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Abstract Development of a vaccine against gonorrhoea is a global priority, driven by the rise in antibiotic resistance. Although Neisseria gonorrhoeae (Ng) infection does not induce substantial protective immunity, highly exposed individuals may develop immunity against re-infection with the same strain. Retrospective epidemiological studies have shown that vaccines containing Neisseria meningitidis (Nm) outer membrane vesicles (OMVs) provide a degree of cross-protection against Ng infection. We conducted a clinical trial (NCT04297436) of 4CMenB (Bexsero, GSK), a licensed Nm vaccine containing OMVs and recombinant antigens, comprising a single arm, open label study of two doses with 50 adults in coastal Kenya who have high exposure to Ng. Data from a Ng antigen microarray established that serum IgG and IgA reactivities against the gonococcal homologs of the recombinant antigens in the vaccine peaked at 10 but had declined by 24 weeks. For most reactive OMV-derived antigens, the reverse was the case. A cohort of similar individuals with laboratory-confirmed gonococcal infection were compared before, during, and after infection: their reactivities were weaker and differed from the vaccinated cohort. We conclude that the cross-protection of the 4CMenB vaccine against gonorrhoea could be explained by cross-reaction against a diverse selection of antigens derived from the OMV component.

ABSTRACT Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome’s co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background—facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Abstract The Saiga are migratory antelopes inhabiting the grasslands of Eurasia. Over the last century, Saiga have been pushed to the brink of extinction by mass mortality events and intense poaching. Yet, despite the high profile of the Saiga as an animal of conservation concern, little is known of its biology. In particular, the gut microbiota of Saiga has not been studied, despite its potential importance in health. Here, we characterise the gut microbiota of Saiga from two geographically distinct populations in Kazakhstan and compare it with that of other antelope species. We identified a consistent gut microbial diversity and composition among individuals and across two Saiga populations during a year without die-offs, with over 85% of bacterial genera being common to both populations despite vast geographic separation. We further show that the Saiga gut microbiota resembled that of five other antelopes. The putative causative agent of Saiga mass die-offs, Pasteurella multocida, was not detected in the Saiga microbiota. Our findings provide the first description of the Saiga gut microbiota, generating a baseline for future work investigating the microbiota’s role in health and mass die-offs, and supporting the conservation of this critically endangered species.

ABSTRACTNeisseria gonorrhoeae causes sexually transmitted infection (STI) gonorrhoea and is on the WHO critical list due to increasing antimicrobial resistance (AMR). The bacterium can carry a conjugative plasmid, pConj, which prevents the use of tetracycline or doxycycline for treating gonorrhea, and is responsible for spread of a β-lactamase plasmid, pbla; over 90% of gonococcal isolates also harbor a small cryptic plasmid, pCryp. We systematically analyzed the presence of these plasmids in other Neisseria spp., including Neisseria meningitidis, which causes sepsis/meningitis. pConj is the most frequently found plasmid in the meningococcus and is in many clonal complexes. The plasmid is associated with meningococcal carriage rather than disease, indicating that pConj imposes fitness costs during systemic disease. Phylogenetic analyses reveal that pConj is genetically diverse in N. meningitidis, indicating that it shares a long evolutionary history with the meningococcus and that the plasmid has been transferred at least twice from N. meningitidis to N. gonorrhoeae. Following the first transfer, gonococcal isolates carrying the plasmid underwent clonal expansion and disseminated pConj to other gonococcal lineages. The second introduction was associated with an altered conjugation machinery which reduces conjugation efficiency. Therefore, in contrast to chromosomal resistance which has evolved through introduction of genes from commensals, gonococcal plasmid-mediated resistance has arisen through transfer from another pathogen, N. meningitidis. Further antibiotic pressure from the use of doxycycline for post-exposure prophylaxis against STIs is likely to promote plasmid-mediated AMR in both N. gonorrhoeae and N. meningitidis.IMPORTANCEHorizontal gene transfer (HGT) is a major influence in driving the spread of antimicrobial resistance (AMR) in many bacteria. A conjugative plasmid which is widespread in Neisseria gonorrhoeae, pConj, prevented the use of tetracycline/doxycycline for treating gonococcal infection. Here, we show that pConj evolved in the related pathogen, Neisseria meningitidis, and has been acquired by the gonococcus from the meningococcus on multiple occasions. Following its initial acquisition, pConj spread to different gonococcal lineages; changes in the plasmid’s conjugation machinery associated with another transfer event limit spread in the gonococcal populations. Our findings have important implications for the use of doxycycline to prevent bacterial sexually transmitted disease which is likely to exacerbate the spread of AMR through HGT in pathogenic bacteria.

