Whole-genome sequencing reveals changes in genomic diversity and distinctive repertoires of T3SS and T6SS effector candidates in Chilean clinical Campylobacter strains

Campylobacter is the leading cause of bacterial gastroenteritis worldwide and an emerging and neglected pathogen in South America. This zoonotic pathogen colonizes the gastrointestinal tract of a wide range of mammals and birds, with poultry as the most important reservoir for human infections. Apart from its high morbidity rates, the emergence of resistant strains is of global concern. The aims of this work were to determine genetic diversity, presence of antimicrobial resistance determinants and virulence potential of Campylobacter spp. isolated from patients with acute gastrointestinal disease at ‘Clinica Alemana’, Santiago de Chile. The study considered the isolation of Campylobacter spp., from stool samples during a 20-month period (January 2020 to September 2021). We sequenced (NextSeq, Illumina) and performed an in-depth analysis of the genome sequences of 88 Campylobacter jejuni and 2 Campylobacter coli strains isolated from clinical samples in Chile. We identified a high genetic diversity among C. jejuni strains and the emergence of prevalent clonal complexes, which were not identified in our previous reports. While ~40% of strains harbored a mutation in the gyrA gene associated with fluoroquinolone resistance, no macrolide-resistance determinants were detected. Interestingly, gene clusters encoding virulence factors such as the T6SS or genes associated with long-term sequelae such as Guillain-Barré syndrome showed lineage-relatedness. In addition, our analysis revealed a high degree of variability regarding the presence of fT3SS and T6SS effector proteins in comparison to type strains 81-176, F38011, and NCTC 11168 and 488. Our study provides important insights into the molecular epidemiology of this emerging foodborne pathogen. In addition, the differences observed regarding the repertoire of fT3SS and T6SS effector proteins could have an impact on the pathogenic potential and transmissibility of these Latin American isolates, posing another challenge in characterizing the infection dynamics of this emergent and neglected bacterial pathogen.

[1]  T. Weitzel,et al.  Campylobacter spp. Prevalence in Santiago, Chile: A Study Based on Molecular Detection in Clinical Stool Samples from 2014 to 2019 , 2023, Pathogens.

[2]  J. Mcdermott,et al.  The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence , 2023, Biomolecules.

[3]  J. Finsterer.  Triggers of Guillain–Barré Syndrome: Campylobacter jejuni Predominates , 2022, International journal of molecular sciences.

[4]  Damion M. Dooley,et al.  CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database , 2022, Nucleic Acids Res..

[5]  S. Huynh,et al.  Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species , 2022, PLoS neglected tropical diseases.

[6]  A. Hachani,et al.  In silico investigation of the genus Campylobacter type VI secretion system reveals genetic diversity in organization and putative effectors , 2022, Microbial genomics.

[7]  S. Huynh,et al.  “Candidatus Campylobacter infans” detection is not associated with diarrhea in children under the age of 2 in Peru , 2022, PLoS neglected tropical diseases.

[8]  Orson Mestanza,et al.  Emergence and Molecular Epidemiology of Campylobacter jejuni ST-2993 Associated with a Large Outbreak of Guillain-Barré Syndrome in Peru , 2022, Microbiology spectrum.

[9]  Konstantinos D. Tsirigos,et al.  DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks , 2022, bioRxiv.

[10]  R. Kaas,et al.  ResFinder – an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes , 2022, Microbial genomics.

[11]  C. A. Conte Junior,et al.  The Distribution of Campylobacter jejuni Virulence Genes in Genomes Worldwide Derived from the NCBI Pathogen Detection Database , 2021, Genes.

[12]  A. Álvarez‐Ordoñez,et al.  Whole Resistome Analysis in Campylobacter jejuni and C. coli Genomes Available in Public Repositories , 2021, Frontiers in Microbiology.

[13]  D. Xia,et al.  Bioinformatic Analysis of the Campylobacter jejuni Type VI Secretion System and Effector Prediction , 2021, Frontiers in Microbiology.

[14]  A. H. V. van Vliet,et al.  A Campylobacter integrative and conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare? , 2021, bioRxiv.

[15]  N. González-Escalona,et al.  Closed Genome Sequence of a Salmonella enterica Serotype Senftenberg Strain Carrying the mcr-9 Gene Isolated from Broken Chicken Eggshells in Trinidad and Tobago , 2021, Microbiology resource announcements.

