Applica ion o UPW _E124 phage cock ail o e ec i e educ ion o a ian
pa hogenic Esche ichia coli in mice and b oile chickens
Da id S´
aez Mo eno
a
, Maciej Kuczkowski
b
, Paweł Ko zeniowski
c
, K zysz o G zymajło
d
,
Anna Wo´
zniak-Biel
b
, Paulina ´
Sliwka
c
, Ani a Rywi´
nska
c
, Ma a Ku´
zmi´
nska-Bajo
c,*
a
Cen e o Biological Enginee ing, Uni e si y o Minho, B aga 4710-057, Po ugal
b
Depa men o Epizoo iology and Clinic o Bi ds and Exo ic Animals, Facul y o Ve e ina y Medicine, W ocław Uni e si y o En i onmen al and Li e Sciences,
G unwaldzki Squa e 45, W ocław 50-366, Poland
c
Depa men o Bio echnology and Food Mic obiology, Facul y o Bio echnology and Food Sciences, W ocław Uni e si y o En i onmen al and Li e Sciences, 37
Chełmo´
nskiego S ., W ocław 51-630, Poland
d
Depa men o Biochemis y and Molecula Biology, Facul y o Ve e ina y Medicine, W ocław Uni e si y o En i onmen al and Li e Sciences, 31 No wida S ., W ocław,
Poland
ARTICLE INFO
Keywo ds:
Bac e iophages
APEC
Phage he apy
Colibacillosis
B oile s
ABSTRACT
A ian pa hogenic Esche ichia coli (APEC) is he main causa i e agen o colibacillosis, causing poul y espi a o y
in ec ions, mo ali y and economic loss. APEC poses a se ious h ea o public heal h and ood sa e y due o i s
mul i-d ug esis ance and capaci y o o m bio ilms. Bac e iophages (phages) ha e eme ged as an al e na i e o
an ibio ics. To cu e APEC-in ec ed chickens, a cock ail consis ing o UPW _E1, UPW _E2 and UPW _E4 APEC-
speci ic phages was de eloped and es ed. In his s udy, we documen ed he main enance o hei ac i i y in
neu alized simula ed gas ic luid (SGF) condi ions and he e iciency o he UPW _E124 phage cock ail in
inhibi ing APEC in bio ilm s uc u es on chicken b eas mea su aces. Fu he , we e alua ed he e icacy o he
UPW _E124 phage cock ail agains APEC in i o in mu ine and chicken in ec ion models. In expe imen ally
in ec ed mice, we e alua ed he in ape i oneal and gas ic ga age adminis a ions o phages. The s udy e ealed
ha gas ic adminis a ion o phages educed bac e ial le els in he espi a o y sys em. Mo eo e , we es ed he
UPW _E124 phage cock ail in a chicken model o in ec ion, whe e phages e ec i ely educed he numbe o
APEC in he lungs, bu sa o Fab icius and blood. These esul s sugges ha he UPW _E124 phage cock ail could
be a po en ial ea men o colibacillosis in he poul y indus y.
1. In oduc ion
One o he bigges challenges o ou wo ld oday is o add ess he
demand o animal p o ein wi hou comp omising animal wel a e. Ac-
co ding o an OECD-FAO epo poul y mea is he mos consumed mea
wo ldwide and i s consump ion has inc eased subs an ially o e he las
decades (OECD, 2024). Ma ke demands in luence in ensi e and
la ge-scale poul y a ming. One o he main challenges in poul y p o-
duc ion is diseases caused by bac e ial pa hogens. A ian colibacillosis,
caused by a ian pa hogenic Esche ichia coli (APEC), is conside ed o be
he mos common in ec ious bac e ial disease ha a ec s in ensi e
poul y p oduc ion p ocesses (Fanche e al., 2021). APEC in ec ion may
occu in b oile chickens, u keys, and egg-laying hens (Ewe s e al.,
2004; K omann e al., 2024). In he b oile chickens, APEC in ec ions a e
conside ed o ypically lead o colibacillosis; a synd ome ha includes
espi a o y ac in ec ion, ai sacculi is, pe ica di is, pe ihepa i is,
splenomegaly, and swollen head synd ome. In ma u e laying hens,
ep oduc i e ac in ec ion leading o salpingi is o salpingo-pe i oni is
synd ome is common (Ewe s e al., 2004; K omann e al., 2024; Col-
lingwood e al., 2014). In all sec o s, APEC in ec ion is synd omic in
na u e, and ul ima ely, leads o educed egg p oduc ion and inc eased
mo ali y a es in b oile s and should be ea ed. An ibio ics a e
commonly used o ea poul y locks a ec ed wi h colibacillosis.
APEC has been iden i ied o possess a ious i ulence ac o s and
esis ance genes in ol ed in pa hogenesis and d ug esis ance (Xu e al.,
2019; Oli ei a e al., 2009a; Mo ow, 2024), which con ibu e o i s
pa hogenici y and make ea men challenging. Mo eo e , APEC is
closely ela ed o human pa hogens collec i ely known as ex ain es inal
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M. Ku´
zmi´
nska-Bajo ).
Con en s lis s a ailable a ScienceDi ec
Ve e ina y Mic obiology
jou nal homepage: www.else ie .com/loca e/ e mic
h ps://doi.o g/10.1016/j. e mic.2025.110398
Recei ed 12 June 2024; Recei ed in e ised o m 15 Janua y 2025; Accep ed 18 Janua y 2025
Ve e ina y Mic obiology 302 (2025) 110398
A ailable online 21 Janua y 2025
0378-1135/© 2025 W oclaw Uni e si y o En i onmen al and Li e Sciences. Published by Else ie B.V. This is an open access a icle unde he CC BY license
( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
pa hogenic Esche ichia coli (ExPEC) (Hu e al., 2022). ExPEC has e ol ed
mechanisms o cause in ec ions beyond he gu , leading o a ange o
synd omes om mild gas oen e i is o se e e ex ain es inal in ec ions
including meningi is caused by newbo n meningi is E. coli (NMEC),
u ina y ac in ec ions a ec ed by u opa hogenic E. coli (UPEC), and
sepsis o igina ed by sep icemic E. coli (SEPEC). Thus, APEC ca ies a
po en ial isk o ho izon al gene ans e and migh be a ese oi o
i ulence genes and an ibio ic- esis an genes o human ExPEC s ains
(Nawaz e al., 2024). One o he enable s o APEC in oduced o he
human ood chain is con amina ed ood o poul y o igin. Raw mea can
se e as an abundan ese oi o APEC, leading o c oss-con amina ion
o o he oods, su aces, and u ensils in he ki chen. The pe sis ence o
APEC in he ood chain is u he enabled due o i s capaci y o o m
bio ilms on di e en su aces such as chicken b eas ille s (Anang e al.,
2010; Possas e al., 2021). In ac , bio ilms play a c ucial ole in he
pe sis ence and su i al o APEC in ad e se condi ions due o i s com-
plex s uc u e, con aining a communi y o mic oo ganisms adhe ing o
su aces, su ounded by a ma ix o ex acellula polyme ic subs ances
which hey p oduce (K omann e al., 2024). These bio ilms p o ide
p o ec ion and esilience o bac e ia, allowing hem o wi hs and a ious
en i onmen al s esses, including nu ien limi a ions and desicca ion
(B idie e al., 2015; Sha ma e al., 2016; Giaou is e al., 2014). This is an
ex a challenge o e ec i e APEC ea men and con ibu es o i s
pe sis ence on mea su aces (Sha ma e al., 2016; Gouda z aleje di
e al., 2022; Chen e al., 2010).
