In this study we explored phenotypic resistance using traditional Kirby-Bauer methods in human and animal derived Campylobacter isolates that were concurrently resistant to both azithromycin and ciprofloxacin to an expanded panel of antimicrobials, including clindamycin, fosfomycin, ampicillin sulbactam and tigecycline. Out of 236 Campylobacter isolates, over 85% of C. jejuni and C. coli were resistant to clindamycin, over 60% were resistant to ampicillin sulbactam and over 30% to fosfomycin. Less than 2% of isolates were resistant to tigecycline and there was no observed resistance to Imipenem.

INTRODUCTION:Enteric bacterial pathogens are a major cause of diarrhoeal disease in low-income and middle-income countries, with complex transmission pathways involving human, animal and environmental reservoirs. Conventional epidemiological and microbiological approaches provide important insights into pathogen burden and distribution but lack the resolution needed to characterise fine-scale diversity, antimicrobial resistance (AMR) and transmission dynamics. Whole-genome sequencing offers high-resolution tools to investigate these processes within a One Health framework. METHODS AND ANALYSIS:The Genomic Epidemiology and Transmission of Campylobacter in Africa (GETCampy-Africa) study uses a multicountry, One Health design to investigate pathogen diversity, source attribution and transmission pathways. The study uses a case-control framework, recruiting children with medically attended diarrhoea and asymptomatic community controls across sites in Burkina Faso, Ghana and The Gambia. Samples were collected from human participants, domestic animals and environmental sources following standardised protocols. Participant enrolment and sample collection have been completed, while laboratory processing, sequencing and genomic analyses are ongoing. Genomic data are analysed to assess population structure, AMR profiles and probabilistic attribution of isolates to potential reservoirs using comparative genomics and machine learning approaches. ETHICS AND DISSEMINATION:Ethical approval was obtained from relevant national and institutional committees in all participating countries. Written informed consent was obtained from participants or their guardians prior to enrolment. Findings will be disseminated through peer-reviewed publications, stakeholder engagement activities and open-access platforms to support public health interventions and policy development.

Background Shigella causes severe diarrheal disease, and S. flexneri and S. sonnei are the targets for multivalent vaccine development. Culture-based agglutination has been the gold standard for serotyping, but it is limited by logistics, subjectivity, and the availability of antisera for emerging serotypes. Newer methods, including a real-time PCR-based approach and whole-genome sequencing offer alternatives, but their performance in Shigella endemic populations are not well documented. Methods Shigella isolates obtained from the Enterics for Global Health (EFGH) study in Iquitos, Peru were simultaneously serotyped using four methods: culture-based agglutination, isolate-based real-time PCR serotyping, stool-based real-time PCR serotyping and WGS using the in-silico tool ShigaPass. The definitive adjudicated serotype was established by an expert analysis of the WGS data, involving the mapping of sequence reads to known O-antigen biosynthesis and modification genes to identify key mutations. Results Results from all four serotyping methods were available for 107/114 isolates. Accuracy for vaccine subtypes S. flexneri 1b, 2a, 3a, 6, and S. sonnei , ranged from 93.3-100% for all methods. Complete concordance between methods was noted in 83/107 isolates, while 24/107 (22.4%) exhibited at least one discrepancy. Most discrepancies derived from S. flexneri serotypes Y, Yv and 1a. Agglutination misclassified eight Y/Yv isolates as 4a, and six isolates correctly classified as 1a by agglutination were classified as 1b by the other methods, a discrepancy associated with a nonsense mutation in the oac gene. Conclusion All four serotyping methods achieved acceptable accuracy for Shigella vaccine efficacy evaluation. Although discrepancies are infrequent, WGS provides information of their genomic basis.

