The Role of Bacterial Biofilms in Antimicrobial Resistance

March 6, 2023

Electron micrograph image of Staphylococcus aureus biofilm.
Staphylococcus aureus biofilm collected from an infected indwelling catheter.
Source: Pixnio
Biofilms have long been recognized in nature and may be found adhering to a variety of surfaces, including rocks in streams (slime), mammalian teeth, roots of plants and even in water pipes. Biofilms may be the most adaptable microbial feature in nature. However, when the associated microbes are pathogenic, this ability to aggregate into biofilms becomes a significant virulence factor. In fact, the vast majority of human infections are actually biofilm mediated. Biofilm infections are often related to medical devices (e.g. knee replacements, catheters, implants, contact lenses, prosthetic valves and joints, screws and pins) or tissue related (e.g. chronic wounds, “staph” skin infections, endocarditis, chronic otitis media, cystic fibrosis lungs). What’s more, biofilms can impact antimicrobial efficacy, as well as the immune response, contributing to antimicrobial resistance and allowing the establishment of persistent/chronic infections.

Since these biofilms are often polymicrobial, they can become even more difficult, if not impossible, to treat. In short, this interaction of microbes (particularly bacteria) with surfaces has far reaching and critical implications in areas including infection and transmission dynamics, biofouling and bioenergy, to name just a few. In order to combat the true cunning of these microbial adversaries, those in healthcare, and other related industries, must work to better understand biofilm makeup and mechanisms of action.

What are Biofilms?

A biofilm is a 3D structure that acts as a microbial battlefront. Biofilms can start forming when a group of microbes sense a given surface and adhere to it. Subsequent colonization and production of an extracellular polysaccharide matrix (EPS) solidify the structure. The EPS matrix substance is sticky, entrenching the bacteria in actual channels—structured, “smart communities”—much like trench warfare. The channeled structure protects the internal surface environment of the bacteria from the external environment; this effectively creates a hidden route to deliver nutrients and waste byproducts and allow for ongoing colonization and maturation of the embedded bacteria. Once the microorganisms mature, they can shed and move from the matured biofilm to join another biofilm community or pioneer a new one.
Formation of a biofilm involves attachment, colonization, growth, production of EPS and attachment of secondary colonizers.
How biofilms are formed.
Source: Openstax

How Biofilms Help Perpetuate Antimicrobial Resistance

The role of biofilms in antimicrobial resistance (AMR) is highly complex and may significantly drive resistance. Bacteria living in a biofilm can exhibit a 10 to 1,000-fold increase in antibiotic resistance compared to similar bacteria living in a planktonic state. For example, in a study examining antibiotic resistance of Staphylococcus epidermidis in biofilms, 100% of isolates were susceptible to the antibiotic vancomycin when tested in a planktonic state. Still, nearly 75% of them were completely resistant to the same antibiotic when tested from a biofilm. The same pattern has been seen for organisms like Klebsiella pneumoniae, which appears to be susceptible when tested from an aqueous solution but becomes highly resistant to certain antibiotics when tested from a biofilm.

In bacteria, common antibiotic resistance mechanisms include point mutations, enzymes and efflux pumps. However, these mechanisms are unlikely to be responsible for the resistance seen in biofilm organisms. Various components work in tandem within a biofilm to reduce, or fully prevent, antibiotic effectiveness and further drive resistance. In combination, these mechanisms allow for the survival of organisms within the biofilm in the presence of high concentrations of antibiotics, a phenomenon known as recalcitrance.

In particular, 3 mechanisms for antibiotic resistance of bacteria in biofilms are important:
  1. Resistance at the Biofilm Surface: The first mechanism occurs at surface levels of the biofilm, when an antibiotic is working to penetrate the sticky, slimy membrane. The biofilm structure’s complexity, which is composed of exopolysaccharide, DNA and protein, makes it challenging for antibiotics to work their way through the matrix and reach the bacterial target within. In addition, due to the slowed diffusion of the antibiotic, it is more likely to be deactivated at the surface level faster than it can diffuse. However, this is not a universal trait of all biofilms, and it is unclear how effective of a driver this mechanism is for AMR.
  2. Resistance Within Biofilm Microenvironments: If an antibiotic is able to move through the first surface layer of the biofilm, it is then faced with a challenging microenvironment that occurs at deeper levels of the biofilm. At this level, metabolic byproducts, waste and nutrients accumulate. Additionally, oxygen may be greatly reduced, creating an anaerobic environment. The combination of these factors has differing impacts on antibiotics, dependent upon each antibiotic’s structure and action. For example, low oxygen levels reduce the bactericidal effects of antibiotics tobramycin and ciprofloxacin, while pH changes can negatively impact aminoglycoside action.
  3. Resistance of Bacterial "Persister" Cells: Once deep inside the biofilm layer where the bacteria reside, even more techniques used to evade antibiotic therapy can be observed. In the act of survival, small subpopulations of bacteria that have avoided antibiotic invasion can enter a “spore-like” state in which they are resistant to extreme conditions, like chemical treatment or antibiotic activity. These cells are known as persister cells. These persisters exist in a dormant state and do not divide in the presence of antibiotics. Importantly, their survival and resistance to antibiotic treatment is not due to any genetic changes, and once the organisms are released from the biofilm or begin dividing again, they return to their pre-persister susceptibility profile.
For bacteria, one of the greatest advantages of the biofilm environment is close proximity of multiple organisms to one another. Not only does this allow for bacterial communication strategies like quorum sensing, it also supports the transfer of mobile genetic elements. In fact, the biofilm environment supports plasmid stability and allows organisms to transmit resistance information more readily. To make matters worse, many of the transposable DNA elements transferred by bacteria encode for biofilm-promoting factors, further contributing to the sustainability of the biofilm and the infection in the patient.
Various mechanisms, including quorum sensing, persister cells and horizontal gene transfer, work in tandem within a biofilm to reduce antibiotic effectiveness and further drive resistance
Mechanisms leading to resistance occur simultaneously within a mature biofilm.
Source: Wikimedia Commons

Diagnosis and Management of Biofilms on Medical Devices

While biofilm-associated infections can be a recurring, life-threatening problem for patients, diagnosis of infections caused by biofilms on medical devices can be difficult for surgeons, physicians and other healthcare professionals. Often, it involves sampling surfaces of medical devices (Table 1) which may require invasive procedures such as aspiration, or biopsy (removal by surgery) of medical devices. However, one may also be able to utilize blood cultures, body fluids or other tissues associated with the infection. Standard microbiological culture for identification, alongside antibiotic susceptibility testing, follows sampling. However, this is often problematic, due to fastidious (difficult to grow) or unusual microbial cultures. Lastly, the biofilm may be polymicrobial. Mixed cultures can be difficult to diagnose making it challenging to identify the correct antibiotic treatment regimen, especially if the drug targets a dominant microbe that grows faster than the polymicrobial community.

Device-associated infections are most commonly caused by Staphylococcus epidermidis and Staphylococcus aureus. However, the list of microbes (especially bacteria and fungi) that can cling to medical devices and cause infection is long and diverse. Research suggests that S. epidermidis accounts for approximately 80% of the bacteria causing medical device-related infections in the hospital setting, but multidrug-resistant gram-negative bacteria like Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, have emerged significantly in catheter-associated urinary tract infection (CAUTI). More recently, fungi like Candida auris have also emerged as dangerous biofilm-associated agents.

Finally, the issue of a recurring infection can often complicate the management of a biofilm on a medical device. This is due to the nature of biofilms itself, and the challenges of fully eradicating biofilm-associated infections. The current prevention (and to some extent therapeutic) methods for these types of infections are placed into 2 broad categories: surface-coating or elution and physical/mechanical/electrical/biological approaches. For example, modifying the surface of medical devices using antimicrobials or antimicrobial agents (silver) has been the focus of much research to reduce microbial colonization and biofilm formation. And high powered sprays and jets have been used as physical–mechanical approaches for biofilm removal (e.g., debridement of surgical-site, exudates or dental biofilms).

Need for Biofilm Awareness in the Fight Against AMR

Given the persistence of biofilms in the hospital setting, and their ability to create an ideal environment for resistance mechanism exchange, greater awareness of these dangers is needed. As antimicrobial stewardship and infection prevention programs continue to evolve, it will be increasingly important to understand the dangers posed by biofilms, and how preventing the transfer and acquisition of biofilm-causing organisms can directly impact the spread of AMR.
Want to learn more about Antimicrobial Resistance and what can be done to combat this pressing threat to global and public health? From drug discovery to antimicrobial stewardship, keep up with the latest AMR reserach. 

Author: Andrea Prinzi, Ph.D., MPH, SM(ASCP)

Andrea Prinzi, Ph.D., MPH, SM(ASCP)
Andrea Prinzi, Ph.D., MPH, SM(ASCP) is a field medical director of U.S. medical affairs and works to bridge the gap between clinical diagnostics and clinical practice.

Author: Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc

Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc
Dr. Rodney Rohde is the Associate Director of the Translational Health Research Initiative at Texas State University.