Thus, no el app oaches o p e en ing and ea ing APEC and hei
bio ilms, such as he de elopmen o accines o an imic obial coa ings,
a e unde de elopmen (Soleymani e al., 2020). Among s a egies
agains APEC s ains, bac e iophages (phages) ha e eme ged as a po-
en ial na u al ool o biocon ol (Nicolas e al., 2023). Phages a e i-
uses ha can speci ically a ge and kill bac e ia and ha e
demons a ed e icacy in he deg ada ion o APEC in bio ilm s uc u es
(Yao e al., 2023; Tang e al., 2023) and in p e en ing and ea ing APEC
in ec ions in b oile chicken (Oli ei a e al., 2010; Jhandai e al., 2024;
So ou e al., 2020). O e all, phages ep esen an app oach o comba -
ing APEC in ec ions in poul y as pa o he b oade One Heal h
s a egy aimed a p omo ing animal heal h, educing an imic obial use,
and sa egua ding public heal h (Sinclai , 2019).
In he cu en s udy, we e alua ed he po en ial o he bac e iophage
cock ail UPW _E124 o he ea men o APEC in an a i icially
con amina ed chicken b eas and in wo di e en in ec ion models in
i o. We compa ed in ape i oneal and gas ic ou es o adminis a ion
in a mu ine model o examine he mode o ac ion o he UPW _E124
phage cock ail. Fu he , o e alua e he e icacy o he UPW _E124
phage cock ail’s an ibac e ial ac i i y agains APEC, we u ilized a
chicken in ec ion model.
2. Ma e ials and me hods
2.1. Bac e ial s ains and cul u e condi ions
Esche ichia coli s ains 158B lux and NCTC 17848 we e used in his
s udy. The E. coli s ain NCTC 17848 possesses a g ea abili y o o m
bio ilms and was used in an i-bio ilm assays. E. coli s ain 158B lux was
ob ained om he S ain Collec ion o he Depa men o Epizoo iology
and Clinic o Bi d and Exo ic Animals, W ocław Uni e si y o En i on-
men al and Li e Sciences. This s ain was isola ed om diseased chicken
wi h colibacillosis, classi ied as a ian pa hogenic E. coli (APEC) and
gene ically modi ied as p e iously desc ibed by Riedel e al. (2007).
B ie ly, E. coli s ain 158B was modi ied wi h he ch omosomal in e-
g a ion ec o p16Slux con aining he lux ope on om Pho o habdus
luminescens consis ing o i e genes, luxCDABE and he e y h omycin
esis ance gene. E. coli 158B lux exhibi s he capaci y o biolumines-
cence, allowing i o be dis inguished om commensal E. coli. These
modi ica ions in G am-nega i e bac e ia did no a ec in asi eness in
mice and chickens (Ku´
zmi´
nska-Bajo e al., 2023). E. coli s ains used in
his s udy we e s o ed wi h 20 % ( / ) glyce ol a −80◦C. Fo p opa-
ga ion, s ains we e cul i a ed in Lu ia-Be ani (LB) b o h (Sigma-Al-
d ich, Ge many), unde ae obic condi ions a 37◦C o e nigh wi h
shaking a 150 pm. E. coli 158B lux cul u e was supplemen ed wi h
e y h omycin (0.2 mg/ml).
2.2. Bac e iophages
The bac e iophages used in his s udy, UPW _E1, UPW _E2 and
UPW _E4, we e isola ed om samples o u ban sewage samples om he
W ocław was ewa e ea men plan and a e desc ibed elsewe e
(´
Sliwka e al., 2025). Taxonomically, UPW _E1 belongs o K isch i us
wi hin he S abo i idae amily and UPW _E2 and UPW _E4 belong o he
genus Tequa o i us wi hin he Te en i inae amily. In i o es s e ealed
lysis e ec i eness a es o 64 % o UPW _E1 and UPW _S4 and 46 % o
UPW _E2 phages on 142 APEC s ains. In silico analysis showed ha hei
genomes a e dep i ed o any known i ulence, oxin, o
pa hogen-associa ed p o ein amily o gene p oduc s o E. coli s ains o
any o he pa hogens. The comple e genome sequences o phages
UPW _E1, UPW _E2 and UPW _S4 we e deposi ed in GenBank unde he
accession numbe s PP418985, PP418986, PP418987, espec i ely. Due
o he cha ac e is ics o hese phages, including hei an i-APEC ac i i y,
hey we e conside ed a p omising ool in comba ing APEC and hence
we e chosen o conduc in i o phage e icacy expe imen s in mice and
chickens.
2.3. Bac e iophage p opaga ion and cock ail p epa a ion
The ampli ica ion o UPW _E1, UPW _E2 and UPW _E4 phages was
pe o med on he APEC 158B lux hos s ain. Phages we e ampli ied
using a me hod desc ibed elsewe e (Ku´
zmi´
nska-Bajo e al., 2021).
