Food Resea ch In e na ional 187 (2024) 114308
A ailable online 18 Ap il 2024
0963-9969/© 2024 The Au ho (s). Published by Else ie L d. 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/).
Pheno ypic and geno ypic cha ac e iza ion o an imic obial esis ances
e eals he e ec o he p oduc ion chain in educing esis an lac ic acid
bac e ia in an a isanal aw ewe milk PDO cheese
Go ka San ama ina-Ga cía
a
,
c
,
d
,
*
, Gus a o Amo es
a
,
c
,
d
, Diego Llamaza es
a
, Igo He n´
andez
a
,
c
,
d
,
Luis Ja ie R. Ba on
b
,
d
, Mailo Vi o
a
,
c
,
d
a
Lac ike Resea ch G oup, Depa men o Biochemis y and Molecula Biology, 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
b
Lac ike Resea ch G oup, Depa men o Pha macy and Food Sciences, 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
c
Bioa aba Heal h Resea ch Ins i u e-P e en ion, P omo ion and Heal h Ca e, 01009 Vi o ia-Gas eiz, Spain
d
Join Resea ch Labo a o y on En i onmen al An ibio ic Resis ance, Depa men o Biochemis y and Molecula Biology, 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
ARTICLE INFO
Keywo ds:
Sheep
Raw ewe milk cheese
An imic obial esis ance
An ibio ic esis ance
An imic obial suscep ibili y es ing
B o h mic odilu ion me hod
High- h oughpu quan i a i e PCR
Resis ance genes
Mobile gene ic elemen s
ABSTRACT
An imic obial esis ance (AMR) is a signi ican public heal h h ea , wi h he ood p oduc ion chain, and, spe-
ci ically, e men ed p oduc s, as a po en ial ehicle o dissemina ion. Howe e , in o ma ion abou dai y
p oduc s, especially aw ewe milk cheeses, is limi ed. The p esen s udy analysed, o he i s ime, he occu -
ence o AMRs ela ed o lac ic acid bac e ia (LAB) along a aw ewe milk cheese p oduc ion chain o he mos
common an imic obial agen s used on a ms (dihyd os ep omycin, benzylpenicillin, amoxicillin and polymyxin
B). Mo e han 200 LAB isola es we e ob ained and iden i ied by Sange sequencing (V1-V3 16S RNA egions);
hese isola es included 8 LAB gene a and 21 species. Signi ican di e ences in LAB composi ion we e obse ed
h oughou he p oduc ion chain (P ≤0.001), wi h En e ococcus (e.g., E. hi ae and E. aecalis) and Bacillus (e.g.,
B. hu ingiensis and B. ce eus) p edomina ing in o ine aeces and aw ewe milk, espec i ely, along wi h Lac o-
coccus (L. lac is) in whey and esh cheeses, while Lac obacillus and Lac icaseibacillus species (e.g., Lac obacillus sp.
and L. pa acasei) p e ailed in ipened cheeses. Pheno ypically, by b o h mic odilu ion, Lac ococcus, En e ococcus
and Bacillus species p esen ed he g ea es esis ance a es (on a e age, 78.2 %, 56.8 % and 53.4 %, espec i ely),
speci ically agains polymyxin B, and we e mo e suscep ible o dihyd os ep omycin. Con e sely, Lac icaseiba-
cillus and Lac obacillus we e mo e suscep ible o all an imic obials es ed (31.4 % and 39.1 %, espec i ely). Thus,
esis ance pa e ns and mul id ug esis ance we e educed along he p oduc ion chain (P ≤0.05). Geno ypically,
h ough HT-qPCR, 31 an imic obial esis ance genes (ARGs) and 6 mobile gene ic elemen s (MGEs) we e
de ec ed, p edomina ing S , S B and aadA-01, ela ed o aminoglycoside esis ance, and he ansposons npA-
02 and npA-01. In gene al, a signi ican educ ion in ARGs and MGEs abundances was also obse ed h oughou
he p oduc ion chain (P ≤0.001). The cu en indings indica e ha LAB dynamics h oughou he aw ewe milk
cheese p oduc ion chain acili a ed a educ ion in AMRs, which has no been epo ed o da e.
1. In oduc ion
An ibio ics a e chemical compounds ha a ack essen ial bac e ial
physiology and biochemis y o cause cell dea h o g ow h cessa ion
(Lade & Kim, 2021). Fo decades, an ibio ics ha e been o e used, bo h
in human medicine and in animal p oduc ion (Sobie ajski e al., 2022),
including he cu en ly o bidden use o sub he apeu ic doses as g ow h
p omo e s (Pa el e al., 2020). As a esul , bac e ial communi ies ha e
been exposed o an ibio ics and ha e de eloped he abili y o wi hs and
o esis he ac ion o one o mo e an imic obial agen s, which is called
* Co esponding au ho a : Lac ike Resea ch G oup, Depa men o Biochemis y and Molecula Biology, 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.
E-mail add ess: [email p o ec ed] (G. San ama ina-Ga cía).
Con en s lis s a ailable a ScienceDi ec
Food Resea ch In e na ional
jou nal homepage: www.else ie .com/loca e/ ood es
h ps://doi.o g/10.1016/j. ood es.2024.114308
Recei ed 17 Janua y 2024; Recei ed in e ised o m 27 Ma ch 2024; Accep ed 16 Ap il 2024
Food Resea ch In e na ional 187 (2024) 114308
2
an imic obial esis ance (AMR) (Konopka e al., 2022; Vi o e al.,
2022). Bac e ia can be in insically esis an o ce ain an imic obial
g oups o agen s, media ed by ch omosomal genes and linked o phys-
iological o ana omical cha ac e is ics. None heless, acqui ed esis ance
also occu s due o ho izon al ansmission be ween bac e ia by means o
mobile gene ic elemen s (MGEs), which can ca y one o mo e esis ance
genes (Iskanda e al., 2022; Nunzia a e al., 2022), o due o gene a-
ional gene ic ansmission by poin mu a ions in genes ha gi e ise o
esis ance o inc eased exp ession o esis ance mechanisms ( e ical
ansmission) (Iskanda e al., 2022; Wall e al., 2016).
The an ibio ics u ilized in human medicine belong o he same
pha macological classes as hose used in e e ina y medicine (De i gi-
liis e al., 2011); consequen ly, acqui ed esis ance o ce ain an imi-
c obial agen s is widesp ead o he poin ha e ec i e ea men o
ce ain a al in ec ions is al eady comp omised (Vi o e al., 2022). In
ac , he p oli e a ion o an imic obial- esis an (AR) mic oo ganisms
has become one o he mos impo an h ea s o human heal h (Wang
e al., 2022) and is classi ied as one o he op 10 h ea s o global public
heal h (WHO, 2022). I causes app oxima ely 700,000 dea hs wo ldwide
pe yea and is p ojec ed o inc ease o 10 million each yea by 2050
(IACG, 2019). Thus, AMR is o u mos impo ance and is included wi hin
he sus ainable de elopmen goals (SDGs) se by he Uni ed Na ions.
