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An imic obial esis ance in he wild : Insigh s om epigene ics
© 2024 The Au ho (s). E olu iona y Applica ions published by John Wiley & Sons L d.
Published e sion
Villalba de la Peña, Ma iana; K onholm, Ilkka
Villalba de la Peña, M., & K onholm, I. (2024). An imic obial esis ance in he wild : Insigh s om
epigene ics. E olu iona y Applica ions, 17(6), A icle e13707.
h ps://doi.o g/10.1111/e a.13707
2024
E olu iona y Applica ions. 2024;17:e13707.  
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1 o 14
h ps://doi.o g/10.1111/e a.13707
wileyonlinelib a y.com/jou nal/e a
1 | INTRODUCTION
The disco e y and widesp ead a ailabili y o an imic obials ha e
g ea ly imp o ed he ea men o bac e ial and ungal in ec ions
in he mode n e a. Howe e , hei excessi e use in a ious sec-
o s, including ag icul u e, li es ock a ming, and human medicine,
has esul ed in a apid inc ease in an imic obial esis ance (AMR)
as he esul o he s ong selec ion p essu e ha an imic obial
subs ances impose on mic obes. This apid p oli e a ion o AMR
poses a se e e h ea o he well- being o humans, animals, and
plan s (Fishe e al., 2022; F ie i e al., 2017; Low & Ro s ein, 2011;
Mu ay e al., 2022). No e ha we use he e m an ibio ics o e e
o subs ances ha ei he kill o inhibi he g ow h o bac e ia, while
an i ungals deno es subs ances a ge ing ungi. The b oade e m
an imic obial encompasses bo h an ibio ics and an i ungals.
Gi en he p essing p oblem ha AMR ep esen s, conside -
able esea ch e o s ha e been di ec ed owa d elucida ing he
mechanisms behind AMR in clinical se ings. Howe e , a signi ican
Recei ed:1Oc obe 2023
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Re ised:4Ma ch2024
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Accep ed:26Ap il2024
DOI: 10.1111/e a.13707
REVIEW
An imic obial esis ance in he wild: Insigh s om epigene ics
Ma iana Villalba de la Peña  | Ilkka K onholm
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
© 2024 The Au ho (s). E olu iona y Applica ions published by John Wiley & Sons L d.
Depa men o Biological and
En i onmen al Science, Uni e si y o
Jy äskylä, Jy äskylä, Finland
Co espondence
Ma iana Villalba de la Peña and Ilkka
K onholm, Depa men o Biological
and En i onmen al Science, Uni e si y
o Jy äskylä, P.O. Box 35, FI- 40014
Jy äskylä, Finland.
Email: ma iana.m. illalba-delapena@jyu. i
and ilkka.k onhol[email p o ec ed]
Funding in o ma ion
Resea ch Council o Finland, G an /Awa d
Numbe : 321584
Abs ac
Sp eading o bac e ial and ungal s ains ha a e esis an o an imic obials poses a se-
ious h ea o he well- being o humans, animals, and plan s. An imic obial esis ance
has been mainly in es iga ed in clinical se ings. Howe e , h oughou hei e olu ion-
a y his o y mic oo ganisms in he wild ha e encoun e ed an imic obial subs ances,
o cing hem o e ol e s a egies o comba an imic obial ac ion. I is well known ha
many o hese s a egies a e based on gene ic mechanisms, bu hese do no ully ex-
plain impo an aspec s o he an imic obial esponse such as he apid de elopmen o
esis ance, e e sible pheno ypes, and he e o- esis ance. Consequen ly, a en ion has
u ned owa d epigene ic pa hways ha may o e addi ional insigh s in o an imic o-
bial mechanisms. The aim o his e iew is o explo e he epigene ic mechanisms ha
con e an imic obial esis ance, ocusing on hose ha migh be ele an o esis ance
in he wild. Fi s , we examine he p esence o an imic obials in na u al se ings. Then
we desc ibe he documen ed epigene ic mechanisms in bac e ia and ungi associa ed
wi h an imic obial esis ance and discuss inno a i e epigene ic edi ing echniques o
es ablish causali y in his con ex . Finally, we discuss he ele ance o hese epigene ic
mechanisms on he e olu iona y dynamics o an imic obial esis ance in he wild, em-
phasizing he c i ical ole o p iming in he adap a ion p ocess. We unde sco e he
necessi y o inco po a ing non- gene ic mechanisms in o ou unde s anding o an i-
mic obial esis ance e olu ion. These mechanisms o e in aluable insigh s in o he
dynamics o an imic obial adap a ion wi hin na u al ecosys ems.
KEYWORDS
adap a ion, an imic obial esis ance, epigene ics, mic obes, na u al en i onmen
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VILLALBA de la PEÑA and KRONHOLM
knowledge gap s ill exis s when i comes o unde s anding an imi-
c obial esis ance in na u al en i onmen s. This is c ucial as wild-
esis an mic obial popula ions se e as en i onmen al ese oi s
ha can ans e o human popula ions, impac he pe sis ence o
wild animals and plan s, and p o oundly a ec en i onmen al heal h
(Polianciuc e al., 2020). Se e al ungal and bac e ial epidemics in
he wild show he g ea isk ha pa hogenic mic obes ep esen
o biodi e si y and en i onmen al heal h ( o example, see Cheng
e al., 2011; Espelund & Kla eness, 2014; Fishe e al., 2009, 2012;
F ick e al., 2010; Sandmeie e al., 2009). Wildli e epidemics can
be in ensi ied by human ac i i ies due o he in oduc ion o alien
pa hogens (Fishe e al., 2012). Howe e , in wildli e, he heal h con-
sequences o he an imic obial- esis an s ains o an imic obial
ea men s emain poo ly unde s ood (A nold e al., 2016).
Mu a ion and ho izon al ans e o esis ance genes ha e
been e y well desc ibed and a e adi ionally conside ed he main
mechanisms h ough which mic obes e ol e esis ance (Hil unen
e al., 2017). Howe e , new e idence sugges s ha esis ance can
also e ol e h ough al e na i e non- gene ic ou es, such as epigen-
e ic mechanisms (Saba ís e al., 2023). We use he e m epigene ics
o e e o changes in gene exp ession pa e ns ha a e no caused
by an unde lying DNA sequence change, and ha can be ansmi -
ed h ough cell di ision. The mos common mechanisms ha medi-
a e epigene ic changes a e DNA me hyla ion, his one modi ica ion,
and small RNAs (K onholm, 2017). These mechanisms a e capable
o gene a ing di e se pheno ypes wi hin an isogenic popula ion by
con olling gene exp ession pa e ns. Also, he e e sible na u e
o hese epigene ic mechanisms makes he genome lexible o e-
spond o en i onmen al changes (Saba ís e al., 2023). T ansc ip ion
is con olled by ansc ip ion ac o s, and in he end hey a e e-
sponsible o egula ion o ansc ip ion ha happens du ing he
li e ime o an o ganism (Da idson, 2006). Epigene ic mechanisms
a e one laye o egula ion; o example, ce ain p o eins can ecog-
nize DNA me hyla ion and p e en he binding o ansc ip ion ac-
o s (Ma ei e al., 2022). Howe e , no all he epigene ic ma ks in
he genome will ha e a ansc ip ional e ec . Wha makes epigen-
e ic egula ion special is he ansmission o he epigene ic s a es
ac oss cell di ision.
