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Gram-negative bacteria belonging to the Xanthomonas genus include plant pathogens representing a major challenge in the field of agriculture for a wide variety of economically important crops, such as tomato, pepper, and lettuce. Due to the massive usage of agrochemicals, Xanthomonas spp. are developing resistance to copper pesticides typically used to control microbial infections. An interesting alternative approach to control bacterial phytopathogens consists of using eco-friendly biocontrol agents, often beneficial microorganisms. Here, following the targeted, broad-spectrum screening of thousands of microorganisms isolated from different environmental locations, we isolated Bacillus velezensis strain 71 and Paenibacillus peoriae strain To99 displaying potent antagonistic activity against Xanthomonas spp. We found that oxydifficidin and polymyxin A secreted by B. velezensis 71 and P. peoriae To99, respectively, are mainly responsible for the anti-Xanthomonas activity. We further evaluated the performance of cell suspensions and cell-free supernatants of these isolates in controlling tomato bacterial spot disease in growth chamber and greenhouse conditions to validate the in vitro results. The overall results demonstrate the potential of treatments based on the secondary metabolites from both isolates and their cells as an alternative to copper-based chemicals to control leaf spot diseases caused by Xanthomonas spp. phytopathogens.

ABSTRACT Pseudomonas aeruginosa, a bacterium causing infections in immunocompromised individuals, regulates several of its virulence functions using three interlinked quorum sensing (QS) systems (las, rhl, and pqs). Despite its presumed importance in regulating virulence, dysfunction of the las system regulator LasR occurs frequently in strains isolated from various environments, including clinical infections. This newfound abundance of LasR-defective strains calls into question existing hypotheses regarding their selection. Indeed, current assumptions concerning factors driving the emergence of LasR-deficient isolates and the role of LasR in the QS hierarchy must be reconsidered. Here, we propose that LasR is not the primary master regulator of QS in all P. aeruginosa genetic backgrounds, even though it remains ecologically significant. We also revisit and complement current knowledge on the ecology of LasR-dependent QS in P. aeruginosa, discuss the hypotheses explaining the putative adaptive benefits of selecting against LasR function, and consider the implications of this renewed understanding.

ABSTRACT Successful colonization of a multitude of ecological niches by the bacterium Pseudomonas aeruginosa relies on its ability to respond to concentrations of self-produced signal molecules. This intercellular communication system known as quorum sensing (QS) tightly regulates the expression of virulence determinants and a diversity of survival functions, including those required for social behaviors. In planktonic cultures of P. aeruginosa, the transcriptional regulator LasR is generally considered on top of the QS circuitry hierarchy; its activation relies on binding to 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), a product of LasI synthase. Transcription of lasI is activated by LasR, resulting in a positive feedback loop. Few studies have looked at the function of QS during surface growth even though P. aeruginosa typically lives in biofilm-like communities under natural conditions. Here, we show that surface-grown P. aeruginosa, including prototypical strain PA14, produces 3-oxo-C12-HSL in the absence of LasR. This phenotype is commonly observed upon surface association in naturally occurring environmental and clinical LasR-defective isolates, suggesting a conserved alternative function for the signal. Notably, in surface-grown cultures, 3-oxo-C12-HSL reaches higher levels than planktonic cells, and there is a delayed timing of its production. Accordingly, 3-oxo-C12-HSL upregulates the autologous expression of pyocyanin and LasR-controlled virulence determinants in neighboring cells even in the absence of the cognate regulator LasR. This highlights a possible role for 3-oxo-C12-HSL in shaping community responses and provides a possible evolutive benefit for mixed populations to carry LasR-defective cells, a common feature of natural populations of P. aeruginosa. IMPORTANCE The bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa, irrespective of the origin of the strains. By investigating the QS circuitry in surface-grown cells, we found an accumulation of QS signal 3-oxo-C12-HSL in the absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.

Biosurfactants are microbial products that have applications as cleaning agents, emulsifiers, and dispersants. Detection and quantification of biosurfactants can be done by various methods, including colorimetric tests, high performance liquid chromatography (HPLC) coupled to several types of detectors, and tests that take advantage of biosurfactants reducing surface tension of aqueous liquids, allowing for spreading and droplet formation of oils. We present a new and simple method for quantifying biosurfactants by their ability, on paper, to reduce surface tension of aqueous solutions, causing droplet dispersion on an oiled surface in correlation with biosurfactant content. We validated this method with rhamnolipids, surfactin, sophorolipids, and ananatoside B; all are anionic microbial surfactants. Linear ranges for quantification in aqueous solutions for all tested biosurfactants were between 10 and 500 µM. Our method showed time-dependent biosurfactant accumulation in cultures of Pseudomonas aeruginosa strains PA14 and PAO1, and Burkholderia thailandensis E264. Mutants in genes responsible for surfactant production showed negligible activity on oiled paper. In summary, our simple assay provides the opportunity to quantify biosurfactant contents of aqueous solutions, for a diversity of surfactants, by means readily available in any laboratory.

Pseudomonas aeruginosa infections are increasingly difficult to treat due to the development of antimicrobial resistance. Here, the authors describe the synthesis, characterisation and efficacy of a quorum sensing inhibitor.

ABSTRACT The ability to generate a subpopulation of small colony variants (SCVs) is a conserved feature of Pseudomonas aeruginosa and could represent a key adaptive strategy to colonize and persist in multiple niches. However, very little is known about the role of the SCV phenotype, the conditions that promote its emergence, and its possible involvement in an adaptive strategy. In the present work, we investigated the in vitro selective conditions promoting the emergence of SCVs from the prototypical strain PA14, which readily forms SCVs in nonagitated standing cultures. We found that O2 limitation, which causes a redox imbalance, is the main factor selecting for the SCV phenotype, which promotes survival of the population via formation of a biofilm at the air-liquid interface to access the electron acceptor. When this selective pressure is relieved by aeration or supplementation of an alternative electron acceptor, SCVs are barely detectable. We also observed that SCV emergence contributes to redox rebalancing, suggesting that it is involved in an adaptive strategy. We conclude that selection for the SCV phenotype is an adaptive solution adopted by P. aeruginosa to access poorly available O2. IMPORTANCE The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that thrives in many environments. It poses a significant health concern, notably because it is a causative agent of nosocomial infections and the most prevalent pathogen found in the lungs of people with cystic fibrosis. In infected hosts, its persistence is often related to the emergence of an alternative phenotype known as small colony variant (SCV). Identification of conditions selecting for the SCV phenotype contributes to knowledge regarding adaptive mechanisms exploited by P. aeruginosa to survive in multiple niches and persist during infections. Hindering this adaptation strategy could help control persistent P. aeruginosa infections.

