Commensal Neisseria Research Articles

Sequence-Based Predictions of Lipooligosaccharide Diversity in the Neisseriaceae and Their Implication in Pathogenicity

Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation.

Neisseria lactamica attenuates TLR-1/2-induced cytokine responses in nasopharyngeal epithelial cells using PPAR-γ

The upper respiratory tract commensal Neisseria lactamica (Nlac) induces protective humoral immunity against pathogenic Nmen serogroup B (Nmen), but whether it also affords anti-inflammatory mucosal protection, as reported for several gut commensals, has not been investigated. Here we demonstrate for the first time that Nlac weakly induces inflammatory responses compared with Nmen in the nasopharyngeal epithelial cell line, Detroit 562, and that Nlac achieves this by attenuation of secretory cytokine (TNF-α and IL-6) and to a lesser extent chemokine (IL-8 and RANTES) responses. Culture of Detroit cells with Nlac inhibited the induction of cytokine-chemokine mRNA by Nmen, reduced Nmen-induced NF-κβ activity and increased constitutive PPAR-γ protein expression. Pretreatment of Detroit cells with a PPAR-γ antagonist abrogated the attenuation of inflammatory IL-6 by Nlac, as did heat-killing of the organisms and preventing their invasion with cytochalasin D. Inflammatory responses from Detroit cells were readily attenuated by Nlac following stimulation with pathogenic Nmen but more specifically following stimulation with the TLR-1/2 agonist Pam3Cys and pro-inflammatory cytokines (IL-1β, TNF-α) but not LTA or LPS. These results indicate that Nlac plays an important role in suppressing pathogen-induced inflammation in the nasopharyngeal mucosa, mediated through TLR-1/2 stimulation, by activating PPAR-γ and inhibiting NF-κβ activity.

Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains isolated from neutropenic patients in Tunisia

We aimed to determine the frequency and the molecular basis of β-lactams resistance in a total of 44 commensal Neisseria strains. Bacterial identification was performed using standard biochemical tests. Genetic diversity of penA gene was studied by penA fingerprinting. Commensal Neisseria strains were represented essentially by Neisseria subflava biovar perflava (75%). Four strains (9%) were β-lactamase producers and had the blaTEM gene. TEM-type β-lactamase was characterized by blaTEM gene sequencing. PenA fingerprinting gave 32 patterns with MspI and 19 patterns with TaqI. Our strains presented an important frequency of β-lactamase production as well as a high rate of reduced susceptibility to β-lactams with an extensive genetic diversity in the penA gene polymorphism.

Molecular detection and confirmation of Neisseria gonorrhoeae in urogenital and extragenital specimens using the Abbott CT/NG RealTime assay and an in-house assay targeting the porA pseudogene

Culture for detection of Neisseria gonorrhoeae (NG) is being replaced by molecular assays, but difficulties are observed with false positive and negatives results, especially for extragenital samples. This study evaluates the Abbott CT/NG Real-Time assay and a real-time porA pseudogene assay. Samples (n = 600) from a mixed prevalence Irish population include 164 male urines with corresponding urethral swabs, 58 endocervical swabs, 173 male pharyngeal swabs, 205 male rectal swabs, 36 NG clinical isolates and 26 commensal Neisseria species isolates. There was a 100% concordance between the Abbott CT/NG Real-Time and the porA assay. The positivity rate was 1.2%, 1.7%, 8.1% and 5.8% for FVU/urethral swabs, endocervical, pharyngeal and rectal swabs, respectively. These results were compared to culture and discrepancies were found with nine pharyngeal and three rectal swabs. Seven of the 12 discrepant positive samples were sequenced and were confirmed "true positives". The sensitivity and specificity of the molecular assays was 100%. The sensitivity of the culture-based testing was 100% for urogenital samples but 36% and 75% for pharyngeal and rectal swabs, respectively. The combined Abbott CT/NG and porA assays provide a valuable alternative to culture and also generate a significant increase in the diagnosis of pharyngeal and rectal NG infection.

Genome Sequencing Reveals Widespread Virulence Gene Exchange among Human Neisseria Species

Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genus Neisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenic Neisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged among Neisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, other Neisseria. Our findings strongly suggest that commensal Neisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.

Neisseria lactamica Arthritis and Septicemia Complicating Myeloma

Neisseria lactamica is a common and normally harmless commensal of the upper respiratory tract found especially in young children. In the July 2006 edition of this journal, Zavascki and colleagues reported the first case of N. lactamica causing cavitatory lung disease in an adult organ transplant recipient (3). The same year, Wang et al. reported a case of N. lactamica bacteremic pneumonia in an adult with liver cirrhosis (2). Apart from a 1991 report of N. lactamica meningitis following skull trauma (1), there are no other published reports of invasive N. lactamica infection in adults or cases listed in selected bacteremia databases from New Zealand, Australia, or North America. We present a case of N. lactamica arthritis and septicemia in a patient immune suppressed by myeloma and corticosteroids. This 60-year-old male from a small mining city in the West Australian outback presented with 1 day of rigors, hypotension, and acute knee pain and swelling. He had been prescribed prednisolone at 50 mg daily, reduced to 15 mg daily, for an infective exacerbation of chronic obstructive airways disease during the 17 days before admission. The myeloma was associated with an IgG kappa-type paraprotein, mild suppression of serum IgA and IgM, and lytic bony lesions. HIV serology was negative. The cloudy knee fluid aspirate had neutrophilic pleocytosis (100 white cells/mm3) and Gram-negative cocci by microscopy. Culture of both joint fluid and blood yielded a Neisseria species, provisionally identified as Neisseria gonorrhoeae. This caused distress for the patient and his wife, who denied sexual activity for 5 years. The isolate was oxidase and catalase positive but Superoxol and Phadebact Monoclonal GC test (Bactus AB, Huddinge, Sweden) negative. API NH (bioMerieux, La Balme-les-Grottes, France) identified N. lactamica (99.9%, profile 5041). BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/) of an amplified 2,098-nucleotide 16S rRNA sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:GU980596,term_id:294874967,term_text:GU980596}}GU980596) showed 98% homology with both N. lactamica (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ239305.1,term_id:5327203,term_text:AJ239305.1}}AJ239305.1) and Neisseria polysaccharea (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ239314.1,term_id:5327212,term_text:AJ239314.1}}AJ239314.1). The isolate was o-nitrophenyl-β-d-galactopyranoside (ONPG) positive and generated acid from sucrose, excluding N. polysaccharea. Our isolate was thus identified biochemically and genetically as N. lactamica but may represent a new virulent strain. The penicillin MIC was 3 mg/liter, and the ceftriaxone MIC was 0.006 mg/liter, both determined by Etest. An echocardiogram showed no endocarditis, and the neisserial infection was cured with 11 days of ceftriaxone therapy. It should not be a surprise that N. lactamica causes septic arthritis, as there are case reports of septic arthritis, discitis, or bursitis caused by other commensal Neisseria spp., including N. sicca, N. subflava, and N. mucosa. Neisseria meningitidis (especially serogroup W-135) and N. gonorrhoeae also cause septic arthritis. Commensal neisserial infections have often been reported in compromised patients, including those who are neutropenic, have invasive devices, or have undergone procedures. Meningococcal infections (especially nonpathogenic strains) have also been reported in compromised patients, including those with complement deficiency, prior corticosteroid use, myeloma, Waldenstrom's disease, and hypogammaglobulinemia. Like the case presented by Zavascki et al. (3), our patient represents a rare example of invasive adult N. lactamica infection and highlights the propensity for commensal neisserial species to cause bloodstream and joint infections and to be of special concern to patients with immune compromises. We encourage others isolating clinically significant N. lactamica strains to deposit the sequences in GenBank to enable further study of this emerging pathogen in compromised patients.

Genomic Content ofNeisseriaSpecies

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.

Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens

ObjectivesThe Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal...

Moraxella catarrhalis,a Human Respiratory Tract Pathogen

Moraxella catarrhalis is an exclusively human pathogen and is a common cause of otitis media in infants and children, causing 15%-20% of acute otitis media episodes. M. catarrhalis causes an estimated 2-4 million exacerbations of chronic obstructive pulmonary disease in adults annually in the United States. M. catarrhalis resembles commensal Neisseria species in culture and, thus, may be overlooked in samples from the human respiratory tract. The prevalence of colonization of the upper respiratory tract is high in infants and children but decreases substantially in adulthood. Most strains produce beta-lactamase and are thus resistant to ampicillin but susceptible to several classes of oral antimicrobial agents. Recent work has elucidated mechanisms of pathogenesis and focused on vaccine development to prevent otitis media in children and respiratory tract infections caused by M. catarrhalis in adults with chronic obstructive pulmonary disease.

