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Daniel C. Spencer

Teagan Paton

Timothy J.J. Inglis

J. Mark Sutton

Hywel Morgan

A fast impedance-based antimicrobial susceptibility test

In this protocol an actively dividing bacterial isolate is incubated in the presence of the antibiotic for 30 minutes, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 minutes. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window.

Cell mediaTryptic soy broth (TSB)&nbsp;[Sigma: 22092]Cation Adjusted Mueller Hinton Broth (MHB) [Sigma: 90922]Hanks media&nbsp;[Sigma: H6648]Agar plates [Sigma L5542] AntibioticsMeropenem [Sigma M2574]Colistin [Sigma 1148001]Gentamicin [Sigma G4918] Ciprofloxacin – [Sigma 17850]Amoxicillin/Clavulanic acid [Sigma A8523 and Sigma 33454]Cefoxitin [Sigma 1098107] Reference ParticlesPolybead® Microspheres 1.50μm [Polysciences 17133-15] Bacterial isolatesKlebsiella pneumoniae strain 43358Klebsiella pneumoniae strain ATCC 700603Klebsiella pneumoniae strain 18397Klebsiella pneumoniae strain 43292Klebsiella pneumoniae strain 44271Klebsiella pneumoniae strain KS1Klebsiella pneumoniae strain KS11Klebsiella pneumoniae strain K1Klebsiella pneumoniae strain 1705Klebsiella pneumoniae strain K14Acinetobacter baumannii strain NCTC 13424Acinetobacter baumannii strain ATCC 17978Klebsiella pneumoniae strain 51851Klebsiella pneumoniae strain NCTC 13368Klebsiella pneumoniae strain CFI-014Pseudomonas aeruginosa strain NCTC 13437Pseudomonas aeruginosa strain PA01Escherichia coli strain CFL 161Escherichia coli strain NCTC 12923Klebsiella pneumoniae strain51851Klebsiella pneumoniae strain NCTC 13438Klebsiella pneumoniae strain 51851Klebsiella pneumoniae strain TW3Klebsiella pneumoniae strain NCTC 13368Klebsiella pneumoniae strain M6Klebsiella pneumoniae strain NCTC 13368Klebsiella pneumoniae strain NCTC 5054Klebsiella pneumoniae strain NCTC 13438Klebsiella pneumoniae strain M6Escherichia coli strain LEC001Escherichia coli strain NCTC 12923Staphylococcus aureus strain NCTC 13616Staphylococcus aureus strain NCTC 6571

--Zurich Instruments HF2IS impedance analyser (Zurich Instruments AG, Zurich, Switzerland)--Shaking incubator set to 36-37oC with an air atmosphere.--Vortex mixer for falcon and eppendorf tubes--Single cell impedance cytometer glass chips. Custom - see procedure section for chip fabrication--Single cell impedance cytometer electronics hardware -see application note here for an example https://www.zhinst.com/sites/default/files/documents/2020-03/zi_hf2is_appnote_microfluidics_2020.pdf--A syringe pump (set to 30uL/min with a 1mL syringe, e.g. KD Scientific 788101, Harvard Apparatus 70-4500)--Adjustable pipettes (P10, P20, P300, P1000)

