Nonpathogenic Neisseria Species Research Articles

Direct Bacterial Profiling by Matrix-Assisted Laser Desorption−Ionization Time-of-Flight Mass Spectrometry for Identification of Pathogenic Neisseria

The present study investigates the suitability of direct bacterial profiling as a tool for the identification and subtyping of pathogenic Neisseria. The genus Neisseria includes two human pathogens, Neisseria meningitidis and Neisseria gonorrhoeae, as well as several nonpathogenic Neisseria species. Here, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling protocol was optimized using a laboratory strain of E. coli DH5alpha to guarantee high quality and reproducible results. Subsequently, mass spectra for both laboratory and clinical strains of N. gonorrhoeae, N. meningitidis, and several nonpathogenic Neisseria species were collected. Significant interspecies differences but little intraspecies diversity were revealed by means of a visual inspection and bioinformatics examination using the MALDI BioTyper software. Cluster analysis successfully separated mass spectra collected from three groups that corresponded to N. gonorrhoeae, N. meningitidis, and nonpathogenic Neisseria isolates. Requiring only one bacterial colony for testing and using a fast and easy measuring protocol, this approach represents a powerful tool for the rapid identification of pathogenic Neisseria and can be adopted for other microorganisms.

Evaluation of commercial kits for the identification of Neisseria gonorrhoeae

Eight identification methods were evaluated against 100 isolates of Neisseria gonorrhoeae and 21 non-gonococcal Neisseria strains. The methods examined included four commercial biochemical kits, API NH, RapID NH, Gonochek II and Neisseria Preformed Enzyme Test (PET), three immunological kits, Phadebact Monoclonal GC test, GonoGen II and MicroTrak, and one in-house carbohydrate-utilization method, cystine trypticase agar (CTA) sugars. The percentage of isolates unambiguously identified as N. gonorrhoeae by each of the methods was as follows: API NH, 66 %; RapID NH, 64 %; GonoChek II, 66 %; Neisseria PET, 66 %; Phadebact Monoclonal GC OMNI test, 99 %; GonoGen II, 100 %; MicroTrak, 100 %; and CTA sugars, 96 %. The low sensitivity of the biochemical kits for the identification of N. gonorrhoeae was due to a lack of the enzyme proline iminopeptidase (Pip) in 34 % of the isolates examined. All the biochemical kits utilized the presence of this enzyme as a marker for N. gonorrhoeae. The Phadebact Monoclonal GC kit, GonoGen II, MicroTrak, CTA sugars and the API NH kit all exhibited high specificity, but non-gonococcal Neisseria were misidentified as N. gonorrhoeae using RapID NH (two strains), Gonochek II (11 strains) and Neisseria PET (11 strains). Whilst the isolates examined in this study may not be truly representative, they do indicate that N. gonorrhoeae isolates lacking the enzyme Pip can give anomalous results when using commercially available biochemical tests and that some non-pathogenic Neisseria species are still being misidentified using some biochemical kits. This further reinforces the recommendation that any dubious biochemical result should be confirmed with an immunological test.

Neisserial pilin genes display extensive interspecies diversity

All Neisseria live in association with host cells, however, little is known about the genetic potential of nonpathogenic Neisseria species to express attachment factors such as pili. In this study, we demonstrate that type IV pilin-encoding genes are present in a wide range of Neisseria species. N. sicca, N. subflava, and N. elongata each contain two putative pilE genes arranged in tandem, while single genes were identified in N. polysaccharea, N. mucosa, and N. denitrificans. Neisserial pilE genes are highly diverse and display features consistent with a history of horizontal gene transfer.

Identification of opcA gene in Neisseriapolysaccharea: interspecies diversity of Opc protein family

The gene encoding the outer membrane adhesin/invasin protein OpcA was previously described in the genomes of two pathogenic Neisseria species, N. meningitidis (Nm) and N. gonorrhoeae (Ng). In order to understand the presence or absence of opcA in nonpathogenic Neisseria species, 13 strains of N. polysaccharea (Np), four strains of N. lactamica, three strains of N. subflava and nine strains of other species were examined by DNA hybridization, polymerase chain reaction (PCR) and nucleotide sequencing. The opcA gene was found in two Np strains (85322 and 89357). The Np-opcA gene is a novel member of this gene family with 93% homology to Ng-opcA. Comparison of opcA-surrounding regions among eight Neisseria strains revealed five types of genetic organization at the opcA locus in Neisseria, which result from insertion or deletion of genetic elements at the upstream region of opcA. Comparison of the deduced peptide sequences from two Np strains, two representative Ng strains, two representative Nm strains and 13 Nm sequence variants demonstrates interspecies diversity of the OpcA protein family with conserved transmembrane regions and species-specific polymorphism at the surface-exposed loops and periplasmic turns. Reverse transcription–PCR analysis and Northern blotting showed that Np-opcA was transcribable. From an alignment of the Np-OpcA and Ng-OpcA sequences against the three-dimensional crystal structure of Nm-OpcA we conclude that there is no obvious structural reason why these proteins would not be able to form stable, folded, outer membrane proteins. The data presented here provide additional information for understanding the distribution, variation and expression of opcA in Neisseria.

A comparative analysis of pilin genes from pathogenic and nonpathogenic Neisseria species

Pathogenic Neisseria species elaborate type IV pili, which are considered important for virulence. In this study, we examined pilin-encoding expression loci (pilE) in nonpathogenic Neisseria species. PCR based screening detected homology to a conserved N-terminal region of pilE in 12 of 15 Neisseria species, including all human commensal isolates. The three species failing to display homology were isolated from nonhuman sources. We have also characterized complete pilE loci from the human commensal species N. lactamica and N. cinerea. As anticipated, the predicted protein sequences from these species display features typical of all type IV pilins. In addition, these commensal pilins possess two highly conserved regions, SV2 and CYS2, which are shared among all neisserial pilins. However, a comparative analysis of pilE loci from pathogenic and nonpathogenic Neisseria species reveals two distinct structural groups, one composed of the pilin genes from N. lactamica, N. cinerea, and the class II pilin-producing subset of N. meningitidis isolates, the other of gonococcal and meningococcal class I pilin-encoding genes. Since both class I and class II pilin-producing meningococci can act as pathogens, structural relationships among neisserial pilin genes do not obviously reflect either species membership or ability to cause human disease.

A clinical study of respiratory infection isolating non-pathogenic Neisseria by transtracheal aspiration

Neisseria species other than N. meningitidis and N. gonorrhoeae are generally regarded as commensal bacterial flora of the oropharynx, and little is known regarding cases of these non-pathogenic Neisseria species in the lower respiratory tract. We clinically examined respiratory tract infections from which non-pathogenic Neisseria species were isolated by transtracheal aspiration (TTA). The incidence of non-pathogenic Neisseria isolated was 54 (15.7%) out of 344 episodes of respiratory tract infections with isolated microorganisms from TTA, and was 17.6%, 15.8%, 14.3% for pneumonia, acute bronchitis, and chronic lower respiratory tract infection, respectively. All 54 episodes were isolated with other microorganisms such as alpha-Streptococcus spp. (75.9%), Haemophilus influenzae (25.9%) and anaerobics (22.2%). The isolation ratio according to the age group increased at 45 years of age or more, but did not increase with the advance of age. Predisposing factors were identified such as overt aspiration, iatrogenic procedure and heavy smoking. Cases without overt aspiration that had fevers of 38 degrees C or more or hypoxemia of less than PaO2 70 torr when detecting non-pathogenic Neisseria were observed more frequently in the aged than the non-aged. The findings suggest the detection of non-pathogenic Neisseria by TTA is influenced by the host state that the fall of microorganisms from the upper to lower respiratory tract cannot be defended or excluded by mucociliary transportation disorder due to underlying disease and smoking, or deterioration of physical status other than overt or silent aspiration.

A novel peptidoglycan-linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae.

A novel peptidoglycan-linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species-related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild-type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild-type strain. comL is a single-copy gene and maps downstream of the previously described comA gene. It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5' end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA'). In E. coli, cloned ComL can be labelled with [3H]-palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseria meningitidis as well as in several nonpathogenic Neisseria species.

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes ( porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.

Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression.

In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins.

Human antibody response to the 70-Kd common neisserial antigen in patients and carriers of meningococci or non-pathogenic Neisseria

A surface antigen of 70 Kd, common to most Neisseria species, was previously described. This antigen is expressed and immunogenic in vivo in humans since 70-Kd-specific antibodies were detected in 22 26 patients with meningococcal meningitis and 12 17 healthy meningococcal carriers. Sera from convalescent patients and from carriers blocked the binding between mouse 70-Kd-specific serum and a strain of N. gonorrhoeae. The correspondence between inhibition-ELISA, using whole gonococci as solid phase, and detection of anti-70-Kd antibodies by Western blotting confirmed the specificity of the mouse serum. Non-pathogenic Neisseria species although possessing the 70-Kd structure elicited less frequently an antibody response in children. The immunogenicity of the 70-Kd antigen in humans supports its potential use as a vaccine component.

