Ci a ion: Salaza -Sánchez, A.;
Baz a ika, I.; Alonso, R.;
Fe nández-As o ga, A.;
Ma ínez-Balles e os, I.;
Ma inez-Malaxe xeba ia, I.
A cobac e bu zle i Bio ilms: Insigh s
in o he Genes Benea h Thei
Fo ma ion. Mic oo ganisms 2022,10,
1280. h ps://doi.o g/10.3390/
mic oo ganisms10071280
Academic Edi o s: Ilana
Kolodkin-Gal and Yun Chen
Recei ed: 15 May 2022
Accep ed: 22 June 2022
Published: 23 June 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
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ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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mic oo ganisms
A icle
A cobac e bu zle i Bio ilms: Insigh s in o he Genes Benea h
Thei Fo ma ion
Ad ián Salaza -Sánchez 1, I saso Baz a ika 1, Rod igo Alonso 1,2, Au o a Fe nández-As o ga 1,
Ila gi Ma ínez-Balles e os 1,2 and I a i Ma inez-Malaxe xeba ia 1,2,*
1
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, Spain;
[email p o ec ed] (A.S.-S.); [email p o ec ed] (I.B.); [email p o ec ed] (R.A.);
[email p o ec ed] (A.F.-A.); [email p o ec ed] (I.M.-B.)
2Bioa aba, Mic obiology, In ec ious Disease, An imic obial Agen s, and Gene The apy,
01006 Vi o ia-Gas eiz, Spain
*Co espondence: [email p o ec ed]; Tel.: +34-945-01-34-71
Abs ac :
A cobac e bu zle i, he mos p e alen species o he genus, has he demons a ed abili y o
adhe e o a ious su aces h ough bio ilm p oduc ion. The bio ilm o ma ion capabili y has been
ela ed o he exp ession o ce ain genes, which ha e no been cha ac e ized in A. bu zle i. In o de
o inc ease he knowledge o his oodbo ne pa hogen, he aim o his s udy was o assess he ole
o six bio ilm-associa ed genes in campylobac e ia ( laA, laB, liS,luxS,p a and spoT) in he bio ilm
o ma ion abili y o A. bu zle i. Knockou mu an s we e cons uc ed om di e en oodbo ne isola es,
and s a ic bio ilm assays we e conduc ed on polys y ene (PS), ein o ced glass and s ainless s eel.
Addi ionally, mo ili y and Congo ed binding assays we e pe o med. In gene al, mu an s in laAB,
liS and luxS showed a dec ease in he bio ilm p oduc ion i espec i e o he su ace; mu an s in spoT
showed an inc ease on s ainless s eel, and mu an s in p a and spoT showed a dec ease on ein o ced
glass bu an inc ease on PS. Ou wo k sheds ligh on he bio ilm- ela ed pa hogenesis o A. bu zle i,
al hough u u e s udies a e necessa y o achie e a sa is ac o y objec i e.
Keywo ds: A cobac e bu zle i; bio ilm; knockou mu an s; Congo ed assay; s a ic bio ilm assays
1. In oduc ion
A cobac e bu zle i is a G am-nega i e bac e ium wi h a wide en i onmen al dis ibu-
ion, classi ied as a oodbo ne pa hogen [
1
] due o i s associa ion wi h human gas oin-
es inal disease. A. bu zle i is he mos p e alen among he species o he genus and is
equen ly isola ed om wild and a m animals’ exc emen s and in es inal egions (boa ,
os ich, Eu asian colla ed do e and accoon), a m animals’ mea (chicken, po k, bee ,
u key, lamb, sheep, abbi and quail mea ), sea ood p oduc s (clam, mussel, cockle, squid
and sh imp), dai y p oduc s ( aw cow milk and esh cheese), ege ables (ca o , spinach,
le uce, cha d, pa sley, a ugula and adish), en i onmen al wa e and human s ool [2–13].
The ansmission o he species o he genus A cobac e h ough he ood chain seems
o be a o ed by hei abili y o o m bio ilms [
14
–
16
]. Many ood- ela ed pa hogens
such as Ae omonas spp., Salmonella Typhimu ium, S aphylococcus au eus,Lis e ia mono-
cy ogenes,L. i ano ii,Esche ichia coli,Bacillus ce eus,C onobac e sakazakii,C. muy jensii,
A. bu zle i
,A. c yae ophilus,Pseudomonas ae uginosa,Klebsiella pneumoniae,Campylobac e je-
juni and C. coli can p oduce hese esis ance s uc u es con o med by cells and ex acellula
compounds [17–25]
. The bio ilm o ma ion abili y o A cobac e species on di e en su -
aces has also been documen ed [
5
,
25
], and i has become appa en ha he e is a no o ious
a iabili y o he adhe ence le el among s ains o he same species. Due o he di icul y o
e adica ing bio ilms and he apidi y wi h which A. bu zle i de elops hem, p e en ing hei
o ma ion is i al o con ol he sp ead o his oodbo ne pa hogen [
25
]. Mo eo e , some
Mic oo ganisms 2022,10, 1280. h ps://doi.o g/10.3390/mic oo ganisms10071280 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2022,10, 1280 2 o 16
au ho s e e ed ha hese s uc u es could also con ibu e o he inc ease in an imic obial
esis ance o A. bu zle i since bio ilm g ow h a o s esis ance gene ansmission [24].
The bio ilm o ma ion p ocess in eg a es di e en s eps [
26
]. When plank onic bac e ia
ind a s ess ul si ua ion (i.e., ca bohyd a e, p o ein, a y acid o o he nu ien de ici s;
an ibio ic exposu e; and un a o able empe a u e/a mosphe e condi ions [
27
–
29
]), cells
s a o adhe e in a smoo h and e e sible manne o an a ailable su ace and o each
o he . Then, he p oduc ion o exopolysaccha ide (EPS) by he loosely adhe ed bac e ia
and/o he exp ession o speci ic adhesins loca ed on pili and imb iae leads o hei
i e e sible a achmen . A his poin , sessile bac e ia begin o o m mic ocolonies, and
he bio ilm ma u es as hey con inue g owing and p oducing EPS [
30
–
32
]. Finally, when
he bio ilm eaches a c i ical mass, cells de ach om he ou e mos lawye o g ow h, and
he dispe sion o plank onic cells om he bio ilm occu s. The composi ion o he bio ilm
o med by A cobac e s ill emains unknown, bu , in gene al, he bio ilm ma ix is a complex
s uc u e ha p esen s channels and po es h oughou nu ien s, oxygen and wa e low.
I is composed o app oxima ely 15% o cells and 85% o EPS; his one is almos en i ely
con o med by wa e and con ains in i s’ solid-phase mainly polysaccha ides, p o eins and
DNA [24,33].
Bio ilm o ma ion is dependen on many ex insic ac o s such as g ow h medium,
a mosphe e, empe a u e, ime, inoculum densi y and su ace, bu also on he in insic
cha ac e is ics o each s ain [
14
,
15
,
25
,
34
]. The bio ilm o ma ion capabili y has been e-
la ed o he exp ession o genes o such di e se unc ions as mo ili y, EPS p oduc ion and
cell signaling in many di e en bac e ia. The exp ession o some o hose genes is cell
densi y-dependen , as he (p)ppGpp syn he ase (i.e., spoT, elA, elP) and Quo um Sensing
(QS) genes (i.e., luxS) [
30
]. In ac , low cell densi ies ha e been ela ed o cell adhesion and
high ones o cell de achmen media ed by (p) ppGpp syn he ases, and high cell densi ies
also o bio ilm o ma ion as a esul o QS au oinduce
p oduc ion [26,35,36]
. In ce ain
bac e ial species, such as E. coli,P. luo escens,P. ae uginosa,B. sub ilis,Ag obac e ium ume-
aciens and Shewanella oneidensis, lagella and o he su aces s uc u es such as imb ia,
ex acellula memb ane p o eins and amyloid-like ib ils a e essen ial in he ini ial bac e ial
a achmen and subsequen bio ilm o ma ion [
24
,
26
,
30
,
37
–
51
]. In ac , i has been demon-
s a ed ha unc ional lagella a e necessa y o maximum bio ilm o ma ion in species
such as Campylobac e jejuni,A. ume aciens and E. coli. This a i ma ion is suppo ed by
s udies whe e he mu a ion o genes implica ed in he syn hesis o he lagella ilamen
( laA, laB, laG, liA and liS in C. jejuni; and liC in E. coli) [
28
,
40
,
50
–
52
] and hook ( lgE in
A. ume aciens
and P. ae uginosa) [
46
,
53
], lagellum mo emen (mo A in
A. ume aciens
,C.
jejuni and
E. coli) [44,46,50,52]
and lagella gene egula ion ( liA and qseB in E. coli, and
liW in C. jejuni) [
44
,
54
] showed educed bio ilm o ma ion o no o ma ion a all. Fu he ,
highe exp ession le els o he laA gene we e epo ed o L. monocy ogenes g owing in
bio ilm compa ed o plank onic o m [55].
The gene spoT encodes he bi unc ional (p) ppGpp syn hase/hyd olase SpoT [
56
].
Among o he s, his ala mone has been ela ed o lagella gene egula ion and bio ilm
o ma ion as pa o he s ingen esponse in many bac e ia such as E. coli,Legionella
pneumophila,Mycobac e ium ube culosis,C. jejuni and Helicobac e pylo i [
56
–
59
]. Lowe
exp ession le els o he lagella genes lgH and lgE we e no iced in spoT mu an s ains
in Vib io spp [
26
,
60
]. The lack o lagella hinde ed he ini ial a achmen and delayed he
bio ilm o ma ion [
26
,
60
]. In H. pylo i,spoT mu an s o med ligh e bio ilms han he wild
ype, showing di e ences in he ma ix con o ma ion [
59
]. This gene has also been di ec ly
ela ed o he up egula ion o bio ilm o ma ion in E. coli,S ep ococcus mu ans,H. pylo i
and C. jejuni [59,61,62].
In 2015, Kim e al. [
63
] no iced ha phospha e ace a e (AcP) could play a ole as a
media o in he exp ession o genes such as elP (a sho RelA/SpoT Homologue (RSH)
wi h ala mone syn hase unc ion [
27
,
64
]) and luxS (implica ed in QS). The AcP is c ea ed
ia he P a-AcK pa hway, which has also shown an implica ion in he bio ilm o ma ion
p ocess o di e en bac e ia such as S. mu an s,E. coli and C. jejuni [
40
,
63
,
65
]. This pa hway
Mic oo ganisms 2022,10, 1280 3 o 16
is composed o he enzymes P a (i) and Ack (ii), encoded by p a and ackA, espec i ely, ha
wo k (i) ans o ming he ace yl-CoA in o AcP and (ii) AcP in o ace a e [
63
,
66
]. In a ecen
ansc ip ional s udy o Campylobac e spp., he p esence o he p a gene was ela ed o
bio ilm p oduc ion and i s absence o weak o no-bio ilm o ma ion [
67
]. In con as , o he
s udies ela ed he absence o p a wi h a bio ilm inc ease in di e en species [
40
,
65
,
68
]. On
he o he hand, he highly conse ed luxS gene [
69
–
71
] encodes he LuxS me allop o ease.