Mechanisms ha media e epigene ic changes, such as DNA
me hyla ion, likely o igina ed as a de ense mechanism agains he
p oli e a ion o ansposable elemen s o i al DNA wi hin genomes,
la e hei oles expanded o encompass a ious genome p ocesses
(Sánchez- Rome o & Casadesús, 2020). These mechanisms a e p es-
en ac oss he domains o li e, bu exhibi a ia ion bo h wi hin and
ac oss axa. Pa icula epigene ic mechanisms a e p esen in di e -
en axa, and e en each species can ha e i s own mechanis ic pecu-
lia i ies ( o examples in highe euka yo es see Bewick e al., 2017;
Klughamme e al., 2023). B oadly, p oka yo es ely on DNA me hyl-
a ion as an epigene ic mechanism, since hey lack his ones. Howe e ,
hey do ha e his one- like p o eins pe o m a compa able unc ion
(Ca abe a, 2020; Sánchez- Rome o & Casadesús, 2020). In euka y-
o ic mic obes, such as mic oscopic ungi, he epigene ic mechanisms
a e mo e di e se. Mos o ou mechanis ic unde s anding comes
om in es iga ions in yeas s such as Schizosaccha omyces pombe and
he ilamen ous ungus Neu ospo a c assa, o which he epigene ic
machine y seems o mainly ely on his one modi ica ions and small
RNAs (Allshi e & Selke , 2009).
In his e iew, we will explo e esea ch conduc ed on AMR wi hin
na u al en i onmen s, wi h a speci ic ocus on he epigene ic mech-
anisms ha may con e esis ance. Ou examina ion will cen e on
bac e ia and mic oscopic ungi, as hey a e among he mos common
pa hogens in humans, animals, and plan s. Fu he mo e, conside ing
he ex ensi e u iliza ion o an ibio ics and an i ungals, which cu -
en ly s and as he p ima y con ibu o s o he de elopmen o AMR
in bo h clinical and na u al en i onmen s (Fishe e al., 2022; F ie i
e al., 2017; Lockha e al., 2023), ea ing hem oge he seems
easonable. Due o space cons ain s, we will no examine p o is s
in de ail, e en i his class con ains impo an pa hogens ha ha e
e ol ed AMR, such as mala ia.
2 | ANTIMICROBIALS IN THE WILD
An imic obial subs ances ha e exis ed na u ally in he wild o long
pe iods in he e olu iona y his o y o mic obes. Mic obes and o he
o ganisms p oduce hem o ou g ow compe i o s o o a oid pa a-
si es. Fo ins ance, a h opods can de elop symbio ic in e ac ions
o gene a e hei own an imic obials o de end agains an agonis s
(Janke e al., 2022; Ke e al., 2021). In a ious an species, gland
sec e ions inhibi he g ow h o en omopa hogenic ungi (Dall
e al., 2012). Bo h bee (Apis melli e a) and an (Pachicondyla gueldi)
enom con ains pep ides wi h po en an imic obial p ope ies e ec-
i e agains bac e ia (O i el e al., 2001). Also, nume ous ins ances o
a h opod- ungal in e ac ions ha e been documen ed; o examples
e e o (Aanen e al., 2002; Holmes e al., 2016; Sco e al., 2008;
Yek e al., 2012). Fo his eason, an imic obial esis ance mecha-
nisms ha e been e ol ing in he wild long be o e he cu en e a
o an imic obial esis ance. Indeed, D'cos a e al. (2011) iden i ied
an ibio ic esis ance genes in 30,000- yea - old sedimen s om he
Be ingian pe ma os . The exis ence o an ibio ic esis ance mecha-
nisms o se e al housand yea s in na u e explains he swi eme -
gence o an ibio ic- esis an s ains in clinical se ings, sugges ing
ha selec ion imposed by new an ibio ics ac s on p e- exis ing e-
sis ance mechanisms ha ha e been p esen in he wild o millennia
(D'cos a e al., 2011).
Al hough an imic obial subs ances occu na u ally in he wild,
an h opogenic ac i i ies ha e inc eased hei p e alence in na u-
al en i onmen s. Leakage in o he en i onmen is mainly h ough
pha maceu ical spillo e , h ough an imic obial p oduc s used in
plan a ions and li es ock, and h ough human exc e ion (Allen
e al., 2010; Hil unen e al., 2017; K aeme e al., 2019; La sson &
Flach, 2022; Wilkinson e al., 2022). Was ewa e ea men plan s
can elimina e some, bu no all an imic obials om ei he wa e o
he sludge (La sson & Flach, 2022). As a esul , hese end up in i -
e s o lakes, and he sludge ends up in ields o be used as e il-
ize . Fo example, Yuan e al. (2021) isola ed en e obac e ia om
17524571, 2024, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/e a.13707 by Uni e si y O Jy äskylä Lib a y, Wiley Online Lib a y on [30/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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VILLALBA de la PEÑA and KRONHOLM
six en i onmen s: hospi al, li es ock manu e, ag icul u al soil, o es
soil, i e sedimen , and was ewa e o unde s and he pe sis ence
o he esis an bac e ial pheno ype in na u e. They ound ha
s ains isola ed om he hospi al and om li es ock manu e had
he highes su i al capabili y when exposed o mul - id ug ea -
men s. This was con i med by PCR ampli ica ion o 12
𝛽
- lac amase
esis ance genes, om which hey ound ha he di e si y o
𝛽
- lac amase genes was highe in he hospi al. Howe e , he highes
abundance o
𝛽
- lac amase was ound in was ewa e , i e sedi-
men s, and ag icul u al soil (Yuan e al., 2021).
In a ecen global s udy pe o med by Wilkinson e al. (2022),
heau ho s epo ed heamoun o 61ac i epha maceu icaling e-
dien sin258 i e sin104coun ies.O  he61pha maceu icals,19
we ean imic obial(13we ean ibio icsand6an i ungals).Fi eo 
he 19 an imic obials we e no de ec ed in any si e (cloxacillin, sul a-
diazine, oxy e acycline, i aconazole, and miconazole). O e all, he
concen a ion o an ibio ics was highe compa ed o an i ungals.
