Prof. Stefani MRSA - Venezia 2010 ppt

MRSA e oltre….Epidemiologia delle resistenze

Problemi diagnostici

Stefania StefaniDipartimento di Scienze Microbiologiche

Universià degli Studi di Catania (I)Email [email protected]

Summary

Venezia - ESCMID conference2010

• Main Gram-positives• Staphylococci• CoNS

• Major problems of resistance• Impact of Reduced Vancomycin

Susceptibility (RVS)• Linezolid resistance• Problems in resistance detection

Gram-positives:

their epidemiological role

• Community-acquired infections – Streptococcus

pneumoniae, Streptococcus pyogenes, CA-MRSA

• Health-care associated infections – CA-MRSA

• Nosocomial infections – HA-MRSA, VRE, MRCoNS,

Corynebacterium striatum


Staphylococcus aureus

1920 1930 1940 1950 1960 1970 1980 1990 2000

Fleming discovers penicillin

First use of

penicillin

Emergence of PRSA

Pandemic MRSA

First use of methicillin

Emergence of MRSA

Toxic shock syndrome

In the USA

Emergence of CA-MRSA

Emergence of VISA,

hVISA and VRSA

Pandemic PRSA

Phage type 80/81

Pandemic HA-MRSA

MRSA-I, followed by II, III

Pandemic CA-MRSA

MRSA-IV + virulence factors

Toxic shock syndrome in the USA

De Leo F, et al J Clin Invest 2009; 119: 2464


Why is MRSA a major concern?

•Frequency

•Virulence•Mortality rate

Methicillin-resistant Staphylococcus

aureus infections: the numbers

1. Approximately 100,000 invasive MRSA infections occurred in 2005 in the USA with 19,000 associated deaths (more or less the same for HIV and AIDS).1

2. In some countries in Europe (Italy, UK, Ireland, Iceland, Greece, Malta, Portugal, Turkey), the incidence of MRSA varied from 7.7 to 24.4 per 100,000 patients/day.2

3. High-virulence organism. Higher mortality rates observed for MRSA BSI than for MSSA BSI in 2 meta-analyses6.

4. MRSA account for ~ 40-60 % of all staphylococcal isolates in Italianhospitals. 3,4,5

5. Community virulent MRSA infections: an increasing problem worldwide

1) Kleven RM et al – JAMA 2007; 298: 1763; 2) Data from EARSS 2007; 3)Stefani S et al –CMI

2003; 9:1179; 4) Raineri E et al – J.Hosp. Inf 2007; 67:308; 5) Lanini S. et L Inf Contr Hosp

Epidemiol 2009; 30: 659; 6) Farr Bm. Infect Control Hosp Epidemiol 2006; 27: 999Venezia - ESCMID conference

2010

HA-MRSA infections: factors driving risk

• Pathogenicity of the microrganism

• General and local patient immunity

• Iatrogenic procedures

• Cross-contamination

• Antibiotic pressure

S.aureus is both a human commensal bacterium present on the

skin and in the nares of c. 30% of healthy people, and one of

the top pathogens in a large range of human HAIs and CAIs,

due to the huge number of enzymes and toxins that this

species can produce.Venezia - ESCMID conference

2010

HA-MRSA vs CA-MRSAin terms of virulence

• There was evidences that the 16 pandemic HA-clones are more resistant to antibiotics, but they are less virulent if compared with the CA-MRSA clones. Specific clones are now emerging with enhancedvirulence and new antibiotic suceptibility profiles1,2;

• A great number of these clones belongs to agrI and agrII groups: in many of them this locus is largely dysfunctional; this means thatthey express adhesins more than toxins3;

• hVISA strains seem to be less virulent4, 5. Clinical data are lacking.

• The epidemiology of HA-MRSA is changing vs more virulentclones1,2

1) Campanile F et al ACMA 2009; 24:8; 2) Dauwalder O et al JCM 2008; 46:

3454 ; 3) Cafiso V et al – FEMS Imm Med Microbiol 2007; 51: 220 4) Peleg AY et

al JID 2009; 199:532; 5) personal observationsVenezia - ESCMID conference2010

Virulence factors of

Staphylococcus aureus

ToxinsToxins::

