Re iew
Biocides as d i e s o an ibio ic esis ance: A c i ical e iew o
en i onmen al implica ions and public heal h isks
Ma iana Sousa
a
,
b
, Idalina Machado
a
,
b
, Lúcia C. Sim~
oes
c
,
d
, Manuel Sim~
oes
a
,
b
,
*
a
LEPABEdLabo a o y o P ocess Enginee ing, En i onmen , Bio echnology and Ene gy, Facul y o Enginee ing, Depa men o Chemical and Biological
Enginee ing, Uni e si y o Po o, 4200-465, Po o, Po ugal
b
ALiCEdAssocia e Labo a o y in Chemical Enginee ing, Facul y o Enginee ing, Uni e si y o Po o, 4200-465, Po o, Po ugal
c
CEBdCen e o Biological Enginee ing, Uni e si y o Minho, 4710-057, B aga, Po ugal
d
LABBELSdAssocia e Labo a o y in Bio echnology and Bioenginee ing and Mic oelec omechanical Sys ems, B aga, Guima ~
aes, Po ugal
a icle in o
A icle his o y:
Recei ed 25 No embe 2024
Recei ed in e ised o m
18 Ma ch 2025
Accep ed 19 Ma ch 2025
Keywo ds:
Adap i e esis ance
An ibio ic esis ance
Biocides
Biofilms
C oss- esis ance
abs ac
The widesp ead and indisc imina e use o biocides poses significan h ea s o global heal h, socioeco-
nomic de elopmen , and en i onmen al sus ainabili y by accele a ing an ibio ic esis ance. Bac e ial
esis ance de elopmen is highly complex and influenced significan ly by en i onmen al ac o s.
Inc eased biocide usage in households, ag icul u e, li es ock a ming, indus ial se ings, and hospi als
p oduces pe sis en chemical esidues ha pollu e soil and aqua ic en i onmen s. Such con aminan s
con ibu e o he selec ion and p oli e a ion o esis an bac e ia and an imic obial esis ance genes
(ARGs), acili a ing hei dissemina ion among humans, animals, and ecosys ems. In his e iew, we
conduc a c i ical assessmen o ou significan issues pe aining o his opic. Specifically, (i) he ole o
biocides in exe ing selec i e p essu e wi hin he en i onmen al esis ome, he eby p omo ing he
p oli e a ion o esis an mic obial popula ions and con ibu ing o he global sp ead o an imic obial
esis ance genes (ARGs); (ii) he ole o biocides in igge ing ansien pheno ypic adap a ions in bac-
e ia, including e flux pump o e exp ession, memb ane al e a ions, and educed po in exp ession, which
o en esul in c oss- esis ance o mul iple an ibio ics; (iii) he capaci y o biocides o dis up bac e ia and
make he gene ic con en accessible, eleasing DNA in o he en i onmen ha emains in ac unde
ce ain condi ions, acili a ing ho izon al gene ans e and he sp ead o esis ance de e minan s; (i )
he capaci y o biocides o dis up bac e ial cells, eleasing in ac DNA in o he en i onmen and
enhancing ho izon al gene ans e o esis ance de e minan s; and (i ) he selec i e in e ac ions be-
ween biocides and bac e ial biofilms in he en i onmen , s eng hening biofilm cohesion, inducing
esis ance mechanisms, and c ea ing ese oi s o esis an mic oo ganisms and ARG dissemina ion.
Collec i ely, his e iew highligh s he c i ical en i onmen al and public heal h implica ions o biocide
use, emphasizing an u gen need o s a egic in e en ions o mi iga e hei ole in an ibio ic esis ance
p oli e a ion.
©2025 The Au ho s. Published by Else ie B.V. on behal o Chinese Socie y o En i onmen al Sciences,
Ha bin Ins i u e o Technology, Chinese Resea ch Academy o En i onmen al Sciences. 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/).
1. In oduc ion
Based on he “One Heal h”concep , which s a es ha “ he
heal h o people is connec ed o he heal h o animals and he
en i onmen ,” he measu es o hal an imic obial esis ance om
sp eading h ough he en i onmen a e imely and much-needed
[1,2]. Biocide pollu ion con ibu es o he e olu ion and dissemi-
na ion o bac e ial esis ance among humans, animals, and he
en i onmen by c ea ing a ou able condi ions o selec ing esis-
an bac e ia and acili a ing he ansmission o gene ic ma e ial
ac oss ecological bounda ies [3e5]. When eleased in o a ce ain
en i onmen , biocides c ea e a selec i e p essu e a ou ing bac e-
ia wi h esis ance mechanisms [6,7]. Fo hese easons, add essing
his challenge equi es a “One Heal h”app oach, inco po a ing
sus ainable biocide use, obus was e managemen p ac ices, and
moni o ing o en i onmen al ese oi s [8]. In addi ion o he
*Co esponding au ho . a : LEPABEdLabo a o y o P ocess Enginee ing, En i-
onmen , Bio echnology and Ene gy, Facul y o Enginee ing, Depa men o
Chemical Enginee ing, Uni e si y o Po o, 4200-465 Po o, Po ugal.
E-mail add ess: [email p o ec ed].p (M. Sim~
oes).
Con en s lis s a ailable a ScienceDi ec
En i onmen al Science and Eco echnology
jou nal homepage: www.jou nals.else ie .com/en i onmen al-science-and-
eco echnology/
h ps://doi.o g/10.1016/j.ese.2025.100557
2666-4984/©2025 The Au ho s. Published by Else ie B.V. on behal o Chinese Socie y o En i onmen al Sciences, Ha bin Ins i u e o Technology, Chinese Resea ch
Academy o En i onmen al Sciences. 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/).
En i onmen al Science and Eco echnology 25 (2025) 100557
exis ing moni o ing p og ams ha ack unnecessa y an ibio ic
p esc ip ions and misuse, i is essen ial o include biocides as hey
may also con ibu e o esis ance sp ead.
Addi ionally, he en i onmen should be conside ed as a po-
en ial sou ce o esis ance [9,10]. In pa icula , biocides, which
exe hei an imic obial ac i i y h ough a mul i- a ge mode o
ac ion, a e essen ial o con ol in ec ious diseases and sa egua d
animal and human heal h [11,12]. Demand o biocides is expec ed
o inc ease in he coming yea s, d i en by hei use in a ious in-
dus ial p ocesses and o he disin ec ion o hospi als and public
spaces [13]. A significan inc ease in pes s and hygiene issues is
expec ed because o global wa ming, clima e change, and associ-
a ed ex eme wea he e en s [14]. This equi es e ec i e esponses
o con ol po en ial pa hogens while ensu ing minimal nega i e
impac s on public heal h and he en i onmen . The socie al
dependence on biocides o disin ec ion was ema kable du ing he
SARS-CoV-2 pandemic [15e18].
