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Genomic cha ac e iza ion o En e o oxigenic E. coli F4 and F18 posi i e s ains om pos -1
weaning dia hea in pigs 2
Vanesa Ga cíaa,b, Michela Gambinoa, Ka l Pede senc, S en Haugegaa dd, John Elme dahl Olsena 3
and Ana He e o-F esnoa,#
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aDepa men o Ve e ina y and Animal Sciences, Facul y o Heal h and Medical Sciences, 5
Uni e si y o Copenhagen, F ede iksbe g, Denma k 6
bLabo a o io de Re e encia de Esche ichia coli (LREC), Depa amen o de Mic obioloxía e 7
Pa asi oloxía, Facul ade de Ve e ina ia, Uni e sidade de San iago de Compos ela (USC), Lugo, 8
Spain 9
cS a ens Ve e inä medicinska Ans al , Uppsala, Sweden 10
dSEGES Danish Pig Resea ch Cen e, Denma k. 11
#Co esponding au ho , Ana He e o-F esno, 12
Email: [email p o ec ed] 13
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Running i le: Genome analysis o E. coli om pos -weaning dia hea 15
Keywo ds: pigs, dia hea, En e o oxigenic E. coli, whole genome sequencing, bioin o ma ics 16
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AEM Accep ed Manusc ip Pos ed Online 18 Sep embe 2020
Appl. En i on. Mic obiol. doi:10.1128/AEM.01913-20
Copy igh © 2020 Ame ican Socie y o Mic obiology. All Righ s Rese ed.
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ABSTRACT 21
This s udy aimed o cha ac e ize in silico En e o oxigenic E. coli F4- and F18-posi i e-isola es 22
(n=90) causing swine pos -weaning dia hea, including pa hogenic po en ial, phylogene ic 23
ela ionship, an imic obial and biocide esis ance (R), p ophage con en and me al ole ance a es. 24
F4-s ains belonged mos ly o he O149 and O6 se og oups and ST100 and ST48 MLST- ypes. F18-25
s ains we e mainly assigned o he O8 and O147 se og oups and ST10, ST23 and ST42. The 26
highes a es o an imic obial-R we e ound agains s ep omycin, sul ame hoxazole, e acycline, 27
ime hop im and ampicillin. No esis ance was ound owa ds cip o loxacin, ce o axime, ce io u 28
and colis in. Genes con e ing ole ance o coppe (showing highes di e si y), cadmium, sil e and 29
zinc we e p edic ed in all genomes. En e o oxins (l cA; 100 % F4, 62 % F18, as A; 100 % F4, 38.1 30
% F18, s a;18.8 % F4, 38.1 % F18, s b; 100 % F4, 76.2 % F18) and imb iae encoding genes yped 31
as F4ac and F18ac we e de ec ed in all he s ains, in addi ion o up o 16 o he i ulence genes in 32
indi idual s ains. Phage analysis p edic ed be ween 7 and 20 di e en p ophage egions in each 33
s ain. High di e si y o plasmid eplicons was shown; IncFII, IncFIB and IncFIC we e p e alen 34
among F4-isola es while IncI1, and IncX1 we e dominan among F18-s ains. In e es ingly, F4-35
isola es om he ea ly 90´s belonged o he same clonal g oup de ec ed o mos o he F4-s ains 36
om 2018-2019 (ONT:H10-A-ST100-CH27-0). The low numbe o SNPs di e ences be ween he 37
oldes and ecen F4-ST100-isola es sugges s a ela i e s able genome. O e all, he isola es 38
analyzed in his s udy showed ema kable di e en gene ic ai s depending on he imb ia ype. 39
IMPORTANCE 40
Dia hea in he pos -weaning pe iod due o En e o oxigenic E. coli (ETEC) is an economically 41
ele an disease in pig p oduc ion wo ldwide. In Denma k, p e en ion is mainly achie ed by Zinc-42
oxide adminis a ion (discon inued by 2022). Besides, a b eeding p og am has been implemen ed 43
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aiming o educe his illness. T ea men wi h an imic obials con ibu es o he p oblem o 44
an imic obial esis ance (AMR) de elopmen . As a no el y, his s udy aims o deeply unde s and 45
he gene ic popula ion s uc u e and a ia ion among isola es dia hea-associa ed by whole genome 46
sequencing cha ac e iza ion. ST100-F4ac is he dominan clonal g oup ci cula ing in Danish he ds 47
and showed high simila i y o ETEC-ST100-isola es om China, USA and Spain. High a es o 48
AMR and high di e si y o i ulence genes we e de ec ed. Cha ac e iza ion o ETEC dia hea-49
ela ed is impo an o unde s anding he disease epidemiology and pa hogenesis, and o 50
implemen a ion o new s a egies aiming o educe he impac o he disease in pig p oduc ion. 51
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INTRODUCTION 54
Pos -weaning dia hea (PWD) a ec s pigs a e weaning, leading o signi ican economic cos s o 55
he pig indus y due o weigh loss, mo ali y, as well as cos o p e en ion (i.e. accina ion), 56
ea men and handling (1, 2). In addi ion o sudden dea h o p o use dia hea, he disease is 57
accompanied by g ow h e a da ion in su i ing pigle s (1, 3). Du ing acu e ou b eaks, mo ali y 58
due o PWD may each 20-30% o e a 1 o 2 mon hs’ ime span among in ec ed pigs (3). Besides, 59
as i is one o he mos common easons o use o an imic obials in pig indus y wo ldwide, PWD 60
signi ican ly con ibu es o he p oblem o an imic obial esis ance (AMR) de elopmen (2, 4, 5). 61
En e o oxigenic E. coli (ETEC) is he main e iological agen in ol ed in PWD wo ldwide. The 62
ETEC pa ho ypes in pigs a e cha ac e ized by he exp ession o speci ic imb ial adhesins, which 63
media e bac e ial coloniza ion o he gu mucosal su ace. The mos commonly de ec ed ypes o 64
imb iae a e F4 (p e iously e med K88) and F18 (F107, 2134P and 8813). Bo h include di e en 65
