METHODS FOR ESTIMATING ANTIBIOTIC SUSCEPTIBILITY AND ANTIBIOTIC SENSITIVITY PROFILE OF MICROORGANISMS  AND DEVICES THEREFOR
FIELD OF THE INVENTION AND USE OF INVENTION
[0001] The invention relates generally to estimating antibiotic susceptibility of microorganisms and more specifically to methods involving preparing sample comprising a predefined concentration of microorganisms and exposing it to at least one antibiotic to obtain its susceptibility  and sensitivity profile and ensuring rapidity of result by minimizing interaction volumes without compromising on cell concentrations.
PRIOR ART AND PROBLEM TO BE SOLVED
[0002] Disease causing microbes that have become resistant to drug therapy are an increasing public health problem. Factors contributing to the rise in antibiotic resistance include widespread and inappropriate prescription of broad spectrum antibiotics and patient non-compliance to antibiotic regimens. It is estimated that about 70 percent of pathogenic bacteria in hospitals are resistant to at least one of the drugs most commonly used to treat infections (Federal Drug Administration (2007)).
[0003] Bacterial drug resistance may be detected in different ways. These include cell or bacterial culture based methods  non-culture techniques that include genetic techniques  immunological techniques  based on the detection of presence of special proteins like the beta-lactamase enzyme  lateral flow test or lateral immunochromatographic assays that are used to detect different compounds in bodily fluids or other biological samples  penicillin detection test (which are not necessarily for antibiotic resistance per se) and the like  and combinations thereof. Exemplary culture based techniques include disk diffusion method (Kirby-Bauer test)  colorimetric methods  and the like. Exemplary non-culture techniques include PCR which enables the detection of DNA/RNA responsible for coding enzyme  bio-sensors based methods  and so on. Exemplary immunological techniques comprise immunoblot reaction  slide agglutination test  latex agglutination etc.
[0004] Generally  the techniques described and used in a variety of situations  such as medical (or veterinary) diagnostics are useful  but expensive and time-consuming. They also require highly qualified staff. According to these disadvantages  there is still a need to develop an antibiotic resistance / sensitivity test and method that could guarantee high sensitivity  reliability and should be achieved in a fast  easy  and cheap manner.
OBJECTS OF THE INVENTION
[0005] The invention provides a cost effective antibiotic resistance / sensitivity technique that can render fast results with high sensitivity and reliability that is extremely useful in resource deficient environments.
[0006] The objects of the invention are listed herein below in the Summary section.
SUMMARY OF THE INVENTION
[0007] In one aspect  the invention provides a method for estimating antibiotic susceptibility of microorganisms in a sample. The method comprises estimating an initial concentration of microorganisms in the sample. Subsequently  the method involves performing a concentration adjustment of the microorganisms in the sample based on the initial concentration to provide an adjusted sample. Then the method includes exposing the adjusted sample to at least one antibiotic to provide an exposed sample. Further  the method comprises estimating a final concentration of microorganisms in the exposed sample; and accordingly estimating the antibiotic susceptibility of the microorganisms based on the initial concentration and final concentration of microorganisms.
[0008] In another aspect  the invention provides a method for obtaining antibiotic sensitivity profile of microorganisms in a sample. The method comprises providing an adjusted sample as described herein. Subsequently  the method includes exposing the adjusted sample to at least one antibiotic to provide a primary exposed sample and independently exposing the adjusted sample to at least one of a different concentration of the at least one antibiotic used to provide a primary exposed sample  or at least one antibiotic that is different from that used to provide a primary exposed sample  and combinations thereof  to provide a secondary exposed sample(s). Then  the method involves estimating a primary final concentration of microorganisms in the primary exposed sample and a secondary final concentration(s) of microorganisms in the secondary exposed sample(s) and obtaining the antibiotic sensitivity profile of the microorganisms based on the initial concentration and primary final concentration and secondary final concentration(s).
[0009] In yet another aspect  the invention provides a kit for estimating antibiotic susceptibility based on the methods described herein.
[0010] In a further aspect  the invention provides a kit for obtaining antibiotic sensitivity profile based on the methods described herein.
[0011] In a yet another aspect  the invention provides a device for estimating antibiotic susceptibility of microorganisms. The device of the invention comprises a sample receptacle to receive samples comprising microorganisms  a sample adjuster to adjust the concentration of microorganisms in the sample within a predetermined level to provide an adjusted sample  an exposure chamber to expose the adjusted sample to at least one antibiotic at one or more concentration ranges to provide an exposed sample(s)  and a sample reader to estimate an initial and final concentrations of microorganisms in the sample from the sample  the adjusted sample  the exposed sample(s)  and combinations thereof. The exposure chamber of the device of the invention is constructed such that the exposure is achieved simultaneously or sequentially  or both.
DRAWINGS
[0012] These and other features  aspects  and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings  wherein:
[0013] FIG. 1 is a block diagrammatic representation of a device of the invention for estimating antibiotic susceptibility;
[0014] FIG. 2 is a plot of the normalized number of live cells against time for Ciprofloxacin;
[0015] FIG. 3 is a plot of the normalized number of live cells against time for Tetracycline;
[0016] FIG. 4 is a plot of the normalized number of live cells against time for Gentamycin;
[0017] FIG. 5 is a plot of the normalized number of live cells against time for Ampicillin; and
[0018] FIG. 6 is a plot of the normalized number of live cells against time for Nalidixic Acid.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
[0020] As used in this specification and the appended claims  the singular forms "a"  "an"  and "the" encompass embodiments having plural referents  unless the content clearly dictates otherwise.
[0021] Unless otherwise indicated  all numbers expressing feature sizes  amounts  and physical properties used in the specification and claims are to be understood as being modified in all instances by the term "about."Accordingly  unless indicated to the contrary  the numerical parameters set forth in the foregoing specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.
[0022] As used in this specification and the appended claims  the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
[0023] As used herein  the term "microorganism" refers to a class of microscopic organisms that comprise single cells  cell clusters or multicellular organisms. The microorganisms of interest in the invention include pathogenic microorganisms  aerobes or anaerobes that are known to cause disease and afflictions among humans and other animals. Such microorganisms include bacteria  fungi  yeast  and the like. A few non-limiting exemplary microorganisms include Enterobacteriaceae  Pseudomonas spp  Staphylococcus spp  Enterococcus spp  Candida  and the like.
[0024] As used herein  the term "antibiotic" refers to any compound that is useful to treat infectious diseases  especially those caused by microorganisms such as bacteria  fungi  yeast  and the like. Antibiotics may be naturally-occurring substances that are produced by microorganisms  such as  for example  fungus or yeast. Alternately  antibiotics may also be semi-synthetic substances wherein a molecular version produced by a microorganism is subsequently modified to achieve desired properties. Further  antibiotics may be a completely synthetic compound produced. Antibiotics known in the art may be classified differently based on the structure. Alternately  antibiotics may be classified based on their function into inhibitors of cell wall synthesis  inhibitors of protein synthesis  inhibitors of membrane function  anti metabolites and inhibitors of nucleic acid synthesis  Folate pathway Inhibitors  and so on. Exemplary antibiotics include  but not limited to  Penicillins  ß-lactam/ß-Lactamase inhibitors  Amidinopenicillin  Cephems Parenteral / Oral (including cephalosporin’s I  II  III  IV and next generation cephalosporin’s  Oral C3G  Anti MRSA Cephalosporin’s  Lipopeptides  Monobactams  Carbapenems  Aminoglycosides  Glycopeptides  Tetracyclines  Fluoroquinolones  Quinolones  Phenicols  Fosfomycins  Nitrofurantoins  Macrolides  Ketolides  Lincosamides  Ansamycins  Streptogramins  Oxazolidinones  and so on  and combinations thereof.
[0025] As used herein  the phrase "antibiotic susceptibility" refers to the degree to which a bacterial cell is affected by an antibiotic. Broadly  the susceptibility may be divided into three different types: (1) the cell may not be affected at all;(2) cell growth and proliferation may have slowed or halted without its being killed; or (3)cells may be killed. Susceptibility also refers to the extent a population of a bacterial species or a particular strain is affected by an antibiotic. In this case  certain highly susceptible cells of the population may be very sensitive and may be killed by very low concentrations of the antibiotic  other  less sensitive cells may have their growth and proliferation slowed while others may not be affected at all.
[0026] As used herein  the phrase "antibiotic sensitivity profile" means identifying susceptibility at a given concentration of one or more antibiotic  wherein the concentration is traceable to any existing standard  or susceptibility to one or more concentrations of one antibiotic. Antibiotic sensitivity profile is a useful measure to identify the extent to which a particular antibiotic is capable of handling a particular microorganism  or alternately  the most potent antibiotic at a given concentration. This is useful in recognizing the best course of treatment under a given set of circumstances. For instance  a patient suffering from some secondary effects such as impaired renal function  may not be capable of handling above a certain concentration of an antibiotic towards treatment of an affliction. Consequently  it is necessary to know the most potent antibiotic within the maximum concentration  or at least whether a particular antibiotic is capable of inhibiting the growth of microorganism within the concentration.
