Patent Application
20150148258
This invention relates to the realm of microbiology—specifically, to the detection and identification of microorganisms in different environments and samples.
A significant number of conventional culture means and methods for detecting and identifying microorganisms are known, whose main disadvantages consist of an extended sample incubation period (at least 18 to 24 hours), along with cumbersome manipulation as it resorts to different means for isolating, enriching and identifying bacteria and yeasts.
Some solutions have been provided in order to reduce the identification time, such as the use of means with chromogenic and fluorogenic substrates, which are likewise too slow for identification needs in terms of availability.
The use of ceramic materials, including nanostructured ones, has been aimed mainly to concentrate microorganisms on samples for their further identification and for detecting or identifying monoclonal antibodies or fragments of genetic structures coupled to nanostructures (Integration of hydroxyapatite concentration of bacteria and seminested PCR to enhance detection of Salmonella typhimurium from ground beef and bovine carcass sponge samples. Elaine D. Berry and Gregory R. Siragusa. United States Department of Agriculture Agricultural Research Service 2. Roman L. Hruska U.S. Meat Animal Research CenterClay Center, NE 689330166. Accepted for Publication Feb. 15, 1999), (Method of manufacturing hydroxyapatite and uses therefore in delivery of nucleic acids. United States Patent Application US20080095820).
On the other hand, silicate nanoparticles have been used instead to inhibit the growth of microbes (Composition comprising aluminum silicates and silver nanoparticles as bactericides. WIPO patent application WO/2011/128488).
On U.S. Pat. No. 6,596,505 B2 from 2003, Ceri et al. proposed a device and methods for testing the effect of materials and surfaces in the formation of biofilms. The method comprises the use of hydroxyapatite and culture media for creating biofilms and the further identification of the characteristics of these microorganisms. The method does not consider identification in one single step, and requires attachments for forming biofilms, thus it is not quite suitable for identification purposes, since it is well known that the metabolic characteristics of microorganisms and their resistance to antimicrobial agents varies when biofilms are formed.
The invention of Hatzmann M. J. et al. (WO 2009/067012 A2) claims a method for detecting microorganisms on different liquid materials, and envisages the concentration of the microorganism on to filter-like device that is connected to other devices and whose filter is formed by to hydroxyapatite structure, to culture medium and chromogenic and fluorogenic substrates. The main limitations of the method are that a sample concentration phase is needed to detect contamination, that it only applies to liquid samples, that it needs further equipment, and that identification response is neither quick nor precise for many microorganisms as it is based on detecting glucuronidase or galactosidase activity.
In brief, the shortcomings of the methods described above on scientific bibliography and on patent documents consist of:
The purpose of this invention consists in promoting a method for detecting, recovering, identifying and simultaneously enumerating a variety of microorganisms in different samples, as well as the devices required for its execution.
The novelty of this invention consists in the following:
The advantages of this method and of the device consist of:
A detailed description of the invention is given below.
The nutritional components of this invention are chosen among a series of mixtures of proteins, carbohydrates, vitamins and minerals that are degraded through chemical or enzyme methods. One or more of these nutritional mixtures that stimulate microbial growth are prepared in aqueous solutions or salt solutions with concentrations of 0.1 to 3 g/L that are inoculated with 0.1 ml of the microorganisms intended to be detected, recovered, identified or enumerated under the concentration of 3×108 UFC/ml and which are incubated at the desired temperature and oxygen tension, measuring the microbial growth kinetics through any known method, preferably determining the increase of optical density over time. Those compounds that ensure a reduction of the lag growth phase, which does not surpass 60-120 minutes for bacteria and 16 hours for yeasts and filamentous fungi, are selected.
Some examples of the nutritional components mentioned above are hydrolyzed enzymes of Spirulina platensis algae described in Cuban Invention Copyright Certificate No. 22310; extract of Saccharomyces cerevisiae obtained through enzyme hydrolysis as described in Cuban Invention Copyright Certificate No. 22221, and hydrolyzed enzymes of Torula fodder yeast (Cuban Invention Copyright Certificate No. 22280); extract of sweet potatoes, as disclosed in Cuban patent No. 23507; tomato extract (Cuban Invention Copyright Certificate No. 22308); hydrolyzed enzymes from beef heart tissue (Cuban Invention Copyright Certificate No. 22442), from bovine blood (Cuban Invention Copyright Certificate No. 22208) and from beef liver (Cuban Invention Copyright Certificate No. 22220); hydrolyzed enzymes of lactoalbumin from rennet whey (Cuban Invention Copyright Certificate No. 22219), hydrolyzed enzymes or casein acids from buttermilk (Cuban Invention Copyright Certificates No. 22166 and 22089, respectively) and hydrolyzed or autolyzed Eudrillus eugeniae (Cuban Invention Copyright Certificate No. 22381). The selected compounds are dissolved or suspended in a first solvent in amounts ranging from 1 to 50 g/L.
To all of the above there can also be added further hydrolyzed enzymes, hydrolyzed chemicals, such as peptones and triptones or commercial protein extracts from algae, microorganisms, vegetables, higher animal tissue and their combinations, such as those obtained from beef meat, brains and potatoes, among others, in quantities ranging from 1 to 10 g/L.
As soon as the compound is prepared, one or more chromogenic, fluorogenic or bioluminescent enzymatic markers compounds in quantities ranging from 0.01 to 2 g/L can be added. Some examples of these markers may be: compounds derived from phenol, such as ortho- and paranitrophenols, paranitroaniline, indolyl derivatives: 5-bromo-4-chloro-3-indolyl, 5-bromo-6-chloro-3-indolyl (magenta), 6-chloro-3-indolyl (salmon), derivatives of methylcoumarin and methylumbelliferyl (MUG) for detecting activity of galactosidase, glucuronidase, decarboxylase, glycosidase and phosphatase, among others.
Other substances can be added to the mixture of nutritional compounds and enzymatic markers, such as microorganism promoters or inhibitors belonging to certain genres, species or groups. Some examples of these substances include vitamins, mineral salts, albumin, antibiotics, dyes, tints, bile salts, beef bile, sugars and amino acids. Likewise, other substances that increase the solubility of enzymatic markers or the permeability of microorganism cells can be added in quantities ranging from 0.01 up to 40 g/L.
If it were intended to determine the sensibility to antimicrobial and antifungal agents or to cleansing or disinfecting solutions, or for proving the particular bactericide or bacteriostatic effect of some substance or product, salts, resins, natural plant extracts, fatty acids, esters, bactericides, bacteriostatics, alcohols, substances with superficial activity or their mixtures in quantities ranging from 0.01 to 2 g/L and/or antibiotics or antifungal agents in quantities ranging from 10 to 100 μg/L may be also added to the nutritional compound selected.
The nutritional compound selected, along with enzymatic markers and other components, is dissolved or dispersed in a first solvent in quantities ranging from 1 to 150 g/L.
The solvent may be distilled or deionised water, aqueous salt solutions (NaCl, phosphate solutions among others), alcohols and alcohol solutions (e.g., 10% p/v basic fuchsine solution in ethyl alcohol), solutions of substances that increase the solubility of enzymatic markers [e.g., dimethyl sulfoxide (DMSO)] or the permeability of microorganisms cells.
Once the nutritional mixture is formed and dissolved or suspended in the first solvent along with the enzymatic markers and other components, they can be sterilized through any known method, except those compounds containing thermolabile substances that cannot be sterilized through heat.
Once the nutritional mixture is formed and dissolved or suspended in the first solvent along with the enzymatic markers and other components, they may come into contact with one or more three-dimensional structures of natural or artificial clays or ceramics.
These three-dimensional structures may be previously sterilized through any known method.
The contact time of the nutritional compound and other components dissolved or suspended in the first solvent and the three-dimensional structure of clay or ceramic ranges in general, from 10 minutes for nanometric or submicrometric (<1000 nm) dimensions, up to 60 minutes for larger structures.
These structures must have a specific surface of 2×103 to 6×108 m2/m3, and be formed by to variety of nano-, micro- and macro-cavities or their combinations.