This enzyme is in ol ed in he p oduc ion o he au oinduce -2 (AI-2), one o he mos
s udied QS signaling molecules [
35
]. In he biosyn hesis o AI-2, S-adenosylhomocys eine
(SAH) is hyd olyzed o S- ibose homocys eine (SRH) by he enzyme P s, hen ans o med
in o 4,5-dihyd oxy-2,3-pen anedione (DPD) by LuxS and inally sel -cycled o o m he
AI-2 [
35
,
72
,
73
]. The LuxS/AI-2 QS pa hway is ela ed o a a ie y o p ocesses such as
bio ilm p oduc ion, plasmid ans e ence, mo ili y, d ug esis ance, adhesion and i ulence-
gene exp ession [
69
,
74
–
77
]. In ac , luxS has been ound necessa y o an e icien bio ilm
o ma ion in S. mu ans,V. chole ae,Salmonella Typhi, L. monocy ogenes,Lac obacillus hamnosus,
H. pylo i,C. jejuni and Po phy omonas gingi alis [28,63,78–83].
A. bu zle i p esen s homologous o he genes laA, laB, liS,luxS,p a and spoT [
5
], bu
hei unc ion ela ed o he bio ilm o ma ion has no been es ablished ye . Ne e he-
less, i is easonable o hink ha hey may also a ec adhe ence and bio ilm o ma ion
in
A. bu zle i
. Unde s anding he mechanism benea h he bio ilm o ma ion is i al o
designing po en ial con ol s a egies. The e o e, in o de o inc ease he knowledge o
A. bu zle i
pa hogenesis, he aim o his s udy was o assess he ole o six bio ilm-associa ed
genes in campylobac e ia ( laA, laB, liS,luxS,p a and spoT) [
28
,
40
,
62
,
67
,
84
] in he bio ilm
o ma ion abili y o A. bu zle i.
2. Ma e ials and Me hods
2.1. Bac e ial S ains, Plasmids and G ow h Condi ions
Fou A. bu zle i s ains we e selec ed o mu agenesis assays o bio ilm-associa ed
genes based on hei di e en abili y o o m bio ilms [
5
,
14
]. All o hem had been p e iously
isola ed om di e en ood p oduc s a e ail [
2
,
5
] and p esen ed bio ilm-associa ed genes,
as con i med by PCR (see he ollowing sec ion). The e e ence s ain A. bu zle i RM 4018
was also included. All he s ains and plasmids used in his s udy a e lis ed in Table 1.
A cobac e s ains we e ou inely g own a 37
◦
C in B ain Hea h In usion (BHI) b o h
(Oxoid, Basings oke, UK) o on Columbia aga base pla es (Oxoid, Basings oke, UK) sup-
plemen ed wi h 5% de ib ina ed ho se blood (Lio ilchem, Rose o degli Ab uzzi, I aly). Es-
che ichia coli s ains we e ou inely g own a 37
◦
C in Lu ia-Be ani (LB) b o h o on LB aga
pla es (Condalab, To ejón de A doz, Spain), supplemen ed wi h ampicillin (
100 µg/mL
)
(CAS: 69-52-3; Sigma-Ald ich, S . Louis, MO, USA) o kanamycin (50
µ
g/mL) (CAS: 25389-
94-0; NZYTech, Lisbon, Po ugal) when necessa y. In bo h cases, he media we e incuba ed
ae obically o 24–48 h.
2.2. G ow h Cu e Measu emen s
O e nigh liquid cul u es we e dilu ed in o esh BHI o each an op ical densi y o
0.05 a 550 nm (OD
550
). Two hund ed mic oli e s o each p epa ed bac e ial suspension
we e inocula ed in o ou wells o a 96-well la -bo om mic o i e pla e (Nes Bio echnology,
Wuxi, China), and he pla es we e placed in a Syne gy
TM
HT pla e eade (BioTek, Winooski,
VT, USA) o moni o he OD
550
o he bac e ia e e y hou o 24 h. The pla es we e
main ained unde ae obic condi ions a 37
◦
C and agi a ed a 17 Hz. The exponen ial
g ow h a e was calcula ed om h ee independen g ow h expe imen s.
2.3. Bio ilm-Associa ed Gene De ec ion
The p esence o six bio ilm-associa ed genes ( laA, laB, liS,luxS,p a and spoT) was
de e mined by indi idual PCRs pe o med on 100 ng o DNA wi h 1.25 U o Sup eme
NZYTaq II DNA Polyme ase (NZYTech, Lisbon, Po ugal), 0.1 mM o each dNTP, 1X
bu e and 0.25
µ
M o each p ime se . All he p ime s used in his s udy a e lis ed in
Mic oo ganisms 2022,10, 1280 4 o 16
Supplemen a y Ma e ials Table S1. The PCR pa ame e s we e 5 min a 95
◦
C; 30 cycles
o 94
◦
C o 30 s, annealing empe a u es anging om 50 o 56
◦
C o 30 s and 72
◦
C o
1 min
; and 10 min a 72
◦
C. DNA om A. bu zle i RM4018 was used as he posi i e con ol
and deionized wa e as he nega i e one.
2.4. Cons uc ion o Knockou Mu an S ains
Knockou (KO) mu an s o bio ilm-associa ed genes we e cons uc ed acco ding o
a p e iously desc ibed me hod [
85
] wi h some modi ica ions. B ie ly, he genes and hei
lanking egions we e ampli ied by PCR using he p oo eading enzyme ACCUZYME
TM
DNA polyme ase (Bioline, Memphis, TN, USA), 5
0
-A ailed using BIOTAQ
TM
DNA poly-
me ase (Bioline, Memphis, TN, USA) and cloned in he comme cial cloning ec o pGEM-T
Easy (P omega, Madison, WI, USA). The esul ing plasmids we e linea ized ei he by
an ou wa d PCR pe o med wi h BamHI cu ing si e con aining p ime s (pG laAB) o by
es ic ion enzyme diges ion wi h MunI, ClaI, Bm I o A lII (pG liS, pGluxS, pGp a and
pGspoT, espec i ely). The linea ized plasmids we e liga ed o a kanamycin (Km) esis-
ance casse e (aph(3
0
)-III) ob ained om he pMW2 plasmid [
86
] ei he by BamHI diges ion
o by PCR ampli ica ion using p ime s ha con ained he app op ia e es ic ion cu ing
si e o each case. The o ien a ion o he casse e and he ORFs was he same in all he
cons uc ed plasmids.
