CLSI AST Update Gram Positive Bacteria - 1

Susan E. Sharp, Ph.D.
, DABMM, FAAM
Director - Regional and Sunnyside Medical Center Laboratories
Director - Regional Clinical Microbiology
Kaiser Permanente

Associate Professor - Department of Pathology
Oregon Health & Sciences University

Portland, OR

Update on the CLSI Standards for
Antimicrobial Susceptibility Testing:

Whats New with the
Gram Positive Organisms?

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OBJECTIVES
Review significant changes made in 2012.

Review this years 2013 CLSI M100-S23 document.

Describe any specific susceptibility testing
methodologies.

Present new antimicrobials.

Discuss new developments for the January 2014 CLSI
M100-S24 guideline.

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3

CLSI AST Standards
January 2013
M100-S23 Tables (2013)*
M02-A11 Disk Diffusion Method (2012)^
M07-A9 MIC Method (2012)^

* M100 updated every year
^ M02, M07 updated every 3 years
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.

Recent breakpoint addition/revision dates are listed in the front of
M100-S23.

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Todays Review:
2012 Significant changes from last year
2013 Changes/New items in these guidelines
2014 Whats on the horizon
5
Significant changes in 2012

6
Staphylococcus species -2012
Penicillin testing
7
Staphylococcus spp. Penicillin - 2012
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

Some Staphylococcus spp. that test S to penicillin by
MIC or disk diffusion may actually possess a -lactamase
(BL) that may cause the patient to fail penicillin therapy

8
Staphylococcus spp. Penicillin - 2012
CLSI Previous recommendation (2011):
Perform induced nitrocefin BL test before reporting
penicillin as S if:
zone diameter 29 mm
MIC 0.12 g/ml

PCR for the blaZ BL gene may be considered

Penicillin Breakpoints
MIC (g/ml) Zone (mm)
S I R S I R
0.12 - 0.25 29 - 28
9
Reference: M100-S21 (2011) - Table 2C. Page 70
Induced -lactamase (BL) Test - 2012
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-Subculture isolate to blood agar

-Drop disk to induce BL production
(e.g., oxacillin or cefoxitin)

-Incubate overnight

-Test cells from periphery of zone

-If BL positive, report penicillin R
Pos Neg

11


-Incubate overnight


Pos Neg

12


-Incubate overnight


Pos Neg

Staphylococcus aureus (BL) - 2012
Induced nitrocefin BL test usually, but not always, detects
staphylococcal BLs

Other BL tests are more sensitive for the detection of 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
all types may not be detected by a single PCR assay

13
Staphylococcus aureus
-lactamase (BL) Study* - 2012
348 MSSA (<4 g/ml PEN MICs) characterized for blaZ by PCR:
303 PCR negative
45 PCR positive (~13%)

Methods:
Phenotypic BL tests
Nitrocefin - Cefinase
Nitrocefin - Dryslide
Cloverleaf assay
Penicillin disk zone edge

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*Statens Serum Institut (Denmark), CDC (Atlanta), MGH (Boston)
Staphylococcus
aureus
BL Study - 2012

Pen MIC
(g/ml)
blaZ functional
Neg Pos
0.008 2
0.016 15
0.032 180 1
0.06 90 5
0.12 15 17
0.25 1 14
0.5 4
1.0
2.0 2
4.0 1
303 45
1 blaZ neg and penicillin R
23 blaZ pos and penicillin S
Reference: CLSI Agenda Book January 2011.
S

R
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Cloverleaf Assay
for BL in S. aureus
5% sheep blood agar

S. aureus ATCC 25923
as the indicator organism

1 unit penicillin disk

Negative (pen-S) strain QC

Some difficulties reading

Reference: CLSI Agenda Book January 2011.
Isolates A-D are all
BL positive
A
B
C
D
BL negative
17
-
lactamase
positive
-
lactamase
negative
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Staphylococcus aureus - 2012
Disk Zone Edge Test (10 U penicillin disk and standard disk diffusion method)
Fuzzy / beach =
-lactamase negative,
Penicillin - S
Sharp / cliff =
-lactamase positive,
Penicillin - R
S. aureus
supplemental QC:

Neg - ATCC 25923

Pos - ATCC 29213

Reference: M100-S22. Table 2C Supplemental Table 1. Page 83
3 Lab BL Study Results (N=348), 2012
Test Sensitivity Specificity
Cefinase 77% 100%
Dryslide 88% 100%
Cloverleaf 100% 100%
Penicillin disk zone edge 96% 100%
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Reference: CLSI Agenda Book January, 2011
20
Penicillin MIC 0.12 g/ml
Nitrocefin -lactamase
positive
Report penicillin R
Nitrocefin -
lactamase negative
Perform penicillin disk zone-edge test
29 mm fuzzy
Report penicillin S
29 mm sharp
Report penicillin R
Penicillin (MIC 0.12 g/ml) Reporting, 2012
Note: If doing disk
diffusion routinely,
just examine zone
edge for those with
zone sizes of >
29mm.
M100-S22. Table 2C Supplemental Table 1. Page 80
A A B
2012 NEW RECOMMENDATION:
Added penicillin disk zone edge test for BL
production in S. aureus

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Staphylococcus spp.
Penicillin 2012
Staphylococcus spp.
Penicillin Optional Strategy - 2012
Report penicillin if R

Suppress penicillin if S and consider a note:
Contact laboratory if penicillin results needed.

If penicillin S and penicillin results needed, perform:
Nitrocefin BL test , and if negative
Penicillin zone edge test
Beach = PEN S
Cliff = PEN R

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S. aureus
Isolates where penicillin zones are 29 mm or penicillin MICs are
0.12 g/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 g/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.

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Action Items - 2012
Staphylococcus Oxacillin 2012
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.
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Staphylococcus Oxacillin 2012
Resistance
Table 2C / Note (12)
If oxacillin-R staphylococci report penicillin as
resistant or do not report.
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Staphylococcus - 2012
Disks per plate clarification
12 disks only on a 150mm plate

Do not measure zone of inhibition of hemolysis
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Enterococcus Vancomycin - 2012
For isolates with MICs of 8-16 g/ml (I)
Perform tests 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)
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Enterococcus Vancomycin 2012
Alternative inoculum method provided for
vancomycin resistance screen test
2D-Supplemental Table 1
OLD: 1-10 L of a 0.5 McFarland suspension spotted onto
agar surface (agar = 6 g/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.

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Streptococcus pneumoniae - 2012
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.

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Streptococcus pneumoniae &
-Streptococcus 2012
Disks per plate clarification

Do not measure zone of inhibition of hemolysis
(for Viridans streptococci as well)
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-Streptococcus - 2012
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.

Use erythromycin & clindamycin for D zone testing, but do not
report erythromcyin.

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** If an isolate is resistant to erythromycin, it might have inducible resistance to clindamycin, even if
it appears susceptible to clindamycin. If a GBS isolate is susceptible to clindamycin, resistant to
erythromycin, and testing for inducible clindamycin resistance is negative, then clindamycin can be
used for GBS intrapartum prophylaxis instead of vancomycin.
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2010 CDC Guidelines
Check for inducible
clindamycin R if:

erythromycin-R
clindamycin-S

But only report
clindamycin!
www.cdc.gov

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ASMs Clinical Microbiology Portal : http://clinmicro.asm.org/
36 | 2011 Kaiser Foundation Health Plan, Inc. For internal use only. January 8, 2014



GPB Protocols jointly developed by ASM (CLP) and CDC.

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protocols


Prenatal Screening for Group B Streptococcus: Non-pigmented broth, subculture only
-Streptococcus - 2012
Table 2H-1
Daptomycin
Disk diffusion testing is not reliable
(previously indicated for the staphylococci)

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New antibiotics - 2012
Doripenem

Ceftaroline

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Doripenem (Doribax)
A broad spectrum injectable antibiotic

A -lactam drug

Belongs to the carbapenem group (imipenem,
ertapenem, meropenem)

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Doripenem
Complicated Intra-Abdominal Infections
Indicated as a single agent for the treatment of complicated intra-abdominal
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.

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Doripenem
Exerts its bactericidal activity by inhibiting bacterial cell wall
biosynthesis.

Inactivates multiple essential penicillin-binding proteins
(PBPs) resulting in inhibition of cell wall synthesis with
subsequent cell death.
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Doripenem
Bacterial resistance mechanisms that affect doripenem
include:
Inactivation by carbapenem-hydrolyzing enzymes
KPC, NDM-1, etc.
Mutant or altered PBPs
Decreased outer membrane permeability
Active efflux
Doripenem is stable to hydrolysis by most BL, including
penicillinases and cephalosporinases produced by GP &GN
bacteria
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Doripenem (Gram positives)

Staphylococcus aureus (MSSA only)
Streptococcus agalactiae
Streptococcus pyogenes
Streptococcus Viridans group
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50
S ( g/ml) I ( g/ml) R ( g/ml)
Streptococcus Viridans group (O) < 1.0

- -
-Streptococcus (O) < 0.12 - -
S.pneumoniae (O)

< 1.0 - -
S.aureus*

- - -
Doripenem (Gram positives)
*Remember -
Only penicillin, oxacillin (cefoxitin), ceftaroline for staph with the -lactam drugs in 2013.
(No disk diffusion criteria)
Ceftaroline (Teflaro)
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.

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Ceftaroline
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.

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Ceftaroline
Staphylococcus aureus (MSSA & MRSA)
Streptococcus pyogenes
Streptococcus agalactiae
Streptococcus pneumoniae

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54
Ceftaroline vs. Other -lactams agents
% Susceptible
Organism N Ceftaroline Ceftriaxone Imipenem
MSSA
186 100 100 100
MRSA
215 100 0 0
S.pneumoniae
894 100 90.8 NA
NA - not available

REF: Jones et al. 2011. J Antimicrob Chemother. 66 (Suppl 3):iii69iii80.
Ceftaroline
S
( g/ml) / mm
I
( g/ml) / mm
R
( g/ml) -mm
S.aureus
(B)
< 1 / > 24 2 / 21-23 > 4 / < 20
-Streptococcus
(C)
< 0.5 / > 26 - -
S.pneumoniae
(nonmeningitis)
(C)
< 0. 5 / > 26 - -
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NEW FOR 2013 !
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All cephalosporins/many penicillins in the 2012 Table 2C
were removed.
Deleted all -lactam breakpoints except penicillin, oxacillin
[cefoxitin], and ceftaroline.

A statement is provided to indicate that results for
cephalosporins and other -lactam antibiotics can be predicted
from the results of penicillin, oxacillin MIC, cefoxitin MIC, or
cefoxitin disk diffusion testing.
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Staphylococcus spp.

M100-S22. Table 2C.
Staphylococcus spp.

2013 - Eliminated breakpoints for:
Penicillins
BL/BL inhibitor combos
Cephalosporins
Carbapenems
M100-S22. Table 2C.
Rationale for deleting breakpoints for -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 2000s.
Can deduce all anti-staphylococcal -lactam results from penicillin
and oxacillin [cefoxitin] results.
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Oxacillin disk diffusion testing has been removed form the
staphylococci charts.

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Oxacillin disk testing is not reliable. For disk
testing see cefoxitin for reporting oxacillin
Staphylococcus spp. - 2013
-Lactam Breakpoints Remaining
Penicillin
Represents penicillinase-labile penicillins
Oxacillin (MIC only; no more DD)
Represents penicillinase-stable penicillins
Cefoxitin
Surrogate for oxacillin
Ceftaroline (added 2013)
Cephem with anti-MRSA activity
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Staphylococcus spp. - 2013
Penicillins and Penicillinases
Penicillinase-labile Penicillins
(penicillin represents)
Penicillinase-stable Penicillins
(oxacillin represents)
amoxicillin
ampicillin
carbenicillin
mezlocillin
penicillin
piperacillin
ticarcillin
cloxacillin
dicloxacillin
flucloxacillin
methicillin
nafcillin
oxacillin

Staphylococcus - -Lactams - 2013
Use Pen and Oxa (FOX) results to predict results for other -lactams
Test Results
Predicts
Penicillin
Oxacillin
(cefoxitin)
S S
Susceptible to:
All penicillins
-lac / -lactamase inhibitor combos
Cephems
Carbapenems
R S
Resistant to:
Penicillinase-labile penicillins
Susceptible to:
Penicillinase-stable penicillins
-lac / -lactamase inhibitor combos
Cephems
Carbapenems
R R
Resistant to:
All -lactams (except cephems with anti-
MRSA activity, e.g., ceftaroline)
Detection of oxacillin resistance:
In most staphylococcal isolates, oxacillin resistance is mediated by mecA-
encoding 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.

If both cefoxitin and oxacillin are tested against S.aureus or S.lugdunensis, and
either result is resistant, the organism should be reported as oxacillin resistant.
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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 .
Table 1A (Test and Report) and Table 2C (Interpretive Standards): 2013
For doxycycline and minocycline: added not to report on organisms isolated from
the urinary tract.
Removed telithromycin from Table 1A due to black box warning from FDA; and
changing Test/Report Group from B to O in Table 2C.
Added footnote to indicate that daptomycin should not be reported for isolates from
the lower respiratory tract.
Removed quinupristin/dalfopristin from Table 1A as it is not FDA cleared for MRSA
or CoNS; 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.
Removed amikacin, kanamcin, netilimicin and tobramycin from Table 1A. Only
gentamicin will remain as C in Table 2C (others changed to O).
Added note to aminoglycoside-S isolates stating that they are to be used only in combination with
other active agents.

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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 either a MIC or disk
method.

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Inducible clindamycin resistance -
S.pneumoniae: 2013

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 is now be included in the 2013 CLSI documents.

The 2013 document includes: IF testing for clindamycin
resistance in S.pneumoniae is performed, it should include
screening for inducible clindamycin resistance.

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NEW AST QC Guidance - 2013
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.
< 1/20 or < 3/30 weekly QC testing
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NEW AST QC: 3x5 (15) Plan - 2013
An alternate QC plan using a two-phase, 15 replicate (3 X 5
day) plan is described in Table 3C as follows:
15 replicate (3 X 5 day) plan
Test three replicates using individual inocula preparations of the
appropriate QC strains for 5 consecutive test days to perform 15 replicates
(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.

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NEW AST QC 3x5 (15) - 2013
Table 3C*

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Number out of range
with initial testing
(based on 15 replicates)

Conclusion from initial
testing

Number out of range
after repeat testing
(based on all 30
replicates)

Conclusion after repeat
testing

0-1

QC plan successful.
Convert to weekly QC
testing.

NA

NA

2-3
Test another 3
replicates for 5 days.

2-3
QC plan successful.
Convert to weekly QC
testing.

4 or greater
QC plan fails.
Investigate and take
corrective action as
appropriate. Continue
QC each test day.

4 or greater
QC plan fails. Investigate
and take corrective action
as appropriate. Continue
QC each test day.
*Assess each QC strain individually
NEW AST QC 3x5 (15) - 2013
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Test 3 replicated of each QC
strain for 5 days using individually
prepared inoculum
Fail.
Continue to
include QC
each test
day. Take
corrective
action.
Test another 3
replicates for 5
days
Pass. Convert
to weekly QC.
Pass. Convert
to weekly
QC.
0-1 of 15 out
of range?
2-3 of 15 out
of range?
> 4 of 15 out
of range?
2-3 of 30 out
of range?
> 4 of 30 out
of range?
Statisticians 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
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Statisticians 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 @ <1/15)
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Conversion from Daily to Weekly QC - 2013
In addition, this testing is required for the
following modifications of existing antimicrobial
susceptibility test system:
Addition of new antimicrobial agent to existing
system
ceftraoline, doripenem

Can use 20-30 day consecutive days of QC testing or
the 3x5 QC plan

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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.
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NEW QC Testing Frequency: Screening Tests
(Table 2D-Supplemental Table 1) 2013 (ex:HLAR)
NEW QC Testing Frequency: Screening Tests - 2013

