A method for rapid detection and quantification of a microorganism responsible for sepsis and antibiotic resistance

Abstract

The present invention relates to a method for the rapid identification and quantification of pathogens in a biological sample, thereby determining whether a subject is infected by pathogens responsible for sepsis. The invention also allows to determine whether said pathogen(s) have antibiotic resistance genes. The invention also relates to uses of the method of the invention and kits.

Description

[0001] A METHOD FOR RAPID DETECTION AND QUANTIFICATION OF A MICROORGANISM RESPONSIBLE FOR SEPSIS AND ANTIBIOTIC RESISTANCE

[0002] FIELD OF INVENTION

[0003] The present invention relates to a method for the rapid identification and / or quantification of at least one microorganism responsible for an infection in a biological sample, in particular a bacterial infection causing sepsis. The present invention also allows to determine whether said microorganism has virulence factors and at least one antibiotic resistance gene. The present invention also relates to uses of the method of the invention and kits.

[0004] BACKGROUND OF INVENTION

[0005] Hospital infections, during or after surgery, are a major healthcare problem, and if not adequately treated properly can be life-threatening, may require re-operations, and generally translate in a prolonged stay in hospital, with a significant cost for the healthcare sector. Sepsis is the consequence of a severe infection that generally stems from a localized infection (peritonitis, pneumonia, urinary tract infection, catheter-related infection, etc.). It often affects patients with weakened immune systems. When sepsis occurs following an invasive procedure (such as major surgery), the infection is referred to as a nosocomial (or healthcare associated) infection. All bacteria, even those naturally present on the skin or in mucous membranes such as the throat, which are normally non-pathogenic, can trigger the onset of sepsis.

[0006] According to the World Health Organization (WHO), sepsis is responsible for one in every five deaths worldwide. There are 49 million cases of sepsis every year, almost half of which occur in children. Around half of patients contract the infection in hospital. In industrialized countries, sepsis is responsible for as many deaths as heart attacks. In developing countries, puerperal sepsis remains a major cause of death for women after childbirth.

[0007] In France, there are thought to be around 250,000-300,000 cases of bacterial sepsis each year, as well as cases of fungal and viral sepsis. Some 25% of patients admitted to hospital for sepsis die, and the death rate from septic shock can reach 50%. In 2017, WHO drew the attention of its member states to the scale of the problem for public health, issuing a resolution encouraging them to improve epidemiological data, preventive strategies, diagnosis and treatment, as well as sepsis research.

[0008] The current standard procedure for sepsis and bloodstream infection diagnosis is based on culture methods. More specifically, the current standard procedure in hospital-based laboratories is as follows: a) Conventional procedure of processing the samples (liquids or solids) by cultivating the bacteria in different growth conditions (agar plates, etc). This normally takes 3 to 5 days and allows for a first broad identification of the suspected bacteria, provided that the culprit bacteria can be cultivated within this time frame. b) If necessary, the bacteria collected on Petri will thereafter be protein sequenced on specialized equipment (MALDI-TOF Mass Spectrometry equipment), allowing to finetune the diagnosis on a species level. MALDI-TOF MS equipment is highly expensive and not all hospitals have this equipment. Hospitals not equipped with MALDI-TOF use other equipment that identifies the bacteria based on enzymatic tests, which are less sensitive and reliable. c) The resistance to antibiotics is checked by conventional methods i.e., culture of the isolated microorganism on different media containing the most used antibiotics. This normally will take up another 2-4 days, after the above-mentioned initial growing step.

[0009] All in all, the standard procedures take at least 5-8 days. Despite their ease of use and high sensitivity, culture methods suffer from slow turnaround failing in a rapid detection and targeted antibiotic administration, drastically decreasing survival rate and increasing the health care expenditure. In addition, standard methods based on culture methods do not allow the quantification of the pathogen present in the biological sample.

[0010] There is thus a need for a method of identification and quantification of a pathogen in a biological sample from a subject which provides a quicker result than the standard procedure. There is also a need for such a method, that would be sensitive and provides reliable results. There is also a need for such a method that would allow to detect and quantify of a pathogen in a biological sample from a subject even at a very low concentration in the biological sample. There is also a need for such a method that would provide information on the antibiotic resistance profile of the pathogen in the biological sample. There is also a need for such a method that would be easy. There is also a need for such a method that would be less expensive than the standard procedures.

[0011] SUMMARY

[0012] The invention fulfills these needs.

[0013] In a first aspect the invention relates to a method for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample from a subject at risk to suffer from and / or suspected to suffer from sepsis, the method comprising the steps of: a) spiking the biological sample with quantified calibrator molecules; b) extract the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from 0.0 Ing to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample. Said method allows the very rapid and reliable identification and quantification of microorganism that are present at a very low concentration in different kind of samples from a subject. Said method further allows to reach up to now unmet Sensitivity, Specificity and Accuracy scores, making it particularly suitable to detect, identify and quantifying microorganisms responsible for sepsis.

[0014] According to other features of the method according to this first aspect, the method includes one or more of the following features, alone or in combination :

[0015] - the DNA polymerase amplifies at least 103CFU / ml and at least of 0.1 ng of genomic DNA, preferably at least 102CFU / ml and at least of 0.01 ng of genomic DNA,

[0016] - the method enables the identification and / or quantification with an accuracy of at least 90%, and / or within a maximum time frame of 24 hours. This is of a particular interest for microorganisms known to cause sepsis, taking into account that current standards of care include time consuming culture and microbiological identification steps . a further step of comparing the quantity of said at least one microorganism to the amount of quantified to quantify said at least one microorganism in the biological sample,

[0017] - the biological sample is selected from blood, urine, synovial fluid, pulmonary wash, plasma, serum, amniotic fluid, cerebrospinal fluid, nasal wash, saliva, semen, vaginal fluid, sputum and the combinations thereof, preferably selected from blood, plasma, serum, urine, synovial fluid, a biopsy, and pulmonary wash.

[0018] - the biological sample is of a volume equal or less than 5ml, equal or less than 4ml, equal or less than 3ml, equal or less than 2ml, equal or less than 1ml equal or even equal or less than 0.5ml.

[0019] - the molecular barcodes of step e) are ligated using 5 ’-phosphated primers. a further step of determination of a viral or fungal infection, which allows to cover all the microorganisms responsible for sepsis. a further comprising a step of detection of at least one gene of antibiotic resistance and / or at least one a virulence factor in the at least one microorganism.

[0020] - when present, the step of detection of at least one gene of antibiotic resistance and / or at least one a virulence factor and / or of determination of a viral or fungal infection is performed using a metagenomic sequencing on the biological sample.

[0021] - the at least one microorganism responsible for sepsis is selected amongst a bacteria, a virus, a fungus and the combinations thereof. Indeed, said method notably allows the detection of possible co infections comprising bacteria, fungus, virus or the mix thereof.

[0022] - the 16S rRNA amplicons of step c) have a length between 0.05-5 kB, preferably 1.5 kB.

[0023] - the ligation of molecular barcodes of step d) enables samples multiplexing.

[0024] - the method provides a result of step g) within a maximum timeframe of 36 hours, preferably of 24 hours. A rapid identification is a particular advantage regarding microorganisms responsible of sepsis.

[0025] - the method provides a result of step g) with a limit of detection of 50 CFU / ml, preferably of 10 CFU / ml.

[0026] - the method identifies and / or quantifies the at least one microorganism present with at least 0.01% relative abundance in the bacterial community of said sample. This is allowed by the yet unmet sensitivity of the method which allow its implementation on various biological samples that can comprise endogenous microbiota.

[0027] - the method does not comprise a growing step to enrich the biological sample with the at least one microorganism. This is of particular interest in case where a rapid identification of the microorganism is desirable as for the microorganisms which can cause sepsis. The absence of a prior step of culture allows to obtain a result in a particularly short limit of time from the sample collection. In a second aspect, the invention relates to a kit of parts for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample according to method according to the first aspect, said kit comprising: i. a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2), ii. a sample of DNA polymerase iii. at least one calibrator molecule, iv. phosphorylated barcoding primers enabling sample multiplexing, and v. at least one collection tube containing glycerol to collect a biological sample, and vi. instructions for use.

[0028] In a third aspect, the invention relates to a method of diagnosing sepsis from a biological sample collected in a subject at risk of, or suspected to suffer from sepsis, the method comprising the steps of: a) Spiking the biological sample with quantified calibrator molecules; b) Extract the genomic DNA from the biological sample of step a); c) Carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from 0.0 Ing to lOOng or 102CFU / ml to 109CFU / ml. d) Further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) Ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) Sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) Comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

[0029] The advantages of the method for the identification and / or quantification of at least one microorganism responsible for sepsis according to the first aspect make it indeed particularly suitable to be included in a diagnostic method of sepsis.

[0030] According to other features of the method of diagnosing sepsis according to this third aspect aspect, said method of diagnosing includes one or more of the following features, alone or in combination : a further comprising a step of determination of a viral or a fungal infection. a step of determination of antibiotic resistance gene of the at least one microorganism.

[0031] - when present, the step of determination of antibiotic resistance gene of the at least one microorganism and / or the determination of viral or fungal infection is determined by a metagenomic sequencing step performed on the biological sample from a subject at risk of or suspected to suffer from sepsis. said method does not comprise a culture step to enrich the biological sample with the at least one microorganism. This allows to save a precious time to obtain the diagnosis and allows the early implementation of treatment or of specific means of care of sepsis.

[0032] All the features according to the method according to the first aspect of the invention are applicable to the sepsis diagnosis method according to the invention.

[0033] DEFINITIONS

[0034] The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims. When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise. Notably, unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by the skilled in the art.

[0035] The term “amplifying” or “amplification” as used herein refers to the production of additional copies of the target sequence.

[0036] “Antibiotic resistance gene” or “gene of antibiotic resistance” or “gene of resistance to an antibiotic” refers to a gene which allows the growing of a microorganism despite the presence of an antibiotic in the culture medium.

[0037] The expression “at least one” means one or more.

[0038] The term “kB” stands for 1000 bp. “Bp” stands for a base pair, which is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds.

[0039] The term “CFU” stands for “colony-forming unit”. This unit estimates the number of microbial cells (bacteria, fungi, viruses etc.) in a sample that are viable and able to multiply via binary fission under controlled conditions.