an igenic a ian s; h ee o F4 (ab, ac and ad) wi h F4ac being he mos p e alen , and wo ypes 66
o F18 (ab and ac), wi h F18ac being he one associa ed wi h PWD (6, 7). In es inal adhesion and 67
subsequen coloniza ion by ETEC depends on F4 o F18 speci ic ecep o s, he p esence o which 68
he e o e is essen ial o ETEC o cause disease (8). ETEC F4 is usually ela ed o PWD o ecen ly 69
weaned pigle s occu ing 2-3 days a e weaning (classical PWD), while F18 is commonly ound 70
associa ed o dia hea 2-6 weeks a e weaning. The age-dependen exp ession o F4 and F18 71
ecep o s in he small in es ine migh explain why ETEC F4 in ec ion mainly akes place igh a e 72
weaning as well as du ing he neona al pe iod, while ETEC F18 in ec ion mainly occu s la e in he 73
pos -weaning pe iod (5). 74
Once ETEC bac e ia ha e adhe ed and colonized he small in es ine, hey can p oduce 75
en e o oxin(s) leading o dia hea. Bo h ETEC F4 and F18 a e epo ed o encode wo classes o 76
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en e o oxins; hea labile (LT) and hea s able (S a, S b, and EAST1 -en e oagg ega i e hea -s able 77
oxin 1) en e o oxins, which induce sec e o y dia hea in he pigs (1, 2, 9-11). The p edominan 78
se og oup o ETEC associa ed wi h classical PWD in pigs wo ldwide is O149-F4 (1). 79
In Denma k, and o he coun ies, zinc oxide (ZnO) in he apeu ic concen a ions has been used 80
du ing he las decades o p e en PWD in he i s 14 days a e weaning. ZnO has been ound o 81
imp o e g ow h pe o mance and educe scou s (ETEC induced) in weaning pigle s (12). Mo eo e , 82
ZnO educes bac e ial adhesion and in lamma o y cy okine exp ession, and p e en s he dis up ion 83
o memb ane in eg i y caused by ETEC (13). Howe e , due o he en i onmen al oxici y and 84
po en ial co-selec ion o AMR, use o ZnO has been banned in pig p oduc ion in EU by 2022 85
(h ps://www.ema.eu opa.eu/en/medicines/ e e ina y/ e e als/zinc-oxide). O he s a egies ha 86
ha en been used o ackle PWD caused by ETEC include a b eeding p og am (DanB ed) which has 87
been implemen ed in some Danish a ms since 2003 and accines. The o me consis s in b eeding 88
pigs which do no exp ess he F4ac speci ic ecep o s on he in es inal mucosa aiming a educing 89
occu ence o dia hea due o ETEC F4 (5). The li e a enua ed accine Colip o ec® F4/F18 90
(h ps://www.ema.eu opa.eu/en/medicines/ e e ina y/EPAR/colip o ec- 4 18) has been de eloped 91
o diminish he incidence o PWD caused by bo h ETEC F4 and F18 bac e ia. Besides hese 92
p e en i e s a egies, neomycin, ap amycin, spec inomycin, e acycline, amoxicillin and 93
sulphadiazine- ime hop im a e he an imic obials commonly used o ea PWD in Denma k (14). 94
In he p esen wo k, we cha ac e ized a collec ion o Danish ETEC F4 and F18 posi i e s ains 95
h ough whole genome sequencing in o de o analyze he pa hogenic po en ial o he s ains 96
h ough iden i ica ion o ele an i ulence ac o s and o de e mine he occu ence o AMR and 97
biocide and me al esis ances. Fu he , he analysis allowed us o unde s and he popula ion gene ic 98
s uc u e and a ia ion among s ains ha a e associa ed wi h PWD. Mo eo e , we also analyzed he 99
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phylogene ic ela ionship be ween he Danish s ains unde s udy and swine ETEC s ains om 100
o he coun ies o he wo ld. 101
RESULTS 102
Phylog oups, sequence ypes, clono ypes and se o ypes 103
Mos o he ETEC isola es belonged o he phylog oup A (74 isola es, 82.2 %), and he emaining 104
we e assigned o ou di e en phylog oups; B1 (one isola e, 1.1 %), C (nine isola es, 10 %), D 105
( i e isola es, 5.5 %) and E (one isola e, 1.1 %). The s ains displayed eigh di e en MLST- ypes 106
(STs) (ST10, ST23, ST42, ST48, ST90, ST100, ST155 and ST3524), wi h ST100 accoun ing o 56 107
(62.2 %) isola es. Based on umC- imH allele combina ions (CH), eigh clono ypes we e iden i ied, 108
wi h CH27-0 as he mos p e alen ype (56 isola es, 62.2 %), co esponding o s ains assigned o 109
ST100 (Supplemen a y Table S2). 110
The Se o ypeFinde ool de ec ed six O se og oups (O6, O8, O29, O141, O147 and O149) and eigh 111
di e en H an igens (H4, H10, H12, H14, H16, H17, H19 and H30). I was no able o p edic he O 112
and H an igen in 14 and 8 isola es, espec i ely. O e all, 13 O:H combina ions (se o ypes) we e 113
ound (Supplemen a y Table S2), wi h O149:H10 being he mos common se o ype iden i ied (49 114
isola es, 54.4 %) ollowed by O6:H16 (11 isola es, 12.2 %). 115
The associa ion be ween clonal g oups (de ined by se o ype, phylog oup, ST and clono ype) and 116
imb ial ype de ec ed among he s ains is shown in Table 1. Two main clonal g oups we e 117
iden i ied among he 69 F4 posi i e isola es, O149/ONT:H10/HNT-A-ST100-CH27-0 and O6:H16-118
A-ST48-CH11-34, ep esen ing 81.2 % (56 s ains) and 15.9 % (11 s ains) o he isola es, 119
espec i ely. The emaining F4 posi i e isola es belonged o he O8:H19-C-ST90-CH4-54 clonal 120
g oup ( wo isola es, 2.9 %) (Supplemen a y Table S2). In e es ingly, i e ou o he six F4 posi i e 121
isola es isola ed in he ea ly 90 ´s also belonged o he ONT:H10-A-ST100-CH27-0 clonal g oup 122
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shown o mos o he F4 isola es collec ed du ing he pe iod 2018-2019 (Supplemen a y Table S2). 123
In con as o he homogenei y obse ed among F4 isola es, he 21 F18 posi i e isola es showed a 124
highe di e si y, and we e assigned o six di e en clonal g oups, o which O141/ONT:H4-A-ST10-125
CH11-24 (33.3 %; se en isola es) and O8:H17-C-ST23-CH4-54 (28.6 %; six isola es) we e he 126
p edominan ones. The emaining F18 posi i e isola es we e assigned o he O147/ONT:H14-D-127
ST42-CH-28-65 ( i e isola es, 23.8 %), O29:H12-B1-ST155-CH4-121, ONT:H19-C-ST90-CH4-0 128
(E. coli Nysø) and O8:H31-E-ST3524-CH23-31 (one isola e each, 4.7 %) clonal g oups. F18 129
posi i e s ains om he same he d belonged o he same clonal g oup including se en, six and i e 130
isola es om he d D, he d B, and he d C, espec i ely (Supplemen a y Table S2). 131
Phylogeny analysis o ETEC isola es 132
The aw- ead mapping o all 90 genomes o he e e ence E. coli K12 genome showed ha 133
3,475,685 ou o 4,641,652 (74.8 %) nucleo ide posi ions in he e e ence genome we e p esen in 134
all he analyzed genomes. A o al o 42,172 a iable nucleo ide posi ions we e de ec ed in his co e 135
genome (Supplemen a y Table S3). 136
Isola es clus e ed in o ou majo clades (I, II, III and IV) and g ouped acco ding o hei ST (Figu e 137
1). Clade I included se en ST10-F18 isola es eco e ed om he same he d and showed 1 o 14 138
SNP di e ences. Isola es belonging o ST48 and encoding F4 imb iae (ele en isola es) g ouped in 139
clade II and showed om 6 (be ween isola es om he same he d) o 633 SNP di e ences. Clade III 140
encompassed isola es assigned o di e en STs (ST155, ST23, ST42, ST3524 and ST90) and all 141
excep wo ST90 isola es we e F18 posi i e. This clus e was di ided in o wo well-de ined 142
subclades: subclade A (including i e ST42 isola es, om he same he d wi h SNP di e ences 143
anging om 4 o 18 and one ST3524 isola e) and subclade B con aining six ST23 isola es (SNP 144
di e ences be ween 3 and 21), h ee ST90 ( wo o hem we e F4 posi i e) s ains (SNPs di e ences 145
be ween 104 and 556) and a single ST155 isola e. Las ly, clade IV consis ed o F4 posi i e isola es 146
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belonging o ST100 (56 isola es) and was spli in o wo subclades, wi h he numbe o di e en 147
SNPs spanning om 16 o 866 be ween all genomes and om 4 o 15 be ween isola es belonging o 148
he same he d. SNP di e ences be ween he i e F4- ST100 isola es om he 90´s and F4-ST100 149
s ains eco e ed du ing 2018-2019 anged om 130 o 699. 150
We in es iga ed he ela ionship be ween ou s ains and ETEC isola es om o he coun ies 151
(Figu e 2, Supplemen a y Table 11). Phylogene ic analysis based on SNP, indica ed ha he Danish 152
ST100 isola es a e closely ela ed o ST100 isola es om China (SNP di e ences be ween 120 and 153
443), Spain (178 o 708) and USA (196 o 466). The lowes numbe o SNP di e ences among 154
ST10 isola es we e de ec ed e sus ST10 s ains om USA (300 and 301), ollowed by Spain (1779 155
o 1852) and China (2023 o 2025). Rega ding ST48, he single isola e om China included o 156
compa isons showed SNP di e ences be ween 4163 and 4291 wi h ega ds o he Danish ST48 157
isola es. Finally, ou ST90 s ains we e mo e simila o he ST90 isola e om China (SNP 158
di e ences be ween 80 and 268) han o hose ST90 s ains om USA (SNP di e ences anging 159
om 388 o 476). 160
An imic obial esis ance pheno ypes and geno ypes 161
The an imic obial suscep ibili y es ing e ealed ha 85 (94.4 %) o he isola es we e esis an o a 162
leas one o he an imic obials in es iga ed, 60 (66.7 %) we e MDR and only i e we e suscep ible 163
o all an imic obials. High a es o esis ance we e ound agains s ep omycin (68.9 % o he 164
isola es), sul ame hoxazole (67.8 %), e acycline (56.7 %), spec inomycin (55.6 %), ime hop im 165
(53.3 %) and ampicillin (48.3 %). Impo an ly, none o he isola es was esis an o ce io u , 166
ce o axime, colis in o cip o loxacin, which a e conside ed highly c i ical d ugs in human medicine 167
(15) (Table 2). In addi ion, one and eigh F4 posi i e isola es showed esis ance o 168
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amoxicillin/cla ulanic acid and nalidixic acid, espec i ely, while none o he F18 isola es es ed 169
posi i e o hese d ugs (Table 2). 170
Resul s om Kappa s a is ical analysis showed ha he e was an almos pe ec ag eemen be ween 171
pheno ypic esis ance and he in silico p edic ion o esis ance geno ype (Table 3), which iden i ied 172
a o al o 39 di e en AMR genes (Supplemen a y Table S2). The genes blaTEM-1B, e (A) and d A1 173
we e he mos commonly de ec ed among ampicillin, e acycline and ime hop im esis an 174
isola es, espec i ely. Simila ly, sul1 and sul2 we e he p edominan genes esponsible o 175
sulphonamide esis ance. A o al o 16 di e en genes encoding o aminoglycoside modi ying 176
enzymes we e iden i ied, wi h he aph (phospho ans e ases: aph(3')-Ia, aph(3')-Ib, aph(3'')-Ib, 177
aph(4)-Ia, aph(6)-Id) and aadA (nucleo idyl an e ases: aadA1, aadA2, aadA5, aadA11, aadA12, 178
aadA17, aadA22, aadA24) genes as he mos common, consis en wi h he high esis ance o 179
spec inomycin and s ep omycin, espec i ely. Rega ding phenicols, ca A1, cmlA1 and loR genes 180
we e ound among he esis an isola es (Table 3, Supplemen a y Table S2). Suscep ibili y o 181
mac olides and lincosamides was no pheno ypically es ed, howe e , genes con e ing esis ance o 182
bo h classes o d ugs we e de ec ed (Table 3, Supplemen a y Table S2). 183
No ably, none o he isola es was ound o ha bo genes ha sugges ed ex ended-spec um be a-184
lac amase (ESBL) p oduc ion o ans e able colis in esis ance (mc -class genes). 185
The ResFinde bioin o ma ics ool also allows he iden i ica ion o ch omosomal mu a ions ela ed 186
wi h an imic obial esis ance. Eigh isola es, pheno ypically esis an o nalidixic acid, showed a 187
single ch omosomal mu a ion in he gy A (S83L) gene. The subs i u ion V161G, associa ed wi h 188
colis in esis ance, was also de ec ed in he pm B gene in i e isola es; howe e , hese isola es we e 189
pheno ypically suscep ible o he an imic obial. In addi ion, one isola e had a nucleo ide change in 190
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isola es om ou di e en coun ies sugges ha he ETEC ST100 isola es a e closely ela ed, 330
since he SNP di e ences we e ela i ely low among hem. In e es ingly, a ela i ely low numbe o 331
SNPs (130-699) we e iden i ied be ween he F4 posi i e isola es om he 90´s and hose eco e ed 332
du ing 2018-2019, indica ing ha e y simila clones ha e been ci cula ing o a leas 30 yea s. To 333
con i m his hypo hesis a la ge numbe o F4 s ains om p e ious yea s ( om he 90 ´s up o 334
nowadays) should be analyzed. The clone O6:H16-ST48, which included ele en isola es eco e ed 335
om i e di e en he ds and di e ing in 6 o 633 SNPs, was he second mos p e alen among F4 336
posi i e isola es. No ably, O6 is one o he mos impo an ETEC se og oups in ol ed in human 337
dia hea globally, pa icula ly among child en unde he age o i e in de eloping coun ies (38), 338
and i is no classically associa ed wi h PWD. A ecen s udy based on he genomic cha ac e iza ion 339
o 40 ETEC O6:H16/HNT human isola es collec ed du ing 1975–2016 showed signi ican genomic 340
di e si y among hem, bu none was assigned o ST48 (38). The occu ence o ETEC O6:H16 341
among pig ETEC isola es may indica e a zoono ic po en ial, howe e , he s ains did no ha bo 342
known imb iae genes in ol ed in adhesion o he human in es ine. 343
The Wo ld Heal h O ganiza ion has de ined AMR a global heal h issue in bo h humans and 344
animals, and has ecommended su eillance o AMR bac e ia in ood-p oducing animals, such as 345
pigs, as hey ep esen a possible sou ce and dissemina o o AMR o humans (39). ETEC om pigs 346
a e no conside ed zoono ic (key i ulence ac o s equi ed o cause disease di e be ween pigs and 347
human (40), bu ea men wi h an imic obials agains PWD may selec o AMR in commensal 348
in es inal bac e ia, and such bac e ia may ans e c i ical esis ances o humans ia he ood chain. 349
To in es iga e his aspec , we de e mined bo h, pheno ypically and in silico, he AMR le els among 350
he ETEC isola es. The highes a es o AMR we e ound agains aminoglycosides and 351
sul ame hoxazole, he use o which has inc eased in li es ock in Denma k du ing las yea s (41). 352
Resis ance o e acycline, ime hop im and ampicillin, which a e among he equen 353
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an imic obials used o ea men o PWD in Denma k, we e b oadly de ec ed. No ably, le els o 354
e acycline and ampicillin esis ance we e simila o hose de ec ed in a p e ious s udy on 355
pa hogenic E. coli om pigs in Denma k (42). All he isola es we e suscep ible o cip o loxacin, 356
ce o axime, ce io u and colis in, which a e c i ically impo an an imic obials o human medicine, 357
(15) bu ea men wi h o he s an imic obial agen s may allow hei co-selec ion. The absence o 358
hese esis ances is possibly linked o he es ic ed use o hese d ug classes in he pig indus y, 359
whe e luo oquinolones, cephalospo ins and colis in all bea a penal y o ac o 10 in he he d le el 360
egis a ion scheme o use o an imic obials ( he Yellow ca d scheme) in he Danish pig p oduc ion 361
(41). Simila indings we e desc ibed in a ecen s udy in Denma k, whe e he highes p opo ions 362
o AMR among ETEC isola es we e ound o ampicillin (60.7 %), sulphame hoxazole (69.7 %), 363
e acycline (47.2 %) and ime hop im (69.7 %), while AMR o cip o loxacin, ce io u and colis in 364
we e no de ec ed (43). MDR in he pig indus y has been linked wi h he wide use o 365
aminoglycosides and be a-lac ams in e e ina y medicine (44). He e, MDR was de ec ed in 60 366
isola es (66.7 %), a om he 94 % de ec ed among ETEC isola es ca ying mc -1 and causing 367
PWD in Spain (36). I is o highligh ha in silico p edic ion o AMR genes showed an almos 368
pe ec ag eemen wi h he pheno ypic analysis acco ding o Kappa s a is ical analysis. In addi ion, 369
genes encoding o mac olides (speci ically o e y h omycin ia Md A) o lincosamides esis ance, 370
which we e no pheno ypically es ed, we e also p edic ed. Bo h d ug classes a e commonly 371
employed in Denma k and o he coun ies o ea men agains Lawsonia in acellula is, an 372
in acellula pa hogen causing en e ic disease in pigs (45-47). The inc ease in he adminis a ion o 373
mac olides du ing las yea s and he s eady use o lincosamides in he pig indus y in Denma k (41) 374
could ha e selec ed o esis ance o he d ugs in E. coli. 375
S udies o ETEC ac oss se e al coun ies wo ldwide, including old s udies om Denma k, desc ibe 376
ETEC F4 as he mos common ype associa ed o PWD, ollowed by F18 (48-52). Howe e , in 377
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o he coun ies such as Poland, Cuba, Japan and Spain (36, 53-55), he highes p e alence was 378
ound o ETEC-F18. In a ecen s udy in Denma k, he numbe o F4 and F18 s ains de ec ed we e 379
simila (annual epo om 2018; h ps://diagnos ik.d u.dk/ aadgi ning/aa s appo e - o -380
diagnos ik_o e aagning/aa s appo e -s in). As demons a ed in p e ious s udies, he 90 s ains 381
unde s udy we e all F4ac o F18ac (6, 7). Denma k has u he had a b eeding s a egy o educe 382
suscep ibili y o pigs agains ETEC F4ac. The s a egy consis ed on he inac i a ion (based on one 383
SNP change) o he candida e gene o he F4ac ecep o MUC4 (5, 56), howe e , acco ding o ou 384
esul s, F4 s ains ype ac a e s ill eco e ed. Since in o ma ion on he a ms is con iden ial, we 385
acknowledge i he he ds unde s udy ha e his s a egy implemen ed and/o pigle s we e 386
accina ed. Thus he eason why F4ac is s ill been de ec ed could be ha some o he isola es es ed 387
we e eco e ed om he ds whe e he s a egy and/o accina ion has no been applied. Besides, 388
some s udies sugges ha MUC13 and no MUC4 is he mos likely gene go e ning suscep ibili y o 389
ETEC F4ac, and his migh explain also why ETEC F4ac is s ill he p edominan causa i e agen o 390
PWD igh a e weaning (57). 391
In wo F4 isola es, F4 and F6 imb iae genes we e de ec ed concu en ly. ETEC isola es encoding 392
o mo e han one imb iae ha e been p e iously desc ibed (28, 48, 49, 58, 59) and such s ains 393
ha e been sugges ed o ha e a pa hogene ic ad an age (27). The mos p e alen en e o oxin 394
de ec ed in ou s udy was STb (85 isola es) consis en wi h p e ious s udies pe o med in o he 395
coun ies (28, 48, 52). All F4-posi i e isola es ca ied as A, l cA and s b genes, in line wi h esul s 396
o p e ious s udies, whe e he F4 gene was s ongly associa ed wi h l and s b (28, 48, 60). 397
Howe e , in ou s udy, only a p opo ion o F18 posi i e isola es (n=8) ca ied as A, l cA and s b 398
genes, while he emaining s ains ha bo ed l cA and/o s b and s a genes. 399
Addi ionally, 16 o he i ulence genes we e p edic ed among s ains, and hei dis ibu ion was 400
associa ed wi h he ype o imb ia, sugges ing ha F4 and F18 posi i e isola es may use di e en 401
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i ulen s a egies o cause disease. As sugges ed, ETEC s ains ha bo ing addi ional imb iae 402
adhesins, could po en ially exploi o he al e na i e pa hways o coloniza ion o he hos (17). 403
O e all, he analysis showed ha ETEC om Danish pigs ha bo o he i ulence ac o s besides 404
hei cha ac e is ic adhesins and oxins, bu cu en ly he ole o such ac o s, i any, in in es inal 405
disease is no known. 406
Plasmids play an impo an ole in he sp ead and dissemina ion o bo h AMR and i ulence genes 407
(61). IncF, IncI and IncX eplicon ypes we e he mos p e alen and a leas one IncF eplicon was 408
de ec ed in all he s ains, as p e iously epo ed in po cine ETEC isola es om Aus alia and Spain 409
(32, 62). In gene al, F4 isola es showed mo e plasmid eplicon di e si y (21 di e en plasmid 410
eplicons) han he F18 isola es (eigh di e en plasmid eplicons). IncF plasmid ype is he mos 411
commonly desc ibed in bac e ia om humans and animals, pa icula ly in E. coli, and is known o 412
ca y i ulence and AMR genes (63, 64). IncI1 plasmids, he mos dominan among F18 s ains, 413
we e also de ec ed in all O141-F18 isola es and in 87.1% o O149:F4 isola es om Aus alia (32). 414
IncI1 ype plasmids a e o en associa ed wi h AMR and known o be ESBL ca ie s (65), howe e , 415
in he cu en s udy, ESBL we e no de ec ed, and he link o AMR was no in es iga ed. IncX 416
plasmids a e na ow hos ange plasmids o En e obac e iceae, which a e known o p o ide 417
addi ional ad an ages, commonly associa ed wi h AMR and bio ilm o ma ion (64, 66). Since 418
ETEC imb iae and oxin genes ha e been epo ed o en o be plasmid loca ed (67), we 419
in es iga ed he pu a i e plasmid localiza ion o hese genes and po en ial co-occu ence wi h AMR 420
de e minan s. F4 and F18 imb ia encoding genes we e ound o be plasmid loca ed in all he 421
s ains), oxins we e plasmid loca ed in mo e han 70% o he isola es, and in addi ion, he majo i y 422
o he AMR genes we e p edic ed o be plasmid encoded. In e es ingly, AMR and i ulence genes 423
we e o en p edic ed o be pa o he same plasmid componen , howe e , u he de ailed s udies 424
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a e needed o con i m his, since he p og am used o p edic ions (plasmidSPAdes) do no sepa a e 425
plasmids o he same ype p esen in a single s ain. 426
Recen s udies ha e epo ed ha AMR migh be associa ed wi h ole ance o hea y me als exis ing 427
na u ally o used in ood animal p oduc ion, such as zinc oxide and coppe (41, 68-70). He e, we 428
p edic ed ha aminoglycoside, e acycline and ampicillin esis ance genes and zinc o coppe 429
esis ance genes we e loca ed on he same plasmid componen , howe e , as men ioned abo e o 430
