Reviewand The very latest information ofAntimicrobial Susceptibility Testing

from CLSI

Piriyaporn Chongtrakool

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

วธีิทดสอบความไวต่อสารตา้นจุลชีพ

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), fastidiousorganism : 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 : ß , 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 forEnterobacteriaceae

Agent Disk content Zone diameter (mm)

ug R I S

AM 10 < 13 14-16 > 17

AK 30 < 14 15-16 > 17

C 30 < 12 13-17 > 18

SXT 1.25/23.75 < 10 11-15 > 16

Zone diameter Interpretive standards forStreptococcus pneumoniae

Agent Disk Zone diameter (mm)

Content R I S

Vancomycin 30ug > 17

การแปลผลการทดสอบความไว

• Susceptible (S)– Á�ºÊ°�nµ�³�°�­ �°��n°�µ¦ ¦ �́¬µ�oª ¥�dose ปกติ

• Resistant (R)– Á�ºÊ° Ťn�nµ�³�°�­ �°��n°�µ¦ ¦ �́¬µ�¤ o�³ Ä�o�dose สูง

• Intermediate (I)– Á�ºÊ° Ū �µ��̈µ��° µ��°�­ �°��n°�µ¦ ¦ �́¬µ�oµÄ�o�dose สูง

– ®¦ º°�ε®�n��·�Á�ºÊ° ¤�̧ª µ¤Á�o¤�o��°�¥µ­ ¼��(โดยคาํนึงถึงผลขา้งเคียง)

• Non-Susceptible (N)– ไม่สามารถจาํแนกไดว้า่เป็น S/I/R Á�ºÉ°��µ��o°�ε�́��°�ª ·�̧��­ °�

– ¥�́ŤnÁ�¥¡ �Á�ºÊ° �̧É�ºÊ°�(�o°�¡ ·­ ¼��r¥º�¥�́Á¡ ·É¤Á�·¤ ) จึงเรียก ไม่ไว

ขอ้จาํกดัของการทดสอบความไว โดยวธีิ Disk Diffusion

• Á®¤µ³ ­ 宦 �́Á�ºÊ° Á�¦ ·�Á¦ Ȫ

• รายงานผลเฉพาะ S I R N

• Serum level = interpretive criteria

• Unreliable to test in some organisms againstsome agents

Unreliable to test by Disk Diffusion=use MIC(Examples)

1. Staphylococcus : Vancomycin, Daptomycin2. Streptococcus viridans group : Penicillin3. Strep. pneumoniae (sterile sites) : P, CTX, CRO, Mem

4. Acinetobacter : Colistin, Netilmicin5. 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 / inducibleresistances

1. Enterobacteriaceae : ESBLs

2. Enterobacteriaceae : KPC

3.Staphylococcus, Beta-Streptococcus,S.pneumoniae : Inducible Clindamycinresistance

Staphylococcus aureus

Penicillin ?Susceptible Staphylococcus aureus

ResistancePhenotype Mechanism

mecA

β-lactamase inhibitor Detection by

OX MIC(ug/ml)

alter resistanceOxacillin agar

screen(6ug/ml)

FOX Diskdiffusion

Classical

homogeneous PBP2a + no yes yes >50

heterogeneous PBP2a + no yes(no) yes >50

BOR-SAhyper-β-lactamase - yes no(yes) No 2-4

MOD-SAmodified PBP1, 2, 4 - no no(yes) No 2-8

Classification of MRSA

MR ? or MS ? --SA

OX FOXunreliable

Staphylococcus : -lactam

• Pen-R FOX-S

• resistant to penicillinase labile pen

• susceptible to penicillinase stable pen, β-lactam/β-lactamase inh., cephems, carbapenems.

• Pen-R FOX-R = resistant to All β-lactams

• Test only Pen and FOX(+OX6ug screen for S.aureusonly)

Cannot detect mecC

Staphylococcus : Vancomycin

Inducible clindamycin resistant(S. aureus, CoNS, Beta-Strep., S. pneumoniae)

(15-26 mm)E DA

12mm

Resistance to macrolides can occur via 2 differentmechanisms with these resulting phenotypes

Mechanism Determinant Erythromycin Clindamycin

(gene)

Efflux msrA R S

Ribosomealteration

erm R R

erm R S*

* = requires induction to demonstrate resistance

Staphylococcus(inducible clindamycin resistance positive)

Agent Test Result Validate

Report

Clindamycin S

Erythromycin R R

“This isolate is presumed to be resistant based ondetection of inducible clindamycin resistance.”“Clindamycin may still be effective in some patients.”

Staphylococcus aureusZone-edge test

FOX+OX 6ug/ml screen

VA MIC

S.aureus MLSB

D-zone test

Streptococcus pneumoniae• ทดสอบความไวต่อ penicillin โดย oxacillin 1ug disk (disk diff. mtd.)

• > 20 mm = Susceptible

• < 19 mm = ??S/I/R , do MIC (to penicillin)

• Different breakpointsS I R

• Non-meningitis <2 4 >8

• Meningitis <0.06 >0.12

• Oral pen <0.06 0.12-1 >2

• ¥µ�̧É��­ °��oª ¥�disk diffusion ไม่ได ้ตอ้งทาํ MIC (βlactam except ceftaroline >26mm/<0.5=S)

• AP,Amox,AUG,CXM,CTX,CRO,FEP,IPM,MEM,ETP

• Meningitis case ควรทดสอบ Pen + CTX or CRO or Mem

Antimicrobial Resistance Mechanism in Enterococcus

High level aminoglycosideTest by GM120 or SM300for synergism withβlactam-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, andassorted related genera [Kocuria, Nesterenkonis,Dermacoccus and Kytococcus spp.], Rothia, Aerococcus andGemella.

• Other existing groups will have additional antibiotics addedor modified.

• Plesimonas shigelloides will be moved to M100 document

• Reevaluate β-lactam breakpoints of Aeromonas to those ofEnterobacteriaceae 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) testconfirmed 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 Carbapenemase Found in: Notes

A KPC1 K. pneumoniae and

other

Enterobacteriaceae

Hydrolyze all β-lactams

Inhibited by clavulanic acid

SME S. marcescens

B Metallo beta-lactamases

(IMP, VIM, GIM,SPM, NDM2)

P. aeruginosaEnterobacteriaceaeAcinetobacter

S. maltophilia

Hydrolyze all β-lactams except aztreonam

Somewhat inhibited byclavulanic acid

Require zinc for enzymaticactivity; inhibited by EDTA

D OXA Acinetobacterbaumannii

Enterobacteriaceae

Less able to hydrolyzecarbapenems

1 Klebsiella pneumoniae carbapenemase – most common carbapenemase in USA.2 New Delhi metallo -lactamase

Adapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.

Modified Hodge Test (MHT)

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

Carba NP test

• Isolated colonies (lyse/centrifuge)

• Incubate with imipenem

• Detect by pH change (red to yellow)

• Within 3 hours

• Microdilution plate or microtube

- +

พฒันาการตรวจหา carbapenemase ใน Gram negative

bacilli โดยใช้ “ CarbaNP “ �¹É�Á�È��rapid test

: a rapid tube test used to indicate the presence

Carbapenemase detection : CLSI2015

CLSI M100-S24 2014Salmonella : 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:

ในกรณีทดสอบ MIC ตอ่ FQs ไมไ่ด้ อาจ screen โดย 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 breakpointsfor this group of organisms in the future.

Salmonella Tips• CLSI 2013 : Some F-quinolones : ciproflox, levoflox, oflox มี breakpoint เฉพาะของ

Salmonella แยกจาก Enterobacteriaceae (มี zone size แปลเฉพาะ ciproflox )

• Salmonella from GI tract

• CLSI ไม่ recommend ให้ทดสอบความไว เพราะไม่จําเป็นต้อง treat Á�ºÉ°��µ��³�εĮoÁ�·��carrier state

• จําเป็นต้องรักษาเฉพาะ severe diarrhoeae หรือ immunocompromised host ¥µ�̧É�ª ¦��­ °��º°�

Ampi, F-quinolone(new 2013 breakpoint), cotrimoxazole

• Salmonella : extraintestinal (blood, CSF,....)

• �o°���­ °��ªµ¤Åª�¨ ³�ª ¦ Á¡ ·É¤�3rd cephalosporin, chloramphenicol (if requested)

• From BSAC(Version12 : May2013): �¦�̧�µ¦�·�Á�ºÊ° Salmonella แบบ systemic(~extraintestinal) และต้องการใช้ ciprofloxacin ในการรักษา ควรทําเป็น MIC หากพบว่า มีค่า MIC ต่อ ciprofloxacin >0.06 ug/ml Ä®o¡ ·�µ¦�µªnµ��ºÊ°��n°�ciprofloxacin ¨ ³�o°�¦ ³ ª�́�µ¦ Ä�o¥µ�̧Ê�Á�ºÉ°��µ�¤�̧o° ¤¼̈�µ��̈·�·��̧É­ �́�­ �»�ªnµ�Salmonella �̧ɤ�̧nµ�MIC ต่อ CIP >0.06 ug/ml มกัตอบสนองไมดี่ต่อการรักษา

• Sal Typhi, ParaTyphi มกัก่อโรคแบบ extraintestinal �́��́Ê�Á�oµÁ���r�extraintestinal

• ¥µ�̧ÉŤn�ª ¦ ¦ µ¥�µ�ªnµ�" ไว " (จริงๆก็คือ ไม่ควรทดสอบ) กบั salmonella คือ aminoglycoside, 1st 2nd

cephalosporins, cephamycin

CLSI Breakpoints Additions/Revisions2014

• Enterobacteriaceae : Cefepime, Cefazolin

• Acinetobacter : Doripenem, Imipenem,Meropenem

Cefepime Breakpoint Change forEnterobacteriaceae 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, β-lactams in urine

• Higher than normal dose (β-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 ormore freq. dose are used.

Enterobacteriaceae Cefepime BreakpointsCLSI / FDA / EUCAST

Breakpoint S SDD I R Dosage

CLSI2014

<2 4-8 - >16 S:1g12hSDD 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 ImplementingNew 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 inHIS

– Report “I” and consult

Enterobacteriaceae : Cefazolin

Test Gr. Agent MIC (ug/ml) Comment

S I R

Cephems (parenteral)

A Cefazolin <2 4 >8 2g/8h

Cephems (oral)

U Cefazolin <16 - >8 (20)

Cefazolin predicts results for oral agents-cefaclor,cefdinir,cefpodoxime,cefprozil,cefuroxime axetil, cephalexin, loracarbef when used for uncomplicated UTI fromE.coli,K.pn,Pt.mira..Cefpodoxime, Cefdinir, cefuroxime axetil may be tested ind. Since some isolates maybe susceptible to these agents while testing resistant to cefazolin. M100S24p53

Enterobacteriaceae : Cephalothin

Test Gr. Agent MIC (ug/ml) Comment

S I R

Cephems (oral)

U Cephalothin <8 16 >32 (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 cephalosporinsfor uncomplicated UTI.

M100S24p52

Non-Enterobacteriaceae

• Disk diffusionPseudomonas aeruginosaAcinetobacter spp.Burkholderia cepaciaStenotrophomonas maltophilia

• Broth dilution• Other non-Enterobacteriaceae

=Pseudomonas spp. (except B.mallei,B.psmallei useM45)

+non-fastidious, glucose non-ferment, gram-negativebacilli

Acinetobacter : carbapenem

Fatal A.baumannii infection with discordant carbapenem susceptibility.Lesho 2005 Clin Infect Dis 41:758

A. baumanniiCarbapenem Resistance Mechanisms

• All A. baumannii produce naturaloxacillinase(OXA51/69) with carbapenemaseactivity (low : may not “R”)

• Carbapenem hydrolysing oxacillinase (Ambler D)(low when compared to MBL, KPC)

• Some metallo β-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 Levofloxacin Meropenem Pip/Tazo

67.9 4.2 22.5 4.3 11.6 1.6

Hawser 2013 ICAAC Abstract#C2-1625

• Minocycline : considerably more active thandoxycycline and tetracycline against A. baumannii

• Recent studies with minocycline clinical efficacyBishburg 2014. Infect Dis Clin Pract 22:26Jankowski 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)