WO2017198240A1 - System for the detection of photosynthetic and non-photosynthetic microorganisms in biological samples by means of controlled photostimulation - Google Patents

System for the detection of photosynthetic and non-photosynthetic microorganisms in biological samples by means of controlled photostimulation Download PDF

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WO2017198240A1
WO2017198240A1 PCT/CU2017/050004 CU2017050004W WO2017198240A1 WO 2017198240 A1 WO2017198240 A1 WO 2017198240A1 CU 2017050004 W CU2017050004 W CU 2017050004W WO 2017198240 A1 WO2017198240 A1 WO 2017198240A1
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photosynthetic
stimulation
growth
detection
microorganisms
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PCT/CU2017/050004
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Spanish (es)
French (fr)
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Nardo RAMÍREZ FRÓMETA
Ángel Regueiro Gómez
Carlos Abel LAMOTHE NUVIOLA
Elier Riverón Rodríguez
Carmen Yamilé Moreno Barrios
Orestes Rolando Contreras Alarcón
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Centro Nacional de Investigaciones Científicas
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
    • G01N21/534—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke by measuring transmission alone, i.e. determining opacity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity
    • G01N21/5907—Densitometers

Definitions

  • This invention constitutes a system (device and method) for the rapid evaluation of the state of microbiological contamination of different biological samples, based on the use of optical stimuli (430 nm ⁇ ⁇ 480 nm and 1 ⁇ ⁇ ⁇ ⁇ p ⁇ 5 ⁇ ⁇ ⁇ /), which increase the growth rate of photosynthetic and non-photosynthetic microorganisms present in a liquid culture medium facilitating their rapid detection.
  • Photo-stimulation is normally used to inhibit or contain bacterial growth (Photostimulation method and photostimulation device, CN 103212161 A; LED multiplex source and method of use of for sterilization, bioactivation and therapy, US 20050256554 A1), or to stimulate photosensitive bacterial species or with fluorescent markers.
  • Photostimulation method and photostimulation device CN 103212161 A; LED multiplex source and method of use of for sterilization, bioactivation and therapy, US 20050256554 A1
  • there is no report of devices that allow for photo-stimulation an increase
  • a light signal from an emitting LED is applied, which allows the stimulation of the growth of bacterial species, and simultaneously, through the measurement of turbidity or bioimpedance of the culture, the detection of the bacteria is possible.
  • optical stimuli can increase the activity of many species from the response of their photo-receptor proteins [Masuda, S (2013), “Light Detection and Signal Transduction in the BLUF Photoreceptors", Plant Cell Physiol. 54 (2): 171-179].
  • US20100099132A1 relates to a new method of preparing bacterial growth inducers based on the use of light, characterized in that the method comprises cultivating Hafnia spp. at a minimum and because the growth inducer is not a self-inducer.
  • the object of the invention US20130190843A1 is to provide a photo-stimulation method in which LEDs are configured to emit red or yellow light in a specific range of illuminance, in order to stimulate the synthesis of collagen in fibroblasts and promote the circulation of the blood; as well as accelerating the removal of dead cells.
  • the LEDs can be configured to emit blue light of a certain intensity to inhibit or kill P. acnes or to suppress or reduce the synthesis of melanin from melanocytes.
  • the invention WO201307491 1 A1 provides methods for light-dependent gene regulation using a photosensitive DNA-binding protein.
  • the invention provides a recombinant nucleic acid molecule comprising a sequence encoding a light sensitive DNA binding protein (LRDP) comprising: a) a LOV domain; and b) a DNA binding domain (DBD) and is operably linked to a polynucleotide.
  • LRDP light sensitive DNA binding protein
  • DBD DNA binding domain
  • the invention WO2014072934A1 describes a method for stimulating the metabolism of non-phototrophic microorganisms involved in biological processes. It claims a biological process that includes a stage of illumination with blue light of non-phototrophic microorganisms to stimulate their metabolism.
  • the main application of this invention is the treatment of municipal wastewater and in biotechnological processes for the bioproduction of molecules of interest.
  • blue light stimulation took into account the intensity of the stimulus but the influence of the waveform and the frequency in stimulation on cellular metabolism are not analyzed.
  • the effect of stimulation on samples of clinical origin with effects on rapid microbiological diagnosis is not analyzed [Tiphlova, O and Karu, T (1991), "Action of low-intensity laser radiation on Escherichia coli", Crit Rev Biomed Eng. 18 (6): 387-412].
  • the objective of the present invention is to provide a system (device and method) that allows the detection of microbial growth early in biological samples in which it is required to detect the growth of microorganisms through the use of micro-samples.
  • This system is based on the rapid detection of turbidimetric or bioimpedance changes produced by the growth of photosynthetic and non-photosynthetic microorganisms present in an optically stimulated liquid culture medium (430 nm ⁇ ⁇ 480 nm and 1 ⁇ ⁇ ⁇ ⁇ p ⁇ 5 ⁇ ⁇ ⁇ /).
  • the novelty of the present technical solution is that it presents a method for rapid diagnosis based on the combined use of optical stimulation and the measurement of turbidity or bioimpedance in biological samples, materialized from the use of a new detection system formed by the integration of hardware and software specially designed for this purpose.
  • the turbidity and / or bioimpedance changes caused by controlled optical stimulation in the culture medium are recorded in the biological sample in liquid medium, which allows the presence of bacteria to be detected, facilitating the study of their microbiological quality.
  • the proposed system is useful for the determination of the antibiogram from isolated colonies or positive samples that are obtained directly from the sources that contain them.
  • the present invention allows to obtain the antibiotic sensitivity scheme of microorganisms either from previously isolated colonies or from positive biological samples, in the latter case saving the time required for isolation and purification processes .
  • the present invention allows, from direct urine samples, to discriminate positive samples from negative ones, whether or not they are contaminated with another germ. With the system of the present invention, results based on 350 examined samples have shown 100% correspondence with the total viable cell count in CLED medium, a method conventionally used for the detection of urinary system infection.
  • the proposed system is characterized by its rapidity, since it allows the determination of urinary infection in less than an hour, and from positive samples it offers reliable results of the antibiogram in less than two hours. It is also a highly accurate system that allows corroborating the results obtained as many times as estimated. From the social point of view it is of great importance, since for those hospitalized people it allows the supply of antibiotics in a rational and timely way, avoiding prolonged hospital stays. Likewise, from the ecological point of view it has a great impact since avoiding the inappropriate use of antibiotics and at the same time limits the development of bacterial resistance.
  • SYSMEX is a reference method for the detection of bacteria and sepsis.
  • the bacteria present must be identified before starting the antibiotic supply, using various methods, from conventional biochemical tests to PCR-based DNA tests, which take an additional 3 to 24 hours.
  • the present invention describes a new measurement system (Fig. 1) that allows to optically stimulate the growth of photosynthetic and non-photosynthetic microorganisms present in a liquid culture medium, which is used as a rapid detection method that decreases the timing of Sample analysis.
  • the proposed invention comprises a device formed by an electronic card with two measurement channels: one with an array of cells for turbidimetric measurements (Fig. 2) in samples with medium or low optical density, and another with an array of cells for impedance measurement with electrodes for high optical density samples, and a common control interface ⁇ software) associated (Fig.
  • the electronic card is structured with a modular design with specific functions and objectives for each module:
  • the Central Processing and Control Unit, UCPC (1) is responsible for generating and synchronizing all control actions on the rest of the blocks.
  • the turbidity and bioimpedance measurement channels are useful for the acquisition and conditioning of the acquired variables
  • the communication module (2) establishes communication between the UCPC processor and the personal computer (3) through commands; and the power supply (4) has the function of supplying power to all devices and blocks of the design.
  • said electronic card is associated with a program tool ⁇ software) installed in a personal computer for automatic reading and storage of the measurement data via USB (communication module), which guarantees the adequate selection of parameters during the process of characterization of the detection of microorganisms in biological samples.
  • the application developed allows the programming of the stimulation parameters of the samples under study through three working windows, also guaranteeing adequate storage and review of the data acquired when requested by the user (Fig. 3).
  • the measurement system consists of a generator of the stimulus signal (5) and reference (6); in addition to a voltage-current converter (7) that supplies the controlled optical stimulus (Sine wave, 430 nm ⁇ ⁇ 480 nm, 1 ⁇ ⁇ ⁇ / ⁇ p ⁇ 5 ⁇ ⁇ ⁇ /) to an array of five LEDs (8 ) properly located in the sample holder (9), and which are responsible for transmitting the signal to stimulate the samples (10) to be analyzed.
  • the generated signal is also used as a synchronous reference for the demodulation process of the signal received from the receivers (photo detectors).
  • the optical stimulus that passes through the sample is received with the help of an opto-electronic integrated circuit (1 1), which contains a photodiode (photodetector) and a monolithic transimpedance amplifier.
  • the signal at the output of the photodetectors is selected with a multiplexer (12) and is transmitted to a conditioning section (13), passing to a synchronous demodulation block (14), filtering (15) and conversion (16) , using a hitch amplifier structure for the synchronization of the demodulation process.
  • a channel for impedance measurements from a cell has been added, using an electrode array Bipolar stainless steel (17) with a length of 10 cm and a diameter of 1 mm, and a high precision impedance converter AD5933 (18) that combines an internal frequency generator with a digital-analog converter.
  • the generator allows the biological sample to be excited with a known frequency in this case of high optical density (19).
  • the signal obtained from the analyzed sample is acquired by the AD5933 internal analog-digital converter and is processed by applying the Fourier Discrete Transform (TDF) by the internal digital processor (PDS).
  • TDF Fourier Discrete Transform
  • PDS internal digital processor
  • the applied algorithm returns a real and imaginary value of the measured impedance, which is transferred by means of a communication protocol (21) to the UCPC and from this to the personal computer (3) for graphic representation in the developed interface.
