Biofilm formation and antimicrobial resistance of coagulase-negative Staphylococci isolated from neonatal intensive care units

Late-onset neonatal sepsis is an obstinate complication frequently accompanying the use of central venous catheters (CVCs) in neonatal intensive care units (NICUs). Coagulase-negative staphylococci (CoNS) are the major cause of catheter-related late-onset neonatal sepsis at the Royal Women’s Hospital (RWH) NICU, Melbourne. In spite of “susceptible” results obtained from conventional in vitro antimicrobial susceptibility tests, failures of CoNS-related neonatal sepsis responding to conventional antimicrobial agents have been repeatedly reported. <br><br>The pathogenesis of CoNS infections is mainly due to their abilities to form biofilms on polymer surfaces and their multi-drug-resistant characteristic. This thesis investigated the antimicrobial susceptibilities of clinical CoNS isolates in different stages of the biofilm formation process; planktonic culture at log phase, early adherent monolayer, mature biofilm and planktonic culture at stationary phase. Some parameters important for antimicrobial susceptibility tests, i.e. initial inocula, bacterial physiological status, and antimicrobial killing outcome, were adjusted for a parallel and comprehensive comparison. Planktonic cells exhibited similar antimicrobial susceptibilities in comparison with early adherent monolayers. A mature biofilm showed similar resistance to antimicrobial eradication to planktonic cells at stationary phase. More importantly, mature biofilms were much more resistant to antimicrobial killing than either planktonic culture at log phase or early adherent monolayers. This finding explains why CoNS biofilm-related infections often fail to respond to conventional antimicrobials. More interesting, oxacillin at a concentration achievable in serum was able to enhance the further growth of established biofilms of some Staphylococcus capitis isolates. <br><br>In order to investigate mechanisms of high resistance of bacterial biofilms to various antimicrobial agents, this study quantitatively examined the percentage of tolerant-but-killable cells and non-killable persister cells in bacterial populations of different growth modes. More tolerant-but-killable cells were found in biofilms than in planktonic cultures at log phase. Persister cells were only present in biofilms, not planktonic cultures at log phase. Both tolerant-but-killable cells and persister cells play important roles in the resistance of biofilms to antimicrobial agents. In addition, both types of cells were found to have normal morphology and were phenotypically resistant to antimicrobial agents. The roles of densely adherent growth mode and bacterial ability to build up extracellular polymer substance (EPS) matrix on biofilm resistance were also assessed, by comparing the antimicrobial susceptibilities of multilayer biofilms formed by biofilm-positive strains, and monolayer biofilms formed by their mutants/variants. Results of this investigation showed that ability of CoNS to adhere to a polymer surface and grow into a highly dense mode, rather than their ability to build up biofilm matrix, contributed to their high resistance to antimicrobial agents.<br><br>Finally, different antimicrobial agents were tested for their efficacies in eradication of biofilm cells as catheter lock solutions (CLS), to find powerful anti-biofilm strategies in the treatment of neonatal sepsis. Ethanol was found to be superior to most conventional antimicrobial agents and their combinations. Exposure to 40% ethanol lock solution for 1 h was found to be sufficient for biofilm eradication.<br>