Intrinsic and selected resistance to antibiotics binding the ribosome: analyses ofBrucella23Srrn, L4, L22, EF-Tu1, EF-Tu2, efflux and phylogenetic implications

  • Shirley M Halling1Email author and

    Affiliated with

    • Allen E Jensen1

      Affiliated with

      BMC Microbiology20066:84

      DOI: 10.1186/1471-2180-6-84

      Received: 16 June 2006

      Accepted: 02 October 2006

      Published: 02 October 2006

      Abstract

      Background

      Brucellaspp. 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 classicalBrucellaspp.

      Results

      Minimum inhibitory concentration (MIC) values ofBrucellareference strains and three marine isolates to antibiotics binding the ribosome ranged from 0.032 to >256 μg/ml for the macrolides erythromycin, clarithromycin, and azithromycin and 2 to >256 μg/ml for the lincosamide, clindamycin. Though sequence polymorphisms were identified among ribosome associated loci 23Srrn,rplV,tuf-1 andtuf-2 but notrplD, 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.Brucellaefflux was examined as an alternative antibiotic resistance mechanism by use of the inhibitor L-phenylalanine-L-arginine β-naphthylamide (PAβN). Erythromycin MIC values of reference and all eryRstrains, except theB. suiseryRmutants, were lowered variably by PAβN. A phylogenetic tree based on concatenated ribosomal associated loci supported separate evolutionary paths forB. abortus, B. melitensis, andB. suis/B. canis, clustering marineBrucellaandB. neotomaewithB. melitensis. ThoughBrucella oviswas clustered withB. abortus, the bootstrap value was low.

      Conclusion

      Polymorphisms among ribosomal loci from the referenceBrucellado not correlate with their highly differential susceptibility to erythromycin. Efflux plays an important role inBrucellasensitivity to erythromycin. Polymorphisms identified among ribosome associated loci construct a robust phylogenetic tree supporting classicalBrucellaspp. designations.

      Background

      Brucellosis is a zoonotic disease caused by the Gram-negative bacteriumBrucella. It is taxonomically related to plant pathogens and other animal symbionts and is transmitted to humans from infected domestic animals and wildlife through contact during animal husbandry practices, meat production, or by ingestion of unpasteurized milk products. The genusBrucellacontains six classical species reflecting host preferences [1,2], and additional species have been proposed to include marine isolates from seal, dolphin, and porpoise [3]. The classical species and their hosts are:B. abortus, bovine;B. melitensis, caprine;B. suis, porcine;B. ovis, ovine;B. canis, canine; andB. neotomae, desert wood rat. However,B. suis and B. canishave similar metabolic profiles [4] and genomic maps [5], supporting their close relationship. Similarly, the metabolic characteristics and phage susceptibility ofB. suisbiovar 5 are more like that ofB. melitensisrather thanB. suis[6,7].

      The classicalBrucellaspp. designations are still widely used to emphasize important pathogenicity, virulence, and host preference differences among theBrucellaeven though similarity among the ribosomal RNA loci led to the designation ofBrucellaas a monospecific species [8,9],B. melitensis.Brucellaspeciation may have arisen as a result of their isolation due to different preferred hosts and to divergence of the host species [10] even though their 16Srrnloci are identical [1114]. In any case, discordant genotype/phenotype may require the use of other widely conserved loci to define bacterial species [15,16].

      Meyer [17] found differences in sensitivity to erythromycin among the classical species ofBrucellaand their biovars by measuring inhibition of growth using high and low concentration antibiotic discs.Brucella abortusbiovars except biovar 2 were resistant to erythromycin, andB. ovis,B. melitensis, andB. caniswere intermediate in resistance betweenB. abortusandB. suis. OnlyB. suisstrains were sensitive to the high concentration antibiotic discs. Meyer argued that investigating the ribosomal structure could explain these differences in sensitivity and generate critical knowledge "to account for and recapitulate the lineage of species and biotypes ofBrucella".

      Bacterial susceptibility to macrolide and lincosamide antibiotics results from their binding to 23S rRNA, inhibiting protein synthesis by blocking the peptide exit tunnel [1820]. Bacteria can become resistant to macrolides and lincosamides by spontaneous mutations of ribosomal associated loci or by increased efflux. Resistance to macrolides and lincosamides is commonly due to (i) mutation of 23Srrncausing a reduction in the binding of the antibiotics to the peptidyl transferase center [21,22], typically nts A2058, A2059, A2062, and C2611,Escherichia coli(Ec) 23S rRNA numbering, (ii) mutation of ribosomal proteins L4 or L22 leading to widening the entrance to the peptide exit tunnel allowing access to the tunnel even in the presence of the antibiotics [18,20,2325], (iii) methylation of ribosomal 23S rRNA [26], or (iv) increased efflux [27,28]. Bacterial resistance to synthetic macrolides or ketolides can be conferred by mutation of ribosome associated factor EF-Tu [29].

      There are several families of efflux pumps, though few non-RND (resistance nodulation division) family efflux pumps cause intrinsic or spontaneous resistance of Gram-negative bacteria to common clinical antibiotics [27,28,30,31]. Inability to demonstrate efflux activity however does not necessarily mean a lack of efflux. Efficiency of efflux of antibiotics is variable, being dependent on the structure of the antibiotic. Antibiotic resistance can be complex as observed forHaemophilus influenzaeL22 mutant HMC-C [32]. For this mutant, an increase in macrolide MIC values was only shown in the presence of efflux [33].

