B acteria exist in the three common forms: free floating or planktonic, colonies, or biofilms. After the planktonic bacteria attach to a surface, they begin to live in groups, forming microcolonies that are embedded in self-produced extracellular matrices of hydrated extrapolymeric substances (EPS). During biofilm formation, the bacteria communicate to one another (through quorum sensing) to multiply. This cell-to-cell signaling also regulates several physiological properties, including the ability to incorporate foreign DNA, formation and maintenance of biofilm structure, and virulent factors. After a biofilm is mature, certain bacteria may be dispersed and become planktonic. It has been shown that some alterations occur in the bacteria’s phenotype after biofilm formation. Various physiological and molecular changes have also been reported. These include reduced growth rates and specific expression of genes and proteins and their regulation by environmental signals.1,2 One of the most important characteristic of biofilms is their ability to demonstrate antimicrobial resistance.1 Several methods to test the effectiveness of antimicrobial agents, both in vitro and in vivo, exist (Figure 1). In-vitro antimicrobial sensitivity data have been helpful in determining the potential utility of an antimicrobial therapy. One of the most frequently used antimicrobial sensitivity tests is a diffusion assay. During a diffusion assay, an antimicrobial agent is placed on an agar plate that has been inoculated with a known concentration of bacteria. After incubating the plate for 24 hours, the zone of inhibition of bacterial growth that develops around the antimicrobial is measured. Minimum inhibitory concentration (MIC) is the most commonly used pharmacodynamic parameter for the evaluation of efficacy of anti-infective agents. It is also used as a pharmacokinetic parameter for in-vitro dose and drug selection. By definition, MIC is the lowest concentration that completely inhibits visible growth of the organism as detected by the unaided eye after an 18–24-hour incubation period with a standard inoculum of approximately 105 CFU/mL.3 However, although MIC is a useful predictor of the potency of the drug-microorganism interaction, it has both pharmacokinetic and pharmacodynamic disadvantages. From the pharmacokinetic point of view, it overlooks two important factors: tissue distribution and protein binding.4 From the pharmacodynamic point of view, the MIC approach does not provide information on the rate of bactericidal activity and whether increasing antimicrobial concentrations can enhance this rate. Time-kill curves are another method of assessment of the antimicrobial activity of agents. Time-kill curves can follow microbial killing and growth as a function of both time and antibiotic concentration. This method has more meaningful information about the interaction between bacteria and antibiotics; however, it does not reflect an in-vivo setting.5 In-vitro methods to assess biofilm bacteria have been introduced, and these assays may be more appropriate to study the true efficacy of antimicrobial therapies as compared to other in-vitro techniques. The authors have utilized a modified staining method for evaluation of biofilm by light microscopy in Pseudomonas aeruginosa.6 In addition, optical density measurements have been used as a tool to assess biofilm formation. In-vitro models are not only useful for the assessment of antibacterial activity but also assist in determining the emergence of resistance. Some of the advantages of in-vitro systems include the ability to control the number of bacteria, extent of antimicrobial-bacteria contact time, as well as the influence of various environmental factors, such as oxygen tension, pH, and temperature. However, in-vitro models do not take into consideration the effect of wound fluid, growth factors, proteases, antimicrobial peptides, etc. that are found in the skin. In-vitro studies also do not account for fluctuations of drug concentrations within the body or the time course of the drug’s in-vivo antibacterial activity. Unfortunately, numerous biological and technical factors can interfere with the performance of the various assays and make the interpretation of the results of in-vitro studies quite difficult. The bactericidal activity of certain antimicrobial agents, notably b-lactams, is directly related to the rate of bacterial growth.7,8 Therefore, in an in-vitro test, some of the bacterial cells may be dormant or replicating slowly and thus are not inhibited by the antimicrobial agent.9 Other factors, such as tolerance,10 phenotypic resistance,11 or growth phase inoculum,12 can affect the result of in-vitro studies. Given the tremendous number of the aforementioned variables that may confound the results of antimicrobial agents, the clinical relevance will ultimately need to be confirmed by in-vivo studies. Many animal models have the ability to describe the pharmacokinetics (PK) and pharmacodynamics (PD) of antimicrobial therapy. In-vivo studies have a distinct advantage over in-vitro models in the ability to determine which PK/PD dosing index is most closely associated with efficacy.13 Animal models are useful in determining the impact of drug concentration on the rate and extent of antimicrobial killing. They can also determine post-antibiotic effects on bacterial growth, which is often of longer duration than when measured with in-vitro techniques. Determining the impact of a number of treatment variables on PK/PD, such as pathogen species, drug-resistant pathogens, site of infection, drugs with similar mechanism of activity, the treatment endpoint, and various immune defects, is another advantage of using in-vivo models.14 Most biofilm research has been performed in-vitro, and currently, there is a growing interest in establishing in-vivo models to study biofilm-associated diseases. Various animal models exist to study biofilms,15,16 and the authors have used a porcine model to demonstrate bacterial biofilm formation in wounds.17 Using a porcine model, the authors examined various topical antimicrobial agents and dressings on colonized wounds.18–21 A complicating factor of these earlier studies was that the wounds were treated within 15–20 minutes of inoculation. In order to establish a model that would better represent a clinical wound infection, the authors included inoculated wounds that were subsequently covered with a polyurethane film for a minimum of 48 hours. This allowed the bacteria to form firmly attached aggregates of microcolonies on the wound bed and facilitated investigation of the efficacy of antimicrobials on both planktonic and biofilm communities. It is also interesting to note that many of the authors’ previous in-vivo studies, which have examined various agents on planktonic bacteria in wounds, have shown that numerous agents were not effective at reducing or eliminating bacteria within the wounds after a short inoculation time. In a recent study, the authors found limited efficacy of two topical antimicrobial agents on partial-thickness wounds containing Staphylococcus aureus biofilms. These antimicrobial agents, however, were able to completely eliminate planktonic S. aureus (106) within 24 hours of treatment.22 In another study, the authors found that various antimicrobial peptides were effective on multi-drug resistant S. aureus (MRSA) in vitro; however, no activity was observed in the authors’ in-vivo biofilm model (unpublished data). Therefore, although in-vitro testing is helpful to determine dose-response studies, the true effectiveness of these agents must be assessed in vivo. Furthermore, data obtained from animal studies may be difficult to interpret and translate into the clinic setting due to the differences in species, wound types, and challenge organisms. Conclusion The use of both in-vitro and in-vivo models is essential in the development of effective therapeutics. Data acquired from both planktonic and biofilm assays are important to help translate results from the bench to the clinical setting. The establishment of new models that better represent the wound environment will facilitate improved understanding of the infection process and development of beneficial therapies.
Development of Antimicrobials for Wound Care: In-Vitro and In-Vivo Assessments
Issue: Volume 16 - Issue 11 - November 2004
Index: WOUNDS. 2004;16(11):344-347.