Kosa is ano ae al. BMC Mic obiology (2023) 23:288
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BMC Mic obiology
Syne gis ic an ibac e ial ac ion o  hei on
complex andampicillin agains S aphylococcus
au eus
Ludmila Kosa is ano a1, Ma in Rihacek1, F an iska Sucha1, Ved an Milosa lje ic1, Pa el S ec1,
Jana Do azilo a2, Lucy Voj o a2, Pe e An al3, Pa el Kopel3, Zdenek Pa ocka4, Voj ech Adam1, Ludek Zu ek1 and
K is yna Dolezeliko a1*
Abs ac
Objec i es Resis ance o an ibio ics among bac e ia o clinical impo ance, including S aphylococcus au eus, is a se i-
ous p oblem wo ldwide and he sea ch o al e na i es is needed. Some me al complexes ha e an ibac e ial p ope -
ies and when combined wi h an ibio ics, hey may inc ease bac e ial sensi i i y o an imic obials. In his s udy, we
syn hesized he i on complex and es ed i in combina ion wi h ampicillin (Fe16 + AMP) agains S. au eus.
Me hods An i on complex (Fe16) was syn hesized and cha ac e ized using spec oscopy me hods. Con i ma ion
o he syne gis ic e ec be ween he i on complex (Fe16) and ampicillin (AMP) was pe o med using ζ–po en ial,
in a ed spec a and FICI index calcula ed om he minimum inhibi o y concen a ion (MIC) om he checke boa d
assay. Cy o oxic p ope ies o combina ion Fe16 + AMP was e alua ed on euka yo ic cell line. Impac o combina ion
Fe16 + AMP on chosen genes o S. au eus we e pe o med by Quan i a i e Real-Time PCR.
Resul s The MIC o Fe16 + AMP was signi ican ly lowe han ha o AMP and Fe16 alone. Fu he mo e, he in a ed
spec oscopy e ealed he change in he ζ–po en ial o Fe16 + AMP. We demons a ed he abili y o Fe16 + AMP o dis-
up he bac e ial memb ane o S. au eus and ha likely allowed o be e abso p ion o AMP. In addi ion, he change
in gene exp ession o bac e ial e lux pumps a he sub-inhibi o y concen a ion o AMP sugges s an insu icien
impo o i on in o he bac e ial cell. A he same ime, Fe16 + AMP did no ha e any cy o oxic e ec s on ke a inocy es.
Conclusions Combined Fe16 + AMP he apy demons a ed signi ican syne gis ic and an imic obial e ec s agains S.
au eus. This s udy suppo s he po en ial o combina ion he apy and u he esea ch.
Keywo ds S aphylococcus au eus, I on complex, Ampicillin, Syne gy, An imic obial ac i i y
*Co espondence:
K is yna Dolezeliko a
k [email p o ec ed]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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Kosa is ano ae al. BMC Mic obiology (2023) 23:288
In oduc ion
The eme gence o an imic obial esis ance is a p ob-
lem causing a global c isis and he a e o de elopmen
o new an imic obial agen s is no adequa e. Conse-
quen ly, in ec ions caused by mul id ug- esis an bac e ia
including S aphylococcus au eus a e o a g ea conce n
wo ldwide [1]. S aphylococcal in ec ions in humans and
animals a e commonly ea ed wi h β-lac am an ibio ics,
including ampicillin (AMP) [2]. A g ea selec i e p essu e
om he in ensi e and ex ensi e use o an ibio ics has
esul ed in β-lac am esis ance based on se e al mecha-
nisms such as e lux pumps, side opho es, and p oduc-
ion o β-lac amases [2–4]. Cu en ly, he majo i y o
clinical s ains o S. au eus a e β-lac amase posi i e [2],
use e lux pumps, and also p oduces side opho es [3, 4].
