Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5

Abstract

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated phiSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The phiSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, phiKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to phiSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, phiSMA5 appears to be the first reported.

FIG. 1.

Adsorption curve of bacteriophage φSMA5 on S. maltophilia T39.

FIG. 2.

One-step growth curve of bacteriophage φSMA5 on S. maltophilia T39. Shown are the PFU per infected cell in cultures at different time points. Each data point is a mean from four experiments.

FIG. 3.

Transmission electron micrographs of phage φSMA5. (A) φSMA5 consists of an elongated isometric head of 90 by 90 nm and a complex tail of 90 by 15 nm. One baseplate and two tail fibers (indicated with arrows) are visible on most phage particles. (B) Shown is a part of the S. maltophilia T39 cell with φSMA5 particles adsorbed (arrows). Scale bars, 50 nm.

FIG. 4.

(A) Agarose gel electrophoresis of the DNA digests from the φSMA5 genome. The restriction enzymes used are labeled above the lanes. (B) PFGE of the φSMA5 genomic DNA. Lanes: 1, size markers; 2, DNA from 1.0 × 10⁹ PFU; 3, DNA from 1.0 × 10⁸ PFU.

FIG. 5.

SDS-PAGE of the φSMA5 virion proteins. About 5.0 × 10⁸ PFU of purified phage particles was boiled in cracking buffer (total of 20 μl) and loaded onto the well. Shown are the 25 bands with estimated molecular masses. Bands P1 to P4 were recovered from the gel and subjected to sequence determination of the N-terminal 10 amino acid residues.