APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2008, p. 6032–6040
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 19
Removal of Antibiotic Resistance Gene-Carrying Plasmids from
Lactobacillus reuteri ATCC 55730 and Characterization of the
Resulting Daughter Strain, L. reuteri DSM 17938?
Anna Rosander,1† Eamonn Connolly,2and Stefan Roos1*
Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden,1and
BioGaia AB, P.O. Box 3242, SE-103 64 Stockholm, Sweden2
Received 1 May 2008/Accepted 4 August 2008
The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibi-
otics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes
when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially
available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this
if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify
the organism before subsequently testing whether the probiotic characteristics were retained. No known ?-lactam
resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and
three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The ?-lactam resistance in ATCC
55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not
transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide
resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by
removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison
of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the
retained probiotic properties of the daughter strain.
The use of probiotic bacteria, generally members of the
genera Lactobacillus and Bifidobacterium, in foods and supple-
ments that promote health or prevent disease, has followed a
long history of safe use of these organisms (8). The spread of
antibiotic resistance determinants in the environment is un-
doubtedly primarily a consequence of indiscriminate use of
antibiotics in both animals and humans. However, this concern
has led to the idea that food-borne lactic acid bacteria may act
as reservoirs of antibiotic resistance genes when purposely
distributed in large doses to the microbiota of the gastrointes-
tinal tract (17, 38). The outcome of the EU PROSAFE project
was to recommend that all future probiotics should not contain
known antibiotic resistance traits (40), and currently the Eu-
ropean Food Safety Authority qualified presumption of safety
proposals seem to be following this recommendation (6).
Bacterial antibiotic resistance can be either intrinsic or ac-
quired (16). Intrinsic resistance is a natural resistance present
in all strains of a bacterial species, while acquired resistance is
often identifiable as a resistance found in only a certain num-
ber of members of a particular species. Although intrinsic
resistance is generally accepted to be nontransferable, ac-
quired resistance may be more easily transferred to other bac-
terial species, particularly if the resistance trait is located on a
plasmid in the carrier strain (37).
Lactobacillus reuteri ATCC 55730, a commercially available,
well-documented probiotic (health-promoting) bacterium (1,
23, 27, 34, 46–48), has been shown to possess a series of
intrinsic antibiotic resistances common to this particular spe-
cies (18, 25, 26). It does, however, carry specific, unusual re-
sistances to tetracycline and lincosamides, as well as a ?-lactam
resistance which appears in approximately half of the members
of this species (18, 24). With a draft genome sequence of L.
reuteri ATCC 55730 determined in our laboratory (7), the aim
of this study was to define the origin, localization, and charac-
ter of these unusual resistances. Subsequent localization of
tet(W) and lnu(A) on plasmids led to the removal of these
plasmids and validation of the properties retained by the
daughter strain generated.
MATERIALS AND METHODS
Bacteria, culturing, DNA preparation, and primers. The bacterial strains and
primers used in this study are listed in Tables 1 and 2, respectively. The primers
were obtained from Invitrogen (Carlsbad, CA). L. reuteri strains were grown in
MRS broth or on MRS agar (Oxoid, Basingstoke, United Kingdom). Plates were
incubated in an anaerobic atmosphere (GasPak; BD, Sparks, MD). For antibiotic
supplementation, 100 ?g ml?1tetracycline (Sigma, St. Louis, MO; MRS-Tet) or
8 ?g ml?1lincomycin (Sigma; MRS-Lin) was used unless otherwise stated.
Bacterial DNA was prepared with the DNeasy tissue kit (Qiagen, Hilden, Ger-
many) according to the manufacturer’s instructions.
Identification of plasmids and putative resistance genes. Identification of
plasmids in L. reuteri ATCC 55730 was achieved by analysis of the draft genome
sequence of this strain (7). The genes encoding putative penicillin resistance
proteins and penicillin-binding proteins (Pbp) were identified by BLAST
searches (3) against the genome sequence with known resistance determinants
(GenBank accession numbers: Bla1, AAK53749; Bla2, AAS42360; BlaZ,
* Corresponding author. Mailing address: Department of Microbi-
ology, Swedish University of Agricultural Sciences, P.O. Box 7025,
SE-750 07 Uppsala, Sweden. Phone: 46 18 67 33 82. Fax: 46 18 67 33
92. E-mail: email@example.com.
† Present address: Department of Biomedical Sciences and Veteri-
nary Public Health, Swedish University of Agricultural Sciences, Box
7009, SE-750 07 Uppsala, Sweden.
?Published ahead of print on 8 August 2008.
CAB94802; FibA, CAB89120; FibB, CAB89121; MecA, CAG39068; MecR1,
NP_720910; Pbp1b, NP_722289; Pbp2a, NP_722252; Pbp2b, NP_721030; Pbp2x,
NP_720898; PbpX, NP_721297). Also, the tetracycline and lincomycin resistance
genes tet(W) and lnu(A) were searched for by using known resistance genes
(GenBank accession numbers ABC18266 and YP_473355, respectively).
