1 Experimental Evolution of Antibiotic Resistance and Quantification of Cross-Resistance and Collateral Sensitivity

Expression Profiling of Antibiotic Resistant Bacteria



265



Table 1

List of antibiotics used for experimental evolution of antibiotic resistance



Antibiotics name Abbreviation Class



Cellular target



Solvent



Concentration of

stock (mg/ml)



Cefotaxime



CTX



Cephalosporin



Cell wall



Water



100



Cefoperazone



CPZ



Cephalosporin



Cell wall



Water



50



Ceftazidime



CAZ



Cephalosporin



Cell wall



Water



50



Cephalexin



CEX



Cephalosporin



Cell wall



Water



10



Cefixime



CFIX



Cephalosporin



Cell wall



EtOH



10



Streptomycin



SM



Aminoglycoside



Protein

synthesis, 30S



Water



10



Kanamycin



KM



Aminoglycoside



Protein

synthesis, 30S



Water



100



Amikacin



AMK



Aminoglycoside



Protein

synthesis, 30S



Water



100



Gentamicin



GM



Aminoglycoside



Protein

synthesis, 30S



Water



50



Neomycin



NM



Aminoglycoside



Protein

synthesis, 30S



Water



100



Tetracycline



TC



Tetracycline



Protein

synthesis, 30S



Water



20



Doxycycline



DOXY



Tetracycline



Protein

synthesis, 30S



Water



100



Minocycline



MINO



Tetracycline



Protein

synthesis, 30S



Water



10



Protein

synthesis, 50S



EtOH



30



Protein

synthesis, 50S



EtOH



100



Chloramphenicol CP

Azithromycin



AZM



Macrolide



Trimethoprim



TP



Folic acid

synthesis



DMSO



50



Rifampicin



RFP



RNA

polymerase



DMSO



100



Nalidixic acid



NA



Quinolone



DNA gyrase



DMF



10



Norfloxacin



NFLX



Quinolone



DNA gyrase



Acetic acid 100



Ofloxacin



OFLX



Quinolone



DNA gyrase



0.5 M

NaOH



Levofloxacin



LVFX



Quinolone



DNA gyrase



Acetic acid 2



100



(continued)



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Shingo Suzuki et al.



Table 1

(continued)

Concentration of

stock (mg/ml)



Antibiotics name Abbreviation Class



Cellular target



Solvent



Enoxacin



ENX



Quinolone



DNA gyrase



0.5 M

NaOH



100



Ciprofloxacin



CPFX



Quinolone



DNA gyrase



Water



20



Lomefloxacin



LFLX



Quinolone



DNA gyrase



0.5 M

NaOH



50



Gatifloxacin



GFLX



Quinolone



DNA gyrase



0.5 M

NaOH



50



­ issolved in water are 0.2 μm filter-sterilized. The antibiotic

d

stock solutions are stored at −80 °C prior to use.

3.Culture plates: 96-well microplates.

4.Incubator.

5.Microplate shaker.

6.Microplate reader.

2.2  Total RNA

Preparation



1.Ice-cold ethanol containing 10 % (w/v) phenol: Dissolve crystalline phenol in 99.5 % ethanol. Keep on ice before use.

2.RNeasy Micro Kit (Qiagen).

3.RNase-Free DNase Set (Qiagen).

4.TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0.

5.Lysozyme in TE buffer: Dissolve lysozyme at 1 mg/ml in TE

buffer.

6.Buffer RLT (RNeasy Micro Kit) containing 1 

% (v/v)

2-­mercaptoethanol: Add 2-mercaptoethanol to Buffer RLT at

1 % (v/v) just before use.

7.Spectrophotometer with microarray analysis mode (e.g.,

NanoDrop ND-1000).

8.(Optional) Agilent 2100 Bioanalyzer (Agilent).

9.(Optional) Agilent RNA 6000 Nano Kit (Agilent).



2.3  DNA Microarray

and Image Analysis



1.Agilent Low Input Quick Amp WT Labeling Kit, one-color

(Agilent, P/N 5190-2943).

2.Agilent One-Color RNA Spike-In Kit (Agilent, P/N 51902943).

3.RNeasy mini Kit (Qiagen).



Expression Profiling of Antibiotic Resistant Bacteria



267



4.NanoDrop ND1000 spectrophotometer (Thermo Fisher

Scientific).

5.Agilent Gene Expression Hybridization Kit (Agilent, P/N

5188-5242).

6.

Agilent SurePrint G3 Custom Microarray (Platform

GPL18948), 8 × 60 K (Agilent, P/N G4863A) (see Note 1).

7.

Agilent Hybridization gasket slide, 8 microarrays/slide

(Agilent, P/NG2534-60014).

8.Agilent Hybridization Chamber, stainless (Agilent, P/N

G2534A).

9.Agilent Hybridization oven (Agilent, P/N G2545A).



10.Agilent Gene Expression Wash Buffer 1 (Agilent, P/N

5188-5325).



11.Agilent Gene Expression Wash Buffer 2 (Agilent, P/N

5188-5326).

12.Agilent Microarray Scanner (Agilent, P/N G4900DA).

13.Agilent Feature Extraction software 9.5.3.1 or later.



3  Methods

Carry out all incubation steps in sterile conditions, and all molecular work in DNase/RNase-free conditions. In Subheading 3.1, the

methods for laboratory evolution of E. coli under antibiotics are

presented. In Subheading 3.2, we explain the protocol of quantification of cross-resistance and collateral sensitivity of the resistant

strains. Subheadings 3.3–3.5 show the protocols of transcriptome

analysis of the resistant strains by using microarray analysis.

Subheading  3.6 presents an example of the integration of transcriptome data and the quantification of cross-resistance and

­collateral sensitivity in Subheading 3.2, by which the change of

resistance to various antibiotics can be quantitatively predicted by

expression levels of a small number of genes.

3.1  Laboratory

Evolution of Antibiotic

Resistance



1. Prepare 100 μl modified M9 medium with eight different concentrations of antibiotics in a 96-well microplate (see Note 2).

2.E. coli MDS42 [10] strain (see Note 3) were pre-cultured

­overnight in 200 μl modified M9 medium without drugs. The

serial transfer culture was started by diluting the pre-culture up

to an OD600 nm of 3 × 10−5.

3. Monitor growth of the cells by measuring the OD600 nm of each

well of the culture plate incubated for 23 h using the microplate reader.



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Shingo Suzuki et al.



(a)



(b)

ENX 1

ENX 2

ENX 3

ENX 4



5



2



3



MIC log2 (µg/mL)



MIC log2 (µg/mL)



4

2

1

0

-1



0

-1

-2

-3

-4



-3



-5

0



10 20 30 40 50 60 70 80 90

Time (days)



1

2

3

4



1



-2

-4



CFIX

CFIX

CFIX

CFIX



3



-6



0



10 20 30 40 50 60 70 80 90

Time (days)



Fig. 1 Examples of laboratory evolution of antibiotic resistance. The time courses of the increase in MIC for

Enoxacin (ENX) and Cefixime (CFIX) over 90 days of experimental evolution, (a, b) respectively. Day 0 corresponds to the parent strain before evolution. Four parallel series of experiments were performed. The figure is

reproduced from [5] with permission



4. Dilute the cells in the well with highest antibiotic concentrations

in which cells can grow with modified M9 medium to an

OD600 nm of 3 × 10−5 (see Note 4).

5. Prepare a second 96-well microplate using 100 μl modified M9

medium per well with eight different concentrations of antibiotics (see Subheading 2.1, item 1) and inoculate 100 μl of the

diluted cells to wells with the corresponding antibiotic.

6. Incubate the culture plate with shaking at 900 rpm on a microplate shaker at 34 °C for 23 h.

7.By repeating the daily propagation, a significant increase of

minimum inhibitory concentration (MIC) can be observed

(Fig. 1 for examples).

8. At appropriate time intervals (e.g., every 3 days), store the cells

after the evolution experiments as glycerol stocks at −80 °C for

further analysis.

3.2  Quantification

of  Cross-­Resistance

and Collateral

Sensitivity



1.Thaw glycerol stocks of evolved and parent strains and add

modified M9 medium to a volume of 210 μl.

2. After mixing the tubes, transfer 200 μl of the cells to a 96-well

microplate.

3.Measure the OD600 nm of each strain using the microplate

reader.

4.Dilute the cells with modified M9 medium to an OD600 nm of

1.5 × 10−5.



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Expression Profiling of Antibiotic Resistant Bacteria



(a)

DNA

gyrase



Cell wall

synthesis



GFLX CTX CPZ

10

CAZ

LFLX

CPFX

CEX

ENX



CFIX



0



LVFX



DNA

gyrase



NFLX



OFLX



AMK



NFLX



GM



NA



NM



RFP

TP



AZM



CP MINO



ENX



KM



-10



TC

DOXY



Protein

synthesis



Cell wall

synthesis



GFLX CTX CPZ

10

CAZ

LFLX

CPFX

CEX

CFIX



0



LVFX



SM



OFLX



Parent

CP1

CP2

CP3

CP4



(b)



Parent

ENX 1

ENX 2

ENX 3

ENX 4



SM

KM



-10



AMK

GM



NA



NM



RFP

TP



AZM



CP MINO



TC

DOXY



Protein

synthesis



Fig. 2 Quantification of cross-resistance and collateral sensitivity. Changes in MICs for 25 antibiotics in

Chloramphenicol (CP) and ENX resistant strains, (a, b) respectively. The radial axis depicts the log2-­transformed

MIC relative to the parent strain. The black thick line indicates MICs of the parent strain, and the colored thick

lines indicate relative MICs of four parallel-evolved resistant strains. The figure is reproduced from [5] with

permission



5.Inoculate 200 μl of the diluted cells to a 96-well microplate.

6. Incubate the culture plate with shaking at 900 rpm on a microplate shaker at 34 °C for 23 h.

7.Prepare 100 μl modified M9 medium with 15 different concentrations of 25 antibiotics (Table 1) and without antibiotics

in 96-well microplates (see Note 5). One microplate set for

each strain to be tested.

8.Measure the OD600 nm of each strain incubated for 23 h using

the microplate reader.

9.Dilute the cells with modified M9 medium to an OD600 nm of

3 × 10−5.

10. Inoculate 100  μl of the diluted cells to the prepared 96-well

microplates (see Subheading 3.2, step 7). One microplate set

for each strain to be tested.

11. Incubate the culture plates with shaking at 900 rpm on a plate

shaker at 34 °C for 23 h.

12.Monitor growth of the cells by measuring the OD600 nm of each

well of the culture plate using the microplate reader (see Note 6).

13.Calculate and plot the MIC of the resistant strain relative to

the parent strain (Fig. 2 for example).

3.3  Total RNA

Preparation

for Transcriptome

Analysis



1.Thaw glycerol stocks of evolved and parent strains and add

modified M9 medium to a volume of 210 μl.

2. After mixing the tubes, transfer 200 μl of the cells to a 96-well

microplate.



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Shingo Suzuki et al.



3.Measure the OD600 nm of each strain using the microplate

reader.

4.Dilute the cells with modified M9 medium to an OD600 nm of

1.5 × 10−5.

5.Inoculate 200  μl of the diluted cells to a new 96-well

microplate.

6.Incubate the culture plate with shaking at 900 rpm on a plate

shaker at 34 °C for 23 h.

7.Measure the OD600 nm of each strain incubated for 23 h using

the microplate reader.

8.Dilute the cells with modified M9 medium to an OD600 nm of

1 × 10−4.

9.Inoculate 200 μl of the diluted cells to a 96-well microplate.

10.Incubate the culture plate with shaking at 900 rpm on a plate

shaker at 34 °C to an OD600 nm in the 0.072–0.135 range

(equivalent of 10 generations) (see Note 7).

11. Harvest 180 μl of the cell solution from the well and place in

an ultracentrifuge tube.

12.Immediately add an equal volume of ice-cold ethanol containing 10 % (w/v) phenol to each cell solution, and mix gently

but thoroughly.

13.Centrifuge it at 20,000 × g at 4 °C for 5 min.

14.Carefully remove and discard supernatant. Store pelleted cells

at −80 °C until use.

15. Add 100 μl of 1 mg/ml lysozyme in TE buffer to the cells and

mix by vortex for 10 s.

16.Incubate the lysozyme reactions at room temperature for

10 min. During incubation, vortex for 10 s every 2 min.

17. Add 350  μl of Buffer RLT containing 1 % (v/v) 2-merca­

ptoethanol, vortex and pulse-spin tubes to collect samples.

18. Add 250  μl of 99.5 % ethanol and pipet up and down several

times. Do not pulse-spin tubes.

19. Load onto RNeasy Micro columns and purify total RNA using

an RNeasy Micro Kit with on-column DNA digestion in accordance with the manufacturer’s instructions.

20. Measure the total RNA concentration and quality using a spectrophotometer and electrophoresis (see Note 8).

21.Store the purified RNA at −80 °C until transcriptome analysis.

3.4  DNA Microarray

Analysis



The total RNA preparation can be applied to other microarray

platforms. Here, we give a concise account of our application to

Agilent SurePrint G3 Custom Microarray, 8 × 60 K.



Expression Profiling of Antibiotic Resistant Bacteria

3.4.1  Preparation

of Spike Mix



271



1.Create an appropriate spike mix dilution using Agilent One-­

Color RNA Spike-In Kit in accordance with the user manual,

“Agilent One-Color Microarray-Based Exon Analysis - Low

Input Quick Amp WT Labeling ver1.0” (see Note 9).

2.Thaw Spike Mix and Dilution Buffer provided in the Spike-In

Kit at 37 °C for 5 min and mix thoroughly on a vortex mixer.

3. Put 2 μl of Spike Mix into a new tube and add 38 μl of Dilution

Buffer provided in the Spike-In kit to make the First Dilution.

Then, mix thoroughly on a vortex mixer.

4. Put 2 μl of the First Dilution into a fresh tube and add 48 μl of

Dilution Buffer to make the Second Dilution. Then, mix thoroughly on a vortex mixer.

5. Put 2 μl of the Second Dilution into a fresh tube and add 38 μl

of Dilution Buffer to make the Third Dilution (10,000-fold

final dilution). Then, mix thoroughly on a vortex mixer.



3.4.2  cDNA Synthesis



1. Add 100 ng of total RNA to a 1.5 ml tube in a final volume of

2.3 μl (see Note 10).

2.Add 2 μl of the Third Dilution of Spike Mix and 1 μl of WT

Primer provided in the Low Input Quick Amp WT Labeling kit.

3.Denature the primer and the template by incubating the reactions at 65 °C for 10 min.

4.Put the reactions on ice for 5 min.

5.Pre-warm the 5× First Strand Buffer provided in the Labeling

kit at 80 °C for 4 min and thaw completely.

6.Prepare cDNA Master Mix containing 2 μl of 5× First Strand

Buffer, 1 μl of 0.1 M DDT, 0.5 μl of dNTP mix, 1.2 μl of

Affinity Script RNase Block Mix provided in the Labeling kit

(see Note 11).

7.Add 4.7 μl of cDNA Master Mix to the sample tube and mix

by pipetting.

8.Incubate the sample at 40 °C in a water bath for 2 h.

9. Move the sample to a 70 °C water bath for 15 min to inactivate

reverse transcriptase.

10.Move the sample on ice and incubate for 5 min.



3.4.3  In Vitro

Transcription



1.Prepare Transcription Master Mix containing 0.75 μl of

Nuclease-­free water, 3.2 μl 5× Transcription Buffer, 0.6 μl of

0.1 M DTT, 1 μl of NTP Mix, 0.21 μl of T7 RNA Polymerase

Blend, and 0.24 μl of Cy3-CTP provided in the Labeling kit

(see Note 11).

2.Add 6 μl of Transcription Master Mix to the sample tube.

3.Incubate the sample in a water bath at 40 °C for 2 h.



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Shingo Suzuki et al.



3.4.4  Purification

of Cy3-Labeled cRNA

Sample Using the RNeasy

Mini Kit



1.Add 84 μl of Nuclease-free water to the sample to bring the

total volume up to 100 μl.

2.Add 350 μl of Buffer RLT to the sample tube and mix by

pipetting.

3.Add 250 μl of 100 % ethanol and mix thoroughly by pipetting.

4.Transfer the 700 μl of the sample to an RNeasy spin column

and centrifuge for 30 s at approx. 16,000 × g in a microcentrifuge at 4 °C. Discard the flow-through.

5.Transfer the RNeasy spin column to a fresh tube and add

500 μl of Buffer RPE containing ethanol. Centrifuge for 30 s

at 16,000 × g at 4 °C and discard the flow-through.

