Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
4Activity
0 of .
Results for:
No results containing your search query
P. 1
JID_Li_acne

JID_Li_acne

Ratings: (0)|Views: 366 |Likes:
Published by scprweb
Why Zits Happen
Why Zits Happen

More info:

Published by: scprweb on Feb 28, 2013
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

08/19/2013

pdf

text

original

 
Propionibacterium acnes 
Strain Populations in theHuman Skin Microbiome Associated with Acne
Sorel Fitz-Gibbon
1,8
, Shuta Tomida
1,8
, Bor-Han Chiu
1
, Lin Nguyen
1
, Christine Du
1,2
, Minghsun Liu
3
,David Elashoff 
2
, Marie C. Erfe
4
, Anya Loncaric
2
, Jenny Kim
2,5
, Robert L. Modlin
2,3
, Jeff F. Miller
3
,Erica Sodergren
6
, Noah Craft
4
, George M. Weinstock
6
and Huiying Li
1,7
The human skin microbiome has important roles in skin health and disease. However, bacterial populationstructure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strainlevel and genome level of
Propionibacterium acnes
, a dominant skin commensal, between 49 acne patients and52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstratedthat although the relative abundances of
P. acnes
were similar, the strain population structures were significantlydifferent in the two cohorts. Certain strains were highly associated with acne, and other strains were enriched inhealthy skin. By sequencing 66 previously unreported
P. acnes
strains and comparing 71
P. acnes
genomes, weidentified potential genetic determinants of various
P. acnes
strains in association with acne or health. Ouranalysis suggests that acquired DNA sequences and bacterial immune elements may have roles in determiningvirulence properties of
P. acnes
strains, and some could be future targets for therapeutic interventions. This studydemonstrates a previously unreported paradigm of commensal strain populations that could explain thepathogenesis of human diseases. It underscores the importance of strain-level analysis of the human microbiometo define the role of commensals in health and disease.
 Journal of Investigative Dermatology 
accepted article preview 21 January 2013; doi:10.1038/jid.2013.21
INTRODUCTION
The diversity of the human microbiota at the strain level andits association with human health and disease are largelyunknown. However, many studies have shown that microbe-related human diseases are often caused by certain strains of aspecies, rather than the entire species being pathogenic.Examples include methicillin-resistant
Staphylococcus aureus 
Escherichia coli 
O157 (Tarr
, 2008). Acne vulgaris (commonly calledacne) is one of the most common skin diseases with aprevalence in up to 85% of teenagers and 11% of adults(White, 1998). Although the etiology and pathogenesis of acneare still unclear, microbial involvement is considered to beone of the main mechanisms contributing to the developmentof acne (Cunliffe, 2002;Bojar and Holland, 2004). In particular,
Propionibacterium acnes 
has been hypothesizedto be an important pathogenic factor (Webster, 1995).Antibiotic therapy targeting
P. acnes 
has been a mainstaytreatment for more than 30 years (Leyden, 2001). However,despite decades of study, it is still not clear how
P. acnes 
contributes to acne pathogenesis while being a majorcommensal of the normal skin flora (Gao
, 2010). Whether
P. acnes 
protects the human skinas a commensal bacterium or functions as a pathogenic factorin acne, or both, remains to be elucidated.Here we compared the skin microbiome at the strain leveland genome level in 49 acne patients and 52 normalindividuals using a combination of metagenomics and gen-ome sequencing. First, for each sample, 16S ribosomal DNA(rDNA) was amplified,
B
400 clones were sequenced, and anaverage of 311 nearly full-length 16S rDNA sequences wereanalyzed. The population structure of 
P. acnes 
strains wasdetermined in each sample. Second, each
P. acnes 
strainwas assigned an ‘‘acne index’’ by calculating its prevalence in
ORIGINAL ARTICLE
1
Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, David Geffen School of Medicine, UCLA, Los Angeles,California, USA;
2
Department of Medicine, David Geffen School of Medicine,UCLA, Los Angeles, California, USA;
3
Department of Microbiology,Immunology and Molecular Genetics, David Geffen School of Medicine,UCLA, Los Angeles, California, USA;
4
Center for Immunotherapeutics Research, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, David Geffen School of Medicine, UCLA, Los Angeles, California,USA;
Department of Dermatology, Greater Los Angeles Healthcare System Veterans Affair, Los Angeles, California, USA;
The Genome Institute at Washington University, St Louis, Missouri, USA and 
UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California, USACorrespondence: Huiying Li, 4339 CNSI, 570 Westwood Plaza, Building 114,UCLA, Los Angeles, California 90095-1770, USA.E-mail:huiying@mednet.ucla.edu 
These authors contributed equally to this work.Received 16 August 2012; revised 19 November 2012; accepted 3 December 2012Abbreviations: CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat; HMP, Human Microbiome Project; rDNA, ribosomal DNA; RT,ribotype 
&
2013 The Society for Investigative Dermatology
1
 
acne patients based on the 16S rDNA metagenomic data. The
P. acnes 
strains associated with the acne patient group wereidentified, as well as the strains enriched in the individualswith normal skin. This metagenomic approach is fundamen-tally different from prior approaches in determining diseaseassociations; it is more powerful and less biased thantraditional methods by bypassing the biases and selection instrain isolation and culturing. To our knowledge, this study hasthe largest number of individual skin microbiomes reported atthe strain level to date. Finally, we sequenced 66 previouslyunreported
P. acnes 
strains and compared 71
P. acnes 
genomes covering the major lineages of 
P. acnes 
found inthe skin microbiota. By combining a metagenomic study of the skin microbiome and genome sequencing of this majorskin commensal, this study provides insight into potentialbacterial genetic determinants in acne pathogenesis andemphasizes the importance of strain-level analysis of thehuman microbiome to understand the role of commensals inhealth and disease.
RESULTS
P. acnes 
dominates the pilosebaceous unit
We characterized the microbiome in pilosebaceous units(‘‘pores’’) on the nose, collected from 49 acne patientsand 52 individuals with normal skin. Nearly full-length 16SrDNA sequences were obtained using the Sanger method,which allowed us to analyze
P. acnes 
at the strain level. Afterquality filtering, our final data set contained 31,461 16S rDNAsequences ranging from position 29 to position 1,483. Of thesequences, 27,358 matched to
P. acnes 
with greater than 99%identity. Our data demonstrated that
P. acnes 
dominates themicrobiota of pilosebaceous units, accounting for 87% of theclones (Figure 1). Other commonly found species in pilose-baceous units included
Staphylococcus epidermidis 
,
Propio- nibacterium humerusii 
, and
Propionibacterium granulosum 
,each representing 1–2.3% of the total clones. A total of 536species-level operational taxonomic units belonging to 42genera and six phyla were identified in the samples(Supplementary Table S1 online).To bypass the potential biases due to PCR amplification anddue to uneven numbers of 16S rDNA gene copies amongdifferent species, we performed a metagenomic shotgunsequencing of the total DNA pooled from the pilosebaceousunit samples of 22 additional normal individuals. Microbialspecies were identified by mapping metagenomic sequencesto reference genomes. The results confirmed that
P. acnes 
wasthe most abundant species (89%) (Figure 1). This is consistentwith the results obtained from 16S rDNA sequencing (87%).
Different
P. acnes 
strain populations in acne
There was no statistically significant difference in the relativeabundance of 
P. acnes 
when comparing acne patients andnormal individuals. We next examined whether there weredifferences at the strain level of 
P. acnes 
by extensivelyanalyzing the
P. acnes 
16S rDNA sequences. We define eachunique 16S rDNA sequence as a 16S rDNA allele type, calleda ribotype (RT). The most abundant
P. acnes 
sequence wasdefined as ribotype 1 (RT1); all other defined ribotypes have
0%10%20%30%40%50%60%70%80%90%100%
   R  e   l  a   t   i  v  e  a   b  u  n   d  a  n  c  e  o   f  s  p  e  c   i  e  s
Other
Staphylococcus capitis 
Unclassified corynebacterineae
Prevotella oris Propionibacterium granulosum Propionibacterium humerusii Propionibacterium acnes 
Pooled normal skinsamples by metagenomicshotgun sequencing
Samples from acne patientsSamples from normal individuals
Escherichia coli Staphylococcus epidermidis 
Figure 1.
Propionibacterium acnes 
was dominant in pilosebaceous units in both acne patients and individuals with normal skin.
By 16S ribosomal DNA (rDNA)sequencing,
P. acnes 
sequences accounted for 87% of all the clones. Species with a relative abundance of 
4
0.35% are listed in order of relative abundance.Species distribution from a metagenomic shotgun sequencing of pooled samples from normal individuals confirmed the high abundance of 
P. acnes 
inpilosebaceous units, as shown in the far right column.
S Fitz-Gibbon 
et al.
The Skin Microbiome Associated with Acne
2
Journal of Investigative Dermatology
 
Z
99% sequence identity to RT1. Similar to the distributionsseen at higher taxonomical levels (Bik
, 2010), at thestrain level a few ribotypes were highly abundant in thesamples with a significant number of rare ribotypes(Supplementary Figure S1 online). After careful examinationof the sequence chromatograms and manual correction of thesequences, a total of 11,009 ribotypes were assigned to the
P. acnes 
16S rDNA sequences. Most of the minor ribotypeswere singletons. On average, each individual harbored 3
±
2
P. acnes 
ribotypes with three or more clones. On the basis of the genome sequences described below, all the sequenced
P. acnes 
strains have three identical copies of 16S rDNA genes(note in Supplementary Information online). This allowed us tocompare the
P. acnes 
strain populations in individuals basedon the 16S rDNA sequences. The top 10 major ribotypes withmore than 60 clones and found in multiple subjects are showninTable 1.Analysis of the top 10 ribotypes showed both disease-specific and health-specific associations. The three mostabundant ribotypes (RT1, RT2, and RT3) were fairly evenlydistributed among acne and normal individuals. However, thenext seven major ribotypes were significantly skewed in theirdistribution (Table 1). Ribotypes 4, 5, 7, 8, 9, and 10 werefound predominantly in acne patients, with four of these sixribotypes being statistically significantly enriched in acne(
o
0.05, Wilcoxon test). Ribotypes 4, 5, and 10 contain anucleotide substitution G1058C in the 16S rDNA sequences,which has previously been shown to confer increased resis-tance to tetracycline (Ross
, 1998a, 2001). However, onlya small percentage of the subjects in our study harboring theseribotypes had been treated with antibiotics (SupplementaryTable S2 online); therefore, enrichment of these threeribotypes in the acne group was not correlated with antibiotictreatment. This is consistent with previous studies, whichshowed that previous use of antibiotics was not alwaysassociated with the presence of antibiotic-resistant strainsand that some patients who were not previously treated withantibiotics harbored strains already resistant to antibiotics(Dreno
, 2002). On the other hand,one ribotype, RT6, although detected in only 11 subjects, wasstrongly associated with normal skin (
¼
0.025, Wilcoxontest;Table 1). Its relative abundance in our normal groupwas similar to that found in the healthy cohort data from theHuman Microbiome Project (HMP) (see SupplementaryInformation and Supplementary Figure S2 online). The per-centage of positive subjects (11/52) was similar as well. Threeof the 14 HMP subjects had RT6 found in the anterior nares,and one additional subject had RT6 in the left retroauricularcrease.On the basis of the distributions of the top 10 ribotypes,statistical analysis using several different tests showed signifi-cant differences in
P. acnes 
population structure between acneand normal skin (Supplementary Table S3 online). This isconsistent with a principal coordinate analysis, in which acnesamples and normal skin samples were separated mostlyby principal coordinates 1 and 2 (Supplementary Figure S3online), explaining 44% and 20% of the variation,respectively.To examine whether different individuals share similar
P. acnes 
population structures, we clustered the samples onthe basis of the relative abundance of the top 10 ribotypes.Five main microbiome types were observed at the
P. acnes 
strain level (microbiome types I to V). Types IV and V, whichare dominated by
P. acnes 
RT4 and RT5, respectively, weremainly found in acne patients (Figure 2and SupplementaryFigure S4 online). The same five main microbiome types wereobserved in the HMP data and the data fromGrice
(2009) (see Supplementary Information and SupplementaryFigure S5 online).
Genome sequence analysis of 71
P. acnes 
strains
All of the top 10 most abundant ribotypes differ from RT1 byonly one or two nucleotide changes in the 16S rDNAsequence (Table 1). To determine whether such small changes
Table 1. Top 10 most abundant ribotypes found in pilosebaceous units
Ribotype (RT)Nucleotidechangesfrom RT1Numberof subjectsNumberof clonesPercentageof clones fromacne patients
1
Percentage of clones fromnormal individuals
2
P-value
3
RT1 90 5,536 48% 52% 0.84RT2 T854C 48 1,213 51% 49% 0.36RT3 T1007C 60 2,104 40% 60% 0.092RT4 G1058C, A1201C 23 275 84% 16% 0.049RT5 G1058C 15 205 99% 1% 0.00050RT6 T854C, C1336T 11 262 1% 99% 0.025RT7 G529A 10 188 99% 1% 0.12RT8 G1004A, T1007C 5 239 100% 0% 0.024RT9 G1268A 4 68 99% 1% 0.29RT10 T554C, G1058C 5 61 100% 0% 0.024
1
The percentage was calculated after the number of clones of each ribotype was normalized by the total number of clones in acne patients (acne index).
2
The percentage was calculated after the number of clones of each ribotype was normalized by the total number of clones in normal individuals.
3
Mann–Whitney–Wilcoxon rank-sum test.
S Fitz-Gibbon 
et al.
The Skin Microbiome Associated with Acne
3

Activity (4)

You've already reviewed this. Edit your review.
1 thousand reads
1 hundred reads
Dr P.R. Raghavan liked this

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->