      Here, we show that the large differences in relative intrinsic susceptibilities of reference strains ofBrucellaand three marine isolates to macrolide antibiotics and a lincosamide do not correlate with ribosomal associated polymorphisms. We establish that antibiotic efflux plays an important role in differential antibiotic susceptibility inBrucella. A robust phylogenetic tree constructed from concatenation of ribosome associated polymorphisms illuminates relationships among theBrucella.

      Results

      MIC determination by Etest

      The relative MIC values of the classicalBrucellaspp. and three marine isolates (Table1) to macrolides and a lincosamide were determined by use of the Etest. Log-fold differences in MIC values were found (Fig.1). The susceptibility ofBrucellawas similar for the three macrolides erythromycin, azithromycin, and clarithromycin. OnlyB. abortus, except biovar 2, andB. melitensishad MIC values of ≥ 16 μg/ml. The pattern of sensitivity ofBrucellato the lincosamide clindamycin differed from that of the macrolides. Generally, MIC values were higher for clindamycin than for the macrolides.Brucella abortus, except biovar 2, was the most resistant to clindamycin, having MIC values of ≥ 128 ug/ml. OnlyB. melitensisbiovars 2 and 3 had lower MIC values for clindamycin than for erythromycin. ForB. suis, clindamycin MIC values ranged from a low of 3 μg/ml to a high of 24 μg/ml. The otherBrucellawith the exception of the seal isolate, ranged from 2 to 64 μg/ml. The seal isolate was resistant to clindamycin.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-6-84/MediaObjects/12866_2006_Article_297_Fig1_HTML.jpg
      Figure 1

      Minimal inhibitory concentration (MIC) of antibiotics toBrucellareference 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.Brucellastrains 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.

      Table 1

      Strains ofBrucellaused in this study.

      Species

      Biovar

      Strain

      Host

      Origin

      Reference

      B. abortus

      1

      544

      Cattle

      England

      ATCC* 23448

       

      1

      9–941

      Cattle

      USA

      11

       

      2

      86/8/59

      Cattle

      England

      ATCC 23449

       

      3

      Tulya

      Cattle**

      Uganda

      ATCC 23450

       

      4

      292

      Cattle

      England

      ATCC 23451

       

      5

      B3196

      Cattle

      England

      ATCC 23452

       

      6

      870

      Cattle

      Africa

      ATCC 23453

       

      9

      C68

      Cattle

      England

      ATCC 23455

      B. canis

       

      RM6/66

      Dog

      USA

      ATCC 23365

      B. melitensis

      1

      16 M

      Goat

      USA

      ATCC 23456

       

      2

      63/9

      Goat

      Turkey

      ATCC 23457

       

      3

      Ether

      Goat**

      Italy

      ATCC 23458

      B. neotomae

       

      5K33

      Wood rat

      USA

      ATCC 23459

      B. ovis

       

      63/290

      Sheep

      Africa

      ATCC 25840

      B. suis

      1

      1330

      Pig

      USA

      ATCC 23444

       

      2

      Thomsen

      Hare

      Denmark

      ATCC 23445

       

      3

      686

      Pig**

      USA

      ATCC 23446

       

      4

      40

      Reindeer

      USSR

      ATCC 23447

       

      5

      513

      Mouse

      USSR

      ***

      Brucellaspp.

       

      2/94

      Seal

      Scotland

      6

      ("maris")

       

      1/94

      Porpoise

      Scotland

      6

        

      14/94

      Dolphin

      Scotland

      6

      *ATCC, American Type Culture Collection, Beltsville, MD; **Isolate from human; ***Reference strain 513 not deposited in ATCC.

      23S rrn sequence comparisons

      Sequences of two regions of theBrucella23Srrnencoding 2498 nts (69 to 1678 and 1920 to 2807), including sites of 23Srrnmutations known to increase bacterial resistance to macrolides and clindamycin were determined and compared (Table2). Mixtures of cells or DNA (1:3) with disparate 23Srrnsequences were amplified to demonstrate that heterogeneity among the three 23Srrncopies would be detectable (data not shown). Though the distal portion of 23SrrnC from the genomicB. suis1330 sequence could not be amplified with either of two primer pairs that were complementary to the publishedB. suis23SrrnC genomic sequence, amplification was successful using primers homologous to internal, conserved genomicrrnC sequences from all threeBrucellagenomes and sequences flankingrrnC fromB. abortusandB. melitensis. The amplified distal portion ofrrnC fromB. suis1330 was identical in sequence to that ofrrnA andrrnB fromB. suis. Among the 23Srrnsequences fromBrucella, three polymorphic and three monomorphic sites were identified. In addition, three monomorphisms were identified in the 23Srrnintervening sequences.
      Table 2

      Brucella23Srrnpolymorphisms&.

      Brucellastrains

      *Nt 1085 934 (Ec)

      *Nt 1564 1423 (Ec)

      *Nt 2632 2610 (Ec)

       

      A

      G

      A

      G

      T

      C

      Ba b1

       

      X

       

      X

       

      X

      Ba b2

       

      X

       

      X

       

      X

      Ba b3

       

      X

       

      X

       

      X

      Ba b4

       

      X

       

      X

       

      X

      Ba b5

       

      X

       

      X

       

      X

      Ba b6

       

      X

       

      X

       

      X

      Ba b9

       

      X

       

      X

       

      X

      Bc

      X

       

      X

       

      X

       

      Bs b1

      X

       

      X

       

      X

       

      Bs b2

      X

       

      X

       

      X

       

      Bs b3

      X

       

      X

       

      X

       

      Bs b4

      X

       

      X

       

      X

       

      Bs b5

       

      X

      X

        

      X

      Bo

       

      X

      X

        

      X

      Bn

       

      X

      X

        

      X

      Bm b1

       

      X

      X

        

      X

      Bm b2

       

      X

      X

        

      X

      Bm b3

       

      X

      X

        

      X

      Dolphin

       

      X

      X

        

      X

      Porpoise

       

      X

      X

        

      X

      Seal

       

      X

      X

        

      X

      &GenBank accession numbers for eachBrucella23Srrnsequences are listed in Methods.#Brucellastrains are as listed in Table 1. *Nt positions as perB. abortus23SrrnA [GenBank:AE017223, BruAb1_rrna_0005, Gene ID: 3339965]. (Ec) denotes position inEscherichia coli23Srrn[GenBank:U00096].

