08/05/2019Ragusa, 3 Ottobre 2015
Antibiogramma e rilevazione
dei meccanismi di resistenza
nei gram positivi
Maria Lina Mezzatesta [email protected]
Dipartimento di Scienze Biomediche e
Biotecnologiche – MicrobiologiaUniversità degli Studi di Catania
HAI – surveillance report EU/EEA
(ECDC PPS) 2011-2012
Ragusa, ottobre 2015
PrologousMany years of Gram-positive
predominance
Few new drugs in the pipeline
Increase of Gram-negative
Enterobacteria
MDR, PDR, XDR
Relative frequency of Staphylococcus aureus as percentage of all microorganisms reported in HAIs, ECDC 2011-2012
Data from the ECDC point prevalence survey of healthcare-associated infections and antimicrobial use in acute care hospitals (ECDC PPS) in the period
2011-2012 as reported to TESSy as of 2013-02-06 14:06:48
Proportion of MRSA isolates in Europe, in 2012-13
Ragusa, Ottobre 2015
S.aureus: a unique organism
Toxins:α-hemolysin (hla)
β –hemolysin (hlb)
γ-hemolisin (hlg)
Exfoliatin (eta)
Superantigen Enterotoxin (sea, sej)
Superantigen TSST-1 (tst)
Superantigen Leukocidin (lukE)
PVl (lukS/F)
Adherence:Binding Proteins for binding to the host:
fibronectin-bp (fnbA)
collagen-pb (cna)
Clumping factors (clfA/B
intercellular adhesion (icaA)
Ser-Asp rich protein (sdrE
autolysin (atlE)
spa (protein A)
Esoenzymes used for invasion:
Coagulase
Staphylokinase
Hyaluronidase
Lipase
AntiphagocytosisCap5/8 (capsular antigens)
spa (protein A)
Adapted from: Lowy. N Engl J Med. 1998;339:520-532.
• The agr-locus controls the
production of many of these
virulance factors (toxins,
adhesins)
• Its effector molecule (RNAIII)
contains hld gene which
encodes δ-hemolisin
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An orchestra of virulence factors
MDRStaphylococcus aureus
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MDR/PDR/XDR
definitions
La tempesta perfetta - 2014
Staphylococcus aureus has propensity to develop resistance to many antimicrobial agents
Ragusa, Ottobre 2015
Acquisition of resistance determinants;
Phenotypic variation;
Profound alteration of its geneticrepertoire;
Compensatory roles of mutations (RAMs) mediating fitness and adptation.
Frequenza ed epidemiologia di S.aureus in Europa ed in Italia, la
nostra esperienza…
Ragusa, Ottobre 2015
S.aureus 3-month Survey, (AMCLI)
. ° Location of the 52 hospital laboratories participating in the survey indicated as dots in the map
Coordinatore:
Gian Maria Rossolini
Segretario:
Mario Sarti
Componenti:
Francesco Luzzaro, Laura
Pagani, Stefania Stefani, Pietro
E. Varaldo, Giovanni Gesu,
Teresa Spanu
Total (n.)