Speci ically, AMR a ec s SDG 3 on good heal h and well-being since i
hinde s he abili y o con ol in ec ious diseases, inc easing mo bidi y
and mo ali y and esul ing heal h ca e cos s (Uni ed Na ions, 2015).
The ood and ood p oduc ion chain is classi ied as a possible ehicle
o he dissemina ion o AR bac e ia and genes (Caniça e al., 2019); and,
speci ically, e men ed p oduc s a e conside ed no able ese oi s
(Wang e al., 2006; Yasi e al., 2022). In his ega d, se e al s udies ha e
been de eloped ecen ly (Zhao e al., 2022), o ins ance, on aw bee ,
sheep and lamb mea (S¸anlıbaba, 2022) and d y- e men ed sausages
(F aqueza, 2015). Howe e , in o ma ion abou dai y p oduc s, espe-
cially aw milk cheeses, is limi ed, wi h mos s udies ocused on aw cow
milk cheeses (Dos San os e al., 2022; Rola e al., 2016) and sca ce in-
o ma ion on aw sheep milk cheeses (Gaglio e al., 2016; Sly ka e al.,
2022). Milk is an ideal g ow h medium o mic oo ganisms due o i s
high nu ien con en (Fusco e al., 2020). Consequen ly, he mic obio a
o aw ewe milk is di e se and is p ima ily composed o lac ic acid
bac e ia (LAB), psycho opic bac e ia and pa hogens (Biçe e al., 2021;
San ama ina-Ga cía e al., 2022a). None heless, he cheese-making and
ipening p ocesses ha e a clea impac on bac e ial communi ies, wi h a
gene al p edominance o LAB (Ca dinali e al., 2021; San ama ina-
Ga cía e al., 2022a). Se e al s udies ha e highligh ed he p esence o
esis an bac e ia in aw ewe milk and cheese, including pa hogenic
Esche ichia coli and S aphylococcus au eus (Im e e al., 2022; Ka ahu o ´
a
& Bujˇ
n´
ako ´
a, 2023; Vý os ko ´
a e al., 2020, 2021). None heless, despi e
he p edominance o LAB (Quigley e al., 2013; San ama ina-Ga cía
e al., 2022a), he e has been limi ed esea ch on AMRs in LAB om aw
ewe milk and de i a e cheeses (Vý os ko ´
a e al., 2020, 2021). In
pa icula , species o he genus En e ococcus, such as E. aecium and
E. aecalis, known as impo an oppo unis ic pa hogens in nosocomial
in ec ions (Conde-Es ´
e ez e al., 2011), ha e been desc ibed as he mos
ema kable AR LAB (Vý os ko ´
a e al., 2021). Add essing AMRs in LAB
is essen ial since hey can se e as po en ial ese oi s o he ans e o
esis ance genes o o he bac e ia, including pa hogenic bac e ia (Caniça
e al., 2019).
Se e al s udies ha e epo ed he p e e ence o consume s o aw
milk cheeses (Colonna e al., 2011; Meunie -Goddik & Wai e-Cusic,
2019), based on hei iche and mo e in ense a oma ic p o iles han
pas eu ized milk cheeses (Ba on e al., 2007; O’Sulli an & Co e ,
2017). Gi en he p essing need o minimize he de elopmen and
dissemina ion o AR LAB o sa egua d public heal h (Vý os ko ´
a e al.,
2021), he p esen s udy is ocused on Idiazabal p o ec ed designa ion o
o igin (PDO) cheese. I is a semiha d o ha d cheese om he Basque
Coun y (sou hwes e n Eu ope) p oduced wi h aw milk om he La xa
and/o Ca anzana au och honous sheep b eeds, and i has a minimum
manda o y ipening pe iod o 60 days (O icial Jou nal o he Eu opean
Communi ies, 1996). Thus, his s udy aimed o cha ac e ize he p e a-
lence o AMRs in LAB om o ine aeces, aw ewe milk, whey, esh
cheeses and 2-mon h-old ipened cheeses by means o pheno ypic and
geno ypic app oaches. Mo eo e , he po en ial di e ences among p o-
duce s p oducing he same kind o aw ewe milk cheese we e also
analysed. To ou knowledge, no s udy has comp ehensi ely analysed he
p e alence o AMRs along he p oduc ion chain o a aw ewe milk
cheese.
2. Me hods
2.1. A ea o s udy
To e alua e he p e alence o AMRs in LAB along he p oduc ion
chain o a isanal aw ewe milk cheeses, his s udy was ca ied ou
wi hin he Eu opean PDO Idiazabal cheese. This pa icula cheese was
selec ed as a case s udy because i s p oduc ion is p ima ily ca ied ou by
small-scale a isanal dai ies ha o e see he en i e p ocess, om he d
managemen o cheese-making. Idiazabal cheese is a semiha d o ha d
cheese made om he aw milk o he au och honous La xa and/o
Ca anzana sheep b eeds and has a manda o y minimum ipening ime
o 2 mon hs. He d managemen and milk p oduc ion o cheese-making
occu in he Basque Coun y, co e ing an a ea o 17,213.06 km
2
in
sou hwes e n Eu ope (43◦27
′
−41◦54
′
N and 1◦5
′
−3◦37
′
W). This
egion co esponds o he na u al habi a o he sheep b eeds (O icial
Jou nal o he Eu opean Communi ies, 1996). He d managemen in-
ol es he use o indoo o age om Oc obe o Ma ch and semi-
ex ensi e o ex ensi e g azing om Ma ch o Oc obe (Aldalu e al.,
2019). Milk collec ion and cheese p oduc ion mainly occu be ween
Janua y and June, ollowing he adi ional seasonal app oach dic a ed
by he biological hy hms o he sheep (Bole ín O icial del Es ado, 1993).
