Old and new methods for detection of emerging resistant organisms

Ellen Jo Baron, Ph.D., D(ABMM) Exec. Director of Medical Affairs, Cepheid Prof. Emerita, Stanford University

Why are some bacteria resistant? Microbes fighting for survival in the environment (soil, human skin, GI tract)

1. Innate (missing mechanism) 2. Prevent uptake; speed exit 3. Modify target site 4. Bind up the antibiotic to prevent activity 6. Inactive the antibiotic (make a new enzyme) 7. Lower importance of target (add new mechanism)

How are bacteria resistant? Rx Target

mecA product

Example: Inactivation - Beta-lactamase

B-lactamase- Serine NCO

H2O

Serine-B-lactamase

O

NCInactive antibiotic

Active ß-lactam antibiotic

Staph aureus (& others)

ACTION OF mecA GENE mecA on transposon

Staph genome Altered genome codes for modified PBP-2a

PBP-1

PBP-2

Meth

PBP-1

PBP-2a

CELL WALL

PBP-1

PBP-2

Methods of detecting resistance 1.  Phenotypic

•  Agar dilution •  Broth dilution (microbroth dilution) •  Disk diffusion and variations •  Gradient diffusion •  Enzymatic detection

2.   Genotypic •  Mutation or other DNA change detection by PCR

(+ microarray) •  Mutation or DNA change detection by line probe •  Sequencing

3.   Other •  Growth and MALDI-TOF facilitated •  Growth and Image analysis

Chromogenic cephalosporin disk

Agar Dilution: Detection of VRSA Staphylococcus aureus •  Do not trust automated systems – they fail to detect VISA

Some VISA = 2 by Etest and 1 by broth MIC •  Use Vanco disk & Vanco screen plate, incubate 24 h •  Growth on Vanco plate & no zone = VRSA •  Growth on Vanco plate & zone ≥7 mm = possible VISA/VRSA Perform Etest or send isolate to reference lab

•  BHI plate with 6 mg/ml Vanco •  Inoculate with swab from 0.5

McFarland suspension 30 mcg Vanco disk

Touch 4-5 isolated colonies of the SAME type with a loop or swab to pick up tiny amount from each one. Pure culture required.

Disk diffusion susceptibility testing (Bauer-Kirby method) For staphylococci, Enterobacteriaceae, Pseudomonas aeruginosa, enterococci, and other organisms that grow well on Mueller-Hinton agar in air at 35°C

# 1 Rub colonies on side of 5 ml saline tube to make very thin smooth suspension

# 2 # 3 Check using lined card & add more organisms or dilute the suspension to match McFarland 0.5 turbidity

# 4 Dip new swab into suspension & squeeze out excess liquid on sides of tube

# 5

1

2

3

# 6 Within 15 minutes, add the antibiotic disks

Rub swab over same Mueller-Hinton plate total surface 3 times in 3 different directions to create smooth layer of inoculum

BAP better than Mac to avoid mixed culture

Draw lines on card

# 7 Tap each disk or tap plate upside down to be sure disks do not fall off

# 8 Incubate 16-18 hours in air at 35°C

# 9 Measure in mm diameter of zone of inhibition across center of disks using ruler or calipers

No zone = 6 mm

# 10 Use CLSI tables to interpret zone size results for each organism & drug combination

Factors that influence results and should be monitored as part of QC Program

1.  Depth of agar (3-5 mm)

2.  pH and cation content of agar

3.  Turbidity of organism suspension (0.5 McF)

4.  Time between steps (15 min maximum)

5.  Incubation temp. (35°C) & time (18-24 h)

6.  Incubation atmosphere (usually air)

7.  Technologist ability to accurately read zones

Detection of MRSA & MRSE

Oxacillin Cefoxitin

Staphylococcus aureus & S. lugdunensis

•  Do not trust automated systems (Vitek, MicroScan) •  Use 30 mcg Cefoxitin disk to screen for MRSA & MRSE •  Read after 24 hrs incubation at 35° C •  Cefoxitin results ≤ 19 mm report as Oxacillin-resistant (Do not

report Cefoxitin)

For all other Coag Neg Staph: Cefoxitin

•  ≥ 25 mm = S •  ≤ 24 mm = R

Stealth MRSA

Cefoxitin

Inducible MRSA

Before induction After induction

PBP2a Latex agglutination

MRSA Chromagar

Before induction

After induction

Gradient diffusion: Detection of Penicillin Resistance in Streptococcus

pneumoniae •  Use Mueller-Hinton + 5% sheep blood agar •  Incubate in CO2 instead of air •  Report actual MIC

Report both Pen & Cefotaxime or Ceftriaxone & Mero MICs for S. pneumo from CSF

Oxacillin no longer will predict Penicillin susceptibility

Detection of Clindamycin resistance in Staph and Strep

Staphylococcus aureus & S. lugdunensis •  Use Erythromycin and Clindamycin disks 15-26 mm apart to detect inducible clindamycin resistance

D-shape = Clinda Resistance

No D shape = Clinda susceptible

20 mm

12 mm E CC

For beta-streptococci place disks 12-15 mm apart

Phenotypic testing of Enterobacteriaceae for some drugs

Amox + Clavulanate

Moxalact

Cefonicid

Cefoperaz

Cefamand

E. coli ATCC 25922

•  Cefamandole •  Cefoperazone •  Cefonicid •  Moxalactam

CRO detection: Not so accurate

Meropenem disk

KPC Neg Control

KPC positive organism

Modified Hodge Test Chromogenic agar

P. aeruginosa

K. pneumoniae

Broth dilution methods

Results dictated by guidelines

Disk zones:

EUCAST CLSI

S ≥ R < S ≥ R < Doripenem 22 17 22 19 Ertapenem - - 23 19 Imipenem 20 17 23 19 Meropenem 24 18 23 19

INH RIF

No abx No abx

2.0 µg/mL

0.4 µg/mL

2.5 µg/mL

7.5µg/mL

6.0 µg/mL

Microscopic Observation Drug Susceptibility Assay (MODS) for Mycobacterium tuberculosis; WHO endorsed

EMB

2.0 µg/mL

STREP 0.1 µg/mL

Results usually by Day 15

Day 7 Day 15

1. Add 100 µl of 20 mM Tris-HCl lysis buffer to two 1.5 ml microcentrifuge tubes

(B-PER II (bacterial protein extraction reagent; Thermo Scientific) 2. Pick growth from around carbapenem disk on

Mueller-Hinton plate 3. Add 1/3 of calibrated loop (10 µl) of growth to each

of the lysis buffer tubes and mix very thoroughly with pipette

4. Add phenol-Zinc sulfate to Tube 1 5. Add phenol-Zinc sulfate + 6 mg/ml Imipenem to

Tube 2 6. Incubate at 37ºC for 2 hours

Nordmann/Poirel Rapid Method for Detection of Carbapenemase

Lysis Buffer Buffer + Imipen

No C’pase Red Red Positive C’pase Red Orange/Yellow Indeterminate Yellow Yellow

BioMx Rapidec Carba NP

Not FDA-cleared yet. Many steps. Takes ~ 2 h total

Molecular methods are MOST accurate Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes. Cole et al. 2009; JCM 47:322.

• Only tiny number of labs able to develop such tests (no such commercial assays)

• New enzymes always being characterized, requiring constant modifications of PCR methods

Check-Points Microarray System

KPC VIM NDM OXA 48

Cartridge detects five classes of resistance genes in any genus • Sample: Isolate, perirectal swabs • Time to result: 48 minutes

Xpert Carba-R Assay

– blaKPC – blaNDM – blaVIM – blaOXA-48 & 181 – blaIMP-1

© Cepheid Proprietary and confidential

Xpert Carba-R Assay

© Cepheid Proprietary and confidential

MALDI-TOF and derivatives

Now: Abbott IRIDICA Delivers identification & some

resistant factors (mecA) in 8 hours

Rapid identification and antimicrobial susceptibility testing of bacteria in bloodstream infections using the Accelerate ID/AST technology. Price, et al. ECCMID 2015

Colorimetric Sensor Array D

iffer

ence

D

rug

C

ontr

ol

Rapid Determination of Antibiotic Influence and Susceptibility by Colorimetric Sensor Array Assessment of Small Molecule Metabolites. Lim et al. ASM GM 2014

K. pneumo in <5 hrs

Controls

rpoB mut’s 96% Sens

katG mut’s 35% Sens

inhA mut’s 35% Sens

HAIN GTMD (~5 hr TAT); WHO endorsed

Hain GenoType® MTBDRplus Assay

WHO endorsed

Example of Rif-Susceptible Profile All 5 probes are positive

Assay design uses RNA polymerase (rpoB) gene targets

Molecular Beacon

Target

Hybrid

Each probe is labeled with a different fluorophore, permitting simultaneous detection of the presence of wild type.

SPC

Improved assay: 4 probes identify rifampin-R mutations in rpoB by shifting their Tm away from a wild type reference value by melt curve analysis

Probes overlap rpoB sequence

A clear change in Tm distinguishes wild type from resistant mutant

Rif S Rif R

rpoB core region. Any mutation = Rifampin resistance

Xpert® MTB/RIF Ultra

July 2011. Vol.8, Issue 7 e1001061

Sequencing includes Mutations in katG, inhA, the inhA locus (inhA regulatory region), the oxyR-ahpC intergenic region (ahpC) and the entire rpoB gene.

•  Mutations in rpoB occur in 95-99% of Rif-R strains •  Rif-R is a good indicator of MDR-TB •  23 discrepancies between phenotype & genotype

S. aureus genome map of Res SNPs

S. aureus results of Sequencing program (Mykrobe) vs phenotype (3 minute computer run-time)

Sequencing Required: Best method = minION

Potential to reduce

sequencing time from 24 h to 7 h

New technologies are revolutionizing the detection of microbial resistance and improving healthcare worldwide