Ultrastructural, metabolic and genetic characteristics of determinants facilitating the acquisition of macrolide resistance by Streptococcus pneumoniae.

AIMS: To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization. METHODS AND RESULTS: Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose. CONCLUSIONS: Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets.

Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C.

Introduction: Outer membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined. Methods: Ten paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays. Results and discussion: Post-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, ß-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design.

Continuing genomic evolution of the Neisseria meningitidis cc11.2 urethritis clade, NmUC: a narrative review.

Neisseria meningitidis (Nm) is a bacterial pathogen responsible for invasive meningococcal disease. Though typically colonizing the nasopharynx, multiple outbreaks of meningococcal urethritis were first reported in 2015-2016; outbreaks originally presumed to be caused by Neisseria gonorrhoeae (Ng). Genomic analysis revealed that the Nm isolates causing these outbreaks were a distinct clade, and had integrated gonococcal DNA at multiple genomic sites, including the gonococcal denitrification apparatus aniA-norB, a partial gonococcal operon of five genes containing ispD, and the acetylglutamate kinase gene argB with the adjacent gonococcal locus NGO0843. The urethritis isolates had also deleted the group C capsule biosynthesis genes cssA/B/C and csc, resulting in loss of capsule. Collectively, these isolates form the N. meningitidis urethritis clade (NmUC). Genomic analysis of recent (2016-2022) NmUC isolates revealed that the genomic features have been maintained in the clade, implying that they are important for NmUC's status as a urogenital pathogen. Furthermore, the analysis revealed the emergence of a sub-clade, designated NmUC-B, phylogenetically separated from the earlier NmUC-A. This sub-clade has integrated additional gonococcal alleles into the genome, including alleles associated with antimicrobial resistance. NmUC continues to adapt to a urethral niche and evolve as a urogenital pathogen.

Acquisition of Gonococcal AniA-NorB Pathway by the Neisseria meningitidis Urethritis Clade Confers Denitrifying and Microaerobic Respiration Advantages for Urogenital Adaptation.

Neisseria meningitidis historically has been an infrequent and sporadic cause of urethritis and other urogenital infections. However, a nonencapsulated meningococcal clade belonging to the hyperinvasive clonal complex 11.2 lineage has recently emerged and caused clusters of urethritis cases in the United States and other countries. One of the genetic signatures of the emerging N. meningitidis urethritis clade (NmUC) is a chromosomal gene conversion event resulting in the acquisition of the Neisseria gonorrhoeae denitrification apparatus-the N. gonorrhoeae alleles encoding the nitrite reductase AniA, the nitric oxide (NO) reductase NorB, and the intergenic promoter region. The biological importance of the N. gonorrhoeae AniA-NorB for adaptation of the NmUC to a new environmental niche is investigated herein. We found that oxygen consumption, nitrite utilization, and NO production were significantly altered by the conversion event, resulting in different denitrifying aerobic and microaerobic growth of the clade. Further, transcription of aniA and norB in NmUC isolates differed from canonical N. meningitidis, and important polymorphisms within the intergenic region, which influenced aniA promoter activity of the NmUC, were identified. The contributions of three known meningococcal regulators (NsrR, FNR, and NarQP) in controlling the denitrification pathway and endogenous NO metabolism were distinct. Overall, transcription of aniA was dampened relative to canonical N. meningitidis, and this correlated with the lower NO accumulation in the clade. Denitrification and microaerobic respiration were bolstered, and protection against host-derived NO was likely enhanced. The acquisition of the N. gonorrhoeae denitrification pathway by the NmUC supports the clade's adaptation and survival in a microaerobic urogenital environment.

Dissemination of Tn916-Related Integrative and Conjugative Elements in Streptococcus pneumoniae Occurs by Transformation and Homologous Recombination in Nasopharyngeal Biofilms.

