Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938

Abstract

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, 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 study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, 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 beta-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 beta-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.

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.

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. Lanes: 1, L. reuteri ATCC 55730; 2, L. reuteri ATCC 55730Tet^s; 3, L. reuteri DSM 17938; 4, L. reuteri DSM 20016 (negative control). (B) Detection of tet(W) (a) 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).

FIG. 3.

(GTG)₅-PCR (rep-PCR)-generated genomic fingerprints of L. reuteri DSM 17938 (lane 1), L. reuteri ATCC 55730Tet^s (lane 2), and L. reuteri ATCC 55730 (lane 3). Lane M, molecular size markers (sizes are shown in base pairs on the right).

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).