summary of day before/ introduction to today’s …...dtu food, technical university of denmark ....

SUMMARY OF DAY BEFORE/ INTRODUCTION TO TODAY’S PROGRAMME

Lina Cavaco

EURL-AR Training Course November 2013

2 DTU Food, Technical University of Denmark EU Reference Laboratory for Antimicrobial Resistance (EURL-AR www.eurl-ar.eu

YESTERDAY

• LECTURES –EFSA presentation on system for data reporting –EUCAST presentation on methods and ressources

available at EUCAST

• LAB WORK –Theme 1B –Theme 2A

• SOCIAL EVENT

http://www.eurl-ar.eu/�





LABORATORY theme 1B

• WHEN – Wednesday

• WHAT?

– Species ID by PCR methods

• WHY?

– For identification of Enterococci (E. faecalis and E. faecium) and Campylobacter spp(C. coli and C.jejuni) of isolates obtained from mixed cultures






Enterococci Mixes to subcultures

Slanetz species strain

mix x E. faecalis ATCC 19433

mix x E. faecium ATCC 19434

mix x E. muntii ATCC 43186




5 DTU Food, Technical University of Denmark

Primer 1 (forward): 1551+1553

Primer 2 (reverse): 1552+1554

DNA polymerase: VWR taq

PCR products: EF (941 bp); EFM (550bp)

Multiplex Enterococci species ID

Remarks: 5 µl of the DNA template. Run: 1.5% agarose gel run at 130V for 30min Reference: Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol. 1995 Jan;33(1):24-7.

1. 5 min at 94 °C 2. 30 Cycles

30 sec at 94 °C 90 sec at 50 °C 60 sec at 72 °C

3. 10 min at 72 °C 4.

hold at

4

°C

1 616,125 96,75

2,5 150,25 1,5

0 00,5 30,5 3

0,125 0,75

20 120

Taq polymerase

Total volume

25 mM MgCl2Primer1 (0,25 μl of each)Primer2 (0,25 μl of each)

No. of reactionsPCR H2O10X PCR Extra BufferdNTP

Please note that in this protocol internal control is not included.

Furthermore that this multiplex PCR protocol was optimized with VWR Taq polimerase and 10X Extra buffer



LABORATORY theme 1C

• WHEN – Thursday

• WHAT?

– Gel pictures of the PCR for species ID on the isolates obtained from the selective agar plates from Tuesday

• WHY?

– For determination of species identification of isolates and cathegorization into relevant species






M: 100 bp Plus

1 E. faecalis ATCC 19433

2 E. faecium ATCC 19434

3 E. durans

4 E. hirae 5 E. munti 6 E. faecalis ATCC 29212

7 E. faecium BM 4147 8 Mastermix

1 2 3 4 5 6 7 8

941 bp E. faecalis

550 bp E. faecium

Theme 1B- Fotos from PCR’s done during preparation-

Enterococci identification multiplex PCR






• Enterococci

Examples of photos from PCR’s done by the groups

Group 1 Group 2

Group 3 Group 4

Group 5 Group 6

Group 7 Group 8






Campylobacter Mixes to subcultures

mix - CCDA Campylobacter coli EURL C-6.8

mix - CCDA Campylobacter jejuni EURL C-6.3

mix - CCDA

Campylobacter lari ATCC 35221






Remarks: 0.5µl of the DNA template. Run: 1.5% agarose gel run at 100V for 45 minutes

Reference: M. Denis, C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat and P. Colin. Development of a m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli. Letters in Applied Microbiology 1999, 29, 406–410

1 69,5 57

12,5 750 00 0

1,5 91,5 90 0

25 150

Taq polymerase

Total volume

25 mM MgCl2Primer F (0,50 μl of each)x3

Primer R (0,50 μl of each)x3

No. of reactionsPCR H2O2xGreen PCR Master MixdNTP

Multiplex PCR Campylobacter species ID

1. 10 min at

95 °C

2. 30 Cycles 30 sec at 94 °C 90 sec at 59 °C 60 sec at 72 °C

3. 10 min at

72 °C

Primer F (forward): 442+3034+3036

Primer R (reverse): 444+3035+3037

DNA polymerase: Dream Taq Green PCR master mix

PCR products: 16S – 800 bp; mapA – 589 bp; ceuE – 462 bp

Please note inclusion of 16S internal control besides the species markers included for DNA amplification control

Furthermore, this multiplex PCR protocol was optimized with Green Master mix from Fermentas in this protocol (easier mix and no need for loading buffer in gel loading)






M: 100 bp Plus

1 C. coli EURL C-6.8

2 C. coli EURL C-6.5

3 C. jejuni EURL C-6.3

4 C. jejuni EURL C-7.6 5 C. jejuni ATCC 33560 6 C. coli CCUG 11283 7 C.jejuni CCUG 11284 8 C. lari Rød 9,3 4-5 9

C. coli ATCC 33559 NB – no band (incl 16S) 10 mastermix

1 2 3 4 5 6 7 8 9 10

Importance of 16S internal control

800bp 16S

589bp mapA

463bp ceuE

Theme 1B- Fotos from PCR’s done during preparation-

Campylobacter jejuni/coli identification multiplex PCR






• Campylobacter


Group 1 Group 2 Group 3 Group 4







• Campylobacter











• Campylobacter



Group 21






LABORATORY theme 2A

• WHEN – Wednesday

• WHAT?

– AST testing of Campylobacter, Enterococci and Salmonella isolates (incl ESBL-producing strains) by microdilution assays

• WHY?

– For determination of MIC and assign susceptibility profiles including ESBL/AmpC confirmation






What you have done Yesterday on theme 2A

Results reading in LAB theme 2B, some already strated, Campylobacter after lunch!






Broth dilution media •Broth dilution

–Use young culture of test strain grown on non selective medium

–Adjust innocullum using Mc Farland standard 0,5 and nephelometer

–Use Cation adjusted Mueller Hinton Broth (not adequate for fastidious organisms)

–Use cation adjusted Mueller Hinton Broth supplemented with lysed blood for Campylobaster AST






Broth dilution procedures •Broth dilution

–Inoculate plate • Use the autoinoculator or a multichannel pipette and

drop a fixed volume into each well (depends on plate design/concentrations)

• Prepare purity control • Seal the plates






Broth dilution procedures •Broth dilution

– Incubate at standard temperature/time (depending on organism)

– Incubate 35+-2C for 16-20h (some exceptions)

• Check purity control • Read plates after incubation to determine end-ponts (observe first control

wells and check plate for skips • Disregard slight growth for sulphonamides and trimethroprim as these

are bacteriostatic, other wise read using manual or autoimatic devices






Broth Microdilution

MIC Drug Concentration

A

0.25 0.5 1 2 4 8 16 32 64 0.03 0.06 0.12

B

C

D

E

F

G

H

2

Repeat (?)

Repeat

.

Controls

8

≤0.03

>64

Repeat

0.06






Dilution Testing • Dilution methods do not

necessarily give exactly the same result each time, despite strict standardization

• The generally accepted methodological variation of dilution methods is + one dilution range of the "true" MIC value






Factors that influence the result of AST:

- Size of inoculum - Contents and acidity (pH) of the broth or

agar - Cation content (Ca, Mg) - Incubation time and temperature - Reading procedures

DTU Food, Technical University of Denmark 22

QC and standardisation – our world

To obtain reliable and reproducible data => standardized methods and quality assurance are required






Frequency of QC testing

• Each test day for organisms tested infrequently –One out of 20 consecutive results may be out of rang –>one result out of range requires corrective action

• Conversion from daily to weekly testing

–Test all applicable QC reference strains for 30 consecutive test days and document results

–Weekly testing may be performed once satisfactory performance is documented





24 DTU Food, Technical University of Denmark EU Reference Laboratory for Antimicrobial Resistance (EURL-AR www.eurl-ar.eu DTU Food, Technical University of Denmark 24

Quality Assurance – inoculum check

• Inoculum should be adjusted to give a final cell number concentration of 5*105 CFU/mL (range 2*105 CFU/mL to 8*105 CFU/mL)- perform checks of CFU at least quarterly

Calculation example for micropanels containing dried antimicrobial: 4mL 0.9% NaCl adjusted to 0.5 McFarland => 1*108 CFU/mL 50µL is added to 10mL broth (CAMH or CAMH+lysed blood) => 5*105

CFU/mL






TODAY

• LECTURES: –ESBL and carbapenem-producing microorganisms, state

of the art- Laurent Poirel

• LAB WORK: –Theme 1C (gel pictures taken this morning) –Theme 2B (reading of MIC panels in the afternoon)






Questions??






On behalf of the EURL-AR team:

Have a very nice day! Before you leave, please remember to fill out and deliver to us the evaluation forms!




summary of day before/ introduction to today’s …...dtu food, technical university of denmark ....

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