Intrinsic and selected resistance to antibiotics binding the ribosome: analyses of Brucella 23S rrn, L4, L22, EF-Tu1, EF-Tu2, efflux and phylogenetic implications

Abstract

Background: Brucella spp. are highly similar, having identical 16S RNA. However, they have important phenotypic differences such as differential susceptibility to antibiotics binding the ribosome. Neither the differential susceptibility nor its basis has been rigorously studied. Differences found among other conserved ribosomal loci could further define the relationships among the classical Brucella spp.

Results: Minimum inhibitory concentration (MIC) values of Brucella reference strains and three marine isolates to antibiotics binding the ribosome ranged from 0.032 to >256 microg/ml for the macrolides erythromycin, clarithromycin, and azithromycin and 2 to >256 microg/ml for the lincosamide, clindamycin. Though sequence polymorphisms were identified among ribosome associated loci 23S rrn, rplV, tuf-1 and tuf-2 but not rplD, they did not correlate with antibiotic resistance phenotypes. When spontaneous erythromycin resistant (eryR) mutants were examined, mutation of the peptidyl transferase center (A2058G Ec) correlated with increased resistance to both erythromycin and clindamycin. Brucella efflux was examined as an alternative antibiotic resistance mechanism by use of the inhibitor L-phenylalanine-L-arginine beta-naphthylamide (PAbetaN). Erythromycin MIC values of reference and all eryR strains, except the B. suis eryR mutants, were lowered variably by PAbetaN. A phylogenetic tree based on concatenated ribosomal associated loci supported separate evolutionary paths for B. abortus, B. melitensis, and B. suis/B. canis, clustering marine Brucella and B. neotomae with B. melitensis. Though Brucella ovis was clustered with B. abortus, the bootstrap value was low.

Conclusion: Polymorphisms among ribosomal loci from the reference Brucella do not correlate with their highly differential susceptibility to erythromycin. Efflux plays an important role in Brucella sensitivity to erythromycin. Polymorphisms identified among ribosome associated loci construct a robust phylogenetic tree supporting classical Brucella spp. designations.

Figure 1

Minimal inhibitory concentration (MIC) of antibiotics to Brucella reference strains and marine isolates. MICs of three macrolides, azithromycin, clarithromycin, erythromycin, and the lincosamide, clindamycin, were determined by Etest. Maximum MIC measurable by the Etest is 256 μg/ml for each of the antibiotics. Brucella strains as listed in Table 1. Ba = B. abortus ; Bs = B. suis; Bm = B. melitensis; Bc = B. canis; Bn = B. neotomae; Bo = B. ovis; numbers following species designate biovar.

Figure 2

Ribosomal protein L22 polymorphisms among Brucella reference strains and three marine isolates. The putative peptide sequence of L22 is underlined, parentheses bracket polymorphic sites and list amino acids found among L22 peptides at that site. In regions where sequence was variable, the sequence for each putative Brucella L22 is given below. Amino acids occurring in the stalk of the β-hairpin appear in bold-italics and amino acids occurring in the loop of the β-hairpin are in bold. Beneath amino acids that are double underlined is a list of Brucella strains containing those aa. The single letter code is used to denote the putative aa sequence of the peptides; b = biovar and numbers following "b" designate biovar numbers. Accession numbers of rplV sequences for each strain are deposited in GenBank and are listed in Materials.

Figure 3

Ribbon diagrams showing predicted secondary structures of ribosomal L22 proteins. The divergent, putative aa sequences from the reference Brucella strains and three marine isolates are found in Fig. 2. The ribbon structures for each group is as follows: (A) B. abortus and B. melitensis; (B) B. canis, B. suis biovars 1, 4, and 5, and the marine isolates, (C) B. ovis; (D) B. suis biovars 2 and 3; and (E) B. neotomae. Structures were predicted based on coordinates of L22 from Thermus thermophilus [52] and prepared using Swiss-Pdb viewer [50, 51]. Region containing β-hairpin loops (→).

Figure 4

Phylogeny of Brucella calculated using highly conserved ribosomal associated loci. Shown is the single optimization alignment tree based on rplV, tuf-1, tuf-2, and 23S rrn sequences from 28 taxa consisting of the 21 Brucella strains (see Table 1), which included the 18 classical Brucella reference strains and three marine Brucella, and seven outgroups of known genomic sequences. Branch lengths (mean number of differences per residue along each branch) are given as well as bootstrap values (percentage of bootstrap support based on 100 replicates). Legionella pneumophila subspecies Pneumophila strain Philadelphia [GenBank:NC_002942] was used to root the tree. Other bacterial outgroups include: Acinetobacter species ADP1 [GenBank:NC_005966], Caulobacter crescentus CB15 [GenBank:NC_002696], Leptospira interrogans serovar Copenhagen strain Fiocruz L1-130 [GenBank:NC_005823], Mesorhizobium loti MAFF303099 [GenBank:BA000012], Agrobacterium tumefaciens C58 circular [GenBank:NC_003062] and linear chromosomes [GenBank:NC_003063], and Xylella fastidosa 9a5c [GenBank:NC_002488].