The s udy de ec ed he highes concen a ion an imic ocbials in
A ica, Asia, and Sou h Ame ica. The e was a nega i e ela ionship
be ween coun y income and concen a ion o an imic obials, coun-
ies wi h he lowes incomes had he highes concen a ions. This
may be associa ed wi h ine icien was ewa e ea men in as uc-
u e in de eloping coun ies (Wilkinson e al., 2022) (Figu e 1). The
lowes cumula i e concen a ion was in Eu ope and No h Ame ica
and Oceania. In he la e , an imic obials we e only de ec ed a one
si e. I is impo an o no e ha he samples we e p ima ily collec ed
om Eu ope; howe e , o he bes o ou knowledge, his s udy ep-
esen s he mos comp ehensi e global analysis wi h compa able
measu emen s.
The s udy by Wilkinson e al. (2022) e ealed ha om he
13 o he an ibio ics de ec ed a leas i e (cip o loxain, cla i h o-
mycin, lincomycin, ime hop im, and me onidazole) exceeded
he sa e a ge concen a ion o AMR selec ion. In A ica and
Asia, he concen a ion o all i e o hese an ibio ics was highe
han he a ge . In Eu ope and Sou h Ame ica, ou an ibio ics ex-
ceeded he a ge , in No h Ame ica only h ee while in Oceania
only one. Howe e , an ibio ic concen a ions in he en i onmen
a e o en o de s o magni ude lowe han he minimum inhibi o y
concen a ion (MIC) (K aeme e al., 2019; La sson & Flach, 2022).
MIC is de ined as he minimum amoun o an ibio ic needed o in-
hibi bac e ial g ow h. I was p e iously assumed ha an ibio ic
esis ance was selec ed a a concen a ion abo e MIC (Kowalska-
K ochmal & Dudek- Wiche , 2021). Un ea ed municipal sewage,
ea ed sewage, i e s, and sea ha e been epo ed o con ain an-
ibio icpollu iona aconcen a ionbelow10 μg/L, which is below
he ypicalMICs(10–10,000 μg/L). While indus ially pollu ed su -
ace wa e and un ea ed hospi al e luen ypically ha e an ibi-
o ic concen a ion abo e MIC (La sson & Flach, 2022). Fo se e al
yea s, a much deba ed ques ion was whe he low amoun s o an i-
mic obials leaked in o he en i onmen could lead o an imic obial
esis ance. Howe e , he e is now much e idence o sugges ha
sub- inhibi o y concen a ions can selec o an imic obial esis ance
(Gullbe g e al., 2011). Fi s , i has been shown, using compe i ion
expe imen s, ha bac e ial s ains ha possess an ibio ic esis an
ma ke s g ow be e han he wild- ype s ain in an ibio ic concen-
a ions below he MIC, demons a ing ha esis an s ains ha e a
selec i e ad an age a low concen a ions (Gullbe g e al., 2011; Liu
e al., 2011). Second, e olu iona y heo y sugges s ha i is easie o
adap o la ge changes in he en i onmen i he o al en i onmen-
al change occu s slowly o in small inc emen s (Lindsey e al., 2013).
Consequen ly, in labo a o y se ings, he p obabili y ha bac e ial
popula ions e ol e an ibio ic esis ance is much highe i an ibio ic
concen a ion is slowly inc eased e sus i he an ibio ic concen a-
ion changes in a single s ep (Lindsey e al., 2013). Mo eo e , when
FIGURE 1 Globalan imic obial
cumula i e concen a ion. We show
he si es and he an imic obials ha
we e de ec ed acco ding o da a
ex ac ed om Wilkinson e al. (2022).
Cumula i e concen a ion is a gene al
measu e o he aqua ic con amina ion.
In his case is calcula ed as he sum o
all he an imic obial pha maceu icals
epo ed a all sampling loca ions in each
con inen (a). Si es whe e an imic obial
ac i e ing edien s we e de ec ed. We
excluded he si es whe e an imic obial
concen a ion was ze o (b). Concen a ion
o each o he an imic obial de ec ed
in each con inen . Ba s ep esen he
s anda d e o . Oceania is no included
in panel b because an imic obials we e
de ec eda alowconcen a ion(80 ng/L),
which is no clea ly isible on he g aph.
(a)
(b)
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VILLALBA de la PEÑA and KRONHOLM
he en i onmen changed slowly, bac e ia could e ol e much highe
le els o esis ance as hey acqui ed mul iple mu a ions, compa ed
o a single esis ance mu a ion ha was selec ed wi h ab up en-
i onmen al change (Lindsey e al., 2013). Mo eo e , u he e i-
dence shows ha an ibio ics can inc ease mu a ion a es, which in
u n can inc ease he p obabili y o esis ance mu a ions (Gu ie ez
e al., 2013).
To da e, a subs an ial body o e idence sugges s ha AMR in he
wild is hugely in luenced by human ac i i y and p esence. Wildli e
popula ions close o humans end o exhibi g ea e pe sis ence and
highe le els o an imic obial esis ance compa ed o popula ions
wi h li le o no con ac wi h human ac i i y (Hwengwe e e al., 2022;
Sku nik e al., 2006; Sousa e al., 2014; Welling on e al., 2013). This
phenomenon has been obse ed in a ious animal species, includ-
ing penguins (Mille e al., 2009), gulls (Wallens en e al., 2011), o he
bi ds (Waldens öm e al., 2005), he wild boa (To es e al., 2020),
and he Ibe ian lynx (Sousa e al., 2014). The esul s sugges ha e-
sis ance in he wild is la gely d i en by human ac i i y. Fo example,
in An a c ica, samples we e aken om wa e , sedimen , and wa e -
il e ing bi al es o isola e esis an s ains (Hwengwe e e al., 2022).
Two ypes o bac e ia we e isola ed: mesophiles, ypically associa ed
wi h humans, and psych ophiles, conside ed na i e o An a c ica.
The s udy e ealed ha mesophiles exhibi ed highe esis ance o a
wide ange o an ibio ics compa ed o psych ophiles. Fu he mo e,
esis ance le els inc eased as sampling si es d ew close o esea ch
s a ions ha ep esen mo e pe sis en human ac i i y in An a c ica
(Hwengwe e e al., 2022). This inding is consis en wi h o he s udies
ha ha e iden i ied a g adien o esis ance le els in animals based
on hei p oximi y o humans. Samples aken om animals in close
p oximi y o humans, including pe s and domes ic animals, showed
he highes p e alence and le els o esis ance compa ed o hose
om wild animals ha eside u he away om human popula ions
(Sku nik e al., 2006). Howe e , i is impo an o no e ha his pa -
e n may di e in wild bi ds and o he mig a o y species, as hey can
ca y esis an s ains o a eas wi h low human ac i i y (Welling on
e al., 2013). Fo example, in emo e loca ions such as Sibe ia, Alaska,
and G eenland, he p e alence o esis ance in bi ds exceeds ha
in local mammal popula ions (Radhouani e al., 2010; Welling on
e al., 2013). Impo an ly, all o hese s udies demons a e ha di ec
use o an ibio ics in he en i onmen is no necessa ily equi ed o
in oduce esis an s ains in o wild popula ions. Ins ead, p oximi y o
humans alone is su icien o ansmission (Hwengwe e e al., 2022;
Sku nik e al., 2006).
