An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics

BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide-1 was achieved by 30 hours of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2 – 1.6 log reduction in biofilm density at 72 hours compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 hours at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies

Optimised chronic infection models demonstrate that siderophore ‘cheating’ in Pseudomonas aeruginosa is context specific

The potential for siderophore mutants of Pseudomonas aeruginosa to attenuate virulence during infection, and the possibility of exploiting this for clinical ends, have attracted much discussion. This has largely been based on the results of in vitro experiments conducted in iron-limited growth medium, in which siderophore mutants act as social ‘cheats:’ increasing in frequency at the expense of the wild type to result in low-productivity, low-virulence populations dominated by mutants. We show that insights from in vitro experiments cannot necessarily be transferred to infection contexts. First, most published experiments use an undefined siderophore mutant. Whole-genome sequencing of this strain revealed a range of mutations affecting phenotypes other than siderophore production. Second, iron-limited medium provides a very different environment from that encountered in chronic infections. We conducted cheating assays using defined siderophore deletion mutants, in conditions designed to model infected fluids and tissue in cystic fibrosis lung infection and non-healing wounds. Depending on the environment, siderophore loss led to cheating, simple fitness defects, or no fitness effect at all. Our results show that it is crucial to develop defined in vitro models in order to predict whether siderophores are social, cheatable and suitable for clinical exploitation in specific infection contexts

Bacteriophages and Biofilms

Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce) enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life

A novel chronic wound biofilm model sustaining coexistence of Pseudomonas aeruginosa

Chronic wounds are a large burden to patients and healthcare systems. Biofilm infections in chronic wounds are crucial factors leading to non‐healing of wounds. It is important to study biofilm in wounds and to develop effective interventions against wound biofilm. This study presents a novel in vitro biofilm model mimicking infected chronic wounds. The novel layered chronic wound biofilm model uses woundlike media and includes both Pseudomonas aeruginosa and Staphylococcus aureus, which have been identified as the most important pathogens in wounds. The model sustains their coexistence for at least 96 h. Microscopy of the model revealed microbial growth in non‐surface attached microcolonies as previously observed in vivo. The model was used to determine log(10)‐reduction for the use of an antimicrobial solution and antimicrobial dressings (containing silver or honey) showing moderate‐to‐low antibiofilm effect, which indicates better concordance with the observed clinical performance of this type of treatment than other widely used standard tests

Recent advances in the study of biocorrosion: an overview Avanços recentes no estudo da biocorrosão: uma revisão

Biocorrosion processes at metal surfaces are associated with microorganisms, or the products of their metabolic activities including enzymes, exopolymers, organic and inorganic acids, as well as volatile compounds such as ammonia or hydrogen sulfide. These can affect cathodic and/or anodic reactions, thus altering electrochemistry at the biofilm/metal interface. Various mechanisms of biocorrosion, reflecting the variety of physiological activities carried out by different types of microorganisms, are identified and recent insights into these mechanisms reviewed. Many modern investigations have centered on the microbially-influenced corrosion of ferrous and copper alloys and particular microorganisms of interest have been the sulfate-reducing bacteria and metal (especially manganese)-depositing bacteria. The importance of microbial consortia and the role of extracellular polymeric substances in biocorrosion are emphasized. The contribution to the study of biocorrosion of modern analytical techniques, such as atomic force microscopy, Auger electron, X-ray photoelectron and Mössbauer spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy and microsensors, is discussed.<br>Processos de biocorrosão na superfície de metais são associados com microrganismos ou com os seus produtos metabólicos, tais como: enzimas, exopolímeros, ácidos orgânicos e inorgânicos, e compostos voláteis como amônio ou sulfeto de hidrogênio. Todos estes produtos podem afetar reações catódicas e/ou anódicas, alterando processos eletroquímicos na interface biofilme/metal. Esta revisão discute diversos mecanismos de biocorrosão causados pelos diferentes atividades fisiológicas associadas com microrganismos e os conhecimentos mais recentes. Estudos modernos da corrosão microbiologicamente influenciada focalizam problemas em ligas de ferro e de cobre. Microrganismos especialmente enfocados são as bactérias redutoras de sulfato e bactérias que depositam metais, destacando aquelas que depositam manganês. A importância de consórcios microbianos e o papel de substâncias poliméricas extracelulares na biocorrosão são enfatizados nesta revisão. Considera-se a contribuição de técnicas analíticas modernas, tais como microscopia de força atómica, espectroscopia Auger, espectroscopia de raio-X, espectroscopia Mössbauer, espectroscopia de infra-vermelho de reflectância total com transformação de Fourier e microsensores