A common gene pool for the Neisseria FetA antigen

Meningococcal FetA is an iron-regulated, immunogenic outer membrane protein and vaccine component. The most diverse region of this protein is a previously defined variable region (VR) that has been shown to be immunodominant. In this analysis, a total of 275 Neisseria lactamica isolates, collected during studies of nasopharyngeal bacterial carriage in infants, were examined for the presence of a fetA gene. The fetA VR nucleotide sequence was determined for 217 of these isolates, with fetA apparently absent from 58 isolates, the majority of which belonged to the ST-624 clonal complex. The VR in N. lactamica was compared to the same region in N. meningitidis, N. gonorrhoeae, and a number of other commensal Neisseria. Identical fetA variable region sequences were identified among commensal and pathogenic Neisseria, suggesting a common gene pool, differing from other antigens in this respect. Carriage of commensal Neisseria species, such as N. lactamica, that express FetA may be involved in the development of natural immunity to meningococcal disease.

A duplex Neisseria gonorrhoeae real-time polymerase chain reaction assay targeting the gonococcal porA pseudogene and multicopy opa genes

Cross-reactions of gonococcal nucleic acid amplification tests (NAATs) with commensal Neisseria strains are well documented. Recent data now indicate that sequence-related false-negative results can occur in gonococcal NAATs, whereby target sequences either are absent or contain several mutations. In this study, a duplex Neisseria gonorrhoeae real-time polymerase chain reaction (PCR) (NGduplex) assay targeting the gonococcal porA pseudogene and multicopy opa genes was developed. The NGduplex was evaluated by testing 596 clinical specimens, including 292 urogenital specimens and 304 throat swab specimens. The results were compared with those obtained using a consensus reference standard comprising 3 monoplex real-time PCR assays. The results show that the NGduplex assay is highly suitable for routine screening for gonorrhea, providing an overall clinical sensitivity and specificity of 100% and 99.3%, respectively, for both urogenital and throat swab specimens. In addition, the 2-target system of the NGduplex assay decreases the potential for sequence-related false-negative results and can provide simultaneous confirmation of positive results.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant

Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

Diagnostic Value of Molecular Confirmation Assays for Neisseria gonorrhoeae

The COBAS Amplicor system (Roche Diagnostics, Rotkreuz, Switzerland) is a widely used screening test for infections with Neisseria gonorrhoeae due to its excellent sensitivity and ease of use. However, it has been found to cross-react with certain commensal Neisseria species (1, 3, 4). Therefore, an independent confirmation assay is mandatory. In the May 2007 issue of the Journal of Clinical Microbiology, Mangold et al. (2) presented a new confirmation assay which is capable of distinguishing between N. gonorrhoeae and Neisseria spp. in comparison to confirmation assays targeting the porA pseudogene and the cppB gene. They obtained positive results in their in-house assay for 102/181 (56%) COBAS Amplicor-positive samples; the porA confirmation assay was positive for 69/181 (38%) samples, and the cppB assay detected 115/181 (64%) positive samples. Additionally, Mangold et al. reported 15%, 2%, and 4% positive results for COBAS Amplicor-negative samples with the cppB, porA, and in-house assays, respectively. The presented data are discrepant from observations at our institution. Upon optimization of published detection protocols, we have compared the performances of three assays targeting the porA pseudogene (5), the cppB gene (6), and the 16S rRNA gene (in-house) for quality assurance. In samples positive for all three targets, the cppB confirmation assay was the most sensitive (mean crossing point [Cp], 25.9), followed by the porA assay (mean Cp, 26.2) and the 16S rRNA gene assay (mean Cp, 32.8). Over a period of 6 months, 398 samples yielded a positive COBAS Amplicor result (optical density [OD] of >0.2, as recommended by the manufacturer). Of these 398 samples, 258 samples (64.8%) were negative in all three confirmation assays. The remaining 140 samples (35.2%) yielded a positive result in at least two of the confirmation assays, with the majority being positive in all three assays (128/140 [91.4%]). Eight of 140 samples (5.7%) were negative in the 16S rRNA gene assay, probably due to small DNA amounts in the sample, as this assay is less sensitive than the other two confirmation assays, which is corroborated by the fact that 2/8 samples showed an OD value lower than 2.0 when initially tested in the COBAS Amplicor system. Four specimens were positive in the 16S rRNA gene and porA detection assays only and were interpreted as gonococci lacking the cppB-harboring plasmid (2.9%); the COBAS Amplicor value was above 2.0 OD in all four samples. Taken together for 136/140 (97.1%) specimens, the porA and cppB confirmation assays were concordantly positive. In contrast to the findings of Mangold et al., we did not obtain positive results for the confirmation assays when applied to COBAS Amplicor-negative samples. The conflicting results might in part be explained by an epidemiological distinct pool of N. gonorrhoeae isolates examined and an unfortunate study design, as modified interpretation guidelines for the COBAS Amplicor assay were used by Mangold et al. Samples were considered positive if two out of three runs gave a positive signal above an OD of 2.0, as opposed to one sample above an OD of 0.2 being sufficient. This decreases the number of positive samples and inevitably has a negative impact on the sensitivity of the screening assay. Presumably, this protocol accounts for the “false”-positive results of the cppB plasmid assay (33/227). It would be interesting if these samples were initially positive in the COBAS Amplicor system (OD of >0.2) by use of the manufacturer's interpretation criteria. Our analysis further cannot confirm the low sensitivity of the porA detection assay, as the majority of our specimens yielded positive results in all confirmation assays. Regarding the in-house NsppID assay published by Mangold et al., the design of the test itself is not completely conclusive: N. gonorrhoeae displays 100% homology with strains of Neisseria polysaccharea at the binding site of the detection probe (referred to as Npolysaccharea1 in the publication). N. polysaccharea was not tested for cross-reactivity in the NsppID assay. By use of an NCBI BLAST search, strains of Neisseria meningitidis also display 100% homology in this region of the 16S rRNA gene. Additionally, coamplification in PCRs should be avoided if no important information is obtained by the coamplified product, as it is likely to decrease sensitivity. A confirmation assay for N. gonorrhoeae that coamplifies apathogenic Neisseria spp. does not provide any additional information of clinical relevance. The studies indicate that the use of molecular diagnostic assays for the detection of N. gonorrhoeae infection remains challenging and that the characterization of the prevalence of target genes in distinct epidemiological pools is important to ensure accurate diagnosis.

Antimicrobial resistance in clinical isolates of Neisseria subflava from the oral cavities of a Japanese population

A recent study indicated that Neisseria subflava, one of the commensal Neisseria species, may play an important role in the emergence of Neisseria gonorrhoeae strains with chromosomally mediated resistance to penicillin or cephalosporin by the horizontal genetic exchange of penA genes encoding the target site for penicillin or cephalosporin. The present investigation examined the antimicrobial susceptibility of 45 isolates of N. subflava from the oral cavities of Japanese men and women to various agents used for the treatment of gonococcal infections. Of the 45 isolates, 40 (88.9%) and 4 (8.8%) were intermediately resistant and resistant to penicillin, respectively, with the minimal inhibitory concentration (MIC)(50) and MIC(90) of penicillin being 0.5 mg/l and 1 mg/l, respectively. Of the 45 isolates, 13 (28.9%) and 14 (31.1%) were resistant to tetracycline and ciprofloxacin, respectively, and 3 (6.7%) showed reduced susceptibility to cefixime (although the susceptibility category was not determined). These results indicate that several isolates of N. subflava have acquired resistance or intermediate resistance to various antimicrobial agents, including penicillin, cephalosporin, tetracycline, and ciprofloxacin. The present study may thus confirm that N. subflava may be involved in the emergence of N. gonorrhoeae strains with either intermediate or total resistance to penicillin or cephalosporin by the horizontal genetic exchange of the penA gene.

Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria.

A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs.

