micb-12-637430 Ma ch 11, 2021 Time: 17:8 # 1
ORIGINAL RESEARCH
published: 17 Ma ch 2021
doi: 10.3389/ micb.2021.637430
Edi ed by:
Mau izio Sanguine i,
Ca holic Uni e si y o he Sac ed
Hea , I aly
Re iewed by:
B unella Pos e a o,
Ca holic Uni e si y o he Sac ed
Hea , I aly
Yun Ki Yeoh,
The Chinese Uni e si y o Hong Kong,
China
*Co espondence:
Ma io López-Pé ez
[email p o ec ed]
Juan Ca los Rod íguez
[email p o ec ed]
†These au ho s ha e con ibu ed
equally o his wo k
Special y sec ion:
This a icle was submi ed o
In ec ious Diseases,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 03 Decembe 2020
Accep ed: 23 Feb ua y 2021
Published: 17 Ma ch 2021
Ci a ion:
Ven e o MP, Cuad a RRC, Vidal I,
And ade BGN, Molina-Pa dines C,
Ha o-Mo eno JM, Cou inho FH,
Me ino E, Regi ano LCA, Sil ei a CB,
A li H, López-Pé ez M and
Rod íguez JC (2021) Nasopha yngeal
Mic obial Communi ies o Pa ien s
In ec ed Wi h SARS-CoV-2 Tha
De eloped COVID-19.
F on . Mic obiol. 12:637430.
doi: 10.3389/ micb.2021.637430
Nasopha yngeal Mic obial
Communi ies o Pa ien s In ec ed
Wi h SARS-CoV-2 Tha De eloped
COVID-19
Ma ia Paz Ven e o1†, Ra ael R. C. Cuad a 2†, Inmaculada Vidal1, B uno G. N. And ade3,4,
Ca men Molina-Pa dines1, Jose M. Ha o-Mo eno5, Felipe H. Cou inho5,
Espe anza Me ino6, Luciana C. A. Regi ano4, Cyn hia B. Sil ei a7, Hai hem A li4,
Ma io López-Pé ez1,5*and Juan Ca los Rod íguez1,5*
1Mic obiology Depa men , Alican e Uni e si y Gene al Hospi al - Alican e Ins i u e o Sani a y and Biomedical Resea ch
(ISABIAL), Alican e, Spain, 2Depa men o Molecula Epidemiology, Ge man Ins i u e o Human Nu i ion
Po sdam-Rehb ücke, Nu he al, Ge many, 3Emb apa Pecuá ia Sudes e, São Ca los, B azil, 4Depa men o Compu e
Science, Muns e Technological Uni e si y (MTU), Co k, I eland, 5E olu iona y Genomics G oup, Di isión de Mic obiología,
Uni e sidad Miguel He nández, San Juan de Alican e, Spain, 6In ec ious Diseases Uni , Alican e Uni e si y Gene al Hospi al,
Alican e Ins i u e o Heal h and Biomedical Resea ch (ISABIAL), Alican e, Spain, 7Depa men o Biology, Uni e si y
o Miami, Miami, FL, Uni ed S a es
Backg ound: SARS-CoV-2 is an RNA i us causing COVID-19. The clinical
cha ac e is ics and epidemiology o COVID-19 ha e been ex ensi ely in es iga ed,
howe e , only one s udy so a ocused on he pa ien ’s nasopha ynx mic obio a.
In his s udy we in es iga ed he nasopha ynx mic obial communi y o pa ien s ha
de eloped di e en se e i y le els o COVID-19. We pe o med 16S ibosomal DNA
sequencing om nasopha yngeal swab samples ob ained om SARS-CoV-2 posi i e
(56) and nega i e (18) pa ien s in he p o ince o Alican e (Spain) in hei i s isi o
he hospi al. Posi i e SARS-CoV-2 pa ien s we e obse ed and la e ca ego ized in mild
(symp oma ic wi hou hospi aliza ion), mode a e (hospi aliza ion), and se e e (admission
o ICU). We compa ed he mic obio a di e si y and OTU composi ion among se e i y
g oups and buil bac e ial co-abundance ne wo ks o each g oup.
Resul s: S a is ical analysis indica ed di e ences in he nasopha yngeal mic obiome o
COVID19 pa ien s. 62 OTUs we e ound exclusi ely in SARS-CoV-2 posi i e pa ien s,
mos ly classi ied as membe s o he phylum Bac e oido a (18) and Fi micu es (25). OTUs
classi ied as P e o ella we e ound o be signi ican ly mo e abundan in pa ien s ha
de eloped mo e se e e COVID-19. Fu he mo e, co-abundance analysis indica ed a
loss o ne wo k complexi y among samples om pa ien s ha la e de eloped mo e
se e e symp oms.
Conclusion: Ou s udy shows ha he nasopha yngeal mic obiome o COVID-19
pa ien s showed di e ences in he composi ion o speci ic OTUs and complexi y o co-
abundance ne wo ks. Taxa wi h di e en ial abundances among g oups could se e as
bioma ke s o COVID-19 se e i y. Ne e heless, u he s udies wi h la ge sample sizes
should be conduc ed o alida e hese esul s.
