Method for producing microbial pellets from biological samples

Abstract

The method according to the invention provides to isolate the microbial fraction from a biological sample. For this purpose, it comprises a first step of sedimentation of the non-microbial cells contained in the biological sample and a second step of sedimentation of the microbial cells contained in the biological sample.

Description

[0001] “METHOD FOR PRODUCING MICROBIAL PELLETS FROM BIOLOGICAL SAMPLES”

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns a method for producing microbial pellets starting from biological samples or their enrichment following tests, for example, but not limited to, from so-called complex biological samples such as blood, urine, screening of biological fluids, blood cultures and suchlike, or from samples of water or other environmental liquids.

[0004] BACKGROUND OF THE INVENTION

[0005] In the presence of infections of the circulatory stream (blood), the speed of response is essential in order to give patients the appropriate drug therapy and lower the likelihood of developing sepsis (Huerta and Rice 2019). Sepsis is a dangerous condition for the patient, it affects more than 30 million people in the world every year - of which at least 1 million children (Purcarea and Sovaila 2020, Schmoch et al. 2021) and has an incidence of 1-2% among hospitalized patients (Huang, Cai, and Su 2019). About 15% of patients with sepsis experience septic shock, a condition that has a mortality rate of more than 50% and accounts for 10% of intensive care admissions (Shankar-Hari et al. 2016, Dugar, Choudhary, and Duggal 2020). Sepsis also plays an important role in hospital budgets (Huang, Cai, and Su 2019; Paoli et al. 2018; Rocheteau et al. 2015). For example, in 2013 the United States spent about $23.6 billion to treat the nearly 1.3 million patients hospitalized with sepsis with an increase rate of 11.5% over the previous year (Gaieski et al. 2013).

[0006] The delay in starting the administration of a targeted drug therapy is correlated to the survival rate of patients with sepsis; if the therapy starts within the first hour of diagnosis, the survival rate is around 80%; in the following 6 hours the survival rate undergoes a linear decrease, decreasing by 7-8% for each hour of delay (Kumar et al. 2006, M. H. Kollef et al. 1999). Delays in the administration of targeted therapies also involve broad-spectrum antibiotic cycles that screen for resistance of the microbes themselves (Sweeney, Liesenfeld, and May 2019).

[0007] The classic method for detecting sepsis is based on blood culture examination, which provides to inoculate blood into a bottle containing a very rich culture medium that promotes bacterial growth, followed by incubation until the presence of microbial growth is confirmed. Microbial growth detection is based on colorimetric or fluorometric methods. Both methods provide that the bottom of the bottle has a pH-sensitive matrix capable of detecting the variations thereof caused by an increase in CO2 inside the bottle following microbial proliferation.

[0008] In the presence of the latter, the matrix modifies color or fluorescence (Lafaurie et al. 2020, Grohs et al. 2007, Fontana et al. 2014; Idelevich, Reischl, and Becker 2018). Once positivity has been confirmed, sowing on a Petri dish and identification are then carried out. Although the above-described method is consolidated and considered as standard in the field, it involves a reporting time of the microbial identification of the order of 24-36h, a time necessary to obtain microbial growth on a Petri dish, which is not optimal for the management of patients suffering from sepsis.

[0009] In recent decades, molecular diagnostic systems have been developed that allow both to detect the microorganism and also to identify it in a single examination, through extraction and subsequent PCR amplification of portions of the genome (Sinha et al. 2018, M. Kollef et al. 2012). The PCR approach is the basis of several products existing on the market, including the BioFire® Film Array® (Biomerieux), SepsiTest™ (molzym), Molecular Mouse (Alifax), which are systems capable of detecting the presence of pathogens and providing a result of microbial identification at the genus and species level, and sometimes even of resistance patterns, within a few hours.

[0010] PCR-based methods generally have excellent reliability and reproducibility; however, the high costs of the test are not sustainable for all hospitals and the number of samples that can be processed in a day is just over a hundred. Finally, PCR’s high sensitivity can identify possible transient bacteremia or the presence of possible contaminants, negatively affecting therapy (Poissy et al. 2020). Consequently, at present, PCR-based methods are not yet able to completely replace classic detection systems and cannot be considered as a laboratory’s only identification system (Liesenfeld et al. 2014).

[0011] A possible strategy to reduce reporting times is to bypass the Petri dish culture step, isolating the microbes directly from the positive blood sample, or from an enrichment thereof such as a positive blood culture. The same consideration can also be extended to samples from other biological fluids and proceed to identify them with infrared spectroscopy techniques.

[0012] Infrared spectroscopy is an inexpensive, reliable and rapid method for microbial identification from Petri dish. However, in order to apply it fruitfully in blood samples or other complex matrices, it is necessary to identify a protocol that allows to obtain an isolate of pure and viable bacteria. In fact, infrared spectroscopy is sensitive to the entire biochemical content of the sample being measured and its three-dimensional organization. The presence of matrix contaminations or the degradation of microbial cells during isolation can heavily alter the spectral profile of microorganisms, preventing their correct identification.

[0013] There are several strategies for isolating the microbial fraction in a complex sample.

[0014] For example, WO-A2-2002 / 021108 discloses a method for separating, purifying and isolating microbes based on a two-dimensional ultracentrifuge of a blood sample, which allows to take advantage of the different sedimentation rates of the blood and microbial components. This method provides a first centrifuge to isolate the solid phase and a second centrifuge, the latter being carried out in tubes containing liquids with a density gradient, allows to obtain a stratification of the different microbes present in a sample. The layers are identified by reading the light scattering and can be recovered and used for other analyses. Although this method is powerful, it is very laborious and requires complex equipment to determine the correct position of the microbial layers.

[0015] As an alternative to this type of method, it is possible to separate microbial cells from blood cells by combining the chemical removal of non-microbial cells, that is, by exploiting the increased resistance of microbes to chemical agents such as surfactants or acidic or basic saponins, with a physical separation using concentration gradients. Examples of these methods are disclosed in W0-A1- 2023009539 and WO-A2-2011 / 006911.

[0016] The use of surfactants is not common in the purification of microbial species because, although in a minor way, these are also sensitive to the action of surfactants and, although some strains tolerate this type of treatment well, others can lose viability. To carry out analyses using mass spectrometry, it is not necessary to preserve the viability of the microbial cells, because they are still lysed with formic acid and acetonitrile in order to extract their proteins. On the contrary, to carry out analyses using infrared spectroscopy it is necessary to prevent any form of cellular damage, since even a single variation in the structure of the cellular constituents is able to alter the spectrum that is recorded. This type of method is therefore incompatible with infrared spectroscopy.

[0017] The article “Culture-free Rapid Isolation and Detection of Bacteria from Whole Blood at Clinically Relevant Concentrations” by M. Henar Marino Miguelez et al., BioRxiv, XP093252517, describes a method for isolating and analyzing bacteria in a blood sample. In this article, the blood is placed in a high density medium (similar to what is done in the state of the art, as disclosed by W0-A2- 2002 / 021108) and subjected to a first centrifuge at 600 g for 5 minutes, and subsequently subjected to a selective lysis (performed by incubating the sample at 37 °C in the presence of saponins and non-denaturing anionic detergents) and a second centrifuge at 1000 g for 13 minutes, in order to reduce the volume of the solution. The sample thus obtained is then used for analysis using a microfluidic device, which allows to isolate the bacteria in a specific region of the fluidic device where they are then analyzed. This document shows how this method is much more effective than a control protocol in which the first centrifuge is carried out at 500 g for 4 minutes. The method described in this article, however, is only effective to isolate only three pathogens: E. colt, K. pneumoniae and E.faecalis, but it does not work to isolate S. aureus. The yield, expressed in terms of percentage of microorganisms recovered, is around 95% for K. pneumoniae, while for the other pathogens it is less than 70%, a value that makes its applicability in a clinical context difficult.

[0018] In addition, the isolate obtained cannot be manipulated by an operator or made available for other analyses, for example with instruments normally present in a clinical microbiology laboratory.

[0019] The doctoral thesis “Rapid detection of Bacteria in Blood” discussed by M. Al- Adhami, the article “Factors affecting sedimentational separation of bacteria from blood” by Pitt et al., Biotechnology progress, American Chemical Society, vol. 36, no. 1, XP072291 169 and US-A 1-2016 / 115520 describe the state of the art without anticipating the idea behind the present invention.

[0020] There is therefore the need to perfect a method for producing microbial pellets from a biological sample (blood, urine, etc.), or its enrichment following a microbiological culture test (positive blood culture, liquid growth media for microbiological screening, etc.) that can overcome at least one of the disadvantages of the state of the art. To do this, it is necessary to resolve the technical problem of providing a method for producing microbial pellets that is compatible with infrared spectroscopy analysis and that can be easily manipulated and analyzed with other instruments commonly found in a microbiology laboratory.

[0021] In particular, one purpose of the present invention is to perfect a method for producing microbial pellets that allows to preserve the integrity and viability of the microbes and makes them available for analysis using conventional clinical instrumentation.

[0022] Another purpose of the present invention is to perfect a method for producing microbial pellets that is rapid, economical, and easy to carry out. The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

[0023] SUMMARY OF THE INVENTION

[0024] The present invention is set forth and characterized in the independent claim. The dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

[0025] In accordance with the above purposes and to resolve the technical problem in a new and original way, also achieving considerable advantages compared to the state of the prior art, a method according to the present invention for producing microbial pellets, in particular starting from a sample of complex biological fluid, for example a blood or urine sample, or its enrichment following a microbiological culture test, for example a positive blood culture sample, exploits the different chemical-physical properties of microbial and non-microbial cells to carry out a complete separation of the microbial fraction from the other components of the sample, without affecting its integrity, viability, and replication capacity. Subsequently, the pellets obtained according to the present invention are subjected as such to analysis using ATR-FTIR spectroscopy in order to determine their microbial identifier, without the need for further processing. Advantageously, the present invention takes advantage of cells’ different sedimentation rate to carry out a separation of the microbial fraction from the corpuscular fraction of a sample (for example blood cells, such as white and red blood cells), through two distinct centrifugation steps without the addition of matrices to promote their separation, or addition of other liquids to reduce the viscosity of the starting sample. The use of two separate and sequential centrifugations allows to easily and economically overcome some of the limitations described in the state of the art such as, for example, the use of two- dimensional and ultra centrifugal separation systems disclosed in W0-A2- 2002 / 021108, which make it very difficult to obtain and recover the sample.

[0026] The corpuscular fraction of a biological sample is composed of particles with larger sizes than those of microbial cells and therefore characterized by a sedimentation rate lower than them. In the invention, a first centrifugation step is defined that allows to obtain a microbial suspension free of corpuscular particles. This centrifugation is carried out with a force smaller than 500 g, preferably smaller than 400 g, to guarantee all microbial species are kept in suspension. Carrying out the centrifugation in the presence of a force greater than 500 g may result in the loss of part of the microbial fraction, limiting the ability to obtain a microbial pellet sufficient to obtain an identification result.

[0027] Preferably, the first centrifugation is carried out with a force greater than 50 g, more preferably greater than 100 g. According to some embodiments, the first centrifugation step is carried out with a force comprised between 300 g and 400 g.

[0028] Advantageously, the first centrifugation step has a duration of less than 10 minutes, preferably less than 7 minutes, more preferably comprised between 1 minute and 5 minutes, for example it can be 1 minute, 2, 3, 4 or 5 minutes. One embodiment provides that the first centrifuge is carried out for a period of time of 3 minutes and with a force equal to 300 g or 400 g.

[0029] After this first centrifugation step, the liquid phase is recovered and subjected to a second centrifugation step that carries out a selective precipitation of the microbial fraction. Since the microbial fraction has a lower weight than the corpuscular one and is characterized by a higher sedimentation rate, the force to which the sample is subjected in this second centrifugation step is greater, advantageously it is comprised between 2000 g and 4000 g, more preferably 3000 g. The choice of the range for the centrifugation derives from the need to be able to sediment the microbial fraction without damaging its cell wall. Any damage to the wall of the cells themselves could alter their vibrational profde to the point of compromising their viability and therefore their compatibility with an infrared analysis.

[0030] The importance of using a low-speed centrifuge to avoid damaging the integrity of microorganisms had already been demonstrated by G.L. Dorn in the late 1970s and was used in the development of the microbial fraction enrichment protocol with Isolator™ tube, initially marketed by DuPont (Wilmington, USA) and later by Oxoid Limited (Basingstoke Hunt, UK). Advantageously, compared to the method with the use of Isolator™ tube, the present invention does not provide to use a plastic liquid immiscible with water that acts as a cushioning in the centrifuge and allows to concentrate the bacteria in a certain region of the isolation tube. Furthermore, the method disclosed in the present invention does not provide a subsequent sowing step, but allows to proceed directly to the microbial identification step.

[0031] For the same reason, the present invention allows to overcome the limitations of the isolation methods described in the state of the art, such as for example WO201 1006911A2, which do not allow to obtain an effective microbial isolation using speeds not lower than 10000 g and cannot therefore be used to obtain a microbial pellet suitable for analysis with techniques based on infrared spectroscopy or which need the microbes to be kept intact and viable.

[0032] The isolation procedure using double centrifuge has been developed in such a way as to allow to isolate the microbial fraction in less than ten minutes. Each of the two centrifuge steps can be implemented so as to have a duration comprised between 30 seconds and 5 minutes, preferably between 1 and 5 minutes, more preferably between 2 and 3 minutes, making the process much faster than what described in the method with Isolator™ tube.

[0033] In the event the sample is blood or a positive blood culture, the second step of sedimentation of the microbial cells can advantageously be carried out in the presence of one or more surfactants and / or one or more saponins. This allows to eliminate any residues of small blood components, such as platelets for example, which have sizes comparable to those of the microbes themselves. Saponins are terpene glycosides of plant origin, which are formed by the union of sugar residues (such as glucose, fructose, galactose, arabinose or others) with a non-sugar molecule called aglycone (in this specific case also sapogenin). Surfactants, such as for example sodium dodecyl sulfate (SDS), Sarkosyl, and sodium lauroylglutamate (SLG), Tween and Triton X-100, are well-known chemical agents with a structure formed by a hydrophilic part, known as head, such as for example a phosphate or sulfate group, and by a hydrophobic part, known as tail, such as for example an aliphatic chain.

[0034] This particular structure, with two distinct regions with opposite polarity, is responsible for the cell lysis activity, based on the ability to bind to cell membranes and compromise their integrity. This mechanism of action also explains why lysis is more effective in the case of non-microbial cells. Single-cell organisms, such as bacteria, are in fact equipped with a much more resistant membrane consisting of peptidoglycans, instead of a double phospholipid layer as in human cells. According to what disclosed in the state of the art (such as for example Bidart, Marie et al., Journal of clinical microbiology vol. 53,5 (2015): 1761-4. doi: 10.1128 / JCM.03600-14 or WO-A2-2011 / 006911), surfactants and / or saponins are added in liquid form to the microbial sample at a final concentration of less than 5%, preferably comprised between 0.01% and 5% by weight, more preferably between 0.02% and 2% by weight of the weight of the solution containing the microbial sample.

[0035] This concentration allows to carry out a complete lysis of the non-microbial cells but also affects the viability of microorganisms, especially Gram positive and yeasts. However, as disclosed in WO-A1-2023009539 and WO-A2-2011 / 006911, this does not present a limitation to the applicability of the method. The analysis technique used in the microbial identification step is based on mass spectrometry, and it is not necessary to guarantee that the integrity and viability of the microorganisms is maintained. The analysis technique used in fact provides a step of extraction of the protein fraction of the microorganisms, which are in any case subjected to lysis in order to promote the recovery of the intracellular proteins.

[0036] In the development of the present invention, the concentration of surfactant or saponin used is advantageously kept below 1%, preferably around 0.1%, and no incubation of the sample in the presence of the agent itself is carried out. Doing so allows to achieve a microbial pellet in which the integrity and viability of the microbes have been preserved. Advantageously, this second sedimentation in the presence of one or more surfactants and / or one or more saponins is carried out at ambient temperature.

[0037] In fact, with this range of concentrations, the surfactants and / or saponins are able to degrade any non-microbial cells remaining in the sample after the first step, but do not degrade the microbial cells due to their low concentration.

[0038] In accordance with another aspect of the present invention, a step of washing the pellets obtained is provided after the second step of sedimentation of the microbial cells. Preferably, the washing step is carried out with physiological solution, that is, an aqueous solution of sodium chloride NaCl at 0.9% by weight of the weight of the solution, avoiding subjecting the microbial cells to osmotic stress, which could cause metabolic stress.

[0039] After washing, the microbial pellet is recovered and can be used as such for analysis with infrared spectroscopy. Advantageously, compared to the analysis with technology based on MALDI-TOF mass spectrometry, this analysis technique does not provide the extraction of a specific fraction of macromolecules from the sample, nor does it require a protocol for its preparation to facilitate its analysis.

[0040] According to another aspect of the invention, a method for identifying microorganisms provides to prepare a microbial pellet as indicated above, and then to use the pellet obtained for analysis, for example using infrared spectroscopy. In particular, the pellet is used as such, that is, as obtained from the method as above, in the analysis step.

[0041] DESCRIPTION OF THE DRAWINGS

[0042] These and other aspects, characteristics and advantages of the present invention will become apparent from the following description of an embodiment, given as a non-restrictive example with reference to the attached fig. 1 which shows the results of an experimental test on the effects of the centrifugal force and of the time on the first centrifugation step of the method according to the invention.

[0043] We must clarify that the phraseology and terminology used in the present description, as well as the figures in the attached drawings also in relation as to how described, have the sole function of better illustrating and explaining the present invention, their purpose being to provide a non-limiting example of the invention itself, since the scope of protection is defined by the claims.

[0044] To facilitate comprehension, the same reference numbers have been used, where possible, to identify identical common elements in the drawings. It is understood that elements and characteristics of one embodiment can be conveniently combined or incorporated into other embodiments without further clarifications.

[0045] DESCRIPTION OF AN EMBODIMENT OF THE PRESENT INVENTION

[0046] The method for producing microbial pellets according to the present invention allows to isolate the microbial fraction of a complex biological sample while keeping it suitable for analysis with any type of analysis whatsoever, in particular infrared spectroscopy.

[0047] A complex biological sample is understood, for example, as a sample of blood, urine, a screening of biological fluids, blood cultures or other potentially infected liquids, such as for example samples of water or other environmental liquids. The microbial fraction is understood as the microorganisms contained in the sample being tested, and the term microorganism includes small sized organisms such as bacteria, yeasts, fungi, etc. We must clarify that the method object of the present invention is provided to isolate and therefore allow to identify any type of microorganism, not only bacteria or yeasts or other.

[0048] The method exploits the different sedimentation rates of microbial species, compared to non-microbial species, and can be applied together with the addition of minimal amounts of surfactants and / or one or more saponins in order to lyse the non-microbial cells.

[0049] Specifically, the method provides a first step of sedimentation of the non- microbial fraction of a sample, that is, the fraction containing the corpuscular cells, such as red and white blood cells, as well as platelets.

[0050] This first sedimentation is carried out using a first centrifuge under mild conditions, that is, at a low rotation speed. More precisely, this first centrifuge is carried out with a force smaller than 500 g, sometimes even smaller than 400 g. Under these centrifugation conditions, the microbial cells remain in solution.

[0051] The supernatant thus obtained is then recovered. A second step of sedimentation of the microbial cells present in the sample is then carried out. This second sedimentation is carried out using a second centrifuge with a higher rotation speed than the first centrifuge, but always at a moderate speed so as not to damage the microbial species to be sedimented.

[0052] In particular, in this second centrifuge, the centrifugal force is greater than 2000 g but not greater than 6000 g, often not greater than 3000 g. Under these conditions, the microbial cells are sedimented, without suffering membrane damage caused by the centrifugal force.

[0053] Also in this second centrifuge, in the event the starting sample is a blood culture, it is advantageously provided to add a minimum concentration of the aforementioned surfactant substances and / or saponins to the sample, that is, at least one surfactant and / or of at least one saponin, such as SDS or Tween for example. These substances are added in liquid form at a final concentration not exceeding 1%, preferably 0.5%, more preferably 0.1% by weight of the weight of the solution containing the microbial sample, that is, of the solution obtained after the first sedimentation step, starting from the starting biological sample.

[0054] The presence of the surfactant and / or saponin allows to avoid a possible cosedimentation of non-microbial cells with comparable sizes, such as the platelets remaining in the sample. In fact, the non-microbial cells are destroyed by these substances, which instead permeate the cell membranes of the non-microbial cells, splitting them. Any damage to the membranes of the microbial cells is avoided thanks to the low concentration of the surfactant and / or saponin.

[0055] This step is carried out, advantageously, at ambient temperature. In fact, it has been observed that the use of an incubator is not required to carry out the lysis at the same time as the second sedimentation. This makes the method according to the present invention suitable to be carried out completely under a biological hood, to the advantage of the safety of the operator, in particular in a hospital context where it is not known in advance which pathogen is being worked with, and consequently what its danger is. It should also be said that the instrumentation to carry out the infrared spectroscopy analysis, since it has small sizes, can also be placed under a biological hood, so as to be able to make the identification in total safety.

[0056] The second centrifuge, which allows to carry out the sedimentation of the microbial fraction of the original sample, leads to obtaining pellets, that is, a solid product in which only the microbial species present in the starting sample are present. The pellet thus obtained is washed with an aqueous solution, preferably a physiological solution, and is then used as such for analysis. A physiological solution is understood as an aqueous solution of sodium chloride at 0.9% by weight of the weight of the solution. Advantageously, infrared spectroscopy is sensitive to the total biochemical content of the sample, and it is therefore not necessary to carry out a protocol for fixing and extracting the microorganisms’ protein fraction. Washing the pellet with physiological solution allows to prevent osmotic stress on the isolated microorganisms, ensuring that the infrared spectrum associated therewith does not suffer alterations which may prevent their identification.

[0057] The entire isolation method can be carried out in a short amount of time, that is, within 15 minutes.

[0058] This method, coupled with the identification procedure carried out using infrared spectroscopy, allows to obtain a microbial identification result in less than 20 minutes, a time not achievable with any of the methods available in the clinical field. In addition, it does not provide for the use of any type of specific support and can be carried out with materials commonly found in a clinical laboratory, by non- highly qualified personnel.

[0059] Test results showing the effect of the first centrifugation on the isolation of microorganisms and the separation of corpuscular cells are shown in fig. 1.

[0060] Samples were centrifuged with the indicated forces, 200 g, 300 g, 400 g and 500 g, for a period of time of 1 minute, 3 minutes and 5 minutes. After this single centrifugation, the solid fraction and the liquid part were analyzed using ATR- FTIR infrared spectroscopy to identify and evaluate the presence of microorganisms or corpuscular cells. The samples were blood samples or urine samples.

[0061] The evaluation of the presence of microorganisms was done by seeding on a Petri dish of 0.5 mL of supernatant (liquid phase) and incubation at 37 °C for 24- 48 h. In the event the supernatant contains microorganisms, a plate growth is obtained, while in the absence of microorganisms the growth is not present. Good growth quality is assessed by comparing the amount of material grown compared to the seeding of the sample in the absence of centrifugation.

[0062] The results show that, by varying the force and time of the first centrifuge, it is possible to eliminate the corpuscular fraction of the blood by precipitation, keeping the microorganisms present in the sample in the liquid phase.

[0063] With a first centrifugation at 200 g it is possible to isolate all the microorganisms present in the sample, with a separation of the corpuscular cells which, although acceptable, can be improved.

[0064] By increasing the force of the first centrifugation to 300 g or 400 g, it is possible to achieve optimal results both in terms of isolation of the largest number of microorganisms, and also in terms of separation of the corpuscular cells. The best results are achieved at a time of 3 minutes, at both 300 g and also 400 g.

[0065] The identification performance was also evaluated using ATR-FTIR, carried out on the pellet obtainable using the method according to the invention. In the performance evaluation, the reference method selected to determine the expected result consists in the seeding of the biological sample on a Petri dish and subsequent identification using MALDI-TOF mass spectrometry.

[0066] In the performance evaluation, the selected samples were evenly distributed across urine and blood samples, possibly enriched.

