The effects of oxygen and reactive oxygen species on antibiotic resistance and microbial communities in chronic wounds

Abstract

Infection is one of the factors that may contribute to non-healing of chronic wounds; the presence of antibiotic resistant bacteria serves to exacerbate the problem due to limited treatment options. Bacteria utilise several mechanisms to survive exposure to antibiotics, including synthesis of deactivating enzymes, target modification or substitution, changes to membrane permeability, upregulation of efflux pumps and the formation of a biofilm. Quorum sensing is a density-dependent mechanism of bacterial cell to cell communication that can be instrumental in co-ordinating biofilm initiation. Hyperbaric oxygen therapy (HBOT) is an option offered to some patients with chronic wounds, including diabetic foot ulcers. Evidence suggests that HBOT can reduce the incidence of major amputation in these patients. As well as the direct toxicity of increased tissue oxygenation on anaerobic bacteria HBOT may also increase levels of reactive oxygen and nitrogen species in the wound environment. This study aimed to investigate the effects of hyperoxia and oxidative damage on three specific mechanisms of antibiotic resistance: the activity of penicillinase, an antibiotic deactivating enzyme synthesised by bacteria; the activity of quorum sensing signalling molecules (AHLs); and biofilms and their associated bacteria. It also analysed the population dynamics of, primarily, bacteria in diabetic foot ulcers during HBOT, by the use of molecular analysis tools such as PCRDGGE. The presence of fungal species was investigated in wounds prior to HBOT and in two wounds at two points during HBOT. This study found that hydrogen peroxide, hypochlorous acid and peroxynitrite reduced the activity of penicillinase in vitro. Hypochlorous acid reduced the activity of a range of AHLs in vitro but not in vivo. Oxygen concentration did not have any impact on biofilm mass, nor did it significantly affect the ability of an oxidant-generating enzyme to reduce live bacterial cells within a biofilm. The population dynamics of bacterial species identified in all the wounds were complex and did not undergo identifiable changes during HBOT. Fungal species were identified in all wounds prior to HBOT, though different profiles were observed in the two wounds investigated during HBOT. These results suggest that oxidants could play a role in the attenuation of antibiotic resistance in chronic wound bacteria. It is unclear whether HBOT alters the population dynamics of non-healing wound microflora