S.aureus/Total(n.-%)
MRSA/Total(n.-%)
MRSA/S.aureus(n.-%)
21873 2541/21873(11.6%)
910/21873(4.1%)
910/2541(35.8%)
Campanile F et al JGAR 2015 in press
Rates of MRSA and MSSA from BSIs, LRTIs and SSTIs and other sources
Source S.aureus n. (%) MRSA n. (%) MSSA n. (%)
BSIs 465 (18.3) 183 (39) 282 (61)
LRTIs 451 (17.7) 184 (41) 267 (59)
SSTIs 768 (30.2) 273 (35.5) 495 (64.5)
other 857 (33.8) 270 (31.5) 587 (68.5)
TOT. 2541 910 (35.8) 1631 (64.2)
BSI (bloodstream infections); LRTI (low-respiratory tract infections); SSTI (skin and soft-tissue infections)
Ragusa, Ottobre 2015
Activity of the main antimicrobials tested by automated methods against S.aureus isolates
MSSA (1044) MIC (mg/L) % RRange MIC50 MIC90 EUCAST
Oxacillin ≤0.25 to >4 ≤0.25 0.5 1.5
Cefoxitin ≤2 ≤2 ≤2 0.0
Teicoplanin ≤0.5 to ≥8 ≤0.5 ≤0.5 0.5
Vancomycin 0.003 to 2 1 2 0.0
Daptomycin ≤0.12 to 2 0.5 0.5 0.8
Linezolid ≤0.12 to ≥8 2 2 0.25
Clindamycin ≤0.25 to >8 ≤0.25 0.5 2.0
Erythromycin ≤0.25 to >8 ≤0.25 >8 15.5Mupirocina ≤1 ≤1 ≤1 0.0
Gentamicin ≤0.12 to >16 ≤0.5 1 7.7Moxifloxacin ≤0.25 to ≥8 ≤0.25 0.5 5.8Levofloxacin ≤0.12 to >8 0.25 1 7.2Tetracycline ≤0.5 to >16 ≤0.5 ≤0.5 3.7
TMP/SMX ≤0.5 to ≥16 ≤0.5 ≤0.5 0.9
Rifampicin ≤0.25 to >32 ≤0.25 ≤0.25 0.8
Tigecyclinea ≤0.12 to 0.25 ≤0.12 ≤0.12 0.0
MRSA (640) MIC (mg/L) %RRange MIC50 MIC90 EUCAST
Teicoplanin ≤0.5 to 8 ≤0.5 1 0.9Vancomycin ≤0.25 to 2 1 2 0.0
Daptomycin ≤0.12 to 2 0.5 1 3.2Linezolid ≤0.25 to ≥8 2 2 0.7Clindamycin ≤0.25 to ≥8 ≤0.25 ≥8 33.1
Erythromycin ≤0.25 to ≥8 ≥8 ≥8 65.4Mupirocina ≤1 to >8 ≤1 ≤1 0.0
Gentamicin ≤0.25 to ≥16 ≤0.25 ≥16 39.5
Moxifloxacin ≤0.25 to ≥8 4 ≥8 72.3Levofloxacin ≤0.5 to ≥8 ≥8 ≥8 85.8Tetracycline ≤1 to ≥16 ≤1 ≥16 12.6
TMP/SMX ≤0.5 to ≥16 ≤0.5 ≤0.5 3.2
Rifampicin ≤0.25 to >32 ≤0.25 4 16.4Tigecyclinea ≤0.12 to 1 ≤0.12 ≤0.12 0.9
CLONE(formerly) ST-MRSA-SCCmec PAP n.strain TrendArchaic ST8-HAMRSA-I hVISA 0 USA500-like ST8-HAMRSA-IV hVISA 8
Iberian ST247-HAMRSA-IA hVISA 0 Rome ST247-HAMRSA-I hVISA 0
Brazilian ST239-HAMRSA-IIIA hVISA 0 Italian ST228-HAMRSA-I hVISA 18 é
Veterinary ST398-LAMRSA-IV hVISA 2 é
EMRSA15(GentaS) ST22-HAMRSA-IV hVISA 4 éUSA100-like ST5-HAMRSA-II hVISA 5 éDNS-VISA-MRSA ST1-HAMRSA-IV VISA 1 é
CLONE(formerly) ST-MRSA-SCCmec PAP n.strain TrendArchaic ST8-HAMRSA-I hVISA 0 USA500-like ST8-HAMRSA-IV hVISA 8
Iberian ST247-HAMRSA-IA hVISA 0 Rome ST247-HAMRSA-I hVISA 0
Brazilian ST239-HAMRSA-IIIA hVISA 0 Italian ST228-HAMRSA-I hVISA 18 é
Veterinary ST398-LAMRSA-IV hVISA 2 é
EMRSA15(GentaS) ST22-HAMRSA-IV hVISA 4 éUSA100-like ST5-HAMRSA-II hVISA 5 éDNS-VISA-MRSA ST1-HAMRSA-IV VISA 1 é
Current MRSA epidemiology
Sometimes they come back….or disappear!
