Review

and The very latest information of

Antimicrobial Susceptibility Testing
from CLSI
Piriyaporn Chongtrakool

[Courtesy Prof. Janet Hindler and Prof. Susan Sharp]


Conventional, Manual, Phenotype
Agar/Broth Dilution (can be automated)
Disk Diffusion
Agar Gradient Diffusion
Detection of Resistance Mechanisms
Phenotype
Direct Detection
Induction
Genotype
Inferred Resistance by Organism species

Disk Diffusion
(Kirby Bauer)
Standard Inoculum size (0.5McFarland = 1.5x108 CFU/ml)
Growth method
Direct Colony Suspension

Inoculate on Mueller Hinton Agar (4 mm depth, pH 7.2-7.4), fastidious

organism : MHA+sheep blood, HTM, GC+supplement
Apply antimicrobial agents disk (paper disks-dia. 6 mm)
Incubate at 35( +2 oC) 16-18 (24) hrs.
Measure Inhibition Zone diameter (mm)
Interprete to
Susceptible (S) , Intermediate susceptible (I)
Resistant (R) ,
Non-susceptible (N)

Pathogens standardized for disk diffusion testing

Enterobacteriaceae
Pseudomonas aeruginosa
Acinetobacter
B. cepacia, S. maltophilia
Staphylococcus
Enterococcus
Streptococcus pneumoniae
Other Streptococcus : , viridans
Haemophilus influenzae, parainfluenzae
Neisseria gonorrhoeae, N. meningitides
Vibrio cholerae
Aeromonas hydrophila complex, P. shigelloides
Moraxella catarrhalis (Aug, SXT, E, T)
Pasteurella multocida

23 mm

6 mm

Zone diameter Interpretive standards for

Enterobacteriaceae

Agent

Disk content

Zone diameter (mm)

ug

AM

10

< 13

14-16

> 17

AK

30

< 14

15-16

> 17

30

< 12

13-17

> 18

SXT

1.25/23.75

< 10

11-15

> 16

Zone diameter Interpretive standards for

Streptococcus pneumoniae
Agent

Disk

Content

Vancomycin

30ug

Zone diameter (mm)

> 17


Susceptible (S)
n n o
dose

Resistant (R)
nn n 

o 
odose

Intermediate (I)

n o
odose
n o
o 
()

Non-Susceptible (N)

S/I/R o
n 
(o r
)

 Disk Diffusion

S I R N
Serum level = interpretive criteria
Unreliable to test in some organisms against
some agents

Unreliable to test by Disk Diffusion=use MIC

(Examples)
1. Staphylococcus : Vancomycin, Daptomycin
2. Streptococcus viridans group : Penicillin
3. Strep. pneumoniae (sterile sites) : P, CTX, CRO, Mem
4. Acinetobacter : Colistin, Netilmicin
5. B. cepacia : Ticar/clav, LvFX, CA
(#CAZ, MEM, Mino, SXT)
6.S. maltophilia : Ticar/clav, CAZ, CA)
(#Mino, LvFX, SXT)
7.Non-enterobacteriacea : most agents

Unreliable to use direct agents

1.Strep. pneumoniae (non-sterile sites) : P (OX 1ug)
2.Staph. : MT, OX (FOX 30ug)

Unreliable to differentiate S/I/R

1.Strep. pneumoniae (sterile sites) : P <20 mm
2.Strep. viridans group : P

Unreliable to detect low level / inducible

resistances
1. Enterobacteriaceae : ESBLs
2. Enterobacteriaceae : KPC
3.Staphylococcus, Beta-Streptococcus,
S.pneumoniae : Inducible Clindamycin
resistance

Staphylococcus aureus

Penicillin ?Susceptible Staphylococcus aureus

Classification of MRSA
Resistance
Phenotype

Mechanism

mec -lactamase
A
inhibitor

Detection by

Oxacillin agar
alter resistance screen(6ug/ml)

OX MIC
(ug/ml)

FOX Disk
diffusion

Classical

homogeneous PBP2a

no

yes

yes

>50

heterogeneous PBP2a

no

yes(no)

yes

>50

BOR-SA

hyper-lactamase

yes

no(yes)

No

2-4

MOD-SA

modified PBP
1, 2, 4

no

no(yes)

No

2-8

MR ? or MS ? --SA

unreliable
OX

FOX

Staphylococcus : -lactam
Pen-R FOX-S
resistant to penicillinase labile pen
susceptible to penicillinase stable pen, lactam/-lactamase inh., cephems, carbapenems.
Pen-R FOX-R = resistant to All -lactams
Test only Pen and FOX(+OX6ug screen for S.aureus
only)

Cannot detect mecC

Staphylococcus : Vancomycin

Inducible clindamycin resistant

(S. aureus, CoNS, Beta-Strep., S. pneumoniae)

DA

(15-26 mm)

12mm

Resistance to macrolides can occur via 2 different

mechanisms with these resulting phenotypes
Mechanism

Determinant

Erythromycin

Clindamycin

(gene)

Efflux

msrA

Ribosome
alteration

erm

erm

S*

* = requires induction to demonstrate resistance

Staphylococcus
(inducible clindamycin resistance positive)
Agent

Test Result

Clindamycin

Erythromycin

Validate
Report

This isolate is presumed to be resistant based on

detection of inducible clindamycin resistance.
Clindamycin may still be effective in some patients.

VA MIC

S.aureus MLSB
D-zone test
FOX+OX 6ug/ml screen
Zone-edge test

Staphylococcus aureus

Streptococcus pneumoniae
penicillin oxacillin 1ug disk (disk diff. mtd.)
> 20 mm = Susceptible
< 19 mm = ??S/I/R , do MIC (to penicillin)
Different breakpoints
Non-meningitis
Meningitis
Oral pen

<2
<0.06
<0.06

>8
>0.12
>2

0.12-1

o
disk diffusion MIC (lactam except ceftaroline >26mm/<0.5=S)
AP,Amox,AUG,CXM,CTX,CRO,FEP,IPM,MEM,ETP
Meningitis case Pen + CTX or CRO or Mem

Antimicrobial Resistance Mechanism in Enterococcus

High level aminoglycoside
Test by GM120 or SM300
for synergism with
lactam-Susc. agent

VA disk + VA 6ug/ml screen

Fastidious pathogens
H. (para)influenzae / N. gonorrhoeae
(other H. spp. use M45)

Disk diffusion with Hemophilus Test Medium (HTM)

/ GC agar+supplement
-lactam resistant by -lactamase or PBP alteration
(-lactamase (nitrocefin) testing is useful)
Few disks/plate (i.e. 4/2)

Fluoroquinolones (FQ)
Next issues : 2015
Table 2F (Neisseria gonorrhoeae) : all FQs from the table
except ciprofloxacin will be removed.
Table 2G (Streptococcus pneumoniae) : all FQs except
gemifloxacin, levofloxacin and moxifloxacin will be
removed. Gatifloxacin will be reevaluated.

Vancomycin
Next issues : 2015
-Two confirmed cases of vancomycin-resistant Group
B Streptococcus
-One confirmed case of vancomycin-resistant Viridans
Group Streptococcus in the United States.
-Currently no resistant breakpoints for vancomycin
with these two organisms. (The committee voted not to add
resistant breakpoints at this time and will follow this issue.)

M45-A3 2016 : Methods for antimicrobial dilution and disk

susceptibility testing of infrequently isolated or fastidious
bacteria; approved guideline
Next issues : 2015
Add Aerococcus, Gemella, Lactococcus, Micrococcus, and
assorted related genera [Kocuria, Nesterenkonis,
Dermacoccus and Kytococcus spp.], Rothia, Aerococcus and
Gemella.
Other existing groups will have additional antibiotics added
or modified.
Plesimonas shigelloides will be moved to M100 document
Reevaluate -lactam breakpoints of Aeromonas to those of
Enterobacteriaceae in M100 document.

Next Issues
New antibiotics:
Oritavancin is a lipoglycopeptide antibiotic for use in acute
soft skin and tissue infections caused by Gram positive cocci.
This drug may be given as a single bolus dose that may work
as well as 7-10 days of twice daily vancomycin therapy for
these organisms.
The committee may investigate if a vancomycin disk testing
might work as a screening test for susceptibility to this new
antibiotic.