[0040] The term “human” refers to a subject of both genders and at any stage of development (z.e., neonate, infant, juvenile, adolescent, adult). An infant refers more specifically to a young child under the age of 1 year. A preterm neonate refers more specifically to an infant born prior the completion of 37 weeks of gestation.

[0041] The “limit of detection” (LoD) is the minimum amount of DNA template successfully amplified or the lowest microbial concentration (CFU / ml) detectable.

[0042] The term “microorganism” as used herein refers to a microscopic living organism, which may be single-celled or multicellular. The microorganism contains RNAs or DNAs. Examples of microorganisms include but are not limited to bacteria, DNA viruses, fungi and parasites. In some specific examples, microorganisms are bacteria and DNA viruses. DNA viruses include but are not limited to DNA viruses with double stranded DNAs and DNA viruses with single stranded DNAs. In some examples, microorganisms are pathogenic. The expression “barcode” refers to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating genome of a nucleic acid fragment.

[0043] The term “pathogenic”, “pathogen” and other grammatical variants as used herein refer to the ability of microorganisms to cause diseases.

[0044] “Polymerase fidelity” is the accuracy with which the DNA sequence is copied. Fidelity comparisons between polymerases can be expressed in absolute terms, often by the number of errors per 1,000 or 10,000 nucleotides, or relative terms by using Taq DNA polymerase as the reference standard (IX). Several methods for measuring polymerase fidelity are available and well-known to the skilled in the art (https: / / www.neb.com / en / tools-and- resources / feature-articles / polymerase-fidelity-what-is-it-and-what-does-it-mean-for-your- Pcr).

[0045] The expression “sample multiplexing” means that the molecular barcodes have been added to link given sequence reads to a given sample, and thus allow for the sequencing of multiple samples in the same reaction.

[0046] The term “sepsis” refers to the illness in which the body has a severe, inflammatory response to bacteria or other germs. It is a life-threatening organ dysfunction caused by a dysregulated host response to infection, the most severe form of which is septic shock. Sepsis is the consequence of a bacterial, viral or fungal infection that generally stems from a localized infection (peritonitis, pneumonia, urinary tract infection, catheter-related infection, etc.). It often affects patients with weakened immune systems. When sepsis occurs following an invasive procedure (such as major surgery), the infection is referred to as a nosocomial (or healthcare-associated) infection. Sepsis is characterized by excessive production of inflammatory mediators, often referred to as a "cytokine storm" because of the overproduction of cytokines (chemical mediators that allow cells to communicate with each other), which have an acute impact on vital organ function and can lead to long-term functional sequelae. All bacteria, even those naturally present on the skin or in mucous membranes such as the throat, which are normally non-pathogenic, can trigger the onset of sepsis. Fungal infections can also induce a similar response, as can some viruses (such as SARS, SARS-CoV-2 which causes COVID-19, H1N1 influenza and hemorrhagic fevers). Meningococcal meningitis (specifically purpura fulminans) is a rare cause of sepsis that can occur in healthy young people, as is staphylococcal toxic shock syndrome due to tampon use. “Sepsis” can be herein referred to as “a bacterial blood infection”.

[0047] The term “sequencing” as used herein refers to sequencing methods for determining the order of the nucleotide bases — adenine, guanine, cytosine, and thymine — in a nucleic acid molecule (e.g., a DNA or RNA nucleic acid molecule).

[0048] The term “NGS” as used herein refers to Next Generation Sequencing methods well known in the art and commercially available. NGS encompasses a variety of advanced techniques which include pyrosequencing, sequencing by reversible terminator chemistry, sequencing by ligation, Illumina (Solexa) sequencing, SOLiD sequencing, DNA nanoball sequencing, Helicos single molecule sequencing, Single Molecule Real Time (SMRT) sequencing, and nanopore sequencing. Each of these methods offers unique advantages in terms of speed, accuracy, and the ability to sequence large volumes of DNA or RNA.

[0049] “Metagenomic sequencing” methods include targeted sequencing and shotgun sequencing. Targeted sequencing, also known as amplicon sequencing, focuses on specific genetic markers or conserved regions within microbial communities, such as 16S rRNA genes for bacteria and ITS regions for fungi. “Shotgun” sequencing, on the other hand, involves sequencing all the DNA present in a sample.

[0050] The “Shotgun” involves randomly breaking up the genome into short DNA fragments that are sequenced individually, using transposases which simultaneously cleave template DNA molecules and attach primer binding sites. A subsequent PCR-mediated reaction further amplifies DNA molecules and attaches unique molecular barcodes, allowing sample multiplexing. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. A computer program looks for overlaps in the DNA sequences, using them to reassemble the fragments in their correct order to reconstitute the genome.

[0051] The term “subject” as used herein, generally refers to an animal or other organism, such as a mammalian species (e.g., human), avian (e.g., bird) species, or plant. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. A subject can be an individual that has or is suspected of having a disease or a pre-disposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. The human can be an adult or a child. A child is understood of any human of age below 18.

[0052] DETAILED DESCRIPTION

[0053] The present invention thus relates to method of identification and / or quantification of a pathogen comprised in a biological sample from a subject, the pathogen being a microorganism responsible for an infection. In a preferred embodiment, the infection is a bacterial infection causing sepsis. The inventors have found out that a specific combination of the following steps allows the quick and accurate identification and / or quantification of at least one microorganism responsible for an infection, such as a bacterial blood infection, in a biological sample.

[0054] The method of the invention comprises the following steps: a) spiking a biological sample with quantified calibrator molecules; b) extract the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step c) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons, wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. genomic DNA extracted in step b) ranging from O.Olng to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample. The bacterial infection can cause sepsis. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, the most severe form of which is septic shock. It is characterized by excessive production of inflammatory mediators, often referred to as a “cytokine storm” because of the overproduction of cytokines (chemical mediators that allow cells to communicate with each other), which have an acute impact on vital organ function and can lead to long-term functional sequelae.

[0055] Sepsis is characterized by excessive production of inflammatory mediators, often referred to as a "cytokine storm" because of the overproduction of cytokines (chemical mediators that allow cells to communicate with each other), which have an acute impact on vital organ function and can lead to long-term functional sequelae.

[0056] The method can enable the identification and / or quantification with an accuracy of at least 90% and / or within a maximum time frame of 48 hours, preferably less than 24 hours, preferably less than 10 hours, more preferably less than 8 hours.

[0057] The present invention thus more particularly relates to a method for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample from a subject at risk to suffer from and / or suspected to suffer from sepsis, the method comprising the following steps : a) spiking the biological sample with quantified calibrator molecules; b) extract the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from 0.0 Ing to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

[0058] The DNA polymerase for carrying out the method of the invention must be able to amplify at least 103CFU / ml and at least 0.1 ng of genomic DNA, preferably at least 102CFU / ml and at least 0.01 ng of genomic DNA, more preferably at least 50 CFU / ml and at least 0.001 ng / ml. Thermal profiles of PCR can be adjusted, according to the polymerase. In particular, the DNA polymerase can amplify at least 103CFU / ml, preferably at least 102CFU / ml, more preferably at least 10 CFU / ml. The DNA polymerase can amplify at least 0.1 ng of genomic DNA, preferably at least 0.01 ng of genomic DNA, more preferably at least 0.001 ng of genomic DNA. Suitable DNA polymerases can be easily identified suitable for the invention can be identified by carrying out tests on serial dilutions of DNA or of cultures of reference strains of microorganisms, for example, but not limited to, a reference strain of Lactobacillus plantarum bacteria, Helicobacter pylori bacteria, Ralstonia sp bacteria, T. radiovictrix, I. halotolerans and A. halotolerans.

[0059] In a particular embodiment, the least one microorganism responsible for sepsis is a bacteria.

[0060] In a preferred embodiment of the invention, the method allows both the identification and the quantification of at least one microorganism in the biological sample.

[0061] In a preferred embodiment, the method does not comprise a prior step of growing step. Indeed, the method of the invention can be performed directly on the biological sample without the need of a culture step to enrich the biological sample with the at least one microorganism, thereby reducing the time of identification and / or quantification of the microorganism in the biological sample. In an embodiment, the method comprises a short prior step of growing, typically of less than 8 hours preferably less than or equal to 6 hours. In some instances, adding such a step can enhance the sensitivity of the method, by allowing the increase of the concentration of genetic material that would be otherwise below or at the limit of detection.

[0062] The cells in the samples can be lysed. The extraction of DNA may be carried out by magnetic bead DNA extraction, phenol -chloroform extraction, ethanol or isopropanol extraction, mini-column purification, or any combination thereof.

[0063] The temperature of the annealing step of the PCR of step c) can be comprised between 55°C and 60°C, and more preferably is about 55°C. The extension temperature of the PCR of step c) can be comprised between 65°C and 72°C. The extension time is adapted to the polymerase used to carry out the PCR.

[0064] Primers 27f and 1492r select and amplify only the full-length 16S rRNA gene of the microorganism(s). Primers 27f and 1492r have the following sequence:

[0065] In the primers 27f and 1492r, the 22 nucleotides 5’ tails were added to the primers to serve as the annealing sites for the barcoding primers while the remaining 20 nucleotides are specific for the 16S gene; Y and M are degenerated bases. In particular, in the context of the present invention, the nucleotides indicated with R, Y, M, H, V or W are indicated with IUPAC nucleotide codes. Therefore, in the herein mentioned primers, R is A or G, Y is C or T, M is A or C, H is A or C or T, V is A or C or G and W is A or T.

[0066] Steps c) and d) may be performed in a single step comprising PCR. High-Fidelity DNA polymerases are highly recommended. This family of polymerases are characterized by superior performance in terms of quality defined as “the ability to insert the correct base during PCR reaction”. Usually, High-Fidelity DNA polymerases share an error rate of 3-6 nucleotides per 106bases. Another important feature is the capability of amplifying long DNA fragments (ex. at least l,500bp). The PCR of step c) is preferably carried out with primers which recognize the sequences of the 5’ tails of the primers 27f and 1492r, anneal to such sequences and amplify the 16S rRNA gene amplicons generated in step (b) plus the same barcode. The 16S rRNA amplicons of step c) can have a length between 1.4-1.6 kB, more preferably about 1.5 kB. The 16S sequencing is a powerful and ease of use tool for taxonomical classification and constitutes a first step to the identification of the microorganism(s) present in the biological sample.

[0067] The molecular barcodes of step d) can be ligated using 5 ’-phosphated primers. The molecular barcodes can be synthetic. The molecular barcodes contain a unique nucleotide combination, which i) has no similarity with any biological sequence and thus avoids the risk of aspecific amplification and ii) has no similarity with any other barcodes included and thus avoids the risk of cross-talk or misidentification of the barcodes / samples. The molecular barcodes are specifically designed to target and amplify only the 16S amplicons previously generated. The ligation of molecular barcodes of step d) enables samples multiplexing. The use of the above mentioned 5 ’-phosphated primers allows to remove a step of enzymatic phosphorylation and a subsequent purification.

[0068] The adding of adapters may comprise the use of PCR, ligation enzymes, transposase enzymes, or any combination thereof.

[0069] The sequencing of step f) can be performed by nanopore sequencing, preferably by Oxford Nanopore Sequencing. Amplicons phosphorylation is a crucial step in Oxford Nanopore library preparation, known as “End-prep step” (Oxford Nanopore Technology), the enzymatic-mediated phosphorylation is required for the subsequent sequencing adapter ligation. The use of 5'-phosphorilated primers allows to skip the “End-prep” step, saving time and reducing the cost of the procedure (as no reaction and clean-up are performed).

[0070] Preferably the sequencing of step f) is performed using a Next Generation Sequencing (NGS) method. The method provides a result of identification and / or quantification within a maximum time frame of 36 hours, preferably of 24 hours, preferably of 12 hours, preferably 10 hours, preferably 8 hours.

[0071] The method of the invention can provide a result of step g) with a sensitivity (i.e. limit of detection) of 100 CFU / ml, preferably 50 CFU / ml, more preferably 10 CFU / ml. The method can identify and / or quantify the at least one microorganism present with at least 0.01% relative abundance in the bacterial community of said biological sample. This low limit of detection is particularly advantageous for the implementation of the method on samples from the subject without any culture step aiming at growing (amplifying) and / or selecting microorganisms or with a short prior step of growing of less than 8 hours, preferably less than or equal to 6 hours as detailed above.

[0072] The method allows the identification and / or quantification of at least one microorganism present in a biological sample with an accuracy of at least 90%, preferably of 95% and more preferably of 99%. Accuracy of the method can be measured by testing the method on a mixture of predefined bacterial species with known amount of each species in the mixture. Then, the results obtained by the method of the invention, both in terms of quantity and quality, are compared to the known quantity of each species in the mixture.

[0073] In an embodiment, the method of the invention identifies and quantifies at least one microorganism responsible for sepsis present in a biological sample, such as blood, urine, pulmonary wash, or synovial fluid, up to a minimum amount of 50 CFU / ml of biological sample with an accuracy of 99% within 24 hours.

[0074] Preferably, the method of the invention comprises a further step of depletion of host DNA. The host DNA is the genomic DNA of the host from which the biological sample comes.

[0075] Advantageously, the method according to the invention can comprise, further to the above- mentioned steps, a step of metagenomic sequencing. Though having a higher limit of detection in term of genome copies, this further step can be used to confirm the identification, or the absence, in the tested sample, of the bacterial infection which can cause sepsis, thereby allowing a gain in the reliability of the diagnostic of said bacterial infection. This redundant detection of at least one microorganism at the phylum level, preferably at the genus level and more preferably at the species level is thus particularly advantageous. In an embodiment, said metagenomic sequencing also allows the identification of possible other fungal or viral infection(s) in the tested subject. Also, it can allow the identification of antibiotic resistance genes or of virulence factor genes associated with the detected bacterial or fungal infection.

[0076] In a preferred embodiment, said metagenomic sequencing step is a Shotgun sequencing.

[0077] Noticeably, even assuming the 16S rRNA gene sequencing and the metagenomic sequencing are performed successively, the total duration of the process remains significantly lower than the Standard of Care (SoC) cultures for patients with sepsis, which usually is around between from 24 hours to 3 - 5 days. In an embodiment, when metagenomic sequencing is used further to the method of the invention, the whole method provides a result of identification and / or quantification within a maximum time frame of 36 hours, preferably of 24 hours even more preferably within a time frame of 16 hours, preferably of 12 hours.

[0078] In an embodiment, the step of metagenomic sequencing is performed concomitantly to the full-length 16S rRNA sequencing of the above steps.

[0079] In an embodiment, the step of metagenomic sequencing can be performed subsequently to the full-length 16S rRNA sequencing of the above steps. This can allow, for example the implementation of precautionary emergency treatments, while waiting for results of Shotgun sequencing on possible co-infection and presence of virulence and / or resistance genes.

[0080] Biological sample

[0081] The biological sample can be selected from blood, urine, synovial fluid, pulmonary wash, bronchoalveolar lavage , plasma, serum, amniotic fluid, cerebrospinal fluid, nasal wash, saliva, semen, vaginal fluid, sputum, a biopsy, rinse liquid of catheters and the like and the combinations thereof.

[0082] According to a preferred embodiment, the biological sample is preferably selected from blood, plasma, serum, urine, synovial fluid, and pulmonary wash. According to a most preferred embodiment, the biological sample is pulmonary wash. According to another preferred embodiment, the biological sample is derived from blood. Accordingly, said blood derived sample is a blood, plasma or serum sample, plasma sample being particularly preferred.

[0083] The up to now unmet sensitivity of the method according to the invention renders possible the implementation of the method even on low quantity of sample from the subject (and possibly low bacteria quantities). This is particularly beneficial, notably but not only, for subjects for whom sample collection is difficult, or only allowed in small quantities compared to, e.g. the commonly analyzed volume of said sample. This is the case, for example, for the blood samples from elderly people, who often present veinous defects and fragility, or from neonates for which only very small volumes of blood can be collected. This low quantity of sample which is needed to carry out the method of the invention allows also more availability of sample to perform other biological analyses if needed.

[0084] In a particular embodiment, therefore, the sample is of a volume equal or less than 5ml, equal or less than 4ml, equal or less than 3ml, equal or less than 2ml, equal or less than 1ml equal or even equal or less than 0.5ml. In a more particular embodiment, the sample is a sample of blood, plasma, or serum of a volume equal or less than 5ml, equal or less than 4ml, equal or less than 3ml, equal or less than 2ml, equal or less than 1ml equal or even equal or less than 0.5ml. In an even more particular embodiment, the sample is or is derived from, a blood sample of a volume equal or less than 5ml, equal or less than 4ml, equal or less than 3ml, equal or less than 2ml, equal or less than 1ml equal or even equal or less than 0.5ml. In another particular embodiment, the sample is a sample of blood, plasma, or serum of a volume equal or less than 0.5ml.

[0085] Calibrator molecules and calibration

[0086] The calibrator molecule can be any microbial species alien to the human microbiome. The calibrator molecule can be selected amongst Lactobacillus plantarum bacteria, Helicobacter pylori bacteria, Ralstonia sp bacteria, T. radiovictrix, I. halotolerans and A. halotolerans, or their combination. Preferably, the calibrator molecule is Lactobacillus plantarum bacteria. Lactobacillus plantarum bacteria are most preferred as they are not associated with sepsis and are difficult to lyse. According to an embodiment, the biological sample is spiked with a fixed amount of the calibrator molecule.

[0087] The method of the invention may comprise a further step of comparing the quantity of said at least one microorganism to the fixed amount of Lactobacillus plantarum and Helicobacter pylori thereby quantifying said at least one microorganism in the biological sample.

[0088] Microorganisms

[0089] According to one embodiment, the method results in redundant detection of at least one microorganism at the phylum level, preferably at the genus level and more preferably at the species level.

[0090] According to one embodiment, at least one microorganism is responsible for sepsis. Sepsis can be caused by any infectious agents such as bacteria, a fungus, a virus, or the combinations thereof. The at least one microorganism responsible for sepsis can be a bacteria. The bacteria can be Gram-positive bacteria or Gram-negative bacteria. The Grampositive bacteria can notably be selected from Methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus spp, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae spp equisimilis (SDSE , Streptococcus anginosus, Streptococcus constellatus, Streptococcus alpha hemolytic, Streptococcus beta hemolytic, Streptococcus pneumoniae, Streptococcus spp, Enterococcus faecalis, Enterococcus faecium, Enterococcus spp, Coagulase negative saphylocci, other gram-positive cocci, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Clostridium spp, Rickettsia spp, and the combinations thereof. The Gram-negative bacteria can notably be selected from Moraxella catarrhalis, Neisseria meningitidis, Neisseria spp, Acinetobacter baumannii, Acinetobacter spp, Aeromonas hydrophila, Aeromonas spp, Bacteroides fragilis, Bacteroides spp, Burkholderia cepacia, Burkholderia spp, Citrobacter freundii, Citrobacter spp, Escherichia coli, Enterobacter spp, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella spp, Legionella pneumophila, Legionella spp, Morganella morganii, Pseudomonas aeruginosa, Pseudomonas spp, Proteus mirabilis, Proteus spp, Rickettsia spp, Salmonella enteritidis, Salmonella spp, Serratia marcescens, Serratia spp, Stenotrophomonas maltophilia, Stenotrophomonas spp, Vibrio vulnificus, Vibrio cholerae. Vibrio spp, and the combinations thereof.

[0091] A fungus responsible for sepsis can notably be selected from Candida species (accounting for the vast majority of invasive fungal infections) and Aspergillus species, or Cryptococcus species. Said fungus can more specifically be selected from Aspergillus spp, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida spp, Mycobacterium tuberculosis, Mycobacterium spp, Chlamydophila pneumoniae, Chlamydophila psittaci, Chlamydophila spp, Mycoplasma pneumoniae, Mycoplasma spp, Pneumocystis jirovecii, and the combinations thereof.

[0092] A Virus responsible for sepsis can notably be selected from cytomegalovirus, Epstein-Barr virus, influenza viruses, coronaviruses and the combinations thereof.

[0093] Subject

[0094] The biological sample can be obtained from an animal. The animal can be a farm animal such as cows, sheeps, goats, pigs and poultry, a zoo animal such as a reptile, a bear, a pet animal such as a dog, a cat, a fish, a bird, a horse, a reptile, a mouse, a rat, a Guinea-pig. . . The animal can be a human.

[0095] The subject can be a patient, i.e., a mammal, preferably a human, who is awaiting the receipt of medical care, or is receiving medical care or has been the object of a medical procedure.

