Influence of quorum sensing and iron on twitching motility and biofilm formation in Pseudomonas aeruginosa

Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl₃ concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl₃ that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

Aeromonas hydrophila biofilm,exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces

Abstract

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.     

Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni

The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.     

Influence of quorum sensing signal molecules on biofilm formation in <Emphasis Type="Italic">Proteus mirabilis</Emphasis> O18

The influence of basis of quorum sensing molecules on Proteus strains is much less known as compared to Pseudomonas or Escherichia. We have previously shown that a series of acylated homoserine lactones (acyl-HSL) does not influence the ureolytic, proteolytic, or hemolytic abilities, and that the swarming motility of Proteus mirabilis rods is strain specific. The aim of the presented study was to find out if the presence of a series of acyl-HSL influences biofilm formation of P. mirabilis laboratory strain belonging to O18 serogroup. This serogroup is characterized by the presence of a unique non-carbohydrate component, namely phosphocholine. Escherichia coli and P. mirabilis O18 strains used in this work contains cloned plasmids encoding fluorescent protein genes with constitutive gene expression. In mixed biofilms in stationary and continuous flow conditions, P. mirabilis O18 overgrow whole culture. P. mirabilis O18 strain has genetically proved a presence of AI–2 quorum sensing system. Differences in biofilm structure were observed depending on the biofilm type and culture methods. From tested acylated homoserine lactones (BHL, HHL, OHL, DHL, dDHL, tDHL), a significant influence had BHL on thickness, structure, and the amount of exopolysaccharides produced by biofilms formed by P. mirabilis O18 pDsRed₂.     

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.     

Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.     

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO₂ sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Prostaglandin E2 from Candida albicans Stimulates the Growth of Staphylococcus aureus in Mixed Biofilms

Background

Previous studies showed that Staphylococcus aureus and Candida albicans interact synergistically in dual species biofilms resulting in enhanced mortality in animal models.

Methodology/Principal Findings

The aim of the current study was to test possible candidate molecules which might mediate this synergistic interaction in an in vitro model of mixed biofilms, such as farnesol, tyrosol and prostaglandin (PG) E₂. In mono-microbial and dual biofilms of C.albicans wild type strains PGE₂ levels between 25 and 250 pg/mL were measured. Similar concentrations of purified PGE₂ significantly enhanced S.aureus biofilm formation in a mode comparable to that observed in dual species biofilms. Supernatants of the null mutant deficient in PGE₂ production did not stimulate the proliferation of S.aureus and the addition of the cyclooxygenase inhibitor indomethacin blocked the S.aureus biofilm formation in a dose-dependent manner. Additionally, S. aureus biofilm formation was boosted by low and inhibited by high farnesol concentrations. Supernatants of the farnesol-deficient C. albicans ATCC10231 strain significantly enhanced the biofilm formation of S. aureus but at a lower level than the farnesol producer SC5314. However, C. albicans ATCC10231 also produced PGE₂ but amounts were significantly lower compared to SC5314.

Conclusion/Significance

In conclision, we identified C. albicans PGE₂ as a key molecule stimulating the growth and biofilm formation of S. aureus in dual S. aureus/C. albicans biofilms, although C. albicans derived farnesol, but not tyrosol, may also contribute to this effect but to a lesser extent.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Background

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/Principal Findings

Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/Significance

These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.     

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.     

Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS_Sm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (R_f value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Isolation and characterization of acyl homoserine lactone–producing bacteria during an urban river biofilm formation

The presence and diversity of acyl homoserine lactone (AHL)-producers in an urban river biofilm were investigated during 60-day biofilm formation. AHL biosensors detected the presence of AHL-producers in 1–60-day river biofilms. Screening for AHL-producers resulted in 17 Aeromonas spp., 3 Pseudomonas spp., 3 Ensifer spp., and 1 Acinetobacter sp. Among these isolates, six of them were closely related to Acinetobacter tjernbergiae, Aeromonas allosaccharophila, Aeromonas aquariorum, Aeromonas jandaei, Pseudomonas panipatensis, and Ensifer adhaerens and represented novel AHL-producing species. Thin layer chromatography revealed that C4-homoserine lactone was prevailing in Aeromonas spp., whereas C6- and C8-homoserine lactones and their derivatives were prevailing in other strains. Using degenerate primers, novel AHL synthetase genes from the three Ensifer spp. were successfully amplified. This study reports for the first time the diversity of AHL-producers from a river biofilm and the variety of novel AHL synthetase genes in Ensifer group.