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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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(a) Morphology of phiAxp-2 plaques. Phages were plated in Luria–Bertani agar and overlain with a liquid culture of A. xylosoxidans A22732. The plates were incubated at 37 °C. Clear, well-defined phiAxp-2 plaques were observed and photographed after 18 h. (b) Phage morphology. Phage was stained with 2% phosphotungstic acid and visualized at 120,000-fold magnification with transmission electron microscopy. Scale bars represent 100 nm. (c) One-step growth curve of the bacteriophage. The phage was grown in an exponential-phase culture of A. xylosoxidans strain A22732. Shown are the pfu per infected cell in the cultures at different time points. Each data point is the mean of three experiments.
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f1: (a) Morphology of phiAxp-2 plaques. Phages were plated in Luria–Bertani agar and overlain with a liquid culture of A. xylosoxidans A22732. The plates were incubated at 37 °C. Clear, well-defined phiAxp-2 plaques were observed and photographed after 18 h. (b) Phage morphology. Phage was stained with 2% phosphotungstic acid and visualized at 120,000-fold magnification with transmission electron microscopy. Scale bars represent 100 nm. (c) One-step growth curve of the bacteriophage. The phage was grown in an exponential-phase culture of A. xylosoxidans strain A22732. Shown are the pfu per infected cell in the cultures at different time points. Each data point is the mean of three experiments.

Mentions: Bacteriophage phiAxp-2 was isolated from A. xylosoxidans strain A22732 and observed with electron microscopy. A. xylosoxidans strain A22732 harbours a conjugative imipenem-encoding plasmid and is resistant to multiple β -lactam antibiotics, including imipenem and meropenem6.Cell lysis was observed after induction (phage yield: 1 × 109 pfu/ml), by propagating the induced lysate on strain A22732. The plaques obtained had a clear pinpoint morphology, with well-defined boundaries (Fig. 1a). Electron micrographs of negatively stained phiAxp-2 virions showed an icosahedral head and a long noncontractile tail (Fig. 1b). The average particle had a capsid of approximately 56 nm in diameter and a tail length of approximately 230 nm, and the phage is therefore morphologically similar to phages in the order Caudovirales and family Siphoviridae. Host range tests suggested that among all the species tested (n = 14), phiAxp-2 was specifically virulent to only four strains of A. xylosoxidans (Table 1). Besides the reported multidrug-resistant strain A22732, the other three clinical A. xylosoxidans strains were shown to be resistant to aztreonam and tobramycin6.


Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans
(a) Morphology of phiAxp-2 plaques. Phages were plated in Luria–Bertani agar and overlain with a liquid culture of A. xylosoxidans A22732. The plates were incubated at 37 °C. Clear, well-defined phiAxp-2 plaques were observed and photographed after 18 h. (b) Phage morphology. Phage was stained with 2% phosphotungstic acid and visualized at 120,000-fold magnification with transmission electron microscopy. Scale bars represent 100 nm. (c) One-step growth curve of the bacteriophage. The phage was grown in an exponential-phase culture of A. xylosoxidans strain A22732. Shown are the pfu per infected cell in the cultures at different time points. Each data point is the mean of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037462&req=5

f1: (a) Morphology of phiAxp-2 plaques. Phages were plated in Luria–Bertani agar and overlain with a liquid culture of A. xylosoxidans A22732. The plates were incubated at 37 °C. Clear, well-defined phiAxp-2 plaques were observed and photographed after 18 h. (b) Phage morphology. Phage was stained with 2% phosphotungstic acid and visualized at 120,000-fold magnification with transmission electron microscopy. Scale bars represent 100 nm. (c) One-step growth curve of the bacteriophage. The phage was grown in an exponential-phase culture of A. xylosoxidans strain A22732. Shown are the pfu per infected cell in the cultures at different time points. Each data point is the mean of three experiments.
Mentions: Bacteriophage phiAxp-2 was isolated from A. xylosoxidans strain A22732 and observed with electron microscopy. A. xylosoxidans strain A22732 harbours a conjugative imipenem-encoding plasmid and is resistant to multiple β -lactam antibiotics, including imipenem and meropenem6.Cell lysis was observed after induction (phage yield: 1 × 109 pfu/ml), by propagating the induced lysate on strain A22732. The plaques obtained had a clear pinpoint morphology, with well-defined boundaries (Fig. 1a). Electron micrographs of negatively stained phiAxp-2 virions showed an icosahedral head and a long noncontractile tail (Fig. 1b). The average particle had a capsid of approximately 56 nm in diameter and a tail length of approximately 230 nm, and the phage is therefore morphologically similar to phages in the order Caudovirales and family Siphoviridae. Host range tests suggested that among all the species tested (n = 14), phiAxp-2 was specifically virulent to only four strains of A. xylosoxidans (Table 1). Besides the reported multidrug-resistant strain A22732, the other three clinical A. xylosoxidans strains were shown to be resistant to aztreonam and tobramycin6.

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.


Related in: MedlinePlus