      23Srrnpolymorphisms were detected at nts 1085 (934, Ec), 1564 (1423, Ec), and 2632 (2610, Ec), clustering theBrucellainto three groups: (1)B. abortus, (2)B. canisandB. suis, except biovar 5, and (3)B. melitensis,B. ovis,B. neotomae,B. suisbiovar 5, and the dolphin, seal and porpoise isolates (Table2). Note that all the 23Srrn Brucellapositions are numbered based onB. abortus23SrrnA, including the intervening sequence. The only polymorphism that occurred in the peptidyl transferase center was nt 2632 (2610, Ec). No correlation could be made between the polymorphisms and relative antibiotic susceptibility. Other sites known to affect susceptibility to macrolides and clindamycin were not polymorphic.

      Monomorphisms were found in both 23Srrnand in the 23Srrnintervening sequences. Monomorphisms were identified in 23S rRNAs from dolphin (A955G);B. neotomae(insertion of a C between nt 1002–1006); andB. suisbiovar 5 (T2090C). Several intervening sequences of theBrucella23Srrnloci varied from the consensus sequence reported by Bricker [34]. The C indel in the intervening sequences ofB. melitensis16 M, forming a string of six Cs instead of five beginning at nt 222, reported by Bricker was confirmed. The other two monomorphisms occurred inB. suisbiovar 5 (C219T) andB. melitensisbiovar 3 (C206T).

      L4 analyses

      Though the GenBankBrucellagenomicrplDsequences encoding ribosomal protein L4, differed due to an indel inrplDfound only in theB. melitensis16 M genomic sequence, we did not observe this indel in our sequence ofrplDfromB. melitensis16 M. We found the threerplDgenes from the genomes were identical. Though no polymorphisms were identified among therplDsequences, three monomorphisms were discovered among their amino termini. Two sequence transitions,B. neotomae(G108A) andB. suisbiovar 2 (C213T), were found. Both of which were silent. A transversion identified in the porpoise isolate (G314T) would replace an Arg, a charged amino acid (aa), with Leu, a noncharged one.

      L22 polymorphisms

      Putative L22 sequences from theBrucellareference strains and three marine isolates (Fig.2) were determined and their tertiary structures predicted and compared by using Swiss-Pdb Viewer (Fig.3). Among theBrucellaputative L22 sequences, all variations except one occurred in the β-hairpin loops or near the carboxy termini.Brucella suisbiovar 5 alone had an alternate Ala codon at aa 44. TheBrucellaβ-hairpin loops were polymorphic and variable in length due to variable copy numbers of a two aa motif, Gly-Arg. The lengths of all the β-hairpin loops of putative L22 peptides except those fromB. neotomaeandB. suisbiovars 2 and 3 were equal, 11 aa. The β-hairpin loops of L22 fromB. suisbiovars 2 and 3 were shorter due to a net two aa (Gly-Arg) deletion, while the β-hairpin loop fromB. neotomaewas longer due to a net two aa (Gly-Arg) insertion. Within the loop of the β-hair pin at aa 101, there was either a Gly, Val, or an Asp. While the variation of sequence at aa 101 of L22 did not greatly affect the predicted tertiary structures, the indels did (Fig.3). A polymorphism was also identified very near the 3'-end ofrplV. L22 polymorphic sites grouped theBrucelladifferently than other loci in this study. Putative L22 sequences fromB. abortusandB. melitensiswere identical.Brucella suisbiovars 1, 4 and 5, and the marine isolates were identical. Putative L22 sequences fromB. suisbiovars 2 and 3 differed from those of biovars 1, 4 and 5 in having one rather than two Gly-Arg motifs. Putative L22 sequences from bothB. ovisandB. neotomaewere unique. No correlation could be made between relative antibiotic susceptibilities of theBrucellastrains and their L22 sequences.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-6-84/MediaObjects/12866_2006_Article_297_Fig2_HTML.jpg
      Figure 2

      Ribosomal protein L22 polymorphisms amongBrucellareference 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 putativeBrucellaL22 is given below. Amino acids occurring in the stalk of the β-hairpin appear inbold-italicsand amino acids occurring in the loop of the β-hairpin are inbold. Beneath amino acids that are double underlined is a list ofBrucellastrains 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 ofrplVsequences for each strain are deposited in GenBank and are listed in Materials.

      http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-6-84/MediaObjects/12866_2006_Article_297_Fig3_HTML.jpg
      Figure 3

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

      EF-Tu sequence comparisons

      Though the nt sequences of EF-Tu loci,tuf-1 andtuf-2, were polymorphic (Table3), the putative peptide sequences of EF-Tu1 and -2 were conserved. In mostBrucellastrains,tuf-1 andtuf-2 sequences were identical. In the cases where they were not identical, they varied by a single nt near either the 5' or 3' termini of the genes, namely nt 12 and nt 1158. Unlike the 23Srrnsequences, the sequences oftuf-1 andtuf-2 fromB. abortuswere more similar to those fromB. suisthan fromB. melitensis. The referenceB. abortusbiovar 1 strain, 544, differed from that of the sequenced strain,B. abortusbiovar 1 strain 9–941 (Table3). Nt 1158 oftuf-2 fromB. abortus544 differed fromB. abortusbiovar 1 strains 2308 and 9–941. Further, nt position 1158 oftuf-1 fromB. abortusstrains 9–941 and 2308 was identical to those fromB. abortusbiovars 2 and 4 but differed from the otherB. abortusbiovars. Whereas a single nt varied among copies oftuf-1 andtuf-2 fromB. abortusbiovar 1 strain 9–941 andB. suis1330, eight nt varied betweenB. suis1330 andB. melitensis16 M. The other classical spp. and the marineBrucellawere intermediate betweenB. abortus/B. suisandB. melitensis(Table3).
      Table 3

      Polymorphic sites amongtuf-1 andtuf-2 fromBrucella.

      $Nt position

      12

      36

      141

      183

      198

      219

      345

      378

      511

      609

      936

      1158

      # Bru↓|Nt→

      T

      C

      C

      T

      C

      T

      C

      A

      G

      A

      A

      G

      C

      T

      C

      T

      C

      A

      G

      A

      C

      T

      C

      T

      Bab 9–941

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      2

      1

      Bab b1

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

        

      X

      Bab b2

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      2

      1

      Bab b3

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      Bab b4

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      2

      1

      Bab b5

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      Bab b6

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      Bab b9

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      B. suis b1

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      B. suis b2

      1

      2

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

        

      X

      X

       

      X

       

      X

       

      B. suis b3

      1

      2

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      B. suis b4

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      B. suis b5

      1

      2

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      Bmel b1

      X

        

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

      X

       

      X

        

      X

      X

       

      Bmel b2

      X

        

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

      X

       

      X

        

      X

      X

       

      Bmel b3

      X

        

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

      X

       

      X

        

      X

      X

       

      Bneo

       

      X

      X

        

      X

       

      X

       

      X

       

      X

      X

        

      X

      X

        

      X

      X

       

      X

       

      B. ovis

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      B. canis

      1

      2

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      X

       

      Seal

      1

      2

      X

       

      X

       

      X

       

      X

        

      X

      X

        

      X

      X

       

      X

       

      X

       

      X

       

      Dolphin

      1

      2

      X

       

      X

       

      X

       

      X

        

      X

      X

        

      X

      X

       

      X

       

      X

       

      X

       

      Porpoise

      X

       

      X

       

      X

       

      X

       

      X

        

      X

      X

        

      X

      X

       

      X

       

      X

       

      X

       

      $Nt = nucleotide;#Brucellastrains, see Table 1; X =tuf-1 andtuf-2 have identical sequences; 1 =tuf-1 sequence only, 2 =tuf-2 sequence only. Polymorphic sites are inbold.tuf-1 andtuf-2 GenBank accession numbers are listed in Materials and methods.

      Erythromycin mutants

      EryRmutants of several referenceBrucellastrains having MIC values less than 2 μg/ml and the three marineBrucellawere selected. Mutant strains were not recovered fromB. ovis,B. abortusbiovar 2, orB. suisbiovar 5. Three ribosomal associated loci, 23S rrn,rplV, andrplD, were analyzed from eryRmutants ofB. suisbiovar 1,B. canis,B. neotomae, and the three marineBrucella(Table4). The spontaneous eryRrate among the classicalBrucellaspp. varied by 100-fold. Rates of mutation to eryRand highest concentration of erythromycin allowing growth for each parental strains of the classicalBrucellaspp. were:B. suis3.7 × 10-7(5 μg/ml erythromycin);B. canis1.5 × 10-8(20 μg/ml erythromycin); andB. neotomae6.6 × 10-8(5 μg/ml erythromycin). All the marine eryRisolates were selected from plates containing 20 μg/ml erythromycin, and mutational rates were 1.9 × 10-6, 5.1 × 10-7, and 2.7 × 10-6for porpoise, seal, and dolphin, respectively.
      Table 4

      Determination and characterization of erythromycin and clindamycin MICs and molecular characterization ofBrucellaeryRmutants.

      Brucella ery R strains

      ERY

      ERY + PAβN

      CL

      CL + PAβN

      Loci

      Mutation

      B. suis

      WT

      1.5

      0.75

      8

      4

      NA

       
       

      a

      6

      6

      12

      12

      -

       
       

      b

      6

      6

      12

      12

      ND

       
       

      c

      4

      6

      8

      8

      -

       
       

      d

      6

      6

      12

      12

      ND

       
       

      e

      6

      6

      8

      8

      ND

       

      B. canis

      WT

      0.5

      0.094

      8

      0.5

      NA

       
       

      a

      2

      0.32

      8

      1.0

      -

       
       

      b

      2

      0.047

      6

      <0.016

      -

       
       

      c

      3

      0.047

      8

      0.38

      -

       
       

      d

      3

      0.047

      4

      0.125

      -

       
       

      e

      2

      0.19

      8

      3

      -

       

      B. neotomae

      WT

      0.75

      0.047

      3

      0.75

      NA

       
       

      a

      4

      0.125

      8

      0.75

      -

       
       

      b

      3

      0.125

      6

      2

      ND

       
       

      c

      2

      0.064

      6

      1.5

      -

       
       

      d

      6

      0.25

      6

      1.0

      L22

      6 bp indel, β-loop

       

      e

      2

      0.38

      4

      2

      -

       

