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://www.cell.com/trends/microbiology/pdf/S0966-842X(04)00264-1.pdf

Survival strategies of infectious biofilms.

▪ Abstract The role of biofilms in the pathogenesis of some chronic human infections is now widely accepted. However, the criteria used to determine whether a given infection is caused by biofilms remain unclear. In this chapter we discuss three infections that are caused by biofilms—infectious kidney stones, bacterial endocarditis, and cystic fibrosis lung infections—and focus on the role of the biofilm in disease pathogenesis. Biofilms are also important as environmental reservoirs for pathogens, and the biofilm growth mode may provide organisms with survival advantages in natural environments and increase their virulence. The consequences of pathogens living in environmental biofilms and an analysis of some specific environmental biofilm systems are presented.

Bacterial Biofilms: An Emerging Link to Disease Pathogenesis

Several pathogens associated with chronic infections, including Pseudomonas aeruginosa in cystic fibrosis pneumonia, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, Staphylococcus aureus in chronic rhinosinusitis and enteropathogenic Escherichia coli in recurrent urinary tract infections, are linked to biofilm formation. Biofilms are usually defined as surface-associated microbial communities, surrounded by an extracellular polymeric substance (EPS) matrix. Biofilm formation has been demonstrated for numerous pathogens and is clearly an important microbial survival strategy. However, outside of dental plaques, fewer reports have investigated biofilm development in clinical samples. Typically biofilms are found in chronic diseases that resist host immune responses and antibiotic treatment and these characteristics are often cited for the ability of bacteria to persist in vivo. This review examines some recent attempts to examine the biofilm phenotype in vivo and discusses the challenges and implications for defining a biofilm phenotype

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1462-5822.2009.01323.x

Evolving concepts in biofilm infections

Acute infections caused by pathogenic bacteria have been studied extensively for well over 100 years. These infections killed millions of people in previous centuries, but they have been combated effectively by the development of modern vaccines, antibiotics and infection control measures. Most research into bacterial pathogenesis has focused on acute infections, but these diseases have now been supplemented by a new category of chronic infections caused by bacteria growing in slime-enclosed aggregates known as biofilms. Biofilm infections, such as pneumonia in cystic fibrosis patients, chronic wounds, chronic otitis media and implant- and catheter-associated infections, affect millions of people in the developed world each year and many deaths occur as a consequence. In general, bacteria have two life forms during growth and proliferation. In one form, the bacteria exist as single, independent cells (planktonic) whereas in the other form, bacteria are organized into sessile aggregates. The latter form is commonly referred to as the biofilm growth phenotype. Acute infections are assumed to involve planktonic bacteria, which are generally treatable with antibiotics, although successful treatment depends on accurate and fast diagnosis. However, in cases where the bacteria succeed in forming a biofilm within the human host, the infection often turns out to be untreatable and will develop into a chronic state. The important hallmarks of chronic biofilm-based infections are extreme resistance to antibiotics and many other conventional antimicrobial agents, and an extreme capacity for evading the host defences. In this thesis, I will assemble the current knowledge on biofilms with an emphasis on chronic infections, guidelines for diagnosis and treatment of these infections, before relating this to my previous research into the area of biofilms. I will present evidence to support a view that the biofilm lifestyle dominates chronic bacterial infections, where bacterial aggregation is the default mode, and that subsequent biofilm development progresses by adaptation to nutritional and environmental conditions. I will make a series of correlations to highlight the most important aspects of biofilms from my perspective, and to determine what can be deduced from the past decades of biofilm research. I will try to bridge in vitro and in vivo research and propose methods for studying biofilms based on this knowledge. I will compare how bacterial biofilms exist in stable ecological habitats and opportunistically in unstable ecological habitats, such as infections. Bacteria have a similar lifestyle (the biofilm) in both habitats, but the fight for survival and supremacy is different. On the basis of this comparison, I will hypothesize how chronic biofilm infections are initiated and how bacteria live together in these infections. Finally, I will discuss different aspects of biofilm infection diagnosis. Hopefully, this survey of current knowledge and my proposed guidelines will provide the basis and inspiration for more research, improved diagnostics, and treatments for well-known biofilm infections and any that may be identified in the future.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/apm.12099

The role of bacterial biofilms in chronic infections

Context.—Otitis media with effusion (OME) can lead to significant hearing loss
in children. Although previous studies have shown that bacterial DNA is present
in a significant percentage of effusions sterile by culture, whether the DNA
represents viable organisms or "fossilized remains" is unknown.Objective.—To determine if bacterial messenger RNA (mRNA), as detected by a reverse
transcriptase–polymerase chain reaction (RT-PCR)–based assay,
is present in chronic pediatric middle ear effusions that contain bacterial
DNA but are sterile by standard cultural methods. Bacterial mRNAs have a half-life
measured in seconds to minutes; therefore, detection of bacteria-specific
mRNAs would be evidence that metabolically active organisms are present.Design.—Blinded comparative study.Patients.—A total of 93 effusions from pediatric outpatients seen for myringotomy
and tube placement for chronic (>3 months) OME (median age of children, 17
months).Setting.—Tertiary care pediatric hospital.Main Outcome Measures.—Percentage of positive test results for RT-PCR–based assays compared
with culture for Haemophilus influenzae and concordance
between RT-PCR and PCR-based findings for bacterial nucleic acids.Results.—Eleven (11.8%) of the 93 specimens tested positive by culture, PCR,
and RT-PCR for H influenzae. A total of 29 specimens
(31.2%) were positive by PCR but negative by culture for H influenzae. All 29 specimens were positive by RT-PCR for H influenzae–specific mRNA.Conclusions.—The RT-PCR–based assay system can detect the presence of bacterial
mRNA in a significant percentage of culturally sterile middle ear effusions,
establishing the presence of viable, metabolically active, intact organisms
in some culture-negative OME.

https://jamanetwork.com/journals/jama/articlepdf/187171/JCI71023.pdf

Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.

The presence of endotoxin (detected by the Limulus amebocyte lysate assay) was compared to the presence of viable Haemophilus influenzae and Moraxella catarrhalis (detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.

Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions

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Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.

Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions