Ultrastructure of Pili and Annular Structures on the Cell Wall Surface of Neisseria meningitidis

In a survey of negatively stained preparations of the prototype strains of Neisseria meningitidis, pili were detected on three strains. However, these pili were detected on fewer than 5% of cells in populations of these three strains. Those individual cells with pili were seldom observed to contain more than two or three pili per cell. In contrast, nearly all cells of the nonprototype group B strain ATCC 13090 had numerous pili on their surfaces. When viewed in frozen-etched replicas, a few pili were observed lying on the cell surface of this latter strain. An annular structure was also found in frozen-etched replicas. This structure usually consisted of a series of concentric rings that were always found on the flat side of these bean-shaped cells. It is concluded that such structures represent a differentiated portion of the cell wall, which is involved in cross-wall formation during synthesis of the cell septum in dividing cells.     

Effects of media, atmosphere, and incubation time on colonial morphology of Arcanobacterium haemolyticum.

Arcanobacterium haemolyticum causes pharyngitis as well as skin and other wound infections. Although it is a beta-hemolytic organism, the hemolysis is less well defined than that of beta-hemolytic streptococci and may be overlooked in cultures with heavy growth of commensal throat flora. To determine whether routine throat culture conditions are sufficient to produce recognizable colonies of A. haemolyticum, the morphology of six distinct strains was studied after various combinations of incubation time, medium, and atmosphere. The agar media, containing 5% sheep blood, were Trypticase soy agar, Columbia agar, and heart infusion agar. Cultures were incubated in ambient air, 6 to 8% CO2, or an anaerobic atmosphere. Cultures were compared after 24, 48, and 72 h of incubation for colony size, clarity and size of hemolytic zone, and macroscopic evidence of agar pitting. A minimum of 48 h was needed for expression of beta-hemolysis and pitting. Trypticase soy agar was the superior medium and CO2 was the superior atmosphere for beta-hemolysis. Agar pitting was not significantly affected by variations in medium or atmosphere. Strains differed in their expression of hemolysis and production of pits at 48 h. After 72 h of incubation, beta-hemolysis and pitting were visible in over 96% of culture observations.     

Organism resembling Neisseria gonorrhoeae and Neisseria meningitidis.

A problem isolate resembling Neisseria gonorrhoeae and Neisseria meningitidis is reported. Growth and biochemical characteristics indicated the organism to be N. meningitidis, whereas serological characteristics indicated it to be N. gonorrhoeae. This vaginal isolate may be a genetically transformed gonococcus with the ability to utilize maltose. Conversely, it may be a meningococcus which has acquired antigenic determinants of N. gonorrhoeae.     

Gelatin neutralization of the inhibitory effect of sodium polyanethol sulfonate on Neisseria meningitidis in blood culture media.

The inhibitory effect of sodium polyanethol sulfonate (0.05%) upon growth of Neisseria meningitidis was found to be neutralized by adding gelatin (l.1%) to the growth medium. The neutralizing effect was demonstrated in solid medium, as well as in nutrient broth for blood cultures. The findings parallel those of Wilkins and West (6) regarding gelatin neutralization of the inhibitory effect of sodium polyanethol sulfonate on Peptostreptococcus anaerobius.     

The Phosphorylcholine Epitope Undergoes Phase Variation on a 43-Kilodalton Protein in Pseudomonas aeruginosa and on Pili of Neisseria meningitidis and Neisseria gonorrhoeae