Characterisation of Branhamella catarrhalis and differentiation from Neisseria species in a diagnostic laboratory.

To distinguish Branhamella catarrhalis from Neisseria species a study of 140 strains was made on simple laboratory media, with particular reference to deoxyribonuclease (DNase) production, superoxol reaction, and growth characteristics. All 97 clinical isolates of B catarrhalis (58 of which were beta-lactamase positive) and eight strains of B catarrhalis from the National Collection of Type Cultures were DNase positive and superoxol positive. None grew on modified New York City medium, modified Thayer Martin medium, MacConkey agar, crystal violet blood agar, nor under anaerobic conditions. Of the 16 different non-pathogenic Neisseria species tested, all were DNase negative, eight (50%) were superoxol reaction negative, and 13 (81%) grew on crystal violet blood agar. Using simple laboratory media, DNase, and superoxol tests, it was possible to identify B catarrhalis and to distingish it from pathogenic and non-pathogenic Neisseria species.

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

Precipitating antibodies against Neisseria meningitidis in normal sera and their possible origin.

Crossed immunoelectrophoresis was used to study precipitating antibodies against Neisseria meningitidis in sera from 214 normal persons and in human gammaglobulin. A polyspecific meningococcal antigen preparation and a corresponding rabbit antiserum constituted the reference system. All the sera contained one to five precipitins against N. meningitidis which could be identified and quantified by means of the reference system. The precipitin score, which expresses the number and titre of precipitins in each serum, increased with age. More than 25 precipitins were found in the gammaglobulin preparation, including antibodies against the groupspecific polysaccharides A, B and C. Absorption of three of the five normally occurring precipitins (nos. 4, 19, 22) with antigens from various bacteria showed that precipitins nos. 4 and 19 could be completely absorbed by antigens from other Neisseria species, but not by antigens from other genera. This indicates that these two precipitins may have been induced by non-pathogenic Neisseria species or by meningococci. The finding of protective antibodies in gammaglobulin might indicate the possibility of its use in prophylaxis or therapy meningococcal infections.

Immunological relationships of some non-pathogenic Neisseria

A collection of nine stock strains of non-pathogenic Neisseria species and one strain of Branhamella catarrhalis has been examined serologically by a simple immunodiffusion technique utilising cultures growing on solid medium.

Relationship of pili to colonial morphology among pathogenic and nonpathogenic species of Neisseria

Growth in colonies with type 1 morphology and the presence of pili are characteristics that have been associated with virulence of gonococci for humans. To determine whether the presence of pili per se might be responsible for colony type 1 morphology, the relationship of pili to colony type was examined in various species of Neisseria. Short pili (175 to 210 nm in length) were seen only on nonpathogenic neisseria, whereas long pili (up to 4,300 nm) were seen on organisms of both nonpathogenic and pathogenic species. Although long pili, similar to those found on organisms from high-domed, type 1 colonies of gonococci, were observed on organisms from high-domed, type 1 colonies of nonpathogenic Neisseria species, they were also observed on low-convex, type 4 colonies of meningococci and nonpathogenic neisseria. Among meningococci there was no difference in the morphology of colonies consisting of organisms with many long pili and colonies consisting of organisms that completely lacked pili. Thus, there was no consistent relationship of pili to colonial morphology. Unless the pili of N. gonorrhoeae are unique among Neisseria species in their influence on colonial morphology, it is likely that factors other than pili determine colony type 1 morphology of gonococci. Whether these same factors, either alone or in conjunction with pili, are also responsible for gonococcal virulence warrants further investigation.

Extracellular carbohydrate antigens from some non-pathogenic Neisseria species.

Thirteen species of Neisseria were examined for the presence of extracellular carbohydrate antigens. Of three media tested for optimum growth of the bacteria, yield of antigens, and ease of isolation of antigens, dialyzed tryptic soy broth was the best. Seven species, N. catarrhalis, N. cinerea, N. flava, N. flavescens, N. lactamicus, N. subflava, and N. winogradski, yielded extracellular antigens that were extractable into phenol and hence were probably proteinaceous. The remaining six species, N. canis, N. caviae, N. cuniculi, N. cuniculi subsp. gigantea, N. denitrificans, and N. sicca, yielded carbohydrate antigens that were purified by a sequence of phenol extraction, chromatography, enzymic digestion, and membrane filtration. These antigens were polymers of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose, the latter being the only constituent of the antigens from N. caviae and N. sicca. Cross-reactivities of the carbohydrate antigens in the homologous antisera showed that there were close relations between the antigens from N. canis and N. cuniculi subsp. gigantea and also between those from N. caviae and N. sicca.