Characterization of the novel cross-genus phage vB_SmaS_QH3 and evaluation of its antibacterial efficacy against Stenotrophomonas maltophilia

Abstract

Background: Bacteriophages, which are natural bacterial predators, demonstrate potential as safe and effective biological control agents against drug-resistant infections. This study aims to characterize the biological properties of the novel lytic phage vB_SmaS_QH3 and comprehensively evaluate its efficacy in preventing and controlling clinically multidrug resistance Stenotrophomonas maltophilia infections using both in vivo and in vitro models.

Methods: The phage was isolated from hospital sewage using the multidrug resistant S. maltophilia no. 3738 as the host. Transmission electron microscopy (TEM) was used to observe phage morphology, and the host range was determined via spot assays. Proliferation kinetics, including multiplicity of infection (MOI), adsorption rate, and one-step growth curves, were analyzed. Stability was assessed under various physicochemical conditions. Based on Illumina whole-genome sequencing data, bioinformatics tools were employed for gene annotation, functional prediction, and phylogenetic analysis. Antimicrobial activity was assessed using in vitro and in vivo models.

Results: A lytic phage vB_SmaS_QH3 was isolated from hospital sewage. TEM revealed that it belongs to the class Caudoviricetes, featuring an icosahedral head (62 ± 3 nm) and a non-contractile long tail (121 ± 5 nm). Although the phage has a narrow host range, it exhibits cross-genus infectivity, lysing S. maltophilia (11/81) and Pseudomonas aeruginosa (3/24). The optimal MOI for phage vB_SmaS_QH3 is 0.01, with an adsorption rate of 49.16% within 20 min, a latent period of 40 min, a lytic period of 50 min, and a burst size of 41.67 plaque-forming units/cell. The phage remained stable at 4-60°C, at pH 3-11, and in chloroform, but it was completely inactivated following 20-min exposure to UV irradiation. Genomic analysis showed a linear double-stranded DNA genome of 43,085 bp with a GC content of 54.2%, containing 54 predicted ORFs, and no virulence or antibiotic resistance genes were detected. In vitro, vB_SmaS_QH3 effectively inhibited bacterial growth within 9 h. In vivo, it significantly improved the survival rate of Galleria mellonella larvae infected with S. maltophilia, regardless of the treatment timing.

Conclusion: vB_SmaS_QH3 is a narrow host range lytic phage with a safe genome and excellent stability. It exhibits significant antibacterial activity both in vitro and in vivo, making it a promising candidate for therapeutic applications.

Keywords: Galleria mellonella; Stenotrophomonas maltophilia; genomic analysis; phage; phage therapy.

Figure 1

vB_SmaS_QH3 physiochemical influences. (A) vB_SmaS_QH3 temperature stability. (B) vB_SmaS_QH3 stability at different pH values. (C) vB_SmaS_QH3 stability against UV radiation. (D) Effect of various chloroform percentages on vB_SmaS_QH3. Figures 1A,B,D: Statistical analysis performed using one-way ANOVA. Significance levels indicated as: ns for non-significance; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Antimicrobial effects of the phage in vitro and in vivo. (A) Lytic activity of vB_SmaS_QH3 against S. maltophilia no. 3738 at different MOIs; (B) Survival curve of G. mellonella after infecting with different S. maltophilia no. 3738 concentrations; (C) Survival rate during phage treatment; (D) Survival rate after phage prophylaxis. ****indicates statistical significance at p < 0.0001 (Mantel-Cox).

Figure 3

Plaques and transmission electron microscopy of phage vB_SmaS_QH3. (A) Plaque morphology of phage vB_SmaS_QH3 on a bacterial lawn of S. maltophilia no. 3738 in a double-layer agar. (B) Transmission electron microscopy of phage vB_SmaS_QH3. Scale bar, 100 nm.

Figure 4

MALDI-TOF MS of clinical strains and host range of vB_SmaS_QH3. Developmental trees were established by cluster analysis using MALDI-TOF fingerprinting. The cleavage spectra were judged as follows: 4+ for completely cleared; 3+ for the entire area clear but with faint haze in the background; 2+ for the entire area clear but with faint haze in the background; 1+ for a few isolated plaques or a severe hazy background; 0 for not cleared (−).

Figure 5

Growth characteristics of vB_SmaS_QH3. (A) The phage titers under different MOIs (phage/bacteria = 0.0001, 0.001, 0.01, 0.1, 1, 10, 100) are depicted on the x-axis. (B) Adsorption rate of vB_SmaS_QH3 (MOI = 0.01). (C) One-step growth curve test of vB_SmaS_QH3 was performed at MOI = 0.01.

Figure 6

Genome map of phage vB_SmaS_QH3. The outermost circle represents the ORFs. Different colors represent genes with different functions: gray, hypothetical protein; red, lysis; deep green, structure; deep blue, DNA replication and modification; light green, packaging.

Figure 7

Phylogenetic analysis of phage vB_SmaS_QH3. (A) A phylogenetic tree was generated using the whole genome sequence through VICTOR analysis, suggesting that phage vB_SmaS_QH3 may represent a novel species. (B) Percentage sequence similarity between phages calculated using VIDIRIC shows that phage vB_SmaS_QH3 could be a novel clade. The horizontal and vertical axes indicate the corresponding phage GenBank number.