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Journal of Clinical Microbiology, December 2004, p. 5439-5443, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5439-5443.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genetic Bases of the Rifampin Resistance Phenotype in Brucella spp.

Dipartimento di Sanità Alimentare ed Animale, Istituto Superiore di Sanità, Rome, Italy

Received 1 April 2004/ Returned for modification 10 July 2004/ Accepted 1 August 2004

	ABSTRACT

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Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the ß subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rif^r) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rif^r in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rif^r vaccine strain Brucella abortus RB51 and of 20 Rif^r clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rif^s) parental strains and to the published B. melitensis strain 16M. All Rif^r strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two "hot" regions were investigated in eight additional Rif^r Brucella laboratory mutants and in 20 reference Rif^s Brucella strains. rpoB mutations were found in all Rif^r mutants. In contrast, no missense mutations were found in any analyzed Rif^s strains. Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rif^r phenotype in prokaryotes. In addition, because of the relationship between Rif^r and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella.

	INTRODUCTION

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Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens and is an important component of effective multidrug therapies for the treatment of brucellosis in humans (2, 5, 26). Previously reported data have indicated that the addition of rifampin to media tends to turn Brucella abortus cultures rough and that organisms with rifampin resistance (Rif^r) are less virulent than rifampin-susceptible (Rif^s) strains (17, 23). Rifampin was therefore utilized to obtain the stable, rough, and attenuated B. abortus strain RB51, currently used in the United States as the official vaccine for brucellosis eradication in cattle. The roughness and attenuation are two important characteristics for the vaccine strain RB51, which is able to induce an adequate, protective cell-mediated response against infection with virulent Brucella strains without interfering with standard serological tests for brucellosis diagnosis (7, 15, 16, 18, 19, 20, 22, 27). Strain RB51 was derived by repeated passaging of the smooth and virulent B. abortus strain 2308 on media supplemented with rifampin (23). Its high level of Rif^r constitutes a useful biochemical marker for its identification. This characteristic was used to develop a selective medium to assist in the recovery of the vaccine strain RB51 from experimental and field samples for its isolation (10).

The genetic bases for Rif^r in RB51 are still unknown. In order to gain insight on genetic relationships between Rif^r and attenuation of virulence, we investigated the genetic bases for Rif^r in Brucella spp.

The rifampin antibiotic target in prokaryotes is the ß subunit of the DNA-dependent RNA polymerase (RNAP) encoded by the rpoB gene (11, 12, 13, 14). The bactericidal activity of rifampin stems from its high-affinity binding to, and inhibition of, the bacterial RNAP. Studies of crystal structure in Thermus aquaticus core RNAP have shown that the inhibitor binds in a pocket of the RNAP ß subunit deep within the DNA-RNA channel and blocks the path of the elongating RNA transcript at the 5' end. In general, amino acid substitutions of the rifampin binding pocket confer Rif^r through small structural distortions of the protein (4, 31). Several missense mutations within the ß subunit have been characterized in Rif^r pathogenic bacteria, such as Escherichia coli and Mycobacterium tuberculosis (6, 9, 11, 12, 16, 29, 30). No data concerning Brucella spp. are available.

In the present study, we have identified the rpoB mutations associated with the Rif^r phenotype in the rough vaccine strain B. abortus RB51 and in 20 stable Rif^r clones derived from two Rif^s rough isolates of Brucella melitensis. The rpoB coding region of 4,134 bp of Rif^r Brucella strains has been amplified, directly sequenced, and compared with that belonging to the respective Rif^s parent strains and to the published genome of B. melitensis strain 16M (8). Results were compared to those of 8 additional Rif^r Brucella laboratory mutants and to 20 reference Rif^s Brucella strains, including B. melitensis, B. abortus, Brucella suis, and Brucella ovis.

	MATERIALS AND METHODS

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Rif^r and Rif^s Brucella strains. The following Rif^r Brucella strains, as described in Table 1, were used: the vaccine strain B. abortus RB51, 20 Rif^r rough mutants derived from two Rif^s rough isolates of B. melitensis named CT1p and CT2p, and 8 Rif^r mutants of smooth or rough morphology derived from reference Rif^s Brucella strains. All Rif^r mutants but RB51 were cloned in our laboratory in accordance with published procedures but with minor modification (23). In brief, they were derived by repeated passaging of their respective Rif^s parental strains on plates containing brucella agar supplemented with 5% sterile horse serum (BAS; Oxoid Ltd., Hampshire, England) and with rising concentrations of rifampin (40, 200, and 400 µg/ml). The putative mutations were then stabilized by passaging the strains seven times on antibiotic-unsupplemented BAS plates. The selected clones were then prepared for PCR tests.