[0096] The human can be an adult or a child. According to one embodiment, the human is at risk of developing sepsis. In an embodiment, said human at risk of developing sepsis is a subject who is or has been hosted in the health facility, notably in intensive care unit or long hospital stay. Risk factors for sepsis include, but are not limited to : age over 65, infancy, lower immune response, chronic disease, immune defect, pregnancy, preterm birth neonate. In a particular embodiment said human is a neonate. In a more particular embodiment, said human is a low birth weight infant or a preterm neonate. According to one embodiment, the subject is at risk of developing sepsis. According to one embodiment, the subject is a patient who has undergone surgery or invasive procedure. According to an embodiment, the subject is a patient who must undergo surgery or invasive procedure. According to an embodiment, the subject is a patient who is immunocompromised. According to an embodiment, the subject is a patient who is or has been connected to devices that go in the body like indwelling intravenous lines or catheters, urinary catheters, breathing tubes and the like. According to an embodiment, the subject is suspected to suffer from sepsis, it is a patient who shows at least one symptom of sepsis. According to an embodiment, the at least one symptom of sepsis is selected with fever, hypothermia, chills, confusion, delirium, low blood pressure, rapid heartbeat, skin rash, mottled skin, lightheadedness, and the combinations thereof. According to another embodiment, the subject suspected to suffer from sepsis is a human with a Sepsis-related Organ Failure Assessment (SOFA) score of at least 2 (Singer et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016 Feb 23;315(8):801-10.).

[0097] Antibiotic resistance gene and virulence factor and / or viral or fungal infection

[0098] The method of the invention can comprise a further step of detection of at least one gene of antibiotic resistance and / or at least one virulence factor in at least one microorganism, and / or at least one gene leading to the identification of a viral or fungal infection possibly (co-) responsible for the sepsis.

[0099] In an embodiment, said further step of detection is implemented through metagenomic sequencing, preferably Shotgun sequencing.

[0100] According to one embodiment, the gene of antibiotic resistance provides resistance to tetracyclines, sulfonamides, P-lactams, macrolides, aminoglycosides, quinolones and fluoroquinolone, colistin, vancomycin, and the combinations thereof. The gene of the at least one antibiotic resistance can be selected from mdr, tetM, W,38 sul!2, mecA,C, vanA,B,C, aad, flo, dfr, eft, bla TEM, bla CTX-M, bla VIM, bla KPC, bla IMP, bla OXA, ermA,B,C,, AAC(6’)-Ie-APH(2 ”)-Ia, ANT(4 )-Ib, arlS-R, blaZ, frmA, fosB, norA, ImrS, mecA,I,Rl, mepA-R, optrA, mgr A, , qnrA,B,S, mefA,E, mrej catAl,B 3, mexA,B,D,X,Y, mprF, mcr-1 and the combinations thereof. The virulence factor is a molecule or structure produced by the at least one bacteria, virus and / or fungus, possibly (co-) responsible for the sepsis, that helps theses pathogen to colonize the host, evade or suppress the host's immune response, enter and exit host cells, and obtain nutrients from the host. The at least one virulence factor can be selected from adsA. cap8A-P, clfA-B. ebp. fnhA. geh, hlb. hlgA-C, hly hla. hysA, icaA-I). icaR, isdA-G, lip, map, sak, sdrC-E, spa, srtB, sspA-C, tsst-1, vWbp and combinations thereof.

[0101] In an embodiment, said further step of detection is used to detect and / or identify a virus or a fungus possibly responsible or co-responsible of a sepsis. Preferably, said detection and / or identification of a virus or a fungus responsible or co-responsible for the sepsis is performed through metagenomic sequencing, preferably Shotgun sequencing.

[0102] In an embodiment, though displaying a higher limit of detection than the 16S rRNA sequencing method according to the invention, said further step is useful to confirm or refute the result obtained by the 16S rRNA sequencing method.

[0103] In an embodiment said further step is adapted to allow the detection and / or identification of a viral or a fungal infection and the detection of at least one gene of antibiotic resistance and / or at least one virulence factor of at least one microorganism.

[0104] Kits

[0105] The present invention also relates to a kit of parts for the identification and / or quantification of at least one microorganism responsible for an infection, preferably a bacterial infection causing sepsis, in a biological sample as disclosed herein: i. a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2), ii. a sample of DNA polymerase iii. at least one calibrator molecule, iv. phosphorylated barcoding primers enabling sample multiplexing, and v. at least one collection tube containing glycerol to collect a biological sample, and vi. instructions for use. According to one embodiment, the DNA polymerase of the sample can amplify at least 103CFU / ml and at least of 0.1 ng of genomic DNA, preferably at least 102CFU / ml and at least of 0.01 ng of genomic DNA.

[0106] According to one embodiment, the kit of parts also comprises reagents for the extraction of genomic DNA selected from a lysis buffer, a PBS buffer, a binding buffer, a washing buffer, an elution buffer, at least one extraction column, and their combinations.

[0107] Instructions for use of a kit of the invention are as follows: collect biological sample from patients, preferably in EDTA tube,

[0108] - transfer the biological sample in a sample tube containing glycerol if the sample need to be stored for future investigations or proceed immediately with the extraction. mix 1 volume of the biological sample with 2 volumes of the at least calibrator molecule provided in the kit of the invention, extract DNA from the biological sample mixed with the at least one calibrator molecule, optionally, deplete the host DNA, for instance the genomic DNA from the patient, add the genomic calibrator molecule provided in the kit, in order to check the quality of the PCR and quantify the eventual pathogen. perform PCR to amplify the 16SRNA using the pair of primers 27f and 1492r provided in the kit of the invention, further amplify the amplified 16SRNA using the phosphorylated barcoding primers for sample multiplexing perform sequencing of the amplified sequence, compare the results of sequencing to a reference database.

[0109] The kit according to the invention is thus particularly useful for use in the method for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample from a subject at risk to suffer from and / or suspected to suffer from sepsis as described herein. In an embodiment, the invention relates to a use of said kit in said method for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample from a subject at risk to suffer from and / or suspected to suffer from sepsis. Also, in another embodiment, the invention relates to the use of the above described kit in a method of diagnosing sepsis from a biological sample collected in a subject at risk of, or suspected to suffer from sepsis as described above.

[0110] Uses

[0111] The present invention also relates to the use of the method of the invention as disclosed herein for detecting an infection and preferably for the diagnosis of sepsis. The method of the invention can be performed to diagnose sepsis in a subject who shows symptoms of sepsis to confirm or infirm a diagnosis of sepsis. In a particular embodiment, the method of diagnosing sepsis is a method of diagnosing bacterial sepsis. The method of the invention can also be used to determine whether the microorganism responsible for sepsis in a subject comprises at least one gene of resistance to an antibiotic and / or at least one gene coding for a virulence factor coding for a virulence factor. The method of the invention can also be used to determine the antibiotic treatment to administer to a subject and / or any other treatment as a function of e.g. the virulence factors that have been identified.

[0112] The method of the invention can be performed in a hospital, a medical laboratory, a microbiology laboratory, a surgical operation room, at bed site for the diagnosis of sepsis.

[0113] The method of the invention can be carried out before a surgical operation to ensure that the patient who must undergo a surgical act is free of sepsis. The method of the invention can also be carried out after a surgical operation to ensure that the patient who has undergone a surgical act did not catch a nosocomial infection. The method of the invention can also be carried out on sample from a patient who is connected to devices that go in the body like indwelling intravenous lines or catheters, urinary catheters, breathing tubes and the like. The method of the invention can be performed to diagnose sepsis in a subject who shows symptoms of sepsis, or to confirm or infirm a diagnosis of sepsis. For example, said subject presents a SOFA score of at least 2. In this instance, the identification, using the method of the invention, of a microorganism in the sample of the patient allows to confirm the patient suffers from sepsis, and therefore allows the rapid implementation of the suitable treatment. The method of the invention can also be used to determine whether the microorganism responsible for sepsis in a subject comprises at least one gene of resistance to an antibiotic. The method of the invention can also be used to determine the antibiotic treatment to administer to a subject, in particular a subject diagnosed as suffering from sepsis.

[0114] According to other embodiments:

[0115] The invention relates to a method for the identification and / or quantification of at least one microorganism responsible for an infection in a biological sample comprising the steps of: a) spiking the biological sample with quantified calibrator molecules; b) extract the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step c) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. genomic DNA extracted in step b) ranging from O.Olng to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16SrRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16SrRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

[0116] In said method, the DNA polymerase can amplify at least 103CFU / ml, preferably at least 102CFU / ml, more preferably at least 10 CFU / ml.

[0117] In said method, the DNA polymerase can amplify at least 0.1 ng of genomic DNA, preferably at least 0.01 ng of genomic DNA, more preferably at least 0.001 ng of genomic DNA. In said method, the DNA polymerase can amplify at least 103CFU / ml and at least of 0.1 ng of genomic DNA, preferably at least 102CFU / ml and at least of 0.01 ng of genomic DNA.

[0118] In said method, the infection is selected amongst a bacterial infection, a viral infection, a fungal infection and the combinations thereof.

[0119] In said method, the infection is a bacterial infection causing sepsis.

[0120] In said method, the method enables the identification and / or quantification with an accuracy of at least 90% and / or within a maximum time frame of 48 hours.

[0121] In said method, the PCR of step c) can comprise an annealing step and the temperature of the annealing step is about 55°C.

[0122] In said method, the molecular barcodes of step d) can be ligated using 5 ’-phosphated primers.

[0123] In said method, the sequencing of step f) can be performed by nanopore sequencing.

[0124] In said method, the 16S rRNA amplicons of step c) have a length between 0.05-5 kB, preferably between IkB to 3 kB, more preferably about 1.5 kB.

[0125] In said method, the ligation of molecular barcodes of step d) can enable sample multiplexing.

[0126] In said method, the method can provide a result within a maximum time frame of 36 hours, preferably of 24 hours, most preferably of 12 hours

[0127] In said method, the calibrator molecule is selected from Lactobacillus plantarum bacteria, Helicobacter pylori bacteria, T. radiovictrix, I. halotolerans and A. halotolerans, and the combinations thereof.

[0128] In said method, the calibrator molecule is a Lactobacillus plantarum bacteria.

[0129] Said method, can further comprise a step of comparing the quantity of said at least one microorganism to the amount of the quantified calibrator molecule thereby quantifying said at least one microorganism in the biological sample. In said method, the biological sample is selected from blood, urine, synovial fluid, pulmonary wash, plasma, serum, amniotic fluid, cerebrospinal fluid, nasal wash, saliva, semen, vaginal fluid, sputum, a biopsy, and the combinations thereof.

[0130] In said method, the biological sample is preferably selected from blood, plasma, serum, urine, synovial fluid, and pulmonary wash.

[0131] In said method, the biological sample is pulmonary wash.

[0132] In said method, the biological sample can be obtained from a subject.

[0133] In said method, the subject is an animal.

[0134] In said method, the animal is a farm animal such as a cow, a sheep, a goat, a pig, and poultry, a zoo animal, a pet animal such as a dog, a cat, a fish, a bird, a horse, a reptile, a mouse, a rat, a Guineapig. . .

[0135] In said method, the animal is a mammal.

[0136] In said method, the mammal is a human.

[0137] In said method, the human is an adult or a child.

[0138] In said method, the subject is at risk of developing sepsis.

[0139] Said method of the invention further comprises a step of detection of at least one gene of antibiotic resistance and / or a virulence factor in at least one microorganism. According to one embodiment, the gene of antibiotic resistance provides resistance to tetracyclines, sulfonamides, P-lactams, macrolides, aminoglycosides, fluoroquinolone, colistin, vancomycin, multidrug (mdr), and the combinations thereof.

[0140] Said method can provide a result of step g) with a sensitivity of 1000 CFU / ml, preferably 100 CFU / ml, more preferably of 50 CFU / ml.

[0141] Said method identifies and / or quantifies at least one microorganism present with at least 0.01% relative abundance in the bacterial community of said sample. Said method results in redundant detection of at least one microorganism at the phylum level, preferably at the genus level and more preferably at the species level.

[0142] The invention also relates to a kit of parts for the identification and / or quantification of at least one microorganism responsible for a bacterial infection, preferably a bacterial infection causing sepsis, in a biological sample as disclosed herein: i. a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2), ii. a sample of DNA polymerase iii. at least one calibrator molecule, iv. phosphorylated barcoding primers enabling sample multiplexing, and v. at least one collection tube containing glycerol to collect a biological sample, and vi. instructions for use.

[0143] In an embodiment the kit of parts also comprises reagents for the extraction of genomic DNA are selected from a lysis buffer, a PBS buffer, a binding buffer, a washing buffer, an elution buffer, at least one extraction column, and the combinations thereof.

[0144] The invention also relates to the use of the method of the invention as disclosed herein for the diagnosis of an infection.

[0145] According to an embodiment, the present invention relates to a method of diagnosing an infection in a biological sample collected from a subject potentially infected with at least one microorganism, the method comprising the steps of: a) spiking the biological sample with quantified calibrator molecules; b) extracting the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from O.Olng to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16SrRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16SrRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

[0146] According to an embodiment, said method of the invention further comprises a step of determination of antibiotic resistance gene and / or a virulence factor of the at least one microorganism.

[0147] In said method the infection is a bacterial infection causing sepsis.

[0148] EXAMPLES

[0149] The present invention is further illustrated by the following examples.

[0150] Example 1: Comparison between 16S rRNA sequencing and Shotgun metagenomic sequencing and extraction protocols

[0151] Two different sequencing-based workflows were tested: 16S rRNA sequencing and Shotgun metagenomic sequencing. The purpose of the example is to assess the limit of detection (LoD) of both sequencing procedures. The LoD is defined as the lowest microbial concentration (CFU / ml) detectable.

[0152] Serial dilutions (1: 10 v / v), ranging from 108to 10° CFU / ml, were prepared using a methicillin-resistant, biofilm-formant Staphylococcus aureus ATCC 43300 strain (LGC Standards). Dilutions were performed using freshly collected human blood: a 99:1 human- microbial cells ratio was maintained to mimic bloodstream infection. In parallel, lOOpl of dilution were plated on 5% Blood Agar Plate to evaluate the real concentration (CFU / ml).

[0153] Two extraction protocols were tested: i) MagMax Microbiome Ultra Nucleic Acid Isolation kit (ThermoFisher) and ii) QIAmp Microbiome kit (Qiagen). While the MagMax kit is based on bead-beating cell lysis enabling total DNA isolation, the QIAmp Microbiome kit includes a chemical-based host DNA depletion followed by a mechanical lysis of microbial cells thus reducing human DNA contamination. DNA extractions were carried out following manufacturers’ instructions.

[0154] Sequencing library preparation is performed as follows. The 16S rRNA amplicon-based sequencing was performed according to the protocol disclosed in M. Szoboszlay et al, Nanopore Is Preferable over Illumina for 16S Amplicon Sequencing of the Gut Microbiota When Species-Level Taxonomic Classification, Accurate Estimation of Richness, or Focus on Rare Taxa Is Required, Microorganisms 2023, 11, 804. https: / / doi.org / 10.3390 / microorganismsl l030804). Briefly, the full-length 16S rRNA gene is amplified through a PCR reaction as described in Example 1. Molecular barcodes are then attached enabling sample multiplexing. Lastly, after an enzymatic T4 ligase-mediated sequencing adapters ligation, 16S amplicons are sequenced. The sequencing run was performed using the up-to-date Nanopore V14 chemistry enabling high-quality data generation: Phred Quality score Q20 roughly corresponding to 99% accuracy.

[0155] On the other hand, Shotgun metagenomics was performed using the previous V10 chemistry (SQK-RPB004). The library preparation is characterized by an easier and user-friendly approach. Using a transposon complex, the input DNA is cleaved into fragments of about 2,000 bp in length and molecular barcodes are then added through a PCR reaction. The reaction is prepared as follows: 25 pl of polymerase are mixed with 20pl nuclease-free water, 4pl tagmented DNA, and I l barcoding primers (RLB1 denaturto 24). The PCR thermal profile consisted of an initial denaturation of 3min at 95°C, followed by 25 cycles of 15s at 95°C, 15s at 56°C and 6min at 65°C, and a final step of 5min at 65°C.

[0156] Results of!6S rRNA sequencing:

[0157] The LoD for 16S rRNA amplicon sequencing was 102CFU / ml, even though the percentage of “other species” (i.e. Ralstonia spp. or spurious classification) exceeds 90%. Notably, the host DNA depletion step had further increased the sensitivity: the percentage of reads correctly identified as S. aureus were 78%, 10-times more compared to MagMax microbiome kit. The increased accuracy can be attributed to a depletion of human DNA increasing the microbial DNA biomass (“pure” microbial DNA).

[0158] Results Shotgun Whole Genome sequencing: Taxonomy

[0159] Results are expressed as percentage (%) of classified reads. Initially, the LoD was set to >106CFU / ml (rapid DNA isolation) but performing a step of host DNA depletion allowed the

[0160] LoD to be further decreased at 104CFU / ml. The analysis is focused on the detection of Antimicrobial Resistance Genes (AMR) and Virulence Factors (VF) as major contributors for pathogenesis and onset of the disease. Results Shotgun Whole Genome sequencing: Genomic Features

[0161] The reference gene list was prepared according to pure S. aureus ATCC 43300 sequencing (positive control). Detected genes are marked with an “X”. In bold are highlighted clinically relevant genes.

[0162] Note: The reference gene list was prepared according to pure S. aureus ATCC 43300 sequencing (positive control). Genes of interest are highlighted in bold. Detected genes are marked with an “X”, while partial genes / operons with

[0163] Example 2: Clinical data Purpose

[0164] The technology was validated on clinical and laboratory samples spiked with Staphylococcus aureus. The aim was i) detection of the aetiologic agent causing disease and determination of the technology’s sensitivity and ii) identification of antimicrobial resistance genes (AMR) and virulence factors (VF).

[0165] Biological samples

[0166] Clinical samples were collected in two different medical centers (Tablet).

[0167] Table 1 - Samples collected Three different sample types were tested: blood, bronchoalveolar lavage (BAL), and plasma.

[0168] All the samples were collected from patients suspected with bloodstream infection and / or pneumonia. Plasma samples were generated by centrifugation of the biological sample at

[0169] 1,500 rpm. Samples should not be centrifugated at speeds higher than 1800 rpm. Once collected the samples were immediately stored at -80° C. Samples of blood and plasma are stored with 10% glycerol (v / v) and the BAL are treated with DTT 0.1% before freezing.

[0170] Serial dilutions of Staphylococcus aureus were performed from diluted (1 : 10 v / v) in sterile water generating reference dilutions ranging from 108to 101CFU / ml. Sterile human blood samples were spiked with known concentrations of S. aureus ranging from 106to 101

[0171] CFU / ml. Samples were analyzed using both full-length 16S rRNA amplicon sequencing and Shotgun sequencing technologies. Method

[0172] Step I: full-length 16S amplicon sequencing.

[0173] Amplification of the full-length 16S rRNA gene was performed pipetting 12.5pl of Q5 HotStart High-Fidelity 2x Master Mix (NEB) DNA polymerase, 2 pl of primers 27f (SEQ ID No. 1) and 2pl of primers 1492r (SEQ ID No. 2), 4.5 pl DNA template, and 4pl water. The PCR thermal profile consisted of an initial denaturation of 3min at 98°C, followed by 30 cycles of 15s at 98°C, 30s at 55°C and Imin at 72°C, and a final step of 2min at 72°C. PCR yields were run on a electrophoretic gel such as E-gel electrophoretic system (Thermo Fisher) in order to evaluate proper amplification.

[0174] Then, samples were processed for Nanopore library preparation. In short, amplicons were purified with 0.6x magnetic beads, barcoded through a second PCR reaction, and adapter ligated prior Nanopore sequencing. The library was sequenced for about 2 hours.

[0175] Step II: Shotgun whole genome sequencing.

[0176] Shotgun whole genome sequencing was performed conventionally, such as using SQK- RPB 114.24 kit (Oxford Nanopore Technologies). 5 pl DNA template were randomly sheared using a transposome complex, generating fragments of about 2,000 Kbp in length (median length). Unique barcodes were ligated through a PCR reaction following manufacturer’s guidelines. The PCR reaction consisted of an initial denaturation of 3min at 95°C, followed by 25 cycles of 15s at 95°C, 15s at 56°C and 6min at 65°C, and a final step of 6min at 65°C. Amplified samples were run on a electrophoretic gel such as E-gel electrophoretic system (Thermo Fisher) to check proper amplification and finally adapter-ligated prior sequencing.

[0177] Step III: host DNA depletion.

[0178] To reduce the impact of the host DNA on the sequencing run and increase sensitivity of the Shotgun technology, a step of host DNA depletion was performed following any conventional method such as using a host depletion kit (QIAamp DNA Microbiome kit, Qiagen). Depletion kits are specifically designed to differentially lyse host and microbial cells, removing unwanted human DNA thus increasing the quantity of recovered microbial DNA. DNA extraction was performed according to manufacturer’s guidelines. Recovered DNA was screened using both 16S and Shotgun sequencing technologies. Results

[0179] Enhanced Sensitivity of Pathogen Detection Technology

[0180] The method of the invention presents a breakthrough in pathogen detection technology, achieving unprecedented levels of sensitivity compared to current state-of-the-art methodologies (such as the one disclosed in Dong M et al; Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens. J Appl Microbiol. 2016 Apr; 120(4): 1119-29. doi: 10.1111 / jam. l3082, in particular in Table 2). The results obtained by the method of the invention demonstrate a sensitivity that is at least 10 times higher than existing methods, revolutionizing pathogen detection in clinical specimens. Current pathogen detection methodologies, as illustrated by Dong M. et al's publication (J Appl Microbiol. 2016), on standardized methods for generating mock clinical specimens indicate success rates of 100% with 10,000 CFU / ml of S. aureus in blood, and a mere 30% success rate with 1,000 CFU / ml of blood. Notably, this study utilized nextgeneration sequencing (NGS) on the 16S rRNA gene for analysis. Despite their reported 100% success rate with 10,000 CFU / ml, the method of the invention attains identical results with a minimum of 100 CFU / ml of S. aureus in blood, evidencing a 100-fold increase in sensitivity compared to the established standards. Where prior studies obtained optimal results with significantly higher pathogen concentrations, the method of the invention outperforms by detecting and identifying pathogens at vastly lower concentrations, signifying its unmatched sensitivity and accuracy. The method of the invention results in a 100-fold increase in sensitivity over existing methods.

[0181] Method of the invention and Shotgun on clinical samples

[0182] The method of the invention showed higher sensitivity compared to Shotgun sequencing in both laboratory and clinical samples. Staphylococcus aureus was correctly identified by the method of the invention, even at lower dilutions (100 CFU / ml). On the contrary, Shotgun sequencing enabled identification of S. aureus only at higher dilutions (reported detection limit of 106CFU / ml) (Table 2 and Table 3).

[0183] A common environmental strain of Ralstonia sp. was spiked in the 16S sequencing analysis as per 105CFU / ml, in order to have a positive control of the PCR reaction and also to have a reference for accurate quantification (Table2). Table 2 - Staphylococcus aureus sequencing results with the method of the invention.

[0184] Table 3 - Staphylococcus aureus sequencing results with the Shotgun technology. Despite having a higher limit of detection, Shotgun sequencing had the potential to detect genomic features such as antimicrobial resistance genes and virulence factors. The method of the invention allows the correct identification of known S. aureus AMR and VF genes (70% and 60% accuracy, respectively) (Table 4 and Table 5).

[0185] Table 4 - Detected Antimicrobial Resistance Genes. antibiotics.

[0186] Table 5 - Detected Virulence Factors - Known S. aureus Virulence Factors (VF) genes

[0187] “nd” = Not Detected gene. Relevance of VFs was determined based on their role in hostpathogen interaction and disease onset.

[0188] Clinical samples were screened following the same library preparation as previously described for laboratory samples. Achieved results were also compared to official diagnostic results (clinicians) to verify the accuracy of the technology. As shown in Table 6, better results were achieved by the method of the invention (higher sensitivity). The sample 110007-BAL was correctly screened with both the method of the invention and Shotgun sequencing, even though <1% of the sequencing reads (in the Shotgun) were from microbial specimens (Legionella maceachernii). For the analysis of pulmonary washes, a step of treatment with 0.1% Dithiothreitol (DTT) is added. Interestingly, this result is in line with the results achieved by the Hospital using classical microbiology approach. Samples AA006, F003, L042, and Y023 failed with both the method of the invention and Shotgun sequencing putatively related to lower microbial loads (ex. below technology detection limit) or to the absence of a pathogen at the time of screening. Sample AA005 was screened for K. pneumoniae with traditional standard workflows and even the method of the invention confirmed the presence of the pathogen. Interestingly, the method of the invention identified another species: S. haemolyticus. This species concentration was also much higher than the K. pneumoniae. However, S. haemolyticus is not a common contaminant (ex. during sample collection procedures) but an occasional nosocomial pathogen. In addition, K. pneumoniae and S. haemolyticus share different 16S profiles excluding classification errors. Another important result achieved from the analysis on clinical samples is that no false positives were reported, indicating a good response of the method of the invention that is detecting no aspecific signals.

[0189] Table 6 - Clinical samples results.

[0190] The huge difference between 16S and Shotgun sequencing sensitivity is putatively related to huge host DNA contamination. Since Shotgun enables the sequencing of total DNA, the number of microbial reads represent only a small fraction (<1%) of the total sequencing yields (Table3). To reduce the impact of the host DNA on the sequencing run and increase sensitivity of the technology, a step of deletion of unwanted human DNA using a commercial kit and following the manufacturer’s instructions was carried out and increase the quantity of recovered microbial DNA. No substantial changes were detected for 16S rRNA sequencing as the reported detection limit was still 102CFU / ml. However, substantial improvements were reported for laboratory samples Shotgun sequencing: S. aureus was successfully detected at 104CFU / ml. No major changes were reported for clinical samples.

[0191] Conclusions

[0192] All data obtained by performing the method of the invention have been compared with results achieved by the original hospitals that used the standard procedures. Moreover, the sensitivity of the technology, validated on human blood, spiked with a reference pathogen such as with S. aureus has been compared to data available in literature.

[0193] No false positive results and no false negative results were reported with the method of the invention: all results were confirmed with the hospital findings, thereby validating the technology. The method of the invention provided results with a LoD of 102CFU / ml, while Shotgun sequencing was only suitable for samples with high microbial load ( at least 106CFU / ml) or at least 104CFU / ml after human DNA depletion step. The sensitivity of the results achieved with the method of the invention has been at least 10 times higher than what is currently available as state of art. In addition, the method of the invention avoids potential diagnosis errors that may occur when using the conventional methods based on growing the pathogen, in case that several pathogens are available in a sample with significant differences in growing rates. The major reported limitations for the Shotgun sequencing are the huge host DNA contamination (which can reach up to 99.9% of the data) and the low microbial loads.

[0194] 16S rRNA sequencing was characterized by a faster workflow than Shotgun as shown in Table 6.

[0195] Table 7 - Workflow turnaround

[0196] Time (hours) required to identify microbial organisms. sequencing time for Shotgun is variable, depending on the amount of host DNA and required sequencing depth. In case of host DNA depletion, 4 hours extra must be included in the turnaround. Despite being characterized by faster library preparation, Shotgun sequencing is a timeconsuming procedure. Shotgun run time is extremely variable, depending on presence of host DNA limiting microbial read sequencing and the required sequencing depth and the detection of AMR and VF genes may require more time. In case that a host DNA depletion step is carried out, an extra 4-hour workflow is included. Due to the different sensitivity and the higher reported LoD, the Shotgun sequencing will generally require a 4 to 8 hours growth in hemocultures to increase the specificity of the assay as more microbial DNA will be recovered. Since Shotgun sequencing suffered the most from the huge host DNA contamination, the availability of depletion kits represents an important technology improvement and thus required further optimization.

[0197] The method of the invention allows for a fast and accurate detection of multiple pathogens in a blood or BAL sample, with a detection limit of LoD of about 102CFU / ml, without prior growing steps. This is much faster that the current conventional method. In addition, the method of the invention also allows the successful species identification in a maximum of 12 hours from collection of the biological sample. This is a huge advancement with respect to standard practice that requires a minimum of 24 hours and a maximum of 5 to 7 days for a successful pathogen identification.

[0198] The findings confirm that Shotgun technology allows for effective determination of the antimicrobial resistance genes and virulence factors at a LoD of at least 106CFU / ml, and therefore generally requires a previous growing step. Even with a growing step, this is much faster than the conventional method.

[0199] Example 3: Study of combined analysis of 16S rRNA sequencing and metagenomic sequencing, comprising a short growing step of blood samples form ICU patients.

[0200] Method

[0201] One hundred (100) patients with suspected sepsis admitted in intensive care units (ICU) were enrolled in the study. Patient enrollment criteria included i) suspicion of sepsis defined by the SOFA (sequential organ failure assessment) score and ii) type of infection referred to community-acquired pneumonia (CAP) or hospital-acquired pneumonia (HAP) or acute ascending cholangitis (AC) or acute pyelonephritis. Presence of sepsis was defined as “the presence of total SOFA scores equal to 2 or more for patients who are admitted with infection at the emergency department or as any increase of admission SOFA by 2 or more points for patients already hospitalized”. Exclusion criteria included patients under 18 years of age, denial for written informed consent, patients already receiving antibiotics, pregnancy or lactation (women of child-bearing potential will be screened by a urine pregnancy test before inclusion in the study).

[0202] The primary endpoint of the study was the assessment of the matching rate between the results of the method according to the invention and the Standard of Care (SoC) cultures for patients with sepsis by assessing the sensitivity, specificity, positive predictive value and negative predictive value of the method of the invention and SoC culture. The results of the SoC cultures were considered as the reference (“gold standard”) for diagnostic performance comparisons. Secondary endpoints included the comparative time for identification of the bacterial pathogen between the method according to the invention and the SoC cultures, and the comparative time to Antimicrobial Susceptibility Testing (AST) between the method according to the invention and the SoC cultures.

[0203] SoC cultures

[0204] The SoC culture analyses were carried out in a hospital facility using Biomerieux devices for cell cultures:

[0205] 15ml of blood were incubated in a 50ml total growing media. Once the Biomerieux equipment gave a positive alarm for a flask, the corresponding culture is seeded on a McConkey agar plate, and if colonies are found, then they are loaded in the BD Phoenix M50 instrument for identification and AST (Antibiotic Susceptibility Testing). time of delivery of samples to the laboratory facilities was around 3 hours. Growth flasks were incubated at 37° C and after 6h of incubation 5.5ml were collected: 0.5ml were stored in sterile Eppendorf tubes used to implement the method according to the invention while the remaining 5ml were refrigerated at -80° C. The remaining volume (~15ml) was recultured to identify the incubated bacterial species and perform AST (SoC cultures).

[0206] Implementation of a method according to the invention Initially a full-length 16S rRNA amplicon sequencing approach is performed allowing accurate bacterial species-level classification and quantification (expressed as pathogen genome copy number) as exposed above, notably using an appropriate polymerase as defined above. Subsequently, Shotgun metagenomic sequencing was applied for the identification and characterization of bacteria, fungi, and viruses, and for providing insights into antimicrobial resistance mechanisms. The Microbiome Ultra II kit (ThermoFisher) and KingFisher Flex device (ThermoFisher) was used to enabling rapid and standardized total DNA isolation from blood samples. Extracted DNAs were quantified using Qubit fluorometer (Invitrogen) and sequencing libraries were prepared as previously described. The sequencing run was performed using the GridlON x5 platform (Oxford Nanopore Technologies). Lastly, sequencing data were analyzed to provide i) pathogen identification, ii) genome copy number detection, and iii) Antimicrobial Resistance (AMR) prediction.

[0207] Results:

[0208] Sepsis was confirmed in fourteen (14) patients out of the 100 patients enrolled.

[0209] Full-length 16S rRNA sequencing enabled the detection of thirteen (13) out of fourteen sepsis cases as identified by SoC cultures as shown in Table 8A below. One sample, patient Bl, was not detected byl6S rRNA sequencing method according to the invention (False Negative result, Table 8 B).

[0210] Accordingly, the 16S rRNA allows a 100% successful identification of pathogens.

[0211] No false positives were reported by 16S rRNA sequencing method according to the invention.

[0212] The procedure showed 92.86% Sensitivity, 100% Specificity and 99% Accuracy.

[0213] Measured positive predictive value and negative predictive value were 100%. The total time to identify the microbial species took less than 10 hours (from 6 to 8 hours) from the time the samples a received by the laboratory. The overall time for SoC culture procedures is in the range of 36 hours. Table 8 A - Numerical 16S rRNA sequencing -based and SoC results

[0214] Table 8 B - Confirmed sepsis cases

[0215] Samples identified as positive by 16S rRNA sequencing were subsequently processed for Shotgun metagenomic sequencing (Table 9). Samples with an estimated genome copy number of 102copies or less failed the sequencing run as they did not contain enough microbial DNA allowing library correct pathogen detection and AMR genes profiling (technology Limit of Detection, LoD of 103). Table 9 - Shotgun sequencing results. bdl#= below detection limit (10E+02 copy number). It was possible to identify the species through 16S rRNA sequencing method of the invention but not to quantify the exact numbers. - = negative result, below Shotgun sequencing detection threshold, that is 10E+03. CoNS= Coagulase-Negative Staphylococci.

[0216] Taxonomy achieved by Shotgun sequencing is found 100% concordant with 16S rRNA sequencing according to the method of the invention. Two distinct bacterial species were detected in patients B5 and B7, species that were not identified by SoC cultures: both 16S and Shotgun sequencing technologies confirmed the presence of a “co-infection”.

[0217] Antimicrobial Resistance (AMR) genes were classified using the Comprehensive Antibiotic Resistance Database (CARD) generating a list of genes putatively linked to resistance phenotypes. A custom proprietary AMR reference database was built harboring genes associated to clinically relevant antibiotics (e.g. antimicrobial employed in hospital / clinical settings) and with a strong literature background regarding their resistance potential. Antimicrobial families of interest (hereafter reported as “clinically relevant”) were defined according to the World Health Organization Medically Important Antimicrobials List for Human Medicine (WHO MIA; https: / / www.who.int / news / item / 08-02-2024-who- medically-important-antimicrobial-list-2024). Identified relevant genes were then associated to antimicrobial families thus predicting a resistance phenotype. All the Shotgun sequencing results were achieved within 8 to 24 hours depending on sample complexity and the measured genome copy number.

[0218] Shotgun NGS predicted antimicrobial resistant profiles were lastly compared to official SoC AST results to assess the assay sensitivity and accuracy of the prediction. For each detected pathogen, different classes of antimicrobial agents were tested by AST evaluating Sensitivity or Resistance profiles. Shotgun NGS successfully predicted most of the resistant profiles identified with AST (Tables 10 A - E).

[0219] Pathogens resistant to penicillins and cephalosporins (Table 10 A) were characterized as Extended-spectrum beta-lactamase (ESBL) producer if in the SoC culture they were found resistant to third-generation cephalosporins while for NGS-based assay it was characterized by detection of any of the following target AMR genes: TEM-1, CTX-M-15, CTX-M-27, CTX-M-139, OXA-1, OXA-IO, OXA-66, SHV-182, or bla_Z. Shotgun NGS showed 100% accuracy, sensitivity and specificity by detecting all resistant strain sequenced. No False negatives were reported.

[0220] Table 10 A - Cross-tabulation of resistance to penicillins and cephalosporins by the assay according to the invention and SoC cultures

[0221] True Positive = Al, B4, B5, B7. True Negative = B6, B8. Carbapenems resistant pathogen were detected if in the SoC culture they were resistant to at least one of the tested carbapenems (ertapenem, imipenem, or meropenem) while for Shotgun NGS-based assay it was characterized by detection of any Metallo-B-lactamases NDM- and VIM or other carbapenemases like KPC and OXA-23, and genes for multi drug efflux pump mexA and mexB (Table 10 B).

[0222] Evaluated NGS Sensitivity, specificity and accuracy were respectively 75%, 100%, and 83.33% respectively. A 100% positive predictive value and 66.67% negative predictive value were reported. Sample B8 was reported as a false negative in the Shotgun NGS assay: in fact, its carbapenem-resistant profile was not characterized by the assay. This can be related to i) low sequencing coverage (low amount of sequencing data generated) or ii) incomplete reference in the AMR database.

[0223] Table 10 B - Cross-tabulation of resistance to carbapenems by Shotgun NGS-based assay and SoC cultures

[0224] True Positive = B4, B5, B7. False Negative = B8. True Positive = Al, B6.

[0225] Aminoglycosides resistance was characterized if in SoC cultures there was resistance to at least one of the tested antimicrobials (amikacin or gentamicin). For Shotgun NGS it was characterized by the presence of AMR genes including ACT-like genes aac(6)-lbl0, aac(6)-lb-cr and NUT like genes aadA22 and aadAll. NGS assay showed 60% sensitivity, 100% specificity and 66.67% accuracy in the detection of aminoglycoside resistance profiles (Table 10 C). Positive predictive value and negative predictive value were respectively 100% and 33.33%. Table 10 C - Cross-tabulation of resistance to aminoglycosides by NGS-based assay and

[0226] SoC cultures

[0227] Footnote: True Positive = B4, B5, B7. False Negative = Al, B8. True Negative = B6.

[0228] Resistance to tetracyclines was evaluated by the presence of genes providing resistance to tetracyclines through efflux pump like mexA, mexB and mexC. Lower sensitivity (33.33%) and specificity (66.67%) were reported. Assay accuracy was 50% while both positive and negative predictive values were measured at 50% (Table 10 D).

[0229] Table 10 D - Cross-tabulation of resistance to tetracyclines by NGS-based assay and SoC cultures

[0230] Footnote: True Positive = B7. False Positive = B4. False Negative = Al, B6. True Negative = B5, B8.

[0231] Lastly, quinolones resistance was evaluated (Table 10 E). Resistant profile was determined if in SoC cultures there was resistance to at least one of the quinolones tested (ciprofloxacin or levofloxacin) while for Shotgun NGS this was detection of AMR like efflux pumps. The NGS assay showed very low Sensitivity (25%) but high Specificity (110%). Average Accuracy was reported: 50% accuracy. Measured positive and negative predictive values were respectively 100% and 40%. Similarly to tetracycline resistance (Table 10 D) the lower detection rate can be addressed to i) poor sequencing coveragen or ii) incomplete AMR reference database. Table 10 E - Cross-tabulation of resistance to quinolones by NGS-based assay and SoC cultures

[0232] Footnote: True Positive = B4. False Negative = B5, B7, B8. True Negative = Al, B6.

[0233] Conclusions:

[0234] The method according to the invention only requires low volume samples. In the experiments as few as 0.5ml of blood samples allows the detection of pathogens from suspected sepsis and the prediction of antimicrobial resistance. This is of particular and crucial importance for preterm neonates and newborns in which no larger blood samples can be drawn, as well as for small frail elderly people.

[0235] It is therefore confirmed, in an experimental set up in real conditions, the method of the invention enables fast microbial pathogen detection which is crucial for sepsis diagnosis), allowing microbial identification within 14 hours (full-length 16S rRNA sequencing, taking into account the 6 hours short growth preliminary step) and resistance prediction in less than 24 hours that remains significantly less than SoC proceedings. Moreover, the method can enable screening of putative co-infections likely not detectable using conventional SoC culture methods because of different bacterial growth condition specificities.

[0236] No false positives were reported, therefore the method according to the invention allows an early diagnosis of sepsis with an accurate identification of its microbial origin and, when including metagenomic analysis, allows control of the diagnosis and the identification of potential resistance profiles.

[0237] The method thus helps the practitioners in making quick decisions for optimal antimicrobial stewardship, reducing costs and increasing chances of treatment of this disease for which the promptness of care is crucial. Example 4: Study of combined analysis of 16S sequencing and metagenomic sequencing, on plasma and broncho-alveolar lavage (BAL) from ICU patients, without a growing step.

[0238] Methods

[0239] This independent study was performed on ICU patients screened according to the SOFA score as exposed above. The primary endpoint included the testing of different samples collected from patients with suspected sepsis (plasma and BAL).

[0240] For each patient admitted, 5ml blood was collected in EDTA tubes and immediately processed (within 1-2 hours from collection). No culture step was applied. The samples were centrifuged for 10 minutes at 1,600 x g (room temperature) and collected plasma, immediately frozen (-20° C). In parallel for each patient, a BAL sample was also collected.

[0241] Full-length 16S rRNA sequencing and Shotgun metagenomics were employed to detect microbial pathogens responsible for sepsis. 16s RNA amplification and sequencing was performed using around 500pl plasma. Library preparation and sequencing were performed as previously specified. Overall, 23 plasma samples and 22 BAL samples were collected from patients with suspected sepsis and tested.

[0242] Results

[0243] Results of BAL and PLASMA samples are presented in Table 11.

[0244] Table 11 - Results from BAL and PLASMA samples

[0245] Detection method

[0246] Sample Sample rRNA sequencing type Number combined with metagenomic analysis result

[0247] Pl A STERILE STERILE

[0248] P2A STERILE STERILE

[0249] P3A STERILE STERILE

[0250] PLASMA P4A STERILE STERILE

[0251] P5A STERILE STERILE

[0252] P6A STERILE STERILE

[0253] P7A Candida glabrata Staphylococcus aureus P9A STERILE STERILE

[0254] P10A STERILE STERILE

[0255] P11A STERILE STERILE

[0256] P12A STERILE STERILE

[0257] P13A STERILE STERILE

[0258] P14A STERILE STERILE

[0259] Pl 5 A STERILE STERILE

[0260] P16A STERILE STERILE

[0261] P17A STERILE STERILE

[0262] P18A STERILE STERILE

[0263] P19A STERILE STERILE

[0264] P20A STERILE STERILE

[0265] P21A STERILE STERILE

[0266] P22A STERILE STERILE

[0267] P23A STERILE STERILE

[0268] P24A STERILE STERILE

[0269] P1B STERILE STERILE

[0270] P2B STERILE STERILE

[0271] P3B STERILE STERILE

[0272] P4B Legionella sp. Legionella maceachernii

[0273] P5B STERILE STERILE

[0274] P8B Staphylococcus aureus Staphylococcus aureus

[0275] P9B STERILE STERILE

[0276] Pl OB Candida albicans Candida albicans

[0277] Aspergillus fumigatus Aspergillus fumigatus

[0278] PUB , , , , uncultured

[0279] Enterobacter cloacae „ , ,

[0280] Enterobacterales

[0281] P12B Citrobacter koseri Citrobacter koseri

[0282] P13B STERILE STERILE

[0283] P14B STERILE STERILE

[0284] P15B yest cells cultivated Haemophilus influenzae

[0285] P16B STERILE STERILE

[0286] P17B STERILE Streptococcus pneumoniae

[0287] P18B Candida albicans STERILE

[0288] P19B STERILE STERILE

[0289] P20B STERILE STERILE

[0290] P21B STERILE Mycoplasma pneumoniae

[0291] P22B STERILE STERILE

[0292] P23B STERILE STERILE

[0293] P24B STERILE STERILE

[0294] The procedure showed 90.91% Sensitivity, 100% Specificity and 97.78% Accuracy. Table 12 -

[0295] 22 out of 23 plasma samples were declared “STERILE” according to SoC culture (absence of growth on culture media).

[0296] Regarding BAL samples, no visible growth was detected in 15 out of 22 (68.2%) while 7 samples (30.43%) were declared positive.

[0297] A controversial result was obtained for the only positive plasma sample (plasma specimen P7A): it was classified positive for Staphylococcus aureus bacterial species using the method combining 16S rRNA sequencing with metagenomic analysis (16S rRNA sequencing and metagenomic sequencing are consistent) whereas SoC method identified fungal C. glabrata species. It is thus likely that the SoC culture led to an erroneous identification.

[0298] Patient P4B (BAL sample) was declared positive for an unspecified Legionella species according to standard SoC cultures. The 16S rRNA sequencing method of the invention combined with metagenomic analysis therefore allows accurate species profiling.

[0299] BAL samples collected from patients P4B, P8B, P10B, PUB, and P12B shared 100% agreement between the 16S rRNA sequencing method of the invention combined with metagenomic analysis and SoC method. Notably, sample PUB was classified positive for the fungal species A. fumigatus and the bacterial species E. cloacae. Sequencing method successfully classified A. fumigatus but did not reach species-level classification for the Enterobacter species being classified as “uncultured” (putatively related to low microbial genome copies) thereby showing the strong complementarity of the 16S rRNA NGS sequencing and the Shotgun NGS sequencing used in the method of the invention.

[0300] Patient P15B was classified using the method of the invention positive for H. influenzae bacterial species (SoC detected yeast cells, putative fungi), while sample P17B was detected positive for S. pneumoniae but classified “STERILE” according to SoC method. Moreover, sample P21B BAL was detected positive for M. pneumoniae. Conclusions:

[0301] These results confirm the increased sensitivity and the rapid diagnostic potential of the method comprising the 16S rRNA sequencing method according to the invention.

[0302] The method allows to detect multiple microbial pathogens, including fungi, in BAL samples and whole blood samples without a previous growing step. The results in regard to the bacterial and / or viral and / or fungal infection were obtained within 8 hours of the sample collection.

[0303] The capability of detecting pathogens from plasma is of particular interest as i) it allows faster diagnosis since no incubation is required, and ii) in general plasma samples are easier to store and process compared to blood samples (can be stored at -20° C).

[0304] Higher sensitivity and accuracy were obtained using the sequencing method of the invention as it allows the correct microbial species detection (patient P4B BAL) and helps clinicians in the early detection of suspected sepsis (P15B, P17B, and P21B), where no microorganism was detected using SoC culture method.

Claims

CLAIMS1. A method for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample from a subject at risk to suffer from and / or suspected to suffer from sepsis, the method comprising the steps of: a) spiking the biological sample with quantified calibrator molecules; b) extract the genomic DNA from the biological sample of step a); c) carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from O.Olng to lOOng or 102CFU / ml to 109CFU / ml. d) further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

2. Method according to claim 1 comprising the DNA polymerase amplifies at least 103CFU / ml and at least of 0.1 ng of genomic DNA, preferably at least 102CFU / ml and at least of 0.01 ng of genomic DNA.

3. Method according to any of the preceding claims wherein the method enables the identification and / or quantification with an accuracy of at least 90%, and / or within a maximum time frame of 24 hours.

4. Method according to any of the preceding claims comprising a further step of comparing the quantity of said at least one microorganism to the amount of quantified to quantify said at least one microorganism in the biological sample.

5. Method according to any of the preceding claims wherein the biological sample is selected from blood, urine, synovial fluid, pulmonary wash, plasma, serum, amniotic fluid, cerebrospinal fluid, nasal wash, saliva, semen, vaginal fluid, sputum and the combinations thereof, preferably selected from blood, plasma, serum, urine, synovial fluid, a biopsy, and pulmonary wash.

6. Method according to any of the preceding claims wherein the biological sample is of a volume equal or less than 5ml, equal or less than 4ml, equal or less than 3ml, equal or less than 2ml, equal or less than 1ml equal or even equal or less than 0.5ml.

7. Method according to any of the preceding claims wherein the molecular barcodes of step e) are ligated using 5 ’-phosphated primers.

8. Method according to any of the preceding claims further comprising a step of determination of a viral or fungal infection.

9. Method according to any of the preceding claims further comprising a step of detection of at least one gene of antibiotic resistance and / or at least one a virulence factor in the at least one microorganism.

10. Method according anyone of claim 8 or 9, wherein the step of detection of at least one gene of antibiotic resistance and / or at least one a virulence factor and / or of determination of a viral or fungal infection is performed using a metagenomic sequencing on the biological sample.

11. Method according to any of the preceding claims wherein the at least one microorganism responsible for sepsis is selected amongst a bacteria, a virus, a fungus and the combinations thereof.

12. Method according to any of the preceding claims wherein the 16S rRNA amplicons of step c) have a length between 0.05-5 kB, preferably 1.5 kB.

13. Method according to any of the preceding claims wherein the ligation of molecular barcodes of step d) enables samples multiplexing.

14. Method according to any of the preceding claims wherein the method provides a result of step g) within a maximum timeframe of 36 hours, preferably of 24 hours.

15. Method according to any of the preceding claims wherein the method provides a result of step g) with a limit of detection of 50 CFU / ml, preferably of 10 CFU / ml.

16. Method according to any of the preceding claims wherein the method identifies and / or quantifies the at least one microorganism present with at least 0.01% relative abundance in the bacterial community of said sample.

17. Method according to any of the preceding claims wherein said method does not comprise a growing step to enrich the biological sample with the at least one microorganism.

18. Kit of parts for the identification and / or quantification of at least one microorganism responsible for sepsis in a biological sample according to any of claims 1-17 comprising: i. a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2), ii. a sample of DNA polymerase iii. at least one calibrator molecule, iv. phosphorylated barcoding primers enabling sample multiplexing, and v. at least one collection tube containing glycerol to collect a biological sample, and vi. instructions for use.

19. A method of diagnosing sepsis from a biological sample collected in a subject at risk of, or suspected to suffer from sepsis, the method comprising the steps of: a) Spiking the biological sample with quantified calibrator molecules; b) Extract the genomic DNA from the biological sample of step a);c) Carrying out a polymerase chain reaction (PCR) to amplify full-length 16S rRNA gene from the mixture of genomic DNA of step b) using a pair of primers 27f (SEQ ID No. 1) and 1492r (SEQ ID No. 2) thereby obtaining 16S rRNA gene amplicons wherein the PCR is performed as follows: i. annealing temperatures between 55°C and 60°C; ii. extension temperatures between 65°C and 72°C; iii. the amount of genomic DNA extracted in step b) ranges from 0.0 Ing to lOOng or 102CFU / ml to 109CFU / ml. d) Further amplifying 16S rRNA gene amplicons of step c) by PCR to ligate molecular barcodes; e) Ligating adapters to the amplified 16S rRNA gene amplicons of step d); f) Sequencing the sequences of adapters-amplicons of step e) generating sequencing reads; g) Comparing the sequences of the sequencing reads of step f) with genomes of microorganisms thereby identifying at least one microorganism present in the biological sample.

20. Method according to claim 19 further comprising a step of determination of a viral or a fungal infection.

21. Method according to any of claim 19 or 20 further comprising a step of determination of antibiotic resistance gene of the at least one microorganism.

22. Method according to any of claims 20 or 21 wherein the step of determination of antibiotic resistance gene of the at least one microorganism and / or the determination of viral or fungal infection is determined by a metagenomic sequencing step performed on the biological sample from a subject at risk of or suspected to suffer from sepsis.

23. Method according to any of claims 19 to 22 wherein said method does not comprise a growing step to enrich the biological sample with the at least one microorganism.