Molecular typing techniques are utilized to determine genetic similarities between bacterial isolates. However, the use of environmental DNA profiling to assess epidemiologic links between patients and their environment has not been fully explored. This work reports the development and validation of two high-throughput short sequence typing (HiSST) schemes targeting the opportunistic pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia, along with a modified SM2I selective medium for the specific isolation of S. maltophilia. These HiSST schemes are based on four discriminative loci for each species and demonstrate high discriminating power, comparable to pairwise whole-genome comparisons. Each scheme includes species-specific PCR primers for precise differentiation from closely related taxa, without the need for upstream culture-dependent methods. For example, the primers targeting the bvgS locus make it possible to distinguish P. aeruginosa from the very closely related Pseudomonas paraeruginosa sp. nov. The selected loci included in the schemes are adapted to massive parallel amplicon sequencing technology. An R-based script implemented in the DADA2 pipeline was assembled to facilitate HiSST analyses for efficient and accurate genotyping of P. aeruginosa and S. maltophilia. We demonstrate the performance of both schemes through in silico validations, assessments against reference culture collections, and a case study involving environmental samples.

Interspecies interactions can drive the emergence of unexpected microbial phenotypes that are not observed when studying monocultures. The cystic fibrosis (CF) lung consists of a complex environment where microbes, living as polymicrobial biofilm-like communities, are associated with negative clinical outcomes for persons with CF (pwCF). However, the current lack of in vitro models integrating the microbial diversity observed in the CF airway hampers our understanding of why polymicrobial communities are recalcitrant to therapy in this disease. Here, integrating computational approaches informed by clinical data, we built a mixed community of clinical relevance to the CF lung composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica. We developed and validated this model biofilm community with multiple isolates of these four genera. When challenged with tobramycin, a front-line antimicrobial used to treat pwCF, the microorganisms in the polymicrobial community show altered sensitivity to this antibiotic compared to monospecies biofilms. We observed that wild-type P. aeruginosa is sensitized to tobramycin in a mixed community versus monoculture, and this observation holds across a range of community relative abundances. We also report that LasR loss-of-function, a variant frequently detected in the CF airway, drives tolerance of P. aeruginosa to tobramycin specifically in the mixed community. Our data suggest that the molecular basis of this community-specific recalcitrance to tobramycin for the P. aeruginosa lasR mutant is increased production of phenazines. Our work supports the importance of studying a clinically relevant model of polymicrobial biofilms to understand community-specific traits relevant to infections.

Driven in part by its metabolic versatility, high intrinsic antibiotic resistance, and a large repertoire of virulence factors, Pseudomonas aeruginosa is expertly adapted to thrive in a wide variety of environments, and in the process, making it a notorious opportunistic pathogen. Apart from the extensively studied chronic infection in the lungs of people with cystic fibrosis (CF), P. aeruginosa also causes multiple serious infections encompassing essentially all organs of the human body, among others, lung infection in patients with chronic obstructive pulmonary disease, primary ciliary dyskinesia and ventilator-associated pneumonia; bacteremia and sepsis; soft tissue infection in burns, open wounds and postsurgery patients; urinary tract infection; diabetic foot ulcers; chronic suppurative otitis media and otitis externa; and keratitis associated with extended contact lens use. Although well characterized in the context of CF, pathogenic processes mediated by various P. aeruginosa virulence factors in other organ systems remain poorly understood. In this review, we use an organ system-based approach to provide a synopsis of disease mechanisms exerted by P. aeruginosa virulence determinants that contribute to its success as a versatile pathogen.

ABSTRACT Interactions between different bacterial species shape bacterial communities and their environments. The opportunistic pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia both can colonize the lungs of individuals affected by cystic fibrosis. Using the social surface behavior called swarming motility as a study model, we noticed intricate interactions between B. cenocepacia K56-2 and P. aeruginosa PA14. While strain K56-2 does not swarm under P. aeruginosa favorable swarming conditions, co-inoculation with a nonmotile PA14 flagellum-less ΔfliC mutant restored spreading for both strains. We show that P. aeruginosa provides the wetting agent rhamnolipids allowing K56-2 to perform swarming motility, while aflagellated PA14 appears to “hitchhike” along with K56-2 cells in the swarming colony. IMPORTANCE Pseudomonas aeruginosa and Burkholderia cenocepacia are important opportunistic pathogens often found together in the airways of persons with cystic fibrosis. Laboratory cocultures of both species often ends with one taking over the other. We used a surface motility assay to study the social interactions between populations of these bacterial species. Under our conditions, B. cenocepacia cannot swarm without supplementation of the wetting agent produced by P. aeruginosa. In a mixed colony of both species, an aflagellated mutant of P. aeruginosa provides the necessary wetting agent to B. cenocepacia, allowing both bacteria to swarm and colonize a surface. We highlight this peculiar interaction where both bacteria set aside their antagonistic tendencies to travel together.

Summary The demand for microbially produced surface‐active compounds for use in industrial processes and products is increasing. As such, there has been a comparable increase in the number of publications relating to the characterization of novel surface‐active compounds: novel producers of already characterized surface‐active compounds and production processes for the generation of these compounds. Leading researchers in the field have identified that many of these studies utilize techniques are not precise and accurate enough, so some published conclusions might not be justified. Such studies lacking robust experimental evidence generated by validated techniques and standard operating procedures are detrimental to the field of microbially produced surface‐active compound research. In this publication, we have critically reviewed a wide range of techniques utilized in the characterization of surface‐active compounds from microbial sources: identification of surface‐active compound producing microorganisms and functional testing of resultant surface‐active compounds. We have also reviewed the experimental evidence required for process development to take these compounds out of the laboratory and into industrial application. We devised this review as a guide to both researchers and the peer‐reviewed process to improve the stringency of future studies and publications within this field of science.