Abstract Background Staphylococcus aureus is a major public health concern and is classified as a priority pathogen by the World Health Organization (WHO) with the global rise of methicillin-resistant S. aureus (MRSA) infections. Community-associated MRSA (CA-MRSA) strains have become increasingly important in both community and healthcare settings. This study aimed to investigate the genomic diversity, evolution, resistome, and virulome of CA-MRSA isolates circulating in Egypt to better understand their persistence, adaptation, and public health implications. Methods A total of 123 CA-MRSA isolates were collected from clinical settings in Alexandria, Egypt. Methicillin resistance was first determined phenotypically using cefoxitin resistance, followed by genotypic confirmation through detection of the mecA gene.Whole-genome sequencing and comparative genomic analyses were performed to characterize sequence types, clonal complexes, SCCmec elements, resistance determinants, and virulence factors. Phylogenetic relationships were reconstructed to assess evolutionary divergence, and network analysis was used to explore associations between resistance and virulence gene profiles. Results Eight distinct clonal complexes (CCs) were identified, dominated by CC121-SCCmecV (15%), CC1-SCCmecV (14%), CC15-SCCmecV (9%), CC1-SCCmecVI (7%), and CC8-SCCmecV (6%). Five novel sequence types (ST8157–ST8161) were discovered and deposited in pubMLST, indicating ongoing local evolution. Within CC8, two divergent lineages (ST239 and ST8) harbored unique SCCmec elements, reflecting significant phylogenetic differentiation. Globally important epidemic clones such as ST239-III-MRSA and ST22-IV-MRSA (EMRSA-15) were also detected. Network analysis revealed broad ecological adaptability, with livestock-associated CC97 and healthcare-associated CC5 harboring genes for immune evasion and biofilm formation. The detection of yopB in CC97 and yscT in CC5, genes typically found in Yersinia species, suggests horizontal gene transfer as a mechanism of adaptation. The high prevalence of fosB (fosfomycin resistance) and elevated fusidic acid resistance (39%) further underscores the emergence of multidrug resistance. Conclusions This large-scale genomic analysis reveals the coexistence of globally disseminated and locally evolved CA-MRSA lineages in Egypt. The findings underscore the adaptive potential of Egyptian MRSA populations and their contribution to regional AMR dynamics. Continued genomic surveillance within a One Health framework is essential for monitoring MRSA evolution, informing control measures, and mitigating the spread of resistance in both community and clinical settings. Clinical trial Not applicable.

Because of the global increase in resistance of Campylobacter to both fluoroquinolones and macrolides, we explored phenotypic resistance using traditional Kirby-Bauer methods in human- and animal-derived Campylobacter isolates that were concurrently resistant to both azithromycin and ciprofloxacin to an expanded panel of antimicrobials, including clindamycin, fosfomycin, ampicillin and sulbactam, amoxicillin and clavulanic, fosfomycin, tigecycline, and imipenem. Of 235 Campylobacter isolates resistant to ciprofloxacin and azithromycin, more than 94.8% of Campylobacter jejuni and Campylobacter coli were resistant to clindamycin, 68.5% were resistant to ampicillin and sulbactam, 2.6% were resistant to amoxicillin and clavulanic, and 36.2% were resistant to fosfomycin. Less than 2% of isolates were resistant to tigecycline, and there was no observed resistance to imipenem. The results suggest that amoxicillin and clavulanic warrant further evaluation for use in Campylobacter enteritis in ambulatory patients, but it points to no clear alternative oral therapies. Study findings were reassuring in demonstrating continued universal susceptibility to carbapenems.

Campylobacter is a leading cause of bacterial gastroenteritis worldwide, with the highest burden among children in low- and middle-income countries (LMICs). Despite global significance, Campylobacter coli remains comparatively under-studied. Here we combine population genomics and phylodynamic analysis of 460 C. coli genomes from the Peruvian Amazon to investigate the emergence of a locally dominant, multidrug-resistant lineage. Genomes were obtained from children with gastroenteritis (n = 136) and asymptomatic carriage (n = 163) in Iquitos, Peru, alongside isolates from poultry in backyard (n = 29) and industrial systems (n = 60), pigs (n = 36), cattle (n = 15), and a small reference collection (n = 21). These genomes represented 102 multilocus sequence types (STs) spanning five distinct clonal complexes (CCs). The globally distributed ST-828 clonal complex accounted for most isolates, whereas a distinct lineage, ST-1150 CC, was significantly over-represented in paediatric diarrhoeal cases yet rare elsewhere globally. Time-scaled phylogenies showed that ST-1150 CC emerged and expanded rapidly from the 1980s, coinciding with intensification of poultry production in the region. Genomic antimicrobial-resistance profiling identified extensive acquired resistance to fluoroquinolones, macrolides, and tetracyclines, underpinned by conserved cmeB efflux-pump variants. Collectively, these findings strongly suggest that agricultural intensification and antimicrobial exposure have driven the ecological emergence and clonal success of a highly adapted C. coli lineage. This work illustrates how regional farming practices can shape bacterial evolution and resistance trajectories, underscoring the need for integrated One Health genomic surveillance to mitigate the spread of zoonotic pathogens that are global in scope.

Abstract Background Staphylococcus epidermidis is a major cause of orthopaedic device-related infections (ODRIs), which are often challenging to treat due to their extensive antimicrobial resistance (AMR) and biofilm formation. It has been hypothesised that S. epidermidis may rapidly adapt to the medical device niche, enhancing persistence, but direct evidence of within-host pathoadaptive evolution remains limited. Results To investigate within-host evolution during chronic infection by S. epidermidis , we analysed isolates from patients with confirmed ODRIs and used a rat infection model to examine the evolution of strains from two distinct epidemic lineages (ST2 and ST23). Our analysis revealed that the replicative transposition of insertion sequence (IS) elements within the accessory genome was the predominant mechanism of genetic diversification. This was largely driven by the IS 256 family, which accounted for approximately 25% of all mutational events. However, other than SCC mec deletions resulting in the loss of mecA , no mutations, including those which exhibited parallel evolution, were predicted or observed to influence AMR or biofilm formation. These findings suggest that the strains investigated in this study, which already exhibited high-level multidrug resistance and biofilm-forming ability, were likely pre-adapted epidemic S. epidermidis clones well suited to establishing persistent ODRIs. Conclusions Our findings highlight the prominent role of IS elements in driving genetic diversification in S. epidermidis , underscoring the need for closer examination of their contribution to pathoadaptation during persistent infection.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) remains a major public health concern. Beyond canonical virulence determinants, biosynthetic gene clusters (BGCs) for specialized metabolites (SMs) and primary metabolic adaptations may shape fitness in community settings. In this study, we analyzed 123 CA-MRSA isolates from Egypt (2020–2021) alongside three reference genomes using a combined genomics workflow: antiSMASH and PRISM for homology-based specialized BGCs and predicted structures, GECCO for de novo BGC detection, BiG-SCAPE for gene-cluster family (GCF) networks and gutSMASH for primary-metabolism clusters detection. Assemblies were generated from Illumina MiSeq data and annotated with Prokka; sequence types and clonal complexes were assigned with FastMLST. antiSMASH identified a total of 934 BGCs (median 8 per genome), dominated by the following BGC types: NRPS-independent siderophores (NIS) (n = 312; IucA/IucC-like siderophores), non-ribosomal peptide synthetase (NRPS) (n = 132), and opine-like metallophores (n = 123). GECCO detected 415 BGCs, including 287 unclassified and 101 NRPS-associated clusters. GutSMASH recovered 582 primary metabolism gene clusters (GCs), with fumarate-to-succinate (n = 96), arginine-to-bicarbonate (n = 89; ADI) and nitrate-reductase (n = 84) energy-capturing modules being the most frequent. NRPS and NIS content varied widely among isolates, exceeding reference genome diversity, and BiG-SCAPE networks suggested multiple isolate-specific GCFs lacking MIBiG matches. Egyptian CA-MRSA displays a rich repertoire of iron-acquisition, peptide, and metallophore BGCs alongside anaerobic/energy-efficiency primary metabolism GCs that may support survival and competitiveness in community niches. While experimental validation is needed, these pathways nominate tractable targets for anti-virulence or metabolism-directed interventions and provide a genomic baseline for CA-MRSA in the North African region.

Background:Oropouche virus (OROV) is an emerging vector-borne pathogen endemic to the Americas, which causes acute febrile illness (AFI) in humans. Starting in late 2023, surges in OROV infections were reported across Latin America, including an outbreak in Iquitos, a city in the Eastern Peruvian Amazon, where RIVERA, an ongoing AFI surveillance program detected and characterized incident OROV cases. Methods:AFI cases presenting to health facilities were screened for OROV using PCR. OROV-positive subjects were compared to AFI OROV negative cases to describe the principal features of clinical disease. Genomes from OROV strains were sequenced and compared using phylogenetic analysis with those from extant samples isolated from other locations in the Americas. Findings:In early 2024, an 8.6% OROV-positivity rate (29 detections in 339 samples) in RIVERA subjects was recorded, a more than 20-fold increase compared with pre-outbreak levels. Illness was characterized by fever, arthralgia, myalgia and dysuria. Genome sequences from strains in this outbreak were phylogenetically distinct from those from a concurrent one in Brazil, but resembled strains from Colombia and Ecuador. The last common ancestor of outbreak strains from Peru and Brazil was 226 years prior to sampling, and that of Peru and Ecuador and Colombia approximately 10 and 8 years prior to sampling, respectively. Interpretation:Genomic analysis suggests that the current outbreak in South America is multifocal in origin and not the result of geographic spread from Brazil. An existing AFI surveillance program successfully documented the emergence and characterized the symptom profile of this emerging arboviral disease. Funding:CDC/HHS U01GH002270; NIH D43TW010913, K43TW012298, K01AI168493, 5T32AI007046-48.

Background:Shigella causes severe diarrheal disease, and S. flexneri and S. sonnei are the targets for multivalent vaccine development. Culture-based agglutination has been the gold standard for serotyping, but it is limited by logistics, subjectivity, and the availability of antisera for emerging serotypes. Newer methods, including a qPCR-based approach and whole-genome sequencing offer alternatives, but their performance in Shigella endemic populations are not well documented. Methods:Shigella isolates obtained from the Enterics for Global Health (EFGH) study in Iquitos, Peru were simultaneously serotyped using four methods: culture-based agglutination, isolate-based qPCR serotyping, stool-based qPCR serotyping and WGS using the in-silico tool ShigaPass. The definitive adjudicated serotype was established by an expert analysis of the WGS data, involving the mapping of sequence reads to known O-antigen biosynthesis and modification genes to identify key mutations. Results:Results from all four serotyping methods were available for 107/114 isolates. Accuracy for vaccine subtypes S. flexneri 1b, 2a, 3a, 6, and S. sonnei, ranged from 93.3-100% for all methods. Complete concordance between methods was noted in 83/107 isolates, while 24/107 (22.4%) exhibited at least one discrepancy. Most discrepancies derived from S. flexneri serotypes Y, Yv and 1a. Agglutination misclassified eight Y/Yv isolates as 4a, and six isolates correctly classified as 1a by agglutination were classified as 1b by the other methods, a discrepancy associated with a nonsense mutation in the oac gene. Conclusion:All four serotyping methods achieved acceptable accuracy for Shigella vaccine efficacy evaluation. Although discrepancies are infrequent, WGS provides information of their genomic basis.

