The Etest was used to determine MIC values of the classical referenceBrucellaspp., their biovars, and three marine isolates to macrolides and a lincosamide. Our results differed somewhat from those reported by Meyer  using antibiotic discs containing low or high concentrations of erythromycin. Meyer foundB. ovisandB. canismore resistant thanB. suisto erythromycin, but Etest MIC values forB. ovisandB. caniswere less than those of the reference strains ofB. suis. The MIC values for the marine isolates were low and more similar to those ofB. suisthan to those of eitherB. melitensisorB. abortus. The patterns of relative sensitivity to macrolides of the referenceBrucellawere similar for erythromycin, clarithromycin, and azithromycin but differed from that for clindamycin. The susceptibility ofB. suisto relatively low concentrations of the macrolide azithromycin suggests that this antibiotic may be a beneficial treatment forB. suisinfections as it has a longin vivohalf-life (50 hours), concentrates in macrophages, and lacks uptake saturation .
Ribosomal associated loci 23Srrn,rplD, rplV, tuf-1, andtuf-2 were analyzed for polymorphisms. Three monomorphisms were identified amongrplDloci, but only one of them resulted in a difference among the putative L4 sequences. Although polymorphism was high among thetuf-1 andtuf-2 loci, all were silent. Sequences among 23SrrnandrplV loci were polymorphic.
The three polymorphic sites identified among theBrucella23Srrnloci separated them into three groups (Table2). The only sequence difference among the 23Srrnpeptidyl transferase centers of the referenceBrucellastrains was at nt 2610 (Ec), where there was either a T or a C. Many nucleotides in the peptidyl transferase center are conserved among bacteria and other organisms, but nt 2610 (Ec) is not. Either a T or C is common in bacteria. In any case, a T2610C (Ec) mutation in 23SrrnfromS. pneumoniaonly slight affected its MIC values for macrolides and clindamycin . Mutation of the peptidyl transferase center of 23S RNA (A2058G, Ec) of porpoise eryRmutant isolate b increased the erythromycin and clindamycin MIC values from 1.5 and 16 μg/ml, respectively, to >256 μg/ml. These MIC values were unaffected by the presence of efflux inhibitor PAβN. Concurrent appearance of resistance to erythromycin and clindamycin by mutation of nt 2058 (Ec) is observed in other bacteria . Methylation of either nt 2059 or 2058 (Ec) of the peptidyl transferase center reduces the sensitivities of bacteria to macrolides and lincosamides . We were unable to identify homologs of any 23Sermmethylation genes by BLAST , but, then, methylation of ribosomal rRNA is much more widely described in Gram-positive clinical isolates .
TherplV sequences of the referenceBrucellastrains and marineBrucellawere polymorphic, resulting in the differences among their putative L22 peptide sequences and lengths of the L22 β-hairpin loops. This was unexpected because L22 peptide sequence is conserved within a bacterial species [18,38] and the length of the L22 β-hairpin loop is highly conserved across biological kingdoms . Differences in β-hairpin loop lengths among theBrucellaL22 peptides were due to variable numbers of Gly-Arg repeats (Fig.2). ThoughB. neotomaeeryRisolate d had four Gly-Arg repeats, due to a six base insertion, the mutant's erythromycin and clindamycin MIC values were only slightly increased.
The single amino acid difference found among the putative L4 sequences of the reference and marine strains could not be correlated with a difference in MIC values. Among the eryRmutants, all but two of the mutations were identified inrplD, and, interestingly, they only occurred among the marine eryRisolates. All erythromycin MIC values that increased among the eryRmarine isolates were lowered by the efflux inhibitor PAβN. Nevertheless, some of the MIC values remained relatively high in the presence of PAβN. The L4 peptides of these mutants may work in conjunction with or be dependent on specific efflux RND pumps as shown forHaemophilus influenzaHMC-C [32,33].
