A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae

Abstract

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

Keywords: Brucella; LPS; Ochrobactrum; evolution; frog; metabolism; motility; virulence.

Figure 1

Phylogenetic placement of B13-0095 relative to other Brucella spp. and Ochrobactrum spp. determined by kSNP. The reference strains for all classical Brucella spp. and novel species were included. The magnified region highlights the divergence point of the “BO clade” in Brucella.

Figure 2

Genetic clusters specific to B13-0095, BO1, and BO2. (A) Schematic representation of a 12.5 kb ectoine utilization genetic cluster conserved in B13-0095 and O. anthropi. (B) Schematic representation of the genes organization in a cluster involved in rhamnose utilization that is conserved in the B13-0095, BO1, BO2, and O. anthropi. (C) Position of the cluster of rhamnose utilization (in blue) relatively to the three loci of flagella related genes (in green) in the BO clade Brucella, compared to O. anthropi and classical Brucella. The red box represents a fragment of 14 kb containing genes involved in several metabolic pathways or cationic antimicrobials resistance that is present in all Brucella.

Figure 3

Motility of Brucella strains. Motility of the indicated strains was visualized using on plate swimming assays. Shown are representative results of at least 3 independent repeats. Scale bar = 2 cm.

Figure 4

Electrophoretic profiles of the LPS produced by different Brucella. Silver staining after SDS-PAGE of proteinase K-digested LPS preparations from the indicated Brucella strains. The bands corresponding to the long, intermediate or short sugar chains of O-PS are indicated.

Figure 5

Schematic representation of the wbk region organization in Brucella. The genes described in classical Brucella and B. inopinata BO1 within or flanking the wbk region are shown in yellow or white, respectively. The genetic features present in both the Pac-Man frog isolate B13-0095 and the B. inopinata-like strain BO2 genomes are shown in green. Features in black are only present in BO2 and features in orange only in B13-0095. The annotation of the genes is indicated, as well as their ID number in PATRIC (peg#) or RefSeq (BIBO2#). The arrows indicate the end/beginning of the contigs in the draft genome sequences of B13-0095 and BO2. ^* indicate predicted pseudogenes. Rectangles represent predicted mobile elements.

Figure 6

Intracellular replication profile in eukaryotic cells. Intracellular replication was determined using gentamycin protection assays. HeLa (top) or J774 (bottom) cells were infected with B. suis bv.1 wild-type (black dotted line), a B. suis virB8 mutant (gray dotted line), or the frog isolate (full line) and lysed at 2, 5, 24, or 48 hpi. The data are shown as Log₁₀ CFU/well and represent the geometric mean for three independent experiments done in triplicate.

Figure 7

Quantification of infection rate by flow cytometry. HeLa cells were infected by mCherry-expressing bacteria, detached, and fixed 24 hpi and analyzed by flow cytometry. The level of infection is given by the % of infected (i.e., mCherry⁺) cells (top). The fluorescence intensity of infected cells was quantified by the MFI (Median Fluorescence Intensity) values (bottom), which is indicative of the intracellular replication rate of the bacteria. The data are mean values (±SEM) calculated on 3 replicates.

Figure 8

Visualization of B13-0095 intracellular replication. HeLa (A) or J774 (B) cells were infected by bacteria expressing mCherry, fixed at 24 hpi and visualized by confocal microscopy using a 60x objective. Shown are representative images for each condition. Bacteria are shown in red. Cells are visualized by transmission in the overlay. Scale bar = 5 μm.

Figure 9

LPS biosynthesis pathways in Brucella. Schematic representation of the O-polysaccharide biosynthesis pathways in Brucella. The A box highlights the reactions previously described for classical Brucella. The B box shows the predicted alternative pathways in the Pac-Man frog isolate B13-0095 and strain BO2. The reactions outside the boxes are common. Dotted lines show speculated reactions. The color code used to highlight some enzymes in this scheme is the same as in Figure 5 and Table S4.