Microbiota around teeth and dental implants in periodontally healthy, partially edentulous patients:is pre-implant microbiological testing relevant?

This study aimed to assess the prevalence of seven periodontal marker pathogens, before implant placement and 1 yr after loading, in periodontally healthy individuals and to assess the long-term effectiveness of pre-implant reduction of pathogens to below threshold levels. In 93 individuals needing single tooth replacement, pooled subgingival microbiological samples from standard sites were cultured and analyzed before implant treatment and 1 yr after loading. Threshold levels commonly used in periodontology to predict periodontal breakdown were applied. Subjects with levels of pathogens above these thresholds received initial periodontal treatment including systemic antibiotics when indicated. At baseline, 49.5% of periodontally healthy subjects harboured one or more marker pathogens above threshold levels. Periodontal treatment reduced the pathogen levels below threshold values in 78.3% of these initially colonized subjects. In all cases Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were reduced to below threshold. At 1 yr after loading, periodontal pathogens were present above threshold levels in 74.1% of all subjects. It is concluded that in almost half of periodontal healthy individuals the subgingival biofilm harbours periodontal pathogens above threshold values. Long-term effectiveness of pre-implant reduction of the selected marker pathogens appeared limited in our patient population, making pre-implant reduction unpredictive for post-implant levels of these pathogens. Thus, considering the applied microbiological criteria, generalized pre-implant microbiological testing is not contributory in periodontally healthy subjects

Quality control for diagnostic oral microbiology laboratories in European countries

Participation in diagnostic microbiology internal and external quality control (QC) processes is good laboratory practice and an essential component of a quality management system. However, no QC scheme for diagnostic oral microbiology existed until 2009 when the Clinical Oral Microbiology (COMB) Network was created. At the European Oral Microbiology Workshop in 2008, 12 laboratories processing clinical oral microbiological samples were identified. All these were recruited to participate into the study and six laboratories from six European countries completed both the online survey and the first QC round. Three additional laboratories participated in the second round. Based on the survey, European oral microbiology laboratories process a significant (mean per laboratory 4,135) number of diagnostic samples from the oral cavity annually. A majority of the laboratories did not participate in any internal or external QC programme and nearly half of the laboratories did not have standard operating procedures for the tests they performed. In both QC rounds, there was a large variation in the results, interpretation and reporting of antibiotic susceptibility testing among the laboratories. In conclusion, the results of this study demonstrate the need for harmonisation of laboratory processing methods and interpretation of results for oral microbiology specimens. The QC rounds highlighted the value of external QC in evaluating the efficacy and safety of processes, materials and methods used in the laboratory. The use of standardised methods is also a prerequisite for multi-centre epidemiological studies that can provide important information on emerging microbes and trends in anti-microbial susceptibility for empirical prescribing in oro-facial infections

High-Throughput Multilocus Sequence Typing: Bringing Molecular Typing to the Next Level

<div><p>Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of <em>Legionella pneumophila</em>, <em>Staphylococcus aureus</em>, <em>Pseudomonas aeruginosa</em> and <em>Streptococcus pneumoniae</em> in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.</p> </div

The workflow for profiling 96 bacterial isolates with HiMLST.

<p>MLST target genes are amplified by a two-step PCR in multi-well plates. These amplicons are pooled, quantified, clonally amplified by emulsion PCR and sequenced by NGS. The workflow ends with the ST profiling of the individual bacterial isolate. Figures in squares indicate the hands-on time for each individual step.</p

Two-Step PCR strategy for HiMLST.

<p>During the first PCR, the targeted MLST gene is amplified and universal tails are incorporated. During the second PCR, the amplicons of each isolate are provided with 454 sequencing-specific nucleotides and a unique DNA barcode, the multiplex identifier (MID).</p

Number of reads per gene for <i>L. pneumophila</i>, <i>S. aureus</i>, <i>P. aeruginosa</i>, and <i>S. pneumoniae</i>.

<p>Box plots show median (middle line), 25^th percentile (lower box limit), 75^th percentile (upper box limit), 10^th percentile (lower whisker) and 90^th percentile (upper whisker) for number of reads per gene.</p

Relation between amplicon size and number of sequence reads.

<p>Mean number of reads (standard error of the mean) are plotted for the 28 different genes from the bacterial species <i>L. pneumophila, S. aureus, P. aeruginosa, and S. pneumoniae</i>. This relationship between size and number of reads is statistical significant (p-value <0.001, linear regression) and shows an R² value of 0.23.</p

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