Update on the 2012-2013 CLSI Standards for Antimicrobial - SWACM

8/28/2012 

<strong>Update</strong> on the 2012-2013 CLSI Standards for 

Antimicrobial Susceptibility Testing: 

What’s New with the 

Gram Positive Cocci 

CLSI AST Standards – 

January 2012 

• M100-S22 Tables (2012)* 

• M02-A11 Disk Diffusion Method (2012)^ 

• M07-A9 MIC Method (2012)^ 

Susan Sharp, Ph.D. 

Director, Kaiser Permanente Laboratory 

Portland, Oregon, USA 

susan.e.sharp@kp.org 

* M100 updated every year 

# M02, M07 updated every 3 years 

1 

2 

Summary of Major Changes 

• Changes to CLSI documents are summarized in the 

front of each document. 

• Information listed in boldface type is new or modified 

since the previous edition of M100 document. 

Today’s Review: 2012-2013 changes 

• Staphylococcus species 

• Streptococcus pneumoniae 

• b-Streptococcus species 

• Enterococcus species 

• Recent breakpoint addition/revision dates are listed in 

the front of M100-S22. 

3 

4 

Staphylococcus species 

• Penicillin testing 

Staphylococcus spp. – Penicillin 

The story….. 

• > 90% of staphylococci are penicillin “R” 

• Penicillin rarely considered for treatment of staphylococcal 

infections 

• …BUT - Penicillin might be considered for infections 

requiring lengthy therapy (e.g., endocarditis, osteomyelitis) 

IF penicillin were known to be “S” 

5 

• Some Staphylococcus spp. that test “S” to penicillin by MIC or 

disk diffusion may actually possess a β-lactamase (BL) that may 

cause the patient to fail penicillin therapy 

6 

jhindler CLSI M100-S22 <strong>Update</strong> 

1



Induced ß-lactamase (BL) Test 

• CLSI Previous recommendation: 

• Perform induced nitrocefin BL test before reporting 

penicillin as “S” if: 

• zone diameter ≥29 mm 

• MIC ≤0.12 µg/ml 

• PCR for the blaZ β-lactamase gene may be considered 

Penicillin Breakpoints 

-Sub isolate to blood agar 

-Induction: Drop disk to induce BL 

production (e.g., oxacillin or cefoxitin) 

-Incubate overnight 

MIC (µg/ml) 

Zone (mm) 

S I R S I R 

≤0.12 - ≥0.25 ≥29 - ≤28 

-Test cells from periphery of zone 

-If BL positive, report penicillin R 

Pos 

Neg 

Reference: M100-S21. Table 2C. Page 70 

7 

8 

Staphylococcus aureus 

β-lactamase (BL) 

• Induced nitrocefin BL test usually, but not always, detects 

staphylococcal BL 

• Other BL tests are more sensitive for BL: 

• Cloverleaf test 

• Penicillin disk zone edge test 

• blaZ gene PCR not optimal for BL 

• blaZ codes for BL production 

• Several types of blaZ genes 


β-lactamase (BL) Study 

• 348 MSSA (low penicillin MICs) characterized for blaZ by PCR: 

• 303 PCR negative 

• 45 PCR positive 

• Methods: 

• Penicillin MICs 

• Phenotypic BL tests 

• Nitrocefin - Cefinase 

• Nitrocefin - Dryslide 

• Cloverleaf assay 

• Penicillin disk zone edge 

9 

*Statens Serum Institut (Denmark), CDC (Atlanta), MGH (Boston) 

10 


BL Study 

• 1 blaZ neg and penicillin “R” 

• 23 blaZ pos and penicillin “S” 

S 

R 

Pen MIC 

(µg/ml) 

blaZ functional 

Neg 

0.008 2 

0.016 15 

Pos 

0.032 180 1 

0.06 90 5 

0.12 15 17 

0.25 1 14 

0.5 4 

1.0 

Cloverleaf Assay for 

β-lactamase + 

S. aureus 

• 5% sheep blood agar 

• S. aureus ATCC 25923 as the 

indicator BL+ organism 

• 1 unit penicillin disk 

• Negative (penicillin-S) strain 

BL negative 

D 

C 

A 

B 

Reference: CLSI Agenda Book January 2011. 

2.0 2 

4.0 1 

303 45 

• Some difficulties reading 

Reference: CLSI Agenda Book January 2011. 

Isolates A-D are all 

BL positive 

12 


2



Disk Zone Edge Test (10 U penicillin disk and standard disk diffusion method) 

β-lactamase 

positive 

β-lactamase 

negative 

Fuzzy / “beach” = 

β-lactamase negative, 

Penicillin - S 

S. aureus QC: 

Neg - ATCC 25923 

Sharp / “cliff” = 

β-lactamase positive, 

Penicillin - R 

Pos - ATCC 29213 

(supplemental QC) 

13 

Reference: M100-S22. Table 2C Supplemental Table 1. Page 83 

14 


3 Lab BL Study Results (N=348) 


Penicillin (MIC ≤0.12 µg/ml) Reporting 

Test Sensitivity Specificity 

Cefinase 77% 100% 

Dryslide 88% 100% 

Penicillin MIC ≤0.12 µg/ml 

A 

Nitrocefin β-lactamase 

positive 

B 

A 

Nitrocefin β-lactamase 

negative 

Note: If doing disk 

diffusion routinely, just 

examine zone edge for 

those with zone sizes 

of > 29mm. 

Cloverleaf 100% 100% 

Penicillin disk zone edge 96% 100% 

Report penicillin “R” 

Perform penicillin disk zone-edge test 

≥ 29 mm fuzzy 

Report penicillin “S” 

≥ 29 mm sharp 

Report penicillin “R” 

Reference: CLSI Agenda Book January, 2011 

15 

M100-S22. Table 2C Supplemental Table 1. Page 80 

16 


Staphylococcus spp. – 

Penicillin Optional Strategy 

• NEW RECOMMENDATION: 

• Added ‘penicillin disk zone edge test’ for BL 

production in S. aureus 

• Report penicillin if “R” 

• Suppress penicillin if “S” and add note “Contact laboratory 

if penicillin results needed” 

• If penicillin “S” and penicillin results needed, perform: 

• S. aureus 

• Nitrocefin BL test , and if negative 

• Penicillin zone edge test 

17 

18 


3


Action Items 

Staphylococcus 

S. aureus 

• Isolates where penicillin zones are ≥29 mm or penicillin MICs are ≤0.12 

µg/ml, perform a penicillin ‘disk zone edge test’ before reporting as 

penicillin susceptible. 

• NOTE: 

• S.lugdunensis isolates where penicillin zones are ≥29 mm or penicillin MICs are 

≤0.12 µg/ml, perform an induced nitrocefin assay or other CLSI reference method on 

isolates before reporting as penicillin susceptible. 

• The penicillin disk zone edge test was shown to be inferior as compared to the 

induced nitrocefin assay and should not be used with S.lugdunensis. 

• Oxacillin – Intermediate 

• Table 2C / Note (13) 

• If oxacillin-I results (disk diffusion testing) are 

obtained for S.aureus, perform testing for mecA or 

PBP 2a, the cefoxitin MIC or cefoxitin disk test, an 

oxacillin MIC test, or the oxacillin-salt agar screening 

test. Report the result of the alternative test rather 

than the oxacillin-I result. 

19 

20 


• Oxacillin – Resistance 

• Table 2C / Note (12) 

• If oxacillin-R staphylococci report penicillin as resistant 

or do not report. 


• Disks per plate – clarification 

• 12 disks only on a 150mm plate 


• Do not measure zone of inhibition of hemolysis 

21 

22 

Enterococcus – Vancomycin (4/32) 

• For isolates with MICs of 8-16 mg/ml 

• Perform tests are listed in 2D-Supplemental Table 1 

• 2D-Supplemental Table 1 

• Motility 

• Pigment 

• E.gallinarum (non-pigmented, motility +) 

• E.casseliflavus (pigmented yellow, motility +) 

• Other Enterococcus (non-pigmented, non-motile) 

23 

Enterococcus - Vancomycin 

• Alternative inoculum method provided for 

vancomycin resistance screen test 

• 2D-Supplemental Table 1 

• OLD: 1-10mL of a 0.5 McFarland suspension spotted 

onto agar surface (agar = 6mg/ml vanco in BHI agar) 

• NEW (added): Alternatively, using a swab dipped in 

the suspension and the excess liquid expressed, spot 

an area 10-15mm in diameter or streak a portion of 

the plate. 

24 


4


Streptococcus pneumoniae 

• Predicting susceptibility to Fluoroquinolones 

• Isolates susceptible to levofloxacin are 

predictably susceptible to gemifloxacin and 

moxifloxacin. 

• Isolates susceptible to gemifloxacin or 

moxifloxacin can not be assumed to be 

susceptible to levofloxacin. 

Streptococcus pneumoniae & 

b-Streptococcus 

• Disks per plate – clarification 



• Do not measure zone of inhibition of hemolysis 

(for Viridans streptococci as well) 

25 

26 


• Table 2H-1 Supplemental Table 1 

(inducible clindamycin resistance) 

• Included new comment regarding CDC 

recommendations: 

• “The 2010 CDC guidelines on prevention of group B 

streptococcal disease in neonates recommend that 

colonization isolates from pregnant women with severe 

penicillin allergy (high risk for anaphylaxis) should be 

tested for inducible clindamycin resistance.” 


• Table 2H-1 

• Daptomycin 

• Disk diffusion testing is not reliable 

(previously indicated for the staphylococi) 

27 

28 

2013 ! ! 

Staphylococcus - 2013 

• All cephalosporins/many penicillins currently in the 

2012 Table 2C will be removed. 

• Deleted all β-lactam breakpoints except penicillin, 

oxacillin [cefoxitin], and ceftaroline . 

• Statements will be made to indicate that results for 

cephalosporins and other b-lactam antibiotics (that 

are appropriate for staphylococci treatment) can be 

predicted from the results of oxacillin MIC, cefoxitin 

MIC, or cefoxitin disk diffusion testing. 

29 

30 


5


Penicillins, 

b-lac inhib combos 

Cephems 

Carbapenems 

31 32 


• Rationale for deleting breakpoints for b-lactams 

(except pencilllin, oxacillin [cefoxitin], & ceftaroline) 

from the CLSI M100 tables for staphylococci: 

• Current breakpoints are most likely inaccurate 

• They were ‘Grandfathered’ into the staphylococcal tables with 

other major table over-hauls in the early 2000’s. 

• Can deduce anti-staphylococcal b-lactam results from penicillin 

and oxacillin [cefoxitin] results. 


• Oxacillin disk diffusion testing will be 

removed form the staphylococci charts. 

33 

34 

35 36 


6




Detection of oxacillin resistance: 

• In most staphylococcal isolates, oxacillin resistance is mediated by mecAencoding 

the penicillin-binding protein 2a (PBP 2a, also called PBP2‘). 

• Other mechanisms of oxacillin resistance are rare and include a novel mecA 

homologue (eg, mecC) REF which may not be detected by tests for mecA or 

PBP 2a. 

• Isolates that test positive for mecA or PBP 2a should be reported as oxacillin 

resistant. 

• Isolates for which either the oxacillin MIC, cefoxitin MIC, or cefoxitin disk 

diffusion test is in the resistant range should also be reported as oxacillin 

resistant. 

REF: Stegger M, Andersen PS, Kearns A, Pichon B, Holmes MA, Edwards G, Laurent F, Teale C, Skov R, Larsen AR. Rapid detection, 

differentiation and typing of methicillin-resistant Staphylococcus aureus harboring either mecA or the new mecA homologue mecA(LGA251). 

Clin Microbiol Infect. 2012;18(4):395-400 . 

37 

Table 1A and Table 2C: 2013 

• Adding for doxycycline and minocycline to not report on organisms 

isolated from the urinary tract. 

• Removing telithromycin due to black box warning from FDA; and 

changing Test/Report Group from ‘B’ to ‘O’ in Table 2C. 

