Growth-independent detection of cells
Disclosed herein are various methods, systems, and compositions for the growth independent detection of cells such as microorganisms including bacteria. While existing cellular detection methodologies benefit from cell growth, the methods, systems, and compositions disclosed herein demonstrate embodiments that are independent of cell growth while still allowing for cell-based detection.
1. A growth-independent method for detecting a microorganism of interest in a sample, comprising:
contacting the sample with a plurality of non-replicative transduction particles (NRTPs), wherein the plurality of NRTPs are produced using a silent mutation/complementation method or a deletion/complementation method, wherein said methods do not produce NRTPs that contain a viral genome, such that the plurality of NRTPs transduces one or more microorganisms of interest in the sample, wherein the plurality of NRTPs comprises a reporter nucleic acid sequence, and wherein the growth rate of the one or more microorganisms of interest is less than logarithmic phase;
providing conditions for activation of the reporter nucleic acid sequence; and
detecting a signal produced by the reporter nucleic acid sequence, wherein the presence of the signal indicates the presence of the one or more microorganisms of interest, and wherein the absence of the signal indicates the absence of the one or more microorganisms of interest.
2. The method of claim 1 , further comprising providing an antimicrobial agent to the sample and detecting the signal produced by the reporter nucleic acid sequence to determine whether the one or more microorganisms of interest is susceptible or non-susceptible to the antimicrobial agent.
3. The method of claim 1 , wherein the one or more microorganisms of interest is in stationary phase; or wherein the one or more microorganisms of interest is undergoing no growth.
4. The method of claim 1 , wherein the one or more microorganisms of interest comprises a Methicillin Resistant Staphylococcus aureus (MRSA) cell, Staphylococcus aureus, Staphylococcus spp., Enterobacteriaceae, Enterococcus spp. Streptococcus spp., Acinetobacter spp., or Pseudomonas spp.
5. The method of claim 1 , wherein the sample is a clinical sample.
6. The method of claim 1 , wherein the signal is a luminescence signal; or wherein the detecting step comprises measuring the relative light units (RLU) emitted by the signal.
7. The method of claim 1 , wherein the providing step comprises contacting the sample with a fatty aldehyde bacterial luciferase substrate reagent, optionally wherein the reagent is tridecanal.
8. The method of claim 2 , wherein the antimicrobial agent is cefoxitin, a β-lactam, an extended-spectrum β-lactam, an Aminoglycoside, an Ansamycin, a Carbacephem, Carbapenems, any generation of Cephalosporin, a Glycopeptide, a Lincosamide, a Lipopeptide, a Macrolide, a Monobactam, a Nitrofuran, an Oxazolidonone, a Penicillin, a Polypeptide, a Quinolone, a Fluoroquinolone, a Streptogramin, a Sulfonamide, a Tetracycline, a Rifampicin, a mycobacterial antibiotic, Chloramphenicol, and/or Mupirocin.
9. The method of claim 1 , wherein the reporter nucleic acid sequence encodes a detectable or selectable marker.
10. The method of claim 1 , wherein the signal can be detected at a limit of detection (LoD) of less than 1-1000, 1-5, 5-10, 10-100, 100-1000, 10, 100, 1,000 colony forming units (CFU).
11. The method of claim 1 , further comprising providing varying pre-determined concentrations of antibiotic to the sample and detecting the amount of the signal to determine the minimum inhibitory concentration of the one or more microorganisms of interest to the antibiotic.