An imic obial p ope ies o me als ha e been known
o se e al decades [5] and hei combina ion wi h an i-
bio ics has been assessed in se e al s udies. Fo exam-
ple, i was shown ha β-lac ams oge he wi h sil e o
zinc oxide nanopa icles had g ea an imic obial ac i -
i y agains a ious mul id ug esis an bac e ia [6, 7].
I on has been also conside ed as a po en ial candida e
o combined ea men wi h an ibio ics [8]. I on is he
mos abundan ansi ion elemen in he human body
and a p omising an imic obial agen in he o m o a
me al complex. I on in he o m o a me al complex can
a ec bac e ial cells whe e hey cause oxida i e s ess,
inhibi espi a o y p ocesses and ATP p oduc ion,
inc ease cell hyd ophobici y, and acili a e hei pen-
e a ion ac oss he cell wall [7–9]. The e o e, combined
ea men s o e se e al ad an ages such as a lowe
po en ial o de eloping esis ance, addi i e o syne gis-
ic e ec s, inc easing he e ec i i y o an ibio ics, and
o e coming d ug esis ance [10]. Combina ion he apy
o an i on complex wi h β-lac ams has been es ed p e-
iously agains Esche ichia coli and i was mo e e ec i e
han an ibio ics alone [9].
The goal o his s udy was o syn hesize he i on
complex Fe16 and es i in combina ion wi h AMP
agains S. au eus. We also aimed o in es iga e he
mechanism o he syne gis ic ac ion o Fe16 + AMP
agains S. au eus and o e alua e whe he i has any
nega i e e ec s on euka yo ic cells ep esen ed by he
HaCaT cell line.
Resul s
Physico‑chemical cha ac e iza ion o Fe16 complex
andin e ac ion be weenFe16 andAMP
The in a ed spec um o Fe16 exhibi ed cha ac e is ic
bands o he Fe(II)- is(diimine) complexes. Also, he
elec onic spec a o Fe16 showed abso p ion bands
cha ac e is ic o Fe(II)- is(diimine) complexes (Fig.
S1Aa,b and Table S2). In e ac ions be ween Fe16 and
AMP we e con i med by he ζ–po en ial and ATR-
FTIR. The ζ–po en ial o Fe16 alone was + 55.8 mV
and AMP alone was -16.5 mV. The ζ–po en ial o
Fe16 + AMP was shi ed owa d sligh ly nega i e al-
ues -4.2mV (Fig. S2) indica ing he o ma ion o new
complexes. To u he s udy he in e ac ions be ween
Fe16 and AMP in he Fe16 + AMP complex, spec a o
Fe16, AMP, and Fe16 + AMP we e eco ded by ATR-
FTIR (Fig. S1C). No majo spec al al e a ions we e
obse ed compa ing he Fe16 spec um and he Fe16
sub ac ed spec a. Wi h he ocus on AMP and AMP
sub ac ed spec a, he wo signi ican band b oaden-
ings co esponding o alence ca boxyla e ib a ions
(COO−) a he wa e numbe alues o 1 590 and 1
370 cm−1 we e obse ed in spec um o Fe16 + A M P.
Theseca boxyla e g oups media e he in e ac ion in
Fe16 + AMP complex.
An an ibac e ial e icacy andsyne gis ic e ec o Fe16,
AMP, andFe16 + AMP onS. au eus by hechecke boa d
assay
The inhibi o y e ec om he checke boa d assay o
Fe16 + AMP agains S. au eus was signi ican ly g ea e
han ha o he indi idual compounds (Fig.1A). The
MIC alue o Fe16 31µg/ml and AMP 0.5µg/ml in com-
bina ion agains S. au eus was signi ican ly lowe han
ha o Fe16 and AMP alone wi h MIC alues o 125µg/
ml and 2µg/ml (Fig.1Ab). This inding also co ela ed
wi h he esul s o he FIC index. Isobolog am showed
he syne gis ic e ec be ween Fe16 (FICFe16 = 0.248)
and AMP (FICAMP = 0.250) (Fig.1Ac) based on he FIC
index calcula ion o he wo es ed componen s. The
an ibac e ial e ec o he Fe16 + AMP and i s syne gis-
ic e ec (≤ 0.5) agains S. au eus was con i med by he
FIC index 0.498.
Obse a ion o mo phological changes o S. au eus
a e Fe16, AMP andFe16 + AMP ea men
SEM mic oscopy o S. au eus ea ed wi h Fe16 + AMP
e ealed ha he in eg i y o he cell wall was comp o-
mised. In con as , un ea ed S. au eus cells e ained
hei coccus mo phology and he cell su ace was com-
pac (Fig. 1Ba). No mo phological change was also
obse ed in S. au eus cells ea ed wi h Fe16 (Fig.1Bb)
and AMP alone (Fig.1Bc). On he o he hand, sig-
ni ican mo phological changes we e obse ed a e
24h on S. au eus cells ea ed wi h Fe16 + AMP a he
sub-inhibi o y concen a ion (0.25 µg/ml) showing
damaged cells wi h dis up ed walls and memb anes
(Fig.1Bd).
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Kosa is ano ae al. BMC Mic obiology (2023) 23:288
Cy o oxici y p ope ies o Fe16, AMP andFe16 + AMP
The cy o oxic e ec o Fe16 on he HaCaT ke a inocy e cell
line was obse ed in he concen a ion ange o 8–125µg/
ml, whe eas o AMP no cy o oxici y was obse ed
(Fig.1C). A he concen a ion o 0.5µg/ml o Fe16 + AMP
he iabili y o ke a inocy es was 90.0% (Fig.1C).
Changes in he egula ion o selec ed genes o e lux
pumps, β‑lac amase andABC anspo e s a e  ea men
wi hFe16, AMP andFe16 + AMP
The exp ession o he e lux pump gene mepA signi i-
can ly inc eased a e all h ee ea men s ( old change in
he ange 3.43—9.33) al hough he e ec o AMP alone
Fig. 1 Aa Visualiza ion o he checke boa d o ma : G een boxes ep esen g ow h and whi e boxes inhibi ion. O ange box shows F ac ional
inhibi ion index, pink boxes demons a e posi ions o minimal inhibi o y concen a ions o Fe16 and AMP. Blue box is no- ea ed S. au eus
(Con ol). Ab image p esen s an ibac e ial and syne gy ac i i y wi h MIC and FIC esul s om checke boa d assay. The alues a e p esen ed
as he a e age om h ee independen expe imen s. Ac Isobolog am showing he syne gy e ec o Fe16 and AMP wi h FIC index ≤ 0.5 and able
demons a ing he he FIC index ca ego y scale. B Ex e nal mo phological changes o S. au eus a e exposu e o Fe16+AMP assessed by SEM.
Figu e Ba shows un ea ed S. au eus as a con ol, Bb demons a es S. au eus ea ed wi h Fe16 and Bc AMP alone a 0.125 ug/ml. Bd is S. au eus
ea ed wi h Fe16+AMP a 0.25 ug/ml. Red a ows indica e S. au eus mo phological changes. C Cy o oxic e ec s o Fe16, AMP and Fe16+AMP
a he di e en concen a ions on he HaCaT ke a inocy e cell line
Page 4 o 8
Kosa is ano ae al. BMC Mic obiology (2023) 23:288
was a signi icance le el p- alue < 0.01. The exp ession
o he no A gene also signi ican ly inc eased a e AMP
(2.8 old change) and Fe16 + AMP (3.1 old change) ea -
men s (Fig.2A). Gene exp ession o blaZ was down egu-
la ed, al hough no signi ican ly (p- alue > 0.2) (Fig. 2A).
