an ibio ics
A icle
Sho Chain Fa y Acids Commonly P oduced by Gu
Mic obio a In luence Salmonella en e ica Mo ili y,
Bio ilm Fo ma ion, and Gene Exp ession
Alexand e Lamas * , Pa icia Regal , Bea iz Vázquez , Albe o Cepeda and
Ca los Manuel F anco
Labo a o io de Higiene Inspección y Con ol de Alimen os, Depa amen o de Química Analí ica, Nu ición y
B oma ología, Uni e sidad de San iago de Compos ela, 27002 Lugo, Spain; pa icia. [email p o ec ed] (P.R.);
[email p o ec ed] (B.V.); [email p o ec ed] (A.C.); [email p o ec ed] (C.M.F.)
*Co espondence: alexand [email p o ec ed]
Recei ed: 14 No embe 2019; Accep ed: 12 Decembe 2019; Published: 13 Decembe 2019
Abs ac :
Sho chain a y acids (SCFAs) a e commonly p oduced by heal hy gu mic obio a and
hey ha e a p o ec i e ole agains en e ic pa hogens. SCFAs also ha e di ec an imic obial ac i i y
agains bac e ial pa hogens by di usion ac oss he bac e ial memb ane and educ ion o in acellula
pH. Due o his an imic obial ac i i y, SCFAs ha e p omising applica ions in human heal h and
ood sa e y. In his s udy, he minimum inhibi o y concen a ions (MICs) o ou SCFAs (ace ic
acid, bu y ic acid, p opionic acid, and ale ic acid) in Salmonella s ains isola ed om poul y we e
de e mined. The e ec o subinhibi o y concen a ions o SCFAs in Salmonella bio ilm o ma ion,
mo ili y, and gene exp ession was also e alua ed. Bu y ic acid, p opionic acid, and ale ic acid
showed a MIC o 3750
µ
g/mL in all s ains es ed, while he MIC o ace ic acid was be ween 1875 and
3750
µ
g/mL. Subinhibi o y concen a ions o SCFAs signi ican ly
(p<0.05)
educed he mo ili y o all
Salmonella s ains, especially in he p esence o ace ic acid. Bio ilm o ma ion was also signi ican ly
(p<0.05)
lowe in he p esence o SCFAs in some o he Salmonella s ains. Salmonella s ain. Salmonella
Typhimu ium T7 showed signi ican
(p<0.05)
up egula ion o impo an i ulence genes, such as
in A and hilA, especially in he p esence o bu y ic acid. The e o e, SCFAs a e p omising subs ances
o he inhibi ion o he g ow h o oodbo ne pa hogens. Howe e , i is impo an o a oid he use o
subinhibi o y concen a ions ha could inc ease he i ulence o oodbo ne pa hogen Salmonella.
Keywo ds:
Salmonella; sho chain a y acids; an imic obial ac i i y; bio ilm; mo ili y; gene exp ession
1. In oduc ion
Sho chain a y acids (SCFAs) a e end me aboli es p oduced by mic obial e men a ion o
undiges ed ca bohyd a es and die a y ibe s. Bu y a e, ace a e, and p opiona e a e he main SCFAs,
bu o he s, such as lac a e and ale a e, a e also p oduced by mic obio a [
1
–
3
]. SCFAs ha e impo an
oles in human gu homeos asis by exe ing se e al e ec s on he hos and i s own mic obio a. SCFAs a e
used as a sou ce o ene gy by in es inal epi helial cells. They also modula e he abso p ion o elec oly es
and inc ease he p oduc ion o an i-in lamma o y cy okines. SCFAs also educe he p oduc ion o
molecules ha ac as p o-in lamma o y subs ances, such as ni ous oxide, in e leukins, and umo
nec osis ac o . SCFAs also ha e a p o ec i e e ec agains bac e ial pa hogens by main aining he
in eg i y o he epi helial ba ie [
4
,
5
]. In addi ion, i has been obse ed ha SCFAs induce he
p oduc ion o an imic obial pep ides by en e ocy es [
6
]. In he same way, i has been obse ed ha
mac ophages ha di e en ia e in he p esence o bu y a e show inc eased an imic obial ac i i y e en
in he absence o an inc eased in lamma o y cy okine esponse [6].
An ibio ics 2019,8, 265; doi:10.3390/an ibio ics8040265 www.mdpi.com/jou nal/an ibio ics
An ibio ics 2019,8, 265 2 o 13
In adequa e concen a ions, SCFAs also ha e di ec an imic obial ac i i y agains pa hogenic
bac e ia. SCFAs can educe mic obial g ow h by modi ying he in acellula pH and he me abolism.
A lowe pH, SCFAs a e commonly p esen in nonionized o ms ha can di use ac oss he bac e ial
memb ane in o he bac e ial cy oplasm. Once in he cy oplasm, SCFAs dissocia e, inc easing he anion
and p o on concen a ions and lowe ing he in acellula pH [7–9].