Rif^s test. All Brucella strains used in this work were tested for drug susceptibility by plating them on BAS plates containing 40 µg of rifampin/ml for 48 h at 37°C. Strains with >1% growth on rifampin-containing media compared to control media were considered resistant.

Sample preparation for PCR. A bacterial pellet was resuspended in 467 µl of Tris-EDTA buffer and digested for 1 h at 37°C by adding 3 µl of a 20-mg/ml concentration of proteinase K and 30 µl of 10% sodium dodecyl sulfate. DNA was purified twice by phenol and chloroform-isoamyl alcohol extractions. One volume of isopropanol and 1/10 volume of sodium acetate (3 M, pH 5.2) were added to precipitate the DNA at –20°C overnight. The recovered DNA was washed with a 70% ethanol solution. The pellet was resuspended in 50 µl of nuclease-free water.

PCR assay of the rpoB gene. The primers used for PCR amplifications were formulated by using the submitted rpoB gene of B. melitensis 16M (accession number AE009516) (8). The whole 4,134-bp gene was amplified by using a forward primer, +1rB (5'-ATGGCTCAGACCCATTCTTTC-3'), and a reverse primer, –4134rB (5'-TTATTCTGCCGCGTCCGGAA-3'), hybridizing, respectively, with the beginning and the end of the published rpoB coding region. PCR amplifications were carried out with an Expand Long Template PCR system kit (Roche, Mannheim, Germany) and by following the enclosed protocol for system 1. Eight hundred nanograms of genomic DNA was added for the reaction. Amplifications were initiated by denaturing the sample for 2 min at 94°C and then subjecting it to 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 3 min; 20 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 3 min; and an elongation step of 20 s for each cycle. After the last cycle, samples were incubated for an additional 7 min at 68°C before they were stored at 4°C. Ten microliters of each reaction mixture was analyzed by electrophoresis through a 1% agarose gel with ethidium bromide.

PCR assays of two "hot " regions within the rpoB gene. Two PCR tests were carried out to amplify two specific regions of the rpoB gene. The first region (named the 5'-end region), located at the 5' end from codons 118 to 240, was amplified by primers +354rB (5'-TGC GAA GTC CAT CAA GGA CAT-3') and –720rB (5'-ACG GGT ATA GGT GAC AGT CTT G-3') to obtain a 367-bp product. The second one (named the central region), located in the center of the gene from codons 473 to 606, was amplified by primers +1418rB (5'-AGT ATC GCG TCG GTC TGC TCC GC-3') and –1818rB (5'-ATC GAC AAC CTT GCG ATA CG-3') to obtain a 401-bp product. PCR amplifications were carried out with an AmpliTaq Gold kit (Applied Biosystems, Foster City, Calif.) and by following the enclosed protocol. Amplifications were initiated by denaturing the samples for 10 min at 95°C and then subjecting them to 30 cycles of 30 s at 95°C, 30 s at 60°C, and 1 min at 72°C. Finally, the samples were held at 72°C for 10 min to ensure complete extension of all PCR products. Ten microliters of each reaction mixture was analyzed by electrophoresis through a 1% agarose gel with ethidium bromide.

Sequence and data analysis. All PCR products were purified by use of Montage PCR centrifugal filter devices (Millipore, Billerica, Mass.) and directly sequenced with ABI PRISM 310 genetic analyzer equipment by use of the PCR cycle sequencing BigDye Terminator protocol (Applied Biosystems). PCR primers +1rB, +354rB, and –4134rB together with sequencing primers were used to sequence the whole rpoB coding region. Sequence primers, chosen within the published rpoB gene of B. melitensis 16M (accession number AE009516), are listed in Table 3. The two hot-region amplifications were done by using the respective PCR primers. The electropherograms were assembled on the basis of the published rpoB sequence of B. melitensis 16M by use of ABI Prism SeqScape software version 2.0 (Applied Biosystems). The generated consensus sequences devoid of PCR primers were then compared for detection of mutations. Mutations were investigated by sequencing the strain twice to confirm the result. To distinguish silent from missense mutations, amino acid sequences were determined.

