In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
A ailable online 9 May 2022
0168-1605/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Gene ic cha ac e iza ion and bio ilm o ma ion o po en ially pa hogenic
oodbo ne A cobac e isola es
I a i Ma inez-Malaxe xeba ia
a
,
b
,
*
, Cecilia Gi bau
a
,
1
, Ad i´
an Salaza -S´
anchez
a
,
I saso Baz a ika
a
, Ila gi Ma ínez-Balles e os
a
,
b
, Lo ena Lao den
a
,
b
, Rod igo Alonso
a
,
b
,
Au o a Fe n´
andez-As o ga
a
a
Mik oIke Resea ch G oup, Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o he Basque Coun y UPV/EHU, Paseo de la
Uni e sidad 7, 01006 Vi o ia-Gas eiz, ´
Ala a, Spain
b
Bioa aba, Mic obiology, In ec ious Disease, An imic obial Agen s and Gene The apy, 01006 Vi o ia-Gas eiz, ´
Ala a, Spain
ARTICLE INFO
Keywo ds:
Food
P e alence
MLST
Vi ulence-associa ed genes
Adhesion o su aces
ABSTRACT
Va ious species o he genus A cobac e a e ega ded as eme ging ood pa hogens and can be cause o human
gas oen e ic illness, among o he s. In o de o gain knowledge on he isk associa ed wi h he p esence o
a cobac e s in e ail oods, his s udy aimed o de e mine hei p esence in a a ie y o p oduc s; o e alua e he
gene ic di e si y and he occu ence o i ulence and bio ilm-associa ed genes in he isola ed s ains; and o
assess hei bio ilm ac i i y on polys y ene, bo osilica e and s ainless s eel. A cobac e s we e de ec ed in he
22.3% o he analysed samples and he 83 eco e ed isola es we e iden i ied as A. bu zle i (n =53),
A. c yae ophilus (n =24), A. ski owii (n =2), A. he eius (n =3) and A. i o iensis (n =1). They we e isola ed om
i ually all es ed ood ypes, bu mos ly om squids and u key mea (con amina ion le els o 60% and 40%,
espec i ely). MLST di e en ia ed 68 STs, mos o which we e no el (89.7%) and ep esen ed by a single s ain
(86.9%). Fi e no el STs we e de ec ed in a ious isola es de i ed om sea ood, and he s a is ical analysis
e ealed hei po en ial associa ion wi h ha ype o ood p oduc (p <0,001). All he isola es excep one
ha bou ed i ulence-associa ed genes and he highes incidence was no ed o A. bu zle i. Nine een isola es
(23.5%) we e able o o m bio ilms on he di e en su aces es ed and, o no e; glass enhanced he adhesion
abili y o he majo i y o hem (84.2%). The esul s highligh he ole ha common ood p oduc s can ha e in he
ansmission o A cobac e spp., he pa hogenic po en ial o he di e en species, and he su i al and g ow h
abili y o se e al o hem on di e en ood con ac su aces. The e o e, he s udy p o ides in e es ing in o ma ion
ega ding he isk a cobac e s may pose o human heal h and he ood indus y.
1. In oduc ion
The genus A cobac e , wi hin he Campylobac e aceae amily, was
i s desc ibed by Vandamme e al. in 1991. The axonomy o his genus
has been unde deba e du ing he las yea s (On e al., 2020, 2021;
P´
e ez-Ca alu˜
na e al., 2018; Wai e e al., 2017) bu , a he ime o w i ing
and acco ding o LPSN, he lis o p oka yo ic names wi h s anding in
nomencla u e (Pa e e al., 2020), he genus comp ises 33 alidly pub-
lished species (h ps://lpsn.dsmz.de/genus/a cobac e ; accessed on
Feb ua y 28 h, 2022) ha ha e been isola ed om a ious di e en
en i onmen s and sou ces. Ce ain species o he genus a e associa ed
wi h human disease. They mainly induce gas oin es inal symp oms
(ch onic wa e y dia hoea and a elle 's dia hoea), bu can also be he
cause o bac e aemia, sep icaemia, pe i oni is and endoca di is (Collado
and Figue as, 2011; Fanelli e al., 2019; Simaluiza e al., 2021).
A. bu zle i is he species mos equen ly associa ed wi h disease, ol-
lowed by A. c yae ophilus, bu in ec ions due o A. ski owii, A. he eius
and A. lan hie i ha e also been epo ed (Ke kho e al., 2021; Ramees
e al., 2017; Ruíz de Aleg ía e al., 2021; Van den Abeele e al., 2014).
Ne e heless, he mechanisms implied in he pa hogenesis o hese
bac e ia emain unclea . Va ious au ho s ha e demons a ed he in i o
cy o oxici y o A cobac e species, along wi h hei abili y o adhe e and
* Co esponding au ho a : Mik oIke Resea ch G oup, Depa men o Immunology, Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o he Basque
Coun y UPV/EHU, Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, ´
Ala a, Spain.
E-mail add ess: [email p o ec ed] (I. Ma inez-Malaxe xeba ia).
1
P esen add ess: Public Heal h Labo a o y o A aba, Basque Go e nmen , A enida San iago 11, 01004 Vi o ia-Gas eiz, Spain.
Con en s lis s a ailable a ScienceDi ec
In e na ional Jou nal o Food Mic obiology
jou nal homepage: www.else ie .com/loca e/ij oodmic o
h ps://doi.o g/10.1016/j.ij oodmic o.2022.109712
Recei ed 7 Augus 2021; Recei ed in e ised o m 1 May 2022; Accep ed 5 May 2022
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
2
in ade di e en human cell lines (Buzzanca e al., 2021; Collado and
Figue as, 2011; Ka adas e al., 2013; Le ican e al., 2013). On he o he
hand, he a ailable A cobac e genomes ha e shown he p esence o
a ious i ulence-associa ed genes ela ed o, among o he s, adap a ion,
cy o oxici y, adhesion, in asi eness and an ibio ic esis ance (Isid o
e al., 2020; Mille e al., 2007; Mülle e al., 2020a, 2020b). The co -
ela ion be ween he epo ed pa hogenic capabili ies and he p esence
o speci ic genes has no been es ablished ye in A cobac e spp. How-
e e , he i ulence-associa ed gene con en o he isola es can be
indica i e o he isk hey may pose o human heal h.
The consump ion o con amina ed d inking wa e and/o unde -
cooked o aw oods seems o be he main human ansmission sou ce o
A cobac e spp. They a e commonly p esen in ood p oduc s including
ege ables, sea ood, e es ial animal ood p oduc s and composi e
oods (G´
onzalez and Fe ús, 2011; Kie si i e al., 2021; Mo ola e al.,
2020; Nie a-Eche a ia e al., 2013); bu also in di e en wa e s
including con inen al, coas al, sea, ec ea ional, d inking and sewage
(Scio ino e al., 2021). Mo eo e , a cobac e s a e o en p esen in ood
p ocessing en i onmen s such as slaugh e houses and dai y a ms and/
o plan s (Fe ei a e al., 2017; Giacome i e al., 2015b; Khodamo adi
and Abi i, 2020) whe e, i he condi ions a e a ou able, hey may
p obably o m bio ilms. A cobac e s ha e he demons a ed abili y o
adhe e o di e en su aces and o o m bio ilms on hem (Fe ei a e al.,
2013; Gi bau e al., 2017; ˇ
Silha e al., 2021). When o med on ood
con ac su aces and/o ma e ials, bio ilms inc ease ood sa e y isk.