The p oduc ion o adequa e and balanced SCFAs by a heal hy gu mic obio a is an impo an
ac o ha p e en s in ec ion by common oodbo ne pa hogens [
10
]. I was ound ha Bac e iodes spp.
media es esis ance o Salmonella coloniza ion by p oducing p opiona e [
9
]. Di e se s udies ha e
obse ed o al SFCAs concen a ions anging be ween 60 and 85 mM wi h le els o ace a e be ween
40 and 50 mM, p opiona e a ound 15 mM, and bu y a e a ound 10 mM [
11
,
12
]. A dys egula ion
o SCFAs le els can acili a e he coloniza ion o in es ine by pa hogens. In his sense, dec eased
concen a ions o bu y a e cause up egula ion o i ulence genes in en e ohemo hagic Esche ichia coli
(EHEC), and di e en spa ial g adien s o SCFAs egula e he exp ession o i ulence and commensal
genes in Campylobac e jejuni [
13
,
14
]. P opiona e dec eases he exp ession o Salmonella genes loca ed in
Salmonella Pa hogenici y Island 1 (SPI-1) [
15
]. In addi ion, p e-incuba ion o Salmonella en e i idis wi h
p opiona e and bu y a e esul s in a educ ion in hos cell in asion [
16
]. I is no ewo hy ha SCFA
concen a ions simila o hose ound in he dis al ileum cause up egula ion o Salmonella i ulence
genes, while concen a ions simila o hose ound in he colon ha e he opposi e e ec [
17
]. The e o e,
he p o ec i e and an imic obial e ec s o SCFAs a e concen a ion dependen .
Unde s anding heinhibi o ye ec so SCFAs on en e ic pa hogens is no only impo an omagu
heal h poin -o - iew, bu his knowledge can also be impo an om a ood sa e y poin -o - iew. In he
las yea s, di e en esea che s ha e been e alua ing al e na i es o inhibi he g ow h o oodbo ne
pa hogens in he ood indus y as an imic obial pep ides [
18
,
19
]. In he same way, SCFAs can be added
o ood and eed as p ese a i es, a oiding he g ow h o bac e ial pa hogens. The SCFAs inges ed
h ough can also ha e posi i e e ec s in gu balance wi h inhibi o y e ec s in en e ic pa hogens. Fo his
pu pose, he aim o his s udy was o de e mine he minimum inhibi o y and biocidal concen a ions
o ou SCFAs (ace ic acid, bu y ic acid, p opionic acid, and ale ic acid) in Salmonella and o de e mine
he e ec o subinhibi o y concen a ions o hese SCFAs in bio ilm o ma ion, mo ili y, and gene
exp ession. As poul y p oduc s a e mainly esponsible o human salmonellosis [
20
], he au ho s
decided o use a o al o 12 Salmonella s ains isola ed om poul y. These Salmonella s ains also
belonged o se en di e en se o ypes and wo Salmonella subspecies in o de o obse e i hey ha e a
simila esponse o he p esence o SCFAs.
2. Resul s and Discussion
2.1. An imic obial Ac i i y o Sho Chain Fa y Acids
Un ilnow, he eha ebeen limi eds udies e alua ing heMIC andminimum biocidalconcen a ion
(MBC) alues o SCFAs in ood-bo ne pa hogens [
21
]. In his s udy, he MICs o he ou SCFAs we e
de e mined. Bu y ic acid, p opionic acid, and ale ic acid had he same MIC alue (3750
µ
g/mL) in all
Salmonella s ains es ed in his s udy. In addi ion, all s ains showed he same MBC (3750
µ
g/mL) wi h
all he SFCAs es ed. Howe e , in he case o ace ic acid, i e o he wel e s ains had a MIC alue o
1875
µ
g/mL, while he o he s ains had he same MIC as hose wi h he o he s SCFAs (3750
µ
g/mL).
The esul s obse ed wi h ace ic acid we e simila o hose obse ed in p e ious wo k whe e he MIC
50
was 1650
µ
g/mL and he MIC
90
was 3280
µ
g/mL in 88 mul id ug esis an Salmonella isola es [
22
].
In he same way, ano he s udy ha e alua ed he an imic obial ac i i y o ace ic acid in di e en
pa hogens, such as E. coli, S aphylococcus au eus, o Acine obac e baumannii, ound ha he MIC alues
we e be ween 0.16% and 0.31% [
23
]. Howe e , ano he s udy ha de e mined he an imic obial ac i i y
o ace ic acid in E. coli and Salmonella sp. s ains ound MIC alues o 1.5% and 1%, espec i ely [
24
].
Campylobac e coli s ains isola ed om pigs had MIC alues o 2048
µ
g/mL in mos o he s ains es ed
in he p esence o bu y ic acid and p opionic acid [
25
], lowe han he MIC alues obse ed in his s udy.
An ibio ics 2019,8, 265 3 o 13
Con e sely, in o he s wo ks wi h Salmonella Typhimu ium ATCC 14028 o Vib io ha eyi, he inhibi ion
alues and p opionic acid o bu y ic acid, espec i ely, we e be ween 500 and 100
µ
g/mL [
26
,
27
].
The an imic obial ac i i y o SCFAs is due o hei di usion ac oss he bac e ial memb ane in o he
bac e ial cy oplasm, modi ying he in acellula pH and he me abolism [7,8].
