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Genus-Wide Assessment of Antibiotic Resistance inLactobacillus spp.
Ilenia Campedelli,a Harsh Mathur,b,c Elisa Salvetti,b,d Siobhán Clarke,c Mary C. Rea,b,c Sandra Torriani,a R. Paul Ross,b,d
Colin Hill,b,d Paul W. O’Tooleb,d
aDepartment of Biotechnology, University of Verona, Verona, ItalybAPC Microbiome Ireland, University College Cork, Cork, IrelandcTeagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Cork, IrelanddSchool of Microbiology, University College Cork, Cork, Ireland
ABSTRACT Lactobacillus species are widely used as probiotics and starter culturesfor a variety of foods, supported by a long history of safe usage. Although morethan 35 species meet the European Food Safety Authority (EFSA) criteria for quali-fied presumption of safety status, the safety of Lactobacillus species and their car-riage of antibiotic resistance (AR) genes is under continuing ad hoc review. Tocomprehensively update the identification of AR in the genus Lactobacillus, we de-termined the antibiotic susceptibility patterns of 182 Lactobacillus type strains andcompared these phenotypes to their genotypes based on genome-wide annotationsof AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were themost common phenotypes. A combination of homology-based screening and man-ual annotation identified genes encoding resistance to aminoglycosides (20 se-quences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol(42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglyco-sides and clindamycin, respectively, were found in Lactobacillus spp. Acquireddeterminants predicted to code for tetracycline and erythromycin resistancewere detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacil-lus amylotrophicus, flanked in the genome by mobile genetic elements with po-tential for horizontal transfer.
IMPORTANCE Lactobacillus species are generally considered to be nonpathogenicand are used in a wide variety of foods and products for humans and animals. How-ever, many of the species examined in this study have antibiotic resistance levelswhich exceed those recommended by the EFSA, suggesting that these cutoff valuesshould be reexamined in light of the genetic basis for resistance discussed here. Ourdata provide evidence for rationally revising the regulatory guidelines for safety as-sessment of lactobacilli entering the food chain as starter cultures, food preserva-tives, or probiotics and will facilitate comprehensive genotype-based assessment ofstrains for safety screening.
KEYWORDS Lactobacillus, antimicrobial resistance, genomics
Antibiotics represent one of the most powerful therapeutic options in human andveterinary medicine for the treatment of infectious diseases caused by bacterial
agents (1, 2). However, overprescribing and misuse of antibiotics in medicine, animalfeed, aquaculture, and agriculture have led to the emergence and spread of antibiotic-resistant bacteria, which constitutes a serious problem for the health of both humansand animals (3). Today, the ever-increasing prevalence of antibiotic resistance (AR) ininfectious microbes is a global public health emergency (4, 5).
Lactic acid bacteria (LAB) have been extensively used as probiotics and starter
Citation Campedelli I, Mathur H, Salvetti E,Clarke S, Rea MC, Torriani S, Ross RP, Hill C,O’Toole PW. 2019. Genus-wide assessment ofantibiotic resistance in Lactobacillus spp. ApplEnviron Microbiol 85:e01738-18. https://doi.org/10.1128/AEM.01738-18.
Editor Johanna Björkroth, University of Helsinki
Copyright © 2018 American Society forMicrobiology. All Rights Reserved.
Address correspondence to Paul W. O’Toole,[email protected].
I.C. and H.M. contributed equally to this article.
Received 17 July 2018Accepted 10 October 2018
Accepted manuscript posted online 26October 2018Published
FOOD MICROBIOLOGY
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cultures due to their long history of safe use, and many species have qualifiedpresumption of safety (QPS) status (6, 7) including members of the genus Lactobacillus,Lactococcus, Leuconostoc, and Pediococcus (8). In Europe, the absence of acquired ortransferable resistance factors must be established for a candidate probiotic or starterculture in order for them to be declared safe for human and animal consumption andto achieve QPS status from the European Food Safety Authority (EFSA) (9).
The significant economic and scientific impact of the members of the genusLactobacillus includes many strains commonly used as probiotics and others marketedas probiotic cosmetics, drug supplements, or even medical devices (10). Furthermore,Lactobacillus species are probably those most widely used as starter cultures forindustrial and agricultural applications (e.g., fermented foods and silage cultures), dueto their long history of safe and technological use (11–13). Despite their safety status,many lactobacilli have been reported as being antibiotic resistant (12, 14, 15), where avancomycin-resistant phenotype is perhaps the best-characterized intrinsic resistancemechanism (16). Most Lactobacillus species are intrinsically resistant to aminoglycosides(gentamicin, kanamycin, streptomycin, and neomycin), ciprofloxacin, and trimethoprim,and they are susceptible to penicillin and �-lactams, chloramphenicol, tetracycline,erythromycin, linezolid, and quinupristin-dalfopristin (14). However, acquired resistanceto tetracycline, erythromycin, clindamycin, and chloramphenicol has been detected inlactobacilli isolated from fermented foods (17–20). Given the widespread use of somespecies of this genus in fermented food production and functional foods/probiotics,lactobacilli could act as donors or reservoirs for AR genes, with the potential risk oftransferring these genes to pathogenic bacteria in food matrices as well as in thegastrointestinal tract (GIT) (21). Thus, even though more than 35 species meet thecriteria of QPS status proposed by the EFSA (8), the safety of Lactobacillus species andtheir possible involvement in the spread of AR determinants along the food chainwarrant investigation. QPS is an attribute of a species rather than a strain, and it isnoteworthy that genome content often varies widely within species, including inlactobacilli (22–24).
The recent determination of the genome sequences of almost all Lactobacillus typestrains (25, 26) allows the safety assessment of the genus Lactobacillus by surveillanceof the presence of AR genes, as well as their potential for transfer to other microor-ganisms. Within the limits of database quality and annotation, whole-genome sequenc-ing (WGS) potentially allows the identification of all possible genetic determinants ofantimicrobial resistance in a microbial genome (27). WGS could revolutionize foodsafety assessments, resulting in a paradigm shift from phenotype-based to genotype-based assays of AR (28).
The aim of the current study was to determine the antibiotic susceptibility patternsof 197 type strains representing the whole Lactobacillus genus and to compare thesephenotypes to their genotypes based on genome-wide annotation of AR genes. Parallelanalysis of phenotype and genotype would provide the definitive knowledge base forthe distribution, origins, and mechanisms of AR in the genus and would facilitaterational discussions about regulating strains or species harboring intrinsic, acquired, ortransmissible AR mechanisms.
RESULTSDetermination of MICs. The MIC values of 16 antibiotics belonging to the most
important antimicrobial classes used in human and veterinary medicine were testedusing broth microdilution VetMIC plates for 197 Lactobacillus strains representing thewhole Lactobacillus genus. The MIC profiles were obtained for 182 strains (because 15strains were not capable of growth in the VetMIC medium) and were analyzed in thecontext of the Lactobacillus phylogroups described in references 25 and 29 and shownin Table 1.
A wide range of MIC values was exhibited by all phylogroups for most antibioticsanalyzed, except for linezolid, quinupristin-dalfopristin, and chloramphenicol (see TableS1 in the supplemental material). In particular, a unimodal MIC distribution was
Campedelli et al. Applied and Environmental Microbiology
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TAB
LE1
Feat
ures
ofth
e19
7ty
pe
stra
ins
ofth
ege
nus
Lact
obac
illus
anal
yzed
,inc
ludi
ngge
nom
eac
cess
ion
num
ber
and
grow
thco
nditi
onap
plie
dfo
rth
ede
term
inat
ion
ofM
ICva
lues
Spec
ies
Stra
inM
etab
olis
mp
hen
otyp
eaPh
ylog
roup
bG
enB
ank
acce
ssio
nn
o.c
Sour
ceN
ich
eca
teg
ory
Gro
wth
con
dit
ion
sTe
mp
(°C
)M
ediu
m
Lact
obac
illus
acet
otol
eran
sD
SM20
749T
FHE
L.de
lbru
ecki
iA
YZC
0000
0000
Ferm
ente
dvi
nega
rb
roth
Food
Ana
erob
ic30
MRS
�0.
05%
cyst
eine
Lact
obac
illus
acid
ifarin
aeD
SM19
394T
OH
EL.
brev
isA
ZDV0
0000
000
Art
isan
alw
heat
sour
doug
hFo
odM
icro
aero
phi
lic30
MRS
�0.
05%
cyst
eine
(pH
5.2)
Lact
obac
illus
acid
ipis
cis
DSM
1583
6TFH
EL.
saliv
ariu
sA
ZFI0
0000
000
Ferm
ente
dfis
hFo
odM
icro
aero
phi
lic37
MRS
Lact
obac
illus
acid
ophi
lus
ATC
C43
56T
OH
OL.
delb
ruec
kii
AZC
S000
0000
0H
uman
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usag
ilis
DSM
2050
9TFH
EL.
saliv
ariu
sA
YYP0
0000
000
Mun
icip
alse
wag
eEn
viro
nmen
tM
icro
aero
phi
lic37
MRS
�0.
05%
cyst
eine
Lact
obac
illus
algi
dus
DSM
1563
8TFH
EL.
saliv
ariu
sA
ZDI0
0000
000
Vacu
um-p
acke
db
eef
Food
Ana
erob
ic20
MRS
(pH
5.7)
Lact
obac
illus
alim
enta
rius
DSM
2024
9TFH
EL.
alim
enta
rius
AZD
Q00
0000
00M
arin
ated
fish
pro
duct
Food
Aer
obic
30M
RSLa
ctob
acill
usam
ylol
ytic
usD
SM11
664T
OH
OL.
delb
ruec
kii
AZE
P000
0000
0A
cidi
fied
bee
rw
ort
Win
ep
rodu
ctPr
efer
ably
anae
rob
ic37
MRS
Lact
obac
illus
amyl
ophi
lus
DSM
2053
3TO
HO
L.de
lbru
ecki
iA
YYS0
0000
000
Swin
ew
aste
-cor
nfe
rmen
tatio
nA
nim
alA
erob
ic30
MRS
�1%
gluc
ose
Lact
obac
illus
amyl
otro
phic
usD
SM20
534T
OH
OL.
delb
ruec
kii
AZC
V000
0000
0Sw
ine
was
te-c
orn
ferm
enta
tion
Ani
mal
Aer
obic
30M
RSLa
ctob
acill
usam
ylov
orus
DSM
2053
1TO
HO
L.de
lbru
ecki
iA
ZCM
0000
0000
Cat
tle
was
te-c
orn
ferm
enta
tion
Ani
mal
Mic
roae
rop
hilic
-ana
erob
ic37
MRS
Lact
obac
illus
anim
alis
DSM
2060
2TO
HO
L.sa
livar
ius
AYY
W00
0000
00D
enta
lp
laqu
eof
bab
oon
Ani
mal
Aer
obic
37M
RSLa
ctob
acill
usan
tri
LMG
2211
1TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sA
ZDK0
0000
000
Gas
tric
bio
psy
spec
imen
s,hu
man
stom
ach
muc
osa
Ani
mal
Ana
erob
ic37
MRS
Lact
obac
illus
apin
orum
DSM
2625
7TO
HE
L.fr
uctiv
oran
sJX
CT0
0000
000
Hon
eyst
omac
hof
hone
ybee
Ani
mal
Ana
erob
ic37
MRS
�2%
fruc
tose
Lact
obac
illus
apis
LMG
2696
4TO
HO
L.de
lbru
ecki
iJX
LG00
0000
00St
omac
hsof
hone
ybee
sA
nim
alA
naer
obic
37M
RSLa
ctob
acill
usap
odem
iD
SM16
634T
OH
EL.
