1. 1 Gram negatives: Mechanisms of Resistance & Lab detection Johann DD Pitout MD, FF Path (SA) University of Calgary Calgary Laboratory Services Calgary, Canada johann.pitout@cls.ab.ca

2. 22 Transparency declaration  Research grants from Merck Frosst, Wyeth and Astra Zeneca  Speaker for Merck Frosst

3. 33 Overview  Introduction  Clones, stones and bones…  Newer ß-lactamases  Laboratory detection  Emergence of clones in Enterobacteriaceae  Summary

4. Am J Med 2006;119(sup1):S62-70 Why Enterobacteriaceae?  NB causes of serious bacterial infections:  Community  Hospital  Several species:  E. coli (ExPEC)  K. pneumoniae  Salmonella spp  Surveillance:  Top 5 community and hospital pathogens

5. Trends Microbiol 2006;14:413-20 Why Resistance?  Resistance is concern:  3 rd GenCephs  Carbapenems  Fluoroquinolones  Empiric treatment  complicates antibiotic selection  Inadequate initial Rx (AAC 07;51:1987)   risk for mortality   health-care costs (AAC 06;50:1257

6. CMI 2007;13:1-46 What is a clone?  Definitions:  Isolate, strain, clone  Clone:  Isolates with identical phenotypic+genotypic characteristics  Different sources/time  Typing methods:  MLST  PFGE  PCR fingerprint (MLVA)

7. clonalclonal

8. AnnRevMicro 2006;60:561-88 MLST  Sequence variation  Housekeeping genes  Evolutionary relationship  Comparing isolates  NOT outbreaks  ST’s and CC’s  E-BURST NOgapAinfBmdhpgiphoErpoBtonBST Kp CG 33111118340 Kp ON 34617438147 Kp SA 23111148569

10. CID 2007;44:418-23 PFGE  Restriction of genomic DNA  Rare-cutting enzyme  +++ discrimination  Excellent outbreaks  Various species  But not portable:  Labour intensive  ? Reproducible  Time consuming

11. ST

12. 1212 Newer ß-lactamases EnzymesInhibitedSpectrumOrganismsExamples ESBLsClavulanateCephalosporinsPenicillins Klebsiella spp. E. coli TEM, SHV, CTX-M Plasmid-AmpCsCloxacillin Boronic acid Cephalosporins Cephamycins Klebsiella spp. E. coli Salmonella spp. CMY, FOX MBLs(CHE-B)EDTACarbapenemsCephalosporins P. aeruginosa Klebsiella spp E. coli IMP, VIM, NDM KPC’s(CHE-A)Clavulanate Boronic acid CarbapenemsCephalosporins Klebsiella spp. E. coli KPC

13. 13 Clinical Case no 1  3 month girl with diarrhoea  Watery with mucus  Abnormal growth parameters  Admitted and Rx  Previous diarrhoea  Other family members  Older sister and Dad  Salmonella enterica serotype Newport

14. 14 Salmonella spp. Susceptibility  CFZ >64  TZP >64/4  CAZ 16  CRO 16  FOX >64  FEP <16  ATM 16  CIP <0.25  GEN <8  IPM <2 CLSI ESBL confirmation test:  CAZ 12mm  CAZ + CLAV 15mm  CTX 16mm  CTX + CLAV 16mm  Neg ESBL test

15. 15 CTT +PBA

16. 16 ClinMicroRev 09;22:161-82 AmpC ß-lactamases Chromosomal  Organisms:  Enterobacter, Serratia, Citrobacter, Pseudo.  cephalosporinases  inducible  not inhibited by c,t,s  eg.: AmpC Plasmid  Organisms:  Klebsiella, P. mirabilis, Salmonella spp.  cephalosporinases  constitutive/inducible  not inhibited by c,t,s  eg.: CMY, FOX, DHA, ACC

17. 1717 DMID 01;41:57-61 E. coli and AmpC’s Weak promoter Strong attenuator Plasmid-mediated AmpC mutations

18. 1818 ClinMicroRev 09;22:161-82 Plasmid-mediated AmpC’s  R to cephamycins  some cephalosporins, penicillins  Not carbapenems  Multiresistant  Organisms: K. pneumoniae, E. coli, Salmonella spp  Origin  Extended-spectrum cephs

