New approaches to the treatment of biofilm-related infections

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Bacteria causing chronic infections predominately grow as surface-attached, sessile communities known as biofilms. Biofilm-related infections including cystic fibrosis lung infection, chronic and recurrent otitis media, chronic wounds and implant- and catheter-associated infections, are a significant cause of morbidity and mortality at great financial cost. Chronic biofilm-based infections are recalcitrant to conventional antibiotic therapy and are often unperturbed by host immune responses such as phagocytosis, despite a sustained presence of host inflammation. The diagnosis of clinically important biofilm infections is often difficult as Koch’s postulates are rarely met. If treatment is required, surgical removal of the infected implant, or debridement of wound or bone, is the most efficient means of eradicating a clinically significant biofilm. New approaches to treatment are under investigation

Cephalosporin-3’-diazeniumdiolate NO-donor prodrug PYRRO-C3D enhances azithromycin susceptibility of non-typeable Haemophilus influenzae biofilms

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Objectives: PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO)-donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against non-typeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. Methods: The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free LC/MS proteomic analyses were performed to identify differentially expressed proteins following NO treatment. Results: PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log-fold reduction in viability (p<0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log reduction was observed (p<0.01). Label-free proteomics showed that NO increased expression of sixteen proteins involved in metabolic and transcriptional/translational functions. Conclusions: NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm associated antibiotic tolerance

Self-Starting Micromotors in a Bacterial Bath

Micromotors pushed by biological entities, like motile bacteria, constitute a fascinating way to convert chemical energy into mechanical work at the micrometer scale. Here we show, by using numerical simulations, that a properly designed asymmetric object can be spontaneously set into the desired motion when immersed in a chaotic bacterial bath. Our findings open the way to conceive new hybrid microdevices exploiting the mechanical power production of bacterial organisms. Moreover, the system provides an example of how, in contrast with equilibrium thermal baths, the irreversible chaotic motion of active particles can be rectified by asymmetric environments.Comment: 4 pages, 3 figure

BEW: Bioinformatics workbench for analysis of biofilms experimental data

Biofilms research has evolved considerably in the last decade and is now generating large volumes of heterogeneous data. MIABiE, the international initiative on Biofilms, is devising guidelines for data interchange, and some databases provide access to biofilms experiments. However, the field is lacking appropriate bioinformatics tools in support of increasing operational and analytical needs. This paper presents a flexible and extensible open-source workbench for the operation and analysis of biofilms experiments, as follows: (i) the creation of customised experiments, (ii) the collection of various analytical results, following community standardisation guidelines and (iii) on-demand reporting and statistical evaluation

Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

© 2017 The Authors Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases

Prophage Spontaneous Activation Promotes DNA Release Enhancing Biofilm Formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population

Transcriptional Regulation of the Capsular Polysaccharide Biosynthesis Locus of Streptococcus Pneumoniae: a Bioinformatic Analysis

The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor, which makes the bacterium resistant to phagocytosis. Expression of capsular polysaccharide must be adjusted at different stages of pneumococcal infection, thus, their transcriptional regulation appears to be crucial. To get insight into the existence of regulatory mechanisms common to most serotypes, a bioinformatic analysis of the DNA region located upstream of the capsular locus was performed. With the exception of serotype 37, the capsular locus is located between dexB and aliA on the pneumococcal chromosome. Up to 26 different sequence organizations were found among pneumococci synthesizing their capsule through a Wzy-polymerase-dependent mechanism, mostly varying according to the presence/absence of distinct insertion elements. As a consequence, only ∼250 bp (including a 107 bp RUP_A element) was conserved in 86 sequences, although only a short (ca. 87 bp) region located immediately upstream of cpsA was strictly conserved in all the sequences analyzed. An exhaustive search for possible operator sequences was done. Interestingly, although the promoter region of serotype 3 isolates completely differs from that of other serotypes, most of the proteins proposed to regulate transcription in serotype 3 pneumococci were also predicted to function as possible regulators in non-serotype 3 S. pneumoniae isolates

Verticalization of bacterial biofilms

Biofilms are communities of bacteria adhered to surfaces. Recently, biofilms of rod-shaped bacteria were observed at single-cell resolution and shown to develop from a disordered, two-dimensional layer of founder cells into a three-dimensional structure with a vertically-aligned core. Here, we elucidate the physical mechanism underpinning this transition using a combination of agent-based and continuum modeling. We find that verticalization proceeds through a series of localized mechanical instabilities on the cellular scale. For short cells, these instabilities are primarily triggered by cell division, whereas long cells are more likely to be peeled off the surface by nearby vertical cells, creating an "inverse domino effect". The interplay between cell growth and cell verticalization gives rise to an exotic mechanical state in which the effective surface pressure becomes constant throughout the growing core of the biofilm surface layer. This dynamical isobaricity determines the expansion speed of a biofilm cluster and thereby governs how cells access the third dimension. In particular, theory predicts that a longer average cell length yields more rapidly expanding, flatter biofilms. We experimentally show that such changes in biofilm development occur by exploiting chemicals that modulate cell length.Comment: Main text 10 pages, 4 figures; Supplementary Information 35 pages, 15 figure

Growth dynamics and the evolution of cooperation in microbial populations

Microbes providing public goods are widespread in nature despite running the risk of being exploited by free-riders. However, the precise ecological factors supporting cooperation are still puzzling. Following recent experiments, we consider the role of population growth and the repetitive fragmentation of populations into new colonies mimicking simple microbial life-cycles. Individual-based modeling reveals that demographic fluctuations, which lead to a large variance in the composition of colonies, promote cooperation. Biased by population dynamics these fluctuations result in two qualitatively distinct regimes of robust cooperation under repetitive fragmentation into groups. First, if the level of cooperation exceeds a threshold, cooperators will take over the whole population. Second, cooperators can also emerge from a single mutant leading to a robust coexistence between cooperators and free-riders. We find frequency and size of population bottlenecks, and growth dynamics to be the major ecological factors determining the regimes and thereby the evolutionary pathway towards cooperation.Comment: 26 pages, 6 figure