Block Biofilm Formation Research Articles

A Selenium-Dependent Xanthine Dehydrogenase Triggers Biofilm Proliferation inEnterococcus faecalisthrough Oxidant Production

Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with (75)Se is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production.

Biofilm Development on Caenorhabditis elegans by Yersinia Is Facilitated by Quorum Sensing-Dependent Repression of Type III Secretion

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.

Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn(2+). The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn(2+)-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.

ANTIBIOTIC RESISTANCE: Biofilm Dispersing Agent Rejuvenates Older Antibiotics

An estimated 75% of bacterial infections involve biofilms, surface-attached colonies of bacteria that are protected by an extracellular matrix.1 Bacteria protected within biofilms are up to 1,000 times more resistant to antibiotics than if they were free-floating (planktonic),2 which severely complicates treatment options. Rather than searching for better antibiotics, researchers have discovered that small molecules3 known as 2-amino-imidazoles disrupt biofilms, making antibiotic-resistant strains of bacteria more vulnerable to conventional drugs.4 Moreover, antibiotics enhance the ability of 2-amino-imidazoles to disrupt biofilms. “Perhaps new antibiotics are not the only way to combat biofilm infections if we could make ineffective older antibiotics active again,” says principal investigator Christian Melander, an associate professor of bio-organic chemistry at North Carolina State University. Melander and his colleagues started with natural 2-amino-imidazoles (isolated from sponges) including oroidin and ageliferin, which are known to block biofilm formation. They synthesized an improved version of oroidin, 2-amino-imidazole/triazole (2-AIT), which featured more powerful antibiofilm activity and less toxicity to surrounding human cells and organs.5 Although 2-AIT alone does not kill bacteria, it disperses biofilms, releasing planktonic cells that are more susceptible to antibiotics than bacterial cells shielded by sticky biofilms. The researchers tested 2-AIT against a variety of clinically relevant biofilm infections along with antibiotics that currently are used or have been used in the past to treat them. Multidrug-resistant strains of Acinetobacter baumannii plague soldiers wounded in the Middle East, and colistin, an older antibiotic with toxic side effects, remains a treatment of last resort due to extensive side effects.6 Staphylococcus aureus infections that colonize catheters and other indwelling medical devices were treated with the antibiotic novobiocin until drug-resistant S. aureus strains arose.7 Tobramycin is an inhaled antibiotic currently used to treat Pseudomonas aeruginosa infections that clog the lungs of cystic fibrosis patients.8 “We chose three antibiotics known to act against certain bacteria circulating in hospital settings that have become drug resistant,” says Melander. The biofilms were grown in culture, then treated with their corresponding antibiotic, all of which produced little dispersion. However, the addition of 2-AIT to the antibiotics produced dramatic dispersion of up to 1,000-fold.4 Like adjuvants that boost the power of vaccines, 2-AIT is “our version of a small molecule adjuvant that allows several classes of older antibiotics to work again,” says Melander. He suspects 2-AIT somehow short-circuits bacterial signaling pathways that regulate biofilm formation; experiments are under way to unravel the details. 2-AIT also was shown to resensitize drug-resistant bacteria to death by antibiotic. When a clinical strain of methicillin-resistant S. aureus (MRSA) was treated with 2-AIT alone, it grew normally. The addition of methicillin, however, reduced its growth by 99%. Additionally, 2-AIT lowered the amount of antibiotics needed to inhibit bacterial growth.4 The combination of 2-AIT with antibiotics could serve as a parallel treatment for antibiotic-resistant infections. The results suggest this cooperative approach may enable “obsolete antibiotics to overcome infections that otherwise would persist if treated with either agent individually,” says Melander. A small molecule adjuvant like 2-AIT potentially could be given orally in pill form, he says. 2-AIT “most remarkably can actually disperse preformed biofilms, something that is much more difficult to do than simply inhibiting their formation,” says Neville Kallenbach, a professor of chemistry at New York University in New York City. Because biofilms are much harder to kill than planktonic bacteria, the combination therapy opens a new avenue for remediating persistent biofilm infections. “The ability to disperse biofilms formed by multidrug-resistant bacteria adds a major new weapon to the limited arsenal of therapies available today,” says Kallenbach—and the impact on human health could be enormous. Agents such as 2-AIT also lend themselves to solving environmental biofilm problems including the biofouling of ship hulls and plugging of waterlines by microbes such as Escherichia coli and Bacillus, Pseudomonas, Proteobacter, and Actinobacteria species. Today’s antifouling paints contain copper, which leaches into seawater, where it inhibits enzymatic activity in brine shrimp larvae9 and impairs sperm quality and larvae development in sea urchins,10 among other effects. Melander is working on a copper-free 2-AIT-based polymer spray to prevent biofouling.

The NanA Neuraminidase of Streptococcus pneumoniae Is Involved in Biofilm Formation

Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccines and effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acid residues from glycoconjugates, is involved in host colonization in animal models of infection and may provide a novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase (NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role in pathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even though we show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formation incorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA block biofilm formation and may provide a novel target for preventative therapy. This work highlights the role played by the neuraminidase in pathogenesis and represents an important step in drug development for prevention of colonization of the respiratory tract by this important pathogen.

The potential of desferrioxamine-gallium as an anti- Pseudomonas therapeutic agent

The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine-gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic gentamicin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

Serum Inhibits P. aeruginosa Biofilm Formation on Plastic Surfaces and Intravenous Catheters

Biofilm formation on medical devices such as intravenous catheters is a serious manifestation of Pseudomonas aeruginosa infections. Serum has bactericidal activity, a function of multiple serum components. In this study, we determined the effect of serum and serum components on the formation of P. aeruginosa biofilm. We examined the effect of adult bovine serum (ABS) or bovine serum albumin (BSA) on biofilm development on plastic coverslips. This was done using both static and continuous flow-through culture systems and P. aeruginosa strain PAO1. Biofilms were quantified using crystal violet assays and visualized using confocal scanning laser microscopy and scanning electron microscopy. We examined the effect of ABS on PAO1 swimming and twitching motilities (both contribute to P. aeruginosa biofilm development). We also analyzed the inhibitory effect of adult human serum (AHS) and plasma (AHP) on PAO1 biofilm development on plastic coverslips and intravenous catheters. Compared with M9 minimal medium (M9), 10% ABS-supplemented medium (M9/ABS-10) caused a significant decrease in biofilm development. Coverslips precoated with M9/ABS-10 failed to develop biofilm when placed in M9. In addition to reduced biofilm formation, adding ABS to M9 reduced an already-developed PAO1 biofilm. Compared with M9, M9/ABS-10 enhanced PAO1 twitching motility considerably, but did not affect swimming motility. Similar to ABS, BSA blocked biofilm formation but did not affect PAO1 twitching motility. Both AHS and AHP blocked PAO1 biofilm formation on plastic coverslips and intravenous catheters. These results suggest that as part of the host innate resistance, serum inhibits P. aeruginosa biofilm formation on plastic surfaces, including intravenous catheters. Two possible scenarios for this inhibition include blocking the direct interaction between P. aeruginosa and the substrates, and the enhancing P. aeruginosa twitching motility.

Iron sequestration by human lactoferrin stimulates P. aeruginosa surface motility and blocks biofilm formation.

This paper summarizes a presentation at the 6th International Conference on Lactoferrin Structure and Function. Some of this data has been previously published.