Protocol 1Step 1. Take a single colony of bacteria from a refrigerated or frozen plate, add to 10mL of Tryptic soy broth (TSB) and place in an incubator overnight at 37oC to revive the cells. This is sample A.Step 2. Add a 1uL aliquot of sample A to an eppendorf containing 1mL Hanks media and vortex for 1 minute to ensure the sample is thoroughly mixed.Step 3. Dilute sample A 1000-fold (1uL in 1mL Hanks) and measure the concentration of cells using the impedance cytometer (see impedance cytometer measurements section later for further details).Step 4. Take an aliquot of sample A and dilute such that the final concentration (called sample B) is 5x105 cells/mL; From step 3, if the concentration of sample A is N cells/mL, add a volume of sample A given by V= N / 5x106 (uL) to 10mL MHB, such that the final concentration (called sample B) is 5x105 cells/mL. Typically sample A will be between 108 and 109 cells/mL, and therefore the volume V will be between 10-100uL.Step 5. Incubate sample B in a shaking incubator for 30 minutes at 37oC.Step 6. Add aliquots (950uL) to 7 pre-warmed test tubes each containing 50uL MHB and Meropenem at a concentration of 0, 5, 10, 20, 40, 80 and 160 mg/L of meropenem (final antibiotic concentration is 20x lower; i.e. 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L.)Step 7. Incubate the tubes for 30 minutes at 37oC.Step 8. Wash the cells and resuspend in Hanks media (spin at 10,000*g for 5 minutes, remove the supernatant and top up to 1mL)Step 9. Dilute each sample 1:10 (add 100uL from each of the 7 eppendorfs to 7 new eppendorfs containing 900uL Hanks).Step 10. Add a volume of concentrated bead suspension that contains approximately 104 polystyrene beads (1.5um diameter) to each eppendorf.Step 11. Load the contents of each tube into a 1mL syringe and measure with the impedance cytometer (see later). Protocol 2.Step 1. Streak out a 10uL loop from a frozen glycerol stock onto an agar plate. Place the plate into an incubator overnight at 37oC.Step 2. Pick three typical colonies from the plate and add to 3mL of MHB - this is sample A.Step 3. Vortex sample A for 1 minute to ensure that it is well mixed.Step 4. Dilute sample A 400-fold (2.5uL in 1mL Hanks) and measure using the impedance cytometer to determine the concentration of the bacteria in sample A (see impedance cytometer measurements section later for further details).Step 5. Assuming the concentration of sample A is N cells/mL, add a volume of sample A given by V=N / 15x105 uL to 3mL MHB, such that the final concentration (called sample B) is 5x105 cells/mL. Typically sample A will be approximately 108 cells/mL, and therefore the volume V will be approximately 15uL.Step 5. Incubate sample B in a shaking incubator for 30 minutes at 37oC.Step 6. Add aliquots (500uL) to pre-warmed test tubes each containing 500uL MHB and an antibiotic. Concentrations of antibiotics are 4mg/L and 32mg/L for Meropenem , 2 mg/L for ciprofloxacin, 16 mg/L for gentamicin, 8 mg/L for Colistin, 16 mg/L for ceftazidime, along with a control (no antibiotic).&nbsp;Final concentrations of antibiotics will be half the above concentrations.Step 7. Incubate the tubes for 30 minutes at 37oC.Step 9. Dilute each sample 1:10 (add 100uL to an new eppendorfs containing 900uL Hanks media).Step 10. Add a volume of concentrated bead suspension that contains approximately 104 polystyrene beads (1.5um diameter) to each eppendorf.Step 11. Load the contents of each tube into a 1mL syringe and measure with the impedance cytometer (see Single cell impedance cytometry measurementsStep 1. Load the sample into a 1mL syringe.Step 2. Connect the syringe to the impedance cytometer tubing.Step 3. Insert the syringe into a syringe pump.Step 4. Start the syringe running at 30uL/min.Step 5. Start the ziControl software which controls the Zurich Instruments impedance analyser, and apply a measurement voltage (see measurement settings below).Step 6. Record data for 2-3 minutes (depending on the required number of bacteria to measure).Step 7. Turn off the measurement voltage.Step 8. Flush through with a suitable cleaning reagent (e.g. buffer, NaOH, NaClO).Repeat steps 1-8 for each remaining sample. Measurement settings (ziControl)Sample rate: 230k samples/secondApplied frequencies: 5MHz and 40MHzApplied voltage: 2v pk-pk Data analysisStep 1. Determine the impedance of each particle by extracting the peak signal amplitude using convolution for each applied frequency.Step 2. Determine the mean signal of the 1.5um beadsStep 3. Normalise the opacity-cell size scatterplot by a single linear multiplier for each axis to ensure the mean of the beads is at Opacity =1 and diameter = 1.5um.Step 4. Define a contour is defined around the population of cells not exposed to antibiotic. This is termed the unexposed contour should include 50% of the cell population.&nbsp;Step 5. For each antibiotic exposed sample, normalise the data (as per step 3) and determine the number of cells that fall within the unexposed contour. Single cell impedance cytometer chip fabricationStep 1. Deposit 10nm titanium and the 200nm platinum onto two 6 inch glass substratesStep 2. Spin-coat a suitable photoresist (e.g. S1813) and pattern by exposure to UV light through a 7 inch photomaskStep 3. Develop the resist by washing with a suitable resist developer.Step 4. Pattern the meta layers using Ion beam milling.Step 5. Strip the photoresist.Step 6. Spin-coat SU-8 to the desired channel thickness (e.g. 30um) on to one of the two patterned wafersStep 7. Expose to UV light through a 7 inch photomask and develop.Step 8. Align and bond the second wafer to the first wafer&nbsp;by vacuum bonding at 180°C, 10kN for 6 hours.Step 9. Carefully scribe and dice the wafer to release the individual cytometer chips.