	RESULTS

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To analyze rpoB mutations associated with the Rif^r phenotype in Brucella spp., the rpoB genes of the vaccine strain B. abortus RB51, of 20 Rif^r B. melitensis clones, and of their respective Rif^s parental strains (B. abortus strain 2308 and B. melitensis isolates CT1p and CT2p) were amplified and sequenced. All samples produced a single 4,134-bp PCR product that served as a suitable template for direct sequencing. A consensus sequence devoid of primers and 4,093 bp in length was obtained, and a 1,364-amino-acid sequence was deduced from all samples. The rpoB consensus sequences of Rif^r Brucella strains were then compared to those of their own Rif^s parental strains and to the published B. melitensis 16M genome (accession number AE009516) in order to detect mutations.

The rpoB nucleotide sequence of the vaccine strain B. abortus RB51 showed one missense mutation in codon 526 (AspTyr) compared to the sequence of its parental strain, B. abortus 2308, as shown in Table 4. The Asp-526-Tyr mutation was confirmed when the RB51 consensus sequence was compared to that of B. melitensis 16M. Five silent mutations at codons 243 (GAT to GAC), 268 (ACG to ACT), 716 (CCG to CCA), 737 (GTT to GTC), and 969 (CGC to CGT) were found in addition. These five silent mutations were also found when B. abortus 2308 was compared to B. melitensis 16M.

Analyzing the above-mentioned Rif^r Brucella strains, we identified two regions within the rpoB gene in which all the mutations found in the gene were present. The 5'-end region, located at the 5' end of the rpoB gene and ranging from codons 118 to 240, included the hot-spot mutation at codon position 154. The central region, located in the center of the gene and ranging from codons 473 to 606, included the hot-spot mutations affecting codons 526 to 574. These two hot regions were then investigated in eight additional Rif^r Brucella mutants (BM1R, BM2R, BM3R, BA3R, BA5R, BS1R, BS2R, and BS3R) and in 20 reference Rif^s Brucella strains. These Rif^r Brucella mutants of smooth or rough morphology were derived from reference Rif^s strains of B. melitensis, B. abortus, and B. suis. Two PCR fragments of 367 and 401 bp in length were obtained from each Brucella strain. The nucleotide sequences of both products were determined and compared to the published B. melitensis 16M sequence. rpoB mutations were found in all additional analyzed Rif^r mutants, as reported in Table 5. Most mutations were found in the central region, affecting codons 536 (His-536-Tyr) (six out of eight) and 539 (Arg-539-His) (one out of eight). Only one mutant carried an amino acid substitution in the 5'-end region, at codon position 154 (Val-154-Phe). The analysis of these two regions in the Rif^s reference Brucella strains did not show any mutations.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The rpoB gene encoding the ß subunit of the RNAP, recognized to be the rifampin antibiotic target in prokaryotes, was amplified and sequenced in the vaccine strain B. abortus RB51 (the only known reference Rif^r Brucella strain), in 20 Rif^r clones (from CT-1 to CT-20) which were derived in our laboratory by repeated passaging of Rif^s B. melitensis isolates on BAS plates supplemented with rifampin, and in their respective detectable parental strains (B. abortus 2308 and B. melitensis CT1p and CT2p). Resistance-associated mutations in rpoB were found in all analyzed Rif^r Brucella strains. The vaccine strain B. abortus RB51, whose high level of Rif^r is a useful biochemical marker for its identification, carried one missense mutation of GAC (Asp) to TAC (Tyr) at codon 526 in comparison to its parental strain B. abortus 2308. Missense mutations affecting the 20 Rif^r clones and leading to substitution of amino acids for Val-154, Asp-526, His-536, Arg-539, Ser-541, and Pro-574 residues are described in Table 4. Mutations involving codon 536 (9 of 20 Rif^r clones) were predominant.

We observed that all mutations found in the above-mentioned Rif^r Brucella strains mapped in two regions located at the 5' end and close to the center of the rpoB gene. To be sure of the association of these molecular events with the development of Rif^r, these regions were investigated in eight additional Rif^r Brucella laboratory mutants and in 20 reference Rif^s Brucella strains. We included B. melitensis strain 16M and B. suis strain 1330, whose complete genomes are available in a data bank (8, 21), to confirm the specificity of our assays by the identity of the sequences to those already published. Two PCR amplifications targeting codon 154 and codons 526 to 574 were carried out for each Brucella strain.

The additional Brucella mutants, derived from reference Rif^s Brucella strains, carried rpoB mutations affecting either the central or the 5'-end region. Most mutations were found in the central region at codons 536 and 539. The amino acid substitution His-536-Tyr was confirmed as the one most involved in Rif^r in Brucella strains. By contrast, no missense mutations were found in these two regions in any analyzed Rif^s strains in comparison to B. melitensis 16M. These data provide an additional proof of the association of specific rpoB mutations with the development of Rif^r in prokaryotes.

High-resolution structural studies of the rifampin-RNAP complex of T. aquaticus demonstrated that the inhibitor binds in a pocket of the RNAP ß subunit deep within the DNA-RNA channel and directly blocks the path of the elongating RNA transcript at the 5' end by a simple steric blocking mechanism (4, 31). Twelve ß subunit residues which surround the rifampin binding pocket are believed to interact directly with the rifampin by van der Waals interactions (six residues) and hydrogen bonds (six residues), and substitutions in these amino acid residues confer Rif^r (4). These 12 residues are identical among E. coli, T. aquaticus, and M. tuberculosis and cluster in an evolutionarily conserved region among all prokaryotic ß subunit sequences (1, 28). This region is also largely mutated in Rif^r E. coli and M. tuberculosis. We searched for this region in our rpoB sequences and found that Asp-526, His-536, Arg-539, and Ser-541 residues cluster in this conserved region and correspond to those residues that interact directly with the rifampin by hydrogen bonds in T. aquaticus. These data strongly suggest that the substitutions in these amino acid residues found in our Rif^r Brucella strains lead to the Rif^r phenotype by preventing the formation of the rifampin-RNAP complex. Amino acid substitutions resulting from mutations at codon 526, 536, or 541 are also indicated to occur frequently in Rif^r M. tuberculosis clinical isolates (22). Val-154 and Asp-574 residues, by contrast, are located in two different evolutionarily conserved regions among all prokaryotes and correspond to two residues whose mutations confer Rif^r in E. coli (11, 12, 24, 25). In T. aquaticus, these last two residues surround the rifampin binding pocket but do not interact directly with the antibiotic (4). Their amino acid substitutions would likely affect the folding or packing of the protein in the vicinity of the substituted residue, causing distortions of the rifampin binding pocket and leading to Rif^r.

Other interesting information emerged from a comparison of the whole rpoB nucleotide sequence of the analyzed Brucella strains to the published sequence of B. melitensis 16M. Five silent mutations at codons 243, 268, 716, 737, and 969 were found in both B. abortus strains, 2308 and RB51. One missense mutation at codon 1249 (Met-1249-Ile) is described for B. melitensis isolates CT1p and CT2p and for all derived clones from CT-1 to CT-20. We believe that this mutation is not involved in the development of Rif^r, because it is present in both Rif^s B. melitensis isolates and their Rif^r derived mutants. Moreover, in comparing the published B. melitensis 16M rpoB gene to that of B. suis 1330 (accession number AE014423), whose complete genome has been determined (21), nucleotide sequence diversity was found. All these data suggest polymorphic characteristics for the rpoB gene, but a further and close investigation is necessary. For this reason, the study of the whole rpoB nucleotide sequence for all reference Brucella strains is in progress.

In conclusion, in the present study, we have characterized the molecular events which lead to the development of Rif^r in Brucella spp., and several Rif^r rpoB genotypes are described. Moreover, virulence studies of Rif^r Brucella mutants may provide additional information about the correlation among the Rif^r phenotype, morphology, and virulence of Brucella spp.

	FOOTNOTES

 
* Corresponding author. Mailing address: Dipartimento di Sanità Alimentare ed Animale, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 0039-06-49902728. Fax: 0039-06-49387077. E-mail: cinzia.marianelli{at}iss.it.

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