Introduction. Antibiotic resistance, particularly in cases of
sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in
sepsis can take as long as 3-5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat
sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common
beta-lactamase genes (Klebsiella pneumoniae
carbapenemase (KPC);
New Delhi metallo-beta-lactamase (NDM);
cefotaximase-Munich (CTX-M);
cephamycin AmpC beta-lactamases (CMY); and
Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial
septicemia.Aim. To develop an assay which can rapidly identify the most common
beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States.Hypothesis/Gap Statement.
Septicemia caused by
carbapenem-resistant bacteria has a death rate of 40-60 %. Rapid diagnosis of
antibiotic susceptibility directly from bacteria in blood by identification of
beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common
beta-lactamase and
carbapenemase genes.Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae
carbapenemase (KPC);
New Delhi metallo-beta-lactamase (NDM);
cefotaximase-Munich (CTX);
cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales
Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay.Results. This pentaplex assay successfully identified
beta-lactamase genes derived from bacteria separated from blood at concentrations of 4-8 c.f.u. ml-1.Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of
septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.