Hyperosmotic Agents and Antibiotics Affect Dissolved Oxygen and pH Concentration Gradients in Staphylococcus aureus Biofilms

Abstract

Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 Ϯ 27.3 m, P ϭ 0.02; and 155.5 Ϯ 27.9 m, P ϭ 0.016, respectively) than in untreated controls (30.1 m). Decreases in pH of ϳ0.6 and ϳ0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P Ͻ 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P ϭ 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

Non‐invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19F MRI: Persistence of an oxygen sink despite prolonged antibiotic therapy

“…The concentration used in the present study of 50 µg/mL, in the middle of this range, was found to inhibit planktonic growth completely (data not shown) but was insufficient to generate even a single log reduction in viable cell counts. These results are consistent with and enrich the long-standing observations in vitro and in vivo that aggressive antibiotic treatment of mature biofilms is often insufficient to eradicate bacterial respiration, [37][38][39] even in cases where viable cell counts are affected, and that following cessation of antimicrobial therapy biofilms often regrow. [40][41][42] Traditional investigations of biofilm oxygen usage are typically conducted on steady-state biofilms wherein oxygen penetration no longer changes with time, and studies examining the effect of antibiotic administration often infer continued oxygen utilization by identifying anoxic conditions at the biofilm base or by probing the 1D oxygen profile, with the exact response of the oxygen sink to antibiotic challenge unknown.…”

“…The concentration used in the present study of 50 µg/mL, in the middle of this range, was found to inhibit planktonic growth completely (data not shown) but was insufficient to generate even a single log reduction in viable cell counts. These results are consistent with and enrich the long‐standing observations in vitro and in vivo that aggressive antibiotic treatment of mature biofilms is often insufficient to eradicate bacterial respiration, even in cases where viable cell counts are affected, and that following cessation of antimicrobial therapy biofilms often regrow …”

Biofilm-Leukocyte Cross-Talk: Impact on Immune Polarization and Immunometabolism

“…Generalizations can be made concerning the direction of a concentration gradient for metabolic products and substrates based on Reaction-Diffusion theory [4]. For example, metabolic products produced by the biofilm, such as carbon dioxide and acids, can quickly diffuse at the biofilm-fluid interface but do so at a slower rate in the interior [4,14]. Conversely, the availability of metabolic substrates, such as glucose, oxygen, and other nutrients, are generally greatest at the fluid-biofilm interface and are slowly depleted as they diffuse towards the center of the biofilm [14,15].…”

“…For example, metabolic products produced by the biofilm, such as carbon dioxide and acids, can quickly diffuse at the biofilm-fluid interface but do so at a slower rate in the interior [4,14]. Conversely, the availability of metabolic substrates, such as glucose, oxygen, and other nutrients, are generally greatest at the fluid-biofilm interface and are slowly depleted as they diffuse towards the center of the biofilm [14,15]. It also follows that the ability of antibiotics to penetrate a biofilm and reach an effective concentration is dependent on diffusion [14].…”

“…Conversely, the availability of metabolic substrates, such as glucose, oxygen, and other nutrients, are generally greatest at the fluid-biofilm interface and are slowly depleted as they diffuse towards the center of the biofilm [14,15]. It also follows that the ability of antibiotics to penetrate a biofilm and reach an effective concentration is dependent on diffusion [14]. Furthermore, biofilms can maintain a zone of relative nutrient depletion and toxin accumulation at their margins that may affect local host cells [4].…”