Prevalence and epidemiology of MRSA: molecular features
Principali problemi di resistenzetests diagnostici
Ragusa, Ottobre 2015
Penicillina:meccanismo inducibile
Misurare Oxacilllina con cefoxitin
hVISA/VISA
VRSA
Linezolid R
Daptomycin non susceptibility
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S.aureus è uno dei maggiori patogeni umani e rappresenta una delle principali cause di infezioni nosocomiali.
MRSA
(Methicillin Resistant Staphylococcus aureus)
1882 – Dr. Oston lo descrisse per la 1° volta
1940 – Penicillina
1950 – Penicillino-resistenti
1960 – Meticillina
1961 – MRSA (UK)
Prima dell’avvento degli antibiotici, il tasso di mortalità dovuto aquesto batterio era abbastanza alto e l’incalzante diffusione diceppi di S.aureus resistenti alle penicilline portò, nel 1960,all’introduzione nella pratica clinica della meticillina, un derivatosemisintetico della penicillina.
La resistenza alla meticillina è dovuta all’acquisizione del gene
mecAche codifica una penicillin-binding- protein addizionale(PBP2 o
PBP2a) a bassa affinità per i B-lattamici.La PBP2a, non venendo saturata, sostituisce le funzioni delle
normali PBPs, producendo resistenza. Il gene mecA si trova
inserito in una particolare regione genica dettalocus mec
che rappresenta il cosidetto “mec complex”, costituito dai geni mecA, mecI e mecR
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MRSA
Crescita confluente fino
al dischetto
Espressione omogenea
Sfumatura interna all’alone
di inibizione
Espressione eterogenea
• mecA viene espresso a livelli più alti in presenza di
Cefoxitin rispetto a Oxacillina
• L’alone di Cefoxitin è più netto (migliore leggibilità)
• Leggere a luce riflessa
Cos’è questo?
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Ragusa, Ottobre 2015
Evaluation of discrepancies between oxacillin and cefoxitin in CoNS
• Many studies have reported that 30-ug cefoxitin disk is more sensitive than 1-ug oxacillin disk for the detection of oxa-resistance in CoNS.
• Conflicting results were published:
• 2.2% of CoNS with mecA displayed cefoxitin disk inhibition zone >25 mm;
• 1.5% of CoNS on a large sample (9017 strains)
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I nuovi mec: mecB mecC
Ragusa, Ottobre 2015
Metodo “gold standard”:
presenza del gene mecA
Rilevazione della resistenza alla meticillina
Test rapido per PBP2a
Ragusa, Ottobre 2015
CLSI EUCAST
Susceptible (VSSA) 2 2
Intermediate (VISA) 4-8 -
Resistant (VRSA) 16 > 2
Clinical and Laboratory Standards Institute - CLSI, 2015;
European Committee on Antimicrobial Susceptibility Testing – EUCAST, 2015
Vancomycin MIC breakpoints
Ragusa, ottobre 2015
VISA(vancomycin-intermediate S.aureus)
hVISA(heterogeneous vancomycin-intermediate S.aureus)
VRSA(vancomycin-resistant S.aureus)
VISA
• Vancomycin MIC: 4-8 mg/L
• Intermediate resistance with 100% of the populationgrowing at 4 mg/L of vancomycin, and also sub.populationat 8 mg/L
• Mechanism: unknown, thickned cell wall is a commonfeature
VRSA
• Vancomycin MIC: 16 mg/L
• Omogeneous resistance
• Mechanism: plasmid mediated vanA gene
hVISA
• Vancomycin MIC: 1 to 2 mg/L
• Heterogeneouse resistance Subpopulations ofcells with varying levels of resistance to VAN;subpopulations of vancomycin intermediate cellswith growth at 8 mg/L at a low frequency of
10-6 CFU/ml.