  • Another fundamental application of this invention is directed towards the determination of the sample antibiogram (susceptibility of microorganisms to antibiotics), which is achieved in a period of time less than 2 hours, using a diagnostic kit designed for these purposes.
  • the culture medium used to follow microbial growth is the modified Mueller-Hinton Broth OXOID medium, pH 7.4 ⁇ 0.2 and sterile, which additionally includes a polymer.
  • the diagnostic kit for the detection of the antibiogram of the sample to be analyzed is characterized by the use of antibiotic discs that are commercially available and that can be used in accordance with schemes that can be varied according to any need.
  • the proposed system allows to face the interferences generated by the contamination of the samples; as well as the infection caused by more than one germ.
  • the contamination interference has been resolved by adjusting the signal in magnitude and time for the detection of internationally accepted infestant levels (> 100,000 CFU / ml), promoting the exclusion of contaminated samples not infested because they generally have bacterial levels below 1,000 CFU / ml, which allows the detection of contaminated samples only when they are also infested.
  • infestant levels > 100,000 CFU / ml
  • the contaminating species attending to the Gram are saprophytes, mostly sensitive to all antibiotics, it is evident that the contaminating strains should not interfere in the detection of the resistance scheme of the infesting strains.
  • Example 1 Influence of wavelength and light intensity in the optical stimulation of bacterial growth
  • Inocula A suspension of Escherichia coli ATCC 25923 was prepared with an initial concentration of 10 4 CFU / ml in DKD culture medium.
  • the culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700.
  • the suspension of Escherichia coli was stimulated with blue wavelength of low intensity continuously, with different light intensities (1, 8; 7 and 1 1 cd respectively) and likewise with red wavelengths with intensities of 7, 1 1 and 21 cd. Turbidity growth curves of the microorganism were recorded using a time between measurements of 5 minutes. The initial concentration of the suspension was corroborated by the plate colony count. Results
  • Example 2 Influence of the frequency of the stimulus signal on the optical stimulation of bacterial growth
  • the culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700.
  • the suspension of Escher ⁇ chia coli was stimulated with a blue wavelength of low intensity continuously, with a light intensity of 1 1 cd.
  • a sweep in frequencies of the stimulus signal from 10 Hz to 10 kHz was performed.
  • Turbidity growth curves of the microorganism were recorded using a time between measurements of 5 minutes. The initial concentration of the suspension was corroborated by the plate colony count.
  • Figure 6 shows the behavior for the different stimulation frequencies analyzed. Each curve represented is the average of five trials. The frequency range that shows a higher level of stimulation within the experimental conditions evaluated was between 10 Hz and 1000 Hz. In general, statistically significant differences in the detection time were obtained in this range (approximately 40% of the average time with respect to the rest of the frequencies used).
  • Example 3 Influence of the intensity of the stimulus signal on the optical stimulation of bacterial growth
  • Inocula A suspension of Escherichia coli ATCC 25923 was prepared with an initial concentration of 10 4 CFU / ml in DKD culture medium.
  • the culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700.
  • the suspension of Escherichia coli was stimulated with a blue wavelength of low intensity continuously, with a light intensity of 1 1 cd, and a given stimulation frequency (10 Hz to 10 kHz). Under these conditions, a current scan from 2.5 mA to 20 mA (maximum stimulation current) was performed, increasing the light intensity applied to the sample.
  • Figure 7 shows the microbial behavior for the different current intensities analyzed where each curve represented is the average of 5 tests. It shows that the growth of Escherichia coli is proportional to the increase in the intensity of the stimulus. The value of the current intensity, in addition to having a significant impact on the percentage of growth stimulation, also has an impact on the duration of the latency phase, associated with a decrease in the detection time.
  • Example 4 Analysis of the growth of microorganisms with and without optical stimulation
  • Inocula Several suspensions were prepared with the different clinical strains at different initial concentrations (from 10 2 to 10 6 CFU / ml in DKD medium).
  • the culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700.
  • Figure 8 shows that for any concentration, the stimulated samples (continuous line) shorten their latency phase and therefore their detection time with respect to the samples taken as a reference without optical stimulation (curves with intermittent lines), which represents for the concentration of 10 6 CFU / ml an average of 18.75% decrease in detection time. This percentage decreases as the concentration of the sample is reduced. However, the most notable difference between both groups of curves is observed in the duration of the exponential phase of the stimulated samples; the percentage of growth stimulation being greater ( ⁇ 256%) compared to the non-stimulated samples. The results obtained (Fig.
  • Example 5 Influence of controlled optical stimulation in the detection of microorganisms in clinical urine culture samples: results of clinical studies conducted in Cuba
  • the CLED reference method standard method
  • the present system was able to detect the same number of negative samples, also in a period of 20 minutes for a 100% effectiveness rate.
  • the general correspondence of the present system with respect to the traditional CLED method was 100%.
  • E.coli To prepare the different concentrations of E.coli, the following procedure was started: Prepare a concentration of 0.5 on the McFarland scale of E.coli using the DKD culture medium and then stimulate the solution for 1 hour. (guarantees that it is in the exponential phase of growth). Then prepare the initial working concentrations (10 8 , 10 7 and 10 6 CFU / ml). To perform the growth kinetics in strips for antibiogram (ATB) 200 ⁇ of each concentration are taken. To perform the susceptibility analysis, an antibiotic disk of nalidixic acid (30 mg) plus 200 ⁇ of the desired working concentration was placed in the well of the ATB strip.
  • ATB antibiogram
  • the culture was carried out at 37 ° C for 2 h with the help of a Memmert incubator model INE 700.
  • the culture of E.coli was continuously stimulated with blue wavelength of light intensity 1 1 cd, in a frequency range of stimulation between 1 Hz and 5000 Hz.
  • Figure 10 shows the temporal record of the diffusion of several antibiotic discs (nalidixic acid, cefepime and clindamycin) in the DKD culture medium. It is observed that the detection is null (all curves overlap), that is, the diffusion of the antibiotic disk in the DKD medium; as well as the DKD medium alone, they do not introduce interference into the measurement system. This behavior guarantees that any variation detected in a growth curve through this system is due solely to the growth of the pathogen of interest.
  • Figure 1 1 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 ⁇ of a concentration of 10 8 CFU / ml). You can see the inhibitory effect of nalidixic acid on the growth of E.coli, which becomes noticeable after 30 minutes of the start of registration, which coincides with the total diffusion of the antibiotic disc in the culture of E.coli . In the case of concentrations 10 7 and 10 6 CFU / ml the antibiotic load is sufficient to completely inhibit the growth of the microorganism during the analyzed recording time (Figs. 12 and 13). List of figures
  • Figure 1 represents the block diagram of the invention. (Drawings)
  • Figure 2 shows an image of the array of cells for turbidimetric measurements with the samples under study inside the MEMMERT incubator.
  • Figure 3 shows an image of the application developed for the configuration of the parameters of stimulation, storage and review of the data acquired from the samples under study.
  • Figure 4 represents the growth behavior of E. coli when stimulated with blue wavelength of various light intensities
  • Figure 5 depicts the effect of optical stimulation with blue wavelength and red wavelength at intensity of 1 1 cd.
  • Figure 6 represents the response of bacterial growth to different frequencies of optical stimulation with blue wavelength and intensity of 1 1 cd.
  • Figure 7 represents the influence of the stimulus signal intensity on the growth curve of E. coli when stimulated with blue wavelength.
  • Figure 8 shows the growth curves of E. coli obtained during the study of the photostimulation effect with blue wavelength for different concentrations (from 1 0 2 to 10 6 CFU / ml).
  • Figure 9 shows the response time of several microorganisms to optical stimulation with blue wavelength.
  • Figure 10 represents the temporal record of the diffusion of several antibiotic discs (nalidixic acid, cefepime and clindamycin) in the DKD culture medium with the use of optical stimulation.
  • Figure 1 1 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 ⁇ of an initial concentration of 10 8 CFU / ml) with the use of optical stimulation.
  • Figure 12 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 ⁇ of an initial concentration of 10 7 CFU / ml) with the use of optical stimulation.
  • Figure 13 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 ⁇ of an initial concentration of 10 6 CFU / ml) with the use of optical stimulation
  • Figure 2 Image of the array of cells for turbidimetric measurements with the samples under study inside a MEMMERT incubator.

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Abstract

The invention relates to the branch of microbiology and food hygiene and comprises a system for the quick evaluation of the state of microbiological contamination of different biological samples based on the combined use of photostimulation and detection of the microorganisms (photosynthetic and non-photosynthetic) present in a liquid culture medium. In the case of photostimulation (430 nm < λ < 480 nm and 1 μW< p < 5 μW) of a biological sample in a liquid medium, the changes in turbidity and/or bioimpedance caused in the culture medium by said growth are registered, which permits the quick detection of the presence of bacteria in biological samples based on the analysis of the culture, facilitating the evaluation of the microbiological quality of same. In addition, it is useful for determining the antibiogram based on isolated colonies or positive samples obtained directly from the sources containing same.

