WHO, HOW AND WHERE? MICROBIAL ECOLOGY USING …Movile Cave. Acknowledgements: Dr Wei Huang...

WHO, HOW AND WHERE?

MICROBIAL ECOLOGY USING

RAMAN SPECTROSCOPY

DR DANIEL READ

CENTRE FOR ECOLOGY & HYDROLOGY,

WALLINGFORD109

55

X (µm)

Y (

µm

)

1 µm

‘‘Progress in science depends on new techniques, new discoveries and new ideas,probably in that order’’. — Sydney Brenner

Outline

• A short intro to spectroscopy

• Raman spectroscopy

• Microbiological applications of Raman

• Phenotyping

• Microbial function

• Mapping with Raman

Who?

How?

Where?

Spectroscopy

Reflection

Absorption

Incident light

Elastic scattering

Inelastic (Raman) scattering

Fluorescence

A

B

C

Molecular Vibrational energy

Virtual state

Inelastic (Raman) scatteringStokes

Scattering of light

Raman spectroscopy:• Identification of

molecules• Bonds• Isotopic composition

•Characteristic spectroscopic patterns results in a “fingerprint” for molecules

•Can be used to identify and characterise molecules of elements and compounds

Data is presented relative to the excitation wavelength

Raman data

Excitationwavelength

Shifted Raman scattered wavelengths

Lactose

Glucose

Glycine

Tyrosine

Phenylalanine

BSA

The Raman microspectrometer

Grating

Notch filter

CCD Camera

Optical image

Monochromatic laser

109

55

X (µm)

Y (

µm

)

1 µm

~300 spectra

109

55

X (µm)

Y (

µm

)

1 µm

Raman applications:

Art

Archaeology

Applications of Raman

Forensics

PharmaceuticalsBiology

Geology

Cancer

Histology

Microbiology

Phenylalanine

Tyrosine

Phenylalanine

Tyrosine +Phenylalanine

Tyrosine +Phenylalanine

Amino acids

Raman applications: Biological sciences

Protein

Amide I

Amide III

Proteins


Guanine

Adenine

Cytosine+ Uracil

Adenine Guanine+ Adenine

Nucleic acids


Lipids and carbohydrates

Lipids

=C-C= (unsaturated fatty acids)

Carbohydrates

Starch


Fingerprint or Phenotype



Raman phenotyping or fingerprinting

• Fast (~30s per sample)

• No sample prep

• Cheap to run (after initial outlay)

• Quantitative (peak height)

• Diverse range of data in single assay

• Non-contact and non-destructive

• Single-cell analysis (sampling down to 1 µm dia)

o microbiology at the level of the individual

Function HOW?

Spatial distribution WHERE?

Phenotype WHO?

Metabolic history HOW?

-8

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2

4

6

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Y (

µm

)

-10 -5 0 5

X (µm)

0.5 µm0.5 µm0.5 µm0.5 µm0.5 µm

Raman applications: Microbial Ecology

Pseudomonas fluorescens

Bacillus subtilis

Escherichia coli

Who?: Raman Phenotyping

-0.08

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0.1

-0.12 -0.1 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1

Bacillus subtilis

Escherichia coli

Pseudomonas fluorescens

Principle Component AnalysisAxis 2

Axis 1

Who?: Raman Phenotyping

Who?: Pathogenic bacteria: linking genotype with phenotype

• Campylobacter jejuni and C. coli – campylobacteriosis

• Accounts for estimated 2.5 million cases (US) and 1,340,000

(UK) each year

• x13 the number of cases caused by Salmonella, E. coli, and

Listeria combined

• Estimated annual economic burden is £500 million in the UK

• Ability to colonize multiple hosts is a key feature of the ecology of

Campylobacter


Sheppard, S.K., Maiden, M.C.J., Falush, D. (2009). Population Genetics of Campylobacter. In: Bacterial

Population Genetics in Infectious Disease. Eds. Robinson, A.D., Falush, D., Feil, E.J. John Wiley &

Sons, New Jersey. Pages 181-194.

Single host lineagesMLST 682 and 177 found only in wild birds

Multi-host lineagesMLST 45 and 21 found in chickens, cattle, wild birds and in clinical samples

We wished to test whether:

1. Campylobacter phenotype is related to genotype (Species, clonal complex and clade)

2. Phenotype is related to species of host organism it was isolated from, independent of MLST (genetic) classification

Multilocus Sequence Typing (MLST)


• Strains used in this study• 108 Strains of Campylobacter

(cultured and analysed in triplicate)

• 2 species - C. jejuni (85) and C. coli

(23)

• 66 Multilocus Sequence Types

• 1,620 Raman spectra

27 Cattle faeces

5 Chicken gut24 Chicken faeces15 Chicken meat

23 Clinical

11 Wild bird

3 Pig


Read, DS., Woodcock, D., Whiteley, AS, Sheppard, SK (In prep) Classification of Campylobacter: linking phenotype, genotype and ecotype. PLOS Pathogens.

Neural network analysis – supervised statistical classification(Dr Dan Woodcock - Systems Biology Centre, University of Warwick)

•Identifies informative peaks and nonlinear relationships between the wavelengths•Distinguish which inputs provide the most information in separating the classes•Discards least informative peak until peak set with most discriminatory powers is left

The two species, C. jejuni and C. coli

C. jejuni ST21 and ST45a) human vs. cattle b) human vs. chicken

Clades 1, & 2 and 3 in C. coli


Phenotypic signature associated with nucleic acids, cytochrome c, and adenine (a nucleobase)


Peaks used in model

89% classification success

Comparison of C. jejuni and C. coli


79% classification success


Phenotypic signature associated with phenylalanine and nucleic acids

Peaks used in model

Campylobacter coli – Clade 1, 2, and 3 separation



C. jejuni ST45 and ST21 human and chicken

Phenotypic signature associated with cytochrome c, nucleic acids and one as yet unidentified peak

3a93% classification success



C. jejuni ST45 and ST21 human and cow

Phenotypic signature associated with cytochrome c, and two as yet unidentified peaks

3b89% classification success


•Links between phenotype and genotype comparatively weak in Campylobacter sp. (compared to other studies)•Why Clade 3 C. Coli phenotypically more similar to C. Jejuni?

•Link between phenotype and host association proved to be stronger

•Possibly due to limitations of MLST technique (only 7 housekeeping genes)

•Campylobacter possibly not a good study organism!


Common ‘light’ isotopee.g. 12C Carbon

‘Heavy’ isotopee.g. 13C Carbon

How?: Microbial function

Phenylalanine

1% 13C

50% 13C

99% 13C

Escherichia coli

ProteinAmino acidNucleic acids

How?: Microbial function

How?: Microbial function (Raman-FISH)

How?: Microbial function (Raman-FISH)

EUB338 (general bacteria) Acidovorax sp. RedPseudomonas sp. Green

•RNA Stable Isotope Probing using 13C labelled Naphthalene in microcosms•Identified Pseudomonas sp. and Acidovorax sp. as main naphthalene degraders•Acidovorax sp. unculturable•Raman-FISH approach

SEquenced REactive BARrier (SEREBAR) •Former manufactured gas plant (FMGP) in the United Kingdom•Groundwater polluted with Polycyclic Aromatic Hydrocarbons (PAHs) including Naphthalene

Huang, W. E., Stoecker, K., Griffiths, R., Newbold, L., Daims, H., Whiteley, A. S., & Wagner, M. (2007). Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization

for the single cell analysis of identity and function. Environmental Microbiology, 9(8), 1878-1889

x10 -3

75

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Inte

nsi

ty (

cnt)

400 600 800 1 000 1 200 1 400 1 600 1 800

Raman Shift (cm-1)

_1

_2

Single Escherichia coli• Mapped over 20x20 grid• 0.5µm between each point• ~400 spectra

Where?: Raman mapping

109

55

X (µm)

Y (

µm

)

1 µm


-10

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1 µm1 µm1 µm1 µm1 µm1 µm

Pigmented cells

Background

Microbial (Eukaryotic?) cell

Fluorescing material

Movile Cave

Acknowledgements:Dr Wei Huang (University of Sheffield)Prof Andrew Whiteley (CEH Wallingford)Dr Sam Sheppard (University of Oxford)Dr Dan Woodcock (University of Warwick)Mr Simon Fitzgerald (Horiba Scientific)Molecular Microbial Ecology Group (CEH Wallingford)

THANK YOU

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WHO, HOW AND WHERE? MICROBIAL ECOLOGY USING …Movile Cave. Acknowledgements: Dr Wei Huang...

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