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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2008, p. 1394–1401 0099-2240/08/$08.00⫹0 doi:10.1128/AEM.01463-07Copyright 2008, American Society for Microbiology. All Rights Reserved.
Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9䌤 Mohammed Salim Ammor,1§ Miguel Gueimonde,1 Morten Danielsen,2 Monique Zagorec,3 Angela H. A. M. van Hoek,4 Clara G. de los Reyes-Gavila Baltasar Mayo,1 and Abelardo Margolles1* Instituto de Productos La ´cteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain1; Chr. Hansen A/S, Bøge Alle ´ 10-12, 2970 Hørsholm, Denmark2; Unite ´ Flore Lactique et Environnement Carne ´, UR309, INRA, Domaine de Vilvert, F-78350 Jouy-en-Josas, France3; and RIKILT – Institute of Food Safety, Wageningen UR, Bornsesteeg 45, Wageningen, The Netherlands4 Received 30 June 2007/Accepted 26 December 2007 Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the
safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants.
However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that
tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is
mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-
carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is
highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to
honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L.
sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC, <32
␮g/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 ␮g/ml,
suggesting that Tet L and Tet M confer different levels of resistance in L. sakei. Remarkably, in the absence of
tetracycline, a basal expression of both genes was detected for L. sakei Rits 9. In addition, subinhibitory
concentrations of tetracycline affected the expression patterns of tet(M) and tet(L) in different ways: the
expression of tet(M) was induced only at high tetracycline concentrations, whereas the expression of tet(L) was
up-regulated at lower concentrations. This is the first time that two different mechanisms conferring resistance
to tetracycline are characterized for the same strain of a lactic acid bacterium.
Lactobacillus sakei is a facultative heterofermentative psy- transfer between industrial bacterial species and food-borne chrotrophic lactic acid bacterium (LAB) that has been isolated pathogens. Therefore, a consensus criterion has been issued from several raw fermented food products of plant and animal for which strains to be used in food systems should be free of origin. It is found in kimchi, silage, cheese, sauerkraut, sour- potentially transferable antibiotic resistance traits (15).
dough, and smoked fish but is mainly found in meat products Tetracyclines are a group of broad-spectrum antibiotics (4, 7, 8). Though some L. sakei strains have been identified as whose general usefulness has been reduced with the onset of responsible for the spoilage of vacuum-packaged meat prod- bacterial resistance. Tetracycline resistance (Tcr) is the most ucts, this bacterium is widely used as a starter culture for the frequent bacterial antibiotic resistance found in nature and is production of fermented sausages and has biotechnological mostly acquired by horizontal gene transfer. Nowadays, 39 potential for biopreservation and food safety (6). Lactobacilli acquired tetracycline determinants are known for bacteria are generally recognized as safe and they are not responsible (37). Usually, these genes code for energy-dependent efflux for human infections in healthy people (46). However, they systems or for proteins that protect the bacterial ribosomes might act as reservoirs of transmissible antibiotic resistance from the blockage of protein synthesis (9, 10, 37). In rare cases, genes that under certain conditions could be transferred to Tcr is mediated through direct inactivation of the antibiotic food or gut microbiota (27). In addition, the emergence of (40) or by mutations in the 16S rRNA that prevent the binding antibiotic-resistant food-borne pathogens originating from of tetracycline to the ribosome (38).
meat products (14) raises the question of the possibility of gene Currently, data on antibiotic resistance in lactobacilli are relatively scarce. However, in recent years a number of studieshave correlated atypically high phenotypic resistances with the * Corresponding author. Mailing address: Instituto de Productos presence of tet genes (11, 17, 18, 19, 20, 26). Tetracycline ´cteos de Asturias, Consejo Superior de Investigaciones Cientı´ficas resistance in Lactobacillus has commonly been associated with (IPLA-CSIC), Ctra. Infiesto s/n, 33300 Villaviciosa, Asturias, Spain.
Phone: 34 985 89 21 31. Fax: 34 985 89 22 33. E-mail: amargolles@ipla the presence of tet(M) (19, 20), but recently the gene coding for the efflux transporter Tet L was also described for some § Present address: Laboratory of Microbiology and Biotechnology of cloacal isolates (5). However, data about the functionality of Foods, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, both genes when they coexist in the same bacterium were not 䌤 Published ahead of print on 11 January 2008.
available until now. In this context, this study reports the iso- TETRACYCLINE RESISTANCE IN L. SAKEI Rits 9 TABLE 1. Primers used in this study Sequence (5⬘–3⬘) Reference or source J. M. Collard, personal tet(M) flanking regions tet(M) flanking regions a R, reverse; F, forward.
b ITS, internal transcribed spacer.
lation of a Tcr L. sakei strain from Italian Sola cheese and the (33). Total RNA was extracted from cells grown up to an OD molecular characterization of both ribosomal protection- and use of an RNeasy mini kit (Qiagen) following the manufacturer's instructionswith the following modifications: the lysis buffer was supplemented with 30 ␮g/ml efflux pump-encoding genes, tet(M) and tet(L), responsible for lysozyme (Sigma) and 100 U/ml mutanolysin (Sigma) and the samples were Tcr in this strain.
incubated for 30 min under gentle stirring. DNA was removed by on-columndigestion using an RNase-free DNase set (Qiagen). Four microliters of RNA MATERIALS AND METHODS
(about 3 ␮g) was reverse transcribed into cDNA by use of a cDNA archive kit(Applied Biosystems, Foster City, CA). The cDNA was stored at ⫺80°C until Bacterial strains and growth conditions. L. sakei Rits 9 was isolated on MRS
agar (Oxoid Limited, Hampshire, United Kingdom) containing 16 ␮g/ml tetra- (ii) Microarray hybridization. DNA microarrays contained 327 oligonucleo-
cycline (Sigma, St. Louis, MO) from an Italian Sola cheese made from raw cow's tides (50 to 60 base pairs long), including control probes and oligonucleotides milk according to International Dairy Federation (IDF) standard 122C:1996. An specific for 250 antibiotic resistance genes, including 28 tet genes (1). Spotting of internal 474-bp fragment of the 16S rRNA gene and an internal 424-bp fragment the oligonucleotides, hybridization conditions, and analysis of the results were as of the katA gene (encoding the L. sakei heme-dependent catalase) were ampli- previously described (43).
fied using primers Y1 (45) and R518 and primers 702-F and 310-R (2), respec- (iii) Real-time PCR conditions. Real-time PCR was used to assess the influ-
tively (Table 1). The resulting nucleotide sequences showed to be identical to the ence of different subinhibitory concentrations of tetracycline (16, 32, and 64 corresponding partial sequences in L. sakei 23K (6). L. sakei 23K, a laboratory ␮g/ml) on the expression levels of tet(L) and tet(M) in L. sakei Rits 9. All the strain originally isolated from sausage and cured of plasmids (3), was used as the primers used in this study are listed in Table 1. Primers TetL-FW-RT and recipient strain for genetic constructions. L. sakei 23K electrocompetent cells TetL-RV-RT and TetM-FW-RT and TetM-RV-RT were designed to amplify were prepared and transformed with pLS55 as described previously (3). After anincubation period of 2 h following electroporation, bacterial suspensions were internal fragments of 70 and 78 bp, respectively. The rRNA 16S-to-23S inter- plated on MRS medium containing 4, 8, 16, or 32 mg/liter tetracycline and genic region was used as the endogenous control by using Lactobacillus-specific incubated for 48 h at 30°C.
primers (24). PCR was performed in an ABI Prism 7500 fast real-time PCR Bacterial strains were stored at ⫺80°C and routinely cultured on MRS agar.
system (Applied Biosystems), and SYBR green I fluorophore was used to cor- All incubations were performed aerobically at 30°C for 48 h.
