SMSB4 reduces phagocytosis of <i>S. aureus</i> by neutrophils.

<p>FITC-labeled bacteria (10⁵ cfu) was opsonized either with 20% NHS or heated serum and challenged with neutrophils (10⁶ cells). Serum samples were pre-treated for 30 min at 37°C with 50 µg/ml SMSB4, 50 µg/ml BSA (<b>A</b>), or various concentrations of SMSB4 or BSA (<b>B</b>). Uptake of FITC by neutrophils was measured at various time points over 1 h (<b>A</b>) or at 40 min (<b>B</b>). Results are shown as means ± SEM from three independent experiments.</p

SMSB4 reduces deposition of C3b (A) and MAC complex (SC5b-9) formation (B).

<p>The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×10⁶ cfu/ml of <i>S. aureus</i>. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments.</p

Effect of SMSB4 on the depositions of C4b (A), C1q, MBL and properdin (B) on <i>S. aureus</i> cells.

<p>The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×10⁶ cfu/ml of <i>S. aureus</i>. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between BSA and SMSB4 treated samples were estimated using two way ANOVA with Sidak’s multiple comparison test. **, <i>p</i><0.01; ***, <i>p</i><0.001; ns, not significant (B).</p

SMSB4 reduces the blood killing of <i>S. aureus</i> strain Xen29 in whole blood (A) and pyoderma isolates of <i>S. aureus</i> (B).

<p>SMSB4 promotes bacteria growth in a concentration dependent manner (C) similarly to CVF (D). <i>S. aureus</i> Xen29 or pyoderma isolates MRSA strains (HS16, M34), MSSA strains (HS56, M5) were harvested from mid-log growth phase culture. Bacteria (1×10⁵ cfu/ml) were challenged with whole blood pre-treated with either 100 µg/ml SMSB4, positive control 10 µg/ml CVF, negative controls 100 µg/ml BSA or GVB²⁺ buffer only. <i>S. aureus</i> cells in PBS only without blood was also included to illustrate that the reduction in bacteria number was due to blood killing (<b>A</b>). Numbers of bacteria were counted as cfu/ml at various time points (<b>A</b>) or at 3 h (<b>B</b>, <b>C</b>, <b>D</b>). Bacterial recovery was calculated as a percentage of the challenge dose. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using two way ANOVA with Tukey’s multiple comparison test. **, <i>p</i><0.01; ***, <i>p</i><0.001; ****, <i>p</i><0.0001, ns, not significant (B).</p

Scabies Mites Alter the Skin Microbiome and Promote Growth of Opportunistic Pathogens in a Porcine Model

<div><p>Background</p><p>The resident skin microbiota plays an important role in restricting pathogenic bacteria, thereby protecting the host. Scabies mites (<i>Sarcoptes scabiei</i>) are thought to promote bacterial infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. Epidemiological studies in humans confirm increased incidence of impetigo, generally caused by <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>, secondary to the epidermal infestation with the parasitic mite. It is therefore possible that mite infestation could alter the healthy skin microbiota making way for the opportunistic pathogens. A longitudinal study to test this hypothesis in humans is near impossible due to ethical reasons. In a porcine model we generated scabies infestations closely resembling the disease manifestation in humans and investigated the scabies associated changes in the skin microbiota over the course of a mite infestation.</p><p>Methodology/Principal Findings</p><p>In a 21 week trial, skin scrapings were collected from pigs infected with <i>S. scabies</i> var. <i>suis</i> and scabies-free control animals. A total of 96 skin scrapings were collected before, during infection and after acaricide treatment, and analyzed by bacterial 16S rDNA tag-encoded FLX-titanium amplicon pyrosequencing. We found significant changes in the epidermal microbiota, in particular a dramatic increase in <i>Staphylococcus</i> correlating with the onset of mite infestation in animals challenged with scabies mites. This increase persisted beyond treatment from mite infection and healing of skin. Furthermore, the staphylococci population shifted from the commensal <i>S. hominis</i> on the healthy skin prior to scabies mite challenge to <i>S. chromogenes</i>, which is increasingly recognized as being pathogenic, coinciding with scabies infection in pigs. In contrast, all animals in the scabies-free cohort remained relatively free of <i>Staphylococcus</i> throughout the trial.</p><p>Conclusions/Significance</p><p>This is the first experimental <i>in vivo</i> evidence supporting previous assumptions that establishment of pathogens follow scabies infection. Our findings provide an explanation for a biologically important aspect of the disease pathogenesis. The methods developed from this pig trial will serve as a guide to analyze human clinical samples. Studies building on this will offer implications for development of novel intervention strategies against the mites and the secondary infections.</p></div

(a) Phylogenetic tree of <i>Staphylococcus</i> indicating tentative species.

