EP3990924A1 - Detection of pathogenic bacteria by chemically functionalized cellulose - Google Patents
Detection of pathogenic bacteria by chemically functionalized celluloseInfo
- Publication number
- EP3990924A1 EP3990924A1 EP20734207.2A EP20734207A EP3990924A1 EP 3990924 A1 EP3990924 A1 EP 3990924A1 EP 20734207 A EP20734207 A EP 20734207A EP 3990924 A1 EP3990924 A1 EP 3990924A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lectin
- adhesin
- type
- pathogenic bacteria
- type adhesin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- 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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
- G01N2400/26—Cellulose
Definitions
- the present invention concerns the rapid and selective detection in a biological environment of pathogenic bacteria expressing a lectin-type adhesin, in particular the phenotype of adherent and invasive E. coli (AIEC), using a cellulose support functionalized with a ligand of a lectin-type adhesin.
- a lectin-type adhesin in particular the phenotype of adherent and invasive E. coli (AIEC)
- the present invention finds notably an application in the diagnosis of infections promoted by Fim-H-expressing pathogenic bacteria, for example in the context of urinary tract infections, osteoarticular infections and inflammatory bowel diseases such as Crohn’s disease.
- references in square brackets ([ ]) refer to the list of references at the end of the text.
- an anti-adhesive strategy consisting of preventing or blocking the adherence of bacteria to host cells through specific inhibitors, could be applied to address the infection in its first stage.
- Pathogenic bacteria expressing a lectin-type adhesin in particular certain E.coli strains expressing the FimH lectin at the top of their type 1 pili (or fimbriae), are involved in urinary tract infections (cystitis), osteoarticular infections (when placing prostheses), colorectal cancer and certain bowel diseases such as inflammatory bowel diseases (i.e. Crohn's disease). In this last pathology, adherent and invasive E.
- FimH lectin abnormally colonize the ileal mucosa of 25-60% of Crohn’s disease patients and are within an ecological community of hundreds of symbiotic microorganisms but, when present, are suspected to play a major role in exacerbated inflammation in some patient cohorts (Carvalho et al. , J. Exp. Med., 206: 2179-2189, 2009; Palmela et al., Gut, 67(3): 574-587, 2018) [1 , 2] The FimH adhesin expressed by pathogenic E.
- coli strains is able to recognize mannosides expressed on the surface of intestinal epithelial cells on CEACAM6 (CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 6) glycoprotein, and is a particularly affine lectin for a-D-mannose.
- CEACAM6 CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 6
- the FimH adhesin has been extensively studied as a target to disrupt the bacterial attachment to the host cells (Hartmann and Lindhorst, Eur. J. Org. Chem., 2011 (20-21 ): 3583-3609, 2011 ) [3] Results were obtained in the context of urinary tract infections (UTI), a prevalent infection generally mediated by the attachment of uropathogenic E.
- UTI urinary tract infections
- this type of strategy for proper stratification of patients harbouring E. coli pathovars before treatment, it would be essential to identify in advance the patients with positive AIEC to personalize such anti-adhesive treatment.
- E. coli can be performed by global analysis of the intestinal bacterial metagenome.
- not all strains of E. coli are classified as pathogenic bacteria, and a large number of healthy subjects are carriers of non-pathogenic commensal E. coli strains.
- no specific biomarkers are currently effective to distinguish certain pathogenic E. coli, such as AIEC from commensal E. coli in the complex gut microbiota.
- Their selective detection is thus carried out by phenotypic analyses of E. coli isolated on selective media on intestinal cell lines in vitro, but this is a time-consuming method which is difficult to apply in clinical practice.
- the Inventors had the idea of trapping pathogenic bacteria expressing a lectin-type adhesin to a cellulosic framework by grafting ligand of lectin-type adhesin onto cellulose paper. It is then sufficient to use a revelation system (e.g. western-blot type using a chromogenic substrate, grinding the paper and extracting the trapped bacteria before spreading in a culture medium, metabolic detection of an enzyme specifically expressed by the trapped bacteria, etc... ) to show that bacteria have adhered to the paper.
- a revelation system e.g. western-blot type using a chromogenic substrate, grinding the paper and extracting the trapped bacteria before spreading in a culture medium, metabolic detection of an enzyme specifically expressed by the trapped bacteria, etc...
- Cellulose fibers are cheap and biocompatible materials composed of b1 -4 linked D-glucose units, provide a heterogeneous support unable to pass through the intestinal barriers thus avoiding possible side effects during interaction with other mannose-binding proteins, and are stable against enzymatic hydrolysis insofar as humans do not possess any enzyme capable of breaking the b-1 ,4- linked glucosyl units.
- modified cellulose papers or nanofibers so that they can specifically trap and accumulate pathogenic bacteria expressing a lectin-type adhesin, in particular AIEC pathogenic bacteria, and therefore allow their specific detection, even in biological fluids containing many bacterial species (e.g . urine, homogenised feces and intestinal tissues).
- the bacterial adhesion of AIEC strains in the intestine being mainly promoted by a lectin-type adhesin recognizing mannose (FimH), the Inventors have grafted synthetic mannosides (HMan) with high affinity for FimH on cellulose papers.
- HMan synthetic mannosides
- the sugar units that make up cellulose have been chemically activated to allow the grafting of functionalized ligands.
- the first in vitro tests performed by the Inventors showed that the adhesion of AIECs to modified cellulose (MC) was dependent on the nature of the ligand grafted.
- the ligands with the highest affinity make it possible, once grafted onto cellulose, to capture more effectively the AIEC.
- MC has also been shown to be superior to bare cellulose (C) in the retention of AIECs.
- the Inventors showed that AIEC mutant bacteria deleted from the fimH gene as well as non-pathogenic and low adhesive E. coli bacteria (strain K12 C600) were very poorly retained on cellulose.
- Paper functionalization with other types of sugars may be extended to other life- threatening pathogen expressing specific adhesins such as Candida albicans, Influenza, Candida glabrata, Burkholderia species, Pseudomonas aeruginosa or Vibrio cholerae.
- specific adhesins such as Candida albicans, Influenza, Candida glabrata, Burkholderia species, Pseudomonas aeruginosa or Vibrio cholerae.
- An object of the present invention is therefore a method for detecting in vitro pathogenic bacteria expressing a lectin-type adhesin in a subject biological sample, comprising:
- the lectin-type adhesin is FimH adhesin.
- the ligand of the lectin-type adhesin is a mannose derivative, preferably a heptylmannoside (HMan), thiazolylmannoside,phenylmannoside, biphenylmannoside, etc... , or a derivative thereof.
- the subject biological sample to be tested is chosen from the group consisting of bacterial culture, feces, urine, intestinal tissues.
- the pathogenic bacteria expressing the lectin-type adhesin are Adherent Invasive E. coli (AIECs).
- Another object of the present invention is a method for identifying a host having a pathology caused by pathogenic bacteria expressing a lectin-type adhesin and mediated by interactions between lectin-type adhesin and host cell surface glycans, comprising using the method for detecting of any one of claims 1 to 5.
- the pathology is chosen in the group consisting of:
- an inflammatory bowel disease such as Crohn’s disease, ulcerative colitis, acute diarrhea
- the lectin-type adhesin is FimH adhesin.
- the host is a human, a domestic mammal (such as dog or cat), or cattle such as pig, poultries (or swine).
- Another object of the present invention is also a kit for detecting pathogenic bacteria expressing a lectin-type adhesin in a subject biological sample, comprising at least a cellulose support functionalized with a ligand to lectin-type adhesin, and at least a mean for detecting trapped pathogenic bacteria expressing the lectin-type adhesin.
- a mean for detecting trapped pathogenic bacteria expressing the lectin-type adhesin is chosen from colorimetric, fluorescence, luminescence, and radioactivity detection.
- a mean for detecting trapped pathogenic bacteria expressing the lectin-type adhesin is colorimetric or fluorescence or luminescence detection.
- the pathogenic bacteria expressing a lectin-type adhesin are Adherent Invasive E. coli (AIECs).
- AIECs Adherent Invasive E. coli
- the lectin-type adhesin is FimFI adhesin.
- Another object of the present invention is also a cellulose support functionalized with a ligand of lectin-type adhesin for use in the treatment or prevention of a pathology caused by pathogenic bacteria expressing a lectin-type adhesin and mediated by interactions between lectin-type adhesin and host cell surface glycans.
- the pathology is chosen in the group consisting of:
- the lectin-type adhesin is FimH adhesin.
- FIG. 1 represents a SEM analysis of HMan-CP showing that the structural integrity of the cellulose fibers is conserved after the chemical grafting.
- mM micromolar
- FIG. 4 represents the decreases of AIEC bacterial loads (logarithmic scale) in feces on day 1 (D1 ) and day 2 (D2) post-infection of CEABAC10 transgenic mice.
- HMan-CN was orally administered 2 h and 24 hours after the bacterial challenge of mice (10 mg/kg, on a mannose basis unit).
- FIG. 5 represents the percentages of bacteria trapped by N3-CP or HMan-CP. from bacterial suspensions prepared at 10 8 bacteria/mL from an overnight culture in Luria Bertani broth medium.
- AIEC LF82-AfimH is a non-piliated mutant, unable to bind to intestinal epithelial cells.
- FIG. 6 represents the percentages of fecal AIEC LF82 bacteria trapped by N3-CP or HMan-CP.
- Fecal samples from LF82-infected CEABAC10 mice were homogenised, centrifuged and the supernatants were incubated with N3-CP or HMan-CP. The percentages of trapped bacteria are defined according to the total number of AIEC bacteria presents in the biological samples.
- FIG. 7 represents the number of AIEC LF82 bacteria from intestinal tissues (in colony forming unit/cm 2 of cellulose) trapped by N3-CP or HMan-CP. Intestinal tissues were homogenised, centrifuged and the supernatants were incubated with N3-CP or HMan-CP. AIEC bacteria bound to the cellulose were quantified on antibiotic selective cultured media. The percentages of trapped bacteria are defined according to the total number of AIEC bacteria presents in the biological samples (indicated on the top of the bars).
- EXAMPLE 1 MANNOSE-FUNCTIONALIZED CELLULOSE FOR THE BINDING AND SPECIFIC DETECTION OF AIEC
- N 3 -CP fibers (disc of 5 mm diameter) were prepared as well as HMan-CP (disc of 5 mm diameter) by chemical functionalization of cellulose paper following four synthesis steps.
- High-resolution mass spectra were recorded with a Thermofisher hybrid LTQ-orbitrap spectrometer (ESI +) and a Bruker Autoflex III SmartBeam spectrometer (MALDI).
- FT-IR spectra were recorded on a Bruker Tensor 27 spectrometer with ATR technic and KBr tablet method. Elemental analyses were performed on a Thermo Fisher Scientific Flash 2000 CHNS organic elemental analyzer. Centrifugations were performed on a Sigma 3-16 Centrifuge. Dialysis were performed with Spectra/Por dialysis membrane MWCO 3500 K. Scanning electron microscopy (SEM) images were recorded with a JEOL 7600 F.
- Glucosyl pentaacetate D 200 mg, 0.51 mmol
- 8-oxaundec-10-yn-1 -ol 131 mg, 0.77 mmol
- silver trifluoroacetate 169 mg, 0.77 mmol
- a solution of SnCU 1 M in dichloromethane (1.53 ml_) was added and the mixture was stirred at room temperature for 3 h under argon atmosphere.
- the solution was diluted with 10 mL of NaHCC (aq) sat. and 10 mL of dichloromethane and the mixture was stirred for 15 min.
- N 3 -CN N 3 -CN.
- the primary hydroxyl groups of the glucose units from cellulose nanofibers (CN) are activated with a tosyl group according to method 1 or 2.
- Method 1 To a mixture of CN (178 mg, 1 mmol U/Glc) in pyridine (10 ml_), p-toluenesulfonyl chloride (1 .144 g, 6 mmol) was added and the mixture was stirred for 40 hours at room temperature under argon atmosphere. The mixture is filtered through Millipore paper and the CN was washed with ethyl actetate and with DCM to give TsO-CN (270 mg, grafting ratio of 50%) as a white solid.
- Method 2 To a mixture of CN (178 mg, 1 mmol U/Glc) in pyridine (10 ml_), tosyl chloride (1 .144 g, 6 mmol) was added and the mixture was stirred for 40 hours at room temperature under argon atmosphere. The mixture is centrifugated for 20 minutes at 40,000 rotations per minute. The supernatant was removed and three other cycles of centrifugation were conducted in DMF. TsO-CN was directly engaged in the next step without drying.
- N3-CN was suspended in water and heated at 80°C under ultrasounds for 20 min. after filtration through Millipore, N 3 -CN was rinsed with acetone and dichloromethane.
- Man-CN was obtained in quantitative yield (IR analysis) by CuAAC of N 3 -CN (19 mg, 0.031 mmol of N3) and propargyl-a-D-mannoside 1 (20 mg, 0.092 mmol) following the general procedure. Elemental analysis: C: 40.23%, FI: 5.44%, N: 5.49%, S: 0.00%; Infra-red analysis (KBr tablet method) cm 1 : 3385 (v(OH)), 2921 (v(C-H)), 1059 (v(C-O-C)). Substitution degree (SD) 20-30%.
- HMan-CN was obtained in quantitative yield (FT-IR) by CuAAC of N 3 -CN (25 mg, 0.040 mmol of N 3 ) and 2 (40 mg, 0.119 mmol) following the general procedure. Elemental analysis: C: 41 .31 %, H: 5.88%, N: 4.34%, S: 0.00%; IR analysis (KBr tablet method) cm 1 : 3396 (v(OH)), 2925 (v(C-H)), 1059 (v(C-O-C)). SD -30%
- HSMan-CN was obtained in quantitative yield (IR analysis) by CuAAC of N 3 -CN (42 mg, 0.067 mmol of N 3 ) and 3 (70 mg, 0.200 mmol) following the general procedure. Elemental analysis: C: 42.21 %, H: 6.08%, N: 4.34%, S: 2.57%; Infra red analysis (KBr tablet method) cm 1 : 3384 (v(OH)), 2924 (v(C-H)), 1059 (v(C- O-C)). SD -30%
- HGIc-CN was obtained in quantitative yield (FT-IR) by CuAAC of N 3 -CN (40 mg, 0.063 mmol of N 3 ) and 4 (63 mg, 0.189 mmol) following the general procedure. Elemental analysis: C: 45.18%, FI: 6.33%, N: 4.80%, S: 0.00%; Infra-red analysis (KBr tablet method) cm 1 : 3377 (v(OH)), 2924 (v(C-H)), 1059 (v(C-O-C)). SD -30%.
- the CP are engaged in tosylation and nitrogenization reactions without being dried (to avoid the re-forming of the hydrogen-bonds network) on an orbital agitator to preserve the CP structure.
- a piece of CP (145 mg, 0.81 mmol Glc) was immersed in pyridine (10 ml_) and treated with p-toluenesulfonyl chloride (464 mg, 2.44 mmol). The mixture was shaken for 20 h at 40°C on an orbital agitator. The paper was sonicated three times with 20 ml_ of DMF and stored in DMF for the next step. For analytical purposes only, Ts-CP was isolated and dried under vacuum. Ts-CP is stored in DMF after three successive washings to remove pyridine and unreacted p- toluenesulfonyl chloride.
- Elemental analysis: C: 46.11 %, H: 4.99%, N: 0.13%, S: 6.18%; IR analysis (ATR) cm 1 : 3377 (v(OH)), 2924 (v(C-H)), 1542 (v(C C)), 1363 (vas(S0 2 )), 1177 (vs(S0 2 )), 1059 (v(C-O-C)).
- Ts-CP (0.81 mmol Glc) was immersed in DMF (10 ml_) and treated with NaN 3 (526 mg, 8.10 mmol). The resulting mixture was shaken for 40 h at 60 °C on an orbital agitator.
- the N 3 -CP paper was sonicated with 20 ml_ of H 2 0, acetone, EtOH and DCM and dried under vacuum. Elemental analysis: C: 41.25%, H: 4.86%, N: 6.65%, S: 0.98%; IR analysis (ATR) cm 1 : 3377 (v(OH)), 2924 (v(C-H)), 2108 (v as (N 3 )), 1059 (v(C-O-C)). SD -30-40%.
- E. coli strain LF82 isolated from a chronic ileal lesion of Crohn’s disease patient was used as the AIEC reference strain expressing the mannose-binding FimH adhesin. Bacteria were grown overnight at 37 °C in Luria-Bertani (LB) broth medium.
- the human intestinal cell line T84 purchased from American Type Culture Collection (ATCC, CCL-248), was maintained in an atmosphere containing 5% CO2 at 37°C in the culture medium recommended by ATCC. T84 cells were seeded in 48-well tissue culture plates at a density of 1.5 x 10 5 cells/well and incubated at 37 °C for 48 h.
- Ns-CN, HMan-CN, HSMan-CN, HGIc-CN, Man-CN and HMan were first incubated at room temperature with AIEC LF82 bacteria for 1 h in DMEM/F12 (50/50) medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) at final concentrations of 100, 10, 1 , or 0.1 mM. Mixtures were then added to the T84 monolayers for 3h at a multiplicity of 10 bacteria/cell, at 37°C, in an atmosphere containing 5% CO2 .
- FCS heat-inactivated fetal calf serum
- N 3 -CN was a poor binder of bacteria because no significant inhibition of adhesion was observed at the high concentration of 100pM.
- HMan- CN or HSMan-CN showed a strong inhibition capacity with less than 5% of bacteria still adhered to the T84 cells at the highest concentration of 100pM.
- the dose-dependent inhibition profile was very similar to the one observed with free HMan meaning that no loss of inhibitory capacity is observed when the HMan or HSMan ligands are grafted on the cellulosic framework.
- HGIc-CN the glucose analog of HMan-CN
- HGIc-CN the glucose analog of HMan-CN
- Glucosides are not recognized by FimH and indeed, HGIc-CN at 100 pM was totally ineffective in preventing AIEC LF82 binding ( Figure 3) further highlighting the extraordinarily control of FimH in the binding process.
- HMan-CN was studied in vivo to evaluate it potency to capture AIEC bacteria in the complex gut microbiota.
- the non-toxic and biocompatible CN is a particularly suited scaffold for developing a potential treatment against Crohn’s disease.
- the high molecular weight of HMan-CN should confine the anti adhesive in the gut, the locus of infection, and preclude its systemic dissemination, lowering the risk of potential side effects.
- the transgenic CEABAC10 mouse model was selected to mimic Crohn’s disease susceptibility to be colonized by AIEC bacteria. Indeed CEABAC10 mice express the highly mannosylated CEACAM6 glycoprotein which is overexpressed at the ileal mucosa of patient with Crohn’s disease. The mannose residues exhibited by CEACAM6 favor the AIEC attachment to the intestinal mucosa.
- Figure 4 represents the fold decreases in AIEC bacterial colonization in feces on day 1 (D1 ) and day 2 (D2), relative to the colonization level on day 1 (D1 ) post-infection of CEABAC10 transgenic mice.
- the results are expressed in box and whiskers (Min to Max) ( ** p ⁇ 0.01 , Mann-Whitney test, relative to LF82- infected mice without any treatment (LF82).
- the results show that the bacterial clearance was more effective in the HMan-CN group compared to the LF82 group with 9,63 x 10 5 vs 3,61 x 10 6 bacteria/g feces at D2 post-infection, despite a higher bacterial level on day 1 for the HMan-CN+LF82 group.
- HMan-CP or N 3 -CP fibers (disc of 6 mm diameter) were pre-incubated in phosphate buffered saline (PBS) at room temperature for 15 minutes with gentle shaking. Fibers were then incubated with bacterial suspensions calibrated at 10 8 bacteria/mL in PBS for one hour with gentle shaking.
- AIEC LF82 reference strain, the non-piliated LF82-AfimH mutant and the laboratory strain Escherichia coli K12 C600 were tested for their abilities to bind HMan-CP or N 3 -CP fibers. Fibers were washed 6 times in PBS and homogenized (Ultra Turrax) in 1 mL of PBS. Homogenized fibers were processed to count captured bacteria.
- HMan-CP Binding ability of bacteria by HMan-CP was assessed on feces and intestinal tissues of mice previously infected with AIEC LF82 bacteria. Briefly, human CEACAM6-expressing mice (CEABAC10 transgenic model) were pretreated for 3 days with streptomycin sulfate (2,5 g/L) and DSS (0,5%) in the drinking water then, they were orally challenged with 3.10 9 bacteria.
- feces were collected and homogenized in PBS at a concentration of 50 mg/ml (Ultra Turrax). Feces suspensions were centrifuged (400 rpm, 5 minutes, room temperature) and the supernatants were collected. HMan-CP or N 3 -CP fibers were incubated with the feces supernatants for one hour at room temperature with gentle shaking. Bound bacteria to the fibers were count as described above.
- the percentages of LF82 bacteria and of enterobacteria captured by the fibers were determined accordingly to the total number of bacteria per gram of feces (determining by plating onto LB agar containing ampicillin (100 pg/ml) and erythromycin (20 pg/ml) to count LF82 bacteria and on Drigalski agar to count enterobacteria) in the initial sample.
- mice were anesthetized with isofluorane and then euthanized by cervical dislocation. Colon was collected, longitudinally opened, washed in PBS and divided into 3 parts (proximal, middle and distal colon). Tissue samples were weighted and homogeneized in PBS at 100 mg/ml.
- Total LF82 bacteria and enterobacteria were quantified in the supernatants after centrifugation as described previously. In parallel, bacteria trapped into the fibers were quantified (in CFU/cm 2 of fibers) and the percentages of bacteria binding the HMan-CP fibers were determined and compared to percentages of bacteria binding the N3- CP fibers.
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Abstract
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EP19305866.6A EP3757572A1 (en) | 2019-06-27 | 2019-06-27 | Detection of pathogenic bacteria by chemically functionalized cellulose |
PCT/EP2020/067795 WO2020260445A1 (en) | 2019-06-27 | 2020-06-25 | Detection of pathogenic bacteria by chemically functionalized cellulose |
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EP3990924A1 true EP3990924A1 (en) | 2022-05-04 |
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EP19305866.6A Withdrawn EP3757572A1 (en) | 2019-06-27 | 2019-06-27 | Detection of pathogenic bacteria by chemically functionalized cellulose |
EP20734207.2A Withdrawn EP3990924A1 (en) | 2019-06-27 | 2020-06-25 | Detection of pathogenic bacteria by chemically functionalized cellulose |
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