Rese oi s o ood spoilage and/o pa hogen bac e ia in ood indus ies
a e an impo an cause o p oduc con amina ion ha can lead no only
o a educed shel li e o oods, bu also o heal h p oblems (Abebe, 2020;
Ade unji e al., 2014). The e o e, he bio ilm o ma ion by ood de i ed
A cobac e spp. can pose a isk o public heal h and a p oblem o he
ood indus y. T acking he in ec ion sou ce and he ansmission ou es
o a cobac e s is one o he necessa y s eps o assess he isk ela ed o
hese pa hogens. Among he molecula sub yping echniques a ailable
o he species, he Mul ilocus Sequence Typing (MLST) scheme p o-
posed by Mille e al. (2009) is a eliable and ep oducible echnique
ha has been success ully u ilized o cha ac e iza ion o A cobac e
isola es om di e en sou ces (Alonso e al., 2014; Ca uso e al., 2020;
Kie si i e al., 2021; Niede meye e al., 2020). Howe e , pa ly due o
he limi ed a ailable da a in he A cobac e MLST da abase (h ps://pub
mls .o g/o ganisms/a cobac e -spp), pa ly due o he g ea gene ic
he e ogenei y shown by A cobac e isola es, no sou ce-associa ed ge-
ne ic ma ke has been epo ed so a o hese species.
In o de o inc ease he knowledge needed o assess he isk ha
a cobac e s pose o human heal h; he pu poses o his s udy we e o
con i m he p esence o di e en A cobac e species in e ail ood
p oduc s; o e alua e, by MLST and i ulence-associa ed gene de ec ion,
he gene ic di e si y o he isola es; and o in es iga e he bio ilm p o-
duc ion o all he eco e ed isola es. We complemen , his way, ou
p e ious su eys on p e alence and cha ac e iza ion o a cobac e s in
oods (Alonso e al., 2014; Gi bau e al., 2014, Gi bau e al., 2017; Nie a-
Eche a ia e al., 2013) by he analysis o ood p oduc s no p e iously
su eyed.
2. Ma e ials and me hods
2.1. Sample collec ion and p ocessing
Two hund ed and wen y samples including cockle, squid, sh imp,
quail mea , abbi mea , u key mea , esh cheese, spinach, Swiss cha d,
le uce and ca o , we e pu chased om di e en local e ail shops and
supe ma ke s in Vi o ia-Gas eiz, Spain, om May o No embe 2015. All
samples, 20 o each ype o ood, we e kep in coole s, anspo ed o he
labo a o y and p ocessed wi hin 2 h o pu chase.
Ten g ams o each sample we e homogenized in o 90 mL (1:10 w /
ol) o A cobac e -CAT b o h (Oxoid) as p e iously desc ibed (Nie a-
Eche a ia e al., 2013), and hen incuba ed ae obically a 30 ◦C o 48
h. A e en ichmen , 0.2 mL o each b o h we e inocula ed by passi e
il a ion wi h 0.45-
μ
m ni ocellulose memb ane il e s (Millipo e) on o
blood aga pla es (Columbia aga supplemen ed wi h 5% sheep blood,
Oxoid) and incuba ed unde he a o emen ioned condi ions o 48–72 h.
A e incuba ion, ou o six suspec A cobac e colonies (small, smoo h
and anslucen o whi ish) we e picked om each pla e and subcul u ed
on o blood aga pla es a leas h ee imes. Upon mic oscopic exami-
na ion, hose isola es p esen ing a cu ed o spi al shape and cha ac-
e is ic mo ili y we e subjec ed o PCR iden i ica ion.
2.2. A cobac e species iden i ica ion
2.2.1. Genomic DNA isola ion
DNA was isola ed using P epMan™ Ul a eagen (Applied Bio-
sys ems) acco ding o he manu ac u e 's speci ica ions. The concen-
a ion was de e mined spec opho ome ically (NanoD op, The mo
Fishe Scien i ic), adjus ed o 20 ng/
μ
L and s o ed a −20 ◦C.
2.2.2. PCR and m-PCR
Suspicious colonies we e iden i ied by he genus-speci ic PCR
desc ibed by Bas yns e al. (1995). To a oid he inclusion o clones in he
collec ion, all A cobac e isola es eco e ed om he same ood sample
we e geno yped using he en e obac e ial epe i i e in e genic
consensus PCR (ERIC-PCR) p o ocol as p e iously desc ibed o A co-
bac e (Hou e al., 2002). Pa e ns wi h a leas one o mo e di e en
bands we e conside ed as di e en geno ypes.
Species iden i ica ion o he isola es was ca ied ou by wo p e i-
ously desc ibed me hods. The m-PCR p oposed by Hou e al. (2000) ha
a ge s A. bu zle i, A. c yae ophilus and A. ski owii was applied i s ,
ollowed by he m-PCR p oposed by Douidah e al. (2010) ha simul-
aneously iden i ies A. bu zle i, A. c yae ophilus, A. ski owii, A. ciba ius
and A. he eius. DNA om A. bu zle i RM4018, A. c yae ophilus CCUG
17801T, A. ski owii CCUG 30483, A. ciba ius CECT 7203 and A. he eius
CCUG 56002, oge he wi h deionized wa e , we e used as posi i e and
nega i e con ols, espec i ely.
2.2.3. Phylogene ic and phylogenomic analysis
In o de o de e mine he axonomic posi ion o one isola e which
could no be iden i ied o he species le el by he a o emen ioned m-PCR
me hods, bo h, phylogene ic and phylogenomic analyses, we e held
(Alonso e al., 2020).
2.3. Gene ic cha ac e iza ion
The 83 s ains eco e ed in his s udy (53 A. bu zle i, 24
A. c yae ophilus, h ee A. he eius, wo A. ski owii and one A. i o iensis)
we e gene ically cha ac e ized by Mul ilocus Sequence Typing (MLST)
and i ulence-associa ed gene de ec ion.
