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Abstract & Commentary
Synopsis: By sequestering iron, lactoferrin enhances twitching motility of P aeruginosa and, as a result, prevents biofilm formation.
Source: Singh PK, et al. A component of innate immunity prevents bacterial biofilm development. Nature. 2002;417: 552-555.
Biofilms play an important role in a variety of infections, including chronic airway infection caused by Pseudomonas aeruginosa in cystic fibrosis patients. It seems likely that normal mucosal surfaces that are commonly colonized by bacteria have mechanisms that prevent such biofilm formation. Singh and colleagues at the University of Iowa examined the hypothesis that a component of the innate immune system, lactoferrin, that is present in high concentrations at mucosal surfaces, is a component of a defense against the production of biofilms by colonizing bacteria.
In vitro studies using flow cell chambers were continuously perfused with "biofilm medium" in the presence and absence of a concentration of lactoferrin (20 mg/mL) that did not affect the growth of a strain of Pseudomonas aeruginosa in a free-swimming state. The typical stages of biofilm development, including attachment, microcolony formation and enlargement, and "towering pillar and mushroom-shaped" biofilm, were observed in the absence of lactoferrin. In contrast, in the presence of lactoferrin, this development pattern was disrupted and differentiated biofilms structures did not develop. There was no effect of lactoferrin, however, upon already mature P aeruginosa biofilms.
Observation with time-lapse video microscopy determined that, while lactoferrin caused only a minimal increase in the dividing time of attached bacterial cells, it markedly altered the movement patterns of those bacteria. In particular, lactoferrin markedly decreased the proportion of "squatters" (stationary cells), while markedly increasing the proportion that were "ramblers" (cells that moved away from the site of bacterial division).
Conalbumin, a lactoferrin-like molecule from chicken eggs that, like lactoferrin, binds iron, also impaired biofilm formation, as did deferoxamine. When lactoferrin was saturated with iron, it failed to prevent such formation. Experiments determined that removal of iron from the environment led to an increase in a specialized form of bacterial surface locomotion mediated by type 4 pile called twitching. Lactoferrin was demonstrated to stimulate twitching motility in P aeruginosa, an effect that was lost when the organisms resided in established biofilms.
P aeruginosa grown in biofilm was highly resistant to H2O2 and to tobramycin, but surface growth in the presence of conalbumin (used in these experiments because of its lesser cost) was associated with a dose-dependent decrease in resistance.
Comment by Stan Deresinski, MD, FACP
It would seem unlikely that the evolution of host defense mechanisms would ignore such an important aspect of infectious disease as the role of bacterial biofilms. The observations in this remarkable set of experiments demonstrate that this problem has not been ignored—evolution has provided us with at least one innate mechanism of defense against this problem in the form of lactoferrin.
Lactoferrin is present in normal airway secretions at concentrations as high as 1 mg/mL and is also present in high concentrations in tears and breast milk. This cationic protein exerts antibacterial activity by 2 mechanisms: binding to lipopolysaccharides, it disrupts bacterial cell membranes, and sequestration of, making it unavailable to microorganisms.
These experiments demonstrate that lactoferrin impairs P aeruginosa biofilm formation by making iron unavailable and consequently stimulating twitching motility of the bacterial cells. Singh et al best summarize the overall conclusions: "Our data indicate that a higher level of iron is required for biofilm formation than is needed for growth. If the iron level is acceptable, P aeruginosa is cued to stop moving, form microcolonies, and eventually develop into biofilms. If iron levels are not sufficient, the P aeruginosa cells keep moving. This response may protect the bacteria from constructing complex, durable biofilm structures in locations where iron, a critical nutrient, is in short supply."