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Relevance. Unencapsulated strains of meningococcus (NmNG) very rarely cause invasive meningococcal disease. A new ciprofloxacin-resistant strain NmNG ST-175 cc175, which has recently caused several cases of invasive meningococcal infection in Europe, has been discovered in the Russian Federation.Aim. To compare the new Russian strains of NmNG ST-175 with the already characterized NmNG ST-175 and to analyze the genetic markers associated with antibiotic resistance.Materials and methods. The nucleotide sequences of NmNG ST-175 strains of more than 2 million base pairs were exported from the PubMLST database. Comparison was carried out for 1605 core genome loci using the N. meningitidis cgMLST v1.0 ". Genetic relationships of 127 NmNG ST-175 strains, including 8 Russian carrier strains, were visualized using the SplitsTree software (version 4.16.2).Results. Of the eight Russian strains, six found themselves in a cluster with German and Swedish isolates that caused invasive meningococcal infection. Two more Russian strains were closest to the isolates of the cluster, including carrier isolates from England. Seven strains showed resistance to ciprofloxacin and possessed the gyrA-187 and gyrA-152 alleles, which, based on the phylogenetic analysis of the alleles, belonged to the genetic branches of Neisseria cinerea and Nm.Conclusion. Protein vaccines alone could potentially provide protection against ST-175 NmNG. It seems promising to study the antigenic characteristics of Russian Nm strains, including NmNG ST-175, to assess the potential vaccination coverage with existing protein vaccines, the possibility of their registration on the territory of the Russian Federation, as well as the development of domestic vaccines.

Objectives To investigate the spread of ceftriaxone-resistant Neisseria gonorrhoeae lineages similar to strains H041 (2009) and FC428 (2015), we characterized 55 strains collected in 2013 from hospitals across Japan. Methods Susceptibility testing and whole-genome sequencing. Results Susceptibility rates were 58% for cefixime and 98% for ceftriaxone. The 55 strains were whole-genome sequenced and classified into nine MLST-STs. MLST-ST1901 was the most prevalent (n = 19) followed by MLST-ST7363 (n = 12) and MLST-ST7359 (n = 11). The most prevalent penA [encoding penicillin binding protein 2 (PBP2)] mosaic types, based on the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) scheme, were 10.001 (n = 20) followed by 34.001 (n = 13). The H041 and FC428 strains were not detected; however, a single ceftriaxone-resistant strain (TUM15748) with a MIC of 0.5 mg/L ceftriaxone was identified. The TUM15748 strain belonged to MLST-ST7359 and N. gonorrhoeae multiantigen sequence typing-ST6771, and had a novel PBP2 (PBP2TUM15748, penA type 169.001). The amino acid sequence of PBP2TUM15748 showed partial similarity to that of PBP2 from N. gonorrhoeae GU140106 and commensal Neisseria perflava and Neisseria cinerea. Natural transformation and recombination experiments using full-length TUM15748 penA showed that the ceftriaxone MICs of transformants increased 16-fold or more compared with the parental ceftriaxone-susceptible recipient strain (NG9807, belonging to MLST-ST7363). No ceftriaxone-resistant MLST-ST7359 strains have previously been reported. Conclusions We showed here that a ceftriaxone-susceptible lineage acquired a mutant PBP2 mosaic type, integrating partial PBP2 sequences from commensal Neisseria species, resulting in the emergence of ceftriaxone-resistant strains.

Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.

Objectives The continuous emergence of ceftriaxone-resistant Neisseria gonorrhoeae strains threatens the effectiveness of current treatment regimens for gonorrhoea. The objective of the present study was to characterize three ceftriaxone-resistant N. gonorrhoeae strains with a novel mosaic penA allele isolated in China. Methods Three ceftriaxone-resistant Neisseria gonorrhoeae strains (GC150, GC161 and GC208) isolated in 2017 were characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST), MLST and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR). Recombination analyses were performed using the SimPlot software. Results Three strains had the same antibiotic resistance profiles, with resistance to ceftriaxone (MIC 0.5 mg/L), ciprofloxacin (MIC 8.0 mg/L), penicillin (MIC 2.0 mg/L) and tetracycline (MIC 2.0–8.0 mg/L). STs were assigned as MLST7360, NG-MAST14292 and NG-STAR1611/NG-STAR1612. The penA gene of these three strains differed from previous ceftriaxone-resistant gonococcal strains and harboured a novel mosaic allele (penA-121.001). Like N. gonorrhoeae FC428, a widely disseminated ceftriaxone-resistant strain that was initially described in Japan in 2015, all strains also possessed substitutions A311V and T483S in PBP2, which are associated with resistance to ceftriaxone. Potential recombination events were detected in penA between N. gonorrhoeae strain FC428 and commensal Neisseria species. Our results provide further evidence that the commensal Neisseria species (Neisseria cinerea and Neisseria perflava) can serve as a reservoir of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains. Conclusions The emergence of such strains may be the result of the interspecies recombination of penA genes between N. gonorrhoeae strain FC428 and commensal Neisseria species.

