Antimicrobial susceptibility testing and microbial identification
Systems and methods for microbe identification (ID) in the context of phenotypic antimicrobial susceptibility testing (AST). Approaches for rapidly identifying polymicrobial samples are introduced that following perform one or more ID methods, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), PCR, or DNA hybridization, following sample incubation in two or more different media, including at least one selective media. Parallel ID and AST workflows are provided that may reduce the time from sample to AST result.
1. A method for assessing the microbial status of a patient sample as monomicrobial or polymicrobial which comprises,
(a) inoculating a growth control reservoir and each of at least eight (8) reservoirs of a selective/differential growth panel (SDGP) with a cultured patient sample, said sample having been confirmed positive for microbial growth, wherein each of the 8 reservoirs comprises a distinct differential or selective growth medium for a selected microorganism or group of microorganisms, wherein the selective growth medium comprises
(i) one or more compounds suitable for promoting growth of one of said selected microorganisms or group of microorganisms,
(ii) one or more compounds that inhibit growth of one of said selected microorganisms or group of microorganisms, or
(iii) both (1) and (ii), and
wherein the differential growth medium comprises a substrate for an enzyme expressed by one of said selected microorganisms or group of microorganisms, and reaction of the substrate catalyzed by said enzyme produces a reaction product;
(b) incubating the SDGP for a time and under conditions for microbial growth to be detectable in the growth control reservoir;
(c) performing at least two different assays with each of the at least 8 reservoirs of the SDGP, wherein the assays are for independently detecting one or more of
(i) the presence, absence or identity of a microorganism or a group of microorganisms in a selective or differential growth media,
(ii) an amount of substrate in a differential growth media, or
(iii) an amount of reaction product in a differential growth media,
(d) determining from the assay results whether one of the selected microorganisms or groups of microorganisms is present in each reservoir, and
(e) assessing (i) the patient sample as at least monomicrobial if all of the assays are negative, as monomicrobial or polymicrobial if at least one assay is positive, and as polymicrobial if two or more different assays are positive and (ii) the identity of the microorganisms or group of microorganisms in the patient sample, if determinable by one of said assays.
2. The method of claim 1 , further comprising performing a coagulase assay and based on the results of the coagulase assay and the results from the selective and differential media, further assessing the microbial status of the patient sample as monomicrobial or polymicrobial.
3. The method of claim 1 , wherein the SDGP comprises a minimum 10, 12, 14, 16, 18, or 20 reservoirs.
4. The method of claim 1 , wherein one or more additional reservoirs on the SDGP comprises non-selective media.
5. The method of claim 1 , wherein said SDGP is configured for detecting a Gram positive microorganism or a group of Gram positive microorganisms or a Gram negative microorganism or a group of Gram negative microorganisms.
6. The method of claim 1 , wherein at least one SDGP reservoir comprises at least one probe compound.
7. The method of claim 6 , wherein said probe compounds is a surface-binding agent, a locked nucleic acid, a peptide nucleic acid, or a fluorescence in situ hybridization probe.
8. The method of claim 7 , wherein said probe compound comprises one or more optical labels.
9. The method of claim 1 , wherein the assay for detecting the identify of a microorganism or a group of microorganisms comprises generating a mass spectrum and comparing the mass spectrum to one or more libraries of standard mass spectra for the microorganism or the group of microorganism.
10. The method of claim 9 , wherein generating a mass spectrum utilizes one or more of the following: a time-of-flight (TOF) detector, a static electric and/or magnetic sector as a mass analyzer, a quadrupole mass analyzer, and an ion trap.
11. The method of claim 9 , wherein generating a mass spectrum utilizes one or more of the following ionization sources: chemical ionization, plasma and glow discharge, electron impact, electrospray ionization, desorption electrospray ionization, fast-atom bombardment, field ionization, laser ionization, liquid-extraction surface analysis, and matrix-assisted laser desorption ionization.
12. The method of claim 9 , wherein generating a mass spectrum comprises spotting a subsample from a reservoir of the SDGP onto a plate for matrix-assisted laser desorption ionization TOF mass spectrometry (MALDI-TOF), and performing MALDI-TOF, thereby generating the mass spectrum.
13. The method of claim 12 , wherein analysis of the mass spectrum identifies proteins and/or glycolipids of the microorganism or the group of microorganisms.
14. The method of claim 12 , wherein the microorganisms in a reservoir are concentrated before spotting on the MALDI-TOF target.
15. The method of claim 12 , wherein one or more biochemical assays are performed before MALDI-TOF is performed.
16. The method of claim 1 , wherein the patient sample is one or more of the following: blood, urine, cerebrospinal fluid, synovial fluid, an aspirate, a lavage, a wound swab, or a respiratory sample.
17. The method of claim 1 , wherein two or more sets of the same SDGP reservoirs are present on a consumable such that two or more patient samples may be inoculated into that consumable.
18. The method of claim 1 , wherein the assay for detecting the identify of a microorganism or a group of microorganisms comprises identifying a nucleic acid of the microorganism or the group of microorganisms.
19. The method of claim 18 , wherein the assay comprises sequencing the nucleic acid, hybridizing a probe to the nucleic acid, or performing an enzyme catalyzed reaction on the nucleic acid.
20. The method of claim 1 , wherein each selective medium comprises one or more media selected from the group consisting of: Streptococcus enrichment broth, Fraser broth, Giolitti Cantoni broth, Streptococcus faecalis media, sodium lauryl sulfate, tellurite, brilliant green media, brain heart infusion media with vancomycin, MacConkey media and purple media, bile esculin media, BCYE selective media with CCVC or CAV, BBE/LKV, Klinger iron media, triple sugar media, indole media, DNAse test media, Mio medium, phenol red media, eosin-methylene blue media, urea media, mannitol salt media with oxacillin, mannitol salt media without oxacillin, selective strep media, cetrimide media, and Leeds Acinetobacter media.