Further Questions Regarding the Role of Mosaic penA Sequences in Conferring Reduced Susceptibility to Ceftriaxone in Neisseria gonorrhoeae

With treatment options for gonorrhea diminishing, the isolation of Neisseria gonorrhoeae strains with reduced susceptibilities to the newer cephalosporins represents a worrying trend that requires monitoring and investigation. Mutations in the penA gene encoding the penicillin-binding protein 2 (PBP2) are a major contributor to the emergence of chromosomally mediated resistance to β-lactam antibiotics in N. gonorrhoeae. Recent studies have identified mosaic-like structures of the PBP2 in clinical isolates of N. gonorrhoeae with decreased susceptibilities to both cefixime and ceftriaxone (1, 2, 4, 5). These mosaic sequences are thought to have evolved from recombination events involving penA gene sequences from several Neisseria species, including N. perflava, N. sicca, and N. cinerea (1, 3). Thus, the reduced susceptibility to the newer cephalosporins is attributed to the acquisition of genetic material from resistant commensal Neisseria spp. by originally susceptible gonococci. We have identified eight clinical isolates of N. gonorrhoeae harboring mosaic PBP2 sequences as part of an ongoing study investigating reduced susceptibility to ceftriaxone in Australian gonococcal isolates (6). DNA sequencing of the complete penA genes from these isolates revealed that seven of these strains possessed PBP2 amino acid sequences (Fig. ​(Fig.1,1, mosaic A) that were identical to the mosaic PBP2 sequence previously described by Ito et al. (2). All seven strains had reduced susceptibility to ceftriaxone (MIC = 0.06 mg/liter). The remaining strain possessed a variation of the mosaic sequence (Fig. ​(Fig.1,1, mosaic B). Briefly, the first 430 amino acids of the mosaic B sequence in this strain were nearly identical to the GenBank reference sequence {type:entrez-nucleotide,attrs:{text:M32091,term_id:150278,term_text:M32091}}M32091, except for an additional Asp-345A codon, whereas the remaining amino acids were similar to those in the mosaic sequence described by Ito et al. but with three substitutions. Notably, the strain possessing this variant mosaic B sequence was fully susceptible to ceftriaxone (MIC ≤0.008 mg/liter). FIG. 1. Mosaic PBP2 amino acid sequences identified in the N. gonorrhoeae isolates in this study. The sequences are aligned with a GenBank N. gonorrhoeae sequence (accession number {type:entrez-nucleotide,attrs:{text:M32091,term_id:150278,term_text:M32091}} ... Takahata et al. recently demonstrated that particular amino acid substitutions in the mosaic PBP2 (G545S, I312M, V316T) were responsible for reduced susceptibility to cefixime in Japanese gonococci (4). All three substitutions were present in our seven strains with the mosaic A sequence but were absent in the variant mosaic B sequence, suggesting that they may also have a role in altered ceftriaxone susceptibility. However, Takahata et al. also found that these substitutions and several others, although impinging on cefixime MICs, did not greatly affect ceftriaxone susceptibility (4). This was also shown in transformation experiments conducted by Takahata et al. in which full-length mosaic penA sequences conferred only very minor reductions in ceftriaxone susceptibility to a susceptible strain and so could not fully explain the reduced susceptibility of the donor strain (4). The demonstration that full susceptibility to ceftriaxone is possible in the presence of this variant mosaic B sequence has been, thus far, detected only in a single isolate. However, this finding adds weight to those of Takahata et al. (4) by suggesting that other mosaic-associated mutations located within the transpeptidase domain have but little impact on ceftriaxone susceptibility. This brings into question the extent of the contribution of mosaic sequences in conferring reduced susceptibility to ceftriaxone in N. gonorrhoeae. These findings would need to be confirmed by additional evidence such as the detection of additional ceftriaxone-susceptible gonococci with mosaic B or similar sequences and adjunctive evidence derived from transformation experiments. Other mechanisms that may confer reduced susceptibility to ceftriaxone, including mutations in the ponA (PBP1) gene (5), have been proposed, and alternatively, as yet undetected second-site mutations may also be responsible for reversion to the susceptibility patterns reported here. Overall, the data currently available suggest that the role mosaic penA sequences play in reduced susceptibility to ceftriaxone, as opposed to cefixime, needs careful and further examination.

A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.

Plasmid Diversity in Neisseriae

Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

Identification of acquired DNA inNeisseria lactamica

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.