Keywo ds: COVID-19, SARS-CoV-2, mic obiome, NGS – nex gene a ion sequencing, co ona i us, P e o ella
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Ven e o e al. Nasopha yngeal Mic obiome Associa ed Wi h COVID-19
IMPORTANCE
This wo k has s udied he mic obio a o he nasopha yngeal
ac in COVID-19 pa ien s using ad anced echniques o
molecula mic obiology. Di e se mic oo ganisms, mos o which
a e ha mless o e en bene icial o he hos , colonize he
nasopha ynx. Howe e , changes in his mic obio a could be
ela ed wi h di e en diseases such as cance , gas oin es inal
pa hologies o e en COVID-19. This s udy in es iga ed he
mic obio a om pa ien s wi h COVID-19, in o de o de e mine
i s associa ions wi h pa hology se e i y. The ob ained esul s
e ealed se e al bac e ial axa wi h di e en ial abundance
among COVID-19 cases. This s udy ep esen s he i s s ep
o unde s and he associa ions be ween mic obio a composi ion
and he se e i y o COVID-19.
BACKGROUND
Se e e Acu e Respi a o y Synd ome Co ona i us-2 (SARS-
CoV-2) is a posi i e-sense single-s anded RNA i us causing
Co ona i us Disease 2019 (COVID-19) (Ande sen e al., 2020).
On Janua y 30, 2020, he Wo ld Heal h O ganiza ion (WHO)
decla ed he COVID-19 ou b eak as “public heal h eme gency
o in e na ional conce n” and 2 mon hs la e on Ma ch 11 h
as a pandemic. The SARS-CoV-2 i us was i s epo ed in
cen al ci y o Wuhan, Hubei p o ince, China, and p esen ed 70%
o sequence simila i y wi h he SARS-CoV-1 i us (Zhu e al.,
2020) and 96% o sequence simila i y wi h a ba co ona i us,
al hough he exac sou ce has ye o be elucida ed. While he
mos common symp oms a e e e , cough and dyspnoea, he
disease can cause o he less equen clinical mani es a ions
such as myalgia, headaches, b ea hlessness, a igue and nausea
(Guan e al., 2020).
Vi uses and bac e ia a e o en p esen in he espi a o y ac
o heal hy and asymp oma ic indi iduals. Mic oaspi a ion o
ae osols and di ec mucosal dispe sal is esponsible o a cons an
in low o mic obes and i uses owa d lowe ai ways. Disease and
in lamma o y p ocesses ha lead o he eme gence o anae obic
zones, o mucus accumula ion in he al eoli can d as ically
change he mic obial communi y o he ai ways (Hu nagle e al.,
2017). Fo example, in diseased indi iduals, he lung mic obio a
composi ion unde goes a dec ease in di e si y (Kalan a e al.,
2019) accompanied by a shi in he dominan axa: om
Bac e oido a o Gammap o eobac e ia, a class ha includes many
espi a o y pa hogens.
Al hough he clinical cha ac e is ics and epidemiology o
COVID-19 ha e been desc ibed (Chen N. e al., 2020;Ro han
and By a eddy, 2020;Wu e al., 2020), s udies ocused on he
associa ions be ween he pa ien ’s mic obio a and he onse o
he disease a e s ill limi ed. This pilo s udy aims o cha ac e ize
he nasopha ynx mic obial communi y o SARS-CoV-2 in ec ed
pa ien s. We in es iga ed samples om a con ol g oup o
SARS-CoV-2 nega i e pa ien s and h ee g oups o SARS-CoV-2
posi i e pa ien s, di ided acco ding o disease se e i y: one g oup
o symp oma ic pa ien s ha did no equi e hospi aliza ion, a
second g oup o pa ien s ha we e admi ed o con en ional
hospi aliza ion acili ies, and a hi d g oup o pa ien s ha
equi ed admission o he ICU.
MATERIALS AND METHODS
Pa ien s and Expe imen al Design
Fi y six nasopha yngeal mic obio a samples om SARS-CoV-
2 posi i e pa ien s and 18 samples om SARS-CoV-2 nega i e
pa ien s we e collec ed du ing Ma ch and Ap il o 2020 in he
Eme gency Se ice o Hospi al Gene al Uni e si a io de Alican e
(HGUA). The samples we e collec ed in a s e ile ube con aining
Vi al T anspo Medium using a nasal swap, and hey we e s o ed
a –80◦C un il he DNA/RNA ex ac ion. Cobas SARS-CoV-2
qPCR Tes o he Cobas 6800 Sys em (Roche Molecula Sys ems,
B anchbu g, NJ, Uni ed S a es) was used o de ec he p esence o
SARS-CoV-2 and o ob ain de C Values (Poljak e al., 2020). The
Table 1 desc ibes he g oups o pa ien s ega ding sex, age, and
clinical a iables.