saliv
ariu
sA
ZFT0
0000
000
Fece
sof
wild
Jap
anes
ew
ood
mou
seA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
aqua
ticus
DSM
2105
1TO
HO
L.sa
livar
ius
AYZ
D00
0000
00Su
rfac
eof
aeu
trop
hic
fres
hwat
erp
ond
Envi
ronm
ent
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
avia
rius
sub
sp.
araf
finos
usD
SM20
653T
OH
OL.
saliv
ariu
sA
YYZ0
0000
000
Inte
stin
eof
chic
ken
Ani
mal
Mic
roae
rop
hilic
37M
RS�
0.05
%cy
stei
ne-h
ydro
chlo
ride
Lact
obac
illus
avia
rius
sub
sp.
avia
rius
DSM
2065
5TO
HO
L.sa
livar
ius
AYZ
A00
0000
00C
hick
enfe
ces
Ani
mal
Mic
roae
rop
hilic
37M
RS�
0.05
%cy
stei
ne
Lact
obac
illus
back
iiD
SM18
080T
OH
OL.
cory
nifo
rmis
NA
Orc
hard
gras
ssi
lage
Plan
tPr
efer
ably
anae
rob
ic28
MRS
Lact
obac
illus
bife
rmen
tans
DSM
2000
3TFH
EL.
cory
nifo
rmis
AZD
A00
0000
00Bl
own
chee
seFo
odA
naer
obic
30M
RSLa
ctob
acill
usbo
mbi
DSM
2651
7TFH
EL.
alim
enta
rius
NA
Dig
estiv
etr
acts
ofb
umb
leb
eequ
eens
Ani
mal
Ana
erob
ic37
MRS
Lact
obac
illus
bom
bico
laD
SM28
793T
FHE
L.de
lbru
ecki
iN
ABu
mb
leb
eegu
tA
nim
alSt
rictl
yan
aero
bic
37M
RS�
0.05
%cy
stei
ne-h
ydro
chlo
ride
Lact
obac
illus
bran
tae
DSM
2392
7TFH
EL.
case
i-L.m
anih
otiv
oran
sA
YZQ
0000
0000
Fece
sof
Can
ada
goos
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
brev
isD
SM20
054T
OH
EL.
brev
isA
ZCP0
0000
000
Fece
sA
nim
alM
icro
aero
phi
lic30
MRS
Lact
obac
illus
buch
neri
DSM
2005
7TO
HE
L.bu
chne
riA
ZDM
0000
0000
Tom
ato
pul
pPl
ant
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usca
caon
umD
SM21
116T
FHE
L.sa
livar
ius
AYZ
E000
0000
0C
ocoa
bea
nhe
apfe
rmen
tatio
nPl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usca
mel
liae
DSM
2269
7TO
HO
L.ca
sei-L
.man
ihot
ivor
ans
AYZ
J000
0000
0Fe
rmen
ted
tea
leav
es(m
iang
)Pl
ant
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
lus
capi
llatu
sD
SM19
910T
FHE
L.sa
livar
ius
AZE
F000
0000
0Fe
rmen
ted
brin
eus
edfo
rst
inky
tofu
pro
duct
ion
Food
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
case
iD
SM20
011T
FHE
L.ca
sei-L
.man
ihot
ivor
ans
AZC
O00
0000
00C
hees
eFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
ceti
DSM
2240
8TFH
EL.
saliv
ariu
sJQ
BZ00
0000
00Lu
ngs
ofa
bea
ked
wha
leA
nim
alSt
rictl
yan
aero
bic
37M
RSLa
ctob
acill
usco
leho
min
isD
SM14
060T
FHE
L.re
uter
i-L.v
acci
nost
ercu
sA
ZEW
0000
0000
Hum
anva
gina
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usco
llino
ides
DSM
2051
5TO
HE
L.co
llino
ides
AYY
R000
0000
0Fe
rmen
ting
app
leju
ice
Food
Ana
erob
ic26
MRS
Lact
obac
illus
com
post
iD
SM18
527T
FHE
Oth
erA
ZGA
0000
0000
Com
pos
ting
mat
eria
lof
dist
illed
shoc
hure
sidu
eW
ine
pro
duct
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
conc
avus
DSM
1775
8TO
HO
Oth
erA
ZFX
0000
0000
Wal
lsof
adi
still
edsp
irit
ferm
entin
gce
llar
Envi
ronm
ent
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
cory
nifo
rmis
sub
sp.
cory
nifo
rmis
LMG
9196
TFH
EL.
cory
nifo
rmis
AZC
N00
0000
00Si
lage
Plan
tM
icro
aero
phi
lic30
MRS
Lact
obac
illus
cory
nifo
rmis
sub
sp.
torq
uens
DSM
2000
4TFH
EL.
cory
nifo
rmis
AZD
C00
0000
00A
irof
cow
shed
Envi
ronm
ent
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
cris
patu
sD
SM20
584T
OH
OL.
delb
ruec
kii
AZC
W00
0000
00Ey
eA
nim
alPr
efer
ably
anae
rob
ic37
MRS
Lact
obac
illus
crus
toru
mLM
G23
699T
OH
OL.
alim
enta
rius
JQC
K000
0000
0W
heat
sour
doug
hFo
odA
erob
ic30
MRS
Lact
obac
illus
curie
aeJC
M18
524T
OH
EL.
buch
neri
CP0
1890
6To
fub
rine
Food
Facu
ltat
ivel
yan
aero
bic
30M
RSLa
ctob
acill
uscu
rvat
usD
SM20
019T
FHE
L.sa
kei
AZD
L000
0000
0M
ilkFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
delb
ruec
kii
sub
sp.
bulg
aric
usD
SM20
081T
OH
OL.
delb
ruec
kii
JQA
V000
0000
0Bu
lgar
ian
yogu
rtFo
odM
icro
aero
phi
lic37
MRS
(Con
tinue
don
follo
win
gp
age)
Antibiotic Resistance in the Genus Lactobacillus Applied and Environmental Microbiology
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TAB
LE1
(Con
tinue
d)
Spec
ies
Stra
inM
etab
olis
mp
hen
otyp
eaPh
ylog
roup
bG
enB
ank
acce
ssio
nn
o.c
Sour
ceN
ich
eca
teg
ory
Gro
wth
con
dit
ion
sTe
mp
(°C
)M
ediu
m
Lact
obac
illus
delb
ruec
kii
sub
sp.
delb
ruec
kii
DSM
2007
4TO
HO
L.de
lbru
ecki
iA
ZCR0
0000
000
Sour
grai
nm
ash
Food
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
delb
ruec
kii
sub
sp.
indi
cus
DSM
1599
6TO
HO
L.de
lbru
ecki
iA
ZFL0
0000
000
Trad
ition
alda
iryfe
rmen
ted
pro
duct
(Dah
ity
pe)
Food
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
delb
ruec
kii
sub
sp.
jako
bsen
iiD
SM26
046T
OH
OL.
delb
ruec
kii
JQC
G00
0000
00D
olo
wor
t(a
lcoh
olic
ferm
ente
db
ever
age)
Win
ep
rodu
ctA
naer
obic
37M
RS
Lact
obac
illus
delb
ruec
kii
sub
sp.
lact
isD
SM20
072T
OH
OL.
delb
ruec
kii
AZD
E000
0000
0Em
men
tal
chee
seFo
odM
icro
aero
phi
lic37
MRS
Lact
obac
illus
dext
rinic
usD
SM20
335T
OH
EO
ther
AYY
K000
0000
0Si
lage
Plan
tA
erob
ic30
MRS
Lact
obac
illus
diol
ivor
ans
DSM
1442
1TO
HE
L.bu
chne
riA
ZEY0
0000
000
Mai
zesi
lage
Plan
tA
erob
ic30
MRS
Lact
obac
illus
equi
DSM
1583
3TO
HO
L.sa
livar
ius
AZF
H00
0000
00H
orse
fece
sA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
equi
curs
oris
DSM
1928
4TO
HO
L.de
lbru
ecki
iA
ZDU
0000
0000
Hea
lthy
thor
ough
bre
dra
ceho
rse
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
useq
uige
nero
siD
SM18
793T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZG
C00
0000
00Fe
ces
ofth
orou
ghb
red
hors
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
fabi
ferm
enta
nsD
SM21
115T
FHE
L.pl
anta
rum
AYG
X00
0000
00C
ocoa
bea
nhe
apfe
rmen
tatio
nPl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usfa
ecis
DSM
2395
6TO
HO
L.sa
livar
ius
NA
Ani
mal
fece
sA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
farc
imin
isLM
G91
89O
HO
L.al
imen
tariu
sA
ZDR0
0000
000
Saus
age
Food
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usfa
rrag
inis
DSM
1838
2TFH
EL.
buch
neri
AZF
Y000
0000
0C
omp
ostin
gm
ater
ial
ofdi
still
edsh
ochu
resi
due
Win
ep
rodu
ctM
icro
aero
phi
lic30
MRS
Lact
obac
illus
ferm
entu
mD
SM20
055T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
JQA
U00
0000
00H
uman
saliv
aA
nim
alA
erob
ic30
MRS
Lact
obac
illus
floric
ola
DSM
2303
7TO
HO
L.de
lbru
ecki
iA
YZL0
0000
000
Flow
erof
Calth
apa
lust
risPl
ant
Aer
obic
30M
RSLa
ctob
acill
usflo
rum
DSM
2268
9TO
HE
L.fr
uctiv
oran
sA
YZI0
0000
000
Peon
y(P
aeon
iasu
ffru
ticos
a)Pl
ant
Ana
erob
ic28
MRS
�1%
fruc
tose
Lact
obac
illus
form
osen
sis
NBR
C10
9509
TO
HO
L.al
imen
tariu
sN
AFe
rmen
ted
soyb
ean
Food
Ana
erob
ic37
MRS
(pH
6.2)
Lact
obac
illus
forn
ical
isJC
M12
512T
?O
ther
NA
Hum
anva
gina
Ani
mal
Aer
obic
37M
RSLa
ctob
acill
usfr
uctiv
oran
sD
SM20
203T
OH
EL.
fruc
tivor
ans
AZD
S000
0000
0N
AU
nkno
wn
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usfr
umen
tiD
SM13
145T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZE
R000
0000
0Ry
eb
ran
sour
doug
hFo
odA
naer
obic
40M
RS(p
H6.
2)La
ctob
acill
usfu
chue
nsis
DSM
1434
0TFH
EL.
sake
iA
ZEX
0000
0000
Vacu
um-p
acka
ged
bee
fFo
odA
erob
ic20
MRS
Lact
obac
illus
furf
uric
ola
DSM
2717
4TO
HO
L.al
imen
tariu
sN
ARi
ceb
ran
pas
tePl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usfu
tsai
iJC
M17
355T
OH
OL.
alim
enta
rius
AZD
O00
0000
00Fu
-tsa
i,a
trad
ition
alfe
rmen
ted
mus
tard
pro
duct
Food
Aer
obic
30M
RS
Lact
obac
illus
galli
naru
mD
SM10
532T
OH
OL.
delb
ruec
kii
AZE
L000
0000
0C
hick
encr
opA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
gass
eri
LMG
9203
TO
HO
L.de
lbru
ecki
iN
C_0
0853
0H
uman
Ani
mal
Pref
erab
lyan
aero
bic
30M
RSLa
ctob
acill
usga
stric
usD
SM16
045T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZF
N00
0000
00G
astr
icb
iop
sysp
ecim
ens,
hum
anst
omac
hm
ucos
aA
nim
alA
naer
obic
37M
RS
Lact
obac
illus
ghan
ensi
sD
SM18
630T
OH
OL.
saliv
ariu
sA
ZGB0
0000
000
Coc
oafe
rmen
tatio
nPl
ant
Ana
erob
ic30
MRS
Lact
obac
illus
gige
rioru
mD
SM23
908T
OH
OL.
delb
ruec
kii
AYZ
O00
0000
00C
hick
encr
opA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
gins
enos
idim
utan
sD
SM24
154T
OH
OL.
alim
enta
rius
CP0
1203
4Ki
mch
iFo
odA
naer
obic
30M
RSLa
ctob
acill
usgo
rilla
eD
SM28
356T
FHE
L.re
uter
i-L.v
acci
nost
ercu
sN
AG
orill
afe
ces
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usgr
amin
isD
SM20
719T
FHE
L.sa
kei
AYZ
B000
0000
0G
rass
sila
gePl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usha
mm
esii
DSM
1638
1TFH
EL.
brev
isA
ZFS0
0000
000
Whe
atso
urdo
ugh
Food
Mic
roae
rop
hilic
30M
RS�
1%m
alto
se�
0.5%
yeas
tex
trac
tLa
ctob
acill
usha
mst
eri
DSM
5661
TFH
EL.
delb
ruec
kii
AZG
I000
0000
0H
amst
erfe
ces
Ani
mal
Ana
erob
ic37
MRS
Lact
obac
illus
harb
inen
sis
DSM
1699
1TFH
EL.
pero
lens
AZF
W00
0000
00C
hine
setr
aditi
onal
ferm
ente
dve
geta
ble
Food
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
haya
kite
nsis
DSM
1893
3TO
HO
L.sa
livar
ius
AZG
D00
0000
00Fe
ces
ofth
orou
ghb
red
hors
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
heilo
ngjia
ngen
sis
LMG
2616
6TO
HO
L.al
imen
tariu
sC
P012
559
Chi
nese
pic
kle
Plan
tM
icro
aero
phi
lic28
MRS
Lact
obac
illus
hels
ingb
orge
nsis
DSM
2626
5TO
HO
L.de
lbru
ecki
iJX
JR00
0000
00H
oney
stom
ach
ofho
neyb
eeA
nim
alA
naer
obic
35M
RS�
fruc
tose
(20
g/lit
er)
Lact
obac
illus
helv
etic
usLM
G22
464
OH
OL.
delb
ruec
kii
JQC
J000
0000
0M
alt
whi
skey
ferm
enta
tion
Win
ep
rodu
ctA
naer
obic
37M
RSLa
ctob
acill
ushe
rbar
umD
SM10
0358
TO
HE
L.pl
anta
rum
LFEE
0000
0000
Ferm
ente
dw
hite
radi
shFo
odM
icro
aero
phi
lic25
MRS
Lact
obac
illus
hilg
ardi
iLM
G68
95T
OH
EL.
buch
neri
AZD
F000
0000
0W
ine
Win
ep
rodu
ctPr
efer
ably
anae
rob
ic37
MRS
Lact
obac
illus
hokk
aido
nens
isD
SM26
202T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
JQC
H00
0000
00Ti
mot
hygr
ass
sila
gePl
ant
Mic
roae
rop
hilic
25M
RSLa
ctob
acill
usho
min
isD
SM23
910T
OH
OL.
delb
ruec
kii
AYZ
P000
0000
0H
uman
inte
stin
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
hom
ohio
chii
DSM
2057
1TFH
EL.
fruc
tivor
ans
JQBN
0000
0000
Spoi
led
sake
Win
ep
rodu
ctM
icro
aero
phi
lic26
MRS
Lact
obac
illus
hord
eiD
SM19
519T
OH
OL.
saliv
ariu
sA
ZDX
0000
0000
Mal
ted
bar
ley
Plan
tA
naer
obic
37M
RSLa
ctob
acill
usin
ers
DSM
1333
5TO
HO
L.de
lbru
ecki
iA
ZET0
0000
000
Hum
anur
ine
Ani
mal
Ana
erob
ic37
MRS
(Con
tinue
don
follo
win
gp
age)
Campedelli et al. Applied and Environmental Microbiology
January 2019 Volume 85 Issue 1 e01738-18 aem.asm.org 4
on October 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
TAB
LE1
(Con
tinue
d)
Spec
ies
Stra
inM
etab
olis
mp
hen
otyp
eaPh
ylog
roup
bG
enB
ank
acce
ssio
nn
o.c
Sour
ceN
ich
eca
teg
ory
Gro
wth
con
dit
ion
sTe
mp
(°C
)M
ediu
m
Lact
obac
illus
ingl
uvie
iD
SM15
946T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZF
K000
0000
0Pi
geon
,cro
pA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
insi
cii
DSM
2980
1TO
HO
L.al
imen
tariu
sN
AFe
rmen
ted
raw
mea
tA
nim
alM
icro
aero
phi
lic30
MRS
Lact
obac
illus
inte
stin
alis
DSM
6629
TFH
EL.
delb
ruec
kii
AZG
N00
0000
00In
test
ine
ofra
tA
nim
alA
erob
ic37
MRS
Lact
obac
illus
iwat
ensi
sD
SM26
942T
OH
OL.
cory
nifo
rmis
NA
Orc
hard
gras
ssi
lage
Plan
tM
icro
aero
phi
lic30
MRS
Lact
obac
illus
jens
enii
DSM
2055
7TFH
EL.
delb
ruec
kii
AYY
U00
0000
00H
uman
vagi
nal
disc
harg
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
john
soni
iLM
G94
36T
OH
OL.
delb
ruec
kii
AZC
Y000
0000
0H
uman
blo
odA
nim
alPr
efer
ably
anae
rob
ic30
MRS
Lact
obac
illus
kalix
ensi
sD
SM16
043T
OH
OL.
delb
ruec
kii
AZF
M00
0000
00G
astr
icb
iop
sysp
ecim
ens,
hum
anst
omac
hm
ucos
aA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
kefir
anof
acie
nssu
bsp
.kefi
rano
faci
ens
LMG
1914
9TO
HO
L.de
lbru
ecki
iA
ZGG
0000
0000
Kefir
grai
nsPl
ant
Ana
erob
ic30
MRS
Lact
obac
illus
kefir
anof
acie
nssu
bsp
.kefi
rgra
num
DSM
1055
0TO
HO
L.de
lbru
ecki
iA
ZEM
0000
0000
Kefir
grai
nsPl
ant
Ana
erob
ic30
MRS
Lact
obac
illus
kefir
iD
SM20
587T
OH
EL.
buch
neri
AYY
V000
0000
0Ke
firgr
ains
Plan
tA
erob
ic30
MRS
Lact
obac
illus
kim
blad
iiD
SM26
263T
FHE
L.de
lbru
ecki
iJX
LH00
0000
00H
oney
stom
ach
ofho
neyb
eeA
nim
alA
naer
obic
30M
RSLa
ctob
acill
uski
mch
icus
JCM
1553
0TFH
EL.
colli
noid
esA
ZCX
0000
0000
Kim
chi
Food
Aer
obic
37M
RSLa
ctob
acill
uski
mch
iens
isD
SM24
716T
OH
OL.
alim
enta
rius
JQC
F000
0000
0Ki
mch
iFo
odM
icro
aero
phi
lic25
MRS
Lact
obac
illus
kiso
nens
isD
SM19
906T
OH
EL.
buch
neri
AZE
B000
0000
0Su
nki,
aJa
pan
ese
trad
ition
alp
ickl
eFo
odM
icro
aero
phi
lic30
MRS
�m
alto
se(1
0g/
liter
)�
L-ar
abin
ose
(10
g/lit
er)
Lact
obac
ilus
kita
sato
nis
DSM
1676
1TO
HO
L.de
lbru
ecki
iA
ZFU
0000
0000
Chi
cken
inte
stin
eA
nim
alM
icro
aero
phi
lic37
MRS
Lact
obac
illus
kore
ensi
sJC
M16
448T
OH
EL.
brev
isA
ZDP0
0000
000
Cab
bag
eki
mch
iFo
odA
erob
ic30
MRS
(pH
5.5)
Lact
obac
illus
kulla
berg
ensi
sD
SM26
262T
FHE
L.de
lbru
ecki
iJX
BY00
0000
00H
oney
stom
ach
ofho
neyb
eeA
nim
alSt
rictl
yan
aero
bic
30M
RSLa
ctob
acill
usku
nkee
iD
SM12
361T
OH
EL.
fruc
tivor
ans
AZC
K000
0000
0C
omm
erci
algr
ape
win
eW
ine
pro
duct
Aer
obic
30M
RS�
0.05
%cy
stei
ne(p
H5.
2)La
ctob
acill
uslin
dner
iD
SM20
690T
OH
EL.
fruc
tivor
ans
JQBT
0000
0000
Spoi
led
bee
rW
ine
pro
duct
Aer
obic
30M
RS�
0.05
%cy
stei
ne(p
H5.
2)La
ctob
acill
usm
alef
erm
enta
nsLM
G11
455T
OH
EL.
colli
noid
esA
ZGJ0
0000
000
Sour
bee
rW
ine
pro
duct
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usm
ali
ATC
C27
304T
OH
OL.
saliv
ariu
sJQ
AR0
0000
000
Win
em
ust
Win
ep
rodu
ctA
erob
ic30
MRS
Lact
obac
illus
man
ihot
ivor
ans
DSM
1334
3TO
HO
L.ca
sei-L
.man
ihot
ivor
ans
AZE
U00
0000
00C
assa
vaso
urst
arch
ferm
enta
tion
Plan
tA
erob
ic30
MRS
Lact
obac
illus
mel
lifer
DSM
2625
4TFH
EL.
alim
enta
rius
JXJQ
0000
0000
Hon
eyst
omac
hof
hone
ybee
Ani
mal
Ana
erob
ic35
MRS
�fr
ucto
se(2
0g/
liter
)La
ctob
acill
usm
ellis
DSM
2625
5TFH
EL.
alim
enta
rius
JXBZ
0000
0000
Hon
eyst
omac
hof
hone
ybee
Ani
mal
Stric
tly
anae
rob
ic30
MRS
�fr
ucto
se(2
0g/
liter
)La
ctob
acill
usm
elliv
entr
isD
SM26
256T
FHE
L.de
lbru
ecki
iJX
LI00
0000
00H
oney
stom
ach
ofho
neyb
eeA
nim
alSt
rictl
yan
aero
bic
30M
RS�
fruc
tose
(20
g/lit
er)
Lact
obac
illus
min
dens
isD
SM14
500T
OH
OL.
alim
enta
rius
AZE
Z000
0000
0So
urdo
ugh
Food
Mic
roae
rop
hilic
30M
RS�
0.05
%cy
stei
ne-h
ydro
chlo
ride
(pH
5.2)
Lact
obac
illus
mix
tipab
uli
DSM
2858
0TO
HE
L.co
llino
ides
NA
Sila
gePl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usm
odes
tisal
itole
rans
NBR
C10
7235
TO
HO
L.pl
anta
rum
NA
Ferm
ente
dfis
hFo
odFa
cult
ativ
ely
anae
rob
ic30
MRS
Lact
obac
illus
muc
osae
DSM
1334
5TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sA
ZEQ
0000
0000
Pig
smal
lin
test
ine
Ani
mal
Pref
erab
lyan
aero
bic
37M
RSLa
ctob
acill
usm
udan
jiang
ensi
sLM
G27
194T
OH
OL.
plan
taru
mN
APi
ckle
Plan
tM
icro
aero
phi
lic28
MRS
Lact
obac
illus
mur
inus
DSM
2045
2TFH
EL.
saliv
ariu
sA
YYN
0000
0000
Inte
stin
eof
rat
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usna
gelii
DSM
1367
5TO
HO
L.sa
livar
ius
AZE
V000
0000
0Pa
rtia
llyfe
rmen
ted
win
eW
ine
pro
duct
Ana
erob
ic30
MRS
Lact
obac
illus
nam
uren
sis
DSM
1911
7TO
HE
L.br
evis
AZD
T000
0000
0So
urdo
ugh
Food
Mic
roae
rop
hilic
30M
RS�
0.05
%cy
stei
ne�
0.7
%m
alto
se(p
H5.
