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

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

<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

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing <em>Escherichia coli</em> Infection in Mice

<div><p>A large outbreak of Shiga toxin (Stx)-producing enteroaggregative <i>Escherichia coli</i> (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing <i>Escherichia coli</i> O157: H- strain E32511 (E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by <i>in situ</i> hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from E32511 <i>in vitro</i>.</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

Microglial activation in i.p. LPS-injected and E32511 model mice.

<p>(A) Microglia in the medulla oblongata and the spinal cord were not activated in the control (uninfected) and E32511-infected mice, but were activated in i.p. LPS-injected mice. Scale bars: 2 µm. (B) The number of activated microglia significantly increased in the LPS-injected mice but not in the control and E32511-infected mice (LPS vs. control, <i>p</i><0.0001; LPS vs. E32511, <i>p</i><0.0001 and control vs. E32511, <i>p</i> = 1). Microglia were observed and counted manually from 5 non-overlapping fields. Statistical difference was measured by student's t test. **<i>p</i><0.001.</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

AZM compared with other antimicrobial agents in the E32511 model.

<p>(A) The concentration of Stx2c in NFLX, KM, OFLX, and CPFX treatment was significantly increased compared with control (no antibiotic) (NFLX vs. control, <i>p</i> = 0.023; KM vs. control, <i>p</i> = 0.02; OFLX vs. control, <i>p</i> = 0.023 and CPPFX vs. control, <i>p</i> = 0.017), while this was not the case for FOM. On the other hand, AZM statistically decreased Stx2c concentration compared with control (AZM vs. control, <i>p</i> = 0.007). Data were collected from the supernatants of cultures with 2-fold serial dilutions of each antimicrobial agent and are shown as mean and standard deviations of two independent experiments. Statistical difference was measured by Student's t test, *<i>p</i><0.05. (B) AZM had a statistically significant effect on the mice survival, with 100% survival; NFLX and KM had a statistically significant effect on the mice survival, with 80% survival (AZM vs. untreated, <i>p</i> = 0.002; AZM vs. FOM, <i>p</i> = 0.003; AZM vs. CPFX, <i>p</i> = 0.014; AZM vs. NFLX and KM, <i>p</i>>0.05). Log Rank and χ² test, *<i>p</i><0.05.</p

Bacterial count at sub-minimum inhibitory concentration (MIC) of AZM and other antimicrobial agents against E32511. AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.

<p>AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.</p

Effectiveness of a single dose of AZM in the E32511 model.

<p>(A) The administration of a single dose of AZM 2 h after infection significantly increased the BW of E32511-infected mice. (B) Mouse BW for AZM 2 h vs. AZM 6 h, <i>p</i> = 0.015 at day 14 after inoculation. Student's t test, *<i>p</i><0.05; **<i>p</i><0.001). (C) The administration of a single dose of AZM was significantly effective in the survival mice when given 2 h after infection, compared with 6 and 24 h after infection, (for AZM 2 h after infection vs. untreated, <i>p</i> = 0.012; AZM 2 h after infection vs. 24 h after infection, <i>p</i> = 0.013 and AZM 6 h and 24 h after infection vs. untreated, <i>p</i>>0.05. Log rank and χ² test, (*<i>p</i><0.05) (D) The administration of a single dose of 200–1.6 µg/g AZM 2 h after infection significantly increased the BW of E32511-infected mice. (E) The correlation between the dose of AZM and the increase in BW of E32511-infected mice was statistically significant, as shown by the Spearman's ρ (rho) = 0.887 and the <i>p</i> value, <i>p</i><0.0001. Black-filled circle = AZM 100 µg/g; grey-filled circle = AZM 25 µg/g; empty circle = AZM 6.25 µg/g. (F) 1.6 µg/g AZM 2 h after infection with E32511 (10¹¹ CFU/ mouse) was still effective in treating the E32511-infected mice, with a significant effect on the survival curve (200–6.25 µg/g AZM vs. untreated, <i>p</i> = 0.002; 3.1 µg/g AZM vs. untreated, <i>p</i> = 0.008 and 1.6 µg/g AZM vs. untreated, <i>p</i> = 0.016. Log rank and χ² test, **<i>p</i><0.01, *<i>p</i><0.05.</p