Rapid and sensitive detection of bacteria in blood products, urine, and other fluids

Abstract

The invention provides methods of detecting bacteria in fluids, including blood, platelets and other blood products for transfusion, and urine. The methods are based on lysing the bacteria to release ATP and detecting the ATP. Eukaryotic cell contamination is a problem to be overcome, because eukaryotic cell contain large amounts of ATP. Thus, some of the methods involve separating intact eukaryotic cells (e.g., platelets) from intact bacterial cells before lysing the bacterial cells to release ATP, contacting the ATP with an ATP-consuming enzyme that catalyzes a reaction, and monitoring the enzyme-catalyzed reaction. Typically, the enzyme is luciferin, and the reaction is monitored by detecting light produced by the luciferin. Other methods of the invention involve contacting a fluid sample with a support surface that binds bacterial cells, lysing the bacterial cells to release ATP, contacting the ATP with an ATP-consuming enzyme, and monitoring the enzyme-catalyzed reaction. Apparatuses for carrying out the methods are also disclosed.

1 . A method of detecting bacteria in a fluid sample suspected of containing bacteria comprising:
 separating intact eukaryotic cells from intact bacterial cells that may be present in the fluid sample;   lysing the bacterial cells to release bacterial ATP into a fluid to generate a bacterial lysate fluid;   contacting the bacterial ATP in the bacterial lysate fluid with an ATP-consuming enzyme that catalyzes a reaction in an ATP assay fluid; and   monitoring the enzyme-catalyzed reaction in the ATP assay fluid;   wherein the fluid sample is urine,   wherein the method detects 10,000 bacterial colony forming units per ml or less of each of the following species of bacteria:  Bacillus cereus, Bacillus subtilis, Clostridium prefringens, Corynebacterium  species,  Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, Propionibacterium acnes, Pseudomonas aeruginosa, Salmonella choleraesuis, Serratia marcesens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes , and  Streptococcus viridans.      
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1  wherein the step of separating intact eukaryotic cells from bacterial cells comprises filtering the eukaryotic cells using a filter that blocks the eukaryotic cells and allows the bacterial cells to pass through, to generate a filtered fluid sample containing the bacterial cells. 
     
     
         4 . The method of  claim 3  wherein the filter has a pore size of 1-10 microns. 
     
     
         5 . The method of  claim 3  further comprising after the step of filtering the eukaryotic cells,
 contacting the filtered fluid sample with a support surface that binds the bacteria.   
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 5 , wherein the support surface does not bind the eukaryotic cells. 
     
     
         8 . The method of  claim 5  wherein the bacterial cells are lysed while bound to the support surface to release bacterial ATP into a fluid to generate a bacterial lysate fluid. 
     
     
         9 . The method of  claim 5  further comprising eluting the bacterial cells from the support surface into an elution fluid before the step of lysing the bacterial cells. 
     
     
         10 . The method of  claim 1  wherein the step of separating intact eukaryotic cells from bacterial cells comprises:
 contacting the fluid sample with a support surface that binds the bacterial cells and does not bind the eukaryotic cells.   
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1  wherein the support surface comprises polycations. 
     
     
         13 . The method of  claim 10  wherein the step of separating intact eukaryotic cells from intact bacterial cells that may be present in the fluid sample further comprises before the step of contacting the fluid sample with a support surface that binds the bacteria:
 filtering the eukaryotic cells from the fluid sample using a filter that blocks the eukaryotic cells and allows the bacterial cells to pass through.   
     
     
         14 . The method of  claim 10  wherein the bacterial cells are lysed while bound to the support surface to release bacterial ATP into a fluid to generate a bacterial lysate fluid. 
     
     
         15 . The method of  claim 10  further comprising eluting the bacterial cells from the support surface into an elution fluid before the step of lysing the bacterial cells. 
     
     
         16 . The method of  claim 1  wherein the volume of the ATP assay fluid is smaller than the volume of the fluid sample. 
     
     
         17 . The method of  claim 1  wherein the bacterial cells are lysed by heat or detergent or both. 
     
     
         18 . The method of  claim 1  wherein the bacterial cells are lysed by acid or base. 
     
     
         19 . The method of  claim 1  wherein the bacterial cells are lysed by sonic energy or contact with particles or both. 
     
     
         20 . The method of  claim 1  wherein the bacterial cells are lysed by organic solvent. 
     
     
         21 . The method of  claim 1  wherein the bacterial cells are lysed by enzyme, freeze-thaw, french press, or a combination thereof. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . The method of  claim 1  wherein the enzyme is luciferase, the method further comprising contacting the bacterial ATP and luciferase with luciferin, wherein the step of minitoring the enzyme-catalyzed reaction comprises monitoring light produced by the reaction. 
     
     
         25 - 32 . (canceled) 
     
     
         33 . The method of  claim 1  wherein the method detects at least three bacterial genera at a level of 1000 bacterial colony-forming units per ml of the fluid sample. 
     
     
         34 . The method of  claim 33  wherein the method detects at least three bacterial genera at a level of 100 bacterial colony-forming units per ml of the fluid sample. 
     
     
         35 - 57 . (canceled) 
     
     
         58 . The method of  claim 1  wherein the step of separating intact eukaryotic cells from bacterial cells comprises filtering the eukaryotic cells using a filter that blocks the eukaryotic cells and allows the bacterial cells to pass through, to generate a filtered fluid sample containing the bacterial cells;
 the method further comprising before lysing the bacterial cells concentrating intact bacterial cells in the filtered fluid sample by passing the filtered fluid sample through a filter that blocks passage of bacterial cells;   wherein the volume of the ATP assay fluid is smaller than the volume of the fluid sample.   
     
     
         59 . The method of  claim 1  wherein the step of separating intact eukaryotic cells from bacterial cells comprises filtering the eukaryotic cells using a filter that blocks the eukaryotic cells and allows the bacterial cells to pass through, to generate a filtered fluid sample containing the bacterial cells;
 the method further comprising after the step of filtering the eukaryotic cells contacting the filtered fluid sample with a support surface that binds most species of bacteria;   wherein the volume of the ATP assay fluid is smaller than the volume of the fluid sample.   
     
     
         60 . The method of  claim 1  wherein the step of separating intact eukaryotic cells from bacterial cells comprises contacting the fluid sample with a support surface that binds the bacterial cells and does not bind the eukaryotic cells; wherein the support surface binds most species of bacteria;
 wherein the volume of the ATP assay fluid is smaller than the volume of the fluid sample.   
     
     
         61 . The method of  claim 58  wherein the method detects at least 3 bacterial genera at a level of 100 bacterial colony-forming units per ml of the fluid sample. 
     
     
         62 . The method of  claim 59  wherein the method detects at least 3 bacterial genera at a level of 100 bacterial colony-forming units per ml of the fluid sample. 
     
     
         63 . The method of  claim 60  wherein the method detects at least 3 bacterial genera at a level of 100 bacterial colony-forming units per ml of the fluid sample.