Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

How does feedback from phage infections influence the evolution of phase variation in Campylobacter?

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.

Selective and non-selective bottlenecks as drivers of the evolution of hypermutable bacterial loci.

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome-wide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci.

Does Blood Transfusion Have an Effect on Outcomes After Aortic Valve Replacement Surgery?

OBJECTIVE: Long-term outcomes following surgical aortic valve replacement (AVR) are excellent. However, there is a significant early morbidity burden. Red cell transfusion is common in the perioperative period and deleterious effects of receiving a transfusion on early postoperative morbidity are well described in observational studies. This study aimed to assess the effect of transfusion on ischaemic or infective outcomes after aortic valve replacement. METHODS: Data from 270 patients enrolled in the Manubrium-limited ministernotomy versus conventional sternotomy for aortic valve replacement (MAVRIC) randomised controlled trial was used to create two cohorts, patients that received red cell transfusions following AVR and those that did not. Propensity score matching was performed to limit the effect of confounding variables. Strict transfusion thresholds were maintained, with patients receiving a transfusion if haemoglobin concentration fell below 80 g/L, or if significant bleeding or haemodynamic instability occurred. The primary outcome was a composite of ischaemic event (myocardial infarction, permanent stroke, gut ischaemia or acute kidney injury) or serious infection (sepsis, endocarditis, respiratory tract or wound infection). Patients were followed for 12 weeks following surgery. RESULTS: Sixty-three (63) of 270 patients received a red cell transfusion (23.3%). Transfused patients had significantly lower body mass index (BMI), a higher proportion of females, a lower preoperative haemoglobin and haematocrit, a higher EuroSCORE II score, worse renal function and were more likely to have undergone urgent surgery compared to the unadjusted control cohort. Once matched, there was no difference in the primary outcome between cohorts. There was a significantly increased length of hospital stay in the transfused group (median 7 days transfused, median 5 days not-transfused, p=0.001). CONCLUSIONS: Red cell transfusion, using a transfusion threshold of 80 g/L, does not appear to be associated with adverse ischaemic or infective outcomes after aortic valve replacement.

Potentiation of Phase Variation in Multiple Outer-Membrane Proteins During Spread of the Hyperinvasive Neisseria meningitidis Serogroup W ST-11 Lineage.

BACKGROUND: Since 2009, increases in the incidence of invasive meningococcal disease have occurred in the United Kingdom due to a sublineage of the Neisseria meningitidis serogroup W ST-11 clonal complex (hereafter, the "original UK strain"). In 2013, a descendent substrain (hereafter, the "2013 strain") became the dominant disease-causing variant. Multiple outer-membrane proteins of meningococci are subject to phase-variable switches in expression due to hypermutable simple-sequence repeats. We investigated whether alterations in phase-variable genes may have influenced the relative prevalence of the original UK and 2013 substrains, using multiple disease and carriage isolates. METHODS: Repeat numbers were determined by either bioinformatics analysis of whole-genome sequencing data or polymerase chain reaction amplification and sizing of fragments from genomic DNA extracts. Immunoblotting and sequence-translation analysis was performed to identify expression states. RESULTS: Significant increases in repeat numbers were detected between the original UK and 2013 strains in genes encoding PorA, NadA, and 2 Opa variants. Invasive and carriage isolates exhibited similar repeat numbers, but the absence of pilC gene expression was frequently associated with disease. CONCLUSIONS: Elevated repeat numbers in outer-membrane protein genes of the 2013 strain are indicative of higher phase-variation rates, suggesting that rapid expansion of this strain was due to a heightened ability to evade host immune responses during transmission and asymptomatic carriage.

Limited Impact of Adolescent Meningococcal ACWY Vaccination on Neisseria meningitidis Serogroup W Carriage in University Students.

Background: In the United Kingdom, rising levels of disease due to Neisseria meningitidis serogroup W clonal complex (cc) sequence type (ST) 11 (MenW:cc11) strains led to introduction of meningococcal conjugate vaccine (MenACWY) for teenagers. We investigated the impact of immunization on carriage of meningococci targeted by the vaccine, using whole-genome sequencing of isolates recovered from a cohort of vaccinated university students. Methods: Strain designation data were extracted from whole-genome sequencing data. Genomes from carried and invasive MenW:cc11 strains were compared using a gene-by-gene approach. Serogrouping identified isolates expressing capsule antigens targeted by the vaccine. Results: Isolates with a W: P1.5,2: F1-1: ST-11 (cc11) designation and belonging to the emerging 2013-strain of the South American-United Kingdom MenW:cc11 sublineage were responsible for an increase in carried group W strains. A multifocal expansion was evident, with close transmission networks extending beyond individual dormitories. Carried group Y isolates were predominantly from cc23 but showed significant heterogeneity, and individual strain designations were only sporadically recovered. No shifts toward acapsulate phenotypes were detected in targeted meningococcal populations. Conclusions: In a setting with high levels of MenACWY use, expansion of capsule-expressing isolates from the 2013-strain of MenW:cc11 but not MenY:cc23 isolates is indicative of differential susceptibilities to vaccine-induced immunity.