[16]  P. Whyte,et al.  Genetic characterisation of a subset of Campylobacter jejuni isolates from clinical and poultry sources in Ireland , 2021, PloS one.

[17]  Daniel J. Wilson,et al.  Machine learning to predict the source of campylobacteriosis using whole genome data , 2021, bioRxiv.

[18]  R. Piro,et al.  Genome-wide insights into population structure and host specificity of Campylobacter jejuni , 2021, Scientific Reports.

[19]  S. Salzberg,et al.  Guillain-Barré Syndrome Outbreak in Peru 2019 Associated With Campylobacter jejuni Infection , 2021, Neurology: Neuroimmunology & Neuroinflammation.

[20]  B. Wren,et al.  Revisiting Campylobacter jejuni Virulence and Fitness Factors: Role in Sensing, Adapting, and Competing , 2021, Frontiers in Cellular and Infection Microbiology.

[21]  A. Hensel,et al.  Campylobacter sp.: Pathogenicity factors and prevention methods—new molecular targets for innovative antivirulence drugs? , 2020, Applied Microbiology and Biotechnology.

[22]  R. Kaas,et al.  ResFinder 4.0 for predictions of phenotypes from genotypes , 2020, The Journal of antimicrobial chemotherapy.

[23]  N. González-Escalona,et al.  Genomic analysis of the diversity, antimicrobial resistance and virulence potential of Campylobacter jejuni and Campylobacter coli strains from Chile , 2020, bioRxiv.

[24]  N. González-Escalona,et al.  Complete Genome Sequences of 17 Clinical Campylobacter jejuni Strains from Chile , 2020, Microbiology Resource Announcements.

[25]  L. Collado.  [Microbial diagnosis and epidemiological surveillance of campylobacteriosis in Chile: Present state and further challenges]. , 2020, Revista chilena de infectologia : organo oficial de la Sociedad Chilena de Infectologia.

[26]  S. Sheppard,et al.  Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon , 2020, medRxiv.

[27]  N. González-Escalona,et al.  Draft Whole-Genome Sequences of 51 Campylobacter jejuni and 12 Campylobacter coli Clinical Isolates from Chile , 2020, Microbiology Resource Announcements.

[28]  Camille Stephan-Otto Attolini,et al.  Differential Distribution of the wlaN and cgtB Genes, Associated with Guillain-Barré Syndrome, in Campylobacter jejuni Isolates from Humans, Broiler Chickens, and Wild Birds , 2020, Microorganisms.

[29]  Laura M. Nolan,et al.  Causalities of war: The connection between type VI secretion system and microbiota , 2019, Cellular microbiology.

[30]  B. Wren,et al.  The Campylobacter jejuni Type VI Secretion System Enhances the Oxidative Stress Response and Host Colonization , 2019, Front. Microbiol..

[31]  Carolina N. Gomes,et al.  Molecular Epidemiology and Resistance Profile of Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Sources in Brazil. , 2019, Microbial drug resistance.

[32]  W. Messens,et al.  The European Union One Health 2018 Zoonoses Report , 2019, EFSA journal. European Food Safety Authority.

[33]  Narmada Thanki,et al.  CDD/SPARCLE: the conserved domain database in 2020 , 2019, Nucleic Acids Res..

[34]  Shaohua Zhao,et al.  Validating the AMRFinder Tool and Resistance Gene Database by Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates , 2019, Antimicrobial Agents and Chemotherapy.

[35]  E. Mcnamara,et al.  Molecular characterization of Campylobacter causing human clinical infection using whole-genome sequencing: Virulence, antimicrobial resistance and phylogeny in Ireland , 2019, PloS one.

[36]  C. Varela,et al.  Genomic Analysis of Chilean Strains of Campylobacter jejuni from Human Faeces , 2019, BioMed research international.

[37]  G. Iraola,et al.  Pathogenomics of Emerging Campylobacter Species , 2019, Clinical Microbiology Reviews.

[38]  F. Mégraud,et al.  Source attribution of Campylobacter jejuni shows variable importance of chicken and ruminants reservoirs in non-invasive and invasive French clinical isolates , 2019, Scientific Reports.

[39]  S. Coulthurst,et al.  The Type VI secretion system: a versatile bacterial weapon. , 2019, Microbiology.

[40]  P. Bork,et al.  Interactive Tree Of Life (iTOL) v4: recent updates and new developments , 2019, Nucleic Acids Res..

[41]  N. Păcală,et al.  Virulence of a T6SS Campylobacter jejuni chicken isolate from North Romania , 2019, BMC research notes.

[42]  Marek Basler,et al.  Clinical impact of the type VI secretion system on virulence of Campylobacter species during infection , 2019, BMC Infectious Diseases.

[43]  L. Comstock,et al.  Type VI Secretion Systems and the Gut Microbiota. , 2019, Microbiology spectrum.

[44]  Jian Yang,et al.  VFDB 2019: a comparative pathogenomic platform with an interactive web interface , 2018, Nucleic Acids Res..

[45]  L. Valinsky,et al.  Genomic Epidemiology of Campylobacter jejuni Transmission in Israel , 2018, Front. Microbiol..

[46]  Keith A Jolley,et al.  Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications , 2018, Wellcome open research.

[47]  M. Buchheim,et al.  Phylogenetic Relatedness Among Plasmids Harbored by Campylobacter jejuni and Campylobacter coli Isolated From Retail Meats , 2018, Front. Microbiol..

[48]  Jiangning Song,et al.  Bastion6: a bioinformatics approach for accurate prediction of type VI secreted effectors , 2018, Bioinform..

[49]  D. Hendrixson,et al.  Campylobacter jejuni: collective components promoting a successful enteric lifestyle , 2018, Nature Reviews Microbiology.

[50]  E. Cascales,et al.  Antibacterial Weapons: Targeted Destruction in the Microbiota. , 2018, Trends in microbiology.

[51]  C. Varela,et al.  Genetic diversity and clonal characteristics of ciprofloxacin-resistant Campylobacter jejuni isolated from Chilean patients with gastroenteritis. , 2018, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[52]  J. Corander,et al.  Convergent Amino Acid Signatures in Polyphyletic Campylobacter jejuni Subpopulations Suggest Human Niche Tropism , 2018, Genome biology and evolution.

[53]  Prateek Shrivastava,et al.  World health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics , 2018 .

[54]  Lukas Zimmermann,et al.  A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. , 2017, Journal of molecular biology.

[55]  João André Carriço,et al.  Origin, evolution, and distribution of the molecular machinery for biosynthesis of sialylated lipooligosaccharide structures in Campylobacter coli , 2017, bioRxiv.

[56]  Qijing Zhang,et al.  Antibiotic resistance trends and mechanisms in the foodborne pathogen, Campylobacter , 2017, Animal Health Research Reviews.

[57]  A. Filloux,et al.  Type VI secretion systems in plant‐associated bacteria , 2017, Environmental microbiology.

[58]  Mirko Rossi,et al.  chewBBACA: A complete suite for gene-by-gene schema creation and strain identification , 2017, bioRxiv.

[59]  O. Lund,et al.  PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens , 2017, The Journal of antimicrobial chemotherapy.

[60]  D. Monack,et al.  T6SS: The bacterial "fight club" in the host gut , 2017, PLoS pathogens.

[61]  Keith A. Jolley,et al.  Core Genome Multilocus Sequence Typing Scheme for Stable, Comparative Analyses of Campylobacter jejuni and C. coli Human Disease Isolates , 2017, Journal of Clinical Microbiology.

[62]  Ryan R. Wick,et al.  Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads , 2016, bioRxiv.

[63]  T. Weitzel,et al.  Impact of changing from staining to culture techniques on detection rates of Campylobacter spp. in routine stool samples in Chile , 2016, BMC Infectious Diseases.

[64]  A. Filloux,et al.  Type VI secretion and anti-host effectors. , 2016, Current opinion in microbiology.

[65]  S. Coulthurst,et al.  Molecular weaponry: diverse effectors delivered by the Type VI secretion system , 2015, Cellular microbiology.

[66]  Si Ming Man,et al.  Global Epidemiology of Campylobacter Infection , 2015, Clinical Microbiology Reviews.

[67]  Andrew J. Page,et al.  Roary: rapid large-scale prokaryote pan genome analysis , 2015, bioRxiv.

[68]  Daniel J. Wilson,et al.  Rapid host switching in generalist Campylobacter strains erodes the signal for tracing human infections , 2015, The ISME Journal.

[69]  J. Platts-Mills,et al.  Update on the burden of Campylobacter in developing countries , 2014, Current opinion in infectious diseases.

[70]  Christian Cambillau,et al.  VgrG, Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors. , 2014, Trends in microbiology.

[71]  Christian Cambillau,et al.  Architecture and assembly of the Type VI secretion system. , 2014, Biochimica et biophysica acta.

[72]  Torsten Seemann,et al.  Prokka: rapid prokaryotic genome annotation , 2014, Bioinform..

[73]  J. Mekalanos,et al.  A view to a kill: the bacterial type VI secretion system. , 2014, Cell host & microbe.

[74]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[75]  J. Rolain,et al.  ARG-ANNOT, a New Bioinformatic Tool To Discover Antibiotic Resistance Genes in Bacterial Genomes , 2013, Antimicrobial Agents and Chemotherapy.

[76]  S. Sheppard,et al.  Elucidating the Aetiology of Human Campylobacter coli Infections , 2013, PloS one.

[77]  L. I. Bouwman,et al.  Identification of a Functional Type VI Secretion System in Campylobacter jejuni Conferring Capsule Polysaccharide Sensitive Cytotoxicity , 2013, PLoS pathogens.

[78]  Alan Bridge,et al.  New and continuing developments at PROSITE , 2012, Nucleic Acids Res..

[79]  Danny S. Park,et al.  Campylobacter jejuni Type VI Secretion System: Roles in Adaptation to Deoxycholic Acid, Host Cell Adherence, Invasion, and In Vivo Colonization , 2012, PloS one.

[80]  M. Maiden,et al.  A Longitudinal 6-Year Study of the Molecular Epidemiology of Clinical Campylobacter Isolates in Oxfordshire, United Kingdom , 2012, Journal of Clinical Microbiology.

[81]  D. Hendrixson,et al.  Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization , 2012, Molecular microbiology.

[82]  Matthew Berriman,et al.  Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data , 2011, Bioinform..

[83]  S. Sheppard,et al.  Niche segregation and genetic structure of Campylobacter jejuni populations from wild and agricultural host species , 2011, Molecular ecology.

[84]  Mitchell J. Sullivan,et al.  Easyfig: a genome comparison visualizer , 2011, Bioinform..

[85]  N. Perna,et al.  progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement , 2010, PloS one.

[86]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[87]  S. O'Brien,et al.  Campylobacters as zoonotic pathogens: a food production perspective. , 2007, International journal of food microbiology.

[88]  T. E. Hickey,et al.  Heterogeneity of a Campylobacter jejuni Protein That Is Secreted through the Flagellar Filament , 2007, Infection and Immunity.

[89]  Matthew Berriman,et al.  ACT: the Artemis comparison tool , 2005, Bioinform..

[90]  Diane E. Taylor,et al.  pVir and Bloody Diarrhea in Campylobacter jejuni Enteritis , 2005, Emerging infectious diseases.

[91]  J. Butzler,et al.  Campylobacter, from obscurity to celebrity. , 2004, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[92]  F. Mégraud,et al.  The seasonal distribution of campylobacter infection in nine European countries and New Zealand , 2002, Epidemiology and Infection.

[93]  B. Allos.  Campylobacter jejuni Infections: update on emerging issues and trends. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[94]  P. Hitchen,et al.  Phase variation of a β‐1,3 galactosyltransferase involved in generation of the ganglioside GM1‐like lipo‐oligosaccharide of Campylobacter jejuni , 2000, Molecular microbiology.

[95]  R. Alm,et al.  Involvement of a Plasmid in Virulence of Campylobacter jejuni 81-176 , 2000, Infection and Immunity.

[96]  P. Hitchen,et al.  Multiple N‐acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo‐oligosaccharide , 2000, Molecular microbiology.

[97]  M. Konkel,et al.  Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells , 1999, Molecular microbiology.

[98]  Jeff H. Chang,et al.  The Agrobacterium Type VI Secretion System: A Contractile Nanomachine for Interbacterial Competition. , 2018, Current topics in microbiology and immunology.

[99]  R. Bustos B,et al.  [Genotyping and antibacterial resistance of Campylobacter spp strains isolated in children and in free range poultry]. , 2011, Revista chilena de infectologia : organo oficial de la Sociedad Chilena de Infectologia.

[100]  Susumu Goto,et al.  The KEGG databases at GenomeNet , 2002, Nucleic Acids Res..