Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain.

The supermucoid Pseudomonas aeruginosa strain PDO300Deltaalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.

A novel porA-based real-time PCR for detection of meningococcal carriage.

Real-time PCR based on the capsule transfer gene (ctrA) is a significant aid in the diagnosis of meningococcal infection but fails to detect a high proportion (60 %) of non-groupable strains associated with nasopharyngeal carriage. This study aimed to design a novel real-time (TaqMan) PCR that would detect more strains of meningococci and be suitable for large-scale carriage studies. Primer and probe sequences were based on the meningococcal porA gene and designed specifically to exclude the highly related porA pseudogene in Neisseria gonorrhoeae. The specificity of the assay was confirmed by testing strains of N. gonorrhoeae known to contain the porA pseudogene together with commensal strains of Neisseria lactamica and Neisseria sicca. None of these was detected in the assay. Neisseria meningitidis strains representing a wide range of serogroups together with non-groupable strains isolated from the nasopharynx were tested by ctrA assay and the novel porA-based TaqMan PCR. All carriage strains were detected by the porA-based assay including four that gave weak or no reaction with the ctrA assay. Comparison of ctrA and porA assays on 71 throat swabs obtained from university students showed that the porA assay detected meningococcal DNA in all samples that were ctrA positive plus three that were ctrA negative but culture positive. This novel porA-based TaqMan assay provides a highly specific method for detecting meningococcal DNA that is more sensitive than the ctrA assay for detecting meningococcal carriage and is particularly suitable for carriage studies where non-groupable strains and other Neisseria are present.

Development of immunity to serogroup B meningococci during carriage of Neisseria meningitidis in a cohort of university students.

Understanding the basis of protective immunity is a key requirement for the development of an effective vaccine against infection with Neisseria meningitidis of serogroup B. We have conducted a longitudinal study into the dynamics of meningococcal acquisition and carriage in first-year university students. The detection of carriage of serogroup B meningococci correlated with an increase in detection of serum bactericidal activity (SBA) against both colonizing and heterologous serogroup B strains. Once induced, SBA remained high throughout the study. Although students showed increases in antibodies reactive with capsular polysaccharide and lipopolysaccharide (LPS), these antibody responses were transitory, and their decline was not accompanied by a corresponding decline in SBA. In contrast, there was a significant correlation between the presence of antibodies to the PorA outer membrane protein and SBA against both homologous and heterologous strains. SBA induced by a PorA-negative mutant confirmed the contribution of PorA to heterologous activity. Increases in SBA against a range of serogroup B strains were also observed in students in whom no meningococcal carriage was detected. This heterologous protection could not be associated with the presence of antibodies reacting with capsule, LPS, PorA, PorB, Rmp, Opa, Opc, or pilin, demonstrating that other, as yet unidentified, antigens contribute to the development of immunity to serogroup B meningococci. Identification of such antigens with the ability to induce an effective cross-reactive bactericidal response to a range of strains would be a major step in the production of a universally effective vaccine against infections caused by serogroup B meningococci.

Detection of meningococcal carriage by culture and PCR of throat swabs and mouth gargles.

The standard method for detecting meningococcal carriage is culture of throat swabs on selective media, but the levels of carriage determined depend heavily on the skills of the individuals taking the swab and interpreting the cultures. This study aimed to determine the most sensitive detection method for meningococcal carriage. Throat swabs and saline mouth gargles, obtained from 89 university students, were processed in parallel by conventional culture and TaqMan ctrA PCR. Carriage of meningococci, as detected by the combined methods, was 20%. The sensitivities of throat swab culture, throat swab PCR, gargle culture, and gargle PCR were 72, 56, 56, and 50%, respectively, and the probabilities that these techniques would correctly identify the absence of carriage (negative predictive value [NPV]) were 93.4, 89.9, 89.9, and 88.8%. Culturing both throat swabs and gargles increased the NPV to 98.6%. The further addition of throat swab PCR increased this to 100%. Testing gargles by both culture and PCR was as sensitive as testing throat swabs by both methods, suggesting that gargles may be a suitable alternative for large-scale screening studies when throat swabs are difficult to obtain, although they required more lengthy laboratory processing. PCR was a useful adjunct to culture for detecting nasopharyngeal carriage, but it failed to detect some nongroupable strains. For maximum sensitivity, a combination of techniques was required. This study indicates the confidence with which health care professionals involved in meningococcal screening can regard laboratory results.