The genus Bordetella includes environmental bacteria as well as human pathogens. Here, the authors present a large database of environmental and clinical Bordetella isolates and genome sequences, and develop genotyping systems to facilitate evolutionary and epidemiological studies.

Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital.

ABSTRACT The bacterial accessory genome provides the genetic flexibility needed to facilitate environment and host adaptation. In Neisseria gonorrhoeae, known accessory elements include plasmids which can transfer and mediate antimicrobial resistance (AMR); however, chromosomal accessory genes could also play a role in AMR. Here, the gonococcal accessory genome was characterized using gene-by-gene approaches and its association with the core genome and AMR were assessed. The gonococcal accessory gene pool consisted of 247 genes, which were mainly genes located on large mobile genetic elements, phage associated genes, or genes encoding putative secretion systems. Accessory elements showed similar synteny across genomes, indicating either a predisposition for particular genomic locations or ancestral inheritance that are conserved during strain expansion. Significant associations were found between the prevalence of accessory elements and core genome multi-locus sequence types (cgMLST), consistent with a structured gonococcal population despite frequent horizontal gene transfer (HGT). Increased prevalence of putative DNA exchange regulators was significantly associated with AMR, which included a putative secretion system, methyltransferases and a toxin-antitoxin system. Although frequent HGT results in high genetic diversity in the gonococcus, we found that this is mediated by a small gene pool. In fact, a highly organized genome composition was identified with a strong association between the accessory and core genome. Increased prevalence of DNA exchange regulators in antimicrobial resistant isolates suggests that genetic material exchange plays a role in the development or maintenance of AMR. These findings enhance our understanding of gonococcal genome architecture and have important implications for gonococcal population biology. IMPORTANCE The emergence of antimicrobial resistance (AMR) against third generation cephalosporins in Neisseria gonorrhoeae is a major public health concern, as these are antibiotics of last resort for the effective treatment of gonorrhea. Although the resistance mechanisms against this class of antibiotics have not been entirely resolved, resistance against other classes of antibiotics, such as tetracyclines, is known to be mediated through plasmids, which are known gonococcal extra-chromosomal accessory elements. A complete assessment of the chromosomal accessory genome content and its role in AMR has not yet been undertaken. Here, we comprehensively characterize the gonococcal accessory genome to better understand genome architecture as well as the evolution and mechanisms of AMR in this species.

Abstract Background Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown. Methods We studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes. Results Based on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996–2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Conclusions Our findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.

ABSTRACT Neisseria meningitidis outer membrane vesicle (OMV) vaccines are safe and provide strain-specific protection against invasive meningococcal disease (IMD) primarily by inducing serum bactericidal antibodies against the outer membrane proteins (OMP). To design broader coverage vaccines, knowledge of the immunogenicity of all the antigens contained in OMVs is needed. In a Phase I clinical trial, an investigational meningococcal OMV vaccine, MenPF1, made from a meningococcus genetically modified to constitutively express the iron-regulated FetA induced bactericidal responses to both the PorA and the FetA antigen present in the OMP. Using peripheral blood mononuclear cells collected from this trial, we analyzed the kinetics of and relationships between IgG, IgA, and IgM B cell responses against recombinant PorA and FetA, including (i) antibody-secreting cells, (ii) memory B cells, and (iii) functional antibody responses (opsonophagocytic and bactericidal activities). Following MenPF1vaccination, PorA-specific IgG secreting cell responses were detected in up to 77% of participants and FetA-specific responses in up to 36%. Memory B cell responses to the vaccine were low or absent and mainly detected in participants who had evidence of preexisting immunity (P = 0.0069). Similarly, FetA-specific antibody titers and bactericidal activity increased in participants with preexisting immunity and is consistent with the idea that immune responses are elicited to minor antigens during asymptomatic Neisseria carriage, which can be boosted by OMV vaccines. IMPORTANCE Neisseria meningitidis outer membrane vesicles (OMV) are a component of the capsular group B meningococcal vaccine 4CMenB (Bexsero) and have been shown to induce 30% efficacy against gonococcal infection. They are composed of multiple antigens and are considered an interesting delivery platform for vaccines against several bacterial diseases. However, the protective antibody response after two or three doses of OMV-based meningococcal vaccines appears short-lived. We explored the B cell response induced to a dominant and a subdominant antigen in a meningococcal OMV vaccine in a clinical trial and showed that immune responses are elicited to minor antigens. However, memory B cell responses to the OMV were low or absent and mainly detected in participants who had evidence of preexisting immunity against the antigens. Failure to induce a strong B cell response may be linked with the low persistence of protective responses.