Pseudomonas aeruginosa (PA) and Aspergillus fumigatus (AF) chronically colonize the airways of patients with cystic fibrosis or chronic immunosuppression and mutually affect each other’s pathogenesis. Here, we evaluated IncuCyte time-lapse imaging and NeuroTrackTM (NT) analysis (Wurster et al., 2019, mBio) as a toolbox to study mycelial expansion and morphogenesis of AF during interaction with PA. Co-incubation of AF with supernatant filtrates of wild-type (WT) PA strains strongly inhibited hyphal growth and branching. Consonant with prior metabolic studies, pyoverdine-deficient PA mutants had significantly attenuated inhibitory capacity. Accordingly, purified PA products pyoverdine and pyocyanin suppressed mycelial expansion of AF in a concentration-dependent way. Using fluorescence-guided tracking of GFP-AF293 mycelia during co-culture with live WT PA cells, we found significant inoculum-dependent mycelial growth inhibition and robust precision of the NT algorithm. Collectively, our experiments position IncuCyte NT as an efficient platform for longitudinal analysis of fungal growth and morphogenesis during bacterial co-infection.

Bioreporter systems based on detectable enzyme activity, such as that of beta-galactosidase or luciferase, are key in novel bacterial promoter discovery and study. While these systems permit quantification of gene expression, their use is limited by the toxicity of the expressed reporter enzymes in a given host. Indeed, the most potent promoters may be overlooked if their activity causes a lethal overproduction of the reporter genes when screening for transcriptional activity of potential promoter sequences with the luxCDABE cassette. To overcome this limitation, a variation of the mini-CTX-lux plasmid has been designed which allows reduction of promoter activity via the addition of an adjacent fluoride riboswitch. The riboswitch adds a layer of regulation between the promoter and the reporter gene, allowing cloning of stronger promoters by weakening expression, while giving the potential to induce with fluoride to provide a good signal for weaker promoters, thus circumventing limitations associated with reporter toxicity. We noticed the riboswitch potential portability issues between species, suggesting caution when using riboswitches non-native to the species where it is being used. This study introduces a new molecular biology tool which will allow for the identification of previously unverifiable or uncharacterized potent promoters and also provides a cloning vector for translational fusion with luciferase in a plasmid compatible with many species such as from the genera Burkholderia and Pseudomonas.

Pseudomonas aeruginosa and Aspergillus fumigatus infections frequently co-localize in lungs of immunocompromised patients and individuals with cystic fibrosis (CF). The antifungal activity of P. aeruginosa has been described for its filtrates. Pyoverdine and pyocyanin are the principal antifungal P. aeruginosa molecules active against A. fumigatus biofilm metabolism present in iron-limited or iron-replete planktonic P. aeruginosa culture filtrates, respectively. Using various P. aeruginosa laboratory wild-type strains (PA14, PAO1, PAK), we found antifungal activity against Aspergillus colonies on agar. Comparing 36 PA14 and 7 PAO1 mutants, we found that mutants lacking both major siderophores, pyoverdine and pyochelin, display higher antifungal activity on agar than their wild types, while quorum sensing mutants lost antifungal activity. Addition of ferric iron, but not calcium or magnesium, reduced the antifungal effects of P. aeruginosa on agar, whereas iron-poor agar enhanced antifungal effects. Antifungal activity on agar was mediated by PQS and HHQ, via MvfR. Among the MvfR downstream factors, rhamnolipids and elastase were produced in larger quantities by pyoverdine–pyochelin double mutants and showed antifungal activity on agar. In summary, antifungal factors produced by P. aeruginosa on agar differ from those produced by bacteria grown in liquid cultures, are dependent on quorum sensing, and are downregulated by the availability of ferric iron. Rhamnolipids and elastase seem to be major mediators of Pseudomonas’ antifungal activity on a solid surface.

ABSTRACT The Burkholderia cepacia complex (Bcc) comprises several species of closely related, versatile bacteria. Some Bcc strains produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously estimated that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intraspecies variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 312 Bcc strains (222 of clinical and 90 of environmental origins) belonging to 18 Bcc species. We confirmed that this operon is not distributed evenly among Bcc species. Among the 30% of strains bearing the hmqABCDEFG operon, we found that 92% of environmental isolates and 82% of clinically isolated Bcc strains produce levels of HMAQs detectable by liquid chromatography-mass spectrometry in at least one of the tested culture conditions. Among the hmqABCDEFG-positive but HMAQ-negative strains, none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g., Burkholderia ambifaria and Burkholderia cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g., Burkholderia cenocepacia and Burkholderia multivorans), suggesting a role for the Hmq system in niche adaptation. We investigated the impact of the Hmq system on plant growth promotion and found that Pisum sativum root development by B. ambifaria required a functional HMAQ system. IMPORTANCE Environmental bacteria belonging to the various closely related species forming the Burkholderia cepacia complex (Bcc) can infect plants and animals, including humans. Their pathogenicity is regulated by intercellular communication, or quorum sensing, allowing them to collaborate instead of acting individually. Bcc organisms generally exploit interacting quorum sensing systems based on N-acyl-homoserine lactones as signaling molecules. Several Bcc strains also carry an hmqABCDEFG operon responsible for the biosynthesis of 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), molecules analogous to the Pseudomonas quinolone signal (PQS) system of P. aeruginosa. Our finding that the prevalences of the Hmq system and HMAQ production are very different between various Bcc species suggests a key role in niche adaptation or pathogenicity. This is supported by a significant reduction in plant growth promotion in the absence of HMAQ production for a beneficial Bcc strain.

