Detection of a microbial biofilm in intraamniotic infection

Abstract

Objective: Microbial biofilms are communities of sessile microorganisms formed by cells that are attached irreversibly to a substratum or interface or to each other and embedded in a hydrated matrix of extracellular polymeric substances. Microbial biofilms have been implicated in >80% of human infections such as periodontitis, urethritis, endocarditis, and device-associated infections. Thus far, intraamniotic infection has been attributed to planktonic (free-floating) bacteria. A case is presented in which "amniotic fluid sludge" was found to contain microbial biofilms. This represents the first report of a microbial biofilm in the amniotic cavity.

Study design: "Amniotic fluid sludge" was detected by transvaginal sonography and retrieved by transvaginal amniotomy. Bacteria were identified with scanning electron microscopy and fluorescence in situ hybridization for conserved regions of the microbial genome; the exopolymeric matrix was identified by histochemistry by the wheat germ agglutinin lectin method. The structure of the biofilm was imaged with confocal laser scanning microscopy.

Results: "Amniotic fluid sludge" was imaged with scanning electron microscopy, which allowed the identification of bacteria embedded in an amorphous material and inflammatory cells. Bacteria were demonstrated with fluorescent in situ hybridization using a eubacteria probe. Extracellular matrix was identified with the wheat germ agglutinin lectin stain. Confocal microscopy allowed 3-dimensional visualization of the microbial biofilm.

Conclusion: Microbial biofilms have been identified in a case of intraamniotic infection with "amniotic fluid sludge."

Figure 1

Two-dimensional transvaginal ultrasound image shows the presence of “amniotic fluid sludge”.

Figure 2

Scanning electron micrograph of a floc of “amniotic fluid sludge” showing the bacterial cells and the exopolymeric matrix material which constitute a biofilm. In the center of the image, cocci are resolved among a fibrous mass of matrix material. The lectin-based evidence for a matrix and a molecular evidence for bacterial presence are demonstrated in Figure 4.

Figure 3

Scanning electron micrograph of “amniotic fluid sludge”. A The biofilm, in the center of the image, and several neutrophils which are identified by arrows. B The coccoid bacteria are on the surface of a fetal epithelial cell, these bacterial cells are seen in the form of individual cells (arrow 1), a chain of cocci (arrow 2), and a biofilm aggregate in which they are partially “buried” in amorphous slime (arrow 3). C Several large aggregates of biofilm, are next to a hair shed from the fetus; most of the bacterial cells are enclosed in amorphous matrix material. D The presence of four very well defined bacterial cells (arrows) on the rugose surface of a fetal epithelial cell, although other bacteria that have colonized this surface have begun to accrete amorphous matrix material.

Figure 4

Demonstration that “amniotic fluid sludge” is a biofilm. These images were generated using confocal laser scanning microscopy. A The structure of a floc of “amniotic fluid sludge” without staining of any component. B The same structure that has been stained with the EUB338-Cy3 probe for eubacteria which reacts with the 16S rRNA component of bacteria. Bacteria and bacterial fragments are seen in red. C The same floc that has been stained with wheat germ agglutinin, which reacts with the N-acetylglucosamine of the component of the matrix material that forms the structural framework of the biofilm. D A superposition of the three images (A, B and C) showing bacteria (red dots), matrix material (green), and some unstained material which is likely to represent host components trapped by the biofilm. The bar represents 100 microns.

Figure 5

Three dimensional reconstruction of sequential Z stack images of a bacterial biofilm in amniotic fluid by confocal laser scanning microscopy. Bacteria are stained in red due to the hybridization with the probe EUB338-Cy3. (A video clip is available online).