Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro

Abstract

There are no examples of stable tetracycline resistance in clinical strains of Chlamydia trachomatis. However, the swine pathogen Chlamydia suis is commonly tetracycline resistant, both in America and in Europe. In tested U.S. strains, this resistance is mediated by a genomic island carrying a tet(C) allele. In the present study, the ability of C. suis to mobilize tet(C) into other chlamydial species was examined. Differently antibiotic resistant strains of C. suis, C. trachomatis, and Chlamydia muridarum were used in coculture experiments to select for multiply antibiotic resistant progeny. Coinfection of mammalian cells with a naturally occurring tetracycline-resistant strain of C. suis and a C. muridarum or C. trachomatis strain containing selected mutations encoding rifampin (rifampicin) or ofloxacin resistance readily produced doubly resistant recombinant clones that demonstrated the acquisition of tetracycline resistance. The resistance phenotype in the progeny from a C. trachomatis L2/ofl(R)-C. suis R19/tet(R) cross resulted from integration of a 40-kb fragment into a single ribosomal operon of a recipient, leading to a merodiploid structure containing three rRNA operons. In contrast, a cross between C. suis R19/tet(R) and C. muridarum MoPn/ofl(R) led to a classical double-crossover event transferring 99 kb of DNA from C. suis R19/tet(R) into C. muridarum MoPn/ofl(R). Tetracycline resistance was also transferred to recent clinical strains of C. trachomatis. Successful crosses were not obtained when a rifampin-resistant Chlamydophila caviae strain was used as a recipient for crosses with C. suis or C. trachomatis. These findings provide a platform for further exploration of the biology of horizontal gene transfer in Chlamydia while bringing to light potential public health concerns generated by the possibility of acquisition of tetracycline resistance by human chlamydial pathogens.

FIG. 1.

Immunofluorescence images of McCoy cells coinfected with C. suis R19/tet^R and C. muridarum MoPn/ofl^R (A) or with C. suis R19/tet^R and Chlamydophila caviae GPIC/rif^R (B). In both panels, C. suis R19/tet^R is labeled red, while the alternate species is labeled green, using species-specific anti-MOMP antibodies. Arrows point to fused inclusions (F) or nonfused inclusions (NF) between the two different infecting strains. Nuclei are labeled blue with 4′,6-diamidino-2-phenylindole (DAPI). Bar, 10 μm.

FIG. 2.

PCR results for parental strains R19/tet^R (lanes 1) and L2/ofl^R-rif^R1 (lanes 2) and for the cloned L2/tet^R1 recombinant (lanes 3). Primers are specific to tet(C) (left), to ompA from parental strain L2/ofl^R-rif^R (middle), and to ompA from parental strain R19/tet^R (right), The molecular size of the tet(C) PCR product is 525 bp, and those of the ompA PCR products are 248 bp for L2/ofl^R and 258 bp for R19/tet^R.

FIG. 3.

Southern blotting of EcoRI-digested genomic DNAs from parental and recombinant strains (Table 2, cross 5). Digested genomic DNAs were hybridized with DNA probes specific to the donor (R19/tet^R) and recipient (L2/ofl^R-rif^R1) 23S rRNA sequences. Lane 1, L2/ofl^R DNA, showing a doublet of two bands (3,024 and 2,973 bp); lane 2, R19/tet^R DNA, showing a predicted band of 13,546 bp; lane 3, cloned L2/tet^R1 progeny from the cross of these two strains, showing appropriately sized fragments of both parental 23S rRNAs. The approximate numbers of base pairs in the observed bands are given on the right.

FIG. 4.

Recombination models for L2/tet^R1 and MoPn/tet^R14. (A) Parental strains, showing the locations of ribosomal operons (rrn) and the tet(C) genomic island. (B) C. suis R19/tet^R donor DNA integrated at the downstream 23S rRNA of C. trachomatis L2/ofl^R-rif^R1, producing a recombinant with three rrn operons and ∼40 kb of donor DNA. The two downstream rrn operons are hybrid genes, each composed of a mosaic of donor and recipient parental DNAs. (C) C. suis R19/tet^R donor DNA recombined at two loci outside of the rrn sequences. The MoPn/tet^R14 recombinant has C. suis R19/tet^R copies of genes TC_0081 through TC_0149, which include the intact C. suis R19/tet^R rrn operons and the tet(C) genomic island. The remainder of MoPn/tet^R14 is identical to the other parental strain, C. muridarum MoPn/ofl^R.

FIG. 5.

Ribosomal operons and recombination sites in the sequenced recombinant L2/tet^R1. (A) The leftmost rRNA operon is completely derived from the parental L2 sequence. (B) The central rRNA operon (see Fig. 4) is a hybrid of the two parental genomes, with a single crossover point identified within the 23S gene. (C) The rightmost rRNA operon is a mosaic of the two parental sequences. The source of each sequence in the L2/tet^R1 genome (the recipient strain, C. trachomatis L2/ofl^R-rif^R1, or the donor strain, C. suis R19/tet^R) is indicated. Identical stretches of DNA sequence in the two parental strains, where the crossovers occurred, are boxed. The lengths (in base pairs) of the identical sequences at the recombination sites are given above each box.