Ci a ion: Cham ou i, M.; Me ghni, A.;
Mi anda-Cadena, K.; Sakly, N.;
Gaddou , N.; de Los Reyes-Ga ilán,
C.G.; Mas ou i, M.; E aso, E.; Quindós,
G. Cha ac e iza ion o Yeas Isola ed
om he Gu Mic obio a o Tunisian
Child en wi h Au ism Spec um
Diso de . J. Fungi 2024,10, 730.
h ps://doi.o g/10.3390/jo 10110730
Academic Edi o : An onella Lupe i
Recei ed: 16 Sep embe 2024
Re ised: 9 Oc obe 2024
Accep ed: 15 Oc obe 2024
Published: 22 Oc obe 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
Fungi
Jou nal o
A icle
Cha ac e iza ion o Yeas Isola ed om he Gu Mic obio a o
Tunisian Child en wi h Au ism Spec um Diso de
Ma iem Cham ou i 1,2 , Abde ahmen Me ghni 3, Ka he ine Mi anda-Cadena 4, Nabil Sakly 5,
Naou el Gaddou 6, Cla a G. de Los Reyes-Ga ilán1,7 , Maha Mas ou i 2, Elena E aso 4,*
and Guille mo Quindós4
1Depa men o Mic obiology and Biochemis y o Dai y P oduc s, Ins i u o de P oduc os Lác eos de
As u ias (IPLA-CSIC), 33300 Villa iciosa, Spain; [email p o ec ed] (M.C.);
[email p o ec ed] (C.G.d.L.R.-G.)
2Labo a o y o T ansmissible Diseases and Biologically Ac i e Subs ances LR99ES27, Facul y o Pha macy,
Uni e si y o Monas i , Monas i 5000, Tunisia; [email p o ec ed]
3
Labo a o y o An imic obial Resis ance LR99ES09, Facul y o Medicine o Tunis, Uni e si y o Tunis El Mana ,
Tunis 1068, Tunisia; [email p o ec ed]
4Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Medicine and Nu sing,
Uni e si y o he Basque Coun y, UPV/EHU, 48080 Bilbao, Spain; [email p o ec ed] (K.M.-C.);
[email p o ec ed] (G.Q.)
5Labo a o y o Medical and Molecula Pa asi ology-Mycology (code LR12ES08), Depa men o Clinical
Biology B, Facul y o Pha macy, Uni e si y o Monas i , Monas i 5000, Tunisia; [email p o ec ed]
6
Uni o Child Psychia y, Monas i Uni e si y Hospi al, Monas i 5000, Tunisia; [email p o ec ed]
7Die , Mic obio a and Heal h G oup, Ins i u o de In es igación Sani a ia del P incipado de As u ias (ISPA),
33011 O iedo, Spain
*Co espondence: [email p o ec ed]; Tel.: +34-946-01-83-71
Abs ac : Resea ch on he mic obio a–gu –b ain axis in au ism has p ima ily ocused on bac e ia,
wi h limi ed a en ion o ungi. The e is a g owing in e es in unde s anding he in ol emen o
ungi, pa icula ly Candida, in pa ien s wi h au ism spec um diso de . The aim o his s udy was
o assess he p e alence, an i ungal suscep ibili y p o iles and i ulence ac o s o Candida isola es
om he gu s o Tunisian child en wi h au ism. Twen y-eigh child en wi h au ism and o y-six
con ols we e en olled. Candida isola es om he aecal samples we e iden i ied using biochemical
and molecula me hods; an i ungal suscep ibili y es ing was de e mined by he EUCAST b o h
mic odilu ion me hod and i ulence ac o s, including bio ilm o ma ion, cell su ace hyd ophobici y
and phospholipase and p o einase ac i i ies, we e assessed
in i o
. As a esul , Candida was de ec ed
in 13 child en wi h au ism (46.4%) and 14 con ol child en (30.4%). Candida albicans was ound o be
he mos common species isola e in he aeces o bo h g oups o child en. An i ungal suscep ibili y
p o iles showed ha one Candida isola e was esis an o ampho e icin B and anidula ungin (3.7%),
six we e esis an o mica ungin (22.2%) and i e we e esis an o luconazole (18.5%). All Candida
isola es we e bio ilm p oduce s. O he wen y-se en isola es, only ou showed phospholipase
ac i i y (14.8%), eigh showed aspa yl-p o einase ac i i y (29.6%) and nine we e hyd ophobic
(33.3%). These esul s highligh he p esence o Candida in he gu s o child en wi h au ism, as well as
he abili y o exp ess mul iple i ulence ac o s and he an i ungal esis ance, and hey emphasize he
need o u he s udies o con i m in es inal Candida coloniza ion and i s po en ial ole in au ism.
Keywo ds: au ism; Candida; gu mic obio a; an i ungal suscep ibili y; i ulence
1. In oduc ion
Au ism spec um diso de (ASD) is a neu ode elopmen al diso de cha ac e ized
by impai ed social in e ac ion and communica ion, as well as es ic i e and epe i i e
beha iou s o in e es s. ASD is one o he mos common childhood men al diso de s,
J. Fungi 2024,10, 730. h ps://doi.o g/10.3390/jo 10110730 h ps://www.mdpi.com/jou nal/jo
J. Fungi 2024,10, 730 2 o 18
wi h a ying deg ees o se e i y. The mos ecen global p e alence o au ism is es i-
ma ed a 0.76%, which accoun s o a ound 16% o he global child popula ion [
1
–
3
]. The
e iopa hogenesis o au ism emains poo ly unde s ood, as his diso de in ol es gene ic
abno mali ies, dys egula ion o he immune sys em, in lamma ion, en i onmen al ac o s
and modi ica ions o gu mic obio a. Gas oin es inal (GI) symp oms, such as cons ipa ion,
abdominal pain, la ulence and dia hoea a e qui e common in pa ien s wi h ASD and a e
s ongly associa ed wi h he se e i y o ASD [
4
–
6
]. The equen ly epo ed GI p oblems
may be associa ed wi h an al e ed gu mic obio a, highligh ing a close connec ion be ween
gu and b ain, he so-called “mic obio a–gu –b ain axis”, a physiological bidi ec ional
ne wo k o communica ion be ween he gu and he b ain [
7
,
8
]. Indeed, he gu mic obio a
could in luence he cen al ne ous sys em h ough mul iple mechanisms, including neu al,
immune and endoc ine ones, and h ough he p oduc ion o mic obial oxins [
4
]. Consis-
en e idence o mic obio a dysbiosis in ASD has been shown in ecen yea s. Despi e he
he e ogenei y o esul s among s udies, mos o hem obse ed an inc ease in he p esence
o gene a such as Clos idium,Su e ella and Faecalibac e ium and a lowe p opo ion o Bi i-
dobac e ium [
9
–
11
]. Fu he mo e, al hough se e al s udies ha e assessed he gu mic obio a
p o ile in ASD child en, o ou knowledge, ew s udies ha e analysed he gu mycobio a
and i s po en ial ole in ASD [4,12]. Some epo s showed an inc eased abundance o Can-
dida species in he aecal samples o child en wi h ASD [
13
–
15
]. Candida coloniza ion can
cause malabso p ion o ca bohyd a es and mine als, and can elease ammonia and oxins,
which ha e been epo ed o be associa ed wi h some au is ic beha iou [
16
,
17
]. These
indings sugges ha Candida may be in ol ed in he ASD pa hogenesis. The e o e, an
ex ensi e unde s anding o he ole o in es inal Candida coloniza ion in ASD de elopmen
is needed.
The p esen s udy aimed o de e mine he p e alence o Candida coloniza ion in
he gu s o Tunisian child en wi h ASD. In addi ion, he dis ibu ion o Candida species,
in i o
an i ungal suscep ibili y o he collec ed isola es and he p oduc ion o se e al
i ulence ac o s, such as bio ilm o ma ion, cell su ace hyd ophobici y and p o einase
and phospholipase ac i i ies, we e e alua ed. We also compa ed he clinical da a and
in es iga ed he associa ion be ween ASD se e i y, GI diso de s and Candida in es inal
coloniza ion among child en wi h ASD and con ol (CC) g oups.
2. Subjec s and Me hods
2.1. Subjec s
A o al o 28 child en wi h ASD aged be ween 4 and 10 yea s old we e ec ui ed a he
Uni o Child and Adolescen Psychia y, Depa men o Psychia y, Fa ouma Bou guiba
Uni e si y Hospi al, Monas i , Tunisia be ween 2019 and 2020. Du ing he same pe iod,
46 age-ma ched child en we e en olled om siblings and child en o he gene al popula ion
as a con ol g oup. ASD pa ien s we e diagnosed acco ding o he diagnos ic and s a is ical
manual o Men al Diso de s (DSM-5) c i e ia [
1
], he Au ism Diagnos ic In en o y-Re ised
(ADI-R) [
18
] and he Au ism Diagnos ic Obse a ion Schedule-2 (ADOS-2) [
19
]. The
se e i y o au ism was assessed by he childhood au ism a ing scale (CARS) [
20
]. Exclusion
c i e ia included associa ed pa hologies ha can lead o isks: neu ological diso de s no
s ic ly associa ed wi h au ism, ype 1 diabe es, gene ic synd omes, coeliac disease, ood
in ole ance o in lamma o y bowel disease. Subjec s in his s udy we e no ea ed wi h
an ibio ics, an i ungal d ugs, p obio ics and/o p ebio ics o a leas one mon h be o e
sampling. The clinical da a o child en wi h ASD and con ol child en we e collec ed in
Table 1.
J. Fungi 2024,10, 730 3 o 18
Table 1. Demog aphic cha ac e is ics and clinical mani es a ions o Tunisian child en om he egion
o Monas i wi h ASD (n= 28) and CC g oup (n= 46).
Cha ac e is ics ASD G oup n(%) CC G oup n(%) p-Value
Gende 0.08
Male 22 (78.6%) 27 (58.7%)
Female 6 (21.4%) 19 (41.3%)
Age (mean ±SD) 7.93 ±2.09 7.39 ±2.02 0.119
4–7 yea s 10 (35.7%) 25 (54.3%)
8–10 yea s 18 (64.3%) 21 (45.7%)
GI diso de s 18 (64.3%) 21 (45.7%) 0.119
Cons ipa ion 17 (60.7%) 8 (17.4%) 0.000 *
Dia hoea 1 (3.6%) 1 (2.2%) 1.000
Vomi ing 1 (3.6%) 0 (0.0%) 0.378
Oesophageal e lux 1 (3.6%) 0 (0.0%) 0.378
Abdominal pain 7 (25.0%) 14 (30.4%) 0.615
CARS sco e
30–36 (mild o mode a e ASD) 11 (39.3%)
37–60 (se e e ASD) 17 (60.7%)
ASD, au ism spec um diso de ; CC, con ol child en; CARS, childhood au ism a ing scale; (*) indica es signi ican
di e ence be ween ASD and CC g oups.