2.2. Sampling
Fo sampling, ou p oduce s a ached o he PDO Idiazabal cheese
we e chosen and iden i ied as A, B, C, and D. Each p oduce came om
one o he dis inc geog aphical p oduc ion a eas (Ala a, Biscay,
Gipuzkoa, and Na a e). All he p oduce s adhe ed o simila lock
managemen and cheese-making p ac ices in acco dance wi h he
speci ica ions ou lined by he Idiazabal PDO egula o y boa d (Bole ín
O icial del Es ado, 1993). The locks consis ed o app oxima ely
350–400 La xa b eed sheep, ollowing he managemen p ac ices
men ioned ea lie . Milking was conduc ed au oma ically, and he milk
was p omp ly e ige a ed (3–4 ◦C) un il cheese-making. Fo he cheese-
making p ocess, he milk was wa med o 25 ◦C, and he comme cial
mesophilic lyophilized s a e cul u e Choozi MM 100 LYO 50 DCU (a
mix u e o Lac ococcus lac is subsp. lac is, Lac ococcus lac is subsp. c e-
mo is, and Lac ococcus lac is subsp. lac is bio a . diace ylac is, DuPon
NHIB Ib´
e ica S.L., Ba celona, Spain) was added. Coagula ion occu ed a
28–32 ◦C o 20–45 min using a isanal enne and/o he comme cial
NATUREN® 195 P emium (Ch . Hansen Holding A/S, Hø sholm,
Denma k). The esul ing cu ds we e cu in o 5–10 mm diame e g ains
and hea ed o 36–38 ◦C. Cheeses we e hen moulded, p essed and sal ed
in sa u a ed b ine, and subsequen ly ipened in chambe s main ained a
80–95 % ela i e humidi y and 8–14 ◦C o 2 mon hs. Thus, o ine aeces,
aw ewe milk, whey, esh cheeses (1-day-old), and 2-mon h-old ipened
cheese samples we e ob ained om each p oduce . Samples we e
collec ed asep ically in quad uplica e, wi h each se o samples co e-
sponding o he same ba ch. The sampling was conduc ed by he p o-
duce s, elimina ing he need o app o al om he E hics Commi ee o
Animal Expe imen a ion. Ve bal consen was ob ained om dai ies
du ing samples collec ion. Samples we e collec ed om heal hy locks,
excluding animals ha unde wen an ibio ic ea men . Samples we e
anspo ed unde e ige a ed condi ions (3 ±1 ◦C) o analysis.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
3
2.3. Reagen s and ma e ials
The pep one wa e was supplied by Pan eac Química (Ba celona,
Spain). De Man, Rogosa and Sha pe (MRS) aga , MRS b o h medium,
sodium ci a e and sodium chlo ide we e pu chased om Scha lab
(Ba celona, Spain). T yp ic soy b o h (TSB) was ob ained om Condalab
(Mad id, Spain). Glyce ol was ob ained om Honeywell Fluka (Mad id,
Spain). Amoxicillin was supplied by Sigma-Ald ich (Mad id, Spain).
Dihyd os ep omycin and polymyxin B we e pu chased om Glen ham
Li e Sciences (Co sham, Uni ed Kingdom). Benzylpenicillin was supplied
by Tokyo Chemical Indus y Co. (Tokyo, Japan). Mag-Bind Bac e ial
DNA 96 Ki was pu chased om Omega Bio-Tek, Inc. (No c oss, Uni ed
S a es). KAPA HiFi Ho S a ReadyMix Ki was ob ained om Roche
Molecula Sys ems, Inc. (B anchbu g, Uni ed S a es). CleanNGS and
CleanDTR ki s we e ob ained om CleanNA (Waddinx een, The
Ne he lands). DNA 5 K Reagen Ki was ob ained om Pe kinElme , Inc.
(Wal ham, Uni ed S a es). BigDye Te mina o 3.1 Cycle Sequencing Ki
and exonuclease I we e pu chased om The mo Scien i ic (Wal ham,
Uni ed S a es). QIAamp® Powe Fecal® P o DNA Ki and QIAGEN®
Mul iplex PCR Ki we e pu chased om Qiagen (Valencia, Uni ed
S a es). Pe i dishes and 96-well pla es we e ob ained om Del alab
(Ba celona, Spain). The Mas e Mix SsoFas TM E aG een® Supe mix Ki
wi h Low ROX was pu chased om Bio-Rad Labo a o ies (He cules,
Uni ed S a es).
2.4. Pheno ypic cha ac e iza ion o AMRs
2.4.1. LAB isola ion and enume a ion
Fo he aeces and cheese samples, 10 g was dilu ed in duplica e 1:10
in pep one wa e and homogenized o 30 s h ee imes in a s omache
(Mas ica o Basic 400, IUL Ins umen s, K¨
onigswin e , Ge many). Se ial
dilu ions we e made in pep one wa e and pla ed on MRS aga media.
Fo he aw ewe milk and whey samples, 100 µL was aken di ec ly and
se ially dilu ed. The pla es we e incuba ed a 37 ±1 ◦C o 48 h. Th ee
p esump i e LAB isola es we e andomly selec ed pe sample based on
colony mo phology di e si y. Then, he isola es we e p een iched in TSB
and subcul u ed on o MRS aga o ensu e pu i y. All he cul u es we e
s o ed a −80 ◦C in 20 % ( / ) glyce ol.
2.4.2. Iden i ica ion o LAB isola es by Sange sequencing
2.4.2.1. DNA ex ac ion. Isola es we e p een iched in TSB and sub-
cul u ed on o MRS aga p io o DNA ex ac ion o ensu e pu i y and
iabili y. Bac e ial DNA was ex ac ed using he Mag-Bind Bac e ial
DNA 96 Ki ollowing he manu ac u e ’s ins uc ions o aga cul u es,
bu he elu ion olume was educed o 60 µL o imp o e he DNA yield.
The quan i y and quali y o he DNA ob ained we e e i ied wi h a
NanoD op ND-1000 spec opho ome e (The mo Scien i ic, Massachu-
se s, USA), in which he abso bance was measu ed a a wa eleng h o
260 nm and he 260/280 and 260/230 a ios we e analysed. DNA
ex ac ion and subsequen Sange sequencing we e conduc ed in he
Sequencing and Geno yping Uni o he Genomic Facili y/SGIke (sup-
po ed by UPV/EHU, MICINN, GV/EJ, FSE) o he Uni e si y o he
Basque Coun y.
2.4.2.2. Sange sequencing. The V1–V3 egions o he 16S RNA gene
we e ampli ied ia PCR wi h he KAPA HiFi Ho S a ReadyMix Ki using
he o wa d p ime 16S–V1–8F: 5
′
- AGAGTTTGSTCCTGGCTCAG-3
′
and
he e e se p ime 16S–V3–534R: 5
′
- ATTACCGCGGCTGCTGG −3
′
. The
PCR p oduc s we e pu i ied by means o he CleanNGS Ki ollowing he
manu ac u e ’s ins uc ions. Amplicon quan i ica ion was pe o med
using a NanoD op ND-1000 spec opho ome e (The mo Scien i ic) and
a LabChip GX Touch Nucleic Acid Analyse (Pe kinElme ) wi h a DNA 5
K Reagen Ki . Sequencing o he pu i ied p oduc was pe o med using
he BigDye Te mina o 3.1 Cycle Sequencing Ki ollowing he
manu ac u e ’s p o ocol. The sequencing p oduc was pu i ied using a
magne ic bead-based CleanDTR ki , and Sange sequencing was pe -
o med on he SeqS udio pla o m (The mo Fishe ).
2.4.2.3. Bioin o ma ic analysis. Quali y il e ing and imming o he
aw eads we e pe o med using SeqS udio Repo e so wa e (The mo
Scien i ic). The sequences we e isualized and edi ed by means o he
BioEdi Sequence Alignmen Edi o so wa e 7.2.5 (Hall e al., 2011).
The esul ing sequences we e app oxima ely 500 bp in leng h. Taxo-
nomic classi ica ion was pe o med agains he Nucleo ide Basic Local
Alignmen Sea ch Tool (NBLAST) 2.14.0+(Zhang e al., 2000), wi h
de aul pa ame e s and aking in o accoun he e- alue, sco e, que y
co e and pe cen age o iden i ica ion as quali y indica o s.
