Previous Article | Next Article 
Journal of Clinical Microbiology, January 2001, p. 235-240, Vol. 39, No. 1
Escuela Nacional de Ciencias
Biológicas, IPN,1 and Centro de
Investigación sobre Fijación de Nitrógeno,
UNAM,3 Cuernavaca, and Servicios de
Salud de Zacatecas, S.S.A., Zacatecas,2 Mexico
Received 30 June 2000/Returned for modification 29 August
2000/Accepted 16 October 2000
Multilocus enzyme electrophoresis (MLEE) of 99 Brucella
isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of
a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a
single electrophoretic type related to Brucella neotomae
and Brucella ovis. Brucella suis and
Brucella canis formed another cluster linked to
Brucella melitensis and related to Rhizobium
tropici. The Brucella strains tested that were
representatives of the six electrophoretic types had the same rRNA gene
restriction fragment length polymorphism patterns and identical
ribotypes. All 99 isolates had similar chromosome profiles as revealed
by the Eckhardt procedure.
Brucellosis is a worldwide zoonosis
that is especially prevalent in northern and central agricultural
regions of Mexico (27). Brucella was once
considered to be related to the genera Bordetella and
Alcaligenes (18). Later on, molecular biology
techniques indicated that Brucella had taxonomic affiliation
with members of the CDC group Vd (8), and an analysis of
16S rRNA gene sequences confirmed its inclusion in the Genetic relatedness within Brucella has been based on a
comparison of omp2 sequences (10) and DNA
restriction maps obtained from various species (30).
Molecular probes have been developed for typing Brucella
strains (6, 14), and PCR methods are available as
diagnostic techniques (22, 23). The high levels of DNA-DNA
relatedness among Brucella species led to the conclusion that Brucella was a monospecific genus (43).
Recently, genes involved in symbiosis in Sinorhizobium
meliloti (the best-studied rhizobium with regard to its symbiotic
determinants) have been found to be homologous to genes implicated in
the pathogenesis of Brucella. Rhizobia (comprising the
genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium,
Rhizobium, and Sinorhizobium) form nitrogen-fixing nodules; Brucella spp., on the other
hand, are intracellular animal pathogens. The rhizobial
bac genes participating in bacteroid differentiation
(12) are homologous to genes which play a role in
Brucella survival in macrophages as well as in mice
pathogenesis (24). A two-component regulatory system BvrR and BvrS (Brucella virulence) is similar to exoS
genes of S. meliloti. Brucella mutants in BvR and
BvrS have a reduced capacity to invade macrophages and do not
replicate intracelullarly (39). The S. meliloti periplasmic protease encoding gene degP is
more similiar to the corresponding Brucella abortus gene
than to that of Escherichia coli (12).
Multilocus enzyme electrophoresis (MLEE) has been frequently used to
determine the genetic relatedness in bacteria, including E. coli (40), Salmonella spp.
(38), and Vibrio cholerae (3). This technique has proven to be valuable in the characterization of
emergent epidemic clones (3). The aim of the present study was to characterize Brucella spp. originating from different
sources by MLEE and to compare the data to data obtained for rhizobia and agrobacteria.
Strains and cultures.
Brucella strains (75 isolates) (Table 1) originating from
Mexico and abroad were isolated from human, animal, and dairy products. Also included in this study were 4 vaccine strains, 20 type strains from all different Brucella species and biovars, and
Rhizobium, Mesorhizobium,
Sinorhizobium, and Bradyrhizobium reference
strains from the Centro de Investigación sobre Fijación de
Nitrógeno (UNAM) collection and Ochrobactrum anthropi
(23) and Agrobacterium spp. reference strains
(36). Ochrobactrum and Brucella
isolates were grown in soybean Trypticase (Difco) at 37°C, in
Brucella agar, or in PY medium. Rhizobium strains
were grown in PY medium (3 g of yeast extract, 5 g of peptone, and 0.7 g of calcium chloride per liter). All Brucella single-colony
isolates were tested for their Gram reaction and for agglutination with
anti-Brucella serum. Growth rates (not shown) were estimated
for Brucella melitensis M16 and B. abortus 544 in
order to determine harvesting times in the logarithmic phase of growth.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.235-240.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Limited Genetic Diversity of
Brucella spp.
