The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species

Abstract

The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51-53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.

Keywords: antibiotic; antimicrobial; intrinsic resistance; non-pathogenic bacteria; resistance evolution; ribosomal protection.

FIGURE 1

The chromosomal region flanking tet(W) in Bifidobacterium animalis subsp. lactis and the same region in Bifidobacterium animalis subsp. animalis. Hypothetical proteins are designated HP. GC content (%) is provided for the genes found in the B. animalis subsp. lactis type strains (TS) DSM 10140. Genes that are present in the majority of the examined B. animalis subsp. lactis strains (represented by DSM 10140) has the same color in all the shown strains [blue colors downstream of tet(W) and green colors upstream of tet(W)].

FIGURE 2

tet(W) protein phylogenetic tree. The tree was built by evolutionary analysis by maximum likelihood method and JTT matrix-based model (Jones et al., 1992; Kumar et al., 2018). The branch lengths are measured in the number of substitutions per site. Strain name and genome or tet(W) gene accession number is provided for the sequences. Type strains (TS) are included for the species, when the type strain encodes tet(W). Clades are defined by the number of SAPs, which can be seen in Table 2. The phylogenetic tree was rooted with the ribosomal protection gene tet(O) from Campylobacter jejuni (M18896) as an outgroup and similar results was obtained with the Streptococcal ribosomal protection gene tet(M) (X04388) (data not shown) (Levy et al., 1999).

FIGURE 3

Core genome phylogenetic tree based on 250 core genes which include B. animalis subsp. lactis strains and other related Bifidobacterium species. Type strain has been included for each species, designated TS and strains both with and without tet(W) are included for each species, except for B. animalis subsp. animalis and B. bifidum. tet(W) positive strains are marked with a green circle. B. animalis subsp. lactis UBBLa 70 exhibit a tet(W) gene with large deletions and is marked with a yellow circle. The tree is rooted with the Bifidobacterium tissieri type strain DSM 100201 as an outgroup (Lugli et al., 2018). Bootstrap percentages are shown at node points.