Campylobacter remains the leading cause of bacterial gastroenteritis worldwide, with C. jejuni accounting for around 90% of infection and C. coli accounting for most of the rest. Seven-locus multilocus sequence typing (MLST) has improved our understanding of host association and population structure, whilst core genome MLST (cgMLST), enables investigation of transmission events at high-resolution. However, the lack of a stable and standardised nomenclature for clustering of cgMLST data has limited reproducibility and long-term comparability between studies. Here we introduce a joint, hierarchical Life Identification Number (LIN) code system that provides reproducible, multi-level genomic identifiers for C. jejuni and C. coli lineages. Using an updated cgMLST v2 scheme (1,142 loci) and globally representative datasets of high-quality genomes selected from over 53,000 assemblies in the Campylobacter PubMLST database (), we firstly defined LIN codes on a dataset of 5,664 genomes. Pairwise allelic distances were computed using MSTclust, and 18 nested thresholds were defined through silhouette, adjusted Wallace and adjusted Rand Index (ARI) statistics to capture the population structure from species to outbreak level resolution. The LIN thresholds were then validated using a second dataset of 1,781 genomes from PubMLST and applied to a large water-associated outbreak dataset from New Zealand in 2016, containing clinical and ecological genomes. Further application of LIN codes was demonstrated by analyses of the C. jejuni ST-21 clonal complex and ST-6175 isolates, as well as the broader population structure of C. coli, using data from PubMLST. Across all datasets, LIN clusters were stable, largely monophyletic, and back-compatible with existing nomenclature, accurately distinguishing host-adapted and outbreak-associated lineages. By embedding cgMLST data within a stable and scalable nomenclature, the Campylobacter LIN system delivers consistent, automated genome-to-lineage assignment. This unified framework bridges population genetics and applied surveillance, enabling robust, real-time comparison of Campylobacter isolates across sources, studies, and time. Impact statement Human cases of Campylobacter worldwide continue unabated. Tracing the source of Campylobacter infection is particularly challenging given the sporadic or multi-source nature of outbreaks, with potential transmission from foodborne, animal or environmental sources. Seven-locus MLST has greatly improved our broad understanding of Campylobacter population structure. However, whilst high-resolution cgMLST alleles and STs themselves do not change, longitudinal cluster analyses of cgMLST data have lacked a stable nomenclature, rendering them unsuitable for robust and comparable surveillance over time. Life Identification Number (LIN) codes provide a solution to this problem, establishing an automated and scalable nomenclature derived directly from cgMLST profiles, that is stable over time. We have implemented a joint C. jejuni and C. coli LIN code scheme in PubMLST, with scripts for real-time lineage assignment. LIN codes are back-compatible with existing MLST nomenclature, and we demonstrate their added practical value for exploring population structure and high-resolution outbreak investigation. LIN codes support surveillance of Campylobacter in a One Health context, by enabling consistent typing at multiple levels across different sources, laboratories and time. Data summary 1. The isolate collections used to develop the LIN codes are publicly available and searchable as individual projects on the PubMLST database (). 2. The software for LIN code development is publicly available as follows: ### Competing Interest Statement The authors have declared no competing interest. His Majesty's Treasury, https://ror.org/03k33gq76, PATH-SAFE program

This study compared the gut microbiota of Thai Indigenous Pradu Hang Dam (PD) chickens and commercial broiler chickens (CC) raised on separate farms under distinct diets and management systems. Cecal contents from 14 PD birds sampled at 16 weeks of age and 15 CC birds sampled at 45 days of age were profiled using 16S rRNA gene sequencing. Alpha-diversity metrics did not differ significantly between groups, whereas beta-diversity analyses showed clear differences in community composition. PD chickens showed broader taxonomic representation and a lower Firmicutes-to-Bacteroidota (F/B) ratio than CC chickens. Several taxa previously associated with short-chain fatty acid production were enriched in PD chickens, although potentially pathogenic taxa were also more abundant in this group. Predicted resistance-associated functional profiles were inferred from the 16S data using PICRUSt2. The two groups showed distinct predicted profiles: aggregate aminoglycoside-associated abundance was slightly higher in CC chickens, whereas several other functional categories were higher in PD chickens; individual aminoglycoside-associated functions varied in direction. These predictions do not demonstrate antimicrobial resistance gene carriage, genomic location, or phenotypic resistance. Overall, the two sampled production settings harbored compositionally distinct gut microbiota and predicted resistance-associated profiles. As breed, age, diet, farm, and management were confounded, multi-farm studies incorporating antimicrobial-use histories and direct metagenomic and phenotypic validation are needed to identify the drivers and functional consequences of these differences.

