Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction
Highlights
► We present a rapid PCR method that is completed in 35 min. ► Sensitivity and specificity of this technique are comparable to conventional PCR. ► A. actinomycetemcomitans detection was similar in 40 human samples tested. ► Twenty seven of 32 rhesus monkeys were shown to harbor A. actinomycetemcomitans.
Introduction
Aggregatibacter actinomycetemcomitans is a Gram negative coccobacillus implicated in localized aggressive periodontitis (Zambon, 1985, Christersson, 1993). A. actinomycetemcomitans colonizes the oral cavities of humans (Slots, 1976, Socransky and Haffajee, 1992) and non-human primates (Eke et al., 1993) and belongs to the HACEK group of organisms believed to be associated with a number of systemic diseases including infective endocarditis (Das et al., 1997, Ellner et al., 1979, Paturel et al., 2004).
In addition to conventional culture based methods used to identify A. actinomycetemcomitans, PCR is now a well-established and widely used technique (Flemmig et al., 1995, Tran and Rudney, 1999). A large number of reports exist in the literature describing PCR based identification of A. actinomycetemcomitans (Flemmig et al., 1995, Goncharoff et al., 1993, Kim et al., 2005). While several studies have aimed at identifying only A. actinomycetemcomitans in the specimen, others have identified additional oral bacteria apart from A. actinomycetemcomitans e.g., using multiplex PCR (Tran and Rudney, 1999). Furthermore, 16S rDNA has been used as the target in PCR in many studies, but other genes such as lktA (Flemmig et al., 1995, Goncharoff et al., 1993, Tonjum and Haas, 1993) have also been used to identify A. actinomycetemcomitans. PCR based detection of bacteria in clinical specimens is sensitive and specific (Ficarra and Eversole, 1992, Olive, 1989). Conventional PCR, however, is time-consuming more often than not. This is mainly due to poor heat transfer on conventional PCR machines, resulting in longer time required to complete the reaction. In a clinical study setting during field screening of patients, generally samples are collected and brought to the laboratory where samples are processed and PCR performed for A. actinomycetemcomitans identification. The overall time for conventional PCR can vary from 2 to 4 h to overnight (Kramer and Coen, 2001). It is advantageous when conducting a screening examination to identify subjects who harbor A. actinomycetemcomitans at chair side within a short time period so that they can be informed that they are carriers of this potentially pathogenic organism. Extended time periods required for conventional PCR are inconvenient and can result in loss of subject interest and participation in ongoing studies. Therefore, rapid attainment of data at chair side during screening examinations could provide a great advantage and should improve recruitment of subjects. In our approach to develop a rapid PCR method for the detection of A. actinomycetemcomitans, we utilized samples from A. actinomycetemcomitans-positive and negative subjects who were involved in a longitudinal study of the relationship of A. actinomycetemcomitans to the initiation of localized aggressive periodontitis. In addition, oral samples from several primate species were used to compare culture to conventional PCR and to a new Super Convection rapid PCR technique. In this report we demonstrate that the new ultrafast PCR technique can be conveniently used as a chair-side tool for rapid A. actinomycetemcomitans detection.
Section snippets
Bacterial culture
Buccal and plaque samples were suspended in A. actinomycetemcomitans Growth Medium (AAGM) broth [trypticase soy broth with 0.8% glucose (8 g/l), 0.6% yeast extract (6 g/l) and 0.4% sodium bicarbonate (4 g/l), 75 μg/ml bacitracin and 5 μg/ml vancomycin] and brought to the laboratory for processing. In some cases, plaque and buccal samples were collected using cytology brushes, and then the brushes were stabbed in half-strength AAGM agar in small glass vials before being sent to our laboratory. Once
Validation of the efficacy of rPCR
In order to test the reliability of the Super Convection rPCR, we performed PCR for lktA of A. actinomycetemcomitans strains in parallel both on the AmpXpress machine as well as the conventional PCR machine. Fig. 1A shows that all A. actinomycetemcomitans strains tested produced an expected 262-bp band of similar intensity from both PCR machines. Sensitivity of the super convection rPCR was also compared with that of the conventional PCR. Genomic DNA from a serial 10-fold dilutions of A.
Discussion
Although microbiological and biochemical tools are an essential part of A. actinomycetemcomitans identification, PCR is used as a routine and common technique. In clinical studies involving subjects, rapid identification of A. actinomycetemcomitans might be of great advantage since chair-side identification could inform patients of the presence of this pathogenic bacterium. In this study, we demonstrated that using a Super Convection rapid PCR technique, A. actinomycetemcomitans could be
Acknowledgements
This study was supported by NIDCR grants R21DE021172 and R01DE017968 to D. H. Fine.
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