These clays and/or natural or artificial ceramics are chosen from kaolinite, halloysite, dickite, nacrite, chrysolite, antigorite, lizardite, vermiculite, mica, hectorite, saponite, hydrotalcite, muscovite, chlorite, diatomaceous earth, bentonites (montmorillonite, sauconite, beidelenite, nontronite) clinoptilotites, hydroxyapatites, zeolites and calcium phosphates or their combinations.
The calcium phosphate structures mentioned in the paragraph above must be chosen between: metaphosphate [Ca(PO3)2], monohydrated monocalcium phosphate [Ca(H2PO4)2H2O], dihydrogen phosphate tetracalcium (Ca4H2P6O20), heptacalcium phosphate [Ca7(P5O16)2], calcium pyrophosphate (Ca2P2O7 and Ca2P2O72H2O), dicalcium phosphate [CaHPO4, CaHPO4.2H2O and Ca(H2PO4)2], tricalcium [Ca3(PO4)2], octacalcium phosphate [Ca8H2(PO4)6.5H2O], calcium-deprived hydroxyapatite [Ca10-x(HPO4)x(PO4)6-x(OH)2-x], hydroxyapatite [Ca10(PO4)6(OH)2], tetracalcium phosphate [Ca4(PO4)2], apatite [Ca10(PO4)6(OH,F,Cl,Br)2], carbonate apatite [Ca5(PO4,CO3)3(OH,F)] or a mixture of them. Clays and/or natural or artificial ceramics and calcium phosphates may have isomorphic ion replacements with cations or previously functionalized with different ions, preferably monovalent, divalent, trivalent or tetravalent, acting as enzyme catalyzers such as Na, K, Ca, Mg, P, Fe and Zn, forming superficial layers or distributed throughout its entire structure.
The three-dimensional structures mentioned above are selected between those whose cavity dimensions correspond to:
Cavities on the structure may appear as pores, channels, tubes, regular or irregular bags of different geometric shapes or their combinations, or if available as layers or sheets.
Three-dimensional structures may form a 5 nm to 1 mm thick layer or film, in particular when used for detection, identification or enumeration of microorganisms using the membrane filtration technique or for detection superficial microorganisms; or a column up to 10 cm high, in particular when it is required to filter large quantities of liquid microorganisms; suspensions that may be found with low concentrations, or when different structure zones are used with different enzymatic markers or with different nutritional mixtures.
Structures may also be used as spheres or pearls with a diameter of 5 nm to 10 mm; hexagons or cubes forming a set of test with different compounds, or they may be added to liquid or suspension sample containing the microorganisms.
Other structures shaped as cylinders or tubes with a very small diameter (5 nm) for small aliquots that join many of these tubes to form a set with a diameter of up to 10 cm, resembling the diameter of a Petri dish for those tests provided by recount standards that require such a surface.
The height of these structures varies depending on the presentation format to use on the method, ranging from 5 nm for nanoaliquots, or microaliquots, or for joining many of these structures in a sandwich-like set of layers that may reach up to 10 cm high.
Three-dimensional structures may appear as fibres or webs that hold the microorganisms, allowing their detection.
Whenever it is desired that structures detect and identify microorganisms throughout the entire volume of a sample, clays are used that are able to swell a lot naturally or by adding jellifying substances that make them adopt the shape of the container holding them.
Three-dimensional structures according to this invention may exhibit multiple zones with different porosities, diameters or clearances of nano-, micro- and macro-cavities throughout their volume, length or diameter, distributing these zones as a gradient or in differentiated zones. Different nutritional compounds and enzymatic markers may be added in each zone, throughout their structure, length or diameter, thus distributing over concentric zones. Distribution of compounds or the concentration of one or more of their components may be ensured through continuous or discontinuous gradients.
To these structures substances may be added that contribute to fixing the nutritional compound and the selected enzymatic markers and other components in those cases in which it is suspected that the sample fluid may drag said compound. Some of these substances can include alginates, such as sodium; natural polysaccharides, such as pectin and quinine; gum Arabic and other gums; starches, such as corn or yam starch, or pre-gelatinized starches; dextran and carboxymethylcellulose and other derivative polymers; carrageenan or sodium carrageenate; agar; agarose and artificial polymer derivatives; vinyl alcohol derivatives, as well as polybutylene and polypropylene; and polyvinylpyrrolidone of different molecular weights in quantities ranging from 0.01 to 0.5 g/g with three-dimensional structure.
In the event that the specific surface of a three-dimensional structure is lower than 500 m2/g, substances may be added that increase its absorption capacity, such as activated carbon and cellulose in quantities ranging from 2 to 4 mg/g, for example, as layers.
When the absorption is completed, the first solvent is eliminated. The most recommended procedure is under ambient temperature and atmospheric pressure with forced air circulation in order to preserve nutrients and prevent their decay, as in the case of thermolabile vitamins.
The first solvent may be eliminated by drying the three-dimensional structure at a temperature between 25 and 110° C. under atmospheric pressure or under pressure lower than the atmosphere, such as a vacuum oven between 30 minutes and 3 hours, or eliminating it through sublimation or aspersion drying at a temperature of 90 to 180° C.
After the first solvent has been eliminated, the structure may be preserved up to the moment when the assays are conducted for periods of up to five (5) years. It is advisable to sterilize these structures with the compounds embedded, preferably but not exclusively by other means of radiation. Other methods may be used, such as autoclave sterilization for those structures that do not contain any thermolabile components.
The capacity of recovering target microorganisms is proven by selecting those structures that have a detection limit of less than 1 UFC/10 L for liquid samples, less than 1 UFC/250 g for solid samples or less than 1 UFC/10 m3 of air and a maximum limit of up to 109 UFC/ml or 109 UFC/g or 103 UFC/m3.
Prior to staring the assay, the three-dimensional structure may be placed over supports shaped as plates, layers or cylinders that are pervious to gases or liquids or impervious to them; or surrounded by impervious materials on at least 90% of its surface.
Later on, those microbial cells that may be formed by a variety of microorganisms may be placed in contact between them in order to be detected, recovered, identified and/or enumerated among a species, genre, group or combinations of them, including nanobacteria, bacteria, moulds and yeasts, as well as spores, hyphae or other propagules with three-dimensional structures in the presence of a second solvent.
This second solvent may be water, a hypotonic, isotonic or hypertonic solution of salts, such as sodium chloride, or the sample itself depending on the nature of those microorganisms to be identified.
Some examples may include biological samples such as blood or food, like milk or sample suspensions.
Another variant of the method consists in placing a microorganism suspension in contact with gaseous carriers such as air or aerosols.
The sample is applied to the structure on the following ratios: 0.05 to 13 ml/g, or from 0.1 to 10 m3/g of the three-dimensional structure.
In order to detect, recover, identify or quantify the diversity of cells, the second solvent or the samples holding them may be placed in contact with the surface of the three-dimensional structure or making it pass through it, down to a certain depth; if cells or the samples that hold them are in the form of a suspension in gas or liquid phase, as a gel or with semi-solid or solid consistency, applying it directly over the structure, or through an application device such as, for example, a swab, a holder or a needle, among others.
The samples or the second solvent holding the microorganisms may be applied over different structures at the same time.
The three-dimensional structure is then subject to temperatures between 20 and 50° C. over a period longer than the greater duration of the lag phase or of the final growth acceleration phase of the microorganism with the slowest development, under variable oxygen tension ranging from aerobic conditions to the total absence of this element, depending on the target microorganisms to be detected.
Growth will be observed from 30 minutes up to 240 minutes for bacteria, and along with detection different species, genres or groups may be identified. For this purpose, structures are maintained under the aforementioned conditions in order to foster microbial growth both inside the cavities and on the surface.
For those microorganisms of very slow growth, such as yeasts and filamentous fungi, growth is observed in only 16 hours, instead of 36-72 hours observed in traditional methods.
Growth is observed on nanoparticles or cavities through nanobacterial activity, and that of other bacteria is observed indirectly over the products from the breakup of enzymatic markers that, under the action of microbial enzymes, may accumulate in them. Smaller-sized bacteria develop in microcavities, while larger bacteria, yeasts and filamentous fungi grow on macrocavities and all other microorganisms grow over the surface.
