Joe J. Harrison

Antimicrobial activity of metals: mechanisms, molecular targets and applications

Metals have been used as antimicrobial agents since antiquity, but throughout most of history their modes of action have remained unclear. Recent studies indicate that different metals cause discrete and distinct types of injuries to microbial cells as a result of oxidative stress, protein dysfunction or membrane damage. Here, we describe the chemical and toxicological principles that underlie the antimicrobial activity of metals and discuss the preferences of metal atoms for specific microbial targets. Interdisciplinary research is advancing not only our understanding of metal toxicity but also the design of metal-based compounds for use as antimicrobial agents and alternatives to antibiotics.

Multimetal resistance and tolerance in microbial biofilms

Geochemical cycling and industrial pollution have made toxic metal ions a pervasive environmental pressure throughout the world. Biofilm formation is a strategy that microorganisms might use to survive a toxic flux in these inorganic compounds. Evidence in the literature suggests that biofilm populations are protected from toxic metals by the combined action of chemical, physical and physiological phenomena that are, in some instances, linked to phenotypic variation among the constituent biofilm cells. Here, we propose a multifactorial model by which biofilm populations can withstand metal toxicity by a process of cellular diversification.

Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms

Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities. Biofilm-forming bacteria such as the opportunistic pathogen Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors affecting surface adhesion, and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as ‘molecular glue’. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface. By combining this technique with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generates positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells becoming extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening

Batch culture of biofilms on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibility. In this paper, we describe a core protocol and a set of parameters (surface composition, the rate of rocking or orbital motion, temperature, cultivation time, inoculum size, atmospheric gases and nutritional medium) that can be adjusted to grow single- or multispecies biofilms on peg surfaces. Mature biofilms formed on peg lids can then be fitted into microtiter plates containing test agents. After a suitable exposure time, biofilm cells are disrupted into a recovery medium using sonication. Microbicidal endpoints can be determined qualitatively using optical density measurements or quantitatively using viable cell counting. Once equipment is calibrated and growth conditions are at an optimum, the procedure requires ∼5 h of work over 4–6 d. This efficient method allows antimicrobial agents and exposure conditions to be tested against biofilms on a high-throughput scale.

The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin

Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.

The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm

ABSTRACT Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a ΔyafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the ΔyafQ strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.

The bacterial response to the chalcogen metalloids Se and Te.

Microbial metabolism of inorganics has been the subject of interest since the 1970s when it was recognized that bacteria are involved in the transformation of metal compounds in the environment. This area of research is generally referred to as bioinorganic chemistry or microbial biogeochemistry. Here, we overview the way the chalcogen metalloids Se and Te interact with bacteria. As a topic of considerable interest for basic and applied research, bacterial processing of tellurium and selenium oxyanions has been reviewed a few times over the past 15 years. Oddly, this is the first time these compounds have been considered together and their similarities and differences highlighted. Another aspect touched on for the first time by this review is the bacterial response in cell-cell or cell-surface aggregates (biofilms) against the metalloid oxyanions. Finally, in this review we have attempted to rationalize the considerable amount of literature available on bacterial resistance to the toxic metalloids tellurite and selenite.

Copper and Quaternary Ammonium Cations Exert Synergistic Bactericidal and Antibiofilm Activity against Pseudomonas aeruginosa

ABSTRACT Biofilms are slimy aggregates of microbes that are likely responsible for many chronic infections as well as for contamination of clinical and industrial environments. Pseudomonas aeruginosa is a prevalent hospital pathogen that is well known for its ability to form biofilms that are recalcitrant to many different antimicrobial treatments. We have devised a high-throughput method for testing combinations of antimicrobials for synergistic activity against biofilms, including those formed by P. aeruginosa. This approach was used to look for changes in biofilm susceptibility to various biocides when these agents were combined with metal ions. This process identified that Cu2+ works synergistically with quaternary ammonium compounds (QACs; specifically benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some cases, adding Cu2+ to QACs resulted in a 128-fold decrease in the biofilm minimum bactericidal concentration compared to that for single-agent treatments. In combination, these agents retained broad-spectrum antimicrobial activity that also eradicated biofilms of Escherichia coli, Staphylococcus aureus, Salmonella enterica serovar Cholerasuis, and Pseudomonas fluorescens. To investigate the mechanism of action, isothermal titration calorimetry was used to show that Cu2+ and QACs do not interact in aqueous solutions, suggesting that each agent exerts microbiological toxicity through independent biochemical routes. Additionally, Cu2+ and QACs, both alone and in combination, reduced the activity of nitrate reductases, which are enzymes that are important for normal biofilm growth. Collectively, the results of this study indicate that Cu2+ and QACs are effective combinations of antimicrobials that may be used to kill bacterial biofilms.

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.

High-throughput metal susceptibility testing of microbial biofilms.

BackgroundMicrobial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated.ResultsThis high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32-) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent.ConclusionThis method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria.