educational workshop - escmid

Educational WorkshopEW05: Antimicrobial susceptibility testing: current and emerging problems and application of expert rules

arranged with EUCAST

(European Committee on Antimicrobial Susceptibility Testing)

Convenors: Gunnar Kahlmeter (Vaxjo, SE)Arne C. Rodloff (Leipzig, DE)

Faculty: Derek Brown (Cambridge, UK)Fred Tenover (Sunnyvale, US)Roland Leclercq (Caen, FR)Christian Giske (Stockholm, SE)Rafael Canton (Madrid, ES)Arne C. Rodloff (Leipzig, DE)

Brown –Lessons from external quality assurance

Defining problems in antimicrobial susceptibility

testing: lessons from external quality assurance

Derek Brown

Austria 43

Belgium 4

Croatia 6

Finland 23

Germany 1

Greece 16

Ireland 45

Italy 124

Participants in UKNEQAS for Microbiology (2008)

Netherlands 18

Poland 1

Portugal 52

Romania 2

Sweden 28

Switzerland 24

United Kingdom 278

Other 82

Breakpoint guidelines used by participants in UKNEQAS 2009

Guideline Number (%) labs

CLSI 368 (53.7)

BSAC (UK) 190 (29.6)

SRGA (Sweden) 36 (4.8)

NWGA (Norway) 6 (0.6)

CRG (Netherlands) 5 (0.7)

EUCAST 4

Other 43

TOTAL 674

3


Methods used by participants in UKNEQAS 2008

Method Number (%) labs

Disk diffusion 352 (51)

Automated 240 (31)

MIC 26 (4)

Breakpoint 23 (3)

Other/not stated 81 (11)

Total 672

Methods related to guidelines used by participants in UKNEQAS 2007

MethodCLSI BSAC SRGA

n (%) n (%) n (%)

Disk diffusion 150 (41) 175 (86) 26 (79)

Automated 199 (54) 14 (7) 2 (6)

MIC 6 (2) 6 (3) 5 (15)

Breakpoint 12 (3) 6 (3) 0 (0)

Other/not stated 1 (1) 2 (1) 0 (0)

Laboratories may perform poorly

4


Zone diameter for K. pneumoniae 9142 with co-amoxiclav (BSAC)

R I S

Some susceptibility tests are more difficult, e.g.

•Some MRSA

•Some penicillin resistant S. pneumoniae

•Some glycopeptide resistant enterococci

•VISA

•BLNAR H. Influenzae

•Borderline susceptibility in general

Performance is affected by breakpoint guidelines

5


Susceptibility testing of P. aeruginosa specimen 8812 to piperacillin-

tazobactam (MIC 32-64 mg/L) by UKNEQAS participants

Method Breakpoints Susceptible Intermediate Resistant

CLSI S<64 R>64 223 37 57

BSAC S<16 R>16 50 12 105

SRGA S<16 R>16 2 9 18

Susceptibility testing of Neisseria gonorrhoeae specimen 8482 to ciprofloxacin (MIC 0.5 mg/L) by

UKNEQAS participants


CLSI S<0.06 R>0.5 73 117 93

BSAC S<0.03 R>0.06 14 4 166

SRGA S<0.03 R>0.06 2 0 28

Changes in breakpoints may affect reportingS aureus 7240, Ciprofloxacin MIC 0.5 mg/L


CLSI S<1 R>2 434 5 4

BSAC S<1 R>1 167 0 1

SRGA S<0.06 R>2 3 19 0

S aureus 7876, Ciprofloxacin MIC 0.25 mg/L


CLSI S<1 R>2 350 0 0

BSAC S<1 R>1 176 0 1

SRGA S<1 R>1 23 2 1

6


Performance is affected by the method used


CLSI S<4 R>16 40 (11%) 52 288

BSAC S<4 R>8 84 (43%) 8 108

SRGA S<4 R>8 14 (38%) 0 23

Detection of VanB glycopeptide resistance in enterococci by UKNEQAS participants

E. faecium 7826Vancomycin MIC 8-16 mg/L, I/R

Methods used for detection of VanB glycopeptide resistance in enterococci

E. faecium 7826Vancomycin MIC 8-16 mg/L, I/R

Method Susceptible Intermediate Resistant

Disk 122 (37%) 37 173

Automated 8 (4%) 12 188

MIC 7 (13%) 9 38

Breakpoint 3 (13%) 2 19

7



CLSI S<8 R>16 105 (31%) 141 92

BSAC S<4 R>4 84 (53%) 8 67

SRGA S<4 R>4 11 (50%) 2 9

Reporting S. epidermidis (specimen 7156) with reduced susceptibility to

teicoplanin (MIC 8-16 mg/L)

Methods used for detection of reduced susceptibility to teicoplanin (MIC 8-16

mg/L) in S. epidermidis (specimen 7156)

Method Susceptible Intermediate Resistant

Disk 148 (30%) 47 52

Automated 31 (19%) 70 62

MIC 9 (11%) 23 52

Breakpoint 2 (11%) 4 13

Detection of methicillin (oxacillin) resistance in S. aureus with cefoxitin

8


Detection of oxacillin/cefoxitin resistance in mecA positive S aureus

OrganismOxacillin

MIC (mg/L)

Oxacillin Cefoxitin

n %R n %R

7240 16->128 535 81 48 98

7538 >128 614 99 77 99

7597 >128 590 96 77 99

7659 >128 647 99 85 100

7703 >128 626 99 106 96

8248 64->128 609 95 162 99

8452 >128 569 99 405 97

8701 32-128 615 96 471 94

8858 >128 604 100 513 99

9141 16-32 600 93 518 95

Penicillinase-hyperproducing S. aureusS aureus 7876oxacillin susceptible(MIC 0.5-1 mg/L)mecA-ve )

OrganismOxacillin Cefoxitin

n %S n %S

7876 619 88 120 100

Routine methods may not be correctly calibrated to MIC

breakpoints

9


Susceptibility testing of S. aureus specimen 8578 to ciprofloxacin (MIC 1

mg/L)


CLSI S<1 R>2 428 20 4

BSAC S<1 R>1 97 3 83

SRGA S<1 R>1 19 1 1

Susceptibility testing of S. haemolyticus specimen 8702 to

gentamicin (MIC 16 mg/L)


CLSI S<4 R>4 64 (18%) 58 224

BSAC S<1 R>1 7 (4%) 2 162

SRGA S<1 R>1 0 (0%) 0 30

Variable application of reporting guidelines

10


Test/reportParticipants results

S I R

Oxacillin screen 2 2 489

Penicillin test 33 193 317

Penicillin report meningitis 12 (1.9%)

61 (9.8%)

549 (88.3%)

Penicillin report pneumonia 190 (30.4%)

152 (24.3%)

284 (45.3%)

Detection and reporting of reduced susceptibility to penicillin in S. pneumoniae

S. pneumoniae 8886 (penicillin MIC 0.5 mg/L)

Variable application of expert rules affects reporting

Agent MIC (mg/L)Participants reporting

S I R

Cefotaxime >128 6 0 603

Ceftazidime 32-64 7 22 661

Piperacillin-tazobactam

8-16 324 (49.3%)

55 (8.4%)

278 (42.3%)

Detection of resistance mediated by ESBL E. coli 8738 (CTX-M-15)

Participants reported ESBL positive 668, negative 8

11



S I R

Cefotaxime 0.12-0.5 534 3 50

Ceftazidime 0.25 629 5 51


1-8 663 1 12

Reporting susceptibility of Serratia marcescens with inducible AmpC

S. marcescens 8859



S I R

Cefotaxime 16-32 16 98 504

Ceftazidime 32->128 2 7 681


4 451 (68.5%)

50 (7.6%)

157 (23.9%)

Detection of resistance mediated by plasmid-borne AmpC

E. Coli 9059 (Cit AmpC)


Defining problems in antimicrobial susceptibility testing: lessons from

external quality assurance

• Some errors are due to poor performance

• Some tests are inherently more difficult

• Breakpoint guidelines affect performance

• Some methods are less reliable with some tests

• Routine methods may not be correctly calibrated to reference MIC tests

• Application of reporting guidelines is variable

• Application of expert rules is variable

12

Tenover - Staphylococci

Antimicrobial Susceptibility Testing Antimicrobial Susceptibility Testing of of Staphylococcus aureusStaphylococcus aureus

Fred C. Tenover, Ph.D., (D)ABMMFred C. Tenover, Ph.D., (D)ABMMSenior Director, Scientific AffairsSenior Director, Scientific Affairs

CepheidCepheid

Consulting Professor of PathologyConsulting Professor of PathologyStanford UniversityStanford University

Which of the following is true about Which of the following is true about Staphylococcal Susceptibility Testing?Staphylococcal Susceptibility Testing?

ØØ Cefoxitin zone diameters are much easier to Cefoxitin zone diameters are much easier to read and interpret than oxacillin zone diameters read and interpret than oxacillin zone diameters for coagulasefor coagulase--negative staphylococci negative staphylococci

ØØ The clindamycin induction test is only The clindamycin induction test is only necessary if the erythromycin MIC or disk result necessary if the erythromycin MIC or disk result is intermediate and clindamycin result is is intermediate and clindamycin result is susceptiblesusceptible

ØØ Susceptibility tests for mupirocin for Susceptibility tests for mupirocin for S. aureusS. aureusmust be tested in the presence of 50 mM Ca++ must be tested in the presence of 50 mM Ca++ to be accurateto be accurate

ØØ The “wild type” distribution was named in honor The “wild type” distribution was named in honor of Derek Brown, Gunnar Kahlmeter, and Rafael of Derek Brown, Gunnar Kahlmeter, and Rafael Canton (the original “wild and crazy guys”)Canton (the original “wild and crazy guys”)

EUCAST Clinical BreakpointsEUCAST Clinical Breakpoints††

Oxacillin MICOxacillin MIC Cefoxitin MIC Cefoxitin MIC ##

S. aureus S. aureus and and S. lugdunensisS. lugdunensis > 2 > 2 µg/mlµg/ml > 4 > 4 µg/mlµg/ml

Coagulase Coagulase --Negative Negative

StaphylococciStaphylococci> 0.25 > 0.25 µg/mlµg/ml Do not useDo not use

† Same breakpoints as CLSI† Same breakpoints as CLSI# Report as # Report as oxacillinoxacillin--resistantresistant

13


Cefoxitin Disk Screening TestCefoxitin Disk Screening Test

ØØ Cefoxitin and oxacillin have Cefoxitin and oxacillin have equivalent equivalent sensitivity for detecting sensitivity for detecting mecAmecA--mediated mediated resistance in resistance in S. aureusS. aureus

ØØ Cefoxitin has Cefoxitin has much better sensitivitymuch better sensitivity for for detecting detecting mecAmecA--mediated resistance in mediated resistance in coagulasecoagulase--negative staphylococci and the zone negative staphylococci and the zone edges are much edges are much easier to readeasier to read

ØØ Most resistant strains can be reported at 16Most resistant strains can be reported at 16--18 18 hours, but the test needs to be held for 24 hours hours, but the test needs to be held for 24 hours to find heteroresistant strains.to find heteroresistant strains.

Swenson et al J Clin Microbiol 37:4051-4058, 1999

CLSI Cefoxitin Disk Diffusion CLSI Cefoxitin Disk Diffusion Screen TestScreen Test

OrganismOrganism Cefoxitin zone Cefoxitin zone 11 Cefoxitin zone Cefoxitin zone 22

S. aureusS. aureus <<19 mm19 mm >>20 mm20 mm

CoNS*CoNS* <<24 mm24 mm >>25 mm25 mm

1-Report as oxacillin-resistant

2-Report as oxacillin-susceptible

EUCAST and CLSI Breakpoints EUCAST and CLSI Breakpoints for for LinezolidLinezolid

EUCASTEUCAST(S / R)(S / R)

CLSICLSI(Susceptible (Susceptible

only)only)

MICMIC << 4 / >4 4 / >4 µg/mlµg/ml <<4 4 µg/mlµg/ml

Disk Disk DiffusionDiffusion Not availableNot available >>21 mm21 mm

14


Where Do You Read a Linezolid Disk Diffusion Result ?

Outer edge

Inner edge

Where should you measure a Where should you measure a linezolid disk diffusion zone?linezolid disk diffusion zone?

ØØ From the outer edge of the zone of inhibitionFrom the outer edge of the zone of inhibition

ØØ At approximately 80% inhibition of growthAt approximately 80% inhibition of growth

ØØ From the inner edge of growth observed with From the inner edge of growth observed with reflectedreflected lightlight

ØØ From the inner edge of growth observed with From the inner edge of growth observed with transmittedtransmitted lightlight

ØØ The linezolid disk diffusion test doesn’t work The linezolid disk diffusion test doesn’t work and should not be usedand should not be used

Testing Linezolid by AgarTesting Linezolid by Agar--Based Based Methods Can Be DifficultMethods Can Be Difficult

Issue:Large zones

with indistinctedges

around disksand Etest strips

MIC <4 µg/ml, Susceptible

Disk >21 mm, Susceptible

15


Accuracy of Linezolid Testing Accuracy of Linezolid Testing by Six Methodsby Six Methods

ØØ Tested 100 isolates (25 Tested 100 isolates (25 S. aureusS. aureus, 25 , 25 CoNS, 25 CoNS, 25 E. faecalisE. faecalis, 25 , 25 E. faeciumE. faecium))

ØØ 32 of 100 organisms were non32 of 100 organisms were non--susceptible or resistantsusceptible or resistant

ØØMajority of problem were in detecting Majority of problem were in detecting linezolid linezolid nonnon--susceptible susceptible S. aureusS. aureus

ØØReading disk diffusion and Etest using Reading disk diffusion and Etest using transmitted lighttransmitted light instead of reflected instead of reflected light improved sensitivitylight improved sensitivity

Tenover FC. J Clin Microbiol 45:2917-22, 2007

Error Rates Testing Linezolid with Error Rates Testing Linezolid with Staphylococci and EnterococciStaphylococci and Enterococci

VM=22/58= 37.9%MA=7/40= 17.5%

Must confirm non-susceptible disk results by MIC

16


EUCAST EUCAST VancomycinVancomycin Breakpoints Breakpoints for for S. aureusS. aureus

SusceptibleSusceptible ResistantResistant

MICMIC <<4 4 µg/mlµg/ml >>8 8 µg/mlµg/ml

Disk diffusionDisk diffusion NANA NANA

The New Challenge: HeteroThe New Challenge: Hetero--Vancomycin Vancomycin Intermediate SIntermediate S. aureus. aureus (hVISA)(hVISA)

Vancomycin Susceptible Intermediate Resistant

MIC (μg/ml) Disk (30 µg)

≤ 2

≥ 15 mm

4 – 8

ND

≥ 16

ND

VISA VRSA

SusceptibleSusceptible HeteroresistantHeteroresistant

<<0.250.25--1 1 µg/mlµg/ml 11--4 4 µg/mlµg/ml

Which statement is true about Which statement is true about vancomycin testing of vancomycin testing of S. aureusS. aureus??

A.A. IsoIso--Sensitest agar is the most sensitive medium for Sensitest agar is the most sensitive medium for detecting heterodetecting hetero--VISA strainsVISA strains

B.B. Disk diffusion will identify Disk diffusion will identify vanAvanA--containing VRSA containing VRSA strains but not VISA strainsstrains but not VISA strains

C.C. MIC results should be interpreted after 16MIC results should be interpreted after 16--18 hours of 18 hours of incubation at 35ºCincubation at 35ºC

D.D. The macroThe macro-- Etest can identify heteroEtest can identify hetero--VISA strains as VISA strains as long as both vancomycin and daptomycin are testedlong as both vancomycin and daptomycin are tested

E.E. Most automated susceptibility testing methods Most automated susceptibility testing methods accurately detect vancomycin MICs of 4 µg/ml in accurately detect vancomycin MICs of 4 µg/ml in S. S. aureusaureus

F.F. None of the above are trueNone of the above are true

17


Disk diffusion Disk diffusion and Etest Zonesand Etest Zones

Even the mostdifficult to detect VRSAstrain wasdetected bydisk diffusion

Tenover et al. Antimicrob Agents Chemother. 48:275-280, 2004.

PAGE | 17

CDC Scattergram of CDC Scattergram of S. aureusS. aureus and and

Vancomycin; To date all VRSA detectedVancomycin; To date all VRSA detected

Former CLSI breakpoint:>15 mm susceptible

vanA-VRSA

18


PAGE | 18

VR-CoNS (Not vanA)

Detection of VISA StrainsDetection of VISA StrainsNo. (%)a categorized as No. of discrepancies in testing method

TestSusceptible(n = 84)

Intermediate(n = 45)

Category agre

e(n = 129)

VISA

called S

VISA

called R

S called

VISA

BMIC BBL 83 (98.9) 40 (88.9) 123 (95.3) 5 1

Agar 82 (97.6) 31 (68.9) 113 (87.6) 14 2

Etest 73 (86.9) 44 (97.8) 117 (90.7) 1 11

MicroScan 74 (88.1) 45 (100) 119 (92.2) 10

Phoenix 64 (76.2) 45 (100) 109 (84.5) 20

Sensititre 84 (100) 29 (64.4) 113 (87.6) 13 1

Vitek 83 (100) 0 (0) 83 (64.8) 35 10

Vitek 2 82 (97.6) 35 (77.8) 117 (90.7) 10 2

aPercentage is based on 85 susceptible strains and 45 vancomycin-intermediate Staphylococcus aureus (VISA) strains.

Population Analysis of hVISA and VISAPopulation Analysis of hVISA and VISA

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

0 1 3 5 7 9

MIC (ug/ml)

Suscept.

hVISA

VISA

VISA

SuscepthVISA

Note scale change

Subpopulation of hVISA isolates, for which

MIC=4-8 μg/ml, are below detection level

Inoculum

19


F ig 1 . P opulation Analysis for Strain 9AJ57 on BHI vs. M HA

Va ncom ycin Con centratio ns (m cg/m L)

0 0 .2 5 0. 5 0. 75 1 2 3 4 6 8 10 12 14 16 32

Lo

g1

0 (

CF

U/m

L)

1

2

3

4

5

6

7

8

9

10

AT CC 29213 9A J57 -M HA 9A J57 -BHI

Subpopulation apparent only on BHI

Mueller-Hinton MIC= 1 µg/mlBHI MIC = 4 µg/ml

MHA

Macro Etest MethodMacro Etest MethodØØ Inoculum: 2.0 McFarland in MuellerInoculum: 2.0 McFarland in Mueller--Hinton Hinton

BrothBroth

ØØ 100 100 µl inoculated onto 90mm BHI agar plateµl inoculated onto 90mm BHI agar plate

ØØ Use vancomycin and teicoplanin Etests; Use vancomycin and teicoplanin Etests; incubate at 35incubate at 35°°CC

ØØ Read at 24 and 48 hoursRead at 24 and 48 hours

ØØ Positive results (hVISA)Positive results (hVISA)

ll Vancomycin and teicoplanin: Vancomycin and teicoplanin: >>8 µg/ml8 µg/ml

ll Teicoplanin: Teicoplanin: >>12 µg/ml12 µg/ml

Novel Etest Novel Etest Method Method -- GRDGRD

Glycopeptide Resistance Detection Strip

TeicoplaninVancomycin

20


hVISA: Clinical RelevancehVISA: Clinical RelevancePopulation Analysis Shows Increasing Population Analysis Shows Increasing

MICsMICs

Vancomycin Concentrations

0 0.25 0.5 0.75 1 2 3 4 6 8 10

Lo

g1

0 (C

F/m

l)

1

2

3

4

5

6

7

8

9

ATCC29213

491-0.5VBHI 492-0.5VBHI

493-0.5VBHI 494-0.5VBHI

522-0.5VBHI

(µg/ml)

Gradual increase

in vancomycinMICs during10 weeks oftherapy with vancomycin

for endocarditis

1 23 4 VISA control

Tenover FC et al. IJAA 2009

Macro Etest

Which statement is NOT true of Which statement is NOT true of daptomycin susceptibility testing?daptomycin susceptibility testing?

A.A. Daptomycin resistance cannot be Daptomycin resistance cannot be detected by disk diffusiondetected by disk diffusion

B.B. Daptomycin MIC testing requires Daptomycin MIC testing requires additional calcium in the mediumadditional calcium in the medium

C.C. Daptomycin tests should be incubated Daptomycin tests should be incubated for a full 24 hours before interpretationfor a full 24 hours before interpretation

D.D. Reduced susceptibility to daptomycin Reduced susceptibility to daptomycin may accompany reduced susceptibility to may accompany reduced susceptibility to vancomycinvancomycin

E.E. All the above are true All the above are true

Development of Daptomycin Development of Daptomycin NonNon--Susceptibility in Same StrainSusceptibility in Same Strain

Daptomycin Concentrations ( g/ml)

0 0.125 0.25 0.5 0.75 1 2 3 4 6 8 10

Lo

g 10

(CF

U/m

l)

1

2

3

4

5

6

7

8

9

ATCC 29213 RWJ 1-D RWJ 2-D RWJ 3-D RWJ 4-D

µ

1 2

3 4

Some hVISA andVISA strains

remainsusceptible todaptomycineven after

vancomycin administration.Susceptibility

testing is criticalfor decision

making

Non-susceptible

21


EUCAST and CLSI Daptomycin EUCAST and CLSI Daptomycin BreakpointsBreakpoints

EUCASTEUCAST(S / R)(S / R)

CLSICLSI(Susceptible (Susceptible

only)only)

MICMIC << 1 / >1 µg/ml1 / >1 µg/ml <<1 1 µg/mlµg/ml

Disk Disk diffusiondiffusion

N/AN/A N/AN/A

Comparison of Comparison of EtestEtest Results to Broth Results to Broth Microdilution for DaptomycinMicrodilution for Daptomycin

1616

88

44 77 2222 99 4343 1212

11 11 22 4141 10100.50.5 7474 1414

0.250.25 6868 770.120.12 22 77 11

<<0.250.25 0.50.5 11 22 44 88 1616

Broth microdilution MIC (µg/ml)

Ete

st

MIC

(µ

g/m

l)

CMI data n=300

False susceptible

False resistance

Etest Results for DaptomycinEtest Results for Daptomycin

ØØOverall, 95.3% of Etest results were Overall, 95.3% of Etest results were within within ++1 1 dilution of broth microdilution dilution of broth microdilution method (n=376) method (n=376)

ØØ 35.3% of Etest results were 1 dilution 35.3% of Etest results were 1 dilution lower; 5% were 2lower; 5% were 2--3 dilutions lower3 dilutions lower

ØØ 90.2% of isolates that were non90.2% of isolates that were non--susceptible by broth microdilution were susceptible by broth microdilution were nonnon--susceptible by Etestsusceptible by Etest

Jevitt, L et al. JCM 2006;44:3098-104.

22


S. aureusS. aureus Mupirocin Susceptibility Mupirocin Susceptibility Phenotypes Phenotypes

PhenotypePhenotypeMIC MIC

Range Range (µg/ml)(µg/ml)

Molecular Molecular MechanismMechanism

SusceptibleSusceptible ≤ 4 ≤ 4 Wild typeWild type

LowLow--level level resistanceresistance

88--256256 Mutations in native Mutations in native isoleucine tRNA isoleucine tRNA

synthetasesynthetase

HighHigh--level level resistanceresistance

≥512≥512 Novel Novel isoleucineisoleucinetRNAtRNA synthetasesynthetase

Mupirocin BreakpointsMupirocin Breakpoints

ØØCLSI has approved both MIC and disk CLSI has approved both MIC and disk diffusion breakpoints for diffusion breakpoints for highhigh--levellevelmupirocin resistance in mupirocin resistance in Staphylococcus Staphylococcus aureusaureus

ØØHighHigh--level mupirocin resistance is level mupirocin resistance is indicated by:indicated by:

ll MIC ≥512 µg /mlMIC ≥512 µg /ml

�&��☺��P�O�&��☺��P�ONo zone of inhibition (6 mm) around a No zone of inhibition (6 mm) around a 200 µg disk200 µg disk

PAGE | 31

CONCLUSIONSCONCLUSIONSØØ Linezolid resistance in staphylococci can be Linezolid resistance in staphylococci can be

hard to detect by agarhard to detect by agar--based methodsbased methods

ØØ Hetero vancomycin resistance is still hard to Hetero vancomycin resistance is still hard to detect although the macro Etest and GRD can detect although the macro Etest and GRD can be usefulbe useful

ØØ Most automated systems are FDAMost automated systems are FDA--cleared to cleared to detect VRSA; detecting VISA may still be a detect VRSA; detecting VISA may still be a problemproblem

ØØ Etest a reasonable approach for detecting nonEtest a reasonable approach for detecting non--susceptibility to daptomycin in staphylococci susceptibility to daptomycin in staphylococci

23

Leclercq –Streptococci, pneumococci and enterococci

Streptococci, pneumococci and enterococci

Roland Leclercq, Caen, France

Enterococci

Enterococci and β-lactams

• E. faecalis is usually susceptible to ampicillin (not bactericidal)

• Resistance to ampicillin is – exceptional in E. faecalis (beta-lactamase producing

isolates not reported in Europe)– frequent in E. faecium (resistance is a marker for

identification); E. hirae and E. raffinosus may be ampicillin-R

– not reported in other species: E. avium, E. gallinarum…

• Intrinsic resistance to all cephalosporins (no need for testing)

25


Cross-resistance to penicillins in E. faecium

• In E. faecium, resistant to ampicillin is due to overproduction of low affinity PBP5 and to mutations near the active site of the PBP (Met485Ala and insertion of serine at position 466)

• Target modificationà cross-resistance to beta-lactams

« If resistant to ampicillin, report as resistant to ureidopenicillins and carbapenems. »

Various susceptibilities to penicillins according to the enterococcal species

JL Mainardi, Antibiogramme, 2006

Species Penicillin G Ampicillin Amoxicillin Piperacillin Imipenem

E. faecalis 2-8 0.25-2 0.25-1 0,5-4 0.5-4E. faecium 2-512 0.5-256 0.25-256 4-512 1-512E. durans 0.25-8 0.12-4 _ NDb NDE. gallinarum 1-4 1-2 _ 16-> 16 1-2E. avium 1-2 0.5-1 _ 16-> 16 0.5-1E. casseliflavus 0.5-4 0.5-2 _ 8-> 16 0.5-4E. hirae 2-8 2-4 _ > 16 2E. raffinosus 32 16 _ > 16 8

Aminoglycosides (Ag) and enterococci

• Low level resistance to Ag (MIC of gentamicin = 8-16 mg/L). However, Ag combined with penicillins or vancomycin produce bactericidal effect against enterococci

àMajor objective of susceptibility testing: To identify loss of bactericidal synergism conferred by

acquisition of Ag resistance mechanisms

AST systems explore bacteriostatic activity of antibiotics… … ..

26


Resistance to aminoglycosides is mostly due to modifying enzymes in enterococciDas Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu wenig Arbeitsspeicher, um das Bild zu öffnen, oder das Bild ist beschädigt. Starten Sie den Co mputer neu, und ö ffnen Sie dann erneut d ie Datei. Wenn weiterhin das ro te x angezeigt w ird, müssen S ie das Bild möglicherweise löschen und dann erneut einfügen.

• Two major classes of enzymes- Phosphotranferases- Acetyltransferases

•Rares mutants (streptomycin)

• Ribosomal methylases that are responsible for Ag resistance in Gram-negatives (ArmA… ) not detected in enterococci

Two major classes of enzymes

Phosphorylation APH(2'')

Phosphorylation APH(3')

AcetylationAAC(6')

• Phosphotransferase: APH(3’)-III• Bifunctional enzyme (acetylase-phosphotransferase): AAC(6’) -APH(2’’)

Modifying enzymes and amikacin

Efficient protection against modification by enzymes from Gram-negative bacilliPoor protection against modification by enzymes from Gram-positive cocci

27


Phenotype conferred by major Ag modifying enzymes in enterococci

Enzyme Resistance phenotype

APH(3’)-III KanAAC(6’) -APH(2’’) Kan Gen

•Generally, presence of a modifying enzyme confers high level resistance to specific Ag (MIC >1000 mg/L )

•High level resistance to kanamycin: alters only weakly bacteriostatic activity of amikacin but suppresses its bactericidal activity and synergism with penicillins and vancomycin•High level resistance to gentamicin: suppresses synergism of all Ag (except streptomycin) with penicillins and vancomycin

Antagonism between amikacin and penicillin against HL kanamycin-resistant enterococci

Thauvin C et al., Antimicrob Agents Chemother, 1985, 28:78-83

APH(3’) producing E. faecalis Non-APH(3’) producing E. faecalis

In vitro detection of Ag resistance

• Screen for high level resistance to kanamycin and gentamicin

• LLR to Ag in enterococci requires testing of highconcentrations of AgRare enzymes found mostly in animal enterococci may confer a

moderate level of resistance to gentamicin (MC=128-256 mg/L) [APH2’’ (b,c,d)]

• Test of kanamycin predicts for amikacin– Generally >40% of enterococci are HLR to Kan…… .

• Test of gentamicin predicts for all Ag (except streptomycin)

28


Acquired resistance to glycopeptidesVanA VanB

Vancomycin MIC (mg/l) >64 4-1000Teicoplanine MIC (mg/l) >32 0.5-2Expression ind indGenetic support Tn1546 Tn1547Espèce E. faecium E. faecium

E. faecalis E. faecalis

E. gall inarum S. bovis

E. casseli flavus

E. avium

E. durans

E. mundtii

E. raffinosus

Modified target D-Ala-D-Lac

Rare other types- Van D (MIC of Van: 64 mg/L, Tei: 4-64 mg/L)- VqnE, Van G, Van L [Serine types (D-Ala-D-Ser)] susceptible to teicoplanin

Europe: Glycopeptide resistance in E. faecium(blood cultures)

2007 2008

Problems with detection of vancomycin resistance in enterococci

Difficulties for detection of vancomycin resistance relate to slow induction of resistance

Control (no antibiotic)

Growth in the presence of 16 mg/L of V

Induced (16 mg/L of V)

Non induced

Brisson-Noël et al. Antimicrob Agents Chemother, 1990, 34:5

29


Difficult to detect VanA enterococci

Naas et al. J Clin Microbiol, 2005, 43:3642

Testing glycopeptides• Intrinsic resistance of E. gallinarum and E.

casseliflavus to vancomycin (MIC 4-16 mg/L)– Phenotypically difficult to distinguish from E.

faecium (ampicillin susceptibility of E. gallinarum/cass.)

