Interkingdom transfer of the acne causing agent, Propionibacterium acnes, from human to grapevine

20 Pages·2014·0.49 MB·English

Interkingdom Transfer of the Acne-Causing Agent,
Propionibacterium acnes, from Human to Grapevine
Andrea Campisano,*,1 Lino Ometto,1 Ste´phane Compant,2 Michael Pancher,1 Livio Antonielli,1,2
Sohail Yousaf,1,3 Claudio Varotto,1 Gianfranco Anfora,1 Ilaria Pertot,1 Angela Sessitsch,2 and
Omar Rota-Stabelli1
1SustainableAgro-EcosystemsandBioresourcesDepartment,ResearchandInnovationCentre,FondazioneEdmundMach,
S.Micheleall’Adige(TN),Italy
2DepartmentofHealth&Environment,BioresourcesUnit,AITAustrianInstituteofTechnologyGmbH,Konrad-Lorenz-Strasse,
Tulln,Austria
3DepartmentofEnvironmentalSciences,FacultyofBiologicalSciences,Quaid-i-AzamUniversity,Islamabad,Pakistan
*Correspondingauthor:E-mail:[email protected].
Associateeditor:ShizhongXu D
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Abstract a
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Here,wereportthesurprisingand,toourknowledge,uniqueexampleofhorizontalinterkingdomtransferofahuman fro
opportunisticpathogen(Propionibacteriumacnes)toacropplant(thedomesticatedgrapevineVitisviniferaL.).Humans, m
h
likemostorganisms,haveestablishedalong-lastingcohabitationwithavarietyofmicrobes,includingpathogensandgut- ttp
s
associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of ://a
bacterialhostswitchesfromdomesticanimalstohumans.Muchlessis,however,knownabouttheexchangeofmicrobial c
a
d
symbiontsbetweenhumansandplants.FluorescentinsituhybridizationlocalizedP.acnesinthebark,inxylemfibers, e
m
and,moreinterestingly,insidepithtissues.Phylogeneticandpopulationgeneticanalysessuggestthattheestablishment ic
.o
ofthegrapevine-associatedP.acnesasobligateendophyteiscompatiblewitharecenttransferevent,likelyduringthe u
p
Neolithic,whengrapevinewasdomesticated. .co
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Keywords:symbiosis,pathogen,endophyte,Propionibacteriumacnes,recA. /m
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e
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Introduction where it feeds on fatty acids secreted by sebaceous glands rtic
le
(Webster et al. 1981; Zouboulis 2004). This species is also -a
Bacteriaarehighlyadaptablemicroorganismsroutinelyfound b
present in the human gastrointestinal tract (Perry and s
assymbiontsorpathogensonplantsandanimalsalike.Some tra
Lambert2011),butitmayexhibitopportunisticpathogenic- c
human bacterial pathogens are even able to colonize plant itybycausingpostsurgicalinfection(Nisbetetal.2007)and t/31
tissues(TylerandTriplett2008),aphenomenonthatinmost /5
has also been occasionally found in other animals (Webster /1
casescanbeconsideredasanopportunisticexploitationofa 0
etal.1981;Lyonsetal.2009).Weherebyreportanewtypeof L5
9
temporaryhabitat.Forexample,somemicroorganismshavea P.acnescharacterizedbyanendophyticlifestyleingrapevine e/9
multihost life cycle and may use plants as alternative/inter- t98
and which we suggest has adapted to the new host to the t7
mediate hosts, as in the case of enteric human pathogens pointofobligateendophytismandpossiblyendocellularsym- er17 b
Escherichia coli, Salmonella enterica, and Klebsiella pneumo- y
biosis.WeproposetonamethisP.acnestypeZappae(hereaf- g
niae (Cooley et al. 2003; Kutter et al. 2006; Bernstein et al. u
ternamedP.Zappae)(NamedaftertheItalianterm“zappa” e
s
2007;Lacavaetal.2007).Notably,mostbacterialendophytes, [hoe]aswellasatributetoeccentriccomposerFrankZappa, t o
n
includingtheaforementionedhumanpathogens,arefaculta- to highlight the unexpected and unconventional habitat of 3
0
tiveendophytes,whichcolonizeplanttissuesonlyduringpart thisP.acnestype.) M
a
of their life time (Klerks et al. 2007). In contrast, obligate We first discovered the endophytic habitat of P. Zappae Frc
ah
endophytes exclusively colonize plants and therefore need whileperforminga16SrDNAsurveyofthegrapevineendo- 2
s0
tobeadaptedtothehostenvironment. microbiome.Specifically,Roche454ampliconsequencingof t19
Although exploiting plants as temporary hosts may be theV5–V916SrDNAhypervariableregionresultedin~6,800 T
common, host switch followed by an adaptation to the sequences ascribable to P. acnes-like bacteria. These se- r
a
newhostisrelativelyrareandoccurspreferentiallybetween quences could be assigned to 54 of the 60 analyzed plants
c
closelyrelatedhostssuchbetweeninsects(RussellandMoran and accounted for 0.5% to 5% of the total endophytic bac- k
2005) or vertebrates (Walter et al. 2011). Here, we report a terialmicrobiota.
strikingcaseofinterkingdombacterialhosttransferinvolving The consistent detection after the accurate peeling and
thehumanpathogenPropionibacteriumacnesandgrapevine sterilizing process adopted strongly suggests that P. Zappae
plants(VitisviniferaL.).Propionibacteriumacnesisthecausing is endophytic. This finding is confirmed by fluorescence in
agent of acnes and is generally associated with human skin, situhybridization(FISH)microscopyobservationsthatmade
(cid:2)TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.Allrightsreserved.Forpermissions,please
e-mail:[email protected]
Mol.Biol.Evol.31(5):1059–1065 doi:10.1093/molbev/msu075 AdvanceAccesspublicationFebruary19,2014 1059
MBE
Campisanoetal. . doi:10.1093/molbev/msu075
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FIG.1. LocalizationofPropionibacteriumacnestypeZappaeingrapevine.CSLMmicrophotographsofgrapevinestemsshowingpresenceofP.acnes(a) .c
o
inbarkand(b)inpith.Arrowspointatwhite/lowbluefluorescentP.acnesduetotheuseofP.acnesprobe-405dylight(bluechannel),EUBmix-FLUOS m
(greenchannel),HGC69a-Cy5probe(redchannel);RGBchannelsweremergedusingImageJ;(c)localizationofP.acnesinbarkand(d)inpithcells; /m
b
(e)P.acnesoccasionaldetectionnearxyleminfibers.In(c–e),P.acnesprobecoupledwithFLUOSwasusedtoshowP.acnesasgreenfluorescentinstead e/a
ofwhitefluorescent. rtic
le
-a
b
useofdifferentfluorochromesandprobes(fig.1;supplemen- sequences are mostly nested within the clade containing stra
tary figs.S1 and S2, Supplementary Material online). In par- P. acnes type I (fig. 2b and c; supplementary fig. S3, c
t/3
ticular, FISH revealed that this bacterium colonizes bark Supplementary Materialonline; for a description of P. acnes 1
/5
tissues (fig. 1a and c), occasionally fibers of xylem vessels population structure see McDowell et al. [2008]; note that /1
0
(fig. 1e), and, more interestingly, pith (fig. 1b and d), where morecloneswereamplifiedfromeachplant,foratotalof51 59
the bacterium might be intracellular (fig. 1d). Notably, FISH sequencespergene).Becauseallofthethreephylogeniesof /99
8
also revealed that only a minority of the other endophytic figure 2 are rooted using P. avidum, which like P. acnes is 71
7
bacteria reside in the pith, whereas the majority, including found mainly on humans, our results support a human b
y
actinomycetes, were found in the vascular tissues, paren- origin for the endophytic P. Zappae. The inconsistency be- g
u
chyma,andfiberssurroundingxylemvessels(supplementary tweenthepresenceoftwodistinctclusters,oneforP.Zappae es
figs.S1andS2,SupplementaryMaterialonline).Thespecific andoneforP.acnes,inthe16SrDNAgenephylogenycom- t on
3
localization of P. Zappae within plant tissues suggests that, paredwiththeinterspersedP.ZappaerecAandtlysequences 0
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while this bacterium has retained its anaerobic habit, it is amongthoseofP.acnessuggestsincompletelineagesorting,a a
subject to distinct selective pressures compared with its typical signature of very recent speciation/diversification rch
2
humancounterpart. (Maddison 1997). Comparison of the three phylogenies fur- 0
1
To investigate the evolutionary history of P. Zappae, we therrevealedgene-specificpatternsofevolution.Forinstance, 9
performedaphylogeneticanalysisofthe16SrDNAgeneby in P.Zappae,recAaccumulatesmanymoremutationsthan
randomlysamplingonerepresentativesequencefromeachof tly and the 16S rDNA gene of P. Zappae (compare red
the54grapevinesamples.Theresultingmaximumlikelihood branches in fig. 2). Second, the starlike shape of P. Zappae
treeshowedthatP.Zappae16SrDNAgenesequencescluster recAphylogeny(fig.2a,seealsoalignmentinsupplementary
in a distinct group, and are closely related to P. acnes ex- fig.S4,SupplementaryMaterialonline)indicatesthatmultiple
tractedfrompublicdatabases(fig.2a).Toconfirmtheaffinity distinctisolatescolonizegrapevine.
