Exploring and modelling the role of environmental biofilms in healthcare-associated infections

The development of nosocomial or healthcare associated infections are a known risk in healthcare. Infection control protocols have been devised to reduce the risk of their development. These protocols include infection control measures, antibiotic stewardship, administrative support, active surveillance, hand hygiene and environmental management. Hand hygiene has been a major focus in reducing nosocomial infection development whilst the investigation into the impact of environmental microbial contamination has been underestimated by comparison. The contamination of hospital environmental surfaces with pathogenic, drug resistant bacteria may act as a microbial reservoir contributing to nosocomial infection rates. One way in which these microorganisms may be able to survive and remain viable on environmental surfaces is through their existence in biofilms. Growth in biofilm is advantageous due to the intrinsic recalcitrant properties it provides such as reduced penetration of antimicrobial agents, the present of dormant ‘persister’ cells to aid in biofilm survival, and a lowered metabolic rate to aid in the evasion of antimicrobial agents targeting active metabolism and transcription. The existence of viable biofilm containing multi-drug resistant organisms (MDROs) has been previously demonstrated on high touch hospital surfaces despite terminal treatment with 1000ppm chlorine as per standard decontamination protocols. Therefore the aims of this study were to 1) sample hospital surfaces within an operational ICU for the presence of multi-drug resistant pathogens important for nosocomial development 2) determine whether a linkage exists between ATP presence and pathogenic bacterial contamination 3) assess the relatedness of the environmental isolates to those from patients admitted to the ICU where environmental sampling took place 4) examine these contaminated surfaces for the existence of biofilm and 5) develop a dry surface biofilm model to mimic the biofilm development observed on hospital surfaces. A total of 18.83% (n=154) of surfaces tested for microbial contamination within the ICU were found to be positive for contamination with at least one MDRO, with 44.44% of these sites found to be contaminated with vancomycin resistant enterococci (VRE). This contamination was not limited to the immediate patient surrounds, but also observed in healthcare worker only areas such as the clinical work station. The examination of sites found to be positive for MDRO contamination for biofilm visualisation was conducted via scanning electron microscopy (SEM). Evidence of extra cellular matrix production was observed in line with previously published literature. Using LIVE/DEAD staining, these biofilm structures were observed to contain viable microbial growth. The relationship of environmental and patient VRE strains was examined first through using ERIC-PCR and PFGE as screening tools. PFGE demonstrated the existence of 3 distinct banding patterns common amongst the environmental and clinical isolates. A single matched pair of each banding pattern was further examined through whole genome sequencing, with in silico sequence typing showing two of the three patterns to be sequence type 80 with the remaining pair remaining untypable due to the deletion of one of seven housekeeping genes. Analysis of core single nucleotide polymorphisms (SNP) demonstrated to the highest resolution the full extent of this relationship, with no more than 7 SNP differences between the pairs, demonstrating a link between this environmental contamination and patient colonisation. Finally, the development of a dry surface biofilm model on both stainless steel and keyboard keys was successful, with biofilm formations observed via SEM. The effects of surface coating as a cofactor for biofilm development was tested, with the presence of a surface coating to mimic sebum (egg yolk and macadamia oil) facilitating higher growth rates in comparison to growth without coating. The presence of both MDRO contaminants almost identical to clinical strains and the presence of biofilm on the same surface provides strong evidence for the importance of environmental contamination in nosocomial development. The future application of FISH techniques during examination of biofilm contamination can be used to provide evidence of the existence of these MDROs within biofilms. The development of a novel dry surface biofilm model has provided a platform for the investigation into the ability of microorganisms to maintain viability in dry conditions. One major benefit of the development of such a model is its future use in the modelling of possible microbial transmission from biofilms and the testing of biofilm removal techniques, providing a way to strengthen and improve infection control policies for environmental management