Because the majority of bacteria in virtually all natural ecosystems grow in biofilms (44
), microbial challenge to humans often comes from this source. We know that natural aquatic biofilms recruit and retain pathogens, such as Salmonella
and Escherichia coli
, and that immunocompetent humans can be infected from these sources. Immunocompromised individuals may be especially susceptible to the nontubercular Mycobacterium avium
complex organisms that are a major component of many natural aquatic biofilms. Horizontal gene transfer is facilitated in biofilms (45
), and G. Ehrlich and colleagues have recently suggested (46
) that these sessile communities may play a major role in the pathogenicity of bacterial species (e.g., Vibrio cholerae
) that alternate between human hosts and natural reservoirs. This group has offered evidence that species of bacteria are composed of multiple strains, each of which contains a unique distribution of contingency genes from a population-based supragenome that is much larger than the genome of any single pathogen. During periods of stress, bacteria upregulate autocompetence and autotransformation systems within biofilms to promote the reassortment of genes that will result in the creation of some strains that may have a selective advantage under prevailing environmental conditions. They further suggest that natural marine and freshwater biofilms constitute a staging area in which the small DNA sequences can be cobbled together to recreate the pathogenicity “islands” necessary for the organisms’ attack on human populations. It is clear that all examinations of communicable enteric diseases must use biofilm models and that investigators of these systems should subscribe to the biofilm concept.
Although the digestive system and the integument may be colonized by bacteria and fungi from environmental biofilms, the human organ system that is by far the most susceptible to attack from environmental biofilms is the pulmonary system. The trachea and the lungs are well designed to resist colonization by planktonic bacteria, and animal experiments have shown the clearance of as many as 1 × 106
bacterial cells in as little as 20 minutes, provided the challenging cells are single and unaggregated (48
). Experiments in the same animal species using the same bacterial species (5
) have shown that the lungs of normal animals are not able to clear bacteria that are introduced in the form of biofilm fragments or of cells enclosed in artificial matrices (e.g., as agar beads). When biofilm fragments or agar beads containing bacteria are introduced into the lung, these aggregates resist phagocytosis by resident phagocytes and killing by both innate and acquired immune factors, and they persist for weeks and even months (48
). Figure a illustrates the mode of growth of the micro-colonies that comprise the Pseudomonas
biofilm in the lung in animal models of CF, and Figure shows how this matrix-enclosed population persists despite the immune reactions of the host. Woods and his colleagues proposed that the lungs may be colonized by the detachment of biofilm fragments from the oropharynx, which becomes overgrown by P. aeruginosa
during periods of stress (49
), and that these fragments cannot be cleared by pulmonary defense mechanisms. These data from animal experiments appear to have been confirmed by clinical examination of CF patients, and they raise the very grim specter of the inevitable colonization of the lungs with biofilm fragments when endotracheal tubes become colonized by mixed-species biofilms. Examination of endotracheal tubes that have been used for assisted ventilation has shown massive aggregations of mixed-species biofilms (Figure ); simple detachment of fragments could lead to chronic colonization of the lungs. In studies of intubated patients in intensive care units, we noted that the biofilms on endotracheal tubes often contained bacteria from the digestive tract when nasogastric tubes were also in use, and that these organisms were often found in the lungs of patients who had died after assisted ventilation. It is well documented that biofilm fragments are aspirated into the lung, and that these matrix-enclosed organisms cannot be cleared by pulmonary defense mechanisms and develop into micro-colonies that persist for months and may give rise to disseminated infection.
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 (more ...)
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 (more ...)