Sequencing of Pbp-encoding genes. For all identified Pbp-encoding genes, one
primer pair located close to the start and end of the gene was designed and for
all but lr1752, one pair located approximately 500 bp from the first pair was also
designed (Table 2). The primers were used for PCR amplification of the genes
encoding the Pbp from ampicillin-sensitive L. reuteri strains DSM 20016, DSM
20015, and ATCC 55148 and ampicillin-resistant strains ATCC 55730, ATCC
55149, and CF48-3A. illustra PuReTaq Ready-To-Go PCR Beads (GE Health-
care, Uppsala, Sweden), primers (10 pmol of each), and DNA (0.5 ?l) were
added to the PCR mixture and amplified with the following program: 95°C for 5
min; 30 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 2 min; and 72°C for
10 min. The PCR products were purified and thereafter sequenced with the same
set of primers. After assembly of the genes (with Contig Express [Invitrogen]),
the corresponding protein sequences were aligned and compared with Lactoba-
cillus Pbp obtained from genomes available in GenBank (L. acidophilus,
CP000033; L. brevis, CP00041; L. casei, CP000423; L. delbrueckii, CR954253; L.
gasseri, CP000413; L. helveticus, CP000517; L. johnsonii, AE017198; L. planta-
rum, AL935263; L. sakei, CR936503; L. salivarius, CP000233).
Curing of plasmids. Plasmid curing was done by protoplast formation and
regeneration essentially as described by Vescovo et al. (43). An overnight culture
of ATCC 55730 was diluted to an optical density at 600 nm (OD600) of 0.1 in a
10-ml MRS culture and grown at 37°C to an OD600of 0.7 to 0.8. Cells were
collected by centrifugation at 3,000 ? g for 10 min, washed in 10 ml Nanopure
water, recentrifuged, and resuspended in 2 ml of protoplast buffer (0.2 M sodium
phosphate, 0.5 M sucrose, 20 mM MgCl2, pH 7.0). The cells were then mixed
with an equal volume of protoplast buffer containing 10 mg ml?1lysozyme
(Sigma) and incubated at 37°C for 1 h. Protoplasts were harvested by centrifu-
gation at 3,000 ? g for 15 min, washed with 20 ml protoplast buffer, recentri-
fuged, and resuspended in 1 ml of protoplast buffer. Dilutions in protoplast
buffer were then plated on MRS agar with 0.5 M sucrose for regeneration.
Dilutions in Nanopure water were plated on MRS agar to assess the number of
remaining whole cells. The number of CFU was determined after 1 and 2 days
of anaerobic incubation at 37°C. Regenerated colonies were picked to MRS and
TABLE 1. L. reuteri strains used in this study
StrainDescription Origin or reference
Alternative name, SD2112; TetrLinrAmpr; human milk isolate
ATCC 55730 cured of plasmid pLR581; TetsLinrAmpr
ATCC 55730 cured of plasmids pLR581 and pLR585; TetsLinsAmpr
Type strain; TetsLinsAmps; human intestinal isolate
Amps; cow manure isolate
Amps; chicken intestinal isolate
Ampr; turkey intestinal isolate
Ampr; human fecal isolate
aDSMZ, German Collection of Microorganisms and Cell Cultures.
TABLE 2. Primers used in this study
Detection of lr1989aon pLR580
Detection of lr1989aon pLR580
Detection of lr2004 on pLR581
Detection of lr2004 on pLR581
Detection of lr2084bon pLR584
Detection of lr2084bon pLR584
Detection of lr2102 on pLR585
Detection of lr2102 on pLR585
Detection of tet(W)
Detection of tet(W)
Detection of lnu(A)
Detection of lnu(A
Sequencing of pbp1a
Sequencing of pbp1a
Sequencing of pbp1a
Sequencing of pbp1a
Sequencing of pbp2b
Sequencing of pbp2b
Sequencing of pbp2b
Sequencing of pbp2b
Sequencing of pbp2a
Sequencing of pbp2a
Sequencing of pbp2a
Sequencing of pbp2a
Sequencing of pbp2x
Sequencing of pbp2x
Sequencing of pbp2x
Sequencing of pbp2x
Sequencing of pbpX
Sequencing of pbpX
5?-ATT TTC CAC CCG CAT ATT CA-3?
5?-TGC ATC ACG AAT CAA ACC AT-3?
5?-AGG TGA AGC ATT TCG AGC AT-3?
5?-GGC TTT CCG TCA TCA TCA GT-3?
5?-TTT GGC TGG CAA AAT CAT TC-3?
5?-TTT TTG CAG CAT TGA AAA CG-3?
5?-GAA CGG AAG CAA CAA CGA AT-3?
5?-CGT TTG GTT GGA GAA GTG GT-3?
5?-TTC GCT GGG ATA CTT GAA CC-3?
5?-TTT TTA CCT GGA CCG TTT CG-3?
5?-TGG AAA ACA ACA AAG AGA ACA CA-3?
5?-CCA GAA TGA AAA AGA AGT TGA GC-3?
5?-ATC AGT CAA CGC GTA GTG AGC-3?
5?-GCA TCG TCC CAA TCA GAT G-3?
5?-AAC GTA AAG CCC AAG AAG CA-3?
5?-TGC AGC AGA AAC TTG GAG TG-3?
5?-CAC GTA CCA AAA AGC GTC AA-3?
5?-AAC CCT TGG GAC TCT GAA CA-3?
5?-GCA GCA ATG AGT GGA GCA TA-3?
5?-GTT TCA CCA GGC AAG TCG AT-3?