Thetuf-1 andtuf-2 loci were the most polymorphic of the ribosomal associated loci examined, yet their putative peptide sequences were identical. Strain sequence differences betweentuf-1 andtuf-2 were confined to the borders. This is consistent with gene conversion occurring more efficiently within conserved sequences rather than near the borders. Given thatB. melitensisandB. abortusgenomes have fewer single nucleotide polymorphisms (SNP) between them than either has withB. suis, tuf-1 and tuf-2fromB. abortusandB. melitensiswere expected to be highly similar. This was not the case.Brucella abortusandB. suis tuf-1andtuf-2 had few sequence differences (Table3). Thetuf-1 andtuf-2 sequences from the marine isolates were intermediate betweenB. abortus/B. suisandB. melitensis/B. neotomae. Thetuf-1 and tuf-2genes encode a core metabolic product and the apparent selective pressure on conserving EF-Tu sequences in the face oftuf-1 andtuf-2 polymorphism supports different evolutionary paths  forB. abortusandB. melitensis.
MIC values and sequences of ribosomal related loci did not correlate with antibiotic susceptibility. To determine if efflux played a part inBrucelladifferential antibiotic resistance, we studied the effect of an RDF efflux inhibitor on MIC values. With the possible exception ofB. abortusbiovar 9, erythromycin MIC values of all the reference strains were reduced by the inhibitor PAβN though MIC values decreases were variable. Even low erythromycin MIC values decreased further in the presence of PAβN, demonstrating that efflux afforded theBrucellaa low level of intrinsic antibiotic resistance similar to that reported forCampylobacter.
Many clinical isolates are resistant to antibiotics due to increased efflux as a result of mutations of efflux promoters and global and physically linked regulator genes or mobilization of insertion sequences (for a review see ). Most of the eryRstrains had increased antibiotic efflux, though the marine eryRstrains had larger increases in efflux than those of the classical reference strains ofB. suisbiovar 1,B. canis, andB. neotomae(Table4). This suggests a fundamental biological difference between these groups. It is known that the marineBrucellahave a high copy number  of the insertion sequence IS711. IS711has been shown to mobilize inBrucellaunder stress or selective pressure [43,44] and could be a source of instability  in marineBrucella.
Brucellaphylogenetic trees and dendrograms have been constructed based on genomics maps [3,5,6], amplified fragment length polymorphisms (AFLP) , multilocus enzyme electrophoresis (MLEE) , and outer membrane proteinsomp2a/omp2b. Now, other universally conserved loci, especially 23S rrn, EF-Tu,rpoB, and gyrase, are increasingly being used to establish relationships among highly similar bacteria with important phenotypic differences to determine their relationships . We constructed a phylogenetic tree based on concatenated sequences of ribosomal associated loci. Most phylogenetic trees and dendrograms, including ours, placeB. abortus,B. suis/B. canis, andB. melitensison separate branches, supporting alternative evolutionary paths. Recently, it was shown thatBrucellaisolates could be identified at the species level using 21 variable number tandem repeats (VNTR) . The neighbor joining tree based on VNTR data produced major clusters that encompassed the classicalBrucellaspp. On this tree, the referenceB. suisbiovar 5 strain, which appears as a unique branch on our tree, was shown to be only distantly related to all other reference strains and isolates by VNTR analyses . ThoughB. ovisformed a single cluster by VNTR analyses, it clustered, albeit with a low bootstrap value, withB. abortuson our tree. Significant sequence differences have been reported betweenB. ovisand other classicalBrucellaspp. reference strains [47,48].Brucella neotomaegrouped withB. melitensishere but was on a separate node. Based on VNTR data,B. neotomaeoccurs on a unique branch but groups withB. abortuson a AFLP generated dendrogram . Marine isolates are not found on manyBrucellaphylogenetic trees. Ours grouped the marineBrucellaandB. neotomaewithB. melitensisbut on separate branches. This is in agreement with the genetic diversity observed among the marine isolates and proposals that marine isolates may comprise more than one species [3,41].