2.2. Clinical Diagnosis
The DSM-5 is a e e ence guide o men al heal h p o essionals o diagnose, classi y and
iden i y men al heal h diso de s. I was published in May 2013 by he Ame ican Psychia ic
Associa ion (APA) [
1
]. The ADI-R is a s uc u ed in e iew adminis e ed o pa en s and/o
ca egi e s o child en wi h suspec ed ASD. I con ains 93 i ems ela ing o social in e ac ion,
communica ion and es ic ed, epe i i e and s e eo yped beha iou s. This exam usually
akes abou 2 o 3 h o comple e [
18
]. The ADOS is a s anda dized p o ocol o diagnosing
ASD. I consis s o a se ies o ac i i ies ha e alua e communica ion, ecip ocal social
in e ac ion, imagina ion and c ea i i y. The ADOS exam las s app oxima ely 40 min [
19
].
The CARS is a 15-i em beha iou al a ing scale used o assessing he se e i y o ASD. The
CARS sco e can ange be ween 15 and 60. Child en who ha e a sco e o less han 30 a e in
he non-au is ic ange. Mild o mode a e au ism is indica ed by a sco e o 30 o 36, whe eas
se e e au ism is indica ed by a sco e o 37 o 60 [20].
2.3. Faecal Sample Collec ion and Yeas Isola ion
A o al o 74 aecal samples we e collec ed om child en in s e ile con aine s by he
i s in es iga o and anspo ed o he labo a o y o p ocessing on he same day. F om
each sample, 1 g o aeces was esuspended in 10 mL o 0.9% s e ile saline solu ion and
homogenized by o exing. Ten
µ
L o his mix u e was inocula ed on Sabou aud dex ose
aga medium wi h chlo amphenicol and incuba ed ae obically a 37 ◦C o 24–48 h.
2.4. Candida Iden i ica ion
Candida iden i ica ion was pe o med by mo phologic, biochemical and molecula
me hods [
21
]. Isola ed colonies wi h a ious mo phologies we e G am s ained and ob-
se ed unde he mic oscope. Each colony ype was subcul u ed o pu i y and s o ed a
−
20
◦
C o u he analysis. Thawed isola es we e cul u ed o 48 h a 37
◦
C on Candida
ch omogenic (Condalab, Mad id, Spain) and Ch omID Candida (bioMé ieux, C aponne,
F ance) aga s and iden i ica ion was ca ied ou conside ing colony colou and mo phology.
Colonies ha appea ed wi h colou s o he han g een and blue on he selec i e media used
we e espec i ely iden i ied using API ID32C (bioMé ieux).
All he isola es ha g ew as g een colonies on Candida ch omogenic aga and blue
colonies on Ch omID Candida we e es ed o dis inguish be ween Candida albicans,Can-
dida dubliniensis and Candida a icana by ampli ica ion o he hyphal wall p o ein 1 gene
J. Fungi 2024,10, 730 4 o 18
(HWP1) based on he di e en amplicon sizes—700 bp o C. a icana, 569 bp o C. dublin-
iensis and app oxima ely 941 bp o C. albicans (Figu e S1) [
21
]. B ie ly, he isola es we e
pla ed on Sabou aud dex ose aga (Di co, Bec on Dickinson, F anklin Lakes, NJ, USA)
and incuba ed o e nigh a 37
◦
C. Then, a single colony was cul u ed in yeas ex ac
pep one dex ose b o h (YEPD, Pan eac, Ba celona, Spain) and incuba ed o 48 h a 30
◦
C
in shaking condi ions. DNA was ex ac ed om Candida isola es using a DNeasy Ul aclean
Mic obial Ki (QIAGEN, Ge man own, MD, USA) ollowing he manu ac u e ’s ins uc-
ions. A Nano D op ND-1000 spec opho ome e (The mo Fishe Scien i ic, Wal ham,
MA, USA) was used o measu e DNA concen a ion and pu i y. Ex ac ed DNA was
s o ed a
−
20
◦
C un il use. The HWP1 gene was ampli ied using he p ime s CRR- :
5
′
-GTTTTTGCAACTTCTCTTTGTA-3
′
and CRR- : 5
′
-ACAGTTGTATCATG TTCAGT-3
′
and he PCR was pe o med acco ding o he p o ocol desc ibed by Romeo e al. [
21
].
PCR eac ion condi ions we e as ollows: dena u a ion a 95
◦
C o 5 min, 30 cycles o
dena u a ion a 94
◦
C o 45 s, p ime annealing a 58
◦
C o 40 s and ex ension a 72
◦
C
o 55 s, ollowed by a inal ex ension a 72
◦
C o 10 min in a C 1000TM The mal Cycle
(Bio-Rad, He cules, CA, USA). The PCR p oduc s we e sepa a ed by elec opho esis on a
1.5% aga ose gel s ained wi h Gel Red (Bio ium, F emon , CA, USA) and hen isualized
on a UV ansillumina o . A DNA ladde (Hype ladde TM 50 bp [50–2000 bp]; Bioline,
London, UK) was used as a molecula weigh s anda d. DNA o C. albicans NCPF 3153, C.
dubliniensis NCPF 3949 and C. a icana ATCC MYA-2669 we e included as posi i e con ols.