Abstract Antibiotics are sprayed on apple and pear orchards to control, among other pathogens, the bacterium Erwinia amylovora, the causative agent of fire blight. As with many other pathogens, we observe the emergence of antibiotic‐resistant strains of E. amylovora. Consequently, growers are looking for alternative solutions to combat fire blight. To find alternatives to antibiotics against this pathogen, we have previously isolated three bacterial strains with antagonistic and extracellular activity against E. amylovora, both in vitro and in planta, corresponding to three different bacterial genera: Here, we identified the inhibitory mode of action of each of the three isolates against E. amylovora. Isolate Bacillus amyloliquefaciens subsp. plantarum (now B. velezensis) FL50S produces several secondary metabolites including surfactins, iturins, and fengycins. Specifically, we identified oxydifficidin as the most active against E. amylovora S435. Pseudomonas poae FL10F produces an active extracellular compound against E. amylovora S435 that can be attributed to white‐line‐inducing principle (WLIP), a cyclic lipopeptide belonging to the viscosin subfamily (massetolide E, F, L, or viscosin). Pantoea agglomerans NY60 has a direct cell‐to‐cell antagonistic effect against E. amylovora S435. By screening mutants of this strain generated by random transposon insertion with decreased antagonist activity against strain S435, we identified several defective transposants. Of particular interest was a mutant in a gene coding for a Major Facilitator Superfamily (MFS) transporter corresponding to a transmembrane protein predicted to be involved in the extracytoplasmic localization of griseoluteic acid, an intermediate in the biosynthesis of the broad‐spectrum phenazine antibiotic D‐alanylgriseoluteic acid.

Antibiotics are used extensively to control animal, plant, and human pathogens. They are sprayed on apple and pear orchards to control the bacterium Erwinia amylovora, the causative agent of fire blight. This phytopathogen is developing antibiotic resistance and alternatives either have less efficacy, are phytotoxic, or more management intensive. The objective of our study was to develop an effective biological control agent colonizing the host plant and competing with Erwinia amylovora. It must not be phytotoxic, have a wide spectrum of activity, and be unlikely to induce resistance in the pathogen. To this end, several bacterial isolates from various environmental samples were screened to identify suitable candidates that are antagonistic to E. amylovora. We sampled bacteria from the flowers, leaves, and soil from apple and pear orchards from the springtime bloom period until the summer. The most effective bacteria, including isolates of Pseudomonas poae, Paenibacillus polymyxa, Bacillus amyloliquefaciens and Pantoea agglomerans, were tested in vitro and in vivo and formulated into products containing both the live strains and their metabolites that were stable for at least 9 months. Trees treated with the product based on P. agglomerans NY60 had significantly less fire blight than the untreated control and were statistically not different from streptomycin-treated control trees. With P. agglomerans NY60, fire blight never extended beyond the central vein of the inoculated leaf. The fire blight median disease severity score, 10 days after inoculation, was up to 70% less severe on trees treated with P. agglomerans NY60 as compared to untreated controls.

Rhamnolipids are surface-active agents of microbial origin used as alternatives to synthetic surfactants. Burkholderia thailandensis is a non-pathogenic rhamnolipid-producing bacterium that could represent an interesting candidate for use in commercial processes. However, current bioprocesses for rhamnolipid production by this bacterium are not efficient enough, mainly due to low yields. Since regulation of rhamnolipid biosynthesis in B. thailandensis remains poorly understood, identifying new regulatory factors could help increase the production of these valuable metabolites. We performed a random transposon mutagenesis screening to identify genes directing rhamnolipid production in B. thailandensis E264. The most efficient rhamnolipid producer we identified harbored an inactivating transposon insertion in the scmR gene, which was recently described to encode as a secondary metabolite regulator in B. thailandensis. We investigated the impact of scmR loss on rhamnolipid biosynthesis and cell growth. Because biosynthesis of rhamnolipids and polyhydroxyalkanoates (PHAs) could share the same pool of lipid precursors, we also investigate the effect of ScmR on PHA production. We found that production of both rhamnolipids and PHAs are modulated by ScmR during the logarithmic growth phase and demonstrate that ScmR downregulates the production of rhamnolipids by affecting the expression of both rhl biosynthetic operons. Furthermore, our results indicate that PHA biosynthesis is reduced in the scmR- mutant, as ScmR promotes the transcription of the phaC and phaZ genes. By studying the relationship between ScmR and quorum sensing (QS) regulation we reveal that QS acts as an activator of scmR transcription. Finally, we pinpoint the QS-3 system as being involved in the regulation of rhamnolipid and PHA biosynthesis. We conclude that ScmR negatively affects rhamnolipid production, whereas it positively impacts PHAs biosynthesis. This could provide an interesting approach for future strain engineering, leading to improved yields of these valuable metabolites.

ABSTRACT The bacterium Pseudomonas aeruginosa has emerged as a central threat in health care settings and can cause a large variety of infections. It expresses an arsenal of virulence factors and a diversity of survival functions, many of which are finely and tightly regulated by an intricate circuitry of three quorum sensing (QS) systems. The las system is considered at the top of the QS hierarchy and activates the rhl and pqs systems. It is composed of the LasR transcriptional regulator and the LasI autoinducer synthase, which produces 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), the ligand of LasR. RhlR is the transcriptional regulator for the rhl system and is associated with RhlI, which produces its cognate autoinducer C4-HSL. The third QS system is composed of the pqsABCDE operon and the MvfR (PqsR) regulator. PqsABCD synthetize 4-hydroxy-2-alkylquinolines (HAQs), which include ligands activating MvfR. PqsE is not required for HAQ production and instead is associated with the expression of genes controlled by the rhl system. While RhlR is often considered the main regulator of rhlI, we confirmed that LasR is in fact the principal regulator of C4-HSL production and that RhlR regulates rhlI and production of C4-HSL essentially only in the absence of LasR by using liquid chromatography-mass spectrometry quantifications and gene expression reporters. Investigating the expression of RhlR targets also clarified that activation of RhlR-dependent QS relies on PqsE, especially when LasR is not functional. This work positions RhlR as the key QS regulator and points to PqsE as an essential effector for full activation of this regulation. IMPORTANCE Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa. PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa. This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections.

Pseudomonas aeruginosa is a motile bacterium able to exhibit a social surface behaviour known as swarming motility. Swarming requires the polar flagellum of P. aeruginosa as well as the secretion of wetting agents to ease the spread across the surface. However, our knowledge on swarming is limited to observed phenotypes on agar-solidified media. To study the surface behaviour and the impact of wetting agents of P. aeruginosa on other surfaces, we assessed surface motility capabilities of the prototypical strain PA14 on semi-solid media solidified with alternative gelling agents, gellan gum and carrageenan. We found that, on these alternative surfaces, the characteristic dendritic spreading pattern of P. aeruginosa is drastically altered. One striking feature is the loss of dependence on rhamnolipids to spread effectively on plates solidified with these alternative gelling agents. Indeed, a rhlA-null mutant unable to produce its wetting agents still spreads effectively, albeit in a circular shape on both the gellan gum- and carrageenan-based media. Our data indicate that rhamnolipids do not have such a crucial role in achieving surface colonization of non-agar plates, suggesting a strong dependence on the physical properties of the tested surface. The use of alternative gelling agent provides new means to reveal unknown features of bacterial surface behaviour.

Abstract Antibiotic resistance is spreading at an alarming rate among pathogenic bacteria in both medicine and agriculture. Interfering with the intrinsic resistance mechanisms displayed by pathogenic bacteria has the potential to make antibiotics more effective and decrease the spread of acquired antibiotic resistance. Here, it is demonstrated that cranberry proanthocyanidin (cPAC) prevents the evolution of resistance to tetracycline in Escherichia coli and Pseudomonas aeruginosa, rescues antibiotic efficacy against antibiotic‐exposed cells, and represses biofilm formation. It is shown that cPAC has a potentiating effect, both in vitro and in vivo, on a broad range of antibiotic classes against pathogenic E. coli, Proteus mirabilis, and P. aeruginosa. Evidence that cPAC acts by repressing two antibiotic resistance mechanisms, selective membrane permeability and multidrug efflux pumps, is presented. Failure of cPAC to potentiate antibiotics against efflux pump‐defective mutants demonstrates that efflux interference is essential for potentiation. The use of cPAC to potentiate antibiotics and mitigate the development of resistance could improve treatment outcomes and help combat the growing threat of antibiotic resistance.

A few Burkholderia species, especially Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia ambifaria, and Burkholderia cepacia, are known to produce and release various 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), a family of molecules analogous to the 4-hydroxy-2-alkylquinolines [aka 2-n-alkyl-4(1H)-quinolones] of Pseudomonas aeruginosa, which include the Pseudomonas quinolone signal (PQS). However, while these exoproducts play several roles in P. aeruginosa virulence and survival, the available literature is very limited on their distribution and function in Burkholderia. In this perspective article, we studied the distribution of the hmqABCDEFG operon, which encodes the enzymes involved in the biosynthesis of HMAQs, in the Burkholderia cepacia complex (Bcc) group. Based on the available sequence data, about one third of Bcc species carry a homolog of the hmqABCDEFG, and not all sequenced strains in a given species possess this operon. Looking at the synteny of genes surrounding the hmqABCDEFG operon, we found that for some species, the operon seems to have been deleted or replaced by other genes. Finally, we review the literature on the possible function of HMAQs. Understanding the Hmq system may provide clues concerning their functions in Bcc.

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens frequently co-inhabiting immunocompromised patient airways, particularly in people with cystic fibrosis. Both microbes depend on the availability of iron, and compete for iron in their microenvironment. We showed previously that the P. aeruginosa siderophore pyoverdine is the main instrument in battling A. fumigatus biofilms, by iron chelation and denial of iron to the fungus. Here we show that A. fumigatus siderophores defend against anti-fungal P. aeruginosa effects. P. aeruginosa supernatants produced in the presence of wildtype A. fumigatus planktonic supernatants (Afsup) showed less activity against A. fumigatus biofilms than P. aeruginosa supernatants without Afsup, despite higher production of pyoverdine by P. aeruginosa. Supernatants of A. fumigatus cultures lacking the sidA gene (AfΔsidA), unable to produce hydroxamate siderophores, were less capable of protecting A. fumigatus biofilms from P. aeruginosa supernatants and pyoverdine. AfΔsidA biofilm was more sensitive towards inhibitory effects of pyoverdine, the iron chelator deferiprone (DFP), or amphothericin B than wildtype A. fumigatus biofilm. Supplementation of sidA-deficient A. fumigatus biofilm with A. fumigatus siderophores restored resistance to pyoverdine. The A. fumigatus siderophore production inhibitor celastrol sensitized wildtype A. fumigatus biofilms towards the anti-fungal activity of DFP. In conclusion, A. fumigatus hydroxamate siderophores play a pivotal role in A. fumigatus competition for iron against P. aeruginosa.

The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule for Pseudomonas aeruginosa that regulates virulence factors, chelates iron, and is an important factor in interactions with eukaryotes, including fungi and mammalian hosts. It was previously shown to inhibit or boost Aspergillus, depending on the milieu iron concentration. We studied several molecular modifications of the PQS molecule, and their effects on Aspergillus biofilm metabolism and growth in vitro, and the effects of iron supplementation. We found that most molecules inhibited Aspergillus at concentrations similar to that of PQS, but with relatively flat dose-responses, and all were less potent than PQS. The inhibition was reversible by iron, suggesting interference with fungal iron metabolism. Stimulation of Aspergillus was not noted. We conclude that the critical Aspergillus-inhibiting moeities of the PQS molecule were partially, but not completely, interfered with by molecular modifications at several sites on the PQS molecule. The mechanism, as with PQS, appears to relate to fungal iron metabolism.

Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is widely occurring in the environment and is recognized for its capacity to form or join biofilms. The present review consolidates current knowledge on P. aeruginosa ecology and its implication in healthcare facilities premise plumbing. The adaptability of P. aeruginosa and its capacity to integrate the biofilm from the faucet and the drain highlight the role premise plumbing devices can play in promoting growth and persistence. A meta‐analysis of P. aeruginosa prevalence in faucets (manual and electronic) and drains reveals the large variation in device positivity reported and suggest the high variability in the sampling approach and context as the main reason for this variation. The effects of the operating conditions that prevail within water distribution systems (disinfection, temperature, and hydraulic regime) on the persistence of P. aeruginosa are summarized. As a result from the review, recommendations for proactive control measures of water contamination by P. aeruginosa are presented. A better understanding of the ecology of P. aeruginosa and key influencing factors in premise plumbing are essential to identify culprit areas and implement effective control measures.

