Robert J. C. McLean

The development of bacterial biofilms on indwelling urethral catheters

Abstract The biofilm mode of growth has been implicated in the majority of human bacterial infections. In the urinary tract, notable biofilm-associated infections include prostatitis, chronic cystitis, struvite urolithiasis, and catheter-associated infections. Biofilms protect the causative organisms from host defences and antimicrobial therapy. Biofilm formation has traditionally been considered to result from adhesion and capsule formation by adherent microorganisms. Recent work has shown that a large number of genes are activated during this process, some of which have been associated with twitching motility, quorum sensing, and slow growth. In this paper, we review some of the recent work on biofilm biology and highlight its role in urinary tract infections, particularly those associated with urinary catheters.

Gram‐negative outer membrane vesicles: beyond the cell surface

Considerable interest has recently mounted regarding the biological roles of Gram-negative outer membrane vesicles (MVs). The first discovery of MVs was made over four decades ago, and it is now clear that most Gram-negative bacteria produce MVs, with Pseudomonas aeruginosa and Escherichia coli as the most extensively studied. Much of our knowledge of the biological roles of MVs and mechanism of MV formation is due to T.J. Beveridge and colleagues. Beveridge pioneered the field of MV research not only by enhancing our understanding of MV function, but also through the application of a wide variety of physical, chemical, and genetic techniques to complement his elegant electron microscopy investigations. Here we review the contributions of Beveridges group to our understanding of MV biology.

Alternative origins for nannobacteria-like objects in calcite

More than 40 calcite-precipitation experiments were performed under sterile conditions in order to investigate the origins of 25–300 nm spherical-, rod-, and ovoid-shaped objects that have been widely interpreted as evidence of nanometer-scale life (i.e., nannobacteria). Individual experiments included the addition of soluble organic compounds, common species of eubacteria, or phage-induced eubacterial lysates. These experiments indicate that many of the nanometer-scale objects have inorganic or nonnannobacterial origins. In the precipitation experiments, calcite formed euhedral crystals 50–800 nm in diameter and smaller (<50 nm) anhedral or rounded particles or protocrystals. The small anhedral or rounded solids resembled nannobacteria. The relative amount of anhedral or rounded calcite was greatest in experiments with a dissolved organic component. These controlled experiments are in accord with observations that rounded nanometer-scale objects are more common in minerals formed in organic-rich environments. Bacterial fragments occur as rounded to irregularly shaped particles that included cell-wall fragments, expulsed cytoplasm, and relict capsules that also closely resembled nannobacteria. Acid etching of the large euhedral crystals produced in the precipitation experiments also resulted in the formation of nanometer-scale features that resembled nannobacteria in natural carbonates. The shapes of the etching artifacts vary as a function of the strength of the acid and the duration of etching. Much caution is advisable in interpreting the origin of rounded features <50 nm.

Indole production promotes Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signaling.

ABSTRACT Indole production by Escherichia coli, discovered in the early 20th century, has been used as a diagnostic marker for distinguishing E. coli from other enteric bacteria. By using transcriptional profiling and competition studies with defined mutants, we show that cyclic AMP (cAMP)-regulated indole formation is a major factor that enables E. coli growth in mixed biofilm and planktonic populations with Pseudomonas aeruginosa. Mutants deficient in cAMP production (cyaA) or the cAMP receptor gene (crp), as well as indole production (tnaA), were not competitive in coculture with P. aeruginosa but could be restored to wild-type competitiveness by supplementation with a physiologically relevant indole concentration. E. coli sdiA mutants, which lacked the receptor for both indole and N-acyl-homoserine lactones (AHLs), showed no change in competitive fitness, suggesting that indole acted directly on P. aeruginosa. An E. coli tnaA mutant strain regained wild-type competiveness if grown with P. aeruginosa AHL synthase (rhlI and rhlI lasI) mutants. In contrast to the wild type, P. aeruginosa AHL synthase mutants were unable to degrade indole. Indole produced during mixed-culture growth inhibited pyocyanin production and other AHL-regulated virulence factors in P. aeruginosa. Mixed-culture growth with P. aeruginosa stimulated indole formation in E. coli cpdA, which is unable to regulate cAMP levels, suggesting the potential for mixed-culture gene activation via cAMP. These findings illustrate how indole, an early described feature of E. coli central metabolism, can play a significant role in mixed-culture survival by inhibiting quorum-regulated competition factors in P. aeruginosa.

Quorum sensing: implications on rhamnolipid biosurfactant production.