Red blood cell (RBC) concentrates remain at risk of bacterial contamination during cold storage. Although infrequent, Yersinia enterocolitica poses a significant blood safety risk. This study aimed to assess Y. enterocolitica bioserotype growth in RBC concentrates, serum sensitivity, and genetic diversity including iron metabolism genes. Ten Y. enterocolitica isolates from bioserotypes 1A, 1B/O:8, 4/O:3, and 2/O:9 were incubated in RBC concentrates and counted on days 3, 7, 14, 21, and 28. After incubation, the isolates were tested in human serum (NHS). Eight genomes were sequenced, analyzed using cgMLST, and screened for iron metabolism genes. The isolates formed two clusters, with 186dz (1A) and Ye8 (1B/O:8) as singletons. After 28 days in the RBC concentrates, the bacterial counts ranged from 1.98 × 10⁵ to 1.2 × 10⁹ CFU/mL, with Ye8 (1B/O:8) achieving the highest growth and one 4/O:3 isolate showing the lowest. All isolates survived 15-30 min in NHS, but the 28s isolate did not survive at 60 min. Serum sensitivity increased in two isolates, decreased in three, and remained unchanged in five. Isolates contained 27-42 iron metabolism genes with multiple allelic variants. The iron metabolism gene content or variants may influence the growth of Y. enterocolitica in RBC.

The Gram-negative bacterium Campylobacter jejuni is part of the commensal gut microbiota of numerous animal species and a leading cause of bacterial foodborne illness in humans. Most complete genomes of C. jejuni are from strains isolated from human clinical, poultry, and ruminant samples. Here, we characterized and compared the genomes of C. jejuni that were isolated from American black bears in three states in the southeastern United States from 2014 to 2016. Despite the limited sample size (n = 9), the isolates displayed substantial genotypic variability, including eight distinct sequence types (STs) and variable gene content encoding surface glycan structures such as capsular polysaccharides (CPS) and lipooligosaccharides (LOS). Phylogenetic analysis identified several C. jejuni host generalist strains among the isolates from bears that clustered with isolates from domestic poultry, cattle, and environmental sources. Three isolates (SKBC94, SKBC3, SKBC5) clustered with wildlife-associated strains, exhibiting mutations or deletions in loci associated with cytolethal distending toxin production and oxidative stress resistance, potentially influencing host-specific colonization. Additionally, strains SKBC3 and SKBC5 harbored distinct Entner-Doudoroff (E-D) loci, suggesting a potential evolutionary fitness advantage. This study provides the first evidence of C. jejuni colonization in American black bears, highlighting their potential role as reservoirs for diverse C. jejuni lineages from both anthropogenic and environmental sources. Further research is needed to determine the prevalence and host specificity of C. jejuni strains in black bears and their potential implications for public and wildlife health.