AMR and i ulence genes, mo e de ailed s udies a e needed o con i m his. Co-localiza ion would 431
imply ha use o ZnO and Cu may co-selec o AMR. In addi ion o hea y me als, biocides, 432
including disin ec an s and an isep ics, widely used in a ms, could also p omo e he sp ead o 433
AMR (68). All isola es analyzed he e we e p edic ed o ca y genes esponsible o biocidal 434
esis ance, bu co-occu ence wi h AMR was no in es iga ed. 435
Each o he 90 isola es unde s udy con ained a leas se en p ophages, mos ly simila o he 436
coliphages P88 ( om 3.9% o 93.3%) and Phi27 ( om 4.8% o 60.9%). Despi e Phi27 is known o 437
encode S x2e, none o he ETEC isola es encoded o Shiga oxins, hus indica ing ha , despi e he 438
simila i y, i is no he same phage. 439
In conclusion, he cu en s udy showed a high clonal di e si y among F18 isola es, while, in 440
con as , simila F4 clonal g oups migh be ci cula ing in Danish he ds. Besides, high a es o AMR 441
agains aminoglycosides, sul ame hoxazole, e acycline, ime hop im and ampicillin as well as 442
high di e si y o i ulence genes we e de ec ed -including oxin genes (l cA, as A, s a, s b), and 443
imb iae encoding genes yped as F4ac and F18ac. 444
MATERIALS AND METHODS 445
Bac e ial s ains and PCR de ec ion o imb iae ypes F4 and F18 446
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ETEC F4 isola es we e collec ed om pigs wi h dia hea in 2018 (n= 34, om 30 di e en a ms), 447
2019 (n=29, om 29 di e en a ms), and 1989-1992 (n=6, om six di e en a ms). P esump i e 448
ETEC F18 isola es (n=20) we e eco e ed om i e di e en a ms (collec ed a he same ime 449
poin in each a m) in 2019. In addi ion, E. coli Nysø, a well cha ac e ized ETEC s ain eco e ed 450
in he 70 ´s (71), was included as F18 his o ical con ol. S ains we e ob ained du ing ou ine 451
diagnos ic p ocedu es and use o esea ch pu poses did no equi e e hical clea ance, as long as 452
a m iden i y was no disclosed. 453
S ains we e con i med posi i e o F4 o F18 imb iae using PCR wi h p ime s and condi ions as 454
p e iously epo ed (52). 455
An imic obial esis ance pheno ype 456
MIC alues o E. coli isola es we e de e mined o amoxicillin–cla ulanic acid (2/1-32/16 µg/ml), 457
ampicillin (1-32 µg/ml), ap amycin (4-32 µg/ml), ce o axime (0.125-4 µg/ml), ce io u (0.5-8 458
µg/ml), chlo amphenicol (2-64 µg/ml), cip o loxacin (0.015-4 µg/ml), colis in (1-16 µg/ml), 459
lo enicol (2-64 µg/ml), gen amicin (0.5-16 µg/ml), nalidixic acid (4-64 µg/ml), neomycin (2-32 460
µg/ml), spec inomycin (16-256 µg/ml), s ep omycin (8-128 µg/ml), sulphame hoxazole (64-1024 461
µg/ml), e acycline (2-32 µg/ml) and ime hop im (1-32 µg/ml) by he b o h mic odilu ion me hod 462
using Sensi i e mic o i e ays (DKMVN4, Sensi i e sys em; The mo Fishe Scien i ic, Wes 463
Sussex, Uni ed Kingdom). E. coli ATCC 25922 was used o quali y con ol. Resul s we e 464
in e p e ed acco ding o EUCAST epidemiological cu -o alues, EUCAST clinical b eakpoin s o 465
amoxicillin-cla ulanic acid (www.EUCAST.o g) and DANMAP o ap amycin (72). Isola es we e 466
de ined as “suscep ible” when classi ied as “wild ype” and “ esis an ” when classi ied as “no wild 467
ype”. Mul id ug- esis an (MDR) s ains we e hose esis an o one agen om h ee o mo e 468
di e en an imic obial classes (73). 469
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PCR o de ec ion o s a and s b oxin genes 470
E. coli DNA was ex ac ed om a single o e nigh -g own colony by he boiling lysis me hod as 471
epo ed (52), and PCR ampli ica ion o s a and s b oxin genes was pe o med using he p ime s 472
and PCR condi ions p e iously desc ibed (52). 473
DNA ex ac ion and whole genome sequencing (WGS) 474
DNA was ex ac ed using he Maxwell® sys em (P omega) ollowing he ins uc ions p o ided by 475
he Maxwell® RSC cul u ed cells DNA ki (P omega). Quali y o he DNA was de e mined by 476
NanoD op-1000 (The mo Fishe Scien i ic) and DNA quan i ica ion was pe o med using dsDNA 477
BR Assay ki wi h he Qubi 2.0 luo ome e (In i ogen, USA). 478
The lib a ies o sequencing we e p epa ed using he Nex e a DNA Flex Lib a y P ep Ki (Illumina, 479
Inc., San Diego, CA, USA) acco ding o he manu ac u e ´s p o ocol and sequenced using Illumina 480
Nex Seq (Illumina). The pai ed-end aw eads we e assembled using SPAdes Genome Assemble 481
.3.13.0 (74) and he quali y o assembly was e alua ed wi h QUAST .5.0.2. (75). The aw 482
sequences we e submi ed o he Eu opean Nucleo ide A chi e (ENA) unde he s udy accession 483
numbe PRJEB38608. 484
Whole genome cha ac e iza ion 485
The assembled con igs, wi h genomic size anging be ween 5.1 and 5.7 Mbp (mean size 5.4 Mbp) 486
(Supplemen a y Table S1), we e analyzed using he bioin o ma ics ools o he Cen e o Genomic 487
Epidemiology (CGE) o he p esence o an ibio ic esis ance (ResFinde .3.2) (76), i ulence 488
genes (Vi ulenceFinde .2.0) (77), plasmid eplicon ypes (PlasmidFinde .2.1) (78), and 489
iden i ica ion o clono ypes (CHType .1.0), sequence ypes (ST) (MLST .2.0) (79) and se o ypes 490
(Se o ypeFinde .2.0) (75). All he CGE p edic ions we e called using de aul se ings. 491
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Iden i ica ion o an ibac e ial biocide- and me al- ole ance genes was assessed using BacMe -Scan 492
