Adipose tissue is the first colonization site of <i>Leptospira interrogans</i> in subcutaneously infected hamsters

<div><p>Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an <i>in vivo</i> imaging system to observe the whole bodies of animals infected with <i>Leptospira interrogans</i> and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 10⁴ bioluminescent leptospires, were analyzed by <i>in vivo</i> imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.</p></div

Treatment adherence and side effects.

<p>Treatment adherence and side effects.</p

Clinical and laboratory characteristics of females.

<p>Clinical and laboratory characteristics of females.</p

Combined oral and topical antimicrobial therapy for male partners of women with bacterial vaginosis: Acceptability, tolerability and impact on the genital microbiota of couples - A pilot study

<div><p>Objectives</p><p>Recurrence following recommended treatment for bacterial vaginosis is unacceptably high. While the pathogenesis of recurrence is not well understood, recent evidence indicates re-infection from sexual partners is likely to play a role. The aim of this study was to assess the acceptability and tolerability of topical and oral antimicrobial therapy in male partners of women with bacterial vaginosis (BV), and to investigate the impact of dual-partner treatment on the vaginal and penile microbiota.</p><p>Methods</p><p>Women with symptomatic BV (Nugent Score of 4–10 and ≥3 Amsel criteria) and their regular male sexual partner were recruited from Melbourne Sexual Health Centre, Melbourne, Australia. Women received oral metronidazole 400mg twice daily (or intra-vaginal 2% clindamycin cream, if contraindicated) for 7-days. Male partners received oral metronidazole 400mg twice daily and 2% clindamycin cream topically to the penile skin twice daily for 7-days. Couples provided self-collected genital specimens and completed questionnaires at enrolment and then weekly for 4-weeks. Genital microbiota composition was determined by 16S rRNA gene sequencing. Changes in genital microbiota composition were assessed by Bray-Curtis index. Bacterial diversity was measured by the Shannon Diversity Index.</p><p>Results</p><p>Twenty-two couples were recruited. Sixteen couples (76%) completed all study procedures. Adherence was high; most participants took >90% of prescribed medication. Medication, and particularly topical clindamycin in males, was well tolerated. Dual-partner treatment had an immediate and sustained effect on the composition of vaginal microbiota (median Bray-Curtis score day 0 versus day 8 = 0.03 [IQR 0–0.15], day 0 vs day 28 = 0.03 [0.02–0.11]). We observed a reduction in bacterial diversity of the vaginal microbiota and a decrease in the prevalence and abundance of BV-associated bacteria following treatment. Treatment had an immediate effect on the composition of the cutaneous penile microbiota (median Bray-Curtis score day 0 vs day 8 = 0.09 [0.04–0.17]), however this was not as pronounced at day 28 (median Bray-Curtis score day 0 vs day 28 = 0.38 [0.11–0.59]). A decrease in the prevalence and abundance of BV-associated bacteria in the cutaneous penile microbiota was observed immediately following treatment at day 8.</p><p>Conclusion</p><p>Combined oral and topical treatment of male partners of women with BV is acceptable and well tolerated. The combined acceptability and microbiological data presented in this paper supports the need for larger studies with longer follow up to characterize the sustained effect of dual partner treatment on the genital microbiota of couples and assess the impact on BV recurrence.</p></div

Bioluminescence changes in hamster organs.

<p>Representative bioluminescence images (ventral view) from M1307-infected hamsters at each phase. Images represent subcutaneous tissues after skin incision and organs after laparotomy, as well as <i>ex vivo</i> organs (blood plus liver and kidney cross sections). The scale is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172973#pone.0172973.g001" target="_blank">Fig 1</a>.</p

Bioluminescence dissemination of <i>Leptospira</i> in hamsters.

<p>(A) The survival rate of Golden Syrian hamsters (n = 8) infected subcutaneously with 10⁴ <i>L</i>. <i>interrogans</i> strain M1307 into the right inguinal region, and representative ventral view photographic images tracking the hamster infections on different days post-infection. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm²/sr as indicated on the scales, where red is the most intense (3×10⁵) and purple is the least intense (3×10⁴). (B,C) Average luminescence intensities in each ROI of injection site (B) and abdominal center (C) at different days post-infection. Data are expressed as the means ± SEM of total flux in photons/second in each ROI in eight infected hamsters (●) and two uninfected controls (◦). <i>p</i> values (*<i>p</i><0.05), between groups.</p

Transmission electron microscopy of adipose tissue blood vessels.

<p>Representative transmission electron microscope images of subcutaneous adipose tissue blood vessels around the injection sites of <i>Leptospira</i>-infected hamsters at phase 4. The framed area in (A) is enlarged in (B). The scale bars represent 5 μm (A) and 1 μm (B). The arrowheads point to <i>Leptospira</i> and the arrows show the red blood cells.</p

Specimen flowchart.

<p>Specimen flowchart detailing number of vaginal and penile skin specimens available for microbiota analysis at baseline, day 8 and day 28. Seventeen women provided vaginal specimens for day 0 and 8 paired comparisons, and 16 provided vaginal specimens for day 0 and 28 paired comparisons. Sixteen males provided cutaneous penile specimens for day 0 and 8 paired comparisons and 15 males provided cutaneous penile specimens for day 0 and 28 paired comparisons. The number of couples providing specimens at each time-point is also shown. ^abaseline specimen was not available for one female; ^btwo d8 penile skin specimens failed to meet the sequence depth threshold and were substituted with day 14 specimens.</p

Behavioural characteristics of couples during study period.

<p>Behavioural characteristics of couples during study period.</p

<i>Leptospira</i> distribution in skin and subcutaneous tissue.

<p>Representative light field (A, C) and fluorescence images (B, D) of the skin and subcutaneous tissue (A, B) or adipose tissue (C, D) around the injection sites of M1307 collected from infected hamsters at phase 4. Fluorescence images (B, D) showing cell nuclei stained with DAPI (blue), autofluorescence of the skin and subcutaneous tissue (green, not shown in panel D), and leptospires stained with rabbit polyclonal antiserum and Cy5-conjugated anti-rabbit monoclonal antibody (red). The framed area in (B) is enlarged at the upper right. Scale bars: 100 μm (A, B), 500 μm (C, D).</p