The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.

Peritonitis can be a lethal outcome of peritoneal dialysis (PD), often leading to significant morbidity and mortality. It is caused mostly by gram-positive organisms. Neisseria cinerea is a gram-negative nasal and oropharyngeal commensal, rarely reported as an etiology of peritonitis in PD patients. Our patient was a 37-year-old female on continuous ambulatory peritoneal dialysis for the last seven years, who developed peritonitis found to be from Neisseria cinerea. She didn't respond to broad-spectrum antibiotics well and had to be switched to intermittent hemodialysis. We highlight this important microorganism that can lead to significant morbidity and an unfortunate change in dialysis modality.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Neisseria cinerea is a commensal bacteria of the human oropharynx, not thought to be pathogenic, and is rarely associated with serious infections, including bacteremia. Case reports involving invasive N. cinerea infections are uncommon in the literature. Retropharyngeal abscesses are unusual in adults, and are usually attributable to local trauma.Based on a review of the literature, Neisseria cinerea bacteremia secondary to a retropharyngeal abscess has not been described. We present a unique case of an elderly female without clear predisposing factors for a retropharyngeal abscess, who presented with a N. cinerea bacteremia and was found to have an asymptomatic retropharyngeal abscess.

Type VI Secretion Systems (T6SS) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp expression on N. cinerea T6SS-dependent killing and show that expression of Tfp by prey strains enhances their susceptibility to T6SS, by keeping them in close proximity of T6SS-wielding attacker strains. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Commensal non-pathogenicNeisseriaspp. live within the human host alongside the pathogenicNeisseria meningitidisandNeisseria gonorrhoeaeand due to natural competence, horizontal gene transfer within the genus is possible and has been observed. Four distinctNeisseriaspp. isolates taken from the throats of two human volunteers have been assessed here using a combination of microbiological and bioinformatics techniques. Three of the isolates have been identified asNeisseria subflavabiovarperflavaand one asNeisseria cinerea. Specific gene clusters have been identified within these commensal isolate genome sequences that are believed to encode a Type VI Secretion System, a newly identified CRISPR system, a Type IV Secretion System unlike that in otherNeisseriaspp., a hemin transporter, and a haem acquisition and utilization system. This investigation is the first to investigate these systems in either the non-pathogenic or pathogenicNeisseriaspp. In addition, theN. subflavabiovarperflavapossess previously unreported capsule loci and sequences have been identified in all four isolates that are similar to genes seen within the pathogens that are associated with virulence. These data from the four commensal isolates provide further evidence for aNeisseriaspp. gene pool and highlight the presence of systems within the commensals with functions still to be explored.

The present study highlights challenges in the veterinary microbiology diagnostic laboratory in the identification of bacteria responsible for infections in veterinary settings, particularly when evidence-based data is lacking. A 1.8-year-old neutered male domestic cat (FIV/FeLV negative) was presented to a veterinary practice in April 2016 with a history of left unilateral mild conjunctivitis that was empirically treated with fusidic acid and chloramphenicol. In January 2017, the same animal was presented with chronic left unilateral conjunctivitis and an eye swab was submitted for microbiological culture and susceptibility testing. Significant growth was not detected in two samples tested. Finally, in February 2017 another eye swab produced a slow growing pure culture identified by VITEK 2 as Neisseria cinerea (94 % confidence). Given the morphology and multidrug resistance profile of the isolate a 16S rRNA PCR was performed for definitive identification. The nucleotide sequence of the PCR amplicon was 99 % homologous to Acinetobacter equi sp. nov. strain 114. Veterinary microbiology diagnostic laboratories play an important role worldwide, not only in preserving animal health and welfare but also in controlling the spread of zoonotic pathogens. The lack of evidence-based information on the ocular microbiome of healthy cats and the complexity of bacterial ecosystems renders the interpretation of results difficult. A further problem for both the laboratory and the clinician is the lack of interpretive criteria for antibiotic susceptibility test results for some types of infections in animals (including those caused by Acinetobacter ) and the complete unavailability of criteria for topical antibiotic preparations.

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces., Author summary Commensal and pathogenic bacteria can establish long term relationships with their hosts. Despite this, very little is known about the processes of attachment, replication and organisation of commensal bacteria at epithelial surfaces. In this work, we have examined how Neisseria cinerea, a typically commensal species that colonises the human nasopharynx similar to the closely-related pathogen Neisseria meningitidis, engages with human epithelial cells. We show that N. cinerea on human epithelial cells mimics some of the behaviour of the meningococcus, but have identified subtle differences that distinguish the two species. Furthermore, we show that the presence of N. cinerea affects the interaction of N. meningitidis with epithelial cells, providing evidence that the interaction between two closely related species can affect pathogen colonisation of the epithelial surface.

