MICROORGANISM FROM POSITIVE BLOOD CULTURES

CROSS-REFERENCE TO RELATED APPLICATIONS

[ 0001 ] This application claims the benefit of the filing date of United States Provisional Patent Application No. 61/604,732, filed February 29, 2012, the disclosure of which is hereby incorporated herein by reference .

RELATED APPLICATIONS

[ 0002 ] This application is related in subject matter to United States Patent Application No. 13/647,072, filed October 8, 2012, which claims the benefit of the filing date of United States Provisional Application No. 61/544,407, filed October 7, 2011, the disclosures of which are herby incorporated herein by reference .

BACKGROUND OF THE INVENTION

[ 0003 ] Sepsis is a serious medical condition caused by an overwhelming response of the host immune system to infection. It can trigger widespread inflammation, which can give rise to impaired blood flow. As sepsis progresses, the body's organs can be starved for oxygen and nutrients, causing permanent damage and eventual failure. Left improperly diagnosed or otherwise untreated, the heart may weaken and septic shock can occur, leading to multiple organ failure and death. Blood cultures are required to detect the presence of bacteria or yeast in the blood of sepsis patients. If a microorganism is present, (positive blood culture ("PBC")) the microorganism ( s ) must be identified and antibiotic susceptibility determined in order to provide appropriate treatment. The PBC samples are used to isolate, identify and perform antimicrobial susceptibility testing ("AST") . The microorganism ( s ) are often identified by methods such as mass spectrometry, including MALDI-TOF/MS or phenotypic growth-based methods, such as Phoenix™ ID.

[ 0004 ] In order to identify the microorganism ( s ) , perform phenotypic analysis on the microorganism, and perform AST testing, intact, viable microorganism ( s ) need(s) to be isolated from the blood cells and other material in the collected sample. For identification of the microorganism by mass spectrometry, the microbial sample needs to be sufficiently free from substances known to interfere with MALDI-TOF/MS identification, such as blood cell components, other cellular debris, and salts. In addition, the microbial sample needs to be of sufficient quantity in order to obtain a reliable identification. Phenotypic identification methods, such as Phoenix™ ID, require intact, viable microorganism free from substances that may interfere with the enzymatic substrates of the assay. For AST testing, such as Phoenix™ AST, the microbial sample needs to contain viable, unaltered microorganism capable of growth in the presence of antibiotic, if resistance mechanisms are present, during performance of the assay. It is important for all methods to be of sufficient quantity and purity as carryover of residual blood or media components will interfere either directly or by falsely increasing the concentration (turbidity) of microorganism.

[0005] Current techniques for isolating viable microorganism from a PBC sample include sub-culturing the microorganism ( s ) , which can take up to 72 hours . This results in the delay of treatment or treatment with inappropriate antibiotics.

[0006] Certain strains of microorganisms are particularly difficult to isolate from a PBC sample while maintaining viability of the organism, such as, for example, Streptococcus pneumoniae ( S .pneumoniae) . Part of this difficulty is traced to the activation of autolysin by S. pneumoniae which causes the microbial cells to "self- destruct". See "Streptococcus pneumoniae Antigen Test Using Positive Blood Culture Bottles as an Alternative Method To Diagnose Pneumococcal Bacteremia", Journal of Clinical Microbiology, Vol. 43, No.5, May 2005, p.2510-2512. The current method for isolating microorganisms from septic patients, including, S. pneumoniae, includes inoculating blood culture bottles. Once a positive signal is achieved, a portion of the PBC sample is removed to perform a gram stain and another portion is used to sub-culture the microorganism. Microbial colonies from the sub-culture are used to perform downstream testing such as identification by MALDI-TOF/MS, phenotypic identification methods, and AST testing. [0007] Additional techniques for isolating viable microorganism ( s ) from a PBC sample often utilize liquid separation methods containing lysis buffers with detergents that lyse the blood cells in the PBC sample. After lysis, the lysed blood cells can be removed while the microorganism ( s ) is/are retained. However, the use of these lysis buffers often result in compromised, damaged, or non-viable microorganism ( s ) which is/are insufficient for performing certain growth-based identification methods such as AST testing.

[0008] One such liquid separation method, the Bruker Sepsityper™ system, allows for direct testing of the microorganism from a PBC sample by MALDI-TOF/MS without the need for sub-culturing the microorganism. This method uses Sodium Dodecyl Sulfate ("SDS") and centrifugation to generate a pellet of microbes. While the Sepsityper™ method will generally support MALDI-TOF/MS testing of the microbial pellet, there is insufficient viability to support growth-based identification methods and AST methods, due to the interaction of the harsh detergents on the microbial cell wall.

[0009] Prod'hom et al., "Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Direct Bacterial Identification from Positive Blood Culture Pellets", Journal of Clinical Microbiology, Vol. 48, No. 4, p. 1481-1483 (February 17, 2010) . discloses a method for lysing erythrocytes in a PBC sample using ammonium chloride to prepare a bacterial pellet for MALDI- TOF/MS analysis. However, these methods are insufficient in obtaining reliable MALDI-TOF/MS data across a panel of microorganism, including S. pneumoniae. In addition, there is no indication that these methods will maintain sufficient viability of the microorganism for use in growth-based identification methods and AST testing.

[0010] Hansson et al., "Microfluidic Blood Sample Preparation for Rapid Sepsis Diagnostics", KTH Engineering Sciences, (2012) suggests the use of detergents for lysing blood cells and selectively lysing certain types of blood cells with ammonium chloride. However, this reference is silent with regard to specific formulations or methods that would allow for the isolation of viable microorganism from a PBC sample that is free from interfering substances and would allow for multiple downstream testing from one PBC sample, such as both MALDI-TOF/MS identification and AST testing.