2.5. In Vi o An i ungal Suscep ibili y Tes ing
The ac i i y o six an i ungal d ugs agains all Candida isola es we e es ed: ampho-
e icin B (Sigma-Ald ich, Mad id, Spain), mica ungin (As ellas Pha ma Inc., Tokyo, Japan),
anidula ungin (P ize SA, Mad id, Spain), luconazole (P ize SA), isa uconazole (Basilea
Pha maceu ica In e na ional, Allschwil, Swi ze land) and ib exa unge p (Scynexis Inc.,
Je sey Ci y, NJ, USA). S ock solu ions o each d ug we e p epa ed in dime hyl sul ox-
ide (DMSO) and s o ed a
−
80
◦
C un il use. The inal d ug concen a ion anged om
0.008 o 4 mg/L o ampho e icin B, anidula ungin, mica ungin and isa uconazole, om
0.125 o 64 mg/L o luconazole and om 0.016 o 16 mg/L o ib exa unge p. The
minimal inhibi o y concen a ions (MICs) o he an i ungal d ugs we e de e mined by
b o h mic odilu ion me hod in 96-well la -bo om mic o i e pla es, in RPMI 1640 medium,
acco ding o EUCAST guidelines [
22
]. P io o he expe imen , an inoculum was p epa ed
in s e ile dis illed wa e o each isola e ob ained om an o e nigh cul u e a 37
◦
C. The
inal inoculum, be ween 0.5–2.5
×
10
5
CFU/mL, was added o he mic o i e pla es. The
pla es we e hen incuba ed a 37
◦
C o 24 h and he abso bance was measu ed a a wa e-
leng h o 450 nm using an In ini e F50 spec opho ome e (Tecan, Männedo ,
Swi ze land
).
Two e e ence s ains Candida k usei ATCC 6258 (cu en ly Pichia kud ia ze ii) and Candida
pa apsilosis ATCC 22019 we e used as quali y con ols. MICs we e ead a 24 h and de ined
as he an i ungal concen a ion ha inhibi ed a leas 50% o Candida g ow h, excep o
ampho e icin B, o which he MIC e alua ed 90% o g ow h inhibi ion. Fo each species
and an i ungal, desc ip i e s a is ics, including MIC anges and geome ic mean MICs
(GM), we e calcula ed. The in e p e a ion o suscep ibili y was pe o med acco ding he EU-
CAST clinical b eakpoin s [
23
]. No in e p e i e b eakpoin s a e a ailable o isa uconazole
and ib exa unge p.
2.6. Bio ilm De elopmen
P io o he expe imen , Candida s ains we e inocula ed in YEPD b o h and we e
incuba ed o e nigh unde o bi al shaking 120 pm a 30
◦
C. Cells we e ha es ed and
washed h ee imes wi h s e ile phospha e bu e ed saline solu ion (PBS, Sigma-Ald ic).
A e cell coun ing by mic oscopy using a Bu ke haemocy ome e , cell suspensions o
each Candida isola e we e adjus ed o a inal concen a ion o 10
6
cells/mL, wi h RPMI
1640 medium supplemen ed wi h L-glu amine and bu e ed wi h mo pholine p opane-
sul onic acid (MOPS, Sigma-Ald ich) o pH 7. Candida bio ilm o ma ion was pe o med on
J. Fungi 2024,10, 730 5 o 18
s e ile, la -bo omed honeycomb 100-well mic o i e pla es (Labsys ems, Van aa, Finland).
One hund ed
µ
L o each adjus ed cell suspension was ans e ed in o he wells o he pla e.
The mic o i e pla es we e hen incuba ed a 37
◦
C. A e 24 h and 48 h, he wells we e
washed wi h 100 µL o s e ile PBS o emo e una ached and weakly a ached cells [24].
Bio ilm biomass was quan i ied using c ys al iole (CV) s aining [
25
]. A e washing,
he bio ilms we e d ied a oom empe a u e o 30 min, hen a olume o 100
µ
L o
0.4% CV solu ion (Me ck, Da ms ad , Ge many) was added o each well and s ained o
20 min. A e wa ds, he excess CV was emo ed by insing he pla es wice wi h 250
µ
L o
s e ile dis illed wa e . Finally, he bound CV was eleased by adding 150
µ
L o 33% ace ic
acid. Bio ilm me abolic ac i i y was measu ed using a colo ime ic me hod based on
he educ ion o 2,3-bis-(2-me hoxy-4-ni o-5-sul ophenyl)-5-[(phenylamino)-ca bonyl]-2H-
e azolium hyd oxide (XTT, Sigma-Ald ich) [
26
]. B ie ly, XTT was p epa ed as a sa u a ed
solu ion a a concen a ion o 0.5 g/L in Ringe ’s lac a e. The solu ion was il e -s e ilized
using a 0.22
µ
m-po e-size il e (Sa s ed , Nümb ech , Ge many) and aliquo s we e s o ed
a
−
70
◦
C un il use. Be o e each assay, 100
µ
L o an aliquo o XTT mixed wi h 1
µ
M
menadione was added o each well and he mic opla es we e incuba ed in he da k a 37
◦
C
o 2 h.
Abso bances we e measu ed a wo ime poin s, 24 and 48 h, using a BioSc een C MBR
mic opla e eade (G ow h Cu es L d., Tu ku, Finland) a a wa eleng h o 600 nm o
bio ilm biomass and 492 nm o bio ilm me abolic ac i i y. Two independen expe imen s
we e pe o med wi h i e eplica es o each condi ion, and he e e ence s ains C. albicans
SC5314 and C. albicans Ca2 hypha-de ec i e mu an (g aciously dona ed by P o esso
An onio Cassone, Ins i u o Supe io e di Sani à, Rome, I aly) we e included as posi i e and
nega i e con ols, espec i ely.
2.7. Cell Su ace Hyd ophobici y Assay
Cell su ace hyd ophobici y (CSH) was de e mined using he mic obial adhesion o
hyd oca bon (MATH) es [
27
,
28
]. B ie ly, he yeas cells g own o e nigh a 30
◦
C in YEPD
b o h we e washed wice wi h s e ile PBS and esuspended in he same bu e o adjus an
abso bance be ween 0.4 and 0.5 a 600 nm (A0). Th ee mL o his yeas cell suspension was
o e laid wi h 0.4 mL o n-hexadecane (Sigma-Ald ich). A e igo ous o exing, aqueous
and o ganic phases we e allowed o sepa a e o 10 min a 30
◦
C and he abso bance o
he aqueous phase was measu ed a 600 nm (A1). The pe cen age o hyd ophobici y was
calcula ed acco ding o he ollowing equa ion: pe cen age o
CSH = [1 −(A1/A0)] ×100
.
The highly hyd ophobic s ains exhibi ed CSH alues o mo e han 50%, and he mode a ely
hyd ophobic s ains had CSH alues anging be ween 20 and 50%. Hyd ophilic s ains
had CSH alues o less han 20% [29].
2.8. De e mina ion o Phospholipase and P o einase Ac i i y
Isola es we e g own on Sabou aud dex ose aga pla es o e nigh a 37
◦
C and cells
we e hen suspended in s e ile saline solu ion o a inal suspension o 10
7
cells/mL. Ten
µ
L
o his suspension was inocula ed on each speci ic medium.
Phospholipase ac i i y was e alua ed ollowing he me hod desc ibed by Polak [
30
],
using mal aga pla es supplemen ed wi h 1 M NaCl, 5 mM CaCl
2
and 8% s e ile egg yolk
emulsion [
31
]. The s ain C. albicans UPV/EHU 04-125 was included as posi i e con ol. As-
pa yl p o einase ac i i y was assessed using bo ine se um albumin (BSA) aga pla es [
32
].
B ie ly, he BSA medium consis ed o 1.17% yeas ca bon base (Di co), 0.01% yeas ex ac
(Condalab) and 0.2% BSA (Sigma-Ald ich). The medium was s e ilized by il a ion and
mixed wi h a s ock solu ion o au ocla ed 2% bac o-aga (Di co). C. dubliniensis UPV/EHU
00-134 was used as posi i e con ol [
33
]. A e inocula ion, he pla es we e incuba ed
a 37
◦
C o 6 days. The phospholipase ac i i y (Pz) alue was de e mined as he a io
o he diame e o he colony o he o al diame e o he colony plus p ecipi a ion zone
and was classi ied as ollows: 0.35–0.5 (high p oduce s);
0.51–0.74 (mode a e
p oduce s);
0.75–0.9 (low
p oduce s) and 1 (non-p oduce s). The aspa yl-p o einase ac i i y was es-
J. Fungi 2024,10, 730 6 o 18
ablished as he diame e o a anspa en halo a ound g owing colonies. Candida isola es
we e classi ied as non-p oduce s (when no isible halo was p esen ), mode a e p oduce s
(when he diame e o he halo was 1–2 mm) and high p oduce s (when he diame e o he
halo was >2 mm) [33]. Each isola e was assayed in iplica e.
2.9. S a is ical Analysis
S a is ical analysis o quan i a i e and quali a i e da a, including desc ip i e s a is ics,
was pe o med. F equencies compa ison o ca ego ical da a was pe o med wi h Chi-
squa e es o Fishe ’s exac es . The in e g oup di e ences o con inuous da a we e
conduc ed by S uden ’s es when da a showed a no mal dis ibu ion and Mann–Whi ney
nonpa ame ic es when da a did no show a no mal dis ibu ion. All es s we e wo
sided and a
p- alue < 0.05
was conside ed as s a is ically signi ican . Da a we e analysed
using SPSS so wa e ( e sion 26) and igu es we e cons uc ed using G aphPad P ism
( e sion 8.0.2).
3. Resul s
3.1. Clinical Cha ac e is ics o he S udy Popula ion
As shown in Table 1, he e we e no s a is ically signi ican di e ences in age and gende
be ween he ASD and CC g oups. Mo eo e , GI diso de s did no signi ican ly di e when
compa ing bo h g oups, excep o cons ipa ion: cons ipa ion was mo e equen in he
ASD g oup han he CC g oup (60.7% s. 17.4%, espec i ely, p= 0.000).
3.2. Associa ion be ween ASD Se e i y and Clinical Da a in he ASD G oup
Acco ding o he CARS sco e, child en wi h ASD we e di ided in o wo g oups:
child en wi h mild–mode a e ASD (n= 11) and child en wi h se e e ASD (n= 17). We
ound signi ican associa ions be ween GI diso de s and ASD se e i y (p= 0.020): a highe
pe cen age o child en su e ing om se e e ASD (82.4%) had GI diso de s. In con as ,
only 36.4% o child en wi h mild o mode a e au ism p esen ed GI diso de s (Figu e 1).
Cons ipa ion was also associa ed wi h ASD se e i y (p= 0.006). Child en wi h se e e
ASD we e mo e likely o p esen cons ipa ion han child en wi h mild o mode a e ASD
(82.4% s. 27.3%) (Figu e 1). No signi ican di e ences we e obse ed be ween ASD
se e i y and o he clinical pa ame e s.
J. Fungi 2024, 10, x FOR PEER REVIEW 6 o 19
ex ac (Condalab) and 0.2% BSA (Sigma-Ald ich). The medium was s e ilized by il a-
ion and mixed wi h a s ock solu ion o au ocla ed 2% bac o-aga (Di co). C. dubliniensis
UPV/EHU 00-134 was used as posi i e con ol [33]. A e inocula ion, he pla es we e in-
cuba ed a 37 °C o 6 days. The phospholipase ac i i y (Pz) alue was de e mined as he
a io o he diame e o he colony o he o al diame e o he colony plus p ecipi a ion
zone and was classi ied as ollows: 0.35–0.5 (high p oduce s); 0.51–0.74 (mode a e p oduc-
e s); 0.75–0.9 (low p oduce s) and 1 (non-p oduce s). The aspa yl-p o einase ac i i y was
es ablished as he diame e o a anspa en halo a ound g owing colonies. Candida iso-
la es we e classi ied as non-p oduce s (when no isible halo was p esen ), mode a e p o-
duce s (when he diame e o he halo was 1–2 mm) and high p oduce s (when he diam-
e e o he halo was >2 mm) [33]. Each isola e was assayed in iplica e.
2.9. S a is ical Analysis
S a is ical analysis o quan i a i e and quali a i e da a, including desc ip i e s a is-
ics, was pe o med. F equencies compa ison o ca ego ical da a was pe o med wi h Chi-
squa e es o Fishe ’s exac es . The in e g oup diffe ences o con inuous da a we e con-
duc ed by S uden ’s es when da a showed a no mal dis ibu ion and Mann–Whi ney
nonpa ame ic es when da a did no show a no mal dis ibu ion. All es s we e wo sided
and a p- alue < 0.05 was conside ed as s a is ically signi ican . Da a we e analysed using
SPSS so wa e ( e sion 26) and igu es we e cons uc ed using G aphPad P ism ( e sion
8.0.2).
3. Resul s
3.1. Clinical Cha ac e is ics o he S udy Popula ion
As shown in Table 1, he e we e no s a is ically signi ican diffe ences in age and gen-
de be ween he ASD and CC g oups. Mo eo e , GI diso de s did no signi ican ly diffe
when compa ing bo h g oups, excep o cons ipa ion: cons ipa ion was mo e equen in
he ASD g oup han he CC g oup (60.7% s. 17.4%, espec i ely, p = 0.000).
3.2. Associa ion be ween ASD Se e i y and Clinical Da a in he ASD G oup
Acco ding o he CARS sco e, child en wi h ASD we e di ided in o wo g oups: chil-
d en wi h mild–mode a e ASD (n = 11) and child en wi h se e e ASD (n = 17). We ound
signi ican associa ions be ween GI diso de s and ASD se e i y (p = 0.020): a highe pe -
cen age o child en suffe ing om se e e ASD (82.4%) had GI diso de s. In con as , only
36.4% o child en wi h mild o mode a e au ism p esen ed GI diso de s (Figu e 1). Con-
s ipa ion was also associa ed wi h ASD se e i y (p = 0.006). Child en wi h se e e ASD
we e mo e likely o p esen cons ipa ion han child en wi h mild o mode a e ASD (82.4%
s. 27.3%) (Figu e 1). No signi ican diffe ences we e obse ed be ween ASD se e i y and
o he clinical pa ame e s.
Figu e 1. P esence o GI diso de s and cons ipa ion in child en suffe ing om mild o mode a e
ASD and se e e ASD. GI, gas oin es inal; ASD, au ism spec um diso de . As e isks (*) indica e
signi ican diffe ences be ween he wo ASD subg oups: mild o mode a e and se e e.
Figu e 1. P esence o GI diso de s and cons ipa ion in child en su e ing om mild o mode a e
ASD and se e e ASD. GI, gas oin es inal; ASD, au ism spec um diso de . As e isks (*) indica e
signi ican di e ences be ween he wo ASD subg oups: mild o mode a e and se e e.
3.3. P e alence o Candida in he Gu Mic obio a o Child en wi h ASD and Con ol Child en
Candida was de ec ed in 13 child en wi h ASD (13 ou o 28, 46.4%) and in 14 con ol
child en (14 ou o 46, 30.4%) (Figu e 2, Table S1). The di e ence in Candida p e alence
be ween he wo g oups o child en was no s a is ically signi ican (p= 0.166). The species
C. albicans was he mos equen in aecal samples o bo h ASD child en (n= 7 ou o
13 isola es; 53.8%) and he CC g oup (n= 10 ou o 14 isola es; 71.4%). The mos common
non-C. albicans species de ec ed in he s ools o he g oup wi h ASD we e Candida glab a a
J. Fungi 2024,10, 730 7 o 18
(cu en ly Nakaseomyces glab a us;n= 2; 15.4%), C. pa apsilosis (n= 2; 15.4%), C. dubliniensis
(n= 1; 7.7%) and Candida guillie mondii (cu en ly Meye ozyma guillie mondii;n= 1; 7.7%).