– Not responsible for outbreaks

• Testing– Disc diffusion not recommended– MICs: read at 24 h… ..

Streptococci

30


β-haemolytic streptococci

• No resistance to β-lactams reported in S. pyogenes

• Some group B streptococci with MIC of penicillin G up to 0.6 mg/L: poor clinical significance

• If susceptible to penicillin à susceptible to aminopenicillins,

cephalosporins and carbapenems.

A Streptococcus pyogenes not fully susceptible to ampicillin?

•If elevated MIC of ampicillin/penicillin check identification and susceptibility.à This is not a group A, C, G streptococcus. It is an E. faecaliswith an haemolysin plasmid

S. pneumoniae and β-lactams• Production of mosaic PBPs leads to various patterns of β-lactam

resistance.

• By disk-diffusion: the screening test of oxacillin predicts susceptibility/resistance to b-lactams à determine MICs of b-lactams.

PBP1a1b

2x2a2b

3

31


S. pneumoniae and β-lactams (2)• MIC of which β-lactam? Generally, linear relationship between

penicillin MICs and other ß-lactams with S. pneumoniae• Same amoxicillin, amox/clav, ceftriaxone, • or 2x lower cefotaxime, cefditoren

• 2x higher cefuroxime, cefpodoxime, cefdinir

• 4x higher cefprozil

• 16x higher cefixime, ceftibuten

• 32x higher cefaclor, loracarbef

• However, there many examples of non-linear relationship: MICs of Amox ≥ Pen ≥ CTX or MICs of CTX ≥ Pen ≥ Amox

à « Determine MIC of Pen, Amp (amox), Cefotaxime (ceftriaxone). Report as interpreted for each of the drugs. »

Resistance to macrolides in streptococci

Target modificationRibosomal methylationplasmid-mediated methylases[erm(B), erm(A) genes]

EffluxPumps encoded by mef genes

Macrolide

MLSB phenotype M phenotype

MLSB cross-resistance due to erm(B)

Cli Lin

Ery

Lin

Ery

Cli

Expression of MLSB resistance

Inducible or constitutive

Inducible

Cross MLSB resistance: erythromycin (and other 14-membered macrolides and azithromycin), clindamycin, (streptogramins B)

32


M resistance due to mef genes

Qui

Lin

Cli

Spi

E

« If R to erythromycin but S to clindamycin or lincomycin, test for inducible MLSB resistance. If negative, report S to clindamycin and lincomycin. If positive, report R to clindamycin and lincomycin »

Dissociated erythromycin resistance in streptococci

Qui

Lin

Cli

Spi

E

Lin

Cli Qui

Spi

E

M Inducible MLSB

Low activity of clindamycin or mutational risk

Percentages of mef(A) and erm(B) in pneumococci vary according to the

countries

0

20

40

60

80

100

Belg

ium

Franc

eSpa

in

Polan

d

Gre

ece

Ger

many

Turke

y

China

South

Africa

USA

Others

erm(B) + mef(A)

mef(A)

erm(B)

Felmingham et al., J Infect, 2007, 55:111-8

2001-2004

33


Variations in prevalence of resistance genes in S. pyogenes: the example of Italy

Creti et al., J Clin Microbiol, 2007, 45:2249-56

S. uberis/mph(B)

ErySpi

Tyl

Some strange new resistance phenotypes

Resistance by phosphorylation only to 16-Md macrolides

Do not infer results for erythromycin to 16 Md-macrolides(tylosin for veterinarians, spiramycin/josamycin)

Achard A, Guérin-Faublée V, Villers C, Leclercq R. Antimicrob Agents Chemother 2008, 52: 2767

Conclusion

• More persistent problems than emerging new resistances– Detection of vancomycin resistance in

enterococci– Various levels of resistance to beta-

lactams in pneumococci– Macrolide resistance

34

Giske –Resistance to beta-lactam drugs

Gram-negative bacilli and resistance to β-lactam antibiotics

Christian G. Giske, MD PhD

Karolinska University Hospital

16 May 2009

16 May 2009Christian G. Giske 2

Principles used when preparing this presentation

§ Be brave (only controversial issues)

§ Be personal (my own comments)

§ Be interactive (main focus on the questions)

§ Be honest (sometimes we just don’t know the correct answer)


ESBL-producers and test results for cephalosporins

35



Personal comments

§ Is there any evidence supporting therapeutic failure with ESBL-producers if the MIC is ≤ 1 mg/L?

�/�"Is there any evidence supporting therapeutic success with ESBL-producers if the MIC is ≤ 1 mg/L?

�/�"PK/PD vs inoculum effect§ Is there something magical about the ESBLs?

Treatment of ESBL producers

0

10

20

30

40

50

60

70

80

1 2 4 8

MIC (mg/L)

% S

uc

ce

ss

Craig et al. ICAAC 2005-35 patients (32 with BSI)-Cephalosporin monotherapy-5 data sources


ESBL-producers and test results for penicillin/β-lactam inhibitor combination


Personal comments

§ Evidence to support the use of piperacillin-tazobactam in the treatment of UTI and pneumonia caused by ESBL-producers exists, although data are limited (Gavin et al. AAC 2006;50:2244)

§ In other infections the use of this combination should probably be approached with caution, especially in critically ill patients

36



Phenotypic detection of KPC


Personal comments

§ Boronic acid inhibits KPC (Tsakris A et al. JCM 2009; 47:362), but also AmpC

Meropenem MIC: KPC- and MBL-isolates

0

2

4

6

8

10

12

14

16

0,032 0,064 0,125 0,25 0,5 1 2 4 8 16 >=32

MIC

No

iso

late

s

MP KPC

MP MBL

SWT


Metallo-β-lactamases and carbapenem test results

37



Personal comment

§ No present data on therapeutic failure with carbapenemase producers if MIC ≤ 2 mg/L

§ Daikos et al AAC 2009 In press: consecutive patients with K. pneumoniae BSI (67 VPKP, 95 non-VPKP)


Risk of selecting AmpC derepressed mutants


Personal comment

§ Schwaber M et al. AAC 2003; 47: 1882

§ Fluoroquinolones are protective against selection of resistance, aminoglycosides are not

§ Piperacillin-tazobactam equally selective as the third generation cephalosporins

38



Concluding remarks

§ Revision of breakpoints is necessary when evidence suggests clinical failures with susceptible isolates

§ Being careful vs. promoting use of antimicrobial agents with broad spectrum of activity

§ Sometimes we should allow ourselves to rethink earlier positions, especially related to the impact of breakpoint revisions

39

Cantón –Resistance to non-beta-lactam drugs

Antimicrobial susceptibility testing: current and

emerging problems and application of expert rules

Gram-negative rods and resistance to

non-ß-lactam drugs

EUCAST EDUCATIONAL WORKSHOP19th ECCMID Helsinki, Finland, 2009

Rafael Cantón

Servicio de Microbiología

Non-ß-lactam drugs and EUCAST Expert Rules

§ Non-ß-lactam antimicrobials are rarely included as a first option in clinical guidelines

§ Less information available on … - correlation of MIC values and clinical outcomes- treatment failure descriptions

§ No new drugs in some of the groups (i.e. aminoglycosides)

§ More difficult to infer non-ß-lactam resistance mechanisms- more experience with ß-lactams and “interpretive reading”- experts prefer ß-lactams than non-ß-lactams

Gram negative rods

but …

Non-ß-lactam drugs and EUCAST Expert Rules

§ Dramatic increase of resistance in some groups (fluoroquinolones)

§ Increasing information of new resistance mechanisms due tothe application of molecular techniques - transferable genetic determinants- low level expressed resistance mechanism

§ Increasing information of Pk/Pd data

§ Revitalization of “old” antimicrobials (colistin, fosfomycin, … )

§ Introduction of new antimicrobials (or variants of previous ones) - glycylcyclines (tigecycline)

§ Different patterns of use: - in hospitals and in the community- geographic areas

Gram negative rods

41


ARPAC (2001) http://www.abdn.ac.uk/arpac/

Antimicrobial use in the hospitals (Europe)

Ferech et al. J Antimicrob Chemother. 2006; 58:401-7

Antimicrobial use in outpatients (Europe)

2002

Antimicrobial use in outpatients (Europe)

Coenen et al. JAC 2009. doi:10.1093/jac/dkp135

Outpatient antibiotic use in Europe (2006)

42

http://www.abdn.ac.uk/arpac/


What do we have in the EUCAST Expert Rules for

Gram-negative rods and resistance to non-ß-lactam drugs?

Intrinsic resistances (Table 1-3)

Exceptional phenotypes (Table 5)

Interpretive rules for aminoglycosides (Table 12)

Interpretive rules for fluoroquinolones (Table 13)

Gram-negative rods and resistance to non-ß-lactam drugs

Intrinsic resistances (Table 1)

Rule no.

Organisms

Am

ino

gly

cos

ides

Tet

racy

clin

es

tig

ecy

clin

e

Po

lym

yxin

B

Co

listi

n

Nit

rofu

ran

toin

1.8 Morganella morganii R R R 1.9 Proteus mirabilis R R R 1.10 Proteus vulgaris R R R 1.11 Proteus penneri R R R 1.12 Providencia rettgeri R2 R R 1.13 Providencia stuartii R2 R R 1.14 Serratia marcescens Note3 R 1.15 Yersinia enterocolitica 1.16 Yersinia pseudotuberculosis R

2 All Providencia spp. produce a chromosomal AAC(2’)-Ia enzyme. Providencia spp. should be considered R to all aminoglycosides except amikacin and streptomycin. Some isolates express the enzyme poorly and can appear S to netilmicin in vitro, but should be reported as R as mutation can result in overproduction of this enzyme 3 All S. marcescens produce a chromosomal AAC(6’)-Ic enzyme that may affect moderate the activity of all aminoglycosides except streptomycin and gentamicin



Rule no.

