METHODS AND COMPOSITIONS FOR THE DETECTION OF BETA-LACTAMASES

Abstract

Presented herein are methods and compositions for the detection of specific beta-lactamases, including class A serine carbapenemases, metallo-beta-lactamases, AmpC beta-lactamases, and extended-spectrum beta-lactamases (ESBLs). The methods presented herein include methods that permit the detection of the presence of specific beta-lactamases in bacterial samples within as few as 2 to 10 minutes.

Claims

1. A method for detecting the presence of a beta-lactamase, comprising: (a) contacting: i. a first bacterial sample with a first composition comprising a detectable beta-lactamase substrate and an AmpC inhibitor, and ii. a second bacterial sample with a second composition comprising a detectable beta-lactamase substrate, an AmpC inhibitor, and a serine beta-lactamase inhibitor in an amount sufficient to inhibit an extended-spectrum beta-lactamase (ESBL) and an original-spectrum beta-lactamase (OSBL) but not a class A serine carbapenemase, wherein the first and second bacterial samples are from the same source; and (b) detecting utilization of the substrate in the first composition and the second composition, such that a beta-lactamase is detected if the substrate has been utilized in the first and second compositions, wherein the beta-lactamase is a class A serine carbapenemase or a metallo-beta-lactamase.

2-28. (canceled)

29. A kit for detecting the presence of a beta-lactamase, comprising, in one or more containers: (a) a first composition comprising a detectable beta-lactamase substrate and an AmpC inhibitor; and (b) a second composition comprising a detectable beta-lactamase substrate, an AmpC inhibitor, and a serine beta-lactamase inhibitor in an amount sufficient to inhibit an ESBL and an OSBL but not a class A serine carbapenemase.

30-48. (canceled)

Description

5. EXAMPLES

[0171] The examples presented herein demonstrate the accuracy and efficiency of using a detectable beta-lactamase substrate and one or more beta-lactamase inhibitors to detect the presence of specific beta-lactamases.

5.1 Carbapenemase Detection

[0172] This example demonstrates that compositions a comprising detectable beta-lactamase substrate and one or more beta-lactamase inhibitors may be used to detect the presence of a carbapenemase in a sample.

5.1.1 Materials & Methods

[0173] Chromogenic Beta-Lactamase Assay

[0174] Representative E. coli bacterial strains were each streaked on a Trypticase Soy Agar with 5% Sheep Blood agar plate and the plate was incubated at 35 C. for 18 hours. Afterwards, a sample from each plate was added to 1 ml sterile water in a BD 2054 Falcon tube to a turbidity of about 1.6 as measured by a Microscan turbidity reader. 50 l of a cell suspension was added to each of the following 150 l compositions to bring the final concentration in each compositions to the following: (i) Composition 1 comprising 50 M of nitrocefin, 2 mM of cloxacillin, and 75 mM pH 7.0 phosphate buffer; and (ii) Composition 2 comprising 50 M of nitrocefin, 2 mM of cloxacillin, 1 mM of clavulanic acid and 75 mM pH 7.0 phosphate buffer. The color of the nitrocefin was assessed visually after 10 minutes (for Klebsiella oxytoca strains) or 1 hour (for E. coli and Klebsiella pneumoniae). The hydrolysis of nitrocefin by a beta-lactamase causes the color of the composition to change from yellow to red.

[0175] Imipenem MIC Assay

[0176] Imipenem MIC Assay was performed by microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute) standard.

5.1.2 Results

[0177] The results of a chromogenic beta-lactamase assay for representative E. coli strains are summarized in Table 7, below. Table 8, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the imipenem MIC assay and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing for the same E. coli strains in Table 7. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the imipenem MIC, IEF and PCR results for the detection of the presence of a carbapenemase.

TABLE-US-00007 TABLE 7 Utilization of Utilization of nitrocefin in nitrocefin in Presence of composition 1 composition 2 carbapenemase 1 No 2 + + Yes 3 + + Yes 4 + No 5 + No 6 + + Yes 7 + No 8 No 9 No 10 + No 11 + No + indicates that nitrocefin is utilized and changes from yellow to red indicates that nitrocefin is not utilized and stays yellow.

TABLE-US-00008 TABLE 8 Presence of Beta-lactamase profile carbapenemase based on reference based on chromogenic Imipenem MIC methods (IEF, PCR, beta-lactamase assay (g/ml) MIC assays) 1 no <=2 C-chromo 2 yes 8 Carb, C-plasmid 3 yes =16 Carb (KPC) 4 no <=2 ESBL, C-chromo 5 no <=2 IRT, C-chromo 6 yes 8 OSBL, Carb 7 no <=2 OSBL, C-plasmid 8 no <=0.25 WT 9 no <=2 WT 10 no <=2 OSBL, C-chromo 11 no <=2 ESBL, C WT means wild-type or without beta-lactamase. C-chromo means chromosomal AmpC beta-lactamase. C-plasmid means plasmid-borne AmpC beta-lactamase. Carb means carbapenemase. Carb(KPC) means carbapenemase encoded by KPCs (KPC-1, KPC-2, KPC-3 or KPC-4). IRT means inhibitor-resistant TEM beta-lactamase. ESBL means extended-spectrum beta-lactamase. OSBL means original-spectrum beta-lactamase. C means either C-chromo or C-plasmid beta-lactamase.

[0178] The results of a chromogenic beta-lactamase assay for representative Klebsiella pneumoniae strains are summarized in Table 9, below. Table 10, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the imipenem MIC assay and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing for the same Klebsiella pneumoniae strains in Table 9. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the imipenem MIC, IEF and PCR results for the detection of the presence of a carbapenemase.

TABLE-US-00009 TABLE 9 Utilization of Utilization of nitrocefin in nitrocefin in Presence of composition 1 composition 2 carbapenemase 1 no 2 + + yes 3 no 4 + + yes 5 + no 6 + no 7 no 8 + no 9 + + yes 10 + no 11 + reduced + no 12 + + yes 13 + + yes 14 + no 15 + + yes 16 + + yes + indicates that nitrocefin is utilized and change from yellow to red. indicates that nitrocefin is not utilized and stays yellow.

TABLE-US-00010 TABLE 10 Presence of carbapenemase based on chromogenic beta- Imipenem MIC lactamase assay (g/ml) Beta-lactamase Profile 1 no <=2 C-plasmid 2 yes =4 ESBL, MBL 3 no <=2 ESBL, C 4 yes =8 ESBL, Carb (KPC) 5 no <=2 ESBL, C-plasmid 6 no <=2 ESBL, C-plamid 7 no <=2 WT 8 no <=2 OSBL, C-plasmid 9 yes >16 OSBL, carb 10 no <=2 WT 11 no <=2 OSBL, C-plasmid 12 yes >16 MBL 13 yes 4 ESBL, MBL 14 no <=2 ESBL, C-plasmid 15 yes =8 Carb(KPC) 16 yes =8 ESBL, Carb(KPC) WT means wild-type or without beta-lactamase. C-plasmid means plasmid-borne AmpC beta-lactamase. C means an AmpC beta-lactamase either plasmid-borne or chromosomal. Carb(KPC) means serine carbapenemase encoded by KPC. ESBL means extended-spectrum beta-lactamase. OSBL means original spectrum beta-lactamase. MBL means metallo-beta-lactamase.

[0179] The results of a chromogenic beta-lactamase assay for representative Klebsiella oxytoca strains are summarized in Table 11, below. Table 12, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the imipenem MIC assay and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing for the same Klebsiella oxytoca strains in Table 11. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the Imipenem MIC, IEF and PCR results for the detection of the presence of a carbapenemase.

TABLE-US-00011 TABLE 11 Utilization of Utilization of Presence of nitrocefin in nitrocefin in carbapenemase based composition 1 composition 2 on Chromogenic Assay 1 + No 2 + no 3 + + yes 4 no + indicates that nitrocefin is utilized and change from yellow to red. indicates that nitrocefin is not utilized and stays yellow.

TABLE-US-00012 TABLE 12 Presence of carbapenemase based on chromogenic beta- Imipenem lactamase assay MIC Beta-lactamase Profile 1 no <=2 K1-high 2 no <=2 OSBL, K1-high 3 yes >16 OSBL, K1-high, Carb 4 no <=2 WT WT means wild-type or without beta-lactamase. Carb means carbapenemase. K1-high means high level of K1 beta-lactamase. OSBL means original-spectrum beta-lactamase.

5.2 Detection of a Metallo-Beta-Lactamase

[0180] This example demonstrates that compositions comprising a detectable beta-lactamase substrate and one or more beta-lactamase inhibitors may be used to detect the presence of a metallo-beta-lactamase in a sample.