Background: Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) compete with each other for nutrients and survival in natural environments, and have been extensively studied because of their intermicrobial interactions in the human microbiome. These are the principal microbes infecting immunocompromised patients and persons with cystic fibrosis, particularly the airways. These intermicrobial studies have largely been conducted in liquid medium or on agar, and thus focus on soluble or diffusible microbial products. Several key inhibitory molecules were defined in such studies. Methods: in the present report, we examine several methodologies which can be conveniently used to study the interaction of microbial volatiles, including capture methods and kinetics. Results: Pa volatiles inhibit Af, and the inhibitory mechanism appears to be the incorporation of the inhibitory molecules into the substrate nourishing the Af, rather than directly onto Af structures. We define by mass spectroscopy some specific volatile Pa products that can inhibit Af. Some of these molecules are selected for interest by the study of gene deletion mutants, producing a few Pa strains that were impaired in inhibition. We presumed the volatiles of these latter strains could be excluded from the search for inhibitors. Conclusion: the Pa inhibition of Af via a gaseous phase could be critical components in their competition, particularly in airways, where more direct contact may not be extensive.

Some pathogens might develop favorable global adaptation in response to certain stress treatments resulting in enhanced virulence and/or resistance to a different stress. β-lactam resistance, as well as ampC and ampG genes involved in this resistance, were studied to evaluate their possible role in Escherichia coli O157:H7 (E. coli) radioresistance. E. coli adapted to 25, 15 or 7 μg/mL of kanamycin or carbenicillin, were produced and treated with sensitization (0.4 kGy) or lethal (1.5 kGy) irradiation doses. In E. coli O157:H7, irradiation treatment at 0.4 kGy dose increased ampC and ampG expression respectively by 1.6 and 2-fold in the wild type strain (Wt) but up to by 2.4 and 3.4-fold when the strain was beforehand adapted to 25 μg/mL of carbenicillin (Carb25). Accordingly, ΔampC and ΔampG mutants and E. coli adapted to 25 μg/mL of kanamycin were more sensitive to 0.4 kGy treatment than Wt. While, E. coli Carb25 or overexpression of ampC and ampG provided complete resistance to 0.4 kGy and were even able to survive and grow after exposure to a normally lethal 1.5 kGy irradiation dose. We further noticed that these strains can tolerate other stresses like oxidative, cold and heat shocks. This demonstrates that carbenicillin adaptation promotes resistance to γ-irradiation and to other stresses, likely at least through increased AmpC and AmpG expression. These results are important for the food industry and particularly when considering the use of irradiation for food preservation of meat obtained directly from animals fed β-lactam antibiotics.

Microbial drinking water quality can be altered in large buildings, especially after stagnation. In this study, bacterial profiles were generated according to the stagnation time and the volume of water collected at the tap. Successive volumes of cold and hot water were sampled after controlled stagnation periods. Bacterial profiles revealed an important decline (> 2 log) in culturable cells in the first 500 mL sampled from the hot and cold water systems, with a steep decline in the first 15 mL. The strong exponential correlation (R2 ≥ 0.97) between the culturable cell counts in water and the pipe surface-to-volume ratio suggests the biofilm as the main contributor to the rapid increase in suspended culturable cells measured after a short stagnation of one-hour. Results evidence the contribution of the high surface-to-volume ratio at the point of use and the impact of short stagnation times on the increased bacterial load observed. Simple faucets with minimal internal surface area should be preferred to minimize surface area. Sampling protocol, including sampling volume and prior stagnation, was also shown to impact the resulting culturable cell concentration by more than 1000-fold. Sampling a smaller volume on first draw after stagnation will help maximize recovery of bacteria.

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.

ABSTRACT The genome of the bacterium Burkholderia thailandensis encodes three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-l-homoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), the primary AHLs produced by this bacterium, was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in QS deletion mutants. This was compared to the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, the levels of expression of btaR1, btaR2, and btaR3 were monitored by quantitative reverse transcription-PCR (qRT-PCR). We observed that C8-HSL, 3OHC10-HSL, and 3OHC8-HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 gene expression profiles, revealing a successive activation of the corresponding QS systems. Moreover, the transcription of the btaR1, btaR2, and btaR3 genes is modulated by cognate and noncognate AHLs, showing that their regulation depends on themselves and on other QS systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a successive regulatory network. IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associated with the establishment of host-pathogen interactions and acclimatization to the environment.

Species from the Burkholderia cepacia complex (Bcc) share a canonical LuxI/LuxR quorum sensing (QS) regulation system named CepI/CepR, which mainly relies on the acyl-homoserine lactone (AHL), octanoyl-homoserine lactone (C8-HSL) as signaling molecule. Burkholderia ambifaria is one of the least virulent Bcc species, more often isolated from rhizospheres where it exerts a plant growth-promoting activity. However, clinical strains of B. ambifaria display distinct features, such as phase variation and higher virulence properties. Notably, we previously reported that under laboratory conditions, only clinical strains of the B. ambifaria species produced 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs) via expression of the hmqABCDEFG operon. HMAQs are the methylated counterparts of the 4-hydroxy-2-alkylquinolines (HAQs) produced by the opportunistic human pathogen Pseudomonas aeruginosa, in which they globally contribute to the bacterial virulence and survival. We have found that unlike P. aeruginosa’s HAQs, HMAQs do not induce their own production. However, they indirectly regulate the expression of the hmqABCDEFG operon. In B. ambifaria, a strong link between CepI/CepR-based QS and HMAQs is proposed, as we have previously reported an increased production of C8-HSL in HMAQ-negative mutants. Here, we report the identification of all AHLs produced by the clinical B. ambifaria strain HSJ1, namely C6-HSL, C8-HSL, C10-HSL, 3OHC8-HSL, 3OHC10-HSL, and 3OHC12-HSL. Production of significant levels of hydroxylated AHLs prompted the identification of a second complete LuxI/LuxR-type QS system relying on 3OHC10-HSL and 3OHC12-HSL, that we have named CepI2/CepR2. The connection between these two QS systems and the hmqABCDEFG operon, responsible for HMAQs biosynthesis, was investigated. The CepI/CepR system strongly induced the operon, while the second system appears moderately involved. On the other hand, a HMAQ-negative mutant overproduces AHLs from both QS systems. Even if HMAQs are not classical QS signals, their effect on AHL-based QS system still gives them a part to play in the QS circuitry in B. ambifaria and thus, on regulation of various phenotypes.