Pa ien s we e i s classi ied based on SARS-CoV-2 p esence,
and hen ega ding on la e de elopmen s (hospi al admission
and se e i y). All samples we e ob ained be o e he onse o
se e e symp oms, and be o e any ea men was adminis e ed
o he pa ien s. Following hese c i e ia, ou g oups we e
es ablished: g oup 0: SARS-CoV-2 nega i e pa ien s (n= 18);
g oup 1: mild COVID-19 symp oms bu no la e hospi al
admission (n= 19); g oup 2: se e e COVID-19 symp oms
ollowed by hospi al admission (n= 18); and g oup 3:
pa ien s wi h se e e COVID-19 symp oms which we e e en ually
admi ed in o in ensi e ca e uni s (ICU) (n= 19). P o ocols
we e de eloped in acco dance wi h he na ional e hical and
legal s anda ds, and ollowing he guidelines es ablished in
he Decla a ion o Helsinki (2000). The esea ch p ojec (Re :
COV20_00236) was conduc ed unde he w i en app o al o he
E hic Commi ee o Clinical Resea ch wi h D ug (in Spanish,
CEIm) o he “Hospi al Gene al Uni e si a io de Alican e
(Spain)”(Re CEIm app o al: PI2020-052), and in collabo a ion
wi h he Biobank o Clinical and Biomedical Resea ch Ins i u e
o Alican e (ISABIAL), which a e included in he Valencian
Ne wo k o Biobanks. All samples and clinical da a o pa ien s
we e managed anonymously.
DNA Isola ion and Sequencing
DNA om nasopha yngeal swab samples was isola ed using
he QIAamp DNA Mini Ki (QIAgen) ollowing he p o ocol
ecommended by he manu ac u e . Sequencing lib a ies we e
p epa ed acco ding o he 16S Me agenomic Sequencing Lib a y
P epa a ion p o ocol dis ibu ed by Illumina. B ie ly, he
sequence spanning he hype a iable egions V3 and V4 o
he 16S RNA gene was ampli ied h ough PCR. Amplicons
we e quan i ied using a Qubi 4 Fluo ome e (Qubi dsDNA HS
Assay Ki ) alida ed by 4200 TapeS a ion (Agilen company).
Amplicons we e sequenced wi h Illumina MiSeq Sys em using
he 2 ×300 bp ca idge. The quali y o aw sequences was
assessed by Fas QC so wa e.
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TABLE 1 | Desc ip i e da a om se e i y g oups.
G oup 0 (N= 18) G oup 1 (N= 19) G oup 2 (N= 18) G oup 3 (N= 19) To al (N= 74)
Age
-Mean (SD) 48.111 (19.244) 59.526 (22.860) 66.833 (13.781) 66.263 (12.458) 60.257 (18.819)
-Range 13.000–86.000 23.000–94.000 46.000–91.000 40.000–85.000 13.000–94.000
Gende
-F 9 (50.0%) 12 (63.2%) 9 (50.0%) 2 (10.5%) 32 (43.2%)
-M 9 (50.0%) 7 (36.8%) 9 (50.0%) 17 (89.5%) 42 (56.8%)
Hospi al admission
-No 18 (100.0%) 19 (100.0%) 0 (0.0%) 0 (0.0%) 37 (50.0%)
-Yes 0 (0.0%) 0 (0.0%) 18 (100.0%) 19 (100.0%) 37 (50.0%)
Diagnosis
-B onchi is 1 (5.6%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (1.4%)
-COPD* 1 (5.6%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (1.4%)
-Fe e 0 (0.0%) 2 (10.5%) 0 (0.0%) 0 (0.0%) 2 (2.7%)
-Pneumonia 2 (11.1%) 4 (21.1%) 14 (77.8%) 15 (78.9%) 35 (47.3%)
-Respi a o y in ec ion 8 (44.4%) 11 (57.9%) 4 (22.2%) 0 (0.0%) 23 (31.1%)
-Respi a o y insu iciency 0 (0.0%) 0 (0.0%) 0 (0.0%) 4 (21.1%) 4 (5.4%)
-RSC-COVID19** 6 (33.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 6 (8.1%)
-To acic pain 0 (0.0%) 1 (5.3%) 0 (0.0%) 0 (0.0%) 1 (1.4%)
-Vomi s 0 (0.0%) 1 (5.3%) 0 (0.0%) 0 (0.0%) 1 (1.4%)
In ensi e uni s
-No 18 (100.0%) 19 (100.0%) 18 (100.0%) 0 (0.0%) 55 (74.3%)
-Yes 0 (0.0%) 0 (0.0%) 0 (0.0%) 19 (100.0%) 19 (25.7%)
C (cycle h eshold)
-Mean (SD) 26.34 (5.17) 27.03 (5.31) 25.86 (4.69) 26.38 (5.07)
*COPD: Ch onic obs uc i e pulmona y disease.
**RSC-COVID19: Respi a o y symp oms compa ible wi h COVID19 disease.
Taxonomic Classi ica ion o Amplicon
Sequences
Pai ed end eads o 300 bp we e gene a ed wi h an a e age
o e lap o 140 bp. Sequences we e immed using immoma ic
(Bolge e al., 2014) o clip ou low-quali y nucleo ides a he
ead ends and o emo e sequences ha , a e imming, one o
he pai ed-end eads was smalle han 150 bp and he esul ing
pai ed eads we e me ged using caspe (Kwon e al., 2014),
gene a ing indi idual agmen s o abou 460 bp. Gi en he
une en co e age be ween samples, he numbe o indi idual
eads was s anda dized o 20,000 pe sample, emo ing samples
ha did no each his sequencing dep h (9 samples). To al
numbe o eads and cycle h eshold (C ) pe sample is shown
in Supplemen a y Table S1. Me ged amplicon sequences we e
g ouped in ope a ional axonomic uni s (OTUs) using cd-hi (Fu
e al., 2012) wi h an iden i y o 97%. Sequences we e que ied
agains small subuni s (16S) RNA genes o he SILVA da abase
( e sion 138, Re e ence subse ) (Quas e al., 2013) using he
SILVA Inc emen al Aligne (SINA) so wa e (P uesse e al., 2012)
o axonomic classi ica ion. Sequences wi h low iden i y (<70%)
o any e e ence 16S RNA gene o classi ied as euka yo ic we e
excluded om u he analysis.
Tes ing o Di e ences in Taxonomic
Composi ion Among Pa ien G oups
We sough o de e mine how di e en samples we e g ouped
acco ding o hei OTU composi ion. To ha end, he
Vegan ( 2.5-6) package a ailable in R ( 3.6.3) was used
o pe o m a non-me ic mul idimensional scaling (NMDS)
analysis on B ay-Cu is dissimila i y measu es among samples
based on ela i e OTU abundances (i.e., pe cen ages). The
ela i e abundances o OTUs we e also used o es o
s a is ically signi ican di e ences among se e i y g oups. G oup
OTU composi ions we e compa ed h ough ANOSIM. Nex ,
Simila i y Pe cen age (SIMPER) analysis was used o de e mine
which OTUs we e esponsible o d i ing he di e ences in
communi y composi ion among g oups. Fo his analysis,
all six possible pai wise combina ions o se e i y g oups
we e es ed.
OTU Associa ion Wi h COVID-19 Se e i y
To in e associa ions be ween he se e i y o COVID-19 and
he nasopha ynx mic obio a, gene al linea models (GLM)
we e buil using he R package MaAsLin2 wi h cen e ed
log- ans o med (CLR) OTUs coun s as he dependen
a iable, and he se e i y g oup (wi h g oup 0 and g oup 1
as e e ences), as independen a iable. Due o he di e ences
o age and sex be ween g oups, we adjus ed by sex and
age. Only OTUs ha p esen ed a p e alence o 20% o e
he sample space we e conside ed. The esul ing p- alues
we e adjus ed o mul iple es ing using he Benjamini-
Hochbe g me hod (BH). The analysis was eplica ed
using QIIME2 (Bolyen e al., 2019) oge he wi h LE SE
(Linea disc iminan analysis E ec Size) and included as a
Supplemen a y Ma e ial.
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Co-abundance Ne wo ks o COVID-19
Se e i y G oups
Fas pa (Wa s e al., 2019), a mul i- h ead implemen a ion
o he Spa CC algo i hm (F iedman and Alm, 2012), was
used o gene a e co-abundance ne wo ks among OTUs o
each o he ou se e i y g oups wi h de aul pa ame e s (50
i e a ions and co ela ion h eshold o 0.2) and 1,000 boo s ap
i e a ions o in e signi icance. Resul s we e p ocessed using an
in-house ipy hon no ebook o gene a e ne wo k ma ices o
isualiza ion wi h Cy oscape 3.8 (Shannon e al., 2003). The
ne wo k ma ices we e loaded in he Cy oscape 3.8 so wa e,
and connec ions il e ed by p- alue (≤0.05) and co ela ion
(≤0.6 o ≥0.6).
RESULTS
S udy Se o COVID-19
Se en y- ou pa ien s we e included in his pilo s udy o
assess associa ions be ween he nasopha yngeal mic obio a
composi ion and he se e i y o he COVID-19 disease. Howe e ,
only 65 samples emained a e quali y co e age con ol
(see sec ion “Ma e ials and Me hods”). Da a including age,
sex, diagnosis, hospi al admission, and disease se e i y we e
egis e ed (Supplemen a y Table S2). Six een pa ien s belonged
o he nega i e con ol (g oup 0, no-SARS-CoV-2), whe eas he
emaining pa ien s we e classi ied in o h ee g oups (g oup 1,
2, and 3) acco ding o he se e i y (see sec ion “Ma e ials and
Me hods”). The a e age age o he pa ien s anged om 48.1 yea s
old (g oup 0) o 66.8 yea s old (g oup 2) and a ound 49% o hem
we e diagnosed wi h pneumonia (Table 1).