Organisms

Cip

rofl

ox

ac

in

Ch

lora

mp

hen

ico

l

Am

ino

gly

co

sid

es

Tri

me

tho

pri

m

Fo

sfo

my

cin

Te

tra

cy

clin

es

T

igec

yc

lin

e

Po

lym

yx

in B

C

olis

tin

2.1 Acinetobacter baumannii, Acinetobacter calcoaceticus

R R

2.3 Burkholderia cepacia complex2 R R R3 R R R 2.4 Chryseobacterium meningosepticum R 2.6 Pseudomonas aeruginosa R Note

4 R5 R 2.7 Stenotrophomonas maltophilia R3 R7 R 3 B. cepacia and S. maltophilia are intrinsically R to al l aminoglycosides. Intrinsic resistance is attributed to poor permeability and putative eff lux. In addition, most S. maltophilia produce AAC(6’)Iz enzyme. On agar plates, resistance to aminoglycosides is more reliably detected after incubation at 30°C or ambient temp. than at 35-37°C. 4 P. aeruginosa is intrinsically R to kanamycin and neomycin due to low level APH(3’)-IIb activity. 5P. aeruginosa typically is R to trimethoprim and moderately S to sulphonamides. Although it may appear S in vitro to co-trimoxazole, it should be considered R. 7S. maltophilia typically is susceptible to co-trimoxazole, but resistant to trimethoprim alone.

43


Tetracyclines and gycylcyclines: Mechanisms of resistance

§ Tetracycline resistance widely distributed in Gram-negatives - high use in humans (70s), still in animals and agriculture

§ Tetracycline resistance genes associated with mobile elements

§ Different resistance mechanism with different epidemiology- efflux pumps - inactivation - ribosomal protection - unknown function

§ Do not equally affect all tetracyclines

- tetracycline > doxycycline > minocycline

§ Glycylcyclines (tigecycline) only affected by certain efflux pumps

Speer et a. Clin Microbiol Rev 1992; 5:387-99Chopra. Drug Resist Updat 2002; 5:119-25

Livermore. J Antimicrob Chemoter 2005; 56:611-4 Shlaes. Curr Opin Investig Drugs 2006; 7:167-71

do not affect tigecycline

Chopra I. Drug Res Updates 2002; 5:119-125

Tetracyclines and gycylcyclines: Mechanisms of resistance

ECOFF

S RECOFF

ECOFF

ECOFF

44



Tigecycline

ECOFF

ECOFF

Tetracyclines and glycylcyclines: Pharmacokinetics

Sabundayo & Standiford. Antimicrobial Agents, 2005Sun et al. Antimicrob Agents Chemother 2009; 49:1629

Meagher et al Clin Infect Dis 2005; 41 (Suppl 5):334Muralidhan et al. Antimicrob Agents Chemother 2005; 49:220

Tetracycline Tigecycline

500 mg oral 100 mg iv 50 mg iv

Cmax (mg/L)

30 min (IV) - 1.4±0.3 0.9±0.2

60 min (IV) - 0.9±0.3 0.6±0.1

2 h (oral) 1.5-5 - -

T1/2 (h) 6-12 27.1±14.3 42.4±35.3

AUC∞ (mg/L.h) - 5.2±1.9 NA

Vd (L) 108 568±244 639±307

Total body

clearance (L/h)

21.8±8.9 23.8±7.8

Tigecycline: Pk/Pd breakpoints

EUCAST Rationale Document (www.eucast.org)

§ Probability of target attainment for tigecycline against E. coli at the CART* identified serum AUC/MIC ratio of 6.96

Susceptiblebreakpoint

*CART: logistic regression or classification and regression tree analysis

45


Tetracyclines and glycylcyclines: Mechanisms of resistance

Proteae: resistance due to hyperexpression of chromosomallyencoded AcrAB efflux pump

Vissalli et al. Antimicrob Agents Chemother 2005; 47:665-669

TET: tetracycline; TGC: tigecycline

Strain: Proteus mirabilis

AcrA hyper-

expression

Comple-

mentation

Tn-

Insertion

MIC (mg/L)

MIN TGC

Wild type + - - 32 4

Laboratory derived ++ + - >64 16

Laboratory derived - - + 1 0.25

Laboratory derived ++ + + >64 16

MIN: minocycline; TGC: tigecycline


Proteae: resistance due to hyperexpression of chromosomallyencoded AcrAB efflux pump

Ruzin et al. Antimicrob Agents Chemother 2005; 49:791-3

TET: tetracycline; TGC: tigecycline

Strain: Morganella morganiiAcrA over-

expression

Comple-

mentation

MIC (mg/L)

TET TGC

S-clinical isolate - - 1 1

R-clinical isolate + - 32 4

R-clinical isolates + insertion in acrA - - 16 0.03

R-clinical isolates + insertion in acrA + + 64 4


P. aeruginosa: resistance due to chromosomally encodedMexXY-OprM efflux pump

Dean et al. Antimicrob Agents Chemother 2003; 47:972-8

DeletionsMIC (mg/L)

TMP CHL GEN CIP CARB TET TGC

Wild type 64 250 1 0.06 160 16 8

mexB 16 ≤8 2 0.06 20 - 8

mexXY 125 125 ≤0.125 0.06 160 4 0.5

mexB/mexXY 4 125 ≤0.125 ≤0.03 1.25 0.5 0.5

mexAB/oprM 4 32 ≤0.125 ≤0.03 1.25 0,25 0.5

TMP: trimethoprim: CHL: chloramphenicol; CIP: ciprofloxacin; CARB: carbenicillin; TET: tetracycline; TGC: tigecycline

46




Rule no.

Organisms

Ma

cro

lid

es

Fu

sid

ic a

cid

Str

ep

tog

ram

ins

Tri

me

tho

pri

m

Na

lid

ixic

ac

id

3.1 Haemophilus influenzae R R 3.2 Moraxella catarrhalis R 3.3 Neisseria spp. R 3.4 Campylobacter fetus R R R R 3.5 Campylobacter jejuni/coli R R R

These bacteria are also intrinsically resistant to glycopeptides, lincosamides, daptomycin and linezolid

Rule no. Organisms Exceptional phenotypes

5.1 Any Enterobacteriaceae (except Proteus spp.)

Resistant to ertapenem, meropenem, imipenem

5.2 Pseudomonas aeruginosa and Acinetobacter spp.

Resistant to colistin.

5.3 Haemophilus influenzae Resistant to any third-generation cephalosporin, carbapenems, fluoroquinolones.

5.4 Moraxella catarrhalis Resistant to ciprofloxacin, any third-generation cephalosporin.

5.5 Neisseria meningitidis Resistant to penicillin (MIC >1 mg/L), third generation cephalosporins, ciprofloxacin.

5.6 Neisseria gonorrhoeae Resistant to third-generation cephalosporins, spectinomycin.


Exceptional phenotypes in Gram-negative bacteria (Table 5)

Fluoroquinolones

Ferech et al. J Antimicrob Chemother 2006; 58:423-7

47


RuleNo.

Organism Agent Rule Exceptions Scientific basis Grade* References

13.6 Enterobacteriaceae Ciprofloxacin If resistant to ciprofloxacin, report as resistant to all fluoroquinolones

Acquisition of at least two target mutations in gyrAor gyrA plus parC

B Komp Lindgren P et al., 2003

13.7 Salmonellaspp. Nalidixic acid If resistant to nalidixic acid, report as resistant to all fluoroquinolones

Evidence for clinical failure of fluoroquinolones in case of resistance to nalidixic acid due to the acquisition of at least one target mutation in gyrA

A Helms et al., 2002Kadhiravan T et al, 2005Slinger et al. 2004

A. There is clinical evidence that reporting the test result as susceptible leads to clinical failuresB. Evidence is weak based only on a few case reports or on experimental models. It is presumed that reporting the test as susceptible my lead to clinical failures C. There is currently no clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged

Expert rules for quinolones & Enterobacteriaceae

RuleNo.


13.6 Enterobacteriaceae Ciprofloxacin If resistant to ciprofloxacin, report as resistant to all fluoroquinolones

Acquisition of at least two target mutations in gyrA plus parC or gyrA

B Komp Lindgren P et al., 2003

13.7 Salmonellaspp. Nalidixic acid If resistant to nalidixic acid, report as resistant to all fluoroquinolones

Evidence for clinical failure of fluoroquinolones in case of resistance to nalidixic ac. due to the acquisition of at least one target mutation in gyrA

A Helms et al., 2002Kadhiravan T et al, 2005Slinger et al. 2004

A. There is clinical evidence that reporting the test result as susceptible leads to clinical failuresB. Evidence is weak based only on a few case reports or on experimental models. It is presumed that reporting the test as susceptible my lead to clinical failures C. There is currently no clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged


Should we apply the Salmonella rule (13.7) for all Enterobacteriaceae?

Fluoroquinolones: EUCAST breakpoints

Fluoroquinolone2 Species-related breakpoints (S</R>) Non-species

related breakpoints1

S</R >

Entero-

bacteriaceae3

Pseudo-

monas/

Acineto-

bacter

Staphylo-

cocc us

Entero-

coccus

Strepto-

coccusA,B,C,G

S.pneu-

moniae6

H.influenzae

M.catarrhalis

N. gonorr-

hoeae

N.menin-

git idis8

Gram-

negat ive anaerobes

Cipro floxacin 0.5/1 0.5/1 1/14 1/15 -- -- 0.125/2 0.5/0.57 0.03/0.06 0.03/0.06 -- 0.5/1

Levofloxacin 1/2 1/2 1/2 1/2 -- 1/2 2/2 1/17 IE IE -- 1/2

Moxifloxacin 0.5/1 -- -- 0.5/1 -- 0.5/1 0.5/0.5 0.5/0.57 IE IE IE 0.5/1

Norfl oxacin 0.5/1 -- -- -- -- -- -- -- IE -- -- 0.5/1

Ofloxacin 0.5/1 -- -- 1/13 -- -- 0.125/4 0.5/0.57 0.12/0.25 IE -- 0.5/1

1. Non-speci es related breakpoin ts have been determinedmainly on the basis of PK/PD data and are independent of MIC dis tributions of spec ific species. They are for use only for species that have not been given a speci es-specific breakpoint and not for those species where susceptibility testing is not recommended (marked wi th -- or IE in the table).

2. For break poin ts for other fluoroquinolones (eg. pefloxacin and enoxacin) - refer to breakpoints determined by national breakpoint committees.

3. Sal mone lla spp- there is clinical evidence for c iprofloxacin to indicate a poorresponse in systemic infections causedby Salmonella spp with low-level fl uoroquinolone resistance(MIC>0.064 mg/L). The availabledata relate mainly to S. typhi but there are also case reports of poor response with other Salmonella species.

4. The S/ I break point has been increased from0.5 to1 mg/L to avoid dividing the wildty pe MIC distribution. Thus there is no intermediate category for Acinetobacter spec ies5. Staphyl ococcus spp - breakpoints for ciprofloxacin and ofloxacin relate to high dose therapy. 6. St reptoc occuspneumoniae - wild type S.pneumoniae are not considered susceptible to ciprof loxacin or of loxacinand are therefo re categorized as in termediate. For ofloxacinthe I/R breakpoint was increased from

1.0 to 4.0 mg/L and for levofloxacin the S/I-breakpoint from 1.0 to 2.0 to avoid dividing the wild type MIC distrib ution. The breakpoints for levofl oxacin relate to high dose therapy.7. St rain s wi thMIC values above the S/I breakpoint are very rare or not yet reported. The identification and antimicrobial susceptibility tests on any such isolate must be repeated and if the result is confirmedthe

isola te sent to a reference laboratory. Until there is evidence regarding clinical response for confi rmed isolates with MIC abovethe current resis tant breakpoint (in it alics) they should be reported resistant. Haemop hil us/Moraxella - fluoroquinolone low-level resistance (ciprofloxacin MIC:s of 0.125 - 0.5 mg/L) may occurin H.influenzae. There isno ev idence that low-level res istance is of c linical importance in respi ratory tract infections with H.influenzae.

8. Neisseria meningitidis - breakpointsapply to the use of ciprofloxacin in the prophylaxis of meningococcal disease.

-- = Susc epti bility testing not recommended as the species is a poor target for therapy with the drug.

IE = There is insufficient evidence that the species in question is a good target for therapy with the drug.RD =Rationale document listing data used for setting EUCAST breakpoints.

48


ECOFF: epidemiological cut-off values

ECOFF S RECOFF

Low level resistance?Decreased susceptibility?

High level resistance


ECOFF: epidemiological cut-off values

ECOFF ECOFF S R

ECOFF S RECOFF

Fluoroquinolone-resistance mechanisms

Relevance/incidenceGene Gram (+) Gram (–) Expression

Topoisomerase modifications

- gyrAmutations Cr + ++++- parC mutations Cr +++/++ + low / high- mosaic gyrA, parC genes Cr + –

Reduction to target access

- porin modification Cr – +- efflux system: AcrAB, … Cr ++ ++ low

QepA Pl – +

Target protection

- Qnr proteins (QnrA, QnrB, QnrS) Pl – +/– low

Quinolone modification

- AAC(6’)-Ib-cr Pl – +/– low

49


§ Development of high level resistance to ciprofloxacin occurs ata higher rate in Enterobacteriaceae that are resistant to nalidixicacid but susceptible to ciprofloxacin than in those isolates that aresusceptible to both nalidixic acid and ciprofloxacin

§ Clinical failure in patients with isolates that are resistant to nalidixic acid has only been adequately documented with Salmonella spp.isolates, most of them with low level resistance levels!

Phenotype Resistance

mechanisms

Resistance

developmentNalidixic acid Ciprofloxacin

S S - +

S(↓) S(↓) Qnr-like ++

R S gyrA mutations +++

Expert rule for quinolones & Enterobacteriaceae

Salmonella spp. and quinolone resistance

§ Single point mutations in gyrA gene confer resistance (MIC>ECOFF)

to nalidixic ac. and low-level resistance to ciprofloxacin

- isolates can appear as susceptible (MICs = 0.12-1 mg/L)

- nalidixic ac.R is a good marker of low-level ciprofloxacin resistance

§ High-level resistance to ciprofloxacin is still scarce comparing

with E. coli and requires ≥2 point mutations in gyrA gene with …

- variably additional mutations in gyrB, parC or

- overexpression of soxR / marA with altered levels of AcrB / OmpF

§Worldwide increment of ciprofloxacin resistance (low- and high-level)

Weinberger . Curr Opin Infect Dis 2005;18: 513-21Parri. Curr Opin Infec Dis 2003; 16: 467-72

Aarestrup et al. Antimicrob Agents Chemother 2003; 47:827-9

Country SerovarNo. of

patients MIC (mg/L)

Denmark Enteritidis 1 Original strain, 0.032; after treatment, 1

Typhim. DT104 27 0.064-0.124

Typhimurium 83 0.06-0.38

Typhi 1 0.19

France Typhi 1 0.12

India Typhi 32 0.06-0.5

Spain Enteritidis 2 Original strains, 0.06; after treatment, 0.5 and 1

United Kingdom

Typhi 1 0.5

Typhimurium 1 Original strain, 0.03; after treatment, 2.0

Typhimurium 2 Original strains, 0.015; 0.03; after treatment, 2; 0.06-1

Bovismorbifica 1 Original isolate, 0.06; after treatment, 2, 16, and 4

Virchow 1 Original strain, 0.016; after initial, treatment, 0.75

Vietnam Typhi 150 0.125-1 (ofloxacin)

Treatment failures in patients infected with Salmonella enterica serovar Typhi and non-Typhi isolates

with decreased susceptibility to fluoroquinolones

Aarestrup et al. Antimicrob Agents Chemother 2003; 47:827-9

50


Suceptible

to ciprofloxacin

(<0.12 mg/L)

Decreased susceptibility

to ciprofloxacin

(0.12-1 mg/L)

Antimicrobial-related

fever clearance time (h) 72 (19-264) 92 (21-373)

Ciprofloxacin-related

fever clearance time (h) 64 (34-204) 90 (9-373)

Treatment failure 4% (2/46) 17% (4/24)

Escherichia coli: fluoroquinolone resistance

Modified from Schedletzky et al. J Antimicrob Chemother, 1999 34 (Suppl B):31-7Saenz et al. J Antimicrob Chemother 2004; 51:1001-5

Mutation in:

Efflux

MIC (µg/ml)

gyrA parC NAL CIP LEV MOX

- - - 2 0.01 0.06 0.06

+ - - 32-256 0.5 0.5 1

- + - 64 0.01 0.03 0.2

+ + - - >1024 1 2 2

+ - + 32->1024 2 4 4

+ + + - >1024 64 32 32

+ + + + >1024 256 64 128

CIP: ciprofloxacin; LEV: levofloxacin; MOX: moxifloxacin

Escherichia coli: low level fluoroquinolone resistance

In vitro effect of the gyrA mutation or qnr presence onthe recovery of ciprofloxacin-resistant mutant

Jacoby GA. Clin Infect Dis. 2005 Jul 15;41 (Suppl 2):S120-6

51


Robisek et al. Nat Med 2006; 12:83-88Robicsek et al. Lancet Infect Dis 2006; 6:629-40

Yamane et al. Antimicrob Agents Chemother 2007; 51:3354-60

Escherichia coli phenotypes

§MICs not always higher thanECOFF (16 mg/L) for nalidixic acid

§ MICs for ciprofloxacin higher than ECOFF (0.032 mg/L) butlower than S breakpoint (0.5 mg/L)

NAL CIP

www.seimc.org

Low level fluoroquinolone resistance

Resistance mechanisms

MIC (mg/L)

NAL CIP LEV MOX

Wild type 2-4 0.008-0.02 0.08-1 0.03

QnrA 8-32 0.12-2 0.25-0.5 0.5-1

QnrB 16 0.25-1 0.5 1-2

QnrS 8-32 0.12-0.5 -- 0.25

AAC(6’)-Ib-cr -- 0.08 0.08 --

QepA 1-2 0.25 0.03-0.06 0.06-0.09

NAL: nalidixic acid; CIP: ciprofloxacin; LEV: levofloxacin; MOX: moxifloxacin

§ MPC are always higher in qnrA1-(+) than qnrA1-(-) isolates

§ Quinolone-R mutants in qnrA1-(+) isolates emerge in a short

period of time and in a low quantity when compared with qnrA1-(-)

qnrA1-(-) qnrA1-(+)

E. coli 0.015-0.125* 2-4

K. pneumoniae 4-8 4-128

*mg/L

Effect of qnr in the ciprofloxacin MPC values in E. coli

Rodríguez-Martínez et al. Antimicrob Agents Chemother 2007; 51;2236-9


52

http://www.seimc.org


Rodríguez-Martínez et al. Antimicrob Agents Chemother 2007; 51;2236-9

Effect of qnr in the ciprofloxacin MPC values in K. pneumoniae


§ Selection of a CIPR E. coli isolate during treatment with norfloxacinof a UTI due to an ESBL (VEB-1) and QnrA1CIPS E. coli isolate

E. coli before treatment

E. coli after treatment

Nalidixic ac. 16* >256

Norfloxacin 2 >256

Ciprofloxacin 0.5 >32

GyrA mutation Wild type Ser83Leu / Asp87Asn

ParC mutation Wild type Ser80Il

*mg/L

Ciprofloxacin: Pk/Pd breakpoints

Monte Carlo simulation, Probabilities of Target Attainment (AUC/MIC = 30-40) for ciprofloxacin (500 mg x 2 oral)

Susceptiblebreakpoint(0.5 µg/ml)

EUCAST rationale document (www.eucast.org)

53




mechanisms

Resistance


S S - +



Expert rules for quinolones and Enterobacteriaceae


mechanisms

Resistance


S S - +



Aminoglycosides

Vander Stichele et al. J Antimicrob Chemother 2006; 58:159-67

Hospital consumptionof antibiotics in 15

European countries(ESAC retrospective data

collection, 1997-2002)

54


Aminoglycosides

Coenen et al. JAC 2009doi:10.1093/jac/dkp135

Outpatient parenteralantibiotic use in 20

European countries, 2006

§ The 3 most commonlyused antibiotic groupswere: - cephalosporins 44.6%- aminoglycosides 25.3% - penicillins 17.8%

§Gentamicin representsnearly 20% of the use

Aminoglycoside resistance mechanisms

1) Decreased permeability and/or accumulation

- passive diffusion or active transport mutations

- porin and/or lipopolisacharide alteration, Gram (-)

- efflux pumps hiperexpression

2) Target (ribosomal) modification

- ribosomal proteins (S3, S4, S5, S6, S12, S17, L6)

- 16S RNA methylation (methylases)

3) Aminoglycoside-modifying enzymes

- acetyltransferases

- phosphotransferases

- nucleotidyltransferases (adeniltransferases)

Expression

+

++

+++

+/+++

Aminoglycosides: EUCAST breakpoints

Aminoglycosides2

Species-related breakpoints (S</R>) Non-species related

breakpoints1

S</R>

Enterobac- teriaceae

Pseudo-monas3

Acineto-bacter4

Staphylo- coccus

Entero- coccus4

Strepto- coccus A,B,C,G

S.pneumoniae

H.influenzae M.catarrhalis

N.gonorrhoeae

N.meningitidis

Gram-negative

anaerobes

Amikacin 8/16 8/16 8/16 8/165 -- -- -- IE -- -- -- 8/16

Gentamicin 2/4 4/4 4/4 1/1 -- -- -- IE -- -- -- 2/4

Netilmicin 2/4 4/4 4/4 1/1 -- -- -- IE -- -- -- 2/4

Tobramycin 2/4 4/4 4/4 1/1 -- -- -- IE -- -- -- 2/4 1. Non-species related breakpoints have been determined mainly on the basis o f PK/PD data and are independent of M IC distributions of specifi c species. They are fo r use only for species that

have not been given a species-specific breakpoint and not for those species where suscepti bility testing is not recommended (marked with -- or IE in the table). 2. The aminog lycoside breakpoints are based on modern once-daily administration of high aminoglycoside dosages. Most often aminog lycosides are given in combinat ion with beta-lactam

agents. For unlisted aminoglycosides refer to breakpoints det ermined by national breakpoint committees. 3. The S/I breakpoint has been i ncreased from 2 to 4 mg/L for agents other than amikacin to avoid dividi ng the wild type MIC distribution. Thus there is no i ntermediate category for

Pseudomonas species and Acinetobacte r species. 4. Enterococcus spp - aminoglycoside monotherapy is ineffect ive against enterococci. There is synergism between aminoglycosides and betalactams in enterococci without acquired resistance

mechanisms. There is no sy nergistic effec t in enterococci with high level aminoglycoside resistance, i.e with gentamicin M IC>128 mg/L. 5. Resistance to amikacin and kanamycin is most reliably determined using kanamycin as test substance.

-- = Susceptibility testing not recommended as the species is a poor target for therapy with the drug. IE = There is ins ufficient evidence that the species in question is a good target for therapy with the drug. RD =Rationale document li sting data used fo r setting EUCAST breakpoints

55


Providencia rettgeriProvidencia stuartii

All Providencia isolates produce a chromosomal AAC(2’)-Ia enzyme Providencia isolates should be considered resistant to all aminoglycosides except amikacin (and streptomycin). The isolates that poorly express the enzyme can appear in vitro susceptible to netilmicinbut should be reported as resistant. Mutations cause overproduction of this enzyme.

Serratiamarcescens

All S. marcescens isolates produce a chromosomal AAC(6’)-Ic enzyme and are intrinsically moderately susceptible to all aminoglycosides excepted streptomycin and gentamicin

Intrinsic resistance (Table 1, footnote)

Expert rules for aminoglycosides & Enterobacteriaceae

§ All S. marcescens constitutively produce a chromosomal acetylase enzyme [ AAC(6’)-Ic ]

- variably expressed (normally low level)

- does not confer resistance to streptomycin and gentamicin

- kanamycin is highly affected but amikacin is only slightly affected

- hyperproducers …

- selected in vitro with amikacin, tobramycin or netilmicin

- are resistant to both tobramycin and amikacin

- associated with superinfection or clinical failures1,2,3 due to the

selection of derepressed mutans when treated with amikacin


Sklaver et al. Arch Intern Med 1978; 138:713-6Honda et al. Drug Intell Clin Pharm 1981; 15:284-6

Torres et al. Chemother 1989; 35(Suppl 1):15-24


≤2*

4*

≤2

Low exp.

MIC (mg/L)

≤2GEN

>8AMK

>8TOB

High exp.

Aminogly-

coside

* Consider as R

Serratia marcescens AAC(6’)-Ic

56


Rule

No.


12.7 AllEnterobacteriaceaeP. aeruginosa A. baumannii

Amikacin If I or R to tobramycin and Sto gentamicin report amikacinas I for Enterobacteriaceae or R for P. aeruginosa

Production of acquired AAC(6’)I may not confer phenotypic resistance despite modification of amikac in.

C Benveniste R, Davies J.

1971a

Galimand et al. 1993

Martin et al., 1988

Shaw KJ et al. 1991

12.8 All Enterobacteriaceae

Gentamicin If I to gentamicin and S to other aminoglycosides report as R to gentamicin

Expression of AAC(3)I enzyme may be low,and isolates may have decreased susceptibility to gentamicin . Resistance to fortimic in may confirm the presence of this enzyme.

C Witchitz JL. et al, 1972

Shaw KJ, et al. , 1993


Tobramycin If I to tobramycin, R to G and S to amikacin report as R to tobramicin

Expression of ANT(2”) enzyme may be low and isolates may have decreased susceptibility to tobramycin

C Benveniste R and Davies

J. 1971b

Shaw KJ et al. , 1993


Netilmicin If I to netilmicin and I or R to gentamicin and tobramycinreport as R to netilmicin

Expression of AAC(3”)II or AAC(3”)V may be low and isolates may appear with decreased susceptibility to netilmicin

C Le Goffic F et al., 1974

Shaw KJ et al., 1993

A. There is clinical ev idence that reporting the test result as sus ceptible leads to clinical failuresB. Ev idence is w eak based only on a few case reports or on ex perimental models. It is presumed that reporting the test as sus ceptible my lead to clinic al failures C. There is currently no clinical ev idence, but microbiological data s uggest that clinical use of the agent s hould be dis couraged

Expert rules for aminoglycosides & Gram (-) bacilli

Interpretive rules: aminoglycosides & Gram-(-) bacilli

Fenotipo Enzima S Sp K A G Nt T Nm

St APH(3’’) R S S S S S S S

St Sp ANT(3’’)(9) R R S S S S S S

G AAC(3)-I S S S S R s/r s/r S

K APH(3’)-I S S R S S S S R

K A APH(3’)-VI S S R R S S S R

G T AAC(3)-VI* S S S S R s/r R S

T K A ANT(4’)-II S S R R S S R S

G T Nt AAC(2’)-I

AAC(3)-IV S S S S R R R R

K G T ANT(2’’)-I S S R S R S r S

K T A Nt AAC(6’)-I S S R r S R R S

K T G Nt AAC(3)-II S S R S R R R S

K T G A Nt Impermeability ±

different enzymes R R R R R R R R

*= AAC(3)-IIa

Kanamycin (R)

Amikacin (I→R)

Gentamicin (S)Tobramycin (R)

Neomycin (S)

Netilmicin (R)

Interpretive rules: aminoglycosides & Gram-(-) bacilli

57


EUCAST interpretive rules in

antimicrobial susceptibility testing for

fluoroquinolones and aminoglycosides

and Enterobacteriaceae

EUCAST EDUCATIONAL WORKSHOP18th ECCMID Barcelona, Spain, 2008

Rafael Cantón

Servicio de Microbiología

58

Rodloff - Anaerobes

Institut für Medizinische Mikrobiologie und Infektionsepidemiologie

AnaerobesAnaerobes

Arne C. RodloffArne C. Rodloff

Antimicrobial Antimicrobial Susceptibility TestingSusceptibility Testing


Increasing Numbers ofIncreasing Numbers ofAnaerobes in BacteremiaAnaerobes in Bacteremia

Lassmann et al., CID 44, 2007Lassmann et al., CID 44, 2007


Increasing Percentage ofIncreasing Percentage ofAnaerobes in BacteremiaAnaerobes in Bacteremia


59

Rodloff - Anaerobes


Increasing Numbers ofIncreasing Numbers ofInfections with Anaerobes in BacteremiaInfections with Anaerobes in Bacteremia



Bacteremia with Anaerobes Bacteremia with Anaerobes

ÜÜ Turku University Central HospitalTurku University Central Hospital

ÜÜ 1000 beds, tertiary referal center1000 beds, tertiary referal center

ÜÜ retrospective analysisretrospective analysis

ÜÜ all blood cultures from 1991all blood cultures from 1991--1996 1996

ÜÜ total ~ 40.000total ~ 40.000

ÜÜ positive: 5%positive: 5%

ÜÜ anaerobes: 4% of positives (n=81)anaerobes: 4% of positives (n=81)

Salonen et al., CID, 1998Salonen et al., CID, 1998


Bacteremia with AnaerobesBacteremia with Anaerobes

clinically relevant

lethal

No clinical signs

B. fragilis 27 5 1 Bacteroides spp. 8 4 0 Prevotella spp. 3 1 0 Clostidium spp. 6 3 3 Pstrept. spp. 7 0 3 Lactobacilli 2 0 0 Propionib. spp. 3 1 12 others 5 0 5


60

Rodloff - Anaerobes


Bacteremia with AnaerobesBacteremia with Anaerobes

Blood culture positive81 patients

Clinically relevant57 patients

No symptoms24 patients

Initial therapy +28 patients

Initial therapy -adjusted

18 patients

Initial therapy -- adjusted11 patients

Lethality 18% Lethality 17% Lethality 55%



ÜÜUntil the 1960s, little was known about the resistance Until the 1960s, little was known about the resistance of of BacteroidesBacteroides

ÜÜBetween 1960 and 1985, a very rapid spread of Between 1960 and 1985, a very rapid spread of resistance to tetracyclin and erythromycin was resistance to tetracyclin and erythromycin was experienced, together with the acceptance that experienced, together with the acceptance that practically all practically all BacteroidesBacteroides are resistant to ampicillinare resistant to ampicillin

ÜÜAfter 1985, increased resistance to clindamycin, After 1985, increased resistance to clindamycin, cefoxitin, metronidazole, imipenem, and recently cefoxitin, metronidazole, imipenem, and recently quinolones was observedquinolones was observed

stolen from Elisabeth Nagystolen from Elisabeth Nagy

Development of ResistanceDevelopment of Resistance


Dilution TestsDilution Tests

61

Rodloff - Anaerobes


AgarAgar--DilutionDilution

1.1. Inoculum in separate tubesInoculum in separate tubes1x101x1088cfu/mL, transfer 1cfu/mL, transfer 1μμLL1x101x1055 cfu/spotcfu/spot

2.2. PlatesPlatesCLSIBrucella agar supplemented with 5 μg hemin and 1 μg vitamin K1 per mL and 5% (v/v) laked sheep bloodDINWilkins-Chalgren-agar without or if necessary with up to 10% sheep blood

1

2


IncubationIncubation

ÜÜ CLSICLSI

35 35 -- 3737°° C, 42 C, 42 -- 48 h48 h

ÜÜ DINDIN

36 36 ±± 11°° C, according to generation timeC, according to generation time



62

Rodloff - Anaerobes


CLSI readingCLSI reading

Reproduced with permission,from NCCLS publication M11-A6Methods for AntimicrobialSusceptibil ity Testing of Anaerobic Bacteria; Approved StandardSixth Edition (ISBN 1-56238-517-8). Copies of the current edition may be obtained from NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.



plusplus

ÜÜ 32 32 strainsstrainson one plateon one plate

ÜÜ good growthgood growth

ÜÜ cost effectivecost effectivefor surveillancefor surveillance

minusminus

ÜÜ labour intensivelabour intensive

ÜÜ requiresrequiresexperienceexperience

ÜÜ not for individualnot for individualisolatesisolates


MicrobrothMicrobroth--DilutionDilution

InoculumInoculum1x101x1077cfu/mL, transfer 10cfu/mL, transfer 10μμLLor 50or 50μμL + 50L + 50μμLL1x101x1055 cfu/wellcfu/wellCLSIBrucella broth supplemented with 5 μg hemin and 1 μg vitamin K1 per mL and 5% (v/v) laked sheep bloodDINWilkins-Chalgren-broth orbrain-heart-infusion-brothwithout or if necessary withup to 10% animal serum

63

Rodloff - Anaerobes


IncubationIncubation

ÜÜ CLSICLSI

35 35 -- 3737°° C, 46 C, 46 -- 48 h48 h

ÜÜ DINDIN

36 36 ±± 11°° C, according to generation timeC, according to generation time


CLSI readingCLSI reading

Reproduced with permission,from NCCLS publication M11-A6Methods for AntimicrobialSusceptibil ity Testing of Anaerobic Bacteria; Approved StandardSixth Edition (ISBN 1-56238-517-8). Copies of the current edition may be obtained from NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.


MicrobrothMicrobroth--DilutionDilution

plusplusÜÜ commercial plates availablecommercial plates available

ÜÜ self prepared plates may be stored frozenself prepared plates may be stored frozen

ÜÜ individual isolates may be testedindividual isolates may be tested

minusminusÜÜ growth?growth?

64

Rodloff - Anaerobes


Growth in Test SystemsGrowth in Test Systems

Rodloff et al., Z.Antimikrob.Antineoplast. Chemother. 3, 1985.


Effect of Media on ResultsEffect of Media on Results



Effect of Method on ResultsEffect of Method on Results


65

Rodloff - Anaerobes


EE--TestTest

ÜÜ some experience necessarysome experience necessary


AgarAgar--DiffusionDiffusion


QCQC

ÜÜAll QCAll QC-- strains need to display MICstrains need to display MIC--values within the given reference rangevalues within the given reference range

Bacteroides fragilisBacteroides fragilis ATCC 25285ATCC 25285

Bacteroides thetaiotaomicronBacteroides thetaiotaomicron ATCC 29741ATCC 29741

Eubacterium lentumEubacterium lentum ATCC 43055 ATCC 43055

Clostridium perfringensClostridium perfringens ATCC 13124ATCC 13124

66

Rodloff - Anaerobes


ßß--LactamaseLactamase--TestTest

ÜÜ Nitrocephin testNitrocephin test

ÜÜ plusplusResult in minutesResult in minutes


Surveillance…Surveillance… Koeth et al., JAC 53, 2004Koeth et al., JAC 53, 2004


Surveillance…Surveillance… Koeth et al., JAC 53, 2004Koeth et al., JAC 53, 2004

67

Rodloff - Anaerobes


MIC 50%/90%...MIC 50%/90%...

010203040

0.25 1 4 16 >32

Distr ibutiva normalia

Rodloffia complicata

MICMIC50%50% MICMIC90%90%


CLSICLSI

breakpointsbreakpoints


EUCAST vs. CLSIEUCAST vs. CLSI

EUCASTEUCAST CLSICLSI

PenicillinPenicillin ≤≤ 0,25/ 0,25/ > 0,5> 0,5 ≤≤ 0,5/ 0,5/ ≥≥ 22

ClindamycinClindamycin ≤≤ 4/ 4/ > 4> 4 ≤≤ 2/ 2/ ≥≥ 88

MetronidazoleMetronidazole ≤≤ 4/ 4/ > 8> 8 ≤≤ 8/ 8/ ≥ ≥ 3232

Piperacillin/Piperacillin/TazobactamTazobactam IE (≤ 8/ >16)IE (≤ 8/ >16) ≤≤ 32/ 32/ ≥≥ 128128

CefoxitinCefoxitin -- ≤≤ 16/ 16/ ≥≥ 6464

ImipenemImipenem ≤≤ 2/ 2/ >> 88 ≤≤ 4/ 4/ ≥≥ 1616

MoxifloxacinMoxifloxacin IE (IE (≤≤ 0,5/ 0,5/ > 1)> 1) --

68

Rodloff - Anaerobes


Surveillance…Surveillance…

Snydman et al., AAC 51, 2007Snydman et al., AAC 51, 2007


USA…USA…

Aldridge + OAldridge + O´́Brien, JCM 40, 2002Brien, JCM 40, 2002

1989/901989/90 1998/991998/99PITPIT ModeMode 50%50% 90%90% ModeMode 50%50% 90%90%B.fragilisB.fragilis 22 22 88 0,120,12 0,120,12 11GroupGroup 44 44 88 0,120,12 0,120,12 11CLICLIB.fragilisB.fragilis 0,50,5 0,250,25 44 0,250,25 0,250,25 1616GroupGroup 0,50,5 0,50,5 3232 0,250,25 0,250,25 1616CFXCFXB.fragilisB.fragilis 88 88 3232 44 44 1616GroupGroup 88 88 3232 44 44 1616


Surveillance…Surveillance… Snydman et al., AAC 51, 2007Snydman et al., AAC 51, 2007

69

Rodloff - Anaerobes


Quinolone Resistance in the USAQuinolone Resistance in the USA

Golan et al., JAC 52, 2003Golan et al., JAC 52, 2003


Moxifloxacin Resistance in the USAMoxifloxacin Resistance in the USA

Golan et al., Golan et al.,

JAC 52, 2003JAC 52, 2003


USA vs. USA…USA vs. USA…

Aldridge + OAldridge + O´́Brien, JCM 40, 2002; Snydman et al., AAC 46, 2002; CID 35, 2002Brien, JCM 40, 2002; Snydman et al., AAC 46, 2002; CID 35, 2002

1998/991998/99 1998/19981998/1998PITPIT ModeMode 50%50% 90%90% MeanMean 50%50% 90%90%B.fragilisB.fragilis 0,120,12 0,120,12 11 -- -- --GroupGroup 0,120,12 0,120,12 11 ~3~3 22 1616CLICLIB.fragilisB.fragilis 0,250,25 0,250,25 1616 1,81,8 0,50,5 256256GroupGroup 0,250,25 0,250,25 1616 ~2~2 11 256256CFXCFXB.fragilisB.fragilis 44 44 1616 14,414,4 1616 3232GroupGroup 44 44 1616 ~20~20 1616 6464

70

Rodloff - Anaerobes


USA vs. Europe…USA vs. Europe…

Aldridge + OAldridge + O´́Brien, JCM 40, 2002; Hedberg + Nord, CMI 9, 2003Brien, JCM 40, 2002; Hedberg + Nord, CMI 9, 2003

1998/991998/99 1999/20011999/2001PITPIT ModeMode 50%50% 90%90% MeanMean 50%50% 90%90%B.fragilisB.fragilis 0,120,12 0,120,12 11 -- 0,50,5 22GroupGroup 0,120,12 0,120,12 11 -- 0,50,5 88CLICLIB.fragilisB.fragilis 0,250,25 0,250,25 1616 -- 11 >64>64GroupGroup 0,250,25 0,250,25 1616 -- 11 >64>64CFXCFXB.fragilisB.fragilis 44 44 1616 -- 88 1616GroupGroup 44 44 1616 -- 88 3232


European European Regions:Regions:Resistance in the Resistance in the B. fragilisB. fragilis GroupGroup

NorthNorth M/WM/W SouthSouth EastEast

AMP (2)AMP (2) 99%99% 99%99% 99,5%99,5% 99,5%99,5%

CFX (64)CFX (64) 11%11% 4%4% 4%4% 4%4%

IMP (16)IMP (16) 0,8%0,8% 1,6%1,6% 0,5%0,5% 0,4%0,4%

PIT (128)PIT (128) 0,8%0,8% 1,6%1,6% 0,5%0,5% 0,4%0,4%

CLI (8)CLI (8) 13%13% 13%13% 31%31% 10%10%

MTR (32)MTR (32) 0,4%0,4% 1%1% 0%0% 0,4%0,4%

MFX (8)MFX (8) 8%8% 10%10% 15%15% 6%6%

Hedberg + Nord, CMI 9, 2003Hedberg + Nord, CMI 9, 2003


B. fragilis, n= 166, Leipzig 2003/4B. fragilis, n= 166, Leipzig 2003/4

0

50

100

0.06 0.25 1 4 16 64

MTR

CLI

IMP

PIT

AMP

71

Rodloff - Anaerobes


B. thetaiotaomicron, n= 46, Leipzig 2003/4B. thetaiotaomicron, n= 46, Leipzig 2003/4

0

10

20

30

0.06 0.25 1 4 16 64

MTR

CLI

IMP

PIT

AMP


Thank youfor yourpatience

Arne C. Rodloff

Antimicrobial Antimicrobial Susceptibility TestingSusceptibility Testing

72

educational workshop - escmid

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