Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis

Abstract

Summary. Non-capsulate strains of Haemophilus injluenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing : (i) the fingerprints of 16 colonies of a single H. injluenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. injluenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. injluenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. injluenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H . injluenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.

Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum

“…One mutator was an invasive blood isolate (C2202) and one was a conjunctivitis isolate (C2475). Pettigrew et al, 2002;van Belkum et al, 1994). We found PCR using only one primer per reaction provided more distinctive and reproducible amplification profiles.…”

Differences in genetic diversity of nonecapsulated Haemophilus influenzae from various diseases

“…All nonencapsulated isolates were X-and Vfactor-dependent for growth, were unable to metabolize 6-aminolevulanic acid, and were nontypable with antisera specific for type a-f by coagglutination (Dirks-Go & Zanen, 1978;Kilian, 1976). The major outer membrane protein patterns and random amplified polymorphic DNA patterns obtained after PCR of these isolates showed a high level of diversity, indicating that the isolates represented different strains (Groeneveld et al, 1990;Lomholt et al, 1993;Moller et al, 1995;van Alphen et al, 1983;Van Belkum et al, 1994). MEE.…”

Comparison of usefulness of randomly amplified polymorphic DNA and amplified-fragment length polymorphism techniques in epidemiological studies on nasopharyngeal carriage of non-typable Haemophilus influenzae

“…Several proposed protocols based on different tools, e.g. outermembrane protein typing, MLEE, PFGE, RAPD or multilocus sequence typing, have been used to evaluate NTHI strain relationships (Fuste et al, 1996;Gazagne et al, 1998;Gilsdorf, 1998;Moor et al, 1999;Saito et al, 1999;St Geme et al, 1994;van Belkum et al, 1994van Belkum et al, , 1997. Of these, some were aimed at finding differences among NTHI strains isolated from patients with persistent infections and from healthy humans (Cerquetti et al, 2000;van Alphen et al, 1991van Alphen et al, , 1997.…”

“…Encapsulated H. influenzae strains are known to be successfully subclassified by serotyping, biochemical testing, outermembrane protein typing, lipopolysaccharide analysis, multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (Fuste et al, 1996;Gazagne et al, 1998;Meats et al, 2003;Moor et al, 1999;Spinola et al, 1986;St Geme et al, 1994;van Belkum et al, 1994). All published data recognize encapsulated H. influenzae population structure as clonal (Moor et al, 1999;Musser et al, 1988;Omikunle et al, 2002).…”