5?-TCA GAT TTG AAA GAG CGA ATC A-3?
5?-ATC GAA CAT TGC TCC ACC AT-3?
5?-GCG GTT GAG GTA GAA AAC CA-3?
5?-CAA AGA CTG CAT AGG CAC GA-3?
5?-AGC CAC AAC GAA CGA AAA AT-3?
5?-TTG AAC GGT TAA AGT TGT TCC A-3?
5?-AAG GCG AAT TTG CTT CTC AA-3?
5?-CAG TTG TGG TCT TTC CAG CA-3?
5?-TTT TTG ACG CTT TGT TCC TTT-3?
5?-AAT TAT GGA GAA TAT CAT CCG AAG C-3?
aGenBank accession number EU620430.
bGenBank accession number EU620435.
VOL. 74, 2008CURING OF ANTIBIOTIC RESISTANCE IN L. REUTERI 6033
MRS-Tet agar for identification of bacteria cured of pLR581 and to MRS and
MRS-Lin agar for bacteria cured of pLR585. Plasmid-cured candidates were
identified by nongrowth on MRS agar supplemented with antibiotics.
PCR detection of plasmids and resistance genes and repetitive-sequence-
based PCR (rep-PCR) typing. The plasmids and resistance genes of strain ATCC
55730 were detected by PCR by the same method as for Pbp-encoding genes.
Primers 1989f and 1989r, 2004f and 2004r, 2084f and 2084r, and 2102f and 2102r
(Table 2), detecting replication protein genes on pLR580, pLR581, pLR584, and
pLR585, respectively (10 pmol of each), and primers 1996f and 1996r and
primers 2105f and 2105r, detecting tet(W) and lnu(A), respectively, were used.
Bacterial isolates were fingerprinted by rep-PCR. PuReTaq Ready-To-Go
PCR Beads, DNA (0.5 ?l), and primer 5?-GTG GTG GTG GTG GTG-3? (20
pmol) were mixed, and the reaction was performed according to the program
described by Versalovic et al. (41): 95°C for 7 min; 30 cycles of 90°C for 30 s, 95°C
for 1 min, 40°C for 1 min, and 65°C for 8 min; and 65°C for 16 min. The PCR
products were separated by electrophoresis (1% agarose gel in 0.5? TBE buffer)
and stained with ethidium bromide. Digitalized images were captured under UV
Determination of MICs. L. reuteri strains were grown in MRS broth for 16 h
at 37°C. After dilution to 105CFU ml?1, 1 ?l of each strain was spotted onto
MRS plates containing lincomycin or clindamycin (Sigma) at concentrations of 0,
0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 ?g ml?1. After adsorption of the drops, the
plates were incubated anaerobically at 37°C for 24 h. The MIC was defined as the
lowest antibiotic concentration at which there was no visible bacterial growth.
MICs of tetracycline were determined by Etest (AB Biodisk) on bacteria growing
on MRS agar plates according to the instructions from the manufacturer.
Colony and cell morphology. L. reuteri strains were grown for 48 h on MRS
plates at 37°C in an anaerobic atmosphere before photographs of single colonies
were taken. The morphology of the bacterial cells was investigated by phase-
contrast microscopy (?400 magnification).
Fermentation pattern and reuterin production. Fermentation patterns were
determined with api 50 CHL (BioMe ´rieux, Marcy l’Etoile, France) according to
the instructions from the manufacturer. Reuterin production (13) was measured
by the reaction between reuterin (3-hydroxy-propinoaldehyde) produced by the
L. reuteri strains with 2,4-dinitrophenylhydrazine to form a red hydrazone. The
bacteria were grown for 48 h on MRS plates (inoculated as streaks). The plates
were then overlaid with 500 mM glycerol agar (1% agar) and incubated at 37°C
for 30 min. Reuterin was detected by the addition of 5 ml 2,4-dinitrophenylhy-
drazine (0.1% in 2 M HCl). After 3 min incubation, the solution was poured off
and 5 ml 5 M KOH was added. Red zones around the colonies demonstrated the
presence of reuterin and the extent of its production.
Growth. Growth was determined by inoculation of an overnight culture to an
OD600of 0.1 in MRS broth in tubes prewarmed to 37°C. The tubes were
incubated at 37°C, and samples were taken for 8 h and the OD600was measured.
Each strain was tested in triplicate.
Binding to mucus. Binding to ovine small intestinal mucus was performed
according to Roos et al. (31). All bacterial strains were analyzed in triplicate.
Acid tolerance. Testing of survival at pH 2 was performed according to Wall et
al. (45) with a synthetic stomach juice described by Cotter et al. (15) that was
modified by adding no enzymes. Duplicate samples were taken on each of two
occasions. Statistical significance of differences was analyzed with Student’s t test.
Bile tolerance. Strains were grown for 16 h in MRS broth at 37°C, and the
stationary-phase suspensions were diluted in phosphate-buffered saline to ap-
proximately 103to 106CFU ml?1. Ten microliters of each dilution was dropped
onto MRS plates containing concentrations of bovine bile of up to 6% (Sigma
B3883). The plates were incubated for 72 h at 37°C in an anaerobic atmosphere,
after which the colonies were counted. Bacteria in exponential phase (OD600of
0.5) were also tested in the same manner. Each bacterium was analyzed on two
Pathogen inhibition. Test strains were grown in MRS broth for 16 h and plated
on MRS agar. The plates were incubated anaerobically (with Anaerogen [Ox-
oid]) for 24 h at 37°C. Isolated single colonies were then streaked in the center
of fresh MRS agar plates and incubated anaerobically for 36 h at 37°C. The
target pathogens were Candida albicans ATCC 28956, Enterobacter sakazakii
ATCC 51329, Salmonella enterica serovar Typhimurium ATCC 14028, and Clos-
tridium difficile ATCC 43593.
C. albicans and E. sakazakii were grown in 10 ml of tryptic soy broth (BBL,
BD) plus 1.5% Lab Lemco (Oxoid) and incubated for 48 h at 37°C (Candida) or
32°C (Enterobacter). The pathogens were then inoculated into freshly prepared
sterile tryptic soy broth plus 1.5% Lab Lemco and 1.5% agar with or without the
addition of glycerol (150 mM) to a target concentration of 105CFU ml?1
(Candida) or 106CFU ml?1(Enterobacter). The pathogen preparations (10 ml)
were overlaid on the streaked test strains growing on the MRS plates. Plates were
incubated anaerobically at 37°C (Candida) or 36°C (Enterobacter) for 48 h. S.
enterica serovar Typhimurium and Clostridium difficile were grown in 10 ml of
brain heart infusion broth (BBL, BD) and reinforced clostridial medium (BD),
respectively, and incubated at 37°C for 16 h. Each pathogen was then inoculated
in brain heart infusion agar or reinforced clostridial medium plus 1.5% agar
(40°C) with or without the addition of glycerol (150 mM) to get a concentration
of 105CFU ml?1. Ten milliliters of this preparation was overlaid on the test
strains growing on the MRS plates. Plates were incubated anaerobically at 37°C
for 48 h. Inhibition halos were measured in triplicate samples on three different
experimental occasions for each strain and pathogen.
Gastrointestinal passage and safety in humans. Sixteen subjects (10 female)
aged 27 ? 7 (mean ? standard deviation) years were recruited after written
informed consent was obtained at the Gastroenterology Unit, Department of
Internal Medicine, Lund University Hospital, Lund, Sweden. The Scientific Eth-
ical Committee of Lund University Hospital approved this study prior to its start.
Inclusion criteria were an age of 18 to 65 years, written informed consent, stated
availability throughout the study period, a lack of any major illnesses (allergy
symptoms were acceptable), and the mental ability to understand and fulfill the
protocol. Exclusion criteria were significant disease, pregnancy, use of oral an-
tibiotics in the 2 weeks prior to ingestion of the study product, and participation
in other clinical trials. Subjects were randomly assigned to receive a placebo (four
subjects), an ATCC 55730 standard dose (8 ? 108CFU/day; three subjects), a
DSM 17938 standard dose (8 ? 108CFU/day; four subjects), or a DSM 17938
high dose (6.5 ? 1010CFU/day; five subjects). The study was double blind with
neither the subjects nor the principal investigator aware of the contents of the
study product in each group.
Subjects were then asked to take a study product each day for 28 days. The
study product (in sealed one-dose sachets) consisted of freeze-dried powder of
the strain mixed with 1.5 g maltodextrin. The placebo contained only maltodex-
trin and was identical in appearance to the active products but had no detectable
bacteria. Sachets were kept refrigerated at all times up to the point of consump-
tion, and L. reuteri levels remained stable in parallel stability testing (data not
shown). The subjects were instructed in writing to open the sachet immediately
before consumption, pour the powder into a glass containing cold water (at least
100 ml), stir well for 15 s, and swallow directly.
Fecal samples (?5 g) were collected at baseline and on days 7, 14, and 28 and
also on days 42 and 56 (after a 2- to 4-week washout period). The samples were
stored for maximally 1 h at room temperature and maximally 1 day in the
refrigerator (not frozen) until they could be collected and delivered to the
laboratory, where they were frozen (?20°C) upon arrival. For analysis of L.
reuteri content, samples were thawed, weighed, diluted in MRS broth (1:10), and
mixed thoroughly. Aliquots (1 ml) were serially diluted and spread on MRS-
vancomycin agar plates (50 ?g ml?1vancomycin) and incubated anaerobically at
37°C for 48 to 72 h. L. reuteri colonies were confirmed by analysis of reuterin
production with the overlay assay described above. Overlay-positive clones were
picked from copied plates and analyzed for specific plasmid content to identify
them as L. reuteri ATCC 55730-like or L. reuteri DSM 17938 strains.
Fasting blood samples were collected at baseline and after 28 days and ana-
lyzed by standard procedures at the Clinical Chemistry Laboratory of Lund
University Hospital for Fe, total iron-binding capacity, hemoglobin, erythrocyte
particle concentration (erythrocytes), blood corpuscle volume, white blood cell
count, blood differential cell counts, low-density lipoprotein cholesterol, high-
density lipoprotein cholesterol, triglycerides, albumin, glucose, calcium, sodium,
potassium, phosphorus, total bilirubin, alanine aminotransferase, aspartate ami-
notransferase, glutamyl transferase, alkaline phosphatase, creatinine, urea,
urate, and C-reactive protein, and even in the absence of overt fever or illness,
a general bacterial analysis was done to detect the possible translocation of any
bacteria into the blood (only on day 28 after last study product dose was taken).
