Biofilm formation by the human pathogen Neisseria meningitidis was analyzed. Biofilm-forming meningococcal strains were identified and quantitated by crystal violet staining. Laser scanning confocal microscopy of the meningococcal biofilm revealed variable layers up to 90 m in thickness. A total of 39 meningococcal isolates were studied; 23 were nasopharyngeal-carriage isolates, and 16 were invasive-disease isolates. Thirty percent of carriage isolates and 12.5% of invasive-disease isolates formed biofilms proficiently on a polystyrene surface. Generally, the strains that formed biofilms showed high-level cell surface hydrophobicity, characteristic of strains lacking a capsule. The inhibitory role of capsule in biofilm formation was further confirmed by comparing the biofilm-forming capabilities of a serogroup B wild-type strain of a disease-associated isolate to those of its capsule-deficient mutant (ctrA). Some strains of meningococci form biofilms, and this process is likely important in menigococcal colonization.Bacterial biofilms are sessile bacterial communities that adhere to each other and solid surfaces and are enclosed in an exopolysaccharide matrix (6). Biofilms are the predominant communities of many bacterial species in numerous ecosystems. Formation of biofilms involves participation of the extracellular-matrix and cellular-surface molecules, including membrane proteins. Biofilm formation also requires considerable bacterial energy and resources. The formation of biofilms begins with the attachment of the planktonic cells to a suitable surface, followed by replication and spreading. Eventually, the biofilms mature to differentiated forms. Exopolysaccharides play a key role in the establishment of biofilm architecture (6).In clinical settings, bacteria in biofilms are less susceptible to antimicrobial agents and host immune responses, thereby becoming persistent colonizers or sources of chronic infections (8). Bacteria are released from biofilms as individual planktonic cells or as a result of the sloughing of the biofilms. While many biofilms form on abiotic surfaces such as medical devices, some also develop on living tissues, as in the case of endocarditis or cystic fibrosis (8).Studies of biofilm formation by the Neisseria species are very limited, and most of those species examined have been oral commensals (4,20,24,26,38). Biofilm formation by Neisseria meningitidis, an etiologic agent of epidemic sepsis and bacterial meningitis, has not been documented. Meningococci are isolated from 5 to 10% of the normal population, and the colonization of the human nasopharyngeal mucosal surface by meningococci is the first step of the host-parasite interaction.Successful meningococcal colonization requires initial attachment facilitated by pili and subsequent interaction of other secondary-surface molecules with the host mucosal surface (12,31,36,43).In this study, the formation of the biofilms by N. meningitidis was assessed. In addition, the roles of the bacterial-surface molecules (pilus, capsule, and ...

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A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2 , and 3 , and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated ␤-(1 3 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amidelinked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, ␤-hydroxybutyrate, is esterified to ( ؊ 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.The family Rhizobiaceae includes the Rhizobium and Sinorhizobium, Gram-negative bacteria able to form nitrogen-fixing symbioses with legumes in a host-specific manner. Sinorhizobium sp. NGR234 is a fast growing, broad-host-range symbiont able to colonize a diverse range of commercially important legumes (1, 2), including both indeterminate and determinate nodule-forming hosts. Partly because of its agricultural role, the molecular genetics of NGR234 are of interest, and the symbiotic plasmid was recently sequenced (3). However, there is little complementary structural information on the cell surface macromolecules or the alterations that occur in these molecules during symbiotic infection and bacteroid differentiation.Lipopolysaccharides (LPS) 1 are the major structural and antigenic components of the rhizobial outer membrane (4 -8) and are proposed to contribute to the biochemical processes that result in symbiotic infection (8 -16). Rhizobial LPS structural mutants typically yield phenotypes with underdeveloped nodules (Ndv Ϫ phenotype) in which nitrogen fixation is absent or diminished (Fix Ϫ ) (5, 9, 15, 17-23).The lipid A moieties of rhizobial LPS are of interest, because of their ...

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Invasive bacterial meningitis caused by Neisseria meningitidis can be prevented by active immunization with meningococcal polysaccharide or polysaccharide-protein conjugate vaccines. In a tetravalent A/C/Y/W-135-DT meningococcal conjugate vaccine vial, or in a final formulated bulk, accurate identification and quantification of each polysaccharide are critical in product release. Determination of sialic acid serogroups (C, W-135, and Y) unambiguously is complex since all these serogroups contribute to the sialic acid monosaccharide peaks that overlap in the high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). We report a quantification method that involves generation of monosaccharide standard plots for respective sugars mannosamine-6-phosphate, sialic acid, galactose- and glucose-derived from hydrolysis of mixtures of the four serogroups A, C, W, and Y reference polysaccharides. These plots were then used to obtain the unknown polysaccharide concentrations of A/C/Y/W-135 in vialed vaccine or from formulated final bulks. We also present our results of the HPAEC-PAD profiles on groups C, W-135, and Y polysaccharides when hydrolyzed individually and/or in mixtures to discuss the individual sialic acid peak contributions.

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We previously reported development of a proteomics and AP-MALDI-MS/MS based system for biological detection and identification, offering fast analysis in minutes. In this work, a direct aerosol sampling module is reported which is interfaced with a small sample preparation module to reduce the overall size and power consumption. Bioaerosol samples are directly collected on a conveyor surface. On the same surface, sample processing is conducted, which include extraction of target-specific proteins using selective solubilization methods and in situ proteolysis. The microorganisms or toxins are identified using an AP-MALDI ion trap MS and MS/MS typing method. Identification for close relatives BA and BC with the MS/MS typing method is described.