Despite continued efforts to improve biosecurity protocols, Campylobacter continues to be detected in the majority of commercial chicken flocks across Europe. Using an extensive data set of Campylobacter prevalence within a chicken breeder flock for over a year, multiple Bayesian models are presented to explore the dynamics of the spread of Campylobacter in response to seasonal variation, species-specificity, bird health, and total colonization prevalence. These models indicated that birds within the flock varied greatly in their response to bacterial challenge, and that this phenomenon had a large impact on the overall prevalence of different species of Campylobacter. Campylobacter jejuni appeared more frequently in the summer, while Campylobacter coli persisted for a longer duration, amplified by the most susceptible birds in the flock. Our study suggests that strains of Campylobacter that appear most frequently likely possess no demographic advantage, but are instead amplified due to the health of the birds that ingest it.

ABSTRACT Streptococcus agalactiae (group B streptococcus; GBS) is a colonizer of the gastrointestinal and urogenital tracts, and an opportunistic pathogen of infants and adults. The worldwide population of GBS is characterized by clonal complexes (CCs) with different invasive potentials. CC17, for example, is a hypervirulent lineage commonly associated with neonatal sepsis and meningitis, while CC1 is less invasive in neonates and more commonly causes invasive disease in adults with comorbidities. The genetic basis of GBS virulence and the extent to which different CCs have adapted to different host environments remain uncertain. We have therefore applied a pan-genome-wide association study (GWAS) approach to 1,988 GBS strains isolated from different hosts and countries. Our analysis identified 279 CC-specific genes associated with virulence, disease, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of particular CCs. In CC17 and CC23, for example, we have identified genes encoding pilus, quorum-sensing proteins, and proteins for the uptake of ions and micronutrients which are absent in less invasive lineages. Moreover, in CC17, carriage and disease strains were distinguished by the allelic variants of 21 of these CC-specific genes. Together our data highlight the lineage-specific basis of GBS niche adaptation and virulence. IMPORTANCE GBS is a leading cause of mortality in newborn babies in high- and low-income countries worldwide. Different strains of GBS are characterized by different degrees of virulence, where some are harmlessly carried by humans or animals and others are much more likely to cause disease. The genome sequences of almost 2,000 GBS samples isolated from both animals and humans in high- and low- income countries were analyzed using a pan-genome-wide association study approach. This allowed us to identify 279 genes which are associated with different lineages of GBS, characterized by a different virulence and preferred host. Additionally, we propose that the GBS now carried in humans may have first evolved in animals before expanding clonally once adapted to the human host. These findings are essential to help understand what is causing GBS disease and how the bacteria have evolved and are transmitted.

ABSTRACT Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis. Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation. IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.

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.

Staphylococcus epidermidis is carried asymptomatically by virtually all humans but is also a major cause of nosocomial infection. Here, the authors study 141 isolates from healthy carriage and 274 isolates from clinical infections, and identify genes and genetic elements associated with pathogenicity.

