Novel antibody assessment method for microbial compositional alteration in the oral cavity

Abstract

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.

Keywords: BHI, brain heart infusion; Dysbiosis; FCM, flow cytometry; LPxTG protein; Oral microbiology; Saliva; Streptococcus mutans; sIgA.

Fig. 1

FCM detection of cultured S. mutans cells with anti-WapA antibodies. Overnight cultures of S. mutans, S. gordonii, S. oralis, and S. sobrinus were incubated with HiLyte Fluor 488-conjugated anti-WapA antibodies and analyzed using FCM (A). FCM density plots of unstained (left panel) and anti-WapA-stained (right panel) S. mutans cells. (B) Representative FCM histograms of the streptococcal cultures stained with anti-WapA antibodies. The percentage of WapA-positive bacteria (intensity greater than unstained S. mutans) is indicated in each panel.

Fig. 2

Detection of WapA-positive bacteria from saliva samples before and after oral cleaning. Unstimulated saliva was collected before and after the subjects cleaned their mouths. The saliva samples were stained with anti-WapA antibodies and analyzed using FCM. (A) Representative FCM density plots of the saliva samples before (left panel) and after (right panel) cleaning. The percentage of WapA-positive bacteria is indicated in each panel. (B) Comparison of the amount of WapA-positive bacteria before and after oral cleaning. The data are mean ± SD from five subjects. *; P < 0.05.

Fig. 3

Detection of IgA-bound bacteria from saliva samples before and after oral cleaning. Unstimulated saliva was collected before and after the subjects cleaned their mouths. The saliva samples were stained with HiLyte Fluor 647-conjugated anti-human IgA antibodies and analyzed using FCM. (A) Representative FCM density plots of the saliva samples before (left panel) and after (right panel) cleaning. The percentage of IgA-bound bacteria is indicated in each panel. (B) Comparison of the amount of IgA-bound bacteria before and after oral cleaning. The data are mean ± SD from five subjects. ***; P < 0.001.

Fig. 4

Analysis of saliva samples by double staining with anti-WapA and anti-IgA antibodies. (A) Schematic representation of the interpretation for the quadrant gates of (B). (B) Unstimulated saliva samples before and after oral cleaning were stained simultaneously with anti-WapA and anti-IgA antibodies and analyzed using FCM. Representative FCM density plots are shown. The numbers in the figure indicate the percentage of bacterial cells in each gate.