Molecular typing of Haemophilus influenzae using a DNA probe and multiplex PCR

The use of new molecular typing methods for the characterization of Haemophilus influenzae strains is reported. Sixty-four isolates of H. influenzae originating from different types of infection and obtained from eight hospitals across Canada were first analysed for restriction polymorphism. Chromosomal DNA fragments generated by two different combinations of restriction endonucleases were electrophoresed and transferred to nylon membranes before hybridization with a species specific 32P-labelled DNA fragment (5 kb) used as a probe. The combinations Bg/II/PstI led to 11 typing groups (A-K) and BamHI/Bg/II/PstI to 14 sub-groups, respectively. Most of the isolates retrieved from cerebrospinal fluids (10/13; 76.9%) were classified in two groups (A and B) and two sub-groups. Isolates from respiratory tract infections were mostly found in groups C and E (24/32; 75.0%), and divided into seven sub-groups. Selected ampicillin-resistant, beta-lactamase-negative strains were also found in groups C and E (11/14; 78.6%). Isolates from conjunctivitis and acute otitis media were classified in various groups. All biotypes (I-VIII) and serotypes (none, a-f) were spread among the typing groups although biotype I prevailed in groups A, B, and G; II in group E (sub-group 6); and III in group C. A PCR approach derived from the typing system was also tested. A set of 25-mer primers was selected from the 5-kb DNA probe for the amplification of a 317-bp region. This set of primers was used concomitantly in a PCR multiplex assay with a set of primers selected from the nucleotide sequence of the gene encoding the H. influenzae P1 protein. This multiplex assay was also able to discriminate the clonal origin of some H. influenzae strains because size polymorphism was observed in PCR products. The PCR approach was then used to determine the genetic relatedness of H. influenzae strains found persistently in sputa of some patients with cystic fibrosis. Genetically related strains could be isolated from some patients even after antibiotherapy and months between visits, whereas other patients showed distinct strains. In summary, our typing system is able to provide new characteristics for strains having identical biotype or serotype. The rapid PCR alternative may prove useful for specific epidemiological and strain-tracking studies.