[0027] The phrase "minimum inhibitory concentration" also sometimes abbreviated in the art as MIC means any concentration of the antibiotic beyond which exposure of the microorganism to the antibiotic will cause 99.9% cell death. One skilled in the art will immediately recognize that obtaining an antibiotic sensitivity profile can also be extended to obtain minimum inhibitory concentration in a facile manner  when done under the right conditions.
[0028] The phrase "clinical breakpoint" also abbreviated in the art as CBP is a discriminating concentration used in the interpretation of results of susceptibility testing to define isolates as susceptible  intermediate or resistant thus obtaining the antibiotic sensitivity profile to guide clinical decision-making as established and defined by existing standards for a testing method  or some other standards such as CLSI  EUCAST or other region specific standards.
[0029] As used herein  "contacting" refers to bringing a substance containing microorganism into contact with a growth medium and the chemicals therein such that the cells of the microorganism proliferate in the absence or  if they are resistant  in the presence of an antibiotic but are killed or inhibited by the antibiotic if they are susceptible.
[0030] As used herein  "incubating" refers to maintaining the physical parameters necessary for growth of a microorganisms  for example  without limitation  the temperature of the medium  the atmosphere (air  carbon dioxide  etc.) in which the medium is placed  pH  nutrients for growth  lighting conditions  and the like.
[0031] As used herein  the phrase "sample" includes a fluid sample obtained from any source. Some examples of a sample include those obtained from a biological source such as sputum  urine  blood  sweat  cerebrospinal fluid (CSF)  ascitic fluid  solid sample such as a tissue biopsy  and the like. Sample may also include those obtained from other sources  such as water for purification testing  sewage sludge  food  beverages and so on. Samples may also include treated and/or processed samples such as solid samples treated to disperse microorganisms into a suitable liquid.
[0032] As noted herein  in one aspect the invention provides a method for estimating antibiotic susceptibility of microorganisms. The method comprises providing a sample comprising the microorganisms. The amount of sample required for the method of the invention ranges from about 1 nanoliter to about 1 milliliter. The small volumes required enables rapid estimation of antibiotic susceptibility as the number of microorganisms needed to achieve the target concentration is very low  and the accuracy of the method is not compromised.
[0033] Subsequently  the method of the invention includes estimating an initial concentration of microorganisms in the sample. Concentration of microorganisms may be expressed in a variety of ways in the art  and one exemplary unit is number of cells per millimeter (cells/mm)  and another exemplary unit is number of cells per milliliter (cells/mL) in a liquid medium. Techniques for estimating concentration of microorganisms are well known in the art  and include methods  such as tagging the microorganisms using colorimetric or fluorescent tags  and measuring the intensity of the light emanating from the sample. Tagging of microorganisms may also involve tagging only live cells  tagging dead cells specifically  or tagging all cells present in a sample. Simultaneously tagging for live cells and dead cells and estimating concentration of both is also encompassed within the scope of the invention. Useful fluorophores known in the art to tag both live and dead cells include the family of bis-benzimides that include commercially available Hoechst stains such as Hoechst® 33342 and the like; DAPI; SYTO®-9; cyanine dyes such as SYBR Green I and the like  CTC  DiOC3  LDS  and so on. Exemplary fluorophores that stain dead cells preferentially include Propidium Iodide; Indole derivatives such as SYTOX® Green commercially available from Invitrogen Inc.  USA. Combinations of fluorescent tags are encompassed within the scope of the invention. In one embodiment  the tags include fluorescent tags. Fluorescence detection techniques are generally popular in the art as they are sensitive enough to be able to obtain relatively good signals with high signal-to-noise ratio thus allowing for accurate and even in some cases quantitative estimations. In one specific embodiment  the estimation of concentration of microorganisms involves the use of fluorescence detection techniques.
[0034] The method of the invention then involves performing a concentration adjustment of the microorganisms in the sample based on the initial concentration to provide an adjusted sample. The ideal concentration of microorganisms in a given sample may be defined as per existing standards for a testing method  or some other standards such as CLSI  EUCAST or other region specific standards. In one specific embodiment  the concentration of live cells of microorganisms in the adjusted sample is held between about 1 x 104cells/mL to about 1 x 107cells/mL. The concentration adjustment may involve reducing the concentration if it is found to be too high. This may be done by dilution with a suitable solvent (such as aqueous or other medium like phosphate buffer) or other substances. If the concentration is determined to be low  then the sample is allowed to be incubated under conditions that allow cell proliferation to increase the concentrations.
[0035] When the concentration adjustment includes an incubation step  the sample is allowed to incubate at some predefined conditions. The time required for incubation may be determined by knowing the initial concentration of microorganisms and a concentration of microorganisms at one or more intermediate time periods in an empirical manner. Further  in some instances  other conditions to accelerate or decelerate growth to achieve the desired concentration may also be suitably employed. One exemplary condition that is varied is temperature. The time required for incubation may also be known to one of ordinary skill in the art based on prior experience  knowledge and the like.
[0036] Subsequently  the method of the invention includes exposing the adjusted sample to at least one antibiotic to provide an exposed sample. Typically  the exposing is conducted under conditions of incubation  such conditions being defined in a variety of different literature sources  and also provided as part of the standards testing procedures given in EUCAST or CLSI  and so on. In this manner  the microorganisms are allowed to grow and proliferate in the presence of the chosen antibiotic. One of ordinary skill in the art will also recognize that the exposure of the microorganisms to multiple antibiotics can also be done using this method. The exact nature of the one or more antibiotics  the concentration of the one or more antibiotics  and other aspects will become apparent to one skilled in the art. In one embodiment  the concentration of the one or more antibiotics is in the range from about 0 micrograms per mL to about 10 milligrams per mL.
[0037] The microorganisms that are subjected to the growth conditions will be affected by the presence of the at least one antibiotics. Thus  depending on the susceptibility of the microorganisms to the at least one antibiotic  the microorganisms will continue to grow  or will show inhibited growth  or will completely die out.
[0038] One skilled in the art will also recognize that it may also be advantageous to independently expose the sample to similar growth conditions as described herein in the absence of the antibiotics to provide a control exposed sample. In some embodiments  the control exposed sample will be provided simultaneously as the exposed sample so as to obtain an instantaneous comparison between the growth patterns of the microorganisms in the presence and in the absence of the at least one antibiotic. Control growth samples also allows for making in situ judgments with regards to many factors  such as effects of extraneous factors  improper growth conditions  artifacts in the data obtained  and so on.
[0039] In some instances  exposing the adjusted sample is done in the presence of a suitable growth inhibitor of non-pathogenic microorganisms. This ensures that no extraneous artifacts affect the data obtained  thus improving efficiency and accuracy of the method. Exemplary growth inhibitors useful in the invention include  but not limited to  sodium azide  dextrose  thallium acetate  brilliant green  and combinations thereof.
[0040] Then  the method of the invention comprises estimating a final concentration of microorganisms in the exposed sample in a similar manner as that for estimating the initial concentration of the microorganisms. The techniques are as described herein  and are generally repeated in the same manner as before to ensure proper comparison of appropriate data.
[0041] In this manner  the method of the invention enables a facile way of estimating the antibiotic susceptibility of the microorganisms based on the initial concentration and final concentration of microorganisms. The final concentration of microorganisms are compared to control samples to account for any artefacts or other extraneous features causing distortion of data. The exact manner of representing the antibiotic susceptibility may vary and includes comparing total cell concentrations in the initial and exposed samples  a ratio of a live cell concentration in the initial and exposed samples  a ratio of dead cell concentrations in the initial and exposed samples  a ratio of the live to total cell concentration in the exposed sample  comparison of a ratio of live to dead cell concentration in the initial and exposed samples  combinations of individual cell event and population response metrics  and so on. Other variations will become apparent to one skilled in the art that includes combinations thereof  and is contemplated to be within the scope of the invention.
[0042] In another aspect  the invention provides an antibiotic susceptibility estimation kit based on the methods of the invention as described herein. The antibiotic susceptibility kit may include all the components for collecting a sample  estimating an initial concentration of microorganisms  performing a concentration adjustment that includes a diluter and/or incubator depending on the necessity  estimating a final concentration of microorganisms  and an indicator of the susceptibility of the microorganisms to the at least one antibiotic. Thus  the kit may also include ports for introducing antibiotics  sealing mechanisms temperature controllers  and any and all related electronics circuitry for smooth operation of such a kit. The kit may further be made available as several independent components  or may be made available as a single piece depending on the requirement of the user and the manufacturer. The level of sophistication may vary on being completely manually operated  including sample transfers from one step to the next and readouts  to being completely automated from the time the sample is introduced to the point results are provided.
[0043] In yet another aspect  the invention provides a method for obtaining antibiotic sensitivity profile of microorganisms. The method for obtaining antibiotic sensitivity profile comprises providing the adjusted sample as described herein.
[0044] Subsequently  the adjusted sample is exposed to at least one antibiotic to provide a primary exposed sample and independently it is also exposed to at least one of a different concentration of the at least one antibiotic used to provide a primary exposed sample  at least one antibiotic that is different from that used to provide a primary exposed sample  and combinations thereof  to provide a secondary exposed sample(s). The exposing may be done in the same manner as already described. In this manner  the susceptibility of the microorganisms to multiple concentrations of the antibiotic  or to multiple antibiotics is obtained.
[0045] Then  the method for estimating antibiotic sensitivity profile comprises estimating a primary final concentration of microorganisms in the primary exposed sample and a secondary final concentration(s) of microorganisms in the secondary exposed sample(s)  wherein the estimating of the final concentrations is done in a similar manner as already described. Based on the initial concentration and primary final concentration and secondary final concentration(s)  the antibiotic sensitivity profile may be obtained in any suitable form. Further  the final concentration of the microorganism(s) in the exposed samples is compared to the control sample to get an accurate measure of the growth of the microorganism(s). The antibiotic sensitivity profile may be in the form of a qualitative estimation of the susceptibility of the microorganisms to different concentrations of a single antibiotic  or to multiple antibiotics  or multiple antibiotics each one at a variety of different concentrations. Alternately  the antibiotic sensitivity profile may be estimated in a quantitative manner  in the form of estimating an MIC for a single antibiotic  or to a plurality of antibiotic combinations. Also  the method as described herein may be used at arrive at CBP for a particular situation in a rapid and facile manner. Further  in this manner  the mechanism of action of antibiotic may also be arrived based on various factors  such as but not limited to  nature of antibiotics used  the growth profile of the microorganisms  the ratio of live to dead cells of microorganisms in the exposed samples  ratio of live cells in the initial sample to the live cells in the exposed sample  and so on  and combinations thereof. The small volumes of sample required for the method of the invention allows for rapid and accurate estimation of the antibiotic sensitivity profile.
[0046] In a further aspect  the invention provides kit for estimating the antibiotic sensitivity profile based on the method for it as described herein. This kit may be constructed and fabricated along the lines for the kit for estimating antibiotic susceptibility as described herein  with the added capability of exposing the adjusted sample to multiple antibiotics  or to different concentrations of a single antibiotic  or combinations thereof. This may be achieved by having a splitter for the adjusted sample into different ports wherein each port has a different concentration of the antibiotic  or different antibiotics  or combinations thereof. In this manner  an antibiotic sensitivity profile is obtained in a facile manner.
[0047] In yet another aspect  the invention provides a device for estimating antibiotic susceptibility of microorganisms. The device of the invention is shown in Fig. 1 in a block diagrammatic form  wherein the device is represented by numeral 10.
[0048] The device 10 comprises a sample receptacle 12 to receive samples comprising microorganisms. The sample receptacle 12 may further comprise sample processing modules (not shown in Fig. 1) to process the sample in a suitable manner. Such sample processing modules may include capabilities to liquefy  dilute  concentrate  and so on  and combinations thereof.
[0049] The device 10 then comprises a sample adjuster 14 to adjust the concentration of microorganisms in the sample within a predetermined level to provide an adjusted sample. Such predetermined levels of concentration of microorganisms may be in the range of from about 1 x 104 cells/mL to about 1 x 107 cells/mL. The concentration may be measured as at least one of live cells or total cells  depending on the choice of method. The sample adjuster may comprise capabilities of diluting the initial sample to provide the sample having the microorganisms within the predetermined levels. Further  the sample adjuster may also include an incubator to incubate the sample under particular conditions to increase the concentration of the microorganisms to be within the predetermined levels.
[0050] The device 10 then includes an exposure chamber 16 to expose the adjusted sample to at least one antibiotic at one or more concentration ranges. When more than one antibiotic is used  or more than one concentration of one antibiotic is used  or combinations thereof  then the exposure of the adjusted sample to each antibiotic concentration may be done simultaneously or sequentially  or both. Thus  the exposure chamber may comprise multiple incubators that comprise slots to introduce antibiotics and the adjusted sample  which can then be sealed and subjected to conditions of incubation that allow for growth and proliferation of microorganisms. Further  the exposure chamber may also include an incubator that serves as a control sample wherein the exposure is done with no antibiotic or a control antibiotic at a predefined concentration that serves as a reference for comparison.
[0051] The device 10 then comprises a sample reader 18 to estimate an initial and final concentrations of microorganisms in the sample from the sample  the adjusted sample  the exposed sample(s)  and combinations thereof. The sample reader may be based on a variety of known techniques in the art  and includes  but not limited to  fluorescence readers  optical readers  colorimetric readers  and the like  and combinations thereof.
[0052] The device 10 may further include a separate staining chamber (not shown in Fig. 1) to provide a detection reagent that is used to tag at least one dead cells  live cells  or total cells present. Alternately  the staining chamber may be provided as part of the exposure chamber and/or the sample adjuster and/or the sample reader  so that every sample that is being made ready for estimation is tagged with the detection reagent.
[0053] The device of the invention further comprises a processing module (not shown in Fig. 1). The processing module may be configured to perform at least one of process the data obtained from the sample reader  estimate nature of actions for the sample adjuster  and combinations thereof. For example  the processing module analyzes the concentration of microorganisms in the sample and assesses it to be lower than the predetermined level  and consequently  sends the sample to the incubator portion of the sample adjuster.
[0054] Further  the processing module of the device is used to obtain at least one of an initial concentration of microorganisms  a final concentration of microorganisms  an initial concentration of live microorganisms  a final concentration of live microorganisms  an initial concentration of dead microorganisms  a final concentration of dead microorganisms  a ratio of initial concentration of microorganisms to final concentration of microorganisms  a ratio of initial concentration of live microorganisms to final concentration of live microorganisms  a ratio of initial concentration of dead microorganisms to final concentration of dead microorganisms  and combinations thereof  including individual event as well as population based metrics. Subsequently  the data can be used to estimate at least one of In this manner  the device of the invention is used to obtain at least one of an antibiotic susceptibility of microorganisms  antibiotic sensitivity profile of microorganisms  and combinations thereof based on at least one of the foregoing calculations.
[0055] Other aspects of the device such as a display module  manual input module to input sample details  memory storage and so on will become apparent to one skilled in the art  and is contemplated to be within the scope of the invention. Further  the device may be operated using software that may include graphic interfaces in an appropriate manner. Also  the results may be displayed in a color coded manner as per instructions executed based on an algorithm to indicate the susceptibility or lack thereof of a microorganism to an antibiotic. Other variations will become obvious to one skilled in the art  and is contemplated to be within the scope of the invention.
EXAMPLES
General procedure for estimating antibiotic sensitivity
[0056] An ATCC® bacterial strain (ATCC® 27853  Pseudomonas aeruginosa) is spread on culture plate with pre-dispensed growth media. The culture plate is incubated at 37 Celsius overnight. After overnight culturing  visible bacterial colonies have grown from which 3-5 colonies are selected and introduced into cation-adjusted Mueller-Hinton Broth (CAMHB) and are incubated at 37°C for acclimatization of bacteria. An optical density (OD) of the bacterial suspension in CAMHB is measured after incubation. The OD of the solution is adjusted to reach 0.5 McFarland Standard by diluting with an appropriate amount of CAMHB. Then  the concentration of the bacteria is adjusted to 1-2x106 CFU/ml  by further dilution in CAMHB. The tubes are then labeled for individual antibiotics at three desired concentration namely  sensitive (Ts)  intermediate (Ti)  resistant (Tr) – and a control sample where no antibiotics are to be introduced (Tc). Then  antibiotics are introduced at appropriate concentrations into the respective tubes Ts  Ti and Tr  and no antibiotics into Tc  following which aliquots of the diluted bacterial suspension in CAMHB is introduced into all the tubes  such that the final concentration is 5-6x105 CFU/ml. All the tubes are incubated at 37º Celsius. An aliquot from each tube is taken every 60 minutes and introduced into separate tubes containing reagents SYTO®-9 from Invitrogen Inc.  USA and Propidium Iodide from Sigma-Aldrich Co  LLC to stain total as well as only dead cells. The staining reagents are allowed to react with the cells for 10 minutes at room temperature. Once stained  a fixed volume of the solution in each staining tube is metered into a capillary or introduced onto a Neubauer’s chamber. The capillary or Neubauer’s chamber is then placed in an epifluorescence microscope. Images of stained total and dead cells are taken at a fixed number of positions for each capillary or Neubauer’s chamber. The total number of cells and number of dead cells is enumerated in each image. This was then used to arrive at a normalized number of live cells which is the ratio of the number of live cells at time = t to the number of live cells in control sample at time = 180 min). This ratio was plotted against time to assess viability  increase or decrease in bacterial events.
EXAMPLE 1
[0057] The procedure described in the general experimental description is used to estimate bacterial resistance to the antibiotic Ciprofloxacin  commercially available from Sigma-Aldrich Co  LLC. Fig. 2 is a plot of the normalized number of live cells against time for Ciprofloxacin. It can be seen that within three hours there is sufficient discrimination between resistant and sensitive strains. Thus the method described herein can be used to make timely decisions for diagnostic and treatment purposes.
