Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/491—Blood by separating the blood components
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
  • G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation

Definitions

• centrifugation makes it more difficult to obtain viable and intact bacteria for further processing and identification.
• gross hemolysis of red blood cells (RBC) and damage to other blood components caused by centrifugation can occlude the samples, making visual or optical interrogation of the samples more difficult. Separation of the microorganisms from other blood components such as RBCs is needed to facilitate downstream testing and to detect the presence or absence of target microorganisms in the sample .
• ID Identification
• AST Antimicrobial Susceptibility Testing
• mass spectrometry Identification by mass spectrometry.
• Alternative methods used to separate bacteria from peripheral blood components require the use of additives that can inhibit or damage the viability/integrity of the target microorganisms for downstream processing and testing methods. More specifically, it is desirable to provide a test sample that has high concentration of bacteria and low concentration of red blood cells for downstream processing and detection of target microorganisms.
• a biological fluid sample e.g., blood
• the described methods concentrate microorganisms without compromising microorganism viability and/or structural integrity for downstream processing and testing methods that require whole/viable microorganisms for testing.
• the described methods concentrate bacteria using a water-soluble polymer, poly-L-glutamic acid (PGA), and do not require centrifugation or additives that destroy microorganism structure and/or viability.
• the described methods preferably concentrate microorganisms in the top layer of the biological sample and sediment the RBCs in the bottom layer without compromising the viability/structure of the microorganisms.
• the top layer of the PGA-treated sample is high in bacterial concentration and, in certain preferred embodiments, low in RBCs by at least two orders of magnitude reduction in the RBC count in the top layer compared to the bottom layer .
• the desired separation is achieved in a maximum of about 60 minutes, preferably within about 30 minutes or less, and most preferably within about 15 minutes or less.
• a biological sample e.g., blood
• PGA Poly-L-glutamic acid
• a top layer of the sample is removed, the top layer containing a higher concentration of microorganisms (if present) than the lower portions of the sample.
• the top layer so obtained is then subjected to additional downstream testing and processing to test for the presence of one or more target microorganisms in the sample.
• Another embodiment of the present invention contemplates obtaining a positive blood culture and adding a PGA solution thereto.
• the sample is then allowed to sediment for about 30 minutes or less.
• a fraction of the sample most likely to contain a higher concentration of microorganisms (if present) is observed, after which a portion of the sample is removed from that fraction. That portion of the sample is then subjected to downstream processing and testing to determine the presence or absence of target microorganisms in the sample.
• the amount of PGA added to the biological sample is selected so that the desired separation can be achieved within about 30 minutes or less, and most preferably within about 15 minutes or less of sedimentation time.
• the presence of up to about 0.1 mL of stock PGA solution in the biological sample provides rapid sedimentation of RBCs within about 10- minutes or less of sedimentation time and significantly increases the sedimentation rate compared to samples without PGA added thereto.
• the sedimentation time required to obtain a test sample having intact/viable bacteria for downstream processing, preferably in the top layer will vary based on the biological sample (i.e., degree or hemolysis, presence of an inflammatory response, hematocrit, etc.), as well as the properties of the PGA additive, including molecular weight of PGA, stock PGA concentration, and the amount of stock PGA solution used to segregate/concentrate bacteria preferably in the top layer of the biological sample.
• FIG. 1A illustrates separated layers in the test tubes obtained after approximately 60 minutes or less of sedimentation with PGA to isolate Staphylococcus aureus (S. aureus) for different molecular weight ranges of PGA polymer.
• FIG. IB illustrates the approximate percentage of bacteria within each separated layer of the test tubes measured by Giemsa and Gram Stains.
• FIGS. 2A and 2B are Box & Whisker plot analysis of bacterial concentration (by percentage) in the top and bottom sedimentation layers using PGA of three different molecular weights to isolate S. aureus and Escherichia coli (E. coli) from the biological sample.
• FIGS. 3A and 3B illustrate visually separated layers and Box & Whisker plot analysis following sedimentation with PGA to isolate bacteria compared to alternative sedimentation additives (i.e., glycerol and sucrose solutions).
• FIGS. 4A and 4B compare the ability of higher molecular weight (50,000-70,000 and 50,000-100,000 molecular weights) PGA to concentrate three microorganisms: E. coll, S. aureus and Streptococcus pneumoniae (S. pneumoniae) in a portion of the sample .
• FIG. 5 compares the ability of PGA with overlapping ranges (50,000-70,000 and 50,000-100,000 molecular weights) to concentrate bacteria in the top portion of a sample at various time intervals for sedimentation.
• the fraction is a top fraction of the sample to ensure that this desired fraction can be easily accessed and used for downstream analysis to determine the presence or absence of microorganisms in the sample.
• the method of concentrating bacteria preferably in the top layer of a biological sample (e.g., in whole blood), does not require conventional protocols (e.g., centrifugation, additives that kill/dissolve microorganisms) to concentrate microorganisms therein.
• a PGA solution is added to a biological sample suspected of containing at least one microorganism. The combined solution is allowed to sediment for approximately 30 minutes or less.
• the top layer of the sedimented sample i.e., the fraction of the sample that contains the most accessible/highest concentration of microorganisms
• the described methods provide rapid sedimentation of a biological sample without the adverse consequences of centrifugation or additives that kill or dissolve the microorganisms.
• the methods described herein provide for a fast and simple aspiration of the PGA-induced segregation of bacteria from a fraction of the sample that has preferably the greatest concentration of microorganisms (if microorganisms are present in the sample) and which fraction is most easily accessed (e.g., the top layer of the sample) .
• the accessed fraction of the sample is the top layer of the sample, which also contains the highest concentration of microorganisms.
• Simply aspirating a portion of the top sample fraction is easier and less hazardous than decanting a biohazardous supernatant and re-suspending the pellet formed by high speed centrifugation .
• test sample is the fraction of the liquid sample that is removed therefrom and subjected to downstream processing to determine the presence or absence of bacteria in the sample that is prepared according to the methods of the present disclosure.
• the test sample is used in methods for downstream detection and/or processing of microorganisms.
• Downstream processing includes, for example, centrifugation, or exposure of the test sample to a solid phase such as capture beads having a bound ligand that would capture bacteria from the test sample.
• a test sample or the microbes in the test sample may be adhered to a solid support .
• a solid support may include microarrays (e.g., DNA or RNA microarrays), gels, blots, glass slides, beads, or ELISA plates. While centrifugation is preferably avoided when separating the test sample from the liquid sample from which it is obtained, centrifugation can be deployed to further process the test sample once obtained.
• the test sample is subsequently processed and used for Identification (ID) and Antimicrobial Susceptibility Testing (AST) in an automated system for large scale testing.
• the test samples can be used for ID by Polymerase Chain Reaction (PCR) or in a Mass Spectrophotometer (e.g., matrix-assisted laser desorption/ionization - time-of-light mass spectrometer, MALDI-TOF) .
• the test sample is directly tested in either a Phoenix ID/AST or MALDI-TOF.
• test sample is subjected to centrifugation and a wash procedure to remove the PGA and extract bacteria from the PGA-concentrated test sample for subsequent testing in a Mass Spectrophotometer (MALDI-TOF).
• MALDI-TOF Mass Spectrophotometer
• the PGA additive to the liquid sample as described herein provides a liquid sample with at least two visually distinct layers stratified by the average molecular weight of the constituents of the liquid sample.
• the bottom layer has a higher concentration of red blood cells and a lower concentration of bacteria than the upper layer.
• the top layer of the stratified liquid sample therefore has the higher concentration of bacteria and a lower concentration of red blood cells.
• the sample is visually stratified into three layers, the top two layers having a higher concentration of bacteria and lower concentration of red blood cells relative to the bottom third layer and the liquid sample prior to PGA-induced sedimentation/stratification.
• the concentration of bacteria is not necessarily higher, but one layer has a visibly lower concentration of other sample constituents. It is that layer from which the test sample is drawn for downstream processing (e.g., ID, AST).
• test sample refers to a liquid sample layer that is same or higher in microbial concentration and lower in the concentration of Red Blood Cells (RBCs) than other portions of the liquid sample from which the test sample remains .
• RBCs Red Blood Cells
• biological sample refers to a sample obtained from a biological subject, including sample of biological tissue or fluid obtained in vivo, for example fresh blood sample or whole banked blood.
• liquid sample refers to the biological sample from which the test sample is obtained.
• positive blood culture refers to a biological sample that consists of a growth media and an anticoagulated whole blood determined to contain bacteria.
• Red Blood Cells are defined as erythrocytes which are formed elements in the peripheral blood.
• Peripheral blood components are defined as the cellular components of blood, consisting of red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow. Red Blood Cells exhibit higher density compared to white blood cells and platelets .