Abstract Background Chlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated. Results WGS data from 157 Ct isolates deposited in the Chlamydiales pubMLST database (http://pubMLST.org/chlamydiales/) were annotated with 902 genes including the core and accessory genome. Plasmid associated genes were annotated and a plasmid MLST scheme was defined allowing plasmid sequence types to be determined. Plasmid allelic variation was investigated. Phylogenetic relationships were examined using the Genome Comparator tool available in pubMLST. Phylogenetic analyses identified four distinct Ct core genome clusters and six plasmid clusters, with a strong association between the chromosomal genotype and plasmid. This in turn was linked to ompA genovars and disease phenotype. Horizontal genetic transfer of plasmids was observed for three urogenital-associated isolates, which possessed plasmids more commonly found in isolates resulting from ocular infections. The pgp3 gene was identified as the most polymorphic plasmid gene and pgp4 was the most conserved. Conclusion A strong association between chromosomal genome, plasmid type and disease was observed, consistent with previous studies. This suggests co-evolution of the Ct chromosome and their plasmids, but we confirmed that plasmid transfer can occur between isolates. These data provide a better understanding of the genetic diversity occurring across the Ct genome in association with the plasmid content.

Abstract Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

Abstract 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%.

Abstract Background Serogroup A Neisseria meningitidis (NmA) was the cause of the 2011 meningitis epidemics in Chad. This bacterium, often carried asymptomatically, is considered to be an “accidental pathogen”; however, the transition from carriage to disease phenotype remains poorly understood. This study examined the role genetic diversity might play in this transition by comparing genomes from geographically and temporally matched invasive and carried NmA isolates. Results All 23 NmA isolates belonged to the ST-5 clonal complex (cc5). Ribosomal MLST comparison with other publically available NmA:cc5 showed that isolates were closely related, although those from Chad formed two distinct branches and did not cluster with other NmA, based on their MLST profile, geographical and temporal location. Whole genome MLST (wgMLST) comparison identified 242 variable genes among all Chadian isolates and clustered them into three distinct phylogenetic groups (Clusters 1, 2, and 3): no systematic clustering by disease or carriage source was observed. There was a significant difference (p = 0.0070) between the mean age of the individuals from which isolates from Cluster 1 and Cluster 2 were obtained, irrespective of whether the person was a case or a carrier. Conclusions Whole genome sequencing provided high-resolution characterization of the genetic diversity of these closely related NmA isolates. The invasive meningococcal isolates obtained during the epidemic were not homogeneous; rather, a variety of closely related but distinct clones were circulating in the human population with some clones preferentially colonizing specific age groups, reflecting a potential age-related niche adaptation. Systematic genetic differences were not identified between carriage and disease isolates consistent with invasive meningococcal disease being a multi-factorial event resulting from changes in host-pathogen interactions along with the bacterium.

Carriage of Neisseria meningitidis, the meningococcus, is a prerequisite for invasive meningococcal disease (IMD), a potentially devastating infection that disproportionately afflicts infants and children. Humans are the sole known reservoir for the meningococcus, and it is carried asymptomatically in the nasopharynx of ~10% of the population. Rates of carriage are dependent on age of the host and social and behavioural factors. In the UK, meningococcal carriage has been studied through large, multi-centre carriage surveys of adolescents in 1999, 2000, and 2001, demonstrating carriage can be affected by immunisation with the capsular group C meningococcal conjugate vaccine, inducing population immunity against carriage. Fifteen years after these surveys were carried out, invasive meningococcal disease incidence had declined from a peak in 1999. The UKMenCar4 study was conducted in 2014/15 to investigate rates of carriage amongst the adolescent population during a period of low disease incidence. The protocols and methodology used to perform UKMenCar4, a large carriage survey, are described here.

Carriage of Neisseria meningitidis, the meningococcus, is a prerequisite for invasive meningococcal disease (IMD), a potentially devastating infection that disproportionately afflicts infants and children. Humans are the sole known reservoir for the meningococcus, and it is carried asymptomatically in the nasopharynx of ~10% of the population. Rates of carriage are dependent on age of the host and social and behavioural factors. In the UK, meningococcal carriage has been studied through large, multi-centre carriage surveys of adolescents in 1999, 2000, and 2001, demonstrating carriage can be affected by immunisation with the capsular group C meningococcal conjugate vaccine, inducing population immunity against carriage. Fifteen years after these surveys were carried out, invasive meningococcal disease incidence had declined from a peak in 1999. The UKMenCar4 study was conducted in 2014/15 to investigate rates of carriage amongst the adolescent population during a period of low disease incidence. The protocols and methodology used to perform UKMenCar4, a large carriage survey, are described here.

