Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus^▿

Bacterial strains and culture conditions.

A total of 69 isolates, collected between 1997 and 2008 by the Servicio Agrícola Ganadero de Chile (SAG) were obtained. They were recovered from milk samples and fetal remains, mainly from regions in southern Chile. Details about the bacterial strains used for this study are provided in Table 1. B. abortus strains 2308, RB51, and 544 were included as references. All strains were characterized by classical microbiological methods as B. abortus biovar 1 and were cultured in brucella agar (Oxoid Limited, Cambridge, United Kingdom) at 37°C with 5% CO₂.

PFGE.

Genomic DNA (gDNA) suitable for pulsed-field gel electrophoresis (PFGE) was prepared according to the method described by Ridler et al. (30), with some modifications. Briefly, B. abortus isolates were grown on brucella agar (Oxoid Limited, Cambridge, United Kingdom) for 48 h at 37°C with 5% CO₂. The colonies were harvested into 3 ml of brain heart infusion broth (Difco Laboratories, Detroit, MI), and the optical density was measured and adjusted to 1.4 at 610 nm. A 100-μl aliquot of cells was pelleted by centrifugation at 13,000 rpm for 5 min. Cells were washed once with 150 ml of Pett IV buffer (1 M NaCl, 10 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0]) and then centrifuged and resuspended in 50 ml of Pett IV buffer. The bacterial suspension was mixed with 100 ml of 1% low-melting-point preparative-grade agarose (Bio-Rad Laboratories, Richmond, CA) and dispensed into plug molds. The agarose plugs were placed on ice for 1 h to solidify. The plugs were lysed overnight at 56°C in a buffer solution (50 mM EDTA [pH 8.0], 50 mM Tris-HCl [pH 8.0], 1% sodium lauroyl sarcosine) containing 1 mg of proteinase K (Sigma Chemical Company, St. Louis, MO) per ml. Following lysis, the plugs were washed five times for 1 h each time with 10 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA) on ice. A 3-mm slice of each plug was equilibrated in 100 μl of XbaI restriction buffer (New England BioLabs, Beverly, MA) for 45 min on ice. The restriction buffer was removed and replaced with 100 μl of fresh restriction buffer containing 30 units of XbaI (New England BioLabs, Beverly, MA). The plug slices were held on ice for an additional 45 min before an overnight incubation at 37°C. The macrorestriction fragments were separated by pulsed-field gel electrophoresis on a CHEF-DRIII system (Bio-Rad Laboratories, Richmond, CA) in a 1% agarose gel (pulsed-field certified agarose; Bio-Rad) in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA [pH 8.0]) at 6 V/cm and 14°C. Macrorestriction fragments produced with XbaI were separated according the method described by Ridler (30), with ramped pulse times of 0.5 to 10 s for 24 h. The lambda ladder PFG marker and low-range PFG marker (New England BioLabs, Beverly, MA) were included as molecular size standards. Gels were stained with ethidium bromide, and images were captured under UV illumination, The macrorestriction patterns were analyzed visually using the criteria of Tenover et al. (35).

MLVA-16 analysis.

In addition to the B. abortus 2308, RB51, and 544 reference strains, MLVA-16 analysis also included the S19 vaccine strain and 67 field strains studied by PFGE (two strains could not be recovered). The genomic DNA was extracted from one loopful of bacterial cells grown for 48 h at 37°C with 5% CO₂ on brucella agar medium using the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA). We studied 16 locus grouped into three panels (panel 1, Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55; panel 2A, Bruce18, Bruce19, and Bruce21; and panel 2B, Bruce04, Bruce07, Bruce09, Bruce16, and Bruce30). The selected primers and PCR amplification protocol for each of the loci comprising panels 1, 2A, and 2B were as described by Le Flèche et al. (20) and Al Dahouk et al. (1), with the addition of Bruce19 in panel 2A. Briefly, the amplification was performed in a MyCycler thermal cycler (Bio-Rad) with an initial step of denaturation at 96°C for 5 min followed by 30 cycles of denaturation at 96°C for 30 s, annealing at 60°C for 30 s, and elongation at 70°C for 1 min. The final extension was performed at 72°C for 5 min. Products were analyzed by gel electrophoresis on a 3% standard agarose gel using 100-bp and 20-bp ladders (Fermentas, Lithuania) as molecular size markers depending on the tandem repeat unit length. Gel images and PCR product sizes were determined using Gel Compar II software (Applied Maths). Allele numbers were established according to product size ranges (see Table 3). Different alleles for each strain were represented as a numerical matrix, and a dendrogram was constructed using the unweighted-pair group method using average linkages (UPGMA) method and Treecon software version 1.3b (36).

