Adam C. Retchless

Whole-Genome Characterization of Epidemic Neisseria meningitidis Serogroup C and Resurgence of Serogroup W, Niger, 2015

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013–2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.

The Establishment and Diversification of Epidemic-Associated Serogroup W Meningococcus in the African Meningitis Belt, 1994 to 2012

Meningococcal disease (meningitis and bloodstream infections) threatens millions of people across the meningitis belt of sub-Saharan Africa. A vaccine introduced in 2010 protects against Africa’s then-most common cause of meningococcal disease, N. meningitidis serogroup A. However, other serogroups continue to cause epidemics in the region—including serogroup W. The rapid identification of strains that have been associated with prior outbreaks can improve the assessment of outbreak risk and enable timely preparation of public health responses, including vaccination. Phylogenetic analysis of newly sequenced serogroup W strains isolated from 1994 to 2012 identified two groups of strains linked to large epidemics in Burkina Faso, one being descended from a strain that caused an outbreak during the Hajj pilgrimage in 2000. We find that applying whole-genome sequencing to meningococcal disease surveillance collections improves the discrimination among strains, even within a single nation-wide epidemic, which can be used to better understand pathogen spread. ABSTRACT Epidemics of invasive meningococcal disease (IMD) caused by meningococcal serogroup A have been eliminated from the sub-Saharan African so-called “meningitis belt” by the meningococcal A conjugate vaccine (MACV), and yet, other serogroups continue to cause epidemics. Neisseria meningitidis serogroup W remains a major cause of disease in the region, with most isolates belonging to clonal complex 11 (CC11). Here, the genetic variation within and between epidemic-associated strains was assessed by sequencing the genomes of 92 N. meningitidis serogroup W isolates collected between 1994 and 2012 from both sporadic and epidemic IMD cases, 85 being from selected meningitis belt countries. The sequenced isolates belonged to either CC175 (n = 9) or CC11 (n = 83). The CC11 N. meningitidis serogroup W isolates belonged to a single lineage comprising four major phylogenetic subclades. Separate CC11 N. meningitidis serogroup W subclades were associated with the 2002 and 2012 Burkina Faso epidemics. The subclade associated with the 2012 epidemic included isolates found in Burkina Faso and Mali during 2011 and 2012, which descended from a strain very similar to the Hajj (Islamic pilgrimage to Mecca)-related Saudi Arabian outbreak strain from 2000. The phylogeny of isolates from 2012 reflected their geographic origin within Burkina Faso, with isolates from the Malian border region being closely related to the isolates from Mali. Evidence of ongoing evolution, international transmission, and strain replacement stresses the importance of maintaining N. meningitidis surveillance in Africa following the MACV implementation. IMPORTANCE Meningococcal disease (meningitis and bloodstream infections) threatens millions of people across the meningitis belt of sub-Saharan Africa. A vaccine introduced in 2010 protects against Africa’s then-most common cause of meningococcal disease, N. meningitidis serogroup A. However, other serogroups continue to cause epidemics in the region—including serogroup W. The rapid identification of strains that have been associated with prior outbreaks can improve the assessment of outbreak risk and enable timely preparation of public health responses, including vaccination. Phylogenetic analysis of newly sequenced serogroup W strains isolated from 1994 to 2012 identified two groups of strains linked to large epidemics in Burkina Faso, one being descended from a strain that caused an outbreak during the Hajj pilgrimage in 2000. We find that applying whole-genome sequencing to meningococcal disease surveillance collections improves the discrimination among strains, even within a single nation-wide epidemic, which can be used to better understand pathogen spread.

Large Cluster of Neisseria meningitidis Urethritis in Columbus, Ohio, 2015

Background Neisseria meningitidis (Nm) is a Gram-negative diplococcus that normally colonizes the nasopharynx and rarely infects the urogenital tract. On Gram stain of urethral exudates, Nm can be misidentified as the more common sexually transmitted pathogen Neisseria gonorrhoeae. Methods In response to a large increase in cases of Nm urethritis identified among men presenting for screening at a sexually transmitted disease clinic in Columbus, Ohio, we investigated the epidemiologic characteristics of men with Nm urethritis and the molecular and phylogenetic characteristics of their Nm isolates. The study was conducted between 1 January and 18 November 2015. Results Seventy-five Nm urethritis cases were confirmed by biochemical and polymerase chain reaction testing. Men with Nm urethritis were a median age of 31 years (interquartile range [IQR] = 24-38) and had a median of 2 sex partners in the last 3 months (IQR = 1-3). Nm cases were predominantly black (81%) and heterosexual (99%). Most had urethral discharge (91%), reported oral sex with a female in the last 12 months (96%), and were treated with a ceftriaxone-based regimen (95%). A minority (15%) also had urethral chlamydia coinfection. All urethral Nm isolates were nongroupable, ST-11 clonal complex (cc11), ET-15, and clustered together phylogenetically. Urethral Nm isolates were similar by fine typing (PorA P1.5-1,10-8, PorB 2-2, FetA F3-6), except 2, which had different PorB types (2-78 and 2-52). Conclusions Between January and November 2015, 75 urethritis cases due to a distinct Nm clade occurred among primarily black, heterosexual men in Columbus, Ohio. Future urogenital Nm infection studies should focus on pathogenesis and modes of sexual transmission.

Expansion of a urethritis-associated Neisseria meningitidis clade in the United States with concurrent acquisition of N. gonorrhoeae alleles

BackgroundIncreased reports of Neisseria meningitidis urethritis in multiple U.S. cities during 2015 have been attributed to the emergence of a novel clade of nongroupable N. meningitidis within the ST-11 clonal complex, the “U.S. NmNG urethritis clade”. Genetic recombination with N. gonorrhoeae has been proposed to enable efficient sexual transmission by this clade. To understand the evolutionary origin and diversification of the U.S. NmNG urethritis clade, whole-genome phylogenetic analysis was performed to identify its members among the N. meningitidis strain collection from the Centers for Disease Control and Prevention, including 209 urogenital and rectal N. meningitidis isolates submitted by U.S. public health departments in eleven states starting in 2015.ResultsThe earliest representatives of the U.S. NmNG urethritis clade were identified from cases of invasive disease that occurred in 2013. Among 209 urogenital and rectal isolates submitted from January 2015 to September 2016, the clade accounted for 189/198 male urogenital isolates, 3/4 female urogenital isolates, and 1/7 rectal isolates. In total, members of the clade were isolated in thirteen states between 2013 and 2016, which evolved from a common ancestor that likely existed during 2011. The ancestor contained N. gonorrhoeae-like alleles in three regions of its genome, two of which may facilitate nitrite-dependent anaerobic growth during colonization of urogenital sites. Additional gonococcal-like alleles were acquired as the clade diversified. Notably, one isolate contained a sequence associated with azithromycin resistance in N. gonorrhoeae, but no other gonococcal antimicrobial resistance determinants were detected.ConclusionsInterspecies genetic recombination contributed to the early evolution and subsequent diversification of the U.S. NmNG urethritis clade. Ongoing acquisition of N. gonorrhoeae alleles by the U.S. NmNG urethritis clade may facilitate the expansion of its ecological niche while also increasing the frequency with which it causes urethritis.

Neisseria genomics: current status and future perspectives

Abstract High-throughput whole genome sequencing has unlocked a multitude of possibilities enabling members of the Neisseria genus to be examined with unprecedented detail, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae. To maximise the potential benefit of this for public health, it is becoming increasingly important to ensure that this plethora of data are adequately stored, disseminated and made readily accessible. Investigations facilitating cross-species comparisons as well as the analysis of global datasets will allow differences among and within species and across geographic locations and different times to be identified, improving our understanding of the distinct phenotypes observed. Recent advances in high-throughput platforms that measure the transcriptome, proteome and/or epigenome are also becoming increasingly employed to explore the complexities of Neisseria biology. An integrated approach to the analysis of these is essential to fully understand the impact these may have in the Neisseria genus. This article reviews the current status of some of the tools available for next generation sequence analysis at the dawn of the ‘post-genomic’ era.

Whole genome sequencing for investigations of meningococcal outbreaks in the United States: a retrospective analysis

Although rare in the U.S., outbreaks due to Neisseria meningitidis do occur. Rapid, early outbreak detection is important for timely public health response. In this study, we characterized U.S. meningococcal isolates (N = 201) from 15 epidemiologically defined outbreaks (2009–2015) along with temporally and geographically matched sporadic isolates using multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), and six whole genome sequencing (WGS) based methods. Recombination-corrected maximum likelihood (ML) and Bayesian phylogenies were reconstructed to identify genetically related outbreak isolates. All WGS analysis methods showed high degree of agreement and distinguished isolates with similar or indistinguishable PFGE patterns, or the same strain genotype. Ten outbreaks were caused by a single strain; 5 were due to multiple strains. Five sporadic isolates were phylogenetically related to 2 outbreaks. Analysis of 9 outbreaks using timed phylogenies identified the possible origin and estimated the approximate time that the most recent common ancestor emerged for outbreaks analyzed. U.S. meningococcal outbreaks were caused by single- or multiple-strain introduction, with organizational outbreaks mainly caused by a clonal strain and community outbreaks by divergent strains. WGS can infer linkage of meningococcal cases when epidemiological links are uncertain. Accurate identification of outbreak-associated cases requires both WGS typing and epidemiological data.

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis linage11.2 urethritis clade: mutations in the pilMNOPQ operon

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non‐encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64–256 µg ml–1). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384–1024 µg ml–1) and colistin (MIC 256 µg ml–1) as well as enhanced LL‐37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA‐mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high‐level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross‐resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High‐level resistance to AMPs may contribute to the pathogenesis of US_NmUC.

