Patent Application
20210373016
The present invention is related to a method for determining Escherichia coli from a sample in particular to a method comprising treating the sample with a reagent comprising a lanthanide(III) ion, transition metal ion and M13 phages.
The diagnosis of urinary tract infection (UTI) is based on the clinical symptoms and determination of a causative pathogen. The golden standard for detection and identification of urinary tract pathogen is culture, which enables estimation of the amount of known uropathogenic bacteria in urine. The most common UTI causing pathogen is Escherichia coli. Growth of 105 colony forming units (cfu)/mL is the most commonly used cutoff for significant bacteriuria, but depending on the isolated bacteria, patient's symptoms and sampling technique it may be significantly lower. Even with modern media and techniques, urinary cultures create a considerable workload for hospital laboratories. UTIs are very common and therefore also lead to substantial amount of antibiotic prescriptions, some of which could be avoided by faster diagnosis.
Rapid urine tests for UTIs have been developed with aim to reduce laboratory workload. Many of them are based on detecting chemical changes in urine. Leukocyte esterase and nitrite dipstick tests are the most commonly used. Leukocyte esterase leaks from white blood cells to the urine and nitrite is produced mainly from gram-negative bacteria. These two parameters are relatively good indicators when used at the same time. It is recommended that dipstick test results should be considered positive if either parameter was positive. However, current diagnostic guidelines state that nitrite and leukocyte esterase tests are unable to eliminate the possibility of bacterial infection.
Flow cytometry is a convenient method for separating particles in different liquids. In case of urine samples, it can detect bacteria and different blood cell types, most importantly leucocytes. Cytometry alone or used with results of dipsticks does not, however, give accurate prediction of UTI with lower cut-offs (104 or even 103 CFU/mL) which are applied on primary uropathogens, especially E. coli, and certain symptomatic patient groups, the percentage of false-negatives may become high with this method, especially if only bacterial counts are considered. However, with locally validated cutoffs flow cytometry can be used to rule out UTI and reduce the number of samples to be cultivated by up to 50% and thus workload in the lab, as well as shorten the time to a negative result.
Kulpakko et al. [Anal. Biochem. 470 (2015) pp. 1-6] have developed a rapid assay for detecting E. coli which is based on lytic phage and bacterial lysis which can be detected via environmentally sensitive lanthanide label. The sensitive label interacts with released molecules from lysed cells. However, specific infection and lysis is a time-consuming process and phage has to be specifically lytic for each bacterial species.
Thus, there is still need for further rapid methods for E. coli detection.
The present invention is based on the observation that E. coli can be determined from a sample simply by admixing a first part of the sample with a reagent comprising a lanthanide(III) ion, a transition metal ion, and a M13 phage, and by admixing a second part of the sample with the lanthanide(III) ion, the transition metal ion and wild type M13 phage followed by detecting signal derived from the lanthanide(III) ions from the two admixtures with time-gated luminescence measurement. When the M13 phage has higher affinity towards the transition metal ion and E coli than the wild type M13 phage, difference between luminescence signals obtained from the two admixtures is an indication of the presence of E. coli in the sample.
According to one aspect the present invention concerns a method for determining E. coli in a sample employing at least a first admixture and a first reference admixture, the method comprising following steps:
According to another aspect the present invention concerns a kit for determining E. coli from a sample, the kit comprising,
Further aspects of the present invention are disclosed in the dependent claims.
Exemplifying and non-limiting embodiments of the invention, both as to constructions and to methods of operation, together with additional objects and advantages thereof, are best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.
The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of unrecited features. The features recited in the accompanied depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of “a” or “an”, i.e. a singular form, throughout this document does not exclude a plurality.
The present invention concerns a method for determining E. coli from a sample employing at least a first admixture and a first reference admixture. The method comprises following steps:
a) for providing the first admixture, admixing a first part of the sample with
b) for providing the first reference admixture, admixing a second part of the sample with
c) detecting at predetermined time point signal derived from the lanthanide(III) ion of the first admixture and signal derived from the lanthanide(III) ion of the first reference admixture with time-gated luminescence measurement,
d) comparing the signal derived from the lanthanide(III) ion of the first admixture to the signal derived from the lanthanide(III) ion of the first reference admixture, and
e) determining the E. coli in the sample based on the comparing.
According to the method, affinity of the M13 phage towards the transition metal ion and E. coli is higher than the affinity of the wild type M13 phage towards the transition metal ion and E. coli.
As defined herein a wild type M13 phage (M13 wt) is a phage that does not interact specifically with transition metal ions, in particular with copper ions and E coli. Binding constant of the wild type M13 phage towards Cu2+ ions is 106 pfu or less, and towards E. coli 105 pfu or less when E. coli concentration is 104 cfu.
Wild type M13 phage is any random phage taken from a library that has not undergone any selection phases of the biopanning procedure. Wild type M13 phage is supposed to contain random DNA-sequence. Its binding to copper and E. coli clearly less than the binding constant of E. coli/transition metal-specific phage. Exemplary differences in the binding constants of wild type M13 phage and biopanned M13 phage towards E. coli and copper(II) ions are shown in table 1.
E. coli
The M13 phages suitable for the method of the present invention can be selected by biopanning. The affinities of the biopanned M13 phages towards E. coli and transition metal ions is preferably 107 pfu/mL, more preferably at least 108 pfu/mL and most preferably at least 109 pfu/mL. According to an exemplary embodiment affinity of the M13 phage towards transition metal ion and E coli is minimum titer of 1.5×106 plaque-forming units/mL when solid transition metal is used and 3×106 plaque-forming units/mL for E. coli.
According to a particular embodiment the transition metal is copper. The affinities of the biopanned M13 phages towards copper ion and E. coli is preferably 107 pfu/mL more preferably at least 108 pfu/mL most preferably at least 109 pfu/mL. According to an exemplary embodiment affinity of the M13 phage towards copper ion and E coli is minimum titer of 1.5×106 plaque-forming units/mL when solid copper is used and 3×106 plaque-forming units/mL for E. coli.
Accordingly, affinity of the M13 phage towards the transition metal ion, in particular copper ion, and E. coli is at least 100, preferably at least 1000, most preferably at least 10 000 times higher than the affinity of the wild type M13 phage towards the transition metal ion and E coli.
It is known that transition metal ions are able to quench luminescence of reagents comprising lanthanide(III) ion, such as lanthanide(III) chelates. This phenomenon was exploited in the present method. Exemplary transition metal ions suitable for the method are Ni2+, Cu2+, Hg2+, Pb2+, Ag+, Cr3+ and Fe3+. A particular transition metal ion is Cu2+. The transition metal ions are introduced to the method as salts. An exemplary salt is transition metal halide, such as chloride. An exemplary transition metal salt is chloride salt such as CuCl2.
The method includes a reagent comprising lanthanide(III) ion, such as a lanthanide(III) chelate. The lanthanide is preferably selected from europium, terbium, samarium and dysprosium, preferably from europium and terbium, most preferably europium. A preferable lanthanide(III) chelate is a luminescent lanthanide(III) chelate. Exemplary lanthanide(III) chelates suitable for the method are Eu:TTA:TOPO and Eu:NTA:TOPO. These chelates can be prepared by admixing EuCl3, NTA and TOPO, and EuCl3, TTA and TOPO, respectively. An exemplary molar ratio is 5:3:3 for Eu, NTA/TTA and TOPO respectively.
Another lanthanide(III) chelate suitable for the method is a terpyridine-Eu of formula (I)
The method of the present invention is suitable for detecting E. coli from a sample, in particular from urine samples. The detection is based on reagent comprising a lanthanide(III) ion in particular to a lanthanide(III) chelate that is sensitive to chemical environment and particularly to transition metals that are able to quench the luminescence of the chelate. The M13 phage in turn interacts both with the metal and the target bacteria. The phage acts as a modulating factor to the achieved time-resolved fluorescence signal. A particular metal is copper as it is well known to quench the fluorescence of lanthanide complexes like Eu3+:TTA:TOPO and Eu3+:NTA:TOPO.