2.1  |  Epigene ic mechanisms o
an imic obial esis ance
The gene ic mechanism unde lying AMR ha e been ex ensi ely
s udied. Some o hese a e inna e, while o he s a e acqui ed h ough
gene ans e o ia de no o bene icial mu a ions. Howe e , hese
gene ic mechanisms alone ail o ully explain all he p ocesses
h ough which mic obes de elop esis ance. Fo example, he apid
eme gence o esis ance: su i al a es obse ed when cells a e
exposed o an imic obials a e oo high compa ed o wha would be
expec ed by gene ic mu a ions alone. Adam e al. (2008) ound ha
20% o E. colicellssu i eup o1 μg/mL ampicillin, and his pe cen -
age is oo high o be explained solely by he appea ance o andom
gene ic mu a ions. E en a educed concen a ions o an ibio ics,
he equencies o gene ic mu a ions a e low and canno accoun
o he obse ed su i al a e. Fo example, i is es ima ed ha he
p obabili y o inding a gene ic mu a ion ha con e s P. ae uginosa
s able esis ance o Quinolone is abou
1.2 ×10
−
6
o
4×10
−
10
de-
pending on he concen a ion used (Adam e al., 2008).
He e o- esis ance is ano he ins ance ha canno be explained
by gene ic changes. He e o- esis ance e e s o he a iable e-
sponse o an imic obial s ess wi hin an isogenic popula ion (Ghosh
e al., 2020). This he e ogenei y can be explained by phase a ia-
ion, which is he quick modi ica ion o gene exp ession pa e ns
by swi ching on and o ce ain esis ance genes. Phase a ia ion
is known o be egula ed by non- gene ic mechanisms such as DNA
me hyla ion (Ghosh e al., 2020; Jiang e al., 2019). Phase a ia ion is
c ucial o adap i e esis ance, cha ac e ized by he empo a y en-
hancemen o mic obes' abili y o su i e an imic obial subs ances
h ough al e a ions in gene exp ession (Hołówka & Zak zewska-
Cze wińska, 2020; Veening e al., 2008). Fo example, e idence
shows ha adap i e esis ance o ce ain an ibio ics in E. coli is co -
ela ed wi h he amoun o a ia ion in he exp ession o he e lux
pump sys em (Fe nández & Hancock, 2012; Mo a e al., 2015).
Adap i e esis ance also includes he apid eme gence o he
esis an pheno ype and he capabili y o e e o he suscep ible
pheno ype upon emo al o an imic obial s ess (Ghosh e al., 2020).
The es o a ion o he suscep ible pheno ype (Day, 2016; Ghosh
e al., 2020) would necessi a e a high numbe o back mu a ions,
which a e known o occu a a e y low a e (Adam e al., 2008;
Le in e al., 2000). Fo adap i e esis ance o be e icien , he i able
pheno ypic a ia ion media ed by epigene ic mechanisms should be
ansmi ed ac oss gene a ions (Fe nández & Hancock, 2012). Once
an imic obial s ess is emo ed, he uns able epigene ic changes a e
no longe ad an ageous, e en ually leading o he es o a ion o he
o iginal suscep ible pheno ype (Ghosh e al., 2020).
An imic obial esis ance can also be explained by he appea ance
o pe sis e cells. When a bac e ial popula ion is challenged wi h
an ibio ics, i s popula ion size will apidly decline due o he high
mo ali y a e. A e he decline, he popula ion size will s abilize
and popula ion g ow h slows down because he una ec ed cells will
en e a do man s a e called pe sis ence (Day, 2016). The pe sis ence
s a e is de ined as a physiological s a e o do mancy ha bac e ia
en e when hey encoun e en i onmen al s esso s (Day, 2016;
Ribe & Hansen, 2021), including low concen a ions o an ibio ics
(Mo a e al., 2015). This s a e is an e ec i e s a egy agains insul -
ing en i onmen s, as i p o ides esis ance in condi ions ha would
be ha m ul o no mally g owing cells. In e es ingly, he pe sis ence
s a e is e e sed in a d ug- ee en i onmen , whe e pe sis e cells
go back o a me abolically ac i e s a e, ees ablishing he o iginal
suscep ible popula ion (Day, 2016; Mo a e al., 2015). This swi ch
17524571, 2024, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/e a.13707 by Uni e si y O Jy äskylä Lib a y, Wiley Online Lib a y on [30/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License

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VILLALBA de la PEÑA and KRONHOLM
be ween ac i e and do mancy s a es seems o be one o he main
causes o adap i e esis ance o an ibio ic ea men , leading o high
a es o bac e ial in ec ion elapses (Ribe & Hansen, 2021).
Gi en his e idence, he e is a g owing ealiza ion ha AMR
la gely depends on he i able pheno ypic a ia ion po en ially caused
by epigene ic changes. In he ollowing sec ion, we will desc ibe he
speci ic epigene ic mechanisms ha ha e been associa ed wi h an i-
bio ic esis ance, emphasizing hose ha ha e been associa ed wi h
below MIC adap a ion and nonclinical en i onmen s.
2.2  |  Epigene ic mechanisms in bac e ia and hei
ole in an ibio ic esis ance
2.2.1  |  DNAme hyla ion
DNA me hyla ion is he addi ion o a me hyl g oup o cy osine o
adenosine wi hin DNA. Bac e ial genomes can hold h ee ypes o
DNA me hyla ion: (1) 5- me hylcy osine (5mC), (2) N4- me hylcy osine
(4mC), whe e a me hyl g oup is added o ei he he posi ion i e o
N4o  hecy osine, espec i ely,and(3)N6-me hyladenosine(6 mA),
which is he mos ho oughly s udied (Sánchez- Rome o e al., 2020).
This modi ica ion occu s when he N6-posi ion o  adenosine is
me hyla ed (Wang e al., 2023). The enzymes esponsible o all
hese modi ica ions a e DNA me hyl ans e ases (MTases). MTases
can be associa ed wi h he es ic ion- modi ica ion (R- M) de ense
sys em which is a de ense agains exogenous DNA. The DNA o he
bac e ial ch omosome con ains me hyla ion, es ic ion enzymes
ecognize i and deg ade only unme hyla ed DNA (Sánchez- Rome o
& Casadesús, 2020). Howe e , bac e ia can also ha bo MTases
ha a e no pa o he de ense sys em ha a e known as o phan
MTases. These o phan MTases pe o m essen ial unc ions wi hin
he cell, including in luencing bac e ial g ow h, pa icipa ing in DNA
epai p ocesses, and egula ing gene exp ession (Sánchez- Rome o
& Casadesús, 2020).