Unde s anding he in ica e connec ions be ween biocides, an-
ibio ics, and an imic obial esis ance is c i ical o de ising s a-
egies o mi iga e he sp ead o esis ance ac oss humans, animals,
and he en i onmen [19,20]. This e iew explo es he e idence
linking biocide exposu e o he eme gence o esis an mic oo -
ganisms, ocusing on he mechanisms by which biocides d i e
esis ance and he en i onmen al pa hways ha acili a e he
dissemina ion o esis ance genes. This s udy unde sco es he u -
gen need o in eg a ed and sus ainable app oaches o manage
biocide use, p o ec public heal h, and p ese e ecosys em
in eg i y.
2. E idence o biocides as d i e s o an imic obial esis ance
Biocides a e a om innocuous o he en i onmen and public
heal h. They can pe sis in hei a ge niches o wo easons [21].
Fi s ly, bac e ia elimina ion by hese agen s has p o en o be
di ficul , and i has become clea ha biocidal e ficacy may be
ques ionable in some ci cums ances. Wi hin his con ex , bac e ia
a e egula ly exposed o sub-bac e icidal concen a ions, which can
lead o bac e ial adap ion and esis ance o in-use biocides [22e24].
Secondly, an imic obial sub-bac e icidal concen a ions a e p esen
in he en i onmen since some biocides ha a e no consumed in
he eac ion o cleaning p ocedu es a e eely eleased in he
discha ge wa e s ( o ins ance, oxida i e species would be ac i ely
quenched by he o ganic load p esen and ha e no legacy e ec s)
[25e27].
Fo ins ance, s udies ha e shown ha Pseudomonas ae uginosa
can de elop esis ance o qua e na y ammonium compounds
(QACs) such as benzalkonium chlo ide (BZK) h ough mechanisms
like e flux pump o e exp ession and al e a ions in memb ane
composi ion [28,29]. This exposu e can also lead o c oss- esis ance
o an ibio ics like cip ofloxacin, as he same e flux sys ems expel
bo h biocides and an ibio ics om he bac e ial cell [28,29].
Mo eo e , Lis e ia monocy ogenes exposed o sub-inhibi o y con-
cen a ions o BZK in ood en i onmen s can adap by al e ing
memb ane composi ion and su ace cha ac e is ics, leading o
inc eased esis ance o BZK and o he un ela ed an ibio ics [29,30].
Conce ning iclosan, esea ch has demons a ed ha low le els o
his biocide, commonly ound in pe sonal ca e p oduc s, can induce
bac e ial esis ance h ough mu a ions in key enzymes in ol ed in
a y acid syn hesis, such as FabI [30]. This esis ance can ex end o
an ibio ics like isoniazid, which also a ge s a y acid syn hesis
pa hways [30]. Fo Acine obac e baumannii, sub-le hal exposu e o
chlo hexidine has been shown o ac i a e e flux pump sys ems and
p omo e biofilm o ma ion, making he bac e ia mo e esis an o
chlo hexidine and an ibio ics like ca bapenems [30].
One o he possible solu ions o his lack o e ficiency is an
inc ease in he ecommended in-use concen a ion [11]. In his
en i onmen , pollu an s a e eleased no only om indus ial and
hospi al discha ges bu also om a wide ange o human ac i i ies,
including consume p oduc s, pe sonal ca e i ems, pha maceu i-
cals, and hei me aboli es. Addi ionally, na u al sou ces, such as
compounds sec e ed by bac e ia, ungi, algae, and plan s,
con ibu e o he pollu ion [31e33]. The inc eased consump ion o
biocides and he ac ha hese a e poo ly biodeg adable make
hem p esen in he en i onmen as pa en compounds o conju-
ga es [31,34e39]. These compounds can be e y pe sis en in he
en i onmen , in a ying ypes and concen a ions ac oss he globe,
since hey adso b s ongly o en i onmen al elemen s such as
sludge and sedimen , showing low deg ada ion po en ial [40e45].
Fig. 1 p esen s a compila ion o some s udies epo ing he wo ld
dis ibu ion o biocides and an ibio ics wi h high ele ance, p e -
alence, and use wo ldwide. Was ewa e ea men plan s
(WWTPs), i e s, and basins ha ecei e ea ed and un ea ed
sewage a e impo an con ibu o s o he dissemina ion o esis an
bac e ia and an ibio ic- esis ance genes (ARGs) [46e49]. The
globaliza ion o ecosys ems, along wi h he ising consump ion o
an ibio ics and biocides (such as an isep ics, p ese a i es, and
biocides), con ibu es o he widesp ead dis ibu ion and accu-
mula ion o hese biopollu an s in ecosys ems wo ldwide [50e53].
Se e al biocides, such as QACs, a e high-p oduc ion olume
chemicals (i.e., p oduc ion >1000 ons pe yea ) and, in many cases,
can also ha e non-biocidal applica ions [54]. In 2017, a Chinese
s udy aimed o examine he p esence and elimina ion o nine een
biocides ac oss en di e en WWTPs [47]. Addi ionally, i sough o
es ima e he pe capi a usage and emissions o hese 19 biocides
using a mass balance analysis app oach.
The mass balance used was:
MIn luen ¼ME luen þMSludge þMLoss (1)
whe e M
Influen
,M
E fluen
, and M
Sludge
(g d
1
) ep esen he mass
loads o he a ge biocide in he influen , e fluen , and excess
sludge o each WWTP, espec i ely. M
Loss
(g d
1
) deno es he loss o
mass loads o he a ge biocide du ing he en i e ea men p o-
cess, p ima ily due o so p ion and deg ada ion.
The loss mass ac ions (M
,Loss%
) o each biocide we e calcu-
la ed using:
M ;Loss% ¼MIn luen ME luen MSludge
MIn luen
100% (2)
The mass ac ions in e fluen (M
,E fluen %)
and excess sludge
(M
,Sludge%
) we e calcula ed as:
M ;E luen % ¼ME luen
MIn luen
100% (3)
M ;Sludge% ¼MSludge
MIn luen
100% (4)
The es ima ed pollu ion loads pe capi a o biocides in he
influen , e fluen , and excess sludge o each WWTP we e gi en by:
Pollu ion load pe capi a ¼
MIn luen =E luen =Sludge
Popula ion se ed (5)
The back-es ima ed usage (U, yea
1
) and es ima ed emissions
(E, yea
1
) o Guangdong P o ince o China we e de e mined as:
U¼LIn luen PTo al 365:25 1012 (6)
M. Sousa, I. Machado, L.C. Sim~
oes e al. En i onmen al Science and Eco echnology 25 (2025) 100557
2
E¼ERu al þEU ban (7)
E¼hLIn luen PRu al þLE luen þLSludgePU bani365:25
1012
(8)
whe e L
Influen
,L
E fluen
, and L
Sludge
(
m
gd
1
pe son
1
) a e he es i-
ma ed pollu ion loads pe capi a o a a ge biocide in he influen ,
e fluen , and excess sludge (mean o en WWTPs). P
To al
,P
Ru al
, and
P
U ban
a e he o al, u al, and u ban popula ions. These alues a e
based on he “China S a is ical Yea book”(2015). Fo Guangdong:
P
To al
¼10724 10
4
,P
Ru al
¼3432 10
4
, and P
U ban
¼7292 10
4
.