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QC Strain Previous New 2013
Positive (resistant) Daily; convert to weekly
after 20-30 days/3x5 plan
Each new
batch/lot/shipment of
testing materials*
Negative (susceptible) Daily; convert to weekly
Daily; convert to weekly
*Applies to disks, agar plates used for agar dilution, or single wells or tubes
used with broth dilution methods.
Intrinsic Resistance Table 2013
Intrinsic Resistance (Appendix B):
Split out to four appendixes as follows:
B.1 Enterobacteriaceae
Added imipenem with note that Proteus species, Providencia species and
Morganella species may have elevated MICs by mechanisms other than by
production of carbapenemases. Isolates that test S should be reported as
S.
Added in a Note 2 information that Enterobacteriaceae are also intrinsically
resistant to clindamycin, daptomycin, fusidic acid, glycopeptides (vancomycin,
teicoplanin), linezolid, macrolides (erythromycin, clarithromycin, azithromycin),
quinupristin-dalfopristin, and rifampin.
New Appendix B.2 Non-Enterobacteriaceae
New Appendix B.3 Staphylococci
New Appendix B.4 Enterococcus spp.
80
Intrinsic Resistance Tables 2013
Staphylococcus (Appendix B) 2013
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
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Note 1: Gram-positive bacteria are also intrinsically resistant to aztreonam, polymyxin B/colistin and
naladixic acid.
Note 2: Oxa-R SA and CoNS are considered R to other BL agents, ie, pen, BL/BL inhibitors, cephems
(exc. ceftraoline), and carbapenems.
Organism/drug
Cephalo-
sporins
Vanco-
mycin
Teico-
planin
Amino-
glycosides
Clinda-
mycin
Q/D
Trimetho-
prim
SXT
Fusidic
acid
E.faecalis
R* R* R* R R* R R
E.faecium
R* R* R* R* R R
E.gallinarum/c
asseliflavus
R* R R* R* R R* R R
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Intrinsic Resistance Tables 2013
Enterococcus (Appendix B) 2013
*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.
NEW FOR 2014 !
I think
83
2014
Not a lot in the way of new Gram Positive
guidelines - but well review the following:
Vancomycin DD with staphylococci
New drugs added
New QC recommendations
(youre gonna like these!)
S-DD

84
Vancomycin / Staphylococci - 2014
Table 2C:
Vancomycin disk diffusion
Previously you could use it for detecting R
VRSA due to the vanA gene will show no zone
of inhibition around a 30- g vancomycin disk
Any isolate with > 7mm zone must be tested
with a MIC method before reporting as S
85
86
- Staphylococcus species
No DD criteria !
87
88
NEW DRUGS - 2014
89
Ceftazidime/avibactam - 2014
Avibactam is a synthetic -lactamase (BL) inhibitor
Addition of avibactam greatly improves the activity of ceftazidime (4-1024x MIC)
against most of the Enterobacteriaceae.

Avibactam also improves the activity of ceftazidime to P.aeruginosa (~4x MIC).

Avibactam does not improve the activity of ceftazidime against Acinetobacter spp.
or to most anaerobic bacteria (exceptions: B.fragilis, C.perfringens, Prevotella and
Porphyromonas spp.).

Data suggest that ceftazidime-avibactam is rapidly active against BL-producing
GNRs that are not inhibited by ceftazidime alone.

Ceftazidime-avibactam is as effective as carbapenems in complicated intra-
abdominal infection and complicated urinary tract infection.

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Ceftazidime/avibactam - 2014

Avibactam serves to broaden the spectrum of ceftazidime against
BL-producing GNRs.

Potential future roles include the treatment of infections caused by
GNR producing ESBLs, KPCs, and/or AmpC BL.

It may be used in combination (with metronidazole) for suspected
polymicrobial infections.
91
NEW DRUGS - 2014
Ceftazidime/avibactam
Ceftaroline/avibactam
Aztreonam/avibactam
Biapenem (a new carbapemen drug)
Ceftolozane/tazobactam
Developing for the treatment of certain serious GNR infections, including those
caused by MDR-P.aeruginosa.
92
Quality Control - 2014
93
Quality Control - 2014
Continuing to look at ways to decrease routine
QC when adding a new drug:
< 1/20
< 3/30
3x15
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Decreasing daily QC testing - 2014
CLSI QC DD Working Group Project
Background CLSI guidance recommends that prior to initiating testing with a
new antibiotic, 20-30 day QC studies must be performed (and now we have
the 3x5 testing as well).
The intent of this current project is to evaluate the necessity of performing a
full 20 or 30 days of testing.
The goal is to assimilate a large amount of 20-30 day QC study data from
different institutions with a variety of drug/bug combinations.
Analyze the data to determine when, during the course of the 20-30 day study,
QC failures occur.
Hypothesize that the systemic problems with QC will reveal themselves early
and that extended testing (ie., 20-30 days) is not necessary.
95

Preliminary Data* 63 total preliminary QC studies have been
accumulated thus far from 2 institutions (with the majority of the data
coming from a single institution).
5 of these studies were conducted for 30 days, 25 for 21 days, and 33 for
19 days.
A total of 6 failures over 1,302 data points have been identified.
day 1 (n=3), day 2 (n=1), day 14 (n=1) and day 22 (n=1).

(*From Chris D. Doern, PhD, D(ABMM) on behalf of ASMs Committee on Laboratory Practices)

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97
0
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
N
u
m
b
e
r

o
f

Q
C

F
a
i
l
u
r
e
s

Day of Testing
QC Failures by Day of Testing
More data is required - but these findings suggest
that it may be possible to reduce the number of
days required for QC studies.
98
Currently = The M2/M7 text and flowchart clearly indicates that you must
perform 5 consecutive days of repeat QC if you can not attribute the out-of-
range result to a specific issue.
If you can contribute it to a particular issue (e.g., contamination, used the wrong
QC strain, etc.), you only have to do one repeat and if in, you may continue
weekly QC testing.

However, the above does not address the 0-5% of out-of-range results which
would be expected due to random error (as ranges are established based on >
95% agreement).
One suggestion is to use retrospective data from the same lot of materials to
satisfy the 5 replicate repeat. This would not be a "statistical" change, only a
change in approach.
Also considering to allow for multiple replicates in one day (up to 3) to determine more
quickly if there is a problem (as laboratories may still be testing patient specimens
while troubleshooting).
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Out-of-Range QC 2014
Add a new section to 3C and 4F (QC frequency) -
Troubleshooting out-of-range results:
If the cause can be identified
Correct the issue, document the reason, and retest the strain on the day
the error is observed (or as soon as possible if need to subculture a new
QC strain first).
If the repeated result is within range, no further corrective action is
required.
If the cause can not be identified -
If the 4 previous QC results have been acceptable using the same lots of
materials (e.g., agar plates, disks, MIC trays/plates/panels), these 4
results, along with the current repeat, may be used to retrospectively
satisfy the requirement for 5 repeats.

100

If the cause can not be identified. Example :
If the 4 previous QC results were acceptable and you now obtain
an out-of-range result, repeat the QC as soon as possible and if
this repeat is within range, the corrective action was successful and
you can resume weekly testing.
If the repeat is out-of-range you must take additional corrective action.
Daily QC must be continued until the problem is resolved.
If 4 previous QC results were not acceptable or not available (e.g.,
new lot recently put into use), test sufficient QC replicates to satisfy
the requirement for a total of 5 results.
Up to 3 QC replicates can be tested / day if individual inocula are used.
101
Week Day Lot # Results Action
1 1 1234 4
2 1 1234 8
3 1 1234 8
4 1 1234 4
5 1 1234 16 Out of range. Repeat QC testing same day.
5 2 1234 8 In range. 5/6 acceptable in range QC tests for
E.coli ATCC 25922 and ampicillin with lot 1234.
Resume weekly QC testing.
102
Using retrospective QC to correct a random error if no error is readily apparent:
If you have an out of range QC result upon weekly QC testing, you will be able to
repeat the QC once if in range and if the last 4 times you previously tested were also
in range you can go back to weekly QC testing. You must have used the same lot of
all materials for all test results (example below).
Example 1 -
E.coli ATCC 25922
ampicillin: range 2-8 ug/ml
1 1 4567 4
2 1 4567 8
3 1 4567 16 Out of range. Repeat QC testing same day.
3 2 4567 4 In range. 3 acceptable in range QC tests for E.coli
ATCC 25922 and ampicillin with lot 4567. Repeat QC for
2 more consecutive days.
3 3 4567 8 In range.
3 4 4567 8 In range. 5/6 acceptable in range QC tests for E.coli
ATCC 25922 and ampicillin with lot 4567. Resume
weekly QC testing.
103
You may also repeat the QC testing prospectively to do as many as you need to
get the 1/6 to be in range using same lots # of all testing materials (e.g.; 2
retrospective and 3 prospective). If all within range, you and can go back to weekly
QC. You must use the same lot of all materials (example below).
Example 2 -
E.coli ATCC 25922
1 1 7891 4
2 1 7891 8
3 1 7891 16 Out of range. Repeat QC testing x3 same day
from different starting dilutions.
3 2x1 7891 4 3 replicates from different starting dilutions
are in range. 5/6 acceptable in range QC tests
for E.coli ATCC 25922 and ampicillin with lot
7891. Resume weekly QC testing.
3 2x2 7891 8
3 2x3 7891 8
104
You may also do up to 3 replicates per day with same lots of
materials using different starting dilutions (example below).
Example 3 -
E.coli ATCC 25922
Using retrospective data to go to weekly testing:

If you have been performing QC testing of a drug each time you test
patient organisms, you will be able to use those contemporary
(within the last year) retrospective results in order to collect data that
can be used for going to weekly QC testing.