Abstract Quorum sensing (QS) has received significant attention in the past few decades. QS describes population density dependent cell to cell communication in bacteria using diffusible signal molecules. These signal molecules produced by bacterial cells, regulate various physiological processes important for social behavior and pathogenesis. One such process regulated by quorum sensing molecules is the production of a biosurfactant, rhamnolipid. Rhamnolipids are important microbially derived surface active agents produced by Pseudomonas spp. under the control of two interrelated quorum sensing systems; namely las and rhl. Rhamnolipids possess antibacterial, antifungal and antiviral properties. They are important in motility, cell to cell interactions, cellular differentiation and formation of water channels that Currently, biosurfactants are unable to compete economically with chemically synthesized compounds in the market due to high production costs. Once the genes required for biosurfactant production have been identified, they can be placed under the regulation of strong promoters in nonpathogenic, heterologous hosts to enhance production. The production of rhamnolipids could be increased by cloning both the rhlAB rhamnosyltransferase genes and the rhlRI quorum sensing system into a suitable bacterium such as E. coli or P. putida and facilitate rhamnolipid production. Biosurfactants can also be genetically engineered for different industrial applications assuming there is a strong understanding of both the genetics and the structure-function relationships of each component of the molecule. Genetic engineering of surfactin has already been reported, with recent papers describing the creation of novel peptide structures from the genetic recombination of several peptide synthetases. Recent application of dynamic metabolic engineering strategies for controlled gene expression could lower the cost of fermentation processes by increasing the product formation. Therefore, by integrating a genetic circuit into applications of metabolic engineering the biochemical production can be optimized. Furthermore, novel strategies could be designed on the basis of information obtained from the studies of quorum sensing and biosurfactants produced suggesting enormous practical applications.

Enhancement of leaf fossilization potential by bacterial biofilms

Terrestrial leaf fossils often form through authigenic preservation in which the leaf surface is coated by a variety of minerals, especially iron oxides. The mechanism of this fossilization is unclear, because the largely hydrophobic leaf surfaces do not readily bind metal ions. Previously proposed mechanisms for mineral encrustation include precipitation of minerals in sediment pore space and precipitation of iron oxides at the surface by decay-produced CO 2 . Here we demonstrate that diverse bacterial species rapidly colonize leaf surfaces and form a biofilm within days of the leaf9s entry into a stream environment. Experimental mineralization of fresh and biofilm-coated leaves indicates that leaves without biofilm do not mineralize, but leaves with biofilms rapidly adsorb metal ions such as Fe 3+ onto the anionic biofilm surface where the ions form ferrihydrite. Once these mineralized leaves are buried by the sediment, they are more likely to be converted to fossils than non-mineralized leaves. Examination of a fossil leaf surface by scanning electron microscopy shows bacteria-sized structures resembling those found in biofilms. These experimental data imply that bacterial colonization of leaves may be an essential prerequisite for authigenic preservation.

Bacteriophage Ecology in Escherichia coli and Pseudomonas aeruginosa Mixed-Biofilm Communities

ABSTRACT Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

Rheinheimera tangshanensis sp. nov., a rice root-associated bacterium.

A Gram-negative, aerobic, rod-shaped bacterium, designated strain JA3-B52(T), was isolated from the roots of fresh rice plants (Oryza sativa). The cells were motile by means of polar single or lateral flagella. The colonies were non-pigmented. On the basis of 16S rRNA gene sequence comparisons, the strain was phylogenetically related to species of the genus Rheinheimera, having the greatest level of sequence similarity with respect to Rheinheimera texasensis A62-14B(T) (97.16 %). The bacterium grew at temperatures from 10 to 37 degrees C, with an optimum at 30 degrees C. The strain exhibited growth with 0-3.0 % (w/v) NaCl and at pH 6.0-8.5. The main cellular fatty acids were C(16 : 1)omega7c, C(17 : 1)omega8c, C (16 : 0), C(18 : 1)omega7c and C(12 : 0) 3-OH. The DNA G+C content was 47.0 mol%. The levels of similarity between the 16S rRNA gene sequence of strain JA3-B52(T) and those of the type strains of Rheinheimera species ranged from 95.38 to 97.16 %. The mean level of DNA-DNA relatedness between strain JA3-B52(T) and R. texasensis A62-14B(T), the strain most closely related to the isolate, was 20.4 %. On the basis of physiological and biochemical characteristics and genotypic data obtained in this work, strain JA3-B52(T) represents a novel species of the genus Rheinheimera, for which the name Rheinheimera tangshanensis sp. nov. is proposed. The type strain is JA3-B52(T) (=CGMCC 1.6362(T) =DSM 19460(T)).

An inexpensive chemostat apparatus for the study of microbial biofilms

Continuous culture is a powerful technique for studying microbial biofilms because it allows for the control of growth rate through nutrient limitation. These conditions offer a realistic view of how microorganisms interact in natural ecosystems. The vast majority of biofilm research is performed with batch cultures, due to the high cost of commercially produced chemostats. We describe a chemostat that could be assembled on a limited budget and could be used in a variety of continuous culture experiments, including biofilm assays. Our design consists of an Erlenmeyer flask with custom-blown ports for aeration and waste removal/direct sampling, and a third port that allows microorganisms in the reaction flask to be circulated through a modified Robbins device and returned via the mouth of the flask. This device enables the formation of highly reproducible biofilm populations, of for example, Aeromonas hydrophila, at various growth rates. As such, it is well-suited for the study of the physiology and genetics of biofilm bacteria.