Ruhl Stefan, Eidt Andreas, Melzl Holger, Reischl Udo, Cisar John O
Department of Oral Biology, School of Dental Medicine, University at Buffalo, The State University of New York, Buffalo, New York, USA Department of Operative Dentistry and Periodontology, Dental School, University Hospital Regensburg, University of Regensburg, Regensburg, Germany
Department of Operative Dentistry and Periodontology, Dental School, University Hospital Regensburg, University of Regensburg, Regensburg, Germany.
Appl Environ Microbiol. 2014 Nov;80(21):6583-90. doi: 10.1128/AEM.01826-14. Epub 2014 Aug 8.
Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method's broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.
目前，由于缺乏一种便捷的检测方法来筛选口腔生物膜中众多的细菌种类，牙菌斑形成过程中的细菌间黏附研究受到了限制。为了克服这一限制，我们开发了一种基于固相荧光的筛选方法，用于检测和鉴定混合物种生物膜中的共黏附伙伴微生物。使用携带2型菌毛黏附素的放线菌和携带受体多糖（RPS）的链球菌的共聚集菌株，证明了该方法的适用性。通过将固定在硝酸纤维素膜上的细菌菌株与悬浮的、荧光素标记的细菌伙伴菌株覆盖，检测特异性黏附素/受体介导的共黏附。无论哪种细胞类型被标记，哪种细胞类型被固定，共黏附情况都是可比的。对悬浮或固定在硝酸纤维素上的细菌进行甲醛处理，可消除放线菌2型菌毛黏附素，但不影响链球菌RPS的功能，从而提供了一种简单的方法来为共黏附对中的成员分配互补的黏附素和聚糖受体。通过将牙菌斑生物膜培养物的菌落印记与荧光素标记的2型菌毛奈瑟放线菌或携带RPS的口腔链球菌菌株覆盖，证明了该方法具有更广泛的适用性。显著的共黏附伙伴不仅包括预期的链球菌和放线菌，还包括先前共聚集研究中未鉴定的其他细菌，如携带黏附素或受体的咽炎奈瑟菌、龋齿罗氏菌和口腔金氏菌。全面筛选复杂微生物群落中特定微生物的共黏附伙伴的能力，为牙菌斑和其他混合物种生物膜的研究开辟了一条新途径。
