Characterisation of non-capsulate Haemophilus influenzae by repetitive extragenic palindromic (REP)-PCR

Abstract

The use of repetitive extragenic palindromic (REP)-PCR to characterise non-capsulateHaemophilus inyuenzae (NCHI) for epidemiological studies was validated by application to four outbreak-associated and three epidemiologically unrelated NCHI strains of the same phenotype which had been well characterised by other methods. The REP-PCR patterns were reproducible and showed the unrelated isolates to be distinguishable from each other, whereas the outbreak-associated isolates were indistinguishable. The results were concordant with those from outer-membrane protein enriched profiles, ribotyping and randomly amplified polymorphic DNA analysis. When applied to six further isolates from two different suspected outbreaks, rapid results were obtained from boiled supernates prepared from one colony and indicated that the isolates in question were not related. REP-PCR provides a rapid method of strain characterisation suitable for NCHI, which is ideal for use in conjunction with other methods.

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

“…Sixty-six NTHI strains gave 62 different patterns (D Ͼ 0.99). These results are in agreement with those obtained by different techniques, showing that NTHI strains are extremely diverse (10,15,17,29,31,32,36). Among the 66 NTHI clinical isolates tested by IDS-PCR, only the epidemiologically related strains (nine strains) and two non-epidemiologically-related strains gave identical or closely related patterns.…”

“…PFGE, which is considered the gold standard, is laborious and time-consuming, and RAPD, which is a rapid and discriminatory technique, lacks reproducibility (15,17,36). PCR-based methods such as rep-PCR, which is based on repetitive REP and ERIC sequences, have been developed to offer the advantages of simple, rapid, reproducible, and discriminatory techniques.…”

“…Nevertheless, for Haemophilus typing, REP-and ERIC-PCR are always carried out under low-stringency conditions to allow primer binding. Thus, an annealing temperature between 25 and 40°C (10,15,28,38,42), 5 mM MgCl 2 (29), or degenerate primers (15) are used. These conditions, which are probably used to compensate for the absence of true REP and ERIC sequences in the Haemophilus genome, result in a lack of reproducibility (10,29).…”

“…rep-PCR assays are quick and inexpensive, are less laborious than ribotyping and PFGE, and generate results that are easier to interpret than those of RFLP and more reproducible than those of RAPD (10,15,29,42). However, Pettigrew et al recently reported that ERIC-PCR was less discriminatory than ribotyping and PFGE, and they recommended that ERIC-PCR be combined with other typing methods (29).…”

Emergence of Extensively Drug-Resistant Haemophilus parainfluenzae in Switzerland

“…Results for all of the above-mentioned genes were compared to the deposited genome of H. parainfluenzae T3T1 (GenBank accession number NC_015964) or homologies were searched for in BLAST (http://blast.ncbi.nlm.nih.gov/Blast .cgi). Repetitive extragenic palindromic PCR (rep-PCR) was per-formed as previously described and interpreted using the Agilent Bioanalyzer (14).…”