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Background  Oral polymicrobial interactions and biofilm formation are associated with initiation and progression of caries, gingivitis, and periodontitis. Transcriptome studies of such interactions, allowing a first mechanistic insight, are hampered by current single-species array designs.    Methodology/Principal Findings  In this study we used 385 K NimbleGene™ technology for design and evaluation of an array covering the full genomes of 5 important physiological-, cariogenic-, and periodontitis-associated microorganisms ( Streptococcus sanguinis ,  Streptococcus mutans ,  Fusobacterium nucleatum ,  Aggregatibacter actinomycetemcomitans , and  Porphyromonas gingivalis ). Array hybridization was done with cDNA from cultures grown for 24 h anaerobically. Single species experiments identified cross-species hybridizing array probes. These probes could be neglected in a mixed-species experimental setting without the need to exclude the whole genes from the analysis. Between 69% and almost 99% of the genomes were actively transcribed under the mono-species planktonic, monolayer, and biofilm conditions. The influence of  Streptococcus mitis  (not represented on the array) on  S. mutans  gene transcription was determined as a test for a dual-species mixed biofilm setup. Phenotypically, under the influence of  S. mitis  an increase in  S. mutans  biofilm mass and a decrease in media pH-value were noticed, thereby confirming previously published data. Employing a stringent cut-off (2-fold, p<0.05), 19  S. mutans  transcripts were identified with increased abundance, and 11 with decreased abundance compared to a  S. mutans  mono-species biofilm. Several of these genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array.    Conclusions/Significance  This new array allows transcriptome studies on multi-species oral biofilm interactions. It may become an important asset in future oral biofilm and inhibitor/therapy studies.

A Five-Species Transcriptome Array for Oral Mixed-Biofilm Studies