Claims: We Claim:
1. A method for a rapid Raman detection of viability of a microorganism, the method comprising:
a preparatory phase, wherein the preparatory phase includes selecting a source containing at least one microorganism, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the microorganism;
a detection phase, wherein the detection phase includes illuminating the dry cast having the microorganism, at a plurality of points around the dry cast, with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering, at a plurality of points including the point of illumination, to obtain a unique biochemical signature specific to each of the microorganism; and
a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism;
wherein the viability index of the microorganism is obtained within a time period of about one hour to about two hours subsequent to preparatory phase.
2. The method as claimed in claim 1, wherein the each of the pre-determined signature profile corresponds to various stages of life cycle of a microorganism.
3. The method as claimed in claim 1, wherein the wavelength of the irradiated electromagnetic radiation is in the range of 200 nm to 1400 nm.
4. The method as claimed in claim 1, wherein the resolution of the Raman spectra for obtaining unique signatures is in the range of 1cm-1 to 8 cm-1.
5. The method as claimed in claim 1, wherein the biochemical signatures are molecular bond specific signatures.
6. The method as claimed in claim 1, wherein the capture of the Raman scattering is independent of angle of illumination.
7. The method as claimed in claim 1, wherein the microorganism is selected from a group comprising of a gram positive bacteria, a gram negative bacteria, an aerobic bacteria, an anaerobic bacteria, a mycobacteria and a virulent species.
8. The method as claimed in claim 1, wherein the source of the microorganism is selected from a group comprising of a body fluid sample, a tissue sample, or a food sample.
9. A method for a Raman screening of antibiotic susceptibility of a microorganism, the method comprising:
a preparatory phase, wherein the preparatory phase includes selecting at least one antibiotic specific to at least one microorganism, culturing the microorganism along with the selected antibiotic, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the antibiotic induced microorganism;
a detection phase, wherein the detection phase includes illuminating the dry cast having the antibiotic induced microorganism at a plurality of points around the dry cast with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering at a plurality of points including the point of illumination to obtain a unique biochemical signature specific to each of the antibiotic bound to a microorganism; and
a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism;
wherein the antibiotic susceptibility is determined as a percentage of viable microorganism.
10. The method as claimed in claim 9, wherein the each of the pre-determined signature profile is a signature specific to a specific antibiotic induced to a specific microorganism.
11. The method as claimed in claim 9, wherein the wavelength of the irradiated electromagnetic radiation is in the range of 200 nm to 1400 nm.
12. The method as claimed in claim 9, wherein the resolution of the Raman spectra for obtaining unique signatures is in the range of 1cm-1 to 8 cm-1.
13. The method as claimed in claim 9, wherein the biochemical signatures are molecular bond specific signatures.
14. The method as claimed in claim 9, wherein the capture of the Raman scattering is independent of angle of illumination.
15. The method as claimed in claim 9, wherein the microorganism is selected from a group comprising of a gram positive bacteria, a gram negative bacteria, an aerobic bacteria, an anaerobic bacteria, a mycobacteria and a virulent species.
, Description:RAPID RAMAN DETECTION OF VIABILITY OF A MICROORGANISM
FIELD OF INVENTION
The invention generally relates to the field of physical chemistry and diagnostic microbiology and particularly to a method for detection of viability of pathogenic microorganisms within a sample.
BACKGROUND OF THE INVENTION
Detection of microorganisms, including, both pathogenic and non-pathogenic, has been both of academic and clinical interest. Specifically, detection and identification of clinically important microorganisms has been evolving continuously, with the need to detect a given microorganism, rapidly and with high specificity.
Examples of sample include but are not limited to those obtained from infectious diseases, food and dairy products. Examples of clinically important conditions include but are not limited to pneumonia, tuberculosis, septicaemia, meningitis, urinary tract infection and gastro-intestinal infections. The diagnostic methods available in the art include but are not limited to nucleic acid/protein based methods, culturing based methods, staining based methods and mass spectrometry based methods.
Culturing based methods require inoculation of the growth medium with the biological sample containing organism. The colonies grown on the growth medium are then identified. The techniques of identification include but are not limited to staining, antibiotic selection and metabolic reactions. One significant disadvantage of the technique is elaborate procedures involved to perform the test. Another disadvantage is time required for completion of the diagnosis, which normally requires 3-4 days but may extend up to 21 days to detect a certain bacteria like Mycobacterium tuberculosis. Furthermore, only live culturable bacteria are detected using standard growth based techniques. Viable but not culturable bacteria (VBNCS) which are metabolically viable but not culturable due to lack of knowledge of their preferred growth conditions or due to dormancy successfully elude culturing dependent detection test, resulting in false negatives.
Nucleic acid based methods use specific Nucleic acid sequences present in various pathogens as signatures for identification. Examples of nucleic acid based methods include but are not limited to PCR, RT-PCR, line amplification assay and serology based assay. One of the significant disadvantages of the technique is the low sensitivity due to paucity of organism in the sample. Another disadvantage of the technique is the false positive results due to contamination of the sample with environmental bacteria. Further disadvantage is created due to the fact that DNA can be isolated from both live and dead organisms and therefore these techniques cannot be employed for viability assessment. Assays based on detection of antibodies against pathogen antigens has major disadvantage of cross reactivity of antibodies to closely related bacterial species. Another disadvantage is that these assays do not provide information regarding the resistance to the antimicrobial agents.
Mass spectrometry uses a mass spectrometer to analyze extracted DNA or RNA or protein or lipids or other biological molecules. The mass signatures obtained are analyzed for bacterial species identification. Limitation of the technique is that it has a poor application as a stand-alone technique for bacterial identification and is often used along with PCR.
Thus, there is a need for a method that has high specificity and sensitivity, is less time consuming and is able to detect the viability of the microorganism in bulk or singly with accuracy.
Raman scattered light has been explored for identifying the structure and chemical nature of the molecules. Examples of known techniques that record Raman scattering include Spatially Offset Raman Spectroscopy (SORS), Surface Enhanced Raman Spectroscopy (SERS) and transmission Raman spectroscopy (TRS).One such method is disclosed in Patent JPH09121889 assigned to Ajinomoto Co. Inc. The method discloses determining the viability of the lyophilized microorganism inside a container based on the ratio of carbon dioxide to oxygen in the container using Raman spectroscopy. The method primarily utilizes the correlation between the carbon dioxide to oxygen ratio inside the container and the microorganism to detect the viability of the freeze dried organisms inside a closed container using Raman spectroscopy without destroying the container. One significant advantage of the method is that it provides the data of survival rate of the freeze dried organisms inside the container. However, the method has a disadvantage of capturing effective readings due to the fact that only gas composition is analyzed and not the microorganism number.
Patent US 2010/0055721A1 assigned to California Institute of Technology mentions a method of quantitative measurement of levels of microorganisms by employing Raman scattering. They have tagged antibodies with SERS nanoparticles as Raman scattering indicators. The primary disadvantage of SERS based detection is that the detection is restricted to a specific experimental geometry, which involves strict description of finite distance between light input and collection of scattered light. Further disadvantage is that SERS relies on metal surface for amplification which is often difficult to reproduce.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method for rapid detection of viability of a microorganism. The method includes preparing a sample, irradiating the prepared sample with an electromagnetic radiation of specific wavelength, capturing the electromagnetic radiation scattered by the sample to obtain a Raman spectra and analyzing the spectra to obtain a unique biochemical signature. The biochemical signature obtained specific to each of the microorganism is compared with a pre-determined signature profiles to obtain a viability index of the microorganism.
Another aspect of the invention provides a method of screening antibiotic susceptibility of the microorganism. The method includes preparation of antibiotic treated/exposed microorganism sample, irradiating the prepared sample with an electromagnetic radiation of specific wavelength, capturing the electromagnetic radiation scattered by the sample to obtain a Raman spectra and analyzing the Raman spectra to obtain a unique biochemical signature specific to the antibiotics present in the microorganism. The unique biochemical signature obtained specific to the microorganism is compared with pre-determined signature profiles to obtain a viability index of the microorganism thereby analyzing the antibiotic exposure status as well as susceptibility index when analyzed in context with live/ dead signatures.
BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG.1 shows a flowchart for identification and viability assessment of a given microorganism, according to an embodiment of the invention.
FIG.2a shows a 2D scatter plot obtained from a PC-Linear Discriminant Viability analysis of the spectra obtained from a single bacterium in the sample containing E. coli, according to one example of the invention.
FIG.2b shows a 2D scatter plot obtained from a PC-Linear Discriminant Viability analysis of the spectra obtained from bulk bacteria in the sample containing E. coli, according to one example of the invention.
FIG.3a shows a 2D scatter plot obtained from a PC-Linear Discriminant Viability analysis of the spectra obtained from a single bacterium in the sample containing Mycobacterium smegmatis (single organism level), according to another example of the invention.
FIG. 3b shows a 2D scatter plot obtained from a PC-Linear Discriminant Viability analysis of the spectra obtained from bulk bacteria in the sample containing Mycobacterium smegmatis, according to another example of the invention
FIG. 4 shows antibiotic susceptibility of M.smegmatis treated with various antibiotics, according to another embodiment of the invention, indicative of efficacy of live-dead analysis.
DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method for detection of the viability of microorganism within a sample. The microorganism is selected from a group comprising of a gram positive bacteria, a gram negative bacteria, an aerobic bacteria, an anaerobic bacteria, a mycobacteria and a virulent species.
The method includes a preparatory phase, wherein the preparatory phase includes, selecting a source containing at least one microorganism, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the microorganism, a detection phase, wherein the detection phase includes illuminating the dry cast having the microorganism, at a plurality of points around the dry cast, with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering, at a plurality of points including the point of illumination, to obtain a unique biochemical signature specific to each of the microorganism, and a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism. The viability index of the microorganism is obtained within a time period about one hour to about two hours. In addition to obtaining the viability index, the method is also capable of identifying the species, through a method, as claimed in the Patent Application no 6468/CHE/2015. The method described herein, briefly shall be discussed in detail, herein below.
The method of rapid detection is based on Raman microspectrometry. Raman microspectrometry uses Raman spectrometry to measure the spectra of microscopic samples. A Raman microspectrometer is used, which allows acquisition of Raman spectra of microscopic samples. The technique captures molecular bond specific vibrations originating from the biochemical constituents of the cell. The spatial resolution required for microbial biochemical fingerprint is achieved with the use of microscopy along with the spectroscopy.
The method described in brief hereinabove shall be described in detail with various embodiments. The method involves the steps of preparing a sample, irradiating the sample with an electromagnetic radiation of specific wavelength, capturing the electromagnetic radiation scattered by the sample to obtain a Raman spectra and analyzing the Raman spectra to obtain a unique biochemical signature. The unique biochemical signature obtained identifies the viability of microbial species within the sample.
As shown in the flowchart of the FIG 1, the steps for identification of microorganism involve the following steps:
First step for detection is preparation of a sample. The sample used for detection includes but is not limited to a body fluid sample, a tissue sample, a food sample. Sample preparation is achieved in two steps; First step is decontamination of the sample. The decontamination of the sample is performed to remove unwanted components including but not limited to mucous, cellular components. Subsequent to decontamination, the sample is subjected to a second step of fixation. Examples of fixation include but are not limited to chemical treatment. In one example of the invention, chemical treatment is performed through 4% PFA. Decontamination is achieved by irradiation or by heat or by chemical treatment. The decontaminated sample is then washed three times with autoclaved Type1 water. Washing is done to remove lysed cellular components. The washed and decontaminated sample is then dried and casted on a substrate. In one embodiment of the invention, prior to casting on the substrate, the decontaminated cells in the sample are fixed by using paraformaldehyde solution.
The substrate is fabricated by depositing a layer of metal on a silicon wafer. The deposition of the metal layer is achieved by first sputter coating and then annealing the metal on the silicon wafer. The thickness of the metal layer is in the range of 100 nm to 1mm. Examples of metal include but are not limited to aluminium, silver and gold. In one embodiment of the invention, aluminium is deposited on the silicon wafer. The prepared sample is then irradiated with an electromagnetic radiation. The wavelength of the electromagnetic radiation is in the range of 200 nm to 1400 nm. In one example of the invention, the source of electromagnetic radiation is a laser source. For irradiation, electromagnetic radiation from a radiation source is focused through a plurality of mirrors and lenses or a microscopic objective on the prepared sample. The electromagnetic radiation scattered by the sample is then dispersed using a grating. The dispersed electromagnetic radiation is then captured by a detector and a Raman spectrum is obtained. The Raman spectra obtained is molecular bond specific and represents biochemical composition of the bacterial species. The Raman spectra obtained is then analyzed to obtain biochemical signatures specific to the microbial species and its viability. The resolution of the Raman spectra obtained is in the range of 1cm-1 to 8 cm-1. Further, the time duration for identification of the bacterium is in the range of one hour to two hour.
Identification and Validation:
The cultured microorganisms are harvested and pelleted by centrifugation at 5000g for 5 minutes. The pelleted microorganisms are then washed thrice with autoclaved Type 1 water. After washing the microorganisms are dry casted on the substrate.