2.2. E ec o Sho Chain Fa y Acids in Salmonella en e ica Mo ili y
Once he MICs we e calcula ed, he e ec o SCFAs on Salmonella mo ili y was de e mined.
Fo his pu pose, he highes subinhibi o y concen a ions o he SCFAs we e used. The mo ili y
was signi ican ly educed in he p esence o SCFAs in compa ison o he con ol semisolid aga in all
s ains es ed in his s udy (Table 1). The e we e also signi ican di e ences (p<0.05) be ween he
di e en SCFAs included in he s udy. The highe educ ion in mo ili y was obse ed in he p esence
o ace ic acid, while ale ic acid caused a lowe educ ion in mo ili y in compa ison o he con ol aga .
In acco dance wi h his s udy, a p e ious s udy also obse ed ha o ganic acids, including ace ic acid,
dec eased he mo ili y o E. coli and Salmonella s ains isola ed om esh ui and ege ables [
24
].
I was obse ed ha Salmonella Typhimu ium ATCC 14,028 mo ili y dec eased as he subinhibi o y
concen a ions o p opionic acid inc eased [
27
]. Deepening in hese e ec s, i has been obse ed ha
he e men a ion p oduc s o Clos idium amosum and SCFAs educe he mo ili y in en e ohemo hagic
E. coli (EHEC), dis u b he lagella o a ion, and he lagella leng h is lowe in compa ison o con ol
condi ions [
28
]. Howe e , he dec ease in in acellula pH could be esponsible o he slow lagella
mo o o a ion and he educed mo ili y [29].
An ibio ics 2019,8, 265 4 o 13
Table 1.
In luence o he ou sho chain a y acids es ed in his s udy (ace ic acid, p opionic acid, bu y ic acid, and ale ic acid) in he mo ili y o Salmonella s ains a
37 ◦C exp essed in millime e s. Di e en le e s in he same ow ep esen s a is ically signi ican di e ences (p<0.05) be ween he di e en aga media.
Mo ili y (mm)
S ain Code Con ol
(n=3) Mean ±SD
Ace ic Acid (n=3)
Mean ±SD
Bu y ic Acid (n=3)
Mean ±SD
P opionic Acid
(n=3) Mean ±SD
Vale ic Acid (n=3)
Mean ±SD
S. en e ica subsp. a izonae AZ3 16.00 ±1.73 a2.33 ±1.53 c6.00 ±1.73 b,c 5.00 ±1.00 b,c 8.67 ±1.15 b
S. en e ica subsp. a izonae AZ26 17.00 ±2.00 a2.00 ±1.00 c5.33 ±1.53 b,c 5.67 ±2.10 b,c 8.00 ±2.00 b
S. B edeney BR1 15.33 ±1.15 a2.00 ±1.73 c4.67 ±1.15 c3.00 ±1.00 c8.33 ±1.53 b
S. In an is I18 15.33 ±2.31 a1.67 ±1.15 c3.67 ±1.15 c3.67 ±0.57 c8.67 ±1.15 b
S. In an is I4 15.33 ±1.53 a3.00 ±1.73 c5.67 ±1.15 b,c 2.67 ±1.53 c7.33 ±2.08 b
S. Isangi IG1 15.67 ±2.08 a1.67 ±0.58 c4.00 ±1.00 c4.67 ±1.15 c9.00 ±1.73 b
S. Mon e ideo M1 20.00 ±2.00 a1.33 ±0.58 c6.33 ±2.08 b5.33 ±0.58 b8.33 ±0.58 b
S. Newpo N6 18.33 ±1.53 a2.67 ±1.53 d7.00 ±1.73 b,c 4.00 ±2.00 c,d 10.00 ±2.00 b
S. Typhimu ium T1 20.00 ±1.00 a3.67 ±1.15 c4.67 ±1.53 c6.00 ±1.73 c10.00 ±1.00 b
S. Typhimu ium T3 17.77 ±2.52 a2.67 ±0.58 c4.33 ±1.53 b,c 4.00 ±2.65 b,c 8.00 ±1.00 b
S. Typhimu ium T7 19.33 ±2.08 a3.33 ±1.15 c5.33 ±1.53 c3.33 ±1.15 c10.67 ±1.53 b
S. Typhimu ium T23 19.67 ±2.10 a2.33 ±2.31 c6.00 ±1.00 b,c 3.67 ±2.10 c9.33 ±2.52 b
To al - 17.36 ±2.41 a2.43 ±1.36 d4.12 ±1.60 c4.24 ±1.69 c8.45 ±1.92 b
An ibio ics 2019,8, 265 5 o 13
2.3. E ec o Sho Chain Fa y Acids on Bio ilm Fo ma ion
The p esence o subinhibi o y concen a ions o SCFAs in luence bio ilm o ma ion in some o
he Salmonella s ains included in his s udy (Figu e 1). The e we e no signi ican di e ences in he
educ ion o bio ilm o ma ion be ween he di e en SCFAs es ed in eigh o he nine s ains. Howe e ,
in Salmonella Typhimu ium T23, he educ ion in bio ilm o ma ion caused by p opionic acid was
signi ican ly lowe (p<0.05) han he educ ion caused by ace ic acid and bu y ic acid. I is also wo h
no ing ha he s ains in which bio ilm o ma ion was no in luenced by he SFCAs we e Salmonella
s ains wi h a low abili y o p oduce a bio ilm a 37 ◦C.
