Eastern PA Branch-ASM, 41st Annual Symposium, Nov 17, 2011

1. New global challenges of Gram
negative cephalosporin and
carbapenem resistance
Robert A. Bonomo, MD
Chief, Medical Service
Director VISN 10 GRECC
Louis Stokes Cleveland VAMC
Vice Chairman, Department of Medicine
University Hospitals Case Medical Center
Professor, Case Western Reserve University School of Medicine
Presented at the 41st Annual Symposium
“Global Movement of Infectious Pathogens and Improved Laboratory Detection”
Eastern PA Branch-American Society for Microbiology
November 17, 2011
Thomas Jefferson University, Philadelphia

2. Disclosures
• Support from VA and NIH
• Steris Foundation
• Pfizer
• Excitement rather than give formulas

3. Objectives
• Overview of the problem of ATBR in
Gram negative bacteria
– A. baumannii, Pseudomonas aeruginosa,
and Klebsiella pneumoniae
• Summarize the rapidly expanding
landscape of resistance determinants
• Use this knowledge to devise effective
treatment strategies

5. Part I
MDR and PDR Ab

6. The clinical challenge of
A. baumannii
• Multi-Drug Resistant (MDR) A. baumannii are
among the most “problematic pathogens”
encountered by clinicians

7. Why? Ab facts…..
• Most common (and emerging) drug
resistant pathogen in the US and world
• 50-70% of Ab clinical isolates are now
eXtensively Drug Resistant (XDR; i.e.
resistant to all antibiotics except
colistin or tigecycline), reflecting a >15-
fold increase since 2000.
• “Pan Drug” Resistant; strains of Ab,
resistant to tige + coli, increasing
Perez et al AAC 2007, Talbot and others, 2006, CID; Talbot ERAIT, 2009, Boucher CID 2009

8. Does resistance matter? Yes
• BSI by XDR Ab cause >50-60% mortality
• In a recent study 13,796 patients in 1,265
ICUs from 75 countries, Ab was one of
only two of 19 microorganisms strongly
linked (p<0.01) to increased mortality by
multivariate analysis;
• Odds ratio for death-1.53
• Resistance + virulence: factors? LPS,
Fe siderophores, PLD, OMPs, biofilm??
McGowan ICHE 2019, Hoffman et al, ICHE 2010, McGowan AJM 2006 Paterson CID 2006, Perez AAC 2007,
Vincent JAMA 2009, Gordon JAC 2009

9. Survey of “Resistance genes” in A. baumannii
bla AMEs QRDR RND OMPs Tet
Efflux pumps
ADC aacC1 gyrA AdeABC HMP-AB tetA
OXA aacC2 parC AdeM OmpA tetB
IMP aacC3 AdeIJK 33-36 kDa tetM
VIM, GIM aacA4 AdeS 25/29 kDa tetX
SIM, SPM, CraS CarO
NDM AdeDE
PER aphA1 Res Is?? OprD PBPs
(43kDA)
TEM* aphA6 AbaR 1-10 OmpW
SHV aadA1 Col R 44, 47kDa, 22 integrons
pmrAB
CTX-M rmt* OMVs

10. “The Resistance Island”
86 Kb, 88 orfs, 82
orfs from another
source and 45
resistance genes
AbaR1-10!
Fournier et al., PLoS Genet. 2006 Jan;2(1):e7. Epub 2006 Jan 13.

11. Threat 1. Carbapenem R
• OXAs and MBLs
• Naturally occurring and acquired
• OXAs- Types and Groups
– Narrow spectrum
– Carbapenem hydrolyzing (CHDLs)
– ES type
• Carbapenemases (Acinetobacter)
– Are not ES; do not have both properties
– Imipenem> meropenem
Poirel et al AAC 2010

12. ENTER NDM-1!!
Thanks Dr. Perez

13. Threat 2. ColistinR
• Polymyxins (E and B) are cationic polypeptide atbs
• Colistin SO4 for PO and Colistimethate Na+ (sodium
colistin methanesulphonate, colistin sulfomethate sodium)
for IV
• Colistin displaces Ca+2 and Mg+2 from PO4-3 groups of
membrane lipids; Insertion of polymyxins disrupts the
OM and LPS is released ; anti-endotoxin activity ;
rapidly bactericidal???
• Urban et al. reported a case of polymyxin BR A. baumannii
Falgas, et al, CID 2005; Urban 2001 AAC; Li et al AAC 2006;
Li et al Int Journal of Antimicrobial Agents, 2005

14. ColistinR
• ColR due to modifications of LPS;
pmr (Adams…Bonomo, AAC US) vs.
lpxA,-C, and -D (Li and Nation,
Australia); Parks lab in S. Korea
found the same locus as we did.
• Heteroresistance (subpopulations
of genetically identical subclones
that are more R than the parent ) by
Li et al; implications for rx?
• “Colistin dependence”. 77 yo
diabetic male with FI and
Moffatt AAC 2010,
Li et al AAC 2006 ;
bacteremia; “increasingly luxuriant
Hawley et al AAC
2007,
growth”
Adams et al AAC 2009

