The application of biofilm science to the study and control of chronic bacterial infections

Abstract

Unequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases.

Figure 1

Electron micrographs of pathogenic bacterial biofilms from a variety of bacterial infections. (a) Transmission Electron Micrograph (TEM) of a section of lung tissue taken (postmortem) from a CF patient. The matrix-enclosed micro-colony of P. aeruginosa cells is surrounded by a prominent electron-dense “crust” of material that reacted very strongly with antibodies directed against IgG. Image published with permission from Infection and Immunity (59). (b) TEM of a section from the affected bone of a patient with very long-term osteomyelitis that had been treated with antibiotics (for four decades) and several debridations. Note the large number of Gram-positive cells (S. aureus was cultured) and the dehydrated remnants of the fibrous matrix of this massive biofilm. (c) Scanning electron micrograph (SEM) of a struvite crystal from the hilus of the kidney of a patient with acute pyleonephritis, who was affected by “staghorn” calculi. Cells of the infecting agent (P. vulgaris) have formed a biofilm whose matrix has become infused with struvite to produce a “petrified” biofilm. (d) TEM of a section from a vegetation formed on the endocardium of a rabbit in an animal model of native-valve endocarditis. Cells of the infecting agent (viridans group streptococci) are seen to have formed this macroscopic biofilm and to have produced very large amounts of fibrous matrix material. (e) SEM of tissue from a culture-negative prostatitis, showing the presence of rod-shaped bacterial cells. (f) SEM of tissue from the prostatitis patient in e, which had been reacted with the patient’s serum, so that the matrix material of this well-developed biofilm was protected from dehydration, and is shown at its full extent.

Figure 2

Diagrammatic representation of the tower- and mushroom-like micro-colonies that constitute the structural elements of biofilms. Sessile cells constitute only approximately 15% of the volume of their matrix-enclosed communities, so the micro-colonies are viscoelastic and deformable in high shear. Well-developed water channels conduct water in convective flow and deliver nutrients to most parts of the community. Figure is reproduced with permission from the American Society for Microbiology (60).

Figure 3

Confocal scanning-laser micrograph showing the invasion of a biofilm by PMNs. The PMNs (large red nuclei) have entered the biofilm via the open water channels and have invaded short distances (1–5 μm) into the biofilm. The bacterial cells have been stained with the live/dead BacLight stain (BacLight Bacterial Viability kit; Molecular Probes, Eugene, Oregon, USA) and living bacterial cells (green) are seen in very close proximity (<1 μm) to PMNs. We conclude that PMNs invade biofilms but are virtually inactive in killing sessile cells and resolving biofilm infections. Reproduced with permission from Infection and Immunity (40).

Figure 4

Fluorescence micrographs of epithelial cells recovered from the vaginas of human volunteers. (a) A single epithelial cell with highly refractile blood cells (the volunteer was menstruating) and colonized by rod-shaped and coccoid bacteria. (b) A similar cell reacted with the fluorescent eubacterial domain (EU 338) probe, whose base sequence reacts with the 16S RNA of all eubacteria, including both lactobacilli and staphylococci. (c) The same cell as in a reacted with a fluorescent probe that reacts only with the 16S RNA of S. aureus. This organism was found in the vaginal flora of all volunteers tested, and this result was confirmed by PCR. Figures reproduced with permission from the Journal of Infectious Diseases (36).

Figure 5

Diagrammatic representation of the defense strategies of the lung. (a) The surface of the alveolar epithelium is “patrolled” by PMNs and macrophages, which phagocytose incoming planktonic bacteria quickly and easily. (b) The alveolar phagocytes are unable to engulf bacteria in matrix-enclosed biofilm fragments, even when these invaders are reacted with specific antibodies. (c) Biofilm fragments grow and burgeon in the colonized lung, and release occasional planktonic cells that react with antibodies and are phagocytosed. (d) The mature biofilm reaches a “standoff” with the immune system, and parts of the microbial community become calcified to form a long-term pulmonary nidus.

Figure 6

SEM of a large multispecies biofilm aggregate that formed on the lumenal surface of an endotracheal tube used to ventilate a patient in a Systems Failure Intensive Care Unit. These uvula-shaped aggregates have a rubbery consistency, and they routinely break off of these surfaces and are aspirated into patients’ lungs.

Figure 7

Confocal scanning-laser micrographs of tissue recovered from the vaginal epithelium of volunteers and stained with fluorescein. (a) The orange nuclei and green cytoplasm of the human cells are clearly seen, as are the bacterial cells in a biofilm aggregate partly detached from human cell surfaces. (b) The green rod-shaped cells of the vaginal biofilms are seen, with the lighter green matrix material that surrounds them, in aggregates at least 30 μm “tall.” The extent and thickness of the vaginal biofilm are indicated by the fact that the orange tissue nuclei appear to be “buried” by this beneficial microbial population.

Figure 8

SEM of the lumenal surface of the intestine of a mouse. The dehydration-condensed residue of the intestinal biofilm is seen to occupy much of the tissue surface, and to be composed of a rich mixture of bacterial and protozoan species. A large Giardia cell is seen to be attached to the surface (P) of the intestinal epithelium, while a detached cell of the same species shows its well-developed sucker structure, and the microvillar surface of opposite side shows the scars of previous protozoan attachment. Attachment plays a large role in the microbial ecology of the intestine, because the intestinal mucus exerts powerful shear forces that tend to remove loosely attached organisms.