Whole-genome sequencing is becoming a leading technology in the typing and epidemiology of microbial pathogens, but the increase in genomic information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method feature frequency profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer and that coupling with in silico genotyping methods gives useful information for comparative and clinical genomic and molecular epidemiology applications. FFP-based phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rRNA genes and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes again showed comparable phylogenetic clustering, consistent with genomotypes assigned by using multilocus sequence typing (MLST). Analysis of 377 H. pylori genomes highlighted the conservation of genomotypes and linkage with phylogeographic characteristics and predicted the presence of an incomplete or nonfunctional cag pathogenicity island in 18/276 genomes. In silico analysis of antibiotic susceptibility markers suggests that most H. pylori hspAmerind and hspEAsia isolates are predicted to carry the T2812C mutation potentially conferring low-level clarithromycin resistance, while levels of metronidazole resistance were similar in all multilocus sequence types. In conclusion, the use of FFP phylogenetic clustering and in silico genotyping allows determination of genome evolution and phylogeographic clustering and can contribute to clinical microbiology by genomotyping for outbreak management and the prediction of pathogenic potential and antibiotic susceptibility.

The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated "cell-train" morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

The Center for Disease Control and Prevention identifies antimicrobial resistant (AMR) Campylobacter as a serious threat to U.S. public health due to high community burden, increased transmissibility, and limited treatability. The National Antimicrobial Resistance Monitoring System (NARMS) plays an important role in surveillance of AMR bacterial pathogens in humans, food animals and retail meats. This study investigated C. coli and C. jejuni from live food animals, poultry carcasses at production, and retail meat in North Carolina between January 2018-December 2019. Whole genome sequencing and bioinformatics were used for phenotypic and genotypic characterization to compare AMR profiles, virulence factors associated with Guillain-Barré Syndrome (GBS) (neuABC and cst-II or cst-III), and phylogenic linkage between 541 Campylobacter isolates (C. coli n = 343, C. jejuni n = 198). Overall, 90.4% (489/541) Campylobacter isolates tested positive for AMR genes, while 43% (233/541) carried resistance genes for three or more antibiotic classes and were classified molecularly multidrug resistant. AMR gene frequencies were highest against tetracyclines (64.3%), beta-lactams (63.6%), aminoglycosides (38.6%), macrolides (34.8%), quinolones (24.4%), lincosamides (13.5%), and streptothricins (5%). A total of 57.6% (114/198) C. jejuni carried GBS virulence factors, while three C. coli carried the C. jejuni-like lipooligosaccharide locus, neuABC and cst-II. Further evidence of C. coli and C. jejuni interspecies genomic exchange was observed in identical multilocus sequence typing, shared sequence type (ST) 7818 clonal complex 828, and identical species-indicator genes mapA, ceuE, and hipO. There was a significant increase in novel STs from 2018 to 2019 (2 in 2018 and 21 in 2019, p<0.002), illustrating variable Campylobacter genomes within food animal production. Introgression between C. coli and C. jejuni may aid pathogen adaption, lead to higher AMR and increase Campylobacter persistence in food processing. Future studies should further characterize interspecies gene transfer and evolutionary trends in food animal production to track evolving risks to public health.

In the United Kingdom, the thermophilic Campylobacter species C. jejuni and C. coli are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate C. jejuni and C. coli from the food chain.

The coupling of environmental sensing to flagella-mediated directed motility allows bacteria to move to optimum environments for growth and survival, either by sensing external stimuli (chemotaxis) or monitoring internal metabolic status (energy taxis). Sensing is mediated by transducer-like proteins (Tlp), either located in the membrane or in the cytoplasm, which commonly influence motility via the CheA-CheY chemotaxis pathway. In this study we have investigated the role of PAS-domain-containing intracellular Tlp-sensors in energy taxis of the food-borne pathogen Campylobacter jejuni, using plate- and tube-based assays utilising the conversion of the redox indicator dyes triphenyl tetrazolium chloride (TTC) and resazurin. Inactivation of the genes encoding the Campylobacter Energy Taxis system (CetA (Tlp9) and CetB (Aer2)) in C. jejuni strain NCTC 11168 resulted in reduced taxis. Inactivation of the cj1191c gene, encoding the CetB homolog CetC (Aer1), did not affect taxis per se, but the cetC gene complemented a cetB mutant in trans, indicating that CetC can form a functional signal transduction complex with CetA in the absence of CetB. Inactivation of both CetB and CetC resulted in greatly reduced taxis confirming the role of CetC in energy taxis. Inactivation of the cj1110c gene, encoding Tlp8 (CetZ), a cytoplasmic sensor with two PAS-domains, resulted in increased taxis, a phenotype opposite to that of CetAB. Inactivation of the cheA gene resulted in the same overall phenotype as the cetAB mutant in both wild-type and cetZ backgrounds, suggesting that both systems use the CheA system for signal transduction. Absence of both CetAB and CetZ resulted in the cetAB taxis phenotype, suggesting that CetZ is subordinate to CetAB. In conclusion, we present evidence that C. jejuni balances the input from two counteracting PAS-domain-containing sensory systems to position itself for optimal usage of energy resources.