An ibio ics 2020, 9, x 5 o 13
2.3. E ec o Sho Chain Fa y Acids on Bio ilm Fo ma ion
The p esence o subinhibi o y concen a ions o SCFAs in luence bio ilm o ma ion in some o
he Salmonella s ains included in his s udy (Figu e 1). The e we e no signi ican di e ences in he
educ ion o bio ilm o ma ion be ween he di e en SCFAs es ed in eigh o he nine s ains.
Howe e , in Salmonella Typhimu ium T23, he educ ion in bio ilm o ma ion caused by p opionic
acid was signi ican ly lowe (p < 0.05) han he educ ion caused by ace ic acid and bu y ic acid. I is
also wo h no ing ha he s ains in which bio ilm o ma ion was no in luenced by he SFCAs we e
Salmonella s ains wi h a low abili y o p oduce a bio ilm a 37 °C.
Figu e 1. In luence o he ou sho chain a y acids es ed in his s udy (ace ic acid, p opionic acid,
bu y ic acid, and ale ic acid) in he bio ilm o ma ion o Salmonella s ains a 37 °C exp essed as
op ical densi y. Resul s a e exp essed as he mean o h ee di e en expe imen s (n = 3) and e o ba s
ep esen he s anda d de ia ion. Di e en le e s in he same Salmonella s ain ep esen s a is ically
signi ican di e ences (p < 0.05) be ween he di e en g ow h media.
Consis en wi h he abo e-men ioned, p e ious s udies ha e also obse ed a educ ion in
bio ilm o ma ion by Salmonella s ains unde he p esence o subinhibi o y concen a ions o ace ic
acid [23,24,27]. The p esence o ace ic acid causes inhibi ion o ex acellula polysaccha ides
p oduc ion in oodbo ne pa hogens such as E. coli o S. Typhimu ium, al hough o a lesse ex en in
he la e [24]. Ace ic acid also showed an i-quo um sensing ac i i y in E. coli and S. Typhimu ium
[24]. Quo um sensing is a cell- o-cell communica ion mechanism ha has g ea impo ance in bio ilm
o ma ion, and inhibi ion o his mechanism can esul in lowe bio ilm o ma ion [30]. Flagella also
ha e impo an oles in he ini ial adhesion o bac e ial cells o a su ace in bio ilm o ma ion [31]. In
his sense, i has been obse ed ha p opionic acid causes changes in ype 1 imb iae, wi h a b i le
and b oken appea ance [27]. The au ho s hypo hesize ha hese e ec s o SCFAs, in combina ion
wi h he sho lagella ha can be syn he ized in he p esence o in acellula acid [29], could be
esponsible o he lowe bio ilm o ma ion obse ed. The esul s o his s udy should be conside ed
as p elimina y and only explo a o y in na u e and migh no ha e ex e nal alidi y. The e a e some
bio ilm o ma ion assays ha a e mo e labo -in ensi e and mo e accu a e han using 96-well
mic opla es in eco e ing he sessile cells using sonica ion o he beads/ o ex me hod [32,33].
The e o e, he p elimina y esul s o his s udy should be con i med in he u u e by using mo e
accu a e assays and including di e en su aces o g ea impo ance in he ood indus y, such as
s ainless s eel.
2.4. E ec o Sho Chain Fa y Acids on Salmonella en e ica Gene Exp ession
The esul s o his and p e ious s udies ha e demons a ed ha SCFAs a e able o inhibi he
g ow h o oodbo ne pa hogens such as Salmonella [9,22,24,27]. Howe e , p e ious s udies also
Figu e 1.
In luence o he ou sho chain a y acids es ed in his s udy (ace ic acid, p opionic acid,
bu y ic acid, and ale ic acid) in he bio ilm o ma ion o Salmonella s ains a 37
◦
C exp essed as
op ical densi y. Resul s a e exp essed as he mean o h ee di e en expe imen s (n=3) and e o ba s
ep esen he s anda d de ia ion. Di e en le e s in he same Salmonella s ain ep esen s a is ically
signi ican di e ences (p<0.05) be ween he di e en g ow h media.
Consis en wi h he abo e-men ioned, p e ious s udies ha e also obse ed a educ ion in
bio ilm o ma ion by Salmonella s ains unde he p esence o subinhibi o y concen a ions o ace ic
acid [
23
,
24
,
27
]. The p esence o ace ic acid causes inhibi ion o ex acellula polysaccha ides p oduc ion
in oodbo ne pa hogens such as E. coli o S. Typhimu ium, al hough o a lesse ex en in he la e [
24
].