2.4.3. An imic obial suscep ibili y es ing (AST) ia he b o h mic odilu ion
me hod
The minimum inhibi o y concen a ion (MIC) o he LAB isola es was
e alua ed by he b o h mic odilu ion me hod o he mos widely used
an imic obial agen s on a ms (namely, amoxicillin, dihyd os-
ep omycin, benzylpenicillin and polymyxin B) acco ding o he
upda ed In e na ional O ganiza ion o S anda diza ion and In e na-
ional Dai y Fede a ion S anda ds (ISO/IDF, 2010) and Eu opean Food
Sa e y Au ho i y guidance (Rychen e al., 2018), wi h mino modi ica-
ions. B ie ly, a 96-well pla e was inocula ed wi h MRS b o h medium
supplemen ed wi h se ial (1:2) concen a ions o an ibio ics (amoxi-
cillin: 0.0313–16 µg/mL; dihyd os ep omycin: 1–512 µg/mL; benzyl-
penicillin: 0.0313–32 µg/mL; and polymyxin B: 2–1024 µg/mL). The
inocula o each isola e we e p epa ed in saline solu ion (0.85 %, m/ ) by
picking up single colonies om p e iously subcul u ed isola es on MRS
aga o ob ain an op ical densi y equi alen o 0.5 on he MacFa land
scale. The inoculum was subsequen ly dilu ed 1:10 in an ibio ic- ee
MRS b o h, and 50 µL o he dilu ed suspension was added o each
well and incuba ed a 37 ±1 ◦C o 48 h. The inoculum in a well wi h
MRS b o h wi hou an ibio ics was used as a posi i e con ol, and an
inoculum- ee well was used as a nega i e con ol. The an imic obial
suscep ibili y o esis ance was in e p e ed using he a ailable mic o-
biological cu -o alues de ined by he Eu opean Food Sa e y Au ho i y
(EFSA) Panel on Addi i es and P oduc s o Subs ances used in Animal
Feed (FEEDAP) (Rychen e al., 2018) and employing he epidemiolog-
ical cu -o alues (ECOFFs) p oposed by he Eu opean Commi ee o
An imic obial Suscep ibili y Tes ing (EUCAST; h ps://www.eucas .
o g).
2.5. Geno ypic cha ac e iza ion o AMRs
2.5.1. DNA ex ac ion
To analyse he p esence o an imic obial esis ance genes (ARGs),
DNA was ex ac ed as p e iously desc ibed (San ama ina-Ga cía e al.,
2022a), wi h some modi ica ions. B ie ly, o he aecal and cheese
samples, 10 g was suspended in 90 mL o 2 % (w/ ) s e ile sodium
ci a e (pH 8.0) and homogenized six imes (20 s ON and 10 s OFF) in a
s omache (Mas ica o Basic 400; IUL Ins umen s, K¨
onigswin e , Ge -
many). The esul ing suspension was cen i uged a 6500 ×g o 8 min
a 4 ◦C, a e which he a -con aining supe na an was disca ded. The
ob ained pelle was washed wi h 50 mL o sodium ci a e and cen i-
uged a 6500 ×g o 8 min a 4 ◦C. The pelle was esuspended in 800 µL
o sodium ci a e and cen i uged h ee imes a 6500 ×g o 8 min a
4 ◦C. The DNA was ex ac ed wi h a QIAamp® Powe Fecal® P o DNA Ki
acco ding o he manu ac u e ’s p o ocol, bu a double DNA elu ion s ep
was ca ied ou wi h 25
μ
L o C6 solu ion o imp o e DNA yields. To
ex ac DNA om he milk and whey samples, 10 mL was p ocessed as
desc ibed abo e, bu wi hou he need o homogeniza ion in he
s omache . The DNA was s o ed a −80 ◦C un il analysis.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
4
2.5.2. High- h oughpu quan i a i e PCR (HT-qPCR)
The de ec ion o ARGs was pe o med by means o HT-qPCR in a
nano luidic qPCR BioMa kTM HD sys em using 96.96 Dynamic A ay
In eg a ed Fluidic Ci cui s (IFCs) (Fluidigm Co po a ion), as p e iously
desc ibed (Jau egi e al., 2021). A o al o 48 p ime se s we e used
(Supplemen a y Table 1) o a ge he ARGs con e ing esis ance
agains he mos commonly used an imic obial agen s on a ms (12
ARGs encoding esis ance o dihyd os ep omycin, 24 ARGs o ben-
zylpenicillin and amoxicillin, 2 ARGs o polymyxin B and 2 mul id ug
ARGs con e ing esis ance o mo e han one o he a o emen ioned
an imic obial agen s), MGE genes (5 genes encoding ansposases and 2
genes encoding in eg ases) and he 16S RNA gene as a e e ence gene.
These genes we e selec ed conside ing he CARD da abase o LAB
(Alcock e al., 2023). The p ime s used o qPCR we e p e iously ali-
da ed (Go ecki e al., 2022; Hu e al., 2016). DNA samples we e p e-
ampli ied using he QIAGEN® Mul iplex PCR Ki and a p ime pool
( inal concen a ion o each p ime pai =50 nM), ollowing he
ampli ica ion p og am (a 95 ◦C o 15 min and 14 PCR cycles a 95 ◦C
o 15 s, 60 ◦C o 4 min and a inal ex ension s ep a 4 ◦C). Then, he
samples we e ea ed wi h exonuclease I (a 37 ◦C o 30 min o
diges ion, 80 ◦C o 15 min o inac i a ion o exonuclease I and kep a
4 ◦C). Subsequen ly, 1:10 dilu ions o speci ic a ge ampli ica ion e-
ac ions we e loaded on o he Dynamic A ay IFCs ollowing he Fluid-
igm’s Fas Gene Exp ession Analysis—E aG een® P o ocol (Fluidigm
Co po a ion). Fo ampli ica ion, he Mas e Mix SsoFas TM E aG een®
Supe mix Ki wi h Low ROX was used, wi h a inal concen a ion o
p ime s o 500 nM, bo h o wa d and e e se. The p og am consis ed o
1 min o dena u a ion a 95 ◦C, ollowed by 30 cycles o 95 ◦C o 5 s and
60 ◦C o 20 s, a mel ing cu e a 60 ◦C o 3 s and a amp a e o 1 ◦C/3 s
up o 95 ◦C. Fou eplica es we e included o each sample. Analyses
we e conduc ed a he Gene Exp ession Uni o The Genomics Facili y/
SGIke (suppo ed by UPV/EHU, MICINN, GV/EJ, FSE) o he Uni e si y
o he Basque Coun y.
2.5.3. Bioin o ma ic analysis
Raw da a we e p ocessed wi h Fluidigm Real-Time PCR Analysis
So wa e ( .3.1.3, Fluidigm Co po a ion), wi h linea baseline co ec-
ion and manual h eshold se ings. A cycle h eshold (CT) alue o 30
was chosen because he highes CT alue ob ained in his s udy was
29.0. The de ec ion o an ARG o MGE gene was conside ed posi i e
when 3 ou o he 4 echnical eplica es o each sample we e abo e he
de ec ion limi . The ela i e abundances o he ARGs we e calcula ed on
he basis o he compa a i e CT me hod (Jau egi e al., 2021), no mal-
ized o he abundance o he 16S RNA con ol gene and exp essed as he
old change (FC).