For example, if you have QC tested a drug approximately 2x/month
for the last year, you can use the most recent 20 QC tests to
document your ability to go to weekly QC testing for the drug.

105
Streamlined QC 2014
Streamlined QC
2014
106
Date
tested
LOT #
disk/MHA
Results OK
(Y/N)
11/13/12 123/258 Y
12/12/12 123/456 Y
12/22/12 123/456 Y
1/1/13 123/003 Y
1/15/13 456/003 Y
1/29/13 456/003 Y
2/5/13 456/225 Y
2.26.13 456/225 Y
3/3/13 456/258 Y
3/26/13 456/258 Y
4/16/13 456/258 Y
4/20/13 788/459 Y
5/1/13 788/459 Y
5/17/13 788/560 Y
6/6/13 788/560 Y
6/18/13 788/777 Y
7/3/13 788/777 Y
7/30/13 788/889 Y
8/16/13 788/889 Y
8/25/13 799/889 Y
20 days consecutive QC testing days
= all acceptable document and
begin weekly QC
Routine QC recommendations/organism:

Those that appear for each organism group will
be streamlined.

These new guidelines can be used for both
weekly QC as well as individual drug QC.

107
Streamlined QC 2014
P.aeruginosa : Table 2B-1
108
CURRENT:
Routine QC Recommendations (See Tables 3A and 4A for acceptable QC ranges.)

Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853
Escherichia coli ATCC 35218 (for -lactam/ -lactamase inhibitor combinations)

NEW for 2014:

Acinetobacter : Table 2B-2
109
CURRENT:

Escherichia coli ATCC 35218 (for -lactam/ lactamase inhibitor combinations)
NEW for 2014:

Escherichia coli ATCC 25922 (for tetracyclines and SXT)
Escherichia coli ATCC 35218 (for -lactam/ lactamase inhibitor combinations)
Burkholderia : Table 2B-3
Stenotrophomonas : Table 2B-4
110
CURRENT:


NEW for 2014:

Escherichia coli ATCC 25922 (for chloramphenicol, minocycline, and SXT)
Other non-Enterobacteriaceae : Table 2B-5
111
CURRENT:


NEW for 2014:

Escherichia coli ATCC 25922 (for chloramphenicol, tetrayclines, sulfonamide and SXT)
Enterobacteriaceae : Table 2A
112
CURRENT:


NEW for 2014:

Pseudomonas aeruginosa ATCC 27853 (carbapenems)

S-DD
Enterobacteriaceae and cefepime

ISSUE: When the cephalosporin breakpoints to the Enterobacteriaceae were change
(lowered) in 2010, cefepime was also reviewed but the committee at that time
left the breakpoint unchanged at < 8 / 16 / > 32 ug/ml.

RESULT: The committee after MUCH discussion voted to change cefepime
breakpoints for the Enterobacteriaceae from current to the below:

S < 2 ug/ml
SDD 4-8 ug/ml (SDD = susceptible, dose-dependent*)
R > 16 ug/ml

S-DD
Enterobacteriaceae and cefepime

This new breakpoints will be published in the 2014 guidelines (I think). The
committee will also attempt to get disk diffusion changes done before end of
year so these new zone sizes can also be published in 2014 as well.

*A SDD indicates that approved, higher therapeutic levels of this drug can
be used to treat serious infections with organisms having an MIC in this
range.
A definition of SDD will be included in the 2014 M100 document; it will read
something like: SDD indicates that this drug may be used in high doses to
achieve the maximum achievable drug doses.
They will also add drug dosages for SDD. For example:
The SDD breakpoint was derived based on a dosage regimens of 1 g Q 8
hr or 2 g Q 12 hr for organisms with a MIC of 4 ug/ml, and 2 g Q 8 hr for
those with a MIC of 8 ug/ml.

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.
Minutes of CLSI AST Subcommittee meetings and other
materials are available at www.clsi.org.

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CLSI
Watch for the January 2014
M100-S24 document!
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CLSI AST Update Gram Positive Bacteria - 1

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