relate the amount of PCR product with the fluorescent signal. Amplification was Determination of the MICs of tetracycline. The MICs of tetracycline for the
carried out in a 25-␮l final volume containing 1 ␮l of cDNA as a template, 200 different strains were determined by microdilution. Briefly, colonies obtained nM of each primer, and 12.5 ␮l of SYBR green PCR master mix (Applied after growth on solid media were picked up and incubated overnight at 30°C in Biosystems). Thermal cycling consisted of an initial cycle of 95°C for 10 min LSM broth (29). The optical density at 625 nm (OD ) of the cultures was followed by 35 cycles of 95°C for 15 s and 60°C for 1 min. The expression levels adjusted to 0.2 in LSM broth, and the suspension was diluted 500-fold in the in the presence of antibiotic were refereed to those obtained for the control same medium. One hundred microliters of this dilution was then transferred to culture (absence of antibiotic). Two independent experiments were carried out 100 ␮l of LSM containing the appropriate amount of tetracycline in serial and each sample was analyzed in duplicate in two independent PCR runs.
twofold dilutions, and the microtiter plates were incubated at 30°C for 24 h. The Negative controls, including all the elements of the reaction mixture except the growth was recorded with a Benchmark plus microplate spectrophotometer template cDNA, were also included.
(Bio-Rad, Hercules, CA). All the experiments were carried out in triplicate.
(iv) Pulsed-field gel electrophoresis (PFGE) and Southern hybridization con-
DNA and RNA techniques. (i) Nucleic acids extractions and labeling.
ditions. The genetic location of tet(L) and tet(M) was assessed by hybridization
Genomic DNA was isolated using the GenElute bacterial genomic DNA kit using as probes 0.7- and 1.5-kb internal segments of the genes obtained by PCR (Sigma). Plasmid DNA was isolated using either the large-scale Qiagen kit and labeled with digoxigenin (Roche Applied Science, Basel, Switzerland). The (Qiagen Inc. Valencia, CA) or the procedure of O'Sullivan and Klaenhammer tet(L) and tet(M) fragments were amplified using primer pairs TetL-FW3/TetL-

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RV3 and DI/TetM-R (9), respectively. Total and plasmid DNAs digested withthe restriction enzymes EcoRI, HindIII, AscI, and PstI (Takara Bio Inc., Shiga,Japan) were hybridized using high-stringency standard conditions at 68°C.
For PFGE analysis, the strain was inoculated in 10 ml MRS supplemented with 20 mM DL-threonine and incubated at 30°C until the OD was 0.5 to 1.0 or above. The cells were harvested by centrifugation, washed in 10 ml 50 mMEDTA, and resuspended in 50 mM EDTA (300 ␮l ⫻ OD ). A 125-␮l cell suspension was mixed gently with 750 ␮l 1% low-melting-point agarose (pre-pared in 50 mM EDTA). The cell-agarose suspension was pipetted into theBio-Rad plug mold. The agarose plugs were incubated at 37°C overnight in alysozyme solution (2 mg/ml lysozyme, 20 units/ml mutanolysin, 0.05% N-lauroylsarcosine in 50 mM EDTA). The lysozyme solution was replaced by a sodiumdodecyl sulfate-proteinase solution (10 mM Tris, pH 8.0, 1% sodium dodecylsulfate, 2 mg/ml proteinase K in 0.5 M EDTA, pH 8.5) and incubated at 50°Covernight. The agarose plugs were washed six times for 30 min in 50 mM EDTAand stored at 4°C in 50 mM EDTA. Slices of 1 to 2 mm of the agarose plugs wereincubated in 200 ␮l of restriction enzyme buffer for 1 to 4 h at 4°C. The buffer wasreplaced with 200 ␮l fresh restriction enzyme buffer, 2 ␮l acetylated bovineserum albumin (10 mg/ml stock), and 20 to 40 units of AscI. The agarose plugswere incubated for 30 to 45 min at 4°C and then at 37°C overnight. The sampleswere loaded on a 1.1% agarose gel prepared in 0.5⫻ Tris-borate-EDTA buffer.
The DNA fragments were resolved on a Bio-Rad contour-clamped homoge-neous electric field mapper using a 24-h program with a linear ramp factor, aninitial switch time of 2 s, and a final switch time of 30 s. The gel was stained inethidium bromide and destained in 0.5⫻ Tris-borate-EDTA buffer.
FIG. 1. (A) Plasmid profile of L. sakei Rits 9 undigested (lane 1) Southern blotting of PFGE gels was performed with DNA probes labeled with and digested with PstI (lane 2). (B) PFGE analysis of total DNA from horseradish peroxidase with the ECL direct nucleic acid labeling kit (Amersham L. sakei Rits 9 undigested (lane 1) and digested with AscI (lane 2).