2)La
ctob
acill
usna
nten
sis
DSM
1698
2TFH
EL.
alim
enta
rius
AZF
V000
0000
0W
heat
sour
doug
hFo
odM
icro
aero
phi
lic30
MRS
�0.
05%
cyst
eine
-hyd
roch
lorid
e�
1%m
alto
se�
0.5%
fres
hye
ast
extr
act
Lact
obac
illus
nasu
ensi
sJC
M17
158T
OH
OL.
case
i-L.m
anih
otiv
oran
sA
ZDJ0
0000
000
Suda
ngra
sssi
lage
sam
ple
Plan
tA
naer
obic
30M
RSLa
ctob
acill
usne
njia
ngen
sis
LMG
2719
2TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sN
APi
ckle
Plan
tM
icro
aero
phi
lic28
MRS
Lact
obac
illus
node
nsis
DSM
1968
2TFH
EL.
alim
enta
rius
AZD
Z000
0000
0Ja
pan
ese
pic
kles
Food
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usod
orat
itofu
iD
SM19
909T
OH
EL.
colli
noid
esA
ZEE0
0000
000
Ferm
ente
db
rine
used
for
stin
kyto
fup
rodu
ctio
nFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
oeni
DSM
1997
2TO
HO
L.sa
livar
ius
AZE
H00
0000
00Bo
bal
win
eW
ine
pro
duct
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usol
igof
erm
enta
nsD
SM15
707T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZF
E000
0000
0Br
oile
rle
gA
nim
alM
icro
aero
phi
lic25
MRS
Lact
obac
illus
oris
DSM
4864
TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sA
ZGE0
0000
000
Hum
ansa
liva
Ani
mal
Ana
erob
ic37
MRS
Lact
obac
illus
oryz
aeD
SM26
518T
OH
EL.
colli
noid
esBB
JM00
0000
00Fe
rmen
ted
rice
grai
nFo
odA
naer
obic
30M
RSLa
ctob
acill
usot
akie
nsis
DSM
1990
8TO
HE
L.bu
chne
riA
ZED
0000
0000
Sunk
i,a
Jap
anes
etr
aditi
onal
pic
kle
Food
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
ozen
sis
DSM
2382
9TO
HE
L.fr
uctiv
oran
sA
YYQ
0000
0000
Chr
ysan
them
um,O
zeN
atio
nal
Park
Plan
tM
icro
aero
phi
lic30
MRS
(Con
tinue
don
follo
win
gp
age)
Antibiotic Resistance in the Genus Lactobacillus Applied and Environmental Microbiology
January 2019 Volume 85 Issue 1 e01738-18 aem.asm.org 5
on October 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
TAB
LE1
(Con
tinue
d)
Spec
ies
Stra
inM
etab
olis
mp
hen
otyp
eaPh
ylog
roup
bG
enB
ank
acce
ssio
nn
o.c
Sour
ceN
ich
eca
teg
ory
Gro
wth
con
dit
ion
sTe
mp
(°C
)M
ediu
m
Lact
obac
illus
pani
sD
SM60
35T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZG
M00
0000
00So
urdo
ugh
Food
Aer
obic
37M
RSLa
ctob
acill
uspa
nthe
risD
SM15
945T
OH
OL.
case
i-L.m
anih
otiv
oran
sA
ZFJ0
0000
000
Jagu
arfe
ces
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
uspa
rabr
evis
LMG
1198
4TO
HE
L.br
evis
JQC
I000
0000
0W
heat
Food
Ana
erob
ic30
MRS
Lact
obac
illus
para
buch
neri
DSM
5707
TO
HE
L.bu
chne
riA
ZGK0
0000
000
Hum
ansa
liva
Ani
mal
Aer
obic
30M
RSLa
ctob
acill
uspa
raca
sei
sub
sp.
para
case
iD
SM56
22T
FHE
L.ca
sei-L
.man
ihot
ivor
ans
AZG
H00
0000
00N
AU
nkno
wn
Mic
roae
rop
hilic
30M
RS
Lact
obac
illus
para
case
isu
bsp
.to
lera
nsD
SM20
258T
FHE
L.ca
sei-L
.man
ihot
ivor
ans
AYY
J000
0000
0Pa
steu
rized
milk
Food
Aer
obic
30M
RS
Lact
obac
illus
para
colli
noid
esD
SM15
502T
OH
EL.
colli
noid
esA
ZFD
0000
0000
Brew
ery
envi
ronm
ent
Envi
ronm
ent
Ana
erob
ic25
MRS
(pH
5.8)
Lact
obac
illus
para
farr
agin
isLM
G24
141T
FHE
L.bu
chne
riA
ZFZ0
0000
000
Com
pos
ting
mat
eria
lof
dist
illed
shoc
hun
resi
due
Win
ep
rodu
ctM
icro
aero
phi
lic28
MRS
Lact
obac
illus
para
kefir
iD
SM10
551T
OH
EL.
buch
neri
AZE
N00
0000
00Ke
firgr
ain
Plan
tA
naer
obic
30M
RSLa
ctob
acill
uspa
ralim
enta
rius
DSM
1323
8TFH
EL.
alim
enta
rius
AZE
S000
0000
0So
urdo
ugh
Food
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
uspa
rapl
anta
rum
DSM
1066
7TFH
EL.
plan
taru
mA
ZEO
0000
0000
Beer
cont
amin
ant
Win
ep
rodu
ctA
erob
ic30
MRS
Lact
obac
illus
past
eurii
DSM
2390
7TFH
EL.
delb
ruec
kii
AYZ
N00
0000
00N
AU
nkno
wn
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
uspa
uciv
oran
sD
SM22
467T
FHE
L.br
evis
JQC
A00
0000
00Ye
ast
stor
age
tank
cont
aini
ngla
ger
bee
rW
ine
pro
duct
Ana
erob
ic28
MRS
�0.
05%
cyst
eine
-hyd
roch
lorid
e�
1%fr
ucto
se(p
H5.
8)La
ctob
acill
uspe
ntos
usD
SM20
314T
FHE
L.pl
anta
rum
AZC
U00
0000
00N
AU
nkno
wn
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
uspe
role
nsD
SM12
744T
FHE
L.pe
role
nsA
ZEC
0000
0000
Ora
nge
lem
onad
eFo
odA
naer
obic
30M
RSLa
ctob
acill
uspl
ajom
iN
BRC
1073
33T
FHE
L.pl
anta
rum
NA
Ferm
ente
dfis
hFo
odFa
cult
ativ
ely
anae
rob
ic30
MRS
Lact
obac
illus
plan
taru
m(f
orm
erly
Lact
obac
illus
ariz
onen
sis)
DSM
2017
4TFH
EL.
plan
taru
mA
ZEJ0
0000
000
Pick
led
cab
bag
eFo
odA
erob
ic37
MRS
Lact
obac
illus
plan
taru
msu
bsp
.ar
gent
orat
ensi
sD
SM16
365T
FHE
L.pl
anta
rum
AZF
R000
0000
0Fe
rmen
ted
cass
ava
root
s(f
ufu)
Plan
tM
icro
aero
phi
lic30
MRS
Lact
obac
illus
pobu
zihi
iN
BRC
1032
19T
FHE
L.sa
livar
ius
JQC
N00
0000
00Po
buz
ih(f
erm
ente
dcu
mm
ingc
ordi
a),C
ordi
adi
chot
oma
Plan
tM
icro
aero
phi
lic37
MRS
�5%
NaC
l
Lact
obac
illus
pont
isD
SM84
75T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZG
O00
0000
00Ry
eso
urdo
ugh
Food
Aer
obic
30M
RSLa
ctob
acill
uspo
rcin
aeLM
G26
767T
FHE
L.ca
sei-L
.man
ihot
ivor
ans
NA
Nem
chua
(fer
men
ted
mea
t)Fo
odM
icro
aero
phi
lic28
MRS
Lact
obac
illus
psitt
aci
DSM
1535
4TO
HE
L.de
lbru
ecki
iA
ZFB0
0000
000
Lung
ofp
arro
tA
nim
alA
naer
obic
37M
RSLa
ctob
acill
usra
piD
SM19
907T
OH
EL.
buch
neri
AZE
I000
0000
0Su
nki,
aJa
pan
ese
trad
ition
alp
ickl
eFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
renn
ini
DSM
2025
3TFH
EL.
cory
nifo
rmis
AYY
I000
0000
0Re
nnin
Ani
mal
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usre
uter
iD
SM20
016T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZD
D00
0000
00In
test
ine
ofad
ult
Ani
mal
Aer
obic
37M
RSLa
ctob
acill
usrh
amno
sus
DSM
2002
1TFH
EL.
case
i-L.m
anih
otiv
oran
sA
ZCQ
0000
0000
NA
Unk
now
nA
naer
obic
37M
RSLa
ctob
acill
usro
dent
ium
DSM
2475
9TO
HO
L.de
lbru
ecki
iN
AD
iges
tive
trac
tof
rode
nts
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usro
ssia
eD
SM15
814T
OH
EO
ther
AZF
F000
0000
0W
heat
sour
doug
hFo
odM
icro
aero
phi
lic30
MRS
�1%
mal
tose
�1%
yeas
tex
trac
t(p
H5.
6)La
ctob
acill
usru
min
isD
SM20
403T
OH
OL.
saliv
ariu
sA
YYL0
0000
000
Bovi
neru
men
Ani
mal
Ana
erob
ic37
MRS
�2%
gluc
ose
Lact
obac
illus
saer
imne
riD
SM16
049T
OH
OL.
saliv
ariu
sA
ZFP0
0000
000
Pig
fece
sA
nim
alA
erob
ic37
MRS
Lact
obac
illus
sake
isu
bsp
.ca
rnos
usD
SM15
831T
FHE
L.sa
kei
AZF
G00
0000
00Fe
rmen
ted
mea
tp
rodu
ctFo
odM
icro
aero
phi
lic37
MRS
Lact
obac
illus
sake
isu
bsp
.sak
eiC
ECT
4591
TFH
EL.
sake
iA
ZDN
0000
0000
“Mot
o”st
arte
rof
sake
Win
ep
rodu
ctA
erob
ic30
MRS
Lact
obac
illus
saliv
ariu
sD
SM20
555T
FHE
L.sa
livar
ius
AYY
T000
0000
0Sa
liva
Ani
mal
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
ussa
nfra
ncis
cens
isLM
G16
002T
OH
EL.
fruc
tivor
ans
AYY
M00
0000
00So
urdo
ugh
Food
Ana
erob
ic28
MRS
�1%
fruc
tose
(pH
5.5)
Lact
obac
illus
sani
viri
DSM
2430
1TFH
EL.
case
i-L.m
anih
otiv
oran
sJQ
CE0
0000
000
Fece
sof
aJa
pan
ese
heal
thy
adul
tm
ale
Ani
mal
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
sats
umen
sis
DSM
1623
0TO
HO
L.sa
livar
ius
AZF
Q00
0000
00Sh
ochu
mas
hW
ine
pro
duct
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usse
calip
hilu
sD
SM17
896T
FHE
L.re
uter
i-L.v
acci
nost
ercu
sJQ
BW00
0000
00So
urdo
ugh
Food
Mic
roae
rop
hilic
37M
RSLa
ctob
acill
usse
lang
oren
sis
ATC
CBA
A66
TO
HO
Oth
erJQ
AT0
0000
000
Chi
lib
oFo
odA
erob
ic30
MRS
Lact
obac
illus
seni
oris
DSM
2430
2TFH
EL.
buch
neri
AYZ
R000
0000
0Fe
ces
ofa
heal
thy
100-
yr-o
ldJa
pan
ese
fem
ale
Ani
mal
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
senm
aizu
kei
DSM
2177
5TFH
EL.
brev
isA
YZH
0000
0000
Senm
aizu
ke,a
Jap
anes
ep
ickl
eFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
shar
peae
DSM
2050
5TO
HO
L.ca
sei-L
.man
ihot
ivor
ans
AYY
O00
0000
00M
unic
ipal
sew
age
Envi
ronm
ent
Mic
roae
rop
hilic
30M
RS�
0.05
%cy
stei
ne
(Con
tinue
don
follo
win
gp
age)
Campedelli et al. Applied and Environmental Microbiology
January 2019 Volume 85 Issue 1 e01738-18 aem.asm.org 6
on October 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
TAB
LE1
(Con
tinue
d)
Spec
ies
Stra
inM
etab
olis
mp
hen
otyp
eaPh
ylog
roup
bG
enB
ank
acce
ssio
nn
o.c
Sour
ceN
ich
eca
teg
ory
Gro
wth
con
dit
ion
sTe
mp
(°C
)M
ediu
m
Lact
obac
illus
shen
zhen
ensi
sD
SM28
193T
OH
EL.
pero
lens
AVA
A00
0000
00Fe
rmen
ted
dairy
bev
erag
eFo
odM
icro
aero
phi
lic37
MRS
Lact
obac
illus
sice
rae
KCTC
2101
2TO
HO
L.sa
livar
ius
NA
Span
ish
natu
ral
cide
rFo
odA
naer
obic
37M
RSLa
ctob
acill
ussi
lage
iD
SM27
022T
OH
EL.
colli
noid
esN
AO
rcha
rdgr
ass
sila
gePl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
ussi
ligin
isD
SM22
696T
OH
EO
ther
JQC
B000
0000
0W
heat
sour
doug
hFo
odA
naer
obic
37M
RSLa
ctob
acill
ussi
mili
sD
SM23
365T
OH
EL.
colli
noid
esA
YZM
0000
0000
Ferm
ente
dca
nem
olas
ses
atal
coho
lp
lant
sW
ine
pro
duct
Mic
roae
rop
hilic
37M
RS
Lact
obac
illus
song
huaj
iang
ensi
sD
SM28
401T
OH
OL.
case
i-L.m
anih
otiv
oran
sN
ASo
urdo
ugh
Food
Mic
roae
rop
hilic
28M
RSLa
ctob
acill
ussp
iche
riD
SM15
429T
FHE
L.br
evis
AZF
C00
0000
00Ri
ceso
urdo
ugh
Food
Mic
roae
rop
hilic
30M
RS(p
H5.
8)La
ctob
acill
ussu
cico
laD
SM21
376T
OH
OL.
saliv
ariu
sA
YZF0
0000
000
Sap
ofan
oak
tree
Plan
tM
icro
aero
phi
lic30
MRS
Lact
obac
illus
sueb
icus
DSM
5007
TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sA
ZGF0
0000
000
Ap
ple
mas
hFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
sunk
iiD
SM19
904T
OH
EL.
buch
neri
AZE
A00
0000
00Su
nki,
aJa
pan
ese
trad
ition
alp
ickl
eFo
odM
icro
aero
phi
lic30
MRS
Lact
obac
illus
taiw
anen
sis
DSM
2140
1TO
HO
L.de
lbru
ecki
iA
YZG
0000
0000
Sila
geca
ttle
feed
Plan
tM
icro
aero
phi
lic37
MRS
Lact
obac
illus
thai
land
ensi
sD
SM22
698T
OH
OL.
case
i-L.m
anih
otiv
oran
sA
YZK0
0000
000
Ferm
ente
dte
ale
aves
(mia
ng)
Plan
tM
icro
aero
phi
lic30
MRS
Lact
obac
illus
tucc
eti
DSM
2018
3TO
HO
L.al
imen
tariu
sA
ZDG
0000
0000
Saus
age
Food
Aer
obic
30M
RSLa
ctob
acill
usul
tune
nsis
DSM
1604
7TO
HO
L.de
lbru
ecki
iA
ZFO
0000
0000
Gas
tric
bio
psy
spec
imen
s,hu
man
stom
ach
muc
osa
Ani
mal
Ana
erob
ic37
MRS
Lact
obac
illus
uvar
umD
SM19
971T
OH
OL.
saliv
ariu
sA
ZEG
0000
0000
Mus
tof
Bob
algr
ape
varie
tyPl
ant
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usva
ccin
oste
rcus
DSM
2063
4TO
HE
L.re
uter
i-L.v
acci
nost
ercu
sA
YYY0
0000
000
Cow
dung
Ani
mal
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usva
gina
lisLM
G12
891T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AZG
L000
0000
0Va
gina
lsw
abA
nim
alA
naer
obic
28M
RSLa
ctob
acill
usve
rsm
olde
nsis
DSM
1485
7TO
HO
L.al
imen
tariu
sA
ZFA
0000
0000
Poul
try
sala
mi
Food
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usve
spul
aeD
SM10
3408
TO
HE
L.fr
uctiv
oran
sN
AG
utof
quee
nw
asp
Ani
mal
Mic
roae
rop
hilic
30M
RSLa
ctob
acill
usvi
niD
SM20
605T
FHE
L.sa
livar
ius
AYY
X00
0000
00M
ust
ofgr
ape
Plan
tA
erob
ic37
MRS
Lact
obac
illus
was
atch
ensi
sLM
G28
678T
OH
EL.
reut
eri-L
.vac
cino
ster
cus
AW
TT00
0000
00C
hedd
arch
eese
Food
Ana
erob
ic25
MRS
Lact
obac
illus
xian
gfan
gens
isLM
G26
013T
FHE
L.pl
anta
rum
JQC
L000
0000
0Pi
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generally observed for these antibiotics, which covered four 2-fold dilutions for moststrains analyzed (89% of strains for quinupristin-dalfopristin and 96% for chloramphen-icol and linezolid), ranging in actual MIC values from 2 to 16 �g/ml for chloram-phenicol, 1 to 8 �g/ml for linezolid, and 0.5 to 4 �g/ml for quinupristin-dalfopristin.
High MIC values were observed for trimethroprim and vancomycin, at �64 and�128 �g/ml, respectively, in the most strains analyzed (81 and 73%, respectively).Interestingly, strains belonging to the Lactobacillus delbrueckii group showed suscep-tibility to low concentrations of vancomycin (concentrations lower than 1 �g/ml inhib-ited the growth of 92% of strains belonging to the L. delbrueckii group), despite thegeneral consensus in the literature that Lactobacillus spp. are not inhibited by vanco-mycin.
The MICs of the remaining antibiotics showed variability across the genus. Inparticular, the MIC values for aminoglycosides covered more than nine 2-fold dilutionsteps, ranging from 2 to �1,024 �g/ml for kanamycin, and from 0.2, 0.5, and 2 to�256 �g/ml for gentamicin, neomycin, and streptomycin, respectively. Bimodal MICdistributions were observed for the Lactobacillus alimentarius, Lactobacillus collinoides,Lactobacillus fructivorans, Lactobacillus plantarum, and Lactobacillus reuteri-L. vaccino-stercus groups for all aminoglycosides tested (Table S1). Moreover, strains of the L.reuteri-L. vaccinostercus phylogroup showed MIC values distributed across the wholeconcentration range tested for gentamicin (0.5 to 256 �g/ml), kanamycin (2 to1,024 �g/ml), and neomycin (0.5 to 256 �g/ml).
The L. alimentarius, L. collinoides, L. delbrueckii, L. fructivorans, L. plantarum, and L.reuteri-L. vaccinostercus groups displayed bimodal MIC value distributions for tetracy-cline, erythromycin, and clindamycin, antibiotics which notably affect the function ofthe ribosome. In contrast, a unimodal MIC distribution was observed for �-lactams,including ampicillin and penicillin, except for some strains belonging to the Lactoba-cillus brevis group (Lactobacillus spicheri DSM 15429T and Lactobacillus zymae DSM19395T), L. collinoides group (Lactobacillus similis DSM 23365T), L. plantarum group(Lactobacillus pentosus DSM 20314T), Lactobacillus salivarius group (Lactobacillusghanensis DSM 18630T), and other (Lactobacillus selangorensis ATCC BAA66T) groups,which displayed MIC values of �16 �g/ml.
The distribution of rifampin and ciprofloxacin MIC values was broad; they trendedtoward the low-end concentration range tested for this antibiotic (0.12 to 16 �g/ml),except for Lactobacillus jensenii DSM 20557T and Lactobacillus oris DSM 4864T, belong-ing to the L. delbrueckii and L. reuteri-L. vaccinostercus groups, respectively, whichshowed MIC values higher than 64 �g/ml.
Identification of resistance phenotypes. Phenotypic resistance was interpreted
based on the epidemiological cutoff (ECOFF) values reported in references 9, 30, and31, classifying a strain as resistant when the MIC value for a specific antibiotic washigher than the corresponding ECOFF. EFSA specified the ECOFF value for the speciesLactobacillus casei/L. paracasei, Lactobacillus plantarum/L. pentosus, and Lactobacillusrhamnosus. For other species of the genus Lactobacillus, EFSA refers to the fermentationmetabolic categories, defining ECOFF values for Lactobacillus obligate heterofermen-tative (OHE), Lactobacillus obligate homofermentative (OHO), and Lactobacillus facul-tative heterofermentative (FHE) metabolic patterns (9). For comparative clarity, thefermentation metabolism for the strains analyzed are thus presented in Table 1.