      Propoise

      WT

      1.5

      0.25

      16

      3

      NA

       
       

      a

      16

      0.75

      4

      3

      L4

      G209A; Gly70Asp

       

      b

      >256

      >256

      >256

      >256

      23S

      A2058G (Ec)

       

      c

      192

      1.5

      4

      3

      L4

      Δ18aa (54–71)

       

      d

      48

      0.38

      6

      3

      L4

      G209A; Gly70Asp

       

      e

      128

      1.5

      4

      3

      L4

      Δ30aa (54–81)

      Seal

      WT

      6

      1.5

      >256

      48

      NA

       
       

      a

      24

      12

      96

      96

      -

       
       

      b

      32

      24

      >256

      >256

      -

       
       

      c

      >256

      >256

      16

      16

      L4

      C217A; Arg73Ser

       

      d

      128

      16

      >256

      >256

      -

       
       

      e

      128

      24

      >256

      >256

      ND

       

      Dolphin

      WT

      3

      0.38

      2

      0.75

      NA

       
       

      a

      >256

      128

      1.0

      1.5

      L4

      G209A; Gly70Asp

       

      b

      >256

      6

      1.5

      1.0

      L4

      C217T; Arg73Cys

       

      c

      >256

      192

      1.5

      1.5

      L4

      G209A; Gly70Asp

       

      d

      >256

      8

      2.0

      1.0

      L4

      C217T; Arg73Cys

       

      e

      >256

      8

      1.5

      1.0

      ND

       

      Mutants examined were isolates ofB. suisbiovar 1, B. canis, B. neotomae, and marine porpoise, seal and dolphin strains as per Table 1; WT, wild type; Ery, erythromycin; CL, clindamycin, PAβN, efflux inhibitor L-phenylalanine-L-arginine-β-naphthylamide; -, no mutation identified in any of three ribosomal loci examined; NA, not applicable; ND, sequence not determined.

      Though MIC values of the eryRmutants of the classical strainsB. suis,B. canis, andB. neotomaeincreased, they did not increase as much as those of the marine isolates (Table4). While MIC values increased for the eryRmutants of the classicalBrucellastrains, the increases were only 2 to 6-fold compared to 15 to > 256-fold for the marineBrucella. Over half of the marine eryRmutants had erythromycin MIC values of 128 μg/ml or higher, and all the dolphin mutants had MIC values greater than 256 μg/ml. The clindamycin MIC values of the eryRmutants were similar to those of the parental strains except for porpoise b which had a mutation in 23Srrnand the seal isolates a and c.

      Mutations among the marine eryRmutants were found in two ribosomal associated loci, 23SrrnandrplD. Only a single mutation was identified in 23Srrn. Porpoise isolate b had a mutation within the peptidyl transferase center of 23Srrn, nt 2058 (Ec), but all three 23Srrncopies were not mutated, as the signal was mixed. This mutant was resistant to both erythromycin and clindamycin, MIC values >256 μg/ml. Most ribosomal associated mutations occurred inrplD(Table4), and these were only found among the marine isolates. The mutations were not random. Several eryRisolates had mutations at nt 209 or nt 217. At nt 209, porpoise isolates a and d and dolphin isolates a and c had an A instead of a G, substituting an Asp for a Gly. Dolphin eryRmutants isolates b and d had a T instead of a C at nt 217 ofrplD, resulting in the incorporation a Cys of rather than an Arg. Seal eryRisolate c had an A instead of a C at nt 217 which resulted in the incorporation of a Ser rather than an Arg. Two of the porpoise eryRisolates c and e had deletions inrplD, resulting in the loss of 18 or 30 codons. The deletion of 18 aa in L4 of porpoise c is consistent with recombination between two copies of 5'GGG-CCG-CGC-3' occurring between nt 153–161 and 207–215.

      Only one ribosomal associated loci mutation was identified among theB. suis,B. canis, andB. neotomaeeryRmutants by analyzes of 23Srrn,rplD, andrplV. A duplication of a six-bp repeat in the β-hairpin loop of L22 ofB. neotomaeisolate d expanded the number of Gly-Arg aa repeats from three to four (Fig.2, Table4). This mutant had a slightly higher MIC value for erythromycin.

      Efflux

      Erythromycin and clindamycin MIC values of the reference strains and the eryRmutants were analyzed in the presence of the efflux inhibitor PAβN (Tables4and5). Using Etest strips, a decrease in MIC values could only be detected if the MIC values >256 μg/ml fell to or below 256 μg/ml. MIC values of the reference strains decreased variably in the presence of the inhibitor (Table5). Though PAβN affected the erythromycin MIC value forB. suisbiovar 1, reducing it two-fold or by two dilutions as per the Etest, the MIC values for theB. suisbiovar 1 eryRmutants were not affected. In the case ofB. abortusbiovar 5, even though its erythromycin MIC value was lowered in the presence of PAβN, the clindamycin MIC value was unaffected.
      Table 5

      Effect of efflux on MICsof Brucellareference strains for erythromycin and clindamycin.

      Brucella

      MIC

       

      Ery

      Ery + PAβN

      CL

      CL + PAβN

      B.abb1

      >256

      96

      >256

      96

      B.abb2

      0.19

      0.0275

      3

      0.25

      B.abb3

      128

      64

      192

      128

      B.abb4

      128

      64

      192

      64

      B.abb5

      >256

      192

      128

      128

      B.abb6

      >256

      256

      >256

      96

      B.abb9

      >256

      >256

      >256

      >256

      B.melb1

      16

      4

      24

      16

      B.melb2

      >256

      48

      64

      12

      B.melb3

      256

      48

      64

      32

      B. suisb1

      1.5

      0.75

      8

      4

      B. suisb2

      0.125

      0.032

      3

      0.047

      B. suisb3

      1.5

      1.0

      24

      48

      B. suisb4

      2

      1.5

      24

      32

      B. suisb5

      0.094

      0.047

      6

      2

      B. canis

      0.50

      0.094

      8

      0.50

      B. ovis

      0.064

      <0.016

      3

      <0.016

      B.neo

      0.75

      0.047

      3

      0.75

      Porpoise

      1.5

      0.25

      16

      3

      Seal

      6

      1.5

      >256

      48

      Dolphin

      3

      0.38

      2

      0.75

      Brucellastrains are as listed in Table 1; MIC, minimum inhibitory concentration; Ery, erythromycin; CL, clindamycin; PAβN, efflux inhibitor L-phenylalanine-L-arginine β-naphthylamide; b, biovar; ab,abortus;mel,melitensis;neo,neotomae.

      Efflux inhibition among the eryRmutants by PAβN (Table4) was variable among the strains. TheB. canisandB. neotomaeeryRmutants had decreases in their erythromycin and clindamycin MIC values in the presence of PAβN. The seal eryRmutants had increased erythromycin MIC values that were variably reduced in the presence of PAβN. For example, isolate a had an erythromycin MIC value of 24 μg/ml which was reduced to 12 μg/ml by PAβN, but isolate d had a MIC value of 128 μg/ml which was reduced to 16 μg/ml. Only the seal eryRclindamycin MIC values were either identical to (>256 μg/ml) or lower than (96 and 16 μg/ml) that of the parental strain, and none were affected by PAβN. The dolphin eryRmutants differed from all the other eryRmutants in that they had uniform erythromycin MIC increases and the highest MIC increases of any of the other groups. Though all the dolphin eryRmutants' erythromycin MIC values increased from 3 μg/ml to >256 μg/ml, their erythromycin MIC values were differentially affected by PAβN. In the presence of PAβN, two dolphin eryRmutants had MIC values equal to or greater than 128 μg/ml while the rest had MIC values of 8 μg/ml or less. Like the porpoise isolates, except isolate b, all the dolphin isolates had lower clindamycin MIC values than the parental strain and the clindamycin MIC values were only slightly affected by PAβN.

      Phylogenetic tree

      A phylogenetic tree was constructed using concatenated 23Srrn,rplV,tuf-1, andtuf-2 (Fig.4).Brucellaformed a node with the closest clades being other α-Proteobacteria,Agrobacterium, Mesorhizobium, andCaulobacterfollowed byLeptospiraand γ-Proteobacteria,Xylella,Acinetobacter, and the facultative intracellular animal pathogenLegionella. The cluster containing theBrucellaspecies is robustly formed (high bootstrap values) into a distinct clade separate from the outgroups and forming four nodes subclustering: (1)B. abortusandB. ovis; (2)B. suisandB. canis;(3)B. melitensis, B. neotomaeand the marineBrucella; and (4)B. suisbiovar 5.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-6-84/MediaObjects/12866_2006_Article_297_Fig4_HTML.jpg
      Figure 4

      Phylogeny ofBrucellacalculated using highly conserved ribosomal associated loci. Shown is the single optimization alignment tree based onrplV,tuf-1,tuf-2, and 23S rrn sequences from 28 taxa consisting of the 21Brucellastrains (see Table 1), which included the 18 classicalBrucellareference strains and three marineBrucella, 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 pneumophilasubspecies Pneumophila strain Philadelphia [GenBank:NC_002942] was used to root the tree. Other bacterial outgroups include:Acinetobacterspecies ADP1 [GenBank:NC_005966],Caulobacter crescentusCB15 [GenBank:NC_002696],Leptospira interrogansserovar Copenhagen strain Fiocruz L1-130 [GenBank:NC_005823],Mesorhizobium lotiMAFF303099 [GenBank:BA000012],Agrobacterium tumefaciensC58 circular [GenBank:NC_003062] and linear chromosomes [GenBank:NC_003063], andXylella fastidosa9a5c [GenBank:NC_002488].

      The tree constructed from a concatenated sequence, i.e. a supergene or supermatrix, was consistent with a concatenated tree calculated from individual loci (data not shown). Both trees supported classical classification, clustered the marine isolates withB. melitensis, and indicated intrinsic differences among marineBrucella. Bootstrap numbers (Fig.4) were robust for all nodes (99 or 100) exceptB. ovis, which clustered withB. abortus; in the additive tree,B. ovisformed a unique branch. ThoughB. suisandB. caniscomposed a node,B. suisbiovars 1 and 4 were on one branch andB. suisbiovars 2 and 3 on another branch withB. canis. Shared 23Srrnpolymorphisms divided theBrucellainto three groups, placingB. melitensisbetweenB. abortusandB. suis(Table2). Thetuf-1 andtuf-2 sequences (Table3) separatedB. abortusandB. suisand placedB. neotomaeand the marine isolates intermediate betweenB. abortusandB. suisandB. melitensis. TherplVfromB. abortusandB. melitensiswere identical. Indels inrplVsplitB. suisbiovars into two groups.

      Discussion

      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 [17] 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 [35].

      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 [36]. 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 [23]. Methylation of either nt 2059 or 2058 (Ec) of the peptidyl transferase center reduces the sensitivities of bacteria to macrolides and lincosamides [26]. We were unable to identify homologs of any 23Sermmethylation genes by BLAST [37], but, then, methylation of ribosomal rRNA is much more widely described in Gram-positive clinical isolates [26].

      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 [38]. 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 [39] 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[40].

      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 [28]). 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 [41] of the insertion sequence IS711[42]. IS711has been shown to mobilize inBrucellaunder stress or selective pressure [43,44] and could be a source of instability [42] in marineBrucella.

      Brucellaphylogenetic trees and dendrograms have been constructed based on genomics maps [3,5,6], amplified fragment length polymorphisms (AFLP) [45], multilocus enzyme electrophoresis (MLEE) [6], and outer membrane proteinsomp2a/omp2b[3]. 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 [16]. 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) [46]. 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 [46]. 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 [45]. 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].

      Conclusion

      Ribosomal associated polymorphisms among the referenceBrucellaspp. did not correlate with differential intrinsic antibiotic resistance to erythromycin or clindamycin. Efflux is an important mechanism of resistance to macrolides and the lincosamide clindamycin inBrucellaand can be inhibited by the RND efflux inhibitor PAβN. A phylogenetic tree constructed based on concatenated ribosomal associated loci supports alternative evolutionary paths forB. melitensis,B. abortus, andB. suis, and clustered the marineBrucellawithB. melitensis, andB. caniswithB. suis. It also supports the doubtful close relationship ofB. suisbiovar 5 withB. suis.

      Methods

      Bacterial strains and growth conditions

      Bacterial strains (Table1) were obtained from our laboratory collection for this study. Bacteria were grown at 37°C on tryptose agar (DIFCO Laboratories, Detroit, MI) containing 5% bovine serum in the presence of 7.5% CO2. Cells were suspended in saline (1010CFU/ml), mixed with two volumes of methanol, and stored at 4°C until needed.

      Etest

      In vitroactivities of azithromycin, clarithromycin, erythromycin, and clindamycin were determined by the Etest (AB Biodisk, Piscataway, NJ). The highest MIC determination for these antibiotics using the Etest is 256 μg/ml. The preformed gradient of the Etest strips covers a continuous MIC range corresponding to 15 two-fold dilutions with a precision of 0.5 dilution. Bacterial inocula were prepared by adjusting the turbidity of a 48 h culture to a 0.5 McFarland standard (5 × 108CFU/ml). The suspension was streaked onto Difco™ Mueller Hinton agar (Becton, Dickinson and Company, Sparks, MD) in the presence or absence of 25 μg/ml of efflux inhibitor PAβN [49] (Sigma Chemical Co., St. Louis, MO) using a cotton swab, and the Etest strips applied. Plates were incubated (37°C) in 7.5% CO2. Results were read after 48 h.

      Selection of erythromycin mutants

      Brucellastrains having erythromycin MIC values <5 μg/ml were suspended in saline and plated (108cfu) in triplicate onto Difco™ Mueller Hinton agar containing 5, 10, or 20 μg/ml of erythromycin (Sigma Chemical Co.) and incubated at 37°C in the presence of CO2. Five colonies were selected from plates with the highest concentration of erythromycin supporting growth, and subsequently streaked onto tryptose serum agar and Difco™ Mueller Hinton agar containing erythromycin.

      PCR amplification

      Master mixes for PCR reactions were prepared by use of the Fast Start Taq DNA polymerase kit (Roche Molecular Biochemicals, Indianapolis, IN) according to manufacturer's instructions. Methanol treated cells were diluted 1/10 in water and used immediately or stored at 4°C up to 2 months. One μL was added per 25 μL of reaction mixture. Reactions were 50 or 100 μL. Cells were disrupted and amplification initiated by heating the reactions to 95°C for 5 min. Melting, annealing, and elongation temperatures and times were 95°C for 15 sec, 60°C for 30 sec, and 72°C for 90 sec, respectively. After amplification for 35 or 40 cycles, elongation was extended by 4 min.

      Primers

      The primer sets for amplification and sequencing of 23Srrngenes annealed torrnA fromB. abortus9–941 [GenBank:AE17223, BruAb1_rrna_0005]: nt 37–62 (CAT-GCA-CAG-GCG-ATG-AAG-GAC-GTG-AT) and nt 540–518 (GGA-TTT-CAC-GTG-TCC-CGC-CCT-ACT-CA) (note that this set amplified intervening sequence); nt 455–481 (AGT-TGG-AAA-ACT-CGA-CCG-AAG-TGG-GTG) and nt 1153-1127 (CCT-TAG-ATG-GTG-GTC-AGG-GTT-GTT-GCC); nt 1013–1039 (GAG-CAC-TGG-ATG-GGC-TAT-GGG-GAC-TCA) and nt 1732-1707 (GTG-CAT-TTT-GCC-GAG-TTC-CTT-CAA-CG); nt 1868–1894 (CCG-GTG-CTG-GAA-GGT-TAA-GAG-GAG-AGG) and nt 2605-2579 (CCC-AAC-TCA-CGT-ACC-GCT-TTA-AAT-GGC); and nt 2505–2529 (CGG-GGT-TGT-TTG-GCA-CCT-CGA-TAT-C) and nt 2850-2825 (CCC-GGC-CTA-TCA-ACG-TGG-TGG-TCT-TC). Primers used in PCR reactions to amplify the 3' end based on the genomic sequence ofrrnC fromB. suis[GenBank:NC_004311, Bs23SC] were forward (GGT-TTC-CCG-CTT-AGA-TGC-CTT-CAG-GA) and reverse 1 (CTT-CAG-AGA-TTA-TCC-CGT-CCG-TAT-ATA-TCT-ACC) and reverse 2 (ATA-GTG-ATC-CGG-TGG-TCC-CGC-GTG). Primers based on 23SB. suis rrnC unique sequences andB. suissequences flankingrrnC, respectively, were: (GGG-TCC-AGG-ACC-GTG-TAT-GGT-GGG-TAG) and (CTT-CCA-TCC-ATG-AGC-GGC-AAA-GGA-AAT-G). Primers for amplification of L4, L22, EF-Tu1, and EF-Tu2 were: L4 forward 1, (ACG-ACC-ACG-ATC-TGC-CGA-AGA-AGG-TTC), and reverse, (GCC-ACG-TTG-AAG-ACG-ACC-TGG-TG); L4 forward 2, (GTG-TTC-AAG-GGC-AAG-AAG-ATG-GCT-GGT-C) and reverse 2, (GAT-CTC-CGC-ACC-GCC-GAT-AAG-AAG-TG); L22 forward, (GCG-GAT-CTT-GAC-ATC-TTC-ATG-CAG-CAG), and reverse, (TTG-TCG-GTC-TGA-CTT-TCG-GCG-TCT-ACA); EF-Tu1, forward, (TCA-AGG-CGA-ATG-CGG-ATG-TTT-TGA-CC) and reverse (GCG-GTC-GCA-CAG-GAA-ATC-CAG-AAG-AAG); and EF-Tu2 forward, (GCG-GGG-AAT-TAT-CTC-GGC-AGC-ACT), and reverse, (CGA-GCG-GTA-TGG-CGT-GTA-AGG-AAT-CAT). Primers internal to the PCR products were synthesized as necessary to obtain sequences of the products.