Biosciences, Buckinghamshire, United Kingdom) according to the manufactur- (C) Southern blot analysis of the PFGE gel with the internal er's instructions.
tet(M) probe. MWM, molecular weight marker.
(v) Sequencing strategy for the tet genes and sequence analysis. Plasmid DNA
was sequenced after serial runs using the first-round primers, which consisted ofthe complementary sequences of TetL-FW3 and TetL-RV3 and then primersdesigned from the DNA sequence newly obtained. The plasmid was thereafterresequenced on the other strand in order to check for sequence accuracy.
signal (data not shown). To verify the presence of both genes, For sequencing the tet(M) region, a pair of primers was designed from the primers derived from known tet gene sequences were used.
tet(M) sequence of Staphylococcus aureus subsp. aureus Mu50 and served for the Amplification of internal fragments of tet(L) and tet(M) with amplification of L. sakei Rits 9 tet(M). Primers tetM-revF and tetM-revR (Table1) were used to amplify regions upstream and downstream of the tet(M) genes.
the primers TetL-FW3 and TetL-RV3 and DI and TetM-R, The sequencing of the flanking regions of tet(M) was carried out using inverse respectively, resulted in amplicons of about 0.7 kb and 1.5 kb, PCR as described elsewhere (16). In short, total genomic DNA was digested with confirming that L. sakei Rits 9 possesses both genes. L. sakei HindIII and self-ligated overnight. The ligated DNA was precipitated, centri- Rits 9 harbors one small plasmid of 5 kb, as revealed by a fuged, dried, and resuspended in 100 ␮l Tris-EDTA prior to use as the template plasmid profile analysis using the O'Sullivan and Klaenham- for PCR amplification. Purified PCR products were sequenced by cycle extensionin an ABI 370 DNA sequencer (Applied Biosystems).
mer method (Fig. 1A), and at least one large plasmid, as Phylogenetic analyses were performed on sequences available in the GenBank revealed by PFGE (Fig. 1B). Southern blots showed tet(L) to database, using the Treetop software (http://www.genebee.msu.su/services be located on the 5-kb small plasmid (data not shown) and tet(M) on a large AscI PFGE chromosomal fragment (⬎450 Nucleotide sequence accession numbers. The nucleotide sequences described
in this paper have been deposited in the GenBank database with the following kb) (Fig. 1C). In order to determine the involvement of those accession numbers: for L. sakei Rits 9 plasmid pLS55, EF605268; and for L. sakei two genes in the resistance phenotype of L. sakei Rits 9, the Rits 9 tet(M) and flanking regions, EF605269.
5-kb plasmid containing tet(L) was totally sequenced, as wasthe chromosomal region encompassing tet(M).
The tet(L) gene is contained by a plasmid, and the tet(M)
gene is flanked by transposon-like regions. The 5-kb plasmid
L. sakei Rits 9 possesses tet(L) and tet(M) resistance genes.
containing tet(L), named pLS55, was sequenced. It was found L. sakei Rits 9 was isolated from an Italian Sola cheese as to be composed of 5,031 bp, consistent with its predicted size.
spontaneously resistant to tetracycline. The presence of genes The plasmid was almost 100% identical to pMA67, a plasmid responsible for such resistance was searched by hybridization recently described for the gram-positive bacterial pathogen of with DNA microarrays containing oligonucleotides character- honeybees Paenibacillus larvae (32). Indeed, only seven of the istic of 28 known tetracycline resistance genes. The results base pairs were found to be different, four of them located in showed the strain to harbor both tet(M) and tet(L). Hybridiza- the tet(L) structural gene (positions 1, 287, 859, and 1197), and tion signals were quite strong for both 50- and 60-mer oligo- the plasmids differ in size by only one nucleotide (5,030 bp for nucleotides used for identifying the respective Tcr genes. Ex- pMA67). Remarkably, a different initiation codon was found cept for positive signals obtained with control probes targeting for tet(L) in pLS55 (ATG instead of GTG), which could sug- lactobacillus tuf genes, no other positive signals were found gest a more efficient translation of the gene in L. sakei (30).
