Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims:  To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages.
Methods and Results:  We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei , Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet (M) and erm (B).
Conclusions:  This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy.
Significance and Impact of the Study:  Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation.     

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3–18

Aims:  The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources.
Methods and Results:  A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum . The examination of proteolytic activity revealed that 28 Lact.   plantarum and two Lact.   paraplantarum hydrolyse β-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3–18 has prtP catalytic domain as well as prtP – prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei , Lact. casei and L. lactis . No presence of prtB , prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains.
Conclusions:  One out of 28 analysed Lact. plantarum strains harbours the prtP -like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s).
Significance and Impact of the Study:  It is the first report about the presence of the prtP –like gene in Lact. plantarum , which illustrates the mobility of this gene and its presence in different species.     

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.
Methods and Results:  A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes . Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively.
Conclusions:  These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells.
Significance and Impact of the Study:  This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum .     

Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species

AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim. The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes. METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact. paracasei, Lact. rhamnosus and Lact. plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1. The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated. E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact. acidophilus. MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact. paracasei, Lact. rhamnosus and Lact. plantarum, respectively. With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact. acidophilus were 0.064-1 microg ml(-1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim. The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.     

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.
Methods and Results:  Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk ( D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica . The three micro-organisms studied varied considerably in their ability to survive to spray drying processes . Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased.
Conclusions:  Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures
Significance and Impact of Study:  It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.     

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

Characterization and Transfer of Antibiotic Resistance in Lactic Acid Bacteria from Fermented Food Products

The study provides phenotypic and molecular analyses of the antibiotic resistance in lactic acid bacteria (LAB) from fermented foods in Xi'an, China. LAB strains (n = 84) belonging to 16 species of Lactobacillus (n = 73), and Streptococcus thermophilus (n = 11) were isolated and identified by sequencing their 16S rRNA gene. All strains were susceptible to ampicillin, bacitracin, and cefsulodin, and intrinsically resistant to nalidixic acid, kanamycin, and vancomycin (except L. bulgaricus, L. acidophilus, and S. thermophilus, which were susceptible to vancomycin). Some strains had acquired resistance for penicillin (n = 2), erythromycin (n = 9), clindamycin (n = 5), and tetracycline (n = 14), while resistance to gentamycin, ciprofloxacin, streptomycin, and chloramphenicol was species dependent. Minimum inhibitory concentrations presented in this study will help to review microbiological breakpoints for some of the species of Lactobacillus. The erm(B) gene was detected from two strains of each of L. fermentum and L. vaginalis, and one strain of each of L. plantarum, L. salivarius, L. acidophilus, L. animalis, and S. thermophilus. The tet genes were identified from 12 strains of lactobacilli from traditional foods. This is the first time, the authors identified tet(S) gene from L. brevis and L. kefiri. The erm(B) gene from L. fermentum NWL24 and L. salivarius NWL33, and tet(M) gene from L. plantarum NWL22 and L. brevis NWL59 were successfully transferred to Enterococcus faecalis 181 by filter mating. It was concluded that acquired antibiotic resistance is well dispersed in fermented food products in Xi'an, China and its transferability to other genera should be monitored closely.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.     

A conjugative macrolide resistance gene, mef(A), in environmental Clostridium perfringens carrying multiple macrolide and/or tetracycline resistance genes

Aims:  To determine if environmental Clostridium perfringens carry antibiotic resistance genes and if the genes are mobile.
Methods and Results:  Clostridium perfringens from water, soil and sewage (2003–2006) were screened for the tetracycline and macrolide resistance genes previously described in animal and human C. perfringens [ erm (B), erm (Q), tetA (P), tetB (P) and tet (M) genes] and the macrolide resistance mef (A) gene. Of the 160 isolates, 108 (67·5%) carried ≥1 of the six antibiotic resistance gene(s). The tetA (P), tetB (P) and tet (M) genes were in 53%, 22% and 8%, and the erm (B), erm (Q) and mef (A) genes in 26%, 1% and 18% of the isolates, respectively. The mef (A) gene and flanking regions were sequenced. The tet (M), erm (B), erm (Q) and mef (A) genes transfer independently from C. perfringens donors to the Enterococcus faecalis recipient.
Conclusions:  Six resistance genes were found in the environmental C. perfringens with the most common being the tetA (P) gene and the erm (Q) gene the least common.
Significance and Impact of the Study:  This is the first time conjugal transfer of macrolide resistance genes and/or the tet (M) gene from C. perfringens has been demonstrated. The data presented supports the hypothesis that antibiotic-resistant environmental C. perfringens are capable of acting as reservoirs for these antibiotic resistance genes.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains

Aims:  To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods.
Methods and Results:  The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g⁻¹. Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus . Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii , Lactobacillus fermentum and Enterococcus faecium . The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly.
Conclusion:  It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step.
Significance and Impact of the Study:  Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.     

Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine

Aims:  To investigate the abilities of various probiotic bacteria to produce volatile sulfur compounds (VSCs) relevant to food flavour and aroma.
Methods and Results:  Probiotic strains ( Lactobacillus acidophilus NCFM, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC55730 and L. reuteri BR11), Lactobacillus delbrueckii ATCC4797, L. plantarum ATCC14917 and Lactococcus lactis MG1363 were incubated with either cysteine or methionine. Volatile compounds were captured, identified and quantified using a sensitive solid-phase microextraction (SPME) technique combined with gas chromatography coupled to a pulsed flame photometric detector (SPME/GC/PFPD). Several VSCs were identified including H₂S, methanethiol, dimethyldisulfide and dimethyltrisulfide. The VSC profiles varied substantially for different strains of L. plantarum and L. reuteri and it was found that L. reuteri ATCC55730 and L. lactis MG1363 produced the lowest levels of VSCs ( P  < 0·05). Levels of VSCs generated by bacteria were found to be equivalent to, or higher than, that found in commercial cheeses.
Conclusions:  Several probiotic strains are able to generate considerable levels of VSCs and substantial variations in VSC generating potential exists between different strains from the same species.
Significance and Importance of the Study:  This study demonstrates that probiotic bacteria are able to efficiently generate important flavour and aroma compounds and therefore has implications for the development of probiotic containing foods.     

Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant

Aims:  To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei .
Methods and Results:  Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk.
Conclusions:  Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making.
Significance and Impact of the Study:  This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Horizontal transfer of tet(M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats

Two wild-type strains of Lactobacillus plantarum previously isolated from fermented dry sausages were analysed for their ability to transfer antibiotic resistance plasmids in the gastrointestinal tract. For this purpose, we used gnotobiotic rats as an in vivo model. Rats were initially inoculated with the recipient Enterococcus faecalis JH2-2 at a concentration of 10(10) CFU mL(-1). After a week, either of the two donors L. plantarum DG 522 (harbouring a tet(M)-containing plasmid of c. 40 kb) or L. plantarum DG 507 [harbouring a tet(M)-containing plasmid of c. 10 kb and an erm(B)-containing plasmid of c. 8.5 kb] was introduced at concentrations in the range of 10(8)-10(10) CFU mL(-1). Two days after donor introduction, the first transconjugants (TCs) were detected in faecal samples. The detected numbers of tet(M)-TCs were comparable for the two donors. In both cases, this number increased to c. 5 x 10(2) CFU g(-1) faeces towards the end of the experiment. For erm(B)-TCs, the number was significantly higher and increased to c. 10(3) CFU g(-1) faeces. To our knowledge, this is the first study showing in vivo transfer of wild-type antibiotic resistance plasmids from L. plantarum to E. faecalis.     

Protoplast formation, regeneration and plasmid curing in Lactobacillus reuteri

Abstract A method for protoplast formation and regeneration suitable for Lactobacillus reuteri strains was developed.
Lysozyme-treated cells formed protoplasts at a high percentage and regenerative ability varied according to the strains considered.
Moreover, production and regeneration of protoplasts promoted the loss of plasmids harboured by the strains.     

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.