Cationic antimicrobial peptides (CAMPs) occur naturally in numerous organisms and are considered as a class of antibiotics with promising potential against multi-resistant bacteria. Herein, we report a strategy that can lead to the discovery of novel small CAMPs with greatly enhanced antimicrobial activity and retained antibiofilm potential. We geared our efforts towards i) the N-terminal cysteine functionalization of a previously reported small synthetic cationic peptide (peptide 1037, KRFRIRVRV-NH2), ii) its dimerization through a disulfide bond, and iii) a preliminary antimicrobial activity assessment of the newly prepared dimer against Pseudomonas aeruginosa and Burkholderia cenocepacia, pathogens responsible for the formation of biofilms in lungs of individuals with cystic fibrosis. This dimer is of high interest as it does not only show greatly enhanced bacterial growth inhibition properties compared to its pep1037 precursor (up to 60 times), but importantly, also displays antibiofilm potential at sub-MICs. Our results suggest that the reported dimer holds promise for its use in future adjunctive therapy, in combination with clinically-relevant antibiotics.

ABSTRACT Chronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR. IMPORTANCE Chronic Pseudomonas aeruginosa infections, such as those in patients with the genetic disease cystic fibrosis, are notable in that mutants with defects in the quorum-sensing transcription factor LasR frequently arise. In laboratory strains of P. aeruginosa, quorum sensing activates transcription of dozens of genes, many of which encode virulence factors, such as secreted proteases and hydrogen cyanide synthases. In well-studied laboratory strains, LasR-null mutants have a quorum-sensing-deficient phenotype. Therefore, the presence of LasR variants in chronic infections has been interpreted to indicate that quorum-sensing-regulated products are not important for those infections. We report that some P. aeruginosa LasR variant clinical isolates are not LasR-null mutants, and others have uncoupled a second quorum-sensing system, the RhlR system, from LasR regulation. In these uncoupled isolates, RhlR independently activates at least some quorum-sensing-dependent genes. Our findings suggest that quorum sensing plays a role in chronic P. aeruginosa infections, despite the emergence of LasR coding variants.

Quorum sensing (QS) is a cell density-dependent mechanism which enables a population of bacteria to coordinate cooperative behaviors in response to the accumulation of self-produced autoinducer signals in their local environment. An emerging framework is that the adaptive significance of QS in the regulation of production of costly extracellular metabolites («public goods») is to maintain the homeostasis of cooperation. We investigated this model using the phytopathogenic bacterium Burkholderia glumae, which we have previously demonstrated uses QS to regulate the production of rhamnolipids, extracellular surface-active glycolipids promoting the social behavior called «swarming motility». Using mass spectrometric quantification and chromosomal lux-based gene expression, we made the unexpected finding that when unrestricted nutrient resources are provided, production of rhamnolipids is carried out completely independently of QS regulation. This is a unique observation among known QS-controlled factors in bacteria. On the other hand, under nutrient-limited conditions, QS then becomes the main regulating mechanism, significantly enhancing the specific rhamnolipids yield. Accordingly, decreasing nutrient concentrations amplifies rhamnolipid biosynthesis gene expression, revealing a system where QS-dependent regulation is specifically triggered by the growth rate of the population, rather than by its cell density. Furthermore, a gradual increase in QS signal specific concentration upon decrease of specific growth rate suggests a reduction in quorum threshold, which reflects an increase in cellular demand for production of QS-dependent target gene product at low density populations. Integration of growth rate with QS as a decision-making mechanism for biosynthesis of costly metabolites, such as rhamnolipids, could serve to assess the demand and timing for expanding the carrying capacity of a population through spatial expansion mechanisms, such as swarming motility, thus promoting the chances of survival, even if the cell density might not be high enough for an otherwise efficient production of rhamnolipids. In conclusion, we propose that the adaptive significance of growth rate-dependent functionality of QS in biosynthesis of costly public goods lies within providing a regulatory mechanism for selecting the optimal trade-off between survival and efficiency.

ABSTRACT Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections.

Burkholderia glumae is a biosafety level 1 bacterium capable of producing rhamnolipid biosurfactant with longer 3-hydroxy fatty acid chains moieties than those produced by the prototypal producer, the opportunistic pathogen Pseudomonas aeruginosa. Although the capacity of production of rhamnolipid, and the parameters affecting this production, are well established for P. aeruginosa, little is known about the factors that may affect their production in B. glumae. Hence, to evaluate and enhance the production of rhamnolipids in B. glumae, following the selection of best carbon and nitrogen sources, a two-level fractional factorial design experiment was performed to identify the limiting factors significantly affecting the production of rhamnolipids in this bacterial species. Effects of six inorganic nutrients and two physical parameters were studied, and mannitol, urea, CaCl2, and potassium phosphate buffer were selected for further optimization by applying a response surface methodology (RSM). Under the identified optimized conditions, a rhamnolipid production of 1.66 g/L was obtained, about five times higher than that of the initial non-optimized conditions. This represents a key step in the development of large-scale production processes.

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.

Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.