• PGA poly-L-glutamic acid
• PGA polymer coats the cellular components of peripheral blood, increasing their respective density, thereby increasing the sedimentation rate of the peripheral blood components in the test sample and decreasing their buoyancy relative to the microorganisms in the sample.
• polymers having similar characteristics for example, alpha-poly-D ( or L) -glutamic acid, gamma-poly-D ( or L) -glutamic acid, alpha-poly-D ( or L)- aspartic acid (PAA), beta-poly-D ( or L)-aspartic acid(PAA), poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate ) (PBM) , polyacrylic acid, poly (methacrylic acid), polysaccharides, polyvinylpyrrolidone, or their analogues, homologues, and their linear, branched or block copolymers are contemplated as suitable in the methods described herein.
• the anti-coagulant used to prepare positive blood is preferably selected from the type that inhibits the antimicrobial systems of blood and is used in blood culture media and in microbiology for clinical specimen processing.
• acid citrate-dextrose solution is not a preferred anti-coagulant for blood culture because it is primarily used to preserve blood specimens required for tissue typing and blood banking.
• the anticoagulant is sodium polyanethole sulfonate (SPS) and the growth media is a BD BACTECTM Standard/10 Aerobic/F media.
• BD BACTECTM Standard/10 Aerobic/F media is a commercially available growth media for use with aerobic culture and recovery of microorganisms (e.g., bacteria, yeast, fungi) from blood .
• the PGA stock solution of various amounts and concentrations is added to about 1.0 mL of positive blood culture.
• the volume and concentration of the PGA added to PBC may vary depending on factors such as the density of the biological sample, the molecular weight of the PGA used to prepare a stock concentration, the volume of the stock PGA concentration used, etc.
• PGA polymers of various molecular weight effect the sedimentation rate, sedimentation quality, visual separation, and viability and intactness of bacteria recovered in the test sample.
• PGA powders of a mixture of PGA up to 100,000 molecular weights are used to prepare a stock solution to concentrate/segregate bacteria in a biological sample.
• Examples of PGA molecular weight ranges for the stock PGA solutions combined with the sample include, but are not limited to about 3,000-15,000, about 15,000- 50,000, about 50,000-70,000 and about 50,000-100,000 molecular weights. Consequently, the range of PGA molecular weights that may be combined to form the PGA solution is about 3,000 to 100,000. In other embodiments the ranges are: 1) about 3,000-15,000; 2) about 15,000-50,000; 3) about 50,000-70,000; and 4) about 50,000-100,000 molecular weights.
• One skilled in the art can determine the molecular weight range most suited for combination with a particular biological sample.
• a stock PGA solution having a final concentration of up to about 26 milligrams per milliliter (mg/mL) is prepared.
• the biological sample which, in certain embodiments, is a positive blood culture.
• the positive blood culture is a biological sample that consists of an anti-coagulated whole blood determined to contain bacteria, upon being combined with a BACTECTM growth media.
• the biological sample is a whole blood sample.
• the biological sample is defibrinated blood.
• the final concentration of PGA, after being combined with the positive blood culture is in the range of about 0.99 mg/mL to about 7.00 mg/mL. In other embodiments, the final concentration of PGA, after being combined with the positive blood culture, is in the range of about 1.1 mg/mL to about 5.8 mg/mL. In the described embodiments, after the PGA solution combined with the positive blood culture is allowed to settle for an amount of time described herein, the sample fraction determined to contain a higher concentration of microorganism, is recovered for subsequent testing. Also contemplated herein are kits for providing PGA to a sample as described herein.
• the PGA stock solution used to obtain the test sample was prepared with PGA having molecular weight of up to 100,000.
• a PGA powder having molecular weight in the range of 3,000 to 100, 000 was used to prepare the stock solution.
• the stock solution was prepared using PGA having a molecular weight of about 50,000-70,000 and about 50,000- 100, 000.
• the positive blood culture for the examples described below was prepared by adding sodium polyanethole sulfonate (SPS) anticoagulated whole blood into a culture vial containing growth media and inoculated with fresh bacteria.
• SPS sodium polyanethole sulfonate
• Various bacteria Gram-positive, Gram-negative), yeast, etc. can be used to inoculate the culture vial containing a growth media and anti-coagulated whole blood.
• Specific examples described herein include inoculated samples with E. coll, S. aureus and S. pneumoniae.