2.5. Mo ili y Assays
The mo ili y o he s ains was assayed by s ab-inocula ion o single colonies in o
hioglycola e semisolid aga pla es ( hioglycola e medium con aining 0.4% aga ) (Scha lau,
Sen mena , Spain). The pla es we e incuba ed unde ae obic condi ions a 37
◦
C o 24 h,
and he diame e o he mo ili y zone was measu ed. The assays we e ca ied ou a leas
on h ee independen occasions.
2.6. Bio ilm Fo ma ion Assays
The bio ilm o ma ion abili y o he s ains on polys y ene (PS), ein o ced glass and
s ainless s eel was e alua ed ollowing p e iously desc ibed p o ocols [
14
] wi h mino
modi ica ions. Fo PS and ein o ced glass assays, he inocula we e adjus ed o an op ical
densi y o 0.2 a 600 nm (OD
600
), he incuba ions we e pe o med a 37
◦
C o 48 h, and
he bio ilms we e s ained wi h 200
µ
L o c ys al iole (1% wa e solu ion) (CAS: 548-62-9;
Sigma-Ald ich, S einheim, Ge many). Fo s ainless-s eel assays, 7 mL o an
OD600 = 0.2 cel
l
suspension was added o each coupon-con aining ube, and he incuba ions we e pe o med
a 37
◦
C o 24 h. A e gen ly washing wi h dis illed wa e , he coupons we e ans e ed
o 15 mL conical plas ic ubes con aining 7 mL o s e ile 0.01 M phospha e-bu e ed saline
(PBS) and 15 glass pea ls. The bio ilms o med on PS and ein o ced glass we e exp essed
by he bio ilm o ma ion index (BFI) acco ding o Niu and Gilbe (2004) [
87
], and he
s ains we e subsequen ly ca ego ized as s ong, mode a e, weak o non-bio ilm o me s
acco ding o Na es e al. (2008) [
88
]. 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, Basings oke, UK), exp essing he
esul s as logUFC/cm2.
2.7. Congo Red Binding Assay
Fo each s ain, isola ed colonies om o e nigh cul u es we e suspended in physio-
logical saline (0.9% (w/ ) NaCl) o 0.5 McFa land. Th ee 10
µ
L d ops o each inoculum we e
added on o Congo Red Aga (CRA) pla es, which we e composed o 37 g/L B ain Hea h
In usion b o h (Oxoid, Basings oke, UK) and 10 g/L o Bac e iological Aga (Scha lau, Sen -
mena , Spain), supplemen ed wi h an au ocla e-s e ilized concen a ed Congo ed (CAS:
573-58-0; Ac os O ganics, Geel, Belgium) solu ion (20 mg/mL) in a inal concen a ion o
0.1 g/L [
89
–
91
]. A e 48 h o incuba ion a 37
◦
C, he CRA pla es we e obse ed agains a
backligh o di e en ia e he colo o he colonies. Fou s ains we e es ed on each pla e,
and he expe imen s we e pe o med a minimum o h ee independen imes. S ains wi h
Mic oo ganisms 2022,10, 1280 5 o 16
a ed pheno ype on CRA pla es we e conside ed cellulose p oduce s and hose wi h a whi e
one as non-p oduce s.
2.8. Da a Analysis
S a is ical analyses we e pe o med using he IBM SPSS S a is ics 26 so wa e (IBM
Co p., New Yo k, NY, USA). The no mali y o he nume ical alues ob ained o each
s ain was de e mined by he Shapi o–Wilk es . The adhesion capaci y, g ow h a e and
mo ili y o he s ains we e compa ed by S uden ’s - es and one-way analysis o a iance
(ANOVA). Signi ican di e ences we e es ablished a p alues o <0.05.
3. Resul s
3.1. Cons uc ion o Knockou (KO) Mu an s and G ow h Analysis
O e all, all he genes we e success ully knocked ou . A o al o 18 KO mu an s ains
we e ob ained om he i e A. bu zle i s udied s ains (Table 1). All he i e expec ed
mu an s we e ob ained om he s ains CCUG 30485 and CH11, ou om CZ6, h ee
om P8 and one om BER7. The mos success ully mu a ed gene was p a, which could be
inac i a ed in all he s udied A. bu zle i s ains, ollowed by liS and luxS in ou . spoT and
laAB we e inac i a ed in h ee and wo s ains, espec i ely. The compa ison o he a ious
mu an s wi h hei co esponden pa en s ain showed no di e ences in bac e ial shape,
colony o ma ion on blood aga pla es o g ow h a e in BHI (ANOVA-based
p> 0.05
)
(Figu e S1).
Table 1. Bac e ial s ains and plasmids used in his s udy.
Bac e ial S ain o Plasmid Sou ce/Func ion Re e ence Bio ilm
Fo ma ion 3
Bio ilm Associa ed
Genes De ec ed
by PCR
A cobac e bu zle i s ains
BER7 Wild s ain isola ed om cockle [5] 2.48 ±1.16
laA, laB, liS,luxS,p a
and spoT
BER7∆p a::Km AB-BER7 de i a i e
∆ABU_RS02465::aph(30)-III
This s udy
CCUG 30485 Human clinical isola e (ATCC 49616;
RM4018) CCUG 1
laA, laB, liS,luxS,p a
and spoT
CCUG 30485∆ laAB::Km CCUG 30485 de i a i e ∆ABU_RS11245-
RS11250::aph(30)-III
This s udy
CCUG 30485∆ liS::Km CCUG 30485 de i a i e
∆ABU_RS01060::aph(30)-III
This s udy
CCUG 30485∆luxS::Km CCUG 30485 de i a i e
∆ABU_RS00560::aph(30)-III
This s udy
CCUG 30485∆p a::Km AB-CCUG 30485 de i a i e
∆ABU_RS02465::aph(30)-III
This s udy
CCUG 30485∆spoT::Km AB-CCUG 30485 de i a i e
∆ABU_RS03230::aph(30)-III
This s udy
CH11 Wild s ain isola ed om squid [5] 0.76 ±0.13
laA, laB, liS,luxS,p a
and spoT
CH11∆ laAB::Km AB-CH11 de i a i e
∆ABU_RS11245-RS11250::aph(30)-III
This s udy
CH11∆ liS::Km AB-CH11 de i a i e
∆ABU_RS01060::aph(30)-III
This s udy
CH11∆luxS::Km AB-CH11 de i a i e
∆ABU_RS00560::aph(30)-III
This s udy
CH11∆p a::Km AB-CH11 de i a i e
∆ABU_RS02465::aph(30)-III
This s udy
CH11∆spoT::Km AB-CH11 de i a i e
∆ABU_RS03230::aph(30)-III
This s udy
CZ6 Wild s ain isola ed om quail [5] 3.00 ±2.90 liS,luxS,p a and spoT
CZ6∆ liS::Km AB-CZ6 de i a i e
∆ABU_RS01060::aph(30)-III
This s udy
CZ6∆luxS::Km AB-CZ6 de i a i e
∆ABU_RS00560::aph(30)-III
This s udy
CZ6∆p a::Km AB-CZ6 de i a i e
∆ABU_RS02465::aph(30)-III
This s udy
CZ6∆spoT::Km AB-CZ6 de i a i e
∆ABU_RS03230::aph(30)-III
This s udy
Mic oo ganisms 2022,10, 1280 6 o 16
Table 1. Con .
Bac e ial S ain o Plasmid Sou ce/Func ion Re e ence Bio ilm
Fo ma ion 3
Bio ilm Associa ed
Genes De ec ed
by PCR
P8 Wild s ain isola ed om chicken [2] 9.44 ±6.07 liS,luxS,p a and spoT
P8∆ liS::Km AB-P8 de i a i e
∆ABU_RS01060::aph(30)-III
This s udy
P8∆luxS::Km AB-P8 de i a i e
∆ABU_RS00560::aph(30)-III
This s udy
P8∆p a::Km AB-P8 de i a i e
∆ABU_RS02465::aph(30)-III
This s udy
Esche ichia coli s ains
E. coli DH5αNCCB2955 Compe en cells o cloning NCCB 2
Plasmids
pGEM-T Easy Cloning ec o , Amp P omega
pG laAB pGEM-T Easy con aining
ABU_RS11245-ABU_RS11250
This s udy
pG liS pGEM-T Easy con aining ABU_RS01060 This s udy
pGluxS pGEM-T Easy con aining ABU_RS00560 This s udy
pGp a pGEM-T Easy con aining ABU_RS02465 This s udy
pGspoT pGEM-T Easy con aining ABU_RS03230 This s udy
pG∆ laAB pGEM-T Easy con aining
∆ABU_RS11245-RS11250
This s udy
pG∆ liS pGEM-T Easy con aining
∆ABU_RS01060
This s udy
pG∆luxS pGEM-T Easy con aining
∆ABU_RS00560
This s udy
pG∆p a pGEM-T Easy con aining
∆ABU_RS02465
This s udy
pG∆spoT pGEM-T Easy con aining
∆ABU_RS03230
This s udy
pG∆ laAB::Km pGEM-T Easy con aining
∆ABU_RS11245-RS11250::aph(30)-III
This s udy
pG∆ liS::Km pGEM-T Easy con aining
∆ABU_RS01060::aph(30)-III
This s udy
pG∆luxS::Km pGEM-T Easy con aining
∆ABU_RS00560::aph(30)-III
This s udy
pG∆p a::Km pGEM-T Easy con aining
∆ABU_RS02465::aph(30)-III
This s udy
pG∆spoT::Km pGEM-T Easy con aining
∆ABU_RS03230::aph(30)-III
This s udy
pMW2 pBluesc ip KS M13 +:KmR (pILL550) [86]
1
CCUG: Cul u e Collec ion Uni e si y o Go henbu g.
2
NCCB: Ne he lands Cul u e Collec ion o Bac e ia.
3Da a
ob ained om Ma inez-Malaxe xeba ia e al. [
5
] and Gi bau e al. [
14
]. Values a e exp essed as mean
±
s anda d e o s.
3.2. Mo ili y Assays
The mo ili y o he s ains, exp essed in nume ical alues, is shown in Table 2. Rep-
esen a i e images can be consul ed in Figu e S2. The i e pa en s ains and mos o he
mu an s (11 ou o 18) we e mo ile. In con as , all hose mu an s in he lagella genes
( laAB and liS) and one in p a (BER 7 de i a i e) we e non-mo ile. This loss o mo ili y e-
sul ed as signi ican in all cases (p
≤
0.001). Among he mo ile s ains, all he ob ained spoT
and luxS mu an s excep P8
∆
luxS::Km showed highe mo ili y han hei co esponding
pa en s ains, and so did he CCUG 30485 and CH11-de i a i e mu an s in p a. In con as ,
CZ6 and P8-de i a i e mu an s in he same gene showed lowe mo ili y han hei pa en
s ains. None o he obse ed di e ences we e s a is ically signi ican .
3.3. Bio ilm Fo ma ion Assays
The abili y shown by he s ains o o m bio ilms on di e en su aces is esumed
in Table 2. Unde he expe imen al condi ions, he majo i y o he s ains (19 ou o
22) o med bio ilms on PS and we e ca ego ized as s ong bio ilm p oduce s, especially
P8 (p< 0.04). The excep ions we e he s ains CZ6, CZ6
∆
luxS::Km and CZ6
∆
liS::Km,
which did no show any adhe ence abili y on his su ace. Almos all mu an s showed
di e en bio ilm o ma ion abili ies om hei pa en s ains bu he only signi ican ly
di e en one (p= 0.023) was ha shown by BER7
∆
p a::Km, which showed a BFI almos
Mic oo ganisms 2022,10, 1280 7 o 16
i e imes highe han BER7. On ein o ced glass, all he s ains o med bio ilms, bu hei
ca ego iza ion di e ed om ha on PS. Among he wild s ains, BER7 was de ined as
weakly adhe en , CZ6 as mode a ely adhe en and CCUG 30485, CH11 and P8 as s ongly
adhe en . On his ma e ial, all he mu an s ains showed di e ences in hei BFI alues
om hei co esponden pa en , and he ANOVA showed a signi ican educ ion in he
bio ilms o med by CZ6
∆
liS::Km and P8
∆
liS::Km (p= 0.033 and 0.001, espec i ely). In
gene al, he bio ilm o ma ion abili y o he s ains was highe on PS han in ein o ced
glass, and acco ding o S uden ’s - es , i was signi ican o CH11 (p= 0.019). Rega ding
s ainless s eel, iable cells could be eco e ed om all he coupons, which indica es he
capabili y o all he s udied s ains o o m bio ilms on his su ace. Based on he ANOVA,
he adhesion o CZ6
∆
spoT::Km was signi ican ly highe han ha o i s pa en al on his
ma e ial. (p= 0.006).
