1

Antibacterial

Susceptibility Testing

Basics

Sandra S. Richter, M.D.

Cleveland Clinic

10/19/18

Disclosures: Research funding from bioMerieux, BD

Diagnostics

Objectives

• Describe antimicrobial susceptibility testing

(AST) methods available for bacteria

• Discuss use of special phenotypic and

molecular methods to detect antimicrobial

resistance

• Determine when AST should be performed

and how to troubleshoot unusual results

Antimicrobial Susceptibility Testing

• A principle function of clinical microbiology lab

• Predict outcome of treatment with agents tested

• 90-60 rule• 90% success when organism is “Susceptible”

• 60% success when organism is “Resistant”

• Intermediate• Higher doses required to ensure efficacy

• Buffer zone that prevents borderline susceptibility from incorrect categorization as R or S

• Guide selection of most appropriate agent • Most narrow spectrum

• Least expensive

Susceptibility Test Methods

• Qualitative (S, I, R)

• Disk diffusion

• Quantitative (MIC)

• Broth macrodilution

• Broth microdilution

• Agar dilution (single concentration per plate)

• Agar gradient diffusion (Etest)

• Automated instrument

Antimicrobial Susceptibility Testing

• MIC = Minimum inhibitory concentration

that inhibits growth over defined interval

(usually 18-24 h)

• MIC is compared to levels of drug

achieved in human body fluids to

determine breakpoints

2

Broth microdilution method M11: Reference Agar Dilution• 2-fold dilutions of

antimicrobial agents added to molten agar (cooled to 48-50ºC), mixed, poured into Petri dish, allowed to solidify

• 0.5 McFarland suspension of isolate inoculated onto each plate in concentration series using replicating device (105

CFU/spot)

• Read after 42-48 h anaerobic incubation

• MIC = lowest concentration inhibiting growth

Commercial AST Systems

• Introduced in 1980s; in most labs since 1990s

• Manual or semiautomated broth microdilution (small

volume)

• Automated broth microdilution (large volume)

• Semiautomated disk diffusion - popular outside of U.S.

• DMS with LIS interface and expert system

analysis

• Epidemiology software

Acceptable performance for FDA

clearance of AST system

• Multicenter comparison of system to CLSI

reference method

• >90% categorical agreement (CA)

• >90% essential agreement (EA)

• <1.5% very major errors (VME)

• <3% major errors (ME)

• >95% reproducibility

• Sufficient no. resistant organisms tested

• <10% growth failure for each organism group

11

FDA clearance of AST systems

• Antimicrobial/organisms without acceptable

performance listed as limitation in the device PI

(alternative method recommended)

• Reporting of results for agents without known clinical

efficacy against organism is discouraged (consult

drug label, CLSI M100 Table 1A, Sanford Guide to

Antimicrobial Therapy)

• Manufacturers required to apply FDA interpretive

standards to AST system results12

FDA clearance of AST systems

• When breakpoints change a comparative study is required

• If new breakpoints affect device performance a new 501(k) submission is required

• If validated, labs may report result using interpretive

criteria other than those published in device label

• FDA regulations and approved devices:

http://www.fda.gov/cdrh/consumer/mda/index.html

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13

Vitek 2

• FDA approval in 2000

• 64-well cards

• Card capacity: 60 or 120 (Vitek 2 XL)

• 1-6 dilutions of 9-20 antimicrobials

• Smart Carrier system

• Auto AST dilution, card inoculation, sealing

• Turbidimetric reading every 15 min

• DMS (Observa) with AES

• MylaTM middleware (remote access, manual AST entry)

Photos courtesy of bioMérieux, Inc.

MicroScan Walkaway

• Available since late 1980s

• 96-well microdilution trays (MIC, breakpt)

• 40 or 96 tray capacity

• Conventional panels (turbidimetric reading, ON incub)

• Rapid panels (turbidimetric reading, 4.5 to 15 h incubation)

www.beckmancoulter.com

BD Phoenix

• Available in U.S. since 2004

• 50 or 100 panel capacity

• AP instrument for automated inoculum standardization

• Turbidimetric and redox readings every 20 min

• Full range MICs <16 h

• 85-well AST / 51-well ID or 136 well AST polystyrene tray

• Growth - redox indicator reduced (oxidized blue →reduced pink)

http://www.bd.com

Sensititre ARIS 2x

• Automated Reading and Incubation System (ARIS)

• Available in U.S. since 1992

• ARIS 2x (2004) hardware, software upgrades

https://www.thermofisher.com

Sensititre Autoinoculator and Vizion

https://www.thermofisher.com

Antimicrobial gradient method

(Etest, MIC Test Strip)

4

Disk diffusion method (Qualititative)

Koneman’s Color Atlas &Textbook of Diag Microbiol 2006

http://www.eucast.orgKoneman’s Color Atlas &Textbook of Diag Microbiol 2006

Factors that influence accuracy of AST

results• Medium (Mueller Hinton, MH-B, HTM)

• pH

• Cations (Ca, Mg)

• Antimicrobial (correct concentration, storage T)

• Inoculum size (5x105 cfu/ml - BMD)• Turbidity 0.5 McFarland (108 cfu/ml - DD)

• 1:100 dilution (106 cfu/ml)

• 1:2 dilution (0.05 ml inoculum added to 0.05 ml broth)

• Incubation atmosphere and time (overnight 16-20 h; detection VRE and MRSA require 24 h)

• Equipment

• Reading and interpretation

Quality Control

• Evaluate precision and accuracy of test procedure

• Monitor reagent performance

• Evaluate personnel conducting test

• Reference strains with acceptable ranges

• Test each new batch and lot plus

• Daily (unless IQCP supports weekly)

• Weekly (if no more than 1 out of 20 or 3 out of 30 consecutive days outside of range)

• If weekly value is outside of range – repeat for 5 consecutive days

CLSI Intrinsic Resistance Tables Troubleshooting Unusual AST Result

• Transcription error?

• Contamination?

• Defective panel, plate, or card?

• Repeat organism ID and AST

• Confirm organism identification with 2nd

method

• Confirm AST result with 2nd method

• CLSI reference method

• FDA cleared commercial method

5

Setting breakpoints

• MIC distributions

• Clinical and bacteriological response rates

• Pharmacokinetics – absorption, distribution, &

elimination of drugs

• Pharmacodynamics – relationship between serum

concentration and pharmacological /toxicological

effects of drugs

PK/PD

• Pharmacodynamics – time course of drug action against organism varies

• Time dependent and no (or short) postantibiotic effect (critical determinant of killing is % time in dosing interval drug conc is above MIC; T>MIC)

• Time dependent and long postantibiotic effect (ratio of area under conc vs. time curve = 24 h AUC/MIC)

• Concentration dependent (Peak conc / MIC)

Pharmacokinetic/Pharmacodynamic

Properties of Agents

Favorable clinical

outcome assoc w\

one of these:

T>MIC for >40%

of dosing interval

AUC/MIC 100-125

Cmax/MIC 8-12

Craig, Clin Infect Dis. 1998; 26:1-12

Relationship between 3 PK/PD

parameters (Cmax/MIC,, T>MIC)

to number of Klebsiella

pneumoniae in lungs of

neutropenic mice after 24 h of

cefotaxime therapy.

The dotted line = number of

bacteria at beginning of therapy.

Target Attainment: Monte Carlo simulations (software)

used to evaluate potential PK-PD cut offs & estimate

probability of attaining target drug exposure c/w efficacy

in the context of pathogen MIC

90% PTA at given MIC usually considered acceptable by CLSI-M23

S 1 Cerexa; CLSI Agenda Book, June 2012

Selecting Antibacterial Agents

for Testing/Reporting

• Organism (efficacy)

• Standardized method with breakpoints

• Site of infection

• Table 1 (CLSI M100)

• Formulary (cost)

6

CLSI Performance Standards for AST

• Enterobacteriaceae

• Pseudomonas aeruginosa

• Acinetobacter spp.

• Burkholderia cepacia complex

• Stenotrophomonas maltophilia

• Other Non-Enterobacteriaceae

• Haemophilius influenzaeand H. parainfluenzae

• Staphylococcus spp.

• Enterococcus spp.

• S. pneumoniae

• β-hemolytic strep

• Viridans gp strep

• Neisseria meningiditis

• Neisseria gonorrhoeae

• Anaerobes

M100-S28

M100, Appx D: Anaerobic Antibiogram: US

Data from 4 Reference Laboratories, 2010-12

• Bacteroides fragilis group (7 spp.), n=2580 for A/S• Ampicillin-sulbactam, 6% R (0-20%)

• Piperacillin-tazobactam, 7% R (1-30%)

• Cefoxitin 7% R (3-21%)

• Ertapenem 1% R (0-2%)

• Imipenem 1% R (0-1%)

• Meropenem 1% R (0-2%)

• Metronidazole 1% R (0-2%)

• Clindamycin 42% R (23-63%)

• Moxifloxacin 36% R (26-76%)

M100, Appx D: Anaerobic Antibiogram: US

Data from 4 Reference Laboratories, 2010-12

• Other anaerobes (not B. fragilis gp), N

= results for A/S

• Ampicillin-sulbactam, 0-9% R

• Piperacillin-tazobactam, 0-16% R (1-30%)

• Cefoxitin 0-97% R (C. difficile 97%R)

• Ertapenem 0-9% R

• Meropenem 0-1% R

• Metronidazole 0-91% R (P. acnes 91%R)

• Clindamycin 0-48% R (Veillonella 34% R)

• Moxifloxacin 0-24% R European Committee on Antimicrobial Susceptibility Testing. Data from the EUCAST MIC

distribution website, last accessed 10/1/2017 http://www.eucast.org

EUCAST penicillin clinical breakpoints for anaerobes: S ≤0.25, R >0.5 mg/L

CLSI penicillin breakpoints for anaerobes: S ≤0.5, I =1, R ≥2 µg/mL

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Confirm ID & AST

1. Check for contamination, transcription errors, or defective panel.

2. Was this AST result confirmed earlier for patient?

3. Repeat ID and AST with initial method.

4. Confirm ID with 2nd method.

5. Confirm AST with 2nd method (ref or FDA cleared method).

Indications for susceptibility testing of

anaerobes

• Serious infections: Brain abscess,

endocarditis, osteomyelitis, joint infection,

prosthetic device, bacteremia

• Normally sterile site

• Organisms considered highly virulent or

unpredictable susceptibility profile

• e.g., B. fragilis gp, Prevotella, Fusobacterium,

Clostridium, Bilophila, Sutterella spp.

• Treatment failure (any anaerobe)

Methods: M100, Table 2J-1. Anaerobes

Staphylococci

Staphylococci % Susceptible

Organism (number tested):

Oxacillin

a

Van

co

my

cin

Lin

ezo

lid

Dap

tom

ycin

Gen

tam

icin

Ery

thro

my

cin

Clin

dam

ycin

Tri

meth

op

rim

/

Su

lfam

eth

oxazo

le

Do

xy

cy

clin

e

Tetr

acy

clin

e

Staphylococcus aureus (5,220) 62 100 99 100 99 44 71 97 97 92

Oxacillin-resistant (MRSA) (1,963) 0 100 100 100 98 13 62 95 96 89

Oxacillin-susceptible (MSSA) (3,257) 100 100 99 100 99 63 76 99 98 93

Staphylococcus lugdunensis (457) 93 100 100 100 100 76 77 99 99 94

Staphylococcus capitis (103) 88 100 100 100 97 75 81 97 100 97

Staphylococcus epidermidis (1,329) 42 100 99 100 85 32 56 56 88 82

Staphylococcus haemolyticus (71) 45 100 100 100 72 24 58 69 83 72

Staphylococcus hominis (89) 61 100 100 100 98 26 60 73 93 82

Staphylococcus simulans (32) 81 100 100 100 100 44 47 100 97 97

Staphylococcus warneri (31) 81 100 100 100 97 61 74 97 100 94

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Oxacillin & cefoxitin AST to detect