19. 19 ERAIT 08;6:657-69 Laboratory detection  No guidelines  Not all cephamycin/3 rd GC R = AmpC  Phenotypic tests  Inhibitor-based approaches  Disks: boronic acid and others  AmpC E-test: cloxacillin  Multiplex PCR

20. 20

22. 22 Clinical case no 2  47 yr F with recent travel  ER with fever, dysuria, frequency  Exam:  T 39.2ºC  supra-pubic and renal tenderness  Diagnosis of UTI  2 sets of BC, urine  Rx ciprofloxacin, referred to HPTP  Next day blood cultures positive

23. 23 Escherichia coli Susceptibility  CFZ >64  TZP 64/4  CAZ 16  CRO >32  FOX <8  FEP 16  ATM 16  CIP >4  GEN >16  IPM <2 CLSI ESBL confirmation test:  CAZ 22mm  CAZ + CLAV 23mm  CTX 8mm  CTX + CLAV 20mm Pos ESBL test

24. 24 AmJMed 97;103:51-9 Extended-spectrum ß-lactamases  Early 1980’s  World-wide  Hydrolyse:  cephalosporins, penicillins, monobact  not: cephamycins, carbapenems  Inhibitor sensitive  clav, sulb, tazo  Types  Enterobacteriaceae  Clinically relevant

25. CLSI guidelines 2010 MIC ≥ 1 CTX/CRO ≥ 2 CAZ Zone ≤ 26mm CTX, ≤ 23mm CRO, ≤ 21mm CAZ No confirmatory test for therapy but useful for infection control/epidemiology

26. 26 Detection of ESBLs E. coli, Klebsiella spp, P.mirablis, Salmonella spp All other Enterobacteriaceace CLSI Screen positive CRO: 1ug/ml and CAZ: 1ug/ml Modified double disk FEP + CLAV CLSI ESBL disk confirmation test: CTX and CTX with Clav CAZ and CAZ with Clav CLSI Screen positive CRO: 1ug/ml and CAZ: 1ug/ml

27. Clinical case no 3  32yr M travelled in Southern India  Admitted city Mysore:  Hyperglycemia  Developed upper UTI  Rx with Ciprofloxacin  Transferred to Alberta  Upper UTI and prostatitis  Rx with ERT

28. 28 Escherichia coli (MH01) Suscept  CFZ >64  TZP >64/4  CAZ >64  CRO >64  FOX >64  FEP >64  ATM >64  CIP >8  GEN >16  IPM 16 ESBL confirmation:  CAZ 6mm  CAZ + CLAV 6mm  CTX 6mm  CTX + CLAV 6mm AmpC boronic test:  CTT 6mm  CTT + PBA 6mm  ERT 6mm

30. ClinMicroRev 2005;18:306-25 Class B CHE’s  Metallo-ß-lactamases (MBLs)  Active site: Zn+  Inhibited by EDTA  R to all ß-lactams except:  monobactams  Chromosomal (Steno)  Types: IMP, VIM, SPM, GIM, SIM, AIM, KHM, DIM, NDM

31. JAC 2011;66:689-92 NDM ß-lactamases  1 st report from Sweden (AAC 2009;53:5046-54)  Worldwide  Widespread in Subcontinent (Lancet ID 2010 10;597-602)  India, Pakistan, Bangladesh  Associated with travel (medical tourism)  Organisms:  E. coli (community)  Klebsiella spp. (hospital)  MultiR  ? Fatal cases

32. 32 Clinical case no 4  CAP external quality assurance program  Klebsiella pneumoniae  Urine; significant colony count  Adult Diabetic patient  Neurogenic bladder  ICU for 2 weeks  Recently hospitalised in New York

33. 33 Klebsiella pneumoniae Susceptibility  CFZ >64  TZP >64/4  CAZ >64  CRO >64  FOX >64  FEP 16  ATM >64  CIP >8  GEN >16  IPM 2 ESBL confirmation:  CAZ 10mm  CAZ + CLAV 13mm  CTX 11mm  CTX + CLAV 14mm AmpC boronic test:  CTT 10mm  CTT + PBA 22mm  ERT 6mm

35. 35 Class A CHEs  Different types (e.g.SME)  KPC ß-lactamases  K. pneumoniae Carbapenemase  1 st reported late 1990’s North Carolina  Hydrolyze all ß-lactams  Including carbapenems  Inhibited by clavulanate  Types: KPC 2-??? Klebsiella Producing Chaos Lancet Infect Dis 2009;9:228-36

36. CLSI June 2010 Carbapenemases & Enterobacteriaceae MIC ≥ 0.25 ERT, ≥ 1 IPM/MER/DOR Zone ≤ 22 mm ERT/IPM/MER/DOR No confirmatory test for therapy if I or R to all carbapenems tested but OK for infection control/epidemiology

37. ++false positives with ERT Use MER Multiplex PCR >0.25

38. Multiplex PCR

39. EuroSurveill 2010;15:pii

40. 4040 Curr Opin Microbiol 2006;9:466-75 CTX-M ß-lactamases  Active CeftoTaXime 1 st Munich   activity CTX   by tazobactam  40% similarity to SHV/TEM  Originate from Kluyvera spp.  Insertion element: ISEcp1  Divided into 5 groups  Groups 1, 2, 8, 9, 25

41. 41 CTX-M-type ESBLs: first reports 1989 Matsumoto et al. AAC 1988; 32:1243 Bauernfeind et al. Infection 1990; 18:294 Power et al. AAC 2002; 46:602 1986 1990s

42. 42 Lancet Infec Dis 08;8:159-66 The CTX-M pandemic: since 2000

43. 43 Lancet Infec Dis 08;8:159-66 The CTX-M-15 pandemic: since 2003

44. 44 Dissemination of CTX-M-15  MLST profile ST131  Broadly disseminated  Homogenous virulence genotype  IncF group plasmids  3 different profiles  OXA-1, aac(6)-Ib-cr, TEM-1 JAC 08;61:273-81, EID 08;14:195-200

45. 45 IJAA 10; 35:316-21 The clone ST131 pandemic: since 2006

46. 46 AAC 10;54:1327-30 MLST clone ST131 (96/209 [46%])

47. 4747 E. coli from blood

48. 48 Distribution of ST131 (69/134[51%]) AAC 09;53:2846-51

49. 49 Characteristics of ST131

50. 50 IJAA 10;35:316-21 ST131 CTX-M-15 What makes ST131 so special? Uropathogenic Phylo B2 Certain VF’s Adherence IncF plasmids R factors Addiction systems

51. CTX-M ESBLs Kluyvera spp E. coli CTX-M-1 CTX-M-15 ST131 ISEcp1

52. 52 Significance of KPCs  Multiple R (Rx options very limited)  Nosocomial clonal outbreaks  Inter-hospital, inter-city, inter-country  Infections  Systemic (invasive devices)  UTIs (indwelling catheters)  Not only in K. pneumoniae  BSIs ↑ mortality (Infect Control Hosp Epidemiol. 2010 Oct 25) Lancet Infect Dis 2009;9:228-36

53. 53 ST258

54. FEMSMicroReviews 2011 Feb 9 KPC and clones  ST258  40% of KPC’s  CTX-M-14  Hypervirulent clone  Other STs  ST438 (Brazil)  Also:  Mobile elements (Tn4401)  Plasmids

55. KPC’s Environmental bugK. pneumoniae KPC-2 Tn4401 KPC-2/3 ST258

56. JAC 2011;66:689-92 Worldwide distribution of NDMs Cricket WC

57. JAC 2011;66:689-92 NDMs and clones  K. pneumoniae   Not clonally related   Broad-host range plasmids   e.g. A/C, N   E. coli   Clonal   ST101 (Canada, UK, Europe, Australia)   ST131 USA (scary)

58. What makes a clone….  International clones  Virulence  Adherence factors  Plasmids  Narrow host range  Broad host range  Clones use plasmids and plasmids use clones  Plasmids as per BioShock: “giving the user what some might call super powers"

59. 59 MLST clone ST131  Understanding how ST131 has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority Neil Woodford, UK JAC 2008;61:233-4

60. 60 Acknowledgements University of Calgary: K. Laupland D. Church D. Gregson Calgary Lab Services: L. Campbell B. Chow G. Peirano NML: M. Mulvey D. Boyd Special Mention: L. Poirel, K. Thomson, N. Hanson, E. S-Molland, J. Johnson, D. Guttman