Fo China: P
To al
¼136782 10
4
,P
Ru al
¼61866 10
4
, and
P
U ban
¼74916 10
4
.
The es ima ed usage o he biocides unde in es iga ion was as
high as 453 ons pe yea , co esponding o 308
m
g pe day pe
pe son (including e fluen and excess sludge) [47]. In Minneso a
(Uni ed S a es),1995 me ic ons o QACs we e sold in 2017 o non-
ag icul u al pu poses [48]. A s udy by A nold e al. [54] epo ed
ha o e 450000 me ic ons o QACs a e p oduced o impo ed
annually in he Uni ed S a es. In 1997, he o al use o QACs in
Ge many was 12349 me ic ons pe yea by indus y and 95.3
me ic ons pe yea by hospi als, while in G ea B i ain, 28852
me ic ons pe yea a e being used [55e57]. Mo eo e , app oxi-
ma ely 75 % o he QACs used each yea a e discha ged in o
was ewa e ea men sys ems, wi h he emainde going di ec ly
in o he en i onmen [58]. Globally, su ace wa e and was ewa e
e fluen con ain concen a ions o QACs om 1 o 100
m
gL
1
, his is
0.0000001 %e0.00001 % [weigh / olume (w/ )], al hough influen
was ewa e some imes con ains 10 imes hese concen a ions
[58,59].
Fu he mo e, he minimum inhibi o y concen a ions (MICs) o
QACs ange om 0.005 % o 0.01 % w/ , while he minimum
bac e icidal concen a ions (MBCs) a e ypically be ween 0.02 %
and 0.05 % w/ [60]. Zheng e al. [61] assessed he bioaccumula ion
po en ial o 18 QACs wi h alkyl chain leng hs anging om C8 o
C18. Tha e alua ion was conduc ed h ough an in i oein i o
Fig. 1. Wo ld dis ibu ion o biocides and an ibio ics (in ng L
1
) in i e s, basins, seas o lakes, and was ewa e ea men plan s (influen ). The in es iga ed biocides and an ibio ics
we e selec ed due o hei high ele ance, p e alence, and use wo ldwide. AMP: Ampicillin; ATMAC: Alkyl ime hylammonium compounds; AZM: Azi h omycin; BZK: Benzy-
lalkyldime hylammonium compounds; CHX: Chlo hexidine; CIP: Cip ofloxacin; CLM: Cla i h omycin; CTAB: Ce yl ime hylammonium b omide;DADMAC: Dia-
lkyldime hylammonium compounds; ERY: E y h omycin; NOR: No floxacin; PhP: O ho-phenylphenol; ROX: Roxi h omycin; SMZ: Sul ame hoxazole; STZ: Sul a hiazole; TCC:
T icloca ban; TCS: T iclosan; TET: Te acycline; TMP: T ime hop im.
M. Sousa, I. Machado, L.C. Sim~
oes e al. En i onmen al Science and Eco echnology 25 (2025) 100557
3
ex apola ion (IVIVE) model, u ilizing da a ob ained om expe i-
men s on human hepa ic me abolism and se um p o ein binding.
O he 18 QACs aimed o de ec ion, 15 we e iden ified in blood,
wi h cumula i e QAC concen a ions eaching a maximum o
68.6 ng mL
1
. Blood samples we e ob ained om wo specific pe-
iods: p e-COVID-19 pandemic ou b eak (2019; n¼111) and du ing
he pandemic (2020; n¼111). The cumula i e QAC concen a ions
we e no ably ele a ed in samples collec ed du ing he pandemic
(6.04 ng mL
1
) compa ed o hose ga he ed be o e (3.41 ng mL
1
)
[61].
In Eu ope, he egula ion o BZK alls unde he pu iew o he
Eu opean Commission (EC). Recen egula ions in he Eu opean
ma ke ha e modified he pe missible maximum esidual le els o
BACs in ood p oduc s, educing he limi om 0.5 o 0.1 mg kg
1
.In
addi ion, he al e a ions in legisla ion, specifically Decision [Eu o-
pean Union (EU)) 2016/1950 and he Biocidal P oduc s Regula ion
(BPR, EU) no. 528/2012 ha e esul ed in he disapp o al o BACs o
applica ion in a ious biocidal p oduc s. No ably, consume hand
and body wash an isep ics a e a ec ed, ma king a depa u e om
he p e ailing egula ions in he Uni ed S a es [62].
Some o hese chemical compounds end up dilu ed in wha we
ea , d ink, and b ea he [25]. This has igge ed conce n because, in
he en i onmen , biocides pe sis and bioaccumula e, being
ha m ul no only o humans bu also o mic oo ganisms [8]. Bio-
cides can modi y he dynamics and ac ion o an ibio ics in na u al
ecosys ems, selec ing esis an o ganisms [3,63,64] wi h inc eased
esis ance and co- esis ance o o he biocides as well as c oss-
esis ance o an ibio ics [24,51].
The ole o biocides in he selec ion, sp ead, and main enance o
esis an bac e ia wo ldwide should be add essed [65], conside ing
ha wa e basins a e globally pollu ed wi h se e al classes o bio-
cides [27,66]. The e o e, he e is an u gency o unde s and he
mechanisms o ac ion o biocides and esis ance and i hei p es-
ence in he en i onmen , e en in educed concen a ions, can cause
esis ance o an ibio ics. This s udy e iews he phenomena o
bac e ial esis ance o in-use biocides and ou cu en knowledge o
how his can igge an ibio ic esis ance, wi h pa icula emphasis
on biocides ha a e con inuously eleased and accumula ed in he
en i onmen [51]. The main ques ion emains: Should biocide sub-
bac e icidal concen a ions be conside ed an impo an d i e o
inc eased an ibio ic esis ance?
3. Mechanisms o ac ion o biocides and hei impac on
bac e ial esis ance
Gene ally, an imic obial agen s can be classified as any molecule
wi h biochemical p ope ies ha kill o p e en he g ow h o
mic oo ganisms, including bac e ia, ungi, and algae, on a bio ic o
abio ic su ace [67]. An imic obial agen s can be di ided in o an-
isep ics (agen s ope a ing on li ing issues), disin ec an s (p od-
uc s applied o inanima e objec s and su aces), and p ese a i es
[68,69]. Howe e , hey can also be classified acco ding o he ype
o mic oo ganisms agains which hey a e e ec i e: an ibio ics,
an i i als, an i ungals, and an ipa asi ics [70,71]. Fu he mo e,
hese molecules a ge c ucial p ocesses in cellula me abolism,
including he p oduc ion o biological mac omolecules, cellula
enzyme ac i i y, and cellula componen s [71,72].
Consequen ly, in con as o an ibio ics ha a e used o ea
specific bac e ial in ec ions, biocides a e a di e se g oup o an i-
mic obial compounds ha , due o hei e sa ili y and b oad-
spec um ac ion, a e used in pe sonal ca e p oduc s (soaps, hand-
washes, oo hpas e, mou hwashes, and cosme ics) [73e75] and as
pa o de e gen o mula ions o su ace cleaning, he p ocess o
emo ing all o eign ma e ial om objec s by using wa e and
de e gen s, and disin ec ion eelimina ion o mos o all pa hogenic
mic oo ganisms (excep spo es) [68,76]. Indus ially, biocides a e
used in high doses o clean ha d su aces efloo s, walls, equipmen
eand ood su aces. Addi ionally, in mos p oduc ion p ocesses, he
wa e used o en has a esidual concen a ion o biocides o ensu e
i s low mic obial load [77,78]. In clinical en i onmen s, biocides a e
undamen al o he an isepsis o he pa ien 's skin as well as o he
disin ec ion o wa e and ha d su aces and s e iliza ion o medical
ins umen s and ooms [77].
Typical biocides used o cleaning and disin ec ion pu poses a e
QACs, alcohols, aldehydes (glu a aldehyde, o ho-ph halaldehyde
[OPA]), bisbiguanides (chlo hexidine), bisphenols ( iclosan), dia-
midines, halogen- eleasing agen s, halophenols, hea y-me al de-
i a i es and pe oxygens (hyd ogen pe oxide) [68,79]. Many ac o s
a ec he e ficacy o biocides in bo h ac ions ecleaning and
disin ec ion e he s abili y o he ac i e molecule, he con ac ime,
he concen a ion o biocide, he age and me abolic s a us o he
mic obial communi y, he exis ence o adhe ed bac e ia o biofilms,
and he p esence o o ganic load and o he en i onmen al ac o s
such as empe a u e and pH [68,80].
Biocides play an impo an ole in limi ing bac e ia p esence and
educing po en ial sou ces o con amina ion and in ec ion and a e
e ec i e due o hei b oad spec um o ac i i y and abili y o ac on
mul iple a ge s [11,68]. Mo eo e , he majo i y o biocides dis up
he cy oplasmic memb ane [68], dissipa ing he p o on mo i e
o ce and inhibi ing memb ane-associa ed enzymes [81]. Unlike
an ibio ics, which ac selec i ely agains specific cell a ge s, he
mechanism o ac ion o biocides occu s a one o se e al o he si es
wi hin he cell [82]. Biocides can in e ac wi h he cell en elope
a ge ing, o ins ance, he cy oplasmic memb ane (which is a
componen o he cell en elope) (e.g., chlo hexidine and QACs)
[83]; c oss-link wi h o he mac omolecules (e.g., glu a aldehyde)
[84]; in e cala e (e.g. ac idines) and in e ac wi h deoxy ibonucleic
acid (DNA; e.g. halogens, sil e ions, and oxidizing agen s) [81]; and
in e ac wi h hiol g oups in enzymes and p o eins (e.g. o gano-
me cu ials and sil e compounds) [81]. Al hough he ou e cell
en elope is he fi s a ge , as i is he ini ial poin o con ac , he
inne cell componen s a e also o en a ec ed. Howe e , he cell is
equen ly inac i a ed h ough his ini ial su ace in e ac ion
[85,86]. O he a ge s inside he cell, such as nucleic acids, enzymes,
and ibosomes, a e mo e di ficul o each since he biocide needs
o pene a e he cell [81]. Unde s anding hese in e ac ions is
essen ial o de eloping e ec i e an imic obial ea men s and
add essing he challenge o bac e ial esis ance. Table 1 p esen s an
o e iew o a ious biocides, hei mechanisms o ac ion agains
bac e ia, and co esponding bac e ial esis ance mechanisms. This
able highligh s how di e en chemical agen s a ge bac e ial cells
and he s a egies employed by bac e ia o esis hei e ec s
[68,87].
Biocides a e usually applied in high concen a ions o imp o e
hei e ficiency, making i impossible o bac e ia o o e come he
damage caused and de elop esis ance [88]. In ac , in hospi als and
clinical se ings, he an imic obial in-use concen a ion is
equen ly wo- o ou - old he MIC o wild- ype s ains, meaning
ha he an imic obials a e e ec i e agains 99 % o he en i on-
men al mic oo ganisms [89,90]. Fu he mo e, as disin ec an s
gene ally con ain mo e han one ype o ac i e componen , each
wi h a di e en an imic obial mode o ac ion, and as hey ha e no
specific a ge , he de elopmen o esis ance a he le el o in-use
concen a ions is hough o be highly unlikely [68,89]. Ne e he-
less, wo ea u es can con ibu e o he esis ance o biocides and
subsequen esis ance: (1) he 1 % o bac e ia ha a e no killed by
he in-use concen a ion can p esen na u ally educed suscep i-
bili y due o he p esence o esis an gene ic in o ma ion [89]; (2) a
cleaning solu ion a a sub-inhibi o y concen a ion can occu , o
example, when o ganic ma e is p esen (since he o ganic ma e
M. Sousa, I. Machado, L.C. Sim~
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4
may physically dilu e he cleaning solu ion, educing he concen-
a ion o ac i e ing edien s a ailable o ac on mic oo ganisms
[91]; can eac chemically wi h disin ec an s [91]; and can c ea e a
physical ba ie ha p o ec s mic oo ganisms om di ec exposu e
o he cleaning agen [92]), i insu ficien cleaning is applied, o
e en in he insing wa e [89].
In he en i onmen , we may hink bo h si ua ions happen:
wa e and land accumula e a mul iplici y o esis an s ains and
gene ic ma e ial, and sub-inhibi o y concen a ions o biocides a e
na u ally p esen as pe sis en o ganic pollu an s [93,94](Fig. 1).
4. C oss- esis ance o an ibio ics
Al hough bac e ial ecalci ance o biocides ( esis ance) was
desc ibed ea ly in he 1950s o 1960s, i appea s o be inc easing,
and nowadays, we a e s ill deba ing on he possible eme gence o
biocide esis ance and i s associa ion wi h he eme gence o an i-
bio ic ecalci ance [95]. The e m “c oss- esis ance”can be defined
as a phenomenon ha occu s when a esis ance mechanism o a
ce ain an imic obial enables a specific s ain o esis se e al o he
an imic obials wi h simila mechanisms o ac ion [96]. In he case
o co- esis ance, he mechanisms con e ing esis ance o bo h
agen s a e un ela ed bu a e gene ically linked, e.g., loca ed on he
same gene ic elemen [97].