Trimethoprim resistance was the most common phenotype observed (84% [152/182strains]), and most of the Lactobacillus strains were not susceptible to vancomycin (77%[141/182 strains]) and kanamycin (61% [111/181 strains]) (Fig. 1). Multidrug resistance,defined as resistance to three or more different antimicrobials, was observed in 152strains (84%). Interestingly, Lactobacillus thailandensis DSM 22698T showed resistanceto all 16 antibiotics tested (Fig. 1). In contrast, Lactobacillus sanfranciscensis LMG 16002T
and Lactobacillus pobuzihii NBRC 103219T were identified as susceptible to all 16antibiotics tested, including vancomycin. Lactobacillus ozensis DSM 23829T, Lactobacil-
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FIG 1 Resistance profiles of 182 type strains of the genus Lactobacillus compared with epidemiologicalcutoff values provided in references 9, 30, and 31. Resistant strains with MIC values higher than the
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lus equigenerosi DSM 18793T, Lactobacillus capillatus DSM 19910T, and Lactobacillus viniDSM 20605T showed resistance only toward vancomycin.
Overall, the 182 type strains showed high susceptibility to gentamicin, erythromycin,penicillin, quinupristin-dalfopristin, linezolid, and rifampin (Fig. 1). In fact, only 5% of thestrains investigated (10 out of 182) were resistant to quinupristin-dalfopristin. Incontrast, the resistance levels toward ampicillin were higher than those identified forpenicillin, and they were mainly detected in members of the phylogroups L. brevis(73%), L. alimentarius (58%), and L. collinoides (57%) (Fig. 2).
Notably, 50% and 49% of the type strains examined in this study were resistant totetracycline and chloramphenicol, respectively, and 31% were resistant to both antibi-otics. Tetracycline resistance phenotypes were mainly observed in species of thephylogroups Lactobacillus buchneri, L. collinoides, L. plantarum, L. reuteri-L. vaccinoster-cus, L. fructivorans, and L. brevis, while members of the phylogroups L. brevis, Lactoba-cillus casei-L. manihotivorans, and Lactobacillus perolens showed the highest preva-lences of resistance to chloramphenicol (Fig. 2). For clindamycin and streptomycin, theresistance levels were low, at 20% and 18%, respectively, out of the 182 Lactobacillusstrains analyzed. Finally, with regard to aminoglycosides, kanamycin resistance oc-curred in at least 50% of the members of all Lactobacillus phylogroups except for L.perolens, which was very susceptible to this antibiotic. Streptomycin resistance wasobserved in 35% of the strains examined.
FIG 1 Legend (Continued)ECOFF are indicated in green, whereas sensitive strains are depicted in gray. Strains are clustered by thephylogroups reported by references 25 and 29 and are demarcated by the colored bar on the left of theheat plot. GM, gentamicin; KM, kanamycin; SM, streptomycin; NM, neomycin; TC, tetracycline; EM,erythromycin; CL, clindamycin; CM, chloramphenicol; AM, ampicillin; PC, penicillin; VA, vancomycin; QD,quinupristin-dalfopristin; LZ, linezolid; TM, trimethoprim; CI, ciprofloxacin; RI, rifampin.
FIG 2 Prevalence of antibiotic-resistant (blue) and antibiotic-susceptible (green) strains within the Lactobacillus phylogroups tested for antimicrobial agentsspecified in reference 9, including inhibitors of cell wall synthesis (ampicillin and vancomycin), inhibitors of protein synthesis (erythromycin, clindamycin,chloramphenicol, and tetracycline), and aminoglycosides (gentamicin, kanamycin, and streptomycin).
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Identification of AR genes. According to the EFSA guidelines, lactobacilli intendedfor human consumption should be tested for their resistance to gentamicin, kanamycin,streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, andvancomycin, and they should be genetically investigated for the absence of acquired ortransferable AR determinants (9). Thus, the genome sequences for 161 out of the 182type strains (21 genome sequences were not available at the time of the study) testedfor phenotypic resistance were aligned against the protein sequences of AR genes inthe Comprehensive Antibiotic Resistance Database (CARD). Based on the selectioncriteria and manual annotation (see Materials and Methods), a total of 146 genesequences were identified among the type strains analyzed, which are predicted toencode resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin(6), clindamycin (60), and chloramphenicol (42) (Fig. S1).
Genes encoding penicillin binding proteins (PBPs) and D-alanine D-alanine ligase(Ddl) involved in ampicillin and vancomycin resistance, respectively, were found in allgenomes investigated. Furthermore, amino acid sequence analysis of the PBPs for the161 type strains confirmed the presence of conserved amino acid residues in the knownbinding site motif for �-lactams. The Ddl enzyme of all vancomycin-resistant typestrains exhibited a conserved phenylalanine (F) residue in the active site of the enzyme,while all members of the phylogroup L. delbrueckii, which were susceptible to vanco-mycin, were characterized by the presence of a tyrosine (Y) residue at this position, withthe exception of strains L. jensenii DSM 20557T, Lactobacillus amylophilus DSM 20533T,and Lactobacillus amylotrophicus DSM 20534T. Their respective proteins carried theY-type motif, even though they were resistant to vancomycin. However, Lactobacillusamylophilus DSM 20533T and Lactobacillus amylotrophicus DSM 20534T harbored spe-cific D-alanine-D-lactate ligase sequences in their genomes, which could explain theirvancomycin resistance phenotypes. In addition, L. sanfranciscensis LMG 16002T, Lacto-bacillus hilgardii LMG 6895T, Lactobacillus composti DSM 18527T, L. pobuzihii NBRC103219T, Lactobacillus farciminis LMG 9189, Lactobacillus ceti DSM 22408T, and Lacto-bacillus algidus DSM 15638T were characterized by the presence of Ddl of the F type,despite their susceptibility to vancomycin (Fig. S2).
Aminoglycoside resistance genes. Twenty different sequences predicted to en-code aminoglycoside-modifying enzymes were identified among the 161 Lactobacillusgenomes, which were mainly acetyltransferases (AACs) (7 sequences), nucleotidyltrans-ferases (ANTs) (8 sequences), and phosphotransferases (APHs) (5 sequences) (Fig. S1). Inparticular, the AAC(3) family N-acetyltransferase was found in five L. brevis phylogroupgenomes and two L. delbrueckii phylogroup members. All these strains showed resis-tance to kanamycin, and some of them were also resistant to streptomycin, such asLactobacillus acidifarinae DSM 19394T, Lactobacillus koreensis JCM 16448T, L. spicheriDSM 15429T, and Lactobacillus hominis DSM 23910T. Moreover, L. zymae DSM 19395T
showed resistance to gentamicin, whereas Lactobacillus namurensis DSM 19117T wassusceptible to aminoglycosides despite harboring the aac(3) gene.
Gene sequences coding for nucleotidyltranferase enzymes, such as ant(6) and ant(9),were identified in 7 type strains, as follows: L. amylophilus DSM 20533T and L. amy-lotrophicus DSM 20534T (L. delbrueckii phylogroup), Lactobacillus fabifermentas DSM21115T (L. plantarum phylogroup), Lactobacillus animalis DSM 20602T, and L. pobuzihiiNBRC 103219T (L. salivarius phylogroup), Lactobacillus sharpeae DSM 20505T (L. casei-L.manihotivorans phylogroup), and Lactobacillus rossiae DSM 15814T (“other” phylo-group). In particular, the ant(9) gene was only found in the genome of L. pobuzihii NBRC103219T, despite its phenotypic susceptibility toward aminoglycosides. Similarly, theant(6) gene was found in L. sharpeae DSM 20505T and L. rossiae DSM 15814T, whichwere susceptible to aminoglycosides. Conversely, the presence of this AR determinantin L. amylophilus DSM 20533T, L. amylotrophicus DSM 20534T, L. fabifermentas DSM21115T, and L. animalis DSM 20602T might explain their resistance to kanamycin andstreptomycin. Interestingly, in these strains, the predicted amino acid sequence en-coded by the ant(6) gene found in the genomes of L. animalis DSM 20602T and L.
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amylophilus DSM 20533T shared 99% similarity with the ANT6 aminoglycoside nucle-otidyltransferases of Streptococcus suis (NCBI RefSeq accession no. WP_044770667.1)and Clostridium difficile (NCBI RefSeq accession no. WP_077726164.1). Regarding phos-photransferases, the aph(3) gene was found in five members of the L. delbrueckiiphylogroup (Lactobacillus acidophilus ATCC 4356T, Lactobacillus gasseri LMG 9203T,Lactobacillus johnsonii LMG 9436T, Lactobacillus kalixensis DSM 16043T, and Lactobacil-lus pasteurii DSM 23907T), which showed resistance to kanamycin, except for ATCC4356T and LMG 9203T.
Clindamycin resistance genes. The lsa gene encoding a lincosamide efflux proteinwas found in 60 strains, 13 of which displayed resistance to clindamycin. Alignment ofthe amino acid sequence of the Lsa efflux protein revealed a truncated carboxyterminus for the predicted Lsa proteins in L. hominis DSM 23910T, Lactobacillus galli-narum DSM 10532T, and Lactobacillus iners DSM 13335T. Four conserved amino aciddomains were found in the remaining 57 sequences, corresponding to two copies ofWalker A and B motifs, which play an important role in ATP binding and hydrolysis thatenergizes efflux (Fig. S3).
Chloramphenicol resistance genes. Among the 161 type strains of the genusLactobacillus, 36 chloramphenicol resistance-related sequences were found coding forchloramphenicol acetyltransferase, like the cat gene, and for specific membrane-associated transporters, like CmlA. The cat gene was detected in 34 lactobacilli, two ofwhich (Lactobacillus kimchicus JCM15530T and L. similis DSM 23365T) carried two copiesof this gene. Moreover, Lactobacillus hammesii DSM 16381T, L. koreensis JCM 16448T, L.namurensis DSM 19117T, and L. zymae DSM 19395T (belonging to the phylogroup L.brevis) were characterized by the presence of either the cmlA or cat gene. Conversely,L. acidifarinae DSM 19394T and L. selangorensis ATCC BAA66T displayed only thepresence of cmlA.
Tetracycline resistance genes. The 18 gene sequences found among Lactobacillus
strains code for ribosomal protection proteins [tet(M), tet(S), tet(Q), and tet(W)] andefflux pumps [tet(L) and tet(P)]. The tet(L) gene was found in the tetracycline-resistantstrains Lactobacillus suebicus DSM 5007T and Lactobacillus ingluviei DSM 15946T. L.ingluviei DSM 15946T was also characterized by the presence of tet(W) and tet(M).Interestingly, tet(M) in DSM 15946T exhibited 99% residue identity with the correspond-ing sequences of Enterococcus faecalis (NCBI RefSeq accession no. WP_049098680.1),Enterococcus faecium (NCBI RefSeq accession no. WP_010777232.1), Streptococcus pneu-moniae PT814 (GenBank accession no. HG799502.1), and Staphylococcus epidermidis(NCBI RefSeq accession no. WP_002403674.1). Similarly, the sequence of tet(L) displayed99% residue identity with those carried by E. faecalis (NCBI RefSeq accession no.WP_002387933.1), E. faecium (NCBI RefSeq accession no. WP_096541192.1), and Strep-tococcus agalactiae (NCBI RefSeq accession no. WP_041974946.1). tet(W) showed 99%residue identity with the sequences of Trueperella pyogenes OX9, Bifidobacteriumlongum subsp. longum F21, and C. difficile CI7. tet(W) was also identified in L. pasteuriiDSM 23907T, while tet(M) was found in L. sharpeae DSM 20505T (L. casei-L. manihotiv-orans group), L. acidophilus ATCC 4356T, Lactobacillus crispatus DSM 20584T, L. gallina-rum DSM 10532T, L. amylophilus DSM 20533T, L. amylotrophicus DSM 20534T (L. del-brueckii group), and L. equigenerosi DSM 18793T (L. reuteri-L. vaccinostercus group).Strains DSM 20584T, ATCC 4356T, and DSM 18793T showed susceptibility to tetracycline.Regarding other ribosomal protection proteins, the tet(Q) and tet(S) genes were foundin L. brevis DSM 20054T and Lactobacillus heilongjiangensis LMG 26166T, respectively,both being resistant to tetracycline, while tet(P) was found in L. gasseri LMG 9203T,Lactobacillus taiwanensis DSM 21401T, Lactobacillus ruminis DSM 20403T, and L. john-sonii LMG 9436T. Only LMG 9436T showed phenotypic resistance to tetracycline.