[ 0011 ] Both M. Drancourt, "Detection of Microorganisms in Blood Specimens Using Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry: A Review, Clinical Microbiology and Infection, 16:1620-1625, (2010) and WO 2010/100612 to Nassif et al. describe methods for isolating microorganism from a PBC sample which include removing red blood cells from a PBC sample by adding saponin and/or ammonium chloride to a portion of the PBC sample, centrifuging the mixture, and washing the resulting pellet with water to remove residual blood proteins. However, these methods produce inconsistent identification of the microorganism at the species level across a panel of microorganisms and/or fail to identify S. pneumoniae. Furthermore, there is no indication that these methods result in a microbial pellet with sufficient viability to perform growth- based testing methods, such as AST.

[ 0012 ] Accordingly, it is desirable to develop reagents and methods that rapidly separate microorganism ( s ) from a PBC sample while maintaining the viability of the microorganism ( s ) , so that analytical growth-based methods that require cell viability, such as AST testing, can be performed. Additionally, it is desirable that the reagents and methods can be used to isolate viable cells from all types of microorganism, including S. pneumoniae.

BRIEF SUMMARY OF THE INVENTION

[ 0013 ] Various embodiments of the disclosed invention provide for reagents and methods for rapidly isolating viable microbial cells from a PBC sample including S. pneumoniae. The resulting microbial pellet obtained using the various reagents and methods is sufficiently free from interfering substances and can be used for identification methods, such as MALDI-TOF/MS, growth-based identification and AST methods. This enables rapid results without the need for sub-culturing the microorganisms . The concentrated mass of viable microbial cells obtained by the various embodiments can be used for both the direct inoculation of rapid ID systems, such as MALDI-TOF/MS, and ID/AST testing (AST) by conventional or automated systems, such as the BD™ Phoenix™ ID/AST system. The various embodiments may also be applicable to other systems, molecular testing methods, such as polymerase chain reaction (PCR) , and other methods known to one skilled in the art.

[ 0014 ] Described herein are various "lyse and wash" buffers that can be used for both lysing mammalian blood cells and washing away cellular debris and other blood components, while maintaining viability of the microorganism ( s ) . The "lyse and wash" buffers described herein contain a base solution comprising, for example, salts, peptones, and other nutrients to protect the microorganism ( s ) , as well as lytic reagents, such as detergents, thiols, and ammonium chloride to remove cellular debris from the mammalian blood cells.

[ 0015 ] The methods for isolating microorganism ( s ) from PBC samples described herein utilize the "lyse and wash" buffers to rapidly produce a pellet of viable microorganism that can be used for various downstream testing methods, for example, MALDI-TOF/MS identification and AST testing. The disclosed methods provide a process for rapidly isolating and concentrating microorganism ( s ) from PBC samples using one sample preparation tube, a single lyse step, a single wash step, and as little as one centrifugation step. These methods are easily adapted to automated systems and do not require sub-culturing of the microorganism in order to maintain cell viability.

[ 0016 ] The methods described herein do not utilize substances that interfere with methods for identifying the microorganism ( s ) , for example mass spectrometry, or with phenotypic methods such as Phoenix™ ID. Furthermore, the methods described herein permit for the rapid isolation of viable microorganism ( s ) from a single PBC sample in sufficient quantity for use in multiple down-stream analysis, such as, identification by mass spectrometry, phenotypic or growth-based identification assays, and AST testing. Furthermore, the methods described herein prepare a sample for downstream analysis that will identify a wide panel of microorganisms, including the most difficult of organisms to characterize, for example, S. pneumoniae.

[ 0017 ] In another embodiment, a kit is provided which may include, for example, one or more of the "lyse and wash" buffers described herein for isolating viable microorganism ( s ) from a sample .

BRIEF DESCRIPTION OF THE DRAWINGS

[ 0018 ] FIG. 1 demonstrates the method of one embodiment described herein for the isolation of viable microorganism ( s ) from a PBC sample.

[ 0019 ] FIG. 2 demonstrates the method of one embodiment described herein for the isolation of viable S. pneumoniae from a PBC sample.

DETAILED DESCRIPTION

[ 0020 ] Isolating and concentrating the microorganism ( s ) from PBC samples using the "lyse and wash" buffers and methods described herein maintains the viability of the microbial cells, and consequently their response to growth-based testing, such as AST testing. The methods described herein also reduce the amount of other substances in the sample, such as cellular debris, that may interfere with multiple identification methods such as MALDI- TOF/MS and Phoenix™ ID. In one embodiment, the buffers described herein are formulated to keep the concentration of salts at a minimum so as to prevent interference with MALDI- TOF/MS identification analysis.

[ 0021 ] In yet another embodiment, the buffers and methods described herein are used to isolate and perform downstream analysis on gram-positive bacteria, gram-negative bacteria, or yeast. In another embodiment, the gram-positive bacteria include S.pneumoniae as well as other species of Streptococci.

[ 0022 ] "Lyse and Wash" Buffer

[ 0023 ] The "lyse and wash" buffers described herein contain a balance of reagents that stabilize the microorganism ( s ) as well as lytic reagents for lysing blood cells. In one embodiment the buffer contains a base solution comprising salts, peptones, and other nutrients for stabilizing the microorganism ( s ) as well as non-ionic detergents, thiols, and optionally, ammonium chloride, for lysing blood cells in the sample.

[ 0024 ] Without being bound by a particular theory, it is believed that while the base solution may help to stabilize the microorganism ( s ) , lytic reagents lyse the blood cells and remove interfering cellular debris. The "lyse and wash" buffers described herein provide for the potential for multiple independent lytic mechanisms: aqueous (osmotic lysis), detergents (membrane solubilization), thiols (disruption of protein-protein interactions in the membranes), and ammonium chloride (red blood cell membranes are effectively permeable to ammonium chloride and cell lysis occurs due to the unbalanced osmotic pressure of their colloid content). The combination of multiple lytic reagents allows for each of the lytic reagents to be less harsh on the microorganism ( s ) and can be used for a shorter duration than if used separately.

[ 0025 ] The base solution may contain a nutrient broth for microorganism ( s ) , an isotonic buffer, peptones, and/or salts. The nutrient broth can be, for example, Trypticase™ soy broth. In one exemplary embodiment the concentration of the nutrient broth in the "lyse and wash" buffer is about 10 g/L to about 50 g/L. Isotonic buffers are clinical buffers known by one skilled in the art to be compatible with whole blood samples. Such buffers are typically mixtures of sodium phosphate, potassium phosphate, and saline, such as sodium chloride or phosphate buffered saline. In one exemplary embodiment, the concentration of isotonic buffer is about 1 g/L to about 85 g/L. In another exemplary embodiment, the isotonic buffer is a phosphate buffer. In one embodiment, the peptones include casein peptones and/or soy peptones. Additional constituents of the base solution contemplated include, for example, sodium pyruvate, yeast extract, sodium citrate, meat peptones, and/or dextrose.

[ 0026 ] In another embodiment, the "lyse and wash" buffer may contain a non-ionic detergent or mixtures of nonionic detergents . In this embodiment, at least one non-ionic detergent in the "lyse and wash" buffer is a non-ionic hemolytic detergent that selectively lyses blood cells but does not lyse the microorganism ( s ) . Exemplary non-ionic hemolytic detergents contemplated include Triton™ X-100 and saponin. Other non-ionic detergents may include, for example, the Tween® family, the Brij® family, octyl-b-Glucoside, the Tergitol® family, the CYMAL® family, the MEGA family (including Noctanoyl-N-methylglucamine, N-nonanoyl, N-decanoyl), PLURONIC® family such as F-68, digitonins, and the CHAP family. In one exemplary embodiment, the concentration of non-ionic detergent (s) in the "lyse and wash" buffer is about 0.01 g/L to about 20 g/L. In another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration of about 6.7 g/L.

[ 0027 ] In one embodiment, the "lyse and wash" buffer contains Triton™ X-100. The Triton™ X-100 concentration is selected to preserve the viability of S. pneumoniae. Exemplary embodiments contemplate concentrations of Triton™ X-100 of up to about 1 g/L. In another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration of about 0.335 g/L. In still yet another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration of about 0.1 g/L to about 1 g/1, about 0.1 g/L to about 0.9 g/1, about 0.1 g/L to about 0.8 g/1, about 0.1 g/L to about 0.7 g/1, about 0.1 g/L to about 0.6 g/1, about 0.1 g/L to about 0.5 g/1, about 0.1 g/L to about 0.4 g/1, about 0.2 g/L to about 1 g/1, about 0.3 g/L to about 1 g/1, about 0.2 g/L to about 0.9 g/1, about 0.2 g/L to about 0.8 g/1, about 0.2 g/L to about 0.7 g/1, about 0.2 g/L to about 0.6 g/1, about 0.2 g/L to about 0.5 g/1, about 0.2 g/L to about 0.4 g/1, about 0.3 g/L to about 0.9 g/1, about 0.3 g/L to about 0.8 g/1, about 0.3 g/L to about 0.7 g/1, about 0.3 g/L to about 0.6 g/1, about 0.3 g/L to about 0.5 g/1, or about 0.3 g/L to about 0.4 g/1. The "lyse and wash" buffers containing a concentration of Triton™ X-100 selected to preserve the viability of S. pneumoniae can also be used to isolate viable microbial cells from an organism other than S. pneumoniae without the need for a different "lyse and wash" buffer for these additional organisms.

[ 0028 ] In one embodiment, the "lyse and wash" buffer may optionally include ammonium chloride. In another embodiment, the concentration of ammonium chloride in the "lyse and wash" buffer is about 0.01 g/L to about 80 g/L.

[ 0029 ] Although the Applicant does not wish to be bound by a particular theory, the addition of ammonium chloride or other lytic components can help mitigate the effects of the other lytic components of the "lyse and wash" buffer on the viability of the microorganism. When using various lytic reagents, a sufficient quantity is needed in order to lyse the blood cells in the PBC sample. This often has the unwanted effect of causing microbial cell death. Detergents, thiols, and ammonium chloride employ different mechanisms of action for lysing blood cells . When combining multiple lytic mechanisms in one "lyse and wash" buffer, each of the different lytic components can potentially be used in a lower amount. This potentiality reduces the negative effect of lysing the microbial cells but may be sufficient for lysing the blood cells, resulting in more efficient blood cell lysis than when using each of the lytic ingredients alone. Therefore, microbial cell viability can be maintained while unwanted blood cell components can be eliminated.

[ 0030 ] In one embodiment the "lyse and wash" buffer contains a base solution comprising phosphate buffered saline, yeast extract, sodium citrate, meat peptones, dextrose, sodium pyruvate; thiols comprising sodium thioglycolate, and L-cysteine HCL; non-ionic detergents comprising saponin and Triton™ X-100; and optionally PBS.

[ 0031 ] In another embodiment the "lyse and wash" buffer contains a base solution comprising Trypticase™ soy broth, yeast extract, sodium citrate, meat peptones, sodium pyruvate; thiols comprising L-cysteine HCL and sodium thioglycolate; and non-ionic detergents comprising saponin and Triton™ X-100.

[ 0032 ] In yet another embodiment the "lyse and wash" buffer contains a base solution comprising Trypticase™ soy broth; thiols comprising L-cysteine HCL and sodium thioglycolate; and non-ionic detergents comprising saponin and Triton™ X-100.