Some s udies in i o confi med his c oss- esis ance concep
(excep ionally cla ified in C^
andido e al. [98]). Sonbol e al. [99]
examined he e ec i eness o se e al an ibio ics, such as
b
-lac ams,
cephalospo ins, mac olides, aminoglycosides, quinolones, sul on-
amides, and e acycline agains Esche ichia coli isola es ha ha e
been exposed o sub-bac e icidal doses o he biocide iclosan. The
ou comes showed ha apa om amikacin and ime hop im/
sul ame hoxazole, cells adap ed o iclosan became mo e esis an
o he an ibio ics es ed and emained suscep ibili y o he biocide,
associa ed wi h dec eased pe meabili y o ou e and inne mem-
b anes, inc eased depola isa ion o memb anes, highe nega i e
ne cha ge o memb anes, and highe e flux ac i i y [84]. As i-
closan ac s as a subs a e o bac e ial mul id ug e flux pumps,
E. coli [99] and P.ae uginosa [100] exposed o iclosan a sub-
bac e icidal doses displayed a la ge ope a ion o e flux pumps,
likely associa ed wi h c oss- esis ance. A s udy by Wand e al. [101]
demons a ed ha , when exposed o chlo hexidine, Klebsiella
pneumoniae acqui ed c oss- esis ance o he an ibio ic colis in. The
au ho s epo ed ha he adap a ion o K. pneumoniae o chlo -
hexidine occu s h ough egula ing genes associa ed wi h e flux
pumps, namely sm R and sm A. Mo eo e , he ope on pm K, which
is esponsible o educing he nega i e cha ge o lipid A, was
up egula ed by chlo hexidine. As a esul , a dec eased binding a -
fini y o colis in o lipid A occu ed, esul ing in c oss- esis ance.
Howe e , he esis ance o chlo hexidine was no inc eased by
exposu e o colis in [101]. A s udy de eloped by Cu iao e al. [102]
showed ha mu an s o Salmonella en e ica Typhimu ium c ea ed
a e exposu e o iclosan, chlo hexidine, and BZK exhibi ed
di e en pheno ypes agains a b oad ange o an ibio ics: ampi-
cillin, ce azidime, cip ofloxacin, e y h omycin, gen amicin, chlo -
amphenicol, and e acycline. Mos o he mu an s ha we e
confi med o be esis an o biocides p esen ed an inc eased sus-
cep ibili y o molecules whose si e o ac ion is he cell wall (
b
-
lac ams) o he cell memb anes (poly-L-lysine, polymyxin B,
colis in, and oxic anions). Mo eo e , bo h biocide mu an s selec ed
in i o and field isola es wi h esis ance o biocides exhibi ed
o e exp ession o genes ela ed o cold-shock esponse (cpeE), i-
bosomal and ansc ip ion p o eins, e flux pumps (sugE), and en-
zymes o mic oae obic me abolism, pa icula ly hose o he
phospho ans e ase sys em [102]. In Sanchez e al. [103], iclosan
p o ed o selec i ely induce he exp ession o he e flux pump
SmeDEF in S eno ophomonas mal ophilia, which in u n igge s a
ansien low-le el esis ance o he biocide and c oss- esis ance.
Compa ed o he wild- ype S. mal ophilia,fi e ou o he 12
iclosan-selec ed mu an s we e mo e esis an o he an ibio ics
e acycline, chlo amphenicol, cip ofloxacin, ob amycin, and i-
closan [103]. Pe ei a e al. [104] conduc ed an adap i e labo a o y
e olu ion o E. coli using cons an and sub-inhibi o y concen a-
ions o en widesp ead biocides: chlo ophene, BZK, glu a alde-
hyde, chlo hexidine, pe oxide, po idone-iodine, isop opanol,
e hanol, sodium hypochlo i e, and pe ace ic acid. In e es ingly, and
as a esul o e olu ion, 43 % o s ains became esis an o h ee
ep esen a i e an ibio ics wi h di e se cellula modes o ac ion:
ampicillin, chlo amphenicol, and no floxacin. The au ho s also
ound mu a ions in genes encoding o mul id ug e flux p o eins
(md A and ac R), po ins (en Z and ompR), and subuni s o E. coli RNA
polyme ase ( poA and poBC). The biocides chlo ophene, BZK,
glu a aldehyde, and chlo hexidine we e he ones ins iga ing highe
an ibio ic suscep ibili y (p obably due o he occu ence o mu a-
ions in memb ane p o eins and hei egula o s, including hose
esponsible o anspo ing chemicals like an ibio ics in o and ou
o he cell), while hyd ogen pe oxide and po idone-iodine we e he
ones con ibu ing leas o an inc ease in suscep ibili y o an ibi-
o ics. No e idence o an ibio ic c oss- esis ance o isop opanol,
e hanol, sodium hypochlo i e, and pe ace ic acid was obse ed
[104]. Salmonella spp. exhibi ed o e exp ession o Ac AB-TolC and
Table 1
O e iew o a ious biocides, hei mechanisms o ac ion agains bac e ia, and co esponding bac e ial esis ance mechanisms, ocusing on how di e en chemical agen s
a ge bac e ial cells and he s a egies bac e ia employ o esis hei e ec s [68,87].
Biocide Mechanism o ac ion Bac e ial esis ance mechanisms
Qua e na y ammonium
compounds
Dis up s cell memb ane in eg i y E flux pumps: Expel qua e na y ammonium compounds ou o he cell
Memb ane modifica ion: Reduces pe meabili y
Alcohols (e.g. E hanol, Isop opanol) Dena u es p o eins, dis up s memb anes Biofilm o ma ion: Limi s pene a ion
Memb ane modifica ion: Al e s lipid composi ion
Chlo ine/Chlo ine-based
compounds
Oxidizes p o eins, lipids, and DNA Enzyma ic deg ada ion: P oduces enzymes o neu alize oxida i e
damage
Reduced up ake: Al e s po in p o eins
Pe oxides (e.g. Hyd ogen pe oxide) P oduces eac i e oxygen species, causing cellula
damage
Enzyma ic deg ada ion: Ca alase and pe oxidase b eak down pe oxides
Biofilm o ma ion: P o ec s agains oxida i e damage
Phenolics Dis up s cell walls and memb anes dena u es p o eins E flux pumps: Ac i ely expel phenolics
Biofilm o ma ion: Shields bac e ia om pene a ion
Hea y me als (e.g. Sil e , Coppe ) Binds o p o eins and enzymes, dis up ing cellula
unc ion
Memb ane modifica ion: Reduces me al ion pe meabili y
E flux pumps: Ac i ely emo e me al ions
Biguanides (e.g. Chlo hexidine) Dis up s cell memb ane, leading o leakage E flux pumps: Expel biguanides
Memb ane modifica ion: Al e s memb ane composi ion o esis
dis up ion
M. Sousa, I. Machado, L.C. Sim~
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5
unde exp ession o ou e memb ane po ins a e exposu e o a
qua e na y ammonium disin ec an dcon aining o maldehyde
and glu a aldehyde. This phenomenon esul ed in an ibio ic esis-
ance o cip ofloxacin, chlo amphenicol, e acycline, and ampi-
cillin [105,106].
Taba a e al. [107] epo ed ha he exp ession le el o Op R was
co ela ed wi h he esis ance le el o P. ae uginosa o QACs. A QAC-
esis an P. ae uginosa s ain exhibi ed a highe exp ession le el o
Op R compa ed wi h a knock-ou mu an [107]. Machado e al.
[108] demons a ed ha P. ae uginosa adap ed o BZK showed di -
e ences ega ding he exp ession o ou e memb ane p o eins
(OMPs) compa ed o a non-adap ed s ain. Some o hese p o eins
a e in ol ed in cip ofloxacin esis ance [108].
One example o a ge al e a ion was demons a ed by She idan
e al. [109], whe e a iclosan- ole an E. coli s ain exhibi ed an
amino acid subs i u ion in he FabI p o ein, whe e a glycine was
eplaced by a aline. This poin mu a ion p e en s he o ma ion o
he FabI-NAD
þ
complex, educing he e ec o iclosan in E. coli
[109]. A s able chlo hexidine- esis an Pseudomonas s u ze i, ob-
ained a e exposu e o chlo hexidine, showed esis ance o i-
closan and BZK and an ibio icsdpolymyxin B, gen amicin, and
ampicillin [110]. Ne e heless, c oss- esis ance phenomena do no
occu exclusi ely in cells in he plank onic s a e, ha ing also been
obse ed in biofilms, as demons a ed in Tabak e al. [111]. The
esul s cla ified ha he exp ession o deg ada i e enzymes,
changes in he bac e ial cell en elope, ac i a ion o he e flux pump,
and mu a ions in he enoyl educ ase enzyme a e all associa ed
wi h he ecalci ance obse ed in plank onic S. Typhimu ium a e
exposu e o iclosan. Addi ionally, he au ho s ound ha ,
ega ding he cells in biofilms o S. Typhimu ium, hei suscep ibili y
o iclosan is associa ed wi h he low ex acellula ma ix di usion
o he subs a e, o e ac i i y o e flux pumps, and exopoly-
saccha ide p oduc ion [111].
In ha sense, he unde lying message o he p e ious s udies is
he same: he e is a di ec ela ionship be ween he p esence o
biocides in he en i onmen and an ibio ic esis ance [112]. How-
e e , o he au ho s a e no ce ain ha his phenomenon can occu ,
as s udies add essing his in i o a e s ill missing [89,97]. The main
mechanisms o esis ance o con en ional classes o biocides and
he possible ela ionship be ween biocide and an ibio ic c oss-
esis ance desc ibed in he li e a u e a e summa ized in Table 2.
Resis ance o an ibio ics due o exposu e o biocides is o public
heal h ele ance when i conce ns pa hogenic o oppo unis ic
bac e ia [24]. Co- esis ance can also be a p oblem wi h non-
pa hogenic and commensal bac e ia when he e is a isk o ho i-
zon al gene ans e o esis ance de e minan s o (oppo unis ic)
pa hogens [113,114]. Mo eo e , he awa eness o isks ela ed o
sub-inhibi o y biocide concen a ions igge ing c oss and co-
esis ance in bac e ia has subs an ially inc eased [97].
In addi ion, c oss and co- esis ance appea when he biocide
and he an ibio ic ac on he same cellula a ge ; he biocide and
he an ibio ic ha e he same anspo mechanism; biocide and
an ibio ic can be accommoda ed by he same esis ance mecha-
nism, and in si ua ions whe e genes con ibu ing owa d biocide
esis ance and an ibio ic esis ance a e ca ied on he same mobile
gene ic elemen [22].
Biocides and an ibio ics may ha e simila and common in-
e ac ions and a ge si es in bac e ia (e.g., e flux pumps, pe me-
abili y changes, and biofilms), which migh exp ess sha ed
esis ance mechanisms [93]. When only sub-bac e icidal concen-
a ions o biocides a e p esen , mino cell damages can occu , and
he exp ession o mul id ug e flux pumps can be induced, selec ing
clones ha con e esis an pheno ypes loca ed on he same mobile
gene ic elemen such as a plasmid, ansposon, o in eg ons
[88,115,116].
Table 3 p esen s a compila ion o he biocides e e ed o in his
s udy, o ganized by hei chemical ca ego y and common applica-
ions: QACs [54], bisbiguanides [117], phenolics compounds [118],
aldehydes [119], alcohols [120], pe oxygens [121], halogen-
eleasing agen s [122], bisphenols [123], hea y-me al de i a i es
[124], and o he s (po idone-iodine and diamidines) [125].
5. Mechanisms induced by biocides in he de elopmen o
esis ance
Recen li e a u e [15,84,126,127] p o ides clea scien ific e i-
dence demons a ing ha he p esence o biocides in he en i-
onmen can be associa ed wi h a ious ypes o esis ance, namely.
(i) he na u al selec ion o mos esis an s ainsdin insic
p ope y [15,126];
(ii) he pheno ypic adap a ion o biocides [84,127];
(iii) he acquisi ion and/o elease o gene ic elemen s om
esis an s ains; ch omosomal gene mu a ion o gene ic
ma e ial acquisi ion o (plasmids o ansposons) [126];
(i ) he ole o biofilm communi ies in esis ance de elopmen
[127].
In he ollowing sec ions, we will c i ically examine hese
di e en ypes o esis ance o biocides.
Fig. 2 p o ides a compila ion o he mechanisms by which
biocides p omo e an ibio ic esis ance, which his s udy add esses.
5.1. Biocides as na u al selec o s o esis an s ains
In insic esis ance is he na u ally g ea e esis ance ela ed o
ce ain mic obial species han o he s. Some mic oo ganisms a e
al eady gene ically p edisposed o esis biocides and an ibio ics
[68,84,128e130](Fig. 3). A ea u e ha is ex ensi ely p esen
wi hin se e al bac e ial species, un ela ed o p io an ibio ic
exposu e and unconnec ed o ho izon al gene ans e is e e ed o
as in insic esis ance [128,131]. Reduced ou e memb ane
pe meabili y and e flux pump ac i i y, namely hose associa ed
wi h mul id ug e flux pumps, a e he wo mos equen bac e ial
mechanisms esul ing in in insic esis ance [128,132].
The cy oplasmic memb ane is p obably he majo a ge o
mos classes o biocides, and since di e en mic oo ganisms ha e
di e en memb ane s uc u es, i is easy o unde s and ha he
ac i i y o biocides a ies be ween di e en ypes o mic oo gan-
isms and e en be ween di e en s ains o he same species
[81,128,133,134]. Based on he memb ane s uc u e, among ege-
a i e bac e ia, mycobac e ia a e conside ed he mos esis an o
biocides, ollowed by G am-nega i e bac e ia, and ege a i e
G am-posi i e he mos suscep ible [81,135]. Some bac e ial species
a e also inna ely mo e esis an o biocides han o he s due o he
p esence o a low le el o e flux pump sys ems ha dec ease
in acellula concen a ion, educe biocide pene a ion, enzyma ic
deg ada ion o biocides, and physiological, s uc u al, and me a-
bolic changes [84,136e139]. In addi ion, i is con enien ly accep ed
ha mos o he in-use biocides, a sub-inhibi o y concen a ions,
a e na u ally selec ing bac e ia acco ding o hei suscep ibili y and
in insic esis ance [130,140,141](Fig. 3).