Description

SISTEMA PARA LA DETECCIÓN DE MICROORGANISMOS FOTOSINTÉTICOS Y NO FOTOSINTÉTICOS EN MUESTRAS BIOLÓGICAS POR FOTOESTIMULACIÓN CONTROLADA DESCRIPCIÓN  SYSTEM FOR THE DETECTION OF PHOTOSYNTHETIC AND NON-PHOTOSYNTHETIC MICROORGANISMS IN BIOLOGICAL SAMPLES BY CONTROLLED PHOTOSTIMULATION DESCRIPTION
Esta invención constituye un sistema (dispositivo y procedimiento) para la evaluación rápida del estado de contaminación microbiológica de diferentes muestras biológicas, a partir del uso de estímulos ópticos (430 nm < λ < 480 nm y 1 μ\Λ < p < 5 μ\Λ/), que incrementan la velocidad de crecimiento de los microorganismos fotosintéticos y no fotosintéticos presentes en un medio de cultivo líquido facilitando su rápida detección. Normalmente se emplea la fotoestimulación para inhibir o contener el crecimiento bacteriano (Photostimulation method and photostimulation device, CN 103212161 A; LED multiplex source and method of use of for sterilization, bioactivation and therapy, US 20050256554 A1 ), o para estimular especies bacterianas fotosensibles o con marcadores fluorescentes. Sin embargo, no existe reporte de dispositivos que permitan por fotoestimulación, un aumento en la velocidad de crecimiento de las bacterias típicas durante la detección de infecciones clínicas.  This invention constitutes a system (device and method) for the rapid evaluation of the state of microbiological contamination of different biological samples, based on the use of optical stimuli (430 nm <λ <480 nm and 1 μ \ Λ <p <5 μ \ Λ /), which increase the growth rate of photosynthetic and non-photosynthetic microorganisms present in a liquid culture medium facilitating their rapid detection. Photo-stimulation is normally used to inhibit or contain bacterial growth (Photostimulation method and photostimulation device, CN 103212161 A; LED multiplex source and method of use of for sterilization, bioactivation and therapy, US 20050256554 A1), or to stimulate photosensitive bacterial species or with fluorescent markers. However, there is no report of devices that allow for photo-stimulation, an increase in the growth rate of typical bacteria during the detection of clinical infections.
En el invento se aplica una señal luminosa proveniente de un LED emisor, que permite la estimulación del crecimiento de las especies bacterianas, y simultáneamente, a través de la medición de turbidez o bioimpedancia del cultivo se posibilita la detección de las bacterias.  In the invention a light signal from an emitting LED is applied, which allows the stimulation of the growth of bacterial species, and simultaneously, through the measurement of turbidity or bioimpedance of the culture, the detection of the bacteria is possible.
El empleo de estímulos ópticos adecuados pueden incrementar la actividad de muchas especies a partir de la respuesta de sus proteínas foto-receptoras [Masuda, S (2013), "Light Detection and Signal Transduction in the BLUF Photoreceptors", Plant Cell Physiol. 54 (2): 171 -179].  The use of suitable optical stimuli can increase the activity of many species from the response of their photo-receptor proteins [Masuda, S (2013), "Light Detection and Signal Transduction in the BLUF Photoreceptors", Plant Cell Physiol. 54 (2): 171-179].
El intervalo de estimulación óptica con 430 nm < λ < 480 nm, permite activar en especial las proteínas del dominio BLUF encontradas en el árbol filogenético de bacterias y eucariotas fotosintéticas, las cuales facilitan la regulación de las actividades biológicas a nivel celular [Jung, A (2005), "Structure of a bacterial BLUF photoreceptor: Insights into blue light-mediated signal transduction", Proc Nati Acad Sci U S A 102 (35): 12350-12355]. A partir de la excitación ocurren cambios estructurales netos en muchos foto-receptores inducidos [Horst, M. (2004), "Photoreceptor proteins star actors of modern times: a review of the functional dynamics in the structure of representative members of six different photoreceptor families", Acc. Chem. Res 37 (1 ): 13-20], que permiten el entendimiento de cómo los organismos responden a determinados estímulos ópticos [Masuda, S (2002), "AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides", Cell 1 10(5): 613-623], [Braatsch, S (2004), "Responses of the Rhodobacter sphaeroides Transcriptome to Blue Light under Semiaerobic Conditions", J. Bacteriol 186 (22): 7726-7735]. The range of optical stimulation with 430 nm <λ <480 nm, allows the activation of BLUF domain proteins found in the phylogenetic tree of bacteria and photosynthetic eukaryotes, which facilitate the regulation of biological activities at the cellular level [Jung, A (2005), "Structure of a bacterial BLUF photoreceptor: Insights into blue light-mediated signal transduction", Proc Nati Acad Sci USA 102 (35): 12350-12355]. From the excitation, net structural changes occur in many induced photo-receptors [Horst, M. (2004), "Photoreceptor proteins star actors of modern times: a review of the functional dynamics in the structure of representative members of six different photoreceptor families ", Acc. Chem. Res 37 (1): 13-20], which allow the understanding of how organisms respond to certain optical stimuli [Masuda, S (2002), "AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides", Cell 1 10 (5): 613-623], [Braatsch, S (2004), "Responses of the Rhodobacter sphaeroides Transcriptome to Blue Light under Semiaerobic Conditions ", J. Bacteriol 186 (22): 7726-7735].
El proceso clave de la fotoestimulacion consiste en la absorción de energía óptica por parte de las moléculas celulares. Cuando una muestra es foto-estimulada, moléculas con dobles enlaces conjugados absorben la luz en una cierta longitud de onda en función de su composición química y entran en un estado más energizado. Estas moléculas energizadas y excitadas pueden pasar su energía añadida a otras moléculas cercanas, lo cual induce una serie de procesos bioquímicos, algunos de ellos relacionados con factores de crecimiento y proliferación celular [Karu, T (1999), "Primary and Secondary Mechanisms of Action of Visible to Near-IR Radiation on Cells", Journal of Photochemistry and Photobiology B: Biology 49(1 ): 1 -17] [Kushibiki, T (2010), "Chondrogenic mRNA expression in prechondrogenic cells after blue láser irradiation", J Photochem Photobiol B. 98(3): 21 1 -215], [Pereira, M (2010), "Influence of 670 nm Low-Level Láser Therapy on Mast Cells and Vascular Response of Cutaneous Injuries", Journal of Photochem. & Photobiology B: Biology 98(3): 188-192]. La invención US20100099132A1 se refiere a un nuevo método de preparación de los inductores del crecimiento bacteriano basado en el empleo de la luz, caracterizado porque el método comprende cultivar Hafnia spp. en medio mínimo y porque el inductor de crecimiento no es un autoinductor.  The key process of photostimulation is the absorption of optical energy by cellular molecules. When a sample is photo-stimulated, molecules with conjugated double bonds absorb light at a certain wavelength depending on their chemical composition and enter a more energized state. These energized and excited molecules can pass their added energy to other nearby molecules, which induces a series of biochemical processes, some of them related to cell growth and proliferation factors [Karu, T (1999), "Primary and Secondary Mechanisms of Action of Visible to Near-IR Radiation on Cells ", Journal of Photochemistry and Photobiology B: Biology 49 (1): 1-17] [Kushibiki, T (2010)," Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation ", J Photochem Photobiol B. 98 (3): 21 1 -215], [Pereira, M (2010), "Influence of 670 nm Low-Level Laser Therapy on Mast Cells and Vascular Response of Cutaneous Injuries", Journal of Photochem. & Photobiology B: Biology 98 (3): 188-192]. The invention US20100099132A1 relates to a new method of preparing bacterial growth inducers based on the use of light, characterized in that the method comprises cultivating Hafnia spp. at a minimum and because the growth inducer is not a self-inducer.
El objeto de la invención US20130190843A1 es proporcionar un método de foto- estimulación en la que se configuran LEDs para emitir luz roja o amarilla en un intervalo específico de iluminancia, a fin de estimular la síntesis de colágeno de los fibroblastos y promover la circulación de la sangre; así como acelerar la eliminación células muertas. Además, en el método de foto-estimulación, los LED pueden configurarse para emitir luz azul de determinada intensidad para inhibir o matar a P. acnés o para suprimir o reducir la síntesis de la melanina de los melanocitos.  The object of the invention US20130190843A1 is to provide a photo-stimulation method in which LEDs are configured to emit red or yellow light in a specific range of illuminance, in order to stimulate the synthesis of collagen in fibroblasts and promote the circulation of the blood; as well as accelerating the removal of dead cells. In addition, in the photo-stimulation method, the LEDs can be configured to emit blue light of a certain intensity to inhibit or kill P. acnes or to suppress or reduce the synthesis of melanin from melanocytes.
La invención WO201307491 1 A1 proporciona métodos para la regulación de genes dependiente de la luz utilizando una proteína fotosensible de unión a ADN. La invención proporciona una molécula de ácido nucleico recombinante que comprende una secuencia que codifica una proteína de unión a ADN sensible a la luz (LRDP) que comprende: a) un dominio LOV; y b) un dominio de unión a ADN (DBD) y está unido operativamente a un polinucleótido. La invención WO2014072934A1 describe un método para estimular el metabolismo de microorganismos no fototróficos implicados en los procesos biológicos. En ella se reivindica un proceso biológico que comprende una etapa de iluminación con luz azul de los microorganismos no fototróficos para estimular su metabolismo. La aplicación principal de esta invención es el tratamiento de aguas residuales municipales y en procesos biotecnológicos para la bioproducción de moléculas de interés. En esta invención la estimulación con luz azul tuvo en cuenta la intensidad del estímulo pero no se analiza la influencia que tiene la forma de onda ni la frecuencia en la estimulación en el metabolismo celular. Por otra parte no se analiza el efecto de la estimulación en muestras de origen clínico con efectos en el diagnóstico rápido microbiológico [Tiphlova, O and Karu, T (1991 ), "Action of low-intensity láser radiation on Escherichia coli", Crit Rev Biomed Eng. 18(6): 387-412]. The invention WO201307491 1 A1 provides methods for light-dependent gene regulation using a photosensitive DNA-binding protein. The invention provides a recombinant nucleic acid molecule comprising a sequence encoding a light sensitive DNA binding protein (LRDP) comprising: a) a LOV domain; and b) a DNA binding domain (DBD) and is operably linked to a polynucleotide. The invention WO2014072934A1 describes a method for stimulating the metabolism of non-phototrophic microorganisms involved in biological processes. It claims a biological process that includes a stage of illumination with blue light of non-phototrophic microorganisms to stimulate their metabolism. The main application of this invention is the treatment of municipal wastewater and in biotechnological processes for the bioproduction of molecules of interest. In this invention, blue light stimulation took into account the intensity of the stimulus but the influence of the waveform and the frequency in stimulation on cellular metabolism are not analyzed. On the other hand, the effect of stimulation on samples of clinical origin with effects on rapid microbiological diagnosis is not analyzed [Tiphlova, O and Karu, T (1991), "Action of low-intensity laser radiation on Escherichia coli", Crit Rev Biomed Eng. 18 (6): 387-412].
El objetivo de la presente invención es proporcionar un sistema (dispositivo y procedimiento) que permite detectar el crecimiento microbiano tempranamente en muestras biológicas en las cuales se requiera detectar crecimiento de microorganismos mediante la utilización de micromuestras. Dicho sistema se basa en la detección rápida de los cambios turbidimétricos o de bioimpedancia producidos por el crecimiento de microorganismos fotosintéticos y no fotosintéticos presentes en un medio de cultivo líquido estimulado ópticamente (430 nm < λ < 480 nm y 1 μ\Λ < p < 5 μ\Λ/).  The objective of the present invention is to provide a system (device and method) that allows the detection of microbial growth early in biological samples in which it is required to detect the growth of microorganisms through the use of micro-samples. This system is based on the rapid detection of turbidimetric or bioimpedance changes produced by the growth of photosynthetic and non-photosynthetic microorganisms present in an optically stimulated liquid culture medium (430 nm <λ <480 nm and 1 μ \ Λ <p < 5 μ \ Λ /).
La novedad de la presente solución técnica radica en que se presenta un método para el diagnóstico rápido basado en el empleo combinado de estimulación óptica y la medición de turbidez o bioimpedancia en muestras biológicas, materializado a partir del empleo de un nuevo sistema de detección formado por la integración de un hardware y un software especialmente diseñados para este fin.  The novelty of the present technical solution is that it presents a method for rapid diagnosis based on the combined use of optical stimulation and the measurement of turbidity or bioimpedance in biological samples, materialized from the use of a new detection system formed by the integration of hardware and software specially designed for this purpose.
En la muestra biológica en medio líquido se registran los cambios de turbidez y/o bioimpedancia originados por la estimulación óptica controlada en el medio de cultivo, lo cual permite detectar la presencia de bacterias facilitando el estudio de la calidad microbiológica de las mismas. Además el sistema propuesto es útil para la determinación del antibiograma a partir de colonias aisladas o de muestras positivas que se obtienen directamente de las fuentes que las contienen.  The turbidity and / or bioimpedance changes caused by controlled optical stimulation in the culture medium are recorded in the biological sample in liquid medium, which allows the presence of bacteria to be detected, facilitating the study of their microbiological quality. In addition, the proposed system is useful for the determination of the antibiogram from isolated colonies or positive samples that are obtained directly from the sources that contain them.
Adicionalmente, y entre otras aplicaciones, la presente invención permite obtener el esquema de sensibilidad a los antibióticos de microorganismos ya bien a partir de colonias aisladas previamente o de muestras positivas biológicas, en este último caso ahorrándose el tiempo requerido para los procesos de aislamiento y purificación. En el caso particular de las infecciones del tracto urinario, la presente invención permite a partir de muestras directas de orina, discriminar muestras positivas de las negativas, estén contaminadas o no con otro germen. Con el sistema de la presente invención, resultados basados en 350 muestras examinadas han mostrado un 100% de correspondencia con el conteo total de células viables en medio CLED, método empleado convencionalmente para la detección de la infección del sistema urinario. Con el empleo de la invención, se logra la obtención de información útil relativa al diagnóstico clínico microbiológico en muy corto tiempo, la cual puede ser utilizada en pacientes para evitar el uso inadecuado de antibióticos, el desarrollo de la resistencia microbiana, largas estadías hospitalarias y desenlaces fatales en infecciones graves. El sistema propuesto se caracteriza por su rapidez, ya que permite la determinación de la infección urinaria en menos de una hora, y a partir de muestras positivas ofrece resultados confiables del antibiograma en un tiempo inferior a dos horas. Igualmente es un sistema altamente preciso que permite corroborar los resultados obtenidos tantas veces como se estime. Desde el punto de vista social es de gran importancia, ya que para aquellas personas hospitalizadas posibilita el suministro de antibióticos de forma racional y oportuna, evitando prolongadas estadías hospitalarias. Igualmente, desde el punto de vista ecológico tiene gran impacto ya que evitar el uso inadecuado de antibióticos y limita al mismo tiempo el desarrollo de la resistencia bacteriana. Additionally, and among other applications, the present invention allows to obtain the antibiotic sensitivity scheme of microorganisms either from previously isolated colonies or from positive biological samples, in the latter case saving the time required for isolation and purification processes . In the particular case of urinary tract infections, the present invention allows, from direct urine samples, to discriminate positive samples from negative ones, whether or not they are contaminated with another germ. With the system of the present invention, results based on 350 examined samples have shown 100% correspondence with the total viable cell count in CLED medium, a method conventionally used for the detection of urinary system infection. With the use of the invention, it is possible to obtain useful information related to the clinical microbiological diagnosis in a very short time, which can be used in patients to avoid the inappropriate use of antibiotics, the development of microbial resistance, long hospital stays and fatal outcomes in serious infections. The proposed system is characterized by its rapidity, since it allows the determination of urinary infection in less than an hour, and from positive samples it offers reliable results of the antibiogram in less than two hours. It is also a highly accurate system that allows corroborating the results obtained as many times as estimated. From the social point of view it is of great importance, since for those hospitalized people it allows the supply of antibiotics in a rational and timely way, avoiding prolonged hospital stays. Likewise, from the ecological point of view it has a great impact since avoiding the inappropriate use of antibiotics and at the same time limits the development of bacterial resistance.
En la actualidad los sistemas automatizados se han convertido en el pilar del diagnóstico para infecciones y entre los sistemas comerciales más utilizados se encuentra: SYSMEX, el cual constituye un método de referencia para la detección de bacterias y sepsis. En el caso de los cultivos positivos la bacteria presente debe ser identificada antes de comenzar el suministro de antibióticos, utilizando varios métodos, desde los test bioquímicos convencionales hasta los análisis de ADN basados en PCR, que demoran de 3 a 24 horas adicionales. Currently, automated systems have become the mainstay of diagnosis for infections and among the most used commercial systems are: SYSMEX, which is a reference method for the detection of bacteria and sepsis. In the case of positive cultures, the bacteria present must be identified before starting the antibiotic supply, using various methods, from conventional biochemical tests to PCR-based DNA tests, which take an additional 3 to 24 hours.
La presente invención describe un nuevo sistema de medición (Fig. 1 ) que permite estimular ópticamente el crecimiento de los microorganismos fotosintéticos y no fotosintéticos presentes en un medio de cultivo líquido, lo cual es utilizado como un método de detección rápida que disminuye los tiempos de análisis de las muestras. La invención propuesta comprende un dispositivo formado por una tarjeta electrónica con dos canales de medición: uno con un arreglo de celdas para mediciones turbidimétricas (Fig. 2) en muestras con mediana o baja densidad óptica, y otro con un arreglo de celdas para medición impedanciométrica con electrodos para muestras de alta densidad óptica, y una interfaz común de control {software) asociada (Fig. 3) para ambos canales. La tarjeta electrónica está estructurada con un diseño modular con funciones y objetivos específicos para cada módulo: La Unidad Central de Procesamiento y Control, UCPC (1 ) se encarga de generar y sincronizar todas las acciones de control sobre el resto de los bloques. Los canales de medición de turbidez y bioimpedancia son útiles para la adquisición y acondicionamiento de las variables adquiridas, el módulo de comunicación (2) establece la comunicación entre el procesador de la UCPC y el ordenador personal (3) a través de comandos; y la fuente de alimentación (4) tiene la función de suministrar la energía a todos los dispositivos y bloques del diseño. The present invention describes a new measurement system (Fig. 1) that allows to optically stimulate the growth of photosynthetic and non-photosynthetic microorganisms present in a liquid culture medium, which is used as a rapid detection method that decreases the timing of Sample analysis. The proposed invention comprises a device formed by an electronic card with two measurement channels: one with an array of cells for turbidimetric measurements (Fig. 2) in samples with medium or low optical density, and another with an array of cells for impedance measurement with electrodes for high optical density samples, and a common control interface {software) associated (Fig. 3) for both channels The electronic card is structured with a modular design with specific functions and objectives for each module: The Central Processing and Control Unit, UCPC (1) is responsible for generating and synchronizing all control actions on the rest of the blocks. The turbidity and bioimpedance measurement channels are useful for the acquisition and conditioning of the acquired variables, the communication module (2) establishes communication between the UCPC processor and the personal computer (3) through commands; and the power supply (4) has the function of supplying power to all devices and blocks of the design.
Además dicha tarjeta electrónica está asociada a una herramienta de programa {software) instalada en un ordenador personal para la lectura y almacenamiento automático de los datos de las mediciones vía USB (módulo de comunicación), que garantiza la adecuada selección de parámetros durante el proceso de caracterización de la detección de microorganismos en las muestras biológicas. La aplicación desarrollada permite a través de tres ventanas de trabajo la programación de los parámetros de estimulación de las muestras bajo estudio, garantizando además el adecuado almacenamiento y revisión de los datos adquiridos cuando sea solicitado por el usuario (Fig. 3). Furthermore, said electronic card is associated with a program tool {software) installed in a personal computer for automatic reading and storage of the measurement data via USB (communication module), which guarantees the adequate selection of parameters during the process of characterization of the detection of microorganisms in biological samples. The application developed allows the programming of the stimulation parameters of the samples under study through three working windows, also guaranteeing adequate storage and review of the data acquired when requested by the user (Fig. 3).
El sistema de medición consta de un generador de la señal de estímulo (5) y referencia (6); además de un conversor tensión-corriente (7) que suministra el estímulo óptico controlado (Onda sinusoidal, 430 nm < λ < 480 nm, 1 μ\Λ/ < p < 5 μ\Λ/) a un arreglo de cinco LEDs (8) ubicados adecuadamente en el portamuestras (9), y que son los encargados de transmitir la señal para estimular las muestras (10) que se desean analizar. La señal generada también es utilizada como referencia sincrónica para el proceso de demodulación de la señal recibida desde los receptores (foto-detectores). En el caso de las celdas para mediciones turbidimétricas el estímulo óptico que logra atravesar la muestra se recepciona con ayuda de un circuito integrado opto-electrónico (1 1 ), el cual contiene un fotodiodo (fotodetector) y un amplificador de transimpedancia monolítico. La señal a la salida de los fotodetectores se selecciona con un multiplexor (12) y es transmitida a una sección de acondicionamiento (13), pasando a un bloque de demodulación sincrónica (14), de filtrado (15) y de conversión (16), empleándose una estructura de amplificador de enganche para la sincronización del proceso de demodulación.  The measurement system consists of a generator of the stimulus signal (5) and reference (6); in addition to a voltage-current converter (7) that supplies the controlled optical stimulus (Sine wave, 430 nm <λ <480 nm, 1 μ \ Λ / <p <5 μ \ Λ /) to an array of five LEDs (8 ) properly located in the sample holder (9), and which are responsible for transmitting the signal to stimulate the samples (10) to be analyzed. The generated signal is also used as a synchronous reference for the demodulation process of the signal received from the receivers (photo detectors). In the case of cells for turbidimetric measurements, the optical stimulus that passes through the sample is received with the help of an opto-electronic integrated circuit (1 1), which contains a photodiode (photodetector) and a monolithic transimpedance amplifier. The signal at the output of the photodetectors is selected with a multiplexer (12) and is transmitted to a conditioning section (13), passing to a synchronous demodulation block (14), filtering (15) and conversion (16) , using a hitch amplifier structure for the synchronization of the demodulation process.
En el diseño propuesto se ha adicionado un canal para mediciones impedanciométricas a partir de una celda, empleando un arreglo de electrodos bipolares de acero inoxidable (17) con longitud de 10 cm y diámetro de 1 mm, y un convertidor de impedancia de alta precisión AD5933 (18) que combina un generador de frecuencia interno con un conversor digital-analógico. El generador permite excitar con una frecuencia conocida a la muestra biológica en este caso de alta densidad óptica (19). La señal obtenida desde la muestra que se analiza es adquirida por el conversor analógico-digital interno del AD5933 y es procesada aplicándose la Transformada Discreta de Fourier (TDF) por el procesador digital interno (PDS). El algoritmo aplicado retorna un valor real e imaginario de la impedancia medida, el cual es transferido mediante un protocolo de comunicación (21 ) hacia la UCPC y de esta al ordenador personal (3) para su representación gráfica en la interfaz desarrollada. In the proposed design, a channel for impedance measurements from a cell has been added, using an electrode array Bipolar stainless steel (17) with a length of 10 cm and a diameter of 1 mm, and a high precision impedance converter AD5933 (18) that combines an internal frequency generator with a digital-analog converter. The generator allows the biological sample to be excited with a known frequency in this case of high optical density (19). The signal obtained from the analyzed sample is acquired by the AD5933 internal analog-digital converter and is processed by applying the Fourier Discrete Transform (TDF) by the internal digital processor (PDS). The applied algorithm returns a real and imaginary value of the measured impedance, which is transferred by means of a communication protocol (21) to the UCPC and from this to the personal computer (3) for graphic representation in the developed interface.
Otra aplicación fundamental de esta invención está orientada hacia la determinación del antibiograma de la muestra (susceptibilidad de los microorganismos a los antibióticos), lo cual se logra en un período de tiempo inferior a las 2 horas, auxiliándose de un juego diagnóstico diseñado para estos propósitos. El medio de cultivo empleado para seguir el crecimiento microbiano es el medio Mueller-Hinton Broth OXOID modificado, PH de 7.4 ± 0.2 y estéril, el cual incluye adicionalmente un polímero. El juego diagnóstico para la detección del antibiograma de la muestra a analizar se caracteriza por el empleo de discos de antibióticos que se encuentran disponibles comercialmente y que pueden utilizarse conformando esquemas que pueden variarse atendiendo a cualquier necesidad.  Another fundamental application of this invention is directed towards the determination of the sample antibiogram (susceptibility of microorganisms to antibiotics), which is achieved in a period of time less than 2 hours, using a diagnostic kit designed for these purposes. . The culture medium used to follow microbial growth is the modified Mueller-Hinton Broth OXOID medium, pH 7.4 ± 0.2 and sterile, which additionally includes a polymer. The diagnostic kit for the detection of the antibiogram of the sample to be analyzed is characterized by the use of antibiotic discs that are commercially available and that can be used in accordance with schemes that can be varied according to any need.
Por otra parte, el sistema propuesto permite enfrentar las interferencias generadas por la contaminación de las muestras; así como por la infección causada por más de un germen. La interferencia de la contaminación ha sido resuelta ajustando la señal en magnitud y tiempo para la detección de los niveles infestantes aceptados internacionalmente (>100,000 UFC/ml), propiciando la exclusión de las muestras contaminadas no infestadas por contar éstas generalmente con niveles bacterianos inferiores a 1 ,000 UFC/ml, lo que permite la detección de muestras contaminadas solamente cuando además éstas están infestadas. En este caso particular, teniendo en cuenta que generalmente las especies contaminantes atendiendo al Gram son saprofitas, en su mayoría sensibles a todos los antibióticos, es evidente que las cepas contaminantes no deben interferir en la detección del esquema de resistencia de las cepas infestantes. Estas constituyen igualmente características distintivas de la presente invención.  On the other hand, the proposed system allows to face the interferences generated by the contamination of the samples; as well as the infection caused by more than one germ. The contamination interference has been resolved by adjusting the signal in magnitude and time for the detection of internationally accepted infestant levels (> 100,000 CFU / ml), promoting the exclusion of contaminated samples not infested because they generally have bacterial levels below 1,000 CFU / ml, which allows the detection of contaminated samples only when they are also infested. In this particular case, taking into account that generally the contaminating species attending to the Gram are saprophytes, mostly sensitive to all antibiotics, it is evident that the contaminating strains should not interfere in the detection of the resistance scheme of the infesting strains. These also constitute distinctive features of the present invention.
Con relación a las infecciones producidas por más de 1 germen, en la práctica se pueden presentar dos situaciones particulares. En la primera, puede existir el predominio de uno de los gérmenes infestantes debido a una mayor velocidad específica de crecimiento transcurrido el tiempo mínimo de incubación, en cuyo caso el antibiograma sería válido. En la segunda, los gérmenes crecen simultáneamente, en cuyo caso el antibiograma podría mostrar uno o varios antibióticos efectivos para ambos gérmenes, o se podría presentar un esquema de resistencia absoluta por complementación, en cuya situación sólo podría indicarse la prueba con otros antibióticos no incluidos en el test, o pasar posteriormente a los necesarios procedimientos de aislamiento y purificación para este caso. Este conjunto de análisis y soluciones inmediatas para cada situación particular, sólo se posibilita por la aplicación del concepto de lecturas sobre muestras directas, con alto nivel de interferencia, a tiempo corto y fijo, aspectos que caracterizan y distinguen el sistema de la presente invención. In relation to infections caused by more than 1 germ, in practice two particular situations can occur. In the first, there may be predominance of one of the infesting germs due to a higher specific growth rate after the minimum incubation time, in which case the antibiogram would be valid. In the second, the germs grow simultaneously, in which case the antibiogram could show one or more effective antibiotics for both germs, or an absolute resistance scheme could be presented by complementation, in which situation only the test with other antibiotics not included could be indicated. in the test, or subsequently move on to the necessary isolation and purification procedures for this case. This set of analyzes and immediate solutions for each particular situation, is only possible by the application of the concept of readings on direct samples, with high level of interference, in a short and fixed time, aspects that characterize and distinguish the system of the present invention.
Ejemplos de realización Examples of realization
A continuación se presentan varios ejemplos relacionados con la determinación de las características de la invención: Below are several examples related to the determination of the characteristics of the invention:
Ejemplo 1 : Influencia de la longitud de onda y la intensidad luminosa en la estimulación óptica del crecimiento bacteriano Example 1: Influence of wavelength and light intensity in the optical stimulation of bacterial growth
Cepa: El experimento se realizó con una cepa de Escherichia coli ATCC 25923. Strain: The experiment was performed with a strain of Escherichia coli ATCC 25923.
Inoculo: Se preparó una suspensión de Escherichia coli ATCC 25923 con una concentración inicial de 104 UFC/ml en medio de cultivo DKD. Inocula: A suspension of Escherichia coli ATCC 25923 was prepared with an initial concentration of 10 4 CFU / ml in DKD culture medium.
Condiciones de Cultivo Growing Conditions
El cultivo se realizó a 37°C durante 18 h con ayuda de una incubadora Memmert modelo INE 700. La suspensión de Escherichia coli fue estimulada con longitud de onda azul de baja intensidad de forma continua, con diferentes intensidades luminosas (1 , 8; 7 y 1 1 cd respectivamente) y de igual forma con longitud de onda roja con intensidades de 7, 1 1 y 21 cd. Se registraron las curvas de crecimiento por turbidez del microorganismo utilizándose un tiempo entre mediciones de 5 minutos. La concentración inicial de la suspensión fue corroborada por el conteo de colonias en placa. Resultados The culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700. The suspension of Escherichia coli was stimulated with blue wavelength of low intensity continuously, with different light intensities (1, 8; 7 and 1 1 cd respectively) and likewise with red wavelengths with intensities of 7, 1 1 and 21 cd. Turbidity growth curves of the microorganism were recorded using a time between measurements of 5 minutes. The initial concentration of the suspension was corroborated by the plate colony count. Results
Cada curva representada en la figura 4 es el promedio de 10 ensayos y en todos los casos, se comprobó mediante la prueba de Kolmogorov-Smirnov que los resultados experimentales no tenían una distribución normal (P < 0,0001 ) y se aplicó el test de Dunn para comparar múltiples muestras (test de Kruskal-Wallis). En los resultados se corroboró la existencia de diferencias estadísticamente significativas con un nivel de confianza del 95 % (P < 0,05).  Each curve represented in Figure 4 is the average of 10 trials and in all cases, it was verified by the Kolmogorov-Smirnov test that the experimental results did not have a normal distribution (P <0.0001) and the test was applied. Dunn to compare multiple samples (Kruskal-Wallis test). The results corroborated the existence of statistically significant differences with a confidence level of 95% (P <0.05).
Cuando se estimula con longitud de onda azul (λ = 470 nm) se observa un comportamiento diferente que el obtenido al estimular con longitud de onda roja. En este caso se aprecia que el crecimiento de Escheríchia coli es proporcional al aumento de la intensidad luminosa. Este efecto no sólo influye en la duración de la fase exponencial sino que también disminuye la duración de la fase de latencia, y por tanto contribuye a la disminución del tiempo de detección.  When stimulated with blue wavelength (λ = 470 nm), a different behavior is observed than that obtained when stimulated with red wavelength. In this case it can be seen that the growth of Escheríchia coli is proportional to the increase in light intensity. This effect not only influences the duration of the exponential phase but also decreases the duration of the latency phase, and therefore contributes to the reduction of the detection time.
En la figura 5 se observa que en las condiciones experimentales estudiadas; el nivel de estimulación del crecimiento microbiano al concluir la fase exponencial es mayor cuando se emplea una longitud de onda azul de baja intensidad correspondiente a λ= 470 nm en comparación con la longitud de onda roja (λ = 625 nm).  Figure 5 shows that in the experimental conditions studied; The level of stimulation of microbial growth at the end of the exponential phase is greater when a blue wavelength of low intensity corresponding to λ = 470 nm is used compared to the red wavelength (λ = 625 nm).
Ejemplo 2: Influencia de la frecuencia de la señal de estímulo en la estimulación óptica del crecimiento bacteriano Example 2: Influence of the frequency of the stimulus signal on the optical stimulation of bacterial growth
Cepa: El experimento se realizó con una cepa de Escheríchia coli ATCC 25923.  Strain: The experiment was performed with a strain of Escheríchia coli ATCC 25923.
Inoculo: Se preparó una suspensión de Escheríchia coli ATCC 25923 con una concentración inicial de 104 UFC/ml en medio de cultivo DKD. Condiciones de Cultivo Inocula: A suspension of Escheríchia coli ATCC 25923 was prepared with an initial concentration of 10 4 CFU / ml in DKD culture medium. Growing Conditions
El cultivo se realizó a 37°C durante 18 h con ayuda de una incubadora Memmert modelo INE 700. La suspensión de Escheríchia coli fue estimulada con longitud de onda azul de baja intensidad de forma continua, con una intensidad luminosa de 1 1 cd. Se realizó un barrido en frecuencias de la señal de estímulo desde 10 Hz hasta 10 kHz. Se registraron las curvas de crecimiento por turbidez del microorganismo utilizándose un tiempo entre mediciones de 5 minutos. La concentración inicial de la suspensión fue corroborada por el conteo de colonias en placa. Resultados The culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700. The suspension of Escheríchia coli was stimulated with a blue wavelength of low intensity continuously, with a light intensity of 1 1 cd. A sweep in frequencies of the stimulus signal from 10 Hz to 10 kHz was performed. Turbidity growth curves of the microorganism were recorded using a time between measurements of 5 minutes. The initial concentration of the suspension was corroborated by the plate colony count. Results
La figura 6 muestra el comportamiento para las diferentes frecuencias de estimulación analizadas. Cada curva representada es el promedio de cinco ensayos. El intervalo de frecuencias que muestra un mayor nivel de estimulación dentro de las condiciones experimentales evaluadas fue el comprendido entre 10 Hz y 1000 Hz. En general, en este intervalo se obtuvieron diferencias estadísticamente significativas en el tiempo de detección (aproximadamente 40% del tiempo promedio respecto al resto de las frecuencias empleadas).  Figure 6 shows the behavior for the different stimulation frequencies analyzed. Each curve represented is the average of five trials. The frequency range that shows a higher level of stimulation within the experimental conditions evaluated was between 10 Hz and 1000 Hz. In general, statistically significant differences in the detection time were obtained in this range (approximately 40% of the average time with respect to the rest of the frequencies used).
Ejemplo 3: Influencia de la intensidad de la señal de estímulo en la estimulación óptica del crecimiento bacteriano Example 3: Influence of the intensity of the stimulus signal on the optical stimulation of bacterial growth
Cepa: El experimento se realizó con una cepa de Escherichia coli ATCC 25923.  Strain: The experiment was performed with a strain of Escherichia coli ATCC 25923.
Inoculo: Se preparó una suspensión de Escherichia coli ATCC 25923 con una concentración inicial de 104 UFC/ml en medio de cultivo DKD. Inocula: A suspension of Escherichia coli ATCC 25923 was prepared with an initial concentration of 10 4 CFU / ml in DKD culture medium.
Condiciones de Cultivo Growing Conditions
El cultivo se realizó a 37°C durante 18 h con ayuda de una incubadora Memmert modelo INE 700. La suspensión de Escherichia coli fue estimulada con longitud de onda azul de baja ntensidad de forma continua, con una intensidad luminosa de 1 1 cd, y una frecuencia de estimulación determinada (10 Hz hasta 10 kHz). Bajo estas condiciones se realizó un barrido en corriente desde 2,5 mA hasta 20 mA (corriente máxima de estimulación), aumentando la intensidad luminosa aplicada a la muestra.  The culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700. The suspension of Escherichia coli was stimulated with a blue wavelength of low intensity continuously, with a light intensity of 1 1 cd, and a given stimulation frequency (10 Hz to 10 kHz). Under these conditions, a current scan from 2.5 mA to 20 mA (maximum stimulation current) was performed, increasing the light intensity applied to the sample.
Resultados Results
La figura 7 muestra el comportamiento microbiano para las diferentes intensidades de corriente analizadas donde cada curva representada es el promedio de 5 ensayos. En ella se puede observar que el crecimiento de Escherichia coli es proporcional al aumento de la intensidad del estímulo. El valor de la intensidad de corriente además de incidir de forma significativa en el porciento de estimulación del crecimiento, también repercute en la duración de la fase de latencia, asociándose a una disminución del tiempo de detección. Ejemplo 4: Análisis del crecimiento de microorganismos con y sin estimulación óptica Figure 7 shows the microbial behavior for the different current intensities analyzed where each curve represented is the average of 5 tests. It shows that the growth of Escherichia coli is proportional to the increase in the intensity of the stimulus. The value of the current intensity, in addition to having a significant impact on the percentage of growth stimulation, also has an impact on the duration of the latency phase, associated with a decrease in the detection time. Example 4: Analysis of the growth of microorganisms with and without optical stimulation
Cepas: Se utilizaron cepas clínicas previamente aisladas en medio Agar sangre.  Strains: Clinical strains previously isolated in blood agar medium were used.
Inoculo: Se prepararon varias suspensiones con las diferentes cepas clínicas a distintas concentraciones iniciales (desde 102 hasta 106 UFC/ml en medio DKD). Inocula: Several suspensions were prepared with the different clinical strains at different initial concentrations (from 10 2 to 10 6 CFU / ml in DKD medium).
Condiciones de Cultivo Growing Conditions
El cultivo se realizó a 37°C durante 18 h con ayuda de una incubadora Memmert modelo INE 700. La suspensiones de microorganismos fueron separadas en dos grupos, un grupo de crecimiento natural (curvas de trazo intermitente) y el otro grupo con estimulación continua (λ= 470 nm, e intensidad de 12 mA, curvas de trazo continuo).  The culture was carried out at 37 ° C for 18 h with the help of a Memmert incubator model INE 700. The suspensions of microorganisms were separated into two groups, a natural growth group (intermittent stroke curves) and the other group with continuous stimulation ( λ = 470 nm, and intensity of 12 mA, continuous line curves).
Resultados  Results
En la figura 8 se aprecia que para cualquier concentración, las muestras estimuladas (trazo continuo) acortan su fase de latencia y por tanto su tiempo de detección con respecto a las muestras tomadas como referencia sin estimulación óptica (curvas con trazos intermitentes), lo cual representa para la concentración de 106 UFC/ml un promedio de 18,75% de disminución del tiempo de detección. Este porciento va disminuyendo en la medida que se reduce la concentración de la muestra. Sin embargo la diferencia más notable entre ambos grupos de curvas se observa en la duración de la fase exponencial de las muestras estimuladas; siendo mayor el porcentaje de estimulación del crecimiento (~ 256%) respecto a las muestras no estimuladas. Los resultados obtenidos (Fig. 9) para otros microorganismos como Stafilococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., Klebsiella Neumoniae y Proteus spp., tuvieron la misma tendencia. Figure 8 shows that for any concentration, the stimulated samples (continuous line) shorten their latency phase and therefore their detection time with respect to the samples taken as a reference without optical stimulation (curves with intermittent lines), which represents for the concentration of 10 6 CFU / ml an average of 18.75% decrease in detection time. This percentage decreases as the concentration of the sample is reduced. However, the most notable difference between both groups of curves is observed in the duration of the exponential phase of the stimulated samples; the percentage of growth stimulation being greater (~ 256%) compared to the non-stimulated samples. The results obtained (Fig. 9) for other microorganisms such as Stafilococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., Klebsiella Neumoniae and Proteus spp., Had the same tendency.
Ejemplo 5: Influencia de la estimulación óptica controlada en la detección de microorganismos en muestras de urocultivo clínico: resultados de estudios clínicos realizados en Cuba Example 5: Influence of controlled optical stimulation in the detection of microorganisms in clinical urine culture samples: results of clinical studies conducted in Cuba
Un total de 350 muestras de orina fueron analizadas para detectar la presencia de un número significativo de uropatogenos empleando el sistema de la presente invención, y comparando los resultados con el método de referencia CLED (método semi- cuantitativo de cultivo en placa de Clarigde). Del total analizado, 90 muestras fueron positivas por el método de referencia CLED y 90 lo fueron empleando el presente sistema, aunque tan sólo en 20 minutos, es decir, mientras que el método CLED da los resultados entre las 24 y 48 horas después de la inoculación del medio de cultivo, el presente sistema fue 100% efectivo en detectar las muestras positivas en tan sólo 20 minutos después de la inoculación de la muestra. De las 260 muestras negativas encontradas por CLED, el presente sistema fue capaz de detectar igual número de muestras negativas, igualmente en un período de 20 minutos para una tasa de efectividad de 100%. La correspondencia general del presente sistema con respecto al método tradicional CLED fue de un 100%. A total of 350 urine samples were analyzed to detect the presence of a significant number of uropathogens using the system of the present invention, and comparing the results with the CLED reference method (semi-quantitative Clarigde plate culture method). Of the total analyzed, 90 samples were positive by the CLED reference method and 90 were using the present system, although only in 20 minutes, that is, while the CLED method gives the results between 24 and 48 hours after inoculation of the culture medium, the present system was 100% effective in detecting positive samples in just 20 minutes after inoculation of the sample. Of the 260 negative samples found by CLED, the present system was able to detect the same number of negative samples, also in a period of 20 minutes for a 100% effectiveness rate. The general correspondence of the present system with respect to the traditional CLED method was 100%.
Se pudo apreciar que el efecto de estimulación óptica continua para una λ=470 nm, afecta el crecimiento de forma similar a todos los microorganismos ensayados, evidenciando la existencia de estructuras fisiológicas blanco similares, que responden a este tipo de estimulación. Un importante número de microorganismos fueron aislados de las muestras positivas, como por ejemplo: E. coli, enterococcus spp, klebsiella spp, klebsiella neumoniae, klebsiella ozaenae, Bacilos no termentadores, pseudomonas aeruginosa, citrobacter freundii, aeromonas, proteus rettgeri y proteus mirabilis. El mayor porcentaje de las infecciones detectadas (78%) estuvo relacionado con la presencia de E. coli como microorganismo patógeno principal.  It was observed that the effect of continuous optical stimulation for a λ = 470 nm, affects growth in a similar way to all the microorganisms tested, evidencing the existence of similar white physiological structures, which respond to this type of stimulation. A significant number of microorganisms were isolated from the positive samples, such as: E. coli, enterococcus spp, klebsiella spp, klebsiella pneumoniae, klebsiella ozaenae, non-tering bacilli, pseudomonas aeruginosa, citrobacter freundii, aeromonas, proteus rettgeri and proteus mirabilis. The highest percentage of infections detected (78%) was related to the presence of E. coli as the main pathogenic microorganism.
En la evaluación práctica con muestras de orina, el tiempo de detección obtenido para cepas de E.coli con concentración de 105 UFC/ml en medio DKD fue inferior a los 20 minutos. Consideramos que el efecto de la fotoestimulación sobre el tiempo de duplicación de la cepa E. coli, cuando se utiliza la colonia aislada en medio de cultivo, en comparación con los resultados que se obtienen a partir de muestras directas, puede deberse a la existencia de factores en la orina o componentes específicos del sitio de infección que se relacionan con los fotoreceptores, cuantitativa o cualitativamente. Igualmente puede deberse a la pérdida de estas cualidades estimulantes del crecimiento en la cepa aislada al ser cultivada en un medio sintético, donde no se encuentran los mismos componentes del medio natural del sitio de la infección, que favorecen su desarrollo. Ejemplo 6: Diagnóstico rápido de la susceptibilidad antimicrobiana (antibiograma) In the practical evaluation with urine samples, the detection time obtained for E.coli strains with a concentration of 10 5 CFU / ml in DKD medium was less than 20 minutes. We believe that the effect of photostimulation on the doubling time of the E. coli strain, when the isolated colony is used in culture medium, compared to the results obtained from direct samples, may be due to the existence of Urine factors or specific components of the infection site that relate to photoreceptors, quantitatively or qualitatively. It may also be due to the loss of these growth-stimulating qualities in the isolated strain when grown in a synthetic medium, where the same components of the natural environment of the infection site are not found, which favor its development. Example 6: Rapid diagnosis of antimicrobial susceptibility (antibiogram)
Procedimiento:  Process:
Para preparar las diferentes concentraciones de E.coli se partió del siguiente procedimiento: Preparar una concentración de 0.5 en la escala McFarland de E.coli utilizando el medio de cultivo DKD y luego estimular la solución durante 1 hora (garantiza que está en la fase exponencial del crecimiento). Luego preparar las concentraciones iniciales de trabajo (108, 107 y 106 UFC/ml). Para realizar la cinética de crecimiento en tiras para antibiograma (ATB) se toman 200 μΙ de cada concentración. Para realizar el análisis de susceptibilidad se colocó en el pocilio de la tira para ATB, un disco antibiótico de ácido nalidixico (30 mg) más 200 μΙ de la concentración de trabajo deseada. To prepare the different concentrations of E.coli, the following procedure was started: Prepare a concentration of 0.5 on the McFarland scale of E.coli using the DKD culture medium and then stimulate the solution for 1 hour. (guarantees that it is in the exponential phase of growth). Then prepare the initial working concentrations (10 8 , 10 7 and 10 6 CFU / ml). To perform the growth kinetics in strips for antibiogram (ATB) 200 μΙ of each concentration are taken. To perform the susceptibility analysis, an antibiotic disk of nalidixic acid (30 mg) plus 200 μΙ of the desired working concentration was placed in the well of the ATB strip.
Condiciones de Cultivo Growing Conditions
El cultivo se realizó a 37°C durante 2 h con ayuda de una incubadora Memmert modelo INE 700. El cultivo de E.coli fue estimulado de forma continua con longitud de onda azul de intensidad luminosa 1 1 cd, en un intervalo de frecuencias de estimulación comprendido entre 1 Hz y 5000 Hz.  The culture was carried out at 37 ° C for 2 h with the help of a Memmert incubator model INE 700. The culture of E.coli was continuously stimulated with blue wavelength of light intensity 1 1 cd, in a frequency range of stimulation between 1 Hz and 5000 Hz.
Resultados Results
La figura 10 muestra el registro temporal de la difusión de varios discos antibióticos (ácido nalidixico, cefepime y clindamicina) en el medio de cultivo DKD. Se observa que la detección es nula (se superponen todas las curvas), es decir, la difusión del disco antibiótico en el medio DKD; así como el medio DKD solo, no introducen interferencias en el sistema de medición. Este comportamiento garantiza que cualquier variación detectada en una curva de crecimiento mediante este sistema se deberá únicamente al crecimiento del patógeno de interés. Figure 10 shows the temporal record of the diffusion of several antibiotic discs (nalidixic acid, cefepime and clindamycin) in the DKD culture medium. It is observed that the detection is null (all curves overlap), that is, the diffusion of the antibiotic disk in the DKD medium; as well as the DKD medium alone, they do not introduce interference into the measurement system. This behavior guarantees that any variation detected in a growth curve through this system is due solely to the growth of the pathogen of interest.
En la figura 1 1 se observa el efecto que introduce un disco de ácido nalidixico en la curva de crecimiento de E. coli (para 200 μΙ de una concentración de 108 UFC/ml). Se puede apreciar el efecto inhibidor del ácido nalidixico sobre el crecimiento de E.coli, el cual se hace notable a partir de los 30 minutos de iniciado el registro, tiempo que coincide con la total difusión del disco antibiótico en el cultivo de E.coli. En el caso de las concentraciones 107 y 106 UFC/ml la carga de antibiótico es suficiente para inhibir completamente el crecimiento del microorganismo durante el tiempo de registro analizado (Figs.12 y 13). Relación de figuras Figure 1 1 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 μΙ of a concentration of 10 8 CFU / ml). You can see the inhibitory effect of nalidixic acid on the growth of E.coli, which becomes noticeable after 30 minutes of the start of registration, which coincides with the total diffusion of the antibiotic disc in the culture of E.coli . In the case of concentrations 10 7 and 10 6 CFU / ml the antibiotic load is sufficient to completely inhibit the growth of the microorganism during the analyzed recording time (Figs. 12 and 13). List of figures
La figura 1 representa el diagrama en bloques de la invención. (Dibujos)  Figure 1 represents the block diagram of the invention. (Drawings)
La figura 2 muestra una imagen del arreglo de celdas para mediciones turbidimétricas con las muestras bajo estudio en el interior de la incubadora MEMMERT. Figure 2 shows an image of the array of cells for turbidimetric measurements with the samples under study inside the MEMMERT incubator.
La figura 3 muestra una imagen de la aplicación desarrollada para la configuración de los parámetros de estimulación, almacenamiento y revisión de los datos adquiridos de las muestras bajo estudio. Figure 3 shows an image of the application developed for the configuration of the parameters of stimulation, storage and review of the data acquired from the samples under study.
La figura 4 representa el comportamiento del crecimiento de E. coli al ser estimulada con longitud de onda azul de varias intensidades luminosas,  Figure 4 represents the growth behavior of E. coli when stimulated with blue wavelength of various light intensities,
La figura 5 representa el efecto de estimulación óptica con longitud de onda azul y longitud de onda roja a intensidad de 1 1 cd. Figure 5 depicts the effect of optical stimulation with blue wavelength and red wavelength at intensity of 1 1 cd.
La figura 6 representa la respuesta del crecimiento bacteriano ante diferentes frecuencias de estimulación óptica con longitud de onda azul e intensidad de 1 1 cd. La figura 7 representa la influencia de la intensidad de la señal de estímulo en la curva de crecimiento de E. coli cuando se estimula con longitud de onda azul.  Figure 6 represents the response of bacterial growth to different frequencies of optical stimulation with blue wavelength and intensity of 1 1 cd. Figure 7 represents the influence of the stimulus signal intensity on the growth curve of E. coli when stimulated with blue wavelength.
La figura 8 muestra las curvas de crecimiento de E. coli obtenidas durante el estudio del efecto de fotoestimulación con longitud de onda azul para diferentes concentraciones (desde 1 02 hasta 106 UFC/ml). Figure 8 shows the growth curves of E. coli obtained during the study of the photostimulation effect with blue wavelength for different concentrations (from 1 0 2 to 10 6 CFU / ml).
La figura 9 muestra el tiempo de respuesta de varios microorganismos ante la estimulación óptica con longitud de onda azul.  Figure 9 shows the response time of several microorganisms to optical stimulation with blue wavelength.
La figura 10 representa el registro temporal de la difusión de varios discos antibióticos (ácido nalidíxico, cefepime y clindamicina) en el medio de cultivo DKD con empleo de estimulación óptica.  Figure 10 represents the temporal record of the diffusion of several antibiotic discs (nalidixic acid, cefepime and clindamycin) in the DKD culture medium with the use of optical stimulation.
La figura 1 1 muestra el efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para 200 μΙ de una concentración inicial de 108 UFC/ml) con empleo de estimulación óptica. Figure 1 1 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 μΙ of an initial concentration of 10 8 CFU / ml) with the use of optical stimulation.
La figura 12 muestra el efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para 200 μΙ de una concentración inicial de 107 UFC/ml) con empleo de estimulación óptica. Figure 12 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 μΙ of an initial concentration of 10 7 CFU / ml) with the use of optical stimulation.
La figura 13 muestra el efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para 200 μΙ de una concentración inicial de 106 UFC/ml) con empleo de estimulación óptica Figure 13 shows the effect introduced by a nalidixic acid disc in the growth curve of E. coli (for 200 μΙ of an initial concentration of 10 6 CFU / ml) with the use of optical stimulation
Figure imgf000016_0001
Figure imgf000016_0001
Figura 2. Imagen del arreglo de celdas para mediciones turbidimétricas con las muestras bajo estudio el interior de una incubadora MEMMERT.  Figure 2. Image of the array of cells for turbidimetric measurements with the samples under study inside a MEMMERT incubator.
Figure imgf000016_0002
Figure imgf000016_0002
Figura 3. Aplicación desarrollada para la configuración de los parámetros de estimulación óptica, almacenamiento y revisión de los datos adquiridos de las muestras bajo estudio.
Figure imgf000017_0001
Figure 3. Application developed for the configuration of the parameters of optical stimulation, storage and review of the data acquired from the samples under study.
Figure imgf000017_0001
Figura 4. Comportamiento del crecimiento de E. colia\ ser estimulada con longitud de onda azul de varias intensidades luminosas.  Figure 4. Growth behavior of E. colia \ stimulated with blue wavelength of various light intensities.
Figure imgf000017_0002
Figure imgf000017_0002
Figura 5. Efecto de fotoestimulación con longitud de onda azul y longitud de onda roja a intensidad de 11 cd.
Figure imgf000018_0001
Figure 5. Photo-stimulation effect with blue wavelength and red wavelength at intensity of 11 cd.
Figure imgf000018_0001
Figura 6. Respuesta del crecimiento bacteriano ante diferentes frecuencias de estimulación (λ = 470 nm.)  Figure 6. Bacterial growth response to different stimulation frequencies (λ = 470 nm.)
Figure imgf000018_0002
Figure imgf000018_0002
Figura 7. Influencia de la intensidad de la señal de estímulo en la curva de crecimiento de E. coli cuando se estimula con longitud de onda azul. Figure 7. Influence of the intensity of the stimulus signal on the growth curve of E. coli when stimulated with blue wavelength.
Figure imgf000019_0001
Figure imgf000019_0001
Figura 8. Curvas de crecimiento de E. coli obtenidas durante el estudio del efecto de estimulación óptica con longitud de onda azul para diferentes concentraciones (desde 106 hasta 102 UFC/ml). Figure 8. Growth curves of E. coli obtained during the study of the optical stimulation effect with blue wavelength for different concentrations (from 10 6 to 10 2 CFU / ml).
Figure imgf000019_0002
Figure imgf000019_0002
Figura 9. Comparación del tiempo de respuesta de varios microorganismos ante la estimulación óptica con longitud de onda azul de baja intensidad (11 cd). Figure 9. Comparison of the response time of several microorganisms to optical stimulation with low intensity blue wavelength (11 cd).
Figure imgf000020_0001
Figure imgf000020_0001
Figure imgf000020_0002
Figure imgf000020_0002
Figura 11. Efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para  Figure 11. Effect that introduces a disk of nalidixic acid in the growth curve of E. coli (for
200 μΙ de una concentración inicial de 108 UFC/ml).
Figure imgf000021_0001
200 μΙ of an initial concentration of 10 8 CFU / ml).
Figure imgf000021_0001
Figura 12. Efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para Figure 12. Effect that introduces a disk of nalidixic acid in the growth curve of E. coli (for
200 μΙ de una concentración inicial de 107 UFC/ml). 200 μΙ of an initial concentration of 10 7 CFU / ml).
Figure imgf000021_0002
Figure imgf000021_0002
Figura 13. Efecto que introduce un disco de ácido nalidíxico en la curva de crecimiento de E. coli (para  Figure 13. Effect that introduces a disk of nalidixic acid in the growth curve of E. coli (for
200 μΙ de una concentración inicial de 106 UFC/ml). 200 μΙ of an initial concentration of 10 6 CFU / ml).

Claims

SISTEMA PARA LA DETECCIÓN DE MICROORGANISMOS FOTOSINTETICOS Y NO FOTOSINTETICOS EN MUESTRAS BIOLÓGICAS POR FOTOESTIMULACION CONTROLADA REIVINDICACIONES: SYSTEM FOR THE DETECTION OF PHOTOSYNTHETIC AND NON-PHOTOSYNTHETIC MICROORGANISMS IN BIOLOGICAL SAMPLES BY CONTROLLED PHOTOSTIMULATION REIVINDICATIONS:
1 . Sistema para la estimulación de microorganismos fotosintéticos y no fotosintéticos caracterizado por una tarjeta electrónica que comprende un generador de señales de estímulo y referencia formado por (5 y 6), un convertidor V/l discreto (7), dispositivo emisor de luz de estado sólido LED (8), las celdas de medición (9), un multiplexor analógico (12) y una etapa de procesamiento análogo-digital (13, 14 y 15) de la señal a la salida del fotodetector (1 1 ), que incluye un demodulador (14), filtro paso bajo con fc=2 Hz (15), y un convertidor A/D (16) en la unidad central de procesamiento y control: UCPC (1 ).  one . System for the stimulation of photosynthetic and non-photosynthetic microorganisms characterized by an electronic card comprising a generator of stimulus and reference signals formed by (5 and 6), a discrete V / l converter (7), solid state light emitting device LED (8), the measuring cells (9), an analog multiplexer (12) and an analog-digital processing stage (13, 14 and 15) of the signal at the output of the photodetector (1 1), which includes a demodulator (14), low pass filter with fc = 2 Hz (15), and an A / D converter (16) in the central processing and control unit: UCPC (1).
2. Sistema para la estimulación de microorganismos fotosintéticos y no fotosintéticos según la reivindicación 1 caracterizado por el empleo de una celda para mediciones turbidimétricas compuesta por un dispositivo emisor óptico de estado sólido LED (8), un portamuestras (9), y además un foto- detector (1 1 ) que mide el grado de turbidez existente en la muestra (10).  2. System for the stimulation of photosynthetic and non-photosynthetic microorganisms according to claim 1 characterized by the use of a turbidimetric measurement cell composed of a solid state LED optical emitting device (8), a sample holder (9), and also a photo - detector (1 1) that measures the degree of turbidity in the sample (10).
3. Sistema según la reivindicación 2 caracterizado por un dispositivo emisor de luz de estado sólido LED (8) que emite señal correspondiente a una longitud de onda comprendida entre 430 nm y 480 nm.  3. System according to claim 2 characterized by a solid state LED light emitting device (8) that emits a signal corresponding to a wavelength between 430 nm and 480 nm.
4. Sistema según la reivindicación 2 caracterizado por la generación de la intensidad del estímulo óptico comprendido entre 1 y 5 μW.  4. System according to claim 2 characterized by the generation of the intensity of the optical stimulus between 1 and 5 μW.
5. Sistema según la reivindicación 2 caracterizado por el empleo de una frecuencia de estimulación sinusoidal comprendida entre 1 y 1000 Hz.  5. System according to claim 2 characterized by the use of a sinusoidal stimulation frequency between 1 and 1000 Hz.
6. Sistema según la reivindicación 1 caracterizado por un canal de medición de bioimpedancia donde una muestra de alta densidad óptica (19) se acopla al arreglo de celdas impedanciométricas (17) a través de electrodos (18) conectados a un medidor de impedancia (20) controlado por la unidad de UCPC (1 ) a través de un bus l2C (21 ). 6. System according to claim 1 characterized by a bioimpedance measuring channel where a high optical density sample (19) is coupled to the array of impedance gauges (17) through electrodes (18) connected to an impedance meter (20) ) controlled by the UCPC unit (1) via a bus l 2 C (21).
PCT/CU2017/050004 2016-05-19 2017-05-18 System for the detection of photosynthetic and non-photosynthetic microorganisms in biological samples by means of controlled photostimulation WO2017198240A1 (en)

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