• Adding footnote to indicate that daptomycin should not be reported for 

isolates from the lower respiratory tract. 

• Removing quinupristin/dalfopristin as it is not FDA cleared for MRSA or 

coagulase negative staphylococci. Stating that for isolates of MSSA 

there are much better drugs to use for treatment with less toxicity. 

Changing Test/Report Group from ‘C’ to ‘O’ in Table 2C. 

• Removing amikacin, kanamcin, netilimicin and tobramycin and their 

breakpoints. Only gentamicin will remain. 

• Added note to gentamicin-S isolates that it is to be used only in combination with 

38 

other active agents 

Streptococcus pneumoniae - 2013 

Streptococcus pneumoniae - 2013 

• New (revised) tetracycline disk diffusion and MIC 

interpretive criteria. 

• New doxycycline disk diffusion and MIC interpretive 

criteria. 

• Clarified that isolates of S. pneumoniae from CSF 

can also be tested against vancomycin using the 

MIC or disk method. 

PENICILLINS 

• For nonmeningitis isolates, a penicillin MIC of ≤0.06 mg/mL (or oxacillin 

zone ≥ 20 mm) can predict susceptibility to the following β-lactams: 

• penicillin (oral or parenteral), ampicillin-sulbactam, amoxicillin, amoxicillinclavulanic 

acid 

• cefaclor, cefdinir, cefditoren, cefepime, cefotaxime, cefpodoxime, cefprozil, 

ceftaroline, ceftizoxime, ceftriaxone, cefuroxime, loracarbef 

• doripenem, ertapenem, imipenem, meropenem 

• Penicillin MICs ≤ 2 μg/mL indicate susceptibility to parenteral penicillin, 

amoxicillin, amoxicillin-clavulanic acid, cefepime, cefotaxime, ceftriaxone, 

and ertapenem. 

39 

40 

b-Streptococcus - 2013 

• Clarified note for erythromycin for testing and reporting on 

isolates from pregnant women with severe penicillin allergies 

• When a Group B Streptococcus is isolated from a pregnant woman 

with severe penicillin allergy (high risk for anaphylaxis), erythromycin 

and clindamycin, (including inducible clindamycin resistance) should 

be tested, and only clindamycin should be reported. 

• Clarified that susceptibility testing of β-hemolytic 

streptococci need not be performed routinely. 

41 

Inducible clindamycin resistance - 

Streptococcus: - 2013 

• b-Streptococcus: 

• If performing susceptibility testing on these organisms, you should 

include inducible-clindamycin resistance testing. 

• S.pneumoniae: 

• The clinical significance of this mechanism of clindamycin resistance 

is not known for S.pneumoniae, but inducible clindamycin resistance 

can be detected using the D-zone test and will now be included in 

the 2013 CLSI documents. 

• If testing S.pneumoniae to clindamycin and the isolate is 

clindamycin-S, a test for inducible clindamycin resistance should be 

performed. 

42 


7


New antibiotics 

• Doripenem 

Doripenem (Doribax) 

• A broad spectrum injectable antibiotic 

• Ceftaroline 

• A b-lactam drug 

• Belongs to the carbapenem group 

(imipenem, ertapenem, meropenem) 

43 

44 

Doripenem 

Complicated Intra-Abdominal Infections 

• Indicated as a single agent for the treatment of complicated intraabdominal 

infections caused by Escherichia coli, Klebsiella pneumoniae, 

Pseudomonas aeruginosa, Bacteroides caccae, Bacteroides fragilis, 

Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides 

vulgatus, Streptococcus intermedius, Streptococcus constellatus and 

Peptostreptococcus micros. 

Complicated Urinary Tract Infections, Including Pyelonephritis 

• Indicated as a single agent for the treatment of complicated urinary tract 

infections, including pyelonephritis caused by Escherichia coli including 

cases with concurrent bacteremia, Klebsiella pneumoniae, Proteus 

mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii. 

45 

Doripenem 

• Exerts its bactericidal activity by inhibiting 

bacterial cell wall biosynthesis. 

• Inactivates multiple essential penicillinbinding 

proteins (PBPs) resulting in inhibition 

of cell wall synthesis with subsequent cell 

death. 

46 

Doripenem 

• Bacterial resistance mechanisms that affect 

doripenem include: 

• Inactivation by carbapenem-hydrolyzing enzymes 

• KPC, NDM-1, etc. 

• Mutant or acquired PBPs 

• Decreased outer membrane permeability 

• Active efflux 

• Doripenem is stable to hydrolysis by most b- 

lactamases, including penicillinases and 

cephalosporinases produced by GP &GN bacteria 

Doripenem (Gram positive’s) 

• Staphylococcus aureus (MSSA only) 

• Streptococcus agalactiae 

• Streptococcus pyogenes 

• Streptococcus Viridans group 

47 

48 


8


Doripenem (Gram positive’s) 

Ceftaroline (Teflaro) 

No disk diffusion criteria 

S (mg/ml) I (mg/ml) R (mg/ml) 

Streptococcus Viridans group (O) < 1.0 - - 

b-Streptococcus (O) < 0.12 - - 

S.pneumoniae (O) < 1.0 - - 

S.aureus* - - - 

Acute Bacterial Skin and Skin Structure Infections 

• Indicated for the treatment of acute bacterial skin and skin structure 

infections caused by susceptible isolates of Staphylococcus aureus 

(including methicillin-susceptible and -resistant isolates), Streptococcus 

pyogenes, S.agalactiae, E.coli, K.pneumoniae, and K.oxytoca. 

Community-Acquired Bacterial Pneumonia 

• Indicated for the treatment of community-acquired bacterial pneumonia 

caused by susceptible isolates of the following microorganisms: 

Streptococcus pneumoniae, Staphylococcus aureus (MSSA only), 

Haemophilus influenzae, Klebsiella pneumoniae, K.oxytoca, and E.coli. 

*Remember: Only penicillin, oxacillin (cefoxitin), ceftaroline for staph with the b-lactams in 2013 

49 

50 

Ceftaroline 

• Ceftaroline is a cephalosporin with in vitro activity against 

GP and GN bacteria. 

• Bactericidal action is mediated through binding to essential 

penicillin-binding proteins (PBPs). 

• Bactericidal against S. aureus due to its affinity for PBP2a 

and against Streptococcus pneumoniae due to its affinity for 

PBP2x. 

• Ceftaroline is not active against Gram negative bacteria 

which produce ESBLs or carbapenemases. 

Ceftaroline 

• Staphylococcus aureus (MSSA & MRSA) 

• Streptococcus pyogenes 

• Streptococcus agalactiae 


51 

52 

Ceftaroline 

NEW AST QC Guidance 

S.aureus 

(B) 


(C) 

S.pneumoniae 

(nonmeningitis) 

(C) 

S 

(mg/ml) - mm 

I 

(mg/ml) - mm 

R 

(mg/ml) - mm 

< 1 / > 24 2 / 21-23 > 4 / < 20 

< 0.5 / > 26 - - 

< 0. 5 / > 26 - - 

Table 3C. Disk Diffusion: 

Reference Guide to QC Frequency 

Conversion from Daily to Weekly QC 

• Routine QC is performed each day the test is performed 

unless an alternative quality control plan has been 

established. 

• CLSI document M02-A11 Section 15.7 describes a QC plan 

using a 20-30 day protocol that if successfully completed 

allows a user to convert from daily to weekly quality control. 

53 

54 


9


NEW AST QC: 3x5 (15) Plan 

• A new QC plan using a two-phase, 15 replicate (3 X 5 day) 

plan is described as follows: 

• 15 replicate (3 X 5 day) plan 

• Test three replicates using individual inoculum preparations of the 

appropriate QC strains for 5 consecutive test days to perform the 15 

replicate (3 x 5 day) plan. 

• Each QC strain tested is evaluated separately according to the 

acceptance criteria and recommended action described below (e.g., 

pass, test another 3 replicates for 5 days, fail). 

• Upon successful completion of the QC plan, the laboratory can convert 

from daily to weekly QC testing. 

• If unsuccessful investigate, take corrective action as appropriate and 

continue daily QC testing. 

55 

NEW AST QC 3x5 (15) 

Table 3C* 

Number out of range Conclusion from Number out of range 

with initial testing initial testing after repeat testing 

(based on 15 

(based on all 30 

replicates) 

replicates) 

QC plan successful. 

0-1 

Convert to weekly QC 

NA 

testing. 

Test another 3 replicates 

2-3 

for 5 days. 2-3 

QC plan fails. Investigate 

4 or greater 

and take corrective 

4 or greater 

action as appropriate. 

Continue QC each test 

day. 

*Assess each QC strain individually 

Conclusion after 

repeat testing 

NA 

QC plan successful. Can 

convert to weekly 

testing. 

QC plan fails. Investigate 

and take corrective 

action as appropriate. 

Continue QC each test 

day. 

56 

NEW AST QC 3x5 (15) 

Test 3 replicated of each QC strain 

for 5 days using individually 

prepared inoculum 


0-1 of 15 out 

of range 

Pass. Convert to 

weekly QC. 

2-3 of 15 out 

of range 

Test another 3 

replicates for 5 

days 

2-3 of 30 out 

of range 

Pass. Convert 

to weekly QC. 

> 4 of 15 out 

of range 

> 4 of 30 out 

of range 

Fail. 

Continue to 

include QC 

each test day. 

Take 

corrective 

action. 

57 

• Statistician’s comments: 

• 3x5 Plan 

• Similar to manufactured product releases 

• ‘Go’ or ‘No-Go’ based on mathematical considerations 

• Two-Stage sampling plan: 

• May be completed in first stage or proceed to a second 

stage 

• Two new plans were considered: 

• 0-1 error allowed in first stage of Plan 1 

• 0 errors allowed in first stage of Plan 2 

58 


• Statistician’s comments 

• Out-of-control results could be due to either 

systemic or random errors 

• Systemic errors = likely to get >2 outliers out of 15 results 

• Random (allowable) errors = very high probability of getting 

1 outlier of 15 results due to random error 

• Plan 1: 0-1 errors allowed: 

• Deemed likely to pick up systematic errors (>2/15) 

• Plan 2: 0 errors allowed: 

• Deemed likely to be problematic and unlikely to improve 

quality of results (no allowance for random errors @


QC Testing Frequency: 

Screening Tests 

NEW QC Testing Frequency: 

Screening Tests* 

QC Strain 

Positive (resistant) 

Negative (susceptible) 

Current 

Daily; convert to weekly after 

20-30 days 

Daily; convert to weekly after 

20-30 days 

QC Recommendations: 

• ‘Routine’ 

• Test negative (susceptible) QC strain: 

• With each new lot/shipment of testing materials (eg, disks, or agar plates used for 

agar dilution, or single wells or tubes used with broth dilution methods) 

• Weekly if the screening test is performed at least once a week and criteria for 

converting from daily to weekly QC testing have been met (see Section 15.7.2.1 in 

M02 or Section 16.7.2.1 in M07) 

• Daily if the screening test is performed less than once per week and/or if criteria 

for converting from daily to weekly QC testing have not been met (see bullet 

above). 

• ‘Lot/shipment’ 

• Test positive (resistant) QC strain at minimum of at least once with each 

new lot/shipment of testing materials 

61 

62 

NEW QC Testing Frequency: 

Screening Tests* 

Intrinsic Resistance Table 

QC Strain Current New 2013 

Positive (resistant) 

Negative (susceptible) 

Daily; convert to weekly 

after 20-30 days 



Each new 

batch/lot/shipment of 

testing materials 



*Applies to disks, or agar plates used for agar dilution (e.g., VRE screen plate), 

or single wells or tubes used with broth dilution methods (inducible clindamycin resistance). 

