Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria.

OBJECTIVE: Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. METHODS: Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. RESULTS: For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. CONCLUSIONS: The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

Effects of bacterial physiological states and bacterial species on host-microbe interactions.

This study investigated how the physiological states of Aggregatibacter actinomycetemcomitans (Aa) and Streptococcus mitis affect their intracellular invasion capabilities and the resulting host cell responses. The physiological states included two forms of planktonic states, floating or sedimented (by centrifugation) and the biofilm state (with centrifugation). Confluent epithelial Ca9-22 cells were challenged with floating or sedimented planktonic cultures, or with 24-h biofilms for 3 h. The results show that intracellular invasion efficiencies were clearly affected by the bacterial physiological states. For both bacterial species, the sedimented-cells displayed 2-10 times higher invasion efficiency than the floating-cells (p < 0.05). The invasion efficiency of Aa biofilms was three fold lower than sedimented cells, whereas those of S. mitis biofilms were similar to sedimented cells. Unlike invasion, the metabolic activities of Ca9-22 were unaffected by different bacterial physiological states. However, Aa biofilms induced higher IL-1ß expression than planktonic cultures. In conclusion, different bacterial physiological states can affect the outcomes of (in vitro) host-microbe interaction in different ways.

prtH in Tannerella forsythensis is not associated with periodontitis.

BACKGROUND: It has been suggested that prtH in Tannerella forsythensis encodes for a cystein proteinase that is associated with its pathogenic potential and can discriminate between periodontal health and disease. The aim of this investigation was to further establish this potentially important observation. METHODS: A group of 33 consecutive adult patients with periodontitis (mean age: 47.6 +/- 10.1 years) harboring T. forsythensis was selected to investigate the presence of prtH by polymerase chain reaction (PCR). The T. forsythensis strains were isolated by anaerobic culture techniques. To investigate the association of this gene with periodontitis, a group of 14 age-matched subjects (mean age: 56.4 +/- 6.9 years) without any signs of periodontal disease (probing depths <3 mm and no radiographic attachment loss) was tested for comparison. Pure isolates and crude subgingival plaque samples were used as a template for the PCR. RESULTS: In the group of 33 T. forsythensis-positive patients, we found two T. forsythensis isolates to be prtH negative. Despite repeated analyses, testing of the whole subgingival plaque samples revealed only 17 of 33 samples to be prtH positive. The T. forsythensis isolates from the 14 periodontally healthy subjects were all prtH positive. The odds ratio of the presence of prtH in T. forsythensis in periodontitis patients versus healthy controls is 1.06 (P >0.05). CONCLUSIONS: On the basis of our data, we conclude that the presence of prtH in T. forsythensis is not discriminative for patients with T. forsythensis-associated periodontitis compared to healthy carriers of T. forsythensis. In addition, the use of whole subgingival plaque samples to test for the prevalence of prtH in bacteria appeared unreliable. Culture of the microorganism is an important condition to receive a sufficient amount of template DNA to detect the specific locus of the genome.