Table 2.
Bio ilm o ma ion abili y, mo ili y and pheno ype on CRA shown by he s ains in his s udy.
Bac e ial S ain PS 1Rein o ced Glass S ainless S eel
(logUFC/cm2)Mo ili y (cm) CRA 4
BFI 2Ca eg 3BFI 2Ca eg 3
BER7 1.679 ±0.609 S 0.462 ±0.093 W 2.285 ±0.292 2.833 ±0.233 Whi e
BER7∆p a::Km 8.263 ±3.108 * S 0.732 ±0.373 M 2.427 ±0.341 0.000 * Whi e
CCUG 30485 5.140 ±2.702 S 2.328 ±0.574 S 2.877 ±0.461 1.233 ±0.145 Red
CCUG
30485∆ laAB::Km 5.144 ±1.981 S 0.965 ±0.278 M 1.832 ±0.113 0.000 * Red
CCUG
30485∆ liS::Km 2.571 ±1.569 S 0.913 ±0.223 M 3.444 ±0.737 0.000 * Red
CCUG
30485∆luxS::Km 3.191 ±0.421 S 1.008 ±0.337 M 2.901 ±0.494 1.233 ±0.186 Red
CCUG
30485∆p a::Km 5.498 ±2.444 S 1.343 ±0.276 S 3.000 ±0.292 1.233 ±0.133 Red
CCUG
30485∆spoT::Km 8.502 ±4.728 S 1.455 ±0.449 S 3.858 ±0.729 0.967 ±0.145 Red
CH11 4.265 ±0.772 S 1.179 ±0.435 †S 4.233 ±0.373 1.067 ±0.033 Whi e
CH11∆ laAB::Km 2.822 ±1.544 S 0.748 ±0.205 M 3.433 ±0.332 0.000 * Whi e
CH11∆ liS::Km 3.171 ±2.154 S 0.542 ±0.187 W 4.322 ±0.260 0.000 * Whi e
CH11∆luxS::Km 2.650 ±1.852 S 0.693 ±0.200 W 3.934 ±0.450 1.167 ±0.067 Whi e
CH11∆p a::Km 4.997 ±3.366 S 0.248 ±0.096 N 4.367 ±0.597 1.433 ±0.203 Whi e
CH11∆spoT::Km 7.271 ±4.163 S 0.331 ±0.211 N 4.458 ±0.456 1.200 ±0.208 Whi e
CZ6 0.000 N 0.851 ±0.191 M 3.419 ±0.213 2.533 ±0.120 Red
CZ6∆ liS::Km 0.007 ±0.007 N 0.176 ±0.102 * N 4.032 ±0.674 0.000 * Red
CZ6∆luxS::Km 0.000 N 0.379 ±0.115 W 3.515 ±0.224 2.800 ±0.115 Red
CZ6∆p a::Km 4.608 ±3.979 S 0.312 ±0.105 N 3.094 ±0.321 2.267 ±0.176 Red
CZ6∆spoT::Km 4.852 ±4.685 S 0.706 ±0.211 M 4.700 ±0.148 * 2.900 ±0.058 Red
P8 17.319 ±3.671 •S 3.825 ±0.257 S 5.989 ±0.140 2.600 ±0.153 Whi e
P8∆ liS::Km 7.961 ±1.448 S 1.752 ±0.270 * S 5.489 ±0.222 0.000 * Whi e
P8∆luxS::Km 13.706 ±2.152 S 4.547 ±0.752 S 5.944 ±0.179 2.167 ±0.088 Whi e
P8∆p a::Km 11.132 ±1.304 S 3.516 ±0.761 S 5.732 ±0.109 2.300 ±0.153 Whi e
1
PS: Polys y ene.
2
BFI, Bio ilm Fo ma ion Index.
3
Ca eg, ca ego iza ion acco ding o Na es e al., [
88
]: S ong (S),
Mode a e (M), Weak (W) and None (N).
4
CRA, pheno ype shown on CRA pla es. * S uden ’s -based s a is ically
signi ican (p< 0.05) di e ences ob ained when compa ing wild- ype s ains wi h hei de i a i es on each su ace.
•
ANOVA-based s a is ically signi ican (p< 0.05) di e ences ob ained when compa ing he BFI alues ob ained
on PS o each wild- ype s ain.
†
S uden ’s -based s a is ically signi ican (p< 0.05) di e ences ob ained when
compa ing bio ilm o ma ion on PS e sus bo osilica e.
3.4. Congo Red Aga Assays
The wild BER7, CH11 and P8 s ains u ned ou o be non-cellulose p oduce s based
on hei pheno ype on CRA pla es (whi e g ow h). In con as , he s ains CCUG 30485 and
CZ6 we e cellulose p oduce s ( ed g ow h). No di e ences we e obse ed be ween wild
and KO mu an s ains. The pigmen a ion acqui ed by he s ains when g own on CRA
pla es can be consul ed in Figu e S3.
4. Discussion
The ansmission and pa hogenici y o many bac e ia a e ela ed o hei capaci y
o o m bio ilms [
14
–
16
]. These s uc u es ha e gained g ea in e es o e he las yea s,
and he mechanisms unde lying hei o ma ion and main enance a e being elucida ed in
many bac e ial species [
25
–
30
,
35
,
40
,
63
,
68
,
92
]. Ne e heless, his knowledge is s ill sca ce
o he oodbo ne pa hogen A cobac e bu zle i. To add ess his i em, in his s udy, we
Mic oo ganisms 2022,10, 1280 8 o 16
aimed o unde s and he ole o laA, laB, liS,luxS,p a and spoT in he bio ilm o ma ion
p ocess o his species. Fo his pu pose, mu an s in he abo emen ioned genes ha a e
associa ed wi h bio ilm o ma ion in o he campylobac e ia we e cons uc ed, and hei
bio ilm o ma ion abili y on a ious su aces o di e en hyd ophobici y was compa ed
o ha o hei pa en s ains. In addi ion, he capabili y o o m bio ilms was also es ed
using he Congo ed binding assay, widely used by o he au ho s in species such as E. coli,
K. pneumoniae,S. en e ica and C. jejuni [29,93,94].
Congo ed indica o binds o cu li/ imb ia and cellulose [
95
,
96
] and conside ing ha A.
bu zle i does no ha e cu li/ imb ia, he assay indica es cellulose p oduc ion in his species.
Ou esul s did no show di e ences be ween mu an and pa en s ains, sugges ing ha
he inac i a ed genes appa en ly do no ake pa in cellulose p oduc ion in A. bu zle i. As
a as we know, his is he i s ime whe e cellulose p oduc ion by A cobac e has been
epo ed. Mo eo e , we a e no awa e o he p esence o genes in ol ed in his p ocess in
A. bu zle i, which lea es he way open o new esea ch lines. On he o he hand, al hough
i has been sa is ac o ily used o de ec bio ilm p oduc ion in campylobac e ia [
29
], he
esul s ob ained on he Congo ed binding assay did no co ela e wi h hose ob ained
on he bio ilm o ma ion assays in ou case. Bo h cellulose-p oducing ( ed g ow h) and
non-p oducing (whi e g ow h) s ains o med bio ilms unde he expe imen al condi ions.
This phenomenon has been p e iously desc ibed in some o he species [
97
]. Consequen ly,
we do no conside he Congo ed binding assay o be eliable o he iden i ica ion o
bio ilm- o ming bac e ia in his species.
In acco dance wi h p e ious s udies held wi h bo h G am-posi i e and nega i e bac e-
ia [
28
,
40
,
98
–
100
], he esul s o his s udy poin o he lagellum as an impo an s uc u e
implied in he bio ilm o ma ion o A. bu zle i. The ob ained mu an s in he laA and laB
genes (CCUG 30485
∆
laAB::Km and CH11
∆
laAB::Km) showed educed bio ilm- o ma ion
abili ies compa ed wi h hei pa en s ains in all he su aces es ed. Simila ly, mu an s o
he liS gene, which encodes he FliS chape one esponsible o lagellin p o ec ion and ans-
po , adhe ed less han wild- ype s ains o PS and ein o ced glass, especially CZ6
∆
liS::Km
and P8
∆
liS::Km o he la e ma e ial (p= 0.033 and p= 0.001, espec i ely). Howe e ,
h ee ou o he ou ob ained liS mu an s, namely CCUG 30485
∆
liS::Km, CH11
∆
liS::Km
and CZ6
∆
liS::Km, showed enhanced adhesion on s ainless s eel. The impo ance o a unc-
ional lagellum o maximum bio ilm o ma ion by campylobac e ia has been p e iously
epo ed. S udies held wi h Campylobac e spp. demons a ed ha mu an s on he laA, laB,
liA, laG and mo A genes showed educed adhesion and bio ilm o ma ion abili y [
28
,
50
,
51
].
Joshua e al. [
40
] obse ed ha a lagella ed C. jejuni liS mu an s we e unable o a ach o
su aces, and Ha h oubi e al. [
101
] ha a lagella ed H. pylo i mu an s p oduced weake
bio ilms. In o he species, such as V. chole ae and P. ae uginosa, mu an s wi h a ec a ion in
lagella showed comp omised bio ilm o ma ion [
102
,
103
]. Despi e no ha ing es ed he
in eg i y o he lagella in ou mu an s in he laA, laB and liS lagella genes, he dec eased
mo ili y obse ed o all o hem could be indica i e o hei non-co ec unc ionali y.
These h ee genes a e essen ial o he syn hesis and anspo o he lagellin subuni s ha
con o m o he lagella ilamen [
24
,
26
,
30
,
37
–
40
,
44
–
48
]. Likely, he inac i a ion o any o
hese genes led o he p oduc ion o lowe lagellin le els and, consequen ly, o abno mal
lagella wi h sho e ilamen o no ilamen a all, as p e iously epo ed elsewhe e [
104
].
Mos ly, he inac i a ion o lagella genes is associa ed wi h a dec ease in he abili y o
adhe e [
28
,
40
,
50
,
51
,
54
,
67
,
98
–
101
,
104
]. In consonance wi h his, di e en ansc ip ional
s udies indica ed ha he exp ession o some lagella genes is highe when bac e ia g ow
on a bio ilm compa ed o he plank onic s a e. L. monocy ogenes seems o o e exp ess laA
when g owing on bio ilm [
55
]. Among campylobac e ia, s ongly adhe en Campylobac e
s ains show highe exp ession le els o laB and liS han weakly adhe en ones [
67
], and
bio ilm g owing H. pylo i up egula es a ious genes ela ed o he o ma ion o he lagella
appa a us [
101
]. Being p ima ily a mobili y s uc u e, he lagellum has impo an unc-
ions o bio ilm o ma ion as mechanosensing o su aces [
105
,
106
] o being a componen
o he bio ilm ma ix [
101
]. E en so, and in line wi h p e ious obse a ions [
107
–
109
],
Mic oo ganisms 2022,10, 1280 9 o 16
he enhanced adhesion obse ed in some o ou lagella mu an s indica es ha , hough
impo an , a unc ional lagellum is no essen ial o bio ilm o ma ion in A. bu zle i. Fu u e
s udies may elucida e whe he a unca ed ilamen (o i s absence) has an in luence on he
composi ion o s uc u e o he bio ilm ma ix. In addi ion, ou esul s poin o he possible
p esence o o he su ace-induced mechanisms in ol ed in he ea ly s eps o he bio ilm
o ma ion p ocesses, as could be he adhesins CadF, PEB1a, JlpA, AcpA and CjaA, p esen
in phylogene ically closely ela ed species [110].