      Sequence determination and comparisons

      Amplified products were purified (QIAquick PCR Purification Kit, Qiagen, Valencia, CA) and sequenced at the Genomics Center at the National Animal Disease Center, Ames, IA (ABI Prism 3700 DNA Analyzer) using primers as listed above. For large products, internal primers were synthesized as needed to obtain coding sequences. Sequences were assembled and aligned, and polymorphisms were identified by use of Sequencer 3.1.2 (Genes Codes Corp., Ann Arbor, MI). In some cases, MacVector (ClustalW) was used for sequence comparisons.

      Protein folding

      The Swiss-Pdb viewer software version 3.7 [50,51] was used to predict folding of L22 based on the coordinates determined for L22 fromThermus thermophilus[52].

      23S rrn base numbering

      The numbering forBrucella23Srrnis based onrrnA fromB. abortus9–941 [GenBank:NC_006932, Gene ID: 3339965]. When the nts refer toE. coli23S rRNArrnG [GenBank:U00096, GI:48994873], the nts are followed by (Ec).

      Accession numbers

      Genome and genomic sequences referred to in this study:B. suis1330 [GenBank:AE014291 and AE014292],B. melitensis16 M [GenBank:AE008917 and AE008918],B. abortus9–941 [GenBank:AE017223 and AE017224],B. abortus2308 [GenBank:AM040264], andB. abortus rrnA sequence [GenBank:NC_006932, Gene ID: 3339965]. Genomic sequences determined in this study: L4 (rplD) [GenBank:DQ289557 to DQ289577], L22 (rplV) [GenBank:DQ227901 to DQ227921], EF-Tu1 (tuf-1) [GenBank:DQ227922 to DQ227942], EF-Tu2 (tuf-2) [DQ227943 to DQ227963], 23Srrnregion 1 (nt 69 to 1678) [GenBank:DQ287886-DQ287906], and 23Srrnregion 2 (nt 1920–2807) [GenBank:DQ287865-DQ287885]. EryRmutant strain ribosomal associated sequences:B. neotomae(rplV) [GenBank:DQ659536], porpoise b (23Srrn) [GenBank:DQ659537], dolphin a-d (rplD) [GenBank:DQ660399-DQ660402], porpoise a, c-e (rplD) [GenBank:DQ6600403-DQ6600406], and seal c (rplD) [GenBank:DQ660407].

      Dendrogram

      Data sets consisted of concatenated genes (rplV,tuf-1,tuf-2) and sequences from 23Srrnfrom 28 taxa consisting of the 18Brucellareference strains, three marine isolates, and seven outgroups of known genomic sequences (see Table1and Fig.4). 23Srrnintervening sequences were eliminated from the comparison. Assembled sequences were aligned using ClustalW v1.83 [53]. Each nucleotide data set was then analyzed under the optimal criteria of maximum likelihood using MrBayes v3.1.2 Baysian analysis and Markov Chain Monte Carlo methods to search tree space and infer posterior distribution of topologies [54]. Settings for MrBayes were the general time reversible substitution model with sites drawn from a gamma distribution. The outgroup for MrBayes wasLegionella pneumophilasubspecies Pneumophila strain Philadelphia [GenBank:NC_002942]. The number of generations was set at 1,000,000, number of chains at 4, print frequencies at 10,000, and sample frequencies at 100. Branch lengths were saved and all other settings were default. The evolutionary tree was displayed using Tree Explorer [55] based on options used to compute or display the phylogeny.

      Abbreviations

      Standard three letter code was used for amino acids and standard one letter code for DNA bases.

      Declarations

      Acknowledgements

      We thank Brooke Petersen-Burch for his assistance in implementation of protein folding software and Kari Loehndorf for advice in the construction of phylogenetic trees. We thank David Alt and Karen Halloum for assistance in DNA sequencing.

      Authors’ Affiliations

      (1)
      Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture,  

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      © Halling and Jensen. 2006

      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.