B ie ly, bac e ial cul u es we e inocula ed on 10 ml o LB b o h wi h a
single colony o APEC 158B lux, ollowed by o e nigh incuba ion a
37◦C wi h shaking a 150 pm. A e incuba ion, 0.5 ml o o e nigh
cul u e was inocula ed in o 10 ml o LB b o h and incuba ed un il he
op ical densi y (OD
600 nm
) eached 0.2. In he nex s ep, he bac e ial
cul u e was cen i uged o 10 min a 5000 ×g o emo e any emaining
cell deb is and il e ed h ough 0.22 µm po e size sy inge il e s. The
5 ml o esul ing phage lysa e, om he i s s ep o p opaga ion, was
added o 150 ml o hos cul u e (OD
600 nm
=0.2) and incuba ed o e -
nigh a 37◦C. As a las s ep, he cen i uga ion and il a ion s eps we e
epea ed. Bac e iophage i e was de e mined using he ou ine es
dilu ion me hod (Adams, 1959). As a phage mix u e, cock ail
UPW _E124 con aining phages UPW _E1, UPW _E2, and UPW _E4 was
o mula ed by combining hem in an equal a io o 1:1:1.
2.4. UPW _E124 su i al in simula ed gas ic luid condi ions
In o de o de e mine he s abili y o bac e iophages in condi ions
imi a ing chicken gas ic juice, simula ed gas ic luid (SGF) was used o
es phage sensi i i y o in agas ic condi ions acco ding o a me hod
desc ibed p e iously (Tang e al., 2013). The expe imen was pe o med
in SGF a pH 2 ep esen ing na u al low pH s omach acidi y and SGF
neu alized by he addi ion o 14 % CaCO
3
as an an acid e lec ing he
neu alizing e ec o he eed p esence in he s omach. One millili e o
he 10
8
plaque- o ming uni s (PFU) pe 1 ml o UPW _E124 phage
cock ail was added o 9 ml o p e-wa med SGF and incuba ed a 42◦C,
ep esen ing he chicken’s body empe a u e, o 2 h. Samples o 100 µl
we e aken e e y 15 minu es o measu e he su i al a e o phages. To
measu e he phage i e , samples we e en old se ial dilu ed in LB me-
dium and spo ed on he lawn o APEC 158B lux o de e mine he
numbe o bac e iophages using he double-laye pla e me hod. The
pla es we e incuba ed a 37◦C o e nigh and obse ed o he o ma ion
o plaques. This expe imen was ca ied ou in iplica e.
D.S. Mo eno e al.
Ve e ina y Mic obiology 302 (2025) 110398
2
2.5. Bio ilm- o ming abili y
To de e mine he abili y o he APEC 158B s ain o o m bio ilms, a
me hod desc ibed elsewhe e (Ko zeniowski e al., 2022) was used and
E. coli s ain NCTC 17848 exhibi ing high bio ilm- o ming capabili y
was used as a con ol. Fo his pu pose, o e nigh APEC 158B and NCTC
17848 cul u es we e dilu ed in LB b o h o OD 0.2 (~2 ×10
8
colony- o ming uni s (CFU/ml) and 200 µl o his suspension was
ans e ed o each well o 96-well polys y ene mic o i e pla es (Sa -
s ed , Ge many). Pla es we e hen incuba ed a 37◦C o 72 h o allow
bio ilm o ma ion. To emo e plank onic cells, wells we e washed wice
wi h s e ile phospha e saline bu e (PBS; Sigma-Ald ich, Ge many).
Cells g owing in bio ilms we e quan i ied by s aining wi h 0.5 % c ys al
iole (Me ck, Ge many) o 20 min ollowed by insing wo imes wi h
PBS. C ys al iole was dissol ed using 96 % e hanol (Sigma-Ald ich,
Ge many). The abso bance o he eleased colo was measu ed using an
au oma ed mic o i e pla e eade (Spa k Tecan, Swi ze land) a
570 nm. The expe imen s we e pe o med independen ly h ee imes.
2.6. Bio ilm o ma ion on chicken b eas
The abili y o APEC 158B lux and E. coli NCTC 17848 bac e ia o
o m a bio ilm on he su ace o poul y mea was de e mined acco ding
o Vik am e al. 2020 wi h mino modi ica ions. Fo his pu pose, he
chicken b eas was s e ilized wi h ul a iole ligh , being u ned wice
du ing he s e iliza ion and es ed o s e ili y ollowing he same p o-
cedu e desc ibed below excep o he imme sion in bac e ial cul u es.
Nex , he chicken b eas was cu in o pieces measu ing 1 cm
3
wi h a
s e ile scalpel. Pieces o chicken b eas we e imme sed o 120 seconds
in 5 ml o he o e nigh cul u es o APEC 158B lux and E. coli NCTC
17848, p e iously dilu ed wi h LB medium o ob ain an op ical densi y
o 0.2 (~2 ×10
8
CFU/ml) measu ed a a wa eleng h o 600 nm
(OD
600 nm
) in a 50 ml Falcon ube. Then, pieces o chicken b eas we e
d ied a oom empe a u e o 20 minu es ollowing imme sion o
2 minu es in a suspension o UPW _E124 phage cock ail wi h a i e o
1 ×10
9
PFU/ml and d ied o 20 minu es a oom empe a u e. Pieces o
mea we e placed in a Pe i dish con aining s e ile pape soaked in PBS
bu e o main ain high humidi y, p o ec ed wi h pa a ilm and incu-
ba ed a 4◦C un il he 7 h day a e inocula ion and placed a 37℃ o
24 hou s p o iding a o able condi ions o bac e ial g ow h. A e
inocula ion, on days 1, 2, 5, 7 du ing incuba ion a 4◦C and on day 8
a e shi ing o g ow h-pe mi ing condi ions, 4 pieces o chicken b eas
we e placed in Falcon ubes con aining 2 ml o PBS bu e and homog-
enized using a Qiagen TissueLyse II. A e homogeniza ion, he numbe
o E. coli was de e mined by pla ing homogena es in en old se ial di-
lu ions on LB aga . Pla es we e incuba ed a 37◦C o e nigh and he
numbe o colonies was coun ed. The esul s a e p esen ed as he
numbe o CFU pe 1 cm
3
o mea .
2.7. In i o an ibac e ial ac i i y assay o phage cock ail UPW _E124 in
he mouse model
The po en ial o educ ion o APEC- a ge ing phage cock ail
UPW _E124 was examined in BALB/c emale mice (Mossakowski Med-
ical Resea ch Cen e Polish Academy o Sciences, Wa saw, Poland)
expe imen ally in ec ed wi h APEC 158B lux. All expe imen al wo k
applying he mouse model o in ec ion was app o ed by he Local
E hical Commi ee o Animal Expe imen a ion (p o ocol code 115/
2015; W ocław, Poland) and pe o med acco ding o he legal e-
qui emen s. Mice a age 6–8 weeks we e di ided in o 7 expe imen al
g oups, as shown in Table 1, and 10 mice we e assigned o each g oup. In
his s udy, 2 ways o APEC and phage cock ail applica ion such as gas ic
ga age di ec ly in o he s omach wi h he ga age needle and in ape -
i oneal injec ion we e examined. Mice in posi i e con ols we e chal-
lenged wi h APEC 158B lux in 100 µl o phospha e bu e saline (PBS) a
doses o 10
9
and 10
7
CFU/mouse by gas ic (g oup 1) and
in ape i oneal (g oup 4) ga age, espec i ely. Two g oups we e ea ed
wi h he UPW _E124 phage cock ail a a dose o 10
9
PFU in 100 µl o PBS
pe animal by gas ic (g oup 2) and in ape i oneal (g oup 5) ga age.