ΔCT(pe eplica e) = CT( a ge gene) − CT(16S RNA gene)
ΔΔCT(pe sample) = ΔCT
FC =2−ΔΔCT
2.6. S a is ical analysis
IBM SPSS s a is ical package e sion 26.0 (IBM SPSS, Inc., Chicago,
IL, USA, 2019) was used o da a p epa a ion and analysis. Plo gene -
a ion was pe o med in RS udio e sion 2023.03.1 and R e sion 4.3.0
(R Co e Team, Vienna, Aus ia, 2023) wi h he “ggplo 2” package (h ps
://gi hub.com/ idy e se/ggplo 2) and in Mic oso O ice P o essional
Plus 2016 Excel® e sion 16.0.5413 (Mic oso , Albuque que, Uni ed
S a es). K uskal–Wallis one-way analysis o a iance (ANOVA) wi h
Bon e oni co ec ion was pe o med wi h he SPSS package o de e -
mine he signi icance (P ≤0.05) o he e ec s o p oduce and p o-
duc ion chain (sample ype) ac o s on he bac e ial coun s and
abundances and pheno ypic and geno ypic esul s. Pe mu a ional
mul i a ia e analysis o a iance (PERMANOVA) was ca ied ou in R
wi h he “ egan” package (h ps://gi hub.com/ egande s/ egan) o
analyse he o e all e ec o p oduce and p oduc ion chain ac o s. The
da a we e log ans o med when necessa y and subjec ed o uni a i-
ance (UV) scaling, and a hea map wi h hie a chical clus e ing analysis
(HCA) was gene a ed wi h he “phea map” package (h ps://gi hub.
com/ ai okolde/phea map) o analyse he clus e ing o he pheno-
ypic and geno ypic esul s. Clus e ing o samples acco ding o pheno-
ypic and geno ypic esul s was also pe o med by means o a
dend og am in R wi h he “ ac oex a” package (h ps://gi hub.com/ka
ssamba a/ ac oex a). T ends in he bac e ial coun s and abundances
and pheno ypic and geno ypic esul s acco ding o p oduce and p o-
duc ion chain ac o s we e explo ed by means o p incipal componen
analysis (PCA), applied o log- ans o med, when necessa y, and UV-
scaled da a and pe o med in SIMCA so wa e e sion 17.0.2.34594
(Ume ics AB, Umeå, Sweden). The numbe o p incipal componen s
(PCs) was de e mined by he eigen alues (g ea e han 1.0) and c oss-
alida ion. Simila ly, o hogonal pa ial leas squa es-disc iminan
analysis (OPLS-DA) was pe o med wi h SIMCA so wa e o analyse
whe he he samples di e ed acco ding o he p oduce and p oduc ion
chain ac o s. Va iable in luence on p ojec ion (VIP) alues and loading
weigh s we e used o analyse he impo ance o each pa ame e in he
model.
3. Resul s
3.1. LAB p e alence and dis ibu ion h oughou he cheese p oduc ion
chain
Fig. 1A shows he p e alence o LAB h oughou he Idiazabal cheese
p oduc ion chain. O e all, la ge di e ences we e ound acco ding o he
sample ype (P ≤0.001). Speci ically, a mean LAB p e alence o 6.45 ±
0.451 log CFU/g was obse ed in he aeces. In he aw ewe milk, he
LAB coun was 3.73 ±0.0659 log CFU/mL, which subsequen ly
inc eased o 5.65 ±0.0623 log CFU/mL in he whey and o 7.99 ±0.172
log CFU/g in he esh cheeses. Howe e , du ing ipening, he LAB coun
sligh ly dec eased o 7.75 ±0.202 log CFU/g, al hough he di e ence
was no signi ican (Fig. 1A). Mo eo e , among p oduce s, signi ican
di e ences we e also obse ed o he whey (P ≤0.01) and esh cheese
samples (P ≤0.05), wi h p oduce A clea ly di e en ia ed om he es
due o he lowe alues. Using mul i a ia e analysis, PERMANOVA
con i med he di e ences among sample ypes (P ≤0.001) and, o a
lesse ex en , among p oduce s (P ≤0.05).
To iden i y he LAB communi ies, 203 isola es we e ob ained om
he aw ewe milk Idiazabal cheese p oduc ion chain. As expec ed, all he
isola es belonged o he phylum Fi micu es and class Bacilli (Fig. 1B).
Two o de s we e iden i ied, p edominan ly Lac obacillales (69.0 %)
and, o a lesse ex en , Bacillales (31.0 %). All he isola es o he Bacil-
lales o de belonged o he Bacillaceae amily and he Bacillus genus,
iden i ying 4 di e en species, B. ce eus (8.37 %), B. hu ingiensis (6.40
%), B. pa amycoides (2.96 %), and B. an h acis (0.99 %), in addi ion o
o he uniden i ied species (Bacillus sp., 12.3 %). The isola es o he o de
Lac obacillales belonged mainly o he En e ococcaceae (37.4 %) and
Lac obacillaceae (22.2 %) amilies and, o a lesse ex en , o he S ep-
ococcaceae (9.36 %). All he En e ococcaceae isola es co esponded o
he genus En e ococcus, iden i ying di e en species, such as E. hi ae
(19.2 %) and E. aecalis (13.3 %), and o a lesse ex en , E. aecium (2.46
%), E. mund ii (1.48 %), E. a ium (0.49 %) and E. du ans (0.49 %). The
Lac obacillaceae isola es belonged o he genus Lac obacillus, wi hou
being able o iden i y species (Lac obacillus sp., 7.88 %); Lac icaseiba-
cillus (7.88 %), wi h he species L. pa acasei (6.40 %) and L. casei (1.48
%); Le ilac obacillus (3.94 %), namely, L. b e is; and Lac iplan ibacillus
(2.46 %), iden i ied as L. plan a um (0.49 %) and L. plan a um subsp.
plan a um (1.97 %). The S ep ococcaceae isola es belonged o he gene a
Lac ococcus (8.87 %), iden i ying L. lac is (4.43 %) and L. lac is subsp.
lac is (3.45 %), in addi ion o uniden i ied s ains (0.99 %), and S ep-
ococcus, o which species could no be iden i ied (S ep ococcus sp.,
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
5
0.49 %). Wi hin all he samples, E. hi ae, E. aecalis, Bacillus sp., B. ce eus
and Lac obacillus sp. we e some o he mos impo an species
h oughou he p oduc ion chain o he Idiazabal cheese.
PERMANOVA con i med he di e ence in LAB composi ion among
he collec ed samples h oughou he p oduc ion chain (P ≤0.001)
(Fig. 1B). E. hi ae clea ly p edomina ed in he aeces (62.5 %), ollowed
by B. hu ingiensis (12.5 %) and o he uniden i ied species (Bacillus sp.)
