TITLE: Protocol for ESBL-EC recovery from stool samples
AUTHORS: Sarah Gallichan

[DESCRIPTION]
Understanding transmission pathways of important opportunistic, drug resistant pathogens, such as extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single nucleotide resolution of E. coli strains as well as E. coli diversity within and between samples is required. However, the microbiological methods to best track these pathogens are unclear. Here we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-hour pre-enrichment in Buffered Peptone Water, plating on cefotaxime supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole genome sequencing. We conclude that our optimised workflow can be applied for single nucleotide variant analysis of an ESBL-EC from stool.



[BEFORE_START]
Before starting, thoroughly clean the Class II MSC with Chemgene (5% solution) and 70% ethanol. Switch the Class II MSC on and leave it running until airflow is safe. All other solid waste is considered contaminated and must be disposed of by autoclaving followed by incineration.

[GUIDELINES]
All the samples should be treated as potentially infectious. Therefore, samples must only be opened in a class II Microbiological Safety Cabinet (MSC) until considered inactivated. The use of lidded, aerosol-tight centrifuge buckets is compulsory. 


[STEPS]
SECTION: Reagent preparation
1. Preparation of Sterile Buffered Peptone Water (BPW) pre-enrichment broth
SECTION: Reagent preparation
1.1. Add 20 g  of BPW powder to 1 L  of distilled water.

SECTION: Reagent preparation
1.2. Mix well and sterilise by autoclaving at 121 °C .

SECTION: Reagent preparation
1.3. Store at room temperature for up to 1 month.
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1.4. Aliquot 5 mL into 15ml falcon tubes in preparation for samples.

SECTION: Reagent preparation
4. Preparation of Stool Solution (adapted from maltodextrin:trehelose method in Burz et al. 2019)
SECTION: Reagent preparation
4.1. Dissolve 9.4 g  of maltodextrin and 3.1 g of trehalose in 450 mL  of PBS.

SECTION: Reagent preparation
4.2. Put into a waterbath set to 40 °C until dissolved.

SECTION: Reagent preparation
4.3. Add 50 mL glycerol (to give a final concentration of 10 % volume glycerol).

SECTION: Reagent preparation
4.4. Filter sterilise.
SECTION: Reagent preparation
4.5. Aliquot 500 µL into cryovials. Cryovials can be stored at Room temperature for up to 6 months. 2 cryovials will be needed for each sample.

SECTION: Sample processing
5. Stool and rectal swab processing
SECTION: Sample processing
5.1. A) Swabs: Transfer the rectal swab into a 15mL falcon tube containing 5 mL  of BPW enrichment broth. Cut the end of the swab and replace the cap of the falcon tube.

B) Stool samples: weigh stool sample pot (use an empty pot to zero the balance) and resuspend in sample in stool solution. Use 5x the volume of stool solution per g of stool (e.g. 0.5g stool = 2.5ml of stool solution). Add a small stirrer bar to the pot and place on a magnetic stirrer to agitate the sample. Vortex sample to resuspend if necessary. Add 100 µL  of resuspended stool into a 15mL falcon tube containing5 mL of BPW.


SECTION: Sample processing
5.2. Incubate the tubes shaking at   for 4 hours.

SECTION: Sample processing
5.3. After 4 hours, transfer samples to the Class II microbiological safety cabinet (MSC) and streak each sample out onto MacConkey agar supplemented with 1 µg/mL cefotaxime using a 1 µL loop. Incubate the plates overnight at 37 °C .

SECTION: Sample processing
6. DNA extraction
SECTION: Sample processing
5.4. Pick several positive morphology colonies (colonies that appear red/pink on MacConkey agar) and streak each colony onto a separate section of a new MacConkey agar plate supplemented with  1 µg/mL cefotaxime. Incubate the plates overnight at 37 °C.
SECTION: Sample processing
6.1.  Transfer a 1 µL loopful of each colony restreak to a 1.5 mL tube. 

SECTION: Reagent preparation
2. Preparation of cefotaxime stock solution
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2.1. Dissolve cefotaxime powder in water to 200 mg/mL. 

SECTION: Reagent preparation
2.3. Store at -20 °C.

SECTION: Reagent preparation
2.2. Dilute this to 10 mg/mL  in water to make working stock solution.
SECTION: Reagent preparation
3. Preparation of MacConkey agar
SECTION: Sample processing
6.2. Extract DNA according to the Lucigen MasterPure Complete DNA and RNA Purification kit manual, available at: https://biosearchtech.a.bigcontent.io/v1/static/manual_NAEXPU-001_MasterPure-Complete
SECTION: Reagent preparation
3.1. Suspend 50 g  in 1 L  distilled water. 

SECTION: Reagent preparation
3.2. Mix well and sterilise by autoclaving at 121 °C.
SECTION: Reagent preparation
3.3. Cool to 50 °C in a water bath and add cefotaxime to a final concentration of 1 µg/mL.

SECTION: Reagent preparation
3.4. Pour into sterile petri dishes and allow to dry, then store at 4 °C  for up to 1 month.  

SECTION: Sample processing
6.3. Measure the concentration of all DNA extracts using a Qubit according to the manufacturer's recommendations (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_dsDNA_BR_Assay_UG.pdf).