Final Technical Report 09 LW 112

Lenhoff, R J

Lawrence Livermore National Laboratory

关键词: Influenza;    Aids Virus;    Lawrence Livermore National Laboratory;    Staphylococcus;    In Vitro;   

DOI  :  10.2172/1018436
RP-ID  :  LLNL-TR-463636
RP-ID  :  W-7405-ENG-48
RP-ID  :  1018436

美国|英语

来源: UNT Digital Library

【 摘 要 】

Since the development of new antibiotics is out-paced by the emergence of bacterial resistance to existing antibiotics, it is crucial to understand the genetic mechanisms underlying resistance existing antibiotics. At the center of this mystery is a poorly understood phenomenon, heteroresistance: the coexistence of multiple subpopulations with varying degrees of antibiotic resistance. A better understanding of the fundamental basis of heteroresistance could result in sorely needed breakthroughs in treatment options. This project proposed to leverage a novel microfluidic (microchemostat) technology to probe the heteroresistance phenomenon in bacteria, with the aim of restoring the efficacy of existing {beta}-lactam antibiotics. The clinically important bacteria Methicillin Resistant S. aureus (MRSA) was used as the test case of bacteria that exhibits antibiotic heteroresistance. MRSA is difficult to treat because it is resistant to all {beta}-lactam antibiotics, as well as other classes of antimicrobials. Whereas {beta}-lactams such as methicillin and oxacillin are the preferred antibiotics to treat S. aureus infections due to their efficacy and low side effects, accurate determination and use of oxacillin/methicillin dosage is hampered by heteroresistance. In fact, invasive MRSA infections now account for about 95,000 deaths per year, a number that exceeds the deaths due to either influenza or HIV (12). In some MRSA strains, two subpopulations of cells may coexist: both populations carry the mecA gene that confers resistance, but mecA is differentially expressed so that only a small number of cells are observed during in vitro testing. Why this occurs is not understood. Prior experiments have sought to explain this phenomenon with conflicting results, with technology being the primary barrier to test the system sufficiently. This is the final report on work accomplished under the Lab-wide LDRD project 09-LW-112. This project was awarded to Frederick Balagadde who has left LLNL for a position at Stanford University. This report is prepared by Raymond Lenhoff who assumed the role of PI on the project for the remaining two months in August of 2010. The project accomplished most of its original objectives despite the fact that numerous biosafety related approvals not envisioned in the original proposal had to be obtained. In addition, the original PI left prior to the last two months of the project. A microfluidic device capable of the culture and optical data collection on microcultures of S. aureus was developed. A simpler chip design was developed and produced. New chip-interface and optical-analysis software was written and tested. S. aureus was successfully cultured and preliminary data (fluorescence and bright field) was collected. The project has provided valuable expertise in microfluidic culture that can be leveraged for host pathogen interaction studies and has been used in a new $9M DARPA proposal which is now being written for submission by Jan 4, 2011.