Virulence and Resistance in Staphylococcus Aureus: 2016 State of the Art Lyon, France

Antimicrobial susceptibility tests for Staphylococcus aureus and EUCAST recommendations

Virulence and Resistance in Staphylococcus aureus: 2016 State of the Art Lyon, France. 28 June – 1 July, 2016 © by author

Dr. Rafael Cantón ESCMIDHospital Universitario Online Ramón y Cajal Lecture Library SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA Departamento de Microbiología II Universidad Complutense. Madrid Antimicrobial susceptibility testing (AST)

CLSI (NCCLS) EUCAST

 CLINICAL BREAKPOINTS  CLINICAL BREAKPOINTS  EPIDEMIOLOGYCAL CUT-OFF © by author ESCMID Online Lecture Library www.eucast.org

Susceptibility MIC

Mechanism of resistance

Mechanism© by author of resistance ESCMID Online Lecture Library Susceptibility MIC Clinical breakpoints: the philosophy

. The aim of clinical breakpoints is to use MIC values …

- to separate strains where there is a high likelihood of treatment success from those where treatment is more likely to fail

- to adequately treat patients but not to detect resistance mechanisms from a microbiological point of view

. Clinical breakpoints have not been created to detect resistance mechanisms

. They are ultimately derived© by from author human clinical studies comparing outcomes with the MICs for the infecting pathogen

. If clinical breakpoints are well established no actions (expert ESCMID rules) are needed Online beyond MIC Lecture interpretation Library (interpretive reading)

.. but this has not been the case in the past! Interpretive reading of AST results

. During more than twenty years interpretive reading of the antibiogram has been used to: - infer resistance mechanisms behind resistant phenotypes - identify resistant organisms for infection control purposes - apply expert rules* and modify (when needed!) previous clinical categorization Courvalin P. ASM News 19921992;58:368-75 Livermore et al. J Antimicrob Chemother 2001;48(Suppl 1):87-102 Cantón R. Enferm Infecc Microbiol Clin 2002; 20: 176-86 Cantón R. Enferm Infecc Microbiol Clin 2010; 28:375-85 © byLeclercq author et al. Clin Microbiol infect 2013; 19:141-60 This approach was partially needed ESCMID Online due to inadequate Lecture breakpoints! Library

*Action to be taken (normally S or I to R), based on current clinical or microbiological evidence, in response to specific AST results Interpretive reading of AST results

. Interpretative reading: the classical example

ESBL positive isolate

ESCMID Online Lectureresistant toLibrary all cephalosporins and aztreonam (irrespective of MICs) Breakpoint definition (ISO 20776-1:2006)

… beyond CLSI and EUCAST

Values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical categories “susceptible”, “intermediate” and “resistant”

Susceptible bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic success

Intermediate bacterial© strain by inhibited author in vitro by a concentration of an antimicrobial agent that is associated with uncertain therapeutic effect ESCMID Online Lecture Library Resistant bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic failure S R CLSI, 2014

S R EUCAST, 2016 ©ECOFF by author ESCMID Online Lecture Library Clin Microbiol Infect, 2013; 19:141-60 © by author ESCMID Online Lecture Library EUCAST: Detection of resistance mechanisms

. EUCAST creates a subcommittee in 2012 to establish criteria for the detection of resistance mechanism of clinical and/or epidemiological importance

www.eucast.org EUCAST breakpoints, 2016 (version 6.0)

MIC (mg/L) brpts* S≤ 2 R>2 mg/L Zone (mm) brpts* S≥ 22 R<22 mm Insufficient evidence IE (Literature: ”not enough evidence for a Can not be substituted. breakpoint” or ”no indication”) Can be supplemented with an MIC without interpretation. Inappropriate drug — (Literature: poor drug – don´t use!© by authorCan be substituted with an automatic ”R”. *whenESCMID numbers are the Online same = no intermediate Lecture category Library S. aureus: EUCAST susceptibility testing

. Intrinsic resistance

. Expert rules

. Antimicrobial susceptibility and breakpoints © by author ESCMID Online Lecture Library Staphylococci: intrinsic resistance

ac.

Organisms Fusidic Ceftazidime Fosfomycin Novobiocin

Staphylococcus saprophyticus R R R R Staphylococcus cohnii R R Staphylococcus xylosus R R Staphylococcus capitis R R Other CNS and Staphylococcus© by author aureus R Also R to aztreonam, temocillin, polymyxin B/colistin, nalidixic ac. R= intrinsic resitance ESCMID OnlineEUCAST Expert Lecture Rules Version 3.0Library (on consultation)

Susceptibility testing is unnecessary and can be reported as R Staphylococci: expert rules

Agent Agent Rule Exceptions, scientific basis, Evidence tested affected and comments grade Oxacillin, All IF R to isoxazolyl-penicillins (as Production of PBP2a (encoded A cefoxitin b-lactams determined with oxacillin, cefoxitin, by mecA) (disk diffusion) or by detection of mecA-gene or of leads to cross-resistance to b- or PBP2a) lactams detection of except ceftobiprole and THEN report as R to all b-lactams mecA gene ceftaroline except those specifically licensed or to treat infections caused by PBP2a methicillin-R staphylococci owing to low affinity for PBP2a

Gentamcin All amino- IF resistant to gentamicin R to gentamicin is generally glycosides caused by the production THEN report© as by reisistant author to all production of ANT(4’)(4”)-I or aminoglycosides bifunctional APH(2’)-AAC(6) enzymes that determine loss of ESCMID Online Lecturesynergism Library of kanamycin, tobramycin and amikacin with b-lactams and glycopeptides irrespective of MIC values S. aureus: EUCAST susceptibility testing

. MIC determination according with the International Standards Organisation (ISO) recommendations (identical to CLSI) ISO 20776-1, 2006

. Disk diffusion (EUCAST standardised disk diffusion method)

- Medium: Mueller-Hinton agar - Inoculum: McFarland 0.5 - Incubation: Air, 35±1ºC, 18±2h - Reading: “Read zone edges as© the by point author showing no growth viewed from the back of the plate against a dark background illuminated with reflected light (except for benzylpenicillin and linezolid). ESCMID Online Lecture EUCAST breakpoint Library table v6, 2016

. Quality control: Staphylococcus aureus ATCC 29213 S. aureus ATCC 29213

QC Tables v6.1 (2016-03-01)

http://www.eucast.org/ast_of_bacteria/qc_tables/ S. aureus and benzypenicillin

MIC breakpoint (mg/L Zone diameter (mm) S ≤ R > S ≥ R < Benzylpenicillin 0.1251 0.1251 26A,B 26A,B

1/A. Most staphylococci are penicillinase producers, which are R to benzylpenicillin, phenoxy- methylpenicillin, ampicillin, amoxicillin, piperacillin and ticarcillin.

Isolates negative for penicillinase and S to methicillin can be reported S to these agents.

Isolates positive for penicillinase and methicillin-S are S to b-lactamase inhibitor combinations and isoxazolylpenicillins (oxacillin, cloxacillin, dicloxacillin and flucloxacillin).

Methicillin- R isolates are, with few exceptions, resistant to all b-lactam agents.

B. Disk diffusion is more reliable© than by MIC determinationauthor for detection of penicillinase producers, provided the zone diameter is measured AND the zone edge closely inspected. If the zone diameter is <26 mm, then report R. If the zone diameter is ≥26 mm AND the zone edge is sharp, then report R. If not ESCMID sharp, then report SOnline and if uncertain, thenLecture report R Library

Chromogenic cephalosporin-based b-lactamase tests do not reliably detect staphylococcal penicillinase S. aureus and benzypenicillin

Examples of inhibition zones with benzylpenicillin

Fuzzy zone edge and Sharp zone edge and ESCMIDzone diameter ≥Online 26 mm Lecturezone diameter Library ≥ 26 mm

Report susceptible Report resistant S. aureus and methicillin resistance

MIC breakpoint (mg/L Zone diameter (mm) S ≤ R > S ≥ R < Oxacillin Note1,2 Note1,2 NoteA NoteA

1/A. Methicillin-R isolates are, with few exceptions, resistant to all b-lactam agents.

2. S. aureus, S. lugdunensis and S. saprophyticus with oxacillin MIC values >2 mg/L are mostly methicillin-R due to the presence of the mecA or mecC gene. The corresponding oxacillin MIC for coagulase-negative staphylococci other than S. saprophyticus and S. lugdunensis is >0.25 mg/L. © by author Cefoxitin is more reliable to detect ESCMID Online Lecturemethicillin resistance Library than oxacillin S. aureus and CNS and methicillin resistance

MIC breakpoint (mg/L) Zone diameter (mm) S ≤ R > S ≥ R < Cefoxitin (screen) S. aureus, S. lugdunenesis, S. saprophyticus Note1 Note1 22A 22A Cefoxitin (screen) CNS other than S. lugdunenesis, S. saprophyticus Note2 Note2 25A 25A Cefoxitin (screen) S. pseudointermedius Note2 Note2 35A 35A

A. Susceptibility of staphylococci to cephalosporins is inferred from the cefoxitin susceptibility except for cefixime, ceftazidime, ceftibuten and ceftolozane-tazobactam, which do not have breakpoints and should not be used for staphylococcal infections. Some methicillin- resistant S. aureus are susceptible© toby ceftaroline author and ceftobiprole

1. S. aureus and S. lugdunensis with cefoxitin MICs >4 mg/L and S. saprophyticus with cefoxitin MICs >8 mg/L are methicillin R, mostly due to the presence of the mecA or ESCMIDmecC gene. Disk diffusion Online reliably predicts Lecture methicillin resistance. Library

2. For staphylococci other than S. aureus, S. lugdunensis and S. saprophyticus, the cefoxitin MIC is a poorer predictor of methicillin resistance than the disk diffusion test. S. aureus and methicillin resistance

1. For breakpoints for other fluoroquinolones (e.g. pefloxacin and enoxacin), refer to breakpoints set by national breakpoint committees. 2. Breakpoints are based on high dose therapy (oral dose of 750 mg x 2, iv dose of 400 mg x 3). © by author 3. Breakpoints are based on high dose therapy (400 mg x 2).

A. The norfloxacin disk diffusion test can be used to screen for fluoroquinolone ESCMIDresistance. Online Lecture Library B. Isolates categorised as S to norfloxacin can be reported S to ciprofloxacin, levofloxacin, moxifloxacin and ofloxacin. Isolates categorised as non- susceptible should be tested for susceptibility to individual agents. S. aureus and fluoroquinolones

Proposed modifications of breakpoints

MIC breakpoints (mg/L) Rationale for breakpoint Agent Current Proposal change S≤ R> S≤ R> Ciprofloxacin 1 1 11 11 - New PK-PD analysis 1 1 Levofloxacin 1 2 1 1 - No changes or marginal Moxifloxacin 0.5 1 0.25 0.25 changes in non-susceptible © by author rates Ofloxacin 1 1 11 11 NA=ESCMID Not applicable; 1Based Online on high dose. Lecture Library S. aureus and macrolides and lincosamides

1/A. Erythromycin can be used to determine susceptibility to azithromycin, clarithromycin and roxithromycin.

2. Inducible clindamycin resistance can be detected by antagonism of clindamycin activity by a macrolide agent. If not detected, then© reportby as authorS. If detected, then report as R and consider adding this comment to the report: "Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy". ESCMID Online Lecture Library B. Place the erythromycin and clindamycin disks 12-20 mm apart (edge to edge) and look for antagonism (the D phenomenon) to detect inducible clindamycin resistance. © by author ESCMID Online Lecture Library S. aureus and glyco- and lipoglycopeptides

1. Glycopeptide MICs are method dependent and should be determined by broth microdilution (reference ISO 20776). S. aureus with vancomycin MIC values of 2 mg/L are on the border of the wild type distribution and there may be an impaired clinical response. The R breakpoint has been reduced to 2 mg/L to avoid reporting "GISA" isolates intermediate as serious infections with "GISA" isolates are not treatable with increased doses of vancomycin or teicoplanin. 2. Non-S isolates are rare or not yet© reported. by The authoridentification and antimicrobial susceptibility test result on any such isolate must be confirmed and the isolate sent to a reference laboratory. 3. MICs must be determined in the presence of polysorbate-80 (0.002% in the medium for broth dilution methods; agar dilution methods have not been validated). Follow the manufacturers' ESCMIDinstructions for commercial Online systems. Lecture Library 4. S. aureus isolates susceptible to vancomycin can be reported susceptible to dalbavancin and oritavancin. 5. MRSA isolates susceptible to vancomycin can be reported susceptible to telavancin.

A. Disk diffusion is unreliable Vancomycin “S” S. aureus with MIC >1 ≤ 2 mg/L

SARM

S (≤2) CLSI, EUCAST R (>2) EUCAST

R (≥16), CLSI © by author SASM ESCMID Online Lecture Library Vancomycin “S” S. aureus with MIC >1 ≤ 2 mg/L

SUSCEPTIBLE RESISTANT hVISA VISA VRSA

MIC (mg/L) ≤ 1 1.5 2 4 - 8 ≥16

. Increment of isolates with glycopeptide tolerance . ©Slightly by increment author of daptomycin MICs but within the susceptible range ESCMID Online. Increment Lecture of failure and lengthLibrary of infection . Well documented in MRSA but less studied in MSSA and CNS . Potential subrogate marker of clinical failure Vancomycin and Staphylococcus aureus

. Decreased bactericidal activity in isolates with MICvancomycin= 2 mg/L

Methicillin susceptible S. aureus

- MICvancomycin: 1 mg/L

- MBCvancomycin: 4 mg/L

- MBCvancomycin: >256 mg/L ESCMID Online Lecture Library

May et al. J Antimicrob Chemother 1998; 42: 189-197 Vancomycin © by author ESCMID Online Lecture Library Vancomycin “S” S. aureus with MIC >1 ≤ 2 mg/L

. MRSA bacteremia:

- higher (P=0.03) mortality with MRSA with vancomycin MICs ≥1.5 mg/L

- patients with hetero-resistant (hVISA) isolates have prolonged (no significant ) duration (≥7-days) of bacteremia and higher mortality

VISA and hVISA

Bacterial evolutionary state favoring persistence due to alterations in susceptibility to: © by author - host immune responses - different antibiotics

ESCMID Online LectureHowden Library et al. Infect Genetic Evol 2014; 21:575-82

Musta et al. J.Clin Microbiol 2009; 47:1640-44 Vancomycin “S” S. aureus with MIC >1 ≤ 2 mg/L

Van Hal et al. Clin Infect Dis 2012; 54:755-71 Van Hal SJ and Fowler VC. Clin Infect Dis 2013; 56:1779-88 Guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance

hGISA and GISA detection . Disk diffusion: unreliable for hGISA or GISA, but useful GRSA

. MIC determination - broth microdilution (ISO 20776-1) is the gold standard - gradient strips (results 0.5-1 two-fold dilution steps higher)

.Specific tests for hGISA - macro gradient test: - does not differentiate between hGISA and GISA - inoculum (2,0 McFarland, higher than the standard) - positive test: ≥8 mg/L© for by both authorVAN and TEI, OR ≥ 12 mg/L for TEI alone - glycopeptide resistance detection (GRD) gradient test: - positive test: ≥8 mg/L for either VAN or TEI ESCMID- teicoplanin screening Online agar plates Lecture: Library - MH agar-5 mg/L TEI, 10 microliters of 2.0 McFarland inoculum - positive test: growth of more than two colonies at 48h - confirmatory test: population analysis profile (area under curve, PAP-AUC) Vancomycin “S” S. aureus with MIC >1 ≤ 2 mg/L

S R

S I R

1. Isolates susceptible to linezolid can be reported susceptible to tedizolid. A. Examine zone edges with transmitted light (plate held up to light). B. Isolates S to linezolid can be reported S to tedizolid. For isolates R to linezolid, perform an MIC test. © by author ESCMID Online Lecture Library Tedizolid: in vitro activity S. aureus

MIC distribution of tedizolid (TR-700) and linezolid against Staphylococcus aureus by phenotype

TEDIZOLID LINEZOLID

CENTER FOR DRUG EVALUATION AND RESEARCH. FDA (205435Orig1s000) Tedizolid vs linezolid: S. aureus

. Linezolid and tedizolid against linezolidS and linezolidR MRSA LINEZOLID % of isolates % of isolates

 Linezolid susceptible

ESCMID Online Lecture % of isolates Library

Livermore et al. JAC 2009; 63:713-5 MIC (mg/L) Tedizolid vs other oxazolidinones

. Oxazolidinone MICs for S. aureus with linezolid-R mechanisms

MIC (mg/L) Ribosomal modifications Linezolid Radezolid Tedizolid - 1-2 0-5-1 0.25-0.5 L4 (Lys68Gln) 2 1 0.5 L3 (ΔSer145) 8 4 1 L3 (ΔPhe127-His46) 8 2 2 23S (T2500A x 2) © by author8 4 2 23S (G2576T x 3) 16 2 2 23S (G2576T / T25717 x 2) 16 2 2 ESCMID23S (G2447T x 3) Online Lecture32 4 Library4

Cfr 8-16 1-4 0.5 S. aureus: tedizolid vs linezolid

7187 Staphylococcus aureus isolates

Zurenko et al. Ann Clin Microbiol Antimicrob. 2014;13:46 www.eucast.org

Virulence and Resistance in Staphylococcus aureus: 2016 State of the Art Lyon, France. 28 June – 1 July, 2016 © by author

Dr. Rafael Cantón ESCMIDHospital Universitario Online Ramón y Cajal Lecture Library SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA Departamento de Microbiología II Universidad Complutense. Madrid