Mic obio a Taxonomic Composi ion
Di e s Among Se e i y G oups o
COVID-19
The bac e ial phylum Fi micu es was he mos abundan
in he nasopha ynx mic obio a among pa ien s om all
se e i y g oups (52.9% ±4.0%), ollowed by Bac e oido a
(22.1% ±6.1%), P o eobac e ia (12.7% ±7.3%), and
Ac inobac e ia (5.4% ±0.6%). A he genus le el, S ep ococcus
was he mos abundan axon (25.2% ±2.0%), ollowed
by P e o ella (16.2% ±5.7%), Veillonella (14.4% ±2.2%),
Haemophilus (5.23% ±4.78%), and Mo axella (3.2% ±3.6%)
(Supplemen a y Figu e S1 and Supplemen a y Table S3).
A o al o 62 OTUs we e ound exclusi ely in SARS-CoV-2
posi i e pa ien s (a a minimum o h ee samples). Mos o hese
OTUs we e classi ied as membe s o he phylum Bac e oido a
(18) and Fi micu es (25). No ably, he mos common gene a
among he OTUs ound exclusi ely on COVID-19 posi i e
pa ien s we e P e o ella (13), ollowed by Lep o ichia (4) and
S ep ococcus (4). Samples we e compa ed based on he ela i e
abundances o OTUs. This analysis e ealed ha samples did no
clus e acco ding o he se e i y g oup nei he by hie a chical
clus e ing (Figu es 1A,B) o NMDS (Figu e 1C). Ne e heless,
he di e ences in OTU composi ion among se e i y g oups we e
signi ican acco ding o ANOSIM (R= 0.046, p= 0.036).
SIMPER analysis e ealed ha 25 OTUs we e esponsible
o app oxima ely 70% (p- alue 0.04) o he di e ences in
communi y composi ion be ween se e i y g oups 1 and 3
(Supplemen a y Table S4). These OTUs we e classi ied as
membe s o he phyla Bac e oido as, Fi micu es, Fusobac e io a,
and P o eobac e ia. Ele en OTUs had highe a e age abundance
among samples om se e i y g oup 1, among which we e
included h ee OTUs classi ied as membe s o he genus
Veillonella. On he o he hand, 14 OTUs we e mo e abundan
among samples om se e i y g oup 3, among which we e
included ou OTUs classi ied as P e o ella.
Mul iple OTUs Display Di e en ial
Abundance Acco ding o COVID-19
Se e i y
Using MaAsLin2 and g oup 0 as a e e ence, we iden i ied a
o al o 10 signi ican associa ions be ween bac e ial OTUs and
pa ien se e i y (p<0.05, q<0.25), co ec ed o age and sex.
Among hose, 9 we e posi i ely associa ed (8 in g oup 2 and
1 in g oup 3 when con as ed wi h g oup 0) and 1 nega i ely
associa ed (in g oup 3 con as ed wi h g oup 0) (Supplemen a y
Table S5 and Figu e 2A). O he OTUs posi i ely associa ed wi h
se e i y, h ee we e classi ied as membe s o he genus P e o ella
(OTUs 4, 14, and 16). Due o he he e ogenei y o g oup 0,
we also decided o in es iga e he di e ences wi hin he SARS-
CoV-2 posi i e pa ien s, using g oup 1 as e e ence. The GLM
model showed jus 1 signi ican OTU (OTU 16), a P e o ella
also ound o be signi ican ly associa ed wi h se e i y in he i s
model (Supplemen a y Table S5 and Figu e 2B). In he same
line, he esul s ob ained wi h QIIME2 using he q2-composi ion
plugin, and wi h LE SE me hod showed ha P e o ella sp. was
associa ed wi h pa ien se e i y (Supplemen a y Figu e S2). We
did no ind any OTUs signi ican ly di e en be ween g oups 1
and 0. Figu e 2A shows he coe icien s o all he signi ican
OTUs ound by bo h GLMs and Figu e 2B shows OTU 16 CLR
ans o med coun s o all se e i y g oups.
Co-abundance Ne wo ks o COVID-19
Se e i y G oups
In o de o in es iga e how OTUs co ela e wi hin he di e en
g oups, we gene a ed a o al o 4 co-abundance ne wo ks, one
o each se e i y g oup. Fo he se e i y g oup 0, he SARS-
CoV-2 nega i e g oup, he ne wo k displayed 118 nodes wi h 179
edges. Rega ding he o he h ee se e i y g oups, anging om
mild o high se e i y, he complexi y o he ne wo k dec eased
wi h he inc ease o se e i y. The ne wo k o pa ien s wi h mild
symp oms (g oup 1) had 137 nodes wi h 457 edges, while he
ne wo k o pa ien s wi h se e e symp oms bu no admi ed in
ICU (g oup 2) had 129 nodes wi h 171 edges. The ne wo k o
se e e pa ien s admi ed in ICU (g oup 3) had 100 nodes and 148
edges. In he ne wo k o se e i y g oup 1, OTU 16 (P e o ella,
associa ed wi h se e i y in wo GLMs) displayed 18 co-abundan
OTUs connec ed in he ne wo k in i s deg ee (Figu e 3). Among
hese connec ions, en we e nega i e associa ions while eigh
we e posi i e. Mos o hese connec ions wi h OTU 16 we e
absen om ne wo ks o se e i y g oups 2 and 3. Only 3 and
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FIGURE 1 | Be a di e si y. Dend og am based on (A) B ay-Cu is dissimila i y and (B) Sø ensen dissimila i y alues. (C) Compa ison o sample axonomic p o iles by
se e i y g oup. Non-me ic mul idimensional scaling was applied o de e mine he clus e ing pa e ns o samples acco ding o hei OTU abundance pa e ns. Each
do ep esen s a sample colo coded acco ding o he se e i y g oup i belongs o. The close he samples a e, he mo e simila was hei OTU abundance
composi ion. No clea clus e ing o samples by se e i y g oup was obse ed.