EXAMPLE 2
[0058] The procedure described in the general experimental description is used to estimate bacterial resistance to the antibiotic Tetracycline  commercially available from Sigma-Aldrich Co  LLC. Fig. 3 is a plot of the normalized number of live cells against time for Tetracycline. It can be seen that within three hours there is sufficient discrimination between resistant and sensitive strains. Thus the method described herein can be used to make timely decisions for diagnostic and treatment purposes.
EXAMPLE 3
[0059] The procedure described in the general experimental description is used to estimate bacterial resistance to the antibiotic Gentamycin  commercially available from Sigma-Aldrich Co  LLC. Fig. 4 is a plot of the normalized number of live cells against time for Gentamycin. It can be seen that within three hours there is sufficient discrimination between resistant and sensitive strains. Thus the method described herein can be used to make timely decisions for diagnostic and treatment purposes.
EXAMPLE 4
[0060] The procedure described in the general experimental description is used to estimate bacterial resistance to the antibiotic Ampicillin  commercially available from Sigma-Aldrich Co  LLC. Fig. 5 is a plot of the normalized number of live cells against time for Ampicillin. It can be seen that within three hours there is sufficient discrimination between resistant and sensitive strains. Thus the method described herein can be used to make timely decisions for diagnostic and treatment purposes.
EXAMPLE 5
[0061] The procedure described in the general experimental description is used to estimate bacterial resistance to the antibiotic Nalidixic Acid  commercially available from Sigma-Aldrich Co  LLC. Fig. 6 is a plot of the normalized number of live cells against time for Nalidixic Acid. It can be seen that within three hours there is sufficient discrimination between resistant and sensitive strains. Thus the method described herein can be used to make timely decisions for diagnostic and treatment purposes.
[0062] While only certain features of the invention have been illustrated and described herein  many modifications and changes will occur to those skilled in the art. It is  therefore  to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

We Claim:
1. A method for estimating antibiotic susceptibility of microorganisms  the method comprising:
providing a sample comprising the microorganisms  wherein the sample has a volume that ranges from about 1 nanoliter to about 1 milliliter;
estimating an initial concentration of microorganisms in the sample;
performing a concentration adjustment of the microorganisms in the sample based on the initial concentration to provide an adjusted sample;
exposing the adjusted sample to at least one antibiotic to provide an exposed sample;
estimating a final concentration of microorganisms in the exposed sample; and
estimating the antibiotic susceptibility of the microorganisms based on the initial concentration and final concentration of microorganisms.
2. The method of claim 1 wherein the estimating the concentration of microorganisms is based on fluorescent methods.
3. The method of claim 1 wherein the estimating the concentration of microorganisms includes live microorganisms  dead microorganisms  total microorganisms  and combinations thereof.
4. The method of claim 1 wherein the concentration of microorganisms in the adjusted sample is in a range from about 1 x 104 cells per milliliter to about 1 x 107 cells per milliliter
5. The method of claim 1 wherein the concentration adjustment comprises a dilution step.
6. The method of claim 1 wherein the concentration adjustment comprises an incubation step.
7. The method of claim 1 wherein the antibiotic is at least one of a natural antibiotic  synthetic antibiotic  semisynthetic antibiotic  or combinations thereof.
8. The method of claim 1 wherein the concentration of the antibiotic is in a range of from about 0 micrograms per milliliter to about 10 milligrams per milliliter.
9. The method of claim 1 wherein the exposing of the adjusted sample is done in the presence of a growth inhibitor of non-pathogenic microorganisms.
10. The method of claim 9 wherein the growth inhibitor is selected from a group consisting of sodium azide  dextrose  thallium acetate  brilliant green  and combinations thereof.
11. An antibiotic susceptibility estimation kit based on the method of claim 1.
12. A method for obtaining antibiotic sensitivity profile of microorganisms  the method comprising:
providing a sample comprising the microorganisms  wherein the sample has a volume that ranges from about 1 nanoliter to about 1 milliliter;
estimating an initial concentration of microorganisms in the sample;
performing a concentration adjustment of the microorganisms in the sample based on the initial concentration to provide an adjusted sample;
exposing the adjusted sample to at least one antibiotic to provide a primary exposed sample;
independently exposing the adjusted sample to at least one of a different concentration of the at least one antibiotic used to provide a primary exposed sample  at least one antibiotic that is different from that used to provide a primary exposed sample  and combinations thereof  to provide a secondary exposed sample(s);
estimating a primary final concentration of microorganisms in the primary exposed sample and a secondary final concentration(s) of microorganisms in the secondary exposed sample(s); and
obtaining the antibiotic sensitivity profile of the microorganisms based on the initial concentration and primary final concentration and secondary final concentration(s).
13. The method of claim 12 wherein the estimating the concentration of microorganisms is based on fluorescent methods.
14. The method of claim 12 wherein the estimating the concentration of microorganisms includes live microorganisms  dead microorganisms  total microorganisms  and combinations thereof.
15. The method of claim 12 wherein the concentration of microorganisms in the adjusted sample is in a range from about 1 x 104 cells per milliliter to about 1 x 107 cells per milliliter.
16. The method of claim 12 wherein the concentration adjustment comprises a dilution step.
17. The method of claim 12 wherein the concentration adjustment comprises an incubation step.
18. The method of claim 12 wherein the antibiotic is at least one of a natural antibiotic  synthetic antibiotic  semisynthetic antibiotic  or combinations thereof.
19. The method of claim 12 wherein the concentration of the antibiotic is in a range of from about 0 micrograms per milliliter to about 10 milligrams per milliliter.
20. The method of claim 12 wherein the exposing the adjusted sample is done in the presence of a growth inhibitor of non-pathogenic microorganisms.
21. The method of claim 21 wherein the growth inhibitor is selected from a group consisting of sodium azide  dextrose  thallium acetate  brilliant green  and combinations thereof.
22. An antibiotic sensitivity profile estimation kit based on the method of claim 12.
23. A device for estimating antibiotic susceptibility of microorganisms  wherein the device comprises:
a sample receptacle to receive samples comprising microorganisms;
a sample adjuster to adjust the concentration of microorganisms in the sample within a predetermined level to provide an adjusted sample;
an exposure chamber to expose the adjusted sample to at least one antibiotic at one or more concentration ranges  wherein the exposure is achieved simultaneously or sequentially  or both  to provide an exposed sample(s); and
a sample reader to estimate an initial and final concentrations of microorganisms in the sample from the sample  the adjusted sample  the exposed sample(s)  and combinations thereof.
24. The device of claim 23 further comprising a processing module to perform at least one of process the data obtained from the sample reader  estimate nature of actions for the sample adjuster  and combinations thereof.
25. The device of claim 23 wherein the sample adjuster comprises an incubator to incubate the sample .
26. The device of claim 23 wherein the sample adjuster comprises a diluter to dilute the sample to an estimated extent.
27. The device of claim 24 wherein the processing module is used to obtain at least one of an initial concentration of microorganisms  a final concentration of microorganisms  an initial concentration of live microorganisms  a final concentration of live microorganisms  an initial concentration of dead microorganisms  a final concentration of dead microorganisms  a ratio of initial concentration of microorganisms to final concentration of microorganisms  a ratio of initial concentration of live microorganisms to final concentration of live microorganisms  a ratio of initial concentration of dead microorganisms to final concentration of dead microorganisms  and combinations thereof.
28. The device of claim 27 wherein the processing module is used to obtain at least one of an antibiotic susceptibility of microorganisms  antibiotic sensitivity profile of microorganisms  and combinations thereof based on at least one of the ratio of initial

concentration of microorganisms to final concentration of microorganisms  a ratio of initial concentration of live microorganisms to final concentration of live microorganisms  a ratio of initial concentration of dead microorganisms to final concentration of dead microorganisms.
Signature of Applicant
Rachna Singh Puri
Authorized Representative
DATE: 13 October 2012

METHODS FOR ESTIMATING ANTIBIOTIC SUSCEPTIBILITY AND ANTIBIOTIC SENSITIVITY PROFILE OF MICROORGANISMS  AND DEVICES THEREFOR
ABSTRACT
[0063] In one aspect  the invention provides a method for estimating antibiotic susceptibility of microorganisms  wherein the method comprises estimating an initial concentration of microorganisms in the sample  followed by adjusting the concentration of the microorganisms such that it is in a predetermined range to provide an adjusted sample. The predetermined range may be from about 1 x 104 cells/mL to about 1 x 107cells/mL. Subsequently  the adjusted sample is exposed to an antibiotic under predetermined conditions to provide an exposed sample following which the concentration of microorganisms is estimated  which is used to estimate the antibiotic susceptibility of the microorganism. The method of the invention is capable of being extended to estimating antibiotic sensitivity profile by exposing the adjusted to different antibiotics at the same concentration or each at various concentrations. Kits and devices based on the methods of the invention are also described herein.