• the positive blood culture contained a microbial count of about 1 x 10 8 colony forming units (CFU) per milliliter by plate count of the total suspension.
• CFU colony forming units
• the microbial plate counts were performed in triplicate and the percentage of microbial recovery was calculated for each individual result. The resulting triplicate values for each sample were used to generate a spread in a Box & Whisker plot.
• Positive blood culture was prepared by adding 10 mL of anti-coagulated whole blood sample into a culture vial containing 30 mL of growth media and inoculating the vial with up to 8 mL of a solution containing bacteria at a concentration of 2 McFarland.
• the growth media was a commercially available BACTECTM Standard/10 Aerobic/F Media (Becton Dickinson) with reactive ingredients consisting of proteins, yeast extracts, amino acids, vitamins and resins to absorb antibiotics.
• Other growth media having similar reagents/ingredients that are well known to those skilled in the art can similarly be used for blood culture without lysing RBCs .
• the PGA stock solution was made by placing a poly-L- glutamic acid (PGA) sodium salt powder of various molecular weights in a container, adding sterile distilled water to the container with PGA powder, placing the container having PGA powder and water on a NutatorTM (TCS Scientific Corp.) or a rocker to mix and dissolve the PGA powder in the water for about 30 minutes and storing at room temperature.
• the dissolved solution having a clear and slightly viscous appearance, was sterilized by passing the solution through a 0.2 ]im filter.
• the poly-L-glutamic acid sodium salt was purchased from Sigma-Aldrich Co. as a solid chemical in various molecular weights and mixed with sterile distilled water in amounts sufficient to achieve the desired stock PGA concentrations .
• Test samples were obtained using the following method. First, the positive blood culture (e.g., 1 mL) was dispensed into a sterile tube. Next, an aqueous PGA solution was added to the sterile tube containing about 1 mL of positive blood culture and capped. The resulting mixture was gently mixed on a flatbed mixer for approximately 5-10 seconds and allowed to rest, undisturbed, in an upright position for about 10 - 30 minutes or until visual separation occurred demarking the PGA-concentrated test sample visually observed in the top layer from the dense bottom layer containing high concentration of red blood cells.
• the positive blood culture e.g., 1 mL
• an aqueous PGA solution was added to the sterile tube containing about 1 mL of positive blood culture and capped. The resulting mixture was gently mixed on a flatbed mixer for approximately 5-10 seconds and allowed to rest, undisturbed, in an upright position for about 10 - 30 minutes or until visual separation occurred demarking the PGA-concentrated test sample visually observed in
• test sample containing a PGA-induced segregation of bacteria from other sample constituents was removed from the top layer using a pipette.
• the volume of the test sample varied depending on the percentage of red blood cells in the test sample (i.e., hematocrit) and the sedimentation time.
• the amount of bacteria present in various layers of the test tube containing a PGA solution and positive blood culture was determined by estimating bacteria per microscopic field using both Giemsa and Gram Stains.
• the test samples were prepared and obtained according to the test method described above.
• the PGA stock solution was prepared using PGA polymers having the following molecular weights: 3,000-15,000 (PGA 1), 15,000-50,000 (PGA 2) and 50,000-70,000 (PGA 3).
• Test tubes 5 and 6 were prepared by adding 0.2 mL and 0.5 mL of PGA 3 having a stock concentration of 12.6 mg/mL to tubes 5 and 6, respectively.
• the final PGA concentrations relative to the total volume (TV) of positive blood culture and stock PGA solutions are summarized in Table 1.
• the tested range of final PGA concentration in tubes 1 through 6 varied based on the amount of PGA added to 1.0 mL PBC at various stock PGA concentrations prepared with low molecular weight PGA (PGA 1 and PGA 2) and high molecular weight PGA (PGA 3) .
• the visually separated layers in the test tubes (shown as 1, 2, and 3 for top layer, middle layer and bottom layer, respectively) obtained after approximately 60 minutes of sedimentation time are illustrated in FIG. 1A. Observations based on visually separated layers were recorded at 30 minutes and at 60 minutes.
• the solution made with PGA having more than 50,000 molecular weight (PGA 3) produced faster separation rate between RBCs and bacteria compared to the solution made with PGA having less than 50, 000 molecular weight (PGA 1 and PGA 2) .