15. Lower expression
of metabolic
proteins
and OmpA
JID 2011

16. Part II
MDR P. aerugoinosa
The resistance challenge of the ages

17. Pa facts
• Colonization rates by Pa are high in the
hospital (50%); immunity and burn
• Seriously ill patients in ICUs.
• Aggregate NNISS and EU data
– 20 to 30% of nosocomial pneumonias
– 10 to 20% of urinary tract infections
– 3% to 10% of bloodstream infections,

18. Mechanisms of resistance
in Pa

19. Pa and ATBR
• ß-lactamases-all classes represented
– Cephalosporinases,
– class A ESBLs (PER),
– OXA ESBLs (OXA-10, -14),
– Carbapenemases (KPC and GES), MbLs
• Loss of permeability (porins and efflux)
• Quinolones and aminoglycosides
– Active antimicrobial efflux
– Alterations in DNA gyrase
– Aminoglycoside-modifying enzymes

20. Antimicrobial resistance
• Efflux pumps
– MFS—major facilitator superfamily
– ABC—ATP-binding cassette family
– RND—resistance nodulation division
– SMR—small multidrug resistance
– MATE—multidrug and toxic compound
extrusion
RND and MFS extrude antibiotics and work by proton motive force; In
GNRs, RND works with MFP (periplasmic membrane fusion protein)
and OEP (outer membrane efflux protein) to get thru both membranes

25. The mysteries of the biofilm..
Trends in Microbiology Jan 2001; 9(1): 34-39

26. Part III
MDR K. pneumoniae
“Killer Klebs”

27. Why should we be afraid of
Klebsiella pneumoniae KPCs?

28. KPC K. pneumoniae
AMIKACIN R
AMPICILLIN R
CEFAZOLIN R
CEFTAZIDIME R
CIPROFLOXACIN R
TRIMETH/SULFA R
IMI/MERO-PENEM 4 ug/ml → (> 64)
GENTAMICIN S
AMPICILLIN/SUL R
CEFOTETAN R
CEFEPIME R
PIP/TAZO R

29. Clinical impact of KPC
carbapenemases
• “The dependability on “last line”
antibiotics is shattered”
• The emergence of KPC
carbapenemase producing Gram-
negatives is a major threat to the
clinician
– K. pneumoniae, Acinetobacter, E.
coli, Enterobacter, Serratia,
Pseudomonas,… the list grows
Patel and Bonomo, 2011, Current Opinion

30. Clinical issues with KPC
• ATB control ?Cephalosporin and b-
Lactam-b-Lactamase Inhibitor restriction
policies? special populations? Imipenem
restriction ??
• How best to implement IC? Carrot or
stick?
• Detection? ESBL identification?
Inoculum effect?
• Colistin-as empiric Rx ??? combined
with aminoglycosides (gent); rifampin

31. Status of the KPC global
epidemic
• Two phenotypes; MIC< 8 and MIC> 32
• ST258> ST384, ST388, others…
• Plasmids from ST258 and other starins
has been transferred to E. coli in
patients (Kreiswirth lab).
• Colistin resistant ST258
• Novel testing methods (ChromAgar,
Boronates, PCR/ESI-MS, Microarray
methods/Checkpoints

32. Mainly KPC-2 and KPC-3 (KPC-2 to KPC- Thanks Dr. Endimia
11)
Poirel L. et al.
Enterobacteriaceae (K. pneumoniae) Outbreaks

33. Dr. End
“Import/Export” of patients carrying
blaKPC
Naas T et al., AAC 2005 Hammerum AM et al. IJAA 2010
Cuzon G et al.

34. Thanks
Dr. Endimiani
Genetic environment of blaKPC (Tn4401) Structure of Tn4401 and insertion sites
P.aeruginosa Isoform B
Tn4401 is at the origin of acquisition and
dissemination of blaKPC
Possible genesis of Tn4401
Isoform A
• Different isoform suggests that this
region is polymorphic

35. Thanks
Dr. Endimiani
Western Blot
No deletion (isoform B)
68-bp deletion
Relative copy number
100-bp deletion (isoform A) with real-time PCR Molecular characterization of
255-bp deletion (versus rpoB gene)
OmpK35 and OmpK36 genes
c Frameshift
(stop codon after aa 88)
Common in ST258

36. The “KPC Tsunami”

37. Worst case scenario!!

38. Is there increased mortality??
The mortality in the IRE group was 33%, compared to
9% among controls.
Being an IRE case was significantly associated with
increased mortality (P 0.043)

39. The tip of the iceberg….

42. The near future
The exciting future Clonal typing
gyrA, parC
mecA, PVL, TSST,
mupA, nucA

45. Options for treatment?
“The basis for a new
research
agenda in Infectious
Diseases”
Can I approach this based upon a knowledge of genetics?

46. Therapy for MDR Ab et al.
Colistin?
Tigecycline?
Minocycline?
Rifampin?
(Teicoplanin? Vancomycin? Are you crazy!)
Do we have enough patients
studied properly? Animal
models may have
(significant) limitations?