Ace ic acid also showed an i-quo um sensing ac i i y in E. coli and S. Typhimu ium [
24
]. Quo um
sensing is a cell- o-cell communica ion mechanism ha has g ea impo ance in bio ilm o ma ion,
and inhibi ion o his mechanism can esul in lowe bio ilm o ma ion [
30
]. Flagella also ha e
impo an oles in he ini ial adhesion o bac e ial cells o a su ace in bio ilm o ma ion [
31
]. In his
sense, i has been obse ed ha p opionic acid causes changes in ype 1 imb iae, wi h a b i le and
b oken appea ance [
27
]. The au ho s hypo hesize ha hese e ec s o SCFAs, in combina ion wi h he
sho lagella ha can be syn he ized in he p esence o in acellula acid [
29
], could be esponsible o
he lowe bio ilm o ma ion obse ed. The esul s o his s udy should be conside ed as p elimina y
and only explo a o y in na u e and migh no ha e ex e nal alidi y. The e a e some bio ilm o ma ion
assays ha a e mo e labo -in ensi e and mo e accu a e han using 96-well mic opla es in eco e ing
he sessile cells using sonica ion o he beads/ o ex me hod [
32
,
33
]. The e o e, he p elimina y esul s
o his s udy should be con i med in he u u e by using mo e accu a e assays and including di e en
su aces o g ea impo ance in he ood indus y, such as s ainless s eel.
2.4. E ec o Sho Chain Fa y Acids on Salmonella en e ica Gene Exp ession
The esul s o his and p e ious s udies ha e demons a ed ha SCFAs a e able o inhibi he
g ow h o oodbo ne pa hogens such as Salmonella [
9
,
22
,
24
,
27
]. Howe e , p e ious s udies also
An ibio ics 2019,8, 265 6 o 13
obse ed ha subinhibi o y concen a ions o SCFAs can induce he exp ession o i ulence genes in
E. coli [
14
], Salmonella [
17
], and C. jejuni [
13
]. In his s udy, he exp ession o 14 genes ela ed o i ulence,
s ess esponse, and ca bon s o age was de e mined in wo Salmonella s ains (S. Typhimu ium T7 and
S. In an is I4).
The exp ession p o iles o he wo s ains we e di e en . Salmonella Typhimu ium T7 showed
o e exp ession o all genes es ed in his s udy in p esence o SCFAs (Figu e 2), especially ace ic acid
and bu y ic acid, in compa ison o con ol g ow h media. Bu y ic acid caused signi ican up egula ion
o all genes e alua ed in his s udy in compa ison o p opionic acid and ale ic acid. Fo gene in A,
he up egula ion in he p esence o bu y ic acid was also signi ican ly highe han in he p esence
o he o he h ee SCFAs. Only he ansc ip ion o poS was no in luenced by SCFAs. Salmonella
In an is I4 (Figu e 3) showed di e en exp ession p o iles acco ding o he gene and he SFCAs. I is
ema kable ha nine genes showed no signi ican di e ences in hei egula ion be ween he di e en
SCFAs and in compa ison o he con ol sample. While gene liC was signi ican ly up egula ed in he
p esence o ale ic acid, gene in A was signi ican ly down egula ed in compa ison o he o he SCFAs.
The exp ession o hilA and spiA was also signi ican ly up egula ed in he p esence o ace ic acid and
bu y ic acid.
An ibio ics 2020, 9, x 6 o 13
obse ed ha subinhibi o y concen a ions o SCFAs can induce he exp ession o i ulence genes in
E. coli [14], Salmonella [17], and C. jejuni [13]. In his s udy, he exp ession o 14 genes ela ed o
i ulence, s ess esponse, and ca bon s o age was de e mined in wo Salmonella s ains (S.
Typhimu ium T7 and S. In an is I4).
The exp ession p o iles o he wo s ains we e di e en . Salmonella Typhimu ium T7 showed
o e exp ession o all genes es ed in his s udy in p esence o SCFAs (Figu e 2), especially ace ic acid
and bu y ic acid, in compa ison o con ol g ow h media. Bu y ic acid caused signi ican
up egula ion o all genes e alua ed in his s udy in compa ison o p opionic acid and ale ic acid.
Fo gene in A, he up egula ion in he p esence o bu y ic acid was also signi ican ly highe han in
he p esence o he o he h ee SCFAs. Only he ansc ip ion o poS was no in luenced by SCFAs.
Salmonella In an is I4 (Figu e 3) showed di e en exp ession p o iles acco ding o he gene and he
SFCAs. I is ema kable ha nine genes showed no signi ican di e ences in hei egula ion be ween
he di e en SCFAs and in compa ison o he con ol sample. While gene liC was signi ican ly
up egula ed in he p esence o ale ic acid, gene in A was signi ican ly down egula ed in
compa ison o he o he SCFAs. The exp ession o hilA and spiA was also signi ican ly up egula ed
in he p esence o ace ic acid and bu y ic acid.
poS cs A liC poE ljB cpx luxS
0
2
4
6
8
Salmonella Typhimu ium T7
Ta ge genes
Ace ic acid
Bu y ic acid
P opionic acid
Vale ic acid
a
a
b
b
a
a,b
b,c
c
a,b
a
b,c
c
a
a,b b,c
c
a
a,b
b
b
a
a,b b,c
c
aaa
a
Figu e 2. Fold change no malized o con ol gene 16s RNA in he ansc ip ion o genes ela ed o
i ulence and s ess in he p esence o sho chain a y acids in compa ison o con ol TSB g ow h
media in Salmonella Typhimu ium T7. Only a ge genes wi h di e en le e s be ween he di e en
g ow h media p esen s a is ically signi ican di e ences a e analysis o a iance (one-way
ANOVA) and he Tukey’s hones ly signi ican di e ence es (p < 0.05).