Data analysis.

Polymorphism was quantified by the Hunter-Gaston diversity index (HGDI) (14).

Genetic characterization of GI-3 by TP-PCR.

A technique called tiling-path PCR scanning (TP-PCR), previously described by Ren et al. (29), was used with some modifications. Briefly, the method consists of several interlocking PCR-based approaches able to characterize large genomic regions. A series of primers was designed to amplify fragments of ∼5 kb spanning the region of interest (Fig. 1) but also having short overlaps of a few hundred base pairs. The DNA sequences of the TP-PCR primers used to amplify regions of the GI-3 were designed using Primer 3 software on the basis of the sequence of chromosome I (GenBank accession no. NC_006932, B. abortus biovar 1 strain 9-941). The primer sequences are listed in Table 2. Long PCRs were performed to amplify the region in question. Any negative results by long PCR for a given primer pair were followed up by amplification of the relevant short overlaps with the same primers and/or a deletion-scanning long PCR that employed primers flanking the lost segments. Long PCRs were performed in 50 μl of reaction mixture containing 1 mM deoxynucleoside triphosphate (dNTP) mix, 10 pmol of each primer, 1.5 mM MgCl₂, 1× reaction buffer supplied by the manufacturer, 0.5 U of High Fidelity DNA polymerase (Fermentas Life Sciences, CA), and 5 μl of DNA as the template. The genomic DNA was obtained with the UltraClean microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA). Long-PCR conditions were 30 cycles of 10 s at 94°C, 30 s at 62°C, and 10 min at 68°C, followed by a 10-min extension at 68°C. For short PCRs, each multiplex PCR assay was performed in 25 μl of reaction mixture containing 1 mM deoxynucleoside triphosphate mix, 10 pmol of each primer, 2.0 mM MgCl₂, 1× reaction buffer (10 mM Tris-HCl, 50 mM KCl), 0.25 U of Taq DNA polymerase (Fermentas Life Sciences, CA), and 2 μl of DNA as the template. The samples were amplified for 35 cycles, and each cycle consisted of 1.5 min at 94°C, 1.5 min at 64°C, and 1.5 min at 72°C. The PCR products were separated by electrophoresis in 1.5% agarose gels in a 0.5× TBE buffer system for 2 h. Following electrophoresis, the gel was stained with ethidium bromide (1 μg/ml) and photographed under UV light.

Restriction analysis of fragments generated by tiling-path PCR.

DNA generated by tiling-path PCR of each fragment was digested with 10 U of restriction enzymes (Promega, Madison, WI) (AvaII to digest fragment A, EaeI to digest fragment B, BanI to digest fragment C, and BspHI to digest fragment D) and incubated at 37°C for 2 h according to the manufacturer's instructions. Restriction DNA fragments were separated by electrophoresis at 80 V in a horizontal gel containing 1.5% agarose in a 0.5× TBE buffer system for 2 h. Following electrophoresis, the gel was stained in ethidium bromide (1 μg/ml) and photographed under UV light.

PFGE analyses shows a low variability in the Chilean B. abortus strains.

We analyzed a total of 69 field strains of B. abortus isolated from milk and fetuses by pulsed-field gel electrophoresis (PFGE) in order to elucidate the genetic diversity of the Chilean strains. We were able to see two patterns, consisting of about 20 or 21 bands with a size ranging from less than 48.5 to 242.5 kb. In this report, a gel with 6 strains (3 reference strains and 3 field strains) is shown (Fig. 2 A). The most prevalent pattern was designated PFGE type A1 (Fig. 2A, lane 3) and was the same as that of B. abortus biovar 1 strain 544 (Fig. 2A, lane 1), being recognized in 67 of 69 isolates. Therefore, all of these 67 strains exhibited a genetic homogeneity that was independent of the city and period of isolation, suggesting that they were of a unique clonal origin. The second pattern was designated PFGE type A2 (Fig. 2A, lane 5 and 6), and it was found in just 2 of the 69 isolates from southern Chile. PFGE types A1 and A2 could be differentiated by the absence of a 109-kb band and the presence of another 104-kb band in type A2, with this pattern being the same for strains RB51 and 2308 (Fig. 2A, lanes 2 and 4). The pulse times used were 0.5 to 10 s for 24 h at 6 V/cm, and these pulses resolved the larger restriction fragments, but RB51 and 2308 strain patterns were indistinguishable. Since RB51 and 2308 showed different rifampin susceptibilities, this feature allowed us to recognize both wild strains (rifampin resistant) as RB51 strains. Restriction analysis with XbaI showed a low variability among strains. These results are consistent with previous findings of other authors (13, 22), However, the genetic homogeneity observed in most B. abortus isolates showed the need to complement this study with a methodology able to provide a higher resolution power, incorporating MLVA analysis and specifically MLVA-16 (comprising 16 loci). MLVA-16 has been shown to have good discriminatory power to determine genetic relationships among strains of the same species of Brucella (19).

When the cutoff value was 51% similarity, cluster analysis of the VNTR data recognized one major group and two minor groups (clusters, I, II and III) (Fig. 2B). When the 60% cutoff value was used, cluster I was divided into two groups, a larger one (named Ia) containing 45 out of 52 B. abortus strains and a minor group (named Ib) including 9 out of 52 strains, with 2 of them being reference strains (B. abortus 544 and S19). When the 80% cutoff value of similarity was applied, the dendrogram showed that subgroup Ia was subdivided into several other small clusters, with variable numbers of members. However, this cutoff highlighted the presence of one group of 11/52 strains of B. abortus, all of them isolated in the metropolitan region, which also contained two clonal strains (Fig. 2B, upper dashed box).

The MLVA-16 analysis allowed us to recognize a genetically heterogeneous population of B. abortus, which could be grouped into three main clusters named I, II, and III. Group I contained the largest number of strains tested (54/71), with individuals from different regions of southern Chile, such as region XIV (11/54), region X (16/54), and region VIII (3/54). It is also possible to observe that a large group of strains in this group originated from the central part of Chile (metropolitan region, 22/54). These 54 field strains belonging to group I in turn could be subdivided into subgroups Ia and Ib. Subgroup Ia included representatives of all the above-mentioned regions, with four pairs of clonally related strains and each of them with a common geographic origin. We also observed that a large group of strains isolated in the central area of Chile (11/22) (Fig. 2B, upper dashed box) were closely related strains (>80% similarity), and the rest of them were related to different groups of strains isolated from southern Chile. In contrast, subgroup Ib (>60% similarity) included mainly individuals from the central area (6/22) (Fig. 2B, lower dashed box) and were genetically less related than those strains belonging to the metropolitan area of group Ia. Interestingly, in this group, there was one isolate from the south of Chile (region XIV) which was genetically more closely related to B. abortus reference strains 544 and S19 and in particular to S19, with more than 80% similarity. In summary, group I included most of the strains studied and could be subdivided into two genetically heterogeneous subgroups (Ia and Ib), with similarity higher than 60%. Only in subgroup Ia were there pairs of clonally related strains, with those bacterial isolates forming clusters mainly corresponding to the same geographic origin. Finally, two reference strains used in this study (544 and S19) were associated with group I (mainly Ib).

Cluster II includes strains from southern (regions VIII and X) and central Chile. It must be noted that this particular group was related to two other reference strains of B. abortus used in this study, strains 2308 and RB51. The number of strains grouped in cluster II was 8/71. The genetic similarities among cluster II strains were also higher than 60%.

Cluster III consisted of 8/71 B. abortus field strains isolates, all of them obtained only from the south of Chile, with similarities higher than 60%. Region VIII has a significant number of representatives (5/8). However, there were no clonally related strains. Finally, this study describes one isolate from the region XIV with a VNTR amplification profile completely different from those of the rest of the strains (1/71). Using MLVA-16, this strain showed a similarity of below 10% to the rest of the strains studied. This strain corresponded to the Sample 24_XIVRegion/Valdivia, and only the Bruce04 and Bruce19 loci were positive for PCR amplification. It was impossible to obtain amplification products for the rest of the loci analyzed.

The different allele sizes found at each locus are shown in Table 3; allele size is given in base pairs for the PCR product. There was little diversity in the loci studied in panel 1, with Hunter-Gaston diversity index (HGDI) values of less than 0.50 and loci such as Bruce06, Bruce11, Bruce42, and Bruce55 having values equal to 0.0. A similar result of little diversity was observed for panel 2A, in which only the locus Bruce19 was highlighted with an HGDI value equal to 0.71. However, the diversity results observed for panel 2B were promising for the analysis of B. abortus strains, where PFGE provided little resolution in establishing genetic relationships. In our study, the average HGDI value obtained was higher than 0.70, highlighting the diversity observed in the loci Bruce07, Bruce09, and Bruce30, with an average value close to 0.75. At the same time, we could see that in addition to the expected sizes of B. abortus amplification products used to characterize panel 2B, an average of four or five different alleles in the reference strain B. abortus 9-941 could be included (Table 3) (20).

GI-3 is present in all isolated Brucella abortus strains.

In order to determine the conservation status of GI-3, we first performed in silico analyses of the region comprising GI-3 gene clusters and the neighboring region, available in the GenBank database, from genome sequences of chromosome I (accession no. NC_006932 for of B. abortus biovar 1 strain 9-941). Hence, 29 ORFs (BruAb1_0245 to BruAb1_0274) were included in GI-3. Based on this genomic sequence, we designed 5 pairs of primers for PCR amplification of 4 fragments (A, B, C, and D) of 4.3 to 7.7 kb (Table 2). Using the external primers neighboring GI-3 BruAb1_0244 (forward) and BruAb1_0274 (reverse), the presence of the island in the genome of the reference strain 544 and all field strains was verified. These results confirmed that all strains possessed the GI-3 gene cluster (data not shown).

To construct a complete tiling path through GI-3 and to determine the genetic organization within the island for the 69 strains, we used the TP-PCR method (29), which exploits short- and long-PCR protocols; this method allowed us to construct a complete tiling path through the GI-3 gene cluster for 69 strains and for type strain 544. Initially, we confirmed that reference strain 544 possessed GI-3 gene clusters by short and long PCR, first amplifying ORFs BruAb1_0251, BruAb1_0256, BruAb1_0266, and BruAb1_0274 by short PCR (data not shown) and then amplifying fragments A, B, C, and D by long PCR. It was also detected by short PCR of gene BruAb1_0244, a neighbor of GI-3.

The next step was to amplify each of the 4 fragments (A, B, C, and D) in the 69 field strains. The result was that 100% of these B. abortus field strains studied (n = 69) showed all genes within the island. In all B. abortus strains (69/69), fragments A, B, C, and D of the expected sizes (5,287 bp, 7,712 bp, 6,804 bp, and 4,312 bp, respectively) were obtained. These results confirmed that the complete GI-3 gene cluster was present in all strains sampled (69 of 69), independently of the city or their period of isolation. Figure 3 shows the products obtained for 5 strains chosen at random and the reference strain 544. This indicates that GI-3 is stable in the Chilean field isolates.

RFLP analysis of fragments generated by TP-PCR of GI-3 from all isolated Brucella abortus strains shows results similar to the B. abortus 544 banding profile.

The next step was to characterize the degree of conservation of the structure of each constituent fragment of GI-3. For this, DNA generated by long PCR of each fragment (A, B, C, and D; 5,287, 7,712, 6,804 and 4,312 bp, respectively) from all 69 samples was digested using restriction enzymes AvaII for fragment A, EaeI for fragment B, BanI for fragment C, and BspHI for fragment D. The results of digestion were analyzed using gel electrophoresis (Fig. 4). The banding patterns of restriction fragment length polymorphism (RFLP)-PCR for all fragments of the isolates were similar to each banding pattern of the B. abortus biotype 1. These results indicate once again that GI-3 is conserved in Chilean field strains.