The practice of modern medicine relies heavily on effective antibiotic therapy to combat bacterial infections. In their absence, many surgeries and chemotherapeutic regimens used to combat cancer and suppress chronic inflammatory conditions would simply not be possible. Development of multidrug-resistant (MDR) bacterial strains threatens to undermine these advances and represents a major global health challenge. A common resistance mechanism in """"""""gram negative rod"""""""" (GNR) bacteria is secretion of a beta-lactamase that hydrolyzes the beta-lactam ring common to all members of the penicillin class. Hundreds of diverse beta-lactamases, which vary in their catalytic mechanism and substrate specificity, have evolved in response to treatment of infections with multiple b-lactam antibiotics Carbapenems represent the last line of effective defense against MDR GNRs expressing extended spectrum beta-lactamases (ESBLs) capable of hydrolyzing all non-carbapenem beta-lactams. However, their effectiveness has been compromised by the recent emergence of plasmid-encoded carbapenemases. To effectively contain and/or prevent outbreaks of carbapenem resistant bacterial infections and to offer optimal patient care, rapid detection of carbapenemase activity in clinical specimens is imperative. We have pioneered a rapid, phenotypic method relying on mass spectrometry to detect carbapenemase activity, by selectively monitoring for the appearance of carbapenem hydrolysis products. In the first aim of this application, we will refine and optimize our current MS- based assay to detect ?-lactam ring hydrolysis of carbapenem antibiotics. We will define necessary kit components and assay protocols for detection of carbapenem hydrolysis activity in bacterial isolates. In the second aim of this application, we will perform a blinded clinical study using selected retrospective and prospective clinical specimens from Yale-New Haven Hospital in order to establish biologic sensitivity and specificity of the overall protocol. Future phases of development will apply the technology to primary specimens, which will be followed by multicenter clinical trials in pursuit o FDA 510(k) clearance of the final product.

Public Health Relevance

Each year, nosocomial (hospital-acquired) infections (HAI) account for 99,000 and 25,000 deaths in the US and Europe, respectively, and place a significant financial burden on the health care system. HAI are an increasing threat to public health because the disease causing organisms are becoming resistant to multiple antibiotics used to treat infections. More specifically, an increasing number of gram negative bacterial infections are resistant to treatment with carbapenems, which previously represented the last line of effective therapy for multi-drug resistant gram negative rod (MDR-GNR). Currently, complete definitive diagnosis of infection by carbapenem resistant bacteria requires approximately 3-4 days, during which the choice of antibiotics used for treatment remains based on empiric guidelines that do not fully incorporate increasing rates of MDR-GNR. Inadequate therapy with ineffective antibiotics leads to increased patient mortality. Therefore, earlier diagnosis will ensure the use of appropriate antibiotics, decrease the length of hospital stays, and relieve the strain on hospitals and health care providers caused by these infections. The proposed research will develop and clinically validate a novel state-of-the-art mass spectrometry (MS) assay that will use patient samples to detect the presence of carbapenem resistant bacteria. Much carbapenem resistance is due to the expression of a specific class of beta-lactamase enzymes that hydrolyze carbapenems and all other beta-lactam antibiotics (i.e. carbapenemases). Using our MS assay, we will detect the presence of carbapenem hydrolysis products thereby confirming the presence of carbapenemase expressing bacteria. Our assay is expected to shorten the time of diagnosis from approximately 3 days to less than 4 hours from the time a bacterial colony is isolated from a patient. This will allow the earlier adoption of an appropriate antibiotic treatment regimen and the implementation of epidemiologic measures to prevent and control outbreaks, saving lives and money. Clinical validation of the assay will be followed by clinical trials and commercialization of a kit for use in clinical microbiology laboratories.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IDM-V (12))
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Ritchie, Alec
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Mz Diagnostics, Inc.
New Haven
United States
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