.2.0 (80).The Cle monTyping ool (h p://cle mon yping.iame- esea ch.cen e /) and PHASTER 493
webse e (h p://phas e .ca/) we e used o p edic he phylog oups and pu a i e p ophage 494
sequences in he bac e ial genomes, espec i ely (81, 82). 495
In silico p edic ion o localiza ion o an imic obial esis ance, i ulence and me al ole ance genes 496
and po en ial co-occu ence on plasmids 497
The pu a i e localiza ion o AMR and i ulence genes (F4, F18, l cA, as A, s a and s b) was 498
p edic ed using a combina ion o plasmidSPAdes .3.13.0 (83), ResFinde and Vi ulenceFinde 499
ools. B ie ly, plasmidSPAdes was used o iden i y con igs mos likely belonging o plasmid DNA 500
and o assign hem o componen s. Each componen is conside ed as a pu a i e plasmid consis ing 501
o one o mo e con igs. This ool is no able o sepa a e simila plasmids ( o example simila 502
plasmids o he same ype p esen in a single s ain), and hus hei con igs may be assigned o he 503
same id (same componen ). ResFinde and Vi ulenceFinde we e used o analyze he p esence o 504
AMR and i ulence genes in all con igs iden i ied as pu a i e DNA egions o plasmids. The ou pu 505
om bo h ools p o ides he con ig ID and componen on which he speci ic genes we e loca ed. 506
Genes con ained in he same con ig and/o componen we e p edic ed o be plasmid loca ed, while 507
hose an imic obial and/o i ulence genes no de ec ed in plasmid DNA con igs we e assumed o 508
be ch omosome encoded. 509
The BacMe da abase (BacMe -Scan .2.0.), which includes me al ole ance and biocide esis ance 510
genes (me al ole ance genes include also hose genes ha a e indi ec ly ela ed o me als) was used 511
o in es iga e he p esence o hese genes in all he genomes. The genome o he E. coli K12 512
subs ain MG1655 (GenBank accession numbe NC_000913.3) was also included in he analysis. 513
Plasmid con igs iden i ied and assigned o componen s wi h plasmidSPAdes .3.13.0 as desc ibed 514
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abo e we e analyzed wi h BacMe -Scan .2.0. Nex , he plasmid con igs we e manually inspec ed 515
o he p esence o me al ole ance genes (zinc, coppe , sil e , me cu y and ellu ium) p e iously 516
iden i ied on he genome assembly by using he BacMe da abase (Supplemen a y Table S4). Co-517
occu ence o AMR genes (de ec ed as indica ed abo e) and he me al ole ance genes on he same 518
plasmid was p edic ed when hey we e ound o belong o he same con ig. 519
Phylogene ic analysis 520
Phylogene ic ela ionships be ween he isola es we e analyzed based on SNP ees cons uc ed using 521
he bioin o ma ics ool CSI Phylogeny .1.4 (84) a ailable a CGE. The genome o he E. coli K12 522
subs ain MG1655 was included as a e e ence s ain, and CGE de aul pa ame e s we e used 523
du ing SNP analysis. The phylogene ic ee was isualized and edi ed by using he bioin o ma ics 524
ool iTOL 5 (85). 525
Besides, he ETEC isola es unde s udy we e compa ed wi h 20 swine ETEC isola es om h ee 526
di e en coun ies (China, USA and Spain) as men ioned abo e. The accession numbe s and 527
MLST- ypes o he 20 ETEC s ains used in his phylogene ic analysis a e indica ed in he 528
Supplemen a y Table S10. 529
S a is ical analysis 530
Di e ences be ween F4 and F18 posi i e s ains ega ding se og oups, an imic obial esis ance, 531
i ulence gene con en and plasmid eplicons we e analyzed using wo- ailed Fishe 's exac es wi h 532
he G aphPad P ism e sion 8.3 so wa e (G aphPad Inc). P alues <0.05 we e conside ed 533
s a is ically signi ican . 534
Cohen's Kappa s a is ical analysis was used o analyze he co ela ion be ween pheno ypic esis ance 535
and in silico gene p edic ions using SPSS e sion 26 (IBM, USA). Kappa alues ≤ 0 indica e no 536
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ag eemen ; 0.01–0.20 none o sligh ; 0.21–0.40 ai ; 0.41– 0.60 mode a e; 0.61–0.80 subs an ial, and 537
0.81–1.00 indica e alues wi h an almos pe ec ag eemen (86). 538
Acknowledgmen s 539
The au ho s a e g a e ul o Joakim La sson and Johan Beng sson-Palme o hei assis ance wi h he 540
use o he Bac-Me da abase as well as K. Aaga d and M. Ca lsen o hei echnical assis ance. 541
Funding 542
V. Ga cía acknowledges he Conselle ía de Cul u a, Educación e O denación Uni e si a ia, Xun a 543
de Galicia o he pos -doc o al g an (g an numbe ED481B-2018/018). This wo k was suppo ed 544
by Inno a ions onden (g an numbe 8088-00032B) and he Danish Minis y o Food o 545
En i onmen (Ve o ligIII). 546
Con lic s o in e es 547
All au ho s decla e ha hey ha e no compe ing in e es s. 548
Da a a ailabili y 549
The d a genome sequences o E. coli isola es om pigs in Denma k in his s udy a e a ailable in 550
he Eu opean Nucleo ide A chi e (ENA) unde he s udy accession numbe PRJEB38608. 551
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799
Legends o Figu es 800
801
Figu e 1. SNP-based phylogeny o he 90 ETEC isola es om pigs. Colou s in he ou e ing 802
co espond o phylog oups, he middle ing o imb iae ype and he inne ing o Sequence Type. 803
804
Figu e 2. SNP-based phylogeny o he 90 ETEC isola es om pigs in Denma k and he 20 ETEC 805
isola es om di e en coun ies. Colou s in he ing co espond o Sequence Type. Isola es 806
highligh ed in ed, o ange and blue co espond o isola es om China, USA and Spain, 807
