Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

Abstract

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.

Keywords: Campylobacter; Campylobacter jejuni; bacteriophages; carrier state life cycle; phage therapy.

Figure 1.

PFGE and Southern blot of genomic DNA from C. jejuni CSLC cultures. PFGE and Southern blot analysis was carried out to establish that the associated phage genomes were not integrated into the host chromosome. (a) PFGE of C. jejuni PT14 and HPC5 carrier strains recovered from biofilms treated with either CP8 or CP30A bacteriophages together with controls not treated with bacteriophages. U, undigested genomic DNA (bacterial genomic DNA remained in the well under these electrophoretic conditions); SmaI, genomic DNA digested with SmaI restriction enzyme. DNA bands of approximately 140 kb (indicated by arrow) occur in the undigested preparation and remain within the SmaI digested DNA fragments; these were identified in the DNA preparations of C. jejuni CSLC strains but not their non-phage-treated progenitors. (b) Southern blot of PFGE above. The Southern blot was hybridized with a CP8 DNA probe, which cross-hybridizes with CP30A DNA, and clearly shows the 140 kb fragments to be phage DNA. Size markers indicate the positions of concatenated lambda DNAs loaded on the PFGE.

Figure 2.

TEM images of CSLC stained with uranyl acetate. (a) Typical C. jejuni HPC5 bacterium showing intact flagella at each end. (b–e) CSLC cultures from BA plates showing bacteriophage particles in association with the surface of Campylobacter cells but with no evidence of infection structures or host lysis. It is also notable that the bacteria have no flagella.

Figure 3.

Growth curves of CSLC isolates and phages incubated under microaerobic conditions with their corresponding motility, phage resistance and carrier state phenotype frequencies. Growth curves were performed to investigate how the CSLC campylobacters behave in the presence of persistent phage infection compared with their parental strains. The phenotypic frequencies for each time point are indicated below the corresponding growth curves, where single colony isolates were scored independently for normal motility, phage resistance and carrier state. (a) Growth curves of C. jejuni PT14, PT14CP8CS, PT14CP30ACS with phage titres of CP8CS and CP30ACS; (b) frequencies of PT14CP8CS culture phenotypes; (c) frequencies of PT14CP30ACS culture phenotypes; (d) growth curves of C. jejuni HPC5, HPC5CP8CS, HPC5CP30ACS with phage titres of CP8CS and CP30ACS; (e) frequencies of HPC5CP8CS culture phenotypes; (f) frequencies of HPC5CP30ACS culture phenotypes. For a full definition of the phenotypes scored, see §3.9.

Figure 4.

Survival of C. jejuni parental strains and CSLC cultures in normal atmospheric oxygen conditions. Cultures were incubated at 42°C under nutrient limitation in normal atmospheric oxygen with shaking. The sampling frequency was optimized according to viability. Change in viability was calculated as the percentage of the original culture that remained at each time point. (a) Campylobacter jejuni PT14, PT14CP8CS, PT14CP30ACS. (b) Campylobacter jejuni HPC5, HPC5CP8CS, HPC5CP30ACS.

Figure 5.

Schematic diagram of how carrier state differs from normal lytic infection in campylobacters. Models accounting for the maintenance of CSLC in Campylobacter envisage an equilibrium between insensitive cells that are able to survive phage attack and divide to produce sensitive host cells that support the replication of bacteriophages.