Dissecting the taxonomic heterogeneity within Propionibacterium acnes

12 Pages·2016·0.46 MB·English

InternationalJournalofSystematicandEvolutionaryMicrobiology(2015),65,4776–4787 DOI10.1099/ijsem.0.000648
Dissecting the taxonomic heterogeneity within
Propionibacterium acnes: proposal for
Propionibacterium acnes subsp. acnes
subsp. nov. and Propionibacterium acnes
subsp. elongatum subsp. nov.
Itaru Dekio,1,2,3 Renata Culak,1 Raju Misra,4 Tom Gaulton,1 Min Fang,1
Mitsuo Sakamoto,3 Moriya Ohkuma,3 Kenshiro Oshima,5
Masahira Hattori,5 Hans-Peter Klenk,6 Dunstan Rajendram,7
Saheer E. Gharbia4 and Haroun N. Shah1
Correspondence 1Proteomics ResearchUnit, CultureCollections,Public HealthEngland, Colindale,London,UK
ItaruDekio 2DepartmentofDermatology,MedicalCentreEast,TokyoWomen’sMedicalUniversity,Tokyo,Japan
[email protected]
3MicrobeDivision/JapanCollectionofMicroorganisms,RIKENBioResourceCenter,Tsukuba,Japan
4GenomicResearchUnit,PublicHealthEngland,Colindale,London,UK
5HattoriLab,TheUniversityofTokyoKashiwaCampus,Kashiwa,Japan
6SchoolofBiology,NewcastleUniversity,NewcastleuponTyne,UK
7GenomicServicesUnit,PublicHealthEngland,Colindale,London,UK
Propionibacteriumacnessubsp.acnessubsp.nov.andPropionibacteriumacnessubsp.
elongatumsubsp.nov.aredescribed.Theseemanatefromthethreeknownphylotypesof
P.acnes,designatedtypesI,IIandIII.Electronmicroscopyconfirmedthefilamentouscellshape
oftypeIII,showingastrikingdifferencefromtypesI/II,whichwereshortrods.Biochemicaltests
indicatedthat,intypesI/II,eitherthepyruvate,L-pyrrolidonylarylamidaseorD-ribose2testwas
positive,whereasallofthesewerenegativeamongtypeIIIstrains.Matrix-assistedlaser-
desorption/ionizationtime-of-flightmassspectrometry(MALDI-TOFMS)spectra,whichprofile
mainlytheirribosomalproteins,weredifferentbetweenthesetwogroups.Surface-enhanced
laser-desorption/ionizationtime-of-flightmassspectrometry(SELDI-TOFMS)spectraofall
phylotypesrevealedaspecificproteinbiomarkerthatwasoverexpressedintypeIIIstrains
comparedwithtypesI/IIonlywhengrownaerobically.Referencestrainshadhighwhole-genome
similaritybetweentypesI(.91%)andII(.75%),butaconsiderablylowerlevelof72%
similaritywithtypeIII.recAandgyrBsequencedendrogramsconfirmedthedistantrelatednessof
typeIII,indicatingthepresenceoftwodistinctcentresofvariationwithinthespeciesP.acnes.
Ontheotherhand,cellularfattyacidprofilesand16SrRNAgenesequencerelatedness
(.99.3%)circumscribedthespecies.Thus,weproposetwosubspecies,Propionibacterium
acnessubsp.acnessubsp.nov.fortypesI/IIandPropionibacteriumacnessubsp.elongatum
subsp.nov.fortypeIII.ThetypestrainofPropionibacteriumacnessubsp.acnesisNCTC737T
(5ATCC6919T5JCM6425T5DSM1897T5CCUG1794T),whilethetypestrainof
Propionibacteriumacnessubsp.elongatumisK124T(5NCTC13655T5JCM18919T).
Propionibacteriumacnesisafacultativelyanaerobicbacteria
that inhabits the human skin as one of the dominant
Abbreviations: FAME, fatty acid methyl ester; MALDI-TOF, matrix-
assistedlaser-desorption/ionizationtime-of-flight;SELDI-TOF,surface- micro-organisms (see, for example, Dekio, 2012; Grice
enhancedlaser-desorption/ionizationtime-of-flight. et al., 2009). It inhabits the hair follicles in order to
avoid the well-aerated surface of the skin (Dessinioti &
The GenBank/EMBL/DDBJ accession numbers for newly determined
Katsambas, 2010). It also exists as a minor microbiome
sequences in this work are available with the online Supplementary
Material. species in the oral cavity (Dewhirst et al., 2010) and
4776 000648G2015IUMS PrintedinGreatBritain
TaxonomicheterogeneitywithinPropionibacteriumacnes
human gut. P. acnes, originally described by Gilchrist length polymorphism and principal component analysis
(1901) as ‘Bacillus acnes’, was given its present name by and confirmed the presence of three clusters.
Douglas & Gunter (1946). P. acnes has long been con-
Inthispaper,wedescribetheresultsofacomparativestudy
sidered as a pathogen of acne vulgaris (common acne),
of types I, II and III and clinical isolates of P. acnes. Our
although this is not proven conclusively (Dessinioti &
results indicated that all strains belong to the species
Katsambas, 2010). In addition to its association with the
P. acnes. However, using morphological, biochemical and
skin, P. acnes hasincreasingly beenreportedin association
proteogenomic properties, two distinct groups were dis-
with various infections and conditions, including prosthe-
cernible, with type III strains representing a novel centre
tic joint infection (Tunney et al., 1999), back pain (Albert
of variation. Genome sequence comparison revealed that
et al., 2013), prostate cancer (Cohen et al., 2005) and sar-
a type III strain JCM 18909 and a type I strain NCTC
coidosis (Negi et al., 2012).