The principle of the present invention is shown in
In order to confirm that M13 phages bind to copper, they were tested in series of different copper chloride concentrations. A comparison was made between wild-type M13 phage and copper/E. coli selected M13 phage in the presence of Eu3+:TTA:TOPO (
The relative fluorescence signal was higher with copper selective phages M13 than wild type M13 phages. This indicates that under these conditions, copper selective M13 phages protect the lanthanide(III) chelate from quenching. Wild type M13 phage was used throughout the experiments as a control because it was purified with exactly the same procedure as copper/E. coli B specific M13 phage. This reduces the possibility that the assay measures irrelevant parameters. In
The assay cross-reactivity was evaluated by comparing E. coli and S. aureus. As shown in
According to an embodiment the method utilizes plurality of admixtures and plurality of reference admixtures. According to this embodiment, each admixture and each reference admixture comprise the reagent comprising lanthanide(III) ion such as Eu:TTA:TOPO, and a transient metal ion, such as Cu(II) ion. Each admixture comprises copper/E. coli B specific M13 phage, and each reference admixture comprises a wild-type M13 phage.
According to a preferable embodiment, each admixture and each reference admixture comprise also one or more modulating agents. A suitable modulating agent has affinity towards one or more of
The modulating agent changes the chemical environment of the admixtures and the reference admixtures and enhances sensitivity and selectivity of the method. Exemplary modulating agent are listed in table 2. Also, exemplary modulator agent concentrations suitable for the method are given.
According to an exemplary embodiment, the method comprises four admixtures and four reference admixtures, namely first admixture, first reference admixture, second admixture, second reference admixture, third admixture, third reference admixture, fourth admixture and fourth reference admixture. All admixtures and all reference admixtures comprise the same reagent comprising lanthanide(III) ion such as Eu:NTA:TOPO, and the same transition metal ion such as Cu(II). The admixtures and the reference admixtures comprise the copper/E. coli specific M13 phage and wild-type M13 phage, respectively. The admixtures and the reference admixtures comprise also one or more modulating agents. For example, the first admixture and the first reference admixture comprise modulating agent A, the second admixture and the second reference admixture comprise modulating agent B, etc. It is also possible that one or more of the admixtures and reference admixtures comprise plurality of modulating agents. For example, the first admixture and the first admixture comprise modulating agents A, C, and D, and the second admixture and the second reference admixture comprise modulating agent B, etc. The plurality of reference admixtures produces a fingerprint of the sample in the presence of wild-type M13 phage and the plurality of admixtures produces a fingerprint of the sample in the presence of copper/E. coli specific M13 phage. E. coli is then determined by comparing TRF signals obtained from these two fingerprints.
Since the response differed between these chemistries (increase/decrease/magnitude) it can be assumed that each of the three modulator agents reveal varying properties of the sample-chelate interaction. Using the three different chemical environments resulted improved the classification precision.
The KNN method was taught by using artificial random noise contaminated averages for each of the categories. The algorithm classified E. coli from sample data with the output of 90% sensitivity and specificity. This is a competitive result when compared with other rapid screening methods for urine. Comparing the categories in
The experiment shown in
E. coli is by far the most prevalent causative agent in urinary tract infection. The assay sensitivity and specificity reached with the method of the present invention is at the same level as current flow cytometric and dipstick methods. The limit of detection was in the range of clinical level (<10 000 cfu/mL). The results could be obtained in 10 minutes from the time reaction was started. Urine samples were not treated before screening them. Nonetheless, this didn't cause interference to the method and clear cross-reactivity to other bacterial species was not seen.
According to another embodiment the present invention concerns a kit for determining E. coli from a sample such as urine, the kit comprising
The binding constant of the wild type M13 phage towards Cu2+ ions is 106 pfu or less, and towards E. coli 105 pfu or less when E. coli concentration is 104 cfu.
According to a preferable embodiment affinity of the M13 phage towards the transition metal ion and E. coli is at least 100 times, preferably at least 1000 times, most preferably at least 10 000 times higher than the affinity of the wild type M13 phage towards the transition metal ion and E. coli.
According to a preferable embodiment the binding constant of the M13 phage towards the transition metal ion is at least titer of 1.5·106 plaque-forming units/mL when solid transition metal is used, and binding constant of the M13 phage towards E. coli is at least titer of 3·106 plaque-forming units/mL, preferably at least titer of 2·108 plaque-forming units/mL.
According to a preferable embodiment the kit comprises also one or more modulating agents, wherein the one or more modulating agent have affinity towards one or more of
The reagent comprising lanthanide(III) ion is preferably a lanthanide(III) chelate preferably selected from Eu:TTA:TOPO, Eu:NTA:TOPO and terpyridine-Eu of formula (I).
The one of more modulating agents are preferably selected from the group consisting of diethyl malonate, 2,3-dichloro-5,6-dicyano-p-benzoquinone, triisopropylsilane, 1-bromonaphthalene, calmagite, chloranil, 1,10-phenanthroline monohydrate, toluidine blue O, crystal violet, murexide, 8-anilino-1-naphthalene sulfonic acid, benzoyl peroxide, creatine hydrate, 2-furoyl chloride, N-hydroxysuccinimide, N-bromosuccinimide, 2,4,6-tribromo-3-hydroxybenzoic acid, malachite green chloride, eosin B, and safarin O, preferably diethyl malonate, 2,3-dichloro-5,6-dicyano-p-benzoquinone, triisopropylsilane.
70 clinical samples were analyzed and cultured at Clinical Microbiology Laboratory of Turku University Central Hospital. Samples were collected in vacutainer Plus C&S boric acid sodium borate/formate tubes (Becton Dickinson). Clearly bloody or smear samples were not excluded from analysis. The urine samples were stored at 4° C. Ethical approval for using the patient samples was not required for the reason that the study was considered as a basic laboratory screening method development and no additional patient information was collected. Microorganisms identified from the samples are shown in table 3.
Escherichia coli
Klebsiella pneumoniae
Enterococcus faecalis
Citrobacter freundii
Proteus mirabilis
Citrobacter koseri
Pseudomonas aeruginosa
enterococcus (non-faecalis species)
Hafnia alvei
Streplococcus agalactiae
Pseudomonas putida
Staphylococcus saprophyticus
Staphylococcus hominis
Raoultella ornithinolytica
Yeast-Tryptone (YT) medium was made of mixing 16 g of tryptone, 10 g of yeast extract and 5 g of NaCl to one liter of MQ water. Diethyl malonate, Bovine Serum Albumin (BSA), Europium (III) chloride hexahydrate, 2,3-Dichloro-5,6-dicyano-p-benzoquinone and (TOPO) tri-n-octyl-phosphine oxide were purchased from Sigma-Aldrich.
Triisopropylsilane was purchased from Fluka, Buchs, Switzerland and (NTA) 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione (NTA) and 2-thenoyltrifluoroacetone (TTA) from Acros Organics. Dimethyl sulfoxide, analytical reagent grade (DMSO) was purchased from Thermo Fisher Scientific, MA, USA. The bacterial reference strains used in the development of the assay were: Staphylococcus aureus ATCC 25923 and E. coli strain B ATCC 11303 as a reference of wild type E. coli because it has no F plasmids that M13 phage requires for infection process.
The method of the present invention utilized a M13 phage which has higher affinity towards the transition metal ion and E coli than wild-type M13 phage. The overall schematics to produce a biosensing phage that is functioning in the method is described in
First phase of creating biosensing phage is to go over biopanning procedure with copper. Even slight phage affinity towards copper is observable with the assay deploying TRF instrument (
Second Affinity Screening Procedure for E. coli B
Enriched phages from previous screening were used for biopanning experiments against E. coli B. Purpose of this biopanning phase is to have affinity against target bacteria without losing too much already obtained affinity towards transition metal quencher. This will be tested with several independent clones obtained from the experiments.
The panning procedure was performed according to the following protocol: E. coli B cells were grown in tryptic soy broth (TSB; Sigma-Aldrich) medium at 37° C. The growth medium was centrifuged, and the pellet washed twice with 1 mL of 4° C. PBST (Phosphate Buffered Saline with Tween 20). The random peptide library (1.5×1011 plaque-forming units) was mixed with washed cells and incubated in 1% BSA (Bovine serum albumin) with the washed infectious E. coli B cells and left on ice for 1 h. After this phage bound cells were washed twice with 1 mL PBST and three times 1 mL PBS. Washed cells were mixed with 1 mL of phage propagating E. coli ER2738 (1.0×109 cells/mL) in 2×YT medium. Before mixing E. coli ER2738 cells were grown in intense shaking (250 rpm) at 37° C. The mixed culture of E. coli ER2738 and E. coli B was incubated for 30 min at 37° C. without shaking and following an incubation period of 30 min with gentle shaking (100 rpm). The enrichment of phages was made according to the manufacturer's protocol (NEB). After third affinity selection phages were ready for to be used in assays. Chosen phages had properties capable of binding copper with minimum titer of 1.5×106 plaque-forming units/mL, and capable of propagating normally in E. coli ER2738. Secondly, phages had properties capable of binding E. coli with minimum titer of 3×106-3×108 plaque-forming units/mL.
Wild type phage used has random 12-mer peptides fused to a minor coat protein (pill) of M13 phage. Basic structure of the wild type phage does not differ from the selected M13-copper-E. coli phage. Only difference is the order of amino acids in the minor coat protein and hence the order of DNA-sequence. The DNA-sequence for functioning biosensing assay can be various combinations of nucleotides.
Each chemical environment consisting a reagent comprising a lanthanide(III) ion, copper chloride and wild type M13 phage or copper/E. coli-specific M13 phage was added 4 μL chemical investigated. A result that enhances copper/E. coli-specific M13 phages signal more than 2-fold compared to situation where wild type M13 was present and was selected for final assay setup.
Tested copper chloride concentration (0-200 mM) in MQ was added in 100 μL volume to 96 well plate. Then each well was added 1012 pfu/mL M13 wild-type (wt-m13) phage or copper selected phage in volume of 10 μL. Finally, 4 μL of 0.1 mM europium chloride, 0.06 mM NTA and 0.06 mM TOPO was added to the microtiter wells. After 10 minutes of incubation, delayed luminescence emission intensities were measured in a 400 μs window after a 400 μs delay time using a Victor 2 multilabel counter (Wallac, Perkin Elmer Life and Analytical Sciences).
Assay for E. coli B and Comparison with S. aureus
A 96 well plate was filled with 100 μL of varying concentrations (0-106 cfu/mL) of E. coli in physiological saline and after this 8 μL of 20 μM copper chloride in MQ was added. Next each well was added 1013 pfu/ml wt-m13 phage or copper/E. coli B selected phage in volume of 10 μL. Finally, 4 μL of 0.1 mM europium chloride, 0.06 mM NTA and 0.06 mM TOPO was added to the microtiter wells. After 10 minutes of incubation, modulated delayed europium luminescence emission intensities were measured again in a 400 μs window after a 400 μs delay time using a Victor 2 multilabel counter. Comparison with S. aureus and E. coli was done with the same protocol but only one concentration of 1 μM copper chloride was used. Finally, 4 μL of 0.1 mM europium chloride, 0.06 mM NTA and 0.06 mM TOPO was added to the microtiter wells. After 10 minutes of incubation, luminescence emission intensities were measured in a 400 μs window after a 400 μs delay time using a Victor 2 multilabel counter.
A 96 well plate was filled with 100 μL of urine sample and right after this, 10 μl of 1013 pfu/ml M13-wt phages or copper/E. coli B selected M13 phages and 8 μL of 20 μM copper chloride in MQ was added. Next 4 μL one of three additional chemicals in DMSO were added (600 mM of diethyl malonate, 50 mM of 2,3-dichloro-5,6-dicyano-p-benzoquinone or 600 mM triisopropylsilane). Finally, 4 μl of 0.1 mM europium chloride, 0.06 mM NTA and 0.06 mM TOPO was added to the microtiter wells. After 10 minutes of incubation, luminescence emission intensities were measured in a 400 μs window after a 400 μs delay time using a Victor 2 multilabel counter.
The samples were analyzed in three chemical environments and with two different phage types: E. coli B/copper specific M13 phage and reference M13 phage. In the analysis the signal from time point 0 min was compared with that of 10 min. This was done to compensate the possible variations in the signal due to unrelated matrix variations. The aim was to obtain and evaluate the statistical difference in signal between E. coli and other samples. These include 14 other bacterial species representing both known uropathogens and normal microbiota of the urogenital area, and negative samples. High signal difference between specific and reference phage indicated positive result for E. coli in urine sample, whereas small signal difference indicated the negative result for E. coli. For screening assay, all selected three chemistries were used as a fingerprint of the sample. The used classification method was K-Nearest Neighbor (KNN), and the averages from each of the class, contaminated with random noise of amplitude equivalent to the noise in the real data, were used in teaching of the algorithm. The KNN analysis was performed with Molegro Data Modeler (Version 2.1), and all plotting and statistics with Prism 6.0 g.
The specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims. Lists and groups of examples provided in the description given above are not exhaustive unless otherwise explicitly stated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20185927 | Nov 2018 | FI | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2019/050742 | 10/17/2019 | WO | 00 |