• the gradient layer (i.e., the middle layer, shown as 2) of the liquid sample was absent in test tubes 4, 5 and 6 (FIG. 1A) after approximately 60 minutes of sedimentation. Only two visually distinct layers were observed in test tubes 4-6, indicating that using PGA of higher molecular weight (PGA 3) produced faster separation and efficiently concentrated the test sample in the top layer (shown as 1) of the liquid sample compared to PGA of lower molecular weight (PGA 1 and PGA 2) .
• test samples were prepared using PBC inoculated with two different bacteria: E. coli and S. aureus.
• the PBC was prepared by adding 10 mL of anti-coagulated whole blood sample into a culture vial containing 30 mL of growth media and inoculating the vial with 8 mL of a solution containing bacteria at a concentration of 2 McFarland to yield an S. aureus count of approximately 8 x 10 8 CFU/ml, confirmed by plate count performed in triplicate, and an E. coli count of approximately 2 x 10 8 CFU/ml, without incubating the samples.
• 1.0 mL of PBC with E. coli was added to three separated tubes.
• Table 2 PGA solutions containing PBC prepared with E. coli and S. aureus
• a Box & Whisker Plot was used to determine the distribution of bacteria within the bottom and top layers of the prepared solution containing PGA and PBC.
• the control used in the exemplary embodiments was a seeded BACTECTM bottle without a PGA solution. More specifically, the concentration of E. coli in the top layer was highest in Solution A (prepared using the lowest molecular weight range PGA 1) and lowest in Solution C (prepared using the highest molecular weight range PGA 3) .
• FIG. 2B which depict the Box & Whisker statistical analysis of the viable bacterial distribution within the top and bottom PGA- concentrated layers achieved after approximately 30 minutes of sedimentation time using seeded BACTECTM bottle as a control.
• the Box represents the lower quartile range of 25% and the upper quartile range of 75% (or 25% and 75% quartiles) .
• the Whiskers represent the minimum and maximum data ranges.
• the values along the Y-axis represent the percentage of bacteria in the top and/or bottom layers.
• the values along the X-axis represent the final PGA concentrations rounded to the nearest whole number (all CFU/mL values are in logio). Solutions A, B, and C that were prepared with PBC containing S. aureus had a higher concentration of S.
• the concentration of S. aureus in the bottom layer was highest in Solution C (prepared using the highest molecular weight range PGA 3) compared to Solution A (prepared using the lowest molecular weight range PGA 1) .
• Solution A from Example 2 was compared to six non- PGA solutions to test alternative sedimentation mechanisms and the visual separation of the sedimentation layers in the absence of PGA (test tube 7) .
• Six test tubes were prepared according to the test methods, except 0.2 mL of glycerol and sucrose solutions were added to the test tubes containing 1 mL of PBC.
• the PBC was prepared by adding 10 mL of anti- coagulated whole blood sample into a culture vial containing 30 mL of growth media and inoculating the vial with 8 mL of a solution containing S. aureus at a concentration of 2 McFarland to yield a microbial count of approximately 2 x 10 8 CFU/ml by plate count without incubating the samples.
• Table 3 summarizes the volume and concentrations of glycerol and sucrose used in the test tubes compared to Solution A which was prepared using PGA 1.
• FIG. 3A The results of visual separation after approximately 30 minutes of sedimentation are shown in FIG. 3A.
• Figure 3B is a Box & Whisker Plot analysis of the percentage of bacteria in the top and bottom layers of the test tubes using seeded BACTECTM bottle as the control. Most of the bacteria were concentrated in the bottom layer for test tubes 1 through 4 and test tube 6.
• Test tube 5 which was prepared by adding 0.2 mL of 1% glycerol to 1.0 mL of PBC, had a higher bacterial concentration in the top layer; however, there was minimal visual separation between the layers compared to Solution A.
• the effects of high molecular weight PGA on the sedimentation and separation rate of the PBC were tested as follows .
• the PBC was prepared according to the protocol described in Example 1.
• the tested bacteria in the PBC were E. coll, S. aureus and S. pneumoniae.
• four test tubes (A, B, C, and D) were prepared by first adding 1.0 mL of PBC into each sterile tube.
• the stock PGA solution added to PBC in tubes A and B was prepared using PGA of 50,000-70,000 molecular weight (PGA 3).
• the stock PGA solution added to PBC in tubes C and D was prepared using PGA of 50,000-100,000 molecular weight (PGA 4).
• the final PGA concentrations in tubes A and B were obtained by adding 0.1 mL and 0.3 mL of PGA 3 at a stock concentration of 12.5 mg/mL to each tube, respectively.
