Department of Microbiology
Policy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis

Pulsed-field Gel Electrophoresis

Table of Contents

I.Introduction

Isolates for Referred-Out PFGE

IV.Procedure

Pulsed-field Bench Workflow

Testing schedule

Test ordering, broth labeling and inoculation in preparation for typing

Cell Extraction Preparation

For MRSA, VRE, GAS, GNB

For Serratia marcescens

Settings for CHEF-DR II/III instruments

CHEF-DR II/III Module

Ethidium bromide for staining

Weekly Ethidium Bromide Disposal

Weekly Ethidium Bromide Preparation

Staining Protocol

How To Use The Gel DOC XR+ Camera

BioNumerics Gel Analysis

BioNumerics Naming CMRSA

IV.Reporting

PFGE Reporting Phrases

Verifying Bionumerics Results

V.Processing a Comparison Request (In-house and for PHL)

Documenting Request

Comparing and Interpreting a Cluster in Bionumerics

Interpretation Example

Sending a Report by E-mail

Appendix I - Documenting Comparison Requests in LIS

Appendix II - Thiourea Use for PFGE protocol

Appendix III - Maintenance and Quality Control

Maintenance and QC CHEF PFGE Machines

Before each run

Weekly

Monthly

Water flush and temperature check.

Maintenance of Gel Run Record

Appendix IV - Preparing PFGE Plugs of the Salmonella ser Branderup H9812 Standard Strain

Appendix V - Preparing PFGE plugs for Serratia marcescens

Preparing the plugs

Appendix VI - Reagents Preparation

PFGE Reagents for Gram Positives

PFGE Reagents for Gram Negative Bacilli

PFGE Reagents for Serratia marcescens

Stock Reagents

TRIS Buffer Chart

Appendix VII - Calculations for Restriction Enzymes, MBI Fermentas Fast Digest enzyme/ buffer Chart

ENZYMES

Appendix VIII - To Send Isolates To NML For: Spa typing

Appendix VIII – Reference Material

Record of Edited Revisions

Appendix VIII – Reference Material: added folder of reference material PDFs

I.Introduction

Pulsed field gel electrophoresis (PFGE) typing or macro-restriction of micro-organisms is performedfor epidemiological purposes to determine the relatedness between the chromosomal DNA of two or more isolates.

PFGE assists the Infection Control department by:

  1. Providing an understanding of the transmission of isolates between patients.
  2. Providing evidence of transmission of isolates from the environment to patients
  3. Determining whether isolates involved in recurrent infections are the same clone or are newly acquired.
  1. Specimen Collection

Isolates for PFGE (Routinely Done) In-House

ISOLATES FOR PFGE IN-HOUSE / UHN / MSH
New MRSA / No / Yes2
New VRE / No / Yes1
New Group A Streptococcus / No / Yes3
Any organism as part of a cluster or upon ICP's request / Yes / Yes
  1. 1st time isolated and once a year there after.
  2. MSH New = 1st time isolated and every 3months thereafter, if no other positive in between isolates.
  3. In-patient onlyor if identified by infection control to be a nosocomial GAS.

Isolates for Referred-Out PFGE

ISOLATES FOR PFGE PHL / Baycrest
New MRSA / Yes1
Any organism as part of a cluster upon ICP's request / Yes

1. 1st time isolated and every 3months thereafter if no other positive in between isolates.

Note: VRE not routinely sent out.

III.Reagents / Materials / Media

See Analytical Process - Bacteriology Reagents/Materials/Media List QPCMI10001

LABORATORY MANUAL

UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY

NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be checked against the document (titled as above) on the server prior to use.

T:\Microbiology\New Manual\Live Manual\IC\Infection Control Pulsed-field Gel Electrophoresis.doc

Department of Microbiology
Policy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis

IV.Procedure

Pulsed-field Bench Workflow

MONDAY / TUESDAY / WEDNESDAY / THURSDAY / FRIDAY
PFGE 1 /
  1. Retrieve incubated PF BHIB isolates from incubator.
  2. Check Bionumerics database to see if done previously. Refer to chart above for testing frequency
  3. Process isolates up until “wash stage"( can be left washing overnight)
  4. Enter isolates in Bionumerics, LIS (plug date and PF done in isolate window)
  5. Create gel legends for each type and log into “Gel Run Record” (in T drive)
  6. Document incubator temperatures, lot number and expiry dates for enzymes on gel legends
  7. Check for new queries Respond ETA for results
  8. Answer queries.
  9. Retrieve any query isolates as necessary.
/
  1. Prepare isolates for digest
  2. Prepare machines (drain, rinse, clean, change buffer, inspect for broken electrodes)
  3. Make agarose for each gel, load plugs onto combs, affix with agarose, chill, pour gel, chill and load
  4. Document machine maintenance
  5. Wash glass wash containers and prepare for autoclaving.
  6. Send green caps for washing.
  7. Send glassware for washing
  8. Check for new queries Respond ETA for results
  9. Answer queries.
  10. Retrieve any query isolates as necessary.
  11. Destain
/
  1. Make fresh stain
  2. Stain/ Destain and photograph gels set up on Tuesday.
  3. File and export data from images photographed into Bionumerics.
  4. Normalize gels from Tuesday's run.
  5. Name MRSA’s.
  6. Report in LIS.
  7. Prepare green caps for autoclaving
  8. Answer queries.
  9. Subculture send out organisms as required
  10. Check for new queries Respond ETA for results
  11. Answer queries.
  12. Retrieve any query isolates as necessary.
/
  1. Check for new queries Respond ETA for result
  2. Answer queries.
  3. Finish reporting queries as required
  4. Make reagents
  5. Prepare aliquots of enzymes
  6. Check inventory
  7. Order enzymes, supplies
  8. Restock supplies
  9. Prepare H9812 plugs as required
  10. Discard plugs > 3 months old
  11. Update Bionumerics database (Cleaning)
  12. Retrieve any query isolates as necessary.
/
  1. Check for new queries Respond ETA result
  2. Answer queries.
  3. Check Didi worklist in LIS for PF ordered but not given to bench
  4. Finish reporting queries as required
  5. Prepare for Mondays run (Monday’s steps 1, 2 & 4.) Labels can than be grouped and placed in box labeled “Labels for PFGE in Progress”
  6. Weekly-check electrodes Monthly- temperature check; Flow rate check and adjustment
  7. Retrieve any query isolates as necessary.

PFGE 1/2 / Verifiy results from Monday's run

LABORATORY MANUAL

UNIVERSITY HEALTH NETWORK/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY

NOTE: This is a CONTROLLED document for internal use only. Any documents appearing in paper form are not controlled and should be checked against the document (titled as above) on the server prior to use.

T:\Microbiology\New Manual\Live Manual\IC\Infection Control Pulsed-field Gel Electrophoresis.doc

Department of Microbiology
Policy & Procedure Manual / Policy # MI\IC\PF\v17 / Page 1 of 51
Section: Infection Control Manual / Subject Title: Infection Control Bench Pulsed-field Gel Electrophoresis

Testing schedule

  • PFGE is run weekly in-house, refrigerate pre-inoculated broths/plates from Monday until Sunday.
  • Incubate broths/plates at 37ºC in walk in incubator on Sunday @ 4:00p.m. for PFGE processing on Monday.
  • The run incubated on Sunday finishes on Wednesday

N.B

A failed or additional run can be done on Thursday (or when possible Wednesday to Friday) if required. It is fine to let it run over the weekend and attend to it on Monday.

Test ordering, broth labeling and inoculation in preparation for typing

Isolates for typing come from different benches, but are handled in the same manner.

Routine Benches

When an isolate qualifies for PFGE:-

  1. Order ^BHIB and ^PF in LIS.
  2. FromPF, select isolate type from keypad(i.e. MRSA, VRE, GNB etc).
  3. Go to the print label screen, and print three(3) labels.
  4. Attach one large label to 10ml BHI broth for gram positive organisms. Alternately, a Mueller Hinton plate can be used for gram negative organisms. Write organism name (i.e. MRSA, VRE, etc) and any other relevant information e.g. (ESBL Class A-LF) on the label.
  5. Touch 1-3 colonies(dependent on colony size) of pure growth with a loop and inoculate the broth/plate.
  6. Put inoculated broth/plate into rack in PFGE fridge for batch testing.
  7. The MRSA bench technologist on the weekend is responsible for placing the BHIB rack into the 37oC walk-in incubator on Sunday @4:00p.m. and the PFGE The only exceptions to this protocol are:
  1. Streptococcucs pneumoniae- must be inoculated just prior to incubation onto 2 BA plates (CO2).

PFGE Bench

  1. Retrieve isolates for PFGE from shaker and plate rack
  2. Put Brain heart infusion tubes / plates in order (alphabetical) within groups
  3. Print required labels
  4. Put labels in the same order within groups
  5. Match labels to isolates
  6. Place cultures @4oC until ready for processing
  7. Using labels check appropriate Bionumerics database MRSA( UHN-MSH MRSA) GNB (UHN-MSH ESBL-MDRO) for repeats(right click on Last Name column and choose arrange by field for alphabetical order)

If an unwarranted repeat, document in LIS (Prev see LIS#...... ) at back of workcard.

  1. Put labels in numerical order with groups. Assign processing numbers to isolates Write processing numbers on labels (1 large label 3 small labels) and on matching BHI tubes

IN LIS

  1. Go to worklist :ZDIDI
  2. Mark all isolates document plug date under PF

(If isolate not on work list order PF)

  1. Go to individual isolates and add isolate letter and pfdone in isolate window
  2. Ensure that the appropriate organism has been ordered in the PF window in LIS. i.e.`MRSA`
  3. Check isolates within date range of order numbers that have not been marked.
  4. Investigate as to why broth is not received.
  5. Document in LIS- PF that broth was not received and request isolate from bench.

IN BIONUMERICS

  1. Add new entries

N.B:

a. Copy and paste demographics from LIS to avoid transcription errors.

b.Choose Organism name and comment (ESBL class A…etc.) from drop down menu for consistency.

Cell Extraction Preparation

Routine Bench

  1. Subculture organism from pure growth to 10mL BHI broth
  2. Refrigerate broths Monday to Sunday. Incubate at 37ºC on Sunday @ 4:00p.m.for PFGE processing on Monday. (For long weekends that have holiday Mondays, incubate on Monday.)

PFGE Bench Monday

  1. Determine isolates for PFGE by checking Bionumerics and LIS databases to eliminate repeats and account for missed isolates.

For MRSA, VRE, GAS, GNB

Step 1 Preparation:

1.Turn on 60ºC, 37 ºC water-bath and adjust the water levels as appropriate.

2.Ensure incubator/shaker on at 55 ºC

3.Calculate how much of each enzyme is necessary for lysis (see Table 1)

TABLE 1: LYSIS ENZYME TABLE

Lysozyme L10 (10mg/mL) / Lysostaphin 1mg/mL (Sigma) / Lysozyme L100
(100mg/mL) / Mutanolysin 5U/µL (Sigma) / Proteinase K 20mg/mL (Roche)
MRSA / 50µL per tube / 10µL per tube / 40µL per tube
VRE or GAS / 40µL per tube / 15 µL per tube / 40µL per tube
GNB / 40µL per tube

4.Remove enzymes from freezer and allow them to thaw. Keep @4 ºC until ready for use.

5.Alphabetize labels for VRE, MRSA, etc and check Bionumerics for previous. Discard repeats. See chart above to determine testing frequency.

6.Put labels in numerical order and BHIB to match.

7.Label BHIB lids and labels 1, 2 etc.

8.Label 2 sets of 1.5ml microfuge tube with the assigned number for each sample to be sub-typed. (i.e. 1, 2,3 etc) (blue for MRSA, black for VRE, red for GNB)

9.Label 1 Vk. Tube for each (1, 2 etc.) (colour coded as above)

10.Aliquot 1.5 mls PIV Buffer (GP) into each tube Vitek tube. (SE buffer for GNB)

11.Label white-capped tubes for each isolate. (colour coded as above) (1, 2 etc.)