Identification of plasmids and resistance genes. Analysis of
the genome sequence of ampicillin-resistant strain L. reuteri
ATCC 55730 identified no known ?-lactam resistance gene,
but five genes encoding putative Pbp were found (Table 3).
The corresponding genes from two other penicillin-resistant
and three penicillin-sensitive L. reuteri strains were PCR am-
plified and sequenced for comparison. Alignment of the de-
duced protein sequences showed few differences, but there
were amino acid differences at four positions that correlated
6034 ROSANDER ET AL.APPL. ENVIRON. MICROBIOL.
with the expression of penicillin resistance. Two of those were
located in Pbp1a, where aspartic acid (Asp, D) in the sensitive
strains was changed to valine (Val, V) at position 399 of the
resistant strains (Asp399Val, exchange 1 [E1]) and glutamine
(Gln, Q) was changed to leucine (Leu, L) at position 479
(Gln479Leu, E2), as shown in Fig. 1. E3 was located in Pbp2a,
where phenylalanine (Phe, F) was changed to valine (Val, V)
at position 147 (Phe147Val), and the last exchange, E4, was
located in Pbp2x, where alanine (Ala, A) was changed to threo-
nine (Thr, T) at position 526 (Ala526Thr). Comparisons with
Pbp from 10 other Lactobacillus species showed that both E1
and E4 are located in conserved regions (Fig. 1). E1 leads to
the substitution of the fully conserved aspartic acid (charged)
to valine, which is an amino acid with fundamentally different
properties (hydrophobic). In the case of E4, the alanine
present in the sensitive strains has properties similar to those
of glycine (small), which is present in all other lactobacilli,
whereas the threonine present in the resistant strains has other
properties (contains a hydroxyl group). Although E2 and E3
are not located in conserved regions, these substitutions also
produce changed properties. E1 to E4 can be explained by
point mutations of the corresponding genes. E1 is caused by a
change of a GAT codon to GTT; E2, CAA to CTA; E3, TTC
to GTC; and E4, GCA to ACA. The Pbp2a- and PbpX-encod-
ing genes did not show any differences that correlated with the
resistance patterns of the strains examined.
The genome sequence of L. reuteri ATCC 55730 showed
four contigs that were found to harbor plasmid-related genes.
Further, these contigs were built up by an elevated number of
sequencing runs, indicating a higher copy number than genes
located on the chromosome. The contigs could be circularized
and therefore concluded to be plasmids, and the sizes were
found to be 8.1 (pLR580), 12.2 (pLR581), 14.2 (pLR585), and
19.1 (pLR584) kb. The genome of ATCC 55730 was also
scanned for the tet(W) and lnu(A) genes, which have previ-
ously been detected in this strain (24), and they were found as
open reading frames lr1996, located on plasmid pLR581, and
lr2105, located on plasmid pLR585, respectively (Table 4).
Removal of plasmids pLR581 and pLR585 from L. reuteri
ATCC 55730. An attempt to cure L. reuteri ATCC 55730 of
plasmids pLR581, harboring tet(W), and pLR585, harboring
lnu(A), was made. Different methods were tested (data not
shown), and a method by which the bacteria first were sub-
jected to protoplast formation with subsequent cell wall regen-
eration was found to be effective in removing the plasmids
from the bacteria. In a first trial, pLR581 was the target. After
incubation in the protoplast buffer, 100-fold more colonies
were obtained on MRS plates with sucrose than on those
TABLE 3. GenBank accession numbers of Pbp compared in this study
GenBank accession no.
Pbp1a Pbp2bPbp2a Pbp2x PbpX
aGenes described in reference 7.
bSequenced in this study.
cSequences obtained from GenBank accession number CP000705.
FIG. 1. Alignment of Pbp1a, Pbp2a, and Pbp2x from penicillin-sensitive and -resistant L. reuteri strains and the consensus sequences of Pbp
from lactobacilli available in GenBank (10 species). Bold characters represent positions where the L. reuteri sequences are identical or have an
amino acid residue with a function similar to that of the consensus sequence. Stars below the consensus sequence show positions where all
Lactobacillus sequences are identical, and dots show positions where all have similar functions. The positions where resistant strains differs from
sensitive strains (E1 to E4) are marked with gray and underlined characters, and the two substitutions (E1 and E4) that are located in conserved
regions and lead to shifts in function are marked with a black background.
VOL. 74, 2008 CURING OF ANTIBIOTIC RESISTANCE IN L. REUTERI6035
without sucrose, indicating efficient formation of protoplasts.
Two hundred colonies from the sucrose plates were examined
for growth or nongrowth on MRS and MRS-Tet agar. Seven
nongrowing colonies were replated and grew on MRS-Tet,
indicating that these colonies were false candidates. After 24 h
of further incubation of the sucrose plates, a few more colonies
appeared. Twelve such colonies were picked to MRS and
MRS-Tet agar and grown overnight. One of these colonies
grew well on the MRS plate but not at all on the MRS-Tet
plate. This colony was replated, stored at ?70°C, and desig-
nated L. reuteri ATCC 55730Tets.