Helicobacter species are among the most successful colonizers of the mammalian gastrointestinal and hepatobiliary tract. Colonization is usually lifelong, indicating that Helicobacter species have evolved intricate mechanisms of dealing with stresses encountered during colonization of host tissues, like restriction of essential metal ions. The recent availability of genome sequences of the human gastric pathogen Helicobacter pylori, the murine enterohepatic pathogen Helicobacter hepaticus and the unannotated genome sequence of the ferret gastric pathogen Helicobacter mustelae has allowed for comparative genome analyses. In this review we present such analyses for metal transporters, metal-storage and metal-responsive regulators in these three Helicobacter species, and discuss possible contributions of the differences in metal metabolism in adaptation to the gastric or enterohepatic niches occupied by Helicobacter species.

Campylobacter is a food-associated bacterium and a leading cause of foodborne illness worldwide, being associated with poultry in the food supply. This is the initial public release of 202 Campylobacter genome sequences as part of the 100K Pathogen Genome Project. These isolates represent global genomic diversity in the Campylobacter genus.

Sialidases, which are widely distributed in nature, cleave the α-ketosidic bond of terminal sialic acid residue. These emerging virulence factors degrade the host glycan. We report here the release of seven sialidase and one sialic acid transporter deletion in Salmonella enterica serovar Typhimurium strain LT2, which are important in cellular invasion during infection.

Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst several individual colonisation and virulence factors have been identified, our understanding of their role in the transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have examined the distribution and predicted functionality of the previously described capC and the newly identified, related capD autotransporter gene families in Campylobacter.

The Type VI Secretion System (T6SS) has important roles relating to bacterial antagonism, subversion of host cells, and niche colonisation. Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis worldwide and is a commensal coloniser of birds. Although recently discovered, the T6SS biological functions and identities of its effectors are still poorly defined in C. jejuni. Here, we perform a comprehensive bioinformatic analysis of the C. jejuni T6SS by investigating the prevalence and genetic architecture of the T6SS in 513 publicly available genomes using C. jejuni 488 strain as reference. A unique and conserved T6SS cluster associated with the Campylobacter jejuni Integrated Element 3 (CJIE3) was identified in the genomes of 117 strains. Analyses of the T6SS-positive 488 strain against the T6SS-negative C. jejuni RM1221 strain and the T6SS-positive plasmid pCJDM202 carried by C. jejuni WP2-202 strain defined the "T6SS-containing CJIE3" as a pathogenicity island, thus renamed as Campylobacter jejuni Pathogenicity Island-1 (CJPI-1). Analysis of CJPI-1 revealed two canonical VgrG homologues, CJ488_0978 and CJ488_0998, harbouring distinct C-termini in a genetically variable region downstream of the T6SS operon. CJPI-1 was also found to carry a putative DinJ-YafQ Type II toxin-antitoxin (TA) module, conserved across pCJDM202 and the genomic island CJIE3, as well as several open reading frames functionally predicted to encode for nucleases, lipases, and peptidoglycan hydrolases. This comprehensive in silico study provides a framework for experimental characterisation of T6SS-related effectors and TA modules in C. jejuni.