mecA-mediated altered PBP2a

Organism Oxacillin

MIC

Cefoxitin

MIC

Cefoxitin

30 µg Disk

Oxacillin

1 µg Disk

S. aureus S ≤ 2 S ≤ 4 S ≥ 22 -

S. lugdunensis S ≤ 2 S ≤ 4 S ≥ 22 -

CoNS S ≤ 0.25 a - S ≥ 25 -

S. pseudintermedius &

S. schleiferibS ≤ 0.25 - - S ≥18

aCoNS other than S. epidermidis: oxacillin breakpoint may overcall resistance, if MIC range 0.5-

2 µg/mL , consider additional testing (mecA or PBP2a) for serious infections.

bCoag-positive staph species, colonizes nares of dogs/cats, vet pathogen (pyoderma)

uncommon human pathogen (animal bites), Wu et al. JCM 2016; 54:535. Cefoxitin not reliable.

Verigene BC-GP

Penicillin

Oxacillin

or

cefoxitinInferred AST result

S S S to penicillins, β-lactam/β-lactamase inhibitor

combinations, cephems, carbapenems

R S R to penicillinase-labile penicillins

S to penicillinase-stable penicillins, β-lactam/β-lactamase

inhibitor combinations, antistaph cephems, carbapenems

R* R R to penicillins, β-lactam/β-lactamase inhibitor combinations,

carbapenems, cephalosporins except those with anti-MRSA

activity (eg, ceftaroline)

*Oxacillin or cefoxitin-resistant staphylococci should always be considered penicillin

resistant

Carbapenemases

• 2009 laboratory detection problem noted: some

Enterobacteriaceae have carbapenem MICs that

are “susceptible” using CLSI breakpoints

• Among 44 Enterobacteriaceae isolates with KPC-2

or KPC-3, 36% had “susceptible” results for

imipenem and 32% had “susceptible” results for

meropenem (MICs of 2 or 4 µg/ml)

Deshpande et al. Diagn Microbiol Infect Dis 2006; 56:367-372.

Modified Hodge Test (detects

carbapenem hydrolyzing enzyme)

• If carbapenem (meropenem,

imipenem, ertapenem) MICs of

2 or 4

• Swab E. coli ATCC 25922 onto

plate to create lawn (1:10

dilution of 0.5 McFarland)

• Place meropenem or ertapenem

disks

• Streak test isolate from edge of

disk to end of plate, incubate ON

• Look for clover leaf of E. coli

growth around test isolate

Positive

Negative48

Changes in CLSI carbapenem breakpoints for

Enterobacteriaceae

aNew breakpoints based on dosage of 500 mg every 8 h.

Antimicrobial

Agent

Jan 2010 June 2010/2012

S I R S I R

Doripenema - - - 1 2 4

Imipenemb 4 8 16 1 2 4

Meropenemc 4 8 16 1 2 4

Ertapenemd 2 4 8 0.5 1 2

cNew breakpoints based on dosage of 1 g every 8 h.

bNew breakpoints based on dosage of 500 mg every 6 h or 1 g

every 8 h.

dNew breakpoints based on dosage of 1 g every 24 h.

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March 2018: CP-CRE must be reported

(& isolates submitted) to ODH

Table 3B, CLSI M100, 2018.

