High epigenetic potential protects songbirds against pathogenic Salmonella enterica infection.

Animals encounter many novel and unpredictable challenges when moving into new areas, including pathogen exposure. Because effective immune defenses against such threats can be costly, plastic immune responses could be particularly advantageous, as such defenses can be engaged only when context warrants activation. DNA methylation is a key regulator of plasticity via its effects on gene expression. In vertebrates, DNA methylation occurs exclusively at CpG dinucleotides and, typically, high DNA methylation decreases gene expression, particularly when it occurs in promoters. The CpG content of gene regulatory regions may therefore represent one form of epigenetic potential (EP), a genomic means to enable gene expression and hence adaptive phenotypic plasticity. Non-native populations of house sparrows (Passer domesticus) - one of the world's most cosmopolitan species - have high EP in the promoter of a key microbial surveillance gene, Toll-like receptor 4 (TLR4), compared with native populations. We previously hypothesized that high EP may enable sparrows to balance the costs and benefits of inflammatory immune responses well, a trait critical to success in novel environments. In the present study, we found support for this hypothesis: house sparrows with high EP in the TLR4 promoter were better able to resist a pathogenic Salmonella enterica infection than sparrows with low EP. These results support the idea that high EP contributes to invasion and perhaps adaptation in novel environments, but the mechanistic details whereby these organismal effects arise remain obscure.

Timing and delivery route effects of cecal microbiome transplants on Salmonella Typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions.

BACKGROUND: Exposure to microbes early in life has long-lasting effects on microbial community structure and function of the microbiome. However, in commercial poultry settings chicks are reared as a single-age cohort with no exposure to adult birds which can have profound effects on microbiota development and subsequent pathogen challenge. Microbiota manipulation is a proven and promising strategy to help reduce pathogen load and transmission within broiler flocks. However, administration of microbiota transplant products in a hatchery setting may prove challenging. Effective administration strategies are dependent on key factors, such as; the age of chicks receiving interventions and mode of delivery. This study aimed to assess these two aspects to provide supporting evidence towards microbiome manipulation strategies for use in commercial hatcheries. RESULTS: Manipulation of the microbiota between 4 and 72 h of hatch markedly reduced faecal shedding and colonisation with the foodborne pathogen Salmonella enterica serovar Typhimurium (ST4/74). Administration of transplant material via spray or gel drop delivery systems had minimal effect on the protection conferred with fewer birds in transplant groups shown to shed ST4/74 in the faeces compared to PBS-gavaged control birds. Analysis of the microbiome following transplantation demonstrated that all transplant groups had higher diversity and species richness than non-transplant groups during the first week of life and the early stages of infection with ST47/4.The relative abundance of the bacterium Faecalibacterium prausnitzii was significantly higher in CMT groups compared to PBS controls. The presence of F. prausnitzii was also shown to increase in PBS-challenged birds compared to unchallenged birds potentially indicating a role of this bacterium in limiting Salmonella infections. CONCLUSIONS: This study demonstrated that administration of microbiome transplants, using methods that would align with hatchery practices, effectively reduced colonisation and shedding of Salmonella in chickens. Age of chicks at microbiome administration had limited effect on the diversity and composition of the microbiome and conferred protection against Salmonella infections. Traditional hatchery delivery systems, such as spray or gel-drop, are sufficient to transfer donor material, alter the microbiome and confer protection against Salmonella. This study helps highlight the opportunity for use of microbiome modification methods within the hatchery.

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BACKGROUND: Invasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. METHODOLOGY: Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment. FINDINGS: 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates. CONCLUSIONS: The finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.

Scalable all-fiber coherent beam combination using digital control.

This paper describes a filled-aperture coherent beam combining (CBC) system based on locking of optical coherence via single-detector electronic-frequency tagging (LOCSET). The sensing and control architecture is implemented using a field-programmable gate array and high-bandwidth electro-optic phase modulators. The all-fiber optical configuration consists of a narrow linewidth 1560 nm seed laser separated into three channels, each containing 7 W erbium-doped fiber amplifiers. The system was demonstrated experimentally, achieving a total stabilized output power of 20 W, a combination efficiency greater than 95%, and an output RMS phase stability of λ/493. As this architecture employs an entirely digital sensing and control scheme based on LOCSET, it presents a highly scalable and cost-effective solution for CBC that is wavelength agnostic and can support an arbitrarily large number of channels.

