Evaluation of Two Commercial Kits and Arbitrarily Primed PCR for Identification and Differentiation of Actinobacillus actinomycetemcomitans , Haemophilus aphrophilus , and Haemophilus paraphrophilus

Abstract

The closely related species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus are common findings in oral microbiota. The aims of this study were to evaluate the applicability of the Rapid NH and API ZYM kits and arbitrarily primed PCR (AP-PCR) in the identification and differentiation of the three species from each other. The material included 62 clinical isolates and three reference strains of A. actinomycetemcomitans representing the 5 serotypes and 18 AP-PCR genotypes. Haemophilus species included 12 clinical isolates and 11 reference strains of H. aphrophilus, H. paraphrophilus, and 5 other species. For the PCR amplification, the oligonucleotide 5-CAGCACCCAC-3 was used as a primer. Contrary to the consistent performance of API ZYM, the Rapid NH system was able to identify only 10 of 65 (15%) A. actinomycetemcomitans isolates, whereas all Haemophilus species were correctly identified. The API ZYM test differentiated A. actinomycetemcomitans from H. aphrophilus and H. paraphrophilus by negative ␤-galactosidase and ␣-glucosidase reactions and a positive esterase lipase reaction. However, the API ZYM test was unable to differentiate H. aphrophilus from H. paraphrophilus, it also could not differentiate A. actinomycetemcomitans serotypes from each other. Among the H. aphrophilus isolates three AP-PCR genotypes and among H. paraphrophilus isolates only one AP-PCR genotype, distinct from those of A. actinomycetemcomitans, were found. The Rapid NH test showed poor ability to identify clinical isolates of all A. actinomycetemcomitans serotypes. Moreover, AP-PCR genotyping proved to be a rapid method for the species differentiation of A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus.

Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in systemic and nonoral infections in Finland

“…Within the species A. actinomycetemcomitans, all the presently detected four serotypes and four arbitrarily primed‐PCR (AP‐PCR) genotypes are commonly found in the oral cavities of Finnish subjects (11, 12). Among the present H. aphrophilus strains, two of the six AP‐PCR genotypes were similar to and four of six AP‐PCR genotypes were different from those previously observed among oral strains (13). This may be due to the limited data on the AP‐PCR genotyping of oral H. aphrophilus comprising only 11 strains (13).…”

“…This could be true in laboratories which routinely discard the plates after 2 days of incubation. In addition, inaccuracy of identification kits (13), obliging the primary laboratories to forward the samples to reference laboratories, may delay the correct diagnosis of the patient. In the present study, all strains were identified by a reference laboratory.…”

“…A. actinomycetemcomitans and H. aphrophilus strains were genotyped by arbitrarily primed polymerase chain reaction (AP‐PCR) with the random sequence oligonucleotide OPA‐13 (5′‐CAGCACCCAC‐3′) as a primer (11). A. actinomycetemcomitans AP‐PCR genotypes were classified according to Asikainen et al (11) and Dogan et al (12), and H. aphrophilus AP‐PCR genotypes according to Dogan et al (13). Minimum inhibitory concentrations (MICs) for A. actinomycetemcomitans and H. aphrophilus strains were determined on Haemophilus test medium (HTM) by the agar dilution method (14), and confirmed by the E test when needed (15).…”

Presence of Aggregatibacter actinomycetemcomitans in young individuals: a 16‐year clinical and microbiological follow‐up study

“…The AP‐PCR genotyping of the A. actinomycetemcomitans isolates was performed as described previously (Saarela et al 1992, Asikainen et al 1995, Doğan et al 1999).…”

Transmission of periodontal bacteria and models of infection

“…The resulting AP‐PCR products will represent a variety of different‐sized DNA fragments that are visualized by agarose gel electrophoresis. This method has been applied to A. actinomycetemcomitans and P. gingivalis (Dogan et al 1999a).…”