1. Evoluzione dell’antibiotico-resistenza: miti e realtà
Gian Maria Rossolini
Dip. Biologia Molecolare
Sezione di Microbiologia
Università di Siena
UO Microbiologia e Virologia
Azienda Osp-Univ Senese .

2. How do we define resistance?
Broth or agar
dilution tests
Inhibition zones
MIC values
Interpretation of results
based on clinical breakpoints

3. The clinical breakpoints
Reference MIC/zone values for interpretation of the results of in vitro susceptibility testing
Definition of susceptibility category (S/I/R) referred to clinical use
Clinical breakpoints are defined by specific committees

4. Breakpoint committees in Europe
Country
BSAC
United Kingdom
CA-SFM
France
CRG
The Netherlands
DIN
Germany
NWGA
Norway
SRGA
Sweden
CLSI
USA

5. with different opinions...
Enterics / cefotaxime
S< / R>
BSAC United Kingdom
1 / 1
CA-SFM France
4 / 32
CRG The Netherlands
4 / 8
DIN Germany
2 / 8
CLSI U.S.A.
8 / 32
NWGA Norway
1 / 2
SRGA Sweden
0.5 / 1
Kahlmeter et al – JAC 2003

6. December 2004

8. The EUCAST Mission to harmonise clinical breakpoints in Europe
to determine breakpoints for new antimicrobials
to provide standardised methodology for AST

9. Setting clinical breakpoints for new drugs in Europe
Co-ordinated process between the Company, EMEA and EUCAST
When a Company applies for registration of a new agent:
EUCAST defines the breakpoints
EMEA decides on all other aspects
EUCAST breakpoints for new drugs are the only ones included in the SPC (Summary of Product Characteristics)

10. EUCAST clinical breakpoints
Freely available on the WEB
Institutional decision body (industry has a consulting role but does not participate in the decisional process)
Defined by consensus
Rationale for decision disclosed (rationale documents available)

11. EUCAST vs. CLSI breakpoints: a remarkable diversity
Courtesy by G. Kahlmeter

12. EUCAST vs. CLSI breakpoints: Pseudomonas aeruginosa
Cefepime 8 16
Ceftazidime
Imipenem 4
Meropenem 2
Pip/Tazo 64
Aztreonam 1
Ciprofloxacin
0.5
Gentamicin
Tobramycin
Amikacin 32
Colistin
For S: EUCAST 5/11 lower
For R: EUCAST 8/11 lower

13. EUCAST vs. CLSI breakpoints: Enterobacteriaceae (beta-lactams & quinolones)
Cefepime 1 8 16
Ceftriaxone 2 32
Ceftazidime
Ertapenem
0.5 4
Imipenem
Meropenem
Pip/Tazo 64
Levofloxacin
Ciprofloxacin
For S: EUCAST 9/9 lower
For R: EUCAST 7/9 lower

14. EUCAST vs. CLSI breakpoints: Enterobacteriaceae (other agents)
Amikacin 8 16 32
Gentamicin 2 4
Tobramycin
Cotrimoxazole
Colistin NA
Tigecycline 1
For S: EUCAST 3/4 lower
For R: EUCAST 3/4 lower, 1/4 higher

15. EUCAST vs. CLSI
Will the change from CLSI to EUCAST breakpoint system significantly affect the epidemiology of antibiotic resistance?

16. EUCAST vs. CLSI
A comparative analysis of AST results interpreted according to CLSI or EUCAST
Data source: historical records from clinical microbiology service, Siena University Hospital (year 2008)

17. Pseudomonas aeruginosa Gentamicin (N = 295)4 8 16 32 64
EUCAST
CLSI
MIC (mg/L)
EUCAST: R CLSI: I

18. Pseudomonas aeruginosa Amikacin (N = 296)4 8 16 32 64
EUCAST
CLSI
MIC (mg/L)
Ricontrollati numeri e grafici
EUCAST: R CLSI: I
EUCAST: I CLSI: S

19. Pseudomonas aeruginosa Ceftazidime (N = 294)1 2 4 8 16 32
EUCAST
CLSI
MIC (mg/L)
EUCAST: R CLSI: I

20. Pseudomonas aeruginosa Meropenem (N = 216)4 8 16 32 64
EUCAST
CLSI
MIC (mg/L)
EUCAST: I CLSI: S

21. Pseudomonas aeruginosa Pip/Tazo (N = 289)4 8 16 32 64
EUCAST
CLSI
MIC (mg/L)
EUCAST: R CLSI: S

22. Pseudomonas aeruginosa Aztreonam (N = 133)2 4 8 16 32
EUCAST
CLSI
MIC (mg/L)
EUCAST: I CLSI: S

23. Pseudomonas aeruginosa Ciprofloxacin (N = 295)
0.25
0.5 1 2 4 8
EUCAST
CLSI
MIC (mg/L)
EUCAST: R CLSI: I
EUCAST: I CLSI: S