L. reuteri ATCC 55730Tetswas then again subjected to proto-
plast formation in order to obtain a strain cured of pLR585. One
hundred colonies from the sucrose plates were examined for
growth or nongrowth on MRS and MRS-Lin agar. One colony
was found to grow well on the MRS plate but not at all on the
MRS plate with lincomycin. This colony was replated, stored at
?70°C, and designated L. reuteri ATCC 55730TetsLins.
Confirmation of the loss of plasmids pLR581 and pLR585
was performed by PCR. In ATCC 55730Tets, lr2004 (gene
encoding a replication protein on pLR581) and lr1996 [tet(W)]
were both missing while the other ATCC 55730 plasmids and
gene lr2105 [lnu(A)] could still be detected (Fig. 2A and B,
lanes 2). Both lr2004 and lr2102 (gene encoding a replication
protein on pLR585) were lacking in L. reuteri ATCC
55730TetsLins, whereas the genes representing the other two
plasmids were still present (Fig. 2A, lane 3). Both tet(W) and
lnu(A) was absent in ATCC 55730TetsLins(Fig. 2B, lane 3).
This clearly showed that L. reuteri ATCC 55730 had been cured
of the tet(W)- and lnu(A)-containing plasmids. The double-
cured ATCC 55730TetsLinsstrain was later deposited at
DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen) as L. reuteri DSM 17938 (L. reuteri Protectis),
and ATCC 55730Tetswas deposited as L. reuteri DSM 17686.
The genes of the removed plasmids and the putative functions
of the encoded proteins are listed in Table 4.
Comparative studies on L. reuteri ATCC 55730 and DSM
17938. The rep-PCR patterns obtained from strains ATCC
55730 and DSM 17938 were not possible to distinguish (Fig. 3),
which shows not only that DSM 17938 is a true variant of
ATCC 55730 and not a contaminant but also that removal of
pLR581 and pLR585 did not affect the rep-PCR fingerprint.
TABLE 4. Genes present on plasmids pLR581aand pLR585band
their putative functions
Putative replication protein
Putative replication protein
Tetracycline resistance protein W ?tet(W)?
Truncated extracellular protein
Arsenite efflux pump ACR3, truncated
Transcriptional regulator, ArsR
Arsenate reductase, truncated
Arsenite efflux pump ACR3
Transcriptional regulator, ArsR
Replication initiator protein
Polyketide antibiotic exporter
Polyketide transporter, ATPase
Putative replication protein
Putative replication protein
Putative transcriptional regulator
Lincomycin resistance protein ?lnu(A)?
Truncated mobilization protein
Transposase, IS30 family
aAccession number EU583804.
bAccession number EU596446.
FIG. 2. (A) Detection of plasmids by PCR. Strains were analyzed for the presence of plasmids as follows: a, pLR580; b, pLR581; c, pLR584; d, pLR585.
and lnu(A) (b) by PCR. Lanes 1 to 4 are as in Fig. 1A. Lane M, molecular size markers (sizes are shown in base pairs on the left).
6036 ROSANDER ET AL.APPL. ENVIRON. MICROBIOL.
The MICs of tetracycline for the DSM 17938 and ATCC
55730Tetsvariants were found to be 12 to 16 ?g ml?1(Table
5), showing the natural intrinsic sensitivity of this species to
tetracycline (18), compared to ?256 ?g ml?1for ATCC 55730.
This shows that the tet(W) gene is responsible for the unusually
high tetracycline resistance of ATCC 55730. Strains ATCC
55730 and ATCC 55730Tetswere both found to be resistant to
lincomycin but not to clindamycin (a derivative of lincomycin).
Removal of pLR585 resulted in a decrease in the lincomycin
MIC for DSM 17938 from ?16 to 0.25 ?g ml?1, while the
sensitivity to clindamycin was unchanged. This confirms that
the lincomycin resistance of ATCC 55730 was mediated by the
pLR585 plasmid-borne lnu(A) gene. All other intrinsic resis-
tances described for ATCC 55730 (18, 24) were unchanged in
DSM 17938 (data not shown).
Fermentation patterns. The fermentation patterns showed
that ATCC 55730 and DSM 17938 fermented L-arabinose,
ribose, galactose, glucose, maltose, lactose, melibiose, saccha-
rose, raffinose, and gluconate and were negative for the rest of
the substrates. The production of reuterin was of the same
magnitude for both strains, and there were no detectable dif-
ferences in either colony or cell morphology or in mucus bind-
ing (data not shown).
Comparison of the growth of strains DSM 17938 and ATCC
55730 showed no difference in generation time, but DSM
17938 did grow to a significantly higher density than ATCC
55730 (P ? 0.01). The final ODs of ATCC 55730 and DSM
17938 were 4.78 ? 0.13 and 6.00 ? 0.26, respectively, after 510
min of growth. Acidic challenge at pH 2.0 showed no difference
after 20 and 90 min, but at the interim 50-min point, DSM
17938 survived significantly better than ATCC 55730, with
survival rates of 41 and 20%, respectively (Fig. 4). Both ATCC
55730 and DSM 17938 tolerated bile well when they were in
stationary phase. In different experiments, 30 to 90% of the
cells grew on MRS agar with 6% bile. Survival and growth of
both strains were lower in the exponential phase (0.1 to 1%
growing cells). However, no difference in bile tolerance could
be detected between the strains (data not shown).