• the final PGA concentrations in tubes C and D were obtained by adding 0.1 mL and 0.3 mL of PGA 4 at a stock concentration of 25 mg/mL into each tube, respectively.
• Table 4 summarizes the PBC and PGA amounts/concentrations used to obtain the test samples in Example 3.
• FIG. 4A-B show the results of the test samples containing viable E. coll, S. aureus and S. pneumoniae that were concentrated using PGA 3 and PGA 4. Viable bacteria were present in all test samples obtained from the top sedimentation layer of the liquid sample. Use of PGA 4 (50,000-100,000) provided more consistent results across all three tested microorganisms by concentrating more bacteria in the top layer of the sample compared to the bottom layer.
• test samples prepared according to the methods described in Example 3 using PBC with E. coli were analyzed at 5 minute intervals starting at time 0 and up to 30 minutes.
• the test samples were obtained after 5, 10, 15, 20, 25 and 30 minutes of sedimentation and analyzed using Box & Whisker Plot to compare the effect of PGA having overlapping molecular weight ranges (PGA 3 and PGA 4) on sedimentation rate.
• the results are summarized in Figure 5.
• the test sample (250 L; 0.25mL) prepared with PGA 4 (50,000-100,000 molecular weight) contained viable E. coli in higher concentrations compared to the test sample prepared with PGA 3 (50,000-70,000 molecular weight).
• test samples prepared according to the methods described herein were tested directly (without first extracting the bacteria) to Phoenix for Antimicrobial Susceptibility Testing (AST) . The results are summarized in Table 5.
• a MALDI ID score of less than 1.699 indicates an unacceptable or not reliable identification. There were no peaks when testing direct samples to MALDI-TOF. The MALDI-TOF spectra generated from direct samples did not match mass spectra currently in the Bruker database. The E. coli isolates in the control sample achieved a reliable quality identification score of more than or equal to 2.289 ( ⁇ 2.289), indicating that use of PGA to concentrate bacteria in a top layer interfered with MALDI-TOF identification when testing direct samples.
• test tubes were prepared according to the methods described in Example 1 using E. coli to prepare PBC .
• the test tubes were prepared by first adding 1.0 mL of PBC into each sterile tube followed by adding 0.1 mL of stock PGA solution prepared using PGA 4 (50,000-100,000 molecular weight) into each tube. All test tubes were subjected to a quick mix using a flatbed mixer to yield a liquid sample having final PGA concentration of 2.27 mg/mL. Following 30 minutes of sedimentation, the test samples were removed from the top layers of the liquid sample in all five test tubes.
• each test sample was combined (800 ]iL x 5) and centrifuged at 16000 x g for 3 minutes, followed by two additional cycles of washing with de- ionized water.
• This pellet was extracted by the Bruker MALDI- TOF extraction protocol.
• duplicate spots from the extracted pellet were used to generate spectra. Recovery of microorganisms was measured by viable plate counts. It would be appreciated by one skilled in the art that methods for identifying bacteria using MALDI-TOF are well known in the art and are not described in detail herein. The results are summarized in Table 6.
• Example 5 The methods in accordance with Example 4 resulted in approximately 54% recovery of E. coli from the PGA- concentrated test samples .
• the extracted E. coli isolates (compared to direct sample testing) achieved a reliable quality identification score of more than or equal to 2.000 ( ⁇ 2.000) .
• Example 5 Example 5
• the BACTECTM culture bottle inoculated with bacteria, placed in a BACTECTM FX automated blood culture instrument and incubated for 12-18 hours or until a positive result was indicated.
• a positive result is indicated when a final bacterial count of approximately lxlO 8 CFU/mL is detected in each BACTECTM bottle.
• 1.0 mL of PBC was placed into a sterile tube with 0.1 mL of stock PGA solution having a final concentration of 25 mg/mL prepared with PGA of 50,000-100,000 molecular weight (PGA 4) resulting in a liquid sample having a final PGA concentration of 2.27 mg/mL.
• the test sample was removed from the top layer of the liquid sample and RBCs were counted in the top layer and the bottom layer of the liquid sample using standard procedures. Compared to the bottom layer (2.89 x 10 6 RBC/ L), there were 3.50 x 10 4 RBC/ L in the top layer indicating that the RBC concentration in the top layer of the liquid sample following PGA-sedimentation was reduced by two logs (all RBC/ L values are in logio ) . The results are summarized in Table 7 below.

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