Enterobacteriaceae
-lactam resistance varies among species
ESBL (extended spectrum -lactamase) test
confirmed in E.coli, K.pneumoniae, K.oxytoca,
Pt.mirabilis) (still important for infection control)
Carbapenem resistant Enterobacteriaceae (CRE) :
Enterobacteriaceae resist all 3rd cep (CAZ,CTX,CRO)
+ non-susceptible >1 carbapenem (IPM,MEM,DOR
+ ETM)
Quinolone resistance : increase
Aminoglycoside resistance : rare

Carbapenem Resistance Mechanisms

Enterobacteriaceae

Cephalosporinase + porin loss

Carbapenemase

P. aeruginosa

Porin loss
Up-regulated efflux
Carbapenemase

Acinetobacter spp.

Cephalosporinase + porin loss

Carbapenemase

Classification of Carbapenemases
Class
A

Carbapenemase

Found in:

Notes

KPC1

K. pneumoniae and
other
Enterobacteriaceae

Hydrolyze all -lactams

Inhibited by clavulanic acid

SME

S. marcescens

Metallo betalactamases
(IMP, VIM, GIM,
SPM, NDM2)

P. aeruginosa
Enterobacteriaceae
Acinetobacter
S. maltophilia

Hydrolyze all -lactams except

aztreonam
Somewhat inhibited by
clavulanic acid
Require zinc for enzymatic
activity; inhibited by EDTA

OXA

Acinetobacter
baumannii
Enterobacteriaceae

Less able to hydrolyze

carbapenems

Klebsiella pneumoniae carbapenemase most common carbapenemase in USA.

2 New Delhi metallo -lactamase
Adapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.
1

Modified Hodge Test (MHT)

http://jac.oxfordjournals.org/content/early/2009/12/08/jac.dkp431/F1.large.jpg

Carbapenemase detection : CLSI2015

carbapenemase Gram negative
bacilli CarbaNP

rapid test

Carba NP test

Isolated colonies (lyse/centrifuge)

Incubate with imipenem
Detect by pH change (red to yellow)
Within 3 hours
Microdilution plate or microtube

CLSI M100-S24 2014

Salmonella : Quinolone
(species-specific breakpoint)

CLSI M100 S24 2014 p56

CLSI M100 S24 2014 p57

Four quinolone susceptibility phenotypes of Salmonella enterica determined by Etest strips

(ciprofloxacin [CI] on the left, nalidixic acid [NA] on the right).

Hakanen A J et al. J. Clin. Microbiol. 2005;43:5775-5778

Humphries CID 2012:55;1107

Salmonella : FQs : CLSI 2015

Salmonella:
MIC FQs screen pefloxacin (5ug) disk
<23mm-R, >24mm-S. ( PFX-5ug disks : not currently available in the
U.S.)
Azithromycin breakpoint with Salmonella Typhi : <16ug/ml
(13mm)-Susceptible; >32 ug/ml (12mm)-NonSusceptible

Enterobacteriaceae: CLSI committee will re-evaluate FQ breakpoints

for this group of organisms in the future.

Salmonella Tips
CLSI 2013 : Some F-quinolones : ciproflox, levoflox, oflox breakpoint
Salmonella Enterobacteriaceae ( zone size ciproflox )
Salmonella from GI tract
CLSI recommend treat o
carrier state
severe diarrhoeae immunocompromised host 
Ampi, F-quinolone(new 2013 breakpoint), cotrimoxazole
Salmonella : extraintestinal (blood, CSF,....)
o 

3rd cephalosporin, chloramphenicol (if requested)
From BSAC(Version12 : May2013):
Salmonella systemic
(~extraintestinal) ciprofloxacin MIC MIC
ciprofloxacin >0.06 ug/ml o n


n 
ciprofloxacin o
o

o

n
Salmonella n
MIC CIP >0.06 ug/ml
Sal Typhi, ParaTyphi extraintestinal

or
extraintestinal
n n
" " ( ) salmonella aminoglycoside, 1st 2nd
cephalosporins, cephamycin

CLSI Breakpoints Additions/Revisions

2014
Enterobacteriaceae : Cefepime, Cefazolin
Acinetobacter

: Doripenem, Imipenem,
Meropenem

Cefepime Breakpoint Change for

Enterobacteriaceae and SDD

New breakpoints will cover all dosage outside urinary tract

SDD : Susceptible Dose Dependent

Intermediate
Imply clinical efficacy in body sites
Drugs are physiologically conc. ( quinolones, -lactams in urine
Higher than normal dose (-lactams) can be used

Buffer zone between S / R

Susceptible Dose Dependent

Multiple dosing options can be applied
Expect same clin. Responses as S if higher dose or
more freq. dose are used.

