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Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans

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ABSTRACT

A novel Achromobacter xylosoxidans bacteriophage, phiAxp-2, was isolated from hospital sewage in China. The phage was morphologically and microbiologically characterized, and its one-step growth curve, host range, genomic sequence, and receptor were determined. Its morphology showed that phiAxp-2 belongs to the family Siphoviridae. Microbiological characterization demonstrated that pH 7 is most suitable for phage phiAxp-2; its titer decreased when the temperature exceeded 50 °C; phiAxp-2 is sensitive to ethanol and isopropanol; and the presence of calcium and magnesium ions is necessary to accelerate cell lysis and improve the formation of phiAxp-2 plaques. Genomic sequencing and a bioinformatic analysis showed that phage phiAxp-2 is a novel bacteriophage, consisting of a circular, double-stranded 62,220-bp DNA molecule with a GC content of 60.11% that encodes 86 putative open reading frames (ORFs). The lipopolysaccharide of A. xylosoxidans is involved in the adsorption of phiAxp-2.

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Resistance of phage phiAxp-2 to physical and chemical agents.(a) Effect of pH on the phage phiAxp-2 titer after incubation for 60 min in LB broth at 37 °C. (b) Inactivation kinetics of phage phiAxp-2 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, and 80 °C. (c) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, 75%, and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, and 95% isopropanol. (e) Viability of phage phiAxp-2 in LB broth with different Ca2+ and Mg2+ concentrations. On all graphs, the values are the means of three determinations.
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f2: Resistance of phage phiAxp-2 to physical and chemical agents.(a) Effect of pH on the phage phiAxp-2 titer after incubation for 60 min in LB broth at 37 °C. (b) Inactivation kinetics of phage phiAxp-2 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, and 80 °C. (c) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, 75%, and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, and 95% isopropanol. (e) Viability of phage phiAxp-2 in LB broth with different Ca2+ and Mg2+ concentrations. On all graphs, the values are the means of three determinations.

Mentions: Figure 2a shows the pH sensitivity of phage phiAxp-2. The phage maintained its infectivity when incubated at 37 °C in a pH range of 4–11. At pH 1 and pH 14, approximately 100% reductions in the phage particle counts were observed. The loss of viability when phage phiAxp-2 was subjected to various temperatures is shown in Fig. 2b. Phage phiAxp-2 displayed heat-sensitivity at 50 °C, 60 °C, 70 °C, and 80 °C. Treatment at 80 °C for 75 min completely inactivated the phage. As shown in Fig. 2c,d, the activity of phage phiAxp-2 was affected by the presence of ethanol and isopropanol. The most effective concentrations of ethanol (95%, v/v) and isopropanol (95%, v/v) reduced the phage titer by 76% and 84%, respectively, after 90 min. Because many phages require divalent ions (such as Ca2+ or Mg2+) for optimal adsorption9, the ion-dependence of phage phiAxp-2 was determined. The most efficient infection was achieved with concentrations of 15 mM Mg2+ and 10 mM Ca2+ (Fig. 2e).


Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans
Resistance of phage phiAxp-2 to physical and chemical agents.(a) Effect of pH on the phage phiAxp-2 titer after incubation for 60 min in LB broth at 37 °C. (b) Inactivation kinetics of phage phiAxp-2 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, and 80 °C. (c) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, 75%, and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, and 95% isopropanol. (e) Viability of phage phiAxp-2 in LB broth with different Ca2+ and Mg2+ concentrations. On all graphs, the values are the means of three determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037462&req=5

f2: Resistance of phage phiAxp-2 to physical and chemical agents.(a) Effect of pH on the phage phiAxp-2 titer after incubation for 60 min in LB broth at 37 °C. (b) Inactivation kinetics of phage phiAxp-2 at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, and 80 °C. (c) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, 75%, and 95% ethanol. (d) Inactivation kinetics of phage phiAxp-2 in the presence of 10%, 50%, and 95% isopropanol. (e) Viability of phage phiAxp-2 in LB broth with different Ca2+ and Mg2+ concentrations. On all graphs, the values are the means of three determinations.
Mentions: Figure 2a shows the pH sensitivity of phage phiAxp-2. The phage maintained its infectivity when incubated at 37 °C in a pH range of 4–11. At pH 1 and pH 14, approximately 100% reductions in the phage particle counts were observed. The loss of viability when phage phiAxp-2 was subjected to various temperatures is shown in Fig. 2b. Phage phiAxp-2 displayed heat-sensitivity at 50 °C, 60 °C, 70 °C, and 80 °C. Treatment at 80 °C for 75 min completely inactivated the phage. As shown in Fig. 2c,d, the activity of phage phiAxp-2 was affected by the presence of ethanol and isopropanol. The most effective concentrations of ethanol (95%, v/v) and isopropanol (95%, v/v) reduced the phage titer by 76% and 84%, respectively, after 90 min. Because many phages require divalent ions (such as Ca2+ or Mg2+) for optimal adsorption9, the ion-dependence of phage phiAxp-2 was determined. The most efficient infection was achieved with concentrations of 15 mM Mg2+ and 10 mM Ca2+ (Fig. 2e).

View Article: PubMed Central - PubMed

ABSTRACT

A novel Achromobacter xylosoxidans bacteriophage, phiAxp-2, was isolated from hospital sewage in China. The phage was morphologically and microbiologically characterized, and its one-step growth curve, host range, genomic sequence, and receptor were determined. Its morphology showed that phiAxp-2 belongs to the family Siphoviridae. Microbiological characterization demonstrated that pH 7 is most suitable for phage phiAxp-2; its titer decreased when the temperature exceeded 50 °C; phiAxp-2 is sensitive to ethanol and isopropanol; and the presence of calcium and magnesium ions is necessary to accelerate cell lysis and improve the formation of phiAxp-2 plaques. Genomic sequencing and a bioinformatic analysis showed that phage phiAxp-2 is a novel bacteriophage, consisting of a circular, double-stranded 62,220-bp DNA molecule with a GC content of 60.11% that encodes 86 putative open reading frames (ORFs). The lipopolysaccharide of A. xylosoxidans is involved in the adsorption of phiAxp-2.

No MeSH data available.