espec i ely. 808
Figu e 3. Dis ibu ion o me al ole ance and biocides esis ance among he 90 ETEC isola es 809
om pigs (A), he F4-posi i e isola es (B) and he F18-posi i e isola es (C). 810
811
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Table 1. Se o ype, phylog oup, Sequence Type and clono ypes associa ion wi h imb ial an igens in he 90 812
ETEC isola es om pigs. 813
Se o ype-PG-ST-CH
Nº (%)
Fimb ial an igen
O149:H10-A-ST100-CH27-0
49 (54.4)
F4
ONT:H10-A-ST100-CH27-0
6 (6.7)
F4
O149:HNT-A-ST100-CH27-0
1 (1.1)
F4
O6:H16-A-ST48-CH11-34
11 (12.2)
F4
O8:H19-C-ST90-CH4-54
2 (2.2)
F4
O8:H17-C-ST23-CH4-54
6 (6.7)
F18
ONT:H4-A-ST10-CH11-24
5 (5.6)
F18
O141:H4-A-ST10-CH11-24
2 (2.2)
F18
O147:H14-D-ST42-CH28-65
3 (3.3)
F18
ONT:H14-D-ST42-CH28-65
2 (2.2)
F18
O29:H12-B1-ST155-CH4-121
1 (1.1)
F18
ONT:H19-C-ST90-CH4-0
1 (1.1)
F18
O8:H31-E-ST3524-CH23-31
1 (1.1)
F18
PG: phylog oup, ST: MLST- ype, CH: clono ype. 814
815
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Table 2. P e alence o an imic obial esis ance among he 90 ETEC isola es om pigs. 816
An imic obiala
Numbe o
esis an
isola es (%)
F4 (%)b
F18 (%)b
p- alue
OR a 95% CI
Ampicillin
47 (48.3)
35 (50.7)
12 (57.1)
0.6282
Amoxicillin/Cla ulanic acid
1 (1.1)
1 (1.5)
0
>0.9999
Chlo amphenicol/ lo enicol
15 (16.7)
9 (13.4)
6 (28.5)
0.1064
Ap amycin
8 (8.9)
7 (10.14)
1 (4.7)
0.6752
Gen amicin
6 (6.7)
8 (11.6)
1 (4.7)
0.6792
Neomycin
23 (25.6)
16 (23.2)
7 (33.3)
0.3964
Spec inomycin
50 (55.6)
35 (50.5)
15 (71.42)
0.1327
S ep omycin
62 (68.9)
53 (76.8)
9 (42.9)
0.0061
4.417 (1.491-12.48)
Sul ame hoxazole
61 (67.8)
46 (66.7)
15 (71.4)
0.7932
Te acycline
51 (56.7)
42 (60.9)
9 (42.8)
0.2084
T ime hop im
48 (53.3)
35 (50.7)
13 (62)
0.4568
Nalidixic acid
8 (8.9)
8 (11.6)
0
0.1901
aThe b eakpoin s used co espond o EUCAST Epidemiological cu -o alues (ECOFFs) o ampicillin (8 µg/ml), 817 chlo amphenicol/ lo enicol (8 µg/ml), gen amicin (2 µg/ml), nalidixic acid (8 µg/ml), neomycin (8 µg/ml), 818 spec inomycin (64 µg/ml), s ep omycin (16 µg/ml), sulphame hoxazole (64 µg/ml), e acycline (8 µg/ml) and 819 ime hop im (2 µg/ml), EUCAST clinical b eakpoin s o amoxicillin/cla ulanic acid (R > 8 µg/ml ) and DANMAP 820 2015 o ap amycin (R >32 µg/ml ) 821 bPe cen age is es ima ed based on he o al numbe o s ains associa ed o each imb ia ype. 822 OR: Odds a io is indica ed when p- alue <0.05; CI: con idence in e al; ∞: in ini y. Signi ican di e ences (p- alue 823 <0.05) a e indica ed in bold 824
825
826
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Table 3. An imic obial esis ance genes de ec ed among he 90 ETEC isola es om pigs. 827
An imic obial
g oup
Genes
To al
(%)
F4 (%)a
F18 (%)a
kappa
p-
alue
Be a-lac ams
bla
TEM-1A
4 (4.4)
4 (5.8)
0
0.933
0.0001
bla
TEM-1B
42 (46.7)
30 (43.5)
12 (57.1)
bla
TEM-30
1 (1.1)
1 (1.4)
0
Aminoglycosidesb
aph (phospho ans e ases)
58 (64.4)
50 (72.5)
8 (38.1)
0.927
0.0001
aadA
(nucleo idyl ans e ases)
57 (63.3)
42 (60.9)
15 (71.4)
aac (ace yl ansphe ases)
9 (10)
8 (11.6)
1 (4.8)
Phenicols
ca A1
3 (3.3)
3 (4.3)
0
1.000
0.0001
cmlA1
8 (8.9)
2 (2.9)
6 (28.6)
loR
5 (5.6)
5 (7.2)
0
Mac olides
md (A)
90 (100)
69 (100)
21 (100)
ND
ND
mph(A)
8 (8.9)
8 (11.6)
0
mph(B)
7 (7.8)
7 (10.1)
0
e m(B)
9 (10)
9 (13)
0
Lincosamides
lnu(F)
5 (5.6)
5 (7.2)
0
ND
ND
lnu(G)
5 (5.6)
5 (7.2)
0
Sulphonamides
sul1
30 (33.3)
28 (40.6)
2 (9.5)
0.898
0.0001
sul2
42 (46.7)
35 (50.7)
7 (33.3)
sul3
9 (10)
3 (4.3)
6 (28.6)
Te acycline
e (A)
40 (44.4)
32 (46.4)
8 (38.1)
1.000
0.0001
e (B)
13 (14.4)
12 (17.4)
1 (4.8)
e (X)
1 (1.1)
1 (1.4)
0
T ime hop im
d A1
34 (37.8)
27 (39.1)
7 (33.3)
0.978
0.0001
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d A5
2 (2.2)
2 (2.9)
0
d A12
8 (8.9)
2 (2.9)
6 (28.6)
d A14
5 (5.6)
5 (7.2)
0
d A17
2 (2.2)
2 (2.9)
0
aPe cen age is es ima ed based on he o al numbe o s ains associa ed o each imb ia ype. A p- alue <0.05 is 828 conside ed signi ican . 829 830
831
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Table 4. Dis ibu ion o i ulence genes (o he han F4 and F18-encoding imb ia) among he 90 ETEC 832
isola es. 833
Func ion
Genes
To al (%)
F4 (%)a
F18 (%)a
p- alue
OD 95% CI
Toxins
as A
77 (85.6)
69 (100)
8 (38.1)
<0.0001
∞ (25.71-∞)
l cA
82 (91.1)
69 (100)
13 (62)
<0.0001
∞ (9.402-∞
S a
21 (23.3)
13 (18.8)
8 (38.1)
0.0820
S b
85 (94.4)
69 (100)
16 (76.2)
0.0005
∞ (5.465-∞)
Fimb iae
lp A
15 (16.7)
2 (2.9)
13 (61.9)
<0.0001
0.01837 (0.003635-
0.08)
asA
2 (2.2)
2 (2.9)
0
>0.9999
Adhesion
iha
77 (85.6)
57 (82.6)
20 (95.2)
0.2854
ai
4 (4.4)
0
4 (19)
0.0023
0 (0.0-0.02885)
Colicin
cba
33 (36.7)
33 (47.8)
0
<0.0001
∞ (4.748-∞)
cma
47 (52.2)
42 (60.9)
5 (23.8)
0.0053
4.978 (1.631-13.29)
celb
10 (11.1)
8 (11.6)
2 (9.5)
>0.9999
Mic ocin
mchB
9 (10)
9 (13)
0
0.1096
mchC
9 (10)
9 (13)
0
0.1096
mchF
9 (10)
9 (13)
0
0.1096
mcmA
9 (10)
9 (13)
0
0.1096
O he s
capU
55 (61.1)
54 (78.3)
1 (4.8)
<0.0001
72 (11.75-759.7)
gad
40 (44.4)
35 (50.7)
5 (23.8)
0.0439
3.294 (1.09-8.811)
sepA
10 (11.1)
10 (14.5)
0
0.1088
iss
20 (22.2)
1 (1.4)
19 (90.5)
<0.0001
0.001548 (0.0001506-
0.0)
eilA
5 (5.6)
0
5 (23.8)
0.0005
0 (0.0-0.1830)
aPe cen age is es ima ed based on he o al numbe o s ains associa ed o each imb ia ype. 834
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