CORR Insights(®): Biofilm Antimicrobial Susceptibility Increases With Antimicrobial Exposure Time.

Abstract

Where Are We Now? Biofilm formation by pathogenic bacteria is a recognized virulence factor important in the persistence of chronic infections. When growing as biofilms, even antibiotic sensitive bacteria (as measured by the planktonic broth minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC] methods) become highly tolerant of antibiotics. In part, this tolerance is due to the presence of a small fraction (< 1 %) of slow-growing “persister” cells and small colony variants which, according to some, cannot be killed by antibiotics alone, regardless of concentration [1, 5]. However, this would contradict other studies which report achieve complete sterilization of biofilms as the basis for the Minimum Biofilm Eradication Concentration (MBEC) assay [2]. The MBEC is based on a 24-hour antibiotic exposure period in accordance with the classic MIC and MBC. However, in device related infections, such as orthopaedic periprosthetic infection, high concentrations of antibiotics may be delivered locally from antibiotic loaded depots (bone cement) during periods of weeks or months. Recent work by Howlin et al. [4] has demonstrated that multiple days of exposure to high concentrations of antibiotics were required to achieve significant reductions or complete killing of biofilms. While a 24-hour MIC or MBC is convenient and may be useful for determining dosages to kill rapidly growing planktonic cultures, it may not be particularly useful for slow growing biofilms. Even the 24-hour MBEC, which is specifically designed to provide guidance in treating bacteria grown in the biofilm phenotype, might reflect the influence of exposure time on an appropriate scale relevant to biofilms. The study by Castaneda and colleagues measures the MBEC of five common biofilm forming pathogens as a function of exposure time to vancomycin and tobramycin alone and in combination. In the United States, these two antibiotics are commonly incorporated into bone cements by surgeons to treat orthopaedic infections. The MBEC was measured at one, three, and five days. The authors found the MBEC of 11 of 14 pathogen antibiotic combinations was lower than the 24-hour assay after Day 3. They also found that on Day 5, 13 of 14 pathogen antibiotic combinations were lower, with the exception attributed to contamination by between a two and eight-fold difference depending on the pathogen and the antibiotic. The authors concluded that a 24-hour assay might actually overestimate the concentration of antibiotic that needs to be administered in order to eradicate a biofilm infection if the exposure time is not taken into consideration. However, this highlights an area of tension that clinicians face, between the need to act quickly in order to suppress or kill a biofilm infection versus the more complex and lengthy culturing methods to determine susceptibility. Further, the MBEC itself, although a useful research tool, is not yet accepted as a “gold standard,” and there is little, if any, indication on how MBEC guided therapeutics may correlate with clinical efficacy. Where Do We Need To Go? The study by Castaneda and colleagues exemplifies many of the biofilm specific issues that need to be addressed in order to provide better guidance to physicians and surgeons on the appropriate application of antibiotics to treat biofilm infections. Despite the fact that biofilms have been widely recognized as critical in many chronic infections for at least a decade, progress on two of the most important issues remains stubbornly mired—diagnostics and treatment. A main reason is the lack of standard methods for growing and testing biofilms. Working with planktonic cultures is convenient since the bacteria can be grown to ensure a homogeneous distribution both physically and by growth phase. They can be readily diluted to achieve standard concentrations. Biofilms are not so domesticated, their surface density is difficult to control and since they are on a surface they cannot be readily divided into defined inoculum. Further, biofilms harbor many different phenotypes including slow growing small colony variants or dormant persisters and will be influenced by the type of surface and age. How Do We Get There? Since biofilm formation is a developmental process the susceptibility will vary with maturity. One of the biggest contributions in advancing biofilm medicine is to develop relevant and robust standard culture and susceptibility methods. Darla M. Goeres PhD and the team at the Center for Biofilm Engineering is pioneering the development of such methods for disinfection and sanitization [3] with the ASTM International, the US Environmental Protection Agency, and Centers for Disease Control & Prevention, including ASTM E2799 for the MBEC method [6]. Encouragingly, they are also working with the FDA. Meaningful advancement will only be made when: (1) Regulatory agencies and bodies such as the Clinical and Laboratory Standards Institute fully recognize the urgent need for it, and (2) government and industry funding is adequate to enable the development of such methods for medical application.