Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone.

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up ß-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

Sodium Dodecyl Sulfate (SDS)-Loaded Nanoporous Polymer as Anti-Biofilm Surface Coating Material.

Biofilms cause extensive damage to industrial settings. Thus, it is important to improve the existing techniques and develop new strategies to prevent bacterial biofilm formation. In the present study, we have prepared nanoporous polymer films from a self-assembled 1,2-polybutadiene-b-polydimethylsiloxane (1,2-PB-b-PDMS) block copolymer via chemical cross-linking of the 1,2-PB block followed by quantitative removal of the PDMS block. Sodium dodecyl sulfate (SDS) was loaded into the nanoporous 1,2-PB from aqueous solution. The SDS-loaded nanoporous polymer films were shown to block bacterial attachment in short-term (3 h) and significantly reduce biofilm formation in long-term (1 week) by gram-negative bacterium Escherichia coli. Tuning the thickness or surface morphology of the nanoporous polymer films allowed to extent the anti-biofilm capability.

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis-dependent release of eDNA.

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.

Dynamics of mutator and antibiotic-resistant populations in a pharmacokinetic/pharmacodynamic model of Pseudomonas aeruginosa biofilm treatment.

Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 µg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms.

Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis.

Multiple bacterial species often coexist as communities, and compete for environmental resources. Here, we describe how an opportunistic pathogen, Pseudomonas aeruginosa, uses extracellular products to interact with the nosocomial pathogen Staphylococcus epidermidis. S. epidermidis biofilms and planktonic cultures were challenged with P. aeruginosa supernatant cultures overnight. Results indicated that quorum-sensing-controlled factors from P. aeruginosa supernatant inhibited S. epidermidis growth in planktonic cultures. We also found that P. aeruginosa extracellular products, mainly polysaccharides, disrupted established S. epidermidis biofilms. Cellulase-treated P. aeruginosa supernatant, and supernatant from pelA, pslF and pelApslBCD mutants, which are deficient in polysaccharide biosynthesis, diminished the disruption of S. epidermidis biofilms. In contrast, S. epidermidis supernatant in overnight cultures had no effect on established P. aeruginosa biofilms and planktonic growth. These findings reveal that P. aeruginosa extracellular products are important microbial competition factors that overcome competition with S. epidermidis, and the results may provide clues for the development of a novel strategy for controlling S. epidermidis biofilms.

Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis.

Staphylococcus epidermidis has become a serious nosocomial pathogen frequently causing infections associated with implanted foreign materials. Biofilm formation is considered a major factor determining S. epidermidis pathogenicity in such device-associated infections. Here, evidence is presented that extracellular DNA is important for the initial phase of biofilm development by S. epidermidis on polystyrene or glass surfaces under static or hydrodynamic conditions. Comparative PCR amplification from S. epidermidis chromosomal and extracellular DNA indicated that the extracellular DNA is similar to chromosomal DNA. Experiments involving the S. epidermidis wild-type and an isogenic atlE mutant indicated that most of the extracellular DNA in S. epidermidis cultures and biofilms is generated through activity of the autolysin AtlE. The presented results suggest that extracellular DNA is generated in S. epidermidis populations through AtlE-mediated lysis of a subpopulation of the bacteria, and that the extracellular DNA promotes biofilm formation of the remaining population.

Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms.

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.

Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates.

Coagulase-negative Staphylococcus epidermidis has become the leading cause of foreign-body infections due to its biofilm formation on all kinds of medical-device surfaces. The biofilm development of S. epidermidis includes two steps: the initial attachment phase and the accumulative phase. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, is the major component mediating intercellular adhesion. However, recent studies have revealed the emergence of biofilm-positive/ica-negative staphylococcal clinical isolates. In this report, two ica-negative S. epidermidis clinical strains, SE1 and SE4, exhibited their heterogeneity in biofilm architecture under static and flow conditions, compared with the biofilm-positive/ica-positive RP62A strain. Strains with this type of absence of PIA from biofilms also displayed intermediate resistance to vancomycin. More importantly, the cells of both SE1 and SE4 strains were more tolerant than those of RP62A to exposure to lysostaphin and vancomycin. Based on the results, it is suggested that the biofilm-positive/ica-negative strain represents a newly emergent subpopulation of S. epidermidis clinical strains, arising from selection by antibiotics in the nosocomial milieu, which displays a survival advantage in its host environment. Recent epidemiological data support this suggestion, by showing a tendency towards an increasing proportion of this subpopulation in staphylococci-associated infections.

Structure-based discovery of inhibitors of the YycG histidine kinase: new chemical leads to combat Staphylococcus epidermidis infections.

BACKGROUND: Coagulase-negative Staphylococcus epidermidis has become a major frequent cause of infections in relation to the use of implanted medical devices. The pathogenicity of S. epidermidis has been attributed to its capacity to form biofilms on surfaces of medical devices, which greatly increases its resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm. RESULTS: In this report, a series of novel inhibitors of the histidine kinase (HK) YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds displayed significant activity in inhibiting S. epidermidis growth. Furthermore, five of them displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds were found to bind to the YycG protein and to inhibit its auto-phosphorylation in vitro, indicating that they are potential inhibitors of the YycG/YycF two-component system (TCS), which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study. CONCLUSION: These novel inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious than traditional screening technology.

The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid.

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.