Phosphorylcholine (ChoP) is a component of the teichoic acids of Streptococcus pneumoniae and has been recently identified on the lipopolysaccharide of Haemophilus influenzae, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of Pseudomonas aeruginosa examined and on several class I and II pili of Neisseria meningitidis. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of H. influenzae, the expression of this epitope was phase variable on these species. In P. aeruginosa, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.Choline, a major constituent of eukaryotic membrane lipids, was previously thought to be an unusual structural feature of prokaryotes. The best-known example is Streptococcus pneumoniae, which accumulates environmental choline and incorporates it in the form of phosphorylcholine (ChoP) into its glycolipid, lipoteichoic acid, as well as its cell wall-associated teichoic acid (8). It has been suggested that ChoP contributes to adherence of the pneumococcus to host cells by binding to the receptor for platelet-activating factor, whose natural ligand also contains ChoP (1). Recently, ChoP has been identified as a unique feature of the lipopolysaccharide (LPS) of Haemophilus influenzae (21, 22). In the case of H. influenzae, choline is also acquired from the growth medium and linked to a glucose residue on the outer core region of the rough LPS (22). The expression of ChoP on the H. influenzae glycolipid undergoes phase variation mediated by a translational switch within the gene licA, a putative choline kinase (20, 22). The only significant homology to licA in protein databases is in a gene found in several species of the genus Mycoplasma, including the common respiratory tract pathogen Mycoplasma pneumoniae. In addition, the ChoP structure has been identified on a polar lipid found in the opportunistic pathogen M. fermentans (2). It appears, therefore, that ChoP is a structure common to several important and distantly related pathogens, including S. pneumoniae, H. influenzae, and various mycoplasma species which reside on the mucosal surface and infect the human respiratory tract. In addition, screening of secretions from the human respiratory tract with a monoclonal antibody (MAb) specific to the ChoP epitope has revealed several other gram-positive species which bind the antibody and may contain the ChoP structure (3).The focus of this study was the identification of the ChoP epitope on pathogenic gram-negative species other than Haemophilus. Results of this screening show that this epitope is present and displays phase variation on protein structures of pathogenic neisseriae, and Pseudomonas aeruginosa. In the case of the neisseriae, the ChoP epitope was found to be present on pili.     

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.     

Relationship of Serogroups of Neisseria meningitidis II. R Variants of Neisseria meningitidis

Meningococci isolated in primary cultures from nasopharyngeal carriers occasionally consisted of mixtures of smooth (S) and rough (R) strains. The R strains were separated from the S strains and their morphological and serological characteristics were studied. Some of these R strains reverted spontaneously to S strains which subsequently produced group-specific polysaccharide. Several R strains, grown in the presence of deoxyribonucleic acid from either an R strain of known parentage or an S strain, formed recombinants with serological group specificity.     

Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae.

A library of Tn916-generated, tetracycline-resistant (Tc) mutants of the group B Neisseri meningitidis strain NMB was screened by using monoclonal antibodies (MAbs) that recognize structural differences in neisserial lipooligosaccharide (LOS). The LOS of parental strain NMB had a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were identified by colony immunoblots, electrophoresis, and Western immunoblots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6E4 but not MAb 3E11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa and reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSs of the R6 and SS3 mutants were predicted to contain different truncations of the core oligosaccharide. The LOS phenotype of each mutant was linked to Tc(r), as determined by transformation of the parent strain with DNA from the mutant. Southern hybridizations and single-specific-primer PCR revealed in each mutant a single truncated tn916 insertion which had lost genes required for mobilization. Tn916 mutagenesis was used to identify two distinct genetic sites in the meningococcal chromosome involved in biosynthesis of the oligosaccharide chain of LOS and to create genetically defined LOS mutants of N. meningitidis and Neisseria gonorrhoeae.     

Enhancement of recovery of Neisseria meningitidis by gelatin in blood culture media.

The efficacy of gelatin for the recovery of Neisseria meningitidis from blood cultures was evaluated in a clinical setting. The organism was isolated from seven patients with meningococcal infections in blood culture media containing 1% gelatin. In contrast, only two blood cultures from these patients were positive in media without gelatin (P less than 0.05). Gelatin did not influence the recovery of other organisms isolated during this study. Conventional blood culture media may be supplemented with gelatin when meningococcemia is suspected.     

