Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host–pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host–pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. Global patterns and mechanistic determinants of bacterial spread in mammalian organisms are difficult to obtain through numerical and topographical mapping of a single bacterial population. Appreciation of the true pathogenetic events during infections needs to be based on the understanding of the fine interactions that control the infection dynamics of individual subpopulations in the same host. We have used molecular techniques to tag individually otherwise identical subpopulations of bacteria. We have used these bacteria, called wild-type isogenic tagged strains (WITS), in simultaneous infections in the same animal to gather insights into the patterns of spread of individual subpopulations of bacteria in the tissues and interactions between bacteria and phagocytes. Combining numerical fluctuation in the WITS populations with mathematical modelling and statistical analysis, we have gathered data on the relative occurrence of bacterial growth and death in different phases of the disease process. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs. The work illustrates the importance of unravelling heterogeneous traits of infections to reconstruct and understand the true nature of the global disease process.

This study characterizes the interaction between Campylobacter jejuni and the 16 phages used in the United Kingdom typing scheme by screening spontaneous mutants of the phage-type strains and transposon mutants of the sequenced strain NCTC 11168. We show that the 16 typing phages fall into four groups based on their patterns of activity against spontaneous mutants. Screens of transposon and defined mutants indicate that the phage-bacterium interaction for one of these groups appears to involve the capsular polysaccharide (CPS), while two of the other three groups consist of flagellatropic phages. The expression of CPS and flagella is potentially phase variable in C. jejuni , and the implications of these findings for typing and intervention strategies are discussed.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics. High quality science has been published concerning the dynamics of infectious disease spread through communities of people or animals, but less work has been done to understand infectious disease dynamics within the host. Many conclusions about how infectious agents work are based on experiments in isolated monocultures of cells or in somewhat crude experiments in whole animals. Understanding this complex process in whole animals is the next major challenge for infectious disease biologists, and is required if intervention strategies to prevent and cure infectious diseases are to be improved and targeted effectively. Bacteria of the species Salmonella enterica are a threat to public health, causing a wide range of life-threatening diseases in humans and animals world-wide. In vitro cell experiments and inference from measuring net growth kinetics in mouse organs suggest that intracellular replication of S. enterica requires proteins delivered by the Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system (T3SS) and that mutants in SPI-2 cannot replicate efficiently intracellularly. However, by observing directly infection dynamics at the single-cell level in vivo, we show that SPI-2 T3SS mutants can replicate to high intracellular densities in phagocytes in the organs of infected animals, but appear unable to leave infected cells.

We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

Campylobacter jejuniis the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes ofC. jejuni(NCTC11168) and itsrpoNmutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent onrpoNfor expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for theCampylobacterresearch community.