If the pulmonary system is in fact susceptible to colonization by aspirated biofilm fragments that cannot be cleared and may act as foci for chronic and/or acute bacterial infection, then other environmental organisms should be able to invade mammalian lungs. For this reason, it is germane to examine a trio of environmental bacteria that are “card-carrying” members of natural ecosystems and that share with P. aeruginosa
the invidious ability to colonize mammalian lungs and cause serious diseases in immunocompetent mammals. Pasteurella haemolytica
is a component of the normal oropharyngeal flora of cattle that proliferates when the animals are shipped to feedlot operations, and the aspiration of fragments of these biofilms into the lungs of these stressed animals results in respiratory infections that kill as many as 2% of these calves (50
). Legionella pneumophila
are the predominant inhabitants of warm littoral zones in freshwater lakes, where they grow in association with green algae (Fisheria
sp.) and avoid predation by free-living amoebae by forming biofilms and secreting antiphagocytic factors. As humans devised air conditioners and elaborate domestic hot water systems, L. pneumophila
found an alternate home in hotels and hospitals, and emerged to kill several dozen elderly gentlemen in a notorious hotel in Philadelphia, as well as many other people in various hospitals and office buildings. An organism that lives in warm water, forms biofilms, and resists phagocytosis by amoeboid enemies survives very well in the condensate trays of air conditioners or in human lungs (52
). The remarkable thing is not the pathogenicity of an environmental organism with no previous history of attacking humans, but the chilling realization that thousands of people must have died of legionellosis before the disease was defined by the Centers for Disease Control. If an individual dies of idiopathic pneumonia, this death causes very little interest, but the cause of this death may have been the mobilization of an acute respiratory infection from niduses of biofilm infection that were acquired by the aspiration of biofilm fragments. The sources of these fragments may be environmental, as in the case of cooling towers that cause seroconversion to L. pneumophila
in people working downwind of the towers, or they may be very focal, as in the case of dental hygienists who breath aerosols for long periods of time every working day.
Perhaps the best example of a pulmonary disease that is caused by biofilms is meloidosis, which affects people in Southeast Asia and Australia, and caused pulmonary infections in US soldiers in Vietnam. The causative pathogen is Burkholderia pseudomallei
, which is a natural component of the freshwater ecosystems in the area; humans make contact with biofilms formed by these organisms when they work in rice paddies or swim in local rivers (53
). Rates of seroconversion indicate that more than 80% of the people in northeast Thailand have been exposed to this pathogen, presumably by aspiration of fragments of its exuberant biofilms, and we have visualized a population with multiple micro-colonies in their lungs (53
). When we set up animal experiments by introducing agar beads containing these organisms into the lower left lobes of the lungs of guinea pigs, we induced an asymptomatic chronic infection that persisted for months (54
). However, when we stressed these chronically infected animals with steroid injections (54
), planktonic bacteria were released from the biofilm micro-colonies, and the animals rapidly succumbed to the resultant acute infections and bacteremias. Several hundreds of people die of acute meloidosis in northeast Thailand when they are stressed by seasonal starvation cycles, so the response to stress seen in the animal model appears to operate in human populations. These data add to the Damoclean image of the pulmonary health of modern humans, because bacteria from biofilm niduses acquired by the aspiration of biofilm fragments from many sources may be mobilized in times of stress and may cause acute pneumonias. So, many among us may carry the seeds of fatal pneumonia (the “old man’s friend”) in their lungs as they enjoy their air-conditioned offices and their homes with spas and hot tubs.
Biofilms may also be involved in the dissemination of disease within the body of an infected individual. When we speak of the hematogenous spread of infection, we must now specify whether the infectious units are planktonic cells or biofilm fragments, because these entities differ radically in important properties such as their antibiotic resistance and their adhesion characteristics. Planktonic cells are shed from virtually all mature biofilms, they are generally susceptible to antibiotics, and they adhere to certain tissues and to inert surfaces with considerable avidity. For this reason it is logical to use prophylactic antibiotic therapy to prevent the colonization of recently installed medical devices by planktonic bacteria introduced into the bloodstream by routine tooth brushing or by dental manipulations. On the other hand, many of the cells that detach from biofilms growing on native heart valves (resulting in endocarditis) or vascular catheters are in the form of matrix-enclosed biofilm fragments (21
) that are very resistant to antibiotics, and they usually circulate until they “jam” in a capillary bed. For this reason, low-dose antibiotic therapy does not prevent the dissemination of bacteria in these biofilm diseases, and the best way of preventing this process is very aggressive high-dose treatment of native valve endocarditis and rapid removal of colonized vascular devices. We have developed animal models of dissemination in biofilm diseases, and we have been amazed at how well sheep lungs can tolerate the small hemorrhages that result from hundreds of biofilm fragments lodging in capillary beds. However, it is clear that one or two large biofilm fragments can cause profound damage if they detach and find their way to critical loci in the lungs or in the brain.