ABSTRACT
A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.
Subject(s)
Bacterial Capsules/classification , Haemophilus influenzae/classification , Polymerase Chain Reaction , Bacterial Capsules/genetics , Base Sequence , DNA, Bacterial/chemistry , Genotype , Molecular Sequence DataABSTRACT
Genes for Haemophilus influenzae type b capsule expression are duplicated to form a potentially unstable structure, cap, of directly-repeated chromosomal regions of approximately 17 kb. Capsule-deficient mutants arise in a two-stage process, initiated by rec-dependent reduction of this region from two copies to one. This recombinational event is usually lethal, only about 1/200 surviving to form slow-growing colonies of organisms that continue to synthesize polysaccharide but are defective in its export. A variety of secondary 'rescue' mutations within cap can occur to reduce polysaccharide synthesis and restore normal organism appearance and colony morphology.
Subject(s)
Bacterial Capsules/genetics , Haemophilus influenzae/genetics , Mutation , Blotting, Southern , Genotype , Haemophilus influenzae/ultrastructure , Microscopy, Electron , Phenotype , Restriction Mapping , Transformation, BacterialABSTRACT
The nucleotide sequence of a 5.1 kb region in the Haemophilus influenzae type b capsulation locus has been determined and found to contain four open reading frames: bexD, bexC, bexB, and bexA. Comparison of the deduced products of bexC, bexB, and bexA to known proteins, and TnphoA mutagenesis, suggests that they form components of an ATP-driven polysaccharide export apparatus. Furthermore, close sequence similarity between BexA and BexB and products of the kpsT and kpsM genes at the Escherichia coli K5 capsulation locus (Smith et al., 1990--accompanying paper) suggests that capsulation genes in these organisms may have a common ancestry.