Bo h g oups 2 and 5 we e nega i e con ols wi hou he bac e ia. Mice in
g oup 3 we e in ec ed wi h APEC 158B lux a a dose o 10
9
CFU/mouse
by gas ic ga age and immedia ely gas ically ea ed wi h 10
9
PFU o
he UPW _E124 phage cock ail. Simila ly, mice in g oup 6 we e in ec ed
by in ape i oneal injec ion wi h 10
7
CFU o APEC 158B lux and 10
9
PFU
o he UPW _E124 phage cock ail pe mouse. G oup 7 did no ecei e
ei he APEC 158B lux o he UPW _E124 phage cock ail. The summa y
o he challenge and ea men o each g oup is shown in Table 1. A e
24 hou s, all animals we e weighed and humanely killed by ce ical
disloca ion, and all es animals we e subjec ed o g oss nec opsy and
dissec ed. Thei lungs, hea s, kidneys, li e s, and spleens we e analyzed
o he p esence o APEC 158B lux and phages. Fo bac e ial numbe
de e mina ion, he o gans we e homogenized in cold PBS, p ope ly
en old dilu ed, and pla ed on LB aga wi h 0.2 mg/ml e y h omycin,
and pla es we e incuba ed o e nigh a 37◦C. The bioluminescen sig-
nals o APEC 158B lux CFU we e coun ed using he Nigh OWL II LB 983
In Vi o imaging Sys em (BERTHOLD TECHNOLOGIES, Ge many). Fo
phage enume a ion issue homogena es we e cen i uged a 4 000 g o
emo e solid pa icles and hen supe na an s we e il e ed h ough
0.22 µm il e s. The phage i a ion was es ima ed using a double aga
laye plaque assay me hod. The mean CFU and PFU pe 1 g o each issue
we e calcula ed.
2.8. Bac e iophage he apy ials in he expe imen al chicken model
Fo he in i o expe imen s on chickens, 7-day-old Ross 308 b oile s
ob ained om a local a m we e di ided in o ou g oups (10 bi ds/
g oup) and housed sepa a ely in wi e cages a an ambien empe a u e
o 30◦C. S e ile chicken eed (B oile G owe II, Tasomix, Poland) and
wa e we e p o ided ad libi um. All expe imen al wo k in ol ing bi ds
was app o ed by he Local E hics Commi ee o Animal Expe imen a-
ion (p o ocol code 114/2015; W ocław, Poland). G oup 1 was a posi i e
con ol o APEC 158B lux in ec ion and bi ds we e challenged in a-
acheally wi h 10
8
CFU o APEC 158B lux in 1 ml o PBS pe bi d. G oup
2 ecei ed only he UPW _E124 phage cock ail a 1 and 5 days o he
expe imen . G oup 3 was in ec ed wi h 10
8
CFU/ml o APEC 158B lux
pe bi d and immedia ely inocula ed wi h 10
9
PFU o UPW _E124 phage
cock ail. A 5 days a e in ec ion bi ds om g oup 3 we e ea ed wi h
he second dose o UPW _E124 phage cock ail. G oup 4 was kep as an
unin ec ed con ol (Table 2). On he 10 h day a e in ec ion, all bi ds
Table 1
G oups o mice ecei ing UPW _E124phage cock ail a e being challenged wi h
APEC 158B lux.
G oup (n ¼10) Doses Ga age
UPW _E124
[PFU]
APEC 158B lux
[CFU]
1
a
- 1 ×10
9
gas ic
2
b
1 ×10
9
- gas ic
3
c
1 ×10
9
1 ×10
9
gas ic
4
d
- 1 ×10
9
in ape i oneal
5
e
1 ×10
7
- in ape i oneal
6
1 ×10
7
1 ×10
9
in ape i oneal
7
g
- - -
a
Posi i e con ol, gas ically in ec ed wi h E. coli 158B lux
b
Nega i e con ol, gas ically bac e iophage- ea ed
c
G oup gas ically in ec ed wi h APEC 158B lux and ea ed wi h UPW _E124
phage cock ail by gas ic ga age
d
Posi i e con ol, in ec ed wi h E. coli 158B lux by in ape i oneal injec ion
e
Nega i e con ol, bac e iophage- ea ed by in ape i oneal injec ion
G oup in ec ed and ea ed by in ape i oneal injec ion wi h APEC 158B lux
wi h UPW _E124 phage cock ail
g
Un ea ed g oup
D.S. Mo eno e al.
Ve e ina y Mic obiology 302 (2025) 110398
3
we e nec opsied and APEC 158B lux and bac e iophages in he in e nal
issues o e e y bi d we e coun ed. The lungs, spleen, li e , and bu sa o
Fab icius we e emo ed asep ically and blood samples we e aken. The
bac e ial and phage loads we e assessed by es ima ing he numbe o
CFU and PFU, espec i ely. The cecal onsils and cecal con en s we e
weighed and homogenized in 0.2 ml o PBS. The bu sa o Fab icius,
spleen, and li e we e weighed a e washing wi h PBS, and each o gan
was homogenized wi h 5, 5 and 50 ml o cold PBS, espec i ely. Ten- old
dilu ions o homogena es we e pla ed on o LB aga con aining e y h-
omycin (0.2 mg/ml) and pla es we e incuba ed o e nigh a 37◦C.
Iden i ica ion o APEC 158B lux was ca ied ou by bioluminescence
imaging and luminescen colonies we e coun ed using bioluminescence
imaging. The i e o he UPW _E124 phage cock ail was de e mined by
coun ing plaques o med on he app op ia e bac e ial s ain. The mean
CFU and PFU pe 1 g o each issue and pe 1 ml o blood we e
calcula ed.