The detection and identification of the variety of cells is basically, but not exclusively, carried out through visual or automatic detection of fluorescence or bioluminescence.
Further methods may be used, such as: for the change of the three-dimensional structure or of its color, consistency, texture, shine, opacity, tone, uniformity or transparency; or through changes of color, shine, tone, transparency or fluorescence of the second solvent or of the sample; or by the appearance of bioluminescence, both inside the cavities and over the surface of the structure; or by observing other morphologic structures; or through metabolic reactions on the three-dimensional structure, in the second solvent or in the sample; or through a combination of some or all identification means.
Cell concentration on the sample is determined over the surface of the structural layers or coats, through visual enumeration, through automatic superficial methods, or by measuring the intensity of the fluorogenic colorimetric or bioluminescent signal under ultraviolet, visible or infrared light, of electrical, thermal or magnetic signals, through pH changes or by quantifying the emission or consumption of gases produced by the activity of microorganisms during the lag phase or the growth acceleration period, such as carbon dioxide, oxygen, hydrogen sulphur, ammonia and hydrogen. Along with the identification of the variety of microorganisms, the resistance or sensibility to antimicrobial agents such as bactericides, bacteriostatics, fungicides, cleaning solutions, added to the mixture of nutritional compound and enzymatic markers may be determined, observing total or partial growth inhibition, its deceleration, the extension of the lag phase, or due to the absence of substrate decay reaction.
The method may be carried out with the aid of devices formed by a nutritional mixture that fosters microbial growth, selected among the hydrolyzed enzymes of alga Spirulina platensis; hydrolyzed enzymes of Saccharomyces cerevisiae and of Torula; extract of sweet potatoes; tomato extract; hydrolyzed enzymes of beef heart and liver tissue and of bovine blood; hydrolyzed enzymes of lactoalbumin from rennet whey, hydrolyzed enzymes or casein acids from buttermilk and hydrolyzed or autolyzed from Eudrillus eugeniae and its combinations and one or multiple chromogenic, fluorogenic or bioluminescent enzymatic markers absorbed and/or adsorbed on a three-dimensional structure of clay or natural or artificial ceramics, selected from kaolinite, halloysite, dickite, nacrite, chrysolite, antigorite, lizardite, vermiculite, mica, hectorite, saponite, hydrotalcite, muscovite, chlorite, diatomaceous earth, bentonites (montmorillonite, sauconite, beidelenite, nontronite) and calcium phosphates or their combinations formed by a variety of nanocavities or particles; submicro-, micro- and macrocavities.
Devices according to this invention contain all the components required for implementing the method, whereas those components are available dehydrated on quantities that ensure the concentrations of each of them as described in the original method.
These devices may be formed by calcium phosphates chosen among: metaphosphate [Ca(PO3)2], monohydrated monocalcium phosphate [Ca(H2PO4)2H2O], dihydrogen phosphate tetracalcium (Ca4H2P6O20), heptacalcium phosphate [Ca7(P5O16)2], calcium pyrophosphate (Ca2P2O7 and Ca2P2O72H2O), dicalcium phosphate [CaHPO4, CaHPO4.2H2O and Ca(H2PO4)2], tricaicium [Ca3(PO4)2], octacalcium phosphate [Ca8H2(PO4)6.5H2O], calcium-deprived hydroxyapatite [Ca10-x(HPO4)x(PO4)6-x(OH)2-x], hydroxyapatite [Ca10(PO4)6(OH)2], phosphate tetracalcium [Ca4O(PO4)2], apatite [Ca10(PO4)6(OH,F,Cl,Br)2], carbonate apatite [Ca5(PO4,CO3)3(OH,F)] or a mixture of them. They may also contain mixtures of the substances mentioned with the phosphates described in the previous paragraph.
Some of these devices may be formed by natural or artificial clays, ceramics and other calcium phosphates with isomorphic ion replacements with cations or be previously functionalized with different ions or monovalent, divalent, trivalent or tetravalent cations, forming superficial layers or distributed throughout their entire structure. These cations may be Na, K, Ca, Mg, P, Fe and Zn that essentially play the role of catalyzers on the marker decay enzyme reaction.
Three-dimensional structures of these devices exhibit cavities in the form of pores, channels, regular or irregular sacks with different geometric shapes or their combinations; or they are available as plates or layers, depending on the type of clay or ceramic used and based on their production technology, these cavities are classified depending on how they will be used in the method as follows:
These devices contain one or more nutritional compounds, whose components are chosen among mixtures of proteins, carbohydrates, vitamins and minerals decayed by chemical or enzyme methods that ensure that the lag phase does not exceed 60-120 minutes for bacteria and 16 hours for yeasts and filamentous fungi in quantities ranging from 0.33 to 20 mg/g of three-dimensional structure.
The devices also contain one or multiple chromogenic, fluorogenic or bioluminescent enzymatic markers inside the cavities or over the surface of the structures in quantities ranging from 0.0033 to 0.66 mg/g of three-dimensional structure. In addition to these substrates, the devices may contain pH and potential redox indicators.
In addition, the nutritional compound inside the device may include further commercial products such as hydrolyzed enzymes, hydrolyzed chemicals or algae protein extracts, microorganisms, vegetable components, higher animal tissue and their combinations in quantities ranging from 0.33 to 4 mg/g of three-dimensional structure. Some examples of these substances include bacteriological peptone, triptone, meat, brain and heart extracts, potato, corn, rice, soy and yeast extracts.
The devices may include other substances such as growth promoters, inhibitors, salts, buffers, carbohydrates and other components used for promoting the growth of those microorganisms belonging to certain genres, species or groups in quantities ranging from 0.003 up to 14 mg/g.
Overall, those components located inside the cavities or over the surface of the devices (mixture of the nutritional compound with enzymatic markers and other components) are found in quantities ranging from 0.33 to 60 mg/g of the three-dimensional structure.
Devices according to this invention have detection limits of less than 1 UFC/10 L for liquid samples, less than 1 UFC/250 g for solid samples or less than 1 UFC/10 m3 and a maximum limit of up to 109 UFC/ml or 109 UFC/g or 103 UFC/m3.
Those devices used for selectively detecting, recovering, identifying or enumerating certain microorganisms inside a sample containing selective microbial growth agents, chosen among salts (e.g. bile salts, sodium desoxycholate), other substances such as resins, natural plant extracts, fatty acids, esters, bactericides, bacteriostatics, alcohols, substances with superficial activity or their mixtures in quantities ranging from 0.0033 to 0.8 mg/g of three-dimensional structures of clays or ceramics and antibiotics (e.g. vancomycin, nadilixic acid), antifungals (e.g. nystatin, ketoconazole, amphotericin B), in quantities ranging from 0.033 to 0.33 μg/g.
Those devices used by passing aqueous samples through their three-dimensional structure, and which have components that are highly soluble in water, may contain substances that contribute to fixating the nutritional compound and the enzymatic markers to three-dimensional structures, such as alginates (e.g. sodium or calcium), natural polysaccharides; pectin, chitin, gum Arabic and other gums, starches such as pregelatinized corn starch, dextran and carboxymethylcellulose and other polymer derivatives of them; carrageenan, agar, agarose and artificial polymer derivatives, derivatives of vinyl alcohol, polybutylene, polyethylene and polypropylene, polyvinylpyrrolidone in quantities ranging from 0.01 to 0.5 gig of three-dimensional structure.
Other compounds may be part of the devices, such as those substances that increase their absorption capacity, like activated carbon and cellulose in the amount of 2 to 4 mg/g, which is used for forming devices whose structures have specific surfaces of less than 3×10 m2/m3.