Lipopolysaccharide banding patterns of Neisseria meningitidis and Neisseria gonorrhoeae.

The lipopolysaccharides of Neisseria meningitidis and Neisseria gonorrhoeae were examined by electrophoresis after whole-cell lysis and proteinase K digestion. The banding patterns observed from clinical isolates and laboratory strains demonstrated lipopolysaccharide which included a small number of smooth high-molecular-weight molecules as well as the previously reported lower-molecular-weight rough lipopolysaccharide.     

Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from lactoferrin.

The ability of 107 Neisseria isolates to compete for iron with human lactoferrin (LF) was examined. Each of 15 meningococci, 53% of 59 selected gonococci, and 24% of 33 commensal Neisseria could use LF-bound iron for growth. Isolates which could not obtain iron from LF were growth inhibited when sufficient LF was added to defined agar medium to bind available free iron. No difference was observed in the ability of colony type 1 and colony type 4 gonococci of the same strain to compete with LF for iron. LF was growth inhibitory for 50% of 22 disseminated disease isolates (DGI strains) and 51% of 35 local urogenital disease isolates (UGI strains). Only 14% of gonococcal isolates requiring arginine, hypoxanthine, and uracil for growth were able to compete with LF for iron, whereas 87% of all other gonococcal isolates could do so (P less than 0.005). Ability to obtain iron from LF does not appear to be required for survival of Neisseria on mucosal surfaces, nor essential for invasion of the bloodstream by gonococci. However, ability to utilize LF as a source of iron may contribute to differences in pathogenicity among certain gonococcal isolates.     

Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae.

The deposition and degradation of human complement component C3 on the cell surfaces of Neisseria meningitidis and Neisseria gonorrhoeae were studied. Bacteria were incubated in human serum, and ester-linked C3 fragments were analyzed by hydroxylamine release and immunoblot detection. Similar patterns of C3 degradation were found for both serum-resistant and serum-sensitive meningococcal strains of serogroups A, B, C, Y, and W135, as well as for serum-sensitive gonococcal strains and their sialylated serum-resistant variants. The predominant fragments in all cases were the 40-kDa alpha' 2 chain of iC3b and the 75-kDa beta chain common to both C3b and iC3b. The 67-kDa alpha' 1 chain of iC3b was also detected. The 105-kDa alpha' chain of intact C3b represented a minor proportion of deposited C3. Capsule-specific immunoglobulin G or immunoglobulin A1 did not alter the observed degradation patterns, nor did incubation of meningococci in properdin-deficient serum. The degradation of C3 in C5-, C6-, or C8-deficient serum was the same as that in normal serum, although the deposition of C3 was severely limited, based as indicated by the intensity of the fragments. With the use of an enzyme-linked immunosorbent assay that measured total iC3b and C3, I found that both iC3b deposition and C3 deposition varied among meningococcal and gonococcal strains and that the amounts of iC3b and C3 were independent of the relative quantities of cell surface sialic acid and of serum sensitivity for meningococci but not for gonococci. I conclude that complement activation on neisserial cell surface results in the formation of an identical repertoire of predominantly iC3b fragments of ester-linked C3b molecules regardless of the presence of sialic acid in either the capsule or the lipooligosaccharide or of the sensitivity of the organism to complement-mediated lysis but that the quantities of both ester- and amide-linked iC3b molecules deposited exhibit strain variability.     

Monoclonal antibody analysis of lipopolysaccharide from Neisseria gonorrhoeae and Neisseria meningitidis.

