Influence of central metabolism disruption on Escherichia coli biofilm formation.

Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often overlooked in biofilm investigations include the examination of metabolic pathways as the assumption might be that interference with central pathways such as glycolysis would only reduce growth and thus not be meaningful. Using the Keio collection of Escherichia coli mutants, we investigated the influence of biofilm formation and planktonic growth in full-strength and diluted Luria-Bertani (LB) broths using strains with a disruption of glycolysis (Δpgi), the Entner-Doudoroff pathway (Δedd), or the pentose phosphate pathway (Δgnd). Unexpectedly, in contrast to the E. coli Keio parent strain (BW25113), planktonic growth was enhanced in full strength and diluted LB broths in the metabolic mutants. Using a microtiter biofilm assay, the E. coli parent strain showed the highest crystal violet staining. However, when analyzed by culture assays, there was an increase in biofilm populations in the mutants in comparison to the parent strain. Fluorescence microscopy showed differences in colonization patterns in the strains. Given the availability of mutant collections in many model organisms, similar metabolic studies are warranted for biofilms, given their importance in nature.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

Cadmium ion inhibition of quorum signalling in Chromobacterium violaceum.

Single-celled bacteria are capable of acting as a community by sensing and responding to population density via quorum signalling. Quorum signalling in Chromobacterium violaceum, mediated by the luxI/R homologue, cviI/R, regulates a variety of phenotypes including violacein pigmentation, virulence and biofilm formation. A number of biological and organic molecules have been described as quorum signalling inhibitors but, to date, metal-based inhibitors have not been widely tested. In this study, we show that quorum sensing is inhibited in C. violaceum in the presence of sub-lethal concentrations of cadmium salts. Notable Cd2+-inhibition was seen against pigmentation, motility, chitinase production and biofilm formation. Cd-inhibition of quorum-signalling genes occurred at the level of transcription. There was no direct inhibition of chitinase activity by Cd2+ at the concentrations tested. Addition of the cognate quorum signals, N-hexanoyl homoserine lactone or N-decanoyl homoserine lactone, even at concentrations in excess of physiological levels, did not reverse the inhibition, suggesting that Cd-inhibition of quorum signaling is irreversible. This study represents the first description of heavy metal-based quorum inhibition in C. violaceum.

Indole production provides limited benefit to Escherichia coli during co-culture with Enterococcus faecalis.

Escherichia coli lives in the gastrointestinal tract and elsewhere, where it coexists within a mixed population. Indole production enables E. coli to grow with other gram-negative bacteria as indole inhibits N-acyl-homoserine lactone (AHL) quorum regulation. We investigated whether E. coli indole production enhanced competition with gram-positive Enterococcus faecalis, wherein quorum signaling is mediated by small peptides. During planktonic co-culture with E. faecalis, the fitness and population density of E. coli tnaA mutants (unable to produce indole) equaled or surpassed that of E. coli wt. During biofilm growth, the fitness of both populations of E. coli stabilized around 100 %, whereas the fitness of E. faecalis declined over time to 85-90 %, suggesting that biofilm and planktonic populations have different competition strategies. Media supplementation with indole removed the competitive advantage of E. coli tnaA in planktonic populations but enhanced it in biofilm populations. E. coli wt and tnaA showed similar growth in Luria-Bertani (LB) broth. However, E. coli growth was inhibited in the presence of filter-sterilized spent LB from E. faecalis, with inhibition being enhanced by indole. Similarly, there was also an inhibition of E. faecalis growth by proteinaceous components (likely bacteriocins) from spent culture media from both E. coli strains. We conclude that E. coli indole production is not a universal competition strategy, but rather works against gram-negative, AHL-producing bacteria.

Indole inhibition of N-acylated homoserine lactone-mediated quorum signalling is widespread in Gram-negative bacteria.

The LuxI/R quorum-sensing system and its associated N-acylated homoserine lactone (AHL) signal is widespread among Gram-negative bacteria. Although inhibition by indole of AHL quorum signalling in Pseudomonas aeruginosa and Acinetobacter oleivorans has been reported previously, it has not been documented among other species. Here, we show that co-culture with wild-type Escherichia coli, but not with E. coli tnaA mutants that lack tryptophanase and as a result do not produce indole, inhibits AHL-regulated pigmentation in Chromobacterium violaceum (violacein), Pseudomonas chlororaphis (phenazine) and Serratia marcescens (prodigiosin). Loss of pigmentation also occurred during pure culture growth of Chro. violaceum, P. chlororaphis and S. marcescens in the presence of physiologically relevant indole concentrations (0.5-1.0 mM). Inhibition of violacein production by indole was counteracted by the addition of the Chro. violaceum cognate autoinducer, N-decanoyl homoserine lactone (C10-HSL), in a dose-dependent manner. The addition of exogenous indole or co-culture with E. coli also affected Chro. violaceum transcription of vioA (violacein pigment production) and chiA (chitinase production), but had no effect on pykF (pyruvate kinase), which is not quorum regulated. Chro. violaceum AHL-regulated elastase and chitinase activity were inhibited by indole, as was motility. Growth of Chro. violaceum was not affected by indole or C10-HSL supplementation. Using a nematode-feeding virulence assay, we observed that survival of Caenorhabditis elegans exposed to Chro. violaceum, P. chlororaphis and S. marcescens was enhanced during indole supplementation. Overall, these studies suggest that indole represents a general inhibitor of AHL-based quorum signalling in Gram-negative bacteria.

Bacterial signaling ecology and potential applications during aquatic biofilm construction.

In their natural environment, bacteria and other microorganisms typically grow as surface-adherent biofilm communities. Cell signal processes, including quorum signaling, are now recognized as being intimately involved in the development and function of biofilms. In contrast to their planktonic (unattached) counterparts, bacteria within biofilms are notoriously resistant to many traditional antimicrobial agents and so represent a major challenge in industry and medicine. Although biofilms impact many human activities, they actually represent an ancient mode of bacterial growth as shown in the fossil record. Consequently, many aquatic organisms have evolved strategies involving signal manipulation to control or co-exist with biofilms. Here, we review the chemical ecology of biofilms and propose mechanisms whereby signal manipulation can be used to promote or control biofilms.

Carbon and clay nanoparticles induce minimal stress responses in gram negative bacteria and eukaryotic fish cells.

We investigated in vitro the potential mutagenic and toxic effects of two clay-based nanoparticles, Cloisite® Na(+) (Cloisite) and halloysite; and multi-walled carbon nanotubes (MWCNT), commonly used in the polymer composite industry. Using the Ames test, the three nanoparticles did not have a true mutagenic effect, although growth of Salmonella enterica var. Typhimurium (S.typhimurium) was diminished at higher nanoparticle concentrations. We investigated the impact of nanoparticles on Escherichia coli and S. typhimurium including oxyR and rpoS mutants, which are susceptible to oxidative stress. The oxyR mutants were inhibited in the presence of nanoparticles, when grown aerobically with light. Toxicity was not observed in the absence of light or during anaerobic growth. E. coli rpoS mutants exhibited some toxicity when cultured with Cloisite and MWCNT only when grown aerobically with light. There was no effect with other nanoparticles, or with S. typhimurium rpoS mutants. MWCNT exhibited a slight toxic effect against Epithelioma papulosum cyprini (EPC) cells only at the highest concentration tested. There was no discernable toxicity to EPC cells caused by the clay nanoparticles. We conclude that clay-based nanoparticles and MWCNT do not exert a mutagenic effect and do not have a general toxic effect across all bacterial species or between prokaryotic and eukaryotic cells. Modest toxicity was only observed in eukaryotic EPC cells against MWCNT at the highest concentration tested. Limited species-specific toxicity to clay based and MWCNT nanoparticles was seen in bacterial strains primarily due to culture conditions and mutations that exacerbate oxidative stress.

Enhancing metagenomics investigations of microbial interactions with biofilm technology.

Investigations of microbial ecology and diversity have been greatly enhanced by the application of culture-independent techniques. One such approach, metagenomics, involves sample collections from soil, water, and other environments. Extracted nucleic acids from bulk environmental samples are sequenced and analyzed, which allows microbial interactions to be inferred on the basis of bioinformatics calculations. In most environments, microbial interactions occur predominately in surface-adherent, biofilm communities. In this review, we address metagenomics sampling and biofilm biology, and propose an experimental strategy whereby the resolving power of metagenomics can be enhanced by incorporating a biofilm-enrichment step during sample acquisition.

Genome Sequences of Bacteria Isolated from the International Space Station Water Systems.

We report draft genomes of five bacteria recovered from the U.S. and Russian water systems onboard the International Space Station. The five genera include Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.

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

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.

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities.

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.

Longitudinal characterization of multispecies microbial populations recovered from spaceflight potable water.

While sequencing technologies have revolutionized our knowledge of microbial diversity, little is known about the dynamic emergent phenotypes that arise within the context of mixed-species populations, which are not fully predicted using sequencing technologies alone. The International Space Station (ISS) is an isolated, closed human habitat that can be harnessed for cross-sectional and longitudinal functional microbiome studies. Using NASA-archived microbial isolates collected from the ISS potable water system over several years, we profiled five phenotypes: antibiotic resistance, metabolism, hemolysis, and biofilm structure/composition of individual or multispecies communities, which represent characteristics that could negatively impact astronaut health and life-support systems. Data revealed a temporal dependence on interactive behaviors, suggesting possible microbial adaptation over time within the ecosystem. This study represents one of the most extensive phenotypic characterization of ISS potable water microbiota with implications for microbial risk assessments of water systems in built environments in space and on Earth.

A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response.

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm(-2)) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).

Quorum sensing: implications on rhamnolipid biosurfactant production.

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 are characteristics of Pseudomonas biofilms. Rhamnolipids have biotechnological applications in the uptake of hydrophobic substrates, bioremediation of contaminated soils and polluted waters. Rhamnolipid biosurfactants are biodegradable as compared to chemical surfactants and hence are more preferred in environmental applications. In this review, we examine the biochemical and genetic mechanism of rhamnolipid production by P. aeruginosa and propose the application of QS signal molecules in enhancing the rhamnolipid production.

Prebiotic, immuno-stimulating and gut microbiota-modulating effects of Lycium barbarum polysaccharide.

The present study was done to evaluate the prebiotic effect of Lycium barbarum polysaccharide (LBP), its effect on murine fecal microbiota composition and innate immune response. Results showed that LBP supports the growth of selective probiotic bacteria with a maximum of 8.23 (log10 cfu/mL) and 6.62 (log10 cfu/mL) for Lactobacillus acidophilus and Bifidobacterium longum respectively. In vivo studies revealed that the administrations of LBP to mice resulted in an increase in the abundance of the phyla Proteobacteria and Firmicutes, while reducing the ratio of the phylum Bacteroidetes. At the genus level, the administration of LBP stimulated the emergence of some potential probiotic genera (Akkermansia, Lactobacillus, and Prevotellaceae). The concentrations of TGF-ß and IL-6 in serum and sIgA in the colon content were enriched significantly after LBP administrations in mice. The thymus index and spleen index of mice treated with LBP displayed significant difference compared to the control group (P < 0.05). These findings suggest that LBP is a good source as a potential prebiotic and can enhance the intestinal microbiota and boost beneficial bacteria levels, modulate innate immune response.

Potential biofilm control strategies for extended spaceflight missions.

Biofilms, surface-adherent microbial communities, are associated with microbial fouling and corrosion in terrestrial water-distribution systems. Biofilms are also present in human spaceflight, particularly in the Water Recovery System (WRS) on the International Space Station (ISS). The WRS is comprised of the Urine Processor Assembly (UPA) and the Water Processor Assembly (WPA) which together recycles wastewater from human urine and recovered humidity from the ISS atmosphere. These wastewaters and various process streams are continually inoculated with microorganisms primarily arising from the space crew microbiome. Biofilm-related fouling has been encountered and addressed in spacecraft in low Earth orbit, including ISS and the Russian Mir Space Station. However, planned future missions beyond low Earth orbit to the Moon and Mars present additional challenges, as resupplying spare parts or support materials would be impractical and the mission timeline would be in the order of years in the case of a mission to Mars. In addition, future missions are expected to include a period of dormancy in which the WRS would be unused for an extended duration. The concepts developed in this review arose from a workshop including NASA personnel and representatives with biofilm expertise from a wide range of industrial and academic backgrounds. Here, we address current strategies that are employed on Earth for biofilm control, including antifouling coatings and biocides and mechanisms for mitigating biofilm growth and damage. These ideas are presented in the context of their applicability to spaceflight and identify proposed new topics of biofilm control that need to be addressed in order to facilitate future extended, crewed, spaceflight missions.

Use of Whole-Cell Bioassays for Screening Quorum Signaling, Quorum Interference, and Biofilm Dispersion.

In most bacteria, a global level of regulation, termed quorum sensing (QS), exists involving intercellular communication via the production and response to cell density-dependent signal molecules. QS 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. N-acylated homoserine lactones (AHLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, commonly found in soil and water, produces the characteristic purple pigment violacein, regulated by AHL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of AHL-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 AHL and QS inhibitors. Due to the inherent low susceptibility of biofilm bacteria to antimicrobial agents, biofilm dispersion, whereby bacteria reenter the planktonic community, is another increasingly important area of research. At least one signal, distinct from traditional QS, has been identified and there are a variety of other environmental factors that also trigger dispersion. We describe a microtiter-based experimental strategy whereby potential biofilm dispersion compounds can be screened.

Molecular modeling, synthesis, and screening of new bacterial quorumsensing antagonists.

Abstract A new series comprising 7 analogs of N-(sulfanyl ethanoyl)-L-HSL derivatives, 2 analogs of N-(fluoroalkanoyl)- L-HSL derivatives, N-(fluorosulfonyl)-L-HSL, and 2,2-dimethyl butanoyl HSL were synthesized using a solid-phase organic synthesis method. Each of the 11 synthesized compounds was analyzed using NMR and mass spectroscopies, and molecular modeling studies of the 11 ligands were performed using SYBYL packages. Thereafter, a bacterial test was designed to identify their quorum-sensing inhibition activity and antifouling efficacy. Most of the synthesized compounds were found to be effective as quorum-sensing antagonists, where antagonist screening revealed that 10 among the 11 synthesized ligands were able to antagonize the quorum sensing of A. tumefaciens.