Pseudomonas aeruginosa ; ; ß ß- -lactamases lactamases… … Pseudomonas aeruginosa and other enzymes enzymes and other Pr. P. Nordmann Nordmann Pr. P. hôpital de Bicêtre, AP Bicêtre, AP- -HP HP hôpital de Faculté é de M de Mé édecine Paris decine Paris- -Sud, Universit Sud, Université é Paris XI, Paris XI, Facult K.- - Bicêtre, France Bicêtre, France K.
Outer membrane PBP • Outermembrane Efflux permeability (100-fold less than that of E. coli ) • Efflux : at least twelve systems... • Naturally-occurring cephalosporinase and oxacillinase Enzymes
Gram- -negatives negatives Gram Outer Membrane Lipopolysaccharide Porin Braun lipoprotein Peptidoglycan Inner (cytoplasmic) membrane Cytoplasm Cytoplasm Cytoplasm ß-Lactam resistance = +++ ß-lactamases
Wild-type P. aeruginosa Natural resistance Natural resistance : : ! ! aminop aminopé énicillines nicillines ; ; aminop aminopé énicilline nicilline/a. /a. clav clav. . ! ! C1G, C2G C1G, C2G ! ! C3G ( C3G (cefotaxime cefotaxime, , ceftriaxone ceftriaxone) ) ! ! ertap ertapé én nè ème me Natural Natural susceptibility susceptibility: : ! carboxypenicillins ! carboxypenicillins ( (ticarcillin ticarcillin) ) ! ! ureidopenicillins ureidopenicillins ( (piperacillin piperacillin) ) ! ! cephalosporins cephalosporins ( (ceftazidime ceftazidime, , cefsulodin, , cefepime cefepime, , cefsulodin cefpirome) ) cefpirome ! monobactam ! monobactam ( (aztreonam aztreonam) ) ! carbapenems ! carbapenems ( (imipenem imipenem, , meropenem) ) meropenem
Acquired resistance to to ß ß- -lactams lactams; ; ß ß- -lactamases lactamases Acquired resistance ! Overexpression ! Overexpression of of cephalosporinase cephalosporinase ! Restricted ! Restricted- -spectrum penicillinases spectrum penicillinases ! Expanded ! Expanded- -spectrum spectrum ß ß- -lactamases lactamases ( (ESBLs ESBLs) ) ! Carbapenemases ! Carbapenemases ( (metallo metallo- -ß ß- -lactamases lactamases ) ) ! Expanded ! Expanded- -spectrum oxacillinases spectrum oxacillinases
In-vitro spectrum of ß-lactamases In-vitro spectrum of ß-lactamases Pénicillinss Cephalosporinss Cephalosporins ß-lactamins/ Carbapenemss 3 rd génération 1st et 2 nd Inhibitors of generation * cefepime**, ß-lactamases cefpirome** Enzyme Class Penicillinases A ESBLs B Carbapenemases (metallo-enzymes) Cephalosporinases C Overexpression of cephalosporinases D Oxacillinases * Cephamycins excluded for ESBLs ** Cefepime, cefpirome excluded for overexpressed cephalosporinase
Overexpressed cephalosporinases β - to all β ! Resistance ! Resistance to all - lactams except to to lactams except carbapenems carbapenems ! 9 ! 9- -12 % of 12 % of the strains the strains
Narrow- -spectrum penicillinases spectrum penicillinases Narrow ! Carbenicillinases ! Carbenicillinases (PSE), oxacillinases (OXA), TEM (PSE), oxacillinases (OXA), TEM resistance to : to : ticarcillin ticarcillin, , piperacillin piperacillin, , cefsulodin cefsulodin resistance intermediated susceptibility to : to : cefepime cefepime, , cefpirome cefpirome, , aztreonam aztreonam intermediated susceptibility susceptibility to : to : ceftazidime ceftazidime, imipenem, , imipenem,meropenem meropenem susceptibility ! 10 % of ! 10 % of P. aeruginosa P. aeruginosa strains strains ( 90 % ( 90 % with with PSE PSE- -1). 1). ! Non ! Non- -inhibitory activity inhibitory activity of of inhibitors against inhibitors against oxacillinases oxacillinases and and PSE PSE- -1 1
OXA-4
Extended- -spectrum spectrum ß ß- -lactamases; ESBL lactamases; ESBL Extended (Ambler class A) (Ambler class A) ! TEM, SHV, VEB, PER, GES, BEL ! TEM, SHV, VEB, PER, GES, BEL ressitance to all ressitance to all ß ß- -lactams except lactams except to to carbapenems carbapenems inhibition by clavulanic acid clavulanic acid, , tazobactam tazobactam, , sulbactam sulbactam inhibition by ! An exception : GES ! An exception : GES- -2 2 Additional resistance to to carbapenems carbapenems Additional resistance
VEB-1 PER-1
BEL-1
ESBLs in P. aeruginosa Detection Distribution in France
P. aeruginosa aeruginosa : GES : GES- -2 2 P. GES-1/GES-2: Gly170Asn 170 170
ESBLs and P. aeruginosa • ESBL ESBL genes associated with genes associated with insertion insertion sequence and sequence and transposons (SHV, transposons (SHV, • PER- PER -1) , 1) ,with with class 1 class 1 integron and specific integron and specific sequences sequences ( (Re Re); VEB, GES, ); VEB, GES, BEL- BEL -1 1 P ant ant intI1 ( P 2) intI1 5'CS 3'CS 5'CS 3'CS Gene- Gene - cassette cassette intI1 intI1 3'CS 3'CS 5'CS 5'CS • Consequences Consequences : : co co- -expression ; co expression ; co- -selection selection; ; co co- -resistance resistance •
ß-lactamases Metallo-
Metallo-ß-lactamases: carbapenemases (Ambler class B) VIM-2 • Resistance to all ß- lactamins except to aztreonam • Genetic background; plasmids, transposons, integrons… • Associated resistance; aminoglycosides ++
Heterogeneous expression of expression of metallo metallo- -enzymes enzymes Heterogeneous ( Senda et al . Antimicrob. Agents Chemother. 1996 ; 40, 349-353) µ g/ml) ( µ MICs ( g/ml) MICs IMP MPM CAZ AZT IMP MPM CAZ AZT P. aeruginosa aeruginosa P. MNA 1456 >128 >128 >128 >128 MNA 1456 >128 >128 >128 >128 MNA 14102 >128 >128 >128 64 MNA 14102 >128 >128 >128 64 MNA 14115 32 64 >128 16 MNA 14115 32 64 >128 16 MNA 1560 2 16 >128 32 MNA 1560 2 16 >128 32
Metallo- β -Lactamases: epidemiology Country Year(s) Prevalence Species Isolates 3700 (17 hosp.) Japan 92-94 P. aeruginosa 0.4% (IMP) 2533 Japan 96-97 1.3% (IMP) P. aeruginosa Nationwide 3222 4.4% (IMP) S. marcescens 200 Japan 98 & 00 P. aeruginosa Regional 0.7% (IMP) 800 Enterobacteriaceae 801 (3 hosp.) Japan 98-02 1.1% (IMP) Gram-negatives Japan 02 P. aeruginosa 1.9% (IMP/VIM) 594 Nationwide 233 (1 hosp.) 0.9% (VIM) Greece 98-00 Pseudomonas spp. 506 (1 hosp.) 0.8% (VIM) Italy 01 P. aeruginosa Tsakris et al ., Clin. Microbiol. Infect. 2003; 9:846 Senda et a l., Antimicrob. Agents Chemother. 1996; 40:349 Luzzaro et al ., Diagn. Microbiol. Infect. Dis. 2004; 48:13 Kurokawa et al ., Lancet 1999; 354:955 Jones et al ., Diagn. Microbiol. Infect. Dis. 2004; 49: 289 Yamasaki et al ., J. Antimicrob. Chemother. 2003; 51:631 Kimura et al ., J. Clin, Microbiol. 2005; 43: 458
Metallo- β -Lactamases: Impact on resistance Country Year(s) Prevalence Species Phenotype IMI-R CAZ-R 11% (IMP) Japan 92-94 P. aeruginosa 25% (IMP) Japan 91-96 P. aeruginosa CAZ-R Enterobacteriaceae 32% (IMP) Japan 01-02 P. aeruginosa 62% (IMP) CAZ-R Other GNNFs PER/SUL-R 11% (VIM) Enterobacteriaceae Pseudomonas spp. 11% (VIM) Korea 00-01 IMI-R/I Acinetobacter spp. 14% ( VIM/IMP ) Greece 01 P. aeruginosa IMI-R/I 62% (VIM) P. aeruginosa IMI-R 39% ( VIM/IMP ) 99-02 Italy Senda et a l., Antimicrob. Agents Chemother. 1996; 40:349 Lee et al ., Emerg. Infect. Dis.. 2003; 9:868 Giakkoupi et al ., J.Clin.Microbiol. 2003; 41:822 Hirakata et al ., Antimicrob. Agents Chemother. 1998; 42:2006 Toleman et al. , J. Antimicrob. Chemother. 2005; 55:61 Shibata et al ., J.Clin.Microbiol. 2003; 41:5407
Associated resistance…. Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes . Clin Microbiol Infect. 2002 . Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo ß-lactamases; the quiet before the storm . Clin Microbol Rev 2005
Extended-spectrum oxacillinases (Ambler class D) ß-Lactamase TIC TCC PIP TAZ CAZ FEP ATM IMP OXA-2 128 64 32 4 16 2 2 4 OXA-15 128 64 32 8 128 4 8 2 OXA-32 256 64 32 16 128 8 32 4 OXA-10 512 64 2 4 16 2 OXA-11 512 64 512 4 128 2 OXA-14 512 256 64 32 512 64 16 2 OXA-16 128 64 32 16 128 32 4 1 OXA-13 256 256 32 32 2 8 1 OXA-19 256 256 64 64 256 16 1 OXA-28 128 128 16 16 256 16 32 0,25 OXA-18 256 64 64 32 128 16 256 2 OXA-31 512 512 256 256 2 256 32 TIC, ticarcillin, TCC, ticarcillin + clav.; PIP, piperacillin; TAZ, piperacillin + tazobactam; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam, IMP, imipenem Detection ? Epidemiology ?
Diversity of oxacillinase resistance patterns OXA-32 OXA-31
Aminoglycoside resistance • Efflux • Outer membrane permeability defect • Modification enzymes • Target alteration
phosphorylation APH(3') P. aeruginosa ; natural resistance APH 3’ OH HO 4' O HO H 2 N OH 2" O H 2 N NH 2 6' HO HO O O NH 2 H 2 N 3 nucleotidylation ANT(2") acetylation AAC(3)
Novel mechanism of resistance to rifampicin
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