Background Urethral infections caused by an emerging nongroupable (NG) urethrotropic clade of Neisseria meningitidis (Nm) were first reported in the United States in 2015 (the "U.S. NmNG urethritis clade"). Here we evaluate for the presence of other urethral pathogens in men with U.S. NmNG urethritis clade infection. Methods: We evaluated 129 urine specimens collected from men at a sexual health clinic, including 33 from patients with culture-confirmed or suspected urethral Nm infection and 96 specimens in which nucleic acid amplification test detected Neisseria gonorrhoeae (Ng), Chlamydia trachomatis (Ct), both pathogens, or neither pathogen. Nm was detected first by real-time PCR, followed by metagenomic shotgun sequencing of 91 specimens to identify coinfections. Nm genomes were sequenced following selective whole genome amplification when possible. Results: Metagenomic sequencing detected Nm in 16 of 17 specimens from culture-confirmed Nm cases, with no coinfection by other conventional urethral pathogens. Metagenomic sequencing also detected Nm in three Ct positive specimens, one specimen positive for both Ng and Ct, and nine specimens with negative Ng and Ct results, eight of which had suspected Neisseria infections. Nm from culture-confirmed Nm cases belonged to the U.S. NmNG urethritis clade, while Nm identified in other specimens belonged to multiple clonal complexes. Additional urethral pathogens were predominant in non-Nm specimens, including Ng, Ct, Mycoplasma genitalium, Ureaplasma urealyticum, and herpes simplex virus type-2. Conclusions: Coinfection with other conventional urethral pathogens is rare in men with culture-confirmed U.S. NmNG urethritis clade infection and points to the strong association of this clade with disease.

BACKGROUND: Cross-protective immunity between Neisseria meninigitidis (Nm) and Neisseria gonorrhoeae (Ng) may inform gonococcal vaccine development. Meningococcal serogroup B (MenB) outer membrane vesicle (OMV) vaccines confer modest protection against gonorrhea. However, whether urethral Nm infection protects against gonorrhea is unknown. We examined gonorrhea risk among men with US Nm urethritis clade (US_NmUC) infections. METHODS: We conducted a retrospective cohort study of men with urethral US_NmUC (N=128) between January 2015 and April 2018. Using diagnosis date as the baseline visit, we examined Ng status at return visits to compute urethral Ng risk. We compared these data to three referent populations: men with urethral Ng (N=253), urethral chlamydia (Ct) (N=251), and no urethral Ng or Ct (N=255). We conducted sensitivity analyses to assess varied approaches to censoring, missing data, and anatomical site of infection. We also compared sequences of protein antigens in the OMV-based MenB-4C vaccine, US_NmUC, and Ng. RESULTS: Participants were primarily Black (65%) and heterosexual (82%). Over follow-up, 91 men acquired urethral Ng. Men with urethral US_NmUC had similar Ng risk to men with prior urethral Ng (adjusted hazard ratio (AHR): 1∙27, 95% CI: 0∙65-2∙48). Men with urethral US_NmUC had insignificantly increased Ng risk compared to men with urethral Ct (AHR: 1∙51, 95% CI: 0∙79-2∙88), and significantly increased Ng risk compared to men without urethral Ng or Ct (AHR: 3∙55, 95% CI: 1∙27-9∙91). Most of the protein antigens analyzed shared high sequence similarity. CONCLUSIONS: Urethral US_NmUC infection did not protect against gonorrhea despite substantial sequence similarities in shared protein antigens.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole genome amplification (SWGA), an isothermal multiple displacement amplification-based method, to efficiently enrich, sequence and de novo assemble Nm DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of Nm reads by 2-3 orders of magnitude enabling identification of at least 90% of the 1605 Nm core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial load were processed by SWGA before sequencing and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the Nm pathogen, therefore enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rate. SWGA produces enough reads for phylogenetic and allelic analysis with a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.

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 microg ml(-1) ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 microg ml(-1) ) and colistin (MIC 256 microg 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.

BACKGROUND: Increased 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. RESULTS: The 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. CONCLUSIONS: Interspecies 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.

National data show an increasing trend of reduced azithromycin susceptibility (AZM-RS) among Neisseria gonorrhoeae (Ng) isolates, especially in the Midwest.1 Sentinel surveillance data from the Gonococcal Isolate Surveillance Project, which examines isolates from men with urethritis from 27 sites across the United States, indicates that the proportion of AZM-RS Ng isolates (minimum inhibitory concentration [MIC] ≥2 μg/mL) increased 4-fold between 2013 (0.6%) and 2014 (2.5%).1 Most AZM-RS Ng infections occur in men who have sex with men.1,2

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 1, which had a different PorB type (2-78). 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.

Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an approximately 20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.

Neisseria meningitidis (Nm) urogenital infections, although less common than infections caused by Neisseria gonorrhoeae (Ng), have been associated with urethritis, cervicitis, proctitis, and pelvic inflammatory disease. Nm can appear similar to Ng on Gram stain analysis (gram-negative intracellular diplococci). Because Nm colonizes the nasopharynx, men who receive oral sex (fellatio) can acquire urethral Nm infections. This report describes an increase in Nm-associated urethritis in men attending sexual health clinics in Columbus, Ohio, and Oakland County, Michigan.