A device may be formed by a three-dimensional structure or by a set of structures. Each three-dimensional structure may form a film or layer with a thickness of 5 nm to 1 mm; or a column up to 10 cm high; or particles with different geometric shapes, such as spheres, pearls, hexagons, cubes, with a diameter of 5 nm to 10 mm; cylinders or tubes with a diameter of 5 nm to 10 cm and a height of 5 nm to 10 cm; fibres, networks, or adopting the shape of the container that holds them. In some cases the structure of the device may increase its size and volume because it “swells” when it absorbs the sample containing the microorganisms, or the second solvent containing the microorganisms and occupying the entire volume of the container that holds it. These may be devices prepared from hydroxyapatite, agar and pregelatinized corn starch.
A device may have a three-dimensional structure that shows different zones with different porosities and different diameters or clearances of nano-, micro- and macrocavities throughout its volume, its length or its diameter, whereas said zones distribute as gradients or in differentiated zones.
The unique three-dimensional structure of each device contains one or more nutritional compounds and enzymatic markers different throughout its volume, length or diameter, whereas those compounds are distributed as gradients or in differentiated zones.
Devices may maintain their three-dimensional structure over supports shaped as sheets, layers or cylinders that may be pervious or impervious to them, or surrounded by impervious materials on at least 90% of their surface.
Some implementation examples are given below
A sample of nutritional bases was taken for the bacterial growth promotion assay (E. coli), such as papain-hydrolyzed heart tissue, according to Cuban Invention Copyright Certificate No. 22442 in the amount of 0.2 g/L of deionised water, extracts of Saccharomyces cerevisiae yeast, according to Cuban Invention Copyright Certificate No. 22221, in the amount of 0.2 g/L, hydrolyzed enzymes of casein (Cuban Invention Copyright Certificates No. 22166) in the amount of 0.2 g/L and 0.2 g/L of pancreatic hydrolysate of heart.
Likewise, a mixture of all of them was formed in the following amount: papain-hydrolyzed heart tissue in the amount of 1 g/L of deionised water, extract of Saccharomyces cerevisiae yeast in the amount of 1 g/L, hydrolyzed enzymes of casein in the amount of 2 g/L and 1 g/L of pancreatic hydrolysate of heart.
Products to be tested were inoculated with 0.1 ml of a suspension of target microorganisms with a concentration of 3×108 UFC/ml.
Bases and the mixture were incubated separately for 8 hours at 37° C. under an aerobic atmosphere, in which the increase of the optical density was monitored with a spectrophotometer at 680 nm.
Of all variants, the nutritional mixture showed a reduction of the E. coli lag growth phase in 30 minutes, while individual bases showed a variable duration on that phase, among them: papain-hydrolyzed heart tissue—45 minutes, extracts of Saccharomyces cerevisiae yeast—80 minutes, hydrolyzed enzymes of casein—60 minutes and pancreatic hydrolysate of heart—50 minutes.
Therefore, the mixture of nutritional components was selected, which hereinafter will be identified as CCL, and was dissolved in 1 L of deionised water as first solvent in the amount of 5 g/L (variant 1) and in the amount of 10 g/L (variant 2).
This nutritional mixture had already been added the pancreatic hydrolysate heart tissue in the amount of 1 g/L (variant 1) and 2 g/L (variant 2), resulting in the already mentioned nutrient concentrations of 5 g/L and 10 g/L, respectively.
Once the compound was prepared, two enzymatic markers were added, a chromogenic one [2-nitrophenyl-β-D-galactopyranoside (C12H15NO6)], in quantities ranging from 0.5 g/L (variant 1) and 1 g/L (variant 2) and another fluorogenic [4-methylumbelliferyl-β-D-glucuronide (C16H16O9.2H2O)] in quantities ranging from 0.075 g/L (variant 1) and 0.15 g/L (variant 2).
Other substances were added to the mixtures of nutritional compounds and enzymatic markers, such as growth promoters, specifically lactose (5 and 10 g/L), sorbitol (0.5 and 1 g/L), L-tryptophan (1 and 2 g/1); inorganic salts, specifically monobasic potassium phosphate (2.75 and 5.5 g/l), dibasic potassium phosphate (2.75 and 5.5 g/L) and sodium chloride (5 and 10 g/L); finally, bile salts were added with a concentration of 1.3 to 2.6 g/L.
The nutritional compound selected, along with the enzymatic markers and other components, were dissolved in the first solvent in quantities ranging from 23.9 g/L for variant 1 and 47.8 g/L for variant 2.
Once the nutritional mixture is formed and the enzymatic markers and other components have been dissolved in the first solvent, they were sterilized by filtration.
Nutritional mixtures along with the enzymatic markers and other components dissolved in the first solvent were put in contact with two three-dimensional structures of ceramics, specifically hydroxyapatite that had been previously sterilized at 180° C. for 60 minutes.
Contact time between the compounds of variant 1 (V1) and of variant 2 (V2) was of 60 minutes.
These structures had a specific surface of 7500 m2/m3.
The dimensions of the three-dimensional structures mentioned above had combinations of all diameters or clearances, corresponding to nano- and microcavities with diameters or clearances ranging from 5 nm to 600 μm in the form of pores. These structures showed cylinder shapes with a 0.5 cm diameter and height of 0.5 cm.
When the absorption stage was completed, the first solvent was eliminated by drying the three-dimensional structures at a temperature of 60° C. in a vacuum oven for 3 hours.
The capacity of recovering the target microorganism (E. coli) was verified, proving that the structures had detection limits of less than 1 UFC/100 ml for both variants.
An E. coli suspension in saline isotonic solution with a concentration of 3×106 UFC/ml was put into contact with 0.1 g of three-dimensional structures in quantities of 0.2 ml (2 ml/g ratio).
Afterwards, the three-dimensional structures were kept under a temperature of 35±2° C., under aerobic conditions throughout a 2-hour period that coincides with the duration of the lag growth phase of E. coli.
When the 2-hour incubation period was completed, the presence of the target microorganism was visually identified on both variants by its fluorescence under UV light at 366 nm over the supernatant liquid, where E. coli could be identified because of its positive reaction to glucuronidase (fluorescence).
Similar to variant 1 of Example 1, although with the following differences:
V3—Hydroxyapatite pearls, with a total weight of 0.2 g and a specific surface of 2×103 m2/m3, impregnated with the nutritional compound according to V2 of Example 1.
V4—Hydroxyapatite pearls, with a total weight of 0.2 g, with specific surface of 3000 m2/m3, impregnated with the nutritional compound according to V2 of Example 1.
The structures were left absorbing the nutritional compounds for 2 hours and were vacuum-dried for 2 hours at 60° C.
Afterwards, they were inoculated with 2 UFC/ml of E. coli in a volume of 0.2 ml (1 ml/g). The fluorescence of E. coli was observed after 120 minutes.
V5—Hydroxyapatite pearls, with a total weight of 0.2 g and a specific surface impregnated with the nutritional compound according to V2 of Example 1.
V6—Hydroxyapatite pearls, with a total weight of 0.2 g and a specific surface of 1.5×103 m2/m3, impregnated with the nutritional compound according to V2 of Example 1.
V7—Cellulose discs with no clays, with a 6-mm diameter, 0.94 cm2 surface and 0.014 g, impregnated with the nutritional compound according to V1 of Example 1.
V8—Nutritional compound according to V1 of Example 1 in a 0.25 ml volume.
Ceramics were impregnated with the nutritional compound for 3 hours and the first solvent was eliminated at a temperature of 70° C.
Afterwards, they were inoculated with 0.1 ml of concentrated suspension (1 colony in 5 ml of saline solution) of E. coli.
As a result, fluorescence was observed after 90 minutes on V6, after 105 minutes on V5 and it was not observed either on V7 or on V8, which proves that the combination of using three-dimensional clays along with the selected nutritional mixtures that reduce the lag growth phase accelerate microbial detection and identification in comparison with using only the compound, or of this with any other kind of structure.
In general, this method was carried out according to Example 1, with the following differences:
The bacterial growth promotion assay was carried out with papain-hydrolyzed heart tissue, according to Cuban Invention Copyright Certificate No. 22442 in the amount of 0.2 g/L of deionised water.
This base was incubated for 8 hours at 37° C. under an aerobic atmosphere and the increase of optical density was monitored with a spectrophotometer at 680 nm.
The nutritional base showed a reduction of the lag growth phase of Enterococcus after 120 minutes.
This hydrolyzed was chosen for the preparation of the device (variant 9) in order to carry out this method, and it was dissolved in 1 L of deionised water as first solvent in the amount of (10 g/L equivalent to 10 mg/g of structure) and salts were added in order to regulate a potential pH change caused by the three-dimensional structure, specifically dipotassium phosphate (3.5 g/L, equivalent to 8.75 mg/g of structure), potassium phosphate (1.5 g/L, equivalent to 3.75 mg/g of structure) and sodium chloride (5 g/L, equivalent to 12.5 mg/g of structure), which makes for a total nutritional mixture quantity of 50 mg/g). To the structure was added methylumbelliferyl-β-glucoside in the amount of 0.075 g/L as fluorogenic marker, equivalent to 0.1875 mg/g of three-dimensional clay structure.
All components were previously sterilized in an autoclave at 121° C. for 15 minutes.
The presence of Enterococcus was observed because of the bluish fluorescence that appeared after 120 minutes.
In general, the method was carried out according to Example 4, with the following differences:
In order to test the bacterial growth promotion (Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 19434, Enterococcus avium ATCC 14025), papain-hydrolyzed heart tissue was used according to Cuban Invention Copyright Certificate No. 22442, in the amount of 0.2 g/L of deionised water.
The bases were incubated for 8 hours at 37° C. under an aerobic atmosphere and the increase of optical density was monitored with a spectrophotometer at 680 nm.
The nutritional bases showed a reduction of the lag growth phase of 2 hours. The devices were prepared for carrying out the method, duplicating the concentrations of each component that was embedded in the structures.
After 90 minutes, a change of color was observed in the second solvent, which turned slightly bluish in regards to the original that was greenish on both devices with three-dimensional clay structures for E. avium.
After 150 minutes, the appearance of fluorescence was observed in the second solvent for the device with 7.5×103 m2/m3 specific surface (variant 10) and slight fluorescence for the device with specific surface of 2×103 m2/m3 (variant 11) for E. avium.
After 210 minutes, E. faecalis was detected in the device of variant 11 due to the appearance of fluorescence.
After 240 minutes, all microorganisms showed fluorescence on their three-dimensional structures.
Carried out according to Example 1, with the following differences:
Three devices were prepared: the one described in variant 11 (V11) with a specific surface of 2×103 m2/m3, the one described in variant 12 (V12) with a specific surface of 3.3×103 m2/m3 and the one described in variant 13 with a specific surface of 1.5×103 m2/m3.
Contact time of the compounds of these variants was 180 minutes.
When the absorption was concluded, the first solvent was eliminated by drying the three-dimensional structures at a temperature of 60° C. in a vacuum oven over a 60-minute period.
An E. coli suspension on a saline isotonic solution was put in contact with a concentration of 1 colony on 5 ml, from which 0.1 ml was taken and applied over the surface of the device.
After 90 minutes, fluorescence was observed on the device with the structure of variant 11 (V11).
After 210 minutes, fluorescence was observed on the other two devices (V12 and V13) with structures of specific surface of 1.5×103 m2/m3 to 3.3×103 m2/m3.
Similar to Variant 1 of Example 1, with the difference that the following variants were formed:
V14—Kaolinite compacted in agglomerates, with a total weight of 0.2 g, impregnated with the nutritional compound according to V2 of Example 1.
Spaces between kaolinite particles form cavities of different shapes, with sizes corresponding to micro- and macrocavities.
V15—Powdered bentonite, ground on a ball mill and later compacted with a total weight of 0.2 g, impregnated with the nutritional compound according to V2 of Example 1. Spaces between kaolinite particles form cavities of different shapes, with sizes corresponding to macrocavities.
Structures are left absorbing the nutritional compounds over 4 hours and vacuum-dried for 3 hours at 60° C.
An E. coli suspension is put in contact with an isotonic sodium phosphate solution with a concentration of 106 UFC/ml with 0.1 g of three-dimensional structures in quantities ranging from 0.2 ml (relation of 2 ml/g).
E. coli fluorescence is observed at 280 minutes.
A strain of E. coli ATCC 25922 is tested as described in Example 1, and which is formed according to V2 of that example, along with a nutritional compound prepared as described in said variant 2 of Example 1, with the difference that only fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) is added in the amount of 0.2 g/L.
The nutritional mixture, along with the enzymatic marker and other components dissolved in the first solvent are put into contact with 3 three-dimensional structures.
The first structure of artificial ceramic nature is formed by a nano-layer of hydroxyapatite with nanoporosity, a specific surface of 50×106 m2/m3 and 20 nm high nanocavities, resting over a lower agaropectin layer in the amount of 0.5 g/g. The structure also rests over the entire surface of a 6-cm diameter disc that is impervious to cellulose derivatives, specifically cellulose nitrate, with which an initial device is formed (variant 16).
The second structure is made of siliceous earth, with a specific surface of 3×105 m2/m3 to 1×106 m2/m3, and nano- and microcavity porosity, on which the nutritional compound is mixed with agar (0.3 g/g) dissolved in the first solvent. The second device is prepared by placing the siliceous earth structure described in a cylinder 3-mm high with a diameter of 90 mm (variant 17).
The third three-dimensional structure is formed by hydroxyapatite with a minimum specific surface of 1.25×103 m2/m3 with irregular macro- and microcavities, followed by a three-dimensional zeolite structure with a specific surface larger than 5×103 m2/m3. The nutritional compounds of Example 1, variant 2 and the fluorogenic substrate described in said variant are absorbed on the first and second structures. The overall height of these three-dimensional structures reaches 10 cm with a 4-cm diameter, forming a device in which this three-dimensional column is placed in a tube made of impervious material with openings on its upper and lower portions, in such a way that the tube of impervious material (PVC) surrounds 90% of the external surface of the structure (variant 18).
An E. coli suspension is put into contact with a saline isotonic solution with a concentration of 102 UFC/ml over the entire surface of the first device (V16), aided with a swab soaked in sodium alginate. The three-dimensional structure is then maintained under a temperature of 35±2° C. and aerobic conditions over a 120-minute period, whereas growth is detected by the emission of fluorescence under light at 366 nm aided by a sensor and identified by glucuronidase activity.
An E. coli suspension is put into contact with a saline isotonic solution with a concentration of 10 UFC/ml over the entire surface of the second device (V17), aided by an automatic pipette and is distributed with a Drigalski spatula. The three-dimensional structure is then maintained at temperatures of 35±2° C., under aerobic conditions over a 240-minute period, whereas growth is detected by the emission of fluorescence under light at 366 nm, aided by a sensor and identified by glucuronidase activity.
A 10-L sample of deionised water, artificially contaminated with E. coli at a concentration of 8 UFC/10 L, is passed throughout the entire volume of the device and is left for incubation under the conditions described in the previous experiments. After 210 minutes, fluorescence was observed inside the device.
Four three-dimensional structures of natural clays or ceramics were evaluated with two nutritional compounds for identifying Gram-negative bacteria.
Nutritional components were selected according to Example 1.
Different devices were prepared with this compound in order to implement the method.
The selected supports were HAP-S(specific surface of 7.5×103 m2/m3) and HAP-56 (specific surface of 3×105 m2/m3) ceramics, as well as siliceous earth purified and calcined (TSC) (specific surface of 5.3×104 m2/m3).
A nutritional mixture was prepared with fluorogenic and chromogenic markers and other components according to V1 of Example 1. In parallel, another mixture of a fluorogenic compound with salts and other components according to the following compound was prepared: ammonium sulfate [(NH4)2SO4] 5.0 g/L of the first solvent; hydrogen potassium phosphate [K2HPO4] 0.45 g/L; dihydrogen potassium phosphate [KH2PO4] 0.31 g/L; hydrogen sodium phosphate [Na2HPO4] 0.92 g/L; sodium chloride [NaCl] 0.1 g/L; calcium chloride [CaCl2] 0.05 g/L; heptahydrated magnesium sulfate [MgSO4.7H2O] 0.2 g/L; L-histidine monohydrochloride 0.005 g/L; L-tryptophan 0.02 g/L; L-methionine 0.02 g/L, dextrose 10.0 g/L and MUG in the amount of 0.2 g/L.
The pH of both compounds was adjusted to 6.8, and they were sterilized by filtration through Nalgene disposable filtration units (0.2 μm, pore size) (Nalge Co., Rochester, N.Y.).
The three-dimensional structures were prepared with the mixtures using the following methodology:
The final devices contained: ammonium sulfate [(NH4)2SO4] 10 mg/g of three-dimensional structure; hydrogen potassium phosphate [K2HPO4] 0.9 mg/g; dihydrogen potassium phosphate [KH2PO4] 0.62 mg/g; hydrogen sodium phosphate [Na2HPO4] 1.84 mg/g; sodium chloride [NaCl] 0.2 mg/g; calcium chloride [CaCl2] 0.1 mg/g; heptahydrated magnesium sulfate [MgSO4.7H2O] 0.4 mg/g; L-histidine monohydrochloride 0.001 mg/g; L-tryptophan 0.04 mg/g; L-methionine 0.04 mg/g and dextrose 20.0 mg/g and MUG in the amount of 0.4 mg/g, for un total of 34.55 mg/g.
The design of the experiment covered the following assay variants:
In order to inoculate all variants of the study, microbial suspensions of approximately 108 UFC/ml were prepared in 9 ml of a sterile dual salt 0.85% (pip) solution from pure cultures of Escherichia coli ATCC 25922 incubated for up to 24 hours. The volume of the inoculum was 0.2 ml for each assay variant, ensuring an inoculum concentration of 2×107. Inoculated devices were incubated at 35±2° C. under aerobic conditions.
Readings were made visually, using a 366 nm UV lamp every 30 minutes.
The results obtained are shown on the following table, stating the period in hours and minutes of positive response to fluorescence.
E. coli
This indicator shows that the microbial species has enzymatic activity that corresponds to the enzymatic marker used on the compounds.
It is also proven that it is not obvious that each nutritional compound combined with a three-dimensional clay structure responds in a shorter time or detects or identifies microorganisms in a shorter period.
It is also proven that the combination of clay or ceramic structures with nutritional mixtures, enzymatic markers and other ingredients accelerates identification in regards to V21 to V23 variants that did not contain nutritional components based on protein hydrolyzed and extracts that were previously protected by the authors of this invention, thus proving that it is paramount to use compounds that allow shortening the lag growth phase of bacteria by reducing the detection and identification time.
This experiment also proved that, surprisingly, the nutritional compounds designed are capable of attenuating or eliminating the inhibitor effect or antibacterial application of this structure, as described by different authors for certain biotechnological processes, in particular that of zeolite (V21) when a nutritional compound especially designed for eliminating this antibacterial effect is added.
The four three-dimensional structures of natural clays or ceramics were evaluated with two nutritional compounds for identifying the Gram-negative bacteria described in the previous example (Example 9).
The selection of nutritional components was likewise carried out in accordance with the methodology of Example 1, but with a strain of Pseudomonas aeruginosa (ATCC 27853). Individual and mixed hydrolyzed enzymes were evaluated, among them papain-hydrolyzed of beef heart tissue, according to Cuban Invention Copyright Certificate No. 22442, and pancreatic hydrolysate heart tissue and their mixtures. Results showed that for these 3 cases the lag phase had a maximum duration of 120 minutes; therefore, both were selected for preparing the nutritional compound.
Different devices were prepared with this compound in order to implement the method.
A nutritional mixture was prepared with fluorogenic and chromogenic markers and other components according to V1 of Example 1. In parallel, another mixture of a fluorogenic compound was prepared with salts and other components according to the description of the previous example (MCS).
All other devices and inocula were prepared according to the descriptions of Example 9, with the difference that the inoculum in the devices of Pseudomonas aeruginosa ATCC 27853 and of Enterococcus faecalis ATCC 29212 were added to the experiment.
The design of the experiment covered the following assay variants:
The results obtained are showed on the following table, stating the period in hours and minutes of positive response to fluorescence.
P. aeruginosa
E. faecalis
The unexpected appearance of greenish fluorescence of Pseudomonas was observed in the three-dimensional ceramic structures after only 3 hours in variant V18; this effect had never been achieved with any other known diagnostic tool.
Secondly, it was proven that in order to detect and identify certain microorganisms in as little time as possible, the specific surface and size of cavities (3 hours for V18 and 24 hours for V19) is of the essence, as well as the dependence on this specific type of clay combined with a mixture for eliminating the inhibitor effect of these clays or ceramics (detected in V18 and not detected in V20 or V21).
It was also proven that the combination of two structures with different compounds in the same device (V19+V22) finally lead to identifying the target microorganisms.
It was proven once more that specific nutritional compounds selected among those that shorten the lag growth phase to a few minutes ensure detection, identification and recuperation in a few minutes (280 minutes).
The selective character of this device was proven, as it inhibited E. faecalis, because the compound embedded in the clay structure, along with it, were able to inhibit it—even with concentrations as high as 107 UFC/ml.
Evaluation of three kinds of three-dimensional structures of natural clays or ceramics (HAP-S, HAP-56 and TSC) with two nutritional compounds for the identification of Gram-positive bacteria.
Different mixtures of nutritional bases were used for testing bacterial growth promotion (Staphylococcus aureus and Streptococcus pyogenes), among them extract of Saccharomyces cerevisiae obtained through enzyme hydrolysis as described in Cuban Invention Copyright Certificate No. 22221, hydrolyzed enzymes of bovine blood (Cuban Invention Copyright Certificate No. 22208), hydrolyzed enzymes of casein (Cuban Invention Copyright Certificates No. 22089, hydrolyzed enzymes of commercial soy and commercial meat extract.
The first mixture (M1) contained: extract of Saccharomyces cerevisiae—3.24 g/L; hydrolyzed enzymes of bovine blood—6.37 g/L; hydrolyzed enzymes of casein—9.97 g/L; commercial soy peptone—3.24 g/L and commercial meat extract—2.43 g/L.
The second mixture (M2) contained: extracts of Saccharomyces cerevisiae—5.0 g/L; hydrolyzed enzymes of bovine blood—5.0 g/L; hydrolyzed enzymes of casein—5.0 g/L; commercial soy peptone—6.0 g/L and commercial meat extract—3.0 g/L.
The third mixture (M3) contained: extracts of Saccharomyces cerevisiae—6.0 g/L; hydrolyzed enzymes of bovine blood—6.0 g/L; hydrolyzed enzymes of casein—3.0 g/L; commercial soy peptone—6.0 g/L and commercial meat extract—4.0 g/L.
The products to be tested were inoculated with 0.1 ml of a suspension of target microorganisms with a concentration of 3×108 UFC/ml.
The mixture and the bases were incubated separately for 8 hours at 37° C. under an aerobic atmosphere, and the increase of optical density was monitored with a spectrophotometer at 680 nm.
All variants shortened the lag phase to only 1 hour for S. pyogenes, and the duration of this phase for S. aureus was only 2 hours. M3 was chosen among these, since the growth of S. pyogenes was slightly more intense in it.
The following nutritional compounds were tested:
Compounds were dissolved in direct proportion with a first solvent (deionised water) whose pH was adjusted to 7.3. They were sterilized by filtration using 0.2 μm disposable filtration units. The devices were prepared following the methodology described in Example 10, and the design of the experiment covered the following assay variants:
Target species Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 19615 were chosen for this experiment. The preparation of their dilutions, inoculation and incubation were conducted as described in Example 10.
The following tables show the fluorescence development period (in hh:mm) for each of the microbial species evaluated against each enzymatic marker, using only those compounds from variants 24 to 27—this is, only those containing the nutritional mixture.
S. aureus
S. pyogenes
The evaluation carried out with this fluorogenic substrate proved that the most satisfactory variants—in terms of response speed—were the combinations of STR-STAP with HAP-S (variant 24) that in only 3 to 5 hours of incubation allowed detection of S. aureus and S. pyogenes.
Variant 25 allowed detecting microorganisms with very high nutritional requirements, such as S. aureus in only 5 hours and S. pyogenes in only 24 hours.
Once more it was proven that only the combination of nutritional mixtures with enzymatic markers and three-dimensional ceramic structures (V24-V25) are capable of accelerating microbial detection when compared to compounds alone (V26), or even achieving their detection (V26 negative for S. pyogenes).
Other combinations were prepared for creating compound devices with two structures and two different compounds: one containing hydrolyzed and extracts, and the other synthetic:
The combination of structures from the studied variants brought better results in terms of detection time for one of the combined devices, so it was not necessary to combine all the others, as described below:
Study of the response from different devices and the method before a urine sample. The devices were formed using four different kinds of three-dimensional structures of natural and artificial clays and ceramics (HAP-S, HAP-56) each combined with the compound described in variant 1 of Example 1 and carrying out the method as described in Example 9.
The inoculum used was 0.2 ml of the sample for each variant.
In parallel, the sample was evaluated using the traditional procedure, using agarized media such as CromoCen CC and bromothymol blue lactose agar (ABL).
In both testing schemes (the new method with the clay device and the traditional method), the inoculated samples were incubated at 35° C. Test readings were taken every 30 minutes after applying the device, using a 366 nm UV lamp.
The following table shows the period (in hh:mm) after which fluorescence was detected, which proved the presence of infection in the clinical sample, related with a positive species on the enzymatic marker used.
The combination of CCL with HAP-56 was the variant that responded faster, in 2 hours and 30 minutes, followed by the combination with the HAP-S structure that was 3 hours. This result indicates that E. coli is the bacteria that caused the infection, considering the selectivity of the nutritional compound and of the device, and that in most cases on record this kind of sample responds to this species.
E. coli was only detected in both media (CromoCen CC and ABL) after 24 hours when using conventional procedures.
This proves how accurate the method and the device are, and that it accelerated the procedure by at least 8 times.
Study of the relationship between the nutritional capacity of the compounds and the development of microbial enzymatic activity over an enzymatic marker.
For this purpose, the nano-compound CCL with HAP-S, prepared as described in Example 9, was selected. On the other hand, a variant using HAP-S as support was prepared, in which an aqueous solution of MUG with a concentration of 02 g/L (p/v) was added and then was dehydrated following the same methodology described in Example 9.
A pure culture of E. coli was used as assay microorganism, from which a 108 UFC/ml suspension was prepared. 0.2 and 0.4 ml volumes were used for the study, producing inocula that were applied on the structures of up to 107, and then both devices were inoculated in parallel: CCL with HAP-S(V35) and MUG with HAP-S(V36).
The variants were incubated at 35° C., observing their response to fluorescence every 30 minutes, using a 366 nm UV lamp.
The results obtained are shown in the following table, highlighting the time in hh:mm after which a positive response to fluorescence was observed.
This experiment proved the significant influence of the combination of hydrolyzed or extracts obtained from nutritional substances of protein nature present on the nutritional compound used. This allowed the microbial species to adapt faster to the conditions of the device and showed enzymatic activity during its development within the lag phase (2 h) that allowed in this case its identification before the enzymatic marker used for a minimum period of 1 hour and 30 minutes. Nevertheless, for the structure that contained only the fluorogenic substrate, its response was detected 2 hours later, since this is not based on the activation of enzymatic mechanisms, but on detecting them at much higher concentrations, which is the method used in the state-of-the-art solutions mentioned above.
Study of the influence of pH of the compound over the action of the fluorogenic substrate for revealing microbial enzymatic activity.
Using the compound CCL described in Example 9, four experimental variants were prepared with different pH values (6.6-6.8-7.0-7.2). Each variant was sterilized through filtration and was independently embedded in the HAP-S ceramic (variant 37) and inoculated and incubated following the procedure mentioned in Example 9.
The results obtained are showed in the following table, stating the period (hh:mm) of positive response to fluorescence as indicator of microbial enzymatic activity over the marker used,
The results show that there is no significant influence of the compound pH on the detection of the enzymatic activity of the (E. coli) microorganism with the enzymatic marker used (MUG).
Positive response was detected in the period between 1 hour and 30 minutes to 2 hours of culture.
Evaluation of four three-dimensional structures of natural clays or ceramics (HAP-S, HAP-56 and TSC) using two nutritional compounds and two enzymatic markers for independent tests aimed to identify a species of the Candida genre.
In order to select the nutritional compound(s), the reduction of the lag phase duration with vegetal extracts of sweet potatoes that was previously developed by the authors of this invention, namely hydrolyzed enzyme of casein, soy peptone, a mixture of yeast peptones and hydrolyzed enzymes, all in quantities ranging from 0.2 g/L was studied. Optical density was monitored every 1 hour with a 380 nm spectrophotometer. The results showed that the extracts of sweet potatoes shortened the lag phase for C. albicans by at least 1 hour from all other comp components, requiring only 16 hours.
The first experimental variants were prepared using the fluorogenic MU-phosphate substrate as the first enzymatic marker. The substrate was added to the CND compound in the amount of 0.2 g, formed by: extract of sweet potatoes 20.0 g; extract of Saccharomyces cerevisiae yeast 10.0 g; potassium phosphate 1.0 g; magnesium sulfate 0.5 g; sodium desoxycholate 0.5 g and nalidixic acid 0.03 g, for one liter of deionised water.
The other compound used was the MCS medium, to which was added the same quantity of MU-phosphate (0.2 g/1).
Similar variants were prepared in parallel, using L-proline methylcoumarin (L-Pro) as enzymatic marker substrate, 0.2 g of which were added to the CND and MCS compounds, respectively.
The nutritional compounds prepared with the enzymatic markers and other ingredients prepared for each experimental variant were proportionally dissolved with one liter of deionised water as first solvent and their pH was adjusted to 6.6. They were sterilized by filtration using 0.2 μm disposable filtration units.
Structures were prepared following the methodology described in Example 9 and the design of the experiment covered the following assay variants:
The microbiological evaluation was conducted with the reference strains: Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019 and Candida glabrata ATCC 15126, just harvested from Sabouraud dextrose agar for 36 hours. Suspensions from these cultures were prepared in 9 ml tubes with a sterile dual salt 0.85% (p/p) solution until a microbial density of 108 UFC/ml was achieved.
A 0.2 ml volume was inoculated with each variant of the devices according to the method and incubated at 35° C. Fluorescent response was recorded every 30 minutes using a 366 nm UV lamp.
The results obtained are shown independently for each enzymatic marker in the following tables, showing the period (hh:mm) in which a positive response to the fluorescent reaction was observed.
C. albicans
C. parapsilosis
C. glabrata
First of all, it should be noted that the three Candida species evaluated exhibited phosphatase activity; therefore, in all cases variants were examined with the purpose of detecting positive response to fluorescence before the MU-phosphate substrate.
On the other hand, once more the evaluation of this fluorogenic substrate (MU-phosphate) proved its decay before the HAP-56 ceramic, because since the nano-compound was hydrated, it revealed the appearance of positive response to fluorescence, which is not related to the enzymatic action of the microbial species. This nullifies the reading from the outset, since it was reported as “not useful.”
On the other hand, TSC clay somehow blocks the response either from the enzymatic activity of the tested microorganisms or of the specific fluorogenic substrate, since the biological functionality of the nano-compound is not observed throughout the entire culture period.
Regarding HAP-S ceramic, a similar response was detected for each tested compound—although it was different for each Candida species.
With C. albicans it was detected that the species was not able to show its activity over the MU-phosphate substrate, while with the C. parapsilosis and C. glabrata species fluorescence appeared after 15 hours of culture—although with very low intensity, along with the appearance of an indicator that did not increase its intensity during a larger incubation period.
In general, the response obtained from using this enzymatic marker (MU-phosphate) as part of the nutritional compounds is closely related to the structure used as nano-structured support; therefore, the response from Z ends up being the most convenient one.
The following table shows the results found with fluorogenic substrate L-Pro.