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2 subdivision
of the Proteobacteria class (31). Since the
position of the nodes in 16S rRNA gene phylogenetic trees is not
without uncertainty (28), the position of
Brucella within the -proteobacteria has not been clearly
determined. Furthermore, ribosomal genes in Brucella have
been implicated in recombination events that promoted the division of a
chromosome into two chromosomes (19).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains
MLEE.
Fresh liquid (1 ml) cultures (at 0.5 turbidity,
MacFarland nephelometer) were used to inoculate 40-ml portions of PY
media, which were shaken for 36 to 48 h at 37°C. Cell pellets
obtained by centrifugation were washed, resuspended in 300 µl of 10 mM MgSO4 containing lysozyme (300 µg per ml) and
incubated at room temperature for 20 min. Cell lysis was achieved by
freezing and thawing at 
70°C for two 15-min cycles, and the
resulting extracts were maintained at 70°C.
x2[n/(n 1)], where
x is the frequency of the ith allele and
n is the number of ETs or isolates in the population;
H is the arithmetic average of all h values.
Amplified ribosomal DNA restriction analysis (ARDRA). PCR products of 16S rRNA genes were synthesized with primers fD1 and rD1 (44) that correspond to positions 8 to 27 and positions 1524 to 1540 of the E. coli gene. PCR products were digested with 5 U of the restriction enzymes MspI, HinfI, HhaI, and Sau3AI, and DNA fragments were separated in 3% agarose gels (21). The PCR product of citrate synthase of B. melitensis M16 DNA was obtained with the Rhizobium tropici primers 512 MAP (TAC-AAG-TAC-CAT-ATC-GGC-CAG-CCC-TT), corresponding to bases 858 to 873, and primer CIR97037 (CCC-ATC-ATG-CGG-AAC-GGA-TC), corresponding to bases 1218 to 1237.
DNA extraction and Southern blot hybridization. DNA was purified, blotted onto nylon filters, and hybridized to the PCR 16S rRNA gene product from B. melitensis M16, to the total DNA from the same strain labeled with 32P by RediPrime (Amersham), or to the PCR-synthesized citrate synthase gene. DNA-DNA hybridization was performed from Southern blots, and washings were performed either at 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (high) or at 1× SSC (low) stringency.
Eckhardt gel electrophoresis. The modified procedure by Hynes and McGregor (16) involving a gentle lysis of the cell pellet with lysozyme and sodium dodecyl sulfate (incorporated into the agarose gel [agarose Sigma Type 1:Low EEO, catalog number A-6013]) was used with early-log-phase bacteria grown in PY medium. Horizontal gels were run at 80 V for 10 h at room temperature. Plasmid sizes were estimated using S. meliloti megaplasmids (1.4 and 1.7 Mb [2]) as references. The miniscreening procedure (4) was also used in order to visualize small plasmids.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Most human isolates from Mexico corresponded to B. melitensis bv. 1, and a majority of dairy product isolates
corresponded to B. abortus bv. 1 based on the traditional
classification methods (27). We did not isolate
Brucella ovis and Brucella neotomae. The
H value among the four Brucella ETs obtained with
10 enzymes was 0.16, and the H value calculated for all
isolates was 0.04. Representatives from each ET and some R. tropici and Ochrobactrum strains were analyzed with an
additional six enzymes in order to reveal further diversity (Table
2). This resulted in two of the
Brucella spp. ETs being split into two related ETs, while the other ETs remained unaltered. Each Brucella species was
distinguishable by its ET with 16 enzymes (Table 2; see Fig. 2). The
total number of ETs obtained with Brucella isolates was six,
and the H value among the six ETs was 0.32. If we consider
that six ETs represent all of the 99 strains tested, then the strain/ET
ratio would be 16.5, a value higher than that encountered (ca. 1) from
single-species Rhizobium populations. The high strain/ET
ratio encountered in Brucella spp. is an indicator of a
limited genetic diversity that is also revealed by the low number of
polymorphic enzymes detected (9 of 16).
|
The MLEE-derived dendrogram obtained with Brucella spp. and
rhizobia confirms their close relationship (Fig.