2.3.1. MLST and minimum spanning ees
MLST was ca ied ou acco ding o he me hod o Mille e al. (2009)
wi h mino modi ica ions. The glyA gene om A. c yae ophilus was
ampli ied wi h di e en annealing empe a u es (55–59 ◦C) and he glnA
gene om A. he eius was ampli ied by using he p ime s glnACR1 and
glnATHF (5′-AAATGGAATGCCTTTTGATGGAG-3′). Allele numbe s and
sequence ypes (STs) we e assigned using he PubMLST da abase (Jolley
e al., 2018) a h ps://pubmls .o g/o ganisms/a cobac e -spp. New
alleles and STs we e submi ed o he da abase cu a o o be assigned
new allele o ST numbe s. In o de o de ec possible ecombina ion
e en s, all he a ailable conca ena ed MLST sequences we e down-
loaded om he A cobac e PubMLST da abase on Janua y 2017 (n =
648) and subsequen ly analysed using i e me hods (RDP, Genecon ,
MaxChi, Chimae a and 3Seq) implemen ed in he RDP3 so wa e
package (Ma in e al., 2010) using de aul pa ame e s. In o de o
isualize he ela ionships be ween he STs and hei dis ibu ion among
di e en ood p oduc s, minimum spanning ees (MST) we e c ea ed by
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
3
he goeBURST algo i hm using he PHYLOViZ 2.0a so wa e (Nasci-
men o e al., 2017). MSTs we e cons uc ed based on he dis ances be-
ween he allelic p o iles o all he A cobac e isola es wi h an assigned
ST a ailable a he PubMLST da abase on Janua y 2021 (n =997),
including hose iden i ied in he p esen s udy.
2.3.2. De ec ion o i ulence genes
The p esence o en pu a i e i ulence genes (cadF, Cj1349, ciaB,
m iN, pldA, lyA, i gA, hecA, hecB and i oE) was de e mined using he
p ime pai s designed by Douidah e al. (2012) and Ka adas e al.
(2013), ollowing he p o ocol desc ibed by Gi bau e al. (2015). DNA
om A. bu zle i RM4018 was used as posi i e con ol and deionized
wa e as nega i e one. Addi ionally, DNAs om A. c yae ophilus CCUG
17801 and Ac-L7 (Gi bau e al., 2015), A. ski owii CCUG 30483 and
A. he eius CCUG 56902 we e also included as con ols whene e any o
hese species we e subjec ed o he a o emen ioned PCRs.
2.4. Bio ilm p oduc ion
The abili y o o m bio ilms o he s ains eco e ed in his su ey
was in es iga ed by bio ilm-associa ed gene de ec ion and in i o
pheno ypic assays.
2.4.1. Bio ilm-associa ed gene de ec ion
Genes o be de ec ed ( laA, laB, liS, luxS, p a, waaF and spoT) we e
selec ed based on hei associa ion wi h adhe ence o abio ic su aces in
o he campylobac e ia. Based on comp ehensi e analyses and align-
men s o he published genome sequences o A. bu zle i RM4018
T
,
A. c yae ophilus ATCC 43158
T
, A. ski owii CCUG 10374
T
, A. he eius
LMG 24486
T
and A. i o iensis F199
T
(GenBank, accession numbe s
GCA_000014025, NZ_CP032823, NZ_VZOH00000000, NZ_CP035926.1
and PDKB00000000, espec i ely), 35 PCR p ime s we e designed using
Clone Manage 9 P o essional Edi ion so wa e (Sci Ed So wa e LLC).
Once designed, he p ime s we e es ed in silico by blas ing hem agains
comple ed A cobac e genome sequences a ailable in he GenBank
da abase. The p ime sequences and he expec ed amplicon sizes
depending on he species a e lis ed in Table S1.
All PCRs we e ca ied ou in inal olumes o 50
μ
L con aining 100 ng
o DNA as empla e, 1.25 U o D eamTaq DNA Polyme ase (The mo
Scien i ic), 0.2 mM o each dNTP, 1×bu e and 0.5
μ
M o each p ime
se . An ini ial dena u a ion s ep a 94 ◦C o 3 min was ollowed by 30
cycles o dena u a ion a 94 ◦C o 45 s; p ime annealing a di e en
empe a u es anging om 50 ◦C o 56 ◦C o 45 s; and elonga ion a
72 ◦C o 1 min. A inal elonga ion s ep a 72 ◦C o 3 min was pe o med.
2.4.2. Mo ili y assay
In o de o es he mo ili y o hose s ains o which lagellin gene
de ec ion ailed, indi idual colonies we e spo ed on o 0.4% hio-
glycola e pla es (Scha lau). The pla es we e incuba ed a 30 ◦C, and he
g ow h and expansion o colonies was examined a e 16–24 h.
2.4.3. S a ic bio ilm assays
The bio ilm o ma ion abili y was i s assessed a 30 ◦C unde ae -
obic condi ions using polys y ene mic o i e pla es, as desc ibed p e i-
ously (Gi bau e al., 2017). Bo osilica e glass ubes and s ainless s eel
coupons we e also used o u he es ing o he abili y o hose s ains
showing adhe ence o polys y ene. Fo each assay, e e y isola e was
examined in h ee eplica es and he expe imen s we e pe o med a
leas on h ee sepa a e occasions.
Those bio ilms o med on polys y ene and bo osilica e we e
exp essed by he bio ilm o ma ion index (BFI) acco ding o Niu and
Gilbe (2004), and subsequen ly ca ego ized as s ong, mode a e, weak
o none bio ilm o ma ion acco ding o Na es e al. (2008). The bio ilms
o med on s ainless s eel we e e alua ed by pla e coun me hod on
Muelle -Hin on aga (Oxoid) a e gen ly washing he coupons wi h
s e ile dis illed wa e (Gi bau e al., 2017).
2.5. S a is ical analysis
Da a we e analysed wi h he SPSS 26 s a is ical package p og am
(SPSS Inc., Chicago, IL, USA). The Chi-squa e and Fishe 's exac es s
we e pe o med in o de o compa e he dis ibu ion o he isola es and
species among samples and o assess possible associa ions be ween
a iables. The isola es de i ed om spinach (n =1), le uce (n =1),
cheese (n =1) and abbi (n =3), along wi h hose iden i ied as
A. he eius (n =3), A. ski owii (n =2) and A. i o iensis (n =1), we e
excluded om he analyses based on hei low ep esen a ion.
Once es ed he no mali y o he nume ical a iable “bio ilm o -
ma ion” using he Kolmogo o -Smi no me hod, he K uskal-Wallis es
was used o compa e he alues ob ained o bio ilm o ma ion abili y on
each su ace among he isola es. S uden - es was used o compa e he
alues ob ained o he o med bio ilms on polys y ene e sus bo osili-
ca e o each s ain. Resul s we e conside ed signi ican a p alues o
<0.05.
3. Resul s
3.1. Occu ence o A cobac e spp. in ood samples
The occu ence o A cobac e spp. in he 220 ood samples analysed
in his s udy is summa ized in Table 1. O e all, hey we e de ec ed in all
he es ed ypes o p oduc s excep cha d, wi h a g oss occu ence o
22.3% (49 ou o 220 samples). The a cobac e s we e mos ly de ec ed in
sea ood p oduc s, which showed a s a is ically signi ican (p <0.001)
con amina ion le el o 43.3%. Foods o e es ial animal and ege able
o igin showed lowe con amina ion le els, 21.3% and 7.5%, espec-
i ely. Speci ically, hose p oduc s om which A cobac e spp. we e
mainly eco e ed we e squid (60.0%) and u key mea (40.0%). The
eco e y om squid was s a is ically signi ican (p <0.001).
