Glossop, Sophie Joanne (2020) The role of growth conditions on the interactions of Pseudomonas extracellular secretions with keratinocytes. Doctoral thesis, University of Huddersfield.

The skin is the largest organ in the body and acts as a barrier to protect from the external environment as well as having an important immunogenic function. Damage to the skin such as wounding results in the loss of protection to the host and can permit the invasion of opportunistic pathogens, which can cause disruption to the healing process resulting in a chronic wound. Pseudomonas aeruginosa is an opportunistic pathogen and a coloniser of chronic wounds and is becoming increasingly recognised as major cause of hospital acquired infections due to its ability to secrete a variety of virulence and pathogenic factors such as toxins, proteases, vesicles and in addition biofilm formation. One of the main controls of preventing infections in hospitals is the use of biocides such as ethanol, which are frequently used in routine hospital cleaning.

Two strains of Pseudomonas (aeruginosa, hospital strain (PS3) isolated from a chronic wound dressing and (fluorescens, a laboratory reference strain (PF) that had no known virulence factors were grown in different media, supplemented with ethanol or glucose and in an additional media, simulated wound fluid for 24 or 80 hours. The effects of culture conditions on the response of the bacteria and their secretions was investigated directly and also by studying their effects on the keratinocyte cell line (HaCaT).

PS3 80 hour cultures showed an increased production of all the virulence factors tested compared to 24 hours cultures. In addition there was some differences between culture conditions with PS3 grown in ethanol producing a greater amount of hemolysin and pyocyanin, however, live bacteria from these cultures had little effect on keratinocytes unlike the corresponding extracellular secretions. The secretions from cultures grown with ethanol for 80 hours produced increased toxicity resulting in greater keratinocyte death and longer healing times in a scratch assay model of wound healing, in addition there was a high pro-inflammatory response from the keratinocytes compared to those exposed to live bacteria grown in the same conditions and also when compared to secretions from glucose grown PS3. Generally the secretion of CXCL8 was higher from cells exposed to PS3 secretions when grown in ethanol, however there was greater expression of MAMP receptors in keratinocytes exposed secretions SWF and glucose grown PS3. PF cultures grown in the same conditions produced no measurable virulence factors and the secretions had no toxic effects on the keratinocytes, however faster healing in the scratch assay occurred for some conditions.

The increase in virulence factors seen from ethanol grown PS3 in addition to the high toxicity in keratinocytes from 80 hour cultures indicates that prolonged exposure to trace amounts of ethanol within the bacterial microenvironment can influence the production of immunogenic and increase virulence factor production from PS3. Considering PS3 is a clinical isolate and there is increasing wide spread use of ethanol based products within clinical environments these studies highlight how the improper use of disinfection products may be enhancing bacterial pathogenicity and virulence within clinical isolates and enhancing microbial fitness potentially leading to an emergence of bactericidal resistant bacteria.

FINAL THESIS - Glossop.pdf - Accepted Version
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