As o he con ol g oup, he iden i ica ion o non-C. albicans species yielded de ec ion o C.
glab a a (n= 2; 14.3%), C. dubliniensis (n= 1; 7.1%) and C. k usei (n= 1; 7.1%). The e we e
no signi ican di e ences in he dis ibu ion o Candida species o in he di e si y o hese
species be ween child en wi h ASD and con ols (p= 0.480). No cul u es wi h mo e han
one species we e de ec ed.
J. Fungi 2024, 10, x FOR PEER REVIEW 7 o 19
3.3. P e alence o Candida in he Gu Mic obio a o Child en wi h ASD and Con ol Child en
Candida was de ec ed in 13 child en wi h ASD (13 ou o 28, 46.4%) and in 14 con ol
child en (14 ou o 46, 30.4%) (Figu e 2, Table S1 Online esou ce 2). The diffe ence in Can-
dida p e alence be ween he wo g oups o child en was no s a is ically signi ican (p =
0.166). The species C. albicans was he mos equen in aecal samples o bo h ASD chil-
d en (n = 7 ou o 13 isola es; 53.8%) and he CC g oup (n = 10 ou o 14 isola es; 71.4%).
The mos common non-C. albicans species de ec ed in he s ools o he g oup wi h ASD
we e Candida glab a a (cu en ly Nakaseomyces glab a us; n = 2; 15.4%), C. pa apsilosis (n = 2;
15.4%), C. dubliniensis (n = 1; 7.7%) and Candida guillie mondii (cu en ly Meye ozyma
guillie mondii; n = 1; 7.7%). As o he con ol g oup, he iden i ica ion o non-C. albicans
species yielded de ec ion o C. glab a a (n = 2; 14.3%), C. dubliniensis (n = 1; 7.1%) and C.
k usei (n = 1; 7.1%). The e we e no signi ican diffe ences in he dis ibu ion o Candida
species o in he di e si y o hese species be ween child en wi h ASD and con ols (p =
0.480). No cul u es wi h mo e han one species we e de ec ed.
Figu e 2. P e alence o Candida in s ools o child en wi h ASD and CC g oup. ASD, au ism spec um
diso de ; CC, con ol child en. C. glab a a (n = 2), C. pa apsilosis (n = 2), C. dubliniensis (n = 1) and C.
guillie mondii (n = 1) we e non-C. albicans species isola ed in s ools o child en wi h ASD. C. glab a a
(n = 2), C. dubliniensis (n = 1) and C. k usei (n = 1) we e non-C. albicans species isola ed in s ools o he
con ol child en g oup.
3.4. Associa ion Be ween Candida Gu Coloniza ion and he Clinical Da a o he ASD G oup
In he g oup o child en wi h ASD, he e we e no s a is ically signi ican diffe ences
be ween aecal specimens wi h posi i e o nega i e Candida cul u es ega ding hei gen-
de o age. Simila ly, no s a is ically signi ican diffe ences in Candida p esence we e ob-
se ed be ween ASD child en wi h o wi hou GI diso de s and wi h o wi hou se e e
ASD (Table S2, Online esou ce 3).
3.5. An i ungal Suscep ibili y P o ile o Candida Isola es
The ac i i ies o he six an i ungal agen s es ed agains he Candida isola es a e p e-
sen ed in Table 2. In he g oup o child en suffe ing om ASD, all C. albicans (n = 7) we e
suscep ible o ampho e icin B, one isola e was esis an o anidula ungin, wo we e e-
sis an o mica ungin and ou we e esis an o luconazole. C. glab a a isola es (n = 2)
we e esis an o mica ungin, suscep ible—dose dependen — o luconazole and suscep i-
ble o ampho e icin B and anidula ungin. The wo isola es o C. pa apsilosis we e suscep-
ible o anidula ungin, mica ungin and luconazole and one isola e was ound o be am-
pho e icin B esis an . The isola e o C. dubliniensis was suscep ible o ampho e icin B and
luconazole.
Figu e 2. P e alence o Candida in s ools o child en wi h ASD and CC g oup. ASD, au ism spec um
diso de ; CC, con ol child en. C. glab a a (n= 2), C. pa apsilosis (n= 2), C. dubliniensis (n= 1) and C.
guillie mondii (n= 1) we e non-C. albicans species isola ed in s ools o child en wi h ASD. C. glab a a
(
n= 2
), C. dubliniensis (n= 1) and C. k usei (n= 1) we e non-C. albicans species isola ed in s ools o he
con ol child en g oup.
3.4. Associa ion Be ween Candida Gu Coloniza ion and he Clinical Da a o he ASD G oup
In he g oup o child en wi h ASD, he e we e no s a is ically signi ican di e ences
be ween aecal specimens wi h posi i e o nega i e Candida cul u es ega ding hei gende
o age. Simila ly, no s a is ically signi ican di e ences in Candida p esence we e obse ed
be ween ASD child en wi h o wi hou GI diso de s and wi h o wi hou se e e ASD
(Table S2).
3.5. An i ungal Suscep ibili y P o ile o Candida Isola es
The ac i i ies o he six an i ungal agen s es ed agains he Candida isola es a e p e-
sen ed in Table 2. In he g oup o child en su e ing om ASD, all C. albicans (n= 7) we e
suscep ible o ampho e icin B, one isola e was esis an o anidula ungin, wo we e esis-
an o mica ungin and ou we e esis an o luconazole. C. glab a a isola es (n= 2) we e
esis an o mica ungin, suscep ible—dose dependen — o luconazole and suscep ible o
ampho e icin B and anidula ungin. The wo isola es o C. pa apsilosis we e suscep ible o
anidula ungin, mica ungin and luconazole and one isola e was ound o be ampho e icin
B esis an . The isola e o C. dubliniensis was suscep ible o ampho e icin B and luconazole.