A hybridoma produced by the polyethylene glycol fusion of the NS-1 variant of the P3x63Ag8 BALB/c plasmacytoma to splenocytes harvested from a BALB/c mouse immunized with whole gonococci was found to be producing antibody to a common region on gonococcal lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay inhibition systems were established by utilizing this antibody, designated 3F11, and 100% inhibition occurred with both LPS and the LPS-LPS and LPS-derived polysaccharides partially inhibited the enzyme-linked immunosorbent assay, whereas similar preparations isolated from Escherichia coli O:111, the J-5 mutant of this strain, and Salmonella minnesota Re595 failed to inhibit the assay. Studies utilizing whole gonococcal strains 4505 and the isogenic variant 4505r, which lacks both the LPS serotype and common determinants as inhibitors, demonstrated that the determinant recognized by the 3F11 antibody was present on the surface of 4505 and absent on 4505r. Inhibition studies were performed with beta-glucose, beta-galactose, D-glucosamine, D-galactosamine, heptose, 2-keto-3-deoxyoctanoate, N-acetylglucosamine, N-acetylgalactosamine, alpha-lactose, and beta-lactose. Complete inhibition of the enzyme-linked immunosorbent assay occurred with D-galactosamine, and partial inhibition was achieved with both alpha-lactose and beta-lactose. Based on these observations, the 3F11 antibody recognizes a site common to gonococcal LPS which is partially shared by meningococcal LPS. The chemical structure of the determinant appears to be a D-galactosamine-O-D-galactopyranosyl-(1-4)-D-glucopyranose. Additional specificity may be conferred by the steric relationship of the determinant on the intact LPS.     

Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from transferrin and iron compounds.

The ability of Neisseria species to use iron compounds and to compete with iron-binding proteins was examined with deferrated defined medium and the iron chelator deferoxamine. All Neisseria species were able to assimilate a variety of ferric and ferrous iron salts. They were not able to efficiently solubilize an inorganic iron salt such as ferric nitrate, but were able to use iron chelated by citrate, oxalacetate, pyrophosphate, or nitrilotriacetate. Each of the 95 Neisseria isolates examined was able to use hemin as a sole source of iron, and most, but not all, of the isolates were able to obtain iron from hemoglobin. Heated human serum stimulated growth of all gonococci, meningococci, and some commensal Neisseria species in iron-deficient medium. All gonococci and meningococci were able to scavenge iron from 25% saturated transferrin, whereas most commensal organisms were inhibited by this iron-binding protein. The ability to compete with transferrin was specific, since partially saturated conalbumin was bacteriostatic for all Neisseria species. Although the pathogenic Neisseria species were able to compete more efficiently with transferrin for iron than were the nonpathogenic Neisseria species, no correlation was observed between the virulence of different strains or colony types of gonococci and the ability to scavenge iron in vitro from transferrin or other chelators.     

Neisseria meningitidis: serogroup Y. Apropos of 38 cases

Thirty-eight cases of infections due to Neisseria meningitidis serogroup Y have been studied. They correspond to the strains sent to the Laboratory (Centre National de Référence since 1987). Their repartition was: meningitidis 9 cases, purpura fulminans (6), septicaemia (5), respiratory syndroms (11), asymptomatic carriage (4), various (3). Patients were 4 months to 86 years old. Mortality was 13.2%. One third or meningitis or meningococcemia happened in correlation with complement deficiency or splenectomy. In most of cases bacterial diagnosis was made by general laboratories but only in two cases over 38 the serogrouping Y was made.     

Inhibitory effect in vitro of sodium polyanethol sulfonate on the growth of Neisseria meningitidis.

The influence of 0.05% sodium polyanethol sulfonate on the growth of 24 strains of Neisseria meningitidis in broth medium was examined; several of the strains were markedly inhibited. A paper disk method was evolved for screening the sensitivity of various bacteria to sodium polyanethol sulfonate on solid medium; the highest sensitivity was observed in N. meningitidis and Neisseria gonorrhoeae. Zones of growth inhibition were also observed in a proportion of strains in certain species of gram-positive cocci, whereas all gram-negative bacilli were uniformly resistant. The implications of these observations for the routine use of sodium polyanethol sulfonate in blood culture media are discussed.     

Neisseria meningitidis; clones,carriage, and disease

Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease.