Detection of Staphylococcus aureus in the Pulp of an Endocarditis Patient

S. aureus by Objective: Dental pulp is acknowledged to be an organic tissue sample on which the microbiological diagnosis of blood-borne pathogens, including those responsible for infectious endocarditis, can be based. Method: Molecular detection of S. aureus was performed in the dental pulp extracted from one tooth collected in a patient firmly diagnosed with S. aureus infectious endocarditis. Result: We report on one patient diagnosed with Staphylococcus aureus endocarditis in whom S. aureus DNA was further detected by PCR in the dental pulp. We advocate not throwing away extracted teeth, appropriate microbial investigations of which may reveal bacteraemic pathogens not otherwise detectable.

gentamycin and acyclovir was then changed for cefazolin, 12g/day and clindamycin 600mg four times/day. A transthoracic echocardiography found vegetation on the native bicuspid aortic valve and a moderate aortic insufficiency. The patient was then diagnosed with S. aureus infectious endocarditis. A total body scan found bilateral renal, splenic and hepatic embolisms. Brain magnetic resonance imaging found several right frontal, left occipital, temporal, thalamic and bilateral cerebellar hyper signals with haemorrhagic organisation in the right frontal and occipital lesion. Five days later, the patient presented with septic shock and was admitted to the cardiologic intensive care unit. Antibiotic therapy was changed for intravenous sulfamethoxazole 4,800mg/day, clindamycin 1,800mg/day, gentamycin 160mg/day and rifampicin 1,800mg/day. Laboratory tests showed leucocytosis at 19G/L, haemoglobin at 10.4g/dL, platelets at 180G/L, and protein C-reactive at 95.5mg/L. 18fluorodeoxyglucose-positron emission tomography/computed tomography showed multiple hypermetabolic foci in the lymph nodes, liver, muscles and bones. Spinal magnetic resonance imaging showed a lumbar L4-L5 and cervical C5-C6 spondylodiscitis.
Initial clinical and biological evolution was favourable, however, three weeks later the cardiac lesion worsened with severe aortic  Table 1). The pulp was extirpated from each tooth using a sterile excavator and cut into three fragments collected in sterile tubes, as previously described [11]. For the bacterial culture, one pulp fragment was crushed in a tube containing 30µL of sterile phosphate-buffered saline (PBS), inoculated on two plates of COS agar (bioMérieux, Mercy l'Etoile, France), and incubated at 37 °C without and/or with 5% CO 2 for 48 hours. Colonies were identified using matrix-associated laser desorption ionization/time of flight mass spectrometry (MALDI TOF MS), as previously described [12]. Two pulp fragments from teeth n°13, n°24 and n°37 were used for S. aureus quantitative qPCR and fast culturomics, as previously described [13]. Table 1: Results and identification of bacteria found by direct culture, real-time PCR (qPCR) and fast culturomics of one negative control patient and one patient with infectious endocarditis. ("+" denotes a positive result; "-" denotes a negative result).

Methods and Result
Teeth n°13, n°25 and n° 37 were negative for the standard bacterial culture, teeth n°24 and n°35 cultured Candida albicans, and tooth n°36 cultured Enterobacter cloacae, despite decontamination.

Discussion
Molecular detection of S. aureus was performed on the dental pulp extracted from one tooth collected from a patient definitively diagnosed with S. aureus infectious endocarditis. The results are presented in Table 1. The absence of contamination of the extracted dental pulp was ensured by the meticulous cleaning of the tooth with UltraPure™ DNase/RNase-Free Distilled Water after using chlorhexidine before the extirpation of the dental pulp. Molecular detection was confirmed by the absence of amplification of the extraction blank and the reaction mix. To our knowledge, this is the second case in which the DNA of the pathogen responsible for infectious endocarditis was found in the dental pulp of the patient. The first case was the detection of Bartonella quintana in a patient who had been bacteraemic six months previously [1].
Aerobic culture of two of the six teeth yielded C. albicans and E.
cloacae whereas culture of the negative control wisdom tooth remained negative, and three teeth from the patient also remained negative. Cultured pathogens may differ from tooth to tooth in the same individual. Moreover, yeasts have already been reported in the dental pulp despite previous decontamination of the manipulated tooth and the presence of antifungal in the culture medium [14]. E. faecium isolated in two of three teeth under at least two rapid culturomics conditions is one of the most prevalent pathogens responsible for IE [7,15]. Its detection in the patient reported on here may result from past E. faecium bacteraemia.
A few weeks prior to tooth extraction, the patient had S. aureus bacteraemia which had remained undocumented in the extracted cardiac valve by culture and PCR-based methods after three weeks of appropriate antibiotic therapy. In this context, S. aureus was detected by PCR in the dental pulp, illustrating the interest of microbial investigations of the dental pulp. This is, therefore, the second ever report of detection of a bacteraemic pathogen in the dental pulp, following our previous report of a similar observation in a patient with B. quintana endocarditis and bacteraemia [1]. These clinical observations may be more than simply fortuitous, as an experimental guinea pig model of C. burnetii bacteraemia also showed the pathogen to be detectable in the dental pulp [5]. These combined observations suggest that dental pulp may be a sanctuary for bacteraemic bacteria in which their elimination is delayed.

Conclusion
In conclusion, we advocate using extracted teeth to search for bacteraemic pathogens rather than disposing of them as is currently the case, as microbiological investigations of the dental pulp may offer diagnoses that are not otherwise achievable; including the very particular situation of post-mortem search for the cause of death in which PCR-based and culture-based investigations may reveal deadly septicaemia and endocarditis.