Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(01)05321-1.pdf

Antibiotic resistance of bacteria in biofilms

▪ Abstract Biofilms can be defined as communities of microorganisms attached to a surface. It is clear that microorganisms undergo profound changes during their transition from planktonic (free-swimming) organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the new phenotypic characteristics developed by biofilm bacteria and occur in response to a variety of environmental signals. Recent genetic and molecular approaches used to study bacterial and fungal biofilms have identified genes and regulatory circuits important for initial cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm transition is a complex and highly regulated process. The results reviewed in this article indicate that the formation of biofilms serves as a new model system for the study of microbial development.

Biofilm Formation as Microbial Development

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

https://hal.archives-ouvertes.fr/hal-00567285/document

Antibiotic resistance of bacterial biofilms

Few discoveries in the history of medicine have had such a profound impact on human life and society as the discovery of antibiotics to combat bacterial infections. In the last few decades, there has been an observed exponential increase in the number of antibacterial compounds with diverse chemical structures introduced clinically to control infectious diseases. The most significant development in antibiotic research has been the ability to extend and improve the action of naturally occurring compounds by chemical modification. An example of this is the chemical modification of the penicillin molecule such that the new derivatives are immune to attack by bacterial enzymes (e.g., ,B-lactamases). Despite efforts in the search for new antibiotics as well as the improvement of existing antibiotic performance, bacterial resistance to antimicrobial agents remains a problem in the treatment of infections. Before a new antibiotic is introduced into clinical practice, extensive laboratory studies are performed to prove that the new compound is superior to existing compounds in killing the many species of pathogens for which the antibiotic is intended. These species of pathogens, which are grown planktonically in test tubes, are often used to test the effectiveness of the compound in their eradication. If the results are promising, then carefully designed in vivo animal models and clinical trials are used to obtain data regarding the efficacy of the compound. Undoubtedly, this approach has been extremely successful, and many new antibiotics are introduced into clinical practice annually. However, resistance to the antibiotic may be detected soon after its release for clinical use. We believe that this is partly due to the inadvertent use of sublethal concentrations of antibiotics in the treatment of chronic infections such as medical device-related infections. It is therefore of great importance to consider the strategies that are being used to deal with medical device-related infections. It is hoped that this minireview will help to stimulate the formulation of effective strategies to combat these chronic infections.

Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy.

In this minireview, we summarize recent studies of the interaction of bacteria in biofilms with antibacterial agents and we discuss the parameters that should be considered in the development of experimental protocols for study of the efficacy of antibacterial agents against pathogenic bacteria in biofilm.

Testing the susceptibility of bacteria in biofilms to antibacterial agents.

Pseudomonas aeruginosa was cultivated at low growth rates under iron-limiting conditions on acrylic tiles Biofilm cells exhibited increased tobramycin resistance compared with that of planktonic cells, and in old biofilms were more resistant than were cells in young biofilms However, on suspension of the biofilm bacteria, glycocalyx-mediated resistance was lost

Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system.

An in vitro chemostat system in which Pseudomonas aeruginosa can be cultivated at a slow growth rate and under iron limitation conditions was used to study the susceptibilities of sessile bacteria of mucoid and nonmucoid P. aeruginosa strains to tobramycin and piperacillin. Planktonic cells of both mucoid and nonmucoid P. aeruginosa strains were susceptible to tobramycin and piperacillin. None of the cells was found to be viable after 2 h of exposure to 200 micrograms of piperacillin plus 10 micrograms of tobramycin per ml. Young sessile bacteria were slightly more resistant to piperacillin or tobramycin than the planktonic cells were. However, eradication of young sessile bacteria could be achieved with a combination of piperacillin and tobramycin. None of these young biofilm bacteria were found to be viable after a 2-h exposure to 200 micrograms of piperacillin plus 10 micrograms of tobramycin per ml. Old sessile bacteria were very resistant to these antibiotics. Eradication of old sessile bacteria could not be achieved with either tobramycin (200 micrograms/ml) or piperacillin (200 micrograms/ml) alone. Combination of higher concentrations of tobramycin with piperacillin resulted in an enhancement of killing of the old sessile bacteria. Exposure of old sessile bacteria to 200 micrograms of piperacillin plus 100 micrograms of tobramycin per ml resulted in the reduction of the viable count to approximately 0.02%. The data suggest that the eradication of biofilm-associated infections is best carried out as early as possible. Enhanced activities against the sessile bacteria were achieved when higher concentrations of aminoglycosides were combined with beta-lactam antibiotics.

Enhanced activity of combination of tobramycin and piperacillin for eradication of sessile biofilm cells of Pseudomonas aeruginosa.

The dynamic interaction of planktonic and biofilm cells of mucoid Pseudomonas aeruginosa with tobramycin and piperacillin was investigated in a chemostat system. The results indicated that planktonic and young biofilm cells of the 2-day-old chemostat culture of P. aeruginosa were susceptible to killing by chemostat-controlled doses of either 250 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml or 500 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml. Complete eradication of the planktonic and young biofilm cells was observed after exposure of the cells to six chemostat-controlled doses of these antibiotic at 8-h intervals for 7 days. Regrowth of the organism was not observed after the termination of antibiotic therapy on day 7. A different picture was observed when antibiotic treatment was initiated on day 10 after inoculation. Viable old biofilm cells were reduced to approximately 20% after exposure to the chemostat-controlled doses of 500 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml. Complete eradication of old biofilm cells could not be achieved, and regrowth of the organism occurred after the termination of antibiotic therapy. These data suggest that young biofilm cells of mucoid P. aeruginosa can be effectively eradicated with the combination of piperacillin and tobramycin, while old biofilm cells are very resistant to these antibiotics and eradication of old biofilm cells is not achievable with the chemostat-controlled doses of piperacillin and tobramycin used in this study.

J W Costerton

Papers

Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy.

Testing the susceptibility of bacteria in biofilms to antibacterial agents.

Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system.

Enhanced activity of combination of tobramycin and piperacillin for eradication of sessile biofilm cells of Pseudomonas aeruginosa.

Dynamic interactions of biofilms of mucoid Pseudomonas aeruginosa with tobramycin and piperacillin.