In gene al, he bio ilms o med by luxS mu an s we e equal o o lowe han hose
o med by he wild- ype s ains on he h ee s udied su aces. The ole o he bac e ial
au oinduce -2 (AI-2) p oduced by luxS has been ela ed o Quo um Sensing (QS), and his
one wi h bio ilm p oduc ion [
111
]. The implica ion o QS in he bio ilm p oduc ion has
been e idenced by he inac i a ion o genes coding o di e en signaling molecules and
he subsequen educ ion o he bio ilm o med, such as he gene lasI in P. ae uginosa [112]
and cep in Bu kholde ia cepacia [
113
]. The gene luxS has been ound necessa y o an e icien
bio ilm o ma ion in S. mu ans,V. chole ae,Salmonella Typhi and P. gingi alis [
63
,
78
]. In
S. mu ans,K. pneumoniae and C. jejuni, he lack o a unc ional luxS led o a dec eased
bio ilm p oduc ion [
28
,
67
,
81
,
114
–
116
], which is qui e in acco dance wi h ou esul s. All
ou mu an s in luxS adhe ed less han hei pa en s ains excep P8 and CZ6 de i a i es
on ein o ced glass and s ainless s eel, espec i ely. Likewise, he absence o di e ences
be ween he bio ilms p oduced by wild- ype and some luxS mu an s ains (CZ6
∆
luxS::Km
in PS and all luxS mu an s on s ainless s eel excep CH11
∆
luxS::Km) epo ed he e had
also been p e iously obse ed in K. pneumoniae [
36
] and S. go donii [
117
]. In con as ,
he inac i a ion o luxS inc eased he bio ilm p oduc ion in H. pylo i [
81
,
116
]. Changes
in bio ilm mo phology [
69
,
114
,
117
], mo ili y educ ion [
52
], dec eased au oagg ega ion
(which con ibu es o bio ilm o ma ion) [
52
], minimized g ow h [
35
] and educed adhesion
o cell lines [
118
–
120
] ha e also been epo ed due o he inac i a ion o luxS. Ne e heless,
ou esul s did no e lec g ow h di e ences be ween pa en and luxS mu an s ains.
B oadly, mu an s o p a showed an inc ease in hei bio ilm o ma ion abili y on
PS, which was s a is ically signi ican o BER7
∆
p a::Km (p= 0.023), bu a educ ion on
ein o ced glass. The e ec o his mu a ion in bio ilm o ma ion on s ainless s eel a ied
among he es ed s ains. The lack o p a has been associa ed wi h bo h inc eased and
dec eased bio ilm o ma ion. The inac i a ion o p a led o hype lagella ed E. coli mu an
s ains in a s udy conduc ed in 2005 [
121
]. Acco ding o he au ho s’ obse a ions, he
inc eased in acellula phospha e ace a e (AcP) pool unde lies he lagella exp ession
change. As men ioned abo e, lagella play an impo an ole in bio ilm o ma ion; he e o e,
mu an s o p a could show enhanced bio ilm p oduc ion i hey o e exp ess lagella genes.
This could be he case wi h ou p a mu an s, which showed a gene al inc ease in mo ili y
and o med highe bio ilms on PS han hei co esponden pa en s ains. Inc eased bio ilm
p oduc ions de i ed om he inac i a ion o p a ha e also been epo ed in E. coli,C. jejuni
and S. mu ans [
40
,
63
,
65
]. Ne e heless, and based on he epo ed e ec o high AcP le els
in he exp ession o luxS and he RelA/SpoT sys em [
63
], we could hypo hesize ha he
inac i a ion o p a and he subsequen AcP inc ease can lead o educed concen a ions
o AI-2 and (p)ppGpp, syn hesized by LuxS and RelA/SpoT sys ems, espec i ely; and,
consequen ly, con ibu e o he dec ease in lagella o ma ion, EPS p oduc ion and bio ilm
gene a ion [
26
,
27
,
35
,
57
,
58
,
60
,
61
,
63
,
64
,
71
]. This would be in acco dance wi h he bio ilm
o ma ion educ ion we epo he e o all ou p a mu an s ains on ein o ced glass and
o hose de i ed om CZ6 and P8 on s ainless s eel, in line wi h he bio ilm o ma ion
educ ion epo ed in S. mu ans [63].
Rega ding he spoT mu an s, when compa ed o hei pa en s ains, all p esen ed
an inc eased abili y o p oduce bio ilms on PS and s ainless s eel and a educed one on
ein o ced glass. This is in ag eemen wi h he educed bio ilm o ma ion on glass e-
po ed o V. chole ae [
61
] and Xan homonas campes is p . Campes is 8004 [
122
] mu an s.
Simila ly, enhanced bio ilms on PS we e no iced in P. pu ida KT2440 [
123
] and V. alginoly i-
cus [
26
]. This gene, which encodes he bi unc ional (p)ppGpp syn hase/hyd olase, has
Mic oo ganisms 2022,10, 1280 16 o 16
122.
Bai, K.; Yan, H.; Chen, X.; Lyu, Q.; Jiang, N.; Li, J.; Luo, L. The Role o RelA and SpoT on PpGpp P oduc ion, S ess Response,
G ow h Regula ion, and Pa hogenici y in Xan homonas campes is P . Campes is. Mic obiol. Spec .
2021
,9, e02057-21. [C ossRe ]
[PubMed]
123.
Liu, H.; Xiao, Y.; Nie, H.; Huang, Q.; Chen, W. In luence o (p)PpGpp on Bio ilm Regula ion in Pseudomonas Pu ida KT2440.
Mic obiol. Res. 2017,204, 1–8. [C ossRe ] [PubMed]
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