with any of the remaining spots, indicating the absence of other The expression of tet(L) seems to depend on the synthesis of a antibiotic resistance determinants. This shows either that other 20-amino-acid leader peptide encoded 22 bp upstream of the resistance genes are absent or that similar genes may be tet(L) ribosome binding site, which is typical of inducible tet present but with a homology too low to get a hybridization genes (25). A phylogenetic analysis performed on all complete

TETRACYCLINE RESISTANCE IN L. SAKEI Rits 9 FIG. 2. Phylogenetic tree of homologs of the deduced Tet(L) and Tet(M) proteins (A and B, respectively). Protein accession numbers are given in brackets. Trees were constructed by the neighbor-joining algorithm and clustered by the unweighted-pair group method using average linkages,and bootstrap values (100 replicates) are given at the branch points. The distances refer to the percentages of different residues. Abbreviations:E. faecium, Enterococcus faecium; B. cereus, Bacillus cereus; M. haemolytica, Mannheimia haemolytica; A. pleuropneumoniae, Actinobacilluspleuropneumoniae; C. difficile, Clostridium difficile; E. coli, Escherichia coli; N. meningitidis, Neisseria meningitidis.
tet(L) sequences available in the GenBank database showed deduced that pLS55 would likely be a mobilizable rolling-circle that both P. larvae and L. sakei Rits 9 tet(L) genes are different replication plasmid in the group II family (also called the from all previously described tet(L) genes and form an inde- pMV158 family).
pendent branch associated with a very strong bootstrap value The sequence of a region encompassing 8,524 bp around the tet(M) gene was obtained by several PCR and sequencing Apart from tet(L), pLS55 contains all the elements for rep- steps. The nucleotide sequence of the L. sakei Rits 9 tet(M) lication control (12, 13, 22, 28) (Fig. 3A). Interestingly, the Rep gene was shown to be identical to the one described for S.
protein is 80% identical to the Rep proteins of L. sakei plasmid aureus subsp. aureus Mu50 and for Streptococcus agalactiae pLS141-1 and of pLC2 identified for Lactobacillus curvatus, a COH1 (Fig. 2B). The tet(M) gene was flanked downstream and lactobacillus species closely related to L. sakei (GenBank ac- upstream by regions with high similarity to the tet(M)-sur- cession no. AB109041 and CAA78602, respectively). It can be rounding regions of several gram-positive bacteria (31, 36, 41),

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FIG. 3. Genetic structure of the tet(L)-containing plasmid pLS55 (A) and L. sakei Rits 9 tet(M) and flanking regions (B). Arrows show the direction of transcription of the open reading frames. Relevant restriction sites and their locations are indicated. The genes which matched thehighest homology scores and the homologies with the partial sequences of different transposons are indicated.
corresponding to transposon-like sequences (Fig. 3B). Up- higher tetracycline concentrations (between 2.74 ⫾ 0.40- and stream of tet(M), a 181-bp region mainly features a sequence 3.07 ⫾ 0.44-fold increases) (Fig. 4).
corresponding to a 28-amino-acid leader peptide. Immediately pLS55 is able to replicate in L. sakei 23K. To determine
upstream the leader peptide sequence, we found a 1,305-bp whether pLS55 replication is possible in another L. sakei strain, sequence that shares more than 99% identity with L. sakeiIS1520, encompassing the transposase subunits A and B of anIS element present in five copies in the L. sakei 23K chromo-some (6).
Regulation and expression levels of tet(M) and tet(L). Real-
time PCR was used to assess the influence of different subin-hibitory concentrations of tetracycline (16, 32, and 64 ␮g/ml)on the expression levels of tet(M) and tet(L) in L. sakei Rits 9.