The specific g ow h a e is ano he significan ac o associa ed
wi h an imic obial esis ance as i is a de e minan ac o o how
bac e ial popula ions espond o molecules, influences he
exp ession o esis ance mechanisms, and can a ec he ou come o
bac e ial ea men [142e144]. Rapidly g owing bac e ia a e o en
mo e suscep ible o molecules ha a ge ac i e cellula p ocesses,
such as cell wall syn hesis and DNA eplica ion, because hese cells
can expe ience mo e mu a ions in a gi en pe iod due o mo e
M. Sousa, I. Machado, L.C. Sim~
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Table 2
Compila ion o s udies es ablishing a possible connec ion be ween se e al biocides and hei esis ance mechanisms wi h c oss- esis ance o an ibio ics in a ious bac e ial
s ains.
Biocides An ibio ics Mic oo ganism Mechanism o esis ance/C oss- esis ance Re e ences
T iclosan Amikacin
Ampicillin
Ampicillin/
sulbac am
Azi h omycin
Ce aclo
Ce azoline
Ce epime
Ce o axime
Chlo amphenicol
Cip ofloxacin
E y h omycin
Gen amicin
Imipenem
Lomefloxacin
Te acycline
T ime hop im/
sul ame hoxazole
Esche ichia coli
clinical isola es
The cells adap ed o iclosan exhibi ed ex ended suscep ibili y o he
biocide and ecalci ance o he in es iga ed an ibio ics associa ed wi h:
Dec eased pe meabili y o ou e and inne memb anes
Inc eased depola isa ion o memb anes
Highe nega i e ne cha ge o memb anes
Highe e flux ac i i y
[99]
T iclosan ePseudomonas
ae uginosa
T iclosan is a subs a e o Pseudomonas ae uginosa e flux pumps, namely
MexAB-Op M, MexCD-Op J, MexEF-Op N, and MexXY, associa ed wi h
c oss- esis ance o as an ibio ics
[100]
Chlo hexidine Colis in Klebsiella
pneumoniae
clinical isola es
The adap a ion o Klebsiella pneumoniae o chlo hexidine occu s h ough he
egula ion o genes associa ed wi h e flux pumps, namely sm R and sm A
The ope on pm K is up egula ed by chlo hexidine, esul ing in a dec eased
binding a fini y o colis in o lipid A, associa ed wi h c oss- esis ance
[101]
Benzalkonium chlo ide
Chlo hexidine
T iclosan
Ampicillin
Ce azidime
Chlo amphenicol
Cip ofloxacin
E y h omycin
Gen amicin
Te acycline
Salmonella
en e ica
Typhimu ium
Resis ance o biocides and c oss- esis ance is associa ed wi h he
exp ession/o e exp ession o :
Genes ela ed o cold-shock esponse (cpeE)
Ribosomal and ansc ip ion p o eins
E flux pumps (sugE)
Enzymes o mic oae obic me abolism
[102]
T iclosan Chlo amphenicol
Cip ofloxacin
Te acycline
Tob amycin
T iclosan
S eno ophomonas
mal ophilia
T iclosan selec i ely induces he exp ession o he e flux pump SmeDEF,
which in u n igge s a ansien low-le el esis ance o he biocide and
c oss- esis ance o an ibio ics
Compa ed o he wild- ype, fi e ou o he wel e iclosan-selec ed mu an s
we e mo e esis an o an ibio ics
[103]
Benzalkonium chlo ide
Chlo hexidine
Chlo ophene
E hanol
Glu a aldehyde
Isop opanol
Pe ace ic acid
Pe oxide
Po idone-iodine
Sodium hypochlo i e
Ampicillin
Chlo amphenicol
No floxacin
Esche ichia coli 43 % o s ains became esis an o he an ibio ics
Resis ance and c oss- esis ance wi h an ibio ics is associa ed wi h he
exp ession o :
Mul id ug e flux p o eins (md A and ac R)
Po ins (en Z and ompR)
Subuni s o Esche ichia coli RNA polyme ase ( poA and poBC)
Fo isop opanol, e hanol, sodium hypochlo i e, and pe ace ic acid, he e was
no e idence o an ibio ic c oss- esis ance
[104]
Qua e na y ammonium compounds
disin ec an con aining o maldehyde
and glu a aldehyde
Ampicillin
Chlo amphenicol
Cip ofloxacin
Te acycline
Salmonella spp. A e exposu e o a qua e na y ammonium disin ec an con aining
o maldehyde and glu a aldehyde, Salmonella spp. exhibi ed o e exp ession
o Ac AB-TolC and unde exp ession o ou e memb ane po ins, esul ing in
esis ance o cip ofloxacin, chlo amphenicol, e acycline, and ampicillin
[105,106]
Qua e na y ammonium compounds ePseudomonas
ae uginosa
A Qua e na y ammonium compound- esis an Pseudomonas ae uginosa
s ain exhibi ed a highe exp ession le el o Op R compa ed wi h a knock-
ou mu an , showing ha he exp ession le el o Op R was co ela ed wi h
he le el o esis ance o qua e na y ammonium compounds.
[107]
Benzalkonium Chlo ide Cip ofloxacin Pseudomonas
ae uginosa
Pseudomonas ae uginosa adap ed o benzalkonium chlo ide showed
di e ences ega ding he exp ession o ou e memb ane p o eins when
compa ed wi h a non-adap ed s ain, and some o hese p o eins a e
in ol ed in cip ofloxacin esis ance
[108]
T iclosan eEsche ichia coli A iclosan- ole an Esche ichia coli s ain exhibi ed an amino acid
subs i u ion in he FabI p o ein, whe e a glycine was eplaced by a aline,
p e en ing he o ma ion o he FabI-NAD
þ
complex and educing he e ec
o iclosan in Esche ichia coli
[109]
Chlo hexidine Ampicillin
Gen amicin
Polymyxin B
Pseudomonas
s u ze i
A chlo hexidine- esis an Pseudomonas s u ze i, ob ained a e exposu e o
chlo hexidine, showed esis ance o iclosan, benzalkonium chlo ide,
polymyxin B, gen amicin, and ampicillin
[110]
T iclosan eSalmonella
en e ica
Typhimu ium
The ecalci ance o plank onic cells o Salmonella en e ica Typhimu ium
a e exposu e o iclosan is associa ed wi h:
The exp ession o deg ada i e enzymes
Changes in he bac e ial cell en elope
The ac i a ion o he e flux pump
[111]
(con inued on nex page)
M. Sousa, I. Machado, L.C. Sim~
oes e al. En i onmen al Science and Eco echnology 25 (2025) 100557
7
equen DNA eplica ion [142,145,146]. In con as , slow-g owing
o do man bac e ia may e ade, leading o ole ance and he
de elopmen o esis ance mechanisms, like pe sis e cells
[147,148]. Slow-g owing o do man cells wi hin a bac e ial popu-
la ion can ac as highly ole an pe sis e cells. These cells can
su i e he ea men and hen esume g ow h once he molecule
is emo ed [147,149]. In addi ion, slow-g owing o s essed bac e ia
may ac i a e s ess- esponse pa hways, which can lead o an
inc eased mu a ion a e and he acquisi ion o esis ance elemen s
[147,150]. The specific g ow h a e can also modula e bac e ial
esis ance mechanisms h ough he exp ession o esis ance genes
and a ec he e olu ion o esis ance [151,152].
5.2. Biocides as induce s o esis an pheno ypes
The concen a ions o biocides in na u al en i onmen s a e
unlikely o be su ficien o kill la ge numbe s o bac e ia [25].
Mo eo e , ch onic sub-bac e icidal exposu e o biocides can cause
Table 2 (con inued )
Biocides An ibio ics Mic oo ganism Mechanism o esis ance/C oss- esis ance Re e ences
Mu a ions in he enoyl educ ase enzyme
The suscep ibili y o biofilms o Salmonella en e ica Typhimu ium o
iclosan is associa ed wi h:
The subs ance's low ex acellula ma ix di usion
O e ac i i y o e flux pumps,
Exopolysaccha ide p oduc ion
Table 3
Compila ion o he biocides e e ed o in his s udy, o ganized by hei chemical ca ego y and common applica ions.
Ca ego y Biocides Common Applica ions Re e ences
Qua e na y ammonium compounds Benzalkonium chlo ide
Ce yl ime hylammonium b omide
Alkyl ime hylammonium compounds
Dialkyldime hylammonium compounds
Disin ec ion in heal hca e, pe sonal ca e p oduc s, and indus ial cleaning [54]
Bisbiguanides Chlo hexidine Hospi al an isepsis, den al ca e, and household cleaning [117]
Phenolics compounds T iclosan
O ho-phenylphenol
Pe sonal ca e p oduc s (e.g., soaps, oo hpas e, cosme ics) [118]
Aldehydes Glu a aldehyde
O ho-ph halaldehyde
Indus ial disin ec ion, hospi al s e iliza ion [119]
Alcohols Isop opanol
E hanol
Hand sani ize s, su ace disin ec an s [120]
Pe oxygens Hyd ogen pe oxide Su ace disin ec ion, wa e ea men [121]
Halogen- eleasing agen s Sodium hypochlo i e
Pe ace ic acid
Wa e disin ec ion, indus ial cleaning, and su ace s e iliza ion [122]
Bisphenols T iclosan
T icloca ban
An imic obial agen s in pe sonal ca e p oduc s [123]
Hea y-me al de i a i es Sil e compounds Medical de ices and su ace coa ings [124]
Po idone-iodine
Diamidines
An isepsis in heal hca e, pe sonal hygiene p oduc s. [125]
Fig. 2. Compila ion o he mechanisms by which biocides p omo e an ibio ic esis ance add essed in his s udy [15,84,126,127].
M. Sousa, I. Machado, L.C. Sim~
oes e al. En i onmen al Science and Eco echnology 25 (2025) 100557
8
ansien mu a ions in hese mic oo ganisms [25,153,154]. Such
mu a ions a e usually no enough o kill he cell, bu mo e likely,
hey jus cause a mino pheno ypic change in he bac e ia ha will
make i mo e o less fi o i s en i onmen and mo e o less sus-
cep ible [155]. These pheno ypic changes a e empo a y, mos o
he ime, bu can some imes become pe manen when mu an s
a ise [156,157]. These esponses ha e e ol ed o pe mi bac e ia o
adap apidly o en i onmen al s esses and may d i e selec i e
en ichmen o an imic obial- esis an s ains o bac e ia [156,158].
The induc ion o such bac e ial esis ance mechanisms is o en
linked wi h he o e -exp ession o e flux pumps [159], he o e -
exp ession o mul igene and p o ein sys ems [128], and al e ed
exopolysaccha ide p oduc ion [151]. Also, al e a ions in su ace
cha ge ha esul in less a fini y o ca ionic an imic obials and
al e a ion in lipid con en ha complica es he di usion o lipid-
soluble an imic obials ac oss he cellula memb ane [160];
educed exp ession o po ins, which limi s an imic obials up ake
[161]; high exp ession o e flux pumps ha expel a wide ange o
oxic molecules educes an imic obials accumula ion inside he cell
[162]. This opic has been comp ehensi ely e iewed ecen ly and
was g aphically summa ized in Fig. 4 [130,159,163,164].
Fu he mo e, al e a ions in memb ane p ope ies can lead o
QAC esis ance and c oss- esis ance o memb ane-ac i e
an ibio ics [29,84,165,166]. In E. coli, spon aneous acquisi ion o
esis ance o ce yl ime hylammonium b omide (CTAB) was e-
po ed o be due o al e a ion in memb ane lipopolysaccha ide
composi ion and educ ion in OmpF, which educes bac e ia
pe meabili y. These changes also con e ed esis ance o a wide
a ie y o an ibio ics [167].
Conce ning he ole o OMP exp ession associa ed wi h QAC
esis ance, some s udies [105,168,169] showed ha esis ance o
QACs was accompanied by educed le els o OmpC,OmpF, and
OmpA.InE. coli, he loss o OmpF and OmpC genes makes cells
esis an o some an ibio ics, namely o hyd ophilic an ibio ics,
such as
b
-lac ams [85,170]. Mac olides, aminoglycosides, i amy-
cins, no obiocin, usidic acid, and ca ionic pep ides di use ac oss
he lipid bilaye , so al e a ions in lipid con en can be ela ed o
esis ance o hese an ibio ics [170].
One o he main con o e sial issues in an imic obial esea ch is
he ac ha in i o condi ions and labo a o y findings do no
mimic pe ec ly he en i onmen al concen a ions o such com-
pounds and so, adap i e esis ance and i s ole in c oss- esis ance
o an ibio ics mus be s udied and epo ed wi h some p ecau ion
[171,172]. The majo i y o suscep ibili y es s a e based on bac e ial
inoculum, which is p epa ed in a e y ich medium, and i is e y
unlikely ha bac e ia in si u ha e such eadiness o nu ien s [171].
Fig. 3. Ascending o de o esis ance o biocides and an ibio ics (based on McDonnell and Russell [68]).
Fig. 4. Non-specific s uc u al and unc ional al e a ions in he bac e ial ou e laye s ollowing biocide exposu e, leading o esis ance o a b oad spec um o an imic obials,
e ol ing he o e -exp ession o e flux pumps, he o e -exp ession o mul igene and p o ein sys ems, al e ed exopolysaccha ide p oduc ion, al e a ions in su ace cha ge and in lipid
con en , and educed exp ession o po ins (based on Lo usso, Ca a a [130], Huang, Wu [159], De Gae ano, Len ini [163], Che i [164]).
M. Sousa, I. Machado, L.C. Sim~
oes e al. En i onmen al Science and Eco echnology 25 (2025) 100557
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