737T(thetypestrainofthespecies)sharedonly72 %simi-
Two phenotypes of P. acnes, types I and II, were distin- larity, together with some biochemical differences.
guished by serological agglutination tests and cell-wall We therefore propose two subspecies, Propionibacterium
sugar analysis (Johnson & Cummins, 1972). Additional acnes subsp. acnes subsp. nov. for types I and II and
studies have shown that these biovars display differences Propionibacterium acnes subsp. elongatum subsp. nov. for
in the fermentation of sugars and sugar alcohols (Higaki type III.
etal.,2000;Kishishitaetal.,1979).Recentmolecularanal-
Seventeen clinical isolates and three culture collection
ysis using the recA gene sequence confirmed their distinc- strains of P. acnes and 14 culture collection strains of
tiveness (McDowell et al., 2005). Unlike types I and II, otherspeciesofthegenusPropionibacteriumwereincluded
with a cell shape of short rods, another phenotype with inthestudy(Table1).TheisolatesofP.acnesfromhuman
filamentous cell shape, type III, was later documented skin werepartofacollection of50strains usedinarecent
(McDowell et al., 2008), dividing the species into three study by the authors, in which the genetic diversity of
clades. Rood et al. (2011) utilized amplified fragment strains of P. acnes from Japan and Europe was compared
Table1.StrainsofP.acnesandothermembersofthegenusPropionibacteriumusedinthestudy
Otherstrainsincludedwere:PropionibacteriumacidifaciensCCUG57100T,P.acidipropioniciDSM4900T,P.australienseCCUG46075T,P.avidum
NCTC 11864T, P. avidum K150 (clinical strain, Japan), P. cyclohexanicum JCM 21245T, P. freudenreichii subsp. freudenreichii NCTC 10470T,
P. freudenreichii subsp. shermanii CCUG 36819T, P. granulosum NCTC 11865T, P. jensenii CCUG 48883T, P. lymphophilum NCTC 11866T,
P.microaerophilumDSM13435T,P.propionicumNCTC12967TandP.thoeniiCCUG28149T.
Strain Culturecollectionnumber(s) Origin Morphology
TypeI
SubtypeIA
1
PAC1T* NCTC737T5JCM6425T5DSM1897T5CCUG1794T Facialacne,UK Shortrods
PAC3 JCM6495 Unknown Shortrods
K107 JCM18916 Normalforeheadskin,Japan Shortrods
B21 2 Plateletbagcontaminant,UK Shortrods
SubtypesIA andIB
2
K51 JCM18907 Normalforeheadskin,Japan Shortrods
K115 JCM18918 Normalforeheadskin,Japan Shortrods
B24 2 Plateletbagcontaminant,UK Shortrods
TypeII
PAC2 JCM6473 Subcutaneousabscess,unknown Shortrods
K127 JCM18920 Eczematousskin,Japan Shortrods
S13 JCM18926 Normalaxillaskin,Japan Shortrods
B23 2 Plateletbagcontaminant,UK Shortrods
TypeIII
K57 JCM18909 Normalforeheadskin,Japan Elongated,mainlyshortrods
K124T* NCTC13655T5JCM18919T Normalforeheadskin,Japan Elongated,branching
K290 JCM18925 Normalforeheadskin,Japan Elongated
B12 2 Plateletbagcontaminant,UK Elongated,branching
B37 2 Plateletbagcontaminant,UK Elongated,branching
B40 2 Plateletbagcontaminant,UK Elongated,mainlyshortrods
B52 2 Plateletbagcontaminant,UK Elongated,branching
*Typestrainsofthenovelsubspecies.
http://ijs.microbiologyresearch.org 4777
I.Dekioandothers
using multilocus sequence typing (Dekio et al., 2012). The Forfattyacidanalysis,eachstrainwasgrownonEGagarat
skinisolateswereidentifiedtospecieslevelusing16SrRNA 37 8Ctostationaryphasebymonitoringchangesinoptical
gene sequencing and further subtyped by multilocus density as described for growth curves. Fatty acid methyl
sequence typing (Dekio et al., 2012). Platelet bag- esters(FAMEs)wereobtainedfrom40 mgcellsbysaponi-
contaminant strains were identified to species level by fication, methylation and extraction using minor modifi-
using the API Coryne kit (bioMe´rieux) and further cations (Kuykendall et al., 1988; Sakamoto & Ohkuma,
subtyped using MALDI Biotyper (Bruker). The culture 2013) of the method of Miller (1982). Cellular fatty acid
collection isolates were obtained from the NCTC, JCM, profiles were determined by using the Sherlock Microbial
CCUG and DSMZ. Identification System version 2.99B (MIDI) with database
BHIBLAversion3.80.Peakswereautomaticallyintegrated,
The morphology of cells was studied using both light and
fatty acids were identified by equivalent chain-length and
electron microscopy. Twenty-one isolates were grown
percentagesofthetotalpeakareawerecalculated.External
anaerobicallyfor5 days,andafewrandomlyselectedcolo-
calibration was done by using MIDI calibration mixture 1
nies were then taken from the agar plates and smeared on
(FAMEsofstraight-chainsaturatedfattyacidsfrom9to20
glassslides.TheywereGramstainedandimagesweretaken
carbons in length and five hydroxy acids).
using a light microscope. Representative strains of types I,
IIandIIIwereselectedforelectronmicroscopy.Thestrains AllstrainsofspeciesofthegenusPropionibacteriumusedin
were: K115, B21 and B24 (type I), K127 and B23 (type II) this study were analysed for meso- or LL-diaminopimelic
and K57, B12, B37, B40 and B52 (type III). They were acid as described by Shah et al. (1976).
grown anaerobically for 2 days and cells were scraped
For proteomic analysis using matrix-assisted laser-
from the agar plates and suspended in a small volume of
desorption/ionization time-of-flight mass spectrometry
PBS before fixation by addition of an equal volume of
(MALDI-TOF MS), the strains were plated on Columbia
Carson’s solution(Carson &Hladik,2009).Thefixed bac-
blood agar (CBA) plates and incubated at 37 8C under
terial suspension was negatively stained with 1 % (w/v)
anaerobic conditions [85 % N , 10 % H , 5 % CO ,
ammonium molybdate on carbon/Formvar-coated, 2 2 2
,0.02 % O in Whitley A35 Anaerobic Workstation
copper electron microscopy grids (Agar Scientific) and 2
(Don Whitley Scientific)], while aerobic growth utilized a
digitalimageswereacquiredwithaJEM1400transmission
standardincubatorsetat37 8C.Allcultureswereincubated
electron microscope (JEOL) equipped with an AMT XR60
for 5 days for both conditions, when they would have
camera (Debben).
reached stationary growth phase. The formic acid-aceto-
For biochemical analysis, 17 bacterial strains (six type I, nitrile extraction method was applied to all isolates and
four type II and seven type III strains) were cultured for mass spectra were acquired as described previously (Khot
2 days under anaerobic conditions. The cells were sus- et al., 2012). Briefly, bacterial cells were suspended in
pended in 3 ml saline in plastic cuvettes (bioMe´rieux). 300 mlwaterandmixedwith900 mlabsoluteethanol.Etha-
UsingtheMcFarlandreadingunit(bioMe´rieux),theden- nolwascompletelyremovedafterthesecondcentrifugation
sity was adjusted to a McFarland reading of 2.7–3.3. The (14 000 g for 2 min) and the pellets were resuspended in
sample cuvettes were placed onto a plastic ‘boat’ with 50 ml70 %formicacidand50 mlpureacetonitrile.Samples
an ‘Anaerobic and Corynebacteria identification card’ were vortexed and centrifuged at 14 000 g for 2 min and
for each of the cuvettes. The ‘boat’ was then placed in a 1 ml of each supernatant was placed in duplicate onto an
VITEK2 system (bioMe´rieux) for analysis. A total of 36 MSP 96 target polished steel (Bruker Daltonics) and air-
biochemical tests were performed on all strains. The dried.Thespotswereoverlaidwith1 mla-cyano-4-hydroxy-
tests used were (acid production unless indicated other-
cinnamic acid (CHCA) (saturated solution of CHCA in
wise): D-galactose, leucine arylamidase, the Ellman reac- 50 % acetonitrile/2.5 % trifluoroacetic acid) was added
tion, phenylalanine arylamidase, L-proline arylamidase, and air-dried to co-crystallize the sample and matrix. The
L-pyrrolidonyl arylamidase, cellobiose, tyrosine aryla- target plate was then processed using a Microflex LT mass
midase, Ala-Phe-Pro arylamidase, D-glucose, D-mannose, spectrometer (Bruker Daltonics; software version 3.4).
maltose, sucrose, arbutin, N-acetyl-D-glucosamine, Data collection was done in automatic mode by collecting
5- bromo-4-chloro-3-indoxyl-b-glucuronide, urease, b-
240 laser shots from six different positions within the spot.
galactopyranosidase indoxyl, a-arabinosidase, 5-bromo-4-
MALDI measurements were recorded in a positive linear
chloro-3-indoxyl-a-galactoside, b-mannosidase, arginine,
mode withmass range 2–20 kDa.
pyruvate, maltotriose, aesculin hydrolysis, b-D-fucosidase,
5-bromo-4-chloro-3-indoxyl b-N-acetyl-glucosamide, 5- For surface-enhanced laser-desorption/ionization time-of-
bromo-4-chloro-3-indoxyl a-mannoside, a-L-fucosidase, flight mass spectrometry (SELDI-TOF) MS, protein
phosphatase, L-arabinose, D-ribose 2, phenylphosphonate, samples were extracted by a standard lysis method using
a-L-arabinofuranosideandD-xylose.Inadditiontobiochemi- 8 M urea, 2 % CHAPS and 40 mM Tris (Shah et al.,
calanalysesusingtheVitek2system,alltestswerecarriedout 2005). The cells were harvested into 500 ml standard lysis
usingconventionalbiochemicaltestsasdescribedpreviously buffer containing 300 mg glass beads. The samples were
(Shah&Collins,1990). placedinaFastPrepdevice(MPBiologicals)anddisrupted
4778 InternationalJournalofSystematicandEvolutionaryMicrobiology65
TaxonomicheterogeneitywithinPropionibacteriumacnes
for a total of 60 s (3620 s with 5 min cooling between Table2. Genome data used in the in silico DNA–DNA
them). The samples were then centrifuged at 21 000 g for hybridizationstudy
30 min and the supernatants were collected. The protein
F,Fullysequenced;C,contig-levelsequence.
concentrationoftheextractswasmeasuredusingtheBrad-
ford assay (Bradford, 1976). The wells of the Q10 Chip
Strain Status(accessionno.)
(Weak Cation Exchanger ProteinChip) arrays were pre-
activated for 15 min with 5 ml 20 mM Tris/HCl, pH 7.4, TypeI
containing 0.1 % Triton X-100. The buffer was decanted SubtypeIA
1
and6 mgtotal protein in pre-activation buffer was applied DSM1897T(5NCTC737T) C(PRJNA169817)
to each spot. The ProteinChip was left in a humidity SK137 F(CP001977)
chamber for 1 h. After the incubation, unbound sample 266 F(CP002409)
and buffer were removed and the spots were washed C1 F(CP003877)
HL096PA1 F(CP003293)
three times with 20 mM Tris/HCl, pH 7.4, containing
0.1 %Triton X-100. The chipwasdried and0.5 ml sinapi- K107(5JCM18916) C(PRJDB916)
SubtypeIA
nic acid (saturated solution, 5 g sinapinic acid in 0.05 % 2
IA2_33 F(CP003195)
trifluoroacetic acid, 50 % acetonitrile) was applied twice
IA2_17 F(CP003196)
to each spot. The ProteinChip arrays were analysed in a
IA2_31 F(CP003197)
linear SELDI-TOF mass spectrometer (Bio-Rad; model
SubtypeIB
PBS II) according to an automated data collection proto-
KPA171202 F(AE017283)
col. The spectra were generated at laser intensity 230,
6609 F(CP002815)
high mass 50 kDa, detector sensitivity 10 and focus mass K115(5JCM18918) C(PRJDB917)
25 kDa. The instrument was operated in positive-ion TypeII
mode and a nitrogen laser emitting at 337 nm was used. ATCC11828 F(CP003084)
CSV files of the spectra were exported for further analysis. K127(5JCM18920) C(PRJDB918)
TypeIII
16S rRNA gene sequences were obtained from the DDBJ/
K57(5JCM18909) C(PRJDB915)
EMBL/GenBankdatabasefortypestrainsandourprevious
work(Dekioetal.,2012).Evolutionary distanceswerecal-
culatedusingCLUSTAL_X softwareandaneighbour-joining
treewasreconstructedusingNJPlotsoftware.recAandgyrB Table3. Comparison of phenotypic test results of the type
gene sequences were extracted from fully sequenced gen- speciesofPropionibacterium,P.freudenreichii,andP.acnes
omes in the NCBI Genome Database. Evolutionary dis-
Alltaxaarepositiveforindoleproduction,catalaseactivityandacid
tances were computed using the JTT matrix-based
productionfromerythritol,fructoseandglucose.Alltaxaarenegative
methodandthetreewasreconstructedinMEGA6software.