The signi ican ly inc eased exp ession was de ec ed o
he anspo e genes pABC and huB a e exposu e
o Fe16 and Fe16 + AMP (p- alue < 0.05) (Fig.2B). Ampi-
cillin alone a ec ed he exp ession o pABC only. The
exp ession o he hi d ABC anspo e gene, h sA, was
down egula ed non-signi ican ly a e all ea men s
(Table S3).
Discussion
In ecen decades, many me al-based complexes ha e
been es ed o hei an imic obial p ope ies [1, 8]. I
has been shown ha i on exhibi s an imic obial e ec s
agains G am-posi i e and G am-nega i e bac e ia in he
o m o me al complex o nanopa icles [8, 11].
In he syn he ized complexes s uc u e [12], he di e -
ence in wa eleng hs obse ed o asymme ic and sym-
me ic ν(COO−) ib a ions indica es ha he compound
Fe16 adop ionic s uc u e and uma a e ions a e no
coo dina ed o he cen al a oms. The s ong bands can
be assigned o asymme ic ν(COO−) ib a ions bu su e
in e e ence wi h C = C and C = N s e ching ib a ions o
a oma ic ing [13]. Conside ing ha pKa alues o AMP
unc ional g oups (2.5/7.3) he ca boxylic acid became
anionic (COO¯), and amine g oup ca ionic (NH34+) [14].
Howe e , wi h inc ease he pH o e he AMP pKa alues
he a ge ed Fe16 complex a e in e ac ion wi h AMP
became dep o ona ed (anionic o m), which explains he
s ong dec ease o he Fe16 complex posi i e cha ge and
sligh inc ease o nega i e su ace cha ge o newly o med
complexes. Due o he shi o ca boxyla e asymme ic
alence ib a ions o highe wa enumbe s and symme -
ic alence ib a ion o lowe numbe s a e obse ed in
FTIR analysis. These spec al a ia ions a e connec ed
o he ca boxyla e g oup passing om he ee zwi e-
ionic COO− g oup o o ganome allic coo dina ion in
he Fe16 + AMP [15]. The band shi s in spec al egion
sugges in e ac ion o AMP phenyl wi h nphen in Fe16
ia π–π elec on dono –accep o complex sys em [16],
while he ib a ion posi ions ela ed o β-lac am ing in
AMP as well as NO2 unc ional g oup in Fe16 emains
unchanged. These da a indica e ha he key an ibac e ial
s uc u e in he complex emains una ec ed [17].
The an imic obial e ec o Fe16 + AMP on S. au eus
showed syne gis ic ac i i y a h e pheno ypic and an-
sc ip omic le el. P e iously, i was shown ha o he me -
als such as coppe , zinc, and i on in combina ion wi h
amoxicillin inc eased e ec i eness agains E. coli [9].
Al hough suscep ibili y o bac e ia o doped an ibio ics
wi h me al complexes has been shown be o e [18], in es-
iga ions on he i on complex a e a e [8]. Cell in eg i y
o S. au eus ea ed wi h Fe16 + AMP was comp omised
leading o cell dea h which co obo a es simila s udy
wi h sil e nanopa icles and an ibio ics [19]. Me al
Fig. 2 Gene exp ession a Log2 ( old change) o he ABC anspo e amily (A) and de ense sys em o S. au eus a e exposu e o AMP, Fe16
and Fe16 + AMP (B). Signi ican changes o Fe16, AMP and Fe16 + AMP in compa ison o ha o con ol a e ma ked wi h as e isks: *p- alue < 0.05,
**p- alue < 0.01, ***p- alue < 0.001
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Kosa is ano ae al. BMC Mic obiology (2023) 23:288
complexes inc ease he lipophilic cha ac e o he ansi-
ion me al ion and he e o e inc ease he hyd ophobici y
and acili a e hei pene a ion h ough he bac e ial cell
wall and memb ane [9]. In ou case, Fe16 + AMP appea s
o acili a e he pene a ion o AMP h ough he cell wall
o S. au eus. S udy o Panacek e al. (2016) showed ha
wi h AgNps sil e nanopa icles in combina ion wi h
an ibio ics including β-lac ams had also syne gis ic e ec
agains S. au eus [20].
I is impo an o no e ha no cy o oxic e ec s agains
he HaCaT cell line a e exposu e o Fe16 + AMP was
obse ed. In ecen yea s, many neu ological diso de s
ha e been a ibu ed o i on o e load; howe e , u he
s udies ha e op imized new complex me al compounds
in combina ion wi h a ious ma e ials o mode a e gen-
e al cy o oxici y [21, 22].
The combina ion o Fe16 + AMP a he sub-inhibi o y
concen a ion also likely led o dis up ion o he e lux
pumps and ha is consis en wi h he MICs esul s. The
s udy o he inhibi o y e ec o e lux pumps unde he
in luence o he i on complex alone and in combina ion
wi h an ibio ics has no been conduc ed p e iously.
Howe e , binding o i on oxide nanopa icles wi h
i ampicin o he ac i e si e o he e lux pump and con-
sequen blocking o i s unc ion has been demons a ed
[23]. Te mina ion o he p o on g adien , ollowed by
dis up ion o he memb ane po en ial and/o loss o
p o on mo i e o ce can lead o ailu e o he d i ing
o ce, which is essen ial o he unc ion o e lux pumps
[24, 25]. I is also assumed ha i on nanopa icles dis-
up he ac i i y o e lux pumps by gene a ing ROS
[25]. La ge me al oxides ha e he abili y o induce as e
elec on ans e kine ics o he ac i e si e o enzymes
[26]. Simila ly, up egula ion o mepA has been epo ed
using sil e nanopa icles and subsequen exposu e o
Ag+ ions [27]. Al hough he highes exp ession o mepA
and no A was eco ded a e he exposu e o he com-
bina ion o Fe16 + AMP, an inc eased exp ession o
hese wo genes was also de ec ed wi h Fe16 and AMP
sepa a ely. Fe16 alone could inc ease he exp ession o
mepA and no A due o he eac i e oxygen species ha
s ess he cells and dis up cellula componen s. Simila
o i ampicin ( unc ionalized i on nanopa icles wi h
an i- ube culosis d ugs), i causes damage o he p o-
ein subuni s and he ch omosome. This could lead o
he ailu e o he e lux pumps [28]. Ampicillin alone
also inc eased he exp ession o mepA and no A e lux
pumps, as shown o an ibio ics simila o β-lac ams
in o he s udies [29]. Howe e , he issue wi h di e en
classes o an ibio ics is he esidual ac i i y on bac e-
ial a ge s and he s eng hening o he selec ion o
esis ance mechanisms [30]. Mo eo e , he esul s o
he Fe16 and AMP componen s alone a e consis en
wi h he MIC esul s. As he down egula ion o blaZ
was demons a ed, he associa ion o an ibio ics wi h
me als supp esses β-lac amase hyd olase and he e-
o e an ibio ics can pass be e h ough he bac e ial
cell wall [31]. Ano he indica o o he ins abili y o he
de ense sys em o S. au eus was a change in he gene
exp ession o he ABC anspo e s. The o e exp es-
sion o he ABC anspo e s unde AMP ea men ,
in ou case he pABC gene, sugges s apid adap a ion
o he eac i e immune sys em and ele a ion o up ake
o i on in o he cell which is consis en wi h he MIC
esul s [32]. On he o he hand, he inc eased exp es-
sion o pABC and huB unde he in luence o Fe16
and Fe16 + AMP and he down egula ion o h sA in all
ea men s indica e insu icien up ake o i on by he
cell. Inac i a ion o in amemb ane p o eolysis o he
ABC anspo e was ound o inc ease he suscep ibil-
i y o S. au eus o se e al an imic obial agen s [33]. In
ano he s udy, he inhibi o y e ec o zinc on he me al
up ake by he ABC anspo e s a physiological con-
cen a ions was demons a ed [34]. Howe e , he num-
be o s udies ocused on his opic is low and u he
esea ch is needed. Based on ou esul s, we p opose
ha he applica ion o Fe16 + AMP causes s ess in S.
au eus, acili a es he pene a ion o AMP h ough he
cell wall, and dis up s he unc ion o e lux pumps and
ABC anspo e s (Fig.3). This oge he wi h insu i-
cien i on in ake leads o he bac e ial cell dea h.
In conclusion, Fe16 + AMP is a p omising new al e -
na i e o ea men o in ec ions caused by S. au eus
and po en ially o he pa hogenic bac e ia. Ou esul s
also demons a e ha his combina ion has no cy o-
oxic side e ec s on euka yo ic cells.
Me hods
Syn hesis o Fe16 = [Fe(nphen)3]( u)·7H2O andp epa a ion
o Fe16 + AMP combina ion
Fe ous uma a e, H2 u = uma ic acid and 5-ni o-
1,10-phenan h oline (nphen) (Sigma Ald ich, USA) we e
used o he syn hesis o he Fe16 = [Fe(nphen)3]( u)·7H2O
complex. The s uc u e o he Fe16 complex was syn he-
sized on he basis o he al eady known s uc u e o he
indi idual componen s men ioned abo e [35, 36]. I on
uma a e was mixed in 40.0ml o wa e wi h nphen dis-
pe sed in he same sol en and s i ed o 6.0h a 40.0°C.
Cha ac e iza ion o Fe16, AMP and Fe16 + AMP was con-
duc ed by spec oscopic me hods. The i on-an ibio ic
complex was p epa ed by mixing Fe16 wi h AMP in Mil-
liQ wa e a a concen a ion o 1.0mg/ml each and s i ed
a oom empe a u e o 24h a 45 pm.

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Kosa is ano ae al. BMC Mic obiology (2023) 23:288
E alua ion o syne gis ic e ec s be weenFe16 andAMP
byζ – po en ial andin a ed spec a (FTIR)
Syne gism be ween Fe16 and AMP was e alua ed by
he ζ–po en ial and in a ed spec a (ATR-FTIR) [37,
38]. Samples we e subjec ed o analysis o DLS o ζ–
po en ial. The Fou ie ans o m in a ed spec ome e
equipped wi h a diamond c ys al was used o eco d
in a ed spec a o Fe16, AMP and Fe16 + AMP ia he
a enua ed o al e lec ance me hod (ATR-FTIR, Ve ex
70 , B uke , Bille ica, MA, USA). De ailed me hodology
is desc ibed in he Supplemen a y ile.
Cul i a ion o  es ed bac e ia
S aphylococcus au eus CCM 4223 (Czech Collec ion
o Mic oo ganisms, Masa yk Uni e si y, B no, Czech
Republic) was cul u ed on 5.0% Columbia blood aga
(LMS, Czech Republic) a 37.0°C o e nigh .
E alua ion o an imic obial ac i i y andan imic obial
syne gis ic e ec o Fe16, AMP andFe16 + AMP
bychecke boa d assay
An imic obial ac i i y and syne gis ic e ec o Fe16,
AMP and Fe16 + AMP was es ed by he checke boa d
assay [39]. B ie ly, Fe16 was placed in 96-well mic opla es
dilu ed wo old in Muelle Hin on b o h (Sigma Ald ich,
USA) along he e ical ows and AMP was c oss-dilu ed
ho izon ally by wo old se ial dilu ion. Bac e ial inocu-
lum o S. au eus was added in o each well o p oduce a
inal concen a ion o 1–2 × 106CFU/ml. The pla es we e
incuba ed a 37 ◦C o 24h. A e incuba ion, he bac-
e ial g ow h was assessed by obse ing he colo and
u bidi y o he solu ion. The es s we e ca ied ou in a
echnical iplica e. The in e ac ions be ween Fe16 and
AMP we e e alua ed by he ac ional inhibi o y con-
cen a ion index (FICI) calcula ed based on he o mula
(MIC o A in combina ion/MIC o A) + (MIC o B in
combina ion/MIC o B) [40].