Complete Genome Sequences of African Salmonella enterica Serovar Enteritidis Clinical Isolates Associated with Bloodstream Infection.

We report the complete genome sequencing and annotation of four Salmonella enterica serovar Enteritidis isolates, two that are representative of the Central/Eastern African clade (CP255 and D7795) and two of the Global Epidemic clade (A1636 and P125109).

Campylobacter Contamination at Retail of Halal Chicken Produced in the United Kingdom.

ABSTRACT: Campylobacter is the leading cause of human bacterial diarrheal disease worldwide, and poultry meat products account for the majority of human cases. Based on recent surveys, the Food Standards Agency has estimated the Campylobacter prevalence in fresh retail chicken in the United Kingdom to be 41.2%. However, such surveys have not distinguished between broiler chickens produced for different consumer demographic groups, such as the Halal market. Campylobacter colonization of broilers is difficult to prevent, especially during routine partial depopulation of flocks. Broilers produced for the Halal market may undergo multiple depopulation events, which may increase the risk of Campylobacter colonization and subsequent contamination of chicken meat. This study was conducted to determine the prevalence and levels of Campylobacter contamination in chicken meat produced for the Halal market in the United Kingdom. Campylobacter was identified and enumerated from the neck skin and outer packaging of 405 Halal chickens. Culture isolates were assigned to species via PCR assays, and disk diffusion assays were used to determine antimicrobial susceptibility. Logistic regression analysis was used to assess risk factors for Campylobacter contamination, the level of Campylobacter contamination among positive carcasses, and antimicrobial resistance. Campylobacter spp. were confirmed in 65.4% of neck skin samples and 17.1% of packaging samples. Neck skin samples had the highest level of contamination; 13.8% of samples had >1,000 CFU/g. Large birds had a significantly higher number of samples with >1,000 CFU/g (P < 0.001). and as chicken carcass weight increased, birds were more likely to be Campylobacter positive (P < 0.05). A high prevalence of resistance was seen to ciprofloxacin (42.0% of samples), and 38.5% of samples contained at least one multidrug-resistant Campylobacter isolate. This study revealed that Halal chicken has a higher Campylobacter prevalence than does non-Halal chicken. Interventions should be introduced to reduce this public health risk.

Changes in the faecal microbiome of pied tamarins (Saguinus bicolor) associated with chronic, recurrent diarrhoea and weight loss.

BACKGROUND: Chronic recurrent diarrhoea and weight loss is a common problem in captive callitrichids. These symptoms are common clinical features of marmoset wasting syndrome (MWS), a chronic enteric inflammation of unknown aetiology associated with mortality in captive marmosets. The unknown aetiology of the condition presents problems for conservation projects where affected colonies present higher mortality and lower birth rates. Since a role for the microbiome has been established in chronic enteric inflammation of other species it is possible that the intestinal microbiome undergoes similar changes during MWS. RESULTS: The faecal microbiome of pied tamarins (Saguinus bicolor) at Jersey Zoo was determined using 16S rRNA gene amplicon sequencing to compare the composition of the faecal microbiome of tamarins affected by chronic recurrent diarrhoea and weight loss with unaffected individuals. Affected individuals had a higher relative abundance of amplicon sequence variants assigned to Lactobacillus and Helicobacter jaachi while unaffected individuals had a higher relative abundance of some Lachnospiraceae and Ruminococcaceae. CONCLUSIONS: Although Helicobacter has been shown to reside in healthy wild and captive marmosets and tamarins and appears to form part of the normal microbiota, the results of this study raise the prospect that certain species of Helicobacter may be associated with chronic, recurrent diarrhoea in captive callitrichids. The presence of Lactobacillus may also play a role in the development of MWS. Since depletion of Lachnospiraceae and Ruminococcaceae have been linked to chronic gastrointestinal inflammation in humans, this feature of the microbiome of affected tamarins provides another avenue of further research in the pathogenesis of MWS.

Integrating Genetic and Genomic Analyses of Combined Health Data Across Ecotypes to Improve Disease Resistance in Indigenous African Chickens.

