Antibiogramma e rilevazione dei meccanismi di resistenza ......08/05/2019 Ragusa, 3 Ottobre 2015...

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

mailto:[email protected]

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


An orchestra of virulence factors

MDRStaphylococcus aureus


MDR/PDR/XDR

definitions

La tempesta perfetta - 2014

Staphylococcus aureus has propensity to develop resistance to many antimicrobial agents


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…


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)


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


Penicillina:meccanismo inducibile

Misurare Oxacilllina con cefoxitin

hVISA/VISA

VRSA

Linezolid R

Daptomycin non susceptibility

08/05/2019 Ragusa, ottobre 2015

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

Ragusa, ottobre 201508/05/2019

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?


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)


I nuovi mec: mecB mecC


Metodo “gold standard”:

presenza del gene mecA

Rilevazione della resistenza alla meticillina

Test rapido per PBP2a


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

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

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 - -


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


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%


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


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


-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.

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


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

08/05/2019 Ragusa, Ottobre 2015

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


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

[email protected]

Lab MMAR (http://www.labmicrobiologia.unict.it/)Dipartimento di Scienze Biomediche e

Biotecnologiche - UNICT

mailto:[email protected]

Antibiogramma e rilevazione dei meccanismi di resistenza ......08/05/2019 Ragusa, 3 Ottobre 2015...

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