Cy o oxic p ope ies o Fe16, AMP andFe16 + AMP
oneuka yo ic cell line
Cy o oxic p ope ies o Fe16, AMP, and Fe16 + AMP
we e e alua ed by he spon aneously ans o med ane-
uploidy immo al ke a inocy e cell line om he adul
human skin (HaCaT). Cell iabili y was quan i ied using
he MTT assay. De ailed me hodology is desc ibed in he
Supplemen a y ile.
Fig. 3 Mechanism o he an imic obial e ec o Fe16 + AMP causing s ess in S. au eus and dis up ion o unc ion o e lux pumps and ABC
anspo e s A. Impo o i on in o cells, needed by S. au eus o g ow h and me abolism, is dis up ed. B No A and MepA e lux pumps a e
no su icien o push Fe16 + AMP ou o cells
Page 7 o 8
Kosa is ano ae al. BMC Mic obiology (2023) 23:288
Cell mo phology o S. au eus a e Fe16, AMP
andFe16 + AMP ea men
Cell mo phology o S. au eus a e Fe16 + AMP ea -
men and Fe16 and AMP alone was obse ed by SEM.
S aphylococcus au eus was mixed wi h Fe16 + AMP in
concen a ion 0.25µg/ml (0.125µg/ml o each Fe16 and
AMP), wi h Fe16 (0.125µg/ml) and AMP (0.125µg/ml)
alone and cul u ed a 37.0°C o e nigh . A e incuba-
ion, samples we e ixed by glu a aldehyde (1.0%) and
incuba ed o 30 min a oom empe a u e. Samples
we e hen dehyd a ed using an ascending e hanol se ies
in ange 40—100% in se e al s eps. Cell mo phology was
examined by SEM on he Tescan MAIA 3 equipped wi h
a ield emission gun (Tescan L d., B no, Czech Republic).
Mo e de ailed me hods a e in he Supplemen a y ile.
RNA ex ac ion, pu i ica ion and e e se ansc ip ion
Fo RNA ex ac ion, S. au eus was cul u ed o e nigh
in Lu ia–Be ani (LB) b o h a 37.0°C and shaking a
120 pm wi h and wi hou sub-inhibi o y concen a-
ions o 0.25µg/ml o Fe16, AMP, and Fe16 + AMP. The
RNA ex ac ion was pe o med using TRIzol eagen ®
(TRIzol Reagen , In i ogen, Ca lsbad, CA) acco ding
o he manu ac u e ins uc ions. The isola ed RNA was
pu i ied by e hanol RNA/DNA p ecipi a ion and e e se
ansc ip ion was pe o med wi h he ansc ip o i s
s and cDNA syn hesis ki o RT-PCR (Roche, Man-
nheim, Ge many) based on he manu ac u e ins uc-
ions using 500.0ng RNA.
Quan i a i e eal‑ ime PCR
Quan i a i e eal- ime PCR analysis was pe o med using
he qTOWER3 sys em (Analy ik Jena, Jena, Ge many)
wi h poB as he housekeeping gene. Resul s we e isu-
alized as log2 old change (ΔΔC ) calcula ions. All p im-
e s we e designed using he IDT sys em. Mo e de ailed
me hods a e in he Supplemen a y ile.
S a is ical e alua ion
The unpai ed - es be ween un ea ed and ea ed sam-
ple om ΔC alues o biological iplica e was used o
de e mine he impac o ea men s on de ense sys em o
S. au eus. All s a is ical analysis and g aphical isualiza-
ions we e done using G aphPad P ism 8.0.1. (G aphPad
So wa e, CA, USA).
Abb e ia ions
AMP Ampicillin
ATR-FTIR A enua ed o al e lec ance-Fou ie - ans o m in a ed spec oscopy
DLS Dynamic ligh sca e ing
Fe16 I on complex
Fe16 + AMP Combina ion o i on complex wi h ampicillin
FIC F ac ional Inhibi o y Concen a ion
MIC Minimum inhibi o y concen a ion
RPM Re olu ions pe minu e
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h ps:// doi.
o g/ 10. 1186/ s12866- 023- 03034-1.
Addi ional ile1: TableS1. P ime s used o Quan i a i e Real–Time PCR.
TableS2. Elec onic spec al da a o aqueous solu ions o Fe16. TableS3.
Exp ession o he all es ed genes o S. au eus shown as Fold change a e
exposu e en i onmen al s ess o AMP, Fe16 and Fe16+AMP ela i e o no
ea ed S. au eus wi h use o poB as housekeeping gene. Figu e S1. Phys-
ico-chemical cha ac e iza ion o he Fe16 complex. Figu e S2. ζ-po en ial
o Fe16, AMP and Fe16+AMP measu ed in MiliQ wa e a pH 8.2.
Acknowledgemen s
This wo k was suppo ed by he ERDF “Mul idisciplina y esea ch o inc ease
applica ion po en ial o nanoma e ials in ag icul u al p ac ice” (No. CZ.02.1.01/
0.0/0.0/16_025/0007314). CzechNanoLab p ojec LM2018110 unded by MEYS
CR is acknowledged o he inancial suppo o he measu emen s a CEITEC
Nano Resea ch In as uc u e.
Au ho s’ con ibu ions
PK and PA pe o med he syn hesis and cha ac e iza ion o he i on complex
and ga e commen s. LV, JD and VM did he expe imen s and e alua ion wi h
commen s o he syne gis ic e ec be ween he i on complex and ampicil-
lins. FS pe o med he cy o oxici y expe imen and PS SEM mic oscopy. MR
designed expe imen o Quan i a i e Real-Time PCR and e alua ion o esul s.
LK pe o med expe imen s, analyzed and commen ed mic obiological and
molecula pa s and comple ion and w i ing o he a icle. ZP designed s a-
is ical e alua ion. VA edi ed he manusc ip . KD and LZ designed he p ojec ,
supe ised he expe imen s and w o e he epo . All au ho s ha e ead and
app o ed he inal manusc ip .
Funding
ERDF “Mul idisciplina y esea ch o inc ease applica ion po en ial o nanoma-
e ials in ag icul u al p ac ice” CZ.02.1.02/0.0/0.0/16_025/0007314 and he
CzechNanoLab p ojec LM2018110 unded by MEYS CR.
A ailabili y o da a and ma e ials
Da a is a ailable on he depa men sha e d i e and can be uploaded when
eques ed. The i s au ho may be con ac ed i someone wan s o eques he
da a om his s udy.
Decla a ions
E hics app o al and consen o pa icipa e
No applicable.
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Au ho de ails
1 Depa men o Chemis y and Biochemis y, Facul y o Ag iSciences,
Mendel Uni e si y in B no, B no, Czech Republic. 2 Cen al Eu opean Ins i u e
o Technology, Uni e si y o Technology, B no, Czech Republic. 3 Depa men
o Ino ganic Chemis y, Facul y o Science, Palacky Uni e si y, Olomouc, Czech
Republic. 4 Depa men o Fo es Managemen and Applied Geoin o ma ics,
Facul y o Fo es y and Wood Technology, Mendel Uni e si y in B no, B no,
Czech Republic.
Page 8 o 8
Kosa is ano ae al. BMC Mic obiology (2023) 23:288
Recei ed: 8 May 2023 Accep ed: 28 Sep embe 2023
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Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub-
lished maps and ins i u ional a ilia ions.