Among he 266 isola es iden i ied as A cobac e spp. by he genus-
speci ic PCR (Bas yns e al., 1995), 83 we e selec ed o u he iden i-
ica ion o he species le el based on he ERIC-PCR esul s (Hou e al.,
2002). Ou o hem, 53 isola es we e iden i ied as A. bu zle i, 24 as
A. c yae ophilus and wo as A. ski owii by bo h m-PCR (Douidah e al.,
2010; Hou e al., 2000); and h ee as A. he eius by one o hem
(Douidah e al., 2010). The iden i ica ion o he emaining A cobac e
isola e equi ed phylogene ic and phylogenomic analyses, which iden-
i ied i as Alia cobac e (now A cobac e ) i o iensis (Alonso e al., 2020).
The dis ibu ion o he eco e ed species acco ding o he ype o ood
di e ed signi ican ly (p =0.009) and i was as ollows: A. bu zle i was
isola ed om all ype o p oduc s excep cha d; A. c yae ophilus om
sea ood and mea p oduc s; and A. ski owii and A. he eius only om
sea ood. A. i o iensis was eco e ed om a ca o sample ha was
simul aneously con amina ed wi h A. bu zle i. Th ee cockle samples
we e doubly con amina ed wi h A. bu zle i and A. c yae ophilus; a sh imp
sample wi h A. bu zle i and A. he eius; and a squid sample wi h
A. c yae ophilus and A. he eius. A. bu zle i was he mos commonly iso-
la ed species om all o he ood p oduc s excep cockle and sh imp,
whe e A. c yae ophilus p e ailed. App oxima ely he hal (50.9%) o he
A. bu zle i isola es we e eco e ed om e es ial animal p oduc s and
he majo i y o he A. c yae ophilus om sea ood (87.5%). Indeed, Fishe
exac es es ablished an associa ion be ween sea ood p oduc s and
A. c yae ophilus (p <0.001).
3.2. Geno yping by MLST
Eigh y wo isola es ou o he 83 analysed we e success ully yped by
MLST and nume ous alleles and STs we e iden i ied (Table S2). The
allele sequences o he A. i o iensis isola e could no be de e mined. A
o al o 351 alleles we e iden i ied ac oss all se en loci, anging om 42
alleles a gl A o 62 a glyA. O e all, 172 ou o he 351 (49%) alleles
we e p e iously un epo ed, anging hei equency om 33.3% (gl A)
o 64.5% (glyA). Six y eigh STs (41 o A. bu zle i, 22 o A. c yae ophilus,
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
4
Table 1
A cobac e species eco e ed om he 220 ood samples pu chased a he e ail le el in Vi o ia-Gas eiz be ween May and No embe 2015.
No. (%) o samples posi i e o : No. o geno ypes iden i ied by ERIC-PCR o :
Type o sample No. A cobac e spp. A. bu zle i A. c yae ophilus A. ski owii A. he eius A. i o iensis A. bu zle i A. c yae ophilus A. ski owii A. he eius A. i o iensis
Sea ood
Cockle 20 7 (35.0)
a
4 (57.1) 6 (85.7) – – – 6 9 – – –
Squid 20 12 (60.0)
b,
* 7 (58.3) 3 (25) 1 (8.3) 2 (16.7) – 9 4 1 2 –
Sh imp 20 7 (35.0)
c
3 (42.9) 3 (42.9) 1 (14.3) 1 (14.3) – 3 8 1 1 –
Sub o al 60 26 (43.3)* 14 (53,8) 12 (46.2) 2 (7.7) 1 (3.8) – 18 21* 2 3
Te es ial animal p oduc s
Tu key mea 20 8 (40.0) 7 (87.5) 1 (12.5) – – – 15 1 – – –
Rabbi mea 20 3 (15.0) 2 (66.7) 1 (33.3) – – – 2 1 – – –
Quail mea 20 5 (25.0)
d
5 (100) 1 (20) – – – 9 1 – – –
F esh cheese 20 1 (5.0) 1 (100) – – – – 1 – – – –
Sub o al 80 17 (21.3) 15 (88,2)* 3 (17,6) – – – 27 3 – – –
Vege ables
Ca o 20 4 (20.0)
e
4 (100) – – – 1 (20) 6 – – – 1
Spinach 20 1 (5.0) 1 (100) – – – – 1 – – – –
Le uce 20 1 (5.0) 1 (100) – – – – 1 – – – –
Cha d 20 – – – – – – – – – – –
Sub o al 80 6 (7.5) 6 (100)* – – – 1 (16.7) 8 – – – 1
To al 220 49 (22.3) 35 (71.4) 15 (30,6) 2 (4.1) 1 (2.0) 1 (2.0) 53 24 2 3 1
a
A. bu zle i and A. c yae ophilus we e simul aneously de ec ed in h ee cockle samples.
b
A. c yae ophilus and A. he eius we e simul aneously de ec ed in a squid sample.
c
A. bu zle i and A. he eius we e simul aneously de ec ed in a sh imp sample.
d
A. bu zle i and A. c yae ophilus we e simul aneously de ec ed in a quail mea sample.
e
A. bu zle i and A. i o iensis we e simul aneously de ec ed in a ca o sample.
*
S a is ically signi ican di e ences (p <0.05) be ween esul s, based on Fishe 's exac es .
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
5
wo o A. ski owii and h ee o A. he eius) we e iden i ied among he 82
geno yped isola es and hei occu ence in sea ood, oods o e es ial
animal o igin and ege ables was 33, 28 and 7, espec i ely (Table 2).
Mos o he STs (89.7%) we e p e iously un epo ed and esul ed om
new allele's sequences (n =50) o new combina ions o known alleles (n
=11). Fi y h ee ou o he 61 no el STs (86.9%) we e ep esen ed by a
single s ain; i e STs ( h ee o A. bu zle i and wo o A. c yae ophilus) by
wo; ST-512 by h ee (A. bu zle i); ST-513 by ou (A. bu zle i) and ST-
517 by i e (A. bu zle i). The analysis o he ela edness and dis ibu-
ion o he iden i ied STs and he o he A cobac e spp. STs a ailable in
de PubMLST da abase (Fig. 1) e ealed ha , o e all, he STs clus e ed by
species. The A. bu zle i geno ypes iden i ied in his s udy dis ibu ed
among all he species-speci ic clades excep one mainly popula ed by
isola es o human o igin, and hey mos ly g ouped in a clus e p inci-
pally composed by isola es de i ed om ood p oduc s. The
A. c yae ophilus geno ypes iden i ied we e mo e closely ela ed o each
o he and hey all excep wo g ouped in he main species-speci ic clade.