• Mechanism: unknown, subpopulation of cells withVISA features
VISA traits
Phenotypic alteration
Slower growth
Smaller colony size
Thickened cell wall
Cell wall thickening
Genotypic alteration
Reduced agr activity
Reduced autolytic activity in part due to lower activity of VISA autolysin
Mutations, inactivation, and altered expression of graRS
vanA, vanB, and vanC1 to vanC3 negative
Ragusa, Ottobre 2015
Choice of easy-to-use detection methods
Vancomycin
heteroresistance
colonies
Sensitivity and specificity are
good
Easy-to-use methods
Tiny colonies around MIC
breakpoints are unambiguously
detected
Campanile et al., Int. J Antimicr. Ag. 2010; 27 th ECCMID, 2011; 28 th ICC, 2013
VISA/hVISA MIC cutoff (48 h: a) MET –
VA ≥8 and TP ≥8 or TP≥12; b) GRD - VA
or TP ≥8 (CLSI 2012; EAS 003)
Methods based on Etest (and MIC-Test-Strip) are more suitable for the
detection ofheteroresistant sub-populations:
Sensitivity is good
Tiny colonies around MIC breakpointof vancomycin and teicoplanin areunambiguously detected
43
Detect ing hVI SA I solates: Etest Clues
Tiny colonies around MIC breakpoint of
vancomycin and teicoplanin
hVISA/VISA detection is tedious and difficult
GRD method
GRD (μg/ml)
positive
MIC
(μg/ml)
hVISA Vancomycin OR
teicoplanin ≥ 8
vancomycin
≤ 4
VISA
Vancomycin OR
teicoplanin ≥ 8
vancomycin
≥ 6
Glycopeptide
Resistance
Detection
(GRD)
Inoculum: 100µl
Suspension: 0.5 McFarland
Medium: MHA with/without 5% sheep
Incubation: 35°C. First reading at 24h. Final
reading at 48h
Vancomycin and teicoplanin Etest (BioMerieux)
and MIC test Strip VA/TEC (Liofilchem®)
GRD positive: 1 or
few tiny colonies
around MIC
breakpoint of
vancomycin or
teicoplanin
S. aureus ATCC
29213; ATCC
700699 (Mu50-
VISA); ATCC
700698 (Mu3-
hVISA)
Method Inoculum Interpretation
Quality
control
Interpretation
Distribution of MIC values from hVISA isolates (n.44) and activity of the main anti-Gram positive antimicrobials, by broth microdilution method (BM).
range MIC50/90 %S/R1 ≤0.016 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16
Vancomycin 0.5-2 1/1 100.0/0.0 - - - - - - 11 31 2 - - -
Teicoplanin 0.25-4 0.5/2 100.0/0.0 - - - - - 5 13 13 10 3 - -
Linezolid 0.125-4 0.5/2 100.0/0.0 - - - - 2 8 15 6 10 3 - -
Daptomycin ≤0.064-2 0.5/1 95.4/- - - - 2 4 4 25 7 2 - - -
Tigecycline 0.016-1 0.125/0.5 97.8/2.2 3 2 3 10 13 9 3 1 - - -
Rifampicin ≤0.016-8 ≤0.016/2 69.5/25.5 28 0 0 0 0 1 2 7 4 2 - -
Distribution of MIC values from VSSA isolates (n.132) and activity of the main anti-Gram positive antimicrobials, by broth microdilution method (BM).
range MIC50/90 %S/R1
≤0.01
6 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16
Vancomycin 0.25/2 1/1 100.0/0.0 - - - - - 7 49 65 11 - - -
Teicoplanin 0.25/2 0.25/1 100.0/0.0 - - - - - 70 33 21 8 - - -
Linezolid 0.125/4 1/2 100.0/0.0 - - - - 2 1 40 59 27 5 - -
Daptomycin 0.016-2 0.5/1 90.9/- - 1 10 24 17 16 33 19 12 - - -
Tigecycline ≤0.016-4 0.125/0.5 95.5/4.5 15 6 12 22 18 30 23 3 2 1 - -
Rifampicin ≤0.016-4 ≤0.016/1 87.9/12.1 70 9 13 14 3 2 5 5 6 5 - -
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Prevalence and epidemiology of hVISA in Italy
S.aureus MRSA hVISA*
BSIs 465 183 45
LRTIs 451 184 66
SSTIs 768 273 49
TOTAL 1684 640 160
25%
hVISA
Prevalence of the hVISA phenotype among S. aureus from BSIs, LRTIs and SSTIs
*hVISA were obtained by GRD
Ragusa, Ottobre 2015
Molecular epidemiology of hVISA in Italy
Prevalence of the main MRSA clones among VSSA and hVISA strains
VSSA
hVISA
E-MRSA15 ST22 - SCCmec IV.h 37%
USA500 like ST8 - SCCmec IV.C 23.5%
USA100 like ST5/105 - SCCmec II 7.7%
Italian clone ST228 - SCCmec I 4.5%
USA300 like ST8 - SCCmec IV.C 3.8%
Brasilian Clone ST241 - SCCmec III 2.3%
Sporadic: ST1-ST5-8-ST22- ST30-ST58- ST217-ST241-ST772 21.2%
Italian clone ST228 - SCCmec I 57%
USA100 like ST5/105 - SCCmec II 16%
USA500 like ST8 - SCCmec IV.C/I 13.5%
E-MRSA15 ST22 - SCCmec IV.h 4.5%
Sporadic: ST5 - ST1446 9%
Ragusa, Ottobre 2015
Dalbavancin Daptomycin Vancomycin Teicoplanin CeftarolineVSSA RANGE 0.06-0.125 0.12-1 0.5-1 0.25-4 0.125-1
MIC50 0.125 0.25 1 0.5 0.25MIC90 0.125 0.5 1 1,00 0.25
hVISA RANGE 0.06-0.25 0.5-2 0.5-2 0.5-4 0.125-1MIC50 0.125 1 1 0.5 0.5MIC90 0.25 2 2 4 1
VISA RANGE 2-4 2-4 8 0.5-4 0.25-0.5MIC50 4 2 8 8 0.25*MIC90 4 4 8 8 0.25*
hVISA behaviour against new drugs
0.12 0.25
1
0.50
0.25
0.120.50
1 1
0.25
VSSA
0.121 1
0.50 0.50
0.25
2 2
4
1
hVISA
4
2
8 8
0.25
4 4
8 8
0.25
VISA
* Possible seesaw effect: Werth BJ, et al., AAC 2013
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Daptomycin: mechanisms leading to attenuated daptomycin activity against S.aureus
Increased Daptomycin MICs in S.aureus:
1. Mutations in genes involved in the regulation of bacterial membranesurface charge: mprF (lysylphsphatidylglycerolsynthetase), yycFG (histidine kynase),rpoB/C, have also been identified in later stage of progression ofdaptomycin resistance in lab derived strains1,2.
2. Several dapto-R clinical isolates, lack these mutations!3. Dapto-non susceptible MRSA have emerged in parallel with VISA via
vancomycin exposure, raising the hypothesis that this mechanisms ofresistance might be due to impaired diffusion through thicker, lesscross-linked cell-wall, possible changes in membrane charge3,4,5
4. MORE WORK IS NEEDED TO FURTHER UNDERSTAND THE MECHANISMSOF THIS LOSS OF SUSCEPTIBILITY TO DAPTOMYCIN
1) Friedman L et al AAC 2006; 50:2137; 2) Mwangi MM et al – PNAS 2007; 104: 9451; 3) Pillai S
et al AAC 2007; 51: 2223; 4) Julian Ket al – AAC 2007; 51: 3445; Jones T et al – AAC 2008; 52:
269
Daptomycin resistance induced by vancomycin therapy
Isolates Dapto MIC Mprf mutations
S.aureus n.1 0.5 mg/L No
S.aureus n.2 1 mg/L No
S.aureus n.3 4 mg/L yes
Ragusa, ottobre 2015
-Autolytic and cell wall charges genes (mprf and dlt) involved
-Both drugs (vancomycin and daptomycin) exerted influence on increasing positive
charges on the cell wall (less binding of drugs)1;
-RpoB mutations involved2.
1) Stefani et al – personal data; 2) Cui L. et al AAC 2010; 54: 5222
In tutti i casi in cui sia
necessario confermare un
fenotipo di R inusuale,
inviare il ceppo ad un centro
di riferimento per la
conferma del fenotipo.
Ragusa, Ottobre 2015
Bacterial resistance to linezolid
• The first reports started to appear shortly after linezolid’s
introduction into the clinic1,2.
• Although the number of strains resistant to linezolid is still
low3,4 there are several reports about linezolid resistance
involving different clinical settings5,6,7.
• In almost all cases, resistance to linezolid affects the large
rbosomal subunit (50S) via a nucleotide mutation resulting in
G2576U (E.coli numbering) for one or more alleles of 23S
rRNA8 . After the first mutation arises, homologous
recombination and gene conversion of the other alleles can
take place.
• Gene dosage effect9, impairment of the biological fitness,
and cross-resistance to quinopristin/dalfopristin and
chloramphenicol.
1) Auckland C et al – JAC 2002; 50:743; 2) Tsiodras SH et al – Lancet 2001; 358:207;
3) Anderegg TR et al IJAA 2005; 26:13; 4) Jones RN et al – JAC 2006; 57:279; 5) Dobbs TE et al
JCM 2006; 44:3368; 6) Roberts SM. Et al –Ped Inf Dis 2006; 25: 562; 7)Seedat J et al- AAC 2006;
50: 4217; 8) Kloss Pl et al – J.Mol Biol 1999; 294:93; 9) Besier S. et al AAC 2008; 52: 1570
Linezolid resistance
The resistance to this antibiotic has been, until now, entirely associated with
distinct nucleotide substitutions in domain V of the 23S rRNA genes. Mutations in
associated ribosomal proteins also affect linezolid activity. Recently
Clinical mutants
G2576T
E.faecalis
E.faecium
S.aureus
CNS
Lab. mutants
G2447T, G2613T…
Enterococcus spp.
S.aureus
CNS
M, 100 bp; Lanes 1 to 6: 23S rRNA internal region
(V1V2); 7, V1V2-NheI restriction
NheI sequence
5'...G|CTAGC...3'
3'...CGATC|G...5'
322bp
98bp
M 1 2 3 4 5 6 7
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La lettura dell’alone di inibizione del Linezolid
sugli Stafilococchi deve essere effettuata
considerando anche le colonie più piccole visibili
ad occhio nudo presenti all’interno dell’alone
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Mutazionale (proteine ribosomiali )
e non mediato da cfr
PFGE No. of isolates ST SCCmec MECHANISM OF LNZR MIC range (mg/liter)
LNZ E L C
A1 /A2 13 23 IV cfr ≥256 1 32-≥256 64 - 256
A2 6 23 IV cfr± L3 (F147L-L94V), L4 (G71D-N158S)
128-256 1 ≥256 64
C1 2 2 VIII cfr ≥256 1-16 ≥256 64
C1/C2 12 2 VIII 23S rRNA (G2576T), L3 (G137A, L94V) 32-64 0.5-256 8-≥256 32-128
C3 8 2 VIII cfr
± 23S rRNA (G2447T/G2576T), L3 (L94V)≥256 8-32 4-16 8-64
F 3 5 IV L3 (H146Q-L94V), L4 (71GGR72, N158S) 64 4 1-2 16-64
cfr-mediated Linezolid resistance
Authors Country Species References
Mendes RE et al Mexico Staphylococci JCM 2010; 48:3041
Sanchez Garcia M et
al
Spain S.aureus JAMA 2010;
303:2260
Bonilla H et al USA S.epidermidis CID 2010; 51: 796
Locke JB et al USA S.aureus AAC 2010; 54: 5352
Mendes Re et al Italy S.epidemidis JAC 2010; 65: 2329
Bongiorno D et al Italy S.epidermidis JAC 2010; 65: 2336
Shore Ac et al USA S.aureus USA300 AAC 2010; 54:4978
Argudin MA et al Germany S.aureus ST398 (non
human)
AEM 2011; 4 ahead
of print
Mendes RE et al USA S.aureus;
S.epidermidis
AAC 2008; 52: 2244
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INTRODUZIONE AGLI ENTEROCOCCHI:
CARATTERISTICHE E PATOGENICITÀ
1899,Thiercelin: Diplococchi Gram+
1933, Lancefield: Steptococchi del gruppo D
1980, Murray: Enterococcus
Infezioni del tratto
urinario
Multiresistenza: fattori intriseci e acquisiti
E. faecalis: 90% - E.faecium: 5-10%
Patogenicità: Endocarditi
Batteriemie di pazienti
ospedalizzati
IL PROBLEMA DELL’ANTIBIOTICO RESISTENZA:
LA PRODUZIONE DI BETA-LATTAMASI
Le penicilline: inibizione della sintesi della parete cellulare
Meccanismi di resistenza:
PBP5
produzione di beta-lattamasi
Impiego di inibitori: “substrati suicida”:
•Acido clavulanico;•Sulbactam;•Tazobactam;
1981: Primo isolamento E.faecalis bla+
1992: Primo isolamento E.faecium bla+
PERCHÉ LE RICERCHE CONDOTTE PER L’IDENTIFICAZIONE
DELLE BETA-LATTAMASI SI SONO RIVELATE POCO SENSIBILI E
INEFFICACI?
Quantità dell’enzimaEffetto inoculoLocalizzazione cellulare
Università di Catania
PRODUZIONE DI BETA-LATTAMASI:
L’ OPERONE BLA
Meccanismo d’azione e resistenza della vancomicina
• Dal 1950 i glicopeptidi, vancomicina e teicoplanina, sono stati considerati come farmaci “salvavita” per il trattamento delle infezioni da enterococchi multiresistenti.
• Già nel 1988, è stata però riscontrata l’insorgenza di ceppi di E. faecium ed E. faecalis vancomicino-resistenti (VRE)
• La resistenza alla vancomicina si basa sulla produzione di un bersaglio alternativo.
• Normalmente il suo bersaglio è la parte terminale del precursore del peptidoglicano, D-ALA-D-ALA (figura A, in rosso), mentre le cellule resistenti non hanno il terminale appena citato bensì, D-ALA-D-LATTATO (o D-ALA-D-SER per il fenotipo VanC), che riduce di molto la sensibilità agli antibiotici glicopeptidici.
Classificazione fenotipica degli enterococchi resistenti alla vancomicina (VRE)
• Da un punto di vista fenotipico, si distinguono sei diverse classi di VRE ma, VanA e VanB, rappresentano i fenotipi maggiormente analizzati in ceppi di Enterococcus spp. isolati da materiali clinici.
Mics
VANCO
Mics
TEICO
Determinante
genico
mobilità specie
VanA 64->1.000 16-512 acquisito Si
(Tn1546)
E. faecium
E.faecalis
VanB 4-1.024 <0.5 acquisito Si
(Tn1549,
Tn1547)
E. Faecium
E.faecalis
VanC 2-32 <0.5 intrinseco no E.Gallinarum
E.caselliflavus
E.flavescens
VanD 128 4 acquisito No E. faecium
BM4339
VanE 16 <0.5 Acquisito No E. Faecalis
BM4405
VanG 16 <0.5 acquisito no E. Faecalis
WCH19
Il cluster genico vanA risiede su un trasposone coniugativo,Tn1546, appartenente alla famiglia dei Tn3 integrabili sul genomao su plasmidi autotrasferibili.
Tn1546
CEPPO Specie
MICs
vanco
MICs
teico Fenotipo PCR
MICs
dapto
MICs
tgc
MICs
q/d
MICs
lnz
5895 E. faecium 256 64 VanA vanA 4 ≤0,25 0,5 32
5903 E. faecium 16 2 VanB vanB 4 ≤0,25 0,5 16
Impallomeni G. E. faecalis >256 >256 VanA vanA 2 0,25 2 2
47634 E. faecalis >256 >256 VanA vanA 1 0,25 8 1
09053 E. gallinarum 8 8 VanC vanC 4 ≤0,06 16,0-64,0 4
C253 E. faecium 256 0,25 VanB vanB 2 0,25 0,25 4
C275 E. faecalis >256 258-128 VanA vanA 1 0,25 8 2
G31 E. faecium 32 ≤0,25 VanB vanB 2 0,25 0,25 4
G37 E. faecalis >256 >256 VanA vanA 1 0,25 4 4
G90 E. faecium 8 1 VanB vanB 4 ≤0,25 ≤0,25 2
Grazie per l’attenzione!Maria Lina Mezzatesta
Lab MMAR (http://www.labmicrobiologia.unict.it/)Dipartimento di Scienze Biomediche e
Biotecnologiche - UNICT