Real-time bacteria detection and identification device

Abstract

The present invention relates to a real-time bacteria detection and identification device (1) comprising a body, said body comprising: an inlet opening (2) and an outlet opening (3), configured for passage of a fluid; a central portion (4) located between the inlet opening (2) and the outlet opening (3), said central portion (4) being configured to allow the visualization of an at least one porous polymer-based matrix (5), said matrix (5) comprising at least one polymer, said polymer comprising at least one entrapped lysis compound, at least one immobilized bacteria attracting compound and at least one immobilized chromogenic substrate, wherein said immobilized chromogenic substrate of the polymer-based matrix (5) is configured to change its colour when contacted with a bacterium containing fluid.

Description

[0001] DESCRIPTION

[0002] "REAL-TIME BACTERIA DETECTION AND IDENTIFICATION DEVICE"

[0003] TECHNICAL FIELD

[0004] The present invention relates to real-time bacteria detection and identi fication device for use with biological and non- biological fluids . This device f inds application in environments prone to trigger infections by pathogenic microorganisms , in particular, hospitals and waste-water systems .

[0005] BACKGROUND OF THE INVENTION

[0006] Pathogenic microorganisms are present in diverse fluids of humans ' daily li fe , which is a reason for concern .

[0007] For instance , two current concerns include pathogens gaining access to the human urinary system, causing urinary tract infections , and accessing water natural reservoirs or even urban water distribution systems leading to water contamination and potential human exposure . Both are of signi ficant interest and are subj ects of ongoing research aimed at prevention and treatment , focusing on the detection and elimination of these pathogens .

[0008] In the hospital context , the environment is a reservoir of pathogens causing Healthcare-associated infections (HCAI s ) . These pathogens are able to persist from hours to months and their circulation can be favoured by healthcare workers and by the environment itself, including water sources. Water exposure in healthcare settings can place patients at risk for infection with water-related organisms and can potentially lead to outbreaks. Statistics say that 1 out of every 10 patients are victim of HCAIs (https: / / www.ncbi.nlm.nih.gov / books / NBK144030 / ) . These infections are responsible for worsening the health status of already debilitated patients, increasing the length of hospital stay, mortality and morbidity, the cost of treatment and specific care and dedication by health professionals.

[0009] The urinary tract infections (UTIs) associated with the use of indwelling urinary catheters (IUC) represent 30% of the total HCAIs, resulting in a cost of 84 Million Euros / Year in Portugal and nearly 300 Million Euros / Year in Spain.

[0010] Approximately 4 million people receive an IUC each year, and 5-20% of hospitalized patients, who receive an IUC, will be diagnosed with a UTI. Despite improvements in nursing care of catheterized patients and the redesign of catheters, UTI remains a problem in catheterized patients.

[0011] The detection of a urinary infection typically occurs when infection is already installed, as physical symptoms become evident in the patient. In some cases, this increases the likelihood of the patient experiencing health complications, which can ultimately progress to a systemic infection and, in many cases, result in death.

[0012] Further, the majority of diagnostic methods for urinary infections detection depend on sample collection and timeconsuming laboratorial procedures for microbiological analyses, by isolation and identification of the pathogenic bacteria. These methods are dependent on the bacteria growth in vitro and additional standard phenotypic and biochemical tests. Therefore, besides being costly, the results of microbiological analyses take from 24 up to 72 h, enabling the deterioration of the patients' clinical status. Although the evolution of diagnostic techniques has reached the molecular technological level, leading to a fast identification of infectious agent (s) in samples, accurate diagnosis is still dependent on the physician' s decision for sampling, sample transportation to the laboratory and the use of expensive equipment and reagents.

[0013] More recently, the development of faster and more selective detection methods of pathogenic bacteria are based on substrates able to provide a naked-eye visible sign for easy detection of urinary infections.

[0014] Santos et al. (2022) , "A review on urinary tract infections diagnostic methods: Laboratory-based and point-of-care approaches", J Pharm Biomed Anal 2022 Sep 20; 219 : 114889. doi: 10.1016 / j .jpba.2022.114889. Epub 2022 Jun 14. PMID: 35724611 , compares and highlights advantages and disadvantages of the traditional and currently most used detection methods, as well as the emerging point-of-care approaches and the relevant advances in on-site detection of pathogens' mechanisms, suitable to be adapted to UTT diagnosis. The main approaches now on use are paper-based tests, lateral flow assays, stack pad assays and hydrogels. However, all of them reveal at least a drawback in the use as continuous monitoring of bacteria in fluids. Bacteria have a remarkable ability to thrive and proliferate in water environments. Water, serving as a universal solvent, provides bacteria with essential nutrients and a conducive habitat for growth. Whether in lakes and rivers, or in human-made systems, like sewers, plumbing and water treatment facilities, bacteria find an array of niches to colonize. The availability of organic matter, moderate temperatures, and oxygen levels in various water sources further support bacterial replication. This proliferation poses significant concerns in contexts such as waterborne serious diseases, leading to death in some cases, and water quality management. Providing a device able to detect bacteria in water and prevent humans' contact and infection is pivotal for safeguarding public health. Contaminated water or water "silently" transporting pathogens or opportunistic bacteria can also harm the economy, causing losses in productivity, agriculture, and tourism. Water quality should be monitored more regularly to detect the presence of bacteria, and conventional methods for water analysis should be complemented with new and innovative techniques to ensure the safety and purity of our water resources (Ramirez-Castillo et al. 2015, "Waterborne pathogens: detection methods and challenges" Pathogens. 2015 Jun; 4 (2) : 307-334, online 2015 May 21, doi: 10.3390 / pathogens4020307 ) .

