Elsevier

Archives of Oral Biology

Volume 74, February 2017, Pages 55-62
Archives of Oral Biology

Detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus oralis in blood samples with different microbiological identification methods: An in vitro study

https://doi.org/10.1016/j.archoralbio.2016.11.007Get rights and content

Highlights

  • Direct anaerobic culture (DAC) reported the best results.

  • DAC, lysis-centrifugation and qPCR were able to detect all concentrations.

  • A detection limit for qPCR at 102 CFU/mL for P. gingivalis is advisable.

  • A detection limit for qPCR at 104 CFU/mL for S. oralis is advisable.

Abstract

Background

Culture-based methods (culture broth bottles or lysis methods) have been the standard for detecting bacteremia. More recently, quantitative polymerase chain reaction (qPCR) was proposed as a more sensitive and specific test although none of them has been validated for the identification of periodontal pathogens (fastidious growing bacteria) in blood samples.

Objective

To compare the ability to detect and quantify Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus oralis (alone or in combination) in blood samples with three culture techniques [direct anaerobic culturing (DAC), haemo-culture (BACTEC), and lysis-centrifugation (LC)] and a non-culture dependent approach (qPCR) in an in vitro study.

Material and methods

Blood samples from 12 periodontally healthy volunteers were contaminated with three concentrations [104,102 and 101 colony forming units (CFU)/mL] of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination. Samples were analysed by DAC, BACTEC, LC and qPCR. Sensitivity, specificity, predictive values, kappa index and Lińs correlation coefficients were calculated.

Results

DAC, LC and qPCR were able to detect the three target species at all concentrations. An excellent concordance (correlation coefficient r: 0.92–1) was observed between DAC and the reference standard (sensitivity raging 93.33–100% and specificity 88.89–100%) values. BACTEC was not able to identify P. gingivalis in any of the performed experiments. qPCR provided false negative results for S.oralis.

Conclusions

DAC showed the best results for the proper identification and quantification of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination, in blood samples.

Introduction

Bacteremia has been defined as the transient, intermittent, or continuous presence of viable bacteria in the bloodstream (Carmona, Diz Dios, & Scully, 2002). Streptococcus oralis, is considered as one of the initial colonizer of the subgingival biofilm (“Consensus report. Periodontal diseases: pathogenesis and microbial factors," 1996) and has been described as the most frequently isolated bacterium in positive blood cultures after toothbrushing (Forner, Larsen, Kilian, & Holmstrup, 2006; Diz, Tobías, Scully, & Donos, 2012) flossing and scaling and root planning (Crasta et al., 2009; Zhang, Daly, Mitchell, & Curtis, 2013). Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are considered specific pathogens associated with periodontitis(“Consensus report. Periodontal diseases: pathogenesis and microbial factors," 1996). They have been found in atherosclerotic plaques, abdominal aortic aneurysms and heart valves (Figuero et al., 2011; Gaetti-Jardim, Marcelino, Feitosa, Romito, & Avila-Campos, 2009; Nakano et al., 2009; Zaremba, Górska, Suwalski, & Kowalski, 2007). Furthermore, these bacteria have been found, although in lower frequency than S. oralis, in blood samples after toothbrushing (Lockhart et al., 2008), flossing (Crasta et al., 2009) or scaling and root planing (Castillo et al., 2011). Therefore, these three target bacteria might represent and adequate representation of bacteremia in periodontitis patients.

However, these studies have reported very heterogeneous results in terms of prevalence (15–90%) and it has been argued that the use of different microbiological techniques may be, at least, in part responsible of this variability (Reyes, Herrera, Kozarov, Roldá, & Progulske-Fox, 2013; Tonetti, Van Dyke, & Working group 1 of the joint EFP/AAP workshop, 2013).

In gingival crevicular samples, direct anaerobe culturing (DAC), in which samples are directly cultivated in enriched medium agar plates, is considered the gold-standard culture method the detection and isolation of periodontal pathogens (Sanz, Lau, Herrera, Morillo, & Silva, 2004). In blood samples the most broadly used approaches are the standard hemoculture methods, using culture broth bottle-based (Crasta et al., 2009; Daly, Mitchell, Highfield, Grossberg, & Stewart, 2001; Kinane, Riggio, Walker, MacKenzie, & Shearer, 2005; Lafaurie et al., 2007, Lockhart et al., 2008; Maestre, Mateo, & Sánchez, 2008), lysis filtration (Forner et al., 2006) or lysis (Zhang et al., 2013). All these culture-based approaches, however, require the proper bacterial growth conditions, the need to identify species based on the colony and cell morphology and suitable specific tests, and time for an appropriate growth. To overcome some of these limitations, microbial diagnosis based on polymerase chain reaction (PCR) has been proposed for detecting bacterial pathogens in blood samples, as it has been considered as a more sensitive and specific method when compared to culture (Castillo et al., 2011, Kinane et al., 2005).

In spite of these bacteremia studies using different microbiological methodologies, there is no published investigation aimed to validate these microbial diagnostic techniques. It is, therefore, the aim of this in vitro investigation to compare and validate different microbiological methods (DAC, BACTEC, LC and qPCR) in the detection and quantification of A. actinomycetemcomitans, P. gingivalis and S. oralis in blood samples. Specifically, it was aimed to determine the diagnostic validity (sensibility, specificity and predictive values), detection limits and the reproducibility of tested microbiological diagnostic approaches.

Section snippets

Study design

This was an in vitro study consisting on twelve experiments (n = 12) where bacteremia was simulated by direct contamination of blood with A. actinomycetemcomitans (n = 3), P. gingivalis (n = 3), S. oralis (n = 3), and all three bacterial species combined (n = 3).

Patients

Samples were obtained from periodontally healthy volunteers at the Faculty of Odontology, University Complutense, Madrid (UCM). All subjects were informed about the aims of the study and signed a consent form, previously approved by the local

Descriptive results

Propionebacterium acnes and Coagulase-negative Staphylococcus were detected with BACTEC® system. However, they were considered contaminants from either the blood sampling procedures or from laboratory procedures and were not considered in data analyses.

DAC, LC, qPCR detected P. gingivalis, A. actinomycetemcomitans and S. oralis in blood samples while BACTEC® did not detect P. gingivalis (alone or in combination with other bacteria) and A. actinomycetemcomitans (in combination with other

Discussion

The present in vitro investigation was designed to compare four microbiological techniques, three of them previously used in bacteremia studies, targeting P. gingivalis, A. actinomycetemcomitans and/or S. oralis in blood samples. Results revealed that: (1) conventional BACTEC® haemoculture was not able to identify P. gingivalis (alone or in combination), or A. actinomycetemcomitans (in combination with P. gingivalis and S.oralis); (2) the use of DAC reported the best results in terms of

Conflicts of interest

The authors report no conflicts of interest related to this study.

Ethical approval

Ethical Approval was given by the local ethical committee (Hospital Clínico San Carlos, Madrid, Spain). Reference number: 13/027-E

Acknowledgements

This work was partially support by a research grant from the ministry of Science and Innovation of Spain (FIS [PI11/00542]).

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