Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria. Results In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units. Conclusion The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results

Genome‑wide insights into population structure and host specifcity of Campylobacter jejuni

The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.Peer Reviewe

Comparison of different technologies for the decipherment of the whole genome sequence of Campylobacter jejuni BfR-CA-14430

Background Campylobacter jejuni is a zoonotic pathogen that infects the human gut through the food chain mainly by consumption of undercooked chicken meat, raw chicken cross-contaminated ready-to-eat food or by raw milk. In the last decades, C. jejuni has increasingly become the most common bacterial cause for food-born infections in high income countries, costing public health systems billions of euros each year. Currently, different whole genome sequencing techniques such as short-read bridge amplification and long-read single molecule real-time sequencing techniques are applied for in-depth analysis of bacterial species, in particular, Illumina MiSeq, PacBio and MinION. Results In this study, we analyzed a recently isolated C. jejuni strain from chicken meat by short- and long-read data from Illumina, PacBio and MinION sequencing technologies. For comparability, this strain is used in the German PAC-CAMPY research consortium in several studies, including phenotypic analysis of biofilm formation, natural transformation and in vivo colonization models. The complete assembled genome sequence most likely consists of a chromosome of 1,645,980 bp covering 1665 coding sequences as well as a plasmid sequence with 41,772 bp that encodes for 46 genes. Multilocus sequence typing revealed that the strain belongs to the clonal complex CC-21 (ST-44) which is known to be involved in C. jejuni human infections, including outbreaks. Furthermore, we discovered resistance determinants and a point mutation in the DNA gyrase (gyrA) that render the bacterium resistant against ampicillin, tetracycline and (fluoro-)quinolones. Conclusion The comparison of Illumina MiSeq, PacBio and MinION sequencing and analyses with different assembly tools enabled us to reconstruct a complete chromosome as well as a circular plasmid sequence of the C. jejuni strain BfR-CA-14430. Illumina short-read sequencing in combination with either PacBio or MinION can substantially improve the quality of the complete chromosome and epichromosomal elements on the level of mismatches and insertions/deletions, depending on the assembly program used.Peer Reviewe

Whole genome sequencing reveals extended natural transformation in Campylobacter impacting diagnostics and the pathogens adaptive potential

Campylobacter is the major bacterial agent of human gastroenteritis worldwide and represents a crucial global public health burden. Species differentiation of C. jejuni and C. coli and phylogenetic analysis is challenged by inter-species horizontal gene transfer. Routine real-time PCR on more than 4000 C. jejuni and C. coli field strains identified isolates with ambiguous PCR results for species differentiation, in particular, from the isolation source eggs. K-mer analysis of whole genome sequencing data indicated the presence of C. coli hybrid strains with huge amounts of C. jejuni introgression. Recombination events were distributed over the whole chromosome. MLST typing was impaired, since C. jejuni sequences were also found in six of the seven housekeeping genes. cgMLST suggested that the strains were phylogenetically unrelated. Intriguingly, the strains shared a stress response set of C. jejuni variant genes, with proposed roles in oxidative, osmotic and general stress defence, chromosome maintenance and repair, membrane transport, cell wall and capsular biosynthesis and chemotaxis. The results have practical impact on routine typing and on the understanding of the functional adaption to harsh environments, enabling successful spreading and persistence of Campylobacter.Peer Reviewe

Phenotype of an oxygen-sensitive mutant <i>sodB</i> lacking superoxide dismutase.

<p><i>H</i>. <i>pylori</i> were grown in BB-FBS at reduced oxygen atmosphere (1% O₂, 10% CO₂, 89% N₂) in the absence and presence of 0.5 mM adenine. A and B, overlay images of DIC/Cy3 of <i>sodB</i> after 10 min of DNA uptake with DNA stained in yellow; arrowheads indicate coccoid formation of the <i>sodB</i> mutant in the absence of adenine (A), while cells kept their rod-shaped morphology in the presence of adenine (B). C, log CFU/ml after 18–24 h of growth of <i>sodB</i>, the wild type and the <i>sodB</i>compl. D, competence development occurred with lower OD₆₀₀ values in the <i>sodB</i> mutant compared to the wild type; the <i>sodB</i>compl showed an intermediate phenotype. Addition of adenine reduced competence development. Data in C and D stem from at least three independent experiments; datapoints of the respective strain/condition were highlighted in D for better visualization.</p

Competence development during microaerobic growth.

<p>Competence development was monitored by the fraction of cells with active outer membrane DNA uptake at distinct growth phases under microaerobic atmosphere at 37°C (n = 16; large graph, circles, with left y-axis, sigmoidal curve fit using Sigma Plot 11.0). pH was monitored during growth (large graph, triangles). <i>H</i>. <i>pylori</i> N6 was grown overnight in BB-FBS to an OD₆₀₀ of 0.19 ± 0.05 (t0) at which only a minor fraction of cells displayed competence (2.2 ± 2.86%). The onset of competence development was defined at t1 at which cells exhibited a mean OD₆₀₀ of 0.39 ± 0.05 and 4 ± 2.8% of competent cells (~ t0 + 3–4 hours). At t2 (~ t0 + 6–8 hours) upon switch into competent state (50.6 ± 15.6% of cells with active outer membrane DNA uptake) cells exhibited a mean OD₆₀₀ of 0.7 ± 0.11. At t0, either effectors (0.5 mM adenine or 0.5 mM glutamine) were added or the medium was exchanged by fresh BB-FBS medium with our without supplementation of 0.125 μg/ml ciprofloxacin or 0.025 μg/ml mitomycin C or temperature was decreased for 5°C or the cell suspension was exposed to aerobic conditions. Differences in fraction of competent cells at t1 or t2 due to change in incubation conditions are shown in the inserted diagram (at least three experiments for each condition; error bars, standard deviation). For aerobic stress conditions data are depicted from time point 1h after t0.</p

Neutral pH opens the opportunity for competence development triggered by oxidative stress.

<p><i>H</i>. <i>pylori</i> N6 was grown microaerobically in BB-FBS overnight until growth phase before competence development (OD₆₀₀~0.2). Cells were exposed to different pH and atmospheric conditions for 1 hour. Data stem from at least three experiments; error bars, standard deviation.</p

Kinetics of competence development under aerobic conditions.

<p><i>H</i>. <i>pylori</i> N6 was grown microaerobically in BB-FBS overnight at t0 before competence development (OD₆₀₀~0.2). Cells were exposed to aerobic conditions at 37°C for 120 min. At the indicated time points the competence fraction of the cells was monitored. Control cells were kept under microaerobic atmosphere. Upper panel, DIC/Cy3 images of bacteria at indicated timepoints, with DNA stained in yellow; lower panel (left), fraction of competent cells; lower panel (right), number of distinct DNA foci per competent cell. Data stem from at least three experiments; error bars, standard deviation.</p

DNA uptake complexes are reversibly shut down at acidic pH values.

<p>Competent bacteria were incubated in BB-FBS that had been titrated with HCl or NaOH to the indicated pH values. Uptake of Cy3-labelled λ DNA occurred for 10 min under aerobic conditions. Cells were washed once in the respective pH medium before DNase treatment for 5 min in TSB-FBS at pH 7.5. The fraction of competent cells relative to the control condition incubated in TSB-FBS at pH 7.5 are depicted. Values stem from at least three experiments; error bars, standard deviation.</p

Transformation rate in <i>H</i>. <i>pylori</i> correlated with the fraction of cells with active outer membrane DNA transport.

<p>Cell suspensions were derived from the experiments illustrated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005626#ppat.1005626.g004" target="_blank">Fig 4</a>. Bacteria with different competent fractions of cells were taken during or after competence development; non-competent cells were taken before competence development. <i>H</i>. <i>pylori</i> N6 were incubated in parallel with either Cy3-labelled λ DNA or a PCR fragment of <i>rpsL</i>(A128G), conferring streptomycin resistance. The data show the principle suitability of measuring the fraction of cells with active outer membrane DNA transport for the evaluation of competence development. Cumulative data from 13 independent experiments (n = 42).</p