C. albicans
C. parapsilosis
C. glabrata
afluorescence with greater intensity
According to other studies done by the authors of this invention for detecting the enzymatic activity of several Candida species, it is well known that the C. albicans and C. parapsilosis species have enzymatic L-proline amidase. On the other hand, C. glabrata does not have the action of said enzyme.
Based on this prior knowledge, a reading was made, looking for a positive response to the fluorescence for the C. albicans and C. parapsilosis species.
It was noticed that the variants produced with the HAP-S, HAP-56 and TSC structures for both culture media (CND and MCS) showed satisfactory results by allowing the detection of C. albicans and C. parapsilosis in only 15 hours. Nevertheless, the greater intensity of the fluorescent response when using the HAP-56 ceramic is noticed in the original case.
Regarding the use of zeolite as support with this fluorogenic substrate, an incompatibility was found since it did not reflect microbial activity nor did it block in any way the decay of the L-Pro substrate, which makes the microbiological functionality of the nano-compound not ideal.
Similar to variant 1 of Example 1, with the difference of the following variants:
V50—set of natural zeolite clays compressed as a tablet, with a total weight of 0.2 g and a specific surface of 3×105 m2/m3 impregnated with the nutritional compound according to V1 of Example 1.
V51 powdered carbonateapatite [Ca5(PO4,CO3)3(OH)], placed on a 1 cm high and 1 cm tall pot, with a weight of 0.5 g and a specific surface of 4×103 m2/m3 and 700 μm cavity size, impregnated with the nutritional compound according to V2 of Example 1.
V52—zeolite cubes with a total weight of 0.2 g and a specific surface of 7.0×103 m2/m3, impregnated with the nutritional compound according to V44 of Example 15.
The structures were left absorbing the nutritional compounds for 1 hour and were vacuum dried for 3 hours at 60° C.
They were inoculated with 106 UFC/ml of E. coli inoculum (V50 and V51) and a 0.2 ml (1 ml/g) volume of C. albicans (V52).
E. coli fluorescence is observed after 180 minutes in V50 and 210 minutes in V51, and that of C. albicans is observed in V52 after 18 hours.
Bacteria such as E. coli, E. coli 0157:117, Aeromonas hydrophila, Enterococcus avium and the filamentous fungi Aspergillus niger were taken for a bacterial growth test.
Each one of these microorganisms is tested with different nutritional bases, such as papain-hydrolyzed heart tissue, according to Cuban Invention Copyright Certificate No. 22442 in the amount of 0.2 g/L of deionised water; extracts of Saccharomyces cerevisiae yeast according to Cuban. Invention Copyright Certificate No. 22221, in the amount of 0.2 g/L; hydrolyzed enzymes of casein (Cuban Invention Copyright Certificates No. 22166); 0.2 g/L of sweet potato extract, as disclosed in Cuban patent No. 23507; tomato extract in the amount of 0.2 g/L (Cuban Invention Copyright Certificate No. 22308); and hydrolyzed enzymes of bovine blood (Cuban Invention Copyright Certificate No. 22208) in the amount of 0.2 g/L. Likewise, a mixture of all of them was formed in quantities ranging from 0.2 g/L of each one of them.
The bases and the mixture were incubated separately for 8 hours at 37° C. under an aerobic atmosphere, and the increase of optical density was monitored with a spectrophotometer at 680 nm.
Of all the variants, the nutritional mixture showed a reduction of the lag growth phase of all microorganisms after 90 minutes, with the exception of Aspergillus while the individual bases showed a variable phase duration, and in some cases higher than 2 hours; therefore, this mixture was chosen for the experiments.
This mixture is dissolved on a saline solution (NaCl with 9.5 g/L) in the amount of 10 g/L.
As soon as the nutritional compound was prepared, it was added different enzymatic markers, one chromogenic [2-nitrophenyl-β-D-galactopyranoside (C12H15NO6)], in quantities ranging from 1 g/L and three fluorogenic (4-methylumbelliferil-β-D-glucoronide, 4-methylumbelliferyl-β-D-galactoside and 4-methylumbelliferyl-β-D-glucoside) in quantities ranging from 0.2 g/L each one.
Other substances were added to the mixtures of the nutritional compounds and enzymatic markers, such as growth promoters, specifically glucose (10 g/L); inorganic salts, specifically monobasic potassium phosphate (5.5 g/L) and dibasic potassium phosphate (5.5 g/L). The nutritional compound selected, along with the enzymatic markers and other components, are found dissolved in the first solvent in quantities ranging from 32.6 g/L.
Once the nutritional mixture is formed and along with the enzymatic markers and other components dissolved in the first solvent, they are sterilized by filtration. Nutritional mixtures along with the enzymatic markers and other components dissolved in the first solvent are put into contact with one and multiple three-dimensional structures of artificial ceramics, specifically the calcined hydroxyapatite that had been previously sterilized at 180° C. for 60 minutes.
Contact time of the compound is of 30 minutes.
This structure has a specific surface of 5×103 m2/m3 and is formed by a variety of nano, micro- and macrocavities.
The three-dimensional structure has cavity dimensions that correspond to different combinations of all cavity diameters or clearances corresponding to nano-, semimicro- and microcavities with diameters or clearances of 5 nm to 10 μm with pore shapes. These structures have a cylinder shape, with a 100 cm diameter and height of 2 cm.
When the absorption was completed, the first solvent was eliminated by drying the three-dimensional structures at a temperature of 60° C. in a vacuum oven over a 3-hour period.
The capacity of recovering target microorganisms is verified after proving that the structures have detection limits of less than 1 UFC/100 ml by filtering 1 L of an artificially inoculated E, Coli suspension with a concentration of up to 6 UFC, which means that there are 0.6 UFC for every 100 milliliters, whereas they can be detected by fluorescence and through the yellowish hue of the structure.
For this assay, the suspensions of target microorganisms in saline isotonic solution with a concentration of 3×106 UFC/ml are put into contact with the three-dimensional structures in quantities ranging from 1 ml and are distributed throughout the entire surface.
The three-dimensional structures are then maintained at temperatures of 35±2° C., under aerobic conditions for a period of up to 4 hours that coincides with the duration of the lag growth phase of E. coli.
When the incubation is completed for 4 hours at the most, the presence of target microorganisms is detected on all variants, on one case inoculated individually with each microorganism and on another case with the mixture of all of them. In the case of E. coli, fluorescence and color change of the structure to a yellowish hue were observed; for E. coli 0157:H7 and Aeromonas hydrophila, only the color change of the structure was observed. In all cases, detection took place after 2 hours.
In the case of Enterococcus avium, blue fluorescence with no color change was observed in the structure after 3 hours, and the filamentous fungi grown like a black structure over the surface of the device, although a yellowish hue was observed first on the growth zone. In the event that the sample is inoculated with the mixture of microorganisms, all reactions can be observed.
The S. aureus strain is tested as described in Example 11, and a nutritional compound and device is prepared according to V26 of that example, with the difference that the three-dimensional structure has the shape of a 0.1 mm high and 60 cm diameter disc. A 3 m3 air volume is passed through the entire the volume, aided by an air filtration device that sucks it due to negative pressure. Contamination is simulated with the assay strain with a concentration of 105 UFC/m3 prior to the air filtration, in order to verify if its flow is any influence on drying the structure or on its operation. When the device is exposed to air, it is moistened with the second solvent, consisting of distilled water and is left to incubate under the temperature and conditions described on Example 11. S. aureus can be identified after 240 minutes.
Similar to Example 1, with the difference that two devices according to V1 of Example 1 are prepared, whereas to one device ciprofloxacin is added in the amount of 0.003 μg and in the other gentamicin in the amount of 0.2 μg. 0.2 ml of the microbial suspension is inoculated with an E. coli strain isolated from a urine culture with a concentration of 3×108 UFC/ml and is incubated for 4 hours. No E. coli growth is observed after 4 hours in the device containing ciprofloxacin, or fluorescence in the device containing gentamicin.
| Number | Date | Country | Kind |
|---|---|---|---|
| /P/2012/0055 | Mar 2012 | CU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CU2013/000002 | 3/27/2013 | WO | 00 |