Background: Expression of a capsule from one of serogroups A, B, C, W, X or Y is usually required for Neisseria meningitidis (Nme) to cause invasive meningococcal disease. The capsule is encoded by the capsule locus, cps, which is proposed to have been acquired by a formerly capsule null organism by horizontal genetic transfer (HGT) from another species. Following identification of putative capsule genes in non-pathogenic Neisseria species, this hypothesis is re-examined. Methods: Whole genome sequence data from Neisseria species, including Nme genomes from a diverse range of clonal complexes and capsule genogroups, and non-Neisseria species, were obtained from PubMLST and GenBank. Sequence alignments of genes from the meningococcal cps, and predicted orthologues in other species, were analysed using Neighbor-nets, BOOTSCANing and maximum likelihood phylogenies. Results: The meningococcal cps was highly mosaic within regions B, C and D. A subset of sequences within regions B and C were phylogenetically nested within homologous sequences belonging to N. subflava, consistent with HGT event in which N. subflava was the donor. In the cps of 23/39 isolates, the two copies of region D were highly divergent, with rfbABC’ sequences being more closely related to predicted orthologues in the proposed species N. weixii (GenBank accession number CP023429.1) than the same genes in Nme isolates lacking a capsule. There was also evidence of mosaicism in the rfbABC’ sequences of the remaining 16 isolates, as well as rfbABC from many isolates. Conclusions: Data are consistent with the en bloc acquisition of cps in meningococci from N. subflava, followed by further recombination events with other Neisseria species. Nevertheless, the data cannot refute an alternative model, in which native meningococcal capsule existed prior to undergoing HGT with N. subflava and other species. Within-genus recombination events may have given rise to the diversity of meningococcal capsule serogroups.

Background: Expression of a capsule from one of serogroups A, B, C, W, X or Y is usually required for Neisseria meningitidis (Nme) to cause invasive meningococcal disease. The capsule is encoded by the capsule locus, cps, which is proposed to have been acquired by a formerly capsule null organism by horizontal genetic transfer (HGT) from another species. Following identification of putative capsule genes in non-pathogenic Neisseria species, this hypothesis is re-examined. Methods: Whole genome sequence data from Neisseria species, including Nme genomes from a diverse range of clonal complexes and capsule genogroups, and non-Neisseria species, were obtained from PubMLST and GenBank. Sequence alignments of genes from the meningococcal cps, and predicted orthologues in other species, were analysed using Neighbor-nets, BOOTSCANing and maximum likelihood phylogenies. Results: The meningococcal cps was highly mosaic within regions B, C and D. A subset of sequences within regions B and C were phylogenetically nested within homologous sequences belonging to N. subflava, consistent with HGT event in which N. subflava was the donor. In the cps of 23/39 isolates, the two copies of region D were highly divergent, with rfbABC’ sequences being more closely related to predicted orthologues in the proposed species N. weixii (GenBank accession number CP023429.1) than the same genes in Nme isolates lacking a capsule. There was also evidence of mosaicism in the rfbABC’ sequences of the remaining 16 isolates, as well as rfbABC from many isolates. Conclusions: Data are consistent with the en bloc acquisition of cps in meningococci from N. subflava, followed by further recombination events with other Neisseria species. Nevertheless, the data cannot refute an alternative model, in which native meningococcal capsule existed prior to undergoing HGT with N. subflava and other species. Within-genus recombination events may have given rise to the diversity of meningococcal capsule serogroups.

