Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction

https://doi.org/10.1016/j.mimet.2012.01.016Get rights and content

Abstract

Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium associated with localized aggressive periodontitis (LAP). Detection of A. actinomycetemcomitans in clinical samples is routinely done by PCR. Our aim was to develop a rapid and reliable PCR method that can be used as a chair-side tool to detect A. actinomycetemcomitans in clinical samples. Sensitivity and specificity assessment was performed on buccal and plaque samples obtained from 40 adolescents enrolled in an ongoing LAP study by comparing 20 A. actinomycetemcomitans-positive subjects and 20 who were negative. In a second study, A. actinomycetemcomitans presence was tested in oral samples from eighty-six primates that included rhesus monkeys, chimpanzees, marmosets, tamarins and baboons. All samples were processed for detection of A. actinomycetemcomitans by means of culture, conventional PCR (cPCR) and rapid PCR (rPCR) using a Super Convection based AmpXpress thermal cycler (AlphaHelix, Sweden). For human samples, culture, cPCR and rPCR showed perfect agreement. Using this method A. actinomycetemcomitans was detected in 27 of 32 rhesus monkeys, 4 of 8 chimpanzees and 1 of 34 marmosets. Rapidity of AmpXpress thermal cycler, combined with Ready-To-Go PCR beads (GE Life sciences), a quick DNA extraction kit (Epicentre Biotechnologies, Madison, Wisconsin, USA) and a bufferless fast agarose gel system, made it possible to obtain results on A. actinomycetemcomitans detection within 35 min. We conclude that AmpXpress fast PCR can be conveniently used as a chair-side tool for rapid detection of A. actinomycetemcomitans in clinical samples.

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.

References (29)

  • G. Jia et al.

    A low-cost, disposable card for rapid polymerase chain reaction

    Colloids Surf. B Biointerfaces

    (2007)
  • L. Paturel et al.

    Actinobacillus actinomycetemcomitans endocarditis

    Clin. Microbiol. Infect.

    (2004)
  • L.A. Christersson

    Actinobacillus actinomycetemcomitans and localized juvenile periodontitis. Clinical, microbiologic and histologic studies

    Swed. Dent. J. Suppl.

    (1993)
  • M. Das et al.

    Infective endocarditis caused by HACEK microorganisms

    Annu. Rev. Med.

    (1997)
  • A.J. deMello

    Microfluidics: DNA amplification moves on

    Nature

    (2003)
  • S. Dreizen et al.

    Monkey models in dental research

    J. Med. Primatol.

    (1977)
  • P.I. Eke et al.

    Sub-gingival microflora in Macaca mulatta species of rhesus monkey

    J. Periodontal Res.

    (1993)
  • J.J. Ellner et al.

    Infective endocarditis caused by slow-growing, fastidious, Gram-negative bacteria

    Medicine

    (1979)
  • G. Ficarra et al.

    Polymerase chain reaction: relevance for oral pathology

    Minerva Stomatol.

    (1992)
  • T.F. Flemmig et al.

    Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR

    J. Clin. Microbiol.

    (1995)
  • O. Gidlof et al.

    Complete discrimination of six individuals based on high-resolution melting of hypervariable regions I and II of the mitochondrial genome

    Biotechniques

    (2009)
  • P. Goncharoff et al.

    Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of lktA-specific sequences

    Oral Microbiol. Immunol.

    (1993)
  • J.B. Kaplan et al.

    Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f

    Infect Immun.

    (2001)
  • S.G. Kim et al.

    Identification of Actinobacillus actinomycetemcomitans using species-specific 16S rDNA primers

    J. Microbiol.

    (2005)
  • View full text