In he CC g oup, all C. albicans isola es (n= 10) we e suscep ible o ampho e icin B,
anidula ungin, mica ungin and luconazole. Two C. glab a a isola es we e suscep ible o
ampho e icin B and anidula ungin and esis an o mica ungin; one isola e was lucona-
zole esis an , and one was suscep ible, dose dependen . The C. dubliniensis isola e was
suscep ible o ampho e icin B and luconazole and he C. k usei isola e was suscep ible o
ampho e icin B and anidula ungin.
Fu he mo e, low MIC alues we e ound o isa uconazole and hey anged om
0.008 o 0.03 mg/L o C. albicans and om 0.008 o 1 mg/L o non-C. albicans. MICs
o ib exa unge p agains Candida isola es we e also es ed
in i o
, anging om 0.016 o
1 mg/L. The lowes ib exa unge p MICs we e obse ed agains C. albicans isola es (GM
0.02 mg/L, MIC ange 0.016–0.03 mg/L) and he highes we e ob ained o he C. guil-
lie mondii isola e (MIC = 1 mg/L). Adop ing wild- ype uppe limi s ecen ly p oposed by
Quindós e al. [
34
] o C. albicans (0.5 mg/L), C. glab a a (1 mg/L), C. pa apsilosis (2 mg/L),
J. Fungi 2024,10, 730 8 o 18
and o C. k usei (4 mg/L), no non-wild- ype pheno ype o ib exa unge p was obse ed,
and all isola es we e conside ed suscep ible o his an i ungal d ug.
J. Fungi 2024,10, 730 9 o 18
Table 2.
In i o
ac i i ies o ampho e icin B, anidula ungin, mica ungin, luconazole, isa uconazole and ib exa unge p agains Candida isola ed om he aecal
samples o child en wi h au ism and con ol child en.
An i ungal
D ugs Species (n)
Child en wi h Au ism Con ol Child en
MIC (mg/L) Isola es n(%) MIC (mg/L) Isola es n(%)
Range MIC GM Mean S I R Range MIC GM Mean S I R
Ampho e icin B
Candida albicans (17) 0.03–0.125 0.16 7 (41.2) 0 0 0.06–0.5 0.16 10 (58.8) 0 0
Candida glab a a (4) 0.125–0.25 0.18 2 (50) 0 0 0.125–0.25 0.18 2 (50) 0 0
Candida pa apsilosis (2) 0.125–4 2.06 1 (50) 0 1 (50)
Candida dubliniensis (2) 0.03 0.03 1 (50) 0 0 0.03 0.03 1 (50) 0 0
Candida guillie mondii (1) 0.125 0.12 ND ND ND
Candida k usei (1) 0.25 0.25 1 (100) 0 0
Anidula ungin
Candida albicans (17) 0.008–4 0.58 6 (35.3) 0 1 (5.8) 0.008–0.03 0.02 10 (58.8) 0 0
Candida glab a a (4) 0.06 0.06 2 (50) 0 0 0.03–0.06 0.05 2 (50) 0 0
Candida pa apsilosis (2) 0.5–4 2.25 2 (100) 0 0
Candida dubliniensis (2) 0.03 0.03 ND ND ND 0.03 0.03 ND ND ND
Candida guillie mondii (1) 0.06 0.06 ND ND ND
Candida k usei (1) 0.03 0.03 1 (100) 0 0
Mica ungin
Candida albicans (17) 0.03–4 0.59 5 (29.4) 0 2 (11.8) 0.016–0.03 0.03 10 (58.8) 0 0
Candida glab a a (4) 0.06–0.125 0.09 0 0 2 (50) 0.06–0.125 0.09 0 0 2 (50)
Candida pa apsilosis (2) 0.008–1 0.5 2 (100)
Candida dubliniensis (2) 0.06 0.06 ND ND ND 0.06 0.06 ND ND ND
Candida guillie mondii (1) 0.06 0.06 ND ND ND
Candida k usei (1) 0.06 0.06 ND ND ND
Isa uconazole
Candida albicans (17) 0.008–0.016 0.01 ND ND ND 0.008–0.03 0.02 ND ND ND
Candida glab a a (4) 0.25–1 0.63 ND ND ND 0.5–1 0.75 ND ND ND
Candida pa apsilosis (2) 0.016–0.03 0.02 ND ND ND
Candida dubliniensis (2) 0.008 0.008 ND ND ND 0.016 0.016 ND ND ND
Candida guillie mondii (1) 0.125 0.125 ND ND ND
Candida k usei (1) 1 1 ND ND ND
Fluconazole
Candida albicans (17) 0.125–64 25.19 3 (16.6) 0 4 (23.5) 0.125–0.25 0.162 10 (58.9) 0 0
Candida glab a a (4) 4–8 6 0 2 (50) 0 4–64 34 0 1 (25) 1 (25)
Candida pa apsilosis (2) 0.5 0.5 2 (100) 0 0
Candida dubliniensis (2) 0.125 0.125 1 (50) 0 0 0.25 0.25 1 (50) 0 0
Candida guillie mondii (1) 1 1 ND ND ND
Candida k usei (1) 32 32 ND ND ND
J. Fungi 2024,10, 730 16 o 18
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