The h ee A. he eius and wo A. ski owii iden i ied geno ypes also
clus e ed close o each o he . No appa en hos -associa ed STs we e
iden i ied. Ne e heless, h ee no el STs om A. bu zle i (ST-512, ST-
513 and ST-517, ep esen ed by wo, h ee and i e isola es, espec-
i ely) and wo no el STs om A. c yae ophilus (ST-521 and ST-596,
ep esen ed by wo isola es each) only included isola es om sea ood
p oduc s (Table 2 and Fig. 1). Mo eo e , he Fishe exac es assessed an
associa ion (p <0,001) be ween sea ood de i ed isola es o hese s udy
and he abo e men ioned STs. Among he p e iously iden i ied STs, all
excep ST-16 we e sha ed wi h isola es de i ed om di e en ood
p oduc s (Fig. 1). The ecombinan analysis (RDP3) held wi h he 648
a ailable conca ena ed MLST sequences (3341 posi ions) de ec ed a
po en ial single ecombina ion e en on ST-599, a he egion be ween
nucleo ide posi ions 1968 and 2081, wi hin he sequence o glyA allele.
ST-609 (glyA-613, A. c yae ophilus) and ST-250 (glyA-263, A. ski owii)
we e iden i ied as he po en ial pa en s (majo and mino , espec i ely).
This e en was s a is ically suppo ed by all he implemen ed me hods:
RDP (1.686 ×10
−06
), Genecon (1.233 ×10
−10
), MaxChi (1.602 ×
10
−04
), Chimae a (2.141 ×10
−04
), and 3Seq (5.106 ×10
−11
).
3.2.1. Pu a i e i ulence genes
The p esence and dis ibu ion o he en pu a i e i ulence genes
in es iga ed is shown in Table 3. The p o iles o i ulence genes iden-
i ied in he di e en species a e shown in Table S2. O e all, all he
genes we e de ec ed among he isola es analysed and, based on he
i ulence gene con en o each isola e, 28 di e en p o iles we e iden-
i ied. None o he isola es ha bou ed all en i ulence genes, and only
one isola e wi h appa en ly no i ulence gene con en was iden i ied.
Among he a cobac e s isola ed ciaB (97.6%) and m iN (94%) we e he
Table 2
Dis ibu ion o he STs iden i ied among A cobac e species.
Sou ce o
isola ion
A. bu zle i A. c yae ophilus A. ski owii A. he eius A cobac e spp.
No. o
s ains
No.
o
ST
Iden i ied ST No. o
s ains
No.
o
ST
Iden i ied ST No. o
s ains
No.
o
ST
Iden i ied
ST
No. o
s ains
No.
o
ST
Iden i ied
ST
No. o
s ains
No.
o
ST
Sea ood 18 9 21 19 2 2 3 3 44 33
Cockle 6 5 475, 513, 517
a
(n =2), 519,
530,
9 9 415, 534,
535, 596
a
,
597, 598,
599, 606, 607
Squid 9 6 18, 172, 512
a
(n
=2), 513
a
(n =
2), 517
a
(n =2),
518
4 4 521
a
, 605,
608, 609
1 1 532 2 2 623, 624
Sh imp 3 3 512
a
, 513
a
, 517
a
8 8 521
a
, 522,
596
a
, 601,
602, 603,
610, 611
1 1 622 1 1 625
Te es ial
animal
p oduc s
27 25 3 3 30 28
Tu key
mea
15 14 16, 452, 514,
515, 516, 520
(n =2), 523,
524, 525, 527,
528, 536, 594,
595
1 1 604
Rabbi
mea
2 2 406, 586 1 1 626
Quail
mea
9 8 3, 506, 507, 508,
509, 510 (n =
2), 511, 587
1 1 600
F esh
cheese
1 1 533
Vege ables 8 7 8 7
Ca o 6 5 526 (n =2),
531, 588, 589,
590
Spinach 1 1 593
Le uce 1 1 529
To al ood
p oduc s
53 41 24 22 2 2 3 3 82 68
Bold ace en ies ep esen STs de ec ed in wo o mo e isola es.
a
Fishe 's exac es based s a is ically signi ican associa ion (p <0.001) be ween STs and sea ood de i ed A. bu zle i and A. c yae ophilus.
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
6
Fig. 1. Minimum spanning ees based on he MLST p o iles o he isola es geno yped in his s udy and all o he A cobac e isola es om di e se sou ces a ailable in
he PubMLST, showing he ela edness and dis ibu ion o he STs among species (A) and di e se sou ces (B). Each ci cle ep esen s an ST ype and he size o he
ci cle co ela es o he numbe o isola es. The numbe nex o he nodes indica es STs in he p esen s udy.
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
7
mos p e alen genes, and i gA (8.4%) he leas one. The p e alence o
he o he genes a ied om 75.9% o cadF o 18.1% o i oE and hecA.
The s a is ical analyses e ealed ha cadF, Cj1349, pldA and lyA genes
we e signi ican ly (p <0.001) mo e p e alen among he s ains de i ed
om e es ial animal p oduc s (100%, 90%, 90% and 93.3%, espec-
i ely) and ege ables (88.9% each gene) han in hose de i ed om
sea ood (56.8%, 40.9%, 40.9% and 47.7%, espec i ely). Speci ically,
cadF was common o all quail and u key de i ed isola es and
signi ican ly mo e common in ca o de i ed ones (85.7%); and Cj1349,
pldA and lyA we e signi ican ly mo e common in quail (90.0% each),
u key (93.8% Cj1349; 93.8% pldA; 100% lyA) and ca o (90.0%
Cj1349 and pldA; 93.3% lyA) de i ed isola es. The genes Cj1349 and
pldA we e also signi ican ly p e alen in hose isola es ob ained om
squids (56.3% each).
Signi ican di e ences (p <0.05) o he gene dis ibu ion we e also
obse ed when he species we e conside ed. All he 53 A. bu zle i isola es
Table 3
P esence and dis ibu ion o pu a i e i ulence genes in A cobac e spp. analysed in his s udy.
