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The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.

The human nasopharynx is colonized by commensal bacteria and pathobionts, which comprise a complex microbial ecosystem capable of generating primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica results in safe, sustained colonization. Herein is described a novel technology to chromosomally transform N. lactamica with heterologous antigen, for the purpose of safe delivery to the mucosal surface and the generation of an antigen-specific immune response. N. lactamica was transformed to express the meningococcal vaccine antigen Neisseria Adhesin A (NadA) and was inoculated intranasally into humans at a dose of 105 colony-forming units. NadA-expressing N. lactamica colonized these individuals and was carried asymptomatically for 3 months. Colonization with NadA-expressing N. lactamica generated NadA-specific IgG-secreting plasma cells within 14 days of colonization and both NadA-specific IgG and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells circulate in the bloodstream of colonized participants for at least 90 days. Genetically transformed N. lactamica has the potential to be a safe bacterial vehicle to generate beneficial immune responses to a wide range of heterologous antigens during sustained pharyngeal carriage.

The enteric, pathogenic spirochaete Brachyspira pilosicoli colonizes and infects a variety of birds and mammals, including humans. However, there is a paucity of genomic data available for this organism. This study introduces 12 newly sequenced draft genome assemblies, boosting the cohort of examined isolates by fourfold and cataloguing the intraspecific genomic diversity of the organism more comprehensively. We used several in silico techniques to define a core genome of 1751 genes and qualitatively and quantitatively examined the intraspecific species boundary using phylogenetic analysis and average nucleotide identity, before contextualizing this diversity against other members of the genus Brachyspira . Our study revealed that an additional isolate that was unable to be species typed against any other Brachyspira lacked putative virulence factors present in all other isolates. Finally, we quantified that homologous recombination has as great an effect on the evolution of the core genome of the B. pilosicoli as random mutation (r/m=1.02). Comparative genomics has informed Brachyspira diversity, population structure, host specificity and virulence. The data presented here can be used to contribute to developing advanced screening methods, diagnostic assays and prophylactic vaccines against this zoonotic pathogen.

OBJECTIVES: Escherichia coli is the leading cause of bacteraemia. In an era of emerging multi-drug-resistant strains, development of effective preventative strategies will be informed by knowledge of strain diversity associated with specific infective syndromes/patient groups. We hypothesised that the number of virulence factor (VF) genes amongst bacteraemia isolates from neutropaenic patients would be lower than isolates from immunocompetent patients.METHODS: Immunocompetent and neutropaenic adults with E. coli bacteraemia were recruited prospectively and the source of bacteraemia determined. VF gene profiles were established in silico following whole genome sequencing.RESULTS: Isolates from individual patients were monoclonal. Strains from immunocompetent patients with urinary tract infective foci (UTIF) harboured more VF genes (median number of VF genes 16, range 8-24) than isolates from both immunocompetent patients with non-UTIF (10, 2-22, p = 0.0058) and neutropaenic patients with unknown focus of infection (NPUFI) (8, 3-13, p < 0.0001). Number of VF genes (OR 1.21, 95% CIs 1.01-1.46, p = 0.039) and urinary catheter/recurrent urinary tract infection (OR 12.82, 95% CIs 1.24-132.65, p = 0.032) were independent predictors of bacteraemia secondary to UTIF vs. non-UTIF in immunocompetent patients. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst UTIF- vs non-UTIF-originating isolates amongst immunocompetent patients, while papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTIF- vs NPUFI-associated isolates.CONCLUSIONS: Bacteraemia-associated E. coli strains originating from UTIF have distinct VF gene profiles from strains associated with non-UTIF- and NPUFI. This diversity must be addressed in the design of future vaccines to ensure adequate coverage of strains responsible for site-specific disease.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution., Carriage of Neisseria lactamica, a harmless coloniser of the human respiratory tract, is inversely correlated with Neisseria meningitidis infection. Here, Pandey et al. provide insights into micro-evolutionary processes in N. lactamica during controlled infection of healthy volunteers.

We present the high quality, complete genome assembly of Neisseria lactamica Y92–1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

Background Escherichia coli is the leading cause of bacteremia with multi-drug-resistant strains proving increasingly problematic. Knowledge of the strain diversity associated with site-specific infections will inform the development of new preventative strategies, e.g. vaccines. We hypothesized that virulence factor (VF) scores of bacteremia strains from neutropenia patients with unknown infective foci (NPUIF—likely due to bowel translocation) would be lower than those from immunocompetent patients. Methods Immunocompetent (n = 49) and neutropaenic adults (n = 8) with E. coli bacteremia were prospectively enrolled and the focus of bacteremia determined. Neutropenia patients were enrolled only if there was no identifiable infective focus. Multi-locus sequence typing and VF score (31 known VFs included) data were derived in silico following whole-genome sequencing and the results compared between patient groups. Results Bacteremia strains from immunocompetent patients with urinary tract infective foci (UTI-foci) harbored significantly more VFs (median VF score 16, range 8–24) than strains from both immunocompetent patients with non-UTI-foci (10, 2–22, P = 0.006) and NPUIF (8, 3–13, P < 0.0001). VF scores of strains from non-UTI-foci were not significantly different to those from NPUIF (10, 2–22 vs. 8, 3–13, respectively. P = 0.28). Logistic regression analysis demonstrated that VF score (OR 1.21, 95% CIs 1.01–1.46, P = 0.039) and recurrent urinary tract infection/urinary tract infection (OR 12.82, 95% CIs 1.24–132.65, P = 0.032) were independent predictors of bacteremia secondary to UTI-foci vs. non-UTI-foci in immunocompetent patients. Hence, for every unit increase in VF score, the odds of a bacteremia strain originating from UTI-foci increased by 1.21. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst strains associated with UTI-foci vs. non-UTI-foci amongst immunocompetent patients. papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTI-foci- vs. NPUIF-associated strains. Conclusion UTI-associated E. coli bacteremia strains have distinct VF profiles from those originating from non-UTI-foci and NPUIF. Future vaccines must consider this diversity to ensure adequate coverage of strains associated with site-specific disease. Disclosures All authors: No reported disclosures.

We report a draft genome sequence for Dichelobacter nodosus ATCC 25549, strain VPI 2340 [11342], a causative agent of ovine footrot. The draft genome shares ~98% gene similarity with the available genome of D. nodosus strain VCS1703A but is differentiated by extensive gene duplication and the absence of 13 particular genes.