1 and
2). With the 16-enzyme analysis, two
subclusters may be distinguished for Brucella isolates, one
with the marine Brucella, B. abortus, B. neotomae, and B. ovis, and the other with B. canis, B. suis, and B. melitensis. R. tropici strains grouped with the second subcluster. O. anthropi and Agrobacterium spp. were related to
Brucella at a genetic distance of 0.9 (Fig. 1).
|
|
A single pattern with three common bands was observed when EcoRI-DNA digestion fragments of several Brucella species (B. melitensis M16 and 84; B. abortus bv. 1, 544, and bv. 3 Tulya; B. neotomae 5K33; B. suis bv. 1, 1330, bv. 4, 40/67; the marine Brucella sp. strain 14/95; B. canis 1226) were hybridized in Southern blottings using the 16S rRNA gene PCR product of B. melitensis M16 as a probe. Three rrn loci have been found in Brucella spp. (30). ARDRA analysis revealed that the Brucella strains listed above shared a common rRNA gene pattern (data not shown).
DNA-DNA homology values indicated that Ochrobactrum and Brucella spp. were more homologous (ca. 30%) than Brucella spp. and rhizobia (ca. 10%). At high stringency, a slightly higher percentage of hybridization is obtained with R. tropici than with S. meliloti, but this may not be significant.
Interestingly, Brucella chromosomes were easily visualized
with the Eckhardt procedure that we normally use to reveal plasmids and
megaplasmids in rhizobia. Most of the strains had the same pattern
corresponding to chromosomes of 2.05 and 1.15 Mb. No large or small
plasmids were encountered in any of the 99 isolates tested. Two
chromosomes of 1.35 and 1.85 Mb were observed with B. suis bv. 2 and bv. 4 in agreement with the data of Jumas-Bilak
(19). The only discrepancy with the reported results was
obtained with B. suis biovar 3 which contained smaller
chromosomes (2.1 and 1.15 Mb) than that (3.2 Mb) observed by
Jumas-Bilak (19). In order to resolve this discrepancy, we
analyzed chromosomes from at least two additional B. suis
bv. 3 strains from different sources, and the data confirmed our
previous findings (Fig. 3). Both
chromosomes from each Brucella strain hybridized to the 16S
rRNA gene probe, while only the larger one hybridized to the citrate
synthase gene (not shown). The megaplasmid of R. tropici,
which is similar in size to the smaller chromosome of B. suis bv. 2 and 4, did not hybridize to the homologous 16S rRNA DNA
gene probe as was reported previously (11).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
MLEE analysis is a useful standard method for evaluating bacterial genetic diversity. In general, a different species is recognized if a genetic distance larger than 0.5 is observed. The limited genetic variation exhibited by the Brucella isolates is congruent with a monospecific genus (43). The fact that only a few clones (ETs) were obtained that are conserved through space and time probably reflects a recent origin of the genus. The H value for all Brucella species was 0.04 or 0.32 (depending if the analysis is on an isolate or ET basis), which is smaller than those normally estimated for a single Rhizobium species (ca. 0.5) (29). A large diversity has been encountered in rhizobia, suggesting that they represent very old lineages. Pathogens, especially those occurring intracellularly, have a narrow diversity which may reflect their habitat constraints (reviewed in reference 29). Thus, limited genetic diversity has also been obtained with Yersinia ruckeri and Salmonella enterica serovar Paratyphi B.
A close relationship of B. suis and B. canis was recognized based on phenotypic characteristics (1), and these organisms were not distinguishable by their physical maps (30). From our data, B. suis is very similar to B. canis and is only distinguishable by 1 enzyme, xanthine dehydrogenase, of 16. In general, our dendrograms obtained by MLEE are in good agreement with the one constructed on the basis of the sequence of omp2 (10), and both methods are in general agreement with the tree derived from genome restriction maps (30). Originally, one isolate from a marine animal was considered related to B. abortus or B. melitensis (9). We could not distinguish the marine Brucella sp. from B. abortus by MLEE; however, we included only a single isolate from marine mammals. An extensive analysis of Brucella spp. in marine mammals showed that they possessed DNA fingerprints that differentiated them from other described Brucella species (5). The two B. suis bv. 5 strains tested had ETs corresponding to B. melitensis (determined with the 16-enzyme analysis) and not to the B. suis ET. The question regarding whether B. suis bv. 5 strains are bona fide B. suis was raised earlier based on metabolic profiles and susceptibility to phages (17).
It is possible that genetic variation may be underestimated by MLEE because different alleles may have identical mobilities (33). Variation within an ET may be revealed by DNA-based fingerprinting methods. Salmonella enterica serovar Typhi was found to have a worldwide limited genetic diversity and a clonal population structure as revealed with MLEE (38). Variation within serovar Typhi clones was shown with ribosomal fingerprinting. These results may be explained if ribosomal gene rearrangements (26) and recombination occurred faster than detectable changes in isoenzymes. Thus, MLEE would allow for the detection of older genetic relationships, as we suppose is the case with R. tropici and Brucella. Mycobacterium tuberculosis, another intracellular human and animal pathogen, which has been found to be genetically very homogeneous, is considered to have evolved relatively recently from a soil bacterium (7, 41). Our hypothesis is that both Brucella and R. tropici have a common ancestor and have conserved the type of inherited alloenzymes. We further suppose that these enzymes were adapted to an acid intracellular environment. R. tropici has been described as highly tolerant to acidity in comparison to many other Rhizobium species, including S. meliloti (13), whereas Brucella spp. must survive low gastric pH, and an adaptive acid tolerance response has been described (20). Tolerance to acidity allows the survival of other bacteria in cheese (25), and Brucella is normally encountered in fermented dairy products. Our suggestion of a common origin of Brucella and Rhizobium spp. certainly agrees with the proposal that a larger chromosome (as in Rhizobium) gave rise to the two smaller chromosomes found in Brucella (19).
The DNA-DNA hybridization results showed that Brucella was more homologous to Ochrobactrum (30% DNA-DNA hybridization) than to R. tropici (12%). It is worth noting that the percentage of total DNA-DNA homology among Brucella spp. and S. meliloti or R. tropici (ca. 11%) is lower than the percentage of nucleotide identity encountered when different homologous genes are compared. For example, Brucella and S. meliloti degP gene sequences are 55.5% identical; a fragment of 400 bp of pckA gene (for phosphoenolpyruvate carboxykinase) is 77% identical among Sinorhizobium sp. strain NGR234 and B. abortus (39, 35), and the citrate synthase gene of Brucella is about 80% identical to the corresponding gene in R. tropici (our unpublished results and reference 15). A fragment of phospholipid N-methyltranferase (pmtA) genes of B. ovis and S. meliloti are 61% identical (O. Geiger, personal communication). This may mean that some parts of the Brucella genome are shared with rhizobia, while others may have been acquired from other sources. The MLEE relationships observed between Brucella and rhizobia may be explained if the common genome encodes the metabolic enzymes we have analyzed. In contrast, similarities in genes coding for a secretion system of B. suis and Bordetella pertussis have been reported (34). It is intriguing that in spite of the fact that Ochrobactrum isolates, especially O. intermedia, are clearly related to Brucella (42), we did not detect a high degree of similarity among Brucella and Ochrobactrum isolates by MLEE. Ochrobactrum has been shown to be highly diverse. This diversity may be the result of extensive interstrain recombination with randomization of enzymatic alleles. Notably, R. tropici type A and type B share only 36% DNA-DNA homology, yet they are considered to constitute a single species. Finally, the easy and clear detection of the chromosomes of Brucella spp. in Eckhardt gels may be useful in Brucella research to determine whether a gene is present on a specific replicon (as we have shown with the citrate synthase gene) and for further characterization or even identification of new isolates.
| |
ACKNOWLEDGMENTS |
|---|
We thank J. Martínez Romero for technical support, L. Barran and M. Dunn for critically reviewing the manuscript, and A. MacMillan for providing the marine Brucella and O. anthropi strains.
B.G.J. was supported by a graduate student scholarship from CONACyT and PIFI-IPN, and A.L.M. was supported by a research scholarship from COFAA-IPN. This work was also supported by grants from CONACyT (27594B) and CGPI-IPN (980367).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Centro de Investigación sobre Fijación de Nitrógeno, Ap. Postal 565-A, Av. Universidad S/N, Col. Chamilpa, Cuernavaca, Mexico. Phone: (52-73)-13-16-97. Fax: (52-73)-17-55-81. E-mail: emartine{at}cifn.unam.mx.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Allardet-Servent, A.,
G. Bourg,
M. Ramuz,
M. Pages,
M. Bellis, and G. Roizes.
1988.
DNA polymorphism in strains of the genus Brucella.
J. Bacteriol.
170:4603-4607 |
| 2. |
Barloy-Hubler, F.,
D. Capela,
M. J. Barnett,
S. Kalman,
N. A. Federspiel,
S. R. Long, and F. Galibert.
2000.
High-resolution physical map of the Sinorhizobium meliloti 1021 pSyma megaplasmid.
J. Bacteriol.
182:1185-1189 |
| 3. |
Beltran, P.,
G. Delgado,
A. Navarro,
F. Trujillo,
R. K. Selander, and A. Cravioto.
1999.
Genetic diversity and population structure of Vibrio cholerae.
J. Clin. Microbiol.
37:581-590 |
| 4. |
Birnboim, H. C., and J. Doly.
1979.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
Nucleic Acids Res.
7:1513-1523 |
| 5. |
Bricker, B. J.,
D. R. Ewalt,
A. P. MacMillan,
G. Foster, and S. Brew.
2000.
Molecular characterization of Brucella strains isolated from marine mammals.
J. Clin. Microbiol.
38:1258-1262 |
| 6. |
Bricker, B. J., and S. M. Halling.
1994.
Differentiation of Brucella abortus bv1, 2 and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv.1 by PCR.
J. Clin. Microbiol.
32:2660-2666 |
| 7. | Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry III, F. Tekaia, K. Badcock, D. Basham, D. Brown, et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544[CrossRef][Medline]. |
| 8. | De Ley, J., W. Mannheim, P. Segers, A. Lievens, M. Denjin, M. Vanhoucke, and M. Gillis. 1987. Ribosomal ribonucleic acid cistron similarities and taxonomic neighborhood of Brucella and CDC group Vd. Int. J. Syst. Bacteriol. 37:35-42. |
| 9. |
Ewalt, D. R.,
J. B. Payeur,
B. M. Martin,
D. R. Cummins, and W. G. Miller.
1994.
Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus).
J. Vet. Diagn. Investig.
6:448-452 |
| 10. |
Ficht, T. A.,
H. S. Husseinen,
J. Derr, and S. W. Bearden.
1996.
Species-specific sequences at the omp2 locus of Brucella type strains.
Int. J. Syst. Bacteriol.
46:329-331 |
| 11. |
Geniaux, E.,
M. Flores,
R. Palacios, and E. Martínez.
1995.
Presence of megaplasmids in Rhizobium tropici and further evidence of differences between the two R. tropici subtypes.
Int. J. Syst. Bacteriol.
45:392-394 |
| 12. |
Glazebrook, J.,
A. Ichige, and G. C. Walker.
1996.
Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP.
J. Bacteriol.
178:745-752 |
| 13. | Graham, P. H., K. J. Draeger, M. L. Ferrey, M. J. Conroy, B. E. Hammer, E. Martínez, S. R. Aarons, and C. Quinto. 1994. Acid pH tolerance in strains of Rhizobium and Bradyrhizobium, and initial studies on the basis for acid tolerance of Rhizobium tropici UMR 1899. Can. J. Microbiol. 40:198-207. |
| 14. | Grimont, F., J. M. Verger, P. Cornelis, J. Limet, M. Lefèvre, M. Grayon, B. Régnault, J. Van Broeck, and P. A. D. Grimont. 1992. Molecular typing of Brucella with cloned DNA probes. Res. Microbiol. 143:55-65[Medline]. |
| 15. | Hernández-Lucas, I., M. A. Pardo, L. Segovia, J. Miranda, and E. Martínez-Romero. 1995. Rhizobium tropici chromosomal citrate synthase gene. Appl. Environ. Microbiol. 61:3992-3997[Abstract]. |
| 16. | Hynes, M. F., and N. F. McGregor. 1990. Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum. Mol. Microbiol. 4:567-574[Medline]. |
| 17. | Jahans, K. L., G. Foster, and E. S. Broughton. 1997. The characteristics of Brucella strains isolated from marine mammals. Vet. Microbiol. 57:373-382[CrossRef][Medline]. |
| 18. |
Johnson, R., and P. H. A. Sneath.
1973.
Taxonomy of Bordetella and related organisms of the families Achromobacteriaceae, Brucellaceae, and Neisseriaceae.
Int. J. Syst. Bacteriol.
23:381-404 |
| 19. | Jumas-Bilak, E., S. Michaux-Charachon, G. Bourg, D. O'Callaghan, and M. Ramuz. 1998. Differences in chromosome number and genome rearrangements in the genus Brucella. Mol. Microbiol. 27:99-106[CrossRef][Medline]. |
| 20. | Kulakov, Y. K., P. G. Guigue-Talet, M. R. Ramuz, and D. O'Callaghan. 1997. Response of Brucella suis 1330 and B. canis RM6/66 to growth at acid pH and induction of an adaptive acid tolerant response. Res. Microbiol. 148:145-151[Medline]. |
| 21. |
Laguerre, G.,
M.-R. Allard,
F. Revoy, and N. Amarger.
1994.
Rapid identification of rhizobia by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes.
Appl. Environ. Microbiol.
60:56-63 |
| 22. | Leal-Klevezas, D. S., A. López-Merino, and J. P. Martínez-Soriano. 1995. Molecular detection of Brucella spp.: rapid identification of B. abortus biovar I using PCR. Arch. Med. Res. 26:263-267[Medline]. |
| 23. | Leal-Klevezas, D. S., I. O. Martínez-Vázquez, A. López Merino, and J. P. Martinez-Soriano. 1995. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J. Clin. Microbiol. 33:3087-3090[Abstract]. |
| 24. |
LeVier, K.,
R. W. Phillips,
V. K. Grippe,
R. M. Roop II, and G. C. Walker.
2000.
Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival.
Science
287:2492-2493 |
| 25. |
Leyer, G. J., and E. A. Johnson.
1992.
Acid adaptation promotes survival of Salmonella spp. in cheese.
Appl. Environ. Microbiol.
58:2075-2080 |
| 26. |
Liu, S.-L., and K. E. Sanderson.
1996.
Highly plastic chromosomal organization in Salmonella typhi.
Proc. Natl. Acad. Sci. USA
93:10303-10308 |
| 27. | López-Merino, A. 1991. Brucelosis humana, p. 4-17. In A. López-Merino (ed.), Brucelosis: avances y perspectivas, México. Publicación técnica del INDRE-SSA no. 6. Secretaría de Salud, Mexico City, Mexico. |
| 28. | Ludwig, W., R. Amann, E. Martínez-Romero, W. Schönhuber, S. Bauer, A. Neef, and K.-H. Schleifer. 1998. rRNA-based identification and detection systems for rhizobia and other bacteria. Plant Soil 204:1-19[CrossRef]. |
| 29. | Martínez-Romero, E., and J. Caballero-Mellado. 1996. Rhizobium phylogenies and bacterial genetic diversity. Crit. Rev. Plant Sci. 15:113-140. |
| 30. |
Michaux-Charachon, S.,
G. Bourg,
E. Jumas-Bilak,
P. Guigue-Talet,
A. Allardet-Servent,
D. O'Callaghan, and M. Ramuz.
1997.
Genome structure and phylogeny in the genus Brucella.
J. Bacteriol.
179:3244-3249 |
| 31. |
Moreno, E.,
E. Stackebrandt,
M. Dorsch,
J. Wolters,
M. Busch, and H. Mayer.
1990.
Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the -2 subdivision of the class Proteobacteria.
J. Bacteriol.
172:3569-3576 |
| 32. |
Nei, M., and W. H. Li.
1979.
Mathematical model for studying genetic variation in terms of restriction endonucleases.
Proc. Natl. Acad. Sci. USA
76:5269-5273 |
| 33. |
Nelson, K.,
T. S. Whittam, and R. K. Selander.
1991.
Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase genes (gapA) in natural populations of Salmonella and Escherichia coli.
Proc. Natl. Acad. Sci. USA
88:6667-6671 |
| 34. | O'Callaghan, D., C. Cazevielle, A. Allardet-Servent, M. L. Boschirolli, G. Bourg, V. Foulongne, P. Frutos, Y. Kulakov, and M. Ramuz. 1999. A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essencial for intracellular survival of Brucella suis. Mol. Microbiol. 33:1210-1220[CrossRef][Medline]. |
| 35. | Osteras, M., T. M. Finan, and J. Stanley. 1991. Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype. Mol. Gen. Genet. 230:257-269[CrossRef][Medline]. |
| 36. | Sawada, H., and H. Ieki. 1992. Phenotypic characteristics of the genus Agrobacterium. Ann. Phytopathol. Soc. Japan 58:37-45. |
| 37. |
Selander, R. K.,
D. A. Caugant,
H. Ochman,
J. M. Musser,
M. N. Gilmour, and T. S. Whittam.
1986.
Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics.
Appl. Environ. Microbiol.
51:873-884 |
| 38. |
Selander, R. K.,
P. Beltran,
N. H. Smith,
R. Helmuth,
F. A. Rubin,
D. J. Kopecko,
K. Ferris,
B. D. Tall,
A. Cravioto, and J. M. Musser.
1990.
Evolutionary genetic relationships of clones of Salmonella serovars that cause human typhoid and other enteric fevers.
Infect. Immun.
58:2262-2275 |
| 39. | Sola-Landa, A., J. Pizarro-Cerdá, M.-J. Grilló, E. Moreno, I. Moriyón, J.-M. Blasco, J. P. Gorvel, and I. López-Goñi. 1998. A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol. Microbiol. 29:125-138[CrossRef][Medline]. |
| 40. |
Souza, V.,
M. Rocha,
A. Valera, and L. E. Eguiarte.
1999.
Genetic structure of natural populations of Escherichia coli in wild hosts on different continents.
Appl. Environ. Microbiol.
65:3373-3385 |
| 41. |
Sreevatsan, S.,
X. I. Pan,
K. E. Stockbauer,
N. D. Connell,
B. N. Kreiswirth,
T. S. Whittam, and J. M. Musser.
1997.
Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination.
Proc. Natl. Acad. Sci. USA
94:9869-9874 |
| 42. |
Velasco, J.,
C. Romero,
I. López-Goñi,
J. Leiva,
R. Díaz, and I. Moriyón.
1998.
Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp.
Int. J. Syst. Bacteriol.
48:759-768 |
| 43. |
Verger, J.-M.,
F. Grimont,
P. A. D. Grimont, and M. Grayon.
1985.
Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization.
Int. J. Syst. Bacteriol.
35:292-295 |
| 44. |
Weisburg, W. G.,
S. M. Barns,
D. A. Pelletier, and D. J. Lane.
1991.
16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol.
173:697-703 |
Journal of Clinical Microbiology, January 2001, p. 235-240, Vol. 39, No. 1
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.235-240.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tiller, R. V., De, B. K., Boshra, M., Huynh, L. Y., Van Ert, M. N., Wagner, D. M., Klena, J., Mohsen, T. S., El-Shafie, S. S., Keim, P., Hoffmaster, A. R., Wilkins, P. P., Pimentel, G.
(2009). Comparison of Two Multiple-Locus Variable-Number Tandem-Repeat Analysis Methods for Molecular Strain Typing of Human Brucella melitensis Isolates from the Middle East. J. Clin. Microbiol.
47: 2226-2231
[Abstract] [Full Text] -
Foster, J. T., Beckstrom-Sternberg, S. M., Pearson, T., Beckstrom-Sternberg, J. S., Chain, P. S. G., Roberto, F. F., Hnath, J., Brettin, T., Keim, P.
(2009). Whole-Genome-Based Phylogeny and Divergence of the Genus Brucella. J. Bacteriol.
191: 2864-2870
[Abstract] [Full Text] -
Kattar, M. M., Jaafar, R. F., Araj, G. F., Le Fleche, P., Matar, G. M., Abi Rached, R., Khalife, S., Vergnaud, G.
(2008). Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Human Brucella Isolates in a Region of Brucellosis Endemicity. J. Clin. Microbiol.
46: 3935-3940
[Abstract] [Full Text] -
De, B. K., Stauffer, L., Koylass, M. S., Sharp, S. E., Gee, J. E., Helsel, L. O., Steigerwalt, A. G., Vega, R., Clark, T. A., Daneshvar, M. I., Wilkins, P. P., Whatmore, A. M.
(2008). Novel Brucella Strain (BO1) Associated with a Prosthetic Breast Implant Infection. J. Clin. Microbiol.
46: 43-49
[Abstract] [Full Text] -
Groussaud, P., Shankster, S. J., Koylass, M. S., Whatmore, A. M.
(2007). Molecular typing divides marine mammal strains of Brucella into at least three groups with distinct host preferences. J Med Microbiol
56: 1512-1518
[Abstract] [Full Text] -
Whatmore, A. M., Shankster, S. J., Perrett, L. L., Murphy, T. J., Brew, S. D., Thirlwall, R. E., Cutler, S. J., MacMillan, A. P.
(2006). Identification and Characterization of Variable-Number Tandem-Repeat Markers for Typing of Brucella spp.. J. Clin. Microbiol.
44: 1982-1993
[Abstract] [Full Text] -
Halling, S. M., Peterson-Burch, B. D., Bricker, B. J., Zuerner, R. L., Qing, Z., Li, L.-L., Kapur, V., Alt, D. P., Olsen, S. C.
(2005). Completion of the Genome Sequence of Brucella abortus and Comparison to the Highly Similar Genomes of Brucella melitensis and Brucella suis. J. Bacteriol.
187: 2715-2726
[Abstract] [Full Text] -
Whatmore, A. M., Murphy, T. J., Shankster, S., Young, E., Cutler, S. J., Macmillan, A. P.
(2005). Use of Amplified Fragment Length Polymorphism To Identify and Type Brucella Isolates of Medical and Veterinary Interest. J. Clin. Microbiol.
43: 761-769
[Abstract] [Full Text] -
Okada, K., Iida, T., Kita-Tsukamoto, K., Honda, T.
(2005). Vibrios Commonly Possess Two Chromosomes. J. Bacteriol.
187: 752-757
[Abstract] [Full Text] -
Baek, S.-H., Rajashekara, G., Splitter, G. A., Shapleigh, J. P.
(2004). Denitrification Genes Regulate Brucella Virulence in Mice. J. Bacteriol.
186: 6025-6031
[Abstract] [Full Text] -
Rajashekara, G., Glasner, J. D., Glover, D. A., Splitter, G. A.
(2004). Comparative Whole-Genome Hybridization Reveals Genomic Islands in Brucella Species. J. Bacteriol.
186: 5040-5051
[Abstract] [Full Text] -
Snoeck, C., Verreth, C., Hernandez-Lucas, I., Martinez-Romero, E., Vanderleyden, J.
(2003). Identification of a Third Sulfate Activation System in Sinorhizobium sp. Strain BR816: the CysDN Sulfate Activation Complex. Appl. Environ. Microbiol.
69: 2006-2014
[Abstract] [Full Text] -
Paulsen, I. T., Seshadri, R., Nelson, K. E., Eisen, J. A., Heidelberg, J. F., Read, T. D., Dodson, R. J., Umayam, L., Brinkac, L. M., Beanan, M. J., Daugherty, S. C., Deboy, R. T., Durkin, A. S., Kolonay, J. F., Madupu, R., Nelson, W. C., Ayodeji, B., Kraul, M., Shetty, J., Malek, J., Van Aken, S. E., Riedmuller, S., Tettelin, H., Gill, S. R., White, O., Salzberg, S. L., Hoover, D. L., Lindler, L. E., Halling, S. M., Boyle, S. M., Fraser, C. M.
(2002). The Brucellasuis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc. Natl. Acad. Sci. USA
99: 13148-13153
[Abstract] [Full Text] -
DelVecchio, V. G., Kapatral, V., Redkar, R. J., Patra, G., Mujer, C., Los, T., Ivanova, N., Anderson, I., Bhattacharyya, A., Lykidis, A., Reznik, G., Jablonski, L., Larsen, N., D'Souza, M., Bernal, A., Mazur, M., Goltsman, E., Selkov, E., Elzer, P. H., Hagius, S., O'Callaghan, D., Letesson, J.-J., Haselkorn, R., Kyrpides, N., Overbeek, R.
(2001). The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA
10.1073/pnas.221575398v1
[Abstract] [Full Text] -
DelVecchio, V. G., Kapatral, V., Redkar, R. J., Patra, G., Mujer, C., Los, T., Ivanova, N., Anderson, I., Bhattacharyya, A., Lykidis, A., Reznik, G., Jablonski, L., Larsen, N., D'Souza, M., Bernal, A., Mazur, M., Goltsman, E., Selkov, E., Elzer, P. H., Hagius, S., O'Callaghan, D., Letesson, J.-J., Haselkorn, R., Kyrpides, N., Overbeek, R.
(2002). The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA
99: 443-448
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»