Multidrug resistance in Streptococcus pneumoniae (or pneumococcus) continues to be a global challenge. An important class of antibiotic resistance determinants disseminating in S. pneumoniae are >20-kb Tn916-related integrative and conjugative elements (ICEs), such as Tn2009, Tn6002, and Tn2010. Although conjugation has been implicated as the transfer mechanism for ICEs in several bacteria, including S. pneumoniae, the molecular basis for widespread dissemination of pneumococcal Tn916-related ICEs remains to be fully elucidated. We found that Tn2009 acquisition was not detectable via in vitro transformation nor conjugative mating with donor GA16833, yielding a transfer frequency of <10-7. GA16833 Tn2009 conjugative gene expression was not significantly induced, and ICE circular intermediate formation was not detected in biofilms. Consistently, Tn2009 transfer efficiency in biofilms was not affected by deletion of the ICE conjugative gene ftsK. However, GA16833 Tn2009 transfer occurred efficiently at a recombination frequency (rF) of 10-4 in dual-strain biofilms formed in a human nasopharyngeal cell bioreactor. DNase I addition and deletions of the early competence gene comE or transformation apparatus genes comEA and comEC in the D39 recipient strain prevented Tn2009 acquisition (rF of <10-7). Genome sequencing and single nucleotide polymorphism analyses of independent recombinants of recipient genotype identified ~33- to ~55-kb donor DNAs containing intact Tn2009, supporting homologous recombination. Additional pneumococcal donor and recipient combinations were demonstrated to efficiently transfer Tn916-related ICEs at a rF of 10-4 in the biofilms. Tn916-related ICEs horizontally disseminate at high frequency in human nasopharyngeal S. pneumoniae biofilms by transformation and homologous recombination of >30-kb DNA fragments into the pneumococcal genome. IMPORTANCE The World Health Organization has designated Streptococcus pneumoniae as a priority pathogen for research and development of new drug treatments due to extensive multidrug resistance. Multiple strains of S. pneumoniae colonize and form mixed biofilms in the human nasopharynx, which could enable exchange of antibiotic resistance determinants. Tn916-related integrative and conjugative elements (ICEs) are largely responsible for the widespread presence of macrolide and tetracycline resistance in S. pneumoniae. Utilizing a system that simulates colonization of donor and recipient S. pneumoniae strains in the human nasopharynx, efficient transfer of Tn916-related ICEs occurred in human nasopharyngeal biofilms, in contrast to in vitro conditions of planktonic cells with exogenous DNA. This high-frequency Tn916-related ICE transfer between S. pneumoniae strains in biofilms was due to transformation and homologous recombination, not conjugation. Understanding the molecular mechanism for dissemination of Tn916-related ICEs can facilitate the design of new strategies to combat antibiotic resistance.

Antibiotic Susceptibility Profile for the US Neisseria meningitidis Urethritis Clade.

The US Neisseria meningitidis urethritis clade (US_NmUC) harbors gonococcal deoxyribonucleic acid alleles and causes gonorrhea-like urogenital tract disease. A large convenience sample of US_NmUC isolates (N = 122) collected between January 2015 and December 2019 in Columbus, Ohio demonstrated uniform susceptibility to antibiotics recommended for gonorrhea treatment and meningococcal chemoprophylaxis.

Ultrastructural, metabolic and genetic determinants of the acquisition of macrolide resistance by Streptococcus pneumoniae.

Aim: Streptococcus pneumoniae (Spn) acquires genes for macrolide resistance, MEGA or ermB, in the human host. These genes are carried either in the chromosome, or on integrative conjugative elements (ICEs). Here, we investigated molecular determinants of the acquisition of macrolide resistance. Methods and Results: Whole genome analysis was conducted for 128 macrolide-resistant pneumococcal isolates to identify the presence of MEGA (44.5%, 57/128) or ermB (100%), and recombination events in Tn916-related elements or in the locus comCDE encoding competence genes. Confocal and electron microscopy studies demonstrated that, during the acquisition of macrolide resistance, pneumococcal strains formed clusters of varying size, with the largest aggregates having a median size of ~1600 µm2. Remarkably, these pneumococcal aggregates comprise both encapsulated and nonencapsulated pneumococci, exhibited physical interaction, and spanned extracellular and intracellular compartments. We assessed the recombination frequency (rF) for the acquisition of macrolide resistance by a recipient D39 strain, from pneumococcal strains carrying MEGA (~5.4 kb) in the chromone, or in large ICEs (>23 kb). Notably, the rF for the acquisition of MEGA, whether in the chromosome or carried on an ICE was similar. However, the rF adjusted to the acquisition of the full-length ICE (~52 kb), compared to that of the capsule locus (~23 kb) that is acquired by transformation, was three orders of magnitude higher. Finally, metabolomics studies revealed a link between the acquisition of ICE and the metabolic pathways involving nicotinic acid and sucrose. Conclusions: Extracellular and intracellular pneumococcal clusters facilitate the acquisition of full-length ICE at a rF higher than that of typical transformation events, involving distinct metabolic changes that present potential targets for interventions.