12.Label disposable plug molds 2 spots per isolate. (colour coded as above)

NB: If unable to proceed with making plugs this is a good place to break off.

13.Prepare 1% Seakem Gold Agarose(SKG) in Sterile de-ionized water(SDH2O) for making all plugs.(0.1gm SKG in 10 mL SDH2O or .2gm SKG in 20mL SDH2O). Use 50mL tube.

14.Place tube in beaker with water and dissolve agarose in microwave for 30 secs to 1 min (check at 30 sections). Keep in 58ºC water-bath until ready for use.

Step 2 Standardization:

  1. Transfer broth from BHIB’s using transfer pipette into labeled microfuge tubes, centrifuge @ 14,000 rpm for 1 minute to pellet.
  2. Pipette off supernatant. Change pipette.
  3. Calibrate Vitek colorimeter.
  4. Re-suspend pellet with small amount of PIV buffer from the pre-labelled Vk. tube and make 20% transmittance suspension for each isolate (SE buffer for Gram negatives) Vortex to make a smooth suspension
  5. Add 200 uls (using 1000ls pipette) of bacterial cell suspension to each labelled microfuge tube.
  6. Add 200 uls (using 200ls pipette) 1% SeaKem Gold agarose . Mix and fill two plug mold spots. (Make sure that there are no bubbles and that the molds have not been overfilled)
  7. Do this for each isolate. Solidify at 4C 5-10 minutes.

Step 3 Lysis & Preparation:

MRSA Lysis Enzymes

Make a master mix for lysis in 50 ml conical tube

  • 2 mLEC Lysis Buffer per test plus 1
  • 50 uls L10 (lysozyme) per test plus 1
  • 10 uL Lysostaphin 1 mg/mL per test plus 1

Document lot # on gel legend sheet.

  • Aliquot 2mlsEC Lysis Buffer/L10 mixture to labeled white-capped tubes for each isolate

VRE Lysis Enzymes

Make a master mix for lysis in 50 ml conical tube

  • 2 mLEC Lysis Buffer per test plus 1
  • 40 uls L100 (lysozyme) per test plus 1
  • 15 uL Mutanolysin(50000 U/mL) per test plus 1

Document lot # on gel legend sheet.

  • Aliquot 2mlsEC Lysis Buffer/L10 mixture to labeled white-capped tubes for each isolate

* NOTE: NO LYSIS STEP FOR GRAM NEGATIVES! (Proceed to step 4.)

1.Remove caps from tubes containing lysis buffer

2.Break off plungers from disposable plug molds. Clean with alcohol and dry with Kim wipe. Remove tape from bottom of wells and dispense plugs into the appropriate tubes by inserting plunger into well and tapping the top to remove plugs. Ensure plugs are immersed in buffer!

3. Incubate 3 hrs at 37C on shaker

TABLE 2:

ORGANISM / LYSIS BUFFER / PK BUFFER
Incubation Temperature / Incubation Time / Incubation Temperature / Incubation Time
MRSA / 37ºC for / 3hrs minimum / 55ºC / 1hr
VRE and GAS / 37ºC for / 3hrs minimum / 55ºC / 1hr
Enterobacteriacae and Other gram negative bacilli / N/A / N/A / 55ºC / 3hrs minimum

4.Make gel legends. (7 tests for 10 well gel and 11 for 15 well) Use small label with barcode for legend

5.Assign GEL ID # T:\Microbiology\BioNumerics\Gel run record

6.Make sure that there is 1 ladder every 5-6 wells. Record date made and date digested.

7.Write organism names on the legends.

8.Label 6-well storage plate for each isolate

9.Dispense adequate amount (2.5mls per isolate) of Gram +ve Wash Buffer (TE buffer) and Gram –ve Wash buffer into two 50 ml. conical tubes

10.Pipette 2.5mls Gram +ve Wash Buffer (TE buffer) or 2.5mls Gram –ve Wash buffer into each well of 6-well storage plate.

11.Pre-warm bottle of SDH2O @ 55C.

12.Pre-warm adequate Wash Buffers (Gram +ve and Gram negative) in 55C incubator.

13.Assemble the required amount of numbered green screen caps for wash step (put in ascending order, lowest numbers on bottom)

14.Label microfuge tubes according to gel legend positions for restriction digest.Include tubes for standards (H9812) also.

15.Aliquot 750 uL SDH2O into prelabeled microtubes

16.Enter plug date and PF in isolate window in LIS and enter into Bionumerics.

Step 4Removal Of Interfering Proteins

1.Calculate PK Buffer needed for each tube (2.0ml PKB and 40ul PK 50) and dispense into pre-labelled white-capped tubes

2.Document Lot # for PK

For Gram negatives:

3.Break off plungers from disposable plug molds. Clean with alcohol and dry with Kim wipe. Remove tape from bottom of wells and dispense plugs into the appropriate tubes by inserting plunger into well and tapping the top to remove plugs. Ensure plugs are immersed in PK solution! Incubate on shaker 55C 3 hours.

For Gram positives:

4.Decant lysis buffer into petri dish, using spatula.

5.Pipette 2 mls PK buffer mixture into each tube. Ensure plugs are immersed in PK solution! Incubate on shaker 55C 30 min.

Step 5 Plug Washing:

  1. Retrieve tubes from incubator
  2. Put green appropriately labeled screw-cap on a 50 ml centrifuge tube labeled “Discard” and decant P K buffer solution for each isolate, stacking one on top of the other as you go. (Don’t exceed 6 caps, before starting a new stack)
  3. Place green cap on top of each stack
  4. Remove stack from discard centrifuge tube
  5. Discard decanted PK buffer
  6. Return stack to the centrifuge tube
  7. Pour 50.0ml pre-warmed SDH2O in a sterile centrifuge tube labeled “SDH2O” and pour through the assembled screw caps to rinse
  8. Place blue cap of a clean 50ml tube on the last cap sealing the top of the last green cap on top of the stack
  9. Invert sideways to rinse through. (3 times)
  10. Decant. Repeat steps 7-9, 2 more times.
  11. Load assembled screw caps intoglass wash dishes.
  12. Pour enough TE buffer (or Gram –ve Wash buffer for Gram negatives) to fully submerge green caps. Seal dishes tightlyand wash for a minimum 60 minutes@ 55C on shaker and then decant. (You can wash overnight at this point.)

After wash is complete:

1.Pour pre-warmed 50.0ml SDH2O in a sterile centrifuge tube and pour through the assembled screw caps to rinse

2.Discard wash buffer in sink

3.Tap green caps on desk to shake plugs into bottom of cap.

4.Transfer plugs from green caps into corresponding 6 well storage plate.

N.B. OPTIONAL: Store plugs overnight or proceed to next step

Step 6 Restriction Enzyme Digestion of Plugs:

1.Calculate the amount of SDH2O, digest buffer and digest enzyme needed for the digest mixture (see chart)(SeeT:\Microbiology\BioNumerics\Protocols\Calculation for Enzyme and buffer chart.doc

2.Put plugs into to pre-labeled 6-well plate (plugs can be stored at 4C at this point)

3. Retrieve 1/2 plug with spatula and place it into the appropriate pre-labeled microfuge tube with 250ul of SDH2O. Rotate for 15 minutes@ RT (25C) to equilibrate plug

4.Pipette off all of the SDH2O from each tube.

TABLE 3.

ENZYME / Fast digest Sma 1 MBI / Fast digest Xba 1 / Fast digest Spe1 for Pseudomonas / Stenotrophomonas / Fast digest Sfi1 Acinetobacter
QUANTITY per sample / 10µL / 5 µL / 5 µL / 10 µL
INCUBATION TIME / 3hrs-O/N / 3hrs- / 3hrs-O/N / 3hrs-O/N
INCUBATION TEMPERATURE / 37ºC / 37ºC / 37ºC / 50 ºC
Buffer / FD buffer / FD buffer / FD buffer / FD buffer

5.Aliquot 200ls of the Enzyme/10x buffer mixture into each tube.