Random sorting of Campylobacter jejuni phase variants due to a narrow bottleneck during colonization of broiler chickens.

Phase variation (PV), involving stochastic switches in gene expression, is exploited by the human pathogen Campylobacter jejuni to adapt to different environmental and host niches. Phase-variable genes of C. jejuni modulate expression of multiple surface determinants, and hence may influence host colonization. Population bottlenecks can rapidly remove the diversity generated by PV, and strict single-cell bottlenecks can lead to propagation of PV states with highly divergent phenotypes. Using a combination of high-throughput fragment size analysis and comparison with in vivo and in silico bottleneck models, we have characterized a narrow population bottleneck during the experimental colonization of broiler chickens with C. jejuni strain 81-176. We identified high levels of variation in five PV genes in the inoculum, and subsequently, massively decreased population diversity following colonization. Each bird contained a dominant five-gene phasotype that was present in the inoculum indicative of random sorting through a narrow, non-selective bottleneck during colonization. These results are evidence of the potential for confounding effects of PV on in vivo studies of Campylobacter colonization factors and poultry vaccine studies. Our results are also an argument for population bottlenecks as mediators of stochastic variability in the propensity to survive through the food chain and cause clinical human disease.

Phase Variation of NadA in Invasive Neisseria meningitidis Isolates Impacts on Coverage Estimates for 4C-MenB, a MenB Vaccine.

A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The meningococcal antigen typing system (MATS) is utilized as a high-throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase-variable changes in transcription due to a 5'TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region (IGR) sequences and 5'TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5'TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and enzyme-linked immunosorbent assay (ELISA). Low-expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or NadA-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with NadA-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS-negative isolates were associated with low-expression repeat numbers, whereas 63% of isolates with MATS relative potency (RP) scores above the 95% confidence interval for the positive bactericidal threshold had high-expression repeat numbers. Analysis of 5'TAAA repeat numbers has potential as a rapid, high-throughput method for assessing strain coverage for the NadA component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied.

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5'CCCGA and 5'CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.

Rise in Group W Meningococcal Carriage in University Students, United Kingdom.

MenACWY conjugate vaccination was recently introduced in the United Kingdom for adolescents and young adults to reduce disease from infection by Neisseria meningitidis group W. We conducted a cross-sectional meningococcal carriage study in first-year UK university students. Despite 71% MenACWY vaccine coverage, carriage of group W increased substantially.

Genome-wide association of functional traits linked with Campylobacter jejuni survival from farm to fork.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, primarily associated with the consumption of contaminated poultry. C. jejuni lineages vary in host range and prevalence in human infection, suggesting differences in survival throughout the poultry processing chain. From 7343 MLST-characterised isolates, we sequenced 600 C. jejuni and C. coli isolates from various stages of poultry processing and clinical cases. A genome-wide association study (GWAS) in C. jejuni ST-21 and ST-45 complexes identified genetic elements over-represented in clinical isolates that increased in frequency throughout the poultry processing chain. Disease-associated SNPs were distinct in these complexes, sometimes organised in haplotype blocks. The function of genes containing associated elements was investigated, demonstrating roles for cj1377c in formate metabolism, nuoK in aerobic survival and oxidative respiration, and cj1368-70 in nucleotide salvage. This work demonstrates the utility of GWAS for investigating transmission in natural zoonotic pathogen populations and provides evidence that major C. jejuni lineages have distinct genotypes associated with survival, within the host specific niche, from farm to fork.

Phage exposure causes dynamic shifts in the expression states of specific phase-variable genes of Campylobacter jejuni.

Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacterjejuni this reversible adaptive process is mediated by mutations in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase for this CPS modification, cj1421, and two other phase-variable CPS genes generated phage resistance in C. jejuni. Here we investigate the population dynamics of C. jejuni NCTC11168 when exposed to phage F336 in vitro using a newly described method - the 28-locus-CJ11168 PV analysis. Dynamic switching was observed in the ON/OFF states of three phase-variable CPS genes, cj1421, cj1422 and cj1426, during phage F336 exposure, with the dominant phage-resistant phasotype differing between cultures. Although loss of the phage receptor was predominately observed, several other PV events also led to phage resistance, a phenomenon that increases the chance of phage-resistant subpopulations being present in any growing culture. No other PV genes were affected and exposure to phage F336 resulted in a highly specific response, only selecting for phase variants of cj1421, cj1422 and cj1426. In summary, C. jejuni may benefit from modification of the surface in multiple ways to inhibit or reduce phage binding, thereby ensuring the survival of the population when exposed to phages.