<p><b>(b)</b> Relative abundance of staphylococcal species in each sample. (<b>a</b>) A phylogenetic tree was constructed for OTUs assigned to the genus <i>Staphylococcus</i> using known reference 16S rRNA genes. The accession is noted in bracket for each reference species. The number in brackets following the OTU name indicates the read counts within the OTU. The tree was rooted with the outlier <i>Bacillus subtilis</i>. Several OTUs (1, 6, 8, 9, 14) are in close proximity to the 16S rRNA gene of <i>S. chromogenes</i>. (<b>b</b>) Bar chart demonstrating the relative abundance of each OTU in the pig cohorts over time. An extensive change of the OTUs is measured in the scabies infected cohort M and MD from week 7 onwards. The change was irreversible despite treatment. In particular, OTU1, OTU6, OTU8, OTU9, OTU14, all related to <i>S. chromogenes</i>, are dominating the skin microbiota.</p

(a) Phylogenetic tree of <i>Streptococcus</i> indicating tentative species.

<p><b>(b)</b> Relative abundance of streptococcal species in each sample. (<b>a</b>) A phylogenetic tree was constructed for OTUs assigned to the genus <i>Streptococcus</i> using known reference 16S rRNA genes. The accession is noted in bracket for each reference species. The number in brackets following the OTU name indicates the read counts within the OTU. The tree was rooted with the outlier <i>Lactococcus lactis</i>. (<b>b</b>) Bar chart demonstrating the relative abundance of each OTU in the pig cohorts over time. The graph depicts that the OTUs are overall stable between the three cohorts within each week and over time, irrespective of the presence or absence of mites.</p

(a) Composition of the microbial communities before, during and after scabies mite infection.

<p>Microbial community structure was analysed by 454 pyrosequencing of the 16S rRNA gene of the total DNA, extracted from the skin scrapings of the pinna of pig ears. Samples were taken before the scabies infection from all cohorts at week 0. Cohorts M and MD were then challenged with an equal dosage of <i>S. scabiei</i> var. <i>suis</i> in both ears. Visible signs of scabies infection developed by week 7, which progressed to severe form of infection by week 10. Scabies infection was treated with an acaricide (Dectomax^®) and skin was allowed to heal. Final skin scrapings were taken at week 21. Bubble volumes correlate with the abundance (%) of the each genus detected in the sample. (<b>b</b>) Effect of scabies mite infection on the microbial community diversity visualised by Shannon index for all samples from the cohorts C (i, mite free), M (ii, mite infested), MD (iii, mite infested and Dexamethasone treated). Community diversity is expressed as the mean Shannon index (OTU level). Paired t-test was carried out for pigs where both time points were available (M.10: n = 2, MD: n = 4, other: n = 5). Significant differences are annotated by *: p<0.05, **:p<0.01, ***: p<0.001.</p

Relative abundance of the bacterial genera at defined time points.

<p>Numbers are presented as the median percentage of the relative abundances measured for five samples in each cohort M (mite infected), MD (Dexamethasone treated and mite infected) and C (mite-free control).</p

Relative abundance of the most predominant bacterial genera in the pinna of porcine ears at various time points in the absence of mites.

<p>At week 0, <i>Streptococcus</i> was the most dominant genus, followed by <i>Lactobacillus, Actinectobacter, Enhydrobacter, Aerococcus</i> and <i>Rothia</i> in decreasing abundance. At week 7, <i>Streptococcus</i> was still the most dominant genus, followed by smaller abundance of <i>Corynebacterium, Chryseobacterium, Rothia, Clostridium</i> and <i>Lactobacillus</i>. Similarly at week 10, <i>Streptococcus</i> was the dominant genus, followed by <i>Corynebacterium, Rothia, Kocouria, Clostridium</i> and <i>Lactobacillus</i>. At week 21, <i>Lactobacillus</i> was the most dominant, followed by <i>Streptococcus</i> and <i>Kocuria</i>. Only genera, with a relative abundance of ≥10% in at least one sample are illustrated.</p