Pathogen inhibition. Strains ATCC 55730 and DSM 17938
were both able inhibit the growth of C. albicans, E. sakazakii,
S. enterica serovar Typhimurium, and C. difficile, and there was
no difference between the strains in strength of inhibition in
the presence or absence of glycerol (Table 6). In the absence of
glycerol, neither strain showed any inhibitory effect on C. al-
Clinical trial. Compliance (participant self-reported) was
99% between days 1 and 14 and 97.5% between days 14 and 28.
General health examinations revealed no alterations in weight,
pulse, blood pressure, or body temperature in any group dur-
ing the supplementation period, and blood safety and meta-
bolic parameters were unchanged in any of the groups (data
not shown). Blood taken directly after supplementation on day
28 was analyzed in 10 subjects, including 4 subjects from the
FIG. 3. (GTG)5-PCR (rep-PCR)-generated genomic fingerprints
of L. reuteri DSM 17938 (lane 1), L. reuteri ATCC 55730Tets(lane 2),
and L. reuteri ATCC 55730 (lane 3). Lane M, molecular size markers
(sizes are shown in base pairs on the right).
TABLE 5. MICs of tetracycline, lincomycin, and clindamycin for
L. reuteri strains
MIC (?g ml?1)
FIG. 4. Acid tolerance of parent strain L. reuteri ATCC 55730
(light bars) and daughter strain L. reuteri DSM 17938 (dark bars). The
columns show the proportion of bacteria surviving at pH 2.0 (mean of
quadruplicates ? standard deviation).***, P ? 0.001 for 55730 versus
17938 (Student’s t test).
VOL. 74, 2008 CURING OF ANTIBIOTIC RESISTANCE IN L. REUTERI6037
high-dose 17938 group. All were negative for bacteremia.
Thus, supplementation with L. reuteri DSM 17938 daily for 28
days at doses of up to 6.5 ? 1010CFU/day was well tolerated
during the trial.
Three of the 16 individuals had detectable L. reuteri in their
baseline fecal samples (Table 7). One of these isolates was
55730 like, while the others were unidentifiable L. reuteri
strains. Standard ingested doses of L. reuteri ATCC 55730 and
DSM 17938 led to similar levels of L. reuteri detection in the
feces (Table 7). Increasing the dose of L. reuteri DSM 17938
100-fold led to 100-fold higher levels in the feces (Table 7).
The levels detected were maximal at day 7 in all L. reuteri-
supplemented groups and remained at this level during the
period of supplementation. After a washout period of at least
2 weeks, there was no evidence of L. reuteri in the feces of any
of the supplemented subjects (Table 7). Identification of fecal
isolates by plasmid analysis obtained from samples taken dur-
ing supplementation were found to correspond to the DSM
17938 plasmid pattern for all of the subjects ingesting this
strain, with the exception of three samples where the strain was
either ambiguous or resembled 55730. Isolates from the 55730
samples were all 55730 like, with the exception of two, which
were ambiguous. Placebo-supplemented subjects showed no
fecal colonization with L. reuteri, except for two observations of
unknown L. reuteri in one individual.
?-Lactam resistance is conferred mainly by two mechanisms:
drug inactivation by ?-lactamases and target site (i.e., Pbp)
alterations. For both types, transferable resistance determi-
nants have been identified, e.g., ampC and mecA, respectively
(21, 30). When the nature of the ?-lactam resistance of L.
reuteri ATCC 55730 was investigated, point mutations in
Pbp1a, Pbp2a, and Pbp2x were identified. Alterations in both
Pbp1a and Pbp2x are often reported to cause resistance to
?-lactams in streptococci, and high-level resistance is often
caused by alterations in more than one of the proteins (14, 22).
The Pbp are “housekeeping” proteins found in all bacteria. In
L. reuteri ATCC 55730, the corresponding genes are all located
on the chromosome and are not coupled to any mechanisms
for transfer to other bacteria. In addition, there is no other
gene present in the genome sequence with similarities to
known penicillin resistance genes. We therefore suggest that
the ?-lactam resistance of L. reuteri ATCC 55730 is caused by
a number of point mutations in the genes encoding Pbp1a,
Pbp2a, and/or Pbp2x and that this resistance can be regarded
The ribosome protection-type resistance gene tet(W) (5, 35)
is one of the most widespread tetracycline resistance genes in
environmental samples (4, 44). Lincosamide nucleotidyltrans-
ferases encoded by lnu (formerly lin) genes inactivate lincos-
amides by adenylation (9, 11), and the gene has been shown to
occur on plasmids (29). Lincosamide antibiotics include linco-
mycin and clindamycin, a semisynthetic derivative of lincomy-
cin (10). Coupled resistances to lincomycin and clindamycin
seem to be associated with the lnu(F) gene but not with the
lnu(A) gene, which only confers lincomycin resistance (2). Our
finding that L. reuteri ATCC 55730 is not resistant to clinda-
mycin is in accordance with others (18, 26) but not with the
result of Kastner et al. (24). The reason for the difference is not
known, but the method validation of Egerva ¨rn et al. (19) in
accordance with the recent ACE-ART project in Europe indi-
cates the reliability of this study. The resistance to lincomycin
in L. reuteri ATCC 55730 is caused by lnu(A), located on
pLR585, but both L. reuteri ATCC 55730 and L. reuteri DSM
17938 are sensitive to clindamycin.
Our data clearly indicate that L. reuteri ATCC 55730 harbors
four plasmids and that two of them harbored the tet(W) and
lnu(A) genes, respectively. The total number of plasmids is not
in agreement with the report of Klein et al. (26), who con-
cluded that the strain harbors six plasmids with sizes in the
range of 5 to 24 MDa, which corresponds to approximately 7.7
to 37 kb. The difference is probably due to the methods used.
Klein et al. detected the plasmids by agarose gel electrophore-
sis, which can give an overestimation of the number of plas-
mids since plasmids can be present in different forms and can
thus give rise to several bands. Different curing techniques
have been used on Lactobacillus species, including chemical
curing with agents like novobiocin, sodium dodecyl sulfate,
TABLE 6. Pathogen inhibition by L. reuteri strains with and without
glycerol in the substrate
Inhibition zone ? SD (mm)
ATCC 55730 DSM 17938
S. enterica serovar
43.7 ? 2.140.0 ? 2.0 42.7 ? 2.139.7 ? 2.1
42.0 ? 1.0
41.0 ? 1.0
41.0 ? 1.0
32.7 ? 1.5
29.7 ? 0.6
41.0 ? 2.0
40.7 ? 1.5
42.3 ? 1.5
33.0 ? 1.0
30.0 ? 1.0
aNI, no inhibition.
TABLE 7. Fecal L. reuteri levels in subjects given either L. reuteri ATCC 55730 or DSM 17938 for 28 days
Group (no. of
Amt of L. reuteria
Day 0Day 7 Day 14Day 28Day 42Day 56
17938 high (5)
17938 standard (4)
aLog10CFU g?1(wet weight) feces. Values are the mean of the samples in which L. reuteri was detected (the number of samples is in parentheses). Variation between
samples was large but similar for all data points.
bL. reuteri was detected in one of the subjects at 3.7 to 6.9 log10CFU g?1.
6038ROSANDER ET AL.APPL. ENVIRON. MICROBIOL.
acridine dyes, and ethidium bromide or using high tempera-
ture or protoplast formation (12, 28, 32, 33, 36, 42, 43). We
have tested all of these methods, and protoplast formation was
the only one that succeeded in curing ATCC 55730 of the
plasmids. Also, protoplast formation was considered to be an
appropriate method since it does not involve genetically mod-
ifying the organism and does not induce mutations in the
bacterial chromosome that might be a consequence of the use
of other curing agents.
Besides the tet(W) gene, eliminated plasmid pLR581 har-
bors 13 other genes. Most of them fall into two categories:
replication protein-encoding genes and arsenic resistance
genes (several of the latter are truncated). The pLR585 plas-
mid also harbors 13 genes besides lnu(A), 2 of which encode
polyketide antibiotic exporters (lr2096 and lr2097). Impor-
tantly, none of the genes on the plasmids have any connection
or function of importance for the known probiotic character-
istics of this strain.
L. reuteri DSM 17938 containing only two plasmids (pLR580
and pLR584) has the same rep-PCR profile, fermentation pat-
tern, reuterin production, morphology, growth rate, adhesion
to mucus, pathogen inhibition profile, and bile tolerance as L.
reuteri ATCC 55730. The abilities of the daughter strain to
grow to a higher density and to survive better under acidic
conditions are probably related to a decreased burden with
respect to DNA replication and thereby a higher competitive
ability compared to the parent strain. L. reuteri ATCC 55730
has earlier been shown to tolerate passage through the human
gastrointestinal tract, as shown by fecal shedding, and attaches
and grows in the stomach, duodenum, and ileum of subjects
ingesting the strain (20, 39). Laboratory analysis of both parent
strain L. reuteri ATCC 55730 and daughter strain L. reuteri
DSM 17938 showed at least similar acid and bile tolerance and
mucus binding, predicting that DSM 17938 should not have a
changed ability to colonize the entire human gastrointestinal
tract compared to ATCC 55730. Fecal analysis after ingestion
confirmed this and shows that L. reuteri DSM 17938 survives
throughout the human gastrointestinal tract in the same way as
L. reuteri ATCC 55730 and further that colonization is only
temporary, as is common in probiotic strains. L. reuteri DSM
17938 could be unequivocally identified by the plasmid pattern,
which showed a direct correlation between the ingested and
shed strains. The blood safety parameters studied demonstrate
that L. reuteri DSM 17938 has a safety profile similar to that
of the L. reuteri ATCC 55730 strain. Furthermore, an analysis
of the L. reuteri DSM 17938 genome annotation did not reveal
any further gene or gene cluster known to be involved in
virulence or antibiotic resistance (not shown).
In conclusion, we have demonstrated that L. reuteri ATCC
55730 could be cured of two independent plasmids carrying
unwanted antibiotic resistance traits and that the resultant L.
reuteri DSM 17938 strain did not lose any of its probiotic
characteristics. This may be a valuable alternative for other
true probiotic strains that are found to harbor plasmid-borne
We thank Noris Carbajal and Uma Nathan for performing the
pathogen inhibition assays, Dan Nilsson for fecal analyses, and Tor
Melin and Karin Diderot for coordination and assistance in the per-
formance of the clinical trial.
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