2 i s deg ee connec ions emained in each o hese ne wo ks,
espec i ely (Supplemen a y Figu e S3).
DISCUSSION
In his p elimina y s udy, he analysis o he axonomic
composi ion o he samples showed di e ences be ween
pa ien s ha de eloped di e en onse s o COVID-19.
P e iously, only one s udy was conduc ed o in es iga e
he nasopha ynx mic obio a o COVID-19 pa ien s (Maio
e al., 2020). This s udy did no ind any di e ences
be ween pa ien s and con ols. Howe e , his s udy was
conduc ed only in wo g oups, he con ol and mild
COVID-19 pa ien s.
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FIGURE 2 | (A) E o ba plo o he GLM coe icien s in e al o each OTU. The Y-axis shows he OTU numbe and Genus classi ica ion. The X-axis ep esen s he
CLR abundance a iance explained by he GLM models. In ed he posi i ely associa ed OTUs wi h se e i y s a e ( anging om g oup 1 o 3) and in blue OTUs he
nega i ely associa ed wi h se e i y s a e OTUS. (B) The cen e ed log- ans o med (CLR) OTU 16 (P e o ella) abundance in he se e i y g oups 0–3, showing i s
highe abundance in se e i y ca ego ies.
FIGURE 3 | Co-abundance ne wo k (se e i y g oup 1) showing only i s -deg ee neighbo s o OTU 16 (P e o ella sp.). OTUs a e ep esen ed by nodes and
signi ican co ela ions by edges. Blue edges ep esen nega i e associa ions and ed, posi i e associa ions. The colo o nodes was de ined by he axonomic
classi ica ion o he OTU a Genus ank.
In ou s udy, we ound sub le changes in nasopha yngeal
communi y composi ion, meaning ha hey a e es ic ed o ew
axa ou o he comple e me a-communi y. Ne e heless, he e
a e de ec able and signi ican changes among OTU abundances.
These changes could be linked o he di e en se e i y g oups,
as we iden i ied bo h axa ha we e p esen exclusi ely among
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COVID-19 posi i e pa ien s as well as hose whose abundance
was signi ican ly highe o lowe among di e en se e i y g oups.
No only his, bu also he complexi y o co-abundance ne wo ks
(which can be aken as a p oxy o po en ial in e ac ions be ween
axa), was dec eased among pa ien s ha de eloped mo e se e e
cases o COVID-19. Below we discuss he mechanisms by which
speci ic mic obes migh play a ole in ei he enhancing o
dec easing he se e i y o COVID-19.
Po en ial Associa ions Be ween
Bac e ial Taxa and COVID-19 Se e i y
Among he OTUs posi i ely associa ed wi h COVID-19 se e i y,
h ee we e classi ied as membe s o he genus P e o ella, and one
o a closely ela ed genus, Allop e o ella. A ecen s udy showed
ha P e o ella p o eins can p omo e i al in ec ion h ough
mul iple in e ac ions wi h NF-κB signaling pa hway, which is
also in ol ed in COVID-19 se e i y (Khan and Khan, 2020).
The genus P e o ella is usually conside ed commensal and, as
such, a ely in ol ed in in ec ions. Howe e , some s ains ha e
been iden i ied as oppo unis ic pa hogens in ch onic in ec ions,
abscesses and anae obic pneumonia (B ook, 1998;B ook, 2004;
Nagy, 2010;La sen, 2017). The ole o some s ains o P e o ella
in ch onic mucosal in lamma ion has been demons a ed. They
a e in ol ed wi h augmen ed T helpe ype 17 (Th17)-media ed
mucosal in lamma ion, h ough ac i a ion o Toll-like ecep o
2, ollowed by p oduc ion o cy okines by an igen-p esen ing
cells, including in e leukin-23 (IL-23) and IL-1 (La sen, 2017).
The se e e symp oms o COVID-19 a e associa ed wi h cy okine
s o ms, many o which a e in ol ed in TH17 ype esponses
(Wu and Yang, 2020). The signi ican associa ion o P e o ella
sp. and disease se e i y obse ed he e sugges s a possible link
be ween P e o ella sp. and he COVID-19 h ough he ac i a ion
o immuni y signaling pa hways ha modula e in lamma ion,
and his link should be u he explo ed.
Reduced Ne wo k Complexi y Among
Pa ien s Who La e De eloped Mo e
Se e e COVID-19
Se e al s udies demons a ed he use ulness o co-abundance
ne wo ks o elucida e changes in he mic obio a associa ed
wi h human diseases (Faus e al., 2012;G eenblum e al.,
2012;Wang e al., 2016;Chen L. e al., 2020). By swi ching
om indi idual OTU associa ions o a communi y in e ac ion
app oach i is possible o a ain a be e unde s anding o
he dynamic o mic obio a/pheno ype associa ions, e ealing
mic obial conso ia (and no only an OTU) ha migh be
collec i ely in luencing he hos pheno ype. Ou linea models
showed OTU 16 (P e o ella sp.) as an impo an OTU associa ed
wi h se e i y. This OTU had he highes numbe o connec ions
in he ne wo k, ollowed by OTU 9 (Veillonella sp.). O
he ou ne wo ks gene a ed, he se e i y g oup 1 ne wo k
showed he highe numbe o in e ac ions wi h his OTU.
Ecological ne wo king, in i o, and clinical s udies showed
ha P e o ella sp. and Veillonella sp. a e keys one species in
he mic obio a du ing ai way disease p og ession, especially
in diseases associa ed wi h mucus accumula ion such as cys ic
ib osis (Flynn e al., 2016;Quinn e al., 2016;Sil ei a e al., 2020).
These anae obes a e e icien a deg ading mucin molecules
on he ai way mucosa, eleasing by-p oduc s ha enable he
coloniza ion and g ow h o pa hogenic bac e ia ha a e poo a
deg ading mucus o g ow h (Flynn e al., 2020). In COVID-19
pa ien s, P e o ella sp. and Veillonella sp. could ha e a simila
ole due o he dec eased mucocilia y clea ance caused by he
i al in ec ion (Robino e al., 2020). Lowe a es o clea ing
inc ease he esidence ime o P e o ella sp. and Veillonella sp.
in he ai ways, likely inc easing hei mucus me abolism and
enabling u he coloniza ion by pa hogenic bac e ia ha may
cause pneumonia.
OTU 96, classi ied as Dolosig anulum sp., was iden i ied in he
g oup 1 ne wo k and displayed a nega i e associa ion wi h OTU
16 (P e o ella sp.) as a i s -deg ee neighbo (Figu e 3). OTU
96 did no pass he q- alue h eshold es ablished o he GLMs
bu shows signi ican p- alue (0.003 in he model compa ing
g oup 2 and g oup 0, and 0.02 in he model compa ing g oup 2
and g oup 1 as e e ence). The only species cu en ly desc ibed
in his genus is Dolosig anulum pig um, which is commonly
ound in he nasopha ynx mic obio a and is p edic ed o bene i
he hos h ough p o ec ion agains pneumococcal coloniza ion
(Biesb oek e al., 2014;Boma e al., 2016) and h ough p o ec ion
agains in lamma ion damage (Moyano e al., 2020). One s udy
also ound a lowe abundance o Dolosig anulum in child en
wi h In luenza A Vi us compa ed o heal hy child en (Wen
e al., 2018). In addi ion, a s udy epo ed ha pa ien s wi h
hei ai way mic obio a domina ed by Co ynebac e ium and
Dolosig anulum expe ienced he lowes a es o ea ly loss o
as hma con ol and ha e a longe ime o de elop a leas 2
episodes (Zhou e al., 2019). We did no iden i y Co ynebac e ium
di ec ly connec ed o OTU 16 (P e o ella sp.), bu OTU 78,
classi ied as Co ynebac e ium is posi i ely associa ed wi h OTU
96 (0.7479, p= 0.001) in he co-abundance ne wo k om g oup
1 (Figu e 3), indica ing ha in asymp oma ic pa ien s hose
wo axa a e o ming a conso ium ha migh p o ec om
disease de elopmen . This “conso ium” was also implica ed in
esis ance o ecu en ea in ec ions and i was p oposed as a
p obio ic candida e o uppe espi a o y ac in ec ions (Lappan
and Peacock, 2019). The eason ha we did no ha e lowe
q- alue in ou GLM o hose wo axa could be he lack o
powe due o he small size o ou s udy. Thus, hese associa ions
wa an u he in es iga ion.
LIMITATIONS
The majo limi a ion o ou s udy is he small sample size.
Wi h only abou 15 samples pe se e i y g oup i is di icul
o ind s a is ically signi ican associa ions be ween mic obio a
composi ion and disease se e i y. Ne e heless, his limi a ion is
mo e likely o lead o alse nega i es han o alse posi i es. We
also canno ule ou con ounding ac o s ha migh explain he
di e ences be ween g oups, and he ac ha many pa ien s o he
con ol g oup we e diagnosed wi h o he espi a o y in ec ions
o pneumonia, which could ha e in luenced hei mic obio a in
a manne un ela ed o COVID-19. Ano he impo an limi a ion
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is he ac ha we pe o med amplicon a he han whole genome
sho gun sequencing. This leads o h ee issues. Fi s , some o
he bac e ial di e si y is los due o he ac ha he selec ed
p ime s do no ampli y he en i e y o bac e ial di e si y. Second,
some genomes ha e mo e han a single copy o he 16S ope on,
which can lead o an o e es ima ion o hei abundance in
he samples. Thi d, wi hou me agenomes (and me agenome-
assembled genomes) we could no make in e ences abou he
p esence o i ulence ac o s and o he ea u es o he genomes
o he mic obes in ou samples. We eso ed o 16S ampli ica ion
because ou non-in asi e app oach o collec samples yields low
DNA amoun s ha a e inadequa e o sequencing. Howe e , as
a as we know, his is a unique pilo s udy in he ield. The aim
is o be able o ans e use ul esul s o help clinical p ac ice
in he igh agains he i us and o op imize all he p o ocols
and analyses o a second analysis in which he sample size will
be much la ge . We a e cu en ly wo king on collec ing mo e
samples and op imizing p o ocols ha will allow us o ob ain
whole genome sho gun sequencing om hem.
