Escherichia coli lethal mouse model 0 6

54. Literature Conclusion: Can be overwhelmed by  lactamases Inoculum effect present Selection of porin less mutants Personal data Recommendations: Serious infections with ESBL producing organisms may not respond

55. Association of ampicillin resistance & ESBL production with resistance to non  lactam antibiotics in invasive E. coli Oteo et. al. 2002. AAC.50:945 - 52 16.7 6.3 9.9 2.5 Gentamicin 77.3 32.9 44 9 Cotrimoxazole 63.3 17.2 24 7.3 Ciprofloxacin + - R (%) S (%) ESBL Ampicillin

56. Potency & spectrum against ESBL+ve E.coli phenotype & all strains in European regions (1997 – 2000) (n 189/1310) MIC 90 (  g/ml) ESBL + All Cefepime 8 1 Piper+ tazo 64 16 Gentamicin 128 8 Tobramycin 128 8 Ciprofloxacin 16 8 Jones et. al. 2003. CMI .9 : 708 - 12

57. Potency & spectrum against ESBL+ve K. pneumoniae phenotype & all strains in European regions (1997 – 2000) (n 306/934) MIC 90 (  g/ml) ESBL + All Cefepime 32 16 Piper+ tazo >128 64 Gentamicin >128 64 Tobramycin 128 64 Ciprofloxacin 16 4 Jones et. al. 2003. CMI .9 : 708 - 12

58. Ciprofloxacin was active in vitro against 21 of 28 isolates, only 21 analyzed 2 of 7 had partial response 5 of 7 cases, treatment failed Isolates had MIC (0.38 ug/ml) close to susceptibility breakpoint, treatment failure ascribed to the inability of the drug to reach therapeutic concentration at infected sites. Endimiani et. al. 2004. CID.38: 243 - 51 Bacteremia due to K.pneumoniae isolates producing the TEM 52 ESBL : treatment outcome of patients receiving Imipenem or ciprofloxacin

59. Therapeutic options in infections caused by ESBL producing E.coli, K.pneumoniae Cephalosporins (3 rd or 4 th generation) Cephamycins  lac +  lac inhibitor combinations Aminoglycosides Fluoroquinolones Carbapenems

60. Potency & spectrum against ESBL+ve E.coli phenotype & all strains in European regions (1997 – 2000) (n 189/1310) MIC 90 (  g/ml) ESBL + All Cefepime 8 1 Piper+ tazo 64 16 Gentamicin 128 8 Tobramycin 128 8 Ciprofloxacin 16 8 Meropenem 0.25 0.12 Jones et. al. 2003. CMI .9 : 708 - 12

61. Potency & spectrum against ESBL+ve K. pneumoniae phenotype & all strains in European regions (1997 – 2000) (n 306/934) MIC 90 (  g/ml) ESBL + All Cefepime 32 16 Piper+ tazo >128 64 Gentamicin >128 64 Tobramycin 128 64 Ciprofloxacin 16 4 Meropenem 1 0.12 Jones et. al. 2003. CMI .9 : 708 - 12

62. Carbapenem

64. Spectrum of activity of carbapenem Susceptible Resistant E. coli including ESBL Stenotrophomonas maltophilia K.pneumoniae ESBL Ps. aeruginosa Acinetobacter spp Proteus spp Haemophilus influenza Staph. aureus (MSSA) MRSA Staph. epidermidis MRSE Strept. pneumoniae Enterococcus faecalis Imipenem better against GPC, active against GNB including Pseudo, anaerobes Meropenem 2x to 4x better against GNB Ertapenem 2x to 4x less active, not very active against GNB

65. Breakpoints Species Imipenem Meropenem Ertapenem S I R Enterobact <4 8 >16 <4 8 >16 <2 4 >8 & staph Anaerobes <4 8 >16 <4 8 >16 <4 8 >16 S.pneumoniae <0.12 >1 <0.25 >1 <1 >4 H. influenzae <4 <0.5 <0.5 S I R S I R

66. Pharmacokinetics 95 2 30% Protein binding 155 49 41 – 83 ug/ml Cmax 4 1 1 hour T ½ IV / IM IV / IM IV Route 24 8 6 – 8 hours Frequency 1 G 500 mg – 1 G 250 mg – 1 G Dose Ertapenem Meropenem Imipenem

67. Carbapenem in clinical practice Suitable for Initial empirical &/or definitive mono-therapy of Febrile neutropenia ICU infections Intra abdominal infections Serious lower respiratory tract infections Pediatric meningitis

68. Infection control Transmitted through contact ICU is hot spot Hands of HCW, thermometer, ultrasound gel, Tag records Education Contact precautions Transfer between wards & hospitals

69. Take home message Detect and report ESBL+ bacteria Use the most appropriate antibiotic Implement effective infection control measures

70. Thank you for your attention