between P. acnes and P. Zappae, we further amplified two Tobetterunderstandtheforcesbehindthefastevolution
widelyusedspecies-specificP.acnesmarkergenes,recAand ofrecAinP.Zappae,weperformedpopulationgeneticanal-
tly, from total DNA isolated from 12 of the tested plants ysesofthisgeneandtheputativelyneutrallyevolvinggenes
(Rossi et al. 1999; McDowell et al. 2008; Sfanos and Isaacs tly and16S. The recA gene is highly polymorphic in the en-
2008; McDowell et al. 2012). Phylogenetic inferences based dophytic P. Zappae (table 1; supplementary fig. S4,
on these two genes revealed that P. Zappae recA and tly Supplementary Material online), with rare variants
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InterkingdomTransferofP.acnes . doi:10.1093/molbev/msu075
er.ndeir
markoreautth
emb
ngonthmulatesthetree,
endiaccuroot
epAto
dP.acnesBank).Recwereused
withinmGeneacteria
DNAgene(),(),and()markers.GrapeendophyticZappae(inred)clusterscloseoratlybrecAcP.hecommonnormallyfoundinhumans(blacktextcorrespondstoentriesdownloadedfroP.acnesassociatedSupportsatnodesaremaximumlikelihoodbootstrapsupports.OtherpropionibP.acnes.andareinsupplementaryfigureS3,SupplementaryMaterialonline.recA Downloaded from https://academic.oup.com/mbe/article-abstract/31/5/1059/998717 by gue
ppaeaccordingto16SrubgrouptypeIandIIoftZappaethaninhumanP.pleterootedtreesfortly st on 30 March 2019
Zaumngtosphyticecom
nibacteriheybeloinendoscale;th
ofPropiondrtecAmutationsalwaysin
F.2.PhylogenyIGAccordingtoatlydifferenttypesofbranchesarenot
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Table 1. Nucleotide Polymorphism in recA and tly Genes.
na Lb L*b Lb L b Rc Rmc Hdd Hnd Se se Se S e pf p f Dg D g
S NS S NS S NS S NS
recA
P. Zappae 55 1,044 985 242 739 0.022 5 0.993 51 124 104 35 92 0.0071 0.0051 (cid:3)2.561*** (cid:3)2.843***
P. acnes 68 1,044 879 220 659 0.001 0 0.627 8 14 3 10 4 0.0124 0.0008 0.808 (cid:3)0.757
tly
P. Zappae 15 696 291 78 213 0.000 0 0.467 4 13 12 78 213 0.0134 0.0038 (cid:3)1.849* (cid:3)1.983*
P. acnes 43 696 696 184 509 0.000 0 0.808 12 34 13 184 509 0.0255 0.0068 0.412 (cid:3)0.188
NOTE.—S,synonymoussites;NS,nonsynonymoussites.
aNumberofsequences.
bAlignmentlengthwith(L)orwithout(L*)sitescontaininggaps.
cRecombinationrate(Hudson1987)estimatedbetweenadjacentsites(R)andminimumnumberofrecombinationevents(Rm).
dHaplotypediversity(Hd)andnumberofhaplotypes(Hn). D
eNumberofsegregatingsites(S),ofwhichsaresingletons. ow
fNucleotidepolymorphism(Tajima1983). nlo
gTajima’sD(Tajima1989). ad
*P<0.05. ed
**P<0.01. fro
***P<0.001. m
h
ttp
contributingtomostofthenucleotidevariability(104ofthe required for the endophytic life of P. Zappae, and that the s
124 single nucleotide polymorphisms are singletons). This resulting lack of functional constraints has favored the fast ://a
c
a
observationisconfirmedbythestarlikephylogeny(fig.2a), accumulationofnonsynonymousmutations.Infact,thede- d
e
m
the high haplotype diversity, and a strong skew in the site generationofrecAhasrarelybeenobservedinotherbacteria, ic
frequencyspectrumofDNApolymorphism(asmeasuredby and it has been suggested that loss of recA function occurs .o
u
p
Tajima’sD;table1).Interestingly,nonsynonymouspolymor- duringtheinitialstagesofendocellularsymbiosis(Daleetal. .c
o
phism in P. Zappae’s recA sites is more than 6-fold higher 2003).Ourdatathereforepointtowardstheestablishmentof m
/m
thaninP.acnesorthologsextractedfromtheNCBInucleotide anobligate(probablyendocellular)symbiosisofP.Zappaein b
e
database((cid:2)=0.0051vs.(cid:2)=0.0008,respectively;table1).We grapevine.Thisconclusionfindsreciprocalconfirmationwith /a
canexcludeaP.Zappae-specificlargemutationratebecause thedirectobservationofP.Zappaebyfluorescentmicroscopy rtic
le
synonymouspolymorphismofrecAandpolymorphismoftly (fig. 1d) and by our inability to cultivate any of the strains -a
b
and16SarelowerinP.ZappaethaninP.acnes(possiblydue carryingamutatedrecA.EffortstocultivateP.acnesfromthe stra
to the bottleneck associated to the establishment of endo- plantendospherewereinfactmostlyunsuccessful,whereby ct/3
phytism; table 1, in the 16S rDNA gene polymorphism was onlytwoP.acnesstrainscouldbeisolatedonNutrientAgar 1
/5
(cid:2)=0.0 and (cid:2)=0.0016 for P. Zappae and P. acnes, respec- medium.ThesetwostrainshadaslowevolvingrecA(inblue /1
0
tively). Rather, such polymorphic patterns suggest that the infig.2c),andtheir16Ssequencesdidnotclusterwiththose 59
/9
accumulation of nonsynonymous mutations is due to the typical of P. Zappae (in blue in fig. 2a). For this reason, we 9
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absence (or strong relaxation) of purifying selection in could not exclude that these isolates originated from 71
7
P.Zappae’srecA,ascorroboratedbyfouradditionalobserva- environmental contaminations occurred after sampling and b
y
tions. First, three of the P. Zappae’s recA sequences contain surface disinfection, and hence did not regard them as g
u
e
frameshiftmutationsthatcausetheappearanceofpremature P.Zappae. s
stopcodons(supplementaryfig.S4,SupplementaryMaterial AsrecAisevolvingneutrally,itcanbesafelyusedtoesti- t on
3
online).Second,themeanrateofnonsynonymoussubstitu- mate the age of P. Zappae endophytism in grapevine. 0
M
tions(0.00244)islargerthantherateofsynonymoussubsti- Assuming a generation time of 5 h as reported for P. acnes a
tutions(0.00239;Wilcoxontest,P=2(cid:2)10(cid:3)8).Third,theratio (Halletal.1994),andassumingamutationratesimilartothat rch
ofnonsynonymoustosynonymouspolymorphismratesinP. ofthecloselyrelatedMycobacteriumtuberculosis(2(cid:2)10(cid:3)10, 20
1
Zappae,whichcanbeusedasaproxyforselectivepressure applied to synonymous sites; Ford et al. 2011), the age of 9
(Schloissnigetal.2013),is0.124/0.145=0.86,veryclosetothe P. Zappae can be set approximately 7,500 years ago, an age
neutral value of 1. Finally, the McDonald–Kreitman test re- highlycompatiblewiththedomesticationofgrapevine.Date
vealed a strong excess of nonsynonymous polymorphisms estimatesare,however,knowntobeextremelydependenton
(P<0.000001). Interestingly, in P. Zappae, recA has also sig- thepriorchoice(e.g.,Omettoetal.2013;Rota-Stabellietal.
naturesofrecombination,whichareabsentintlyaswellasin 2013);therefore,wehaverepeatedourageestimatesassum-
recAofP.acnes(table1). ing varying generation times (to account for the fact that
Takentogether,ourresultssuggestalackofselectivecon- grape endosymbiotic P. Zappae may have a slow life cycle
straintsinrecAofP.Zappaeassociatedtolossofitsfunction. during cold winter season) as well as alternative mutation
In bacteria, loss of function may be beneficial whenever it rates for bacteria (i.e., Drake 1991; Lee et al. 2012, see
entails a change, in the gene network, which turns out to Materials and Methods for more details). Date estimates
be adaptive for specific metabolic needs (Koskiniemi et al. vary substantially, but in the majority of the cases they are
2012; Hottes et al. 2013). We hypothesize that recA is not compatible with the timescale of grapevine domestication
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and cultivation (supplementary fig. S5, Supplementary Supplementary Material online), and phylogenetic analysis
Material online). As a further proof, we estimated the age performed by the maximum likelihood method in Phyml
of P. Zappae directly on the tree using Beast (Drummond (Guindon et al. 2010) employing a nonparametric boot-
etal.2012)andapermissivemutationratedistribution,which strap procedure (100 pseudoreplicates) and the GTR + G
settheoriginofP.Zappaeatapproximately6,300yearsago modelofnucleotidereplacement.Populationgeneticanalyses
(supplementaryfig.S6,SupplementaryMaterialonline).Our were done using DnaSp (Libradoand Rozas 2009). Levelsof
concordant age estimatesreinforce the view thatP. Zappae nucleotide diversity were estimated using p (Tajima 1989),
originated from human-associated P. acnes. We speculate and recombination rate using the method proposed by
that P. Zappae has exploited common agronomic practices, Hudson (1987). Neutral equilibrium was tested by the
suchasgraftingandpruning,totransfertothenewhost. Tajima’s D test (Tajima 1989). To capture the evolution-
Our results represent the first evidence of human to ary pattern following the endophytization, rates of nonsyn-
plant horizontal transfer of an obligate symbiont and open onymousandsynonymoussubstitutionswereestimatedonly
new perspectives on the occurrence and significance of in each of the type IB sequences (n=51; fig. 2) using the D
o
bacteria host transfer between humans and domesticated humanasn1sequenceasoutgroup.Inaddition,weestimated w
n
plants. Moreover, our findings underline how the extreme the (non)synonymous polymorphism rate as the ratio be- loa
d
adaptability of bacteria and their ability to exploit and tween the number of variable (non)synonymous sites in e
d
taking advantage of novel habitats can have unpredictable our sample and the total number of (non)synonymous fro
m
impact on host–pathogen evolution and eventually human sites. To test for the occurrence of positive or relaxed h
health. The exploration of endophytic communities, selection, we also used the McDonald–Kreitman test ttp
s
using metagenome-based community analyses (Bulgarelli (Mcdonald and Kreitman 1991), which compares the poly- ://a
etal.2012;Lundbergetal.2012)willcertainlyallowprecious morphism (p) and substitution (k) pattern in ca
d
insights in the occurrence and significance of bacterial synonymous (S), and nonsynonymous (NS) sites: em
endophytes. Likewise, the possible functional significance of BecauseP.Zappaehasnofixedsubstitutionwhenusingacnes ic.o
the P. Zappae—grapevine interaction and the conse- asoutgroup,weusedP.aviduminsteadandobtainedp =35, up
quences for plant growth and health merits further p =92,k =112,k =6. S .co
NS S NS m
investigation. /m
b
e
Materials and Methods FISH Microscopy /a
Planttissueswereharvestedandfrozeninliquidnitrogenand rtic
Plant Handling, DNA Isolation, and Polymerase Chain le
stored at (cid:3)20(cid:4)C for long-term storage. Samples (0.5-mm -a
Reaction long sections) were fixed overnight at 4(cid:4)C in a paraformal- bstra
Gfrorampefiveinlde-gsraomwpnleasnudsegdrefeonrhDouNsAe ceuxtttriancgtsioinnwnoerrtehoerbntaIitnaelyd. ddreahtyeddeinsoalnuteiothnan(4o%lseinriePsB(S2)5,%ri,n5s0ed%,t7w5i%ce,ainndP9B9S.9a%n;d15demhiyn- ct/31
/5
Plantsweresurfacesterilizedwithethanolandsodiumhypo- each step). A lysozyme 1mg/ml(cid:3)1 solution (in PBS) treat- /1
0
chloriteasdescribedbefore(Pancheretal.2012).Twigsused ment was then applied to the samples for 10min at 37(cid:4)C. 59
forDNAextractionweredecorticatedwithascalpeltoavoid FISH was carried out using probes bought at Genecust /99
8
contamination from superficial DNA in following reactions. (Luxembourg). EUB mix (equivalent mixture of EUB338, 71
Total DNA was isolated by freeze-crushing plant stems, fol- EUB338II, EUB338III; Amann et al. 1990; Daims et al. 1999) 7 b
y
lowedbyFastDNAspinkitforsoil(MPBio,USA)extraction coupled with a FLUOS fluorochrome, a HGC69a probe (for g
u
kit.Theidentityofbacterialcolonieswasconfirmedbypoly- actinobacteria;Manzetal.1992coupledwithCy5,andaPAC es
merasechainreaction(PCR)usingstandard16SrDNAgene 16S 598 probe (for P. acnes/P. Zappae; Poppert et al. 2010) t on
primers16S-27F/16S-1492R(Lane1991).Roche454GSFLX+ 3
coupled with a 405 dylight fluorochrome. NONEUB probe 0
system technology was used for pyrosequencing using pri- M
(Wallner et al. 1993) coupled with Cy5 was used indepen- a
mers 799f/1520R (Chelius and Triplett 2001). dently as a negative control. Hybridization was also carried rch
Propionibacterium acnes-specific recA and tly genes were 2
out with only PAC 16S 598 probe coupled with FLUOS. 0
1
PCR amplified using primer pairs PAR-1/PAR-2 and PAT-1/ Hybridizationwascarriedoutat46(cid:4)Cfor2hwith10–20ml 9
PAT-2,respectively(McDowelletal.2005).Pyrosequencingof
of solution (containing 20mM Tris–HCl pH 8.0, 0.1% w/v
16SrDNAampliconswasobtainedusingprimers799f/1520r
SDS, 0.9M NaCl, 25% formamide, and 10ng/ml of each
with 454 adaptors and a sample-specific barcode. PCR was
probe) applied on each plant samples placed on slides in a
performed using High Fidelity FastStart DNA polymerase
50ml moist chamber (with a piece of tissue inside imbibed
(Roche, USA) and 454 pyrosequencing was carried out on
with5mlofhybridizationbuffer).Posthybridizationwascar-
the GS FLX+ system using the new XL+ chemistry dedi- ried out at 48(cid:4)C for 30min with a post-FISH prewarmed
catedtolongreads.
solution containing 20mM Tris–HCl pH 8.0, 0.1% w/v SDS,
5mMethylenediaminetetraaceticacid(EDTA)pH8.0,anda
Evolutionary Analyses NaClconcentrationcorrespondingtoformamideconcentra-
Sequences of 16s rDNA, recA and tly from grapevine DNA tion. Samples were then rinsed with ice-cold distilled water
extracts were aligned to all available homologs in before air drying for at least one day in the dark. The sam-
GenBank (May 2013) (see the list in supplementary fig. S7, ples were covered with cover slips and mounted
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Campisanoetal. . doi:10.1093/molbev/msu075
withCitifluorAF1(CitifluorLtd.,London,UK)beforeobser- Acknowledgments
vation under confocal microscope (Olympus Fluoview
A.C.conceivedthestudy.A.C.,O.R.-S.,andL.O.designedthe
FV1000 with multiline laser FV5-LAMAR-2 HeNe(G)laser
research. S.C. performed FISH. M.P., A.C., and L.A. collected
FV10-LAHEG230-2). Pictures were taken at 405-, 488-, 633-
samplesandgeneratedandassembledgenedata.O.R.-S.per-
nm wavelengths and under normal light and then merged
formedphylogeneticanalyses.L.O.performedpopulationge-
(RGB) using the image J software. Propionibacterium acnes/
netic analyses. I.P., G.A., C.V., S.C., and A.S. contributed
P.Zappaeappearsaslowblue/whitefluorescent,actinobac-
reagentsandimprovedthemanuscript.A.C.draftedtheman-
teria (other than P. acnes) appear as low orange and other
uscript with O.R.-S. and L.O. This work was funded by
bacteria as green fluorescent. In case of use of the PAC 16S
Provincia Autonoma di Trento, progetto PAT – Call 2
598probecoupledwithFLUOS,P.acnes/P.Zappaeappeared
Team 2009 – Incoming – Mecagrafic to A.C., and progetto
asgreenfluorescent.
PAT Call 3 COFOUND 2010 to O.R.S. The authors thank
COSTActionFA1103forsupportingthiswork.
D
Estimation of P. Zappae Age ow
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