[0067] The evaluation was made on microorganisms commonly isolated in clinic, such as Acinetobacter baumannii, Candida albicans, Citrobacter spp., Corynebacterium spp., Enterobacter cloacae complex, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Micrococcus luteus, Moraxella catarrhalis, Morganella morganii, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Salmonella spp., Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes. The epidemiology of the pathogens included in the validation of the method depended on the pathogens found at the sites chosen for validation at the time of the study, although data obtained during the development of the method demonstrate that the method can be applied to any microbial isolate whatsoever. For each of them, the concordance, balanced accuracy, sensitivity and specificity of the identification were determined using infrared spectroscopy.

[0068] In terms of concordance, apart from the Enterobacter cloacae complex which amounts to 75%, all the microorganisms tested have a value greater than 90%. For example, S. aureus has a value of 98.4%. The total concordance, assessed on all samples tested, is 98.0%. Similarly, the total balanced accuracy is 98.9%.

[0069] It should also be mentioned that the pellet obtained can be used in any method of spectroscopic analysis of the sample in addition to infrared spectroscopy indicated above, such as for example mass spectroscopy, in particular MALDI TOF spectroscopy, or other types of spectroscopy.

[0070] It is clear that modifications and / or additions of parts may be made to the method as described heretofore, without thereby departing from the field and scope of the present invention, as defined by the claims.

[0071] It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art will be able to achieve other equivalent forms of a method for producing microbial pellets from biological samples, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.

[0072] In the following claims, the sole purpose of the references in brackets is to facilitate their reading and they must not be considered as restrictive factors with regard to the field of protection defined by the claims.

Claims

CLAIMS1. Method for producing microbial pellets from a biological sample or its enrichment following a microbiological culture test, comprising a first step of sedimentation of the non-microbial cells contained in said biological sample and a second step of sedimentation of the microbial cells contained in said biological sample, characterized in that said first step is carried out by means of a first centrifuge of said biological sample with a force smaller than 500 g, and that said second step is carried out by means of a second centrifuge of said biological sample with a centrifugal force greater than the centrifugal force of said first centrifuge and smaller than 6000 g.

2. Method as in claim 1, characterized in that said first centrifuge is carried out with a centrifugal force comprised between 300 and 400 g.

3. Method as in claim 1 or 2, characterized in that said second centrifuge is carried out with a centrifugal force comprised between 2000 g and 4000 g.

4. Method as in claim 1, 2 or 3, characterized in that each centrifuge is carried out for a duration comprised between 1 and 5 minutes, preferably 3 minutes.

5. Method as in any claim hereinbefore, characterized in that said first centrifuge is carried out for a duration of 3 minutes and with a force equal to 300 g or 400 g.

6. Method as in any claim hereinbefore, characterized in that said second step can be carried out in the presence of at least one surfactant and / or at least one saponin added in liquid form, at a total concentration of between 0.01% to 5% by weight, preferably 0.02% to 2% by weight.

7. Method as in claim 6, characterized in that the total concentration of the at least one surfactant and / or of the at least one saponin is at most equal to 0.1% by weight of the weight of the solution containing the microbial sample, that is, the solution obtained after the first sedimentation step, starting from the starting biological sample.

8. Method as in any claim hereinbefore, characterized in that after said second step of sedimentation of the microbial cells, there is provided a step of collection of said sedimented microbial cells and of formation of a pellet.

9. Method as in claim 8, characterized in that after the formation of the pellet, there is provided a washing step thereof, using a washing liquid.

10. Method as in claim 9, characterized in that said washing liquid is anaqueous solution, in particular a physiological solution.

11. Method as in claim 10, characterized in that the sample is subsequently used as such for the analysis.

12. Method as in claim 11, characterized in that the analysis technique used to carry out the microbial identification is based on infrared spectroscopy.

13. Method as in any claim hereinbefore, characterized in that it is carried out on a biological sample enriched following a biological fluid screening.

14. Method as in any claim hereinbefore, characterized in that it is carried out on a biological sample enriched following a microbiological culture test.

15. Method according to claim 14, characterized in that it is carried out on a blood culture, preferably a positive blood culture.

16. Method for identifying microorganisms, comprising preparing microbial pellets using the method as in any claim hereinbefore and using the pellets thus obtained for analysis.

17. Method as in claim 16, characterized in that said analysis is a spectroscopic analysis, preferably an analysis using infrared spectroscopy.

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