Genomic Analysis of Serogroup Y Neisseria meningitidis Isolates Reveals Extensive Similarities Between Carriage-Associated and Disease-Associated Organisms.

BACKGROUND: Neisseria meningitidis is a frequent colonizer of the human nasopharynx, with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High-resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors. METHODS: Whole-genome sequence data were obtained for 99 MenY carriage isolates recovered in the United Kingdom during 1997-2010. Sequences were compared to those of 73 MenY invasive isolates recovered during 2010-2011, using a gene-by-gene approach. RESULTS: Comparisons across 1605 core genes resolved 91% of isolates into one of 8 clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated during 1997-2001, suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into one subcluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in 5 core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB), and 2 hypothetical proteins. CONCLUSIONS: High-resolution genetic analyses detected long-term temporal stability and temporally overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone.

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, "phasotypes," was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.

Phase variable genes of Campylobacter jejuni exhibit high mutation rates and specific mutational patterns but mutability is not the major determinant of population structure during host colonization.

Phase variation of surface structures occurs in diverse bacterial species due to stochastic, high frequency, reversible mutations. Multiple genes of Campylobacter jejuni are subject to phase variable gene expression due to mutations in polyC/G tracts. A modal length of nine repeats was detected for polyC/G tracts within C. jejuni genomes. Switching rates for these tracts were measured using chromosomally-located reporter constructs and high rates were observed for cj1139 (G8) and cj0031 (G9). Alteration of the cj1139 tract from G8 to G11 increased mutability 10-fold and changed the mutational pattern from predominantly insertions to mainly deletions. Using a multiplex PCR, major changes were detected in 'on/off' status for some phase variable genes during passage of C. jejuni in chickens. Utilization of observed switching rates in a stochastic, theoretical model of phase variation demonstrated links between mutability and genetic diversity but could not replicate observed population diversity. We propose that modal repeat numbers have evolved in C. jejuni genomes due to molecular drivers associated with the mutational patterns of these polyC/G repeats, rather than by selection for particular switching rates, and that factors other than mutational drift are responsible for generating genetic diversity during host colonization by this bacterial pathogen.

Navigating the infodemic: A qualitative study of university students' information strategies during the COVID-19 pandemic.

Objectives: We aimed to study the strategies which university students developed for vetting information during the COVID-19 pandemic and associated infodemic. Methods: We conducted semi-structured interviews with 34 students, using a piloted topic guide which explored several areas of pandemic experiences, including students' use of media. Transcripts were analysed inductively following the thematic approach. Higher order themes were finalised following a coding exercise undertaken by two of the authors. Results: Participants were acutely aware of misinformation during the pandemic. They rated legacy news media (print and broadcast media with pre-Internet origins) higher than social media for reliable information about the pandemic. However, strikingly, not all legacy media were automatically trusted and not all social media were uniformly distrusted. Participants identified a set of mechanisms for establishing whether a piece of information was truthful and accurate. These mechanisms had four main focal points: (1) the source, (2) the message, (3) individual media literacy and (4) the trustworthiness of others. Despite possessing a critical awareness of misinformation, participants avoided posting anything in relation to the pandemic for fear of becoming the target of online abuse. Conclusions: In addition to underscoring the role of media literacy, our research foregrounds the need to attend to the importance of fostering media confidence. We define media confidence as the ability of digital media users to challenge and interrogate questionable or inaccurate information safe in the knowledge that there are adequate regulatory mechanisms in place to curb abuse, trolling and intimidation.

Antithrombotic therapy after heart valve surgery: contemporary practice in the UK.

OBJECTIVES: There is a lack of high-quality data informing the optimal antithrombotic drug strategy following bioprosthetic heart valve replacement or valve repair. Disparity in recommendations from international guidelines reflects this. This study aimed to document current patterns of antithrombotic prescribing after heart valve surgery in the UK. METHODS: All UK consultant cardiac surgeons were e-mailed a custom-designed survey. The use of oral anticoagulant (OAC) and/or antiplatelet drugs following bioprosthetic aortic valve replacement or mitral valve replacement, or mitral valve repair (MVrep), for patients in sinus rhythm, without additional indications for antithrombotic medication, was assessed. Additionally, we evaluated anticoagulant choice following MVrep in patients with atrial fibrillation. RESULTS: We identified 260 UK consultant cardiac surgeons from 36 units, of whom 103 (40%) responded, with 33 units (92%) having at least 1 respondent. The greatest consensus was for patients undergoing bioprosthetic aortic valve replacement, in which 76% of surgeons favour initial antiplatelet therapy and 53% prescribe lifelong treatment. Only 8% recommend initial OAC. After bioprosthetic mitral valve replacement, 48% of surgeons use an initial OAC strategy (versus 42% antiplatelet), with 66% subsequently prescribing lifelong antiplatelet therapy. After MVrep, recommendations were lifelong antiplatelet agent alone (34%) or following 3 months OAC (20%), no antithrombotic agent (20%), or 3 months OAC (16%). After MVrep for patients with established atrial fibrillation, surgeons recommend warfarin (38%), a direct oral anticoagulant (37%) or have no preference between the 2 (25%). CONCLUSIONS: There is considerable variation in the use of antithrombotic drugs after heart valve surgery in the UK and a lack of high-quality evidence to guide practice, underscoring the need for randomized studies.

