Microbiological Characterization And Genomic Analysis Of Achromobacter Xylosoxidans Bacteriophage

Achromobacter xylosoxidans is Gram-negative bacillus,which mostly distributed in aquatic environment and was found in various clinical specimens.With a large number and long-term use of antibiotics,A.xylosoxidans is resistant to multiple antibiotics.As the prevention and control become more difficult,the development of new strategies for the control of A.xylosoxidans is urgently needed.Phage is bacterial viruses that only infect its specific hosts,with several unique advantages,including rapid proliferation,no residue and more safely.Thus,phage shows great potential as substitute for antibiotics.Research ideas and methods might be further applied in research and development of food science technologies.Three virulent phages,named phiAxp-1,phiAxp-2 and phiAxp-3,were isolated when Achromobacter xylosoxidans A22732 strains were used as hosts.In this article,we investigated the microbiological characterization,the best preservation methods and genomic characteristics of phiAxp-1,phiAxp-2 and phiAxp-3.The contents and results are shown as follows:1.The microbiological characterization of phages?1?The phiAxp-1 produced plaques of 2-3mm in diameter on a lawn of A.xylosoxidans A22732,transmission electron microscopy observation showed that it belonged to the family of Siphoviridae.One-step growth curve analysis of phiAxp-1revealed that latent period was 75 min and rise period was 15 min.The average bust size was estimated about 1742 pfu/cell.The titer of phiAxp-1 can be maintained at the same order of magnitude when incubated at 50?for 75min,the activity of phi Axp-1 will lose when the temperature rises.More than 40%of phiAxp-1 can survive at pH5-pH10.The livability of phiAxp-1 is largest at 10mmol/L Ca²⁺and 15mmol/L Mg²⁺.The tolerance of phiAxp-1 was strong when treated in low concentration?10%?of ethanol or isopropanol,whereas it became unstable with medium or high concentration of ethanol or isopropanol.The receptor of phiAxp-1 was the lipopolysaccharide of A.xylosoxidans,12.5?g/mL of lipopolysaccharide from A.xylosoxidans was sufficient to inhibit the binding activity of50%of phiAxp-1,while lipopolysaccharide at 800?g/mL resulted in 89.8%inactivation of phiAxp-1.?2?The phiAxp-2 produced plaques of 0.5-1mm in diameter on a lawn of A.xylosoxidans A22732,transmission electron microscopy observation showed that it belonged to the family of Siphoviridae.One-step growth curve analysis of phiAxp-2revealed that latent period was 180 min and rise period was 60 min.The average bust size was estimated about 2985 pfu/cell.The titer of phiAxp-2 decreased an order of magnitude when bathed at 50?for 75min,the activity of phiAxp-2 will lose when the temperature rises.More than 50%of phiAxp-2 can survive at pH5-pH10.The livability of phiAxp-2 is largest at 10mmol/L Ca²⁺and 15mmol/L Mg²⁺.The tolerance of phiAxp-2 was strong when treated in low concentration?10%?of ethanol or isopropanol,whereas it became unstable with medium or high concentration of ethanol or isopropanol.The receptor of phiAxp-2was the lipopolysaccharide of A.xylosoxidans,19?g/mL of lipopolysaccharide from A.xylosoxidans was sufficient to inhibit the binding activity of 50%of phiAxp-1,while lipopolysaccharide at 800?g/mL resulted in 82.6%inactivation of phiAxp-2.?3?The phiAxp-3 produced plaques of 2-3mm in diameter on a lawn of A.xylosoxidans A22732,transmission electron microscopy observation showed that it belonged to the family of Podoviridae.One-step growth curve analysis of phiAxp-3revealed that latent period was 80 min and rise period was 40 min.The average bust size was estimated about 9000 pfu/cell.The titer of phiAxp-3 decreased an order of magnitude when bathed at 50?for 75min,the activity of phiAxp-3 will lose when the temperature rises.More than 50%of phiAxp-2 can survive at pH6-pH9.The livability of phiAxp-2 is largest at 15mmol/L Ca²⁺and 10mmol/L Mg²⁺.The tolerance of phiAxp-3 was poor when treated in different concentrations of ethanol;the tolerance was strong when treated in low concentration?10%?of isopropanol,whereas it became unstable with medium or high concentration of isopropanol.The receptor of phiAxp-3 was the lipopolysaccharide of A.xylosoxidans,25?g/mL of lipopolysaccharide from A.xylosoxidans was sufficient to inhibit the binding activity of 50%of phiAxp-1,while lipopolysaccharide at 800?g/mL resulted in 88.77%inactivation of phiAxp-3.2.The preservation of phageThe titers of phiAxp-1 and phiAxp-2 maintained at the initial 10¹⁰ pfu/mL,and that of phiAxp-3 decreased slightly from 10¹¹ pfu/mL to 10¹⁰ pfu/mL after 16 months when the three Achromobacter xylosoxidans bacteriophages were stored in glycerol and dimethyl sulfoxide at-80?,-20?and 4?.When stored in chloroform at 4?for 16 months,the titers of all of the three phages decreased slightly but was higher than the other temperatures?-80?,-20?,room temperature,37??.Three preservatives were ineffective when the phage was stored at 37?.3.Genomic analysis of phage?1?The genome of phiAxp-1 contains 45,045bp of circular double-stranded DNA with an average GC content of 56.02%and encodes 64 open reading frames?ORFs?;however,only 21 ORFs share similarity at the protein level with other sequences deposited in GenBank.12 virion proteins have been assigned putative functions through liquid chromatography-electrospray ionization-tandem mass spectrometry?LC-ESI-MS/MS?.Multiple genome alignments showed the weak homology of phiAxp-1 with multiple phages at the whole genome level.Therefore we propose phiAxp-1 was a novel phage.Phylogenetic analysis of the available phage terminase proteins demonstrated that phiAxp-1belongs to a separate branch.The annotated genomic sequence of phage phiAxp-1 has been submitted to Genbank,accession number:KP313532.?2?The genome of phiAxp-2 contains 62,220bp of circular double-stranded DNA with an average GC content of 60.11%and encodes 86 open reading frames?ORFs?;however,only 28 ORFs share similarity at the protein level with other sequences deposited in GenBank.Multiple genome alignments showed the homology of phiAxp-2 with Burkholderia phage AH2,and BcepNazgul at the whole genome level.Thus we propose phiAxp-2 was a novel phage.A phylogenetic analysis of the DNA polymerase and the terminase large subunit predicted that phiAxp-2 is most closely related to AH2.The annotated genomic sequence of phage phiAxp-2 has been submitted to Genbank,accession number:KT321316.?3?The genome of phiAxp-3 contains 72,825bp of liner double-stranded DNA with an average GC content of 55.2%and 416-bp terminal redundant ends,encodes 80 open reading frames?ORFs?;however,only 22 ORFs share similarity at the protein level with other sequences deposited in GenBank.10 virion proteins have been assigned putative functions through liquid chromatography-electrospray ionization-tandem mass spectrometry?LC-ESI-MS/MS?.Multiple genome alignments showed the homology of phiAxp-3 with Achromobacter phage JWAlpha and JWDelta,and the Enterobacter phage,N4,indicating that phiAxp-3 is an N4-like phage.A phylogenetic analysis of the RNA polymerase and the terminase large subunit placed phiAxp-3 within the branch of JWAlpha and JWDelta.The annotated genomic sequence of phage phiAxp-3 has been submitted to Genbank,accession number:KT321317.