Detecting and Quantifying Bacteriophage M13 on Human Skin

Transcription

1 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

2 Rapid evolution of antibiotic resistant bacteria : Non-Resistant : Resistant Population of Pathogenic Bacteria

3 Rapid evolution of antibiotic resistant bacteria : Non-Resistant : Resistant Antibiotics Selection of Antibiotic Resistant Bacteria Population of Pathogenic Bacteria

4 Rapid evolution of antibiotic resistant bacteria : Non-Resistant : Resistant Antibiotics Selection of Antibiotic Resistant Bacteria Population of Pathogenic Bacteria Carbapenem-Resistant Enterobacteriaceae

5 Bacteriophages: natural viruses that infect bacteria : Non-Resistant : Resistant Antibiotics Selection of Antibiotic Resistant Bacteria Population of Pathogenic Bacteria Bacteriophages

6 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

7 Characterizing the microbial community of wounds

8 Characterizing the microbial community of wounds Diabetic Foot Ulcers Surgical Wounds

9 Characterizing the microbial community of wounds Diabetic Foot Ulcers Surgical Wounds

10 Characterizing the microbial community of wounds Acinetobacter baumannii Escherichia coli Streptococcus pneumoniae Klebsiella pneumoniae Staphylococcus aureus Diabetic Foot Ulcers Surgical Wounds Burkholderia cepacia

11 Using phage M13 to develop the skin swab protocol Bacteriophage M13

12 Using phage M13 to develop the skin swab protocol Bacteriophage M13 Escherichia coli

13 Using phage M13 to develop the skin swab protocol Bacteriophage M13 Escherichia coli How much phage can we recover by swabbing a human skin??

14 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection

15 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Phage M13

16 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Cotton Swab Phage M13

17 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers Cotton Swab Phage M13

18 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Cotton Swab Cotton Phage M13 Vortex

19 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Cotton Swab Phage M13 Cotton Vortex Centrifuge

20 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Cotton Swab Sterilized Tweezers PBS Buffer Phage M13 in supernatant Phage M13 Cotton Vortex Centrifuge

21 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Phage M13 in supernatant Cotton Swab Phage M13 Cotton Vortex Centrifuge Transfer the supernatant

22 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Phage M13 in supernatant Cotton Swab Phage M13 Cotton Vortex Centrifuge Transfer the supernatant DNase

23 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Phage M13 in supernatant Cotton Swab Phage M13 Cotton Vortex Centrifuge Transfer the supernatant DNase Remove contaminating sequences

24 Recovering phage M13 from human skin swab 1. Skin Swab Sample Collection Sterilized Tweezers PBS Buffer Phage M13 in supernatant Cotton Swab Phage M13 Cotton Vortex Centrifuge Transfer the supernatant DNase Isolated Phage M13 Remove contaminating sequences

25 Recovering phage M13 from human skin swab 2. Phage DNA Extraction Isolated Phage M13

26 Recovering phage M13 from human skin swab 2. Phage DNA Extraction Phage M13 Phage DNA Isolated Phage M13 PureLink Viral DNA Extraction Mini Kit

27 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

28 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

29 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 Phage M13 Lysis DNA Extraction

30 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 Quantitative-PCR DNA Extraction Phage M13 Lysis

31 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 Quantitative-PCR qpcr Principles DNA Extraction Phage M13 Lysis

32 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 Quantitative-PCR qpcr Principles DNA Extraction Phage M13 Lysis n=1

33 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 Quantitative-PCR qpcr Principles Phage M13 Lysis DNA Extraction... 2 n n = number of cycles n=1 n=2

34 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 Quantitative-PCR qpcr Principles Phage M13 Lysis DNA Extraction... 2 n n = number of cycles n=1 n=2

35 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 Quantitative-PCR Data Analysis qpcr Principles Phage M13 Lysis DNA Extraction... 2 n n = number of cycles n=1 n=2

36 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold M13KE...

37 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold Control M13KE...

38 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold Control M13KE...

39 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold Control M13KE... Skin Swab

40 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold Control M13KE... Skin Swab

41 Identifying qpcr limit of detection for phage M13 DNA Samples 10-fold Control M13KE... Skin Swab Samples were assayed in triplicate

42 10 Y (# of DNA Copies) Samples Identifying qpcr limit of detection for phage M13 DNA 10-fold 12 Absolute Quantification of M13KE Skin Swab Samples Control M13KE R 2 = 0.98 Skin Swab 6 Samples were assayed in triplicate Cq (# of cycles) No M13

43 10 Y (# of DNA Copies) Samples Identifying qpcr limit of detection for phage M13 DNA 10-fold 12 Absolute Quantification of M13KE Skin Swab Samples Control M13KE R 2 = 0.98 Skin Swab qpcr baseline of detection = 2.36 x 10 4 copies Cq (# of cycles) No M13

44 10 Y (# of DNA Copies) Samples Identifying qpcr limit of detection for phage M13 DNA 10-fold 12 Absolute Quantification of M13KE Skin Swab Samples Control M13KE R 2 = 0.98 Skin Swab qpcr baseline of detection = 2.36 x 10 4 copies Cq (# of cycles) No M13 No M13

45 10 Y (# of DNA Copies) Samples Control Identifying qpcr limit of detection for phage M13 DNA M13KE 10-fold Absolute Quantification of M13KE Skin Swab Samples Phage M13 detectable by qpcr 8 R 2 = 0.98 Limit of Detection = 1.00 x 10 6 copies Skin Swab qpcr baseline of detection = 2.36 x 10 4 copies Limit of detection for human skin swab = 1.00 x 10 6 copies Contaminating sequences on human skin Phage M13 not detectable by qpcr Cq (# of cycles) No M13 No M13

46 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies Human skin wounds

47 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies More than 10 6 copies Human skin wounds

48 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies Human skin wounds More than 10 6 copies Can quantify using qpcr

49 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies Human skin wounds More than 10 6 copies Less than 10 6 copies Can quantify using qpcr

50 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies Human skin wounds More than 10 6 copies Less than 10 6 copies Can quantify using qpcr Cannot quantify using qpcr

51 Establishing a protocol to quantify phages from wounds qpcr limit of detection for human skin swab = 1.00 x 10 6 copies Human skin wounds More than 10 6 copies Less than 10 6 copies Can quantify using qpcr Cannot quantify using qpcr?

52 Extracted Phage DNA Sequencing phage M13 genome for quantification

53 Sequencing phage M13 genome for quantification Mix with PhiX control plasmid Extracted Phage DNA

54 Sequencing phage M13 genome for quantification Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation

55 Sequencing phage M13 genome for quantification Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing

56 Sequencing phage M13 genome for quantification Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing alaxy enome Assembly & Mapping

57 Sequencing phage M13 genome for quantification Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing alaxy enome Assembly & Mapping Data Analysis

58 Applying the protocol to analyze human skin wounds Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing alaxy enome Assembly & Mapping Data Analysis Human skin wounds

59 Applying the protocol to analyze human skin wounds Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing alaxy enome Assembly & Mapping Data Analysis Apply the phage detection protocol Human skin wounds

60 Applying the protocol to analyze human skin wounds Mix with PhiX control plasmid Extracted Phage DNA Sequencing Library Preparation Illumina Next eneration Sequencing alaxy enome Assembly & Mapping Data Analysis Apply the phage detection protocol Analyze the metagenomics data in the microbiome Human skin wounds

61 Acknowledgments Chen roup: Celia Blanco regory Campbell Irene Chen Michael Devano Evan Janzen Coulter Keeler MARC U-STAR: Arica Lubin Stephanie Mendes Joel Rothman Thomas Nguyen Abe Pressman Ranajay Saha Allison Tam Claire Tran Samuel Verbanic Baoqing Zhou Research reported here was supported by the National Institute Of eneral Medical Sciences of the National Institutes of Health under Award Number T34M

62 Log Starting Quantity Quantifying phage M13 DNA by qpcr Standard Curve Serial dilution of M13mp18 8-fold M13mp18 Expected Sample 100x 10x 1x 0.1x 0.01x 0.001x x x Negative Control (dh 2 O) Total # of DNA 6.7 x x x x x x x x Observed Total # of DNA 6.7 x x x x x x x x x 10 5 Mean Cq Standard Error x 10x Baseline = 1.3 x x y = x R² = Cq (# of cycles) Baseline of detection = 1.3 x 10 5 copies/ul Linear equation, R 2 = x Standard This standard curve was used in the absolute quantification of our samples. 0.01x 0.001x x NC x

63 Log Starting Quantity Quantifying phage M13 DNA by qpcr Control Samples 10-fold serial dilution of M13KE 10uL 10uL x x Control x 0.1x y = x R² = x M13KE 90 ul dh 2 O Sample Expected 100x 10x 1x 0.1x 0.01x 0.001x x Negative Control (PBS) Total # of DNA 5.0 x x x x x x x Observed Total # of DNA 4.63 x x x x x x x x 10 4 Mean Cq Standard Error x x NC Cq (# of cycles) Baseline of detection for M13KE = 2.36 x 10 4 copies Control samples were compared to skin swab samples

64 Log Starting Quantity M13KE Quantifying phage M13 DNA by qpcr Swab Samples 10-fold serial dilution of M13KE 10uL Sample 100x 10x 1x 0.1x 0.01x 0.001x x Negative Control (PBS) 10uL 90 ul dh 2 O Expected Total # of DNA 1.0 x x x x x x x Observed Total # of DNA 2.86 x x x x x x x x 10 6 Mean Cq Standard Error x 100x 10x Limit of Detection = x 1x 1x 0.1x y = x R² = Cq (# of cycles) Skin Swab Control Limit of detection for human skin swab = 1.0 x 10 6 copies Contaminating sequences on human skin 0.1x 0.01x 0.01x 0.001x x NC 0.001x x NC