Poultry play an important role in the agriculture of many African countries. The majority of chickens in sub-Saharan Africa are indigenous, raised in villages under semi-scavenging conditions. Vaccinations and biosecurity measures rarely apply, and infectious diseases remain a major cause of mortality and reduced productivity. Genomic selection for disease resistance offers a potentially sustainable solution but this requires sufficient numbers of individual birds with genomic and phenotypic data, which is often a challenge to collect in the small populations of indigenous chicken ecotypes. The use of information across-ecotypes presents an attractive possibility to increase the relevant numbers and the accuracy of genomic selection. In this study, we performed a joint analysis of two distinct Ethiopian indigenous chicken ecotypes to investigate the genomic architecture of important health and productivity traits and explore the feasibility of conducting genomic selection across-ecotype. Phenotypic traits considered were antibody response to Infectious Bursal Disease (IBDV), Marek's Disease (MDV), Fowl Cholera (PM) and Fowl Typhoid (SG), resistance to Eimeria and cestode parasitism, and productivity [body weight and body condition score (BCS)]. Combined data from the two chicken ecotypes, Horro (n = 384) and Jarso (n = 376), were jointly analyzed for genetic parameter estimation, genome-wide association studies (GWAS), genomic breeding value (GEBVs) calculation, genomic predictions, whole-genome sequencing (WGS), and pathways analyses. Estimates of across-ecotype heritability were significant and moderate in magnitude (0.22-0.47) for all traits except for SG and BCS. GWAS identified several significant genomic associations with health and productivity traits. The WGS analysis revealed putative candidate genes and mutations for IBDV (TOLLIP, ANGPTL5, BCL9, THEMIS2), MDV (GRM7), SG (MAP3K21), Eimeria (TOM1L1) and cestodes (TNFAIP1, ATG9A, NOS2) parasitism, which warrant further investigation. Reliability of GEBVs increased compared to within-ecotype calculations but accuracy of genomic prediction did not, probably because the genetic distance between the two ecotypes offset the benefit from increased sample size. However, for some traits genomic prediction was only feasible in across-ecotype analysis. Our results generally underpin the potential of genomic selection to enhance health and productivity across-ecotypes. Future studies should establish the required minimum sample size and genetic similarity between ecotypes to ensure accurate joint genomic selection.

Salmonella identified in pigs in Kenya and Malawi reveals the potential for zoonotic transmission in emerging pork markets.

Salmonella is a major cause of foodborne disease globally. Pigs can carry and shed non-typhoidal Salmonella (NTS) asymptomatically, representing a significant reservoir for these pathogens. To investigate Salmonella carriage by African domestic pigs, faecal and mesenteric lymph node samples were taken at slaughter in Nairobi, Busia (Kenya) and Chikwawa (Malawi) between October 2016 and May 2017. Selective culture, antisera testing and whole genome sequencing were performed on samples from 647 pigs; the prevalence of NTS carriage was 12.7% in Busia, 9.1% in Nairobi and 24.6% in Chikwawa. Two isolates of S. Typhimurium ST313 were isolated, but were more closely related to ST313 isolates associated with gastroenteritis in the UK than bloodstream infection in Africa. The discovery of porcine NTS carriage in Kenya and Malawi reveals potential for zoonotic transmission of diarrhoeal strains to humans in these countries, but not for transmission of clades specifically associated with invasive NTS disease in Africa.

Development of the Ileal Microbiota in Three Broiler Breeds.

The development and succession of the microbiota in ileal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 3 and 42 days post hatch (d.p.h). Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 d.p.h, chicks were kept in three circular brooder pens ensuring a mixture of breeds in each brooder. From 22 d.p.h, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of ileal mucus and luminal contents taken from five birds of each breed at 3, 7, 14, 21, 28, and 42 d.p.h. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The initial microbiota in the ileum varied between breeds. The common features were a low diversity and general dominance by one or two taxa such as Enterococcus or Escherichia with relatively low numbers of Lactobacillus. Escherichia became the most abundant genus in samples where Enterococcus was previously the dominant taxa. The next phase of development was marked by an increase in the abundance of Candidatus Arthromitus in the mucus and Lactobacillus in the lumen. The high abundance of Candidatus Arthromitus persisted between 7 and 14 d.p.h after which Lactobacillus became the most abundant genus in both the mucus and lumen. Dominance of the ileal microbiota by Lactobacillus was a transient feature. By 42 d.p.h, the relative abundance of Lactobacillus had fallen while a range of other taxa including Escherichia, Turicibacter, and members of Clostridiales increased. This general pattern was followed by all breeds, however, the rate at which succession occurred differed as Ross matured quicker than Cobb with Hubbard as an intermediate.

