Methods for predicting risk of developing pulmonary colonization / infection by pseudomonas aeruginosa

Abstract

To provide a method for predicting the risk of developing pulmonary colonization / infection by Pseudomonas aeruginosa.SOLUTION: There is provided a method for predicting the risk of developing pulmonary colonization / infection by Pseudomonas aeruginosa in a subject suffering from cystic fibrosis (CF), the method comprising measuring the abundance of Porphyromonas genus bacteria in a biological sample obtained from the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for predicting the expression risk of lung colonization / infection by Pseudomonas aeruginosa.

[0002] Background Art Multiple bacterial infections of the respiratory tract play an important role in the progression of cystic fibrosis (CF), and the acquisition of pathogens during the course of the disease is described below. Indeed, the pulmonary microbiota in CF typically has either Haemophilus influenzae or Pseudomonas aeruginosa as the dominant species (Rogers GB, van der Gast C, Serisier DJ (2015) Predominant pathogen competition and core microbiota divergence in chronic airway infection. ISME J 9:217-225). Pseudomonas aeruginosa has a negative impact on lung function, promoting more frequent acute exacerbations (Rosenfeld M, Ramsey BW, Gibson RL (2003) Pseudomonas acquisition in young patients with cystic fibrosis: pathophysiology, diagnosis, and management. Curr Opin Pulm Med 9(6):492-497). After ~25 years of age, the establishment of CF pathogens is usually complete, and Pseudomonas aeruginosa becomes the most dominant species in the CF lung (Coburn B, Wang PW, Diaz Caballero J, Clark ST, Brahma V, Donaldson S, Zhang Y, Surendra A, Gong Y, Tullis DE, Yau YCW, Waters VJ, Hwang DM, Guttman DS (2015) Lung microbiota across age and disease stage in cystic fibrosis. Sci rep 5:10241). The colonization of Pseudomonas aeruginosa is considered an important turning point in the disease course of CF patients.

[0003] The current challenge is to elucidate the factors involved in this turning point. Demographic and environmental factors have been shown to increase the risk of Pseudomonas aeruginosa acquisition (Maselli JH, Sontag MK, Norris JM, MacKenzie T, Wagener JS, Accurso FJ (2003) Risk factors for initial acquisition of P. aeruginosa in children with cystic fibrosis identified by newborn screening. Pediatr pulmonol 35(4):257-262). Most anaerobic bacteria such as Prevotella and Veillonella are also detected in CF sputum samples and are presumed to act detrimentally on respiratory function (Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, Cavalcoli JD, Van Devanter DR, Murray S, Li JZ, Young VB, LiPuma JJ (2012) Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci USA 109(15):5809-5814).In addition to bacteria, fungi and viruses also colonize the upper and lower respiratory tracts of CF patients (Wat D, Gelder C, Hibbitts S, Cafferty F, Bowler I, Pierrepoint M, Evans R, Doull I (2008) The role of respiratory viruses in cystic fibrosis. J Cyst Fibros 7(4):320-328) (Mounier J, Gouello A, Keravec M, Le Gal S, Pacini G, Debaets S, Nevez G, Rault G, Barbier G, Hery-Arnaud G (2014) Use of denaturing high-performance liquid chromatography (DHPLC) to characterize the bacterial and fungal airway microbiota of cystic fibrosis patients. J Microbiol 52(4):307-314.), and may play an important role in its pathogenesis.

[0004] Summary of the Invention: The present invention relates to a method for predicting the risk of manifestation of Pseudomonas aeruginosa lung colonization / infection. Specifically, the present invention is defined by the claims.

[0005] Modes for Carrying Out the Invention The object of the present inventors was to identify predictive biomarkers for Pseudomonas aeruginosa colonization / infection.

[0006] For 88 months, 34 CF patients (mostly pediatric) were followed and divided into two groups; one group became infected with Pseudomonas aeruginosa during follow-up, and the other group remained uninfected. Considering potential host factors involved in the progression of lung disease, airway microbiota were analyzed from 65 sputum samples through 16S rRNA gene sequencing and RT-PCR screening of 18 respiratory viruses. The inventors compared microbiota data between the two groups of patients. The inventors further examined the presence of "pulmotypes" across the CF cohort. Multiple statistical approaches were implemented and cluster strength was tested.

[0007] The Porphyromonas genus (P. catoniae and P. endodontalis), which was more abundant (p-value < 0.001) in patients not infected with Pseudomonas aeruginosa, was a biomarker for a non-permissive airway microbiota.

[0008] In the era of personalized medicine, the inventors intended to provide close monitoring for CF patients at high risk of early Pseudomonas aeruginosa colonization / infection and to find biomarkers to improve the clinical utility of successful early Pseudomonas aeruginosa clearance.

[0009] Accordingly, a first aspect of the present invention is a method for predicting the risk of development of lung colonization / infection by Pseudomonas aeruginosa in a subject suffering from cystic fibrosis (CF), comprising: - measuring the abundance of bacteria of the genus Porphyromonas in a biological sample obtained from the subject; - when the abundance of bacteria of the genus Porphyromonas is measured to be high, concluding that the subject has a low risk of developing lung colonization / infection by Pseudomonas aeruginosa, or when the abundance of bacteria of the genus Porphyromonas is measured to be low, concluding that the subject has a high risk of developing lung colonization / infection by Pseudomonas aeruginosa and related to a method comprising.

[0010] As used herein, the term "cystic fibrosis (CF)" has its ordinary meaning in the art and refers to a genetic disorder that affects various parts of the body, particularly not only the lungs but also the pancreas, liver, kidneys, and intestines. Long-term problems include breathing difficulties as a result of frequent lung infections. Other signs and symptoms may include rhinitis, poor growth, fatty stools, clubbing of the fingers and toes, and male infertility. Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the white population.

[0011] As used herein, the term "lung colonization / infection by Pseudomonas aeruginosa" refers to any infectious disease related to the lungs caused by Pseudomonas aeruginosa.

[0012] As used herein, the term "Pseudomonas aeruginosa" has its ordinary meaning in the art and refers to a common Gram-negative bacillus.

[0013] As used herein, the term "genus Porphyromonas" has its ordinary meaning in the art and refers to a Gram-negative, non-spore-forming, anaerobic, and non-motile genus of the family Porphyromonadaceae, which is recognized as a distinct taxonomic group based on ribosomal DNA homology and 16S rRNA data.

[0014] As used herein, the term "abundance" refers to the amount or concentration of the bacterium at a particular location / sample.

[0015] In one embodiment, the abundance is an absolute abundance.

[0016] As used herein, the term "absolute abundance" refers to the concentration of the bacterium at a particular location / sample, expressed, for example, as the number of CFU per mL or the genome equivalent per mL.

[0017] In one embodiment, the abundance is a relative abundance.

[0018] As used herein, the term "relative abundance" refers to the component percentage of a bacterial genus relative to the total number of bacterial genera at a given location / sample.

[0019] As used herein, the term "subject" means a mammal. In a preferred embodiment of the invention, the subject according to the invention refers to any subject (preferably human) suffering from or at risk of suffering from cystic fibrosis. The methods of the present invention have been revised, for example, in the World Health Organization classification of cystic fibrosis, and can be carried out for any type of cystic fibrosis selected from group E84: cystic fibrosis, cystic fibrosis with pulmonary symptoms, cystic fibrosis with intestinal symptoms, and cystic fibrosis with other symptoms.

[0020] In one embodