The dynamics of biofilm development and dispersal should be taken into account when quantifying biofilm via the crystal violet microtiter plate assay.

The crystal violet microtiter plate biofilm assay is often used to compare the amount of biofilm formed by a mutant versus wild-type or a compound-treated biofilm versus the non-treatment control. In many of these studies the amount of biofilm is assessed only at one single time point. However, if the dynamics of biofilm development of the mutant (or compound-treated biofilm) is different than that of the wild-type (or non-treatment control), then biofilm quantification at a single time point may give misleading results.

Upscaling and Risk Evaluation of the Synthesis of the 3,5-Diamino-1H-Pyrazole, Disperazol.

This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound , as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.

Inhibition of quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen , a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in .

Challenges in using transcriptome data to study the c-di-GMP signaling network in clinical isolates.

In the type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding DGCs.

The role of individual exopolysaccharides in antibiotic tolerance of aggregates.

The bacterium is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria.

A genetic switch controls Pseudomonas aeruginosa surface colonization.

Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR-hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces.

Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms.

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa.

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases.

Redox Protein OsaR (PA0056) Regulates and the Oxidative Stress Response in Pseudomonas aeruginosa.

Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of the oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation.

Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation.

Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics.

High levels of cAMP inhibit Pseudomonas aeruginosa biofilm formation through reduction of the c-di-GMP content.

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P.

High-Throughput Screening for Compounds that Modulate the Cellular c-di-GMP Level in Bacteria.

Bacteria in the biofilm mode of growth cause numerous problematic infections due to their resistance to antimicrobials and the immune system. Because conventional antimicrobial compounds cannot efficiently eradicate biofilm infections, we urgently need new efficient anti-biofilm drugs. The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle.

Real-Time Monitoring of Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin.

Biofilm infections caused by are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in , encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of mutants in the wild-type PAO1 flow cell biofilm model.

The anti-cancerous drug doxorubicin decreases the c-di-GMP content in Pseudomonas aeruginosa but promotes biofilm formation.

Current antibiotic treatments are insufficient in eradicating bacterial biofilms, which represent the primary cause of chronic bacterial infections. Thus, there is an urgent need for new strategies to eradicate biofilm infections. The second messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria.

C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth.

Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO3(2-)) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa.

Xylo-oligosaccharides inhibit pathogen adhesion to enterocytes in vitro.

We previously reported that the non-digestible carbohydrates inulin and apple pectin promoted Listeria monocytogenes infection in guinea pigs, whereas xylo- and galacto-oligosaccharides (XOS and GOS), prevented infection by this pathogen. In the present study, mechanisms that could explain the previous in vivo observations were explored. Mixing bacterial cultures with XOS significantly (P < 0.

Quantification of specific E. coli in gut mucosa from Crohn's disease patients.

We here present a method based on qRT-PCR to quantify E. coli LF82 in intestinal human samples. Two different primer-probe sets were designed to detect LF82, and a third to target total E.

Comparison of three Listeria monocytogenes strains in a guinea-pig model simulating food-borne exposure.

Three different Listeria monocytogenes strains, LO28 (a laboratory strain with truncated InlA), 4446 (a clinical isolate) and 7291 (a food isolate), were compared in a guinea-pig model designed to mimic food-borne exposure. The objectives were (1) to verify the applicability of the animal model for distinguishing between Listeria with different virulence properties and (2) to explore whether it was possible to reduce the required number of animals by dosing with mixed cultures instead of monocultures. Consistent with in vitro observations of infectivity in Caco-2 cells, faecal densities and presence in selected organs were considerably lower for LO28 than for the other two strains.

Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs.

Background: Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis. The infectivity of Listeria monocytogenes ScottA, cultivated with and without oxygen restriction, was compared in vitro and in vivo.

Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

Biofilms are thought to play a key role in the occurrence of lung infections by Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In this study, 20 nonmucoid P. aeruginosa isolates collected during different periods of chronic infection from eight CF patients were assessed with respect to phenotypic changes and in vitro biofilm formation.

Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model.

Background: In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF.

Involvement of bacterial quorum-sensing signals in spoilage of bean sprouts.

Bacterial communication signals, acylated homoserine lactones (AHLs), were extracted from samples of commercial bean sprouts undergoing soft-rot spoilage. Bean sprouts produced in the laboratory did not undergo soft-rot spoilage and did not contain AHLs or AHL-producing bacteria, although the bacterial population reached levels similar to those in the commercial sprouts, 10(8) to 10(9) CFU/g. AHL-producing bacteria (Enterobacteriaceae and pseudomonads) were isolated from commercial sprouts, and strains that were both proteolytic and pectinolytic were capable of causing soft-rot spoilage in bean sprouts.

Identity and effects of quorum-sensing inhibitors produced by Penicillium species.

Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps.