Preventing and combating Campylobacter infections: On track towards a One Health approach

C. jejuni exhibits outstanding genetic diversity primarily due to frequent natural transformation. Therefore, in this project, the mechanism of natural transformation was investigated. Specifically, a single-cell-based assay was established in which the DNA uptake of Campylobacter was visualized under the fluorescence microscope. Using this assay, parameters were examined for their stimulatory or inhibitory effects in competence development, i.e., capacity for natural transformation. Slightly acidic pH and oxygen stress inhibited competence development, which occurred mainly in motile and growing C. jejuni. Furthermore, the DNA uptake mechanism was further characterized by producing mutants with defects in individual proteins. This identified the function of known homologous proteins, but also a new protein with function in transporting DNA across the outer membrane. Neither of these genes played a role in biofilm formation, although extracellular DNA is important as a binding partner for biofilm initialization. The influence of natural transformation in the expression of gene variants was evident in C. coli field isolates that had substantial sequence insertion of C. jejuni. These isolates were no longer diagnosable as C. coli by conventional routine PCR. Effects on multi-locus sequence typing were also detected. The data and selected strains have been incorporated for validation of the molecular annex of ISO 10272 and demonstrate that monitoring of genome diversity is essential for continued food safety. Thermotolerant C. spp. can, besides adaptation by genetic adaptation, undergo a viable but non culturable status (VBNC) under stress conditions. Therefore, in cooperation with Freie Universität, the chicken model was used to investigate the colonization ability of these no longer cultivable Campylobacter, which could be visualized by an established live/dead discriminating real-time PCR.

To better understand the process of natural transformation, i.e. the uptake of free external DNA and the integration into the genome, and to be able to propose more targeted reduction strategies in the future, an alternative to the classical transformation approach in Campylobacter jejuni was established in funding phase I. The approach is based on DNA containing a resistance marker. The classical approach is based on transformation with DNA containing a resistance marker. The transformation rate is determined by selecting the transformed cells. This approach is time-consuming and labor-intensive, and due to the selection process of the transformants, other factors play a role that may bias the direct result. For these reasons, a single-cell-based assay was established to visualize DNA uptake using fluorescently labeled DNA and analysis by fluorescence microscopy. By using this assay, stimulatory or inhibitory effects of different parameters on competence development and DNA uptake could be tested. This showed that competence development, i.e. the ability to take up external DNA of other Campylobacter, was strongly pH-dependent. A slightly acidic pH led to complete inhibition of DNA uptake in the single cell assay as well as to a drastic reduction in transformability by a factor of 10,000 compared to the optimal conditions tested. These results are in agreement with the results obtained in IP5, where organic acids were suggested as reducing agents.To better understand the process of DNA uptake and to propose more targeted reduction strategies in the future, mutants with potential defects in natural transformation were produced and tested for their competence development using the single cell-based DNA uptake assay established in this project. This involved inactivating genes using antibiotic insertion cassettes that play a potential role in the direct transport of the DNA macromolecule across both membranes of the Gram-negative bacterium. Mutants were successfully produced that did not exhibit uptake of labeled DNA across the outer membrane into the periplasm of C. jejuni (pilQ) and were therefore not transformable with a classical selection marker. As expected, a mutant that had an inactivated gene for the structural unit of the inner membrane transporter (comEC) did not show reduced DNA uptake into the periplasm, but further transport of DNA was not possible and therefore no transforming activity was evident. The findings are valuable since they increase the understanding of Campylobacter spp. genetic exchange and highlight potential steps for inhibition of natural transformation that may improve its sustainability in the future when combined with the application of reduction strategies. These results were published in the journal "Biomolecules" (Golz et al 2023). This paper also investigated a potential pilus protein that plays a major role in the natural transformation of C. jejuni. Deletion of this gene (cj0683) resulted in significantly reduced DNA uptake and transformation ability, which was only slightly above the detection limit. Using the established single cell assay, in which DNA uptake is visualized by fluorescently labeled DNA, DNA uptake events were very rarely detected in the mutant. However, the amount of DNA taken up in these rare events, as measured by the fluorescence intensity of DNA foci in the bacteria, was not substantially different from wild type. This indicated that the DNA uptake complexes transported infrequently but then fully functioned. Therefore, the newly-identified protein Cj0683, which is unique to Campylobacter, may be a competence pilus protein involved in the initialization of DNA uptake. The protein could thus represent a good target for the development of strategies to reduce the adaptive potential of Campylobacter.Already in funding phase I, a screening revealed Campylobacter isolates for which routine species differentiation by quantitative real-time PCR was no longer possible (Golz et al.short foret alii (lat. "and others"), 2020). These isolates were analyzed by whole-genome sequencing. This revealed that they were Campylobacter coli strains that had received substantial sequence introgression from C. jejuni. Upon closer analysis, it was apparent that the strains shared a common set of genes, with C. jejuni sequence introgression. Interestingly, the gene products had functionality in stress response. Among them were functions in oxidative, osmotic, and general stress response, but also in chromosome stability and repair, membrane transport, cell wall and capsule biosynthesis, and chemotaxis. In funding phase II, additional C. coli/C. jejuni hybrids were identified from the current submissions and studied in more detail by sequencing followed by K-mer analysis. The similar set of genes with C. jejuni sequence introgression was also observed in these strains. The results from the overall project suggest that the C. jejuni sequence insertion may be an adaptation to harsh environmental conditions. Experimental approaches to demonstrate increased stress tolerance of these strains relative to C. coli or C. jejuni have not yet yielded reproducible results, so a possible fitness advantage of these hybrid strains remains unclear. Nevertheless, this shows that natural transformation can lead to immense transfer of genetic material between different species. This has an impact on routinely applied fine typing and may lead to greater survivability and thus persistence of Campylobacter. For this reason, it is essential to look at genetic exchange when searching for reduction strategies for Campylobacter, so that the measures are effective in longer term. This is the only way to achieve a stable and sustainable reduction of germs on carcasses.The NRL for Campylobacter is a member of CEN/TC463/WG3. The issue of C. coli/C. jejuni hybrid strains, which can lead to ambiguous species differentiation, was taken into account in the validation of the molecular annex of ISO 10272-1/2 AMD 2023 in order to be able to correctly detect the mutable pathogen within the ÖGD but also in other laboratories in the future. In addition, an animal experiment to determine the infectivity of Campylobacter in "viable but non culturable" (VBNC) status was conducted in funding phase II. At this stage, Campylobacter are viable and thus potentially infectious, but can no longer be cultured using current methods. They are presumably survival forms for surviving unfavorable environmental conditions. In our experiments, we observed two VBNC states. VBNC-1 forms cannot be grown under standard ISO 10272-1:2017 conditions, but exhibit a fully intact cell envelope according to live qPCR. Evidence for viability of these VBNC-1 was obtained by adjusting the incubation atmosphere outside the ISO standard to very low oxygen concentrations with the addition of hydrogen and extending the incubation time by 2 days (Wulsten et al.short foret alii (lat. "and others") 2020). As time progresses, VBNC-1 change to the VBNC-2 state, where even the special atmosphere cannot restore growth in the laboratory. These VBNC-2 cells were produced under controlled conditions in funding phase II and subsequently applied to groups of 10 chickens each in the crop. In parallel, exponentially growing C. jejuni actively dividing in vitro were tested in a chicken model to identify the minimal infective dose. The results showed a higher minimum infective dose of exponentially dividing bacteria than currently published. It underpins the need for improved, more robust diagnostics of C. spp. This could be realized, for example, by using live qPCR, which provides better results than the microbiological gold standard, at least for fresh samples (Stingl et al.short foret alii (lat. "and others") 2021). The VBNC-2 forms could not lead to colonization of the chicken in the doses presented. Here, further experiments are important to also investigate the VBNC-1 forms, which are also not detected in routine laboratories, for colonization ability. In addition, it is conceivable that the form and duration of administration would also have to be designed more naturally in future experiments, e.g. by application via drinking water, so that the risk of transmission of C. jejuni via VBNC forms can be realistically assessed.