Phase variation of PorA, a major outer membrane protein, mediates escape of bactericidal antibodies by Neisseria meningitidis.

Several outer membrane proteins of Neisseria meningitidis are subject to phase variation due to alterations in simple sequence repeat tracts. The PorA protein is a major outer membrane protein and a target for protective host immune responses. Phase variation of PorA is mediated by a poly-G repeat tract present within the promoter, leading to alterations in protein expression levels. N. meningitidis strain 8047 was subjected to serial passage in the presence of P1.2, a PorA-specific bactericidal monoclonal antibody. Rapid development of resistance to bactericidal activity was associated with a switch in the PorA repeat tract from 11G to 10G. Phase variants with a 10G repeat tract exhibited a 2-fold reduction in surface expression of PorA protein. A mutS mutant of strain 8047, with an elevated phase variation rate, exhibited a higher rate of escape and an association of escape with 10G and 9G variants, the latter having a 13-fold reduction in surface expression of PorA. We conclude that graduated reductions in the surface expression of outer membrane proteins mediated by phase variation enable meningococci to escape killing in vitro by bactericidal antibodies. These findings indicate how phase variation could have a major impact on immune escape and host persistence of meningococci.

Distribution and diversity of the haemoglobin-haptoglobin iron-acquisition systems in pathogenic and non-pathogenic Neisseria.

A new generation of vaccines containing multiple protein components that aim to provide broad protection against serogroup B meningococci has been developed. One candidate, 4CMenB (4 Component MenB), has been approved by the European Medicines Agency, but is predicted to provide at most 70-80 % strain coverage; hence there is a need for second-generation vaccines that achieve higher levels of coverage. Prior knowledge of the diversity of potential protein vaccine components is a key step in vaccine design. A number of iron import systems have been targeted in meningococcal vaccine development, including the HmbR and HpuAB outer-membrane proteins, which mediate the utilization of haemoglobin or haemoglobin-haptoglobin complexes as iron sources. While the genetic diversity of HmbR has been described, little is known of the diversity of HpuAB. Using whole genome sequences deposited in a Bacterial Isolate Genome Sequence Database (BIGSDB), the prevalence and diversity of HpuAB among Neisseria were investigated. HpuAB was widely present in a range of Neisseria species whereas HmbR was mainly limited to the pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae. Patterns of sequence variation in sequences from HpuAB proteins were suggestive of recombination and diversifying selection consistent with strong immune selection. HpuAB was subject to repeat-mediated phase variation in pathogenic Neisseria and the closely related non-pathogenic Neisseria species Neisseria lactamica and Neisseria polysaccharea but not in the majority of other commensal Neisseria species. These findings are consistent with HpuAB being subject to frequent genetic transfer potentially limiting the efficacy of this receptor as a vaccine candidate.

Novel method for prediction of combinatorial phase-variable gene expression states.

Short sequence repeat mediated phase variation results in diverse phenotype presentation in many bacteria including Campylobacter and Neisseria species. Current methods for identifying the expression states of phase-variable genes involve taking a high number of single colonies. This approach is subject to bias, sampling effects and high workloads that reduce the ability to perform intermediary sampling. The use of high concentration colony sweeps provides a work around but reduces the resolution of combinatorial expression profiles (termed phasotypes). A parsimonious approach combining both single colony and sweep data was developed to overcome these limitations. The critical methodological advance is the use of an algorithm that utilises the experimental data from the two sample types and a parsimonious, iterative mathematical analysis that outputs the phasotype distribution with the highest likelihood of underpinning the experimental data sets. The advantages of this unified method are increased resolution and accuracy of gene expression state combinations as compared to conventional single colony sampling, reduced requirement for sampling large numbers of colonies leading to reduced costs, and a higher capacity for collecting samples and replicates.•Inputting of sweep and single colony data into an algorithm for a rapid determination of the combinatorial phase variation states (phasotypes) for repeat-mediated phase-variable bacterial genes•This method reduces the number of single colony samples required to produce accurate estimates of phasotypes•This method will reduce the costs of phasotype analyses and increase potential to analyse more time points or sample sites leading to an improved understanding of how phase variation contributes to bacterial host persistence and the ability to cause disease.