Biofilm formation and biocide resistance of acinetobacter baumannii

Multi drug resistant (MDR) strains of Acinetobacter baumannii have emerged as a major cause of nosocomial infections associated with significant morbidity and mortality. Over the last 20 years a worldwide expansion in Acinetobacter infections has been observed associated with intensive care units (ICUs), long term care facilities and wounded armed forces personnel. The developing resistance patterns seen in Acinetobacter sp suggest that the number of effective antibiotics may shortly be exhausted. The ability of Acinetobacter sp to form biofilms, resist desiccation and persist on hospital surfaces has played a critical role in the emergence of this bacterium as a human pathogen. The ability of clinical strains of A. baumannii to form strong adherent biofilms has also been recognised as a key virulence factor for this pathogen.

This thesis has investigated the ability of a range of A. baumannii strains to form biofilms and resist the impact of common biocides. In order to facilitate this research a carbohydrate free minimal media employing glucose or alcohols as sole carbon sources was developed. Considerable variations in the sensitivity of strains to ethanol or IPA was observed with the Type strain being more sensitive and less able to use alcohols to support growth than many of the clinical strains investigated. Alcohols as sole carbon sources had an impact on bacterial adherence, with 71% of strains being highly adherent when fed on alcohol as the sole carbon source. Scanning electron microscopy and fluorescent microscopy indicated that highly adherent strains were able to establish biofilms on both hydrophobic (plastic) and hydrophilic (glass) surfaces, forming carbohydrate based extracellular polymeric substances (EPS) during biofilm formation. EPS generation occurred alongside the transient generation of lactate, the latter being degraded during the stationary phase. Biofilm forming strains generated high MW EPS when grown on mineral media with ethanol as a sole carbon source, extracted EPS was shown to contain repeating units of both galactose and rhamnose sugars.

The Bioscreen system was used to determine the MICs of a range of quaternary ammonium compounds (QACs) and PHMB against the highly adherent strains. MIC values were below 35 mg/l for all biocides tested and MBC for planktonic cells were from 6 to 100 x greater than the MIC values. MBC values for biofilms were orders of magnitude greater than MBC values for planktonic cells with little variation between biocides or carbon source. Planktonic cells were able to form biofilms at concentration considerably greater than the 24 hour MBC for planktonic cells, demonstrating that biofilm formation provided additional protection against the biocides investigated.

A range of antimicrobial wound dressings (NSCD, ISCD, Honey and PHMB) were evaluated for their impact on commonly occurring wound pathogens i.e. A. baumannii, P. aeruginosa and S. aureus (MRSA). Dressings were evaluated against both planktonic cells and cells immobilised in a collagen matrix. In all cases there were significant differences (p<0.05) between strains of the same species when treated with the same dressing, indicating that significant variations in the susceptibility of wound pathogens to antimicrobial dressings were present at the sub species level. The diffusion barrier provided by the collagen matrix generated lower reduction values than the planktonic approach with a few exceptions. Broadly speaking the NSDC dressing was the most effective, PHMB least effective and the Honey dressing was most affected by the diffusion barrier provided by the collagen matrix.