Topical Application of Adult Cecal Contents to Eggs Transplants Spore-Forming Microbiota but Not Other Members of the Microbiota to Chicks.

The intestinal microbiota plays an essential role in the metabolism and immune competence of chickens from the first day after hatching. In modern production systems, chicks are isolated from adult chickens, instead hatching in a clean environment. As a result, chicks are colonized by environmental bacteria, including potential pathogens. There is a need to investigate methods by which chicks can be exposed to a more appropriate microbial community at hatching. Such methods must be easy to apply in a hatchery and produce consistent results. The development of the intestinal microbiota of chicks hatched from eggs sprayed with dilute adult cecal content during incubation was observed at 0, 3, 7, and 14 days posthatching (dph) across two experiments. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. A topical treatment of dilute adult cecal content was sufficient to transplant spore-forming bacteria such as Lachnospiraceae and Ruminococcaceae However, this treatment was not able to transplant other taxa that are considered to be core elements of the chicken cecal microbiota, such as Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, and Burkholderiaceae The topical treatment significantly altered the microbiota of chicks immediately posthatching and accelerated the normal development of the microbiota with earlier colonization by Ruminococcaceae in the cecum and "Candidatus Arthromitus" in the ileum. The effect of the treatment on the cecal microbiota was maximal at 3 dph but diminished over time.IMPORTANCE Over the last 60 years poultry production has intensified in response to increased demand for meat. In modern systems, chicks hatch without contacting chickens and their gut bacteria. Consequently, they are colonized by environmental bacteria that may cause disease. The normal bacteria that live in the gut, or intestinal microbiota, play an important role in the development of the immune system. Therefore, it is essential to find easy ways to expose chicks to the more appropriate bacteria at hatching. This experiment investigated whether spraying eggs with adult cecal contents was sufficient to transfer an adult microbiota to chicks. Our findings show that spore-forming bacteria were transplanted, but other members of the microbiota were not. In this respect, the spray application was partially successful, but the timing of the spray needs to be modified to ensure that more bacteria are transferred.

Development of the Caecal Microbiota in Three Broiler Breeds.

The development of the caecal microbiota plays a role in the metabolism and immune competence of chickens. A detailed understanding of normal succession in the caecal microbiota can inform the use of probiotics and other interventions to optimize the caecal microbiota. The development of the microbiota in caecal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 0 and 42 days post hatch. Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 days post hatch, chicks were kept in brooder pens ensuring a mixture of breeds in each brooder. From 22 days post hatch, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of caecal mucus and luminal contents from five birds of each breed at 0, 3, 7, 14, 21, 28, and 42 days post hatch. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The early caecal microbiota was characterized by poor diversity and dominance by one or two bacterial species. Early colonizers of the caecum included Bifidobacteriaceae, Lachnospiraceae, Bacteroidaceae and Burkholderiaceae with some amplicon sequence variants (ASVs) assigned to Ruminococcaceae. Later colonizers of the caecal microbiota were most apparent from 14 d.p.h and included Ruminococcaceae, Clostridiales vadin BB60 group, Christensenellaceae and Bacillaceae. The caecal microbiota continued to change until 42 d.p.h when the microbiota was characterized by a high abundance of Bacteroidaceae, Lachnospiraceae and Ruminococcaceae. The lumen microbiota was significantly different to the mucus with some ASVs assigned to Lachnospiraceae, Ruminococcaceae, Christensenellaceae and Bacillaceae showing increased abundance in the mucus. ASVs assigned to Bacteroidaceae, Lactobacillaceae and Burkholderiaceae showed a preference for the lumen. Analysis of five caecal mucus samples from each breed at 42 days post hatch showed differences in microbiota composition between Ross and Cobb as well as between Ross and Hubbard. Since performance data was not collected no functional inferences as to the significance of this finding can be made.

The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.

Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans.

The Genomic Architecture of Fowl Typhoid Resistance in Commercial Layers.

Salmonella enterica serovar Gallinarum causes devastating outbreaks of fowl typhoid across the globe, especially in developing countries. With the use of antimicrobial agents being reduced due to legislation and the absence of licensed vaccines in some parts of the world, an attractive complementary control strategy is to breed chickens for increased resistance to Salmonella. The potential for genetic control of salmonellosis has been demonstrated by experimental challenge of inbred populations. Quantitative trait loci (QTL) associated with resistance have been identified in many genomic regions. A major QTL associated with systemic salmonellosis has been identified in a region termed SAL1. In the present study, two outbreaks of fowl typhoid in 2007 and 2012 in the United Kingdom were used to investigate the genetic architecture of Salmonella resistance in commercial laying hens. In the first outbreak 100 resistant and 150 susceptible layers were genotyped using 11 single nucleotide polymorphism (SNP) and 3 microsatellite markers located in the previously identified SAL1 region on chromosome 5. From the second outbreak 100 resistant and 200 susceptible layers, belonging to a different line, were genotyped with a high-density (600 K) genome-wide SNP array. Substantial heritability estimates were obtained in both populations (h 2 = 0.22 and 0.26, for the layers in the first and second outbreak, respectively). Significant associations with three markers on chromosome 5 located close to AKT1 and SIVA1 genes, coding for RAC-alpha serine/threonine protein kinase, and the CD27-binding protein SIVA1, respectively, were identified in the first outbreak. From analysis of the second outbreak, eight genome-wide significant associations with Salmonella resistance were identified on chromosomes 1, 6, 7, 11, 23, 24, 26, 28 and several others with suggestive genome-wide significance were found. Pathway and network analysis revealed the presence of many innate immune pathways related to Salmonella resistance. Although, significant associations with SNPs located in the SAL1 locus were not identified by the genome-wide scan for layers from the second outbreak, pathway analysis revealed P13K/AKT signaling as the most significant pathway. In summary, resistance to fowl typhoid is a heritable polygenic trait that could possibly be enhanced through selective breeding.

The role of local adaptation in sustainable village chicken production.

Village chickens are ubiquitous in smallholder farming systems, contributing to household, local and national economies under diverse environmental, economic and cultural settings. However, they are raised in challenging environments where productivity is low while mortality is high. There is much interest in utilizing indigenous genetic resources to produce a chicken resilient to its environment, whilst providing the basis of an economically sustainable enterprise. Globally, however, a wide variety of interventions have so far proved unable to deliver sustainable improvements. Here, we show that regional differences in trait preferences and parasite burden are associated with distinct chicken genepools, likely in response to interacting natural and human-driven (economic and social) selection pressures. Drivers of regional differences include marketing opportunities, cultural preferences, agro-ecologies and parasite populations, and are evident in system adaptations, such as management practices, population dynamics and bird genotypes. Our results provide sound multidisciplinary evidence to support previous observations that sustainable poultry development interventions for smallholder farmers, including breeding programs, should be locally tailored and designed for flexible implementation.

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella.

Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.

Campylobacter jejuni transcriptome changes during loss of culturability in water.

BACKGROUND: Water serves as a potential reservoir for Campylobacter, the leading cause of bacterial gastroenteritis in humans. However, little is understood about the mechanisms underlying variations in survival characteristics between different strains of C. jejuni in natural environments, including water. RESULTS: We identified three Campylobacter jejuni strains that exhibited variability in their ability to retain culturability after suspension in tap water at two different temperatures (4°C and 25°C). Of the three, strains C. jejuni M1 exhibited the most rapid loss of culturability whilst retaining viability. Using RNAseq transcriptomics, we characterised C. jejuni M1 gene expression in response to suspension in water by analyzing bacterial suspensions recovered immediately after introduction into water (Time 0), and from two sampling time/temperature combinations where considerable loss of culturability was evident, namely (i) after 24 h at 25°C, and (ii) after 72 h at 4°C. Transcript data were compared with a culture-grown control. Some gene expression characteristics were shared amongst the three populations recovered from water, with more genes being up-regulated than down. Many of the up-regulated genes were identified in the Time 0 sample, whereas the majority of down-regulated genes occurred in the 25°C (24 h) sample. CONCLUSIONS: Variations in expression were found amongst genes associated with oxygen tolerance, starvation and osmotic stress. However, we also found upregulation of flagellar assembly genes, accompanied by down-regulation of genes involved in chemotaxis. Our data also suggested a switch from secretion via the sec system to via the tat system, and that the quorum sensing gene luxS may be implicated in the survival of strain M1 in water. Variations in gene expression also occurred in accessory genome regions. Our data suggest that despite the loss of culturability, C. jejuni M1 remains viable and adapts via specific changes in gene expression.

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.

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.