TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

Abstract

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 μg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.

Keywords: Staphylococcus aureus; antibiotic-resistant bacteria; antimicrobial drug; bacteriophage; endolysin; skin infection; thermostable lytic protein.

Figure 1

Identification of TSPphg by sequencing the complete genome sequence of Thermus phage TSP4. (A) Genome map of TSP4, from inside to outside: circle 1 displays the GC skew ((G − C)/(G + C)); circle 2 shows the GC percentage plot; circles 3 and 4 denote the ORFs on the minus (‘−’) strand (green) and plus (‘+’) strand (red), respectively; circle 5 demonstrates the numbered scale with an interval of 4 kb. (B) The conserved domain analysis of TSPphg at both nucleotide and protein levels. (C) SDS-PAGE analysis of the purified TSPphg in this study. The black arrowhead denotes the band of recombinant TSPphg lysin.

Figure 2

Time-kill assays. Antimicrobial activity of TSPphg against Salmonella paratyphi B (A), Escherichia coli O157 (B), Klebsiella pneumoniae (C), Bacillus subtilis (D) and Staphylococcus aureus (E). S. paratyphi B, E. coli O157 and K. pneumoniae are Gram-negative bacteria while B. subtilis and S. aureus are Gram-positive bacteria. Bacterial cell numbers were counted as Log₁₀ (CFU/mL) at different time points as denoted. CFU means colony-forming unit. Data were presented as mean ± standard deviation (n = 3) and were analyzed using one-way analysis of variance (ANOVA) with a Bonferroni correction. *, p < 0.05; ***, p < 0.001. The dotted line shows the limit of detection (<10 CFU/mL).

Figure 3

TSPphg promotes wound healing in a mouse model of a multidrug-resistant S. aureus-infected skin wound. (A) Photographs of the infected wound sites treated with 100 μL PBS, 100 μL of 50 μg/mL kanamycin, or 100 μL of 50 μg/mL TSPphg, and the group without infection. (B) Wound healing percentage in all groups. Data were presented as mean ± standard deviation (n = 4 mice). p values were calculated using one-way analysis of variance (ANOVA) with a Bonferroni correction. **, p < 0.01; ***, p < 0.001. (C) In vivo decolonization activity of TSPphg in experimental mice infected with multidrug-resistant S. aureus (1 × 10⁵ CFU/mouse). At 24 h after colonization, the mice were continuously treated once a day with 100 μL PBS, 100 μL of 50 μg/mL kanamycin or 100 μL of 50 μg/mL TSPphg. At day 7, the bacterial loads in the wounds were examined. Data were presented as mean ± standard deviation of n = 4 mice, and p values were determined using a Student’s t-test. ***, p < 0.001; N.D., non-detectable; N.S., not significant (p > 0.05).

Figure 4

Scanning electron microscopy showing the effects of exogenous TSPphg treatment on the bacterial cell lysis of both Gram-negative (S. paratyphi B and E. coli O157) and Gram-positive (S. aureus) strains. For all tests, the 10⁵ CFU/mL of bacterial cells was treated with or without 50 μg/mL TSPphg dissolved in PBS at 37 °C for 1 h. The magnification is 10,000 times the original size.