Erythromycin resistance genes. Across the 161 Lactobacillus genomes, six genesequences predicted as contributing to erythromycin resistance were detected, whichinclude the erm(B) gene coding for a predicted rRNA methylase, and two variants of themef, mef(E), and mef(B) genes, encoding macrolide efflux pumps. These genes were
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identified in three erythromycin-susceptible strains, namely L. delbrueckii subsp. lactisDSM 20072T [mef(E)], L. casei DSM 20041T, and Lactobacillus paracasei subsp. paracaseiDSM 5622T [mef(B)]. erm(B) was found in the erythromycin-resistant strains L. amylo-philus DSM 20533T, L. amylotrophicus DSM 20534T, and L. ingluviei DSM 15946T, whichalso showed the presence of tetracycline resistance determinants.
Analysis of ORFs in regions flanking tetracycline and erythromycin resistancegenes. Tetracycline and erythromycin are two of the most widely used antimicrobialsin human and animal therapy, and genetic determinants involved in the resistance tothose antibiotics are usually associated with mobile genetic elements. Therefore,upstream and downstream regions of the tetracycline and erythromycin resistancegenes were further investigated in L. ingluviei DSM 15946T, L. amylophilus DSM 20533T,and L. amylotrophicus DSM 20534T, the only three strains that simultaneously harborederythromycin and tetracycline resistance genes within the data set. Sequence analysisof the L. ingluviei DSM 15946T genome revealed that tet(M) and tet(L) were closelylocated in the same genomic region, separated by 146 nucleotides. Upstream of tet(M),12 open reading frames (ORFs) encoding conjugation transfer elements were identifiedthat shared 99% residue identity with the corresponding sequences of the Tn5251transposon carried by S. pneumoniae DP1322. Moreover, a sequence predicted toencode a transposase was found downstream of the tet(L) determinant (Fig. 3). This isa common configuration for a Tn916-like transposon. The analysis of the distribution ofthese genetic elements in the available genome sequences of Lactobacillus isolatesrevealed that the predicted transposon carried by L. ingluviei DSM 15946T (GenBankaccession no. CP016400.1) is essentially identical to that harbored by L. johnsonii BS15,except for the presence of a recombinase downstream of the tet(L) gene. In contrast,L. salivarius JCM 1046 (GenBank accession no. CP007650.1) and L. salivarius JCM 1047(GenBank accession no. NBEF01000044.1) lack the tet(L) determinant, and the regula-tion region has been retained, similar to that of the Tn5251 transposon in S. pneu-moniae DP1322. These two transposons are essentially identical except for the presenceof ORF 23 in L. salivarius JCM 1047, which was not identified in the transposon harboredby L. salivarius JCM 1046. Interestingly, L. iners UMB1051 (GenBank accession no.PNGO01000001.1) was characterized by the presence of a Tn916-like transposon inwhich a Tn917 carrying an erm(B) gene was inserted, resulting in a Tn3872-like trans-poson (Fig. S4). The tet(W) and erm(B) genes were located in the same genomic regionand shared 99% similarity with the corresponding sequences of the strain S. suis SsCA.Moreover, these AR determinants were flanked by regions with high similarity (99%) toreplication proteins and to the integrase of the plasmid pLR581 of Lactobacillus reuteriSD2112 (GenBank accession no. CP002845.1) (Fig. 4).
L. amylophilus and L. amylotrophicus harbored the tet(M) and erm(B) genes in twodifferent genomic regions, and they shared 99% residue identity with the correspond-ing sequences of S. agalactiae SG-M4 and Staphylococcus hyicus HW17, respectively. Theflanking region structures of these AR determinants were identical at the amino acidlevel in the two type strains. In particular, the up- and downstream sequences sur-rounding tet(M) were characterized by the presence of several genes predicted toencode conjugation proteins and transposases (Fig. 5A). For erm(B), the upstreamregion showed an 83-bp sequence corresponding to a 27-amino-acid leader peptide. Agene encoding a mobilization protein was found upstream of the leader peptide
FIG 3 Diagram showing the genetic organization of the Tn916-like transposon (transp) identified in L. ingluviei DSM15946T. Red, AR genes; yellow, genes involved in genetic transfer; gray, ORFs involved in the conjugation process;green, regulatory sequences. Numbers above the diagram refer to loci in Tn916; numbers below are a base pairscale.
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sequence, sharing 99% residue identity with the corresponding sequence in the S.hyicus plasmid pSTE1 (GenBank accession no. HE662694.1) (Fig. 5B). Unfortunately, thelocation of erm(B) at the 3= end of the contig did not allow for the characterization ofthe downstream region.
Phenotype-genotype correlation. Overall, phenotypic resistance correlated withgenotypes for 67% of the cases examined with genomic data. In detail, the genotypewas in accordance with the phenotype for 892 out of 1,449 phenotypic tests investi-gated, which included 782 cases representing a susceptible phenotype toward aspecific antibiotic linked to the absence of AR determinants and 110 cases for which theresistance phenotype correlated with the presence of one or more AR genes. Mostnotably, the F-type Ddl enzyme was found in 99% of the vancomycin-resistant Lacto-bacillus strains, revealing the high relationship between genotype and phenotype forthis antibiotic. For aminoglycosides, the 20 AR determinants identified in this studymight explain the resistance phenotype for 13.7%, 14.2%, and 20.8% of the strains forkanamycin, streptomycin, and gentamicin, respectively. For chloramphenicol, the catand cmlA genes were found in 20 strains out of 79 chloramphenicol-resistant lactoba-cilli and in 59 strains out of 82 lactobacilli susceptible to this antibiotic. However, thegenetic basis of chloramphenicol resistance was not revealed for 74.7% of the resistantstrains. The presence of the lsa gene positively correlated with the resistance pheno-type for 40.6% of the strains. However, 47 strains harbored the lsa gene in theirgenomes even though they showed susceptibility to clindamycin. For tetracycline anderythromycin, the presence of AR genes explained the resistance phenotype for 12.7%and 10.3% of the strains, respectively. However, in a significant number of examples(364 cases), a genetic basis was not detected for a demonstrated resistance phenotype(Fig. 6).
DISCUSSION
The data reported in the present study provide evidence at phenotype and geno-type levels for rationally revising the regulatory guidelines for safety assessment of
FIG 4 Genetic organization of sequences surrounding the tet(W) and erm(B) genes identified in L.ingluviei DSM 15946T. Red, AR genes; yellow, genes involved in genetic transfer; pink, genes encodingplasmid-associated replication proteins; gray, gene coding for hypothetical proteins. Numbers above thediagram refer to loci; numbers below are a base pair scale. Rep, plasmid replication; int; plasmidintegration.
FIG 5 Genetic organization of sequences surrounding the tet(M) (A) and erm(B) (B) genes identified in L. amylophilus DSM 20533T and L.amylotrophicus DSM 20534T. Red, AR genes; yellow, genes involved in genetic transfer; green, genes encoding regulatory proteins; gray, genecoding for hypothetical proteins. int, integrase. Numbers below the diagram are a base pair scale.
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FIG 6 Phenotype-genotype correlation analysis for the 161 type strains of the genus Lactobacillus. Positivecorrelations between genomic data and phenotypes observed are presented in green and gray, whereasnegative correlations are indicated in yellow and blue. GM, gentamicin; KM, kanamycin; SM, streptomycin; TC,tetracycline; EM, erythromycin, CL, clindamycin; CM, chloramphenicol; AM, ampicillin; VA, vancomycin.
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lactobacilli, showing that certain resistances are widespread within the genus. Forexample, low-level resistance to linezolid, rifampin, and quinupristin-dalfopristin wasgenerally observed for type strains, in line with previous reports (30–35). We alsoconfirmed low-level resistance to rifampin, as previously reported for Lactobacillusspecies isolated from the human GIT (36), traditional dairy products (37), and inprobiotic strains in marketed foods and drugs (38). Generally, we found Lactobacillusspecies to be susceptible to low concentrations of �-lactams, including penicillin andampicillin (16), even though atypical insensitivity to higher concentrations of these cellwall inhibitors has been reported for some L. crispatus and L. johnsonii strains isolatedfrom healthy chickens (39). Higher levels of resistance to ampicillin compared topenicillin were detected for the type strains analyzed, in line with some previous studies(40–42). Interestingly, ampicillin resistance in L. reuteri strains has been attributed topoint mutations in the genes encoding the penicillin binding proteins (PBPs) (43), butthe PBP sequences for the 161 type strains in this study all retained conserved aminoacid residues (i.e., linked to sensitivity) in the binding sites for these antibiotics.
Conversely, the Lactobacillus strains showed high intrinsic (as opposed to acquired)resistance to trimethoprim and vancomycin. Folate auxotrophic lactobacilli have beenreported as being intrinsically resistant to trimethoprim (44), including L. johnsonii, L.acidophilus, L. salivarius, L. brevis, L. casei, L. gasseri, L. rhamnosus, L. delbrueckii,Lactobacillus fermentum, Lactobacillus helveticus, L. plantarum, L. reuteri, Lactobacillussakei, and L. crispatus (45), where the vancomycin-resistant phenotypes are perhaps thebest-characterized resistance mechanisms described in lactobacilli (16), with manyspecies being intrinsically resistant (30). Exceptions to this are L. delbrueckii, L. acidoph-ilus, L. johnsonii, and L. crispatus, where the vancomycin-susceptible phenotype hasbeen associated with the presence of a Y-type Ddl enzyme (46). In this genus-wideanalysis, the vancomycin-susceptible strains were mainly represented by almost allmembers of the L. delbrueckii phylogroup, which were characterized by the presence ofa Y-type Ddl. The substitution of tyrosine 261 in the Ddl enzyme by a phenylalanineresidue (F-type enzyme) is associated with the synthesis of peptidoglycan precursorscontaining a D-Ala-D-Lac residue conferring vancomycin resistance (46). Notably, almostall vancomycin-resistant strains in this study carried a Ddl of the F type. However, wenoted inconsistencies between phenotypes and genotypes for vancomycin in 8 of thespecies (L. jensenii, L. sanfraciscensis, L. hilgardii, L. composti, L. pobuzihii, L. farciminis, L.ceti, and L. algidus), which could be due to the presence of alternative resistancemechanisms or to alteration of gene expression.