If the microcytometer chip gets blocked, try flushing with buffer from outlet back to the inlet. If this is unsuccessful, replace with a new chip. If the cytometer is frequently clogging, filter all reagents with a 0.22um (sterile) filter.

A populations of beta-lactam exposed cells from an isolate that is sensitive to the antibiotic are expected to increase in low-frequency impedance and decrease in electrical opacity relative to an unexposed sample of the same isolate. A populations of beta-lactam exposed cells from an isolate that is resistant to the antibiotic are expected to not change significantly in low-frequency impedance or electrical opacity relative to an unexposed sample of the same isolate. A populations of aminoglycoside exposed cells from an isolate that is sensitive to the antibiotic are expected to decrease in low-frequency impedance and increase in electrical opacity relative to an unexposed sample of the same isolate. The bacterial concentration is expected to decrease relative to the unexposed aliquot. Populations of aminoglycoside exposed isolates that are resistant to the antibiotic are expected to not change or decrease slightly in low-frequency impedance and not change significantly in electrical opacity relative to an unexposed sample. A populations of Colisitin exposed cells from an isolate that is sensitive to the antibiotic are expected to decrease in low-frequency impedance and increase in electrical opacity relative to an unexposed sample of the same isolate. The bacterial concentration is expected to decrease relative to the unexposed aliquot. Populations of Colistin exposed isolates that are resistant to the antibiotic are expected to not change or decrease slightly in low-frequency impedance and not change significantly in electrical opacity relative to an unexposed sample. A populations of fluoroquinolone exposed cells from an isolate that is sensitive to the antibiotic are expected to increase in low-frequency impedance and not change significantly in electrical opacity relative to an unexposed sample of the same isolate. Populations of fluoroquinolone exposed isolates that are resistant to the antibiotic are expected to not change significantly in low-frequency impedance or in electrical opacity relative to an unexposed sample.

We thank Katie Chamberlain for fabricating the cytometer chips.

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There is an urgent need to develop simple and fast antimicrobial susceptibility tests that allow informed prescribing of antibiotics. In this protocol we describe a method for a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material.

Method Article

Daniel C. Spencer, Teagan Paton, Timothy J.J. Inglis, J. Mark Sutton, Hywel Morgan

Daniel C. Spencer

Teagan Paton

Timothy J.J. Inglis

J. Mark Sutton

Hywel Morgan

Corresponding Author

DOI:

10.21203/rs.3.pex-1088/v1

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antimicrobial susceptibility test, impedance, cytometry, single cell analysis

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Daniel C. Spencer, Teagan Paton, Timothy J.J. Inglis, J. Mark Sutton, Hywel Morgan

Daniel C. Spencer

Teagan Paton

Timothy J.J. Inglis

J. Mark Sutton

Hywel Morgan

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