[ 0033 ] In one embodiment the "lyse and wash" buffer comprises: a base solution comprising casein peptone at a concentration of about 8 g/L to about 35 g/L, sodium chloride at a concentration of about 2 g/L to about 10 g/L, soy peptone at a concentration of about 1.5 g/L to about 15 g/L, and potassium phosphate at a concentration of about 0.5 g/L to about 5 g/L; thiols comprising L-cysteine at a concentration of about 0.01 g/L to about 2.5 g/L and sodium thioglycolate at a concentration of about 0.01 g/L to about 2.5 g/L; and non-ionic detergents comprising saponin at a concentration of about 0.01 g/L to about 10 g/L and Triton™ X- 100 at a concentration about 0.01 g/L to about 20 g/L.

[ 0034 ] In one embodiment the "lyse and wash" buffer comprises a base solution comprising casein peptone at a concentration of about 8 g/L to about 35 g/L, sodium chloride at a concentration of about 2 g/L to about 10 g/L, soy peptone at a concentration of about 1.5 g/L to about 15 g/L, and potassium phosphate at a concentration of about 0.5 g/L to about 5 g/L; thiols comprising L-cysteine at a concentration of about 0.01 g/L to about 2.5 g/L and sodium thioglycolate at a concentration of about 0.01 g/L to about 2.5 g/L; non-ionic detergents comprising saponin at a concentration of about 0.01 g/L to about 10 g/L and Triton™ X- 100 at a concentration of about 0.01 g/L to about 20 g/L; and ammonium chloride at a concentration of about 0.01 g/L to about 80 g/L.

[ 0035 ] In one embodiment, an antifoaming agent can be optionally added to the "lyse and wash" buffer for manufacturing convenience . Preferably, the antifoaming agent has no demonstrable impact on performance of the "lyse and wash" buffer. Antifoaming agents are well known to those skilled in the art and are not described in detail herein. Selection of an antifoaming agent, and the amount, is largely a matter of design choice, and well within the capabilities of those skilled in the art based on the "lyse and wash" buffer requirements described herein. In one embodiment that antifoaming agent in the "lyse and wash" buffer is at a concentration of 0.1 g/L.

[ 0036 ] The concentrations of the various constituents of the "lyse and wash" buffers described herein represent the final concentrations of each constituent in the "lyse and wash" buffer. Often, a 1:1 volume ratio of PBC sample is mixed with the "lyse and wash" buffer; however, other volume ratios are contemplated. Accordingly, the concentrations of each constituent in the "lyse and wash" buffer can be adjusted to account for changes in the volume ratio of "lyse and wash" buffer to PBC sample in order to achieve a desired final concentration of the constituents of the "lyse and wash" buffer when mixed with the PBC sample.

[ 0037 ] In one embodiment, the "lyse and wash" buffer contains both saponin and Triton™ X-100 but does not contain an antifoaming agent. Without being bound by theory, the combination of saponin and Triton™ X-100 results in much less foaming of the treated PBC samples relative to using saponin as the sole non- ionic detergent in the "lyse and wash" buffer. This reduced foaming improves workflow, makes removal of the supernatant after centrifugation easier, and reduces or eliminates the need for antifoaming reagents (i.e., silicone fluids).

[ 0038 ] In one embodiment the "lyse and wash" buffer includes a choline-containing solution, for example, those choline- containing solutions as described in United States Patent Application No. 13/647,072, the contents of which are hereby incorporated herein by reference in their entirety. Although Applicant does not wish to be bound by a particular theory, the addition of a choline-containing solution to the "lyse and wash" buffer may inhibit, prevent, and/or mitigate autolysis of the microorganism, particularly S. pneumonia, in the presence of lytic components of the "lyse and wash" buffer.

[ 0039 ] In another embodiment, a kit is provided which may comprise, for example, one or more of the "lyse and wash" buffers described herein for isolating viable microorganism ( s ) from a PBC sample .

[ 0040 ] Methods For Isolating Microorganism ( s ) From A PBC Sample

[ 0041 ] The methods for isolating microorganism (a) from a sample suspected of containing at least one microorganism, for example a PBC sample, described herein may utilize the various "lyse and wash" buffers contemplated to rapidly produce a viable microbial pellet that can be used for various downstream testing methods, such as, identification by MALDI-TOF/MS, growth-based phenotypic assays and AST testing. In one embodiment, the method includes adding a portion of a PBC sample with the "lyse and wash" buffer to form a mixture. In one embodiment the volume ratio of PBC sample to "lyse and wash" buffer is 1:1. The mixture is incubated for a period of time to lyse the blood cells in the BPC sample.

[ 0042 ] In one embodiment the "lyse and wash" buffer mixed with the portion of the PBC sample comprises at least one non-ionic detergent at a concentration of about 0.01 g/L to about 20 g/L. In another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration of about 6.7 g/L. In yet another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration up to about 1 g/L. In yet another embodiment, the "lyse and wash" buffer contains Triton X-100 at about 0.335 g/L. In still yet another embodiment, the "lyse and wash" buffer contains Triton™ X-100 at a concentration of range of about 0 .1 g/L to about 1 g/i, about 0 .1 g/L to about 0 .9 g/i, about 0. 1 g/L to about 0. 8 g/i, about 0 .1 g/L to about 0 , .7 g/i, about 0. 1 g/L to about 0. 6 g/i, about 0 .1 g/L to about 0 , .5 g/i, about 0. 1 g/L to about 0 .4 g/i , about 0. 2 g/L to about 1 g/i, about 0. 3 g/L to about 1 g/i, about 0. 2 g/L to about 0. 9 g/i, about 0. 2 g/L to about 0. 8 g/i, about 0 .2 g/L to about 0 , .7 g/i, about 0. 2 g/L to about 0. 6 g/i, about 0 .2 g/L to about 0 , .5 g/i, about 0. 2 g/L to about 0. 4 g/i, about 0 .3 g/L to about 0 , .9 g/i, about 0. 3 g/L to about 0. 8 g/i, about 0 .3 g/L to about 0 , .7 g/i, about 0. 3 g/L to about 0. 6 g/i, about 0 .3 g/L to about 0 , .5 g/i, or about 0.3 g/L to about 0.4 g/1.