αα--hemolysinhemolysin ((hlahla))ββ ––hemolysinhemolysin ((hlbhlb))δδ--hemolisinhemolisin ((hldhld))γγ--hemolisinhemolisin ((hlghlg))ExfoliatinExfoliatin ((etaeta) ) SuperantigenSuperantigenEnterotoxinEnterotoxin ((seasea, , sejsej) ) SuperantigenSuperantigenTSSTTSST--1 (1 (tsttst) ) SuperantigenSuperantigenLeukocidinLeukocidin ((luklukEE))PVlPVl ((luklukSS/F) /F)

AdherenceAdherence

BindingBinding ProteinsProteins forfor

bindingbinding toto the the hosthost::

fibronectinfibronectin--bpbp ((fnbfnbAA) )

collagencollagen--pbpb ((cnacna))

clumpingclumping factorsfactors ((clfclfAA/B)/B)

intercellularintercellular adhesionadhesion ((icaicaAA))

SerSer--AspAsp richrich proteinprotein ((sdrsdrEE))

autolysinautolysin ((atlatlEE))

spaspa ((proteinprotein A)A)

EsoenzymesEsoenzymes usedused forforinvasioninvasion::

CoagulaseCoagulase,,

StaphylokinaseStaphylokinase,,

HyaluronidaseHyaluronidase,,

LipaseLipase

ProteaseProtease ((splsplBB))

IgGIgG boundbound byby the the

FcFc portionportion

ProteinProtein AA

AntiphagocytosisAntiphagocytosis

Cap5/8 (Cap5/8 (capsularcapsular antigens)antigens)

spaspa ((proteinprotein A)A)


HA-MRSA vs CA-MRSA

Two distinct organisms?

CA-MRSA vs HA-MRSA: distinction is nowblurred!

Characteristics CA-MRSAFirst isolates

CA-MRSACurrent strains

HA-MRSABefore 2007

HA-MRSAAfter 2007

Clinical Infections SSTIs Pneumonia, BSI, endocarditis

Respiratory,Urinary, BSI,

others

Respiratory,Urinary, BSI,

others

Epidemiology Multiple clonalorigin

Multiple clonal origin

Fewer clones

healthcareassociated

Fewer clones

Heathcareassociated

Underlyingconditions

? ? healthcareassociated

Risk factors

healthcareassociated

Risk factors

Age Younger ? Older Older

Antibioticsusceptibility

Many classes

Including AG, FQ

Resistantclones are appearing

Few classes

MDR

More susceptibleclones

Genotypiccharacteristics

SCCmectypeIV – PVL +

SCCmectypes IV, and variants V, VI

SCCmec I, II, III, PVL -

SCCmec IV

PVL? Othervirulent traits.Venezia - ESCMID conference

2010


CA-MRSA in

Italy n.19

ST 8 (t0008)

ST 80 (t044,

2453)

ST88 (t2526,

002)

ST772 (t345)

ST5 (t319, 002)

ST30 (t755,

Italian CA-MRSA (16) susceptibility testing

Range (mg/L) MIC 50 MIC 90

Ceftobiprole 0.25-1 1 2

Vancomycin 1-2 1 2

Teicoplanin 0.5-2 1 1

Daptomycin 0.12-1 0.25 0.5

Linezolid 0.25-4 1 2

Q/D*6 ceppi resistenti

0.25-4 1 2

Tigecycline 0.12-0.5 0.25 0.25

Stefani S et al JAC 2009Venezia - ESCMID conference

2010

What is going on withMRSA…

CC ST spa-type ST-MRSA-SCCmec Epidemic clones Geographic spread

1 1 t127, t128, t174, t175, t176, t386, t558 ST1-CA-MRSA-IV USA400 pvl + South Africa;USA; Chn, Ids, Lat, Leb; Tai; NZ;Aus, Bel, Cro, Cyp, Den, Fin, Fra, Ger, Ice, Net, Nor, Pol, Rom, Spa, Swe, Swi, UK.

5 5 t001, t002, t003, t010, t045, t053, t062, t105, t178, t179, t187, t214, t311, t319, t389, t443 ST5-HA-MRSA-II New York/Jap or USA100 Can, Mex, Uru, USA; Chn, Isr, Jap, Kor,SA, Sin, Tai; Ast; Bel, Den, Fin, Fra, Ger, Hun, Ire, Nor, Ger, Swe, Swi,

UK.

5 ST5-HA-MRSA-I UK-EMRSA3 Arg, Par;Jap, Tai; Den, Ger,Nor, Pol, Ger, Slo, UK.

5 t001, t002, t003, t010, t045, t053, t062, t105, t178, t179, t187, t214, t311, t319, t389, t443 ST5-HA-MRSA-IV Paediatric or USA800 Alg; Arg, Bra, Col, Uru, USA;Kor, Tai;Ast; Aus, Den, Fin, Fra, Ger, Gre, Nor, Pol, Ger, Spa, Swe, UK.

5 t002 ST5-VISA/hVISA-II Mu50(VISA); Mu3(hVISA) Jap

228 t001, t023, t041, t188, t201 ST228-HA-MRSA-I Southern German or Italian

Aus , Bel, Den, Fin, Ger, Hun, Ita, Pol, Slo, Spa, Swi.

250 t008, t009, t194 ST250-HA-MRSA-I COL; Archaic Colindale (UK); Uga; Can, USA; Ast; Den, Ger, Swi, UK.

8 t008, t024, t064, t190, t206, t211 ST8-HA-MRSA-IV UK-EMRSA2/6 or USA500 Can, USA; Isr, Jap,Tai; Ast; Aus, Bel, Den, Fin, Fra, Ger, Hun, Ire, Net, Nor, Swi.

8 t008, t024, t064, t190, t206, t211 ST8-HA-MRSA-II Irish-1 Can, USA; Ast;Ire, UK.

8 t008, t024, t064, t190, t206, t211 ST8-CA-MRSA-IV USA300 pvl + ACME + South Africa; Gab; Can, USA; Est, Ind, Isr, Jap, Jor, Leb; NZ; Aus; Bel, Bul, Cro, Cze, Den, Fin, Fra,, Ger, Hun, Ice, Ita, Net, Nor, Pol, SR, Spa, Swe, Swi, UK.

247 t008, t051, t052, t054, t200 ST247-HA-MRSA-I Iberian or UK-EMRSA5; Rome

USA; Isr; Aus, Bel, Cro, Cze, Den, Fin, Fra, Ger, Hun, Ita, Net, Nor, Pol, Ger, Slo, Spa, Swe, Swi, UK.

239 t030, t037, t234, t387, t388 ST239-HA-MRSA-

III Brazilian; Hungarian Alg; Arg,Bra, Can, Chi, Par, Uru, USA; Chn, Ind, Ids, Kor, Mon,RoG, Rus, SA, Sin, Sri, Tai Tha,Vie; Ast; Aus, Cze, Den, Fin, Ger, Gre, Hun, Net, Nor, Pol, Ger, Slo, Spa, Swe, UK.

22 22 t005, t022, t032, t223, t309, t310, t417, t420 ST22-HA-MRSA-IV UK-EMRSA15, Barnin or GS-MRSA

Can; Chn, Kuw, Sin, Ast, NZ; Aus, Bel, Cze, Den, Fin, Ger, Hun, Ire, Mal, Nor, Ger, Spa, Swe, UK

30 30 T021 ST30-MSSA Phage-type 80/81 pvl + Pandemic, during the 1950s

30 t012, t018, t019, t021, t138, t268, t276, t318, t338, t391 ST30-CA-MRSA-IV Southwest Pacific or USA1100 pvl +

South Africa; Can, USA; Jor, Lat, Leb; NZ; Aus, Bel, Cyp, Cze, Den, Fin, Fra, Ger, Ice, Ita, Net, Nor, Pol, Spa, Swe, Swi, UK.

36 t018, t253, t418, t419 ST36-HA-MRSA-IV UK-EMRSA16 or USA200 Can, Mex, USA; Ast; Aus, Bel, Den, Fin, Ger, Gre, Ire, Nor, Ger, Spa, Swe, Swi, UK.

45 45 t004, t015, t026, t031, t038, t050, t065, t204, t230, t390 ST45-HA-MRSA-IV Berlin or USA600 Arm, Chn, Isr; USA; Ast; Aus, Bel, Den, Fin, Ger, Hun, Net, Nor, Spa, Swe, Swi.

59 59 t199, t216, t444 ST59-CA-MRSA-IV Taiwan or USA1000 pvl + USA; NZ; Aus , Bel, Cze, Den, Fra, Ger, Ice, Net, Nor, Pol, Spa, Swe, Swi, UK.

80 80 t044, t131, t376, t416, t436, t455, t1109 ST80-CA-MRSA-IV European pvl + Jor, Leb; Aus , Bel, Bul, Cro, Cyp, Cze, Den, Fin, Fra, Ger, Hun, Ice, Ita, Net, Nor, Spa, Swe, Swi, UK

Molecular details and geographic distribution of the major HA- and CA-MRSA clones.

8

Campanile F. et al – European Inf Dis 2010; 4(1): 70-76Venezia - ESCMID conference

2010

MSSA and MRSA - BSIs

• Six-month period: 25 BSIs, 72% MSSA and 28% MRSA.

• Vancomycin MICs: 1-2 mg/L

• 12% of the strains were hVISA (1 strain was MS)

• MRSA strains: no MDR, only one was a nosocomial isolate (ST228-HA-MRSA-I), all others were CA-MRSA: ST22-CA-MRSA-IV; ST5-CA-MRSA-IV; ST88-CA-MRSA-IV.3

Stefani et al – unpublished data 2009

AMCLI Rimini (I) November 2009


The hazard of animalassociated MRSA

• In animal food a new clone CC398 has emerged.• Carriage of this clone has been found in intensively

reared production animals (primarily pigs, but also cattleand poultry) in several countries around the world.

• CC398 can be transmitted from food producing animalsto humans.

• Animals in food production and their products are therefore a potential source of MRSA for humans.

Joint scientific report of ECDC, EFSA and EMEA on MRSA in livestock, companion animals and foods. EFSA

scintific report 2009; 301: 1-10 and EMEA/CVMP/SAGAM/62464/2009


Host specificity• Different states of interactions: 1) infections, 2) carriage

or colonization, and 3) contamination.

• Animal and human isolates (cow) differ in the carriage of particular MGE carrying genes responible for hostimmune evasion (scin, chip, and/or salk) and proven tointeract specifically with the human immune system1,2,3:

these MGE are only present in human isolates;

• CC151 (found in cow in the UK) have double mutationsin hsdS gene: more susceptible to LGT

1) Gladysheva et al PNAS 2003; 100: 9168; de Haas et alJ.Exp Med 2004; 199: 687; Rooijakkers et al Nat

Immunol 2005; 6: 920


Resistance to antibiotics and antiseptics

• MRSA are always MDR to a wide range of antibiotics and alsosometimes, antiseptics and heavy metals, due to resistant genesencoded on MGE, or mutations in housekeeping genes; they are nearly always sensitive to glycopeptide antibiotics;

• VISA strains possess a partial resistance due to synthesis of a thicker cell wall that absorbs the antibiotic and allows sufficientcross-linking of newly synthesized cell wall for survival; howeverthese strains are rare, slow-growing and unfit;

• VRSA – few strains


RSV strains


• Main Gram-positives• Staphylococci• CoNS


Susceptibility (RVS)• Linezolid resistance• Problems in resistance detection


Antibiotic deployment – antibioticresistance observed

Clatworthy A - Nature Chem Biol 2007; 3: 541Venezia - ESCMID conference

2010

Vancomycin MIC creep• Change in the vancomycin MIC distribution in S. aureus

over time such that strains with higher MICs become more common

Types of vancomycin resistance• MIC ≤2 µg/ml1–4

Vancomycin tolerance (MIC/MBC ratios, time–kill curves)

• VISA (Mu50-like): MIC 4–8 µg/ml1,3,5

• hVISA (Mu3-like): MIC 2–4 µg/ml3,5

• VRSA (vanA+): MIC 16 µg/ml1–3,5

1. CLSI. Performance standards for antimicrobial susceptibility testing; 18th informational supplement. 2010. M100–S20

2. EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. April 2010

3. Gould I. Int J Antimicrob Agents 2008;31S2:1–9

4. May J et al. J Antimicrob Chemother 1998;42:189–197

5. Tenover FC, Moellering RC. Clin Infect Dis 2007;44:1208–1215

Venezia - ESCMID conference 2010


EUCAST web site, accessed April 2010

MIC creep!

GlycopeptidesEUCAST4/2010

CLSI2010

MIC breakpoints(mg/L)


Staphylococcus aureus S R S I R

Vancomycin ≤2 >2 ≤2 4-8 ≥16

Teicoplanin ≤2 >2 ≤8 16 ≥32

CoNS MIC breakpoints(mg/L)


Vancomycin ≤2 >2 ≤4 8-16 ≥32

Teicoplanin ≤4 >4 ≤8 16 ≥32

San Francisco ICAAC 2009


MRSA MIC creep in other antibiotics?

2001 Median

MIC, µg/ml

Isolates with MIC ≤ 2001 median MIC (%), by year

P-value2001 2002 2003 2004 2005

Vancomycin 0.75 84 76 48 29 24 <0.0001

Linezolid 0.5 72 82 77 60 41 <0.0001

Daptomycin 0.25 54 55 53 51 66 0.1361

Oxacillin 128 67 48 38 28 36 <0.0001

Proportion of isolates with MICs less than or equal to 2001 median MIC

Steinkraus G et al. J Antimicrob Chemother 2007;60:788–794


What is hidden behind a MIC between 1 and 2 mg/L?

hVISAhVISA


PAP PAP analysisanalysis

of MRSA of MRSA isolatesisolates


Sensitivity and specificity values for the three screening methods usedwith respect to PAP/AUC

METHOD TIME PAP PAP hVISAhVISANN°°3636 % SENS PAP VSSA NPAP VSSA N°°7878 % SPECVSSA hVISAhVISA VSSAVSSA hVISA

BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF

Table 3. Sensitivity and specificity values for the three screening metho ds used, with respect to PAP/AUC


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF

Table 2. Sensitivity and specificity values for the three screening methods used, with respect to PAP/AUC


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF

Table 3. Sensitivity and specificity values for the three screening metho ds used, with respect to PAP/AUC


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF


BHI T52MF 48h 9 2727 75% 3434 44 43.5%

BHI V4 48h 12 2424 88.8% 4848 30 61.5%2MF

E-testmacro 48h 9 2727 75% 7878 0 100%2MF

Table 2. Sensitivity and specificity values for the three screening methods used, with respect to PAP/AUC


Haemolytic activity

Reduced haemolytic activity in

hVISA and VISA strains (personal

observations)


•hGISA were determined by macroEtest with the following criteria TP>12mg/L or

VA>8 mg/L

•All strains were confirmed by PAP analysis

•** agr is disfunctional in these strains – work is in progress in our lab

•All hVISA were susceptible to linezolid and daptomycin

Stefani S. et al – abs 1487 ICAAC – San Francisco 2009 ; ms submitted 2010Venezia - ESCMID conference

2010

Italian hVISA (n.36) susceptibility testing

Range (mg/L) MIC 50 MIC 90

Ceftobiprole 1-4 4 4

Vancomycin 1-2 1 2

Teicoplanin 2-4 2 2-4

Daptomycin 0.25-1 0.25 0.5

Linezolid 0.5-4 1 2

Q/D 0.25-1 0.5 1

Tigecycline 0.12-0.5 0.5 0.5

Stefani S et al 2010 submittedVenezia - ESCMID conference

2010

• Main Gram-positives• Staphylococci• Enterococci• CoNS


Susceptibility (RVS)• Problems in resistance detection


LinezolidDaptomycin

cfr methyltransferase confersPhLOPSa* resistance


Methylation of A2503 at the C8 position

Verde –cloramphenicol

Verde scuro – clyndamycin

Grigio – Q/D

Grigio scuro – tyamulin

Blu -linezolid

•phenicols, lincosamides,

oxazolidinones. Pleuromutilins, and

streptograminA

Susceptibility testing methods fordaptomycin, vancomycin and linezolid

MethodsMethods DAPTODAPTO VANCOVANCO LINEZLINEZ

BrothBroth microdilutiodmicrodilutiod (BMD)(BMD) YESYES YESYES YESYES

EtestEtest YESYES YESYES YESYES

Agar Agar dilutiondilution NONO YESYES YESYES

Disk Disk diffusiondiffusion NONO YES?*^YES?*^ YES*YES*°°

AutomatycAutomatyc systemssystems YESYES YESYES YESYES

•Transmitted light

•^ diameters < 14mm should be tested by a ref MIC method

•° nonsusceptible results: confirmed by MICs

•? Failure in detection for hVISAVenezia - ESCMID conference

2010

• Staphylococus aureus is the “highest profile pathogen”among all MDR nosocomial pathogens;

• Among the antibiotic resistance mechanisms developedby bacteria, RSV seems to be the most dangerousbecause affects the global physiology of the organisms; the extent of this problem is still far to be explained and solved;

• Issues surrounding laboratory detection and the clinicalimpact of RVS continue to trouble clinicalmicrobiologists and infectiuos diseases specialists: weneed to reach a diagnostic consensus


Prof. Stefani MRSA - Venezia 2010 ppt

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