for starch hydrolysis and acid production from glycogen, mannitol,
FourofouroriginalisolatesofP.acnes,K57(5JCM18909; mannose,melezitose,melibiose,raffinose,rhamnose,salicin,trehalose
type III), K107 (5JCM 18916; type IA ), K115 (5JCM and xylose. d, Strain-dependent. Most strains of types I/II (P. acnes
1
18918; type IB) and K127 (5JCM 18920; type II), had subsp. acnes) were sorbitol-positive and, using the VITEK 2 system
their chromosomal DNA extracted using a Genomic-tip (bioMe´rieux), were positive for the pyruvate, L-pyrrolidonyl
100/G (Qiagen) for whole-genome analysis. Sequencing arylamidase andD-ribose 2 tests. Strains oftype III (P.acnes subsp.
was performed to contig level by using Ion Torrent PGM elongatum) were negative for these tests. See Table 5 for further
Systems (Life Technologies). Low-quality and short reads differentiationofthesephylotypes.
were filtered and the remaining reads were assembled
using Newbler version 2.8 (454 Life Sciences). In the next Test P.freudenreichii P.acnes
step, 15 genomes of strains of P. acnes, including ten
TypesI/II TypeIII
fully sequenced genomes on the NCBI Genome Database
(ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/), one scaf- Aesculinhydrolysis + 2 2
fold genome of the type strain in the DSMZ (DSM Gelatinliquefaction 2 + 2
1897T) and four contig-level genomes from our clinical Acidproductionfrom:
strains, were analysed (Table 2). The FASTA files of the Arabinose + 2 2
genome sequences were analysed to calculate genome Inositol + + +
sequence similarities using the GGDC 2.0 algorithm Lactose +/2* 2 2
Maltose + 2 2
(Meier-Kolthoff et al., 2013).
Ribose d d +
Sugar fermentation results of P. acnes types I, II and III Sorbitol 2 d 2
with Propionibacterium freudenreichii, the type species of Sucrose + 2 2
genusPropionibacterium,areshowninTable3.Theresults
of the cellular fatty acid analysis are shown in Table 4. *P. freudenreichii subsp. freudenreichii is lactose-negative, whereas
In keeping with its identity, all strains designated P. acnes Propionibacteriumfreudenreichiisubsp.shermaniiislactose-positive.
http://ijs.microbiologyresearch.org 4779
I.Dekioandothers
Table4.Long-chaincellularfattyacidprofilesof24strainsofP.acnes(fivetypeIA ,fourtypeIA ,fourtypeIB,eighttypeII,three
1 2
typeIII)andsixotherspeciesofPropionibacterium
Strains:1,P.acnestypeIA (3strains);2,P.acnestypeIA JCM6495;3,P.acnestypeIA NCTC737T;4,P.acnestypeIA (4strains);5,P.acnestype
1 1 1 2
IB (4 strains); 6, P. acnes type II (7 strains); 7, P. acnes type II JCM 6473; 8, P. acnes type III (3 strains); 9, P. avidum NCTC 11864T; 10,
P.cyclohexanicum JCM 21245T; 11, P.freudenreichii subsp. freudenreichii NCTC 10470T; 12, P.granulosum NCTC 11865T; 13, P.lymphophilum
NCTC11866T;14,P.propionicumNCTC12967T.Thefirstandsecondmostabundantfattyacids(inbold)wereidenticalinstrainsofP.acnes,
except for culture collection strains (listed separately), which suggests that components may alter with repeated subculturing. ALDE, Aldehyde;
DMA,dimethylacetal.
Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14
12:0FAME 0.9 0.4 1.2–0.0 0.3–0.0 0.2–0.0 0.2 1.8
13:0FAME 0.3 0.4–0.0
14:0FAME 1.3–1.0 1.2 1.1 1.4–0.9 2.3–1.3 1.7–0.0 1.0 1.2–0.0 0.8 2.5 1.3 0.9 9.1 1.4
14:0DMA 0.6–0.3 0.6 0.7–0.4 0.7–0.0 0.4 0.5–0.0 0.4 0.6 0.4
15:0isoFAME 39.4–33.8 37.9 38.8 36.3–30.0 34.2–23.3 35.4–28.2 39.3 38.1–35.4 25.8 3.5 34.9 33.4 21.5
15:0anteisoFAME 8.1–5.4 7.7 8.9 7.1–5.4 7.6–5.3 7.0–4.7 7.3 5.9–4.9 7.6 24.4 7.6 5.5 14.1 40.9
16:0ALDE 1.0–0.6 0.5 1.1 0.7–0.0 0.9–0.6 1.4–0.0 0.7 0.6–0.0 0.7 0.4 0.7 0.4
15:0FAME 1.0–0.7 0.7 0.4 3.8–0.9 1.6–0.8 2.5–0.0 0.7 2.6–2.2 1.9 3.8 0.7 3.7 11.3
15:0isoDMA 6.4–5.7 8.0 6.5 4.7–3.5 5.3–3.3 6.5–2.9 5.3 5.5–3.0 4.3 0.3 5.4 5.0
15:0anteisoDMA 0.8–0.6 1.0 0.9 0.6–0.0 0.8–0.7 1.1–0.0 0.7 0.7–0.0 0.9 0.6 0.7 0.6
16:0FAME 7.3–5.7 6.3 5.6 8.2–7.2 8.7–7.5 11.4–6.9 5.5 9.6–5.6 5.5 6.5 7.4 4.6 25.2 2.8
16:0DMA 4.6–3.2 2.6 4.8 3.0–2.3 4.6–3.1 6.9–4.0 3.3 3.3–2.4 3.3 2.6 3.8 1.9
17:0isoFAME 6.4–5.3 6.7 4.6 7.4–5.8 7.4–5.0 6.6–3.4 7.3 8.9–3.5 4.1 1.4 6.1 9.5 1.1
17:0anteisoFAME 0.7–0.5 0.7 0.7 1.0–0.8 1.1–0.7 0.8–0.0 1.1 1.2–0.4 1.1 7.1 1.0 0.5 1.3 2.1
17:0FAME 0.4–0.3 0.6 0.4–0.0 1.1–0.0 0.6–0.0 0.3 0.5–0.0 0.5 2.2 0.3 3.1 3.5
17:0isoDMA 3.1–1.8 3.2 3.4 2.3–1.5 3.5–2.5 4.2–0.0 3.5 2.0–1.3 2.3 0.3 2.6 2.6
17:0anteisoDMA 0.6–0.4 0.6 0.7 0.4–0.0 0.8–0.5 0.9–0.0 0.6 0.3–0.0 0.8 0.9 0.5
17:0DMA 0.2 0.3 0.8 0.6
18:2cis9,12FAME 3.8–1.8 0.7 1.5–1.0 1.7–0.6 5.9–2.5 1.0 2.5–2.3 2.9 0.8 0.9 2.3 2.2 0.8
18:1cis9FAME 19.0–11.8 5.3 8.4 23.1–18.9 18.3–15.0 29.1–11.8 6.8 23.6–15.8 21.2 11.0 16.2 17.8 31.8 5.8
18:0FAME 2.2–1.5 2.2 1.2 2.5–1.2 3.9–1.6 4.0–1.3 1.1 2.3–1.5 1.3 0.8 1.8 1.0 3.9 0.7
18:1cis9DMA 0.9–0.0 1.1 2.3 1.1–0.0 1.9–1.4 2.1–0.0 2.0 2.6–0.0 3.0 0.4 1.0 0.5 3.6
18:1cis11DMA 0.3–0.0 1.1 2.0 2.0–0.0 2.6–0.0
19:0FAME 0.4 0.6–0.0 0.5
20:0FAME 1.1–0.0 3.5 5.0–0.0 2.2–0.0 1.9–0.0
shared common long-chain fatty acid profiles among the but were taxonomically more distantly related to other
phylotypes, with iso-C FAME and cis9-C FAME species of the genus Propionibacterium (approx. 96–
15:0 18:1
beingthecommonpredominantfattyacidsamongisolates. 91 %).However,othertestsusedinthisstudyshowedsig-
However, there were minor differences among strains; for nificant intraspecies heterogeneity.
example, the three culture collection strains NCTC 737T
Housekeeping gene analysis confirmed the considerable
(type IA ), JCM 6495 (type IA ) and JCM 6473 (type II)
1 1 genomic distance between types I/II and type III. While
haddifferent fattyacidsastheir secondmajor component.
Thus, NCTC 737T possessed anteiso-C FAME, JCM the gyrB dendrogram divided the species into seven
15:0
clades, the recA tree clearly separated the three types into
6495 had iso-C DMA, while JCM 6495 had iso-
15:0
three clades, separating type III into an individual branch
C FAME (Table 4). The ratio of the major iso-C
17:0 15:0
FAME and the second major component was between (Fig. 2).
0.18 and 0.92. All strains of Propionibacterium used in
Gram-stainsmearsofcellsrevealedtwobroadcelltypesby
this study possessed LL-diaminopimelic acid. lightmicroscopy.All14strainsbelongingtotypesI/IIwere
Comparative analysis of 800 bp-long contig consensus seenas shortrods,whileall seven type IIIstrainsappeared
sequences of the 16S rRNA gene generated by assembling as predominantly elongated cells (not shown). Electron
contigs of forward and reverse sequences revealed a high microscopy confirmed these two cellular forms among
degree of relatedness among strains designated P. acnes the phylotypes, although no distinctive ultrastructural fea-
(Fig. 1). Strains shared 99.3–100 % sequence similarity, tures were apparent (Fig. 3). The proportion of elongated
4780 InternationalJournalofSystematicandEvolutionaryMicrobiology65
TaxonomicheterogeneitywithinPropionibacteriumacnes
P. freudenreichii subsp freudenreichii DSM 20271T (CP010341)
P. propionicum JCM 5830T (AB971802)
P. granulosum JCM 6498T (AB638444)
P. granulosum DSM 20700T (AJ003057)
P. avidum DSM 4901T (AJ003055)
P. avidum ATCC 25577T (KF906606)
P. avidum K150 (LC098768)
K51_IA2 (AB777852)
P. acnes
K72_IA2 (AB777854)
K56_IA2 (AB777853)
K107_IA1 (AB777860)
K282_IA1 (AB777865) Type I
K280_IB (AB777864)
P. acnes JCM 6425T
K114_IB (AB628069)
K115_IB (AB628070)
K80_II (AB777855)
K127_II (AB628071)
K106_II (AB777859)
Type II
K96_II (AB777857)
K104_II (AB777858)
0.01
K145_II (AB777862)
K124_III_T (AB777861)
Type III
K57_III (AB628068)
Fig. 1. Phylogenetic tree, based on 16S rRNA gene sequence comparisons of ,1400 aligned bases and reconstructed
using the neighbour-joining method, showing the relationship of types of P. acnes and other representative species of the
genusPropionibacterium.Bar,0.01substitutionspernucleotideposition.
cells varied among the latter group. For example, five shared by all members of the genus Propionibacterium
strains (K124, K290, B12, B37 and B52) showed more including P. acnes, and 14 were common to all members
than 50 % of cells with an elongated form, whereas two of P. acnes. However, 10 reactions (Table 5) were differ-
strains, K57 and B40, possessed only around 20 % of the ent among members of P. acnes as follows. Positive
cells as elongated forms. The finding was consistent with results were obtained with pyruvate and 5-bromo-4-
a previous report by McDowell et al. (2008). chrolo-3-indoxyl a-mannoside tests among type I/II
strains. Type III strains were less reactive to these 10
A panel of 36 biochemical reactions was assessed using
tests, with only one or two positive reactions, such as
ten strains of P. acnes and the type strains of four
deamidation of arginine (see Table 5).
other species of the genus Propionibacterium,
P. freudenreichii, P. acidipropionici, P. avidum and MALDI-TOF MS revealed prominent mass ions within
P. granulosum, as controls. Twelve reactions were the range 2000–11 000 Da. The predominant mass
http://ijs.microbiologyresearch.org 4781
I.Dekioandothers
10 Propionibacterium acnes JCM18916 (BAVN00000000)
Propionibacterium acnes J165 (ADJL00000000) recA
Propionibacterium acnes HL030PA1 (ADYB00000000)
0.001
Propionibacterium acnes TypelA2P.acn31 (CP003197)
Propionibacterium acnes HL027PA1 (ADYT00000000)
Propionibacterium acnes C1 (CP003877)
Propionibacterium acnes TypelA2P.acn17 (CP003196)
1 Propionibacterium acnes HL078PA1 (ADZG00000000)
Propionibacterium acnes DSM1897 (AWZZ00000000)
Propionibacterium acnes JCM18918 (BAVO00000000) Type I
Propionibacterium acnes KPA171202 (AE017283)
29 Propionibacterium acnes 266 (CP002409)
Propionibacterium acnes HL020PA1 (ADYO00000000)
Propionibacterium acnes FZ1/2/0 (AOGR00000000)
34
Propionibacterium acnes SK137 (CP001977)
Propionibacterium acnes 6609 (CP002815)
50
Propionibacterium acnes HL097PA1 (ADWI00000000)
63 Propionibacterium acnes PRP-38 (AIJP00000000)
Propionibacterium acnes TypelA2P.acn33 (CP003195)
Propionibacterium acnes ATCC11828 (CP003084)
64 Propionibacterium acnes JCM18920 (BAVP00000000)
Type II
Propionibacterium acnes HL110PA3 (ADZE00000000)
62 Propionibacterium acnes J139 (ADFS00000000)
Propionibacterium acnes JCM18909 (BAVM00000000) Type III
Propionibacterium acnes HL030PA1 (ADYB00000000)
50Propionibacterium acnes KPA171202 (AE017283) Type IB gyrB
58 Propionibacterium acnes 6609 (CP002815)
Propionibacterium acnes JCM18918 (BAVO00000000)
Propionibacterium acnes TypeIA2P.acn17 (CP003196)
79 Propionibacterium acnes TypelIA2P.acn33 (CP003195)
Type IA
Propionibacterium acnes HL027PA1 (ADYT00000000) 2
Propionibacterium acnes TypelA2P.acn31 (CP003197)
Propionibacterium acnes HL078PA1 (ADZG00000000)
13Propionibacterium acnes SK137 (CP001977)
35 Propionibacterium acnes FZ1/2/0 (AOGR00000000) Type IA1 (Clade 1)
Propionibacterium acnes C1 (CP003877)
Propionibacterium acnes J165 (ADJL00000000)
19 45 Propionibacterium acnes HL020PA1 (ADYO00000000) Type IA (Clade 2)
Propionibacterium acnes 266 (CP002409) 1
Propionibacterium acnes DSM1897 (AWZZ00000000)
Propionibacterium acnes JCM18916 (BAVN00000000)
40
Propionibacterium acnes HL110PA3 (ADZE00000000)
66 Propionibacterium acnes JCM18920 (BAVP00000000) Type II
47 Propionibacterium acnes J139 (ADFS00000000)
69
Propionibacterium acnes ATCC11828 (CP003084)
Propionibacterium acnes HL097PA1 (ADWI00000000)
Type IC
0.0005 58 49 Propionibacterium acnes PRP-38 (AIJP00000000)
Propionibacterium acnes JCM18909 (BAVM00000000) Type III
Fig.2.PhylogenetictreebasedonrecAandgyrBhousekeepinggenesequenceanalysis.Genesequencesfromgenomesof
strains of P. acnes were extracted in silico and the computed evolutionary distances were expressed in neighbour-joining
dendrograms. The symbol ‘[]’ means that the branch is not shown to scale. Bars, 0.001 (recA) and 0.0005 (gyrB) substi-
tutionspernucleotideposition.
ions were around 3500 and 7100 Da. Utilizing a combi- type III strains. Prominent mass ions at 3617 and
nation of both major and less intense peaks, four dis- 7240 Da were characteristic for type III strains. The
tinct mass spectral profiles were evident (Fig. 4). Type mass ion detected at 7240 Da is consistent with work
I and II strains shared three very similar profiles, reported by Nagy et al. (2013). Type I and II strains
whereas a unique mass spectrum was associated with showed a broader distribution of mass ions around
4782 InternationalJournalofSystematicandEvolutionaryMicrobiology65
TaxonomicheterogeneitywithinPropionibacteriumacnes
(a) (b)
(c) (d)
Fig. 3. Cell shapes of the three types of P. acnes under the electron microscope. (a) Type I (strain B21). (b) type II (strain
B23).(c,d)TypeIII(strainB37),showingoverallcellshape(c)andbranchingfeatures(d).Bars,500mm.
