Primers were designed to amplify the rpoB gene of Neisseria meningitidis. The region of the gene amplified covered clusters I and II of the rifampin resistance (Rifr) mutation sites identified in Escherichia coli. DNAs from six Rifr isolates and 21 rifampin-susceptible isolates from the United Kingdom representing a number of serogroups were amplified and sequenced. All six Rifr isolates had identical DNA sequences and the same amino acid change, a His to an Asn change at position 35 (H35N). This His residue is equivalent to the His residue at position 526 in E. coli, one of the known Rifr mutation sites. DNAs from an additional six Rifr mutations generated in vitro were amplified and sequenced. Three had H35Y changes, one had an H35R change, one had an H35N change and one had an S40F change. The predominance of mutations at the His residue at position 35 in Rifr N. meningitidis isolates suggests that it plays a critical role in the selection of antibiotic-resistant variants. All six Rifr isolates belonged to the same clonal group when analyzed by restriction enzyme analysis and pulsed-field gel electrophoresis. These data suggest that a single clone of Rifr N. meningitidis is present and widespread throughout the United Kingdom.
Neisseria rneningifidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningifidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods -PFGE, MLEE and MLST -is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.
The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial antiserum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.
Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.
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