5.2.1 Materials & Methods

[0181] Chromogenic Beta-Lactamase Assay

[0182] Representative Klebsiella pneumoniae strains were each streaked on a Trypticase Soy Agar with 5% Sheep Blood agar plate and the plate was incubated at 35 C. for 18 hours. Afterwards, a sample of Klebsiella pneumoniae from each plate was added to 1 ml of sterile water in a BD 2054 Falcon tube to a turbidity of about 1.6 as measured by a Microscan turbidity reader. 50 l of a cell suspension was added to each of the following 150 l compositions to bring the final concentration in each composition to be the following (i) Composition 1 comprising 50 M of nitrocefin, 2.5 mM of cloxacillin, and 50 mM phosphate buffer pH 7.0; (ii) Composition 2 comprising 50 M of nitrocefin, 2.5 mM of cloxacillin, 1 mM of clavulanic acid and 50 mM phosphate buffer pH 7.0; and (iii) Composition 3 comprising 50 M of nitrocefin, 2.5 mM of cloxacillin, 1 mM of clavulanic acid, DEDTC at a concentration of 5 mM or 15 mM, DPC at a concentration of 1.5 mM or 4.5 mM or TPEN at a concentration of 9 mM and 50 mM phosphate buffer pH 7.0. The color of the nitrocefin after 30 minutes or 1 hour was assessed visually. The hydrolysis of nitrocefin by a beta-lactamase causes the color of the composition to change from yellow to red.

[0183] Imipenem MIC Assay

[0184] Imipenem MIC Assay were performed by microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute) standard.

5.2.2 Results

[0185] The results of a chromogenic beta-lactamase assay for representative Klebsiella pneumoniae strains are summarized in Table 13, below. In the assay, the affect of two different metal chelators at two different concentrations was assessed. Each composition was incubated for 30 minutes with a representative strain before the results of the assay were assessed by visual inspection. Table 14, below, compares the conclusions based on the chromogenic beta-lactamase assay described herein to the results obtained by the imipenem MIC assay, and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing for the same Klebsiella pneumoniae strains in Table 13. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the imipenem MIC, IEF and PCR results for the detection of the presence of a metallo-beta-lactamase in a sample.

TABLE-US-00013 TABLE 13 Comp. #3 Comp. # 3 Comp. #3 Comp. #3 (5 mM (15 mM (1.5 mM (4.5 mM Presence of Comp. #1 Comp. #2 DEDTC) DEDTC) DPC DPC) MBL based Utilization Utilization Utilization Utilization Utilization Utilization on of of of of of of Chromogenic nitrocefin nitrocefin nitrocefin nitrocefin nitrocefin nitrocefin Assay 1 + + reduced + Yes 2 No 3 + + + + + + No 4 + No

TABLE-US-00014 TABLE 14 Conclusion from Imipenem beta-lactamase Chromogenic Beta- Organism MIC profile Lactamase Assay 1 KLEPNEP =4 ESBL, MBL MBL 2 KLEPNEP <=2 WT WT 3 KLEPNEP >16 OSBL, serine Carbapenemase carbapenemase 4 KLEPNEP <=2 ESBL, Beta-lactamase but C-plasmid not carbapenemase MBL means metallo-beta-lactamase. WT means wild-type or without beta-lactamase. Bold letters indicate the presence of a metallo-beta-lactamase or a serine carbapenemase which is consistent with the conclusion from the chromogenic beta-lactamase assay.

[0186] The results of a chromogenic beta-lactamase assay for representative Klebsiella pneumoniae (KLEPNEP) and Pseudomonas aeruginosa (PSEAER) strains are summarized in Table 15, below. In the assay, the affect of two different metal chelators was assessed. Each composition was incubated for 60 minutes with a representative strain before the results of the assay were assessed by visual inspection. Table 16, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the imipenem MIC assay, and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing for the same bacterial strains in Table 15. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the Imipenem MIC, IEF and PCR results for the detection of the presence of a metallo-beta-lactamase or a serine carbapenemase in a sample.

TABLE-US-00015 TABLE 15 Comp. #3 Comp. #3 Comp. #3 (4.5 mM (15 mM (9 mM Presence of Comp. #1 Comp. #2 DPC) DEDTC) TPEN) MBL based Utilization Utilization Utilization Utilization Utilization on of of of of of Chromogenic Organism nitrocefin nitrocefin nitrocefin nitrocefin nitrocefin Assay 1 KLEPNEP + + reduced + Yes 2 KLEPNEP + + Yes 3 KLEPNEP + + + + + No 4 KLEPNEP + reduced + No 5 KLEPENEP No 6 PSEAER + + reduced + Yes

