Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa.
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Journal: 2003/June - Applied and Environmental Microbiology
ISSN: 0099-2240
PUBMED: 12676715
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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 free-swimming 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.
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Appl Environ Microbiol 69(4): 2313-2320
PMID: 12676715

Heavy Metal Resistance of Biofilm and Planktonic <em>Pseudomonas aeruginosa</em>

Department of Civil and Environmental Engineering, Northwestern University, Evanston, Illinois 60208
Corresponding author. Mailing address: Department of Civil and Environmental Engineering, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Phone: (847) 467-7445. Fax: (847) 491-4011. E-mail: ude.nretsewhtron@kesrap-m.
Received 2002 Sep 27; Accepted 2003 Jan 21.
Copyright © 2003, American Society for Microbiology

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 free-swimming 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.

Abstract

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 to volatile Hg, 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 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 is found in other gram-positive bacteria, that pumps out Cd and Zn by using a phospho-aspartate intermediate (30, 32, 36).

The genetics and biochemistry of heavy metal resistance mechanisms have been carefully studied in free-swimming organisms; however, many bacteria in the environment exist in surface-attached communities called biofilms. Biofilm bacteria are usually embedded in an extracellular polymeric substance (EPS) matrix composed of polysaccharides, proteins, and nucleic acids (11, 34, 38, 44, 47). A hallmark trait of biofilms is increased resistance to antimicrobial agents compared to the resistance of free-swimming organisms (7, 13). A proposed mechanism that contributes to this increased resistance is binding and sequestration of antimicrobial agents by EPS components, such as negatively charged phosphate, sulfate, and carboxylic acid groups (17). Another factor that may contribute to the resistance of biofilms is that many antimicrobial agents target metabolically active cells. Biofilms are subject to a wide range of chemical gradients that result in decreased metabolic activity within the depths of a biofilm. In a recent study, Spoering and Lewis examined the relative effects of antimicrobial agents on stationary- and logarithmic-phase cells of P. aeruginosa and found that stationary-phase cells were more resistant to a variety of different antimicrobial agents (37). In the same study the researchers suggested that the resistance of biofilms to antimicrobial agents can be primarily attributed to the stationary phase or slow growth and the presence of a small resistant subpopulation of cells termed “persistors.”

Previous studies of biofilm and heavy metal interactions have mainly focused on the sorption of heavy metals. Several researchers have reported that biofilms are capable of removing heavy metal ions from bulk liquid (10, 16, 22, 25), and the use of biofilms to remove heavy metals from wastewater has been investigated (40, 45). Electron microscopy revealed that a P. aeruginosa biofilm was capable of sequestering heavy metals and that there was surface-associated precipitation of lanthanum by biofilm cells (23), while mercury-reducing Pseudomonas putida biofilms were found to accumulate elemental mercury on the exterior of the biofilms (41). Burkholderia cepacia biofilms on hematite and alumina surfaces were found to preferentially accumulate Pb at concentrations higher than 1 μM, implying that the chemical nature of the attachment surface affects metal sequestration (39). Within a biofilm it has been found that EPS, specifically the polysaccharide components, binds heavy metals (5, 19-21, 27, 28).

In this study we sought to assess the effects of heavy metal toxicity on biofilm and free-swimming P. aeruginosa. The relative toxicities of the heavy metals zinc, copper, and lead for biofilm and free-swimming cells were examined. In addition, we compared the relative susceptibilities of logarithmic- and stationary-phase P. aeruginosa liquid cultures. Surprisingly, logarithmically growing P. aeruginosa was found to be more resistant than stationary-phase cells. However, biofilms were found to be less susceptible than free-swimming cultures. Our results also indicate that the exterior of a biofilm is preferentially killed after heavy metal treatment, which suggests that a biofilm is protected by sorption of heavy metals to the EPS matrix.

Acknowledgments

Gail Teitzel is supported by grant CHE 9810378 from the Institute of Environmental Catalysis and by the National Science Foundation.

We thank Grant Balzer for assistance with SCLM, Jean-François Gaillard and Amy Dahl for assistance with working with heavy metals, and David Chopp for assistance with programming the BacLight subroutine in COMSTAT.

Acknowledgments

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