Identifying environmental sources of Pseudomonas aeruginosa (Pa) related to hospital-acquired infections represents a key challenge for public health. Biofilms in water systems offer protection and favorable growth conditions, and are prime reservoirs of microorganisms. A comparative genotyping survey assessing the relationship between Pa strains recovered in hospital sink biofilm and isolated in clinical specimens was conducted. Environmental strains from drain, faucet and sink-surface biofilm were recovered by a culture method after an incubation time ranging from 48 to 240 h. The genotyping of 38 environmental and 32 clinical isolates was performed using a multiple-locus variable-number of tandem repeats analysis (MLVA). More than one-third of Pa isolates were only cultivable following ≥48 h of incubation, and were predominantly from faucet and sink-surface biofilms. In total, 41/70 strains were grouped within eight genotypes (A to H). Genotype B grouped a clinical and an environmental strain isolated in the same ward, 5 months apart, suggesting this genotype could thrive in both contexts. Genotype E grouped environmental isolates that were highly prevalent throughout the hospital and that required a longer incubation time. The results from the multi-hospital follow-up study support the drain as an important reservoir of Pa dissemination to faucets, sink surfaces and patients. Optimizing the recovery of environmental strains will strengthen epidemiological investigations, facilitate pathway identification, and assist in identifying and controlling the reservoirs potentially associated to hospital-acquired infections.

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens in airway infections of cystic fibrosis (CF) patients. We studied how these pathogens coexist and interact with each other. Clinical isolates of both species were retrieved from adult CF patients. Culture supernatants from 63 P. aeruginosa isolates triggered a wide range of biofilm-stimulatory activities when added to the culture of a control S. aureus strain. The extent of biofilm formation by S. aureus was positively correlated to the levels of the 2-alkyl-4-(1H)-quinolones (AQs) Pseudomonas Quinolone Signal (PQS) and 2-heptyl-4-hydroxy quinoline N-oxide (HQNO) produced by the P. aeruginosa isolates. Supernatants from P. aeruginosa isogenic mutants deficient in PQS and HQNO production stimulated significantly less biofilm formation by S. aureus than that seen with the parental strain PA14. When studying co-isolated pairs of P. aeruginosa and S. aureus retrieved from patients showing both pathogens, P. aeruginosa supernatants stimulated less biofilm production by the S. aureus counterparts compared to that observed using the control S. aureus strain. Accordingly, some P. aeruginosa isolates produced low levels of exoproducts and also some of the clinical S. aureus isolates were not stimulated by their co-isolates or by PA14 despite adequate production of HQNO. This suggests that colonization of the CF lungs promotes some type of strain selection, or that co-existence requires specific adaptations by either or both pathogens. Results provide insights on bacterial interactions in CF.

SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.

Pseudomonas aeruginosa is an important opportunistic human pathogen that can establish bacterial communication by synchronizing the behavior of individual cells in a molecular phenomenon known as "quorum sensing". Through an elusive mechanism involving gene products of the pqs operon, the PqsE enzyme is absolutely required for the synthesis of extracellular phenazines, including the toxic blue pigment pyocyanin, effectively allowing cells to achieve full-fledged virulence. Despite several functional and structural attempts at deciphering the role of this relevant enzymatic drug target, no molecular function has yet been ascribed to PqsE. In the present study, we report a series of alanine scanning experiments aimed at altering the biological function of PqsE, allowing us to uncover key amino acid positions involved in the molecular function of this enzyme. We use sequence analysis and structural overlays with members of homologous folds to pinpoint critical positions located in the vicinity of the ligand binding cleft and surrounding environment, revealing the importance of a unique C-terminal α-helical motif in the molecular function of PqsE. Our results suggest that the active site of the enzyme involves residues that extend further into the hydrophobic core of the protein, advocating for a lid-like movement of the two terminal helices. This information should help design virtual libraries of PqsE inhibitors, providing means to counter P. aeruginosa virulence acquisition and helping to reduce nosocomial infections.

Bacterial cells have evolved the capacity to communicate between each other via small diffusible chemical signals termed autoinducers. Pseudomonas aeruginosa is an opportunistic pathogen involved, among others, in cystic fibrosis complications. Virulence of P. aeruginosa relies on its ability to produce a number of autoinducers, including 4-hydroxy-2-alkylquinolines (HAQ). In a cell density-dependent manner, accumulated signals induce the expression of multiple targets, especially virulence factors. This phenomenon, called quorum sensing, promotes bacterial capacity to cause disease. Furthermore, P. aeruginosa possesses many multidrug efflux pumps conferring adaptive resistance to antibiotics. Activity of some of these efflux pumps also influences quorum sensing. The present study demonstrates that the MexEF-OprN efflux pump modulates quorum sensing through secretion of a signalling molecule belonging to the HAQ family. Moreover, activation of MexEF-OprN reduces virulence factor expression and swarming motility. Since MexEF-OprN can be activated in infected hosts even in the absence of antibiotic selective pressure, it could promote establishment of chronic infections in the lungs of people suffering from cystic fibrosis, thus diminishing the immune response to virulence factors. Therapeutic drugs that affect multidrug efflux pumps and HAQ-mediated quorum sensing would be valuable tools to shut down bacterial virulence.

Colonization with bacterial species from the Burkholderia cepacia complex (Bcc) is associated with fast health decline among individuals with cystic fibrosis. In order to investigate the virulence of the Bcc, several alternative infection models have been developed. To this end, the fruit fly is increasingly used as surrogate host, and its validity to enhance our understanding of host-pathogen relationships has been demonstrated with a variety of microorganisms. Moreover, its relevance as a suitable alternative to mammalian hosts has been confirmed with vertebrate organisms.The aim of this study was to establish Drosophila melanogaster as a surrogate host for species from the Bcc. While the feeding method proved unsuccessful at killing the flies, the pricking technique did generate mortality within the populations. Results obtained with the fruit fly model are comparable with results obtained using mammalian infection models. Furthermore, validity of the Drosophila infection model was confirmed with B. cenocepacia K56-2 mutants known to be less virulent in murine hosts or in other alternative models. Competitive index (CI) analyses were also performed using the fruit fly as host. Results of CI experiments agree with those obtained with mammalian models.We conclude that Drosophila is a useful alternative infection model for Bcc and that fly pricking assays and competition indices are two complementary methods for virulence testing. Moreover, CI results indicate that this method is more sensitive than mortality tests.

Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.