Concentrations higher than 64 ␮g/ml affected the growth rateof the strain and therefore were not included in the study. Abasal constitutive expression of both genes was observed inde-pendent of the presence of tetracycline. Remarkably, we no-ticed that tet(M) expression was gradually induced by exposureto increasing amounts of tetracycline. Indeed, tet(M) inductionwas about 13% increased at low tetracycline concentration (16␮g/ml) and up to 100% (relative induction was 2.095 ⫾ 0.215)after exposure to 64 ␮g/ml compared to the control conditions FIG. 4. Relative expression levels of tet(L) and tet(M) in L. sakei (absence of antibiotic) (Fig. 4). On the contrary, the tet(L) Rits 9 grown in the presence of different tetracycline concentrations gene was induced up to 2.74-fold at the lower tetracycline refereed to those obtained for the control culture (absence of anti- concentration, and its relative expression remained similar at TETRACYCLINE RESISTANCE IN L. SAKEI Rits 9 and to assess the functionality of the tet(L) gene, the transfor- pLS55 and the 80% identity between the Rep protein of pLS55 mation of the plasmid into L. sakei 23K was attempted, and and some Rep proteins described for other L. sakei or L.
transformants were plated with different tetracycline concen- curvatus plasmids suggest that pLS55 can be transferred and trations (4, 8, 16, and 32 ␮g/ml). When 4 ␮g/ml was used, a stably maintained in L. sakei. Indeed, we could electroporate it background was quite visible, but the background disappeared in the plasmid-free laboratory strain L. sakei 23K, in which it when 8-, 16-, and 32-␮g/ml concentrations of tetracycline were used. Several transformants were obtained on plates with 8 and On the other hand, tet(M) was shown to be located on a 16 ␮g/ml of tetracycline. No transformants were obtained at 32 transposon-like region. Upstream of tet(M), a fragment of ␮g/ml. Plasmid preparations of four clones confirmed the pres- 1,305 bp identical to L. sakei IS1520 was also present. This ence of a 5-kb plasmid in all of them. Then, one of them, suggests that the acquisition of tet(M) by L. sakei Rits 9 oc- named L. sakei 23K-TL, was selected to analyze its MIC to curred through an insertion event, although a more detailed tetracycline in comparison with the control L. sakei 23K and L.
study is necessary to corroborate this.
sakei Rits 9. While the MIC of the Rits 9 strain was found to The high Tcr level in L. sakei Rits 9 and the absence of be ⬍256 ␮g/ml and that of the 23K strain ⬍1 ␮g/ml of tetra- positive hybridization results other than the ones obtained with cycline, the MIC of L. sakei 23K-TL was ⬍32 ␮g/ml.
the tet(L) and tet(M) oligonucleotides in the microarray anal-ysis suggest that Tcr in this strain is linked to the presence of one or both genes. In order to ascertain the functionalities ofboth genes and the partial contribution of each to the Tcr Tetracyclines have been extensively used in the prophylaxis phenotype, we have transformed the plasmid-free laboratory and treatment of human and animal infections. Furthermore, strain L. sakei 23K with the tet(L)-containing plasmid pLS55.
they have been administered at subtherapeutic concentrations The resulting strain displayed an intermediate Tcr level com- as growth promoters in animal feeds (34, 44). This intensive pared with the Rits 9 strain, which displayed a much higher and extensive use has caused Tcr to spread to a large number MIC. Thus, the higher Tcr level of L. sakei Rits 9 could be due of commensal bacteria (9, 37). In fact, different Tcr genes are to the presence of tet(M) or to a synergistic effect of both present in the fecal microbiota of babies not previously ex- genes. Furthermore, these data indicate that both tet genes are posed to the antibiotic (23). At present, there is great concern functional in L. sakei, with tet(L) conferring a moderated re- that animal and human commensal bacteria, such as LAB, sistance level, whereas tet(M) confers a high Tcr level to this could act as a reservoir for antibiotic resistance genes. These bacteria. In relation to this, it has been shown that tet(L) and microorganisms may subsequently contaminate the raw milk tet(M) can contribute differently to the Tcr phenotype depend- and meat produced from these animals, and the foods pre- ing on the Enterococcus or Streptococcus strain (39). It is also pared from those raw materials can therefore be considered as likely that the resistance level conferred by these two genes is potential vehicles for the spread of antibiotic-resistant LAB species dependent and probably strain dependent.
along the food chain to the consumer (42). Resistances could Finally, expression studies were carried out to go more ultimately be transferred to human pathogenic and opportu- deeply into the functionality of tet(L) and tet(M) in L. sakei nistic bacteria, hampering the treatment of infections (27).
