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 .

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

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

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

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

December 2004

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

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)

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)

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

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

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

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

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

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)

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

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

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

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

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

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

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

Pseudomonas aeruginosa CLSI vs. EUCAST % susceptibility Antibiotics

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

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

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

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

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

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

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

Resistant to 3rd gen. ceph. 2003: Proteus mirabilis (isolates from UTIs and ulcers) Ampicillin >128 R Amoxi/Clav 32 R Pip/Tazo 4 S Cephalotin 32 R Cefotaxime 64 R Ceftazidime 32 R Cefepime 2 Ertapenem 0.12 S Amikacin 2 S Gentamicin 4 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

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

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

P. mirabilis CMY+ spreading in Italy 2007-2008: 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

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)

Nationwide clonal spread (ST37) 2007-2008: 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 1 - 2 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

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 2-4 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 1 - 2 S Colistin <1 MIC (mg/L) Vitek-2 AES: changes to resistance to all carbapenems

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

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

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

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

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

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

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

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

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

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