# Patent

## Title
Method for separation, characterization and/or identification of microorganisms using spectroscopy

## Abstract

The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation step. The method may be useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The invention further provides for spectroscopic interrogation of the separated microorganism sample to produce measurements of the microorganism and characterizing and/or identifying the microorganism in the sample using said spectroscopic measurements.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Patent Application No. 61/110,187, entitled, “Method and System for Detection and/or Characterization of a Biological Particle in a Sample”, filed Oct. 31, 2008, which is incorporated herein.


FIELD OF THE INVENTION
The present invention relates to methods and systems for detecting, isolating and/or identifying microorganisms in a sample. In particular, the present invention is directed to a method for rapid characterization and/or identification of a microorganism using spectroscopic techniques.
BACKGROUND OF THE INVENTION
The detection of pathogenic microorganisms in biological fluids should be performed in the shortest possible time, in particular in the case of septicemia for which the mortality remains high in spite of the broad range of antibiotics which are available to doctors. The presence of biologically active agents such as a microorganism in a patient's body fluid, especially blood, is generally determined using blood culture bottles. Bloodstream infections are associated with high morbidity and mortality, yet current diagnostic methods, of culture followed by biochemical identification and antibiotic susceptibility testing, can take several days to perform. Typically, empiric therapy is initiated based on clinical symptoms, and test results only impact clinical decisions when the initial therapy fails. The ability to characterize bloodstream infections within the first few hours, preferably within an hour, after a positive blood culture result would significantly boost the clinical relevance of the diagnostic information provided. Molecular amplification methods have been proposed to fill this need, but serious challenges to this approach remain. The positive blood culture broth itself represents a naturally amplified population of microorganisms with potential for use in a variety of rapid, identification (ID) tests.
Traditional automated phenotypic ID tests, such as the Vitek®, PhoenixTM and Microscan® systems, or manual phenotypic tests such as API require that microorganisms be in an appropriate growth phase and free of interfering media and blood products in order to provide robust results. These systems use colonies grown from the positive broth for 18-24 hours on plated media. However, in an effort to obtain faster results, some laboratories have reported using these systems with microorganisms isolated from positive blood culture bottles. These direct-from-the-bottle tests are not appropriate for all microorganisms (e.g., Gram-positive cocci), are not validated by the test manufacturers, and generally take 3-8 hours to provide results. Faster and more broadly specific tests are urgently needed in order to provide the physician with clinically relevant results within the first few hours, preferably within an hour, after a positive culture result.
Optical spectroscopy methods, such as intrinsic fluorescence (IF), infrared spectroscopy (FTIR), or Raman spectroscopy, and mass spectrometry methods such as MALDI-TOF, have the potential to allow for identification of microorganisms very quickly, but may encounter interference from the many highly fluorescent and absorptive compounds present in liquid microbiological culture media and in clinical samples such as blood or combinations thereof. The most commonly employed methods for recovering microorganisms directly from positive blood culture broth are two-step differential centrifugation and centrifugation in a serum separator tube.
Other methods for separation, characterization and/or identification of microorganisms have been described, include:
U.S. Pat. No. 4,847,198 discloses a method for the identification of microorganisms using UV excited Raman spectroscopy. According to the '198 patent, a bacterial suspension is contacted by a single wavelength in the ultra-violet range. A portion of the light energy used is absorbed and a portion of the light energy is emitted. The emitted light energy, resonance enhanced Raman scattering, is measured as backscattered energy. The energy is processed to produce spectra which are characteristic of the bacteria.
U.S. Pat. No. 5,938,617 to Vo-Dinh is directed to a system which identifies biological pathogens in a sample by exciting a sample with light at several wavelengths and synchronously sampling the emission intensities. The system includes mechanisms for exposing the sample to excitation radiation and thereby generating an emission radiation. The biological pathogens may be viruses and bacteria.
U.S. Pat. No. 6,177,266 discloses a method for the chemotaxonomic classification of bacteria with genus, species and strain specific biomarkers generated by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of either cellular protein extracts or whole cells.
In U.S. Pat. No. 7,070,739 a method is presented to extract, separate, and purify microbes including viruses by two-dimensional ultra-centrifuging directly from body fluids or homogenized tissue. In a first centrifuging step, all particles are removed having a sedimentation speed higher than those of the microbes to be identified. In the second ultra-centrifuging step, isopycnic banding is used in liquids filled in to form a wide-range density gradient, using special serrated centrifuge tubes. According to the patent, the separation technique can be used for detecting banded particles by light scatter or fluorescence using nucleic acid specific dyes, and for recovering the banded particles in very small volumes for characterization by mass spectrometry of viral protein subunits and intact viral particles, and by fluorescence flow cytometric determination of both nucleic acid mass and the masses of fragments produced by restriction enzymes.
U.S. Pat. Appl. Pub. No. 2007/0037135 discloses a system for the identification and quantification of a biological sample suspended in a liquid. The system includes a fluorescence excitation module with at least one excitation light source; a sample interface module optically coupled to the fluorescence excitation module for positioning a biological sample to receive excitation light from the at least one excitation light source; a fluorescence emission module optically coupled to the sample interface module and comprising at least one detection device for detecting fluorescence excitation-emission matrices of the biological sample; and a computer module operatively coupled to the fluorescence emission module. The computer module performs multivariate analysis on the fluorescence excitation-emission matrices of the biological sample to identify and quantify the biological sample. However, the '135 application does not discuss identification and quantification of microorganisms from complex biological samples, such as blood.
U.S. Pat. Appl. Pub. No. 2007/0175278 describes using a liquid culture medium for culturing a sample of interest, including for example, blood, urine, feces, intravenous catheters etc., industrial production lines, water systems, a food product, a cosmetic product, a pharmaceutical product and a forensic sample. Subsequently, the microorganisms can be harvested from the liquid medium by methods known in the art, e.g. by centrifugation. The concentrated microorganisms may then be transferred to carrier material, optionally after drying, for obtaining a vibrational spectrum. The patent application discusses various methods for identifying and classifying microorganisms, including vibrational spectroscopy, such as Raman spectroscopy.
However, these methods have several drawbacks when attempting to separate and characterize microorganisms from complex samples such as blood-containing culture media. The resultant microbial preparations often contain contaminating red blood cells, platelets, lipid particles, plasma enzymes and cellular debris, which can cause poor results. These methods are also very labor-intensive and unsafe due to steps which can result in aerosol exposure of potentially dangerous pathogens to the user. Simple, safe and reliable methods are needed to isolate microorganisms from clinical samples (e.g., blood culture broth) and other complex samples that are free of these interfering materials and compatible with rapid identification technologies.
SUMMARY OF THE INVENTION
The present invention provides methods for isolating, characterizing and/or identifying microorganisms in a sample. The methods allow for the characterization and/or identification of microorganisms more quickly than prior techniques, resulting in faster diagnoses (e.g., in a subject having or suspected of having septicemia) and identification of contaminated materials (e.g., foodstuffs and pharmaceuticals). The steps involved in the methods of the invention, from obtaining a sample to characterization and/or identification of microorganisms, can be carried out in a very short time frame to produce clinically relevant actionable information, e.g., in less than about 120 minutes. Additionally, the methods of the invention can be fully automated, thereby reducing the risk of handling infectious materials and/or contaminating samples.
In one aspect, the present invention is directed to a method of characterizing and/or identifying a microorganism from a test sample, comprising:
(a) obtaining a test sample known to contain or that may contain microorganisms;
(b) selectively lysing non-microorganism cells in said test sample to produce a lysed sample;
(c) separating microorganisms from other components of said lysed sample to form an isolated sample of microorganisms;
(d) spectroscopically interrogating said isolated microorganisms to produce spectroscopic measurements of said microorganism; and
(e) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In one aspect, the present invention is directed to a method of characterizing and/or identifying a microorganism from a blood culture, comprising:
(a) obtaining a sample from a blood culture known to contain or that may contain microorganisms;
(b) selectively lysing non-microorganism cells in said sample to produce a lysed sample;
(c) layered said lysed sample on a density cushion in a sealed container;
(d) centrifuging the container to separate microorganisms from other components of said sample and form a pellet of microorganisms;
(e) spectroscopically interrogating said isolated microorganisms to produce spectroscopic measurements of said microorganism; and
(f) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In another aspect, the present invention is directed to a method of characterization and/or identifying a microorganism, comprising:
(a) obtaining a test sample known to contain or that may contain microorganisms;
(b) placing said test sample in a sealed container;
(c) separating microorganisms in situ from other components of said test sample to form an isolated sample of microorganisms of microorganisms in said sealed container;
(d) spectroscopically interrogating said isolated microorganisms in situ to produce spectroscopic measurements of said microorganism; and
(e) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In one embodiment, the separation is carried out by layering the test sample over a density cushion in a sealed container (e.g., a hermetically sealed container) and centrifuging the container to pellet the microorganisms while the test sample medium remains on top of the density cushion. In another embodiment, the container has an optical window at the bottom and/or sides so that the microorganism pellet can be interrogated spectroscopically. The microorganisms can be identified by comparing the spectrum of the pellet to a spectrum or spectra, or spectroscopic properties predicted, of known microorganisms. The ability to identify microorganisms directly in the pellet without further handling significantly improves the safety value of this microbial identification method.
In one embodiment, the spectroscopic interrogation is based on intrinsic characteristics of the microorganisms (e.g., intrinsic fluorescence). In other embodiments, the spectroscopic interrogation is based in part on signals obtained from additional agents that are added during the methods of the invention and interact with specific microorganisms or groups of microorganisms.
In another embodiment, the methods further comprise a step of recovering the microorganism pellet, resuspending the microorganism and performing further identification or characterization tests (e.g., drug resistance, virulence factors, antibiogram).
The present invention is explained in greater detail in the figures herein and the description set forth below.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1B shows photographs of separation containers after carrying out the lysis, separation steps of the present invention on a S. pneumoniae culture and a negative culture broth.
FIGS. 2A-2C show example excitation/emission spectra of isolated microorganisms using lysis buffers A, B and C in the lysis step preceding interrogation.
FIG. 3 shows photographs of separation containers after carrying out the lysis- and separation steps of the present invention using five different lysis buffers and two density cushions.
FIG. 4 is a photograph of a separation device showing a post-centrifugation of lysed microorganism-containing blood culture broth. Clearly visible in the photograph are the lysed blood culture, density cushion and microorganism pellet.
FIGS. 5-8 show examples of excitation/emission spectra for various microorganisms read in a sealed separation container.
FIG. 9 is a graphic showing the average intrinsic fluorescence signal of pellets obtained from ten S. mitis (circles) and for ten S. pneumoniae (triangles) cultures.
FIG. 10 is a graphic showing the average pellet size for pellets obtained from ten S. mitis (circles) and ten S. pneumoniae (triangles) cultures.



DETAILED DESCRIPTION OF THE INVENTION
The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
DEFINITIONS
As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
As used herein, the term “microorganism” is intended to encompass organisms that are generally unicellular, which can be multiplied and handled in the laboratory, including but not limited to, Gram-positive or Gram-negative bacteria, yeasts, molds, parasites, and mollicutes. Non-limiting examples of Gram-negative bacteria of this invention include bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, Brevundimonas, Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella, Pasteurella, Providencia, and Legionella. Non-limiting examples of Gram-positive bacteria of this invention include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria. Non-limiting examples of yeasts and molds of this invention include those of the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces and Trichosporon. Non-limiting examples of parasites of this invention include those of the following genera: Trypanosoma, Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, and Naegleria. Non-limiting examples of mollicutes of this invention include those of the following genera: Mycoplasma and Ureaplasma. 
In one embodiment, as described in further detail herein, microorganisms from a sample or growth medium can be separated and interrogated to characterize and/or identify the microorganism present in the sample. As used herein, the term “separate” is intended to encompass any sample of microorganisms that has been removed, concentrated or otherwise set apart from its original state, or from a growth or culture medium. For example, in accordance with this invention, microorganisms may be separated away (e.g., as a separated sample) from non-microorganism or non-microorganism components that may otherwise interfere with characterization and/or identification. The term may include a layer of microorganisms sandwiched between two other layers, e.g., microorganisms collected on top of a high-density cushion after centrifugation, or a layer of microorganisms collected on a solid surface (e.g., a filter membrane). The term may also include a collection of microorganisms that has passed partially through a layer (e.g., a density cushion). As such, a separated microorganism sample may include any collection or layer of microorganisms and/or components thereof that is more concentrated than, or otherwise set apart from, the original sample, and can range from a closely packed dense clump of microorganisms to a diffuse layer of microorganisms. Microorganism components that can be comprised in a separated form or sample include, without limitation, pilli, flagella, fimbriae, and capsules in any combination. Non-microorganism components that are separated away from the microorganisms may include non-microorganism cells (e.g., blood cells and/or other tissue cells) and/or any components thereof.
In yet another embodiment, as described in further detail herein, microorganisms from a sample or growth medium can be isolated and interrogated to characterize and/or identify the microorganism present in the sample. As used herein, the term “isolated” is intended to encompass any sample of microorganisms that has been at least partially purified from its original state, or from a growth or culture medium, and any non-microorganisms or non-microorganism components contained therein. For example, in accordance with this invention, microorganisms may be isolated away (e.g., as an isolated sample) from non-microorganisms or non-microorganism components that may otherwise interfere with characterization and/or identification. Non-microorganism components that are separated away from the microorganisms may include non-microorganism cells (e.g., blood cells and/or other tissue cells) and/or any components thereof.
In yet another embodiment, as described in further detail herein, microorganisms from a sample or growth medium can be pelleted and interrogated to characterize and/or identify the microorganism present in the sample. As used herein, the term “pellet” is intended to encompass any sample of microorganisms that has been compressed or deposited into a mass of microorganisms. For example, microorganisms from a sample can be compressed or deposited into a mass at the bottom of a tube by centrifugation, or other known methods in the art. The term includes a collection of microorganisms (and/or components thereof) on the bottom and/or sides of a container following centrifugation. Microorganism components that can be comprised in a pellet include, without limitation, pilli, flagella, fimbriae, and capsules in any combination. In accordance with this invention, microorganisms may be pelleted away (e.g., as a substantially purified microorganism pellet) from non-microorganism or non-microorganism components that may otherwise interfere with characterization and/or identification. Non-microorganism components that are separated away from the microorganisms may include non-microorganism cells (e.g., blood cells and/or other tissue cells) and/or any components thereof.
As used herein, the term “density cushion” refers to a solution having a homogenous density throughout.
The present invention provides methods for isolating, characterizing and/or identifying microorganisms in a sample. Moreover, the method may be particularly useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The rapid methods also allow for the characterization and/or identification of microorganisms more quickly than prior techniques, resulting in faster diagnoses (e.g., in a subject having or suspected of having septicemia) and characterization/identification of contaminated materials (e.g., foodstuffs and pharmaceuticals). The steps involved in the methods of the invention, from obtaining a sample to characterization/identification of microorganisms, can be carried out in a very short time frame to obtain clinically relevant actionable information. In certain embodiments, the methods of the invention can be carried out in less than about 120 minutes, e.g., in less than about 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 minute. The tremendous rapidity of the methods of the invention represents an improvement over prior methods. The methods can be used to characterize and/or identify any microorganism as described herein. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a yeast. In another embodiment, the microorganism is a mold. In a further embodiment, the microorganism is a parasite. In another embodiment, the microorganism is a mollicute. Additionally, the methods of the invention can be fully automated, thereby reducing the risk of handling infectious materials and/or contaminating the samples.
In one aspect, the present invention is directed to a method of characterizing and/or identifying a microorganism from a test sample, comprising:
(a) obtaining a test sample known to contain or that may contain microorganisms;
(b) selectively lysing non-microorganism cells in said test sample to produce a lysed sample;
(c) separating microorganisms from other components of said test sample to form an isolated sample of microorganisms;
(d) spectroscopically interrogating said isolated microorganisms to produce spectroscopic measurements of said microorganism; and
(e) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In one aspect, the present invention is directed to a method of characterizing and/or identifying a microorganism from a blood culture, comprising:
(a) obtaining a sample from a blood culture known to contain or that may contain microorganisms;
(b) selectively lysing non-microorganism cells in said sample to produce a lysed sample;
(c) layered said lysed sample on a density cushion in a sealed container;
(d) centrifuging the container to separate microorganisms from other components of said sample and form a pellet of microorganisms;
(e) spectroscopically interrogating said isolated microorganisms to produce spectroscopic measurements of said microorganism; and
(f) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In yet another aspect, the present invention is directed to a method of characterization and/or identifying a microorganism, comprising:
(a) obtaining a test sample known to contain or that may contain microorganisms;
(b) placing said test sample in a sealed container (e.g., a hermetically sealed container);
(c) separating microorganisms in situ from other components of said test sample to form an isolated sample of microorganisms of microorganisms in said sealed container;
(d) spectroscopically interrogating said isolated microorganisms in situ to produce spectroscopic measurements of said microorganism; and
(e) characterizing and/or identifying said microorganism in said isolated sample by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms.
In another embodiment of the invention, the methods involve recovering the pellet of microorganisms formed during the separation step or a portion thereof from the separation container prior to interrogation of the microorganisms. For example, after formation of the pellet, the fluids can be aspirated way from the pellet and the pellet resuspended in a suitable medium (e.g., a medium in which the microorganisms are viable). The resuspended microorganisms can be removed from the separation container. The microorganisms can then be interrogated for characterization and/or identification, e.g., in the suspension or after they have been repelleted. In other embodiments, the resuspended microorganisms can be interrogated in the separation container, e.g., in the suspension or after they have been repelleted. In a further embodiment, microorganisms recovered from the pellet can be used directly for further interrogation (e.g., Raman spectroscopy, mass spectrometry) without being resuspended.
Samples
Samples that may be tested (i.e., a test sample) by the methods of the invention include both clinical and non-clinical samples in which microorganism presence and/or growth is or may be suspected, as well as samples of materials that are routinely or occasionally tested for the presence of microorganisms. The amount of sample utilized may vary greatly due to the versatility and/or sensitivity of the method. Sample preparation can be carried out by any number of techniques known to those skilled in the art although one of the advantages of the present invention is that complex sample types, such as, e.g., blood, bodily fluids, and/or other opaque substances, may be tested directly utilizing the system with little or no extensive pretreatment. In one embodiment, the sample is taken from a culture. In another embodiment, the sample is taken from a microbiological culture (e.g., a blood culture). In another embodiment, the sample is suspected of, or known to, contain microorganisms therein.
Clinical samples that may be tested include any type of sample typically tested in clinical or research laboratories, including, but not limited to, blood, serum, plasma, blood fractions, joint fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and swab rinsates, other body fluids, and the like. In another embodiment, the clinical sample can be cultured, and a culture sample used.
The present invention finds use in research as well as veterinary and medical applications. Suitable subjects from which clinical samples can be obtained are generally mammalian subjects, but can be any animal. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. Subjects from which samples can be obtained include, without limitation, mammals, birds, reptiles, amphibians, and fish.
Non-clinical samples that may be tested also include substances, encompassing, but not limited to, foodstuffs, beverages, pharmaceuticals, cosmetics, water (e.g., drinking water, non-potable water, and waste water), seawater ballasts, air, soil, sewage, plant material (e.g., seeds, leaves, stems, roots, flowers, fruit), blood products (e.g., platelets, serum, plasma, white blood cell fractions, etc.), donor organ or tissue samples, biowarfare samples, and the like. The method is also particularly well suited for real-time testing to monitor contamination levels, process control, quality control, and the like in industrial settings. In another embodiment, the non-clinical sample can be cultured, and a culture sample used.
In one embodiment of the invention, samples are obtained from a subject (e.g., a patient) having or suspected of having a microbial infection. In one embodiment, the subject has or is suspected of having septicemia, e.g., bacteremia or fungemia. The sample may be a blood sample directly from the subject. The sample may be from a blood culture grown from a sample of the patient's blood, e.g., a BacT/ALERT® blood culture. The blood culture sample may be from a positive blood culture, e.g., a blood culture that indicates the presence of a microorganism. In certain embodiments, the sample is taken from a positive blood culture within a short time after it turns positive, e.g., within about 6 hours, e.g., within about 5, 4, 3, or 2 hours, or within about 60 minutes, e.g., about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 minute. In one embodiment, the sample is taken from a culture in which the microorganisms are in log phase growth. In another embodiment, the sample is taken from a culture in which the microorganisms are in a stationary phase.
The present invention provides high sensitivity for the detection, characterization and/or identification of microorganisms. This enables detection, characterization and/or identification without first having to go through the steps of isolating microorganisms by growing them on a solid or semisolid medium, and sampling the colonies that grow. Thus, in one embodiment of the invention, the sample is not from a microbial (e.g., bacteria, yeast, or mold) colony grown on a solid or semisolid surface. Thus, in one embodiment of the invention, the sample is not from a microbial (e.g., bacteria, yeast, or mold) colony grown on a solid or semisolid surface.
The volume of the sample should be sufficiently large to produce an isolated sample of microorganisms or a pellet of microorganisms which can be interrogated after the separation/isolation step of the methods of the invention is carried out. Appropriate volumes will depend on the source of the sample and the anticipated level of microorganisms in the sample. For example, a positive blood culture will contain a higher level of microorganisms per volume than a drinking water sample to be tested for contamination, so a smaller volume of blood culture medium may be needed as compared to the drinking water sample. In general, the sample size can be less than about 50 ml, e.g., less than about 40, 30, 20, 15, 10, 5, 4, 3, or 2 ml. In certain embodiments, the sample size can be about 1 ml, e.g., about 0.75, 0.5, or 0.25 ml. In certain embodiments in which the separation is carried out on a microscale, the sample size can be less than about 200 e.g., less than about 150, 100, 50, 25, 20, 15, 10, or 5 μl. In some embodiments (e.g., when the sample is expected to comprise a small number of microorganisms), the sample size can be about 100 ml or more, e.g., about 250, 500, 750, or 1000 ml or more.
Optional Lysis Step
In some embodiments, after obtaining a sample, the next step in the method of the present invention is to selectively lyse undesired cells that may be present in the sample, e.g., blood cells and/or tissue cells. Cells may be lysed to permit separation of microorganisms from other components of the sample. The separation of microorganisms from other components prevents interference during the interrogation step. If non-microorganism cells are not expected to be present in the sample or not expected to interfere with the interrogation step, the lysis step may not need to be carried out. In one embodiment, the cells to be lysed are non-microorganism cells that are present in the sample and no microorganism cells that may be present in the sample are lysed. However, in some embodiments, the selective lysing of specific classes of microorganisms may be desirable and thus can be carried out according to the methods described herein and as are well known in the art. For example, a class of undesired microorganisms can be selectively lysed, e.g., yeast are lysed while bacteria are not or vice versa. In another embodiment, the desired microorganisms are lysed in order to separate a particular subcellular component of the microorganisms, e.g., cell membranes or organelles. In one embodiment, all of the non-microbial cells are lysed. In other embodiments, a portion of the non-microbial cells are lysed, e.g., enough cells to prevent interference with the interrogation step. The lysing of cells may be carried out by any method known in the art to be effective to selectively lyse cells with or without lysing microorganisms, including, without limitation, addition of a lysis solution, sonication, osmotic shock, chemical treatment, and/or a combination thereof.
A lysis solution is one that is capable of lysing cells, e.g., non-microorganism cells (e.g., by solubilizing eukaryotic cell membranes) and/or microorganism cells. In one embodiment, the lysis solution can comprise one or more detergents, one or more enzymes, or a combination of one or more detergents and one or more enzymes, and can further include additional agents. In one embodiment, the detergent can be a non-denaturing lytic detergent, such as octylphenol ethoxylate (e.g., Triton® X-100 Triton® X-100-R, Triton® X-114), NP-40, Genapol® C-100, Genapol® X-100, (Octylphenoxy)polyethoxyethanol (e.g., Igepal® CA 630, ArlasolveTM200, polyoxyethylene 10 oleoyl ether (e.g., Brij® 96/97), CHAPS, octyl β-D-glucopyranoside, saponin, and nonaethylene glycol monododecyl ether (C12E9, polidocenol). Optionally, denaturing lytic detergents can be included, such as sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO. Optionally, solubilizers can also be included, such as polyoxyethylene (20) oleyl ether (e.g, Brij® 98), polyoxyethylene 20 cetyl ether (e.g., Brij® 58), polyoxyethylene (23) lauryl ether (e.g., Brij® 35), polyoxyethylenesorbitan monooleate (e.g., Tween® 80), polyoxyethylene sorbitol ester (e.g., Tween® 20), polyoxyalkylene ether (e.g., Pluronic® L64, Pluronic® P84), non-detergent sulfobetaines (NDSB 201), amphipols (PMAL-C8), and methyl-β-cyclodextrin. Typically, non-denaturing detergents and solubilizers are used at concentrations above their critical micelle concentration (CMC), while denaturing detergents may be added at concentrations below their CMC. For example, non-denaturing lytic detergents can be used at a concentration of about 0.010% to about 10%, e.g., about 0.015% to about 1.0%, e.g., about 0.05% to about 0.5%, e.g., about 0.10% to about 0.30% (final concentration after dilution with the sample). In another embodiment, polyoxyethylene detergent detergents may be preferred. The polyoxyethylene detergent can comprise the structure C12-18/E9-10, wherein C12-18 denotes a carbon chain length of from 12 to 18 carbon atoms and E9-10 denotes from 9 to 10 oxyethylene hydrophilic head groups. For example, the polyoxyethylene detergent can be selected from the group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol® X-100, nonaethylene glycol monododecyl ether (polidocanol), or a combination thereof.
Enzymes that can be used in lysis solutions include, without limitation, enzymes that digest nucleic acids and other membrane-fouling materials (e.g., proteinase XXIII, DNase, neuraminidase, polysaccharidase, Glucanex®, and Pectinex®). Other additives that can be used include, without limitation, reducing agents such as 2-mercaptoethanol (2-Me) or dithiothreitol (DTT) and stabilizing agents such as magnesium, pyruvate, and humectants. The lysis solution can be buffered at any pH that is suitable to lyse the desired cells, and will depend on multiple factors, including without limitation, the type of sample, the cells to be lysed, and the detergent used. In some embodiments, the pH can be in a range from about 2 to about 13, e.g., about 6 to about 13, e.g., about 8 to about 13, e.g., about 10 to about 13. Suitable pH buffers include any buffer capable of maintaining a pH in the desired range, e.g., about 0.05 M to about 1.0 M CAPS.
In one embodiment, the sample and the lysis solution are mixed and then incubated for a sufficient time for lysis and solubilization of cell membranes to occur, e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 60 seconds, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 minutes or longer, e.g., about 1 second to about 20 minutes, about 1 second to about 5 minutes, or about 1 second to about 2 minutes. The incubation time will depend on the strength of the lysis solution, e.g., the concentration of the detergent and/or enzymes. In general, milder lysis buffers will require more time and a greater dilution of the sample to fully solubilize non-microbial cells. The strength of the lysis solution can be selected based on the microorganisms known to be or suspected to be in the sample. For microorganisms that are more susceptible to lysis, a mild lysis solution can be used. The lysis can take place at a temperature of about 2° C. to about 45° C., e.g., about 15° C. to about 40° C., e.g., about 30° C. to about 40° C. In one embodiment, the lysis solution can be loaded into a syringe and the sample can then be aspirated into the syringe such that mixing and incubation occurs within the syringe. In one embodiment, the lysis solution can be loaded into a syringe and the sample can then be aspirated into the syringe such that mixing and incubation occurs within the syringe.
In some embodiments, the lysis conditions (e.g., the solution or the incubation time), as well as the separation and/or interrogation steps, can be sufficient to kill some or all of the microorganisms in the sample. The methods of the present invention are highly versatile and do not require that all microorganisms be alive for the isolation and identification to occur. In certain embodiments, some or all of the microorganisms may be dead, with death occurring before, during, and/or after the steps of the methods being carried out.
Separation Step
The next step in the method of the present invention (e.g., the step after the sample has been lysed, if a lysing step is performed) is a separation step. The separation step can be carried out to separate the microorganisms from other components of the sample (e.g., non-microorganisms or components thereof) and to concentrate the microorganisms into a pellet that can be interrogated for identification and characterization purposes. The separation does not have to be complete, i.e., it is not required that 100% separation occur. All that is required is that the separation of the microorganisms from other components of the sample be sufficient to permit interrogation of the microorganisms without substantial interference from the other components. For example, the separation can result in a microorganism pellet that is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% pure or higher.
In one embodiment, the separation is carried out by a centrifugation step in which the sample (e.g., a lysed sample) is placed on top of a density cushion in a separation container and the container centrifuged under conditions which allow the microorganisms to be isolated (e.g., the microorganisms can form a pellet at the bottom and/or sides of the container). In accordance with this embodiment, other components of the sample (e.g., non-microorganisms or components thereof that may be present in the sample medium) stay on top of the density cushion or within the top portion of the density cushion. In general, any known container may be used for the separation step. In one embodiment, the separation container is the separation device disclosed in related U.S. patent application Ser. No. 12/589,969, entitled “Separation Device for Use in the Separation, Characterization and/or Identification of Microorganisms”, filed Oct. 30, 2009. This separation step isolates the microorganisms away from materials in the sample, such as medium, cell debris, and/or other components that might interfere with interrogation of the microorganisms (e.g., by intrinsic fluorescence). In one embodiment, the density cushion also serves to separate live microorganisms from dead microorganisms (which do not pass through the density cushion). In another embodiment the density cushion does not comprise a density gradient, either before or after the centrifugation. In other words, the separation container is not centrifuged for a sufficient amount of time and/or acceleration for the material making up the density cushion to form a density gradient.
The density of the cushion is selected such that the microorganisms in the sample pass through the cushion while other components of the sample (e.g., blood culture broth, cell debris) remain on top of the cushion or do not pass all of the way through the density cushion. The density cushion may also be selected to separate live microorganisms (which pass through the cushion) from dead microorganisms (which do not pass through the cushion). Suitable densities will depend on the material used in the density cushion and on the sample to be separated. In one embodiment, the density of the cushion is in the range of about 1.025 to about 1.120 g/ml, e.g., about 1.030 to about 1.070 g/ml, about 1.040 to about 1.060 g/ml or any range between about 1.025 to about 1.120 g/ml. In another embodiment, the density of the cushion is about 1.025, 1.030, 1.035, 1.040, 1.045, 1.050, 1.055, 1.060, 1.065, 1.070, 1.075, 1.080, 1.085, 1.090, 1.095, 1.100, 1.105, 1.110, 1.115, or 1.120 g/ml.
The material for the density cushion can be any material that has the appropriate density range for the methods of this invention. In one embodiment, the material is colloidal silica. The colloidal silica may be uncoated (e.g., Ludox® (W.R. Grace, CT)) or coated, e.g., with silane (e.g., PureSperm® (Nidacon Intl, Sweden) or Isolate® (Irvine Scientific, Santa Ana, Calif.)) or polyvinylpyrrolidone (e.g., PercollTM, PercollTM Plus (Sigma-Aldrich, St. Louis, Mo.)). In one embodiment, the colloidal silica exhibiting the least interference with spectroscopic interrogation is selected, e.g., the material with the lowest intrinsic fluorescence. The colloidal silica may be diluted in any suitable medium to form the proper density, e.g., balanced salt solutions, physiological saline, and/or 0.25 M sucrose. Suitable densities can be obtained with colloidal silica at a concentration of about 15% to about 80% v/v, e.g., about 20% to about 65% v/v. Another suitable material for density cushions is an iodinated contrast agent, e.g., iohexyl (OmnipaqueTM NycoPrepTM, or Nycodenz®) and iodixanol (VisipaqueTM or OptiPrepTM). Suitable densities can be obtained with iohexyl or iodixanol at a concentration of about 10% to about 25% w/v, e.g., about 14% to about 18% w/v, for blood culture samples. Sucrose can be used as a density cushion at a concentration of about 10% to about 30% w/v e.g., about 15% to about 20% w/v, for blood culture samples. Other suitable materials that can be used to prepare the density cushion include low viscosity, high density oils, such as microscope immersion oil (e.g., Type DF; Cargille Labs, New York), mineral oil (e.g., Drakeol® 5, Draketex 50, Peneteck®; Penreco Co., Pennsylvania), silicone oil (polydimethylsiloxane), fluorosilicone oil, silicone gel, metrizoate-Ficoll® (LymphoPrepTM), e.g., at a concentration of about 75% to about 100% for blood culture samples, diatrizoate-dextran (PolymorphoPrepTM), e.g., at a concentration of about 25% to about 50% for blood culture samples, carboxymethyl cellulose, hydroxypropylmethyl cellulose, polyethylene oxide (high molecular weight), polyoxyalkylene ether (e.g., Pluronic® F127, Pluronic® F68), mixtures of Pluronic® compounds, polyacrylic acid, cross-linked polyvinyl alcohol, cross-linked polyvinyl pyrrolidine, PEG methyl ether methacrylate, pectin, agarose, xanthan, gellan, Gellan Gum (e.g., PhytagelTM), sorbitol, a sucrose and epichlorohydrin copolymer (Ficoll® (e.g., Ficoll® 400 at a concentration of about 10% to about 15% for blood culture samples)), glycerol, dextran (e.g., at a concentration of about 10% to about 15% for blood culture samples), glycogen, cesium chloride (e.g., at a concentration of about 15% to about 25% for blood culture samples), perfluorocarbon fluids (e.g., perfluoro-n-octane), hydrofluorocarbon fluids (e.g., Vertrel XF), and the like as are well known in the art. In one embodiment, the density cushion is selected from one or more of colloidal silica, iodixanol, iohexyl, cesium chloride, metrizoate-Ficoll®, diatrizoate-dextran, sucrose, Ficoll® 400, and/or dextran in any combination. The density cushion can also be made up of a combination of materials, e.g., a combination of colloidal silica and oil. Certain combinations of the above compounds may be beneficial for the separation and reading steps of the present invention. For example, combinations of compounds with different UV-quenching properties, such as cesium chloride and iohexyl.
The volume/height of the density cushion should be sufficient to achieve separation of the microorganisms from other sample components. The volume will depend on the size and shape of the separation container. In general, a volume of about 0.1 to about 5 ml can be used, e.g., about 0.2 to about 1 ml, e.g., about 0.2 ml to about 0.5 ml. If the separation is performed on a microscale, the volume of the density cushion can be about 1 μl to about 100 μl e.g., about 5 μl to about 50 μl. The volume of sample laid or layered on top of the density cushion should be sufficient to provide enough microorganisms to produce a pellet suitable for interrogation. In general, any volume that fits into the container can be used. For example, a volume of about 0.1 ml to about 5 ml can be used, e.g., about 0.2 ml to about 1 ml, e.g., about 0.2 ml to about 0.5 ml. If the separation is performed on a microscale, the volume of sample can be about 1 μl to about 100 μl, e.g., about 5 μl to about 50 μl. The available space in the container for sample will depend on the size and shape of the container. In some embodiments, an intermediate layer (liquid or solid) can be placed on top of the density cushion before the sample is laid or layered on top in order to prevent any mixing of the density cushion and the sample. In one embodiment, the intermediate layer can be polyethylene beads. In another embodiment, a small air bubble can be positioned between the density cushion and the sample to prevent mixing. In a further embodiment, the density cushion can be layered on top of a high density material (e.g., a perfluorocarbon fluid) such that the microorganisms pass through the density cushion during the separation and collect at the interface between the density cushion and the high density material.
In one embodiment of the invention, the separation container is centrifuged in a swing out rotor so that the microorganisms form a pellet directly on the bottom of the container. The container is centrifuged at a sufficient acceleration and for a sufficient time for the microorganisms to be separated (e.g., a pellet formed) from other components of the sample. The centrifugation acceleration can be about 1,000×g to about 20,000×g, e.g., about 2,500×g to about 15,000×g, e.g., about 7,500×g to about 12,500×g, etc. The centrifugation time can be about 30 seconds to about 30 minutes, e.g., about 1 minute to about 15 minutes, e.g., about 1 minute to about 5 minutes. The centrifugation can be carried out at a temperature of about 2° C. to about 45° C., e.g., about 15° C. to about 40° C., e.g., about 20° C. to about 30° C. In one embodiment, the separation container comprises a closure, and the closure is applied to the container to form a hermetic seal prior to centrifugation. The presence of a closure decreases the risks from handling microorganisms that are or may be infectious and/or hazardous, as well as the risk of contaminating the sample. One of the advantages of the methods of the invention is the ability to carry out any one or more of the steps of the methods (e.g., lysis, separation, interrogation, and/or identification) with the microorganisms in a sealed container (e.g., a hermetically sealed container). The present methods, involving the use of automated systems, avoid the health and safety risks associated with handling of highly virulent microorganisms, such as occurs with recovery of microorganisms from samples for direct testing. In one embodiment, the container is not centrifuged for a sufficient time and/or force for a density gradient to form within the density cushion. The present invention does not involve ultracentrifugation of samples, e.g., centrifugation at forces greater than about 100,000×g. Further, the present invention does not involve isopycnic (equilibrium) sedimentation or banding.
The separation container may be any container with sufficient volume to hold a density cushion and a sample. As noted herein, the separation device disclosed in related U.S. patent application Ser. No. 12/589,969, entitled “Separation Device for Use in the Separation, Characterization and/or Identification of Microorganisms”, filed Oct. 30, 2009, may be used in the practice of this invention. In one embodiment, the container fits or can be fitted into a centrifuge rotor. The volume of the container can be about 0.1 ml to about 25 ml, e.g., about 1 ml to about 10 ml, e.g., about 2 ml to about 8 ml. If the separation is done on a microscale, the volume of the container can be about 2 μl to about 100 μl, e.g., about 5 μl to about 50 μl. In one embodiment, the container has a wide internal diameter in an upper portion to hold the sample and the majority of the density cushion, and a more narrow internal diameter in a lower portion where the pellet of microorganisms is collected. The narrow portion can have an internal diameter of about 0.04 to about 0.12 inches, e.g., about 0.06 to about 0.10 inches, e.g., about 0.08 inches. The wide portion can have an internal diameter of about 0.32 to about 0.40 inches, e.g., about 0.34 to about 0.38 inches, e.g., about 0.36 inches. For microscale separations, the internal diameters can be even smaller. For example, the internal diameter of the narrow portion can be about 0.001 to about 0.04 inches, e.g., about 0.002 to about 0.01 inches. A tapered internal diameter portion can connect the upper and lower portions. The tapered portion can have an angle of about 20 to about 70 degrees, e.g., about 30 to about 60 degrees. In one embodiment, the lower narrow portion is less than half of the total height of the container, e.g., less than about 40%, 30%, 20%, or 10% of the total height of the container. The container can have a closure device attached or may be threaded to accept a closure device (e.g., a cap) such that the container can be hermetically sealed during centrifugation. In certain embodiments, the container is designed such that the microorganism sample or pellet can be readily recovered, or otherwise obtained or removed from the container after separation, either manually or in an automated manner (so that technicians are not exposed to the container contents). For example, the container can comprise a removable portion or a break-away portion which contains the pellet and which can be separated from the rest of the container. In another embodiment, the container comprises means for access to the pellet after separation, such as one or more ports or permeable surfaces for insertion of a syringe or other sampling device or for drawing off the pellet. In one embodiment, the container can be a tube, e.g., a centrifuge tube. In another embodiment, the container can be a chip or a card. In one embodiment, the container is a stand alone container, i.e., a device for separating a single sample. In other embodiments, the container is part of a device that comprises two or more separation containers such that multiple samples can be separated at the same time. In one embodiment, the device comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 36, 42, 48, 60, 72, 84, 96, or more separation containers.
The container can comprise an optical window through which the interrogation can occur. The optical window may be on the bottom, top, and/or sides of the container. The window can be composed of any material that is transparent to light (e.g., at least a portion of the near infrared (NIR; 700 nm-1400 nm), ultraviolet (UV; 190 nm-400 nm) and/or visible (VIS; 400 nm-700 nm) light spectrum. Examples of suitable materials include, without limitation, acrylic, methacrylate, quartz, fused silica, sapphire, and/or a cyclic olefin copolymer (COC). In one embodiment, the entire container is made of optical window material. In another embodiment, the container may be prepared (e.g., molded) from two or more separate parts, such as an optical UV-VIS-NIR transparent component for the optical window and another material (e.g., a lower-cost standard molding plastic) to make up the rest of the container. In one embodiment, the optical window is thin enough to permit spectroscopic interrogation, which will depend on the material of the window. In another embodiment, the optical window is as thin as possible to reduce interference with spectroscopic interrogation. For example, the window can have a thickness of less than about 0.20 inches, e.g., less than about 0.15, 0.10, or 0.05 inches.
In another embodiment, the separation is carried out by a filtration step in which the sample (e.g., a lysed sample) is placed in a device fitted with a selective filter or filter set with pore sizes that retain the microorganisms. The retained microorganisms may be washed by gently passing a suitable buffer through the filter. The washed microorganisms may then be interrogated directly on the filter and/or recovered for interrogation by directly sampling the surface of the filter or by back-flushing the filter with suitable aqueous buffer.
Interrogation Step
Once the microorganisms have been separated, isolated and/or pelleted, the separated sample, isolated sample or pellet can be interrogated to identify and/or characterize the microorganisms in the sample or pellet. In one embodiment, the interrogation takes place in a non-invasive manner, that is, the pellet is interrogated while it remains in the separation container. In another embodiment, the separation container remains sealed throughout the interrogation. The ability to identify the microorganisms in a non-invasive manner, optionally coupled with keeping the container sealed throughout the separation and identification/characterization process and automating some or all of the procedure avoids the constant handling of contaminated and/or infectious samples and greatly increases the safety of the entire process. Furthermore, the ability to characterize and/or identify microorganisms by direct interrogation without further processing of the sample or pellet (e.g., resuspension, plating, and growth of colonies), greatly increases the speed with which identification/characterization can be made. In one embodiment, the sample or pellet is recovered and/or resuspended and optionally removed from the separation container prior to interrogation. In another embodiment, the sample or pellet is recovered and/or resuspended after in situ interrogation and further interrogation is then carried out. For example, techniques such as latex agglutination tests or automated phenotypic identification tests that can be applied to isolated microorganisms but not a pellet of microorganisms can be carried out on the recovered and/or resuspended microorganisms.
In some embodiments, the isolated sample or pellet can be interrogated spectroscopically. In one embodiment, optical spectroscopic methods can be used to analyze one or more intrinsic properties of the microorganisms, e.g., a property present within the microorganism in the absence of additional agents, such as stains, dyes, binding agents, etc. In other embodiments, the optical spectroscopic methods can be used to analyze one or more extrinsic properties of the microorganisms, e.g., a property that can only be detected with the aid of additional agents. The interrogation can be carried out using, for example, fluorescence spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, terahertz spectroscopy, transmission and absorbance spectroscopy, Raman spectroscopy, including Surface Enhanced Raman Spectroscopy (SERS), Spatially Offset Raman spectroscopy (SORS), transmission Raman spectroscopy, and/or resonance Raman spectroscopy. To enhance Raman (SERS) and fluorescence signals, microorganisms could either be coated with gold and/or silver nanoparticles prior to centrifugation, and/or the inner optical surface could be pre-coated with metal colloids of particular size and shape (refs: Lakowicz, Anal. Biochem. 337:171 (2005) for fluorescence; Efrima et al., J. Phys. Chem. B. (Letter) 102:5947 (1998) for SERS). In another embodiment, the nanoparticles are present in the density cushion prior to centrifugation and associate with microorganisms as the microorganisms pass through the density cushion. In other embodiments, the microorganisms in the pellet can be interrogated using mass spectrometry techniques, such as MALDI-TOF mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, GC mass spectrometry, LC mass spectrometry, electrospray ionization (ESI) mass spectrometry, and Selected Ion Flow Tube (SIFT) spectrometry. In one embodiment, the isolated sample or pellet is interrogated while it remains in the separation container. The container can be interrogated through an optical window in the container. The optical window may be on the bottom and/or any side or sides and/or on the top of the container. In one embodiment, the separation container fits into or can be fitted into a holder in a spectrometer in a suitable position for interrogation. The spectroscopic interrogation can be carried out by any technique known to those of skill in the art to be effective for detecting and/or identifying one or more intrinsic or extrinsic properties of microorganisms. For example, front face fluorescence (where the exciting and emitted light enters and leaves the same optical surface, and if the sample is generally optically thick, the excitation light penetrates a very short distance into the sample (see, e.g., Eisinger, J., and J. Flores, “Front-face fluorometry of liquid samples,” Anal. Biochem. 94:15 (1983)) can be used for identification of microorganisms in pellets. Other forms of measurement, such as epifluorescence, reflectance, absorbance, and/or scatter measurements, can also be employed in the present invention. In another embodiment, as described herein, the isolated sample or pellet can be removed for interrogation (e.g., the isolated sample or pellet can be removed and prepared for interrogation by mass spectrometry, as is well known in the art). In still further embodiments, the isolated sample or pellet can be interrogated using more than one means. For example, the isolated sample or pellet can be interrogated using fluorescence spectroscopy and Raman spectroscopy. In accordance with this embodiment, these interrogation steps may be carried out sequentially or simultaneously.
The sample illumination source, or excitation source, may be selected from any number of suitable light sources as known to those skilled in the art. Any portion of the electromagnetic spectrum that produces usable data can be used. Light sources capable of emission in the ultraviolet, visible and/or near-infrared spectra, as well as other portions of the electromagnetic spectrum, can be utilized and are known to those skilled in the art. For example, light sources may be continuum lamps such as a deuterium or xenon arc lamp for generation of ultraviolet light and/or a tungsten halogen lamp for generation of visible/near-infrared excitation. These light sources provide a broad emission range and the spectral bandwidth for specific excitation wavelengths may be reduced using optical interference filters, prisms and/or optical gratings, as are well known in the art.
Alternatively, a plurality of narrowband light sources, such as light emitting diodes and/or lasers, may be spatially and/or temporally multiplexed to provide a multi-wavelength excitation source. For example, light emitting diodes are available from 240 nm to in excess of 900 nm and the sources have a spectral bandwidth of 20-40 nm (full width at half maximum). Lasers are available in discrete wavelengths from the ultraviolet to the near-infrared and can be employed using multiplexing methods well known to those skilled in the art.
The spectral selectivity of any of the light sources may be improved by using spectral discrimination means such as a scanning monochromator. Other methods of discrimination may be utilized, as known to those of skill in the art, such as an acousto-optic tunable filter, liquid crystal tunable filter, an array of optical interference filters, prism spectrograph, etc., and in any combination. A consideration in selecting the spectral discriminator takes into the account the range of tunability as well as the level of selectivity. By way of illustration, for example, a discriminator might utilize the wavelength range of 300-800 nm with a selectivity of 10 nm. These parameters generally determine the optimum technology necessary to achieve the tunability range as well as the selectivity.
Typically, the light source results in the excitation of the sample, followed by measurement of the emission of fluorescence of the sample at predetermined time points or continuously. Similarly, the reflected light from interaction of the excitation source with the sample may be measured to provide pertinent data for detection and/or characterization.
The emission from the sample may be measured by any suitable means of spectral discrimination, most preferably employing a spectrometer. The spectrometer may be a scanning monochromator that detects specific emission wavelengths whereby the output from the monochromator is detected by a photomultiplier tube and/or the spectrometer may be configured as an imaging spectrograph whereby the output is detected by an imaging detector array such as a charge-coupled device (CCD) detector array. In one embodiment, a discriminator allows the observation of the fluorescence and/or scattering signal by a photodetection means (such as a photomultiplier tube, avalanche photodiode, CCD detector array, and/or electron multiplying charge coupled device (EMCCD) detector array).
The spectroscopic technique is used to obtain measurements that are preferably provided as Excitation-Emission Matrix (EEM) measurements. As used herein, EEM is defined as the luminescent spectral emission intensity of fluorescent substances as a function of both excitation and emission wavelength, and includes a full spectrum or a subset thereof, where a subset may contain a single or multiple excitation/emission pairs(s). Additionally, a cross section of the EEM with a fixed excitation wavelength may be used to show the emission spectra for a specific excitation wavelength, and a cross section of the EEM with a fixed emission wavelength may be used to show the excitation spectra for a sample. In one embodiment, multiple EEMs are measured at more than one specific excitation-emission wavelength pair, e.g., at least at 2, 3, 4, 5, 6, 7, 8, 9, 10, or more specific excitation-emission wavelength pairs.
In accordance with one embodiment of the invention, it has been found that a front-face fluorescence spectroscopy provides an advantage in measuring the fluorescence and/or reflectance properties of highly scattering and highly quenching samples. In one embodiment, the front-face method may be particularly useful. For example, front-face fluorescence may be particularly useful in highly absorbent samples because the excitation and emission beam does not need to travel through the bulk of the sample, and thus, may be less affected by the interfering components that may be contained therein (e.g., blood cells and microbiological culture media). The optical surface of the container may be illuminated at such an angle as to provide acceptable results as known to those skilled in the art, (e.g., Eisinger, J., and J. Flores, “Front-face fluorometry of liquid samples,” Anal. Biochem. 94:15-21 (1983)). In one embodiment, the system is designed such that the spectroscopic system measures diffuse reflected light at a minimum of one fixed angle in addition to measuring emitted fluorescence at a minimum of one fixed angle.
According to the invention, control measurements are taken for known microorganisms, thus allowing for correlation of measured test data with characterization of the microorganisms of interest using various mathematical methods known to those skilled in the art. For example, the data from samples may be compared with the baseline or control measurements utilizing software systems known to one skilled in the art. More particularly, the data may be analyzed by a number of multivariate analysis methods, such as, for example, General Discriminant Analysis (GDA), Partial Least Squares Discriminant Analysis (PLSDA), Partial Least Squares regression, Principal Component Analysis (PCA), Parallel Factor Analysis (PARAFAC), Neural Network Analysis (NNA) and/or Support Vector Machine (SVM). These methods may be used to classify unknown microorganisms of interest into relevant groups based on existing nomenclature, and/or into naturally occurring groups based on the organism's metabolism, pathogenicity and/or virulence in designing the system for monitoring, detecting and/or characterizing the organism as described previously.
In yet another embodiment, non-spectroscopic measurements from the detection system, such as detection times and growth rates can be used to assist in the characterization and/or identification of microorganisms from the isolated sample or pellet. Additionally, measurements taken from a photographic image of the lower region of the separation device can provide valuable information on the identity of the isolate, such as pellet size, shape, color and density.
In some embodiments of the invention, characterization and/or identification of the microorganisms in the isolated sample or pellet need not involve identification of an exact species. Characterization encompasses the broad categorization or classification of biological particles as well as the actual identification of a single species. Classification of microorganism from an isolated sample or pellet may comprise determination of phenotypic and/or morphologic characteristics for the microorganism. For example, characterization of the biological particles may be accomplished based on observable differences, such as, composition, shape, size, clustering and/or metabolism. In some embodiments, classification of the biological particles of interest may require no prior knowledge of the characteristics of a given biological particle but only requires consistent correlations with empiric measurements thus making this method more general and readily adaptable than methods based on specific binding events or metabolic reactions. As used herein “identification” means determining to which family, genus, species, and/or strain a previously unknown microorganism belongs to. For example, identifying a previously unknown microorganism to the family, genus, species, and/or strain level.
In some instances, characterization encompasses classification models which provide sufficient useful information for action to be taken. As used herein, the preferred classification models comprise grouping into one or more of the following: (1) Gram Groups; (2) Clinical Gram Groups; (3) Therapeutic Groups; (4) Functional Groups; and (5) Natural Intrinsic Fluorescence Groups.
(1) Gram Groups: Within the Gram Groups classification, microorganisms may be placed into one of three broad classification categories based on their Gram staining reaction and overall size, said groups selected from one or more of the following: (a) Gram positive microorganisms that stain dark blue with Gram staining; (b) Gram negative microorganisms that stain red with Gram staining; and (c) yeast cells that stain dark blue with Gram staining, but are very large rounded cells that are distinguished from bacteria by their morphological characteristics and size.
(2) Clinical Gram Groups: The Gram Groups may be further divided into several sub-categories representing distinguishing morphological features. These sub-categories comprise all the relevant clinical information reported by an experienced laboratory technologist, and thus provide a higher level of identification than a positive or negative Gram reaction. This particular classification is very helpful because it eliminates concerns about relying on the quality of a Gram stain and/or the skill level of the technician reading the smear by providing the equivalent clinically relevant information with an automated system. More specifically, subcategories of microorganisms based on this classification model may be selected from one or more of the following: (a) cocci, which are small rounded cells; (b) diplococci, which are two small rounded cells joined together; (c) rods, which are rectangular shape; and (d) bacilli, which are rod shaped. Examples of these sub-categories that can be ascertained by additional morphological information include: (i) Gram positive cocci; (ii) Gram positive cocci in chains; (iii) Gram positive cocci in clusters (i.e., “grape-like” clusters); (iv) Gram positive diplococci; (v) Gram positive rods; (vi) Gram positive rods with endospores; (vii) Gram negative rods; (viii) Gram negative coccobacilli; (ix) Gram negative diplococci; (x) yeast; and (xi) filamentous fungi.
(3) Therapeutic Groups: The therapeutic groups comprise multiple microbial species that, when isolated from particular specimen types, are treated with the same class of antibiotics or mixture of antibiotics (e.g., as described in “Sanford Guide to Antimicrobial Therapy 2008”). In many cases, identity to the species level is not required by the clinician to enable a change from initial empiric therapy to a more targeted therapy because more than one species can be treated with the same choice of antibiotic(s). This classification level correctly places these “same-treatment” microorganisms into single therapeutic categories. Examples of this characterization level include the ability to distinguish highly resistant Enterobacteriacae (EB) species from sensitive EB species (Enterobacter spp. from E. coli), or fluconazole-resistant Candida species (C. glabrata and C. kruzei) from sensitive Candida species (C. albicans and C. parapsilosis), and so on.
(4) Functional Groups: According to the invention, microorganisms may also be placed into several groups based upon a mixture of metabolic, virulence and/or phenotypic characteristics. Non-fermentative organisms may be clearly distinguished from fermentative ones. Furthermore, microorganism species that produce hemolysins may be grouped separately from non-hemolytic species. In some cases, these groups represent broader categories than genus level (e.g., coliforms, Gram negative non-fermentative rods), some at the genus level (e.g., Enterococcus, Candida), and some with closer to species-level discrimination (e.g., coagulase-negative staphylococci, alpha-hemolytic streptococci, beta-hemolytic streptococci, coagulase-positive staphylococci, i.e., S. aureus).
(5) Natural Intrinsic Fluorescence (“IF”) Groups: Microorganisms may also be placed into categories based on their natural tendency to group together by their innate and/or intrinsic fluorescence characteristics. Some of these groups may be common to Therapeutic and Functional Group categories. These groupings may comprise individual species, such as E. faecalis, S. pyogenes, or P. aeruginosa that have characteristic IF signatures and/or may contain small groups of organisms with relatively conserved IF signatures such as the K. pneumoniae-K. oxytoca or E. aerogenes-E. cloacae groups.
In addition to measuring intrinsic properties of microorganisms (such as intrinsic fluorescence) for identification purposes, the methods of the present invention can further comprise the use of additional identifier agents to aid in the separation and/or identification process. Agents that bind to specific microorganisms, such as affinity ligands, can be used to separate microorganisms, to identify a class or species of microorganism (e.g., through binding to a unique surface protein or receptor) and/or to identify a characteristic of the microorganism (e.g., antibiotic resistance). Useful identifier agents include, without limitation, monoclonal and polyclonal antibodies and fragments thereof (e.g., anti-Eap for S. aureus identification), nucleic acid probes, antibiotics (e.g., penicillin, vancomycin, polymyxin B), aptamers, peptide mimetics, phage-derived binding proteins, lectins, host innate immunity biomarkers (acute phase proteins, LPS-binding protein, CD14, mannose binding lectin, Toll-like receptors), host defense peptides (e.g., defensins, cathelicidins, proteogrins, magainins), bacterocins (e.g., lantibiotics, such as nisin, mersacidin, epidermin, gallidermin, and plantaricin C, and class II peptides), bacteriophages, and dyes selective for nucleic acids, lipids, carbohydrates, polysaccharides, capsules/slime or proteins, or any combination thereof. If the agent does not itself give out a detectable signal, the agent can be labeled to provide a detectable signal, such as by conjugating the agent to a marker (e.g., visible or fluorescent). Markers include, without limitation, fluorescent, luminescent, phosphorescent, radioactive, and/or colorimetric compounds. The agent can be added to the microorganisms at any step in the methods of the invention, e.g., when the sample is obtained, during lysis, and/or during separation. In some embodiments, the presence of the agent in the pellet can be determined during interrogation of the pellet. Other useful identifier agents include substrates for microbial enzymes, chelating agents, photosensitizing agent, quenching agent, reducing agent, oxidizing agent, buffer, acid, base, solvent, fixative, detergents, surfactants, disinfectants (eg. alcohols, bleach, hydrogen peroxide) and toxic compounds (eg. sodium azide, potassium cyanide) and metabolic inhibitors such as cyclohexamide, etc. Similarly, many fluorescent compounds for measuring microbial cell viability, metabolism and/or membrane potential may be used as an identifier agent in the present invention. As would be readily appreciated by one of skill in the art, the sensitivity of a particular microorganism to any compound affecting its physical state or metabolism, such as an antibiotic, could be rapidly ascertained by adding the compound to the sample, lysis buffer, density cushion or any mixture thereof.
In one aspect of the invention, the method can further comprise a step of recovering the pellet of microorganisms and performing additional tests. In one embodiment, the pellet can be recovered by aspirating off the sample medium and density cushion. In another embodiment, the pellet can be recovered by inserting a syringe into the container and aspirating out the pellet while the sample medium and density cushion remain intact. The recovered pellet can then be resuspended in a suitable medium, e.g., saline. Once resuspended, the microorganisms can be subject to any further tests that are desired, as would be known to those of skill in the art and as described above. In particular, any test requiring clean samples of microorganisms can be carried out with the resuspended microorganisms. In some embodiments, additional identification tests can be performed. Examples of identification tests include Vitek® 2, amplified and non-amplified nucleic acid tests (NAT), chromogenic and latex agglutination assays, immunoassays, (e.g., employing labeled antibodies and/or other ligands), mass spectrometry (e.g., MALDI-TOF mass spectrometry) and/or other optical techniques such as infrared spectroscopy (FTIR) or Raman spectroscopy. Additional characterization tests can also be performed, such as resistance to antibiotics and/or other drugs. The additional characterization may be part of a test that was started during the initial separation and identification steps of the method. For example, the detection of methicillin resistant S. aureus can begin by adding fluorescently-labeled penicillin to the sample prior to separation of the microorganisms. Once the pellet has been recovered and resuspended, the level of bound penicillin can be determined.
In one aspect of the invention, some or all of the method steps can be automated. Automating the steps of the methods allows a greater number of samples to be tested more efficiently and reduces the risks of human errors in handling samples that may contain harmful and/or infectious microorganisms. Of greater importance, however, automation can deliver critical results at any time of the day or night without delay. Several studies have shown that faster identification of the organisms causing sepsis correlates with improved patient care, shorter hospital stays and lower overall costs.
In certain embodiments of the invention, the methods can also be used to detect the presence of microorganisms in a sample. In these embodiments, the methods comprise the steps of:
(a) obtaining a sample;
(b) optionally lysing cells in said sample to produce a lysed sample; and
(c) separating microorganisms from other components of said sample to form a pellet of microorganisms;
wherein the presence of a pellet indicates that microorganisms are present in the sample. In one embodiment, the pellet is detected with the naked eye. In other embodiments, the pellet is detected by interrogation, e.g., spectroscopically.
In some embodiments, the detection methods can be used to monitor samples for contamination by microorganisms, e.g., foodstuffs, pharmaceuticals, drinking water, etc. In one embodiment, the methods can be carried out in a repetitive fashion for constant monitoring for contamination, e.g., once a month, once a week, once a day, once an hour, or any other time pattern. In another embodiment, samples can be tested as needed, e.g., when contamination is suspected. In further embodiments, the detection methods can be used to look for the presence of microorganisms in clinical samples, e.g., blood cultures. For example, a sample can be removed from a blood culture at certain time points and the detection method carried out on the sample to determine if the blood culture is positive. In one embodiment, a sample may be taken at a set time point after inoculation of the culture, e.g., 24 hours after inoculation, to determine if the blood culture is positive. In another embodiment, samples can be taken from the blood culture regularly, e.g., every 12, 6, 4, or 2 hours or every 60, 50, 40, 30, 20, 15, 10, or 5 minutes, to identify positive blood cultures within a short time of being detectably positive. In certain embodiments of the detection methods, the detection step can optionally be followed by identification methods as described herein. In other embodiments, the detection methods are partially or fully automated, particularly for the embodiments involving repetitive monitoring of samples.
The present invention is further detailed in the following examples, which are offered by way of illustration and is not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
EXAMPLES
Example 1
Rapid Microbial Separation and Identification Method
A. Lysis-Centrifugation Separation Procedure
A suspension of colloidal silica (0.2-0.5 mL; 1.040-1.050 gm/mL density) was added to several conical microcentrifuge tubes. Lysed positive BacT/ALERT® SA blood culture broth samples (0.5-1.0 mL) were overlaid onto the colloidal silica suspension. Alternatively, the colloidal silica solution can be added underneath the lysed blood culture broth using a needle or cannula.
Positive broth from cultures containing the following microorganisms were tested:

    
    
        E. coli, ATCC 25922
        E. faecalis, ATCC 29212
        S. aureus, ATCC 12600
        P. aeruginosa, ATCC 10145
    
    


The tubes were capped, and then spun in a microcentrifuge for 2 min at about 10,000 g at room temperature (20-25° C.). The supernatants were aspirated, then the purified microbial pellets were resuspended in 0.45% w/v NaCl to an optical density @ 660 nm of 0.40.
One portion of each suspension was transferred to an acrylic cuvette and scanned in a spectrofluorimeter (Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey)) to measure microbial intrinsic fluorescence (MIF).
A second portion was loaded into Vitek® 2 ID/AST cards (bioMérieux Inc., Missouri). The “direct” Vitek® 2 results were compared with those from suspensions of overnight grown colonies sub-cultured from the positive broth (traditional method). All 4 species gave excellent identification confidence levels with both the direct-from-blood culture and standard Vitek® methods, demonstrating that the density-based separation method provided microorganisms substantially free of blood and/or broth-derived particles and proteins.
B. Rapid Procedure for In Situ Identification by Intrinsic Fluorescence
Positive blood culture bottles were removed from the BacT/ALERT® Microbial Detection System (bioMérieux Inc., Missouri) within 1 hour of flagging positive, preferably within 10 minutes of flagging positive. A 2.0 mL sample of positive blood culture broth was added to 0.5 mL of Lysis Solution (0.75% Triton® X-100-Reduced (Rohm and Haas, Pennsylvania)+0.375% Proteinase XXIII) in a sterile, screw-capped tube. The tube was vortexed briefly to mix and incubated for 5 minutes at room temperature. The lysed broth sample (0.5 mL) was added to a custom-built microcentrifuge tube (having a 0.5 mm thick quartz optical window at the base) containing the separating solution (Isolate® colloidal silica 30% v/v in 0.15 M NaCl) at a density of 1.045 mg/ml. The tube was spun in an Eppendorf® 5417R micrcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York) for 2 minutes at about 10,000 rpm at room temperature (20-25° C.). The tube was removed from the centrifuge and transferred to a custom-built front face adapter for the Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey) spectrofluorimeter. The fluorescence of the pelleted microorganisms in the bottom of the tube was immediately read from below. The data was exported to Excel and Statistica software for multivariate analysis.
Example 2
Evaluation of the Rapid Microbial Purification and Identification Method
To assess the potential of the rapid identification concept described in Example 1, twenty-four isolates (6 strains of 4 species comprising C. albicans, E. coli, S. aureus and S. epidermidis) recovered from positive blood cultures were tested in the method.
SPS-anticoagulated blood was collected from three donors and pooled. Ten mL of fresh human blood was added to BacT/ALERT® (BTA) SA blood culture bottles (bioMérieux Inc., Missouri). Suspensions of each of the isolates were prepared in tryptic soy broth (TSB). Each bottle was inoculated with 0.4 ml of a 103/mL suspension and the bottles were incubated at 36° C. in a BTA cabinet. Four BacT/ALERT® SA bottles containing 10 mL blood but no organisms were included as negative controls.
Positive bottles were removed from the BTA cabinet within 3 hours of flagging positive. A negative control bottle was removed with each set of positive bottles (by species). One bottle at a time was removed from the BTA cabinet in the order they became positive. A 2.0 mL sample of positive blood culture broth was removed using a 3 mL syringe and an 18G needle, and immediately added to 0.5 mL Lysis Buffer (0.75% w/v Triton® X-100-Reduced (Fluka)) in a sterile screw-capped glass tube. The tube was vortexed briefly to mix and incubated for 5 minutes at room temperature. A 0.5 mL portion of the lysed broth sample was added to a custom-built capillary microcentrifuge tube (containing a 0.5 mm quartz optical window at the base of the capillary section) previously loaded with 55 Separating Solution (30% v/v Isolate® in 0.15 M NaCl). The microcentrifuge tube was centrifuged in an Eppendorf® 5417R microcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York) for 2 minutes at 10,000 rpm at 22° C. The tube was removed from the centrifuge and transferred to a custom-built 30-degree front face adapter for the Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey) spectrophotometer. The fluorescence of the pelleted purified microorganisms in the bottom of the tube was immediately read using a 5-minute custom-built program. The data was exported to Excel and Statistica for chemometric analysis and classification of the isolate.
Optimization of the classification model was carried out on the data with and without normalization and with leave-one-out cross-validation. The results are shown in Table 1. In the table, the “# steps” refers to the number of Discriminant Analysis steps that resulted in highest sensitivity using a “leave-one-out” cross-validation approach. In the absence of data normalization, the percentage of correctly identified strains was 82.6%. The best results (approximately 96% correct identification) were obtained when the data was normalized to the Rayleigh scattering point, the collagen region or the pyridoxamine peak, although improved results were still obtained by normalizing to the NADH, tryptophan, and flavin peaks.












TABLE 1







Data
Principle/
% Correct




Normalization
Fluorophore
on X-Val
# steps





















None
n/a
82.6
2



320_320
Scattering
95.7
2



320_405
Collagen
95.7
6



300_400
Pyridoxamine
95.7
8



345_460
NADH
91.0
10



285_350
Tryptophan
87.0
1



465_515
Flavin
87.0
3










Example 3
Assessment of Lysis Buffers
Experiments were carried out to assess the separation efficiency and microbial intrinsic fluorescence (MIF) profiles of freshly positive S. pneumoniae WM-43 blood culture broth treated with harsh and mild lysis buffers. The following lysis buffer formulations were tested: (A) 2.0% TX100-R in 0.5M CAPS, pH 11.7 (harsh=LB-A); (B) 0.75% TX100-R in 0.5M CAPS, pH 11.7 (mild=LB-B); and (C) 0.45% TX100-R in 0.3M CAPS, pH 11.7 (mild=LB-C). 1.0 mL of lysis buffers A and B and 2.0 mL of lysis buffer C were placed in screw-capped tubes and the tubes were placed in a rack in a 37° C. water bath. Samples of broth (4.0 mL) were removed from a S. pneumoniae WM43-positive BacT/ALERT® SA culture bottle using a 5 mL syringe and 18G needle within 5 minutes of flagging positive in the BTA system. The broth was quickly dispensed to each lysis buffer-containing tube and the tubes were capped and vortexed for approximately 5 seconds. A test broth from overfilled (15 mL blood) negative control BacT/ALERT® SA bottles was also sampled to determine the efficacy of the lysis and separation steps. The tubes were returned to the 37° C. water bath for 1 minute. The tubes were removed from the water bath and 0.5 mL of lysate was overlayed into a preloaded separation tube containing 200 μL of a 14% w/v iohexyl density cushion. The tubes were centrifuged for 2 min at 10,000 rpm (approx. 10,000×g) at 25° C. in an Eppendorf® 5417R micrcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York). Photographs of the lower regions of the separation tubes using LB-B are shown in FIG. 1. Note the absence of any pellet when an overfilled (15 mL blood) negative control broth was processed. Immediately after completion of centrifugation, the tubes were read in the Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey) spectrofluorimeter using a dual 30-degree tube adapter, PMT detector and the “Full EEM” scan file (20.8 min scan). Examples of the EEM spectra from the individual samples are shown in FIGS. 2A-2C.
The intrinsic fluorescence EEM profiles of the S. pneumoniae pellets shown in FIGS. 2A-2C correlated well with the cell viability results. Treatment of positive broth with the 0.40% TX100-containing (final concentration) lysis buffer (LB-A; harsh buffer) resulted in a 30-fold drop in pneumococcal viability and a 5-fold decrease in peak NADH fluorescence compared to treatment with both milder buffers containing 0.15% TX100 (final concentration), despite similar tryptophan signals in all three pellets. Another change associated with decreased microbial viability was an increase in the peak flavin fluorescence (Table 2). Decreased NADH and increased flavin fluorescence is clearly evident in FIG. 2A.







TABLE 2







S. pneumoniae Pellet Intrinsic Fluorescence Signals














Lysis
TX100-R
CAPS,
Broth
Tryptophan
NADH
Flavin



Buffer
(final)
pH 11.7
Diln.
peak
peak
peak
Viability

















LB-A
0.40%
0.10 M
1 pt LB:
699,975
52,123
119,622
1.0 × 107 CFU/ml





4 pts Broth


LB-B
0.15%
0.10 M
1 pt LB:
824,886
245,880
54,594
3.7 × 108 CFU/ml





4 pts Broth


LB-C
0.15%
0.10 M
1 pt LB:
799,015
283,701
49,548
3.2 × 108 CFU/ml





2 pts Broth









Example 4
Improved Devices and Methods for the In Situ Identification of Purified Microbial Pellet
To further explore the potential of the rapid in situ separation and identification method described in Example 2, several proprietary devices were designed and molded from UV-transparent plastic. These devices are disclosed in related U.S. patent application Ser. No. 12/589,969, entitled “Separation Device for Use in the Separation, Characterization and/or Identification of Microorganisms”, filed Oct. 30, 2009, and which is incorporated herein by reference. These devices contained several common features, including a closure, sample reservoir and a tapered optical quality lower region to enable spectroscopic interrogation of the sedimented microbial pellet from below and/or the side, and features that facilitated the coupling of the device to a spectrofluorimeter. The devices must also be capable of withstanding relatively high g-forces during the separation step. Several iterations of this tube were designed to improve microbial recovery, fluorescence reproducibility and reduce contamination by stray scattered light. The tube was also designed to be hermetically sealed.
Optical interrogation of the sedimented microbial pellet was achieved by either inserting the separation device into a custom-built adapter placed within the sample compartment of the spectrofluorimeter or by coupling the separation device directly to a bifurcated six-around-one 300-400 micron fiber optic cable (Ocean Optics, Florida) attached to the spectrofluorimeter (Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey)). A three-mirror fiber optic adapter was built to enable the use of both the systems detectors (PMT and CCD). Full Excitation-Emission Matrix (EEM) spectra were collected on each microbial pellet (scan range: Excitation 260-800 nm; Emission 260-1100 nm; increments of 5 nm).
Gage reproducibility and reliability studies were performed on the disposable device-fiber optic cable configuration using purified tryptophan and riboflavin solutions. Target CV's of <2.5% were obtained for both fluorophores, confirming the quality of the disposable and the research platform.
Example 5
Identification of Microorganisms Using Measurements of Microbial Pellets and Comparison to Microbial Suspensions
Several investigators have previously described using right-angle fluorescence measurements of dilute suspensions of microorganisms in order to identify them. We compared the effectiveness of this traditional method with our novel approach of front face measurement of a sedimented microbial pellet within the base of a proprietary UV-transparent separation device, or optical tube. Further, we compared the effectiveness of two detectors for the front face measurements; a photomultiplier tube (PMT) detector connected to a double grating spectrometer, and a charge-coupled device (CCD) detector connected to a single grating spectrometer. These experiments were conducted using the two-piece separation device design and microbial colonies grown on agar plates. A panel of 42 strains representing 7 species (S. aureus, S. epidermidis, E. coli, K. oxytoca, C. albicans, C. tropicalis and E. faecalis) were tested in each of the following three optical configurations:

    
    
        1. A 0.40 OD at 660 nm suspension of each microorganism was prepared in 0.45% NaCl, added to a UV-transparent cuvette and full EEM's were collected at right angle using a PMT detector (traditional method)
        2. A 2-3 mm thick microbial pellet was prepared in the custom-built separation device by centrifuging a suspension. Full EEM's of the resultant pellet was collected in front face mode using the PMT detector.
        3. A 2-3 mm thick microbial pellet was prepared in the custom-built separation device by centrifuging a suspension. Full EEM's of the resultant pellet was collected in front face mode using the CCD detector.
    
    


The intrinsic fluorescence data in the EEM's were analyzed using commercially available multivariate analysis software (General Discriminant Analysis; Statistica). The results of the analyses are depicted in Table 3.









TABLE 3






Detector



Optical Configuration
Type
% Correct to Species Level







1. Suspension in cuvette
PMT
83.3 (35/42 strains correct)


2. Pellet in custom optical tube
PMT
97.6 (41/42 strains correct)


3. Pellet in custom optical tube
CCD
97.6 (41/42 strains correct)









Surprisingly, scanning microbial pellets in front face mode significantly improved the ability to identify them using known multivariate analysis methods. Further analysis of the fluorescence EEM data revealed that the main discriminatory region for the traditional suspension-in-cuvette configuration was within the tryptophan region of the spectrum. In contrast, front face measurements of the microbial pellets resulted in several additional regions of the EEM spectrum that provided strong discriminatory power, particularly within the 360-440 nm Excitation wavelengths. This experiment also demonstrated the functional equivalency of the PMT and CCD detectors.
The additional intrinsic fluorescence spectral information provided by front face interrogation of a microbial pellet is both an unexpected and advantageous result.
Example 6
Development of Selective Lysis Buffers
We set out to design selective Lysis Buffers capable of dissolving human blood components within seconds but leaving the majority of sepsis-causing microorganisms intact and metabolically active. The most common sample used for these studies was BacT/ALERT® SA culture medium containing human blood. Bottles were seeded with 10-15 mL of human blood with and without a small inoculum of test microorganism, then loaded into a BacT/ALERT® Microbial Detection System. Positive or negative-to-date bottles were removed and a sample of the broth treated as described below.
The earliest lysis solutions used in the present invention were unbuffered (Examples 1-2) and used primarily in combination with colloidal silica density cushions. One of the interesting properties of colloidal silica is its ability to separate incompletely-solubilized blood cell components, which collect at the top of this density cushion, and intact microorganisms, which pass through the cushion and form a pellet. As more broadly applicable density cushions were sought (refer to Example 7), many failed to prevent sedimentation of this non-microbial debris, so improved lysis buffer formulations were needed. We determined that the non-microbial debris was rapidly dissolved by potassium hydroxide but not by acid, so a number of alkaline pH buffers were tested in the presence of a variety of detergents. During these studies we discovered that a mixture of Triton X100-R detergent and CAPS buffer at pH 11.7 resulted in complete solubilization of blood cell components from a sterile blood culture broth sample.
A preliminary assessment of the effect of Triton X100-R detergent concentration and high pH lysis conditions on the viability of S. pneumoniae strain WM-43 was described in Example 3. Conditions resulting in lower microbial viability caused a significant decrease in NADH fluorescence with a concomitant increase in flavin fluorescence (see Table 2; LB-A).
A series of anionic, neutral and zwitterionic detergents were screened for inclusion in a selective lysis buffer. Anionic and zwitterionic detergents were found to be too denaturing for blood proteins, so we focused on non-denaturing neutral detergents, such as the selection given in Table 4. Detergents with the strongest blood cell lysis activity at the alkaline pH tested are listed as #1-6. These detergents completely solubilized blood cells in a negative control blood culture broth within 20 seconds, as assessed by an increase in % Transmittance at 660 nm. A second group of detergents (#7-10) demonstrated a slower, more controlled lysis of the blood cells, giving maximal solubilization in 30-40 seconds. A third group (#11-12) took 8-10 minutes for full lysis at comparable detergent concentrations.
Lysis buffers were prepared containing detergents of differing structure and lytic activity in a base buffer formulation of 0.3M CAPS, pH 11.7. The detergent concentrations are given in Table 4. These lysis buffers were functionally assessed using positive blood culture broth containing a clinical microorganism strain known to be sensitive to detergents and alkaline pH conditions, Streptococcus pneumoniae, strain WM-43. Parameters evaluated were microbial intrinsic fluorescence levels (Excitation-Emission Matrix spectra), separation efficiency, appearance of isolated microbial pellet, and Gram stain characteristics and viability of microorganisms recovered from the pellet beneath the density cushion.
The procedure was performed as follows:
Mix 2 parts of positive broth with 1 part of test lysis buffer, incubate in a 37 C water bath for 1 minute to lyse blood cells, then layer 0.5 mL of lysate over 0.2 mL of density cushion (14% w/v Iohexyl+1.93% w/v NaCl+10 mM Hepes, pH 7.4) dispensed into a 2-piece optical separation tube which was then hermetically sealed with a screw-cap. After centrifuging the tube in an Eppendorf® 5417R micrcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York) for 2 minutes at 10,000 rpm, the tube was placed into a custom-built adapter which coupled the base of the tube directly to a 400 micron fiber optic probe connected to a spectrofluorimeter (Fluorolog® 3 (HORIBA Jobin Yvon Inc., New Jersey), and a complete EEM scan was taken (Ex 260-850 nm; Em 260-1100 nm; every 5 nm). Following the scan, the supernatant was removed and the microbial pellet resuspended in 0.5 mL of Tryptic Soy Broth (TSB). Smears of the suspensions were prepared for Gram staining and twenty microliters of a 1:100 dilution were plated onto Sheep Blood Agar plates for viability estimation on a scale of NG (no growth) to 4+ (maximal growth).
The results of this experiment demonstrated that, under the desired lysis conditions described above, the test detergents fell into three categories corresponding broadly to their lytic properties:

    
    
        1. Rapidly lytic detergents (#1-6) had some effect on the Gram reaction of this S. pneumoniae isolate. The two last detergents in this group (Genapol® X-080 (Hoechst AG, Frankfurt, Germany) and polidocenol) had a relatively minor effect, while the first three completely altered Gram reactivity and reduced viability.
        2. A group of milder lytic detergents (#7-10) that effectively solubilized blood cells within the 60 second lysis period and did not alter the Gram reactivity or viability of the S. pneumoniae isolate.
        3. A group of detergents (#11-12) that also did not alter the Gram reactivity or viability of the S. pneumoniae isolate, but had slower lytic activity.
    
    









TABLE 4







Effect of Detergent Type on Gram Reactivity and Viability of S. pneumoniae











Formula or
Gram Stain of Pellet
Viability












No.
Detergent
Class
Bacteria (cocci)
Debris
of Pellet















1
3.0% n-octyl
Alkyl
No organisms seen
3+
No growth



glucoside
glucoside


2
3.0% CHAPS
Bile acid-like
Rare Gram
3+
Scant





negative

growth


3
0.45% Triton
PEO ether
2+ Gram negative
2+
2+



X100-R


4
0.45% Igepal CA-
PEO ether
2+ Gram positive
1+
3+



630

and 2+ Gram





negative


5
0.45% Genapol ®X-
C13E8
4+ Gram positive
1+
3+



080

and 1+ Gram





negative


6
0.45% Polidocenol
C12E9
4+ Gram positive
<1+ 
4+





and +/− Gram





negative


7
0.45% Genapol ® C-
C12E10
3+ Gram positive
1+
3+



100


8
0.45% Genapol ® X-
C13E10
3+ Gram positive
1+
4+



100


9
0.45% Brij ® 96V
C18E10
3+ Gram positive
1+
3+


10
0.45% Brij ® 97
C18E10
3+ Gram positive
1+
4+


11
1.5% Arlasolve 200
Isoceteth-20
3+ Gram positive
2+
3+


12
a1.5% Brij ® 98
C18E20
3+ Gram positive
1+
3+





aRequired a 5 min lysis step for full solubilization of blood cells.






The effect of the test detergents on the level of intrinsic fluorophores present within the S. pneumoniae cell pellet is shown in Table 5. As taught by the results presented in Example 3, the ratio of Flavin to NADH fluorescence is an excellent indicator of this sensitive microbe's health. Low ratios are associated with reduced microbial viability, such as observed with n-octyl glucoside and CHAPS detergents. The four groups represented in Table 5 fall into categories that directly correlate with the alterations observed in Gram reactivity of the S. pneumoniae cells recovered from the pellet (Table 4). The data demonstrates that detergents 7, 8, 9, 10 and 12 show significant improvement in the Flavin/NADH surrogate marker for viability.
Interestingly, these five detergents are all of the polyoxyethylene class. Surprisingly, the four milder detergents that solubilize blood cells readily (#7-10), which have optimal intrinsic fluorophore levels suggestive of unaltered viability and have expected Gram reactivity, all share a common chemical nature. Each has a polyoxyethylene hydrophilic head group with an average chain length of 10 (E) and average hydrocarbon chain lengths ranging from 12-18 (C).
We propose that this particular group of detergents represent excellent candidates for inclusion in lysis buffers designed to selectively solubilize mammalian blood cells while maintaining the viability of nearby microorganisms.







TABLE 5







Effect of Detergent Type on Level of Intrinsic Fluorophores of S. pneumoniae.















Formula or



NADH/


No.
Detergent
Class
Tryptophan
NADH
Flavin
Flavin
















1
3.0% n-octyl
Alkyl
606,171
33,268
71,678
0.5



glucoside
glucoside


2
3.0% CHAPS
Bile acid-
607,343
55,976
77,403
0.7




like


3
0.45% Triton X-100-R
PEO ether
895,915
593,178
48,262
12.3


4
0.45% Igepal CA-630
PEO ether
1,019,649
1,300,815
38,703
33.6


5
0.45% Genapol ® X-
C13E8
985,085
1,469,240
36,361
40.4



080


6
0.45% Polidocenol
C12E9
825,892
1,295,956
37,611
34.5


7
0.45% Genapol ® C-
C12E10
974,429
1,571,416
26,537
59.2



100


8
0.45% Genapol ® X-
C13E10
926,102
1,604,789
28,109
57.1



100


9
0.45% Brij ® 96V
C18-1E10
1,075,022
1,235,051
23,306
53.0


10
0.45% Brij ® 97
C18E10
1,054,839
1,310,612
25,383
51.6


11
1.5% Arlasolve 200
Isoceteth-20
1,079,990
1,178,954
37,115
31.8


12
1.5% Brij ® 98
C18E20
1,047,866
1,703,221
29,781
57.2









A screening model was developed to enable testing the sensitivity of a larger number of microbial isolates to several detergents. Briefly, suspensions of test fastidious microorganisms were spiked into human blood-containing BacT/ALERT® SA culture media at 103-105 CFU/mL. Samples were treated with test Lysis Buffers for 1-5 minutes at 37° C., then if required, diluted 1:100 into TSB and plated for viable counts.
A summary of the colony counts, given in Table 6, shows that Brij® 97 demonstrated minimal toxicity, however, the alkaline conditions of the CAPS buffer alone rapidly killed N. meningitidis, H. influenzae and A. actinomycetemcomitans. Genapol® C-100 had similar activity to Brij® 97, but was slightly more toxic towards H. parainfluenzae and C. hominis in this model. In a follow up experiment, the viability of S. pneumoniae, S. pyogenes, S. agalactiae, S. mitis, P. mirabilis, K. pneumoniae and E. coli remained unaffected following a 5-minute contact time with both Brij® 97 and Genapol® C-100 (data not shown).







