Burkholderia contaminans Bacteriophage CSP3 Requires O-Antigen Polysaccharides for Infection

Abstract

The Burkholderia cepacia complex is a group of opportunistic pathogens that cause both severe acute and chronic respiratory infections. Due to their large genomes containing multiple intrinsic and acquired antimicrobial resistance mechanisms, treatment is often difficult and prolonged. One alternative to traditional antibiotics for treatment of bacterial infections is bacteriophages. Therefore, the characterization of bacteriophages infective for the Burkholderia cepacia complex is critical to determine their suitability for any future use. Here, we describe the isolation and characterization of novel phage, CSP3, infective against a clinical isolate of Burkholderia contaminans. CSP3 is a new member of the Lessievirus genus that targets various Burkholderia cepacia complex organisms. Single nucleotide polymorphism (SNP) analysis of CSP3-resistant B. contaminans showed that mutations to the O-antigen ligase gene, waaL, consequently inhibited CSP3 infection. This mutant phenotype is predicted to result in the loss of cell surface O-antigen, contrary to a related phage that requires the inner core of the lipopolysaccharide for infection. Additionally, liquid infection assays showed that CSP3 provides suppression of B. contaminans growth for up to 14 h. Despite the inclusion of genes that are typical of the phage lysogenic life cycle, we saw no evidence of CSP3's ability to lysogenize. Continuation of phage isolation and characterization is crucial in developing large and diverse phage banks for global usage in cases of antibiotic-resistant bacterial infections. IMPORTANCE Amid the global antibiotic resistance crisis, novel antimicrobials are needed to treat problematic bacterial infections, including those from the Burkholderia cepacia complex. One such alternative is the use of bacteriophages; however, a lot is still unknown about their biology. Bacteriophage characterization studies are of high importance for building phage banks, as future work in developing treatments such as phage cocktails should require well-characterized phages. Here, we report the isolation and characterization of a novel Burkholderia contaminans phage that requires the O-antigen for infection, a distinct phenotype seen among other related phages. Our findings presented in this article expand on the ever-evolving phage biology field, uncovering unique phage-host relationships and mechanisms of infection.

Keywords: Burkholderia cepacia complex; O-antigen; bacteriophage; characterization; phage; phage characterization; phage therapy; receptor.

FIG 1

Morphological features of phage CSP3. (A) Transmission electron microscope images of CSP3 virions stained with 2% uranyl acetate. CSP3 has a noncontractile tail (15.5 ± 0.79 nm) and an icosahedral head (62 ± 0.95 nm). Tail fibers could not be visualized. Scale bar, 100 nm in the overlaid image. (B) Spot plaque assay of CSP3 filtrate on a double agar overlay with host, B. contaminans. Decreasing concentrations of CSP3 show small, individual plaques.

FIG 2

Circular genome map of CSP3. The tRNA^ser is colored in red, and predicted ORFs are colored in blue and numbered, with the name of the protein they encode.

FIG 3

Whole-genome and phylogenetic relationships between CSP3 and related phages. (A) Intergenomic similarities between CSP3 and the top phage matches on the NCBI database. The blue and orange dashed-line boxes indicate the two separate phage groups. (B) Whole-genome (amino acid) phylogenetic tree using GBDP d₆ formula with suggested taxa collated with data from the ICTV. (1) Family-level classification; (2) subfamily-level classification; (3) genus-level classification; (4) species-level classification.

FIG 4

Gene sharing and placement of CSP3 and relatives. A reticulate network of CSP3 and over 17,000 phage genomes (nodes) currently deposited on the GenBank database with key bacterial host genera color coded to highlight their placement. The zoomed in panel shows CSP3 with the Burkholderia phage group, Ralstonia phage group, and other relatives using an edge-weight spring-embedded layout model to highlight their gene sharing, indicated by the lines (edges) connecting the nodes.

FIG 5

Whole-genome comparison of the Lessievirus group and CSP3. (A) Whole-genome map alignments of the Lessievirus genus and CSP3, aligned from the small terminase gene. Key genes are color coded, and identity of shared percentage is shown by the grayscale bar. (B) Heatmap comparison of the shared amino acid identities of the Lessievirus group tail fiber genes (TFGs). Each TFG is numbered 1 to 3 or 4 in order of how they appear within the genome (i.e., CSP3 TFG 1 is labeled as CSP3 1).

FIG 6

Biological properties of CSP3. (A) pH and thermal stability of CSP3. (B) Growth curve of CSP3. (C) Bacterial growth reduction assays using multiple MOI ratios of B. contaminans (Bco) and CSP3 over 18 h. Panels A to C are presented as the means of three biological replicates with error bars representing SEM.

FIG 7

B. contaminans waaL gene mutations and predicted modification of the LPS. (A) WaaL mutations found in four CSP3-resistant mutants. (B) Schematic of the changes to the cell wall LPS upon mutation of the waaL gene and effects on CSP3 infection. The left panel depicts regular cell wall and LPS composition, compared to the right panel, which depicts the putative phenotype in the mutant B. contaminans. Mutation to WaaL and loss-of-function results in the O-antigen and, potentially, the outer core polysaccharide unable to attach to the lipid A and outer core polysaccharide. This results in CSP3 unable to attach and, ultimately, unable to lyse its host.