Enterobacteriaceae Cefepime Breakpoints

CLSI / FDA / EUCAST
Breakpoint

SDD

Dosage

CLSI
2014

<2

4-8

>16

S:1g12h
SDD MIC=4:1g8h, 2g12h
SDD MIC=8:2g8h

FDA

<8

16

>32

See info.

EUCAST

<1

2-4

>8

1-2g/8h

Kahlmeter G.2008. Clin Microbiol Infect 14 Suppl 1:169

Practical Considerations for Implementing

New Cefepime Breakpoints
Only for Enterobacteriaceae
FDA BPs not revised (lab must verify)
SDD on LIS or HIS reporting system
Report 1 character D in LIS explodes to SDD in
HIS
Report I and consult

Enterobacteriaceae : Cefazolin
Test Gr.

Agent

MIC (ug/ml)
I

Cephems (parenteral)
Cefazolin
A
<2

>8

2g/8h

Cephems (oral)
Cefazolin
U

>8

(20)

<16

Comment

Cefazolin predicts results for oral agents-cefaclor,cefdinir,cefpodoxime,cefprozil,

cefuroxime axetil, cephalexin, loracarbef when used for uncomplicated UTI from
E.coli,K.pn,Pt.mira..
Cefpodoxime, Cefdinir, cefuroxime axetil may be tested ind. Since some isolates may
be susceptible to these agents while testing resistant to cefazolin. M100S24p53

Enterobacteriaceae : Cephalothin
Test Gr.

Agent
S

MIC (ug/ml)
I

Comment
R

<8

16

>32

Cephems (oral)
U

Cephalothin

(11)

Cephalothin can be used only to predict susceptibility to oral agents-cefadroxil,

cefpodoxime, cephalexin, loracarbef.
Testing cefazolin preferred over cephalothin to predict activity of oral cephalosporins
for uncomplicated UTI.

M100S24p52

Non-Enterobacteriaceae
Disk diffusion
Pseudomonas aeruginosa
Acinetobacter spp.
Burkholderia cepacia
Stenotrophomonas maltophilia
Broth dilution
Other non-Enterobacteriaceae
=Pseudomonas spp. (except B.mallei,B.psmallei use
M45)
+non-fastidious, glucose non-ferment, gram-negative
bacilli

Acinetobacter : carbapenem

Fatal A.baumannii infection with discordant carbapenem susceptibility.

Lesho 2005 Clin Infect Dis 41:758

A. baumannii
Carbapenem Resistance Mechanisms
All A. baumannii produce natural
oxacillinase(OXA51/69) with carbapenemase
activity (low : may not R)
Carbapenem hydrolysing oxacillinase (Ambler D)
(low when compared to MBL, KPC)
Some metallo -lactamase (IMP, VIM)
Some KPC
Non-carbapenemase (porin loss, efflux)
Poirel, Nordmann. 2006. Clin Microbiol Infect.12:826

Acinetobacter : % susceptible
(N=1660)
Minocycline

Cefepime

Amikacin

67.9

4.2

22.5

Levofloxacin Meropenem
4.3

11.6

Pip/Tazo
1.6

Hawser 2013 ICAAC Abstract#C2-1625

Minocycline : considerably more active than

doxycycline and tetracycline against A. baumannii
Recent studies with minocycline clinical efficacy
Bishburg 2014. Infect Dis Clin Pract 22:26
Jankowski 2012. Infect Dis Clin Pract 20:184

Next Issues
SDD Susceptible Dose-Dependent:
develop SDD breakpoints for the following b-lactam
antibiotics: aztreonam, cefotaxime, ceftriaxone, cefoxitin,
and ceftazidime for the Enterobacteriaceae.
develop SDD breakpoints for Pseudomonas aeruginosa
with ceftazidime, aztreonam, and cefepime in the future.

Next Issues
remove 12 drugs (that are no longer available in USA) from
Table 1 in the M100 document
CLSI will clarify information (with pictures!) for reading
trailing endpoints in broth microdilution microtiter tray
assays.
Develop standard methods for colistin MIC testing(+0.002%
Tween)
The 2015 M100 document will have an updated anaerobe
antibiogram table (from isolates testing from 2010-2012)