Background: Protein-conjugate capsular polysaccharide vaccines can potentially control invasive meningococcal disease (IMD) caused by five (A, C, W, X, Y) of the six IMD-associated serogroups. Concerns raised by immunological similarity of the serogroup B capsule to human neural cell carbohydrates, meant that ‘serogroup B substitute’ vaccines target more variable subcapsular protein antigens. A successful approach using outer membrane vesicles (OMVs) as major vaccine components had limited strain coverage. In 4CMenB (Bexsero®), recombinant proteins have been added to ameliorate this problem. Methods: Scalable, portable, genomic techniques were used to investigate the Bexsero® OMV protein diversity in meningococcal populations. Shotgun proteomics identified 461 proteins in the OMV, defining a complex proteome. Amino acid sequences for the 24 proteins most likely to be involved in cross-protective immune responses were catalogued within the PubMLST.org/neisseria database using a novel OMV peptide Typing (OMVT) scheme. Results: Among these proteins there was variation in the extent of diversity and association with meningococcal lineages, identified as clonal complexes (ccs), ranging from the most conserved peptides (FbpA, NEISp0578, and putative periplasmic protein, NEISp1063) to the most diverse (TbpA, NEISp1690). There were 1752 unique OMVTs identified amongst 2492/3506 isolates examined by whole-genome sequencing (WGS). These OMVTs were grouped into clusters (sharing ≥18 identical OMVT peptides), with 45.3% of isolates assigned to one of 27 OMVT clusters. OMVTs and OMVT clusters were strongly associated with cc, genogroup, and Bexsero® antigen variants, demonstrating that combinations of OMV proteins exist in discrete, non-overlapping combinations associated with genogroup and Bexsero® Antigen Sequence Type. This highly structured population of IMD-associated meningococci is consistent with strain structure models invoking host immune and/or metabolic selection. Conclusions: The OMVT scheme facilitates region-specific WGS investigation of meningococcal diversity and is an open-access, portable tool with applications for vaccine development, especially in the choice of antigen combinations, assessment and implementation.

Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a “supercore” genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population.

Background: Protein-conjugate capsular polysaccharide vaccines can potentially control invasive meningococcal disease (IMD) caused by five (A, C, W, X, Y) of the six IMD-associated serogroups. Concerns raised by immunological similarity of the serogroup B capsule, to human neural cell carbohydrates, has meant that ‘serogroup B substitute’ vaccines target more variable subcapsular protein antigens. A successful approach using outer membrane vesicles (OMVs) as major vaccine components had limited strain coverage. In 4CMenB (Bexsero®), recombinant proteins have been added to ameliorate this problem. Methods: Here, scalable, portable, genomic techniques were used to investigate the Bexsero® OMV protein diversity in meningococcal populations. Shotgun proteomics identified 461 proteins in the OMV, defining a complex proteome. Amino acid sequences for the 24 proteins most likely to be involved in cross-protective immune responses were catalogued within the PubMLST.org/neisseria database using a novel OMV peptide Typing (OMVT) scheme. Results: Among these proteins there was variation in the extent of diversity and association with meningococcal lineages, identified as clonal complexes (ccs), ranging from the most conserved peptides (FbpA, NEISp0578, and putative periplasmic protein, NEISp1063) to the most diverse (TbpA, NEISp1690). There were 1752 unique OMVTs identified amongst 2492/3506 isolates examined by whole-genome sequencing (WGS). These OMVTs were grouped into clusters (sharing ≥18 identical OMVT peptides), with 45.3% of isolates assigned to one of 27 OMVT clusters. OMVTs and OMVT clusters were strongly associated with cc, genogroup, and Bexsero® antigen variants, demonstrating that combinations of OMV proteins exist in discrete, non-overlapping combinations associated with genogroup and Bexsero® Antigen Sequence Type. This highly structured population of IMD-associated meningococci is consistent with strain structure models invoking host immune selection. Conclusions: The OMVT scheme facilitates region-specific WGS investigation of meningococcal diversity and is an open-access, portable tool with applications for vaccine development, especially in the choice of antigen combinations, assessment and implementation.