Adenosine me hyla ion occu s in bac e ia mainly in palind omic
sequences (5′-GATC-3′) (Sánchez- Rome o & Casadesús, 2020), bu
can occu in o he mo i s as well (B uneaux e al., 2022). Palind omic
mo i s allow he inhe i ance o DNA me hyla ion pa e ns. MTases
ecognize hemime hyla ed DNA o med a e DNA eplica ion and
e- me hyla e he unme hyla ed DNA s and. Fo adenosine me h-
yla ion, he hemime hyla ed s a e is usually sho - li ed, bu s able
hemime hyla ed GATC si es can o m i a DNA- binding p o ein blocks
MTase ac i i y. I his s a e pe sis s h ough DNA eplica ions, a non-
me hyla ed si e is o med. Such compe i ion be ween MTases and
DNA binding p o eins is esponsible o he i able epigene ic changes
in bac e ia, which a e c ucial o adap i e esis ance o be e ec i e
(He nday e al., 2003; Phillips e al., 2019). Such changes be ween
me hyla ed and non- me hyla ed s a es u n ansc ip ion o and
on and a ec many impo an pheno ypes, such as pa hogenici y,
phage esis ance, g ow h, an ibio ic esis ance, and gene exp ession
he e ogenei y (A ack e al., 2015; Co a e al., 2015; Sánchez- Rome o
e al., 2020; T am e al., 2021; an de Woude e al., 1996).
While he genome wide a es o spon aneous adenosine me h-
yla ion changes a e no known, some s udies ha e looked a a es o
adenosine me hyla ion changes a indi idual loci. Fo example, Blyn
e al. (1989) in es iga ed he swi ching a e o he pap- ope on in E.
coli. When E. coli we e g own wi h glyce ol as a ca bon sou ce, he
a e o change om OFF o ON s a e was
1.57 ×10
−
4
pe cell pe
gene a ion, and a e om ON o OFF s a e was
2.60 ×10
−
2
. When
cells we e g own wi h glucose as he ca bon sou ce, he a e o an-
si ion om OFF o ON s a e was
4.51 ×10
−
6
. Glucose is a be e
ca bon sou ce han glyce ol, so s ess may inc ease a es o spon-
aneous adenosine me hyla ion change. Mo eo e , we do know ha
swi ching e en s a di e en loci happen independen ly om each
o he (Sánchez- Rome o e al., 2020).
Empi ical e idence has shown ha a ious s ains o E. coli mu-
an s, lacking he MTase DNA adenine me hylase, exhibi ed inc eased
sensi i i y and lowe EC50 alues when exposed o be a- lac ams,
quinolones, and nalidixic acid. EC50 ep esen s he an ibio ic con-
cen a ion ha induces a biological esponse hal way be ween he
baseline and he maximum esponse (Adam e al., 2008; Chen &
Wang, 2021; Cohen e al., 2016). Toge he , all o his e idence sug-
ges s a c ucial ole o adenosine me hyla ion in an ibio ic esis ance.
The e ec s o cy osine me hyla ion in bac e ia a e no well cha -
ac e ized, al hough some e idence sugges s ha cy osine me hyl-
a ion is in ol ed in ansc ip ional egula ion du ing he s a iona y
phase (Kah amanoglou e al., 2012). Fu he mo e, high le els o
DNA cy osine me hyla ion ha e been associa ed wi h an ibio ic
esis ance. Fo example, s udies ha e iden i ied a posi i e co ela-
ion be ween 5mC and an ibio ic esis ance in en e obac e ia (Yuan
e al., 2021; Yugend an & Ha ish, 2016). Fu he mo e, DNA cy osine
me hylase knockou mu an s o E. coli exhibi ed lowe EC50 alues
when exposed o 20 di e en an ibio ics (Chen & Wang, 2021).
Howe e , con adic o y esul s ha e been epo ed in o he s udies
in which E. coli mu an s lacking 5mC did no show any signi ican
e ec on an ibio ic su i al (Adam e al., 2008).
Cy osine DNA me hyla ion has been desc ibed o a ec he eg-
ula ion o he e lux pump sys em in E. coli and En e obac e clocae
(Fe nández & Hancock, 2012; Mili ello e al., 2014). E lux pump sys-
ems a e ene gy- dependen sys ems ha allows he cell o expel oxic
compounds om he inne - cell en i onmen o p e en he accumu-
la ion o oxins (Mo a e al., 2015). Se e al e lux pump sys ems ha e
been highly associa ed wi h an ibio ic esis ance; hey ha e also been
pa icula ly cha ac e ized as being in ol ed in mul i- d ug esis ance,
as mos e lux pump sys ems can anspo mo e han one subs ance
(Fe nández & Hancock, 2012; Mo a e al., 2015). Fo example, he
memb ane anspo e sugE, which is classi ied as a mul i- d ug esis-
ance anspo e has DNA cy osine me hylase ecogni ion si es in
he gene body and ups eam o he ansc ip ion s a si e (Mili ello
e al., 2014). The in luence o cy osine DNA me hyla ion on sugE ex-
p ession was con i med by using DNA cy osine me hylase knockou
mu an s. Knockou mu an s exp ess sugE a le els se en imes highe
compa ed o wild ype, p o iding e idence ha 5mC in luences he
sensi i i y o an an imic obial compound h ough changes in gene ex-
p ession (Mili ello e al., 2014).
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VILLALBA de la PEÑA and KRONHOLM
2.2.2  |  His one-likep o eins
The bac e ial ch omosome is ci cula and is no packed inside a nu-
cleus a ound his ones, as in euka yo es. Howe e , bac e ial DNA
s ill needs o be compac ed. Bac e ia pack hei genomes a ound
nucleoid- associa ed p o eins (NAPs) (Wang e al., 2023). NAPs a e
small p o eins ha old and condense DNA and egula e gene ex-
p ession. They pa icipa e in p ocesses such as eplica ion, ans-
la ion, and epai o he bac e ial genome (Amemiya e al., 2021;
S ojko a e al., 2019; Wang e al., 2023). Also, unde s ess condi-
ions, hey can ei he help p o ec he DNA o induce ansc ip omic
changes o ac i a e s ess- ela ed genes (Amemiya e al., 2021;
Hołówka&Zak zewska-Cze wińska,2020).
NAPs di e be ween species, wi h hose in E. coli being be e
desc ibed. The main NAPs a e HU (hea - uns able p o ein), IHF (in e-
g a ion hos ac o ), H- NS (his one- like nucleoid s uc u ing p o ein),
L p (leucine- esponsi e egula o y p o ein), Fis ( ac o o in e sion
s imula ion), and Dps (DNA- binding p o ein om s a ed cells)
(Hołówka&Zak zewska-Cze wińska,2020).
NAPs can unde go pos ansla ional modi ica ions (e.g., ace yl-
a ion o phospho yla ion o lysines). These modi ica ions in luence
DNA- binding e iciency. Due o he c ucial ole ha NAPs play in
DNA condensa ion and s ess esponse h ough phase a ia ion, i
is likely ha NAPs and hei chemical pos ansla ional modi ica-
ions emain s able h ough se e al di isions (Ghosh e al., 2016).
Addi ionally, p o ein pos ansla ional modi ica ions ha e been
p o en o be a memo y mechanism in bac e ia (Lisman, 1985;
Veening e al., 2008).
The i s e idence o he ole o NAPs in an ibio ic esis-
ance came om a s udy using Mycobac e ium smegma is (Saka os
e al., 2018). P e ious s udies wi h his bac e ium had shown ha
subpopula ions o pe sis e cells would a ise a a ela i ely high
equency wi hin an isogenic popula ion (Muhammad e al., 2022).
Th ough ansc ip omics and li e cell imaging,c Saka os e al. (2018),
dis inguished hese pe sis en cells wi hin a popula ion and de-
sc ibed hei unique ansc ip omic signa u es. The pe sis e pheno-
ype was inhe i ed by daugh e cells and emained s able o sho
pe iods o ime, al hough i was e en ually los in he absence o
an ibio ics. The au ho s we e able o de e mine ha he his one-
like p o ein HupB played a c ucial ole in he he e ogeneous e-
sponse, since dele ing he HupB p o ein made he cell popula ion
mo e suscep ible o an ibio ics. Fu he mo e, hey also disco e ed
ha mu a ing si es wi h pos - ansc ip ional modi ica ions in HupB
dec eased he pe sis e subpopula ions (Saka os e al., 2018). This
s udy was he i s o demons a e ha p oka yo es u ilize pos -
ansla ional modi ica ions o egula e an ibio ic esis ance.
Fu he mo e, s udies in mul i- d ug- esis an bac e ia
Acine obac e baumannii showed ha he his one- like nucleoid s uc-
u ing p o ein (H- NS) egula es he exp ession o genes in ol ed in
esis ance o se e al an ibio ics (Rodge s e al., 2021). Impo an ly,
his his one- like p o ein and o he well- known NAPs (IHF and HU)
a e c ucial o bio ilm o ma ion. Bio ilms a e sys ems o mic obial
cells ha a e s ongly associa ed wi h a su ace embedded in a ma ix
o mic obial o igin (Dias e al., 2018). Bio ilms highly enhance AMR in
clinicalandna u alse ings.In ac ,mo e han65%o mic obialin-
ec ions a e caused by bac e ia g owing in bio ilms (Dias e al., 2018;
Wang e al., 2023). Fo example, wild p o icien bio ilm p oduce s
bac e ia (Acine obac e spp., Klebsiella pneumoniae, Pseudomonas lu-
o escens, and Shewanella pu e aciens) we e highly esis an o mul i-
d ug ea men s in hei bio ilm o m (Dias e al., 2018).
2.2.3  |  RNAmodi ica ions
The inhe i ance o ac o s such as RNAs and p o eins du ing cell
di ision has p omp ed he s udy o RNA molecule modi ica ions
as po en ial mechanisms o p o iding an ibio ic esis ance (AMR)
ac oss gene a ions. In bac e ia, a ious o ms o me hyla ion (5mC,
6 mA, and N1-me hyladenosine) ha e been iden i ied on di e en 
ypes o RNA molecules, including ans e RNA ( RNA), messenge
RNA (mRNA), ibosomal RNA ( RNA), and non- coding RNA (ncRNA)
(Ma baniang & Vogel, 2016; Shi e al., 2019). These modi ica ions
play a ole in egula ing and s abilizing RNA molecules, con ibu -
ing o di e si y in ansla ion and c ea ing apid pheno ypic a ia ion
(E ans e al., 2019).
No ably, RNA me hyla ion has been associa ed wi h an ibio ic
esis ance by p e en ing an ibio ics om binding o hei a ge
si es (Liu e al., 2015; Tada e al., 2013). Addi ionally, he knockou
o RNA me hyl ans e ase a ec s he biosyn hesis o he double
memb ane in g am- nega i e bac e ia, weakening he cell en elope
s uc u e, which se es as a pe meabili y ba ie and an ancho o
e lux pumps (Hou e al., 2020; Masuda e al., 2019).
Recen esea ch by Babosan e al. (2022), iden i ied RNA
modi ica ion genes no p e iously linked o an ibio ic esis ance
as ele an o i ness in Vib io chole ae. Pa icula ly in iguing is
he obse a ion o hese mechanisms in bac e ia g owing in sub-
minimal inhibi o y concen a ion (sub- MIC), making hem espe-
cially pe inen o esis ance in he wild. T ansposon sequencing
e ealed di e en ial ac i a ion o inac i a ion o genes unde sub-
MIC an ibio ic s ess ( ob amycin and cip o loxacin), wi h RNA
modi ica ion genes being en iched di e en ly no only in he p es-
ence o absence o an ibio ics bu also when exposed o di e en
an ibio ics. This s udy sheds ligh on he b oade ole o RNA mod-
i ica ions in an ibio ic esis ance. Ne e heless, u he esea ch is
essen ial, o e ing nume ous oppo uni ies o explo a ion in his
ield (Babosan e al., 2022).
Whe he RNA modi ica ions can be conside ed epigene ic e-
mains o be seen. To be inhe i ed, a pa icula ype o RNA has o
be s able enough o pe sis h ough cell di ision. Fo mRNAs his
is unlikely o be ue, as measu ed mRNA hal - li es in bac e ia a e
a ound a ew minu es (Selinge e al., 2003). Ribosomal RNA is likely
s able enough as ibosomes a e s able in g owing bac e ial cells (Pii
e al., 2011). Mo eo e , he RNA modi ica ions would likely ha e o
be p esen in la ge enough numbe s o ha e an e ec , which would
likely equi e ha he modi ica ion is o iginally igge ed by an en i-
onmen al signal.
17524571, 2024, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/e a.13707 by Uni e si y O Jy äskylä Lib a y, Wiley Online Lib a y on [30/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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7 o 14
VILLALBA de la PEÑA and KRONHOLM
2.3  |  Epigene ic mechanisms o an imic obial
esis ance in ungi
2.3.1  |  His onemodi ica ions
In euka yo ic cells, genomic DNA is packed in ch oma in, which
is made up o nucleosomes. Each nucleosome comp ises app oxi-
ma ely146basepai so DNAwounda oundeigh his onep o eins,
including wo subuni s each o he his ones H2A, H2B, H3, and H4
(F ei ag, 2017). These his ones can ha e pos ansla ional modi i-
ca ions on ce ain esidues on hei N- e minal ails, such as me h-
yla ion, ace yla ion, phospho yla ion, and ubiqui ina ion. These
modi ica ions se e as key egula o s o ch oma in s uc u e, by
making DNA mo e o less accessible o he ansc ip ion and epai
machine y (F ei ag, 2017).
Mos unc ional desc ip ions o ungal his one modi ica ions
come om model species, such as budding (Saccha omyces ce e i-
siae) and ission (Schizosaccha omyces pombe) yeas . The landscape
o his one modi ica ions among ungal species is gene ally well con-
se ed. Howe e , i is impo an o no e ha ce ain his one mod-
i ica ions a e no p esen in all ungal species (F ei ag, 2017). Fo
example, SET enzymes, he p o eins esponsible o he me hyla ion
o he N- e minal his one ails, a e p esen among mos , bu no all
ungal species. Budding yeas lacks me hyla ion a lysine 9 o his-
one 3 (H3K9) (F ei ag, 2017; O'Kane & Hyland, 2019), while i oc-
cu s in he ission yeas and se e al o he ilamen ous ungi (e.g.,
Muco , Rhizopus, and Aspe gillus; B osch e al., 2008). Fu he mo e,
me hyla ion on lysine 27 o his one 3 (H3K27) is absen in he bud-
ding yeas , ission yeas , and se e al ilamen ous ungi (B osch
e al., 2008; O'Kane & Hyland, 2019), bu o he ilamen ous ungi
such as Neu ospo a c assa and Fusa ium g aminea um do exhibi
me hyla ion a his posi ion (B osch e al., 2008).
In ission yeas Schizosaccha omyces pombe, he mechanisms
o epigene ic inhe i ance h ough his one modi ica ions a e bes
unde s ood. Yeas gene icis s ha e known o some ime ha S.
pombe can exhibi a “cul u e memo y,” whe e p e ious en i onmen-
al condi ions can a ec he g ow h o a popula ion (Pe e sen &
Russell, 2016). The mechanism behind hese e ec s can be he o -
ma ion o he e och oma in and associa ed ansc ip ional changes.
This p ocess is guided by small RNAs (Yamanaka e al., 2013), in-
deed epigene ic inhe i ance has been demons a ed in ission yeas
(Aude gon e al., 2015; Raguna han e al., 2014; Yu e al., 2018). The
main silencing epigene ic ma k in S. pombe is H3K9me and sho in-
e e ing RNAs (siRNAs) which a e equi ed o main ain he silenced
epigene ic s a e ac oss cell di isions (Yu e al., 2018).
Ch oma in ea angemen s and his one modi ica ions can a-
cili a e he exp ession o di e en pheno ypes, p o iding addi-
ional mechanisms h ough which o ganisms cope wi h an i ungal
d ugs. Fo example, his one ace yla ion has been demons a ed
o play a ole in an i ungal esis ance in Candida albicans (Chang,
Yada , e al., 2019; Ga naud e al., 2016). Deace ylase p o eins
ha e been shown o be c ucial in an i ungal esis ance. Genes
encoding deace ylases, such as HDA1 and RPD3, exhibi highe
exp ession le els in s ains esis an o azoles (Ga naud e al., 2016).
Fu he mo e,deple iono H3K56ace yla ionleads oa educ ion
in i ulence. This is also ue o an al e na i e deace ylase complex
composed o Se 3, Hos2, SNT1, and Si 2, which media e an i un-
gal esis ance in C. albicans bio ilms (Nobile e al., 2014). Simila ly, in
C yp ococcus neo o mans, he dele ion o his one deace ylase genes
weakens pa hogenici y and a ec s sensi i i y o a ious en i on-
men al s esso s. Deace ylase p o eins, in addi ion o hei ac ion on
his ones, can also egula e o he p o eins, including he hea shock
p o ein 90, which is essen ial o s ess esponse, i ulence, and d ug
esis ance (Lamo h e al., 2015).
I is impo an o no e ha e en when wo species sha e he
same epigene ic pa hways, hei unc ions may di e . Fo example,
me hyla ion o lysine 4 in his one h ee (H3K4me) plays a c ucial
ole in an i ungal esis ance in bo h budding yeas Saccha omyces
ce e isiae and Candida glab a a (Bake e al., 2022). Howe e , he
an i ungal esis ance con e ed by H3K4me may be a ibu ed o
he egula ion o di e en pa hways. In he case o budding yeas ,
he absence o H3K4 inc eases suscep ibili y o azoles by p e en -
ing o e exp ession o e lux pumps. These e lux pumps, simila o
hose desc ibed in bac e ia, expel oxins om he cell. In he o he
case, he same epigene ic mechanism in C. glab a a inc eases sus-
cep ibili y o azoles by a ec ing he exp ession o genes in ol ed
in he e gos e ol biosyn hesis pa hway, which helps o main ain cell
memb ane in eg i y in ungi (Bake e al., 2022).
In ission yeas , i was shown ha he e och oma in silencing,
o ches a ed by he H3K9me epimu a ion, can allow adap a ion o
ca eine (To es- Ga cia e al., 2020). Yaseen e al. (2022) dissec ed
he unde lying molecula mechanism by which H3K9me con e s
esis ance o ca eine. Exposu e o ca eine a ec s he egula ion
o Epe1, which con ains a conse ed domain ha p omo es his one
deme hyla ion and is c ucial o he o ma ion o he e och oma in-
euch oma in bounda ies (So ida & Mu akami, 2020). Exposu e o
en i onmen al insul s esul s in he accumula ion o a unca ed
o m o Epe1, which, in u n, inc eases H3K9me in se e al egions
o he genome, educing he exp ession o he unde lying genes and
imp o ing esis ance (To es- Ga cia e al., 2020; Yaseen e al., 2022).
This esea ch is ele an because i un angles he molecula mech-
anisms behind esis ance ha is con e ed exclusi ely by epigene ic
mechanisms. Fu he mo e, ca eine- esis an s ains show c oss-
esis ance o an i ungal agen s. Simila he e ochoma in silencing
mechanisms a e highly conse ed in se e al pa hogenic ungi, sug-
ges ing ha simila silen mechanisms could be behind an i ungal
esis ance also in he wild.