6.Add 500 μl of Buffer RPE containing ethanol. Centrifuge for

60 s at 16,000 × g at 4 °C and discard the flow-through.

7.Transfer the RNeasy spin column to a fresh tube and elute the

cRNA sample. Add 30 μl RNase-Free water onto the filter

membrane in the column. Wait 60 s and then centrifuge for

30 s at 16,000 × g at 4 °C.

8.Keep the cRNA sample on ice.



3.4.5  Assessment

of Cy3-Labeled cRNA

by Spectrophotometry



1. Start the NanoDrop software and set to Microarray Measurement

mode.

2.Select RNA-40 as the Sample type.

3.Load 1.0–2.0  μl of nuclease-free water and click Blank

button.

4.Load 1.0–2.0  μl of the sample and click Measure button

to quantify (1) Cy3 dye concentration (pmol/μl), (2) RNA

absorbance ratio (260 nm/280 nm), and (3) cRNA concentration (ng/μl).

5.Calculate specific activity as (concentration of Cy3)/(concentration of cRNA) × 1000 = pmol Cy3 per μg cRNA.

6.Examine the yield and specific activity results. The recommended cRNA yield and specific activity for hybridization are

6 μg and 15 pmol Cy3 per μg cRNA, respectively.



3.4.6  Hybridization



1.Add 600 ng Cy3-labeled cRNA, 5 μl of 10× Gene Expression

Blocking Agent provided in the Gene Expression Hybridization

Kit, nuclease-free water up to 24 μl into a 1.5 ml fresh tube.

2.Add 1 μl of 25× Fragmentation Buffer provided in the Gene

Expression Hybridization Kit and incubate at 60 °C for exactly

30 min to fragment RNA. Immediately, cool on ice for

one min.

3. Add 25 μl of 2× Hi-RPM Hybridization Buffer provided in the

Gene Expression Hybridization Kit to stop the fragmentation

reaction.



Expression Profiling of Antibiotic Resistant Bacteria



273



4.Mix the sample well by careful pipetting. Do not introduce

bubbles by the mixing.

5.Centrifuge for 1 min at room temperature at 16,000 × g.

6.Put sample on ice and apply it onto the array as soon as

possible.

7.Load a clean gasket slide into the Agilent SureHyb chamber

base.

8.Slowly apply 40 μl of hybridization sample onto the one of 8

gasket wells. Repeat this sample application eight times for the

8 × 60  K slide.

9.Grip the slide and slowly put it down on the SureHyb gasket

slide. Make sure that the sandwich-pair is properly aligned.

10.Put the SureHyb chamber cover onto the sandwiched slides

and firmly hand-tighten the clamp onto the chamber.

11.Load the chamber into Agilent Hybridization oven. Then

hybridize at 65 °C for 17 h.

3.4.7  Washing

Microarray Washing Slide



1. Fill slide-staining dish #1 with Gene Expression Wash Buffer 1

at room temperature.

2.Put a slide rack into slide-staining dish #2 containing a magnetic stir bar. Fill the dish with Gene Expression Wash Buffer 1

to cover the slide rack at room temperature. Put the dish on a

magnetic stir plate.

3. Put the empty dish #3 containing a magnetic stir bar on the stir

plate.

4.Remove one hybridization chamber from incubator. Then,

slide off the clamp assembly and remove the chamber cover.

5. Remove the array-gasket sandwich from the chamber and submerge it into slide-staining dish #1 containing Gene Expression

Wash Buffer 1.

6.Open the array-gasket sandwich in slide-staining dish #1 and

remove the slide. And then put it into the slide rack in the

slide-staining dish #2 containing Gene Expression Wash Buffer

1 at room temperature.

7. When all slides are put into the slide rack, stir the slide-staining

dish #2 for 1 min.

8.Pour pre-warmed Gene Expression Wash Buffer 2 at 37 °C

into the slide-staining dish #3.

9.Transfer the slide rack to slide-staining dish #3 containing

Gene Expression Wash Buffer 2. Stir it using a moderate speed

setting for 1 min.

10.Remove the slide rack from the slide-staining dish #3 slowly

and put the slides into a slide holder.



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Shingo Suzuki et al.



3.4.8  Microarray

Scanning



1.Carefully put the microarray slide into the slide holder. Then,

put assembled slide holders into the scanner cassette.

2.Select the scanner protocol “AgilentG3_GX_1color.”

3.Click Start Scan. After scanning, a .tif image file is created.



3.5  Acquisition

of Expression Levels

and Normalization



1. To analyze the data, signal intensities of each probe are quantified from the .tif image file using Agilent Feature Extraction

software 9.5.3.1 or later.

2. Acquisition of expression levels: In the microarray we designed

for E. coli transcriptome analysis, 12 different probes are prepared for each coding region (see Note 1). The expression levels of each coding region are calculated as the median of signal

intensities of these 12 probes (see Note 12).

To compare gene expression profiles among multiple samples,

the expression levels should be normalized in an appropriate

way (see Note 13) as such as quantile normalization [11]. The

goal of the quantile normalization method is to make the distribution of expression for each sample the same.

3.Given n expression profiles with m genes, form X of dimension

m × n where each sample is a column.

4.Sort each column of X to give Xsort.

5. Take the averages across rows of Xsort and assign this average to

each element in the row to get X'sort.

6.Obtain Xnormalized by rearranging each column of X'sort to realize

the same ordering as original X.

7.After this procedure, each sample has the same distribution of

gene expression.



3.6  Example of Data

Analysis: Prediction

of Antibiotic

Resistance

from Transcriptome

Data



The purpose of transcriptome analysis is to extract genes whose

expression changes contribute to the change of antibiotic resistance. Here, we show one example of such analyses, in which resistance levels (MIC) to various antibiotics are quantitatively predicted

based on a small number of gene expression changes. For the

details of the results of this analysis, see [5].

1.Prepare strains resistant to a single antibiotic by laboratory

evolution (Subheading 3.1).

2.For each resistant strain, analyze cross-resistance and colla­

teral sensitivity, i.e., quantify MICs to various antibiotics

(Subheading 3.2).

3.For each resistant strain, quantify the expression profile by

microarray analysis. To standardize the culture condition

among the resistant strains, all transcriptome data should

be obtained in a synthetic medium without addition of

antibiotics.



Expression Profiling of Antibiotic Resistant Bacteria



275



4. In this analysis, we assumed that the drug resistance quantified

by the MICs is determined as a function of gene expression

levels. Also, for simplification, we neglected nonlinear effects

and cross terms of the changes in gene expression. Thus, we

assumed the following simple linear model to predict the MICs

by the expression levels of N genes:

N







MICkj = åaik X ij + b k

i =1







MICjk is the log2-transformed relative MIC of the j th strain

for the k th antibiotic, Xij is the log10-transformed expression

level of the i th gene in the j th resistant strain after standardization to zero mean and unit variance, and αik and βk are fitting parameters.

5.Use the cross-validation method to avoid overfitting and to

seek the number of genes with the highest predictive accuracy.

For example, the resistant strains were randomly partitioned

into four equally sized subgroups; one subgroup was used as

the test data set for validation and the remaining three subgroups were used for the fitting of αik and βk.

6. Sets of N genes that have high prediction accuracy for test data

are obtained by a genetic algorithm (GA). First, prepare 1000

sets of randomly chosen N genes as an initial population.

7.Using each gene set, obtain αik and βk by multiple regression

method (see Note 14). The correlation coefficient between the

predicted and observed MICs of the training data sets is used

as the fitness of each gene set.

8.Among the 1000 gene sets, select those with fitness in the top

5 % as the parent sets of the next generation. Then, generate

mutant sets by randomly replacing a single gene without

changing N.

9. Repeat more than 300 cycles of the generation and selection of

mutant sets to obtain sets of N genes whose expression levels

could represent the MICs for training data sets.

10.Repeat the selection of gene sets using 10,000 different training data sets prepared randomly by partitioning the total data

set to obtain the frequency of genes selected after the GA. The

expression levels of the frequently selected genes provide the

most relevant information for predicting MIC changes.

11. To obtain the number of genes N which has highest prediction

accuracy of test data set (see Note 15), perform the GA screening (more than 300 cycles for each) by changing N, for example, from 2 to 18.

12.After obtaining the optimal number of N, choose the appropriate gene set to represent the prediction accuracy of a test

data set (Fig. 3 for example).