The PubMLST.org website hosts a collection of open-access, curated databases that integrate population sequence data with provenance and phenotype information for over 100 different microbial species and genera. Although the PubMLST website was conceived as part of the development of the first multi-locus sequence typing (MLST) scheme in 1998 the software it uses, the Bacterial Isolate Genome Sequence database (BIGSdb, published in 2010), enables PubMLST to include all levels of sequence data, from single gene sequences up to and including complete, finished genomes. Here we describe developments in the BIGSdb software made from publication to June 2018 and show how the platform realises microbial population genomics for a wide range of applications. The system is based on the gene-by-gene analysis of microbial genomes, with each deposited sequence annotated and curated to identify the genes present and systematically catalogue their variation. Originally intended as a means of characterising isolates with typing schemes, the synthesis of sequences and records of genetic variation with provenance and phenotype data permits highly scalable (whole genome sequence data for tens of thousands of isolates) means of addressing a wide range of functional questions, including: the prediction of antimicrobial resistance; likely cross-reactivity with vaccine antigens; and the functional activities of different variants that lead to key phenotypes. There are no limitations to the number of sequences, genetic loci, allelic variants or schemes (combinations of loci) that can be included, enabling each database to represent an expanding catalogue of the genetic variation of the population in question. In addition to providing web-accessible analyses and links to third-party analysis and visualisation tools, the BIGSdb software includes a RESTful application programming interface (API) that enables access to all the underlying data for third-party applications and data analysis pipelines.

ABSTRACT Neisseria meningitidis is a common cause of bacterial meningitis in children and young adults worldwide. The 4CMenB vaccine (Bexsero), developed to combat meningococcal serogroup B (MenB) disease, contains subcapsular antigens that may induce immunity against strains of N. meningitidis, regardless of serogroup. Owing to differential levels of expression and peptide diversity in vaccine antigens across meningococcal strains, the meningococcal antigen typing system (MATS) was developed to estimate the potential MenB strain coverage of 4CMenB. Prior to introducing the 4CMenB vaccine into routine use, we sought to estimate the potential 4CMenB coverage against invasive MenB strains isolated in the Republic of Ireland (RoI) over four consecutive epidemiological years. MATS was applied to a panel of 105 invasive MenB strains isolated during July 2009 to June 2013. Sequence data characterizing the multilocus sequence typing (MLST) alleles and the major 4CMenB target peptides were extracted from isolate genome sequence data, hosted in the Bacterial Isolate Sequencing database (BIGSdb). MATS data indicated that 4CMenB may induce protective immunity against 69.5% (95% confidence interval [CI95%], 64.8% to 84.8%) of circulating MenB strains. Estimated coverage was highest against the most prevalent disease-causing lineage, cc41/44, where the most frequently observed sequence types, ST-154 and ST-41 (21% of isolates, collectively), were typically covered by three antigens. No significant temporal trends were observed. Overall, these data provide a baseline of strain coverage prior to the introduction of 4CMenB and indicate that a decrease in invasive meningococcal disease (IMD) is predicted following the introduction of 4CMenB into the routine infant immunization schedule in the RoI. IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland.

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Correction After publication of the original article [1] it was identified that an incorrect version of the manuscript has been published. This was caused by a technical error which led to a discrepancy between the editorially-accepted version of the manuscript, and the published version.

Campylobacter­ spp.–related gastroenteritis in diners at a catering college restaurant was associated with consumption of duck liver pâté. Population genetic analysis indicated that isolates from duck samples were typical of isolates from farmed poultry. Campylobacter spp. contamination of duck liver may present a hazard similar to the increasingly recognized contamination of chicken liver.

Subtypes, defined by variation in the outer membrane protein PorA, are an integral part of the characterization scheme for Neisseria meningitidis. Identification of these variants remains important as the PorA protein is a major immunogenic component of several meningococcal vaccines under development, and characteristics of PorA are used to provide detailed epidemiologic information. Historically, serosubtypes have been defined by reactivity with a set of monoclonal antibodies. However, nucleotide sequence analyses of porA genes have established that the panel of serosubtyping monoclonal antibodies is not exhaustive and many porA variants cannot be detected. In addition, the nomenclature system used to define subtypes is inadequate. We examined all available nucleotide sequences of the porA VR1 and VR2 regions to identify and define subtype families. A revised nomenclature scheme, compatible with the previous serologic nomenclature scheme, was devised. A Web-accessible database describing this nomenclature and its relationship to previous schemes was established (available from: http://neisseria.org/nm/typing/pora).

Supplementing Campylobacter spp. multilocus sequence typing with nucleotide sequence typing of 3 antigen genes increased the discriminatory index achieved from 0.975 to 0.992 among 620 clinical isolates from Oxfordshire, United Kingdom. This enhanced typing scheme enabled identification of clusters and retained data required for long-range epidemiologic comparisons of isolates.

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified >20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.