Species/sou ce No. o s ains No. (%) o s ains gene a ing speci ic gene amplicon
cadF ciaB Cj1349 hecA hecB i gA m iN pldA lyA i oE
By species
A. bu zle i 53 53 (100)* 53 (100) 53 (100)* 9 (17) 16 (30.2)* 6 (11.3) 52 (98.1) 53 (100)* 53 (100)* 12 (22.6)
A. c yae ophilus 24 8 (33.3) 22 (91.7) 0 6 (25) 0 1 (4.2) 22 (91.7) 0 3 (12.5) 3 (12.5)
A. ski owii 2 0 2 (100) 0 0 0 0 2 (100) 0 1 (50) 0
A. he eius 3 2 (66.7) 3 (100) 0 0 0 0 1 (33.3) 0 0 0
A. i o iensis 1 0 1 (100) 0 0 0 0 1 (100) 0 0 0
A cobac e spp. 83 63 (75.9) 81 (97.6) 53 (63.9) 15 (18.1) 16 (19.3) 7 (8.4) 78 (94) 53 (63.9) 57 (68.7) 15 (18.1)
By sou ce o isola ion
Sea ood
Cockle 15 11 (73.3) 13 (86.7) 6 (40.0) 3 (20.0) 3 (20.0) 0 14 (93.3) 6 (40.0) 7 (46.7) 2 (13.3)
Squid 16 10 (62.5) 16 (100) 9 (56.3)* 5 (31.3) 5 (31.3) 1 (6.3) 14 (87.5) 9 (56.3)* 10 (62.5) 3 (18.8)
Sh imp 13 4 (56.8) 13 (100) 3 (23.1) 3 (23.1) 1 (7.7) 0 12 (92.3) 3 (23.1) 4 (30.8) 0
Sub o al 44 25 (56.8) 42 (95.5) 18 (40.9) 11 (25) 9 (20.5) 1 (2.3) 39 (88.6) 18 (40.9) 21 (47.7) 5 (11.4)
Te es ial animal p oduc s
Rabbi mea 3 3 (100) 3 (100) 2 (66.7) 2 (66.7) 1 (33.3) 1 (33.3) 3 (100) 2 (66.7) 2 (66.7) 2 (66.7)
Quail mea 10 10 (100)* 10 (100) 9 (90.0)* 1 (10.0) 0 3 (30.0) 10 (100) 9 (90.0)* 9 (90.0)* 1 (10.0)
Tu key mea 16 16 (100)* 16 (100) 15 (93.8)* 1 (6.3) 4 (25.0) 2 (12.5) 16 (100) 15 (93.8)* 16 (100)* 3 (18.8)
F esh cheese 1 1 (100) 1 (100) 1 (100) 0 1 (100) 0 1 (100) 1 (100) 1 (100) 1 (100)
Sub o al 30 30 (100)* 30 (100) 27 (90.0)* 4 (13.3) 6 (20) 6 (20.0) 30 (100) 27 (90)* 28 (93.3)* 7 (23.3)
Vege ables
Spinach 1 1 (100) 1 (100) 1 (100) 0 0 0 1 (100) 1 (100) 1 (100) 0
Le uce 1 1 (100) 1 (100) 1 (100) 0 0 0 1 (100) 1 (100) 1 (100) 1 (100)
Ca o 7 6 (85.7)* 7 (100) 6 (85.7)* 0 1 (14.3) 0 6 (85.7) 6 (85.7)* 6 (85.7)* 2 (28.6)
Sub o al 9 8 (88.9)* 9 (100) 8 (88.9)* 0 1 (11.1) 0 8 (88.9) 8 (88.9)* 8 (88.9)* 3 (33.3)
To al all ood p oduc s 83 63 (75.9) 81 (97.6) 53 (63.9) 15 (18.1) 16 (19.3) 7 (8.4) 78 (94) 53 (63.9) 57 (68.7) 15 (18.1)
*
S a is ically signi ican di e ences (p <0.05) be ween esul s, based on Fishe 's exac es .
Table 4
P esence and dis ibu ion o he bio ilm-associa ed genes in A cobac e spp. analysed in his s udy.
Species/sou ce No. o s ains No. (%) o s ains gene a ing speci ic gene amplicon
liS luxS p a waa Spo laA laB
By species
A. bu zle i 53 53 (100) 53 (100) 53 (100) 53 (100) 53 (100) 36 (67.9) 36 (67.9)
A. c yae ophilus 24 24 (100) 24 (100) 24 (100) 24 (100) 24 (100) 24 (100) 17 (58,3)
A. ski owii 2 2 (100) 2 (100) 2 (100) 2 (100) 2 (100) 2 (100) 2 (100)
A. he eius 3 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 0
A. i o iensis 1 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100)
A cobac e spp. 83 83 (100) 83 (100) 83 (100) 83 (100) 83 (100) 66 (79.5) 56 (67.5)
By sou ce o isola ion
Sea ood
Cockle 15 15 (100) 15 (100) 15 (100) 15 (100) 15 (100) 13 (86.7) 13 (86.7)
Squid 16 16 (100) 16 (100) 16 (100) 16 (100) 16 (100) 14 (87.5) 9 (56.3)
Sh imp 13 13 (100) 13 (100) 13 (100) 13 (100) 13 (100) 12 (92.3) 9 (69.2)
Sub o al 44 44 (100) 44 (100) 44 (100) 44 (100) 44 (100) 39 (88.6) 31 (70.5)
Te es ial animal p oduc s
Rabbi mea 3 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 3 (100) 2 (66.7)
Quail mea 10 10 (100) 10 (100) 10 (100) 10 (100) 10 (100) 7 (70) 6 (60)
Tu key mea 16 16 (100) 16 (100) 16 (100) 16 (100) 16 (100) 9 (56.3) 10 (62.5)
F esh cheese 1 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 0 0
Sub o al 30 30 (100) 30 (100) 30 (100) 30 (100) 30 (100) 19 (63.3) 18 (60)
Vege ables
Ca o 7 7 (100) 7 (100) 7 (100) 7 (100) 7 (100) 6 (85.7) 6 (85.7)
Spinach 1 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 0 0
Le uce 1 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100)
Sub o al 9 9 (100) 9 (100) 9 (100) 9 (100) 9 (100) 7 (7.8) 7 (7.8)
To al all ood p oduc s 83 83 (100) 83 (100) 83 (100) 83 (100) 83 (100) 66 (79.5) 56 (67.5)
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
8
we e posi i e o cadF, ciaB, Cj1349, pldA and lyA, while he de ec ion
a e o he o he genes anged om 11.3% o i gA o 98.1% o m iN.
The mos equen combina ion o genes in A. bu zle i was cadF, ciaB,
Cj1349, m iN, pldA and lyA, which was de ec ed in 47.2% o he iso-
la es. The gene con en o he A. c yae ophilus isola es was no ably lowe :
only i e ou o he 24 es ed s ains showed ou genes o mo e. The
mos p e alen genes in his species we e ciaB and m iN (91.7% each),
whose combina ion was no ed in 37.5% o he isola es. Cj1349, hecB and
pldA we e no de ec ed and he p e alence o he emaining genes was
a iable, anging om 4.2% (i gA) o 33.3% (cadF). Bo h A. ski owii
isola es we e posi i e o ciaB and m iN, being one o hem also posi i e
o lyA. All he h ee A. he eius isola es possessed ciaB, wo o hem cadF
and he hi d one also m iN. A cobac e i o iensis was posi i e jus o
ciaB and m iN.
3.3. Bio ilm p oduc ion
All he A cobac e isola es we e posi i e o liS, luxS, p a, waaF and
spoT genes, bu 17 ou o he 83 (20.5%) isola es esul ed nega i e o
laA and laB and en (12%) o laB (Table 4). Among hese, all we e
mo ile upon examina ion on hioglycola e so aga pla es (da a no
shown).