Staphylococci are the most common cause of orthopaedic device-related infections (ODRIs), with Staphylococcus aureus responsible for a third or more of cases. This prospective clinical and laboratory study investigated the association of genomic and phenotypic variation with treatment outcomes in ODRI isolates. Eighty-six invasive S. aureus isolates were collected from patients with ODRI, and clinical outcome was assessed after a follow-up examination of 24 months. Each patient was then considered to have been ‘cured’ or ‘not cured’ based on predefined clinical criteria. Whole-genome sequencing and molecular characterization identified isolates belonging to globally circulating community- and hospital-acquired lineages. Most isolates were phenotypically susceptible to methicillin and lacked the staphylococcal cassette chromosome mec cassette [methicillin-susceptible S. aureus (MSSA); 94%] but contained several virulence genes, including toxins and biofilm genes. Whilst recognizing the role of the host immune response, we identified genetic variance, which could be associated with the infection severity or clinical outcome. Whilst this and several other studies reinforce the role antibiotic resistance [e.g. methicillin-resistant S. aureus (MRSA) infection] has on treatment failure, it is important not to overlook MSSA that can cause equally destructive infections and lead to poor patient outcomes.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis globally, presenting with either watery or bloody/inflammatory diarrhea. Despite this clinical variability, the underlying mechanisms driving these distinct diarrheal manifestations remain unclear. The neonatal piglet model provides a unique opportunity to investigate these differences using strains with defined disease manifestations. We evaluated ten C. jejuni strains previously shown to cause consistent diarrheal manifestations in neonatal piglets: five strains associated with watery diarrhea and five associated with bloody/inflammatory diarrhea. The study found significant strain to strain variation among most assays, but overall strains associated with bloody/inflammatory diarrhea exhibited significantly higher levels of epithelial cell invasion, intracellular survival, macrophage survivability, and disruption of intestinal barrier integrity compared to watery diarrhea strains. Notably, both groups had similar levels of adherence, produced similar levels of cytolethal distending toxin (CDT) and elicited comparable IL-8 responses. While both groups facilitated translocation of commensal E. coli , only bloody/inflammatory strains caused increased paracellular permeability, as shown by TEER and FITC-dextran assays. This study reveals that there is significant variation in the virulence phenotype of C. jejuni strains and identifies several characteristics consistently associated with a specific diarrheal manifestation group. These findings provide new insights into the strain-specific pathogenic mechanisms of C. jejuni , with implications for understanding host-pathogen interactions and informing targeted diagnostic and therapeutic strategies. Author Summary Campylobacter jejuni is one of the most common bacterial causes of diarrhea worldwide, affecting millions each year. Infected individuals may experience either watery or bloody/inflammatory diarrhea, but the reasons why different strains cause different types of illness have remained a mystery. In this study, we used a neonatal piglet model, which accurately mimics human diarrheal disease, to investigate why certain C. jejuni strains cause more severe symptoms than others. We compared strains known to cause either watery or bloody diarrhea and found key differences in their ability to invade intestinal cells, survive inside immune cells, and damage the intestinal barrier. While both groups of strains could trigger inflammation and translocate E. coli through the intestinal lining, only the strains linked to bloody diarrhea caused significant barrier disruption and had increased intracellular survival. These findings reveal that different C. jejuni strains follow distinct paths during infection, which helps explain why they produce different symptoms. Unraveling these differences improves our overall understanding of Campylobacter pathogenesis that could lead to improved diagnostics and/or treatments to reduce the burden of campylobacteriosis around the world. ### Competing Interest Statement The authors have declared no competing interest. University of Arizona, https://ror.org/03m2x1q45, Start-up funds Technology and Research Initiative Fund (TRIF), University of Arizona, CALS\_ACBS\_Cooper_2101696

Introduction . Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide. Infections with C. jejuni can result in two different diarrhoeal manifestations in humans: watery diarrhoea or bloody/inflammatory diarrhoea.Hypothesis/Gap Statement. Currently, little is known about C. jejuni and/or host factors associated with the elicitation of these two distinct diarrhoeal manifestations. We hypothesize that these factors may include growth and metabolic trait differences between C. jejuni strains associated with watery diarrhoea and bloody/inflammatory diarrhoea.Aim. Using C. jejuni strains with a defined diarrhoeal manifestation in the neonatal piglet model, we aimed to assess differences in temperature-dependent growth rates, motility, biofilm production and carbon utilization between diarrhoeal manifestation groups.Methodology. Strains were initially assessed for 192 different carbon sources using phenotypic microarrays followed by specific carbon utilization, growth, motility and biofilm assays at 37 and/or 42 °C.Results. We found that at 37 °C, watery diarrhoea-associated C. jejuni strains grew significantly faster compared with bloody/inflammatory diarrhoea-associated C. jejuni strains. However, there was no significant growth difference at 42 °C between the groups, due to bloody/inflammatory diarrhoea-associated strains growing faster at 42 °C compared with 37 °C. Additionally, at 37 °C, we found that l-fucose utilization was significantly higher among watery diarrhoea-associated strains, while l-glutamine utilization was significantly higher among bloody/inflammatory diarrhoea-associated strains.Conclusion. The results indicate there are distinct metabolic adaptations between watery and/or bloody/inflammatory diarrhoea-associated C. jejuni strains particularly at 37 °C, which may be one of the factors associated with differing diarrhoeal manifestations.