Neisseria cinerea is a commensal species capable of colonizing the upper respiratory tract of humans. Bacteremia associated with this organism is very uncommon, with only seven previous cases described. We report a case of Neisseria cinerea bacteremia in a patient maintained on indefinite eculizumab therapy. It is the eighth reported case of Neisseria cinerea bacteremia and represents the first case in a patient receiving eculizumab.

Summary Background Non-meningococcal, non-gonococcal Neisseria spp. are typically commensal and rarely cause invasive disease. Eculizumab is a terminal complement inhibitor that increases susceptibility to meningococcal disease, but data on disease caused by typically-commensal Neisseria spp. are lacking. This series describes postmarketing reports of typically-commensal Neisseria spp. disease in patients receiving eculizumab. Methods We searched the FDA Adverse Event Reporting System (FAERS) and medical literature for reports of commensal Neisseria spp. disease in patients receiving eculizumab, from eculizumab U.S. approval (2007) through January 31, 2018. Results We identified seven FAERS reports (including one case also reported in the literature) of non-meningococcal, non-gonococcal Neisseria disease, including N. sicca (mucosa)/subflava (n = 2), N. cinerea (n = 2), N. sicca (mucosa) (n = 1), N. mucosa (n = 1, with concurrent alpha-hemolytic Streptococcus bacteremia), and N. flavescens (subflava) (n = 1). Four cases had sources of patient immunosuppression in addition to eculizumab. Three patients had sepsis (n = 2) or septic shock (n = 1). Five patients were bacteremic. All patients were hospitalized; the infections resolved with antibiotics. Conclusions Our search identified seven cases of disease from typically commensal Neisseria spp. in eculizumab recipients. These findings suggest that any Neisseria spp. identified from a normally sterile site in an eculizumab recipient could represent true infection warranting prompt treatment.

Background Microbial flora in several organs of HIV-infected individuals have been characterized; however, the palatine tonsil bacteriome and mycobiome and their relationship with each other remain unclear. Determining the palatine tonsil microbiome may provide a better understanding of the pathogenesis of oral and systemic complications in HIV-infected individuals. We conducted a cross-sectional study to characterize the palatine tonsil microbiome in HIV-infected individuals. Results Palatine tonsillar swabs were collected from 46 HIV-infected and 20 HIV-uninfected individuals. The bacteriome and mycobiome were analyzed by amplicon sequencing using Illumina MiSeq. The palatine tonsil bacteriome of the HIV-infected individuals differed from that of HIV-uninfected individuals in terms of the decreased relative abundances of the commensal genera Neisseria and Haemophilus. At the species level, the relative abundances and presence of Capnocytophaga ochracea, Neisseria cinerea, and Selenomonas noxia were higher in the HIV-infected group than those in the HIV-uninfected group. In contrast, fungal diversity and composition did not differ significantly between the two groups. Microbial intercorrelation analysis revealed that Candida and Neisseria were negatively correlated with each other in the HIV-infected group. HIV immune status did not influence the palatine tonsil microbiome in the HIV-infected individuals. Conclusions HIV-infected individuals exhibit dysbiotic changes in their palatine tonsil bacteriome, independent of immunological status. Electronic supplementary material The online version of this article (10.1186/s12866-018-1274-9) contains supplementary material, which is available to authorized users.

Neisseria cinerea is a commensal which usually resides in the human respiratory tract. Very rarely, the organism finds its way into the bloodstream causing severe bacteremia. So far, very few cases of Neisseria bacteremia have been reported. We report a case of a 78-year-old male, post-splenectomy, who presented with high fever, cough and shortness of breath. The patient was initially managed for septic shock with fluid resuscitations, vasopressors and broad-spectrum antibiotics. Later, the blood cultures grew gram-negative coccobacilli, Neisseria cinerea. The patient was successfully treated with intravenous ceftriaxone. This is the first case ever of Neisseria cinerea bacteremia in a post-splenectomy patient and ninth case overall. This case illustrates that the physicians should maintain heightened awareness for Neisseria cinerea bacteremia in post-splenectomy patients.

We report a case of a 3-day-old boy with Neisseria cinerea conjunctivitis, originally misidentified as Neisseria gonorrhoeae conjunctivitis. Neonates are at increased risk for disseminated gonococcal infection, and physicians should be cognizant of N cinerea and its potential to be mistaken for N gonorrhoeae.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae , but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 μg/ml) caused ceftriaxone resistance (MIC, 1 μg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3′-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae, but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 μg/ml) caused ceftriaxone resistance (MIC, 1 μg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3′-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W, and Y. More recently two protein-based vaccines, Bexsero and Trumenba, against meningococcal serogroup B strains have been licensed; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface-exposed lipoprotein that binds the negative complement regulator complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the functions of these genes and how immunization with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here, we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and that it is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH with affinity similar to that of meningococcal fHbp and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea. We found that immunization with Bexsero elicits serum bactericidal activity a gainst N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis and cross-reactive responses elicited by Bexsero suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W and Y. More recently two protein based vaccines, Bexsero® and Trumenba®, have been licenced against meningococcal serogroup B strains; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface exposed lipoprotein which binds the negative complement regulator, complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the function of these genes and how immunisation with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH at similar affinity as meningococcal fHbp, and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea We found that immunisation with Bexsero® elicits serum bactericidal activity against N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis, and cross reactive responses elicited by Bexsero® suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W, and Y. More recently two protein-based vaccines, Bexsero and Trumenba, against meningococcal serogroup B strains have been licensed; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface-exposed lipoprotein that binds the negative complement regulator complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the functions of these genes and how immunization with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here, we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and that it is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH with affinity similar to that of meningococcal fHbp and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea. We found that immunization with Bexsero elicits serum bactericidal activity against N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis and cross-reactive responses elicited by Bexsero suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

SinceNeisseria gonorrhoeaeandNeisseria meningitidisare obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensalNeisseriagenome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. OnlyNeisseria mucosacontained a nitrate reductase (Nar), and onlyNeisseria lactamica,Neisseria cinerea,Neisseria subflava,Neisseria flavescensandNeisseria siccacontained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested exceptN. mucosahad a two- to fourfold lower nitrite reductase activity thanN. gonorrhoeae, whileN. meningitidisand most of the commensalNeisseriaspecies had a two- to fourfold higher nitric oxide (NO) reductase activity. ForN. meningitidisand most of the commensalNeisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared withN. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that ofN. gonorrhoeae. The greatest difference between theNeisseriaspecies was the lack of a functional Nos system in the pathogenic speciesN. gonorrhoeaeandN. meningitidis.

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1β and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1β/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1β production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1β secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.

In Neisseria gonorrhoeae , the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava , has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae , and its sensitivity was ≥10 1 copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of ≥0.5 μg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.

Objectives In Neisseria gonorrhoeae, the mosaic structure of penicillin-binding protein 2 (PBP 2), composed of fragments of PBP 2 from Neisseria cinerea and Neisseria perflava, was significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to determine the affinity of mosaic PBP 2 for cephalosporins in N. gonorrhoeae. Methods Two types of non-mosaic PBP 2 from the type strain of N. gonorrhoeae (ATCC 19424) and a clinical strain (GU01-29), as well as the mosaic PBP 2 from a clinical strain (GU01-89), were expressed in insect cells, and recombinant PBP 2s were purified. ATCC 19424 and GU01-29 were susceptible to cephalosporins. GU01-89 showed decreased susceptibility to cephalosporins. Bindings of fluorescent penicillin to PBP 2 were characterized by the Scatchard plot analysis. The affinity of the recombinant PBP 2s for cefdinir, cefixime and ceftriaxone was determined by PBP 2 competition assays with fluorescent penicillin. Results The K(d) value of mosaic PBP 2 for fluorescent penicillin was higher than that of non-mosaic PBP 2s. The affinity of mosaic PBP 2 for cefdinir or cefixime was lower than that of the non-mosaic PBP 2s. The affinity of the mosaic PBP 2 for ceftriaxone was not changed, compared with that of the non-mosaic PBP 2s. Conclusions Other mechanisms may be involved in clinical isolates with decreased susceptibility to cephalosporins, but this study suggests that the decreased affinity of mosaic-structure recombinant PBP 2 for oral cephalosporins may contribute to decreased susceptibility to these antibiotics in N. gonorrhoeae.

Within the frame of our study we investigated Microccocus luteus, Neisseria cinerea, and Pseudomonas fluorescens by means of capillary zone electrophoresis (CZE). They form chains and clusters on a different scale, which can be reflected in the electropherograms. A low buffer concentration of Tris-borate and Na2EDTA containing a polymeric matrix of 0.0125% poly(ethylene) oxide (PEO) was used. Key factors were the standardization and optimization of CE conditions, buffer solution, and pretreatment of bacterial samples, which are not transferable to different bacterial strains, in general. The different compositions of the cell wall of on the one hand Gram-positive (M. luteus) and Gram-negative (N. cinerea) cocci and on the other hand Gram-negative, rod-shaped bacteria (P. fluorescens), are probably responsible for the different pretreatment conditions.

Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-kappaB) nuclear binding activity. In some experiments, NF-kappaB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-kappaB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-kappaB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 μg/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene ( penA ) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 μg/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava ( N. sicca ), Neisseria cinerea , Neisseria flavescens , and Neisseria meningitidis . These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae .