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Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage.

Ma Y, Li E, Qi Z, Li H, Wei X, Lin W, Zhao R, Jiang A, Yang H, Yin Z, Yuan J, Zhao X - Sci Rep (2016)

Bottom Line: Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles.Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3.Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

View Article: PubMed Central - PubMed

Affiliation: College of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China.

ABSTRACT
Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

No MeSH data available.


Related in: MedlinePlus

Resistance of phage phiAxp-3 to physical and chemical agents.(a) The effect of pH on the adsorption of phage phiAxp-3 to A. xylosoxidans A22732 in LB broth. (b) Inactivation kinetics of phage phiAxp-3 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C. (c) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50%, 75% and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50% and 95% isopropanol. (e) Effect on phage phiAxp-3 titre of incubation in LB broth with and without CaCl2 or MgCl2 (0, 5, 10, 15, 20, 25 and 30 mmol/l) at 37 °C. For all the graphs, the values represent the mean of three determinations.
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f2: Resistance of phage phiAxp-3 to physical and chemical agents.(a) The effect of pH on the adsorption of phage phiAxp-3 to A. xylosoxidans A22732 in LB broth. (b) Inactivation kinetics of phage phiAxp-3 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C. (c) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50%, 75% and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50% and 95% isopropanol. (e) Effect on phage phiAxp-3 titre of incubation in LB broth with and without CaCl2 or MgCl2 (0, 5, 10, 15, 20, 25 and 30 mmol/l) at 37 °C. For all the graphs, the values represent the mean of three determinations.

Mentions: Figure 2a shows the pH sensitivity of phage phiAxp-3. The phage titres decreased to different extents when the pH was above or below 7. At pH 4 and pH 10, reductions of 90.25% and 75.76% in phage particle counts were observed, respectively. Almost no viral particles were detected at pH 1 and pH 14. The viability loss when phiAxp-3 was subjected to temperatures of 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C is shown in Fig. 2b. A control at temperature of 4 °C was also included. The phage titres reduced dramatically at 50 °C, 60 °C, 70 °C and 80 °C. After 75 min at 50 °C, the phage titre reduced by 91.2%. At 80 °C, a 99.86% reduction in viral particles was recorded after 15 min, and compared with the control, after 75 min only 0.0002% of the viral particles were detected. Scarcely any reduction in the phage titres were observed at 4 °C, 25 °C and 37 °C after 75 min of treatment. The survivor curves for phiAxp-3 in different biocides are shown in Fig. 2c,d. The results show that the presence of ethanol at low (10%) and high (95%) concentrations reduced the phage titres (Fig. 2c). The phage titres reduced by 20.75%, 69.76% and 99.62% after 75 min of treatment with isopropanol at 10%, 50% and 95%, respectively (Fig. 2d). Divalent ions such as Ca2+ or Mg2+ are necessary for phage attachment and intracellular growth18. phiAxp-3 showed divalent cation dependency for plaque development, but the concentration of Ca2+ or Mg2+ had to be less than or equal to 20 mM (Fig. 2e).


Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage.

Ma Y, Li E, Qi Z, Li H, Wei X, Lin W, Zhao R, Jiang A, Yang H, Yin Z, Yuan J, Zhao X - Sci Rep (2016)

Resistance of phage phiAxp-3 to physical and chemical agents.(a) The effect of pH on the adsorption of phage phiAxp-3 to A. xylosoxidans A22732 in LB broth. (b) Inactivation kinetics of phage phiAxp-3 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C. (c) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50%, 75% and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50% and 95% isopropanol. (e) Effect on phage phiAxp-3 titre of incubation in LB broth with and without CaCl2 or MgCl2 (0, 5, 10, 15, 20, 25 and 30 mmol/l) at 37 °C. For all the graphs, the values represent the mean of three determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837373&req=5

f2: Resistance of phage phiAxp-3 to physical and chemical agents.(a) The effect of pH on the adsorption of phage phiAxp-3 to A. xylosoxidans A22732 in LB broth. (b) Inactivation kinetics of phage phiAxp-3 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C. (c) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50%, 75% and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-3 in the presence of 10%, 50% and 95% isopropanol. (e) Effect on phage phiAxp-3 titre of incubation in LB broth with and without CaCl2 or MgCl2 (0, 5, 10, 15, 20, 25 and 30 mmol/l) at 37 °C. For all the graphs, the values represent the mean of three determinations.
Mentions: Figure 2a shows the pH sensitivity of phage phiAxp-3. The phage titres decreased to different extents when the pH was above or below 7. At pH 4 and pH 10, reductions of 90.25% and 75.76% in phage particle counts were observed, respectively. Almost no viral particles were detected at pH 1 and pH 14. The viability loss when phiAxp-3 was subjected to temperatures of 25 °C, 37 °C, 50 °C, 60 °C, 70 °C and 80 °C is shown in Fig. 2b. A control at temperature of 4 °C was also included. The phage titres reduced dramatically at 50 °C, 60 °C, 70 °C and 80 °C. After 75 min at 50 °C, the phage titre reduced by 91.2%. At 80 °C, a 99.86% reduction in viral particles was recorded after 15 min, and compared with the control, after 75 min only 0.0002% of the viral particles were detected. Scarcely any reduction in the phage titres were observed at 4 °C, 25 °C and 37 °C after 75 min of treatment. The survivor curves for phiAxp-3 in different biocides are shown in Fig. 2c,d. The results show that the presence of ethanol at low (10%) and high (95%) concentrations reduced the phage titres (Fig. 2c). The phage titres reduced by 20.75%, 69.76% and 99.62% after 75 min of treatment with isopropanol at 10%, 50% and 95%, respectively (Fig. 2d). Divalent ions such as Ca2+ or Mg2+ are necessary for phage attachment and intracellular growth18. phiAxp-3 showed divalent cation dependency for plaque development, but the concentration of Ca2+ or Mg2+ had to be less than or equal to 20 mM (Fig. 2e).

Bottom Line: Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles.Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3.Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

View Article: PubMed Central - PubMed

Affiliation: College of Food Science, Henan Institute of Science and Technology, Xinxiang, 453003, China.

ABSTRACT
Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

No MeSH data available.


Related in: MedlinePlus