24. Pseudomonas aeruginosa CLSI vs. EUCAST
% susceptibility
Antibiotics

25. MRSA impact (USA) a in hospital deaths
Boucher & Corey Clin Infec Dis 2008

26. Resistance trends in major pathogens Europe
E. coli R to 3GC
23/30
1/30
6/30 =
MRSA
8/30
14/30 =
Countries
2008:
2/31
10/31
19/31 =
2008:
21/30
0/30
9/31 =
EARSS annual report, 2007

27. Resistance trends in major pathogens Europe
MDR E. coli
(R to 3GC,AG,FQ)
24/30
0/30
6/30 =
E. coli R to 3GC
23/30
1/30
6/30 =
Countries
EARSS annual report, 2007

28. The growing challenge of resistant Gram-negatives
MRSA and VRE rates have leveled off or decreasing in several European countries
Resistant Gram-negatives are increasing in most European countries
Major challenges:
Enterobacteriaceae
ESBL/AmpC, MDR, XDR (ESC/FQ/AG/NEM)
Pseudomonas aeruginosa and Acinetobacter
MDR, XDR (COL-S only)
Rossolini & Mantengoli, CMI 2008

29. ESBLs: increasing trends
K. pneumoniae
% resistant to 3GC
Year
EARSS database

30. ESBLs: increasing trends
E. coli
% resistant to 3GC
Year
EARSS database

31. ESBL-producing Enterobacteriaceae , Italy%
Spanu et al. - AAC 2002; Luzzaro et al. - JCM 2006; OASIS study, data on file

32. Resistant to 3rd gen. ceph.
2003: Proteus mirabilis (isolates from UTIs and ulcers)
Ampicillin >128 R
Amoxi/Clav R
Pip/Tazo S
Cephalotin R
Cefotaxime R
Ceftazidime R
Cefepime
Ertapenem S
Amikacin S
Gentamicin S
Ciprofloxacin >32 R
Levofloxacin >32 R
MIC (mg/L)
Suspect ESBL
Aztreonam
Ceftazidime
Cefotaxime
Ceftriaxone
Amoxi/Clav R S
Resistant to 3rd gen. ceph.
but ESBL-negative

33. P. mirabilis resistant to
Same clone detected
in LTCFs
P. mirabilis resistant to
3rd gen. cephalosporins .
By clonal expansion
Luzzaro et al – IJAA 2009

34. Clinical features of infections caused by the P. mirabilis CMY-16+
Mean age: 75±15 yrs
(76±16 for ESBL+; 57±28 for susceptible strains)
Female/male ratio: 1.1
(1.6 for ESBL+; 2.1 for susceptible strains)
80%
37%
51%
Patients
Sources
Luzzaro et al – IJAA 2009

35. P. mirabilis CMY+ spreading in Italy:
P. mirabilis CMY+ spreading in Italy
>30 cases from BSIs
Clonally related isolates detected in Greece and Poland: an internationally spreading clone
D’Andrea et al – unpublished

36. ESBLs/AmpC and carbapenem overuse
Increased # ESBL/AmpC cases
Increased Carb-R strains
Increased carbapenem use
Cross transmission of Carb-R strains
Selection of Carb-R strains
Courtesy of Vincent Jarlier, Sept 2009 (modified)

37. Nationwide clonal spread (ST37)
: emergence of MDR Klebsiella pneumoniae ERT-R from several hospitals …
Ampicillin >16 R
Amoxi/Clav >16 R
Pip/Tazo >64 R
Cefotaxime >16 R
Ceftazidime >32 R
Cefepime >16 R
Aztreonam >16 R
Imipenem ≤1
Meropenem 2-4
Ertapenem >4 R
Amikacin >32 R
Gentamicin >8 R
Tobramycin >8 R
Ciprofloxacin >32 R
Levofloxacin >32 R
TMP/SXT >2 R
Tigecycline
Colistin <1
MIC (mg/L)
Nationwide
clonal spread
(ST37)
No carbapenemase act.
Production of:
CTX-M-15 ESBL
SHV-11
- (OXA-9) (TEM-1)
Reduced porin expression
D’Andrea et al. – 19th ECCMID

38. changes to resistance to all carbapenems
Klebsiella pneumoniae with reduced carbapenem
susceptibility due to ESBL prod. + porin loss:
detection and reporting issues
Ampicillin >16 R
Amoxi/Clav >16 R
Pip/Tazo >64 R
Cefotaxime >16 R
Ceftazidime >32 R
Cefepime >16 R
Aztreonam >16 R
Imipenem ≤1 R
Meropenem R
Ertapenem >4 R
ESBL positive
Amikacin >32 R
Gentamicin >8 R
Tobramycin >8 R
Ciprofloxacin >32 R
Levofloxacin >32 R
TMP/SXT >2 R
Tigecycline S
Colistin <1
MIC (mg/L)
Vitek-2 AES:
changes to resistance to all carbapenems

39. KPC-2
K. oxytoca
Yigit et al. AAC 2003

40. Klebsiella pneumoniae Due to spread of KPC carbapenemases
Brooklyn, New York …
22% of isolates resistant to:
Aminoglycosides
Fluoroquonolones
3rd 4th gener. Cephems
Carbapenems
Klebsiella pneumoniae
Due to spread of KPC carbapenemases
Susceptibility only to:
Colistin
Tigecycline
Landman et al – JAC 2007

41. KPC-type carbapenemases in Israel: a major problem
Nationwide outbreak
Carbapenem resistance rates in K. pneumoniae from Israel:
2006: 11%
2007: 22%
2008: 19%
EARSS database

42. KPC-type carbapenemases: a new pandemic?
Two cases, one with Israel connection
Clonally related
7 cases
4 from Greece
2 from Israel
Clonally related
Literacka et al. AAC 2009
Tsakris et al. JAC 2008
Hawser et al. IJAA 2009
Intercontinental spread of ST258 KPC+ clone
Villegas et al. AAC 2007
Nordmann et al. Lancet ID 2009

43. KPC-type carbapenemases: emerging in Italy
Giani et al - JCM 2009
Florence
Oct 2008: KPC-3 positive K. pneumoniae ST258 isolated from a cIAI (high-level carbapenem resistance)
No epidemiological link with areas of endemicity, but patient cared for by a trainee from Israel
May 2009: KPC positive K. pneumoniae
Lecco
Patient transferred from another hospital
Large outbreak ongoing in that hospital (26 patients colonized or infected), variable carbapenem resistance
Mostly by clonal spread (ST258), but at least two clones and also in Enterobacter
Luzzaro et al - unpublished
Santoriello et al - unpublished
Additional reports

44. Often susceptible only to colistin and tigecycline
Carbapenem-resistant K. pneumoniae, Greece
Vatopoulos, p.2
Production of
VIM-1 MBL
Multiple clones
Often susceptible only to colistin and tigecycline
(XDR)
Vatopoulos et al. - Eurosurveillance 2008
Psichogiou et al. – JAC 2008

45. Carbapenemases of clinical relevance
KPC-type (active-site serine, class A)
Metallo-β-lactamases (class B)
OXA-type (active-site serine, class D)

46. VIM-1 MBL-producing index strain
VR-143/97 (ser. O11; ST227) GM
COL AK
TOB
FEP
IPM
MEM
CAZ
TZP
ATM
CIP
PRL
VERONA
1997
Lauretti et al. – AAC 1999
Cornaglia et al. – CID 2000

47. 1.3% 7% Acquired MBLs in Pseudomonas aeruginosa
first Italian nationwide survey
2004:
Overall prevalence
1.3%
VARESE 2.6%
PAVIA 1.3%
CREMONA 0.6%
PERUGIA 1.1%
SASSARI 0.9%
ROME 0.3%
AVELLINO 1.4%
NEAPLES 9.2%
FOGGIA 1.2%
GENOA 0%
TURIN
L’AQUILA
2008:
Overall prevalence 7%
Rossolini et al. AAC 2008
Luzzaro et al. - unpublished

48. Conclusions
CLSI soon replaced by EUCAST: resistance rates will be affected in some cases
Resistance in Gram-negatives: now a major problem
XDR phenotypes not only in Pseudomonas and Acinetobacter but also among enterobacteria
Multiple resistance mechanisms: not easily deducible from the antibiotype
Open issues in: lab detection, reporting, infection control and treatment