Rits 9. The fact that tet(M) expression was mainly induced at Several Tcr LAB have been isolated from raw milk dairy high Tcr levels, whereas tet(L) induction was achieved at lower products, e.g., Lactobacillus fermentum ROT1 (21) and Lacto-coccus lactis subsp. lactis K214 (34), and from raw meat-based concentrations, sheds some light onto the physiological func- fermented products, such as L. alimentarius, L. curvatus, L.
tion of both genes. These data indicate that, at a low tetracy- plantarum, and L. sakei (19). The Tcr has been found to be cline concentration, the activity of the efflux pump Tet L is mediated mainly by tet(M), which could be plasmid encoded enough for L. sakei Rits 9 to cope with antibiotic challenge; and transferred through interspecies and intergenus conjuga- however, at concentrations higher than 16 ␮g/ml, the cells need tion mechanisms (17, 27). In this study, we show that L. sakei an extra input, which is supplied by a higher amount of the Rits 9, a Tcr strain isolated from a dairy product, harbors two ribosomal protection protein Tet M. These findings also sup- Tcr genes, namely, the ribosomal protection tet(M) gene fre- port the previous results just discussed above, indicating that quently encountered in lactobacilli and the efflux pump-encod- Tet M is responsible, to a larger extent than Tet L, for the high ing tet(L) gene. This combination of tet(L) and tet(M) genes is Tcr phenotype of L. sakei Rits 9.
very frequently found for Streptococcus spp. and Enterococcus In conclusion, the results of the current study indicate that sp. strains (35, 39) and also for cloacal Lactobacillus salivarius Lactobacillus species from raw milk cheese can harbor ac- subsp. salivarius isolates (5). However, to the best of our quired Tcr determinants associated with mobile elements, po- knowledge this is the first report on the coexistence of two tentially enabling them to spread to other LAB or potentially genes encoding different mechanisms of Tcr in the same L.
pathogenic bacteria. We also demonstrated, for the first time, that two different Tcr mechanisms, active efflux and ribosomal The gene tet(L) was found to be associated with the plasmid protection, are functional when they are together in the same pLS55, which is highly similar to pMA67, a plasmid described strain. Remarkably, our data suggest that the two genes are for the honeybee-pathogenic species P. larvae (32). As L. sakei dedicated to cope with two different physiological conditions, and P. larvae are not known to share a common ecological low and high tetracycline concentrations. This functional niche, it is therefore plausible that such a plasmid has been complementarity of both mechanisms and their involvement in horizontally transferred in these two hosts through different the physiology of L. sakei under tetracycline challenge will microorganisms. The presence of a Mob protein encoded by contribute to an understanding of how a bacterium makes use AMMOR ET AL.
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of different resistance determinants and of how they are en- tet genes along the process line of fermented dry sausages. Syst. Appl.
gaged to fight against the deleterious action of antimicrobials.
20. Gevers, D., M. Danielsen, G. Huys, and J. Swings. 2003. Molecular charac-
terization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl. Environ. Microbiol. 69:1270–1275.
21. Gfeller, K. Y., M. Roth, L. Meile, and M. Teuber. 2003. Sequence and genetic
Work on antibiotic resistance at our laboratories was supported by organization of the 19.3-kb erythromycin- and dalfopristin-resistance plas- an EU project within the Sixth Framework Programme (ACE-ART, mid pLME300 from Lactobacillus fermentum ROT1. Plasmid 50:190–201.
reference no. FP6-506214). M. S. Ammor was awarded a postdoctoral 22. Grohmann, E., G. Muth, and M. Espinosa. 2003. Conjugative plasmid trans-
fellowship from the Secretarı´a de Estado de Universidades e Investi- fer in gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67:277–301.
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antibiotic (tet) resistance genes in infant faecal microbiota. FEMS Immunol.
no. SB2004-0165). Miguel Gueimonde is the recipient of a Juan de Med. Microbiol. 48:21–25.
la Cierva contract from the Spanish Ministry of Education and 24. Haarman, M., and J. Knol. 2006. Quantitative real-time PCR analysis of
fecal Lactobacillus species in infants receiving a prebiotic infant formula.
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