Rapid, label-free antibiotic susceptibility determined directly from positive blood culture.

Bacterial bloodstream infections are a significant cause of global morbidity and mortality. Constrained by low bacterial burdens of 1-100 colony-forming-units per ml blood (CFU/ml), clinical diagnosis relies on lengthy culture amplification and isolation steps prior to identification and antibiotic susceptibility testing (AST). The resulting >60-h time to actionable treatment not only negatively impacts patient outcomes, but also increases the misuse and overuse of broad-spectrum antibiotics that accelerates the rise in multidrug resistant infections. Consequently, the development of novel technologies capable of rapidly recovering bacteria from blood-derived samples is crucial to human health. To address this need, we report a novel bacterial recovery technology from positive blood cultures that couples selective hemolysis with centrifugation through a sucrose cushion to perform rapid, background-free cytometric ASTs without long subculturing steps. Demonstrated on the most common bloodstream infection-causing bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria are rapidly recovered (≤15 min) with minimal user intervention. Susceptibilities of recovered bacteria are readily performed via high throughput flow cytometry with excellent agreement with much slower, standard microbroth dilution assays. Altogether, this novel direct-from-positive blood culture AST technology enables susceptibility determinations within as little as 5 h, post blood culture positivity.

Infection With the US Neisseria meningitidis Urethritis Clade Does Not Lower Future Risk of Urethral Gonorrhea.

BACKGROUND: Cross-protective immunity between Neisseria meningitidis (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 3 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: .65-2.48). Men with urethral US_NmUC had nonsignificantly increased Ng risk compared with men with urethral Ct (aHR: 1.51; 95% CI: .79-2.88), and significantly increased Ng risk compared with 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.

A Narrative Review of the W, X, Y, E, and NG of Meningococcal Disease: Emerging Capsular Groups, Pathotypes, and Global Control.

Neisseria meningitidis, carried in the human nasopharynx asymptomatically by ~10% of the population, remains a leading cause of meningitis and rapidly fatal sepsis, usually in otherwise healthy individuals. The epidemiology of invasive meningococcal disease (IMD) varies substantially by geography and over time and is now influenced by meningococcal vaccines and in 2020-2021 by COVID-19 pandemic containment measures. While 12 capsular groups, defined by capsular polysaccharide structures, can be expressed by N. meningitidis, groups A, B, and C historically caused most IMD. However, the use of mono-, bi-, and quadrivalent-polysaccharide-conjugate vaccines, the introduction of protein-based vaccines for group B, natural disease fluctuations, new drugs (e.g., eculizumab) that increase meningococcal susceptibility, changing transmission dynamics and meningococcal evolution are impacting the incidence of the capsular groups causing IMD. While the ability to spread and cause illness vary considerably, capsular groups W, X, and Y now cause significant IMD. In addition, group E and nongroupable meningococci have appeared as a cause of invasive disease, and a nongroupable N. meningitidis pathotype of the hypervirulent clonal complex 11 is causing sexually transmitted urethritis cases and outbreaks. Carriage and IMD of the previously "minor" N. meningitidis are reviewed and the need for polyvalent meningococcal vaccines emphasized.

Neisseria meningitidis Urethritis Outbreak Isolates Express a Novel Factor H Binding Protein Variant That Is a Potential Target of Group B-Directed Meningococcal (MenB) Vaccines.

Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.

Repeat Episodes of Symptomatic Urethritis Due to a Uropathogenic Meningococcal Clade.

In 2015, we identified a non-groupable clade of Neisseria meningitidis that causes urethritis in men (the US_NmUC). Because repeat infection is common with Neisseria gonorrhoeae, we examined whether reinfection also occurs with the US_NmUC. We provide evidence that men are susceptible to repeat episodes of urethritis from the US_NmUC.

Author Correction: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.

Change history: We could not replicate the results in Fig. 2a and g of this Letter, and new information has revealed a flaw in the interpretation of Fig. 2h. As a result, we do not have evidence to support RNA degradation as the mechanism that underlies Cas9-mediated regulation of FTN_1103 mRNA expression; see accompanying Amendment. This has not been corrected online.

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 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.

FAST: Rapid determinations of antibiotic susceptibility phenotypes using label-free cytometry.

Sepsis, a life-threatening immune response to blood infections (bacteremia), has a ∼30% mortality rate and is the 10th leading cause of US hospital deaths. The typical bacterial loads in adult septic patients are ≤100 bacterial cells (colony forming units, CFU) per ml blood, while pediatric patients exhibit only ∼1000 CFU/ml. Due to the low numbers, bacteria must be propagated through ∼24-hours blood cultures to generate sufficient CFUs for diagnosis and further analyses. Herein, we demonstrate that, unlike other rapid post-blood culture antibiotic susceptibility tests (ASTs), our phenotypic approach can drastically accelerate ASTs for the most common sepsis-causing gram-negative pathogens by circumventing long blood culture-based amplification. For all blood isolates of multi-drug resistant pathogens investigated (Escherichia coli, Klebsiella pneumoniae, and Acinetobacter nosocomialis), effective antibiotic(s) were readily identified within the equivalent of 8 hours from initial blood draw using <0.5 mL of adult blood per antibiotic. These methods should drastically improve patient outcomes by significantly reducing time to actionable treatment information and reduce the incidence of antibiotic resistance. © 2018 International Society for Advancement of Cytometry.

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

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.

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.

Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen.

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 ∼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.

Notes from the Field: Increase in Neisseria meningitidis-Associated Urethritis Among Men at Two Sentinel Clinics - Columbus, Ohio, and Oakland County, Michigan, 2015.

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) (1-5). Because Nm colonizes the nasopharynx, men who receive oral sex (fellatio) can acquire urethral Nm infections (1,3,5). This report describes an increase in Nm-associated urethritis in men attending sexual health clinics in Columbus, Ohio, and Oakland County, Michigan.

Regulation of capsule in Neisseria meningitidis.

Neisseria meningitidis, a devastating pathogen exclusive to humans, expresses capsular polysaccharides that are the major meningococcal virulence determinants and the basis for successful meningococcal vaccines. With rare exceptions, the expression of capsule (serogroups A, B, C, W, X, Y) is required for systemic invasive meningococcal disease. Changes in capsule expression or structure (e.g. hypo- or hyper-encapsulation, capsule "switching", acetylation) can influence immunologic diagnostic assays or lead to immune escape. The loss or down-regulation of capsule is also critical in meningococcal biology facilitating meningococcal attachment, microcolony formation and the carriage state at human mucosal surfaces. Encapsulated meningococci contain a cps locus with promoters located in an intergenic region between the biosynthesis and the conserved capsule transport operons. The cps intergenic region is transcriptionally regulated (and thus the amount of capsule expressed) by IS element insertion, by a two-component system, MisR/MisS and through sequence changes that result in post-transcriptional RNA thermoregulation. Reversible on-off phase variation of capsule expression is controlled by slipped strand mispairing of homo-polymeric tracts and by precise insertion and excision of IS elements (e.g. IS1301) in the biosynthesis operon. Capsule structure can be altered by phase-variable expression of capsular polymer modification enzymes or "switched" through transformation and homologous recombination of different polymerases. Understanding the complex regulation of meningococcal capsule has important implications for meningococcal biology, pathogenesis, diagnostics, current and future vaccine development and vaccine strategies.