(6.25 %). In aw ewe milk, ins ead, E. aecalis (36.4 %) p edomina ed,
ollowed by B. hu ingiensis (13.6 %), Bacillus sp. (9.09 %) and E. hi ae
(9.09 %). Du ing cheese-making, Bacillus species, such as B. ce eus (15.8
%) o Bacillus sp. (13.2 %), domina ed he whey; oge he wi h En e o-
coccus, such as E. hi ae (10.5 %) o E. aecalis (7.89 %); and Lac ococcus,
L. lac is (7.89 %) and L. lac is subsp. lac is (7.89 %), o L. b e is (7.89 %).
In esh cheeses, a simila end was main ained, wi h a p edominance o
uniden i ied Bacillus species (22.0 %), along wi h En e ococcus species,
such as E. hi ae (14.6 %) and E. aecalis (14.6 %), and also L. lac is (12.2
%). Howe e , a e ipening, Lac obacillus species p edomina ed (20.8
%), ollowed by Lac icaseibacillus, speci ically L. pa acasei (16.7 %). In
gene al, he abundance o o he species, such as E. hi ae (10.4 %) and
Bacillus sp. (10.4 %), dec eased du ing ipening. The g ea es di e ences
among sample ypes along he p oduc ion chain we e mainly obse ed
o E. aecalis, L. pa acasei, L. lac is and Lac obacillus sp. (P ≤0.05).
PERMANOVA co obo a ed he lack o di e en ia ion among p oduce s
(P >0.05).
3.2. Pheno ypic p o ile o an imic obial esis ance
Subsequen ly, an imic obial suscep ibili y was es ed by he b o h
mic odilu ion me hod o mo e han 200 LAB isola es. The dis ibu ions
o MICs a e shown in Table 1. Clea di e ences we e obse ed in he
AMR pheno ypes among he LAB communi ies (P ≤0.05) (Table 1 and
Fig. 2A), which was con i med by an OPLS-DA model (Supplemen a y
Fig. 1). O e all, Lac ococcus and S ep ococcus species had he g ea es
esis ance a es (on a e age, 78.2 % and 75.0 % o esis ance o all
isola es o all an ibio ics, espec i ely), ollowed by Le ilac obacillus,
En e ococcus and Bacillus (65.6 %, 56.8 % and 53.4 %, espec i ely).
Lac iplan ibacillus, Lac icaseibacillus and Lac obacillus species, ins ead,
we e he mos suscep ible bac e ia (31.3 %, 31.4 % and 39.1 %,
espec i ely).
In mo e de ail, clea di e ences we e obse ed in he AMR pheno-
ypes among LAB species om he same gene a and amilies (P ≤0.05).
Wi hin he Bacillaceae and Bacillus genus, B. an h acis species clea ly
di e ed om he o he species because o hei low esis ance (12.5 %),
wi h he emaining species, B. ce eus, B. pa amycoides, B. hu ingiensis
and Bacillus sp., exhibi ing g ea e esis ance (69.1 %, 58.3 %, 71.2 %
and 56.0 %, espec i ely). Fo he En e ococcaceae and En e ococcus
isola es, di e ences we e also de ec ed among he species, wi h E. du ans
and E. aecium being he mos esis an (100 % and 80.0 %, espec i ely);
E. hi ae, E. mund ii and E. aecalis p esen ing g ea e suscep ibili y (57.1
%, 58.3 % and 45.4 %, espec i ely); and E. a ium isola es being sensi-
i e o all he an ibio ics es ed. On he o he hand, mos Lac obacillaceae
gene a and species showed simila low esis ance a es, including un-
iden i ied Lac obacillus species (39.1 %), Lac icaseibacillus species,
namely, L. pa acasei and L. casei (21.2 % and 41.7 %, espec i ely), and
Lac iplan ibacillus species, speci ically L. plan a um and L. plan a um
subsp. plan a um (25.0 % and 37.5 %, espec i ely). The Le ilac obacillus
genus and he L. b e is species we e unique excep ions o hei highe
le els o esis ance. Finally, S ep ococcaceae isola es also showed low
di e ences among gene a and species. All Lac ococcus species, including
L. lac is, L. lac is subsp. lac is and o he uniden i ied species (72.2 %,
75.0 % and 88.0 % on a e age, espec i ely), exhibi ed high esis ance,
simila o uniden i ied S ep ococcus species (75.0 % on a e age). Thus,
he HCA and dend og am di ided he LAB communi ies in o wo clus e s
(Fig. 2A and B). E. a ium, L. plan a um, L. plan a um subsp. plan a um and
Fig. 1. Mean coun s (log CFU/g o mL) and ela i e abundance (%) o lac ic acid bac e ia h oughou he Idiazabal cheese p oduc ion chain ( aeces, aw milk, whey,
esh cheese and ipened cheese samples). The di e en lowe case le e s o each ype o sample indica e s a is ically signi ican di e ences.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
6
B. an h acis we e he mos di e en ia ed LAB, as hey we e he mos
sensi i e bac e ia o all he an ibio ics es ed (on a e age, 18.8 % o all
he isola es we e esis an o all he an ibio ics) (clus e 1). On he o he
hand, he emaining LAB species exhibi ed highe esis ance a es
(clus e 2). L. casei, L. pa acasei, E. aecalis and Lac obacillus sp. we e
closely ela ed, as hey showed highe bu s ill lowe esis ance a es (on
a e age, 36.8 %) (clus e 2.3). E. du ans, Lac ococcus sp., E. mund ii and
S ep ococcus sp. s ood ou (clus e s 2.1 and 2.2), showing he highes
esis ance a es o all he an imic obial agen s es ed (93.8 %). The
esis ance a e agains dihyd os ep omycin was he main eason o he
Table 1
Dis ibu ion o minimum inhibi o y concen a ion (MIC) alues o he 202 isola es ob ained h oughou he p oduc ion chain (o ine aeces, aw ewe milk, whey, esh
cheese and 60-day-old ipened cheese) o aw ewe milk Idiazabal cheese.
1
The ange o dilu ions es ed o each an imic obial agen is indica ed in whi e. The e ical lines indica e he epidemiological cu -o (ECOFF) alues acco ding o he
Eu opean Commi ee on An imic obial Suscep ibili y Tes ing (EUCAST) o Eu opean Food Sa e y Au ho i y (EFSA). MICs lowe han he lowes concen a ion es ed
a e indica ed in he closes concen a ion o he g ey ange.
2
n.d. =no de ec ed.
3
MIC
50
(µg/kg) =MIC eque ied o he inhibi ion o he g ow h o he 50% o he isola es.
4
MIC
90
(µg/kg) =MIC eque ied o he inhibi ion o he g ow h o he 90% o he isola es.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
7
di e ence be ween hese wo clus e s (2.1 and 2.2). The emaining
bac e ial species belonging o he Bacillus, En e ococcus, Lac ococcus o
Le ilac obacillus gene a exhibi ed simila high-in e media e esis ance
(66.7 % and 67.2 %, espec i ely) (clus e s 2.4 and 2.5).