TABLE 6







Viable Counts of Fastidious Organisms Following Lysis Buffer Treatment













CAPS

Genapol ®



Water
buffer
Brij ® 97
C100














Microorganism
1 min
1 min
5 min
1 min
5 min
1 min
5 min

















S. pneumoniae
37
39
38
18
7
30
19


WM43


S. pneumoniae
19
29
17
14
11
17
12


WM48


N. meningitidis
249
2
0
0
0
0
0


H. influenzae
>200
1
0
0
0
0
0


A. actinomycetecomitans
>200
0
0
0
0
0
0


H. parainfluenzae
183
117
6
124
19
0
0


C. hominis
50
92
91
74
28
0
0









Many lysis buffers, particularly those used for molecular methods, contain chelating agents such as ethylenediaminetetraacetic acid (EDTA) to assist in the solubilization step. We assessed the impact of adding EDTA to the TX100-CAPS lysis buffer base using a panel of Gram-negative and Gram-positive microorganisms. Table 7 shows the rapidly toxic effect of EDTA on P. aeruginosa and A. baumanii, but not on two other Gram-negative rods, B. cepacia and K. pneumoniae, or the Gram-positive S. aureus. Note that while EDTA was inhibitory to both P. aeruginosa and A. baumanii, the changes in major intrinsic fluorophores were very different between these two species. P. aeruginosa was the only organism tested that had a significant drop in both NADH and tryptophan fluorescence following EDTA treatment.
These experiments represents a good example of how certain compounds or identifiers can be added to the lysis buffer to rapidly alter the base microbial intrinsic fluorescence profile of a particular microorganism and present opportunities for enhanced identification and further characterization of the isolate. As would be readily appreciated by one of skill in the art, the sensitivity of a particular microorganism to any compound affecting its physical state or metabolism could be rapidly ascertained by adding the compound to the sample, lysis buffer, density cushion or any mixture thereof. Similarly, alterations to the lysis conditions or the formulation of the selective lysis buffer (e.g. buffer pH, detergent type and its concentration) can produce characteristic changes in microbial intrinsic fluorescence, as explemlified in FIGS. 2A and 2B. (See, e.g., co-assigned U.S. patent application Ser. No. 12/589,985, entitled “Method for the Separation and Characterization of Microorganisms Using Identifier Agents”, filed Oct. 30, 2009, and which is incorporated herein by reference).







TABLE 7







EDTA in Lysis Buffer as an “Identifier Agent” for P. aeruginosa















+20 mM



NADH/


No.
Microorganism
EDTA
Tryptophan
NADH
Flavin
Flavin
















1
P. aeruginosa
No
2,266,444
4,483,691
62,491
72


2
P. aeruginosa
Yes
802,291
132,304
29,964
4


3
A. baumanii
No
1,274,164
2,157,065
47,163
46


4
A. baumanii
Yes
1,204,811
207,628
120,395
2


5
B. cepacia
No
2,199,711
2,577,516
42,135
61


6
B. cepacia
Yes
1,945,836
1,639,781
48,185
34


7
K. pneumoniae
No
2,770,779
2,691,451
151,112
18


8
K. pneumoniae
Yes
2,840,377
3,326,047
126,217
26


9
S. aureus
No
1,422,810
6,173,521
80,550
77


10
S. aureus
Yes
1,279,566
5,396,721
93,038
58









Example 7
Development of Density-Based Separation Buffers
The purpose of the separation buffers, also known as density cushions, was to reliably separate and partition metabolically active microorganisms from lysed blood components and culture media within a few minutes. The cushion material was selected to have a density between the intact microorganisms and that of the lysed positive blood culture broth sample. Separation was accomplished by centrifugal force.
We determined initially (Examples 1-2) that colloidal silica had satisfactory properties for rapidly isolating microorganisms from the highly fluorescent blood and media components found in positive blood culture broth. However, some microbial species, such as S. pneumoniae, did not satisfactorily sediment through colloidal silica at the density required to form an effective phase barrier between sedimented microorganisms and the contaminating blood and media components. Therefore, a search for additional, more broadly effective density cushions was conducted.
A series of compounds were screened for inclusion in a separation buffer. Preferable compounds for the density cushion have the following characteristics:

    
    
        1. Low viscosity to enable rapid sedimentation of microorganisms through the cushion
        2. Low fluorescence to minimize interference with microbial-derived intrinsic fluorescence
        3. Preferably be optically clear throughout the wavelengths of light being interrogated
        4. Be non-toxic to microorganisms
        5. Be inexpensive and readily available
        6. Be broadly applicable to a wide range of bacteria and samples
    
    









TABLE 8







List of Compounds Tested for Density Cushion












Useful
Preferable


Compound
Trade Names/Comment
concentration
concentration





Colloidal silica
Percoll ®, Percoll Plus ®,
15-80% of stock
20-65% of stock



Isolate ®


Iohexol
Omnipaque ®, Nyco-
10-30% w/v
12-22% w/v



Prep ®, Nycodenz ®


Metrizoate-Ficoll
LymphoPrep ®
70-100% of stock
75-90% of stock


Diatrizoate-
PolymorphoPrep ®
20-60% of stock
30-50% of stock


Dextran


Cesium chloride

10-30% w/v
15-25% w/v


Sucrose

10-30% w/v
15-20% w/v


Polyvinyl alcohol
Type 4-88
10-20% w/v
12-18% w/v


Ficoll 400

 5-13% w/v
10-12% w/v


Dextran 70

 5-12% w/v
10-12% w/v


Iodixanol
Visipaque ®, Opti-Prep ® -



similar properties to



Iohexol


Perfluorocarbon
Useful as a high density


fluid
fluid bed


Mineral oils
Can be blended with



denser oils


Silicone oils
Can be blended with



denser oils


Microscope
Type DF = low


immersion oils
fluorescence and high



density


Pluronic F127
Temperature sensitive



gelling agent


Polyethylene


oxide


Hydroxypropylm


ethyl cellulose


Xanthan gum









Potential density compounds were screened using positive blood culture broths containing S. pneumoniae (a low-density capsulated microorganism), E. coli (a medium-density microorganism) and S. aureus (a high-density microorganism). Broth samples were lysed under the conditions described in Example 6, by mixing one part of lysis buffer (0.45% Triton X100-R+0.3M CAPS, pH 11.7) with two parts of sample and incubating for 1 minute in a 37 C water bath. 0.7 mL of the lysate was then carefully overlayed onto 0.5 mL of test density cushion in a standard 1.5 mL microcentrifuge tube. The tubes were spun for 2 minutes at approximately 10,000 g and the separation results recorded. Excellent separation was noted when there was a solid microbial pellet sedimented at the base of two distinct liquid layers with no visual hemoglobin contamination present in the lower layer.
Excellent separation results were obtained using colloidal silica, Iohexyl, cesium chloride, LymphoPrep® and PolymorphoPrep® as the density cushion. Satisfactory results were obtained with dextrose T70, Ficoll 400, sucrose and polyvinyl alcohol. Iodixanol is expected to have similar properties to Iohexyl. We also discovered that making the density cushions hyperosmotic, for example by the addition of sodium chloride, improved the sedimentation of low-density microorganisms such as S. pneumoniae and K. pneumoniae. 
Further testing was performed with 14% w/v Iohexyl, 18% w/v cesium chloride and 30% PolymorphoPrep® stock in combination with Lysis Buffers prepared with Triton X100-R, Polidocenol, Brij® 97, Genapol® C-100 and Genapol® X-100 using positive broth containing a sensitive S. pneumoniae strain, WM43. The combination of five Lysis Buffers and four Density Cushions gave the expected excellent separation. The results for the Iohexyl and cesium chloride density cushions are shown in FIG. 3. As discussed in more detail in Example 6, viability of the recovered S. pneumoniae cells was not as high with buffers containing Triton X100-R and polidocenol, irrespective of the makeup of the density cushion.
Example 8
Evaluation of Several Lysis Buffers and Density Cushions
To determine the broad applicability of the concept of the present invention, we compared the effectiveness of two selective lysis buffer and two density cushion formulations using a seeded blood culture study to develop a mini classification model. Microorganisms used in the model were K. pneumoniae, K. oxytoca, S. aureus, S. epidermidis, C. tropicalis and S. pneumoniae (6 strains of each). The following combinations of lysis buffers and density cushions were tested:
Set A=Brij®-97 lysis buffer+24% CsCl cushion containing Pluronic F-108
Set B=Genapol® C-100 lysis buffer+24% CsCl cushion containing Pluronic F-108
Set C=Brij®-97 lysis buffer+14% Iohexyl cushion containing Pluronic F-108
Set D=Brij®-97 lysis buffer+24% CsCl cushion (no Pluronic F-108)
Samples of positive broth were treated under each of the four conditions described above, as follows:

    
    
        1. A 2.0 mL sample of positive broth was mixed with 1.0 mL of selective lysis buffer, then placed in a 37° C. water bath for 1 minute.
        2. A 1.0 mL sample of lysate was overlayed onto 0.5 mL of density cushion contained in a custom-built optical separation tube. A polypropylene ball was present on the surface of the density cushion to facilitate loading without disturbing the two aqueous phases.
        3. The optical separation tube was sealed with a screw-cap and centrifuged for 2 minutes at 10,000 rpm (Eppendorf® 5417R micrcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York); FIG. 4).
        4. The sealed tube was then transferred to a custom-built adapter which coupled the base of the tube directly to a 300 micron fiber optic probe connected to a spectrofluorimeter (Fluorolog® 3 from HORIBA Jobin Yvon Inc., New Jersey)
        5. A full EEM scan was taken using the CCD detector configuration (Ex 260-850 nm, every 5 nm; Em 260-1100 nm)
        6. The EEM data was exported to Excel and a mini classification model was built for each reagent set using General Discriminant Analysis (Statistica).
    
    


The separation results were equivalent for all four sets of reagents with one exception. Set D, which did not contain the mild surfactant Pluronic F-108 in the density cushion, had significantly reduced biomass of several C. tropicalis strains due to strong adherence of these organisms to the sidewall of the separation tube. This phenomenon also occurred when the Iohexyl cushion was used (data not shown). Subsequent data analysis showed that while the presence of a mild surfactant in the density cushion was preferable, it was not essential. In fact, GDA results of all four reagent combinations revealed satisfactory classification performance (Table 9). Any differences between the four reagent sets were relatively minor. The most common mis-classifications occurred between the two closely related species on the panel, K. pneumoniae and K. oxytoca.









TABLE 9







% Correct to


LB Detergent
Density Cushion
Species Level







A. Brij ® 97
Cesium chloride
>90%


B. Genapol ® C-100
Cesium chloride
>90%


C. Brij ® 97
Iohexol
>90%


D. Brij ® 97
Cesium chloride (no F108)
>90%









One of the potential disadvantages of the Iohexyl-containing density cushion over the cesium chloride-containing cushion is the strong adsorptive properties of Iohexyl, a medically-used contrast agent, which results in significant quenching of fluorescence at excitation wavelengths below about 380 nm. In a surprising finding however, the analytical differentiation of K. pneumoniae and K. oxytoca was improved when the Iohexyl cushion was used, suggesting that partial quenching of the more prominent fluorophores, such as NADH and tryptophan, may uncover differences in lower level cellular fluorophores with different quenching properties.
Brij® 97 and Genapol® C-100 containing lysis buffers delivered similar classification results for this limited panel of microorganisms. Cesium chloride and Iohexyl density cushions also performed similarly. In an intriguing finding, Iohexyl can be used to selectively aid the differentiation of some microbial species.
Example 9
Improved Method for the Rapid Identification of Blood Culture Isolates by Intrinsic Fluorescence
The culmination of advances made in the design and validation of the optical separation tubes and the associated optical platform, improved classification ability of front face interrogation of microbial pellets, and optimization of rapid selective lysis buffers and a density-based separation step, has resulted in a novel method with the potential to identify microorganisms within minutes from complex samples such as blood culture broth.
The method has advantages in simplicity and safety as the separation and read steps take place within a sealed device. To further establish the utility of this method, we built a database of 373 strains of microorganisms representing the most prevalent 29 species known to cause sepsis. These organisms were “seeded” at a low inoculum into BacT/ALERT® SA bottles containing 10 mLs of human blood. Blood culture broth samples were removed from bottles within a few minutes of being flagged positive by the BacT/ALERT® 3D Microbial Detection System. The samples were treated as follows:

    
    
        1. A 2.0 mL sample of positive broth was mixed with 1.0 mL of selective lysis buffer (0.45% w/v Brij® 97+0.3M CAPS, pH 11.7), then placed in a 37° C. water bath for 1 minute.
        2. A 1.0 mL sample of lysate was overlayed onto 0.5 mL of density cushion (24% w/v cesium chloride in 10 mM Hepes ph 7.4+0.005% Pluronic F-108) contained in a custom-built optical separation tube. A polypropylene ball was present on the surface of the density cushion to facilitate loading without disturbing the two aqueous phases.
        3. The optical separation tube was sealed with a screw-cap and centrifuged for 2 minutes at 10,000 rpm (Eppendorf® 5417R micrcentrifuge fitted with a A-8-11 swing out rotor (Eppendorf, New York); FIG. 4).
        4. The sealed tube was then transferred to a custom-built adapter which coupled the base of the tube directly to a 300 micron fiber optic probe connected to a spectrofluorimeter (Fluorolog® 3 from HORIBA Jobin Yvon Inc., New Jersey)
        5. A full EEM scan of the purified microbial pellet was taken using the CCD detector configuration (Ex 260-850 nm, every 5 nm; Em 260-1100 nm)
        6. The EEM data was exported to Excel.
        7. The entire process, which took less than 20 minutes from the bottle flagging event, was repeated using positive broth that had been stored at 2-8 C for 4-5 hours. The stored broth was warmed for 5 minutes before processing.
    
    


Some representative examples of the EEM spectra of microbial pellets isolated from positive blood culture broth are given in FIGS. 5-8. Differences are visually evident between the species depicted, in both the magnitude and shapes of the various cellular fluorophores present.
The data was analyzed by a variety of multivariate analysis methods with the purpose of building a microbial classification database. Each scan file contained over 9,000 individual fluorescence readings, so a variety of approaches were used to minimize and normalize the data prior to analysis. As an example, Table 10 shows some preliminary results using a General Discriminant Analysis tool (Statistica). Additional input variables, such as Time-to-Detection and growth rates obtained from the BacT/ALERT® Microbial Detection System, and the amount of biomass present in the cell pellet, may be used to aid in the identification and/or characterization of the sepsis-causing isolate (see Example 10).
While the data in Table 10 presents identification results to the species level, any grouping level which provide the attending physician with clinically-relevant actionable information can be delivered. An example of such would be “Therapeutic” groups, where microbial species are grouped according to the antibiotics used to treat them.
The method described in the present invention satisfies the urgent need to rapidly identify microorganisms from a positive blood culture bottle in a safe and reliable manner. The results exemplified in Table 10 rival those of other identification methods that rely on growth or molecular characteristics of the microorganism, but without the time delay or cost. Furthermore, the method can be fully automated so the ID result can be sent directly to the physician via an electronic device anytime of the day or night.
The method of the present invention is also compatible with multiple diagnostic techniques due to the inbuilt separation and read sections of the custom-built disposable device, with intact microorganisms representing the “solid phase” (FIG. 4). Examples of supplemental tests that are being developed using the concept of the present invention include, but are not limited to, measurement of microbial enzymes, cell-surface markers, nucleic acid probes and inhibitors of microbial metabolism. The method is amenable to automation and miniaturization. This potential is described in detail in co-pending U.S. patent application Ser. No. 12/589,985, entitled “Methods for Separation and Characterization of Microorganisms Using Identifier Agents”, filed Oct. 30, 2009, which is incorporated herein by reference.







TABLE 10







Rapid Microbial ID Method


Database was built with combined Fresh and Stored sample data


(746 scans; 373 strains; 29 species)


Leave-one-out cross-validation results of Fresh samples only












Top Choice
aLow Discrim.
Within Top Two
Misclassified as:





C. albicans
10/10
10/10
10/10



C. tropicalis
11/11
11/11
11/11


C. parapsilosis
11/11
11/11
11/11


C. krusei
12/12
12/12
12/12


C. glabrata
10/10
10/10
10/10


All Yeasts:
54/54
54/54
54/54


% correct:
100.0 
100.0 
100.0 


S. aureus
*29/30 
*29/30 
30/30
*S. epidermidis


S. epidermidis
23/23
23/23
23/23


S. mitis
10/10
10/10
10/10


S. pneumoniae
*9/10
*9/10
10/10
*S. mitis


S. pyogenes
11/11
11/11
11/11


S. agalactiae
10/10
10/10
10/10


E. faecalis
14/14
14/14
14/14


E. faecium
*12/13 
*12/13 
12/13
*S. mitis


All GPC:
118/121
118/121
120/121


% correct:
97.5
97.5
99.2


A. baumanii
10/10
10/10
10/10


P. aeruginosa
20/20
20/20
20/20


S. maltophilia
9/9
9/9
9/9


All GNNF:
39/39
39/39
39/39


% correct:
100.0 
100.0 
100.0 


H. influenzae
12/12
12/12
12/12


N. meningitidis
*11/12 
11/12
11/12
*P. aeruginosa


All Fastidious:
23/24
23/24
23/24


% correct:
95.8
95.8
95.8


E. aerogenes
*8/10
10/10
10/10
*E. cloacae and K. pneumoniae


E. cloacae Cpx
*8/10
 9/10
 9/10
*E. aerogenes (2)


C. freundii
10/10
10/10
10/10


E. coli
*23/24 
23/24
23/24
*K. oxytoca


S. enteritidis
11/11
11/11
11/11


K. oxytoca
*11/12 
11/12
11/12
*K. pneumoniae


K. pneumoniae
*13/16 
13/16
15/16
*K. oxytoca and E. aerogenes (2)


S. marcescens
9/9
9/9
9/9


M. morganii
11/11
11/11
11/11


P. mirabilis
*8/11
 8/11
10/11
*P. vulgaris (3)


P. vulgaris
11/11
11/11
11/11


All GNR:
123/135
126/135
130/135


% correct:
91.1
93.3
96.3


All 29 species
357/373
360/373
366/373


n = 373 strains
95.7
96.5
98.1





a= within top 2 choices and a posterior probability >0.10






Example 10
Non-Spectroscopic Measurements to Aid in Characterization and/or Identification of Microorganisms
Non-spectroscopic measurements such as the detection time and microbial growth rates obtained from the detection system algorithms, and the size, shape, color and density of the isolated microbial pellet can be used as additional variables for the characterization and/or identification of a microorganism.
The examples given in FIGS. 9 and 10 demonstrate that measurement of pellet size, detection time and average intrinsic fluorescence can be used to assist in the differentiation of, or to confirm the identification of, two closely-related species, S. pneumoniae and S. mitis. Each symbol represents a separate clinical isolate recovered from positive blood culture broth by the method of the current invention. The S. pneumoniae isolate circled in FIGS. 9 and 10 is the one incorrectly identified as S. mitis in the preliminary results given in Table 10 (Example 9).
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and any other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

## Claims

1. A method of identifying an unknown microorganism from a test sample, comprising:
(a) obtaining a test sample known to contain or that may contain an unknown microorganism;
(b) selectively lysing non-microorganism cells in said test sample to produce a lysed sample;
(c) layering said lysed sample on a density cushion in a container, wherein said density cushion has a homogenous density of from about 1.025 to about 1.22 g/ml;
(d) centrifuging the container to separate said unknown microorganism from other components of said lysed sample, said unknown microorganism passing through said density cushion to form a microorganism pellet at the bottom of said container;
(e) spectroscopically interrogating said pellet in situ to produce an excitation-emission matrix (EEM) of said unknown microorganism, wherein said spectroscopic interrogation comprises intrinsic fluorescence, and wherein said intrinsic fluorescence is measured in front face mode; and
(f) identifying said unknown microorganism in said pellet by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms, wherein said unknown microorganism is identified to the family level, genus level, species level, and/or strain level.

2. The method of claim 1, wherein steps (d) and (e) are carried out in a sealed container and wherein said interrogation step (e) is non-invasive.

3. The method of claim 1, wherein said spectroscopic interrogation further comprise Raman spectroscopy.

4. The method of claim 1, wherein said spectroscopic interrogation further comprises fluorescence spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, terahertz spectroscopy, or any combination thereof.

5. The method of claim 1, wherein said EEM is compared to a database of EEMs of known microorganisms.

6. The method of claim 1, wherein said identification further comprises characterization of said microorganisms based on one or more phenotypic and/or morphologic characteristics.

7. The method of claim 1, wherein said identification further comprises characterization of said microorganisms based on one or more measurements of detection time, growth rate and microorganism pellet size, shape, color and/or density.

8. The method of claim 1, wherein said selective lysis step (b) is performed by sonication, osmotic shock, chemical treatment, or a combination thereof.

9. The method of claim 1, wherein said selective lysis step (b) is performed using a lysis solution comprising one or more detergents.

10. The method of claim 9, wherein said one or more detergents is selected from the group consisting of octylphenol ethoxylate, NP-40, polyoxyethylene detergent, (Octylphenoxy)polyethoxyethanol, polyoxyethylene 10 oleoyl ether, CHAPS, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether, sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, polyoxyethylene (20) oleyl ether, polyoxyethylene 20 cetyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylenesorbitan monooleate, polyoxyethylene sorbitol ester, polyoxyalkylene ether, non-detergent sulfobetaines, amphipols, and methyl-β-cyclodextrin.

11. The method of claim 9, wherein said detergent is a polyoxyethylene detergent comprising the structure C12-18/E9-10.

12. The method of claim 11, wherein said polyoxyethylene detergent is selected from the group consisting of polyoxyethylene 10 oleoyl ether, polyoxyethylene detergent, and polidocenol.

13. The method of claim 9, wherein said lysis solution further comprises an enzyme composition comprising one or more proteinases and one or more nucleases.

14. The method of claim 9, wherein said lysis solution comprises one or more buffering agents.

15. The method of claim 1, wherein said density cushion is selected from the group consisting of colloidal silica, iodinated contrast agents, sucrose, microscope immersion oil, mineral oil, silicone oil, fluorosilicone oil, silicone gel, diatrizoate-dextran, carboxymethyl cellulose, hydroxypropylmethyl cellulose, polyethylene oxide (high molecular weight), polyoxyalkylene ether, polyacrylic acid, cross-linked polyvinyl alcohol, cross-linked polyvinyl pyrrolidine, PEG methyl ether methacrylate, pectin, agarose, xanthan, gellan, Gellan Gum, sorbitol, a sucrose and epichlorohydrin copolymer, glycerol, dextran, glycogen, cesium chloride, perfluorocarbon fluids, hydrofluorocarbon fluid, and combinations thereof.

16. The method of claim 1, wherein said test sample is a culture sample known to contain microorganisms.

17. A method of identifying an unknown microorganism from a blood culture, comprising:
(a) obtaining a sample from a blood culture known to contain or that may contain an unknown microorganism;
(b) selectively lysing non-microorganism cells in said sample to produce a lysed sample;
(c) layering said lysed sample on a density cushion in a container, wherein said density cushion has a homogenous density of from about 1.025 to about 1.22 g/ml;
(d) centrifuging the container to separate said unknown microorganism from other components of said sample, said unknown microorganism passing through said density cushion to form a microorganism pellet at the bottom of said container;
(e) spectroscopically interrogating said pellet in situ to produce an excitation-emission matrix (EEM) of said unknown microorganism, wherein said spectroscopic interrogation comprises intrinsic fluorescence, and wherein said intrinsic fluorescence is measured in front face mode; and
(f) identifying said unknown microorganism in said pellet by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms, wherein said unknown microorganism is identified to the family level, genus level, species level, and/or strain level.

18. The method of claim 17, wherein said spectroscopic interrogation further comprise Raman spectroscopy.

19. The method of claim 17, wherein said spectroscopic interrogation further comprises fluorescence spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, terahertz spectroscopy, or any combination thereof.

20. The method of claim 17, wherein said EEM is compared to a database of EEMs of known microorganisms.

21. The method of claim 17, wherein said density cushion is selected from the group consisting of colloidal silica, iodinated contrast agents, sucrose, microscope immersion oil, mineral oil, silicone oil, fluorosilicone oil, silicone gel, diatrizoate-dextran, carboxymethyl cellulose, hydroxypropylmethyl cellulose, polyethylene oxide (high molecular weight), polyoxyalkylene ether, polyacrylic acid, cross-linked polyvinyl alcohol, cross-linked polyvinyl pyrrolidine, PEG methyl ether methacrylate, pectin, agarose, xanthan, gellan, Gellan Gum, sorbitol, a sucrose and epichlorohydrin copolymer, glycerol, dextran, glycogen, cesium chloride, perfluorocarbon fluids, hydrofluorocarbon fluid, and combinations thereof.

22. The method of claim 17, wherein said identification further comprises characterization of said microorganisms based on one or more phenotypic and/or morphologic characteristics.

23. The method of claim 17, wherein said identification further comprises characterization of said microorganisms based on one or more measurements of detection time, growth rate and microorganism pellet size, shape, color and/or density.

24. The method of claim 17, wherein said identification further comprises characterization of said microorganisms into on one or more classification models selected from the group consisting of Gram Groups, Clinical Gram Groups, Therapeutic Groups, Functional Groups, and Natural Intrinsic Fluorescence Groups.

25. The method of claim 17, wherein said test sample is a culture sample known to contain microorganisms.

26. The method of claim 17, wherein steps (d) and (e) are carried out in a sealed container and wherein said interrogation step (e) is non-invasive.

27. A method of identifying an unknown microorganism from a test sample, comprising:
(a) obtaining a test sample known to contain or that may contain an unknown microorganism;
(b) selectively lysing non-microorganism cells in said test sample to produce a lysed sample;
(c) layering said lysed sample on a density cushion in a container, wherein said density cushion comprises cesium chloride or iohexyl;
(d) centrifuging the container to separate said unknown microorganism from other components of said lysed sample, said unknown microorganism passing through said density cushion to form a microorganism pellet at the bottom of said container;
(e) spectroscopically interrogating said pellet in situ to produce an excitation-emission matrix (EEM) of said unknown microorganism, wherein said spectroscopic interrogation comprises intrinsic fluorescence, and wherein said intrinsic fluorescence is measured in front face mode; and
(f) identifying said unknown microorganism in said pellet by comparison of the spectroscopic measurements with spectroscopic measurements taken, or spectroscopic properties predicted, of known microorganisms, wherein said unknown microorganism is identified to the family level, genus level, species level, and/or strain level.

28. The method of claim 27, wherein steps (d) and (e) are carried out in a sealed container and wherein said interrogation step (e) is non-invasive.

29. The method of claim 27, wherein said spectroscopic interrogation further comprises fluorescence spectroscopy, diffuse reflectance spectroscopy, infrared spectroscopy, terahertz spectroscopy, or any combination thereof.

30. The method of claim 27, wherein said EEM is compared to a database of EEMs of known microorganisms.

31. The method of claim 27, wherein said identification further comprises characterization of said microorganisms based on one or more phenotypic and/or morphologic characteristics.

32. The method of claim 27, wherein said identification further comprises characterization of said microorganisms based on one or more measurements of detection time, growth rate and microorganism pellet size, shape, color and/or density.