Modified Carbapenem Inactivation

Method (mCIM) in Enterobactericeae• Emulsify 1-µl loop of bacteria in 2 ml

TSB & vortex 15 s

• Add 10 µg meropenem disk to tube

• Incubate at 35 ± 2C for 4 h ± 15 m

• Remove mero disk & place on MHA

plate previously inoculated with 0.5

McFarland suspension of mero

susceptible E. coli 25922

• Incubate at 35 ± 2C for 18-24 h

• Carbapenemase positive: 6-15 mm zone

(negative if zone >=19 mm) Left: Negative & Positive QC (narrow ring of

growth around negative results from carryover

of test organism in TSB & should be ignored)

Positive mCIM:

Rapid Carbapenemase Gene Detection

• Detects and differentiates the most

prevalent carbapenemase gene families:

KPC, NDM, VIM, IMP-1, OXA-48, OXA-

181, OXA- 232

• Rectal swab specimens

• 48 min TAT (culture CRE screen: 72 h)

• Bacterial isolates

Organism N CTX-M KPC1 NDM VIM IMP OXA

Acinetobacter spp. 43 13

Citrobacter spp. 26

Enterobacter spp. 103 1 3

Proteus spp. 111 6 5

E. coli 845 125 2

K. pneumoniae 306 36 10 1

K. oxytoca 53 1 2

Klebsiella spp. 3

Klebsiella spp. or

Enterobacter spp.2

P. aeruginosa 150

NEGGNB 319

Total 1961 169 (8.4) 18 (0.9) 0 0 0 19 (1)

Verigene BC-GN 2017 CLSI M45 Guideline

• The extensive microbiological, clinical, and

pharmacodynamic databases employed for setting

CLSI breakpoints does NOT exist for the collection

of “orphan” organisms described in the document

• Consultation with an ID physician is recommended

for assistance in determining the need for testing

and the interpretation of results

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CLSI M45-A2 Guideline (2010) New M45-A3 Tables (2015)

CAMHB-LHB, 35 °C

5% CO2

Aerococcus spp. Gemella spp.

Incubation time 20-24 h 24-48 h

Penicillin* S ≤ 0.12 S ≤ 0.12

Ceftriaxone* S ≤ 1 S ≤ 0.25

Vancomycin* S ≤ 1 S ≤ 1

Erythromycin - S ≤ 0.25

Meropenem S ≤ 0.5 S ≤ 0.5

Levofloxacin S ≤ 2 S ≤ 2

New M45-A3 Tables (2015)

Ambient air,

20-24 h, 35 °C

Lactococcus

spp.

Micrococcus

spp.

Rothia

mucilaginosa

Medium CAMHB-LHB CAMHB CAMHB-LHB

Penicillin S ≤ 1 S ≤ 0.12 S ≤ 0.12

Ceftriaxone S ≤ 1 - -

Vancomycina S ≤ 2 S ≤ 2 S ≤ 2

Erythromycina S ≤ 0.5 S ≤ 0.5 S ≤ 0.5

Clindamycina S ≤ 0.5 S ≤ 0.5 S ≤ 0.5

Levofloxacin S ≤ 2 - S ≤ 1

a Breakpoints adapted from staphylococci

M45: Indications for AST

• Species potentially R to common agents

• Normally sterile sites (eg, blood, CSF, joint, bone, prosthetic devices)

• Serious wound infections (eg, Aeromonas and Vibrio spp.)

• Refractory diarrhea: Campylobacter jejuni/coli

• Refractory gastritis: H. pylori

• Not recommended for superficial or non-sterile sites

• Skin/mucous membrane microbiota: Aerococcus, Corynebacterium, Abiotrophia, Granulicatella, Lactobacillus, Micrococcus, Pediococcus, Leuconostoc spp.

• Environmental: Bacillus spp.

Conclusion

• Revision of breakpoints will continue

• Emergence of resistance

• Need to apply new PK/PD analyses

• Need more timely availability of new agents

and new breakpoints on FDA cleared panels

• Opportunity for new technology to shorten

TAT & improve patient outcomes