The MIC
50
and MIC
90
, de ined as he MIC equi ed o he inhibi ion
o he g ow h o 50 % and 90 %, espec i ely, o he isola es con i med
he high esis ance o some o he bac e ial species (Table 1). Howe e ,
some ends we e obse ed along he p oduc ion chain (Supplemen a y
Table 2). B. ce eus, E. hi ae, E. mund i, L. lac is subsp. lac is and L. b e is
main ained simila MIC
50
and MIC
90
alues h oughou he p oduc ion
chain, while o B. pa amycoides, E. aecium, L. pa acasei and Lac obacillus
sp. inc eased, indica ing a g ea e p e alence o esis an bac e ia
h oughou he p oduc ion chain. Finally, he MIC
50
and MIC
90
o
B. hu ingiensis, Bacillus sp., E. aecalis, L. casei, L. plan a um subsp.
plan a um and L. lac is dec eased, which indica ed an inc ease in he
abundance o sensible bac e ia du ing he cheese p oduc ion p ocess.
In gene al, 24.1 % (49/203) o he LAB isola es we e suscep ible o
all he an imic obial agen s es ed (Fig. 2C). Thus, 75.9 % (154/203)
we e esis an o a leas one o he an ibio ics es ed, wi h esis ance o 3
o 4 an imic obial agen s being he mos common (46/203, 22.7 % and
50/203, 24.6 %, espec i ely) (Fig. 2C). Howe e , di e ences we e
obse ed h oughou he cheese p oduc ion chain and we e mainly
ela ed o di e ences in LAB composi ion (P ≤0.001) (Fig. 2C). In
aeces, LAB isola es esis an o 3 an imic obial agen s p edomina ed
(25.0 %), ollowed by hose esis an o 4 an imic obial agen s (21.9 %),
since he p edominan E. hi ae was mainly esis an o 2 o 3 an imi-
c obial agen s. O he mino species, such as L. b e is o S ep ococcus sp.,
we e p incipally esis an o 3 o 4 indi idual compounds. In aw ewe
milk, esis ance o 4 an imic obial agen s p edomina ed (34.1 %), ol-
lowed by esis ance o 3 an imic obial agen s (20.5 %), since he p e-
dominan species, E. aecalis, B. hu ingiensis, and E. hi ae, and mos
mino species we e mainly esis an o 4 and, o a lesse ex en , o 3
an imic obial agen s. In whey, a simila end compa ed o milk was
obse ed, al hough esis ance o 3 an imic obial agen s p edomina ed
(31.6 %), ollowed by esis ance o 4 an imic obial agen s (28.9 %). In
his case, he p edominan B. ce eus was equally esis an o 3 o 4
an imic obial agen s, while Bacillus sp. was mainly esis an o 4 an i-
mic obial agen s, E. hi ae was esis an o 3 an imic obial agen s, and
mos mino species we e esis an o 3 o 4 an imic obial agen s. In esh
cheeses, esis ance o 4 an imic obial agen s was dominan (26.8 %),
ollowed by esis ance o 1 an imic obial agen (22.0 %). The p edom-
inan Bacillus sp. species we e equally esis an o 3 o 4 an imic obial
agen s, while o he dominan species such as E. hi ae being mainly
esis an o 1 an imic obial, E. aecalis o 2 an imic obial agen s and
L. lac is o 4 an imic obial agen s. A la ge p opo ion o he mino spe-
cies we e also esis an o 1 o 4 an imic obial agen s. Finally, in he
ipened cheeses, esis ance o 2 o 3 an imic obial agen s p edomina ed
(25.0 % in bo h cases), since he p edominan Lac obacillus sp. and
L. pa acasei we e esis an o 2 an imic obial agen s, and, o a lesse
ex en , o he mino impo an species (B. ce eus o Bacillus sp.) we e
esis an o 3 an imic obial agen s. O e all, he p opo ion o LAB
esis an o 1 o 2 an imic obial agen s inc eased h oughou he p o-
duc ion chain, while he p opo ion o s ains esis an o 3 o 4 an i-
mic obial agen s dec eased. No di e ences in e ms o abundance we e
Fig. 2. HCA hea map (A), dendog am clus e ing (B), box plo ep esen a ions (C, D and E) based on an imic obial suscep ibili y es ing (AST) esul s acco ding o he
bac e ial species (A and B), numbe o an imic obials (C) and sample ype along p oduc ion chain (D and E). Abb e ia ions: DHS: dihyd os ep omycin; PB: poly-
myxin B; PG: benzylpenicillin; AMX: amoxicillin.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
8
obse ed o he suscep ible bac e ia h oughou he p oduc ion chain
(P >0.05), al hough he bac e ia di e ed axonomically. In aeces, aw
ewe milk and whey, suscep ible LAB belonged, mainly, o E. hi ae and/o
E. aecalis species, while in esh cheese, hey we e, mainly, uniden i ied
Bacillus species; and in ipened cheeses, hey co esponded o
L. pa acasei; and, o a lesse ex en , o L. plan a um subsp. plan a um and
Lac obacillus sp.
Rega ding indi idual compounds, he esis ance agains polymyxin B
was he mos common (67.0 %), ollowed by benzylpenicillin (54.7 %)
and amoxicillin (51.2 %), and being clea ly mo e sensi i e o dihy-
d os ep omycin (37.4 %) (Table 1, Fig. 2D-E). The p edominance o
esis ance o polymyxin B was obse ed in all he samples excep o
ipened cheeses, whe e a highe p e alence o LAB isola es esis an o
benzylpenicillin was obse ed. Resis ance o benzylpenicillin was p i-
ma ily obse ed in ipened cheeses, while amoxicillin esis ance was
mainly de ec ed in aeces and whey, and dihyd os ep omycin esis ance
was mainly de ec ed in aw ewe milk and esh cheeses (Fig. 2D-E). This
di e en ia ion along he p oduc ion chain was ela ed o he LAB
composi ion o each sample ype (P ≤0.05) (Table 1). E. du ans o
Lac ococcus sp., ollowed by E. aecium, we e he mos esis an o
dihyd os ep omycin (Fig. 2A), and he mos sensi i e species we e
B. an h acis, E. a ium, E. mund ii, L. plan a um o S ep ococcus sp. In he
case o benzylpenicillin, L. plan a um subsp. plan a um, E. du ans, Lac-
ococcus sp. and S ep ococcus sp. we e he mos esis an , while
B. an h acis o E. a ium we e he mos sensi i e. E. du ans, Lac ococcus
sp., L. lac is subsp. lac is and S ep ococcus sp. we e he mos esis an o
polymyxin B, while E. a ium and L. plan a um subsp. plan a um we e he
mos suscep ible. Finally, E. du ans, E. mund ii o S ep ococcus sp. we e
he mos esis an o amoxicillin, and B. an h acis, E. a ium o
L. plan a um we e he mos sensi i e.
Rega ding he AMR p o iles o LAB (Fig. 3A-B), which esul s om all
possible combina ions o all an ibio ics es ed, esis ance o all an ibi-
o ics p edomina ed (24.6 %), ollowed by polymyxin B-benzylpenicillin-
amoxicillin (16.7 %), polymyxin B (6.90 %) and dihyd os ep omycin-
polymyxin B (5.42 %). These pa e ns we e mainly obse ed o
E. hi ae and, o a lesse ex en , o Bacillus sp., Bacillus ce eus and
Fig. 3. Box plo ep esen a ions based on he esis ance pa e ns acco ding o he sample ype along p oduc ion chain and bac e ial species (A and B, espec i ely),
and box plo ep esen a ion (C), HCA hea map (D) and dendog am clus e ing (E) based on he esis ance agains he an imic obial classes es ed acco ding o he
sample ype along p oduc ion chain (C) and bac e ial species (D and E). Abb e ia ions: DHS: dihyd os ep omycin; PB: polymyxin B; PG: benzylpenicillin; AMX:
amoxicillin.