2.3.2  |  RNA-basedmechanisms
The ansc ip ional p oduc s o non- coding genes can be b oadly
classi ied as small non- coding o long non- coding RNAs (lncRNAs),
bo h o hese ha e been shown o impac an i ungal esis ance
(Chang, Yada , e al., 2019). Small in e e ing RNA molecules (siR-
NAs) a e one o he bes unde s ood mechanisms o gene silencing
17524571, 2024, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1111/e a.13707 by Uni e si y O Jy äskylä Lib a y, Wiley Online Lib a y on [30/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
8 o 14
|
VILLALBA de la PEÑA and KRONHOLM
in ungi. siRNAs a e 20–30 base pai long RNA agmen s ha e-
p ess gene exp ession (Dang e al., 2011). Muco ci cinelloides esis -
ance o heFK506an i ungalagen isawell-knownexampleso how
epimu a ions can con e an imic obial esis ance ia siRNAs (Chang,
Yada , e al., 2019). Calo e al. (2014) desc ibed ha in M. ci cinel-
loides, endogenous exp ession o siRNAs con e ed esis ance by si-
lencing he exp ession o he kbA gene. kbA encodes he subs a e
on which he FK506 an i ungal ac s. The au ho s also ound ha 
when he ungus was e u ned o a d ug- ee en i onmen , wild- ype
esis ance was es o ed as siRNA epimu a ion was no longe p e-
sen . I was also desc ibed ha his same ungus can use he same
mechanism o gain esis ance o he an i ungal 5- luo oo o ic acid
(5- FOA) by siRNAs (Chang, Billmy e, e al., 2019). The accumula ion
o siRNAs silences he exp ession o he py F o py G genes, which
p oduce necessa y enzymes ha con e 5- FOA in o a oxic agen
o he cell (Chang, Billmy e, e al., 2019; Chang, Yada , e al., 2019).
In he ission yeas , i has been shown ha lncRNAs can egula e
an i ungal esis ance. A d e al. (2014) demons a ed ha by dele ing
he lncRNA ncRNA.1343, hey could inc ease he sensi i i y o a b oad
spec um o an i ungals. In his same in es iga ion, hey desc ibed
ha his is possible because ncRNA.1343 con ols he egula ion o
a neighbo ing gene, gp1, which encodes a glyce ophosphodies e
memb ane anspo e . The lncRNA inc eases nucleosome densi y,
impeding ansc ip ion ac o binding, esul ing in he down egula ion
o gp1. Finally, hey demons a ed ha he dele ion o he lncRNA
induces he exp ession o gp1, demons a ing ha ncRNA.1343 can
egula e an imic obial esis ance in he ission yeas .
3 | EDITING THE EPIGENOME
Due o he subs an ial body o e idence ha poin s o he signi i-
can ole o epigene ics in AMR, i becomes impe a i e o es ablish
a causal ela ionship be ween epigene ic mechanisms and an imi-
c obial esis ance. The comp ehension and disen angling o complex
egula o y sys ems ega ding he ela ionship be ween epigene ics
and AMR a e i al o undamen al and applied esea ch, o e ing
p omising a enues o comba he g owing challenge o an imic obial
esis ance.
The esea ch e iewed he e e eals a obus co ela ion be ween
epigene ic s a es and an imic obial esis ance h ough gene egula-
ion. Howe e , demons a ing a di ec causal ela ionship be ween
epigene ic s a es and gene exp ession has p o en o be a challeng-
ing ask. Ne e heless, his could be made possible hanks o he
a ailabili y o nume ous epigene ic edi ing ools. Allegedly, nea ly
e e y locus in he genome can be a ge ed using hese ools o mod-
i y exp ession pa e ns. Achie ing his in ol es making si e- speci ic
al e a ions in he epigenome h ough he use o p og ammable
DNA- binding domains (Thako e e al., 2016). Among hese domains,
zinc inge s, ansc ip ion ac i a o - like e ec o s (TALEs), and ype
II CRISPR a e he mos used (Thako e e al., 2016). These p og am-
mable DNA- binding domains ha e been success ully u ilized o a -
ge ed ansc ip ional ac i a ion and ep ession, p o iding e idence
o causali y, unc ionali y, and c oss- alk among epigene ic ma ks.
Jus a ew s udies ha e e ec i ely subs an ia ed he causal ela-
ionship be ween epigene ic s a es and pa e ns o gene egula ion
(Polica pi e al., 2021).
The e icacy o epigene ic edi ing in he ealm o an imic obials
emains a nea - e m objec i e. Howe e , he e is a lack o esea ch
in es iga ing epigene ic edi ing in mic obes wi hin he con ex o an-
imic obial esis ance. To da e, he epigene ic edi ing ad ancemen s
a e expe imen al. While hese ools con inue o ad ance, ques ions
ega ding hei speci ici y pe sis . Se e al s udies ha e iden i ied
subs an ial o - a ge e ec s associa ed wi h he h ee p ima y
ypes o p og ammable DNA- binding domains (Polica pi e al., 2021;
Thako e e al., 2016). I is c ucial o e ine and imp o e hese ech-
niques, gi en ha , in many cases, epigene ic edi ing is p e e ed
o e gene ic edi ing. This p e e ence elies on he e e sible na u e
o epigene ic changes and hei inducibili y in speci ic issues, de-
elopmen al s ages, o en i onmen al condi ions, o en acili a ed
h ough he u iliza ion o chemically inducible p omo e s (Thako e
e al., 2016; Veley e al., 2023).
Epigene ic edi ing in hos o ganisms has al eady demons a ed
he po en ial o epigene ic edi ing in comba ing mic obial in ec-
ions. A no able example o his is he case o he cassa a bac e ial
bligh disease. Cassa a is widely cul i a ed o nume ous pu poses,
including human and animal consump ion, he p oduc ion o lou ,
alcohol, s a ches, swee ene s, and ex iles, and i is suscep ible o a
disease caused by he bac e ium Xan homonas phaseoli p . maniho is
(Veley e al., 2023). Resea ch has shown ha he pa hogenic bac e-
ia uses he TAL20 ( ansc ip ion ac i a o - like e ec o ) o induce
exp ession o he suscep ibili y gene MeSWEET10a, which belongs
o he suga anspo e amily. Ac i a ion o MeSWEET10a leads
o obse able symp oms, such as lea lesions and po en ial plan
dea h. Mu a ing MeSWEET10a is undesi able, as i plays a c ucial
ole in no mal plan de elopmen . Ins ead, Veley e al. (2023) con-
duc ed a ge ed me hyla ion o he TAL20- binding si e wi hin he
MeSWEET10a p omo e using a syn he ic zinc- inge DNA- binding
domain used o a componen o he RNA- di ec ed DNA me hyla-
ion pa hway. DNA me hyla ion p e en s he binding o he e ec o
o he MeSWEET10a p omo e , blocking ansc ip ional ac i a ion.
Thei indings demons a ed ha his a ge ed me hyla ion educed
he plan 's symp oms wi hou in e e ing wi h egula plan de el-
opmen . Fu he mo e, epigene ic edi ing has been applied o o he
c ops o comba bac e ial in ec ions wi hou he need o an ibio ics
(Selma & O záez, 2021).
4 | EVOLUTION OF ANTIMICROBIAL
RESISTANCE AND EPIGENETICS
4.1  |  Epigene ics and adap a ion
Gi en ha epigene ic a ia ion is likely o be p e alen in mi-
c obes and can con ibu e o he e olu ion o an imic obial esis -
ance, wha is he expec ed con ibu ion o epigene ic changes?
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