2.9. S a is ical analysis
All s a is ical es s we e pe o med on log
10
ans o med da a using
STATISTICA e sion 13 so wa e (TIBCO So wa e Inc.). S uden ’s - es
was employed o s a is ical analysis o da a ob ained om in i o assay
in simula ed gas ic juice. Da a ob ained om he chicken b eas mea
bio ilm assay we e analyzed by applying ANOVA along wi h he leas
signi ican di e ence (LSD) pos -hoc es . Coloniza ion o in e nal o -
gans in bo h mouse and chicken models was analyzed using he Man-
n–Whi ney U es wi h he non-Gaussian dis ibu ion. A p- alue o
<0.05 was conside ed o be signi ican . All da a we e analyzed using
wo- ailed es s. To compa e bac e ial load (CFU) and phage load (PFU)
eco e ed om chickens’ in e nal o gans he nonpa ame ic Mann-
Whi ney U- es was employed.
3. Resul s
3.1. Deg ada ion o E. coli bio ilm o med on chicken mea
The e ec o he UPW _E124 phage cock ail in he deg ada ion o
bio ilm o med on chicken b eas was es ed using he bac e ial s ains
APEC 158B lux and E. coli NCTC 17848. These s ains we e classi ied as
mode a e and s ong bio ilm p oduce s, espec i ely, acco ding o he
classi ica ion sugges ed by S epano ic e al. (2004). E. coli NCTC 17848
was ound o ha e app oxima ely 2.8 imes g ea e abili y o o m a
bio ilm han APEC 158B lux (Supplemen a y Ma e ial 1).
As depic ed in Fig. 1, a e ea men o he bio ilms wi h he
UPW _E124 phage cock ail, he iabili y o bo h s ains was signi ican ly
educed ela i e o un ea ed con ols (p <0.01) a 1, 2, 5, 7 and 8 days.
The un ea ed bio ilm o med by APEC 158B lux was s able om day
1–7 and was es ima ed a a ound 5.1 and 5.2 log
10
CFU/cm
3
, espec-
i ely (Fig. 1). A e ea men om he i s day o he se en h day, he
UPW _E124 phage cock ail educed he numbe o APEC 158B lux by
1.0–1.4 log
10
CFU/cm
3
. On he eigh h day, he pieces o chicken b eas
we e incuba ed a 37◦C o allow APEC e-g ow h. On ha day, un ea ed
bio ilm biomass inc eased o 5.9 log
10
CFU/cm
3
, while he phage-
ea ed bio ilm biomass was main ained a 4.2 log
10
CFU/cm
3
Table 2
Chicken expe imen al design. UPW _E124 phage cock ail and APEC 158B lux
we e adminis e ed di ec ly o he c op.
G oup
(N ¼10)
Doses T ea men schedule o
bac e iophages [dpi]
e
UPW _E124 APEC 158B
lux
1
a
- 2 ml
1 ×10
8
CFU/ml
-
2
b
1 ml
10
9
PFU/ml
- 1, 5
3
c
2 ml
3 ×10
10
PFUml
2 ml
1 ×10
8
CFU/ml
1, 5
4
d
- - -
a
Posi i e con ol, in ec ed wi h APEC 158B lux
b
G oup in ec ed wi h APEC 158B lux and bac e iophage ea ed
c
Nega i e con ol, bac e iophage ea ed
d
Un ea ed g oup
e
dpi – days pos -in ec ion
Fig. 1. E ec o UPW _E124 phage cock ail on bio ilm on chicken b eas mea . E ec o UPW _E124 phage cock ail on educ ion o bio ilm o med by APEC 158B,
and e ec o UPW _E124 phage cock ail on educ ion o bio ilm o med by E. coli NCTC 17848. The b eas mea was s o ed a 4◦C o 7 days and hen a 37◦C o
24 hou s. * ep esen s p<0.01 and indica es a signi ican di e ence. Values ep esen he mean wi h a s anda d de ia ion o h ee eplica es.
D.S. Mo eno e al.
Ve e ina y Mic obiology 302 (2025) 110398
4
(p <0.01). In he absence o he UPW _E124 phage cock ail, he numbe
o iable E. coli NCTC 17848 was s able o 5 days and was es ima ed o
be be ween 4.9 and 5.0 log
10
CFU/cm
3
(Fig. 1). A e 7 days o s o age a
4◦C he numbe o E. coli NCTC 17848 inc eased o 5.8 log
10
CFU/cm
3
.
In phage- ea ed E. coli NCTC 17848 bio ilm, he numbe o bac e ia was
e ec i ely main ained a a lowe le el be ween 3.7 (day 1) and 3.8 (day
7) log
10
CFU/cm
3
in compa ison o un ea ed bio ilm. On he eigh h day,
when pieces o un ea ed chicken b eas we e ans e ed o 37◦C, he
numbe o un ea ed bac e ia apidly inc eased by 1.8 log
10
CFU/cm
3
.
Unde he same condi ions, he numbe o E. coli NCTC 17848 on mea
ea ed wi h he UPW _E124 phage cock ail was signi ican ly lowe han
ha obse ed in he un ea ed con ol, wi h a di e ence o 1.3 log
10
CFU/cm
3
. S e ili y es s o chicken b eas con i med he s e ili y o he
assay and he s e iliza ion p ocess. No bac e ial g ow h was de ec ed in
chicken b eas s ha we e no inocula ed wi h bac e ia.
3.2. Assessmen o UPW _E124 phage cock ail su i al in he SGF model
The UPW _E124 phage cock ail’s abili y o endu e in agas ic con-
di ions was es ed in i o wi h and wi hou he addi ion o an an acid, a
42◦C, co esponding o he chicken’s deep body empe a u e. The
phages we e a ec ed by he low pH and he pepsin ac i i y o SGF wi h
he educ ion o 3.6 log
10
CFU a e 15 minu es o incuba ion and we e
comple ely inac i a ed ollowing 30 min o incuba ion (Supplemen a y
ma e ial 2). Howe e , he addi ion o he an acid 14 % CaCO
3
esul ed
in comple e neu aliza ion o he inac i a ion e ec o SGF condi ions on
phages, which we e s able o e he 120 minu es es ed (p<0.01).
3.3. UPW _E124 e icacy in he mouse model
To e alua e he in i o e ec i eness o he UPW _E124 phage cock-
ail, a mouse model in ec ed wi h APEC by gas ic ga age and in a-
pe i oneal injec ion was used. Rega dless o he ou e o adminis a ion,
o all eu hanized mice in ec ed wi h APEC 158B lux, no clinical
symp oms o bac e ial in ec ion we e de ec ed. Acco ding o all he e-
sul s p esen ed in Fig. 2, in compa ison o gas ic ga age (g oups 1 and
2), in ape i oneal injec ion o APEC 158B lux (g oups 3 and 4) esul ed
in a signi ican ly highe numbe o colonized o gans such as lungs, hea ,
kidney, li e and spleen, accompanied by he la ge inc eased bac e ial
load o all es ed o gans (p>0.05).
Using gas ic ga age o he adminis a ion o he UPW _E124 phage
cock ail, we ound a signi ican educ ion in he coloniza ion o lungs by
APEC in mice ha ecei ed he UPW _E124 phage cock ail (g oup 3)
(Fig. 2A) compa ed o he un ea ed g oup 1. I also esul ed in a
educ ion o he numbe o o gans colonized by APEC 158B lux
including a dec eased mean numbe o bac e ia isola ed om o gans
such as he hea , kidney, li e and spleen by 0.6–1.1 log
10
CFU/g.
Howe e , hese di e ences we e no s a is ically signi ican (p>0.05).
The highes o gan coloniza ion was eco ded o spleens and he lowes
in li e s, as depic ed in Fig. 2B.
Adminis e ing he UPW _E124 phage cock ail in ape i oneally, he
bac e ial bu den in all es ed o gans showed compa able le els o APEC
158B lux load and no s a is ically signi ican di e ence was de ec ed in
ea ed o un ea ed mice (p>0.05) (Fig. 2B).
Phages composing he UPW _E124 phage cock ail we e de ec ed
only in o gans aken om mice in ec ed and phage- ea ed in ape i o-
neally (g oup 2) and he PFU we e es ima ed o be 1.9 ±1.36 log
10
PFU
in lungs, 1.8 ±1.67 log
10
PFU in hea s, 3.6 ±0.24 log
10
PFU in kid-
neys, 2.3 ±0.45 log
10
CFU in spleens and 48.1 ±1.97 log
10
CFU in
li e s wi h 4, 1, 4, 2 and 10 phage-posi i e o gans, espec i ely.
3.4. UPW _E124 phage cock ail e ec i eness in expe imen al chicken
model
To e alua e he e ec i eness o he UPW _E124 phage cock ail in
poul y we used b oile chickens expe imen ally in ec ed wi h APEC
158B lux. We examined he APEC 158B lux load in he lungs, li e and
lymphoid o gans such as he spleen, he bu sa o Fab icius, and in he
blood a 10 days a e in ec ion. In he phage- ea ed g oup, a signi ican
educ ion in APEC numbe was de ec ed in he li e , bu sa o Fab icius
and blood (p<0.05) when compa ed wi h un ea ed bi ds (Fig. 3A).
The APEC numbe s in he lungs and spleen o phage- ea ed chickens
we e lowe by 3.04 and 2.88 log
10
CFU/g, espec i ely, in compa ison o
he un ea ed g oup, bu his di e ence was no s a is ically signi ican
(p>0.05).
In compa ison o chickens om he unin ec ed g oup ecei ing only
phages, he numbe o phages isola ed om bi ds om he phage- ea ed
g oup was signi ican ly highe in he li e (p<0.05), bu no in he
o he o gans (p>0.05). In blood samples, we also de ec ed he phage
i e in samples aken om bi ds om he APEC-in ec ed g oup and
unin ec ed g oup ecei ing he UPW _E124 phage cock ail, wi h mean
alues o 1.06 and 0.42 log
10
PFU/g, espec i ely, bu hese di e ences
we e no s a is ically signi ican (p>0.05) (Fig. 3B). In e nal o gan
samples o con ol bi ds om unin ec ed and un ea ed g oups we e
Fig. 2. E ec o phage cock ail UPW _E124 on APEC 158B lux numbe in in e nal o gans in an expe imen al mu ine model. Bac e ial loads a e shown as coun s o
indi idual animals ea ed by gas ic ga age plus he mean (n=10 pe g oup) o con ol g oup 1 (ci cles) in ec ed wi h APEC 158B lux and g oup 2 (diamonds)
in ec ed wi h APEC 158B lux ea ed wi h phage cock ail UPW _E124 (A). Bac e ial loads a e shown as coun s o indi idual animals ea ed by in ape i oneal
injec ion plus he mean (n=10 pe g oup) o con ol g oup 3 (ci cles) ea ed wi h phage cock ail UPW _E124 and g oup 4 (diamonds) in ec ed wi h APEC 158B lux
ea ed wi h phage cock ail UPW _E124 (B). * ep esen s p<0.05 and indica es a signi ican di e ence be ween g oups, n.s., no signi ican .
D.S. Mo eno e al.
Ve e ina y Mic obiology 302 (2025) 110398
5
nega i e o bo h bac e iophages and APEC, indica ing ha no
con amina ion occu ed du ing he expe imen al se -up.
4. Discussion
Add essing he economic and public heal h impac s o APEC in-
ec ions and hei p esence on poul y p oduc s equi es a comp ehen-
si e app oach ha in eg a es p e en i e measu es, su eillance, and
a ge ed in e en ions such as he use o phages as a species-speci ic
na u ally exis ing an ibac e ial ool (Oli ei a e al., 2010; Hu e al.,
2004; Mosimann e al., 2021). In his s udy, we i s in es iga ed he
abili y o he UPW _E124 phage cock ail o inhibi APEC g owing in
bio ilm s uc u es on he su ace o chicken b eas mea . We obse ed a
signi ican educ ion in he le el o li e APEC using he UPW _E124
phage cock ail. This phage cock ail no only dec eased he numbe o
APEC 158B lux a e phage applica ion bu also p e en ed u he
g ow h o bac e ia, bo h in e ige a ed condi ions and g ow h- a o ing
empe a u es. The UPW _E124 phage cock ail exhibi ed highe e ec-
i eness in he educ ion o E. coli on he mea su ace in compa ison o
he comme cial EcoShield PX phage cock ail agains he E. coli O157:H7
s ain p esen on he su ace o he chicken ille by 0.7 log
10
CFU
(Vik am e al., 2020). UPW _E124 educ ion is compa able o phages
PBL66-CL1 and PBL116-CS6, which educed he numbe o E. coli s ain
EBL116 on he su ace o poul y mea by 2.02 and 1.67 log
10
CFU/4
cm
2
o mea pieces a e 6 h a 25◦C and 5◦C, espec i ely (Hoang Minh
e al., 2016). Impo an ly, he e a e esul s indica ing ha an i-E. coli
phages e ec i ely educe he bac e ia on mea . Phage CEH-162 was
used agains E. coli EH-162 on he su ace o minced chicken mea ,
demons a ing no inhibi ion o bac e ial g ow h a 4◦C o 7 hou s
(Jassim e al., 2012). These esul s could be explained by he speci ic
s uc u e o minced mea ha could a o he g ow h o bac e ia, o by
he lowe phage e ec i eness. In summa y, we demons a ed he po-
en ial o he UPW _E124 phage cock ail and he use o ly ic phages as an
e ec i e bac e icide o E. coli con olling on he su ace o aw chicken
mea .
Fo success ul phage he apy ea men , phages need o each bac-
e ia a he in ec ion si e and es ablish a p oduc i e in ec ion in su i-
cien numbe s o educe bac e ial load. While many in i o phage s udies
ha e shown g ea an i-APEC po en ial (Oli ei a e al., 2010, 2009b; Hu
e al., 2003), he e ha e been some ailed a emp s (Tsonos e al., 2014)
ha highligh he impo ance o p ope selec ion o APEC- a ge ing
phages ha e ec i ely comba hese bac e ia in i o. Thus, i is
ex emely impo an o comple e in i o esul s wi h in i o da a.
In i o, phages should wi hs and he p o eoly ic en i onmen and
low pH o he gas ic juice (Colom e al., 2017). Howe e , phages in he
UPW _E124 cock ail comple ely lose hei abili y o in ec APEC 158B
lux a e incuba ion in simula ed gas ic condi ions. Simila esul s we e
ob ained by o he phages eco e ed a e exposu e o SGF
(Ku´
zmi´
nska-Bajo e al., 2023; Mhone e al., 2022). To p e en hei
inac i a ion, we added calcium ca bona e, which esul ed in a p o ec-
i e e ec on phage ac i i y, con i ming o he s udies (Koo e al., 2000;
Yongsheng e al., 2008). In ac , we ha e p e iously epo ed ha he
p esence o chicken eed in SGF had a compa able neu alizing e ec on
phage su i abili y o calcium ca bona e (Ku´
zmi´
nska-Bajo e al., 2023),
sugges ing ha he p esence o eed in he s omach e lec ing na u al
condi ions allows he main enance o he UPW _E124 phage cock ail’s
ac i i y when adminis e ed o ally.
To unequi ocally es ablish he sa e y and sui abili y o using APEC-
a ge ing phages in poul y-in ensi e p oduc ion, comp ehensi e and
mul i ace ed s udies a e essen ial. To assess he e ec i eness o he
phage cock ail in i o, we sough o de ine he e ec i eness o he
me hod o adminis a ion pe o ming in i o s udies on mice expe i-
men ally in ec ed wi h APEC 158B lux and ea ed wi h he UPW _E124
phage cock ail by in ape i oneal injec ion and by gas ic ga age. The
mos impo an inding is ha he UPW _E124 phage cock ail adminis-
e ed by gas ic ga age comple ely e adica ed APEC 158B lux om he
lungs, an APEC- a ge ed o gan in colibacillosis. These esul s suppo
ou assump ion ega ding he abili y o phages o o e come ha sh
gas ic condi ions. In o gans such as he hea , spleen, li e , and kidney
he impac o he UPW _E 124 phage cock ail on bac e ial load was
insigni ican in bo h models o phage adminis a ion es ed. Mice
in ec ed wi h APEC 158B lux showed no clinical signs o in ec ion
ega dless o he ou e o adminis a ion o he bac e ia, wi h he bac-
e ial load o in e nal o gans es ima ed o be signi ican ly highe o
in ape i oneal in ec ion compa ed o gas ic ga age. The coloniza ion
ha we ound in in e nal o gans o mice a e in ape i oneal APEC
adminis a ion is compa able o ha ound by Wang e al. (2021) and
he APEC load in blood, b ain, and lungs, a le els es ima ed o be
10
8
–10
9
CFU/g a 12 hou s a e in ec ion. Con a y o APEC 158B lux,
in his s udy, such accumula ion o APEC was accompanied by clinical
signs o u ina y ac in ec ion con i ming he close ela edness o APEC
wi h UPEC. Yao e al. (2023) epo ed high mouse mo ali y (83.3 %)
Fig. 3. E ec o phage cock ail UPW _E124 on APEC 158B lux numbe in in e nal o gans in an expe imen al chicken model. Resul s o bac e ial load a e shown as
coun s o indi idual animals plus he mean (n=10 pe g oup) o con ol g oup 1 in o gans (emp y ci cles) and blood (black ci cles) in ec ed wi h APEC 158B lux and
g oup 3 in o gans (emp y diamonds) and blood (black diamnds) in ec ed wi h APEC 158B lux ea ed wi h phage cock ail UPW _E124 (A). Resul s o phage load a e
shown as coun s o indi idual animals plus he mean (n=10 pe g oup) o o gans (emp y ci cles) and blood (black ci cles) o con ol g oup 2 ea ed wi h phage
cock ail UPW _E124 and o gans (emp y diamonds) and blood (black diamonds) o g oup 3 in ec ed wi h APEC 158B lux ea ed wi h phage cock ail UPW _E124 (B).
* ep esen s p<0.05 and indica es a signi ican di e ence be ween g oups, n.s., no signi ican .
D.S. Mo eno e al.
Ve e ina y Mic obiology 302 (2025) 110398
6
wi hin 24 hou s a e in ape i oneal injec ion o APEC s ain AH50,
sugges ing a g ea e in ec ion po en ial han ha o APEC 158B lux.
Mice mo ali y was signi ican ly educed by 66.7 and 83.3 % by ea -
men wi h 10
8
PFU/ml phage PEC9 a MOI=1 a 6 and 12 hou s a e
in ec ion, espec i ely. Bac e ial load in he spleens o in ec ed and
phage- ea ed mice was signi ican ly lowe by 1 log
10
CFU/g in com-
pa ison o un ea ed mice. These disc epancies in APEC’s abili y o
in ade mouse in e nal o gans and induce clinical symp oms could be
explained by he s ong dependency o he in ec ion on pa icula APEC
s ains, especially conside ing ha APEC s ains speci ically in ec
poul y and canno be expec ed o beha e iden ically in mice and
chickens.
Se e al s udies ha e managed o ansla e hei in i o indings o in
i o condi ions and ha e epo ed he success ul ea men o APEC
(Oli ei a e al., 2010; Jhandai e al., 2024; So ou e al., 2020). The mos
common modes o adminis a ion o phages agains APEC ha e been
sp aying phages on su aces, o al adminis a ion, and injec ion,
including in a enous, in amuscula , and in ape i oneal ou es. We
u he de eloped ou esea ch on applica ion o he UPW _E124 phage
cock ail o assess a ious aspec s o phage ac i i y, pa icula ly i s
e ec i eness in a ge ing APEC in chickens. Ou esul s om in i o
mouse s udies indica ed ha he UPW _E124 phage cock ail po en ially
can elimina e APEC om he espi a o y sys em. In ac , we con i med in
chickens he esul s om s udies on mice, whe e he bac e ial load in he
lungs o APEC-posi i e b oile s was signi ican ly educed in he
phage- ea ed g oup o1.5 ±0.7 CFU/g in compa ison o 8.8 ±3.0
CFU/g in he un ea ed g oup. The numbe o chickens posi i e o APEC
was simila in ea ed and un ea ed g oups, bu s ill UPW _E124 phage
cock ail ea men o chickens esul ed in a signi ican educ ion o
bac e ial load in he li e , bu sa o Fab icius and blood. These esul s
show he e icien inhibi ion o APEC eplica ion in lungs and o he
o gans by he UPW _E124 phage cock ail. This e ec is compa able o
o he s udies using phage cock ails adminis e ed h ough an
in a- acheal ou e ha also signi ican ly educed APEC load in he
li e , lungs, and hea in b oile s (Oli ei a e al., 2009b; Tawakol e al.,
2019). O he au ho s ha e also epo ed e ec i e APEC educ ion by
phages a e applica ion ia in a enous (Hu e al., 2005) and in a-
muscula (Tang e al., 2023) injec ion. Local applica ion o bac e io-
phages di ec ly a he si e o APEC in ec ion, pa icula ly h ough
in a-ai sac adminis a ion, could be an e ec i e s a egy o
comba ing he disease. This app oach allows a high concen a ion o
bac e iophages o a ge he bac e ial in ec ion a he si e o in ec ion, in
a eas ha may be ela i ely inaccessible ia he ci cula o y sys em,
inc easing he e ec i eness o ea men (Hu e al., 2006). This local-
ized deli e y me hod could po en ially imp o e ea men ou comes and
educe he isk o sys emic side e ec s and is applicable in la ge-scale
poul y p oduc ion. None heless, he e a e se e al limi a ions o he
s udy ha could ha e biased ou in e p e a ion o he esul s. One key
limi a ion is he use o a mu ine model o s udying a ian pa hogens,
which may no ully eplica e he APEC in ec ion and APEC-phage in-
e ac ions seen in a ian species. This migh ha e a ec ed he obse ed
immune esponses o APEC beha io and phage e ec i eness, po en-
ially leading o esul s ha di e om hose seen in a mo e sui able
a ian model. Tha is why we also es ed he phages in chicken. Addi-
ionally, as a p oo o concep in his s udy, UPW _E124 phage cock ail
was used o ea chickens in ec ed wi h APEC 158B lux. Howe e , APEC
and phages we e adminis e ed in a acheally o ully con ol he con-
cen a ion o bac e ia and phages. A he a m le el, an i-colibacillosis
phages should be adminis e ed by sp ay o wi h d inking wa e .
Despi e he limi a ions o he s udy, we belie e ha ou animal ial da a
a e impo an o unde s anding he po en ial o use o phages agains
colibacillosis in b oile chickens as an al e na i e o an ibio ic ea -
men . Fu he mo e, in es iga ing he impac o phage on p e en ing
e ical ansmission o APEC wi hin comme cial locks is essen ial o
assessing hei u ili y in chicken b eeding.
5. Conclusions
This s udy e ealed he UPW _E124 phage cock ail o be an e ec i e
ool in comba ing APEC in b oile chickens. The poly alen UPW _E124
phage cock ail can main ain ac i i y a e neu aliza ion o he acid pH
o SGF. We ound ha he phage cock ail UPW _E124 was e ec i e in
comba ing APEC in chicken mea bio ilms in s o age condi ions and a
empe a u es a o ing bac e ial g ow h. We p o ed he UPW _E124
phage cock ail’s e icacy in educing he APEC bu den in he lungs in he
mu ine model o in ec ion. High an i-APEC e ec i eness was also
co obo a ed in expe imen ally in ec ed b oile chickens. Thus, hese
obse a ions suppo he inclusion o he UPW _E124 phage cock ail
among e ec i e biocon ol agen s agains APEC.
CRediT au ho ship con ibu ion s a emen
Da id S´
aez Mo eno: W i ing – o iginal d a , Me hodology, In es-
iga ion. Kuczkowski Maciej: Visualiza ion, So wa e, Me hodology,
In es iga ion. Ko zeniowski Paweł: In es iga ion. G zymajło K zysz-
o : Me hodology, In es iga ion. Wo´
zniak-Biel Anna: In es iga ion.
´
Sliwka Paulina: In es iga ion. Rywi´
nska Ani a: In es iga ion.
Ku´
zmi´
nska-Bajo Ma a: W i ing – e iew & edi ing, P ojec admin-
is a ion, Me hodology, Funding acquisi ion, Da a cu a ion,
Concep ualiza ion.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgmen s
We hank Jus yna Dubiel-Pieczy´
nska o he echnical assis ance.
This s udy was unded by he Na ional Cen e o Resea ch and De el-
opmen , LIDER p og am no. LIDER/378/L-6/14/NCBR/2015. The APC/
BPC is co- inanced by W ocław Uni e si y o En i onmen al and Li e
Sciences.
Au ho s’ con ibu ions
DSM p o ided assis ance h oughou he s udy on he mouse model
and conduc ed mic obiological analysis o e mina ed mice and was a
majo con ibu o o w i ing he manusc ip ; MK pe o med and su-
pe ised in i o ials in he chicken model and pe o med he s a is ical
analysis; PK pe o med he analysis o phages in he SGF model on
bio ilm, and p o ided assis ance h oughou he s udy on he chicken
model; KG pe o med and supe ised he s udy on he mouse model;
AW-B pe o med mouse nec opsy and ma e ial sampling; PS assis ed in
he s udy on mice; AR conduc ed mic obiological analysis o e mina ed
chickens; MK-B concei ed and supe ised he s udies, had subs an ial
inpu s in o he analysis and all d a s, ob ained unding, and was a
con ibu o o w i ing he manusc ip . All au ho s con ibu ed o he
a icle and app o ed he submi ed e sion.
Appendix A. Suppo ing in o ma ion
Supplemen a y da a associa ed wi h his a icle can be ound in he
online e sion a doi:10.1016/j. e mic.2025.110398.
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