The bio ilm ac i i y could only be es ed wi h 81 o he 83 s ains
included in he s udy (we we e unable o eco e wo A. c yae ophilus
isola es, Ac-BER3 and Ac-CH1, om he s ain collec ion). The ini ially
measu ed adhe ence by mic o i e assay is shown in Table S3. Table 5
summa izes he dis ibu ion and ca ego iza ion o he es ed isola es
among ood p oduc s and su aces. O e all, 19 isola es (23.5%) we e
able o o m bio ilms on polys y ene su aces unde he expe imen al
condi ions. Among hem, eigh (42.1%) we e ca ego ized as weakly
adhe en , ano he eigh (42.1%) as mode a e, and h ee (15.8%) as
s ongly adhe en . Ne e heless, based on K uskal-Wallis, no signi i-
can ly highe adhesion abili y was iden i ied among hese adhe en
isola es. The p opo ion o adhe en isola es di e ed signi ican ly (p =
0.037) among species: 32.1% (17 isola es) in A. bu zle i, 9.1% (2 iso-
la es) in A. c yae ophilus and 0% in A. ski owii, A. he eius and
Table 5
Dis ibu ion and ca ego iza ion o he isola es based on hei adhesion abili y among ood sou ces and su aces.
Polys y ene Bo osilica e
No. () o es ed s ains No. (%) o adhe en s ains on: No. (%) o s ains ca ego ized as: No. (%) o adhe en s ains on polys y ene
ca ego ized as:
WA
b
MA
c
SA
d
NA
a
WA
b
MA
c
SA
d
By species
A. bu zle i (53) 17 (32.1)* 8 (47.1) 6 (35.3) 3 (17.6) 1 (5.9) 2 (11.8) – 14 (82.3)
A. c yae ophilus (22) 2 (9.1) – 2 (100) – – – 1 (50) 1 (50)
A. ski owii (2) – – – – – – – –
A. he eius (3) – – – – – – – –
A. i o iensis (1) – – – – – – – –
A cobac e spp. (81) 19 (23.5) 8 (42.1) 8 (42.1) 3 (15.8) 1 (5.3) 2 (10.5) 1 (5.3) 15 (78.9)
By sou ce o isola ion A cobac e Ab
e
Ac
As
g
A
h
A
i
Sea ood (42)
Cockle (6 Ab, 8 Ac) 4 (9.5) 4
(66.7)*
– – – – 1 Ab
(25)
2 Ab
(50)
1 Ab
(25)
– 1 Ab
(25)
– 3 Ab (75)
Squid (9 Ab, 3 Ac, 1 As,
2 A )
5 (11.9) 5
(55.6)*
– – – – 3 Ab
(60)
2 Ab
(40)
– 1 Ab
(20)
– – 4 Ab (80)
Sh imp (3 Ab, 8 Ac, 1
As, 1 A )
3 (7.1) 1 (33.3) 2 (25) – – – 1 Ab
(100)
2 Ac
(100)
– – – 1 Ac
(50)
1 Ab (100), 1
Ac (50)
Sub o al (18 Ab, 19 Ac,
2 As, 3 A )
12 (28,6) 10
(55.6)*
2
(10.5)
– – – 5 (41.7) 6 (50) 1 (8.3) 1 (8.3) 1 (8.3) 1 (8.3) 9 (75)
Te es ial animal p oduc s (30)
Rabbi mea (2 Ab, 1 Ac) 1 (3.3) 1 (50) – – – – 1 Ab
(100)
– – – – – 1 Ab (100)
Quail mea (9 Ab, 1 Ac) 3 (10) 3 (33.3) – – – – 2 Ab
(66.7)
– 1 Ab
(33.3)
– – – 3 Ab (100)
Tu key mea (15 Ab, 1
Ac)
1 (3.3) 1 (6.7) – – – – – – 1 Ab
(100)
– 1 Ab
(100)
– –
F esh cheese (1 Ab) – – – – – – – – – – – – –
Sub o al 5 (16,7) 5 (18.5) – – – – 3 (60) – 2 (40) – 1 (20) – 4 (80)
Vege ables (8)
Spinach (1) 1 (12.5) 1 (100) – – – – – 1 Ab
(100)
– – – – 1 Ab (100)
Le uce (1) – – – – – – – – – – – –
Ca o (6 Ab, 1 A ) 1 (12.5) 1 (16.7) – – – – – 1 Ab
(100)
– – – 1 Ab (100)
Sub o al 2 (25) 2 (25) – – – – – 2 (100) – – – – 2 (100)
To al all ood p oduc s (81) 19 (23.5) 17
(32.1)*
2 (9.1) – – – 8 (42.1) 8 (42.1) 3 (15.8) 1 (5.3) 2 (10.5) 1 (5.3) 15 (78.9)
a
NA, no adhe en .
b
WA, weakly adhe en .
c
MA, mode a ely adhe en .
d
SA, s ongly adhe en .
e
Ab, A. bu zle i.
Ac, A. c yae ophilus.
g
As, A. ski owii.
h
A , A. he eius.
i
A , A. i o iensis.
*
S a is ically signi ican di e ences (p <0.05) be ween esul s, based on Fishe 's exac es .
I. Ma inez-Malaxe xeba ia e al.
In e na ional Jou nal o Food Mic obiology 373 (2022) 109712
9
A. i o iensis. Rega ding he sou ce o isola ion, adhe en isola es we e
de ec ed in all ype o ood p oduc s excep le uce and esh cheese. The
dis ibu ion o he A. bu zle i adhe en isola es a ied signi ican ly (p =
0.03) among sou ces, and was as ollows: en adhe en isola es de i ed
om sea ood (58.8%), i e om oods o e es ial animal o igin
(29.4%) and wo om ege ables (11.8%). Speci ically, he numbe o
adhe en s ains among cockle and squid-de i ed isola es was signi i-
can ly highe (p =0.034). Bo h A. c yae ophilus adhe en isola es
de i ed om sea ood.
The in luence o he ma e ial on he adhesion abili y o he 19
adhe en s ains was also es ed, and he esul s a e shown in Table 6.
Eigh een isola es (94.7%) we e able o adhe e o bo osilica e su aces,
and all o hem o s ainless s eel. The adhesion abili y o he majo i y o
he s ains (84.2%) was highe on bo osilica e han on polys y ene,
anging he inc ease in BFI alues om app oxima ely double o mo e
han 14 imes highe . In ac , six weakly and se en mode a ely adhe en
isola es we e ca ego ized as s ongly adhe en when es ed on bo osili-
ca e. The inc eased bio ilm o ma ion capabili y on bo osilica e su ace
was signi ican o Ab-CH8 (p =0.02), Ab-CH11 (p =0.037) and Ac-G2
(p =0.044) acco ding o he S uden - es . Based on K uskal-Wallis, no
s ain was signi ican ly mo e adhe en han o he on bo osilica e.
Rega ding s ainless s eel, he numbe o iable cells ha adhe ed o he
coupons anged om 0.69 ±0.43 o 3.66 ±0.26 log CFU pe cm
2
, and
based on K uskal-Wallis, he adhesion abili y o Ab-CH11 was signi i-
can ly highe (p =0.002).
4. Discussion
Va ious membe s o he genus A cobac e a e ega ded as eme ging
ood and wa e bo ne pa hogens (Collado and Figue as, 2011; Ramees
e al., 2017); and hei dis ibu ion in oods has been widely s udied
wo ldwide (C uzado-B a o e al., 2020; Fe n´
andez e al., 2015;
G´
onzalez e al., 2017; Hsu and Lee, 2015; Kie si i e al., 2021; Laish am
e al., 2016; Mo ola e al., 2016a, 2016b, 2020, 2021; Uljano as e al.,
2021; Zhang e al., 2019). Howe e , hese s udies a e no so abundan in
Spain, being e en less common hose combining a ious p oduc s in he
same su ey. In his s udy, di e en ypes o sea ood, mea , ege ables
and esh cheese pu chased in local ma ke s in he ci y o Vi o ia-Gas eiz
we e examined o A cobac e spp., and he eco e ed isola es we e
gene ically cha ac e ized by MLST and i ulo yping. Addi ionally, he
bio ilm ac i i y o he isola es was s udied.
A cobac e spp. was isola ed om i ually all he es ed ood p od-
uc s wi h an o e all p e alence o 22.3%. The mos equen ly isola ed
species among he i e iden i ied was A. bu zle i (71.4%), bu he
en ichmen b o h employed o he p ocedu e may ha e a ou ed his
esul , as i is known o bene i he eco e y o his species o e o he s
(Le ican e al., 2016).
In line wi h a p e ious s udy ca ied ou in he same geog aphical
a ea (Nie a-Eche a ia e al., 2013), he mos highly con amina ed
p oduc s (p <0.001) we e hose coming om he sea (43.3%), especially
he squids (60%). This esul con i ms he cephalopods o be an
impo an ese oi o A cobac e spp. (Ra hla a h e al., 2017; Zhang
e al., 2019) and highligh s he impo ance o squids as a po en ial
sou ce o human in ec ion i consumed aw o poo ly cooked. I is known
ha shell ish such as bi al es cons i u e na u al ese oi s o a ious
ma ine A cobac e species (Collado and Figue as, 2011). The sea ood
de i ed isola es o his s udy we e iden i ied as A. bu zle i,
A. c yae ophilus, A. ski owii and A. he eius. The isola ion o h ee
A. he eius s ains om squid and sh imp samples was an in e es ing
inding ha , in acco dance wi h p e ious obse a ions (Le ican e al.,
2014; Zhang e al., 2019), con i ms ha A. he eius can also be isola ed
om o he sou ces apa om animal aeces and abo ions. No in line
wi h o he epo s (Le ican e al., 2014; Mo ej´
on e al., 2017; Mo ola
e al., 2016b; Nie a-Eche a ia e al., 2013; Ra hla a h e al., 2017;
Zhang e al., 2019), A. c yae ophilus p e ailed abo e A. bu zle i among
sea ood de i ed isola es.
In con as , A. bu zle i was he mos common species isola ed om
mea (90%) and ege able (88.9%) p oduc s, especially om poul y
and ca o s. The p edominance o hese species in mea p oduc s has
been equen ly epo ed (Collado and Figue as, 2011; Khodamo adi
and Abi i, 2020; Kim e al., 2019; Nie a-Eche a ia e al., 2013; Ohnishi
and Ha a-Kudo, 2021). In addi ion, he obse ed p e alence o a co-
bac e s in u key (40%) and abbi (15%) mea s is consis en wi h ha
epo ed by Collado e al. (2009) o he same p oduc s in he same
coun y (33.3% and 10%, espec i ely); ega dless o he di e ences
no ed in compa isons be ween ou esul s and hose ob ained
Table 6
Bio ilm o ma ion abili y o he adhe en A. bu zle i and A. c yae ophilus isola es on di e en abio ic su aces.
Isola e Polys y ene Bo osilica e S ainless s eel
BFI
a
Classi ica ion
b
BFI
a
Classi ica ion
b
log CFU/cm
2
Ab-BER1 0.57 ±0.12 Weak 0.69 ±0.42 Weak 0.69 ±0.43
Ab-BER4 1.05 ±0.15 Mode a e 1.86 ±1.06 S ong 1.92 ±0.16
Ab-BER6 0.96 ±0.05 Mode a e 3.29 ±2.21 S ong 2.12 ±0.3
Ab-BER7 2.48 ±1.16 S ong 10.56 ±8.75 S ong 1.92 ±0.23
Ab-CH8 0.46 ±0.04 Weak 3.67 ±0.17* S ong 1.79 ±0.24
Ab-CH9 0.74 ±0.22 Mode a e 1.94 ±0.25 S ong 1.59 ±0.03
Ab-CH10 0.41 ±0.34 Weak 1.95 ±1.1 S ong 3.28 ±0.23
Ab-CH11 0.76 ±0.13 Mode a e 2.55 ±0.48* S ong 3.66 ±0.26
•
Ab-CH12 0.41 ±0.22 Weak 0.04 ±0.503 None 2.29 ±0.2
Ab-CZ3 0.53 ±0.26 Weak 2.74 ±3.04 S ong 1.11 ±0.03
Ab-CZ5 0.62 ±0.62 Weak 1.96 ±1.46 S ong 1.31 ±0.34
Ab-CZ6 3.00 ±2.9 S ong 1.90 ±0.77 S ong 2.99 ±0.66
Ab-PV7 1.50 ±1.01 S ong 0.40 ±0.44 Weak 1.83 ±0.15
Ab-CN1 0.65 ±0.64 Weak 3.78 ±1.76 S ong 1.07 ±0.26
Ab-E1 0.74 ±0.39 Mode a e 1.82 ±0.83 S ong 2.84 ±0.42
Ab-G1 0.65 ±0.44 Weak 2.96 ±2.45 S ong 1.99 ±0.48
Ab-Z7 0.78 ±0.13 Mode a e 11.18 ±11.45 S ong 2.06 ±0.18
Ac-G2 0.73 ±0.13 Mode a e 5.11 ±1.35* S ong 3.15 ±2.66
Ac-G4 0.81 ±0.23 Mode a e 0.83 ±0.9 Mode a e 3 ±2.68
a
BFI, bio ilm o ma ion index. Values a e exp essed as means ±s anda d e o s.
b
Bio ilm o ma ion pa e n acco ding o Na es e al. (2008).
•
K uskal-Wallis based s a is ically signi ican (p =0.002) di e ences ob ained when compa ing he alues ob ained o bio ilm o ma ion abili y on s ainless s eel
o each isola e.
*
S uden –based s a is ically signi ican (p <0.05) di e ences ob ained when compa ing bio ilm o ma ion on polys y ene e sus bo osilica e. The highe BFI alue,
ep esen ing he mos sui able su ace o bio ilm o ma ion, is indica ed.
I. Ma inez-Malaxe xeba ia e al.
