Detecting and Quantifying
Bacteriophage M13 on Human Skin
Young Hun (Colin) Kim
Baoqing Zhou
Biochemistry, College of Creative Studies
Chen Lab
Department of Chemistry and Biochemistry, UCSB
Rapid evolution of antibiotic resistant bacteria
: Non-Resistant
: Resistant
Population of
Pathogenic Bacteria
Rapid evolution of antibiotic resistant bacteria
: Non-Resistant
: Resistant
Antibiotics
Selection of Antibiotic Resistant Bacteria
Population of
Pathogenic Bacteria
Rapid evolution of antibiotic resistant bacteria
: Non-Resistant
: Resistant
Antibiotics
Selection of Antibiotic Resistant Bacteria
Population of
Pathogenic Bacteria
Carbapenem-Resistant
Enterobacteriaceae
Bacteriophages: natural viruses that infect bacteria
: Non-Resistant
: Resistant
Antibiotics
Selection of Antibiotic Resistant Bacteria
Population of
Pathogenic Bacteria
Bacteriophages
Bacteriophages: natural viruses that infect bacteria
: Non-Resistant
: Resistant
Antibiotics
Selection of Antibiotic Resistant Bacteria
Population of
Pathogenic Bacteria
Bacteriophages
Successful Treatment of Pathogenic Bacteria
Characterizing the microbial community of wounds
Characterizing the microbial community of wounds
Diabetic Foot Ulcers
Surgical Wounds
Characterizing the microbial community of wounds
Diabetic Foot Ulcers
Surgical Wounds
Characterizing the microbial community of wounds
Acinetobacter baumannii
Escherichia coli
Streptococcus pneumoniae
Klebsiella pneumoniae
Diabetic Foot Ulcers
Staphylococcus aureus
Surgical Wounds
Burkholderia cepacia
Using phage M13 to develop the skin swab protocol
Bacteriophage M13
Using phage M13 to develop the skin swab protocol
Bacteriophage M13
Escherichia coli
Using phage M13 to develop the skin swab protocol
Bacteriophage M13
Escherichia coli
How much phage can we recover by swabbing a human skin?
?
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
Phage M13
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
Cotton Swab
Phage M13
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
Sterilized Tweezers
Cotton Swab
Phage M13
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Cotton Swab
Cotton
Phage M13
Vortex
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Cotton Swab
Cotton
Centrifuge
Phage M13
Vortex
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Phage M13 in
supernatant
Cotton Swab
Cotton
Centrifuge
Phage M13
Vortex
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Phage M13 in
supernatant
Cotton Swab
Cotton
Centrifuge
Phage M13
Vortex
Transfer the
supernatant
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Phage M13 in
supernatant
Cotton Swab
Cotton
Centrifuge
Phage M13
Transfer the
supernatant
Vortex
DNase
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Phage M13 in
supernatant
Cotton Swab
Cotton
Centrifuge
Phage M13
Transfer the
supernatant
Vortex
DNase
Remove
contaminating
sequences
Recovering phage M13 from human skin swab
1. Skin Swab Sample Collection
PBS Buffer
Sterilized Tweezers
Phage M13 in
supernatant
Cotton Swab
Cotton
Centrifuge
Phage M13
Transfer the
supernatant
Vortex
DNase
Isolated
Phage M13
Remove
contaminating
sequences
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
Isolated
Phage M13
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
Phage M13
Phage DNA
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
Phage M13
Phage DNA
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
Phage M13 Lysis
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
Phage M13
Phage DNA
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
DNA Extraction
Phage M13 Lysis
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
Extracted Phage DNA
DNA Extraction
Phage M13 Lysis
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
Phage M13 Lysis
G G
G
G G
G
G
Extracted Phage DNA
DNA Extraction
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
qPCR Principles
Phage M13 Lysis
G G
G
G G
G
G
Extracted Phage DNA
DNA Extraction
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
G
G G
G
G G
G
G
Extracted Phage DNA
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
qPCR Principles
DNA Extraction
Phage M13 Lysis
n=1
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
G
G G
G
G G
G
G
Extracted Phage DNA
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
qPCR Principles
… ... 2n
DNA Extraction
n = number of cycles
Phage M13 Lysis
n=1
n=2
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
G
G G
G
G G
G
G
Extracted Phage DNA
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
qPCR Principles
DNA Extraction
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Phage M13 Lysis
n=1
n=2
… ... 2n
n = number of cycles
Recovering phage M13 from human skin swab
2. Phage DNA Extraction
3. Quantification of Phage DNA
Phage M13
Phage DNA
G
Isolated
Phage M13
PureLink
Viral DNA
Extraction
Mini Kit
G
G
G G
G
G G
G
G
Extracted Phage DNA
G
G
G
G
G
G
G
G
G
G
Quantitative-PCR
qPCR Principles
DNA Extraction
G
G
G
G
G
G
G
G
G
G
G
G
Data Analysis
G
G
G
G
G
G
G
G
G
G
G
G
Phage M13 Lysis
n=1
n=2
… ... 2n
n = number of cycles
Identifying qPCR limit of detection for phage M13 DNA
Samples
10-fold
… ...
M13KE
Identifying qPCR limit of detection for phage M13 DNA
Samples
10-fold
… ...
Control
M13KE
Identifying qPCR limit of detection for phage M13 DNA
Samples
10-fold
… ...
Control
M13KE
Identifying qPCR limit of detection for phage M13 DNA
Samples
… ...
Control
M13KE
Skin Swab
10-fold
Identifying qPCR limit of detection for phage M13 DNA
Samples
… ...
Control
M13KE
Skin Swab
10-fold
Identifying qPCR limit of detection for phage M13 DNA
Samples
10-fold
… ...
Control
M13KE
Skin Swab
Samples were assayed in triplicate
Identifying qPCR limit of detection for phage M13 DNA
Samples
12
10-fold
… ...
Control
Absolute Quantification of M13KE Skin Swab Samples
10
Skin Swab
Samples were assayed in triplicate
10Y (# of DNA Copies)
M13KE
R2 = 0.98
8
6
4
2
No M13
0
0
5
10
15
20
Cq (# of cycles)
25
30
35
Identifying qPCR limit of detection for phage M13 DNA
Samples
12
10-fold
… ...
Control
Absolute Quantification of M13KE Skin Swab Samples
10
Skin Swab
• qPCR baseline of detection =
2.36 x 104 copies
10Y (# of DNA Copies)
M13KE
R2 = 0.98
8
6
4
2
No M13
0
0
5
10
15
20
Cq (# of cycles)
25
30
35
Identifying qPCR limit of detection for phage M13 DNA
Samples
12
10-fold
… ...
Control
Absolute Quantification of M13KE Skin Swab Samples
10
Skin Swab
• qPCR baseline of detection =
2.36 x 104 copies
10Y (# of DNA Copies)
M13KE
R2 = 0.98
8
6
4
2
No M13
No M13
0
0
5
10
15
20
Cq (# of cycles)
25
30
35
Identifying qPCR limit of detection for phage M13 DNA
Samples
12
10-fold
Phage M13 detectable by qPCR
… ...
Control
Absolute Quantification of M13KE Skin Swab Samples
10
Skin Swab
• qPCR baseline of detection =
2.36 x 104 copies
• Limit of detection for human
skin swab = 1.00 x 106 copies
• Contaminating sequences on
human skin
10Y (# of DNA Copies)
M13KE
R2 = 0.98
8
Limit of Detection = 1.00 x 106 copies
6
Phage M13 not detectable by qPCR
4
2
No M13
No M13
0
0
5
10
15
20
Cq (# of cycles)
25
30
35
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
Human skin wounds
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
More than 106 copies
Human skin wounds
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
G
G
More than
106
copies
G
G G
G
G G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Can quantify using qPCR
Human skin wounds
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
G
G
More than
106
copies
G
G G
G
G G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Can quantify using qPCR
Human skin wounds
Less than 106 copies
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
G
G
More than
106
G
G G
G
G G
G
copies
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Can quantify using qPCR
Human skin wounds
G
Less than 106 copies
G
G
G G
G
G G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Cannot quantify using qPCR
Establishing a protocol to quantify phages from wounds
qPCR limit of detection for human skin swab = 1.00 x 106 copies
G
G
More than
106
G
G G
G
G G
G
copies
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Can quantify using qPCR
Human skin wounds
G
Less than 106 copies
G
G
G G
G
G G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
Cannot quantify using qPCR
?
Sequencing phage M13 genome for quantification
Extracted Phage DNA
Sequencing phage M13 genome for quantification
Mix with PhiX
control plasmid
Extracted Phage DNA
Sequencing phage M13 genome for quantification
Mix with PhiX
control plasmid
Extracted Phage DNA
Sequencing
Library
Preparation
Sequencing phage M13 genome for quantification
Mix with PhiX
control plasmid
Extracted Phage DNA
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Sequencing phage M13 genome for quantification
Mix with PhiX
control plasmid
Extracted Phage DNA
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Galaxy
Genome
Assembly &
Mapping
Sequencing phage M13 genome for quantification
Mix with PhiX
control plasmid
Extracted Phage DNA
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Galaxy
Genome
Assembly &
Mapping
Data Analysis
Applying the protocol to analyze human skin wounds
Mix with PhiX
control plasmid
Extracted Phage
DNA
Human skin wounds
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Galaxy Genome
Assembly &
Mapping
Data Analysis
Applying the protocol to analyze human skin wounds
Mix with PhiX
control plasmid
Extracted Phage
DNA
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Apply the
phage detection
protocol
Human skin wounds
Galaxy Genome
Assembly &
Mapping
Data Analysis
Applying the protocol to analyze human skin wounds
Mix with PhiX
control plasmid
Extracted Phage
DNA
Sequencing
Library
Preparation
Illumina Next
Generation
Sequencing
Apply the
phage detection
protocol
Human skin wounds
Galaxy Genome
Assembly &
Mapping
Data Analysis
Analyze the
metagenomics
data in the
microbiome
Acknowledgments
Chen Group:
Celia Blanco
Gregory Campbell
Irene Chen
Michael Devano
Evan Janzen
Coulter Keeler
Thomas Nguyen
Abe Pressman
Ranajay Saha
Allison Tam
Claire Tran
Samuel Verbanic
Baoqing Zhou
MARC U-STAR:
Arica Lubin
Stephanie Mendes
Joel Rothman
Research reported here was supported by the National Institute Of General Medical Sciences of the
National Institutes of Health under Award Number T34GM113848.
Quantifying phage M13 DNA by qPCR – Standard Curve
Serial dilution of M13mp18
12.000
8-fold
100x
Standard…
10x
10.000
1x
… ...
M13mp18
Sample
Expected
Total # of DNA
Observed
Total # of DNA
Mean Cq
Standard
Error
6.7 x 1010
1.79
0
100x
6.7 x
10x
8.4 x 109
8.4 x 109
6.02
1.59
1x
109
109
8.80
1.32
13.33
1.59
1.0 x
1010
1.0 x
0.1x
1.3 x 108
1.3 x 108
0.01x
107
107
18.41
0.96
1.6 x
1.6 x
0.001x
2.0 x 106
2.0 x 106
21.72
1.00
0.0001x
105
105
24.25
0.20
3.2 x 104
3.2 x 104
25.22
0.05
0
1.3 x 105
25.29
0.155
0.00001x
Negative
Control
(dH2O)
2.6 x
2.6 x
Log Starting Quantity
0.1x
8.000
0.01x
0.001x
6.000
y = -0.2483x + 11.379
R² = 0.9827
Baseline = 1.3 x 105
0.0001x
NC
0.00001x
4.000
2.000
0.000
0
5
10
15
20
25
30
Cq (# of cycles)
• Baseline of detection = 1.3 x 105 copies/uL
• Linear equation, R2 = 0.98271
This standard curve was used in the absolute quantification of our samples.
Quantifying phage M13 DNA by qPCR – Control Samples
10-fold serial dilution of M13KE
12.000
100x
10uL
10uL
Control
10x
10.000
… ...
90 uL dH2O
M13KE
Expected
Observed
Sample
Total # of DNA
Total # of DNA
Mean Cq
Standard
Error
100x
5.0 x 1010
4.63 x 1010
2.87
0
10x
5.0 x 109
1.50 x 109
8.87
0.54
1x
5.0 x 108
3.57 x 108
11.38
0.76
0.1x
5.0 x 107
3.58 x 107
15.41
0.57
0.01x
106
106
19.99
1.31
5.0 x
2.60 x
Log Starting Quantity
1x
y = -0.2483x + 11.379
R² = 0.9827
0.1x
8.000
0.01x
0.001x
6.000
0.0001x
4.000
2.000
0.001x
5.0 x 105
5.70 x 105
22.65
0.30
0.0001x
5.0 x 104
1.84 x 105
24.62
0.17
Negative
Control
(PBS)
0
NC
0.000
0
5
10
15
20
25
30
Cq (# of cycles)
• Baseline of detection for M13KE = 2.36 x 104 copies
Control samples were compared to skin swab samples
2.36 x
104
28.22
2.82
35
Quantifying phage M13 DNA by qPCR – Swab Samples
10-fold serial dilution of M13KE
10uL
12.000
100x
10.000
… ...
1x
90 uL dH2O
Expected
Observed
Sample
Total # of DNA
Total # of DNA
Mean Cq
Standard
Error
100x
1.0 x 1010
2.86 x 1010
7.75
0
10x
1.0 x 109
1.12 x 109
9.38
0.89
1x
1.0 x 108
1.31 x 108
13.13
0.65
0.1x
1.0 x 107
2.56 x 107
15.99
0.26
0.01x
106
106
18.09
0.13
1.0 x
7.68 x
1.0 x 105
4.61 x 106
18.99
0.71
0.0001x
104
7.20 x 106
18.21
0.24
7.04 x 106
18.24
0.13
Negative
Control
(PBS)
0
y = -0.2483x + 11.379
R² = 0.9827
0.1x
8.000
1x
0.01x
Limit of Detection = 106
0.1x
6.000
0.001x
0.01x
0.001x
0.0001x
4.000
0.0001x
2.000
0.001x
1.0 x
10x
10x
Log Starting Quantity
M13KE
Skin Swab
Control
100x
10uL
NC
NC
0.000
0
5
10
15
20
25
30
Cq (# of cycles)
• Limit of detection for human skin swab = 1.0 x 106 copies
• Contaminating sequences on human skin
35