RESULTS:

Antibiotic resistance has become a serious threat to human health (WHO Antibacterial Agents in Clinical Development: an Analysis of the Antibacterial Clinical Development Pipeline Including Tuberculosis. Geneva: World Health Organization; 2017) and the ability to predict antibiotic resistance from genome sequencing has become a focal point for the medical community. With this genocentric prediction in mind we were intrigued about two particular findings for a collection of clinical Pseudomonas aeruginosa isolates (Marvig et al. Nature Genetics 2015;47:57–64; Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629): (i) 15 out of 52 genes found to be frequently targeted by adaptive mutations during the initial infection stage of cystic fibrosis airways (‘candidate pathoadaptive genes’) (Marvig et al. Nature Genetics 2015;47:57–64) were associated with antibiotic resistance (López-Causapé et al. Fronters in Microbiology 2018;9:685; López-Causapé et al. Antimicrobal Agents and Chemotherapy 2018;62:e02583-17); (ii) there was a parallel lack of resistance development and linkage to the genetic changes in these antibiotic-resistance-associated genes (Frimodt-Møller et al. Scientific Reports 2018;8:12512; Bartell et al. Nature Communications 2019;10:629). In this review we highlight alternative selective forces that potentially enhance the infection success of P. aeruginosa and focus on the linkage to the 15 pathoadaptive antibiotic-resistance-associated genes thereby showing the problems we may face when using only genomic information to predict and inform about relevant antibiotic treatment.

The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics and reports of peptide resistance are rare in the clinical setting. Paradoxically many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF–PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc which produces levan and alginate and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme levansucrase occurred mainly during early exponential growth of both planktonic and sessile cells. Thus accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell–cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2 and are readily phagocytosed by PMNs in contrast to bacteria with functional QS systems. In contrast to the wild-type QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

N-Acyl-l-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition it reduces the production of important virulence factors indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does however affect the architecture of the biofilm and enhances the process of bacterial detachment leading to a loss of bacterial biomass from the substratum.

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method two defined mutants H111-I and H111-R were constructed in which cepI and cepR respectively had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment but rather controls the maturation of the biofilm. Furthermore it is shown that B. cepacia is capable of swarming motility a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.

The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.

The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors many of which are controlled by two N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P. aeruginosa in vivo. Mice were challenged intratracheally with alginate beads containing P. aeruginosa and E. coli and killed on different days after the challenge. By means of confocal scanning laser microscopy GFP-expressing E. coli bacteria could be detected in the lung tissues indicating production and excretion of AHL molecules in vivo by the infecting P. aeruginosa. AHL signals were detected mainly in lung tissues exhibiting severe pathological changes. These findings support the view that expression of AHL molecules by P. aeruginosa during infection coincides with its pathogenesis.

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratoy failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs) which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung P. aeruginosa PAO1 a typical non-mucoid strain was grown in a biofilm. This was treated with low levels of H2O2 as if released by the PMNs and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA which encodes an anti-σ factor; this leads to the deregulation of an alternative σ factor (σ22 AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation the mucA22 allele a common allele seen in CF isolates. The mucoid mucA22 variants when compared to the smooth parent strain PA01 produced 2--6-fold higher levels of alginate|ii) exhibited no detectable differences in growth rate|iii) showed an unaltered LPS profile|iv) were ~72% reduced in the amount of inducible-β-lactamase and (v) secreted little no LasA protease and only showed 44% elastase activity. A characteristic ~54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus the common phenotype of the mucoid variants which included a genetically engineered mucA22 mutant suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly expose to activated PMNs in vitro mucoid variants were also observed mimicking in vivo observations. Thus PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals similar to that found in plants and mammalian cells may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients