Results of anaerobic culture and detection of <i>P</i>. <i>acnes</i> genome counts in disc tissue samples.

<p>Prevalence rate of the bacteria isolated from disc tissue samples (A), stratification of the samples accordingly to bacterial counts reached by culture (B) and differences in the bacteria genome counts detected by real-time PCR in group of patients with abundant <i>P</i>. <i>acnes</i> by culture, non-abundant <i>P</i>. <i>acnes</i> and culture-negative cases (C).</p

Distribution of <i>P</i>. <i>acnes</i> colony counts in culture-positive disc tissue specimens.

<p>Distribution of <i>P</i>. <i>acnes</i> colony counts in culture-positive disc tissue specimens.</p

Summary of the microorganisms isolated in 162 cases from 368 patient intervertebral disc specimens by anaerobic culture.

<p>Summary of the microorganisms isolated in 162 cases from 368 patient intervertebral disc specimens by anaerobic culture.</p

Visualization of bacterial biofilm in the disc tissue by CSLM and confirmation of <i>P</i>. <i>acnes</i> by FISH.

<p>A. Three dimensional reconstructed CSLM image of biofilm bacteria stained with a DNA stain (SYTO9, green) in a disc tissue sample (#4, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174518#pone.0174518.t002" target="_blank">Table 2</a>). B-C. The presence of <i>P. acnes</i> biofilms in this sample verified using FISH. Epifluorescence micrographs of a biofilm cluster showing red fluorescence from the CY5-labeled EUB338 general eubacterial probe (B) and green fluorescence from the CY3-labled <i>P. acnes</i>-specific probe (C). Co-localization of the red and green fluorescence indicates that all of the bacteria in this biofilm were <i>P. acnes</i>.</p

<i>Propionibacterium acnes</i> biofilm is present in intervertebral discs of patients undergoing microdiscectomy

<div><p>Background</p><p>In previous studies, <i>Propionibacterium acnes</i> was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of <i>P</i>. <i>acnes</i> as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate <i>P</i>. <i>acnes</i> prevalence in resected disc cultures, while providing microscopic evidence of <i>P</i>. <i>acnes</i> biofilm in the intervertebral discs.</p><p>Methods</p><p>Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and <i>P</i>. <i>acnes</i> phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH.</p><p>Results</p><p>Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with <i>P</i>. <i>acnes</i>. In 89 cases, <i>P</i>. <i>acnes</i> was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative <i>Staphylococcus spp</i>.) Among positive specimens, the median <i>P</i>. <i>acnes</i> bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight <i>P</i>. <i>acnes</i> isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA₁, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed <i>P</i>. <i>acnes in situ</i>. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. <i>P</i>. <i>acnes</i> bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013).</p><p>Conclusions</p><p>This study confirms that <i>P</i>. <i>acnes</i> is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of <i>P</i>. <i>acnes</i> biofilms within such specimens, consistent with infection rather than microbiologic contamination.</p></div

Characteristics of the disc samples evaluated by microscopic methods.

<p>Characteristics of the disc samples evaluated by microscopic methods.</p

Visualization of <i>P</i>. <i>acnes</i> biofilm in the disc tissue by use of FISH.

<p>A. This color-combined image shows the “pocket” of green fluorescent <i>P. acnes</i> cells (biofilm) near the center right of the image (disc tissue sample #8, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174518#pone.0174518.t002" target="_blank">Table 2</a>). The presence of <i>P. acnes</i> biofilms in this sample was verified using FISH. B-C. Red fluorescence is the general eubacterial probe (B) and green is the <i>P. acnes</i> probe (C). The B/C image is a zoom of A showing fluorescence from the red and green channels separately. Almost all of the cells in A are emitting both red and green fluorescence indicating that they are <i>P. acnes</i>.</p