Table5.PhenotypiccharacteristicsthatdifferentiateP.acnestypeIIIfromotherstrainsofP.acnes
Tests: pyruvate, pyruvate degradation test; L-pyrrolidonyl arylamidase, pyrrolidonyl degradation test; D-ribose 2, ribose acidification reaction.
CommonamongallstrainsofP.acnesbutnotamongallmembersofthegenusPropionibacterium:positiveforEllmanreactionandphenylalanine
arylamidase;negativeforD-galactose,Ala-Phe-Pro-arylamidase,maltose,sucrose,N-acetyl-D-glucosamine,b-galactopyranosidaseindoxyl,malto-
triose,aesculinhydrolysis,b-D-fucosidase,phosphatase,L-arabinose,phenylphosphonateanda-L-arabinofuranoside.Commonamongallmembers
of the genus Propionibacterium: Positive for leucine arylamidase, L-proline arylamidase and D-glucose, and negative for cellobiose, D-mannose,
arbutin, urease, a-arabinosidase, b-mannosidase, a-L-fucosidase, 5-bromo-4-chloro-3-indoxyl b-N-acetyl-glucosamide, 5-bromo-4-chloro-3-
indoxyl a-mannoside and D-xylose. +, Positive; (+), usually positive but occasionally negative in repeated analysis; (2), usually negative but
occasionallypositiveinrepeatedanalysis;2,negative.
Reaction TypeI TypeII TypeIII
NCTC737T K51 K107 K115 B21 B24 JCM6473 K127 S13 B23 K57 K124T* K290 B12 B37 B40 B52
Pyruvate + 2 + + (+) + + + + (+) 2 2 2 2 2 (2) 2
L-Pyrrolidonyl 2 (+) (+) (+) + + 2 2 2 + 2 2 2 2 2 2 2
arylamidase
D-Ribose2 2 + 2 2 2 (+) + 2 (2) + 2 2 2 2 2 2 2
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I.Dekioandothers
m/z
3488 3500 3516 3589 3617 3628 6991 7033 7179 7240 7258
Type IA + + + +
1
Type IA /IB + + + +
2
Type II + + + +
Type III + +
3516.6 7033.1
Type IA 3589.1 7179.2
1
3487.8 3617.1 6976.7 7226.1
3559.87 7106.02
3588.0 6999.4 7175.2
3500.0
3589.1
Type IA /IB
2
3491.13513.3 3617.1 6983.7 7037.8 7232.5
3559.87 7106.02
3628.4 7258.0
3488.9 6978.2
Type II
3501.7 3619.4 7007.2 7240.4
3588.5 7181.6
3559.87 7106.02
3617.1 7233.3
Type III
3500.5 3630.7 7260.4
3491.1 3584.6 6982.9 7037.8 7185.5
3559.87 7106.02
Fig. 4. Predominant peaks in MALDI-TOF MS spectra were determined around 3500 and 7100Da in all types. In both of
these mass ranges, types I and II presented two major peaks, whereas type III presents only one peak in each of the mass
ranges. The molecular masses of the peaks were slightly different between types IA , IA /IB and II, which can be a markers
1 2
forrapididentificationusingMALDI-TOFMS.
the two prominent mass ions (see Fig. 4). To assess the (reported by Dekio et al., 2013). In accordance with the
reliability of this approach, a panel consisting of 40 % dataobtainedbyMALDI-TOFMS,theSELDI-TOFspectra
of the strains used in this study was typed initially againrevealedtwouniquebiomarkersforstrainsbelonging
using these parameters and the strains were correctly to type III (Fig. 5).
identified. These data reinforce the potential of
MALDI-TOF MS to provide key biomarkers for identi- In silico DNA–DNA hybridization based on BLAST analysis
fying unknown strains. showed relatedness values of 91–100 % among genomes of
typeIstrainsand96 %amongtypeIIstrains.Valuesbetween
SELDI-TOF MS has the capacity to extend the mass range
typeIandtypeIIwere74–78.5 %,whilethosebetweentype
ofproteinsnormallyobtainedusingMALDI-TOFMS.Fur-
I/II and type III were 72–73 %, suggesting that type III is
thermore, the selective capture of proteins using different
more distantly related to types I and II (Table 6).
ProteinChip arrays enables different classes of proteins to
be analysed. To expand the classes of proteins further, Theresultsofthisstudythereforeconfirmthatthetwocell-
cells were cultured both aerobically and anaerobically. structure types long observed among isolates of P. acnes
Despite the range of methods used to maximize available represent two centres of variation within the species. Bio-
biomarker proteins, the spectra among all strains showed chemical and genomic analyses are consistent with this
considerable homogeneity. However, differences were morphological grouping of isolates. Furthermore,
observed in expression profiles as visualized by heat maps MALDI-TOF MS and SELDI-TOF MS provide reliable
4784 InternationalJournalofSystematicandEvolutionaryMicrobiology65
TaxonomicheterogeneitywithinPropionibacteriumacnes
10000 15000 20000 25000 30000 M
An 127,JC ypeIII M18909 ¡22.9¡02.9¡52.9¡22.9 ¡22.9¡02.9¡22.9 ¡82.9¡82.9 ¡92.9 ¡02.9
Type I 5K T JC 72.72.72.72. 72.72.72. 72.72. 72. 6 72.
0
O2 1892 8920 2.92.92.92.9 2.92.92.9 2.82.8 1.1 2.9 2.9
M 1 ¡¡¡¡ ¡¡¡ ¡¡ ¡ ¡ ¡
JC II JCM 75.175.375.475.2 76.576.276.4 78.178.1 96.3 73.2 75.4
An K115, Type 828
Type II 518 C11 ¡2.9¡2.9¡2.9¡2.9 ¡2.9¡2.9¡2.9 ¡2.8¡2.8 ¡2.9 ¡2.9
O2 189 ATC 75.275.575.575.3 76.676.476.6 78.478.5 6 72.9 75.6
M
An Type III 5K107,JC JCM18918 ¡91.01.6¡91.12.0¡91.32.0¡91.02.0 ¡91.61.9¡91.12.0¡91.71.9 ¡100.00.0¡100.00.0 ¡76.82.9 ¡72.52.9 ¡91.32.0
6
1
O2 189 IB 9 1.71.71.71.7 1.61.71.6 ¡0.1 2.8 2.9 1.7
e 0 ¡¡¡¡ ¡¡¡ ¡ ¡ ¡
M p 6 0
C Ty 6 3.23.33.53.1 3.63.43.8 00.6 8.5 2.8 3.4
(J 9999 999 1 7 7 9
PIFwrigoht.ee5inn.csSueElttsLuDrbeIe-dtTwaOenFeaneMr1oS5bipacnraodllfiy1l;e7ssukocDfhatohwveeetrrheerxeopevreetsryespxioepnsreiosnfsaePnd.aiaencrotnybepisec. ourstrains KPA ¡93.01.7¡93.11.7¡92.21.7¡92.21.7 ¡93.51.2¡93.31.7¡93.61.6 6¡100.00.1 ¡78.42.8 ¡72.82.9 ¡93.21.7
of
culture in this mass range didn’t occur in types II and III. The 3
peak patterns differed between types I/II and type III (circled). data 2_31 ¡1.0¡1.0¡0.9¡1.0 ¡0.3¡00. ¡1.6¡0.8 ¡2.9 ¡2.9 ¡1.0
Mexoprreeosvseior,naoefrocbeirctaignropwrothteionfstbypeetwIeIIeynie1ld8edansdig2n0ifickaDnatly(bhoixgehde)r. affold IA 97.096.897.297.0 99.4100.6 93.693.8 76.6 72.2 96.9
An,Anaerobicculture;O2,aerobicculture. dsc IA2 17 1.01.11.01.0 0.4 ¡0.3 1.70.9 2.9 2.9 1.1
an pe 2_ ¡¡¡¡ ¡ 0 ¡¡ ¡ ¡ ¡
ata Ty IA 96.896.696.996.9 99.26100. 93.393.4 76.4 72.0 96.6
protein biomarkers to rapidly distinguish both subtypes, d
ftsohtrraatiwnshtirocahfinwsPe.opfarctonypepose,ssebItewaonasdnsiogIvIne,eldwsuhbtiocshpePcirnioecpsl.uioWdneiebtaphcretoerptiyuopmsee gorithm sequence IA2_33 ¡96.81.0¡96.71.0¡97.01.0¡96.91.0 6¡99.20.4¡99.40.3 ¡93.51.7¡93.61.6 ¡76.72.9 ¡72.22.9 ¡96.71.1
acnes subsp. acnes subsp. nov. We consider that type III al me 6
ipPslraocpseuimofnfieicnbiatencittnelryiaumdseispataicnrnacettsessufrubobsmspp.eectliohenissg,awttuahmxicohnsuwbtseopd.ewnsoaigvrnr.aantet GDC2.0 hole-geno JCM1891 ¡96.91.0¡98.40.6¡97.20.9¡96.81.0 ¡95.01.4¡94.91.4¡95.11.4 ¡91.61.9¡91.71.9 ¡74.12.9 ¡71.92.9 ¡98.50.6
G w
Description of Propionibacterium acnes singthe atedwith HL096PA1 ¡100.00.1¡98.30.7¡98.80.46 ¡96.90.4¡96.91.0¡97.01.0 ¡92.91.7¡93.11.7 ¡75.32.9 ¡72.22.9 ¡98.30.6
subsp. acnes subsp. nov. u ul
MaPrp.oLpo.iiongnet,inb.ianncct.oearricrunemcetslayocfntraeacsnnsseul)ibt.espra.taecdneass(Mac.9Ln.esn..Garc.nne.aaccnmee; dizationby entage,calc TypeIA1 C1 ¡98.80.5¡98.80.56¡98.80.5 ¡97.01.0¡96.91.0¡97.20.9 ¡93.21.7¡93.51.7 ¡75.62.9 ¡72.52.9 ¡98.80.5
Tg(GmmA4Gbc9uilhfurvmoiottloneheaneotmwnerritdld,deeeti,erdahenisawgnniiic(tnnatti1rihiisrodia9rBiphntle(4meenthi)6iris,tnooogo)ocanecn,rssanysutt-pineu’dsnobsupdrofablaoaytvtrMpethid.rihorcnieedaadoiCn-asnbifmo,tteobueiaslrcore,uliatisotmngblt)tli3rosaaniaat0onpnrnoran–fegee,ils3ce,clppDisdG7uhaeiuecnensaarusntearCrkacefnacemeorr.tctomrilil-tpolfSreteosoitbnoutrniworasiotramcriiofsncntatapihtun-ctvtoahbpaloegeaqtyeoear0ucs,Bewd.iDoie5rntam,eeolioocsrvmwt3ticeuneonerhm,grwiluideeiilponattesr,eteelaestocioenkooingttrr&stnnooy---.. Table6.InsilicoDNA–DNAhybri Theindexscoreinthistableisaperc518909K57). Strain SK137266 TypeIA16¡SK13798.30.6¡626698.30.6¡¡C198.80.598.80.5¡¡HL096PA1100.00.198.30.7TypeIA2¡¡IA2_3396.81.096.71.1¡¡IA2_1796.81.096.61.0¡¡IA2_3197.01.096.81.0TypeIB¡¡KPA17120293.01.793.11.7¡¡660993.21.793.21.7TypeII¡¡ATCC1182875.22.975.52.9TypeIII¡¡JCM1890972.22.972.02.9Typestrain(typeIA)1¡¡TDSM189798.40.6100.00.0
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