High-throughput metagenome analysis of the Sarcoptes scabiei internal microbiota and in-situ identification of intestinal Streptomyces sp

Abstract

Multiple parasitic arthropods of medical importance depend on symbiotic bacteria. While the link between scabies and secondary bacterial infections causing post infective complications of Group A streptococcal and staphylococcal pyoderma is increasingly recognized, very little is known about the microbiota of Sarcoptes scabiei. Here we analyze adult female mite and egg metagenome datasets. The majority of adult mite bacterial reads matched with Enterobacteriaceae (phylum Proteobacteria), followed by Corynebacteriaceae (phylum Actinobacteria). Klebsiella was the most dominant genus (78%) and Corynebacterium constituted 9% of the assigned sequences. Scabies mite eggs had a more diverse microbial composition with sequences from Proteobacteria being the most dominant (75%), while Actinobacteria, Bacteroidetes and Firmicutes accounted for 23% of the egg microbiome sequences. DNA sequences of a potential endosymbiont, namely Streptomyces, were identified in the metagenome sequence data of both life stages. The presence of Streptomyces was confirmed by conventional PCR. Digital droplet PCR indicated higher Streptomyces numbers in adult mites compared to eggs. Streptomyces were localized histologically in the scabies mite gut and faecal pellets by Fluorescent In Situ Hybridization (FISH). Streptomyces may have essential symbiotic roles in the scabies parasite intestinal system requiring further investigation.

Figure 1

Comparison of cleaning procedures of scabies eggs. Concentrations of 16S rDNA amplicon copy numbers generated from scabies egg samples treated with four different cleaning solutions. Statistically significant differences between treated samples and compared to unwashed control are indicated by *. Statistical differences were determined using 1-way ANOVA, Dunnett’s multiple comparisons test (**p ≤ 0.005) and (***p ≤ 0.0005).

Figure 2

Barchart showing the relative abundances of bacteria in the metagenomes of adult female scabies mites and scabies eggs. Only the top 10 genera in each sample are listed. The remaining genera including Streptomyces are categorized to “others”. For more detail see S2_Fig. 1. Sequence data were annotated using Kraken and visualized using the software package R.

Figure 3

The number of (a) Streptomyces sp. and (b) K. pneumoniae present in washed vs. unwashed adult female scabies mites and eggs respectively. One ng of gDNA from 50–100 adult female mites or eggs was used as a template for duplex ddPCR using Streptomyces genus-specific and K. pneumoniae specific primers. Results are shown as means ± SEM from three independent experiments. The statistical significance of the differences between the numbers of bacteria in washed vs unwashed samples was estimated using 2 way ANOVA with Sidak’s multiple comparison test, ns: not significant.

Figure 4

Visualization by Fluorescent In Situ hybridization (FISH) of Streptomyces sp. and K. pneumoniae within (a) an adult female scabies mite, (b) mite feces within epidermal burrows and (c) an egg. An adjacent section (i) was stained with Hematoxylin for orientation. The universal EUB338 probe (red, ii) was labelled with CalFlour590 while Streptomyces sp. (fuchsia, iii) and K. pneumoniae probes (green, iv) were both labelled with Cy5. Positive staining is visible in the gut area of the mite indicated as ‘g’ in section ‘i’ of panel ‘a’, and in the feces indicated as ‘f’ of panel (b).