Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

Abstract

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and freeswimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.Heavy metals are ubiquitous and persistent environmental pollutants that are introduced into the environment through anthropogenic activities, such as mining and smelting, as well as through other sources of industrial waste. In fact, over one-half the superfund sites in the United States are contaminated with at least one heavy metal (www.atsdr.cdc.gov). Heavy metals contaminate drinking water reservoirs and freshwater habitats and can alter macro-and microbiological communities (18, 24). The known mechanisms of heavy metal toxicity include inducing oxidative stress and interfering with protein folding and function (30).Bacteria have developed a variety of resistance mechanisms to counteract heavy metal stress. These mechanisms include the formation and sequestration of heavy metals in complexes, reduction of a metal to a less toxic species, and direct efflux of a metal out of the cell (30-33). Pseudomonas aeruginosa is a ubiquitous, environmentally important microbe that may employ many resistance mechanisms, such as the mer operon that reduces toxic Hg 2ϩ to volatile Hg 0 , which then diffuses out of the cell (33). However, in bacteria, efflux systems are a more common resistance mechanism for dealing with heavy metals. One such system is the cop system of Pseudomonas syringae, which contains the structural genes copABCD and is homologous to the pco system in Escherichia coli. The copB and copD genes are involved in the transport of copper across the membrane, while the products of the copA and copC genes are outer membrane proteins that bind Cu 2ϩ in the periplasm, protecting the cell from copper (6, 36). Other types of efflux systems simply pump toxic metal ions out of the cell; these systems include the P-type ATPase cadA, which was first identified in Staphylococcus aureus and...

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria

“…The upload of the mercury pools of the cells and the biofilm are competitive processes. Another possible explanation for increased resistance to mercury in biofilms is that the negatively charged extracellular polysaccharides can effectively bind Hg 2+ from the bulk solution (Teitzel and Parsek 2003). This view is further supported by our experiment where the natural biofilm is replaced by a similar artificial polysaccharide network produced by Ficoll.…”

Post-Outbreak Investigation of Pseudomonas aeruginosa Faucet Contamination by Quantitative Polymerase Chain Reaction and Environmental Factors Affecting Positivity

“…Viable but nonculturable P. aeruginosa cells can persist in the presence of copper concentrations up to 2 mM, which is orders of magnitude higher than copper concentrations found in drinking water. 33 Copper concentrations observed in drinking water would therefore affect culturability without affecting viability, with the capacity to recover culturability once copper stress is reduced. 22 Mean residual chlorine (Fig.…”

Quorum sensing inhibitors: a bargain of effects

“…For the clinician, biofilms pose a major problem, as they are highly tolerant to antimicrobial compounds. It has been found that bacteria living in the biofilm mode of growth are often up to 1000-fold more tolerant to antibiotics, biocides and heavy metals than planktonic-growing cells (Allison et al, 1993;Anwar et al, 1990;Teitzel & Parsek, 2003). Consequently, the concentrations required to eradicate bacterial biofilms often exceed the highest deliverable dose, thus precluding efficient treatment based on conventional antibacterials (Costerton et al, 1987(Costerton et al, , 1999Drenkard, 2003;Hoiby et al, 2001).…”

“…In fact, a recent transcriptome analysis performed at different stages during biofilm development and maturation demonstrated that the bulk of biofilm cells, as judged from their gene expression profiles, must have adopted stationary-phase physiology (Hentzer et al, 2005). This explains, at least in part, why most antimicrobials which target active, growing cells are unable to fully eradicate the bacterial biofilms (Campanac et al, 2002;Drenkard, 2003;Lewis, 2001;Teitzel & Parsek, 2003). Genes that are differently expressed in biofilms might further contribute to this tolerance.…”