Outbreak of Neisseria meningitidis serogroup C outside the meningitis belt—Liberia, 2017: an epidemiological and laboratory investigation

BACKGROUND On April 25, 2017, a cluster of unexplained illnesses and deaths associated with a funeral was reported in Sinoe County, Liberia. Molecular testing identified Neisseria meningitidis serogroup C (NmC) in specimens from patients. We describe the epidemiological investigation of this cluster and metagenomic characterisation of the outbreak strain. METHODS We collected epidemiological data from the field investigation and medical records review. Confirmed, probable, and suspected cases were defined on the basis of molecular testing and signs or symptoms of meningococcal disease. Metagenomic sequences from patient specimens were compared with 141 meningococcal isolate genomes to determine strain lineage. FINDINGS 28 meningococcal disease cases were identified, with dates of symptom onset from April 21 to April 30, 2017: 13 confirmed, three probable, and 12 suspected. 13 patients died. Six (21%) patients reported fever and 23 (82%) reported gastrointestinal symptoms. The attack rate for confirmed and probable cases among funeral attendees was 10%. Metagenomic sequences from six patient specimens were similar to a sequence type (ST) 10217 (clonal complex [CC] 10217) isolate genome from Niger, 2015. Multilocus sequencing identified five of seven alleles from one specimen that matched ST-9367, which is represented in the PubMLST database by one carriage isolate from Burkina Faso, in 2011, and belongs to CC10217. INTERPRETATION This outbreak featured high attack and case fatality rates. Clinical presentation was broadly consistent with previous meningococcal disease outbreaks, but predominance of gastrointestinal symptoms was unusual compared with previous African meningitis epidemics. The outbreak strain was genetically similar to NmC CC10217, which caused meningococcal disease outbreaks in Niger and Nigeria. CC10217 had previously been identified only in the African meningitis belt. FUNDING US Global Health Security.

Molecular characterization of invasive meningococcal isolates in Burkina Faso as the relative importance of serogroups X and W increases, 2008–2012

BackgroundNeisseria meningitidis serogroup A disease in Burkina Faso has greatly decreased following introduction of a meningococcal A conjugate vaccine in 2010, yet other serogroups continue to pose a risk of life-threatening disease. Capsule switching among epidemic-associated serogroup A N. meningitidis strains could allow these lineages to persist despite vaccination. The introduction of new strains at the national or sub-national levels could affect the epidemiology of disease.MethodsIsolates collected from invasive meningococcal disease in Burkina Faso between 2008 and 2012 were characterized by serogrouping and molecular typing. Genome sequences from a subset of isolates were used to infer phylogenetic relationships.ResultsThe ST-5 clonal complex (CC5) was identified only among serogroup A isolates, which were rare after 2010. CC181 and CC11 were the most common clonal complexes after 2010, having serogroup X and W isolates, respectively. Whole-genome phylogenetic analysis showed that the CC181 isolates collected during and after the epidemic of 2010 formed a single clade that was closely related to isolates collected in Niger during 2005 and Burkina Faso during 2007. Geographic population structure was identified among the CC181 isolates, where pairs of isolates collected from the same region of Burkina Faso within a single year had less phylogenetic diversity than the CC181 isolate collection as a whole. However, the reduction of phylogenetic diversity within a region did not extend across multiple years. Instead, CC181 isolates collected during the same year had lower than average diversity, even when collected from different regions, indicating geographic mixing of strains across years. The CC11 isolates were primarily collected during the epidemic of 2012, with sparse sampling during 2011. These isolates belong to a clade that includes previously described isolates collected in Burkina Faso, Mali, and Niger from 2011 to 2015. Similar to CC181, reduced phylogenetic diversity was observed among CC11 isolate pairs collected from the same regions during a single year.ConclusionsThe population of disease-associated N. meningitidis strains within Burkina Faso was highly dynamic between 2008 and 2012, reflecting both vaccine-imposed selection against serogroup A strains and potentially complex clonal waves of serogroup X and serogroup W strains.

Automated Next Generation Sequencing Bioinformatics Pipelines for Pathogen Discovery and Surveillance

Next-generation sequencing (NGS) has become a vital tool in clinical microbiology, with numerous applications in infectious disease diagnostics, outbreak investigations, and public health surveillance. Although the NGS technology enables comprehensive pathogen detection in a relatively short time at a low cost, the enormous amount of genomics data generated creates a critical challenge of effectively organizing, archiving, analyzing, and reporting the results within a clinically relevant timeframe. Automated pipelines provide the first step in standardizing NGS data processing and reporting, thus eliminating the common bottlenecks in bioinformatics analyses, and providing rapid turnaround. Here, we present the Viral NGS Pipeline optimized for identification and whole genome assembly of viruses, and the Bacterial Meningococcus Genome Analysis Platform (BMGAP), designed for genotypic characterization of meningitis pathogens. These respective pipelines have been used to analyze more than 11,000 clinical samples and isolates. The pipelines are deployable on both standalone and cloud-based servers, enabling their accessibility to internal CDC users, as well as external partners, including state public health laboratories and other collaborators worldwide. These automated pipelines have the potential to contribute to the development of unbiased NGS-based clinical assays for pathogen detection that demand rapid turnaround times, and are expected to play a key role in infectious disease surveillance in the future.