P e ious s udies ha e shown ha ace a e can be used by Salmonella as a signal o in asion gene
exp ession by up egula ion o genes such as hilA and si A [17]. On he o he side, i has been obse ed
ha p opiona e down egula es in asion genes loca ed in SPI-1 [15]. The esul s o his s udy showed
Figu e 2.
Fold change no malized o con ol gene 16s RNA in he ansc ip ion o genes ela ed o
i ulence and s ess in he p esence o sho chain a y acids in compa ison o con ol TSB g ow h
media in Salmonella Typhimu ium T7. Only a ge genes wi h di e en le e s be ween he di e en
g ow h media p esen s a is ically signi ican di e ences a e analysis o a iance (one-way ANOVA)
and he Tukey’s hones ly signi ican di e ence es (p<0.05).
An ibio ics 2019,8, 265 7 o 13
P e ious s udies ha e shown ha ace a e can be used by Salmonella as a signal o in asion
gene exp ession by up egula ion o genes such as hilA and si A [
17
]. On he o he side, i has been
obse ed ha p opiona e down egula es in asion genes loca ed in SPI-1 [
15
]. The esul s o his s udy
showed ha up egula ion o hilA o spiA genes caused by p opiona e was lowe in he p esence o
p opionic acid han in he p esence o o he SCFAs, such as ace ic acid o bu y ic acid. In his ega d,
i is wo h men ioning ha mos o he genes es ed in his s udy we e signi ican ly up egula ed in
S. Typhimu ium T7 in he p esence o ace ic acid and especially bu y ic acid (Figu e 2). In conco dance
wi h hese esul s, i was obse ed ha en e ohemo hagic E. coli inc eased he exp ession o i ulence
genes in he p esence o subinhibi o y concen a ions o SFCAs and especially in he p esence o bu y ic
acid [
14
]. Consequen ly, subinhibi o y concen a ions o SCFAs can p oduce an o e exp ession o
i ulence genes in common en e ic pa hogens, such as Salmonella and E. coli, esul ing in inc eased
i ulence o hese pa hogens.
An ibio ics 2020, 9, x 7 o 13
ha up egula ion o hilA o spiA genes caused by p opiona e was lowe in he p esence o p opionic
acid han in he p esence o o he SCFAs, such as ace ic acid o bu y ic acid. In his ega d, i is wo h
men ioning ha mos o he genes es ed in his s udy we e signi ican ly up egula ed in S.
Typhimu ium T7 in he p esence o ace ic acid and especially bu y ic acid (Figu e 2). In conco dance
wi h hese esul s, i was obse ed ha en e ohemo hagic E. coli inc eased he exp ession o
i ulence genes in he p esence o subinhibi o y concen a ions o SFCAs and especially in he
p esence o bu y ic acid [14]. Consequen ly, subinhibi o y concen a ions o SCFAs can p oduce an
o e exp ession o i ulence genes in common en e ic pa hogens, such as Salmonella and E. coli,
esul ing in inc eased i ulence o hese pa hogens.
poS cs A liC poE ljB cpx luxS
-4
-2
0
2
4
6
8
Salmonella In an is I4
Ta ge genes
Fold change in ansc ip ion
Ace ic acid
Bu y ic acid
P opionic acid
Vale ic acid
a
bb
b
a
a
a
a
aa
aaaaa
a
aaaaaa
a
a
aa
aa
Figu e 3. Fold change no malized o con ol gene 16s RNA in he ansc ip ion o genes ela ed o
i ulence and s ess in p esence o sho chain a y acids in compa ison o con ol TSB g ow h media
in Salmonella In an is I4. Only a ge genes wi h di e en le e s be ween he di e en g ow h media
p esen s a is ically signi ican di e ences a e analysis o a iance (one-way ANOVA) and he
Tukey’s hones ly signi ican di e ence es (p < 0.05).
In p e ious wo k, o ganic acids, such as ace ic acid, showed an i-quo um sensing ac i i y in
Salmonella and E. coli [24]. Quo um sensing is an impo an mechanism ha egula es he exp ession
o i ulence de e minan s o e ec i ely colonize he hos [34]. In his s udy, he exp ession o luxS,
which codi ies he Au oinduce -2 (AI-2), was signi ican ly up egula ed in S. Typhimu ium T7 in he
p esence o SCFAs. The au ho s conside ha SCFAs can inhibi he quo um sensing mechanism by
blocking AI-2 ins ead o down egula ing he genes implica ed in he p oduc ion o he signal
molecule. Finally, i is necessa y o e-emphasize he di e en ansc ip ional p o iles obse ed in S.
Typhimu ium T7 and S. In an is I4. This is due o he s ain a iabili y in he beha io o oodbo ne
pa hogens [35]. I is impo an o conside ha S. Typhimu ium is one o he mos pa hogenic
se o ypes o S. en e ica [36]. As a consequence, s ains o his se o ype could ha e an enhanced
i ulence esponse in he p esence o subinhibi o y concen a ions o SCFAs.
Figu e 3.
Fold change no malized o con ol gene 16s RNA in he ansc ip ion o genes ela ed o
i ulence and s ess in p esence o sho chain a y acids in compa ison o con ol TSB g ow h media
in Salmonella In an is I4. Only a ge genes wi h di e en le e s be ween he di e en g ow h media
p esen s a is ically signi ican di e ences a e analysis o a iance (one-way ANOVA) and he Tukey’s
hones ly signi ican di e ence es (p<0.05).
In p e ious wo k, o ganic acids, such as ace ic acid, showed an i-quo um sensing ac i i y in
Salmonella and E. coli [
24
]. Quo um sensing is an impo an mechanism ha egula es he exp ession
o i ulence de e minan s o e ec i ely colonize he hos [
34
]. In his s udy, he exp ession o luxS,
which codi ies he Au oinduce -2 (AI-2), was signi ican ly up egula ed in S. Typhimu ium T7 in he
p esence o SCFAs. The au ho s conside ha SCFAs can inhibi he quo um sensing mechanism
by blocking AI-2 ins ead o down egula ing he genes implica ed in he p oduc ion o he signal
molecule. Finally, i is necessa y o e-emphasize he di e en ansc ip ional p o iles obse ed in
An ibio ics 2019,8, 265 8 o 13
S. Typhimu ium T7
and
S. In an is I4
. This is due o he s ain a iabili y in he beha io o oodbo ne
pa hogens [
35
]. I is impo an o conside ha S. Typhimu ium is one o he mos pa hogenic se o ypes
o
S. en e ica [36]
. As a consequence, s ains o his se o ype could ha e an enhanced i ulence esponse
in he p esence o subinhibi o y concen a ions o SCFAs.
3. Ma e ials and Me hods
3.1. Salmonella en e ica S ains and Sho Chain Fa y Acids
A o al o wel e Salmonella s ains belonging o se en di e en se o ypes and wo Salmonella
subspecies we e included in his s udy (Table 2). All he s ains we e p e iously isola ed om poul y
houses and chicken mea in ou labo a o y acco ding o ISO 6579:2003 [
37
]. The s ains we e kep a
−
20
◦
C in yp ic soy b o h (TSB, Oxoid, UK) supplemen ed wi h 20% glyce ol un il use. Salmonella
s ains we e p e iously g own in nu ien aga (Applichem Pan eac, Ba celona, Spain) and incuba ed
o 24 h a 37
◦
C be o e use. The ollowing ou di e en SCFAs we e es ed in his s udy: ace ic acid
(
≥
99%, Sigma Ald ich, Schnelldo , Ge many), bu y ic acid (
≥
99%, Al a Aesa , The moFishe Scien i ic,
Massachuse s, Wal ham, MA, USA), p opionic acid (
≥
99%, Sigma Ald ich), and ale ic acid (
≥
99%,
Sigma Ald ich).
Table 2. Lis o Salmonella s ains included in his s udy and i s sou ce.
S ain Code Sou ce
S. en e ica subsp. a izonae AZ3 Poul y a m
S. en e ica subsp. a izonae AZ26 Poul y a m
S. B edeney BR1 Poul y a m
S. In an is I18 Poul y a m
S. In an is I4 Poul y a m
S. Isangi IG1 Poul y a m
S. Mon e ideo M1 Poul y a m
S. Newpo N6 Poul y a m
S. Typhimu ium T1 Chicken mea
S. Typhimu ium T3 Chicken mea
S. Typhimu ium T7 Poul y a m
S. Typhimu ium T23 Poul y a m
3.2. Minimum Inhibi o y Concen a ion and Minimum Biocidal Concen a ion o Sho Chain Fa y Acids
Minimum inhibi o y concen a ions (MICs) o SCFAs we e de e mined acco ding o he
mic odilu ion b o h me hod desc ibed by he Clinical and Labo a o y S anda ds Ins i u e (CLSI)
guidelines. An ini ial concen a ion o SCFAs a 20% in Muelle –Hin on b o h was used o pe o m
se ial dilu ions o he es ed SCFAs. Salmonella s ains we e ini ially g own in nu ien aga o 24 h a
37
◦
C, and isola ed colonies we e used o p epa e a saline suspension wi h a u bidi y equi alen o a
0.5 McFa land s anda d. Two se ial dilu ions o he ini ial inoculum in Muelle –Hin on b o h we e
pe o med o ob ain a inal concen a ion o 1
×
10
6
CFU/mL. In a 96-well mic o i e pla e, 100
µ
L o
each dilu ion was mixed wi h 100
µ
L o he inal inoculum, and mic opla es we e incuba ed o 24 h
a 37
◦
C. The MIC was de ined as he lowes concen a ion in which no isual bac e ial g ow h was
obse ed. The liquid o wells wi h no isual bac e ial g ow h obse ed was ans e ed o nu ien
aga pla es o de e mine he minimum bac e icidal concen a ion (MBC). Pla es we e incuba ed o
24 h a 37 ◦C.
3.3. Mo ili y Assays
Mo ili y assays we e ca ied ou in nu ien semisolid aga pla es composed o 8 g/L o nu ien
b o h (AppliChem, Pan eac, Ba celona, Spain), 4g/L o aga (Lio ilchem, Ab uzzi TE, I aly),
and 0.05 g/L
o 2,3,5- iphenyl e azolium chlo ide (Sigma Ald ich). Fi e di e en ypes o aga we e used: a con ol
An ibio ics 2019,8, 265 9 o 13
aga and ou semisolid aga s, each supplemen ed wi h one SCFA a he highes subinhibi o y
concen a ion obse ed in his s udy. A saline suspension wi h a u bidi y equi alen o 0.5 McFa land
was p epa ed o each s ain om he isola ed colonies o nu ien aga pla es incuba ed o 24 h a
37
◦
C. An inocula ing loop was imme sed in he Salmonella saline suspension, and hen he s ains
we e inocula ed in he semisolid aga pla es by s abbing. Mo ili y aga pla es we e incuba ed a 37
◦
C
o 48 h. A e incuba ion, he a io be ween he s abbing poin and he end o he g ow h ci cle was
measu ed. The expe imen s we e ca ied ou in iplica e.
3.4. Bio ilm Fo ma ion on Polys y ene
The e ec o SCFAs on he abili y o p oduce a bio ilm in polys y ene o Salmonella s ains included
in his s udy was e alua ed acco ding o he me hod desc ibed by S epano i´c e al. [
38
]. Fi e di e en
g ow h media we e used: con ol TSB media and ou g ow h media, each supplemen ed wi h one o he
SCFAs es ed in his s udy o a inal concen a ion o he highes subinhibi o y concen a ion obse ed
he MICs e alua ion. Polys y ene, 96-well mic opla es we e illed wi h 200
µ
L o he co esponding
g ow h media and hen inocula ed wi h 20
µ
L o saline solu ion wi h a Salmonella concen a ion o
10
4
CFU/mL. Mic opla es we e incuba ed o 24 h a 37
◦
C and hen washed h ee imes wi h 250
µ
L o
dis illed wa e . Salmonella cells ha we e adhe ed o he su ace o he wells we e ixed wi h 250
µ
L o
me hanol o 15 min. Then, wells we e emp ied, ai d ied, and illed wi h 250
µ
L o 0.1% c ys al iole
solu ion (Pan eac, Ba celona, Spain) o 5 min. Excess c ys al iole was emo ed unde ap wa e ,
and he c ys al iole ha was adhe ed o he wells was esolubilized wi h 250
µ
L o 33% ace ic acid
solu ion. The abso bance o he mic opla es was ead a 630 nm using a pla e eade (das, Palomba a
Sabina, I aly). The expe imen s we e ca ied ou in iplica e.
3.5. RNA Isola ion and RT-qPCR
The ansc ip ion o 14 Salmonella genes (Table 3) in he p esence o subinhibi o y concen a ions
o SCFAs was e alua ed in wo Salmonella s ains (S. Typhimu ium T7 and S. In an is I4). A o al o
i e g ow h media we e used: TSB media ha was used as con ol media and ou g ow h media, each
supplemen ed wi h he highes subinhibi o y concen a ions o one o he SFCAs es ed in his s udy.
Fi een-millili e plas ic ubes we e illed wi h 10 mL o he co esponding g ow h media and 100
µ
L o
a saline solu ion wi h a concen a ion o 10
4
CFU/mL o he co esponding Salmonella s ains. The ubes
we e incuba ed o 12 h a 37
◦
C and hen cen i uged a 2000
×
g o 10 min. The supe na an was
disca ded, and he pelle was esuspended in 1 mL o NZYol (nzy ech, Lisboa, Po ugal), and he RNA
was isola ed acco ding o he manu ac u e ’s ecommenda ions. Immedia ely, RNA was quan i ied
using a luo ome e (Qubi , In i ogen, Ca lsbad, CA, USA, The mo ishe Scien i ic) and e e sed
ansc ibed using he NZY Fi s -S and cDNA Syn hesis ki (nzy ech) ollowing he manu ac u e ’s
p o ocol. The cDNA was s o ed a −20 ◦C un il use.
The 10
µ
L RT-qPCR eac ions, composed o 5
µ
L o NZYSpeedy qPCR g een mas e mix 2
×
ROX
(nzy ech), 0.4
µ
L o each p ime , 1
µ
L o he sample, and 3.2
µ
L o RNase- ee wa e , we e ca ied ou
in a Quan S udio 12 k Flex eal- ime PCR sys em (Applied Biosys ems, The moFishe Scien i ic, Fos e ,
CA, USA). The condi ions we e as ollows: ini ial dena u a ion a 95
◦
C o 20 s, ollowed by 40 cycles
o dena u a ion a 95
◦
C o 1 s, annealing a 60
◦
C o 20 s, and a inal mel ing cu e p og am o 15 s a
95
◦
C, 60 s a 60
◦
C, and ollowed by a dissocia ion s ep o 15 s a 95
◦
C. The exp ession o a ge genes
in compa ison o he con ol gene 16 s RNA was e alua ed using he 2
∆∆C
me hod, whe e
∆∆
C
=
(C a ge genes −C 16s RNA) ea men −(C a ge genes −C 16s RNA)con ol.
