Typing of Nonencapsulated Haemophilus Strains by Repetitive-Element Sequence-Based PCR Using Intergenic Dyad Sequences

Abstract

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of Haemophilus.Nontypeable Haemophilus influenzae (NTHI) strains are human commensal bacteria that are responsible for a number of respiratory tract infections such as otitis media, bronchitis, and sinusitis. NTHI strains are also responsible for genital, motherinfant, and neonatal infections. A group of genital strains usually identified as biotype IV NTHI constitutes a cryptic genital genospecies that shows remarkable genetic homogeneity and forms a monophyletic unit with H. influenzae and Haemophilus haemolyticus (31,32).NTHI strains are extremely diverse, and many typing methods have been proposed for epidemiological studies and pathogenesis investigations. Biotyping and more-discriminatory phenotypic typing methods such as outer membrane protein analysis (2,25,26,33) and lipooligosaccharide analysis (6, 13, 26) have gradually been replaced by genotypic techniques. Multilocus enzyme electrophoresis (MLEE) (27, 30, 31) is a discriminatory technique but is also very laborious. DNA restriction fragment length polymorphism (RFLP) analysis (4, 20, 42) has a high discriminatory capacity but gives overly complex banding patterns. Analysis of rRNA gene restriction patterns (ribotyping) (4, 16, 32, 46) is a discriminatory but time-consuming method. Analysis of pulsed-field gel electrophoresis (PFGE) patterns in which genomic DNA is digested with a rare cutting restriction enzyme (1, 36) is currently considered to be the "gold standard" for H. influenzae typing but is also a laborious and time-consu...

Polymerase Chain Reaction Amplification of Repetitive Intergenic Consensus and Repetitive Extragenic Palindromic Sequences for Molecular Typing of Pseudomonas anguilliseptica and Aeromonas salmonicida

“…The REP-PCR and ERIC-PCR analyses have been successfully used to type several other genera of bacteria (Hulton et al 1991;Rademaker et al 2000;da Silveira et al 2002;Bruant et al 2003); however, previous studies consider ERIC-PCR best for typing Aeromonas spp. (Soler et al 2003;Szczuka and Kaznowski 2004;Figueras et al 2005).…”

“…Amplification by PCR of the DNA repetitive elements is used to obtain strainspecific DNA fingerprints that can easily be analyzed with pattern recognition computer software. Previously, Rep-PCR based on ERIC and REP sequences have been successfully used to differentiate strains within several bacterial species (Georghinu et al 1994;Woods et al 1996;Bennasar et al 2000;Bruant et al 2003;Maluping et al 2005).…”

Capsule Gene Analysis of Invasive Haemophilus influenzae : Accuracy of Serotyping and Prevalence of IS 1016 among Nontypeable Isolates

“…Chromosomal DNA was extracted from NTHi strains (as determined by PCR assay) and capsule a to f control strains as previously described (5). Ten micrograms of H. influenzae chromosomal DNA was digested to completion with EcoRI (New England Biolabs, Beverly, MA) overnight at 37°C and separated by 0.7% agarose (Bio-Rad, Hercules, SC) gel electrophoresis and transferred to nylon membranes (Osmonics, Westborough, MA).…”

Molecular intraspecific characterization ofPhotobacterium damselaessp.damselaestrains affecting cultured marine fish

“…1991; Martin et al. 1992), and for this reason, they are useful for the discrimination of bacterial species and/or strains (Bruant et al. 2003; Rodriguez et al.…”

“…The most promising are the random amplified polymorphic DNA (RAPD), the repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) techniques, because they are fast and simple (Gillings and Holley 1997). These repetitive elements located in the intergenic regions of many bacterial genomes are highly conserved (Hulton et al 1991;Martin et al 1992), and for this reason, they are useful for the discrimination of bacterial species and ⁄ or strains (Bruant et al 2003;Rodriguez et al 2006). The use of appropriate outward-facing PCR primers designed to detect these repeated sequences generates multiple amplification products, which reflect distance polymorphisms between adjacent DNA repeat.…”