IADR Abstract Archives
Characterization of Aggregatibacter Phage S1249: Lysogenic and Lytic Phenotype Switches
Objectives: Antimicrobial resistance is a rising global public health threat and resistant strains have been also identified from the Gram-negative, oral pathobiont Aggregatibacter actinomycetemcomitans, which necessitates alternative antimicrobial strategies. Aggregatibacter phage S1249 was originally identified as a prophage linked to increased sensitivity of its lysogen to serum killing. This study was developed to understand phage biology in A. actinomycetemcomitans.
Methods: Phage S1249 was isolated using mitomycin C induction. Briefly, mid-logarithmic bacterial cell was exposed to mitomycin for 30 min for induction, washed thoroughly and recovered in the broth for 18h. Phages was harvested by low-speed centrifugation to remove the un-lysed cells, followed by high-speed centrifugation to harvest phages. The morphology of isolated phage was examined under transmission electron microscopy (TEM) and the isolated phage was used to infect strains across different serotypes to determine the infection specificity. Briefly, the isolated phage was incubated with bacteria at room temperature, followed by spreading the mixture on agar plates to recover. Phage-infected cells were screened by colony PCR using phage specific primers, and further characterized using molecular microbiologic and biochemical approaches.
Results: The bacteriophage S1249 demonstrated contractile tail structures under TEM, which were composed of inner rigid tubes and outer contractile sheaths, features that attributed to phages belonging to the family of Myoviridae. This phage mainly infected serotypes a, c and f. Interestingly, infection of this phage in different strains resulted in different phenotypes. Obvious lytic activity was observed in an S1249-infected serotype a strain, with 90% inhibition of bacterial growth when grown in broth with human serum. Concomitantly, an increase in the protein level of the bacterial pyruvate dehydrogenase complex (PDHc), including E1, E2, and E3 units, were also observed.
Conclusions: Our data suggest that the metabolic requirement of ATP is important for phages switching to lytic life cycle and the up-regulation of PDHc is critical during this process.
Methods: Phage S1249 was isolated using mitomycin C induction. Briefly, mid-logarithmic bacterial cell was exposed to mitomycin for 30 min for induction, washed thoroughly and recovered in the broth for 18h. Phages was harvested by low-speed centrifugation to remove the un-lysed cells, followed by high-speed centrifugation to harvest phages. The morphology of isolated phage was examined under transmission electron microscopy (TEM) and the isolated phage was used to infect strains across different serotypes to determine the infection specificity. Briefly, the isolated phage was incubated with bacteria at room temperature, followed by spreading the mixture on agar plates to recover. Phage-infected cells were screened by colony PCR using phage specific primers, and further characterized using molecular microbiologic and biochemical approaches.
Results: The bacteriophage S1249 demonstrated contractile tail structures under TEM, which were composed of inner rigid tubes and outer contractile sheaths, features that attributed to phages belonging to the family of Myoviridae. This phage mainly infected serotypes a, c and f. Interestingly, infection of this phage in different strains resulted in different phenotypes. Obvious lytic activity was observed in an S1249-infected serotype a strain, with 90% inhibition of bacterial growth when grown in broth with human serum. Concomitantly, an increase in the protein level of the bacterial pyruvate dehydrogenase complex (PDHc), including E1, E2, and E3 units, were also observed.
Conclusions: Our data suggest that the metabolic requirement of ATP is important for phages switching to lytic life cycle and the up-regulation of PDHc is critical during this process.