The ability to form biofilms is a critical factor in chronic infections by Pseudomonas aeruginosa and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 g/ml, far below that required to kill or inhibit growth (MIC ‫؍‬ 64 g/ml). LL-37 also affected existing, pregrown P. aeruginosa biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.

In addition to exhibiting swimming and twitching motility, Pseudomonas aeruginosa is able to swarm on semisolid (viscous) surfaces. Recent studies have indicated that swarming is a more complex type of motility influenced by a large number of different genes. To investigate the adaptation process involved in swarming motility, gene expression profiles were analyzed by performing microarrays on bacteria from the leading edge of a swarm zone compared to bacteria growing in identical medium under swimming conditions. Major shifts in gene expression patterns were observed under swarming conditions, including, among others, the overexpression of a large number of virulence-related genes such as those encoding the type III secretion system and its effectors, those encoding extracellular proteases, and those associated with iron transport. In addition, swarming cells exhibited adaptive antibiotic resistance against polymyxin B, gentamicin, and ciprofloxacin compared to what was seen for their planktonic (swimming) counterparts. By analyzing a large subset of up-regulated genes, we were able to show that two virulence genes, lasB and pvdQ, were required for swarming motility. These results clearly favored the conclusion that swarming of P. aeruginosa is a complex adaptation process in response to a viscous environment resulting in a substantial change in virulence gene expression and antibiotic resistance.Swarming motility is a multicellular phenomenon involving the coordinated and rapid movement of a bacterial population across a semisolid surface (14). It is widespread among flagellated bacteria, including Salmonella, Vibrio, Yersinia, Serratia, and Proteus (9,18). Swarming is highly dependent on bacterial cell density, nutrient growth medium, and surface condition moistness (53). In addition to physical changes such as an increase in the number of flagella or cell elongation, swarmer cell differentiation results in substantial alterations in metabolic bias and gene expression, indicating that swarming represents a complex lifestyle adaptation in response to particular medium conditions rather than merely a form of locomotion (18,45).Swarming of Pseudomonas aeruginosa is often typified by a dendritic colonial appearance. This gram-negative bacterium is a major cause of hospital-acquired bacterial infections and the most significant pulmonary pathogen in cystic fibrosis patients (17,20,44). It is one of the most difficult infections to treat due to its high natural (intrinsic) antibiotic resistance. It possesses three types of movement depending on medium viscosity, namely, swimming in aqueous environments, twitching on solid surfaces or interfaces, and swarming on semisolid, viscous media, such as those containing 0.4 to 0.7% (wt/vol) agar. It has been previously shown that swarming of P. aeruginosa is dependent on both flagella and type IV pili, which mediate actual movement, as well as on rhamnolipids, which are proposed to enable swarming cells to overcome the strong surface tension of the water surrounding swarmi...

During a screening of a mini-Tn5-luxCDABE transposon mutant library of Pseudomonas aeruginosa PAO1 for alterations in swarming motility, 36 mutants were identified with Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes in P. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.Pseudomonas aeruginosa is an opportunistic human pathogen that can cause nosocomial pneumonia, catheter and urinary tract infections, and sepsis in burn wound and immunocompromised patients (14,18,23,28,34). Moreover, P. aeruginosa is the most prevalent and significant pulmonary pathogen and the most common cause of eventually fatal lung disease in patients with cystic fibrosis (16,29). In addition to its intrinsic resistance to a broad spectrum of antimicrobial compounds, this organism is also noted for its metabolic diversity and extremely versatile lifestyle, which allows it to colonize a large number of different environments (33).Motility has been strongly implicated in the virulence of P. aeruginosa. It plays an important role in mobilization to and colonization of different environments, attachment of the bacteria to surfaces, and biofilm formation (27). P. aeruginosa is unusual in that it is capable of three different types of motility: flagellum-mediated swimming in aqueous environments and at low agar concentrations (Ͻ0.3% [wt/vol]); type IV pilus-mediated twitching on solid surfaces or interfaces; and, most recently observed, swarming on semisolid (viscous) media (0.5 to 0.7% [wt/vol] agar) (7,20,30). Swarming is described as a social phenomenon involving the coordinated and rapid movement of bacteria across a semisolid surface, often typified by a dendritic-like colonial appearance. It is widespread among flagellated bacteria, including Salmonella, Vibrio, Yersinia, Serratia, and Proteus (4,12,31).Recently, it was shown that swarming of P. aeruginosa is dependent on both flagella and type IV pili as well as the presence of rhamnolipids (2,7,20). It is induced under nitrogen limitation and in response to certain amino acids (e.g., glutamate, aspartate, histidine, or proline) when provided as the sole source of nitrogen (20). Like other swarming bacteria that differentiate from short, motile, vegetative swimmer cells into longer, hyperflagellated swarmer cells (10, 13), P. aeruginosa swarmer cells are elongated and can possess two polar flagella (20,30). In addition to these physical changes, swarmer differentiation can also be coupled to increased expres...

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarmingassociated genes functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25 regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient phenotype of this mutant.

Pseudomonas aeruginosa offers substantial therapeutic challenges due to its high intrinsic resistance to many antibiotics and its propensity to develop mutational and/or adaptive resistance. The PA14 comprehensive mutant library was screened for mutants exhibiting either two-to eightfold increased susceptibilities (revealing genes involved in intrinsic resistance) or decreased susceptibilities (mutational resistance) to the fluoroquinolone ciprofloxacin. Thirty-five and 79 mutants with increased and decreased susceptibilities, respectively, were identified, as confirmed by broth dilution.

No abstract

The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine-or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH 2 ) and Sub6 (RWWKIWVIR-WWR-NH 2 ), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.

The Core Content for Clinical Informatics defines the boundaries of the discipline and informs the Program Requirements for Fellowship Education in Clinical Informatics. The Core Content includes four major categories: fundamentals, clinical decision making and care process improvement, health information systems, and leadership and management of change. The AMIA Board of Directors approved the Core Content for Clinical Informatics in November 2008.