CONCLUSION
Ou da a p o ides p elimina y e idence o signi ican di e ences
in he composi ion o he uppe ai way mic obio a acco ding o
COVID-19 se e i y, sugges ing po en ial bioma ke s o disease
se e i y. While he ichness and di e si y indexes did no
show signi ican di e ences among g oups, speci ic axa we e
signi ican ly associa ed wi h disease de elopmen . We also
demons a ed ha he complexi y o he co-abundance ne wo k
is dec eased in pa ien s who came o de elop se e e cases o
he disease, indica ing ha he in e ac ions be ween he axa a e
also ele an o his p ocess. Fu he s udies will be necessa y o
shed ligh on he molecula mechanisms ha gi e ise o hese
associa ions. Finally, we make no claim ha he di e ences in
mic obio a composi ion epo ed he e a e he cause o COVID-
19 se e i y. Ne e heless, he signi ican associa ions ound
be ween hese a iables sugges s ha he ole o he mic obio a
on he onse o disease se e i y wa an s u he in es iga ion.
DATA AVAILABILITY STATEMENT
The da ase s p esen ed in his s udy can be ound in
online eposi o ies. The names o he eposi o y/ eposi o ies
and accession numbe (s) can be ound in he a icle/
Supplemen a y Ma e ial. Raw da a was deposi ed o he
Na ional Cen e o Bio echnology In o ma ion Sequence Read
A chi e unde BioP ojec accession numbe PRJNA673585.
ETHICS STATEMENT
The s udies in ol ing human pa icipan s we e e iewed and
app o ed by E hic Commi ee o Clinical Resea ch o Alican e
Uni e si y Gene al Hospi al (Re . CEIm: PI2020-052). W i en
in o med consen o pa icipa ion was no equi ed o his
s udy in acco dance wi h he na ional legisla ion and he
ins i u ional equi emen s.
AUTHOR CONTRIBUTIONS
JR concei ed he s udy. MPV, IV, CM-P, and EM collec ed he
da a. RRCC, BGNA, CS, JH-M, and FHC analyzed he da a.
MPV, RRCC, ML-P, FHC, BGNA, LCAR, HA, and JR w o e he
manusc ip . All au ho s e iewed and app o ed he inal e sion
o he manusc ip .
FUNDING
This wo k was suppo ed by a g an om Ins i u o de Salud
Ca los III (ISCIII; g an no. COV20/00236). This esea ch
ecei ed unding om he Eu opean Union’s Ho izon 2020
Resea ch and Inno a ion P og amme unde he Ma ie
Skłodowska-Cu ie g an ag eemen no. 801522, by Science
Founda ion I eland and co- unded by he Eu opean Regional
De elopmen Fund h ough he ADAPT Cen e o Digi al
Con en Technology g an numbe 13/RC/2106.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h ps://www. on ie sin.o g/a icles/10.3389/ micb.
2021.637430/ ull#supplemen a y-ma e ial
Supplemen a y Figu e 1 | Rela i e abundance o bac e ial popula ions, a genus
le el, in he mic obiome o pa ien s wi hin COVID-19 se e i y g oups. Only
mic oo ganisms wi h a ela i e abundance g ea e han 0.5% a e shown in
he legend.
Supplemen a y Figu e 2 | Rela i e abundance o P e o ella sp. wi hin se e i y
g oups. Class 1: Se e i y g oup 1; Class 2: Se e i y g oup 2; Class 3
Se e i y g oup 3. The da a was ob ained using QIIME2 oge he wi h LE Se (Linea
disc iminan analysis E ec Size).
Supplemen a y Figu e 3 | Co-abundance ne wo k showing only i s -deg ee
neighbo s o OTU 16 (P e o ella sp.). (A) Se e i y g oup 0, (B) Se e i y g oup 2,
and (C) Se e i y g oup 3.
Supplemen a y Table 1 | To al numbe o eads and cycle h eshold pe samples.
Supplemen a y Table 2 | Clinical ea u es o pa ien s.
Supplemen a y Table 3 | OTUs axonomic classi ica ion.
Supplemen a y Table 4 | Lis o OTUs and hei pe cen age o a iance
explained when compa ing communi y composi ion among all se e i y (SIMPER).
Supplemen a y Table 5 | MaAsLin2 esul s (GLM) o OTUs associa ions
(q<0.25) o bo h models.
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F on ie s in Mic obiology | www. on ie sin.o g 9Ma ch 2021 | Volume 12 | A icle 637430
