Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform

Abstract

Although biofilms have been shown to be reservoirs of pathogens, our knowledge of the microbial diversity in biofilms within critical areas, such as health care facilities, is limited. Available methods for pathogen identification and strain typing have some inherent restrictions. In particular, culturing will yield only a fraction of the species present, PCR of virulence or marker genes is mainly focused on a handful of known species, and shotgun metagenomics is limited in the ability to detect strain variations. In this study, we present a single-cell genome sequencing approach to address these limitations and demonstrate it by specifically targeting bacterial cells within a complex biofilm from a hospital bathroom sink drain. A newly developed, automated platform was used to generate genomic DNA by the multiple displacement amplification (MDA) technique from hundreds of single cells in parallel. MDA reactions were screened and classified by 16S rRNA gene PCR sequence, which revealed a broad range of bacteria covering 25 different genera representing environmental species, human commensals, and opportunistic human pathogens. Here we focus on the recovery of a nearly complete genome representing a novel strain of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis JCVI SC001) using the single-cell assembly tool SPAdes. Single-cell genomics is becoming an accepted method to capture novel genomes, primarily in the marine and soil environments. Here we show for the first time that it also enables comparative genomic analysis of strain variation in a pathogen captured from complex biofilm samples in a healthcare facility.

Figure 1.

Distribution of 78 candidate 16S rRNA sequences found in single-cell sorted wells from the sink biofilm sample. 16S rRNA sequences from single-cell amplifications observed for this FACS sorted sample.

Figure 2.

Circular representation of the draft JCVI SC001 genome. The assembled draft genome is the SPAdes assembly of MDA3 with the contigs ordered to the TDC60 reference genome. From the inner to the outer ring: coordinates in the assembled and concatenated JCVI SC001genome, G+C content, GCskew, ordered contigs, predicted CDS, TBLASTN alignment showing percent identity against P. gingivalis TDC60, W83, ATCC 33277, and Prevotella buccae ATCC33574 (near neighbor) reference genomes.

Figure 3.

Single nucleotide polymorphisms and read coverage across fimA of the reference strain TDC60. (Row 1) Reference gene fimA; (row 2) shared SNPs across the three single-cell genomes with 100% frequency at a coverage of 10×; (row 3) shared SNPs at a coverage of 30×; (rows 4–8) SNPs at 10× and 30× for each single-cell amplification; (rows 9–10) mapped deletions; (rows 11–13) mapped reads; (row 14) mapped 454 reads.

Figure 4.

Comparison of the polysaccharide capsule locus found in MDA3 (bottom) with W83, ATCC 33277, and TDC60. Genes of the same color are from the same orthologous group.

Figure 5.

Comparison of a clustered regularly interspaced short palindromic repeat region (CRISPR). Successful multiple displacement amplification and de novo assembly of the repeats in CRISPR region 36-30. This region was first identified in strain W83, and all three genomes have 100% identical repeat sequences. The regions vary in the number of repeats, number of spacer sequences, and spacer identity.