TABLE-US-00016 TABLE 16 Conclusion from Imipenem Beta-lactamase chromogenic beta- Organism MIC profile lactamase assay 1 KLEPNEP 4 ESBL, MBL MBL 2 KLEPNEP >16 MBL MBL 3 KLEPNEP =8 ESBL, Class A serine Carbapenemase carbapenemase 4 KLEPNEP <=2 ESBL, C- Beta-lactamase but plasmid not carbapenemase 5 KLEPENEP <=2 WT WT or no beta- lacatamase 6 PSEAER >16 MBL MBL WT means wild-type. MBL means metallo-beta-lactamase. C-plasmid means a plasmid-borne AmpC beta-lactamase. Bold letters indicate the presence of a metallo-beta-lactamase or a serine carbapenemase which is consistent with the conclusion from the chromogenic beta-lactamase assay

5.3 Detection of an Ampc Beta-Lactamase

[0187] This example demonstrates that compositions comprising a detectable beta-lactamase substrate and one or more beta-lactamase inhibitors may be used to detect the presence of an AmpC.

5.3.1 Materials & Methods

[0188] Chromogenic Beta-Lactamase Assay

[0189] Representative E. coli strains were each streaked on a Trypticase Soy Agar with 5% Sheep Blood agar plate and the plate was incubated at 35 C. for 18 hours. Afterwards, a sample of E. coli from each plate was added to 1 ml of sterile water in a BD 2054 Falcon tube to a turbidity of about 1.6 as measured by a Microscan turbidity reader. l of a cell suspension was added to each of the following 150 l compositions to bring the concentration in each composition to be the following: (i) Composition 1 comprising 50 M of nitrocefin and 50 mM of phosphate buffer pH 7.0; (ii) Composition 2 comprising 50 M of nitrocefin, 5 mM of cloxacillin, and 50 mM of phosphate buffer pH 7.0; (iii) Composition 3 comprising 50 M of nitrocefin, 1 mM of clavulanic acid and 50 mM of phosphate buffer pH 7.0; and (iv) Composition 4 comprising 50 M of nitrocefin, 5 mM of cloxacillin, 1 mM of clavulanic acid and 50 mM of phosphate buffer pH 7.0. The color of the nitrocefin after 60 minutes was assessed visually. The hydrolysis of nitrocefin by a beta-lactamase causes the color of the composition to change from yellow to red.

[0190] Cefoxitin MIC Assay

[0191] Cefoxitin MIC Assay were performed by microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute) standard.

5.3.2 Results

[0192] The results of a chromogenic beta-lactamase assay for representative E. coli strains are summarized in Table 17, below. Table 18, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the cefoxitin MIC assay and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing using the same E. coli strains in Table 17. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the cefoxitin MIC, IEF and PCR results for the detection of the presence of an AmpC in a sample.

TABLE-US-00017 TABLE 17 Conclusion based on Comp. #1 Comp. #2 Comp. #3 Comp. #4 Chromogenic Assay 1 WT 2 + + + AmpC, OSBL or AmpC, ESBL 3 + + AmpC 4 + OSBL or ESBL 5 + + + AmpC, OSBL or AmpC, ESBL 6 + + OSBL or ESBL 7 + Reduced + OSBL or ESBL 8 + + AmpC 9 + + + + carbapenemase 10 + + AmpC 11 + + + AmpC, OSBL or AmpC, ESBL WT means wild-type or no beta-lactamase. OSBL means original-spectrum beta-lactamase. ESBL means extended-spectrum beta-lactamase. AmpC means AmpC beta-lactamase either plasmid-borne or chromosomal.

TABLE-US-00018 TABLE 18 Conclusion based on Conclusion based on chromogenic beta- Cefoxitin MIC reference methods lactamase assay 1 <=4 WT No detectable - lactamase 2 >32 OSBL, AmpC, OSBL C-plasmid or AmpC, ESBL 3 >32 OSBL, C-chromo AmpC 4 32 OSBL ESBL or OSBL 5 >32 ESBL, AmpC, OSBL C-plasmid or AmpC, ESBL 6 =32 ESBL, ESBL or OSBL C-chromo 7 =8 ESBL ESBL or OSBL 8 >32 C-chromo AmpC 9 >32 Carba- Carbapenemase penemase 10 >32 C-plasmid AmpC 11 >32 OSBL, AmpC, OSBL C-plasmid or AmpC, ESBL WT means wild-type or beta-lactamase. OSBL means original-spectrum beta-lactamase. ESBL means extended-spectrum beta-lactamase. AmpC means an AmpC beta-lactamase that is either plasmid-borne or chromosomal. C-plasmid means plasmid-borne.

5.4 Detection of an Esbl

[0193] This example demonstrates that compositions comprising a detectable beta-lactamase substrate and one or more beta-lactamase inhibitors may be used to detect the presence of an ESBL.

5.4.1 Materials & Methods

[0194] Chromogenic Beta-Lactamase Assay

[0195] Representative E. coli and Klebsiella pneumoniae strains from BD collection were each streaked on a Trypticase Soy Agar with 5% Sheep Blood agar plate and the plate was incubated at 35 C. for 18 hours. Afterwards, a sample of E. coli or Klebsiella pneumoniae from each plate was added to 1 ml of sterile water in a BD 2054 Falcon tube to a desired turbidity (reading 1.5 for sample 2-5, 8-10, reading 1.0 for sample 1-2 and reading for sample 6-7) as measured by a Microscan turbidity reader. 50 l of a cell suspension was added to each of the following 150 l compositions to bring the final concentration in each composition to be the following: (i) Composition 1 comprising 50 M of nitrocefin and 50 mM of phosphate buffer pH 7.0; (ii) Composition 2 comprising 50 M of nitrocefin, mM of clavulanic acid, and 50 mM of phosphate buffer pH 7.0; (iii) Composition 3 comprising 50 M of nitrocefin, 25 mM of cefotaxime and 50 mM of phosphate buffer pH 7.0; and (iv) Composition 4 comprising 50 M of nitrocefin, 1.25 mM of ceftriaxone (Toku-E, Japan) and 50 mM of phosphate buffer pH 7.0. The color of the nitrocefin after minutes or 60 minutes was assessed visually. The hydrolysis of nitrocefin by a beta-lactamase causes the color of the composition to change from yellow to red.

[0196] MIC Assays

[0197] MIC Assays of CTX (cefotaxime), CAZ (cefotaxime/clavulanic acid), CAZ (ceftazidime) and CCZ (ceftazidime/clavulanic acid) were performed by microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute) standard.

5.4.2 Results

[0198] The results of a chromogenic beta-lactamase assay for representative E. coli (ESCCOL) and Klebsiella pneumoniae (KLEPNEP) strains are summarized in Table 19, below. Table 20, below, compares the conclusions based on the beta-lactamase assay described herein to the results obtained by the cefoxitin MIC assay and the beta-lactamase profile determined previously by isoelectric focusing (IEF) and PCR testing using the same strains in Table 19. The conclusions based on the chromogenic beta-lactamase assay results are consistent with the conclusions based upon the cefoxitin MIC, IEF and PCR results for the detection of the presence of an ESBL in a sample.

TABLE-US-00019 TABLE 19 Con- Organism Comp. #1 Comp. #2 Comp. #3 Comp. #4 clusion 1 ESCCOL + ESBL 2 ESCCOL + ESBL 3 ESCCOL + + + OSBL 4 ESCCOL + + + OSBL 5 ESCCOL WT 6 KLEPNEP + reduced + reduced + ESBL 7 KLEPNEP + ESBL 8 KLEPNEP + + OSBL 9 KLEPNEP + + + OSBL 10 KLEPNEP WT WT means wild-type or without beta-lactamase. ESBL means extended-spectrum beta-lactamase. OSBL means original-spectrum beta-lactamase.

TABLE-US-00020 TABLE 20 Conclusion based on Conclusion chromogenic based on beta- CTX CCX CAZ CCZ reference lactamase Organism MIC, g/ml MIC, g/ml MIC, g/ml MIC, g/ml methods assay 1 ESCCOL =32 <=0.25 >128 <=0.25 ESBL ESBL 2 ESCCOL >64 <=0.25 16 <=0.25 ESBL ESBL 3 ESCCOL <=0.25 <=0.25 <=0.25 <=0.25 OSBL OSBL 4 ESCCOL <=0.25 <=0.25 <=0.25 <=0.25 OSBL OSBL 5 ESCCOL <=0.25 <=0.25 <=0.25 <=0.25 WT WT 6 KLEPNEP >64 <=0.25 >128 =4 ESBL ESBL 7 KLEPNEP >64 <=0.25 8 <=0.25 ESBL ESBL 8 KLEPNEP <=0.25 <=0.25 <=0.25 <=0.25 OSBL OSBL 9 KLEPNEP <=0.25 <=0.25 <=0.25 <=0.25 OSBL OSBL 10 KLEPNEP =0.5 =0.5 <=0.25 <=0.25 WT WT WT means wild-type or without beta-lactamase. OSBL means original-spectrum beta-lactamase. ESBL means extended-spectrum beta-lactamase. Bold letters indicate the presence of an ESBL, which is consistent with the conclusion from the chromogenic beta-lactamase assay. Note that assay for sample 6 was finished at 20 minutes and the rest samples finish at 1 hour.

5.5 Detection of Beta-Lactamases in the Presence of Lysis Reagents

[0199] This example demonstrates that the in situ lysis of bacterial cells and the detection of beta-lactamases provides a high level of sensitivity of the beta-lactamase assay, which can be particularly advantageous when detecting certain beta-lactamases expressed by certain types of bacteria (e.g., gram negative bacteria).

5.5.1 Materials and Methods

[0200] Panel production. Stock solutions with nitrocefin, trehalose, and/or reagents used to lyse the bacterial cells in pH6 MES buffer were prepared and dispensed into wells on the bottom part of Phoenix panel (BD, USA), with one stock solution in one well. Panels were then dried in an oven for approximately 30 min at approximately 70 C. The top part of Phoenix panel was then attached to the bottom part forming a panel system ready for inoculation. The concentrations of reagents after rehydration following Phoenix panel inoculation were 3 mg/ml lysozyme, 1 mM EDTA, and 1% trehalose in 0.1 M pH6 MES buffer.

[0201] Inoculation and lysis testing. Representative strains of bacteria, such as E. coli and Klebsiella pneumoniae, were each streaked on a Trypticase Soy Agar with 5% Sheep Blood agar plate (BD, USA) and the plates were incubated at 35 C. for 18 hours. Afterwards, colonies from pure culture on each plate were inoculated into BD Phoenix ID broth (BD, USA) and adjusted to a desired turbidity of approximately 0.5 MacFarland. The bacterial cell suspension was then poured into the ID side of the Phoenix panels that were specifically made for cell lysis testing. Once inoculated, the panel was loaded into Phoenix instrument, where nitrocefin hydrolysis rate was monitored by colorimetric signals changes every 20 minutes for 16 hours.

[0202] Microorganism. The presence of beta-lactamase in the strains tested herein was previously characterized by isoelectric focusing (IEF) gel electrophoresis, PCR testing, or MIC assays, which were performed by microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute) standard.

5.5.2 Results

[0203] As shown in Table 21, below, some strains that harbor intracellular beta-lactamase have relatively low nitrocefin activity (<=4 within 10 hrs on Phoenix panel) in the absence of lysis reagents. The addition of lysis reagents in Phoenix wells significantly improved the nitrocefin hydrolysis rate by those cells. Some strains exhibited relatively high nitrocefin activity (>4 within 10 hrs on Phoenix panel) in the absence of lysis reagents. With respect to those strains, the presence of lysis reagents did not result in a change or only resulted in a slight improvement in the nitrocefin activity. The lysis reagents did not inhibit the nitrofecin activity of the beta-lactamases expressed by the bacterial strains. Neither lysozyme nor EDTA, alone or in combination, hydrolyzed nitrocefin. Without being bound by any theory, it is believed that the improvement in the nitrocefin hydrolysis rate is primarily because more beta-lactamase is released from periplasmic space, and thus, more beta-lactamase is accessible to its substrate, nitrocefin.

TABLE-US-00021 TABLE 21 Presence of intracellular NCF activity Sample Organism beta-lactamase NCF activity with lysis reagent 1 E. coli + 1.9 7.2 2 E. coli + 2.1 7.7 3 E. coli + 2.3 8.1 4 E. coli + 1.9 6.8 5 E. coli + 8.2 9.1 6 E. coli + 7.2 8.1 7 E. coli + 6.8 8.4 8 E. coli 1.7 1.8 9 Klebsiella + 1.3 4.6 pneumoniae 10 Klebsiella + 3.3 6.3 pneumoniae 11 Klebsiella + 3.9 6.1 pneumoniae 12 Klebsiella + 2.6 5.6 pneumoniae 13 Klebsiella + 6.7 6.8 pneumoniae 14 Klebsiella + 7.5 8.8 pneumoniae 15 Klebsiella + 9.1 9.5 pneumoniae 16 Klebsiella 1.2 1.8 pneumoniae NCF activity, the value shown is the maximum nitrocefin hydrolysis rate within 10 hrs following Phoenix panel loading into the instrument. The unit of activity is calculated based on Phoenix signals.

Equivalents

[0204] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0205] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

[0206] Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.