Life and Death in Biofilms
First of all: bacteria can withstand much higher concentrations of antimicrobial when they live in biofilms compared to living as single, planktonic cells. This phenomenon is frequently termed "resistance", although in the strict sense, resistance is heritable with a genomic basis. In biofilms, they may show signs of resistance, but when the biofilm is disrupted, and the cells grow planktonically, they can return to being antibiotic susceptible - this is understood as "tolerance". This is one of the emergent properties of biofilms.
But when are bacteria actually dead? The most common sign is that they don´t grow any more. The question when bacteria are dead or still alive is of enormous relevance in any field of medicine, hygiene, pharmacy, food and beverage industries. It is also crucial for microbiological research, e.g., in testing disinfectants, antibiotics or novel antimicrobial strategies.
However, it is surprisingly difficult to really define bacterial death - it completely depends upon the method of determination. Although cultivation and colony counting represent the most common means, in many cases bacteria refuse to grow, but they are not dead. This can be understood as a response to stress, entering a "viable-but-non-culturable" state (VBNC). Many hygienically relevant organisms are reported to be capable entering the VBNC state. Cultivation-independent methods can reveal life signs including presence of DNA, membrane integrity, ribosomal activity, ATP-content, and molecular biological parameters which all provide different perspectives of microbial life. In most real-world situations, the culture-based methods fulfil their purpose. Problems arise if bacteria enter the VBNC state from which they can resuscitate and return to growth and virulence, e.g., in cases of recurring regrowth in water systems. It is pointed out to be aware of the limitations of the cultivation method, consider possible VBNC states and to embrace advanced methods in cases of failing or inconsistency of cultivation approaches.
Biocides and Antimicrobial Resistance in Domestic Biofilms: Realism Based Studies
The control of microorganisms is a key objective in medical, industrial, domestic, and food-production environments. Whilst the effectiveness of biocides in these contexts is well-evidenced, debate continues about the resistance risks associated with their use. Much of the evidence for resistance generation by biocides is based on correlation analysis, where reports of treatment failure are generally uncommon, or laboratory studies that do not necessarily represent real biocide applications. This presentation will consider a series of studies that were designed to increase realism in the assessment of biocide resistance risk.
Antimicrobial Drug Discovery: Current Status and Emerging Strategies
Infectious diseases have been traditionally controlled by antibiotics and vaccines, and these approaches have greatly improved health and longevity. However, the looming antibiotic-resistance crisis, together with the alarmingly low rate of newly approved antibiotics for clinical usage, is jeopardizing our ability to prevent and treat infections. Although warnings have been issued, current treatment options have not been improved. This bleak picture of the current state of antibiotics pipeline reveals the need for increased basic research efforts and antibiotic discovery programs.
Historically, antibiotic discovery has been closely related to systematic procedures (platforms) that catalysed the antibiotic golden age. This journey was changed during the genomics era with the arrival of target-based approaches. Even so, they were considered a failure due to limitations in translating drugs to the clinic, and as such, phenotypic screenings were re-instituted. Whatever happens in the post-genomic era, the antibiotic pipeline is still lacklustre. Despite herculean efforts, practically no new classes of antibiotics have been translated into new medicines for decades, a problem that is related to the stringent chemical, biological and pharmacological requirements. Understanding the causes of the failures and successes of clinical trials can generate a supportive database and, hopefully, a starting point to increase the productivity in antimicrobial drug discovery.
Current research, focused for instance in microbial metabolism and transport mechanisms, and allied to the technical advances in structure-based and fragment-based drug discovery approaches offers many promising new concepts and ideas to work with. Exploring multiple paths, namely the use of polypharmacology models, pathblockers, small molecule disruptors and adjuvants, can be a turning point and act as a blueprint for future antimicrobial drugs.
This holistic overview can be translated into novel databases, platforms and task forces, composed by multidisciplinary groups, which will hopefully result in a sustainable discovery
and development of new and better antibiotics.
Funding: Antimicrobial drug discovery research is supported by the grants: BIOFILM- PTDC/BII-BTI/30219/2017; ABFISH - PTDC/ASP-PES/28397/2017 and UIDB/ 04469/2020.
Structure-based Computational Approaches Targeting the Inhibition of Bacterial Biofilm Formation
Sérgio F. Sousa
Biofilms play an important role in health, being associated with >80% of all microbial infections in the body and in the development of antibiotic resistance. Research in this field has produced large volumes of data. Being able to take advantage of this information is paramount for the progress in this field, which coupled with the developments in computers offers unparallel opportunities.
This presentation will illustrate some of our recent advances in this field by combining bioinformatics data, structural information, virtual screening, structure-based docking, molecular dynamics simulations and free energy calculations to target the inhibition of bacterial biofilm formation. Special emphasis will be given to the Biofilm Structural Database developed and maintained at BioSIM (https://biofilms.biosim.pt)  and to the specialized multi-level computational approach that we have been optimizing for different biofilm associated targets .
 Magalhães RP, Vieira TF, Fernandes HS, Melo A, Simões M, Sousa SF. The Biofilms Structural Database. Trends in Biotechnology. 2020; 38(9): 937
 Martins FG, Melo A, Sousa SF. Identification of New Potential Inhibitors of Quorum Sensing through a Specialized Multi-Level Computational Approach. Molecules. 2021; 26(9):2600
Bacterial Biofilm Structure and Stress Responses
On surfaces, bacteria grow into multicellular communities termed biofilms, which are the most abundant form of life on Earth. Such biofilms cause many chronic and acute infections, which are difficult to treat because the cells inside these communities display strongly increased tolerance to antibiotics and disinfectants. I will first present imaging and image analysis techniques we recently developed to follow dynamical processes in biofilms at the single-cell level. Using these techniques, combined with physical models, I will show how we can reveal the key cell-cell interaction processes during biofilm growth across different species, and how biofilms respond to environmental stresses at the unicellular and multicellular level.
Biofilms and the Challenges of Mass Transfer
Life does not exist without mass transfer. In complex communities such as biofilms, such phenomena interfere with the rates of microbial growth and dispersal, the matrix structure and the resistance to biocides and antibiotics.
The talk will give a very brief overview of the fundamentals of mass transfer and discuss a few illustrative examples of how mass transfer relates to biofilm properties and function.
Biofilm Disinfection: Key Aspects & Insights into Comparative Studies Using Phytochemicals
Biofilms are the default mode of microbial growth in most environments. Within biofilms, microorganisms may be well protected from various stresses and antimicrobials. This enhanced survival and persistence may provoke significant problems within various areas including the industry, clinical settings, agriculture, water distribution, and marine traffic. Due to the great recalcitrance of biofilms, currently applied antimicrobial protocols do not always sufficiently deal with biofilm-related problems, while these may be also toxic and harmful to the environment. Considering the great impact biofilms may have, alternative eliminations methods that will be efficient, cost-effective, and safe are needed. These should focus not only on the successful removal of formed biofilms but also prevent/delay their formation, if possible, with the lowest possibility for resistance development. In this presentation, some key aspects determining the resistance of biofilms to antimicrobials, and as thus the efficacy of disinfection will be initially highlighted, while the second part will consist of the results of some representative studies on the use of phytochemicals to control biofilms and their efficiency in comparison to planktonic cells and some common chemical disinfectants.