Overall, Lactobacillus type strains displayed higher resistance toward kanamycin andstreptomycin than to gentamicin and neomycin. Aminoglycoside resistance has beendescribed as an intrinsic feature for some Lactobacillus species (i.e., L. rhamnosus, L.acidophilus, L. delbrueckii subsp. bulgaricus, and L. helveticus) (47, 48) due to the lack ofcytochrome-mediated drug transport (15). In contrast, high-level susceptibility to gen-tamicin is most likely linked to the superior ability of this antibiotic to cross themembrane compared to other aminoglycosides (49).
We found genes for nucleotidyltransferases, such as ant(6) and ant(9), in 7 typestrains, 4 of which showed resistance to kanamycin and streptomycin (L. amylophilus,L. amylotrophicus, L. fabifermentas, and L. animalis), in accordance with the high affinityof ANT6 for streptomycin (50). Moreover, the nucleotide sequence of the ant(6) geneharbored by L. animalis DSM 20602T and L. amylophilus DSM 20533T was 99% identicalto the sequences in S. suis and C. difficile, of which S. suis is usually associated with theanimal GIT (51–53), from which L. animalis strains can be isolated (54). The GIT ischaracterized by a high cell density, which can lead to microbial interactions andfacilitates horizontal gene transfer (HGT) events (55, 56). These findings suggest that theant(6) gene is undergoing HGT between commensal/food bacteria and pathogenicspecies, which in and of itself is a particular concern. As such, these lactobacilli couldbe acting as vectors for AR genes in the food chain and the gut. Furthermore, the ant(6)and aph(3) genes are usually found on plasmids or transposons, increasing the risk ofresistance dissemination between different bacteria (57). We found the aph(3) and
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aac(3) genes in 9 Lactobacillus strains conferring resistance to kanamycin, in accordancewith the substrate specificity of these AR determinants for aminoglycosides (46).Moreover, the aph(3) gene has been identified in Lactobacillus strains resistant tokanamycin, streptomycin, and gentamicin in a previous study (50).
Although the majority of Lactobacillus species are susceptible to antibiotics thatinhibit protein synthesis, including erythromycin, tetracycline, clindamycin, and chlor-amphenicol (14), resistance to tetracycline and chloramphenicol was the most commonphenotype detected. Even though this Lactobacillus type strain panel was characterizedby low-level resistance to clindamycin, some strains carried the lsa gene, which in E.faecium plays a key role in resistance to clindamycin and quinupristin-dalfopristin (58).The ABC transporter Walker A and B motifs (59) are conserved in the Lsa proteinpredicted for the strains in this study, suggesting that it is functional.
Chloramphenicol (cat genes) and tetracycline resistance determinants [tet(M), tet(S),tet(W), tet(O), and tet(Q)] are the most commonly acquired resistance genes found inlactobacilli (15, 19, 39, 60, 61). In particular, the most widespread resistance genes aretet(M) and tet(S) in foodborne and probiotic bacteria (12, 14, 62) due to the frequentassociation of tet(M) with conjugative transposons, such as Tn916 (63), which wascorroborated by this study. Furthermore, our data revealed the association of tet(M)with a Tn916-like transposon in the genome of L. ingluviei DSM 15946T. This transposonsequence is 99% identical to the Tn5251 of S. pneumoniae DP1322 (64), except for thepresence of tet(L) in Tn916. Notably, this study reports a Tn916-like transposon in L.ingluviei, and the transferability of this element warrants further investigation. ATn916-like transposon has been previously identified in L. paracasei and L. sakei isolatedfrom Italian traditional cheese (65, 66) and in L. salivarius JCM 1046 (22).
Notably, we detected the simultaneous presence of tet(M) and erm(B) genes in L.amylophilus DSM 20533T and L. amylotrophicus DSM 20534T, which were flanked bytransposases and conjugative proteins in both organisms. This is probably reflectiveof/arising from the close phylogenetic relatedness between L. amylophilus and L.amylotrophicus (67). The erm(B) gene (associated with erythromycin resistance) hasbeen reported in Lactobacillus species in several studies (19, 30, 60, 68–70), where it isgenerally found in conjugative transposons located in chromosomes, in plasmids, andin nonconjugative transposons, such as Tn917 and Tn551 (15). These observationssuggest that L. ingluviei, L. amylophilus, and L. amylotrophicus could act as dissemina-tion vectors for tetracycline and erythromycin resistance. Strains of L. ingluviei and L.amylophilus are commonly isolated from animal GIT (71), whereas L. amylotrophicus hasbeen found in swine waste (67); these are considered potential hotspots for promotingthe dissemination of AR genes in the environment. Indeed, the transfer of erm(B) andtet(M) from lactobacilli to other microorganisms has been previously demonstrated invitro (62, 68, 72). The present study reveals that in many cases, the genetic basis for ARremains unknown, as no known genes were identified which could explain the asso-ciated phenotype. This could be due to the inherent insensitivity of some of the strainsto particular antibiotics due to intrinsic factors, such as cell envelope (wall/membrane)structure or the ability to produce polysaccharides. Indeed, it would be very difficult topredict these phenotypes based on just database comparisons alone, and as such,functional studies would be required to probe the basis of these resistances moredeeply. Overall, this pangenomic study reveals that while AR is widespread in lactoba-cilli, the level of resistance to widely used antibiotics in human clinical medicine is lowor not so common (with the possible exception of erythromycin and tetracycline).However, the vast majority (88%) of these strains were found to fail the EFSA guidelinesfor AR and as such might encounter problems in obtaining regulatory approval for fooduse unless the resistance is proven to be nontransferable. As such, opportunities todevelop new applications for novel Lactobacillus strains could be delayed or lost, whichhighlights the urgent need to fundamentally understand AR and its spread withinmembers of the genus. Only then can a proper framework to regulate their use forhuman and animal purposes be implemented.
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MATERIALS AND METHODSBacterial strains and growth conditions. The 197 type strains of the Lactobacillus genus used in this
study are listed in Table 1 and were obtained from the American Type Culture Collection (ATCC,Manassas, VA), BCCM/LMG Bacteria Collection (Ghent, Belgium), the Spanish Type Culture Collection(CECT, Valencia, Spain), the German Collection of Microorganisms and Cell Cultures (DSMZ, Braun-schweig, Germany), the Korean Collection for Type Cultures (KCTC, Jeollabuk-do, South Korea), the JapanCollection of Microorganisms (JCM, Koyadai Tsukuba, Japan), and the NITE Biological Resource Centre(NBRC, Nishihara, Japan). Lactobacillus strains were grown in de Man-Rogosa-Sharpe (MRS) medium(Thermo Fisher Scientific, Waltham, MA, USA) under specific conditions reported in Table 1 and kept inliquid cultures with 20% (w/vol) glycerol at �80°C for long-term storage.
Antimicrobial susceptibility testing. The MICs of several antibiotics were determined using brothmicrodilution methods according to the Clinical and Laboratory Standards Institute (CLSI; www.clsi.org),the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast.org), and ISOstandards. In particular, VetMIC plates (National Veterinary Institute, Uppsala, Sweden) for LAB were usedcontaining serial 2-fold dilutions of 16 antibiotics (ampicillin, ciprofloxacin, clindamycin, chlorampheni-col, erythromycin, gentamicin, kanamycin, linezolid, neomycin, penicillin, quinupristin-dalfopristin, rifam-pin, streptomycin, tetracycline, trimethoprim, and vancomycin). These antibiotics represent the mainclasses of antimicrobials employed in human and veterinary treatments and overlap the main antibioticsof interest to the EFSA.
MICs were evaluated in LAB susceptibility test medium (LSM) (73), a mixed formulation containing90% Iso-Sensitest broth and 10% MRS Difco broth (Beckton, Dickinson and Company, Le Pont-de-Claix,France) supplemented with 0.05% (wt/vol) L-cysteine, as described in the ISO 10932 (IDF 223) document(74) and recommended by the EFSA (9). L. paracasei LMG 12586 was used as a control strain. Briefly,individual Lactobacillus strains were grown on MRS agar (with supplements as required for the specificstrains and incubation for 24 to 48 h depending on the strain). One-microliter sterile loops with biomassfrom approximately 3 to 5 colonies were suspended in 4 ml maximum recovery diluent (MRD; Oxoid Ltd.,Basingstoke, Hants, UK) sterile saline solution to obtain a concentration of approximately 3 � 108 CFU/mlin each case. This suspension was diluted 1:1,000 in LSM broth (final concentration, approximately3 � 105 CFU/ml), and then 100 �l was added to each well of the VetMIC plate. This test was performedusing a minimum of three biological replicates for each strain. Plates were incubated under anaerobicconditions at the recommended temperatures for 48 h. MICs were read as the lowest concentration ofan antimicrobial agent at which visible growth was inhibited. Epidemiological cutoff (ECOFF) values wereretrieved from reference 9. Breakpoints for antibiotics not covered by EFSA were adopted from refer-ences 30 and 31.
Identification of resistance genes. The annotated sequences of the available genomes for the typestrains of the genus Lactobacillus (25) were downloaded from NCBI using the accession numbersreported in Table 1. These sequences were employed to query the Comprehensive Antibiotic ResistanceDatabase (CARD, version 1.0.6; http://arpcard.mcmaster.ca) (75) through the Basic Local AlignmentSearch Tool (BLAST; https://blast.ncbi.nlm.nih.gov) in order to identify all AR genes involved in theresistance phenotypes observed. A gene was annotated as a putative AR determinant according to itsbest BLASTP hit in CARD, with a threshold of amino acid sequence identity of �30% and query coverageof �70%. In addition, the amino acid sequences of all AR genes retrieved from CARD, resulting in areference data set of 2,163 amino acid sequences, were aligned against the annotated genomesequences of the collection, and the best BLASTP hits were filtered as described above. In order tominimize putative false-negative or false-positive outputs, only the putative AR determinants obtainedfrom both approaches were considered for subsequent analyses. Specifically, each putative AR deter-minant was manually annotated querying the NCBI nonredundant (NR) protein database to verify itsputative function in the resistome and to determine its involvement in acquired phenotypes.
Phenotype-genotype correlation. By focusing on the nine antibiotics for which the EFSA definedreference ECOFFs (9) and the genome sequences available for 161 Lactobacillus strains, a total of 1,449phenotypic tests were considered for the phenotype-genotype correlation. Each interpretation of aresistant or susceptible phenotype to a given antimicrobial agent was compared with the presence orabsence of a known corresponding resistance gene(s) manually annotated and/or structural genemutations identified through genome sequence analysis (76–78). The overall correlation betweenphenotype and genotype was classified as positive when genomic data agreed with phenotypic testing;thus, resistance and susceptible phenotypes correlated with the presence or absence of one or more ARgenes, respectively. Otherwise, the correlation was considered negative.
Flanking regions of the AR genes. The genetic composition of upstream and downstream se-quences flanking tetracycline and erythromycin resistance genes was characterized by performing aBLASTN and BLASTX alignment of the contigs carrying the AR genes against the NCBI NR database. Thisanalysis was carried out for L. ingluviei DSM 15946T, L. amylophilus DSM 20533T, and L. amylotrophicusDSM 20534T and allowed us to identify mobile genetic elements which might be involved in the spreadof AR determinants.
SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM
.01738-18.SUPPLEMENTAL FILE 1, PDF file, 2.9 MB.
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ACKNOWLEDGMENTSWork in the APC Microbiome Ireland was supported by a centre award from Science
Foundation Ireland, grant SFI/12/RC/2273. E.S. has received funding from the EuropeanUnion’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 659801. I.C. has received funding from the University ofVerona under the CooperInt Internalization Program.
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