[ 0043 ] In one embodiment, after the mixture is prepared from combining a portion of the PBC sample with "lyse and wash" buffer, the mixture is incubated for up to about 5 minutes. In another embodiment the mixture is inverted several times during incubation to ensure even mixing of the PBC sample and "lyse and was" buffer. After incubation, the mixture is centrifuged to form a pellet containing viable microorganism ( s ) and a supernatant containing blood cell debris. The range of speed and time of centrifugation may be optimized in order to achieve a readily discernible pellet in order to easily remove the supernatant without disturbing the pellet. Various variables will affect the speed and time of centrifugation including, for example, volume of the mixture, size and shape of the sample tube, and type of rotor. In one embodiment the mixture is centrifuged at a speed of about 100 x g to about 5000 x g. In another embodiment the mixture is centrifuged for up to and including about 10 minutes .

[ 0044 ] After centrifugation the supernatant is discarded while the pellet is washed with a second volume of the "lyse and wash" buffer. The "lyse and wash" buffer used for the washing step can be the same or different formulation than the "lyse and wash" buffer used for lysing the blood cells in the lysing step. After washing the pellet, the mixture is centrifuged for a second time. The supernatant, containing blood cell debris, is again discarded while the pellet, containing viable microorganism is retained. Trace liquids from the top of the pellet can be removed. Lastly, the pellet may be resuspended in a solution for downstream analysis. In one embodiment, the pellet is resuspended in a solution at a concentration of at least about 4 McFarland.

[0045] The viable microbial pellet resulting from the various embodiments described herein can be used to prepare a common sample for various downstream testing methods including identification by mass spectrometry, for example, MALDI-TOF/MS identification, phenotypic growth-based identification, for example, Phoenix TM ID, and AST testing, for example, PhoenixTM AST testing. In addition, the entire method can be performed in one sample tube without the need for transferring sample between multiple tubes. Therefore, the methods described herein are readily adaptable to automated systems .

[0046] Techniques such as higher PBC sample volume, multiple aliquots of PBC sample, multiple spins, etc., described herein can be deployed to increase the number of microorganism ( s ) in the starting volume to improve yield. In addition, these methods provide a rapid sample preparation method and are easily automated. Furthermore, the methods and buffers described herein subject the blood cells to lysis, remove interfering substances from the PBC sample, and provide high yields of viable microorganism ( s ) . In one embodiment, the yield of viable microbial pellet can be increased by increasing the starting volume of PBC sample and/or by performing the isolation method on several aliquots from one PBC sample and combining the resulting microbial pellets into one sample.

[0047] FIG. 1 illustrates one embodiment of the methods described herein. A portion of a PBC sample, for example 5 ml, is transferred (100) to a sample tube. A volume of "lyse and wash" buffer, for example 5 ml, is added (105) to the PBC sample and mixed (110), for example for 5 minutes. The mixture is centrifuged (115), for example at 2200 x g for 10 minutes, resulting in pellet A containing viable microorganism ( s ) and supernatant A containing blood cell debris. Supernatant A is decanted and discarded (120) while pellet A is retained (125) . A second volume of "lyse and wash" buffer, for example 5 ml, is added (130) to pellet A to resuspend the pellet. Resuspended pellet A is mixed (135) to create a second mixture. The second mixture is centrifuged (140), for example at 2200 x g for 10 minutes, resulting in pellet B containing viable microorganism ( s ) and supernatant B containing additional blood cell debris not removed by the first centrifugation step (115) . Supernatant B is decanted and discarded (145) while pellet B is retained (150) . At least a portion of pellet B is resuspended (155) in a liquid, for example, with 600 μΐ of sterile deionized water, to achieve a dense microbial suspension, for example a suspension of approximately 4 McFarland. The resulting microbial suspension can be used for various downstream testing methods (160) such as, for example, identification by MALDI/TOF-MS and phenotypic growth- based identification, for example, identification by the

Phoenix TM system, and growth-based AST methods such as PhoenixTM AST testing.

[0048] In one embodiment of the methods for isolating viable microorganism described herein, the PBC sample is incubated with a choline solution prior to or simultaneously with the "lyse and wash" buffers as described in United States Patent Application No. 13/647,072, the contents of which is incorporated herein by reference in its entirety.

[0049] In another embodiment, methods are provided for the isolation of viable S. pneumoniae from a PBC sample. These methods include obtaining a blood sample suspected of containing at least one microorganism. A portion of the blood sample is added to an anaerobic blood culture bottle while a second portion of the blood sample is added to an aerobic blood culture bottle. The bottles are incubated until a positive signal is obtained. An early indication of the presence of S. pneumoniae in the blood sample is obtained by combining a portion of a aerobic PBC sample with the "lyse and wash" buffer described herein to form a mixture, incubating the mixture for a period of time to lyse the blood cells, and centrifuging the mixture to form a pellet. A pellet that is green in color provides an early indication of the presence of S. pneumoniae in the blood sample. In the alternative an early indication of the presence of S. pneumoniae in the blood sample is obtained by visually observing the aerobic positive blood culture bottle for a solution green in color.

[ 0050 ] After this early indication of S. pneumoniae, the anaerobic PBC sample may be used to isolate viable S. pneumoniae for downstream analysis using any of the methods described herein for the isolation of viable microorganism. In one embodiment, the method includes adding a portion of an anaerobic PBC sample with the "lyse and wash" buffer described herein to form a mixture. The mixture is incubated for a period of time to lyse the blood cells in the BPC sample.

[ 0051 ] In one embodiment, the methods for isolating viable S. pneumoniae include the use of a "lyse and wash" buffer containing Triton™ X-100. The Triton™ X-100 concentration is selected to preserve the viability of the S. pneumoniae. A concentration of up to about 1 g/L is contemplated. In another embodiment, the methods for isolating viable S. pneumoniae include the use of a "lyse and wash" buffer containing Triton™ X-100 of about 0.335 g/L.

[ 0052 ] In one embodiment, after the mixture is prepared from combining a portion of the PBC sample with "lyse and wash" buffer, the mixture is incubated for up to and including about 5 minutes. In another embodiment the mixture is inverted several times during incubation to ensure even mixing of the PBC sample and "lyse and was" buffer. After incubation, the mixture is centrifuged to form a pellet containing viable microorganism ( s ) and a supernatant containing blood cell debris.

[ 0053 ] After centrifugation the supernatant is discarded while the pellet, containing viable S. pneumoniae, is washed with a second volume of the "lyse and wash" buffer. The "lyse and wash" buffer used for the washing step can be the same or different formulation than the "lyse and wash" buffer used for lysing the blood cells in the lysing step. After washing the pellet, the mixture is centrifuged for a second time. The supernatant, containing blood cell debris, is again discarded while the pellet, containing viable S. pneumoniae, is retained. Lastly, the pellet may be resuspended in a solution for downstream analysis . [0054] One embodiment of the method for isolating viable S. pneumoniae from a PBC sample is illustrated in FIG. 2. A blood sample that is suspected of containing S. pneumoniae is obtained

(200) . A portion of the blood sample (for example 10 ml) is added (205) to an anaerobic blood culture bottle while a second portion of the blood sample (for example 10 ml) is added (210) to an aerobic blood culture bottle. A positive signal is obtained

(215) in both the anaerobic and aerobic PBC bottles indicating the presence of a microorganism. A portion of the aerobic PBC sample, for example 5 ml, is transferred (220) to a tube. A volume of the "lyse and wash" buffer described herein containing a concentration of Triton™ X-100 selected to preserve the viability of S. pneumoniae, for example 0.335 g/L Triton™ X-100, for example 5 ml, is added (225) to the PBC sample to form a mixture. The mixture is incubated (230) for a period of time

(for example 5 minutes) to lyse the blood cells. The mixture is then centrifuged (235) to form a pellet green in color which provides an early indication (240) of the presence of S. pneumoniae in the blood sample. After an early indication of S. pneumoniae (240) a portion of the anaerobic PBC sample is prepared (245) for downstream analysis using any of the methods described herein for isolating viable microorganism from a PBC sample, including, for example, the methods described in FIG. 1 using a "lyse and wash" buffer containing a concentration of Triton™ X-100 selected to preserve the viability of S. pneumoniae, for example 0.335 g/L of Triton™ X-100.

[0055] Examples

[0056] Example 1

[0057] Positive blood culture samples were obtained by inoculating a blood culture bottle with Enterobacter aerogenes

(ATCC 13048) or Staphylococcus aureus (ATCC 43300 MRSA) and incubating until a positive signal was indicated. A portion to the PBC sample (5 ml) was directly withdrawn from the bottle and placed into a 15 ml conical tube. Choline chloride (0.5 ml of 20% choline chloride solution) was added to the PBC sample, incubated at room temperature for 20 minutes, and centrifuged at 132 x g for 5 minutes in a Beckman J6-MI centrifuge with a JS-4.2 rotor. The supernatant was transferred to a second 15 ml conical tube and mixed with 5 ml of a "lyse and wash" buffer containing 16 g/L ammonium chloride 6.7 g/L Triton™ X-100, and BD Bactec Lytic 10 (catalog number 442265) . The mixture was incubated for 5 minutes at room temperature. The lysed sample was then centrifuged at 1855 x g for 10 minutes. After removing the supernatant, the remaining bacteria pellet was resuspended in 4.5 ml BD Phoenix™ ID broth (Catalog number 246001) . A portion of the recovered microorganism was used to determine viability of the organism by plate count. The results of the viability testing by plate count indicated 1.85 x 10 ⁹ cfu/ml of E. aerogenes after lysis compared to 1.65 x 10 ⁹ cfu/ml for the PBC sample prior to lysis. Viability testing also demonstrated 1.25 x 10 ⁸ cfu/ml for S. aureus after lysis compared to 2.00 x 10 ⁸ cfu/ml for the PBC sample prior to lysis .

[ 0058 ] A second portion of the recovered bacterial suspension was then further diluted with the BD Phoenix™ ID broth to a concentration of 0.5 McFarland and used for Phoenix™ ID/AST testing. The BD Phoenix™ ID/AST System is described in, e.g., U.S. Patent Nos. 5,922,593, 6,096,272, 6,372,485, 6,849,422, and 7,115,384, the contents of which are hereby incorporated herein by reference. A portion of the inoculum prepared above was used for phenotypic identification by Phoenix™ ID and was poured into the identification portion of a BD Phoenix™ ID/AST panel (Becton, Dickinson and Company) and sealed with a plastic closure. A second portion of the inoculum prepared above was used for AST testing by Phoenix™ AST and was poured into the AST portion of a BD Phoenix™ ID/AST panel (Becton, Dickinson and Company) and sealed with a plastic closure. The antimicrobial susceptibility minimal inhibitory concentration (MIC) for a series of antibiotics was calculated for each of the isolated microbial pellets. The MIC results from the microbial pellets were compared to the MIC results from the same strains obtained from plated pure cultures ("Control"). This comparison indicates whether the Test Method provides substantially equivalent results to the Control Method, i.e., the rate of essential agreement (EA) between the Test Method and the Control Method. A dilution difference of 0 indicates exact agreement between the Test Method and the Control Method. A dilution difference of -1 or +1 indicates that the Test Method is considered within EA to the Control Method, i.e., within normal test variation. Dilution differences outside this range, such as for example -4, +4, -3, +3, -2 and +2, indicates the results are not within essential agreement for the antibiotic/microorganism tested. The methods and verification of AST results are further described in "Verification and Validation of Procedures in the Clinical Microbiology Laboratory", Cumitech 31, (Feb. 1997, ASM Press).

[0059] Recovered bacteria from both PBC samples resulted in a correct Phoenix™ identification as E. aerogenes and S. aureus with both samples having a 99% confidence value. The Phoenix™ AST results showed 100% essential agreement (EA) when compared with those samples prepared from plated colonies, i.e., the dilution difference was within -1 to +1 dilution difference. The S. aureus AST results included correct identification of the four (4) resistance markers expected for this MRSA strain, including methicillin resistent staphylococcus (MRS), mecA-mediated resistent staphylococcus (mecA) , beta-lactamase-producing staphylococcus (BLACT), and staphylococcus MLSb phenotype (STAMLS) .

[0060] These results demonstrate that the "lyse and wash" buffers described herein can be used to isolate viable microorganism from a PBC sample for downstream identification and AST testing.

[0061] Example 2

[0062] Four positive blood culture samples were obtained by inoculating a blood culture bottle with either Enterococcus faecium (ATTC 700221), Enterococcus faecalis (ATCC 51299), Staphylococcus aureus (ATCC 43300), or Proteus mirabilis (ATCC 29906). Four different "lyse and wash" buffers were prepared comprising the formulations summarized in Table 1 below.

Table 1 :

Buffer #1 Bactec Lytic 10 media and

6.7 g/L Triton™ X-100

Buffer #2 Bactec Lytic 10 media,

3.35 g/L Triton™ X-100, and

16 g/L ammonium chloride Buffer #3 Bactec Lytic 10 media,

3.35 g/L Triton™ X-100, and

40 g/L ammonium chloride

Buffer #4 Bactec Lytic 10 media,

3.35 g/L Triton™ X-100, and

80 g/L ammonium chloride

[ 0063 ] Each of the four "lyse and wash" buffers were used to prepare a microbial pellet from each of the four PBC samples for a total of 16 samples prepared. To isolate viable microorganism, a portion of each PBC sample (5 ml) was pre-treated with 0.5 ml of a 20% choline chloride solution for 20 minutes in a 15 ml conical tube. After incubation with the choline chloride solution, 8 ml of each "lyse and wash" buffer was added to the sample and mixed for 10 minutes. The sample was then centrifuged at 1855 x g for 10 minutes. The supernatant was decanted and discarded while the pellet was washed by resuspending the pellet in the same tube with 5 ml 3.4 g/L Triton™ X-100 in BD Bactec Lytic 10 media (catalog number 442265) without ammonium chloride. The resuspended pellet was allowed to mix for 5 minutes followed by centrifugation at 3000 rpm 1855 x g for 10 minutes. The supernatant was removed and discarded while the pellet was used for both MADLI-TOF/MS identification and Phoenix™ ID/AST testing as described below.

[ 0064 ] A portion of the bacterial pellet prepared above was resuspended in a microcentrifuge tube containing 0.6 ml sterile deionized water. A portion of the suspension (1 μΐ) was spotted onto a MALDI-TOF/MS target plate for bacterial identification analysis and allowed to air dry. Formic acid (1-2 μΐ of a 70% aqueous solution) was overlaid onto the dried, spotted sample and allowed to air dry. The dried sample was overlaid with 1 μΐ of MALDI-TOF/MS matrix solution (MALDI-MS matrix solution prepared by dissolving 2.5 mg of HCCA in 250 μΐ of 2.5% trifluoroacetic acid and 47.5% acetonitrile in de-ionized water) and allowed to air dry before identification by MALDI-TOF mass spectrometry. All mass spectrometry data was recorded on a Bruker Microflex™ LT with Biotyper 2.0 software. The methods of preparing the isolated microbial pellet and identifying the microorganism by the mass spectrometry are further described in U.S. Application Number 13/636,944, filed August 31, 2012, which is hereby incorporated herein by reference. A MALDI-TOF/MS score greater than or equal to 2.0 indicates identification to the species level. A MALDI-TOF/MS score between 1.7 and 1.999 indicates identification to the genus level. And a MALDI-TOF/MS score less than 1.7 indicates no identification or a non-reliable identification. The results are summarized in Table 2 below. A "yes" indicates that a correct identification was obtained at least to the genus level, i.e., with a MALDI score of at least 1.7.

[0065] A second portion of the suspension prepared for MALDI- TOF/MS as described above was further diluted with Phoenix™ ID broth to approximately 0.5 McFarland and used to inoculate a Phoenix™ AST panel as described in Example 1. The results are summarized in Table 2 below. For the Phoenix™ AST results, a "yes" represents those samples that were in essential agreement between the control sample (plated colonies), i.e., those samples with a dilution difference of only +1 or -1.

Table 2 :

Buffer #3 Yes Yes

P. mirabilis

Buffer #4 Yes Yes

E. faecium

Buffer #4 Yes Yes

E. faecalis

Buffer #4 Yes Yes

S. aureus

Buffer #4 No Yes

P. mirabilis

Total 15/16 16/16

[ 0066 ] The results demonstrate that various "lyse and wash" buffers can be used to isolate viable microorganism from a PBC sample for downstream identification by mass spectrometry and AST testing across a panel of microorganisms.

[ 0067 ] Example 3

[ 0068 ] Three positive blood culture samples were obtained by inoculating a blood culture bottle with either Enterococcus faecalis (VRE ATCC 51299), Klebsiella pneumoniae (ATCC 700603), or Staphylococcus aureus (ATCC 43300) . Two different "lyse and wash" buffers were prepared comprising the formulations summarized in Table 3 below. The only difference between buffer #1 and buffer #2 was the addition of 16 g/L ammonium chloride in buffer #2.

Table 3 :

[ 0069 ] Each of the "lyse and wash" buffers of Table 3 were used to prepare a microbial pellet from each of the three PBC samples for a total of six samples prepared. A portion of each PBC sample (5 ml) was mixed with 5 ml of each "lyse and wash" buffer. The samples were then centrifuged at 2200 x g for 10 minutes . The supernatant was decanted and discarded while the pellet was washed by resuspending the pellet in the same tube with the same "lyse and wash" buffer. The resuspended pellet was centrifuged at 2200 x g for 10 minutes. The supernatant was removed and discarded while the pellet was used for both MADLI- TOF/MS identification and Phoenix™ ID/AST testing as described below .

[0070] A portion of the bacterial pellet was resuspended in a microcentrifuge tube containing 0.6 ml sterile water. A portion of the suspension (1 μΐ) was spotted onto a MALDI-TOF/MS target plate for bacteria identification analysis as described in Example 2. The results are summarized in Table 4 below and indicate whether a correct identification was obtained at least to the genus level, i.e., with a MALDI score of at least 1.7. A second portion of the suspension prepared for MALDI-TOF/MS above was further diluted with Phoenix™ ID broth to approximately 0.5 McFarland and used to inoculate a Phoenix™ ID panel as described in Example 1. The results are summarized in Table 4 below.

Table 4:

[0071] The results demonstrate that various "lyse and wash" buffers, with or without ammonium chloride, can be used to isolate viable microorganism from a PBC sample for downstream identification across a panel of microorganisms.

[0072] Example 4 [ 0073 ] Tests were performed to determine the viability of several different strains of S. pneumoniae upon exposure to various "lyse and wash" buffers described herein. Additional tests were performed to determine whether processing a PBC sample with these various "lyse and wash" buffers sufficiently removes substances that interfere with identification of the microorganism.

[ 0074 ] Cell Viability

[ 0075 ] Three different "lyse and wash" buffers were prepared as summarized in Table 5 below, each with a different amount of Triton™ X-100, i.e., 6.7 g/L, 0 g/L, or 0.335 g/L.

Table 5 :

[ 0076 ] A volume of each of these buffers (10 ml) was inoculated with 1 X 10 ³ cfu/ml of either S. pneumoniae non- mucoidal strain (POS3092) or S. pneumoniae mucoidal strain (POS650) and incubated for up to four hours. As a positive control, a 10 ml volume of anaerobic nutrient broth was also inoculated with each of the S. pneumoniae strains, as the microorganisms would be expected to grow in this nutrient broth. After inoculation at the time points of 0 hour, 1 hour, 2 hour, and 4 hour, a 100 μΐ volume of the mixture was plated onto agar plates containing 5% sheep blood at a density of approximately 1 X 10 ² cfu/plate. The plates were incubated overnight at 35°C/5% C0 ₂ at which time the plates were manually counted for bacterial colonies. The results of the manual plate count are summarized in Table 6 below.

Table 6 :

[ 0077 ] As Table 6 demonstrates, viable S. pneumoniae were not obtained following a 1 hour exposure to a high concentration of Triton™ X-100 (buffer #1). However, comparable amounts of viable S. pneumoniae were obtained using the no Triton™ X-100 (#2) and low Triton™ X-100 (#3) buffers.

[ 0078 ] Identification by MALDI-TOF/MS

[ 0079 ] The various "lyse and wash" buffers of Table 5 were used to prepare a bacterial pellet from a PBC sample to be used for identification by MALDI-TOF/MS to determine whether the various "lyse and wash" buffers sufficiently cleaned the PBC sample from interfering cellular debris. PBC samples were prepared with five different strains of S. pneumoniae including non-mucoidal strain POS3092, non-mucoidal strain POS3996, non- mucoidal strain POS3999, mucoidal strain POS650, and mucoidal strain POS532 by inoculating both an aerobic and anaerobic bottle for each of the strains . The microorganism was isolated from each of the PBC samples using each of the "lyse and wash" buffers of Table 5 by adding to each PBC sample a volume of the lyse and wash" buffer at a ratio of 1:1. The mixtures were mixed on a Nutator™ for 5 minutes. The mixture was centrifuged at 2200 x g for 10 minutes followed by decanting and discarding of the supernatant. To wash the pellet of residual blood cell debris, an additional 5 ml of the same "lyse and wash" buffer was added to the tube and vortexed to resuspend the pellet. The mixture was centrifuged at 2200 x g for 10 minutes. The supernatant was decanted and excess liquid removed from the resulting microbial pellet using a cotton applicator swab. The microbial pellet was resuspended in 600 μΐ sterile deionized water to achieve an inoculum of microbial suspension of approximately 4 McFarland.

[0080] The resulting pellet was prepared for identification by MALDI-TOF/MS by the methods described in Example 2 above. The MADLI-TOF/MS scores are summarized in Table 7 below.

Table 7:

[0081] The results indicate that preparation of a viable microbial pellet from an anaerobic PBC bottle produced significantly better MALDI scores for various strains of S. pneumoniae compared to the pellets prepared from the aerobic PBC bottle. In addition, the "lyse and wash" buffers described herein containing low amounts of Triton™ X-100 produce significantly better MALDI scores across the entire panel of S. pneumoniae strains compared to samples prepared with a high concentration of Triton™ X-100.

[ 0082 ] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.