G. San ama ina-Ga cía e al.
Food Resea ch In e na ional 187 (2024) 114308
9
E. aecalis. Resis ance pa e ns we e signi ican ly ela ed o he LAB
communi ies (P ≤0.01), consequen ly leading o di e en ia ion in he
cheese p oduc ion chain. Resis ance o all an ibio ics was mainly
obse ed o E. hi ae and Bacillus sp., ollowed by L. lac is and E. aecalis,
while he pa e n o esis ance o polymyxin B-benzylpenicillin-amoxi-
cillin was mos common o E. hi ae, B. ce eus and Bacillus sp. (Table 1,
Supplemen a y Fig. 2). Resis ance o polymyxin B was ela ed o E. hi ae,
E. aecalis and Lac obacillus sp., and dihyd os ep omycin-polymyxin B
combina ion was obse ed in E. hi ae, E. aecalis and L. pa acasei
(Table 1, Supplemen a y Fig. 2). Thus, conside ing LAB communi ies
h oughou he p oduc ion chain, esis ance o polymyxin B-benzylpe-
nicillin-amoxicillin (25.0 %) and o all an ibio ics p edomina ed (21.9
%) in aeces. In aw ewe milk, a simila end was obse ed, al hough
esis ance o all an imic obials was no ably g ea e (34.1 %) han ha o
polymyxin B-benzylpenicillin-amoxicillin (9.09 %), which was main-
ained in whey (28.9 % and 23.7 %, espec i ely) and esh cheeses
(26.8 % and 7.32 %, espec i ely), albei a di e en p opo ions. In he
ipened cheeses, he esis ance o all he an imic obial agen s was
clea ly lowe (12.5 %), and he esis ance o polymyxin B-benzylpeni-
cillin and benzylpenicillin was also no able (12.5 % and 10.4 %,
espec i ely). No ably, no isola e esis an o he combina ion o
dihyd os ep omycin-amoxicillin o dihyd os ep omycin-benzylpeni
cillin-amoxicillin was ound in any ype o sample.
In ela ion o mul id ug esis ance (MDR) (Fig. 3C-E), which is
de ined as esis ance o 3 o mo e classes o an ibio ics, i was obse ed
in 30.5 % o he LAB isola es. The MDR di e ed h oughou he p o-
duc ion chain (P ≤0.05). Speci ically, he p opo ion o bac e ia esis-
an o 1 class o an imic obial did no di e h oughou he p oduc ion
chain (12.9–29.0 %) (P >0.05). Howe e , he numbe o isola es
esis an o 2 classes di e ed signi ican ly acco ding o he sample ype
(P ≤0.05), wi h he highes p e alence ound in ipened cheeses (32.8
%) and aeces (21.3 %) and he lowes in aw ewe milk and esh cheeses
(13.1 % in bo h cases) (Fig. 3C). The p e alence o mul i esis an
bac e ia also di e ed h oughou he p oduc ion chain (P ≤0.01), wi h
he highes a e obse ed in aw ewe milk (32.3 %), which dec eased
h oughou cheese-making and ipening p ocesses (12.9 %) (Fig. 3C).
These dynamics we e ela ed o he LAB communi ies (P ≤0.05)
(Supplemen a y Fig. 3), since MDR was mainly obse ed in E. hi ae
(17.2 %), E. aecalis (15.6 %), B. hu ingiensis (15.6 %) and Bacillus sp.
(15.6 %). Resis ance o 2 classes was de ec ed mainly in E. hi ae (23.7
%), B. ce eus (15.3 %) and Bacillus sp. isola es (13.6 %), and esis ance o
1 class was mo e common in Lac obacillus sp. (19.4 %) and E. hi ae (16.1
%). Mo eo e , he HCA and dend og am di ided LAB species in o 4
clus e s acco ding o he p edominan pheno ype wi hin each species.
Thus, he MDR pheno ype was p edominan wi hin E. du ans, Lac o-
coccus sp., E. aecium, L. lac is, B. hu ingiensis and B. pa amycoides.
3.3. Geno ypic p o ile o an imic obial esis ance
Rega ding he ARGs and MGEs, 37 ou o he 47 genes s udied we e
de ec ed (Fig. 4A, Supplemen a y Table 3). Among all he samples, he
p edominan ARGs we e S (a e age ela i e abundance o 387), ol-
lowed by S B (39.3) and aadA-01 (19.3), while among he MGEs, npA-
02 and npA-01 p edomina ed (71.3 and 26.5, espec i ely). The ARGs
aph, aph6ia, blaZ, blaTEM, blaGES, blaCTX-M−03, blaOKP and pbp2x and
he MGEs in I1 and npA-03 we e no de ec ed. In gene al, amino-
glycoside ARGs p esen ed g ea e abundances han β-lac am, polymyxin
and mul id ug ARGs. Among he an imic obial agen s, he S gene
exhibi ed he highes ela i e abundance wi hin aminoglycosides, ol-
lowed by S B and aadA-01, while aacA/aphD and aadA5-01 p esen ed
he lowes abundances. Fo β-lac ams, bla-ACC-1, pbp and blaCMY2-01
domina ed, while ampC, blaIMP-01 and pbp5 we e mino ARGs. Fo
polymyxins, mc -2 p esen ed he g ea es abundance compa ed o mc -1.
Among he mul id ug ARGs, wo genes we e also de ec ed, namely, olC-
01 and mexD, he i s p esen ing he g ea es abundances. Among he
MGEs, ansposons p esen ed he g ea es abundance, especially npA-02
and npA-01, while npA-07 was he leas abundan . A simila abundance
was obse ed o in eg ons, namely, in I and IS613. O e all, p edomi-
nan ansposons p esen ed highe abundances han p edominan ami-
noglycoside ARGs, wi h he excep ion o S , while lowe abundances
we e obse ed o in eg ons, simila o β-lac ams, polymyxins and
mul id ug ARGs.
PERMANOVA indica ed ha he e we e no di e ences among p o-
duce s in e ms o he de ec ed ARGs o MGEs (P >0.05). None heless,
Fig. 4. HCA hea map (A) and Venn diag am (B) showing he dis ibu ion o ARGs and MGEs along cheese p oduc ion chain ( aeces, aw milk, whey, esh cheeses
and ipened cheeses).
G. San ama ina-Ga cía e al.
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