[0015] In the field of water systems, several methods for water microbiological analysis have been developed and are being commercialized, but even those consistently rely on sample collection and processing at laboratory, and in most cases are dependent on microorganisms' growth revealing specific metabolic activity . Also, versatile equipment as well as on-site detection methods have been developed and improved for the identification and quantification of a wide range of microorganisms (i.e., viruses, bacteria, protozoa) in water samples (Samendra et al. 2014, "Rapid Detection Technologies for Monitoring Microorganisms in Water" Biosensors Journal. 2014 August; 109. doi : 10.4172 / 2090- 4967.1000109; Jung et al. ,2014, "Microbial Contamination Detection in Water Resources: Interest of Current Optical Methods, Trends and Needs in the Context of Climate Change" Int. J. Environ. Res. Public Health 2014, 11, 4292-4310. https: / / doi.org / 10.3390 / ijerphll0404292) , but not in real-time.

[0016] Therefore, there is a need for providing a device able to continuous monitoring fluids for the presence of bacteria, that simultaneously provides a rapid and easy way of bacterial identification .

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides a real-time bacteria detection and identification device (1) comprising a body, said body comprising :

[0019] - an inlet opening (2) and an outlet opening (3) , configured for passage of a fluid;

[0020] - a central portion (4) located between the inlet opening (2) and the outlet opening (3) , said central portion (4) being configured to allow the visualization of an at least one porous polymer-based matrix (5) , said matrix (5) comprising at least one polymer, said polymer comprising at least one entrapped lysis compound, at least one immobilized bacteria attracting compound and at least one immobilized chromogenic substrate, wherein said immobilized chromogenic substrate of the polymer-based matrix (5) is configured to change its colour when contacted with a bacterium containing fluid.

[0021] In a preferred embodiment of the device (1) of the invention, the at least one porous polymer-based matrix (5) has 42-72% of at least one polymer, 4-28% of at least one entrapped lysis compound, 12-41% of at least one immobilized bacteria attracting compound and 0.1-5% of at least one immobilized chromogenic substrate.

[0022] Preferably, the at least one polymer of the device (1) of the invention is selected from the group consisting of polyesters, polyamides, polyacrylates, polyurethanes, cellulose and combinations thereof. More preferably, the polymer is polyurethane foam.

[0023] Also preferably, the at least one entrapped lysis compound of the device (1) of the invention is a solution containing one of a detergent, salt, enzyme, alkaline chemicals and the like. More preferably, the entrapped lysis compound is a solution containing Octyl-p-D-glucopyranoside .

[0024] Preferably, the at least one bacteria-attracting compound of the device (1) of the invention is selected from the group comprising sugar, nitrogen sources and carbon sources. More preferably, the bacteria-attracting compound is glucose. Preferably, the at least one immobilized chromogenic substrate of the device (1) of the invention is selected from the group comprising 5-Bromo-3-indolyl p-D-galactopyranoside (Blue- Gal) , 6-Chloro-3-indolyl-p-D-galactopyranoside (Red-Gal) , N- Methylindolyl-p-D-galactopyranoside (Green-Gal) , Chlorophenol Red-p-D-galactopyranoside (CPRG) , 5-bromo-4-chloro-3-indolyl-p-D- glucuronide sodium salt (X-Glu) and the like. More preferably, the immobilized chromogenic substrate is 5-bromo-4-chloro-3-indolyl- p-D-glucuronide sodium salt (X-Glu) .

[0025] In one preferred embodiment of the invention, the porous polymer-based matrix (5) has 71.66% of polyurethane foam, 6.44% of a solution containing Octyl-p-D-glucopyranoside, 18.83% of glucose and 3.07% of X-Glu.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In the following, a detailed description of the invention is provided making reference to the appended drawings:

[0028] Fig. 1 represents one embodiment of the device (1) of the invention with one porous polymer-based matrix (5) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate.

[0029] Fig. 2 represents one embodiment of the of the device (1) of the invention with a first porous polymer-based matrix (5) containing one entrapped lysis compound and one immobilized bacteria attracting compound and a second porous polymer-based matrix (5' ) containing one immobilized chromogenic substrate.

[0030] Fig. 3 represents one embodiment of the device (1) of the invention with a first porous polymer-based matrix (5) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying one bacterium and a second porous polymer-based matrix (5' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying another and different bacterium.

[0031] Fig. 4 represents one embodiment of the device (1) of the invention with a first porous polymer-based matrix (5) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a first bacterium, a second porous polymer-based matrix (5' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a second bacterium and a third porous polymer-based matrix (5' ' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a third bacterium.

[0032] Fig. 5 represents one embodiment of the device (1) of the invention with a first porous polymer-based matrix (5) containing one entrapped lysis compound and one immobilized bacteria attracting compound and a second porous polymer-based matrix (5' ) containing one immobilized chromogenic substrate for detecting and identifying a first bacterium, and a third porous polymer-based matrix (5' ' ) containing one entrapped lysis compound and one immobilized bacteria attracting compound and a fourth porous polymer-based matrix (5' ' ' ) containing one immobilized chromogenic substrate for detecting and identifying a second bacterium.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] To solve the problems identified above, the present invention provides a new real-time bacteria detection and identification device (1) comprising a body, said body comprising:

[0035] - an inlet opening (2) and an outlet opening (3) , configured for passage of a fluid;

[0036] - a central portion (4) located between the inlet opening (2) and the outlet opening (3) , said central portion (4) being configured to allow the visualization of an at least one porous polymer-based matrix (5) , said matrix (5) comprising at least one polymer, said polymer comprising at least one entrapped lysis compound, at least one immobilized bacteria attracting compound and at least one immobilized chromogenic substrate, wherein said immobilized chromogenic substrate of the polymer-based matrix (5) is configured to change its colour when contacted with a bacterium containing fluid.

[0037] It should be noted that independently of the explicit presentation of the quantitative expression "about X" , any X value presented in the course of the present description is to be interpreted as an approximate value of the true X value , since such an approximation to the true value would reasonably be expected due to experimental and / or measurement conditions that introduce deviations from the true value .

[0038] In the context of the present description, the term "comprising" and its verbal variations are to be understood as "including, among others" . As such, the term should not be interpreted as "consisting only of" .

[0039] In the context of the present invention, all the values referred as percentages are meant as percentages weight by weight ( % w / w) .

[0040] In the context of the present invention, "bacteria-attracting compounds" also known as "compounds to attract bacteria" means sources of bacterial nutrients ( carbon, nitrogen, mono- or polysaccharides , etc . ) that promote bacterial chemotaxis , which is the process / movement in which the bacteria respond / move according to a chemical gradient present in the environment , until retained / held at some point . In some embodiments of the device of the invention, bacteria-attracting compounds are chemically immobi li zed through its incorporation as a monomer in the polymer synthesis . However, in another embodiments of the invention, when the polymer of the matrix is cellulose , bacteria-attracting compounds are physically immobili zed to the paper .

[0041] These compounds are selected from the group comprising sugar, nitrogen sources and carbon sources . Preferably, bacteria- attracting compounds are saccharides such as glucose , fructose , lactose, sucrose and starch and nitrogen sources such as alanine, glutamine, glycine and peptones. More preferably, the bacteria- attracting compound is glucose.

[0042] "Lysis compounds" also known as "Compounds for bacterial cells lysis" (BCL) are meant as compounds capable of disrupting bacteria cell membranes by breaking down the cell wall and the lipidic bilayer of cell membranes, exposing the internal cell content .

[0043] In the present invention, lysis compounds are entrapped in the polymeric matrix, which means that they are physically enclosed in the polymeric matrix without being chemically bounded.

[0044] Further, the lysis compounds are a solution containing one of a detergent, salt, enzyme, alkaline chemicals and the like. Preferably, the lysis compound is a solution containing a detergent such as Octyl-p-D-glucopyranoside (OGP) , Sodium Dodecyl Sulfate (SDS) or Sodium Lauroyl Sarcosinate. More preferably, the lysis compound is a solution containing Octyl-p-D-glucopyranoside, marketed as B-PER™ (obtained from Thermo Scientific, USA (Lot TH269908) which uses a proprietary, mild nonionic detergent Octyl- b-D-glucopyranoside ) in a 20 mM Tris HC1, pH 7.5 buffer, with no enzymatic components) .

[0045] "Immobilized chromogenic substrate", in this context, means a substrate linked to the polymer-based matrix. "Immobilized" means that the chromogenic substrate is chemically linked to the matrix. However, when the polymer of the matrix is cellulose, "immobilized" relative to the chromogenic substrate means that it is physically linked to the matrix. Chromogenic substrates are compounds that typically remain colourless until they are cleaved by the target enzyme , which triggers a chemical reaction resulting in the release of a chromophore . The presence of the chromophore produces a visible colour change, allowing the rapid bacteria identi fication .

[0046] The chromogenic substrate immobili zation is performed in order to keep its stability and concentration during the use of the device . Otherwise , the continuous passage of the fluid would lixiviate the chromogenic substrate which could lead to the mal function of the device , since its absence in the device , caused by leaching, would prevent its degradation by enzymes of the lysed bacterial cells and consequently their detection and identi fication .

[0047] Some examples of these compounds can be selected from the group comprising 5-Bromo-3-indolyl p-D-galactopyranoside (Blue- Gal ) , 6-Chloro-3-indolyl- p-D-galactopyranoside (Red-Gal ) , N- Methylindolyl- p-D-galactopyranoside ( Green-Gal ) , Chlorophenol Red- p-D-galactopyranoside ( CPRG) , 5-bromo-4-chloro-3-indolyl- p-D- glucuronide sodium salt (X-Glu) and the like . Preferably, the chromogenic substrate is 5-bromo-4-chloro-3-indolyl- p-D- glucuronide sodium salt (X-Glu) .

[0048] The immobili zation of the chromogenic agent is performed using the l-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide (EDC ) / N- hydroxysuccinimide (NHS ) chemistry where the amidation between the X-Glu carbonyl group and the primary amine of functionali zed polyurethanes ( PU) occurs . The polymer-based matrix (5) must be capable of absorbing liquid medium and transport this liquid from the base of the matrix to its top, in particular to the central portion, (visible from outside the device and where it will be possible to see the colour change) . Thus, the polymer-based matrix (5) must be porous. Preferably, the polymer-based matrix (5) is in the form of a gel or a sponge.

[0049] The polymers of the matrix (5) may be selected from the group consisting of polyesters, polyamides, polyacrylates, polyurethanes, cellulose and combinations thereof. The porosity of the selected polymer is very important because it has an important role on enhancing the absorption of the medium and its diffusion through capillary action. Preferably, the polymer is polyurethane foam.

[0050] The at least one porous polymer-based matrix (5) of the present invention has 42-72% of at least one polymer, 4-28% of at least one entrapped lysis compound, 12-41% of at least one immobilized bacteria attracting compound and 0.1-5% of at least one immobilized chromogenic substrate.

[0051] In a preferred embodiment of the present invention, the porous polymer-based matrix (5) has 71.66% of polyurethane foam (polymer) , 6.44% of B-PER™ (entrapped lysis compound) , 18.83% of glucose (bacteria attracting compound) and 3.07% of X-Glu (immobilized chromogenic substrate) .

[0052] In one embodiment of the invention, the device (1) comprises at least one polymer-based matrix (5) . In one aspect of the invention, the at least one bacteria- attracting compound, the at least one lysis compound and the at least one single chromogenic substrate can be mixed in a single polymer-based matrix (5) .

[0053] In another aspect of the invention, the at least one bacteria- attracting compound, the at least one lysis compound and the at least one single chromogenic substrate can be present in distinct polymer-based matrices (5, 5' , 5' ’ , 5' ’ ’ ) . For example: a first matrix (5) with a bacteria-attracting compound and a second matrix (5' ) with a lysis compound and one single chromogenic substrate; a first matrix (5) with a bacteria-attracting compound and a lysis compound and a second matrix (5' ) with one single chromogenic substrate .

[0054] In a preferred embodiment, the device (1) of the invention may comprise two or more polymer-based matrices (5, 5' , 5' ’ , 5' ’ ’ ) , where each one will be sensitized to detect and identify a different bacterium. This identification will depend on the specificity of each chromogenic substrate for the intended bacteria .

[0055] Thus, in a more preferred embodiment, the device (1) of the invention has a first porous polymer-based matrix (5) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying one bacterium and a second porous polymer-based matrix (5' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying another and different bacterium. In another preferred embodiment, the device (1) of the invention has a first porous polymer-based matrix (5) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a first bacterium, a second porous polymer-based matrix (5' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a second bacterium and a third porous polymer-based matrix (5' ' ) containing one entrapped lysis compound, one immobilized bacteria attracting compound and one immobilized chromogenic substrate for detecting and identifying a third bacterium.

[0056] Yet in another preferred embodiment, the device (1) of the invention has a first porous polymer-based matrix (5) containing one entrapped lysis compound and one immobilized bacteria attracting compound and a second porous polymer-based matrix (5' ) containing one immobilized chromogenic substrate for detecting and identifying a first bacterium, and a third porous polymer-based matrix (5' ' ) containing one entrapped lysis compound and one immobilized bacteria attracting compound and a fourth porous polymer-based matrix (5' ' ' ) containing one immobilized chromogenic substrate for detecting and identifying a second bacterium.

[0057] In this way, each matrix will detect and identify different pathogenic bacteria in the same physical device. So, the device (1) of the invention allows multiple bacteria detection and identification . The functionalization of the polymer composing the polymer- based matrix involves the insertion of specific groups, in this case, primary amines, into the polymer structure in order to subsequently enable the chemical bonding of the chromogenic agent to these groups.

[0058] The insertion of these amine group may be performed through different methodologies, such as:

[0059] - Modification of the polymer matrix through an alkaline hydrolysis reaction that leads to the formation of amine groups in the polymer;

[0060] - Incorporation of a branched polymer containing amine groups (polyethyleneimine (PEI) through a phase inversion mechanism;

[0061] - Deposition of a polymer containing amine groups (chitosan) on the surface of the polymer-based matrix.

[0062] The device (1) of the invention will be used for analysis of a fluid that may be contaminated by bacteria. So, the fluid under analysis contacts the device (1) by entering the inlet opening (2) . Then, the fluid runs through the porous polymer-based matrix (5) and bacteria are attracted by compounds selected to attract them. This allows bacterial cells migration and accumulation / concentration up to the area containing the compounds for bacterial cell lysis, where the cells will be disrupted, and enzymes released. The bacteria's enzymes will react with the immobilized chromogenic substrate, thus causing the colour change of the polymer-based matrix (5) when the fluid contains bacteria.

[0063] The device (1) is useful in the detection of bacteria infections in several biological and non-biological fluids. Examples of biological and non-biological fluids are urine and water .

[0064] The device (1) of the present invention is useful to detect pathogenic bacteria causing human infections. Examples of such bacteria are Gram-negative bacteria such as Enterobacteriaceae (Escherichia coli, Enterobacter spp . , Klebsiella spp . and Citrobacter spp., also known as coliforms) , Proteus spp., Pseudomonas aeruginosa and Aci netobacter baumannii, and Grampositive bacteria such as those belonging to genera Staphylococcus and Enterococcus .

[0065] Coliforms are a group of bacteria used as indicators of fluids faecal contamination. Those pertaining to the group of coliform bacteria are known for being easy to isolate and identify. The device (1) of the invention is useful to detect Escherichia coli (E. coli) contaminated fluids. Furthermore, E.coli is one of the most common bacterium responsible for infections and contaminations and so, one with the most interest.

[0066] The device (1) of the invention allows an easy and rapid way for detecting bacteria present in a fluid due to the visible change in the colour of the polymer-based matrix (5) by reaction of the chromogenic substrate with bacterial enzymes upon lysis.

[0067] This device (1) allows continuous detection and identification of pathogenic microorganisms in different biological and non-biological fluids, by conducting the fluid to be monitored through the device (1) that contains the polymer- based matrix (5) with specific compounds for bacteria detection and for converting this detection in an easy-to-read signal. Further, with the device (1) of the invention, there is no need to collect a fluid sample and transport it to any Laboratory Facilities for microbiological analyses in order to detect and identify pathogenic bacteria, as both results are given by the colour indication in the polymer-based matrix.

[0068] Podkovik, S. et al. (2019) "Prevalence of Catheter-Associated Urinary Tract Infections in Neurosurgical Intensive Care Patients - The Overdiagnosis of Urinary Tract Infections", Cureus . 2019 Aug 26; 11 (8) : e5494. doi: 10.7759 / cureus .5494. PMID: 31667030; PMCID: PMC6816532, refers that a catheter-associated urinary tract infection (CAUTI) is defined by 3 criteria, being one of them an urine culture with more than 10A5 colony forming units (CFU) / mL of one bacterial species. Surprisingly, with the device (1) of the invention, a number of bacterial cells much lower than the reference value of 10A5 CFU / mL allows obtaining confirmation of a bacterium presence in a fluid. In most cases, a minimal number of bacterial cells of 10A2 CFU / mL is enough for obtaining confirmation of a bacterium presence in a fluid.

[0069] This device (1) continuously monitors the presence of microorganisms and so it is not dependent on the sample collection for detection and identification.

[0070] The device (1) of the invention may be continuously used up to 2 weeks, without compromising its function.

[0071] The use of the device (1) will reduce the costs associated with the specialized human resources required for sampling and analysis . In the context of healthcare and hospitali zation, the assembly of this device to a urinary catheter, bladder probes , etc . , promotes an early stage of bacteria detection and therefore it helps to reduce the infection cases and their severity, by increasing the treatment success rate while decreasing the complications associated with a late diagnostic, thus reducing the use of antibiotics , hospitali zation days and costs , to avoid outbreaks as well as contributes to decreasing the transmission of these agents throughout the general population .

[0072] Another application of the device ( 1 ) of the invention within the hospitali zation context is when assembled to a respiratory ventilator to monitor the water used for the humidi fier unit of the system . The humidi fier is used to warm and moisture the forced air before it is delivered to the patient and can constitute a hotspot for nosocomial infection . The use of the device of the invention will allow the continuous monitoring of the aqueous interface of the humidi fier system and determine i f it hosts potential pathogens .

[0073] The device ( 1 ) of the invention eliminates the decisionmaking step of analysing the fluid, thus ef fectively preventing possible cases of infection .

[0074] Furthermore , the device ( 1 ) is user- friendly for both healthcare professionals and patients . It provides crucial "on time" information to the health professional , allowing an early intervention on patient status , which will save financial resources , for example , by reducing the need of longer hospitali zation associated to acquired infections . In particular, if the device reveals a colour change, the healthcare professional (or the patient himself or a caregiver) will be alerted for an early-stage urinary infection and, consequently, adequate procedures may be early undertaken, starting with the catheter removal. Moreover, this alert will also provide a way for a medical prescription of a judicious treatment for a condition often asymptomatic in early stages.

[0075] Due to its wide applicability, the device (1) may also be applied to monitor several different fluids, such as, but not limited to urine. For example, the monitoring of water supply, respiratory ventilators, venting systems or air-conditioned systems with a device of the present invention may detect the presence of bacteria and therefore anticipate cleaning and decontamination actions and preventing propagation of said microorganisms to the population.

[0076] The device (1) of the invention may further comprise other features such as digital result reading, sound alerts, the addition of other healthcare parameters that would benefit with 24 h / 7 days monitorization of any biological or non-biological fluid, as well as the constant monitoring of bacteria concerning other biological or non-biological fluids.

[0077] The device (1) may assume different configurations upon the intended application. Furthermore, there are also other elements that may be present. For example:

[0078] - a first one-way valvule disposed between the inlet opening

[0079] (2) and the central portion (4) and a second one-way valvule disposed between the central portion (4) and the outlet opening (3) that allows the fluid to flow in one direction while preventing the reflux against the current;

[0080] - a transparent cover to protect the device while allowing a continuous visualization of the polymer-based matrix (5) ,

[0081] - a coupling site for the attachment of the digital reader and signal transmitter, fixing means, such as straps or adhesives, for allowing to fasten the device to other adjacent devices, around the patient's leg or even to the skin.

[0082] EXAMPLES

[0083] The examples below refer to the preparation of different polymer-based matrixes of a device of the present invention. The size of the matrix is variable and so, taking Example 1 as reference, each component is given with the amount used in the preparation as well the corresponding %w / w in the final matrix. For the remaining Examples, %w / w of each component is generally used.

[0084] EXAMPLE 1

[0085] A polymer-based matrix of approximately 19 cm3of a device of the present invention was prepared as follows: the polyurethane foam used in this formulation was synthesized using 1 g of glucose (bacteria-attracting compound) as a polyol. It was first solubilized in dimethyl sulfoxide (DMSO) and stirred. 2 pL of Triethylamine were added as a catalyst and 10 pL of water as a blowing agent. Then it was added 0.2 g of surfactant TEGOSTAB B8406 (obtained from Evonik, Germany (Batch E119947724) , which is a polyether polydimethylsiloxane copolymer) and finally, 0.99 mL of tolylene-

[0086] 2 , 4-diisocyanate . The mixture was stirred again and poured into a plastic mold where it was allowed to expand and cure overnight at room temperature. The samples were then cut into 2x2x1 cm and dried in a vacuum chamber until constant weight. The percentages (w / w) of each component of the matrix in the final foam were: 41.50% glucose, 0.06% catalyst, 0.42% blowing age st, 8.30% surfactant and 49.72% isocyanate .

[0087] The polyurethane foams were then functionalized by alkaline hydrolysis by immersion in 15 mL of a 5% (w / w) sodium hydroxide (NaOH) aqueous solution at 30 °C for 30 min and 140 rpm. Then, the samples were washed with ultrapure water and dried in a vacuum oven until constant weight.

[0088] For the immobilization of the chromogenic agent (X-Glu) , the functionalized polyurethane foams were subjected to an ( l-etnyl-3- ( 3-dimethylaminopropyl ) carbodiimide hydrochloride (EDC) / N- hydroxysuccinimide (NHS) coupling reaction. Each sample was immersed in 10 mL of a Phosphate-buff ered saline (PBS) solution (pH 7.4) containing 4.50 mM of X-Glu, 6.70 mol EDC and NHS / mol X-Glu for 6 h at 25 °C and 100 rpm. The samples were then washed with PBS for 2 h and once again dried in the vacuum oven until constant weight. Then, 42 pL of B-PER™ reagent was placed on top of each foam.

[0089] Upon the above steps, the polymer-based matrix is ready to be used on a device of the invention.

[0090] The polymer-based matrix of a device according to present invention has 8.54% of B-PER™ (entrapped lysis compound) , 36.43% of glucose (chemically immobilized bacteria attracting compound) and 4.07% of X-Glu (immobilized chromogenic substrate) , being the remaining 50.96% the polyurethane foam.

[0091] EXAMPLE 2

[0092] This Example was carried out following example 1 except for the functionalization conditions.

[0093] The polyurethanes foams were prepared as in example 1. Functionalization by alkaline hydrolysis was carried out by immersing the samples in a 10% (w / w) sodium hydroxide (NaOH) aqueous solution at 30 °C for 30 min and 140 rpm. Then, the samples were washed with ultrapure water and dried in a vacuum oven until constant weight .

[0094] The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to the example 1.

[0095] The polymer-based matrix of a device according to present invention has 9.78% of B-PER™ (entrapped lysis compound) , 32.61% of glucose (chemically immobilized bacteria attracting compound) and 4.66% of X-Glu (immobilized chromogenic substrate) , being the remaining 52.95% the polyurethane foam.

[0096] EXAMPLE 3:

[0097] This Example was carried out following example 1 except for the functionalization method. Polyurethanes foams were prepared as in the example 1. The modification was carried out using a phase inversion technique to promote the incorporation of branched polyethylenimine (Mw= 10 000 g / mol) . Each foam was submersed in 10 mL of a modification solution containing 5% of branched polyethyleneimine (PEI) , 81.3% of N,N- dimethyl acetamide (DMAc) and 13.7% of H2O at 55 °C for 2 days without stirring. The modification solution was then replaced by ultrapure water, and the samples were subsequently washed with 80 °C distilled water before drying in a vacuum oven until constant weight.

[0098] The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to example 1.

[0099] The polymer-based matrix of a device according to present invention has 5.24% of B-PER™ (entrapped lysis compound) , 32.77% of glucose (chemically immobilized bacteria attracting compound) and 2.49% of X-Glu (immobilized chromogenic substrate) , being the remaining 59.50% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0100] EXAMPLE 4 :

[0101] Formulation 4 was similar to formulation 3 except for the glucose amount in the polyurethane synthesis resulting in the following final mass percentages: 23.79% glucose, 0.08% catalyst, 0.54% blowing agent, 10.82% surfactant and 64.77% isocyanate.

[0102] The modification with PEI was performed as in the example 3. The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were also similar to the example 1. The polymer-based matrix of a device according to present invention has 6.44% of B-PER™ (entrapped lysis compound) , 18.83% of glucose (chemically immobilized bacteria attracting compound) and 3.07% of X-Glu (immobilized chromogenic substrate) , being the remaining 71.66% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0103] EXAMPLE 5:

[0104] Formulation 5 was similar to formulation 3 except for the B-

[0105] PER™ amount added: 200 pL per sample.

[0106] The polymer-based matrix of a device according to present invention has 20.83% of B-PER™ (entrapped lysis compound) , 27.38% of glucose (chemically immobilized bacteria attracting compound) and 2.08% of X-Glu (immobilized chromogenic substrate) , being the remaining 49.71% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0107] EXAMPLE 6:

[0108] Formulation 6 was similar to formulation 1 except for the functionalization method. In this example, 400 pL of a 1.5 wt% chitosan solution in 1 wt% acetic acid was added on top of each foam. The solvent evaporated at a temperature in a range of 20-25 °C.

[0109] The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to the example 1. The polymer-based matrix of a device according to present invention has 5.98% of B-PER™ (entrapped lysis compound) , 37.44% of glucose (chemically immobilized bacteria attracting compound) and 2.85% of X-Glu (immobilized chromogenic substrate) , being the remaining 53.73% the polyurethane foam, namely chitosan- functionalized polyurethane foam.

[0110] EXAMPLE 7:

[0111] Formulation 7 was similar to formulation 6 except for the glucose amount in the polyurethane synthesis resulting in the following final mass percentages: 23.80% glucose, 0.08% catalyst, 0.54% blowing agent, 10.81% surfactant and 64.77% isocyanate.

[0112] The functionalization with chitosan was similar to example 5. The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to the example 1.

[0113] The polymer-based matrix of a device according to present invention has 7.34% of B-PER™ (entrapped lysis compound) , 21.45% of glucose (chemically immobilized bacteria attracting compound) and 3.50% of X-Glu (immobilized chromogenic substrate) , being the remaining 67.71% the polyurethane foam, namely chitosan- functionalized polyurethane foam. EXAMPLE 8:

[0114] In formulation 8, the polymer-based matrix was cellulose in the form of a Whatman™ gel-blotting-paper with 1,5 mm thickness. In this example 100 pL of 0.1 M aqueous glucose solution was added on top of each 2x2 cm filter paper. After drying, 400 pL of a 1.5 wt% chitosan solution in 1 wt% acetic acid was also added. Samples were then dried under a temperature in a range of 20-25 °C.

[0115] The immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to the previous examples.

[0116] The polymer-based matrix of a device according to present invention has 79.67% of chitosan-functionalized filter paper, 13.39% of B-PER™ (entrapped lysis compound) , 0.57% of glucose (attracting compound physically immobilized to the paper) and 6.37% of X-Glu (immobilized chromogenic substrate) .

[0117] EXAMPLE 9:

[0118] Formulation 9 was similar to formulation 8 except for the concentration of glucose solution. In this example 100 pL of 1 M aqueous glucose was added to each Whatman™ gel-blotting-paper s amp 1 e .

[0119] The addition of chitosan, immobilization of the chromogenic agent and the addition of the B-PER™ reagent were similar to the prior example. The polymer-based matrix of a device according to present invention has 75.76% of chitosan-functionalized filter paper, 12.73% of B-PER™ (entrapped lysis compound) , 5.45% of glucose (attracting compound physically immobilized to the paper) and 6.06% of X-Glu (immobilized chromogenic substrate) .

[0120] EXAMPLE 10:

[0121] The polyurethane synthesis in formulation 10 is similar to formulation 3 except for the amounts of blowing agent and surfactant. The resultant mass percentages are: 43,46% glucose, 0.04% catalyst, 0.09% blowing agent, 4.35% surfactant and 52.06% isocyanate.

[0122] The modification with PEI is performed as in the example 3.

[0123] The immobilization of the chromogenic agent is similar to example 3, except for the concentration of X-Glu on immobilization solution. The samples are immersed in 10 mL of a PBS solution (pH 7.4) containing 2.25 mM of X-Glu, 6.70 mol EDC and NHS / mol.

[0124] The addition of the B-PER™ reagent is similar to the previous examples .

[0125] The polymer-based matrix of a device according to present invention has 6.32% of B-PER™ (entrapped lysis compound) , 40.65% of glucose (chemically immobilized bacteria attracting compound) and 0.15% of X-Glu (immobilized chromogenic substrate) , being the remaining 52.88% the polyurethane foam, namely PEI-functionalized polyurethane foam. EXAMPLE 11:

[0126] Formulation 11 is similar to formulation 10 except for the B- PER™ reagent amount added to the foam. The amount of B-PER™ reagent added is 200 pL .

[0127] The modification with PEI and the immobilization of the chromogenic agent is similar to the example 3.

[0128] The polymer-based matrix of a device according to present invention has 24.32% of B-PER™ (entrapped lysis compound) , 32.84% of glucose (chemically immobilized bacteria attracting compound) and 0.12% of X-Glu (immobilized chromogenic substrate) , being the remaining 42.72% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0129] EXAMPLE 12:

[0130] Formulation 12 is similar to formulation 3 except for the amounts of blowing agent and isocyanate in the polyurethane synthesis. The resulting mass percentages are: 31,86% glucose, 0.03% catalyst, 0.06% blowing agent, 6.37% surfactant and 61.68% isocyanate .

[0131] The modification with PEI, the immobilization of the chromogenic agent and the addition of B-PER™ reagent are similar to the example 3. The polymer-based matrix of a device according to present invention has 4.62% of B-PER™ (entrapped lysis compound) , 29.69% of glucose (chemically immobilized bacteria attracting compound) and 2.20% of X-Glu (immobilized chromogenic substrate) , being the remaining 63.49% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0132] EXAMPLE 13:

[0133] Formulation 13 is similar to formulation 12 except for the concentration of X-Glu in the immobilization solution and the amount of B-PER™ reagent added to the foam.

[0134] The modification with PEI, the immobilization of the chromogenic agent and the B-PER™ reagent addition is similar to the example 11.

[0135] Thus, the polymer-based matrix of a device according to present invention has 19.07% of B-PER™ (entrapped lysis compound) , 25.75% of glucose (chemically immobilized bacteria attracting compound) and 0.10% of X-Glu (immobilized chromogenic substrate) , being the remaining 55.08% the polyurethane foam, namely PEI- functionalized polyurethane foam.

[0136] EXAMPLE 14 :

[0137] The polyurethane synthesis in formulation 14 is similar to the one in formulation 3 except for the amount of glucose and isocyanate, resulting in the following final mass percentages: 16.44% glucose, 0.04% catalyst, 3.74% blowing agent, 7.47% surfactant and 72.31% isocyanate .

[0138] The modification with PEI and the immobilization of the chromogenic agent is similar to the example 3.

[0139] The amount of B-PER™ reagent to be added is 200 pL .

[0140] Thus, the polymer-based matrix of a device according to present invention has 21.21% of B-PER™ (entrapped lysis compound) , 12.62% of glucose (chemically immobilized bacteria attracting compound) and 2.12% of X-Glu (immobilized chromogenic substrate) , being the remaining 64.05% the polyurethane foam, namely PEI- functionalized polyurethane foam.

[0141] EXAMPLE 15:

[0142] The formulation 15 is similar to the formulation 14 except for the amount of glucose, blowing agent and surfactant. The resultant mass percentages are: 32.82% glucose, 0.03% catalyst, 0.33% blowing agent, 3.28% surfactant and 63.54% isocyanate.

[0143] The modification with PEI, the immobilization of the chromogenic agent and the addition of B-PER™ reagent is similar to the example 14.

[0144] The polymer-based matrix of a device according to present invention has 19.18% of B-PER™ (entrapped lysis compound) , 25.89% of glucose (chemically immobilized bacteria attracting compound) and 1.92% of X-Glu (immobilized chromogenic substrate) , being the remaining 53.01% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0145] EXAMPLE 16:

[0146] The formulation 16 is similar to the formulation 15 except for the amount of glucose and blowing agent. The resultant mass percentages are: 17.75% glucose, 0.04% catalyst, 0.08% blowing agent, 4.03% surfactant and 78.10% isocyanate.

[0147] The modification with PEI, the immobilization of the chromogenic agent and the addition of B-PER™ reagent is similar to the example 15.

[0148] The polymer-based matrix of a device according to present invention has 22.49% of B-PER™ (entrapped lysis compound) , 13.36% of glucose (chemically immobilized bacteria attracting compound) and 2.25% of X-Glu (immobilized chromogenic substrate) , being the remaining 61.90% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0149] EXAMPLE 17:

[0150] The formulation 17 is similar to the formulation 10 except for the amount of surfactant and blowing agent. The resultant mass percentages are: 40.02% glucose, 0.04% catalyst, 4.00% blowing agent, 8.00% surfactant and 47.94% isocyanate. The modification with PEI, the immobilization of the chromogenic agent and the addition of B-PER™ reagent is similar to the example 15.

[0151] The polymer-based matrix of a device according to present invention has 5.85% of B-PER™ (entrapped lysis compound) , 37.62% of glucose (chemically immobilized bacteria attracting compound) and 0.14% of X-Glu (immobilized chromogenic substrate) , being the remaining 56.39% the polyurethane foam, namely PEI-functionalized polyurethane foam.

[0152] EXAMPLE 18:

[0153] The formulation 18 is similar to the formulation 13 except for the amount of glucose and blowing agent. The resultant mass percentages are: 22.69% glucose, 0.05% catalyst, 5.16% blowing agent, 10.32% surfactant and 61.78% isocyanate.

[0154] The modification with PEI, the immobilization of the chromogenic agent and the addition of B-PER™ reagent is similar to the example 13.

[0155] The polymer-based matrix of a device according to present invention has 27.61% of B-PER™ (entrapped lysis compound) , 28.92% of glucose (chemically immobilized bacteria attracting compound) and 0.14% of X-Glu (immobilized chromogenic substrate) , being the remaining 43.33% the polyurethane foam, namely PEI-functionalized polyurethane foam. EXAMPLE 19:

[0156] The formulation 19 is similar to formulation 8, except for the chromogenic agent and its immobilization method. The base matrix is Whatman™ gel-blotting-paper with 1,5 mm thickness. In this example Red-Gal is used as chromogenic agent. Red-Gal is physically immobilized in the chitosan solution: 1.5 wt% chitosan solution in 1 wt% acetic acid and 7.5 wt% of Red-Gal. 400 pL of this solution is added to the paper matrix. Samples are then dried under a temperature in a range of 20-25 °C.

[0157] The addition of the B-PER™ reagent is similar to the previous examples .

[0158] The polymer-based matrix of a device according to present invention has 77.21% of chitosan-functionalized filter paper, 12.97% of B-PER™ (entrapped lysis compound) , 0.56% of glucose (attracting compound physically immobilized to the paper) and 9.26% of Red-Gal (physically immobilized chromogenic substrate) .

[0159] EXAMPLE 20:

[0160] The formulation 20 is similar to formulation 19, except for the chromogenic agent used. In this example Green-Gal is used as chromogenic agent, being physically immobilized in the chitosan solution: 1.5 wt% chitosan solution in 1 wt% acetic acid and 8.75 wt% of Green-Gal. 400 pL of this solution is added to the paper matrix. Samples are then dried under a temperature in a range of 20- 25 °C. The addition of the B-PER™ reagent is similar to the previous examples .

[0161] The polymer-based matrix of a device according to present invention has 75.99% of chitosan-functionalized filter paper, 12.76% of B-PER™ (entrapped lysis compound) , 0.61% of glucose (attracting compound physically immobilized to the paper) and 10.64% of Red-Gal (physically immobilized chromogenic substrate) .

[0162] EXAMPLE 21:

[0163] The formulation 21 is similar to formulation 20, except for the chromogenic agent used. In this example CPRG is used as chromogenic agent, being physically immobilized in the chitosan solution: 1.5 wt% chitosan solution in 1 wt% acetic acid and 3.75 wt% of CPRG. 400 pL of this solution is added to the paper matrix. Samples are then dried under a temperature in a range of 20-25 °C.

[0164] The addition of the B-PER™ reagent is similar to the previous examples .

[0165] The polymer-based matrix of a device according to present invention has 80.91% of chitosan-functionalized filter paper, 13.59% of B-PER™ (entrapped lysis compound) , 0.65% of glucose (attracting compound physically immobilized to the paper) and 4.85% of Red-Gal (physically immobilized chromogenic substrate) .

Claims

1. EXAMPLE 22:According to this example, a device of the present invention has formulations 8 and 19 as two different polymer-based matrices.In this example two matrices, each containing different chromogenic agents (X-Glu and Red-Gal) are placed side by side on the device, allowing the identification of different bacteria through different color changes. If Escherichia coli is present in the fluid under analysis, both matrices change color: the one containing X-Glu changes to blue and the one containing Red-Gal changes to red / pinkish. If the bacteria present is Klebsiella spp. , only the matrix containing Red-Gal will change color.CLAIMS1. Real-time bacteria detection and identification device (1) characterized by comprising a body, said body comprising:- an inlet opening (2) and an outlet opening (3) , configured for passage of a fluid;- a central portion (4) located between the inlet opening (2) and the outlet opening (3) , said central portion (4) being configured to allow the visualization of an at least one porous polymer-based matrix (5) , said matrix (5) comprising at least one polymer, said polymer comprising at least one entrapped lysis compound, at least one immobilized bacteria attracting compound and at least one immobilized chromogenic substrate, wherein said immobilized chromogenic substrate of the polymer-based matrix (5) is configured to change its colour when contacted with a bacterium containing fluid.

2. Device (1) according to claim 1, characterized in that the at least one porous polymer-based matrix (5) has 42-72% of at least one polymer, 4-28% of at least one entrapped lysis compound, 12-41% of at least one immobilized bacteria attracting compound and 0.1-5% of at least one immobilized chromogenic substrate.

3. Device (1) according to any of claims 1-2, characterized in that the at least one polymer is selected from the groupconsisting of polyesters, polyamides, polyacrylates, polyurethanes, cellulose and combinations thereof.

4. Device (1) according to claim 3, characterized in that the polymer is polyurethane foam.

5. Device (1) according to any of claims 1-4, characterized in that the at least one entrapped lysis compound is a solution containing one of a detergent, salt, enzyme, alkaline chemicals and the like.

6. Device (1) according to claim 5, characterized in that the entrapped lysis compound is a solution containing Octyl-0-D- glucopyranoside .

7. Device (1) according to any of claims 1-6, characterized in that the at least one bacteria-attracting compound is selected from the group comprising sugar, nitrogen sources and carbon sources.

8. Device (1) according to claim 7, characterized in that the bacteria-attracting compound is glucose.

9. Device (1) according to any of claims 1-8, characterized in that the at least one immobilized chromogenic substrate is selected from the group comprising 5-Bromo-3-indolyl p-D- galactopyranoside (Blue-Gal) , 6-Chloro-3-indolyl-p-D- galactopyranoside (Red-Gal) , N-Methylindolyl-p-D- galactopyranoside (Green-Gal) , Chlorophenol Red-p-D-galactopyranoside (CPRG) , 5-bromo-4-chloro-3-indolyl-p-D- glucuronide sodium salt (X-Glu) and the like.

10. Device (1) according to claim 9, characterized in that the immobilized chromogenic substrate is 5-bromo-4-chloro-3- indolyl-p-D-glucuronide sodium salt (X-Glu) .

11. Device (1) according to any of claims 1-10, characterized in that the porous polymer-based matrix has 71.66% of polyurethane foam, 6.44% of a solution containing Octyl-0-D- glucopyranoside, 18.83% of glucose and 3.07% of X-Glu.

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