63 

Intrinsic Resistance (Appendix B): 

Split out to four appendixes as follows: 

• B.1 Enterobacteriaceae 

• Deleted 'R' for Citrobacter koseri with amoxicillin-clavulanate and ampicillin-sulbactam 

• P. mirabilis – clarified that there is no intrinsic resistance to penicillin and 

cephalosporins 

• Added imipenem with note that Proteus species, Providencia species and Morganella 

species may have elevated MICs by mechanisms other than by production of 

carbapenemases 

• Added information that Enterobacteriaceae are also intrinsically resistant to 

clindamycin, daptomycin, fusidic acid, glycopeptides (vancomycin, teicoplanin), 

linezolid, macrolides (erythromycin, clarithromycin, azithromycin), quinupristindalfopristin, 

and rifampin. 

• New Appendix B.2 Other Non-Enterobacteriaceae 

• New Appendix B.3 Staphylococci 

• New Appendix B.4 Enterococcus spp. 64 

Intrinsic Resistance Tables – 

Staphylococcus (Appendix B) 

Intrinsic Resistance Tables – 

Enterococcus (Appendix B) 

Organism / Drug Novobiocin Fosfomycin Fusidic Acid 

S.aureus/S.lugdunensis There is no intrinsic resistance in these species. 

S.epidermidis 

There is no intrinsic resistance in this species. 

S.haemolyticus 

There is no intrinsic resistance in this species. 

S.saprophyticus R R R 

S.capitis 

R 

S.cohnii 

R 

S.xylosus 

R 

Organism/drug 

Cephalo 

-sporins 

Vancomycin 

Teicoplanin 

Aminoglycosides 

Clindamycin 

Q/D 

Trimethoprim 

E.faecalis R* R* R* R R* R R 

E.faecium R* R* R* R* R R 

E.gallinarum/ 

casseliflavus 

SXT 

Fusidic 

acid 

R* R R* R* R R* R R 

*Warning: For Enterococcus spp., cephalosporins, aminoglycosides (except for 

high-level resistance screening), clindamycin, and SXT may appear 

active in vivo, but are not effective clinically and should not be reported 

as susceptible. 

Note 1: 

Gram-positive bacteria are also intrinsically resistant 

to aztreonam, polymyxin B/colistin and naladixic acid. 

65 

NOTE 1: Gram-positive bacteria are also intrinsically resistant 

to aztreonam, polymyxin B/colistin and naladixic acid. 

66 


11


Summary 

• CLSI updates AST tables (M100) each January. 

• CLSI updates documents that describe how to perform reference disk 

diffusion (M02) and reference MIC (M07) tests every 3 years. 

• Changes to CLSI documents are summarized in the front of each 

document. 

• Information listed in boldface type is new or modified since the 

previous edition of M100. 

• Recent breakpoint addition/revision dates are listed in the front of 

M100-S22. 

• Minutes of CLSI AST Subcommittee meetings and other materials 

are available at www.clsi.org. 

67 68 

Case A 

• 32 year old pregnant woman had a vaginal-rectal 

specimen sent for GBS culture. 

• The culture was positive and results were sent to 

the doctor. 

• Two days later the doctor’s office calls and requests 

suceptibility testing because the patient is very 

allergic to penicillin and the doctor needs the results 

for a non b-lactam antibiotic for this patient. 

• You subculture the isolate for susceptibility testing. 

Case A 

• When testing GBS from a prenatal screen culture, 

the most important drugs to test and report are 

• Drugs to Test (and why): 

• Drugs to Report (and why): 

69 

70 

Case B 

• You want to implement a new Staphylococcus panel 

with ceftraoline (not previously tested in any panel) 

on your AST system. What will you do 

• i) Test QC strains on new panel concurrently with patient 

isolates for 20-30 days and then go to weekly testing 

• ii) Test QC strains on new panel before testing patient isolates 

in the 3x5 replicate plan and then go to weekly testing 

• iii) Test 10 clinical isolates on new panel and compare 

ceftaroline results to a reference methods before testing 

patients isolates 

• iv) Something else 

71 

Case C 

• SPECIMEN: Joint Fluid 

• DIAGNOSIS: Septic Arthritis 

• ORGANISM: Staphylococcus aureus 

MIC (mg/ml) 

clindamycin < 0.5 “S” 

erythromycin < 0.5 “S” 

oxacillin < 0.5 “S” 

penicillin 

“R” 

vancomycin < 0.5 “S” 72 


12


Case C 

• SPECIMEN: Joint Fluid 

• DIAGNOSIS: Septic Arthritis 


Case C 

• Physician calls and asks that ceftriaxone (not on 

your panel) be tested. What do you do 

clindamycin 

erythromycin 

oxacillin 

penicillin 

MIC (mg/ml) 

< 0.5 “S” 

< 0.5 “S” 

< 0.5 “S” 

“R” 

Physician calls with 

an additional 

request… 

vancomycin < 0.5 “S” 73 

74 

Case D 

• SPECIMEN: Blood culture 

• DIAGNOSIS: Endocarditis 


Case D 

• SPECIMEN: Blood culture 

• DIAGNOSIS: Endocarditis 


MIC (mg/ml) 

clindamycin 8 “R” 

erythromycin 16 “R” 


vancomycin < 0.5 “S” 

MIC (mg/ml) 

clindamycin 8 “R” 

erythromycin 16 “R” 


vancomycin < 0.5 “S” 

Physician calls with 

an additional 

request… 

75 

76 

Case D 

• The physician would like to treat this patient with 

penicillin as it will be a long and protracted course 

of therapy for this patient. 

• They notice that penicillin is not resulted on the 

patient’s report. 

• What do you tell the physician about the penicillin 

result on this patient’s isolate 

• What further steps do you take regarding this 

request 

Case E 

25 y/o woman with acute cystitis. 

• UR culture grows >100,000 Staphylococcus 

species 

• The physician wants additional identification 

and AST done. 

• What laboratory tests do you do next 

• What do you tell the physician 

77 

78 


13


Case F 

• Young boy 3 y/o present with pneumonia. 

• The suctioned sputum grows out the 

pathogen: Streptococcus penumoniae. 

• You do AST and report out…… … … 

• Doc wants to use clindamycin for this patient. 

• What antibiotics do you test (and how do you 

test) and how do you report the susceptibility 

results 

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Case G 

• Patient develops pain and swelling in the 

abdomen. 

• Ascetic fluid is collected and sent for culture. 

• The specimen shows many 

polymorphonuclear cells on initial GS along 

with moderate GPC in chains. 

• The culture grows a pure culture of 3+ 

Streptococcus anginosus group. 

80 

Case G 

• You report out your normal AST of the following for this 

organism: 

• pencillin (R), ceftriaxone (S), vancomycin (S), 

clindamycin (S) 

• Is there anything suspect about the above susceptibility 

results 

• The physician calls and asks for doripenem to be tested. 

You do have doripenem disks and it is on your 

streptococci microtiter panels. What do you do 

Case H 

• 78 year old man with signs of pneumonia is 

admitted through the Emergency 

Department. 

• Sputum is collected and grows many 

Streptococcus pneumoniae with a few oral 

flora (GS was significant for many PMNS and 

GPC in short chains). 

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82 

Case H 

• The doctor calls and ask for a 

‘fluoroquinolone to be tested’ other than 

levofloxacin (which is in your current pneumo 

panel). 

• Here is your antibiotic panel results: 

• Penicillin (nonmeningitis) – S 

• Penicillin (oral) – S 

• Erythromycin – R 

• SXT – S 

• Levofloxacin - S 

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Case H 

• What antibiotics do you test 

• What do you report to the physician 

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Today’s Review: 2012-2013 changes 

• Staphylococcus species 


• b-Streptococcus species 

• Enterococcus species 

CLSI Review 

• Changes to CLSI documents are summarized in the 

front of each document. 

• Information listed in boldface type is new or modified 

since the previous edition of M100 document. 

• Recent breakpoint addition/revision dates are listed in 

the front of M100-S22. 

• Go to CLSI website for up-to-date information. 

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86 

CLSI 

Watch for the 2013 

M100 document! 

Thank you for 

attending ! 

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Update on the 2012-2013 CLSI Standards for Antimicrobial - SWACM

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