47. Colistin is King???

48. “After an exhaustive review of much the
available evidence….”
Perez et al, AAC 2007

49. CID 2010

50. Colistin + rif?
Colistin + minocycline?
Not enough data Antagonistic?
Synergy? Sometimes???
Meta-analysis (Falagas IJAA)--no statistical
difference in cure rates when colistimethate sodium
alone was compared with the combinations with
meropenem, piperacillin/tazobactam or
ampicillin/sulbactam

51. The colistin “bottom line”
• “Efficacy rate” of 57-76% in IV form;
“microbiological eradication” of 67-90.9%Renal
tox 0-37%
• Nebulized colistin (CF studies + others)
effective; FDA warning; impact of shift to more
resistant strains ; use with IV!!
• 32 cases “microbiological eradication” in the
CNS with ITh/IVe colistin (safe e 1) (2.5 mg/kg,
10-20 mg ITh)
• Colistin was independently associated with
higher mortality vs. treatment with sulbactam in
patients with A. b infections

52. Colistin dosing
• Administration of a loading dose (300
mg)
• Colistin exposure during the first 12 h
“may be beneficial, providing enough
net killing such that the immune system
may be able to eradicate any remaining
colistin- resistant cells”

53. Major concerns…real ?
1. Rapid resistance
Tigecycline?
can emerge;
2. Cases of Patients % Improvement
breakthrough 25 84
bacteremia
reported; 18 50
3. Adequacy of 17 82.4
blood levels??
29 30
Pachon and Vila Curr Opin 75 70
Investig Drugs. 2009
Feb;10(2):150-6. 34 68
Giamarellou & Poulakou, Drugs. 45 78-90%
2009
bacteremic patients treated with tige failed to
Michalopoulos A, Falagas ME. clear their bacteremia 10-fold more commonly
Expert Opin Pharmacother. than patients treated with comparator drugs
2010 Apr;11(5):779-88. Gordon JAC 2009, Gardiner CID

54. My recommendations
• Susceptible strains
1. A/S, 3 q6; higher doses?
2. Imipenem; meropenem is worrisome;
dori?? Cephalosporins are tricky.
3. Colistin loading dose 5 mg/kg not to
exceed 300 mg then (4.5 mg/kg/day) and
split it tid (1.5 mg/kg q8).
4. Colistin and rifampin, tigecycline, or
minocycline, doripenem?)

55. Colistin and vanco??

56. E-129
GSK2251052…Comparative In Vitro
Activity Against Pseudomonas
aeruginosa from a Global Population.
Bouchillon et al.
Drug MIC range MIC50 MIC90 % Susceptible
GSK2251052
0.06 – 64 2 4 NA
Imipenem ≤0.5 - >16 2 16 72.9
Meropenem
≤0.12 - >16 0.5 16 79.3
Cefepime ≤0.5 - >32 4 32 75.3
Pip./tazob.
≤0.5 - >128 8 >128 81.3
Amikacin ≤0.5 - >64 4 16 90.2

57. Combination therapy for
PSDA?

58. If NDM-1? Treatment options
►Aztreonam + NXL?
►BAL30072, meropenem
and ??
►Tige?

59. E-722
Activity of the Novel Sulfactam BAL30072,
Alone and in Combination with
Meropenem, Against Diverse Gram-
negative Isolates Carrying NDM-1 β- Dihydropyridone
lactamase Gene. T. R. Walsh et al. siderophore
monocyclic
sulfactam
Organism (N) Ceftazidime Meropenem Aztreonam BAL BAL30072:
30072 Meropenem
A. baumannii (23) >256 256 16 4 1
P. aeruginosa (2) 256 32 16 0.5 <0.125
S. maltophilia (1) 256 64 64 4 1
Escherichia coli (3) 256 32 64 32 1
K. pneumoniae (2) 256 128 64 32 2
C. freundii (3) 128 128 64 8 2
Thanks to Dr. F. Perez

60. KPC Rx
68 blaKPC-possessing K. pneumoniae including
23 tigecycline- and/or colistin-nonsusceptible strains.
By agar dilution, 93% of the overall
KpKPC were susceptible (MIC50/90 of 16/64 g/ml, respectively).
Notably, 5 out of 6 extremely drug-resistant (tigecycline
and colistin nonsusceptible) KpKPC were susceptible
to fosfomycin. Compared to agar dilution, disk diffusion
was more accurate than Etest.

61. Summary
• Extraordinary challenge against cunning
pathogens
• Basic understanding of molecular biology is
needed (the complexities of resistance
genes will only increase)
• Research is needed in therapeutics and
infection control
• CALL TO ARMS: Coordinate scientific and
clinical trials to answer these important
questions

62. Acknowledgments
• NIH, VA Merit Review
• Drs. Alan Evangelista and Linda Miller
• Dr. Barry Kreiswirth
• Chris Bethel, Steve Marshall, Magda Taracila,
Kristine Hujer and Andrea Hujer
• Drs. Krisz Papp-Wallace, Marisa Winkler,
Federico Perez, Curtis Donskey, Dror
Marcham