The substrate is fabricated by first sputter coating/thermal evaporation and then annealing aluminium as a 200 nm layer on a silicon wafer. The dry-casted microbial sample are then mounted on a microscope, focused to the appropriate location and then irradiated by a laser source having a wavelength of 633nm. The electromagnetic radiation scattered by the microbial sample are focused and collected using a 100x, 0.8NA objective. The scattered radiation after passing the notch filter is focused on to a monochromator with 1200lines/mm grating and detected using a Peltier cooled CCD camera at 256x1048 pixels resolution.
During spectroscopic evaluation, for the lower wave number region 500-1900cm-1 the microorganisms are exposed for 15 seconds and the spectra is accumulated 5 times to get a good signal to noise ratio. For the higher wave number region 2800-3000cm-1 the microorganisms are exposed for 10 seconds and the spectra is accumulated three times. After data collection, the spectra is subjected to pre-processing which included, cosmic ray removal, multipoint base line correction, Savitzky- Golay smoothing and vector normalization using Renishaw wire 4.2 and OriginPro 8.5 software. The dataset is then is used for multivariate analysis.

EXAMPLE 1: Viability analysis for E.coli
Freshly inoculated overnight cultures of E.coli grown in Luria broth (LB), is treated with Ampicillin at 40µg/ml concentration. After overnight exposure to the antibiotic at 37°C, cells are pelleted down to discard LB containing ampicillin and are re-suspended in double distilled water. To validate the viability, the cells are plated on LB plates and are incubated overnight. The incubated plates confirms that the untreated E.coli show growth wherein ampicillin treated cells do not show any signs of growth. The Raman analysis performed as per the described method gives the spectra that are collected for further analysis.
FIG. 2a shows the results of single bacterial level assessment. PC-LDA gives an accuracy of 96.77% which is evident in confusion matrix as shown in table 1 where 4 spectra of live are misclassified as dead as the bacteria can die due to multiple other factors in a culture which contribute to the misclassification. But in actual condition it is possible to have cells that are live but non-culturable and hence they give spectra signature of live bacteria, which incidentally cannot be recorded using any other technique. Raman can identify live and dead even when both are mixed in one sample. It is possible to evaluate the percentage of microbial load that is alive or dead from the above described method. FIG.2b and table 2 shows the bulk bacterial analysis in which the PC-LDA gives an accuracy of 100% in identifying the viability.

Actual ?
Predicted ? E.coli Live E.coli Dead
E.coli Live 51 0
E.coli Dead 4 69
Table 1: Confusion matrix for E.coli (single bacterial level)

Actual ?
Predicted ? E.coli Live E.coli Dead
E.coli Live 55 0
E.coli Dead 0 65
Table 2: Confusion matrix for E.coli (Bulk bacterial analysis)
EXAMPLE 2: Viability analysis for Mycobacterium smegmatis
The method also includes, a preparatory phase, wherein the preparatory phase includes selecting at least one antibiotic specific to a microorganism, culturing the microorganism induced with the selected antibiotic, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the antibiotic induced microorganism, a detection phase, wherein the detection phase includes illuminating the dry cast having the antibiotic induced microorganism at a plurality of points around the dry cast with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering at a plurality of points including the point of illumination to obtain a unique biochemical signature specific to each of the antibiotic bound to a microorganism; and a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism, wherein the antibiotic susceptibility is determined as a percentage of viable microorganism.
M. smegmatis is treated with the various antibiotics viz. isoniazid, ethambutol, rifampicin and streptomycin with concentrations of 32 ?g/ml, 42 ?g/ml, 20 ?g/ml, 10 ?g/ml respectively, known to be sufficient to kill Mycobacterial cells. In order to mimic the tuberculosis treatment protocol, M.smegmatis is treated with combination of all above drugs to achieve complete killing. Viability assessment is performed using plate based culture assay. Only combination of all the antibiotics is successful in killing the mycobacteria completely.
FIG.3a and 3b shows a 2D scatter plot obtained from a PC-Linear Discriminant Analysis of the spectra of live or dead bacteria, according to one example of the invention. Each spectrum is colour coded according to the viability (live/dead) of the bacteria. The PC-LDA scatter plot for single and bulk M.smegmatis as shown in FIG. 3a and 3b gives 100% accuracy, which is further evident in the confusion matrix as shown in table 3 obtained for predicting the efficiency and reliability of the detection method.

Table 3: Confusion matrix for M.smegmatis (a) Single bacterial analysis (b) Bulk bacterial analysis.
EXAMPLE 3: Antibiotic Susceptibility Analysis
The method proposed for viability assessment can also be applied to antibiotic susceptibility. The untreated M.smegmatis cells shows maximum growth, the ethambutol and rifampicin treated M.smegmatis has the second highest growth, the isoniacied treated M.smegmatis has the third highest growth and fourth highest is the streptomycin treated M.smegmatis. In order to predict the viability the PC-LDA model built in FIG. 3a and 3b is referred. FIG.4 shows the result of the prediction. In single bacterial level, ethambutol and rifampicin shows more than 30% live mycobacterium whereas isoniazid and streptomycin showed less than 30% live mycobacterium. In bulk bacterial analysis, more than 80% are predicted live for ethambutol treated mycobacterium. Rifampicin shows 30% live and streptomycin shows 2% live mycobacterium. The streptomycin shows less than 5% live mycobacterial cells in both single and bulk bacterial analysis. Thus the presence of both live and dead can be simultaneously identified in case of mixed culture analysis. Thus, we can see a correlation of Raman spectral analysis with the plate assay.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

We Claim:
1. A method for a rapid Raman detection of viability of a microorganism, the method comprising:
a preparatory phase, wherein the preparatory phase includes selecting a source containing at least one microorganism, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the microorganism;
a detection phase, wherein the detection phase includes illuminating the dry cast having the microorganism, at a plurality of points around the dry cast, with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering, at a plurality of points including the point of illumination, to obtain a unique biochemical signature specific to each of the microorganism; and
a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism;
wherein the viability index of the microorganism is obtained within a time period of about one hour to about two hours subsequent to preparatory phase.
2. The method as claimed in claim 1, wherein the each of the pre-determined signature profile corresponds to various stages of life cycle of a microorganism.
3. The method as claimed in claim 1, wherein the wavelength of the irradiated electromagnetic radiation is in the range of 200 nm to 1400 nm.
4. The method as claimed in claim 1, wherein the resolution of the Raman spectra for obtaining unique signatures is in the range of 1cm-1 to 8 cm-1.
5. The method as claimed in claim 1, wherein the biochemical signatures are molecular bond specific signatures.
6. The method as claimed in claim 1, wherein the capture of the Raman scattering is independent of angle of illumination.
7. The method as claimed in claim 1, wherein the microorganism is selected from a group comprising of a gram positive bacteria, a gram negative bacteria, an aerobic bacteria, an anaerobic bacteria, a mycobacteria and a virulent species.
8. The method as claimed in claim 1, wherein the source of the microorganism is selected from a group comprising of a body fluid sample, a tissue sample, or a food sample.
9. A method for a Raman screening of antibiotic susceptibility of a microorganism, the method comprising:
a preparatory phase, wherein the preparatory phase includes selecting at least one antibiotic specific to at least one microorganism, culturing the microorganism along with the selected antibiotic, decontaminating the source, washing the decontaminated source, and dry casting the decontaminated source to obtain a dry cast of the antibiotic induced microorganism;
a detection phase, wherein the detection phase includes illuminating the dry cast having the antibiotic induced microorganism at a plurality of points around the dry cast with an electromagnetic radiation of specific wavelength, and capturing the Raman scattering at a plurality of points including the point of illumination to obtain a unique biochemical signature specific to each of the antibiotic bound to a microorganism; and
a validation phase, wherein the obtained unique biochemical signature specific to each of the microorganism is compared with a plurality of pre-determined signature profiles to obtain a viability index of the microorganism;
wherein the antibiotic susceptibility is determined as a percentage of viable microorganism.
10. The method as claimed in claim 9, wherein the each of the pre-determined signature profile is a signature specific to a specific antibiotic induced to a specific microorganism.
11. The method as claimed in claim 9, wherein the wavelength of the irradiated electromagnetic radiation is in the range of 200 nm to 1400 nm.
12. The method as claimed in claim 9, wherein the resolution of the Raman spectra for obtaining unique signatures is in the range of 1cm-1 to 8 cm-1.
13. The method as claimed in claim 9, wherein the biochemical signatures are molecular bond specific signatures.
14. The method as claimed in claim 9, wherein the capture of the Raman scattering is independent of angle of illumination.
15. The method as claimed in claim 9, wherein the microorganism is selected from a group comprising of a gram positive bacteria, a gram negative bacteria, an aerobic bacteria, an anaerobic bacteria, a mycobacteria and a virulent species.