Campylobacter jejuni and Campylobacter coli are important bacterial causes of human foodborne illness. Despite several years of reduced antibiotics usage in livestock production in the United Kingdom (UK) and United States (US), a high prevalence of antimicrobial resistance (AMR) persists in Campylobacter. Both countries have instigated genome sequencing-based surveillance programs for Campylobacter, and in this study, we have identified AMR genes in 32,256 C. jejuni and 8,776 C. coli publicly available genome sequences to compare the prevalence and trends of AMR in Campylobacter isolated in the UK and US between 2001 and 2018. AMR markers were detected in 68% of C. coli and 53% of C. jejuni isolates, with 15% of C. coli isolates being multidrug resistant (MDR), compared to only 2% of C. jejuni isolates. The prevalence of aminoglycoside, macrolide, quinolone, and tetracycline resistance remained fairly stable from 2001 to 2018 in both C. jejuni and C. coli, but statistically significant differences were observed between the UK and US. There was a statistically significant higher prevalence of aminoglycoside and tetracycline resistance for US C. coli and C. jejuni isolates and macrolide resistance for US C. coli isolates. In contrast, UK C. coli and C. jejuni isolates showed a significantly higher prevalence of quinolone resistance. Specific multilocus sequence type (MLST) clonal complexes (e.g., ST-353/464) showed >95% quinolone resistance. This large-scale comparison of AMR prevalence has shown that the prevalence of AMR remains stable for Campylobacter in the UK and the US. This suggests that antimicrobial stewardship and restricted antibiotic usage may help contain further expansion of AMR prevalence in Campylobacter but are unlikely to reduce it in the short term.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

Enteropathogenic Escherichia coli (EPEC) constitutes one of the main causes of mortality in children in low- to medium-income countries. Diverse animal species have been linked as reservoirs, including birds. The aim of this study was to describe the genomic and phylogenetic features of an EPEC recovered from a pet macaw and further characterizing the macro and microscopic lesion in a rabbit ileal loop experimental model. The isolate was whole-genome sequenced (WGS) obtaining its genotypic and phenotypic in silico characteristics and inoculated in a rabbit experimental model with subsequently evaluating the strain's pathogenicity by scanning electron microscopy (SEM) and histopathology. The isolate was characterized as O109:H21-B1-ST40 typical EPEC, harboring several virulence factors of diarrheagenic E. coli. The macaw EPEC genome was located in a monophyletic clade of human and animal ST40 EPEC sequences. In vivo inoculation demonstrated severe hemorrhage with SEM and histopathological analysis confirming these lesions to be associated with intra-epithelial lymphocytes. Therefore, the isolate not only shared several genotypic and phylogenetic similarities with EPEC that affects humans and animals, but was able to induce severe tissue injury in a mammal model. These findings highlight the underrated role of pet birds as zoonotic reservoirs and the diversity in virulence factors being unraveled by new WGS studies.

Salmonella enterica is a significant foodborne pathogen, which can be transmitted via several distinct routes, and reports on acquisition of antimicrobial resistance (AMR) are increasing. To better understand the association between human Salmonella clinical isolates and the potential environmental/animal reservoirs, whole genome sequencing (WGS) was used to investigate the epidemiology and AMR patterns within Salmonella isolates from two adjacent US states.

The genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, Δfur, ΔperR and ΔfurΔperR isogenic deletion mutants under both iron-replete and iron-limited conditions.

Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide. Despite the microaerophilic nature of the bacterium, C. jejuni can survive the atmospheric oxygen conditions in the environment. Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of reactive oxygen species. The MarR-type transcriptional regulators RrpA and RrpB have recently been shown to play a role in controlling both the C. jejuni oxidative and aerobic stress responses. Analysis of 3,746 C. jejuni and 486 C. coli genome sequences showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with specific MLST clonal complexes (predominantly ST-21 and ST-61). C. coli strains in contrast lack both rrpA and rrpB. In C. jejuni rrpB+ strains, the rrpB gene is located within a variable genomic region containing the IF subtype of the type I Restriction-Modification (hsd) system, whilst this variable genomic region in C. jejuni rrpB- strains contains the IAB subtype hsd system and not the rrpB gene. C. jejuni rrpB- strains exhibit greater resistance to peroxide and aerobic stress than C. jejuni rrpB+ strains. Inactivation of rrpA resulted in increased sensitivity to peroxide stress in rrpB+ strains, but not in rrpB- strains. Mutation of rrpA resulted in reduced killing of Galleria mellonella larvae and enhanced biofilm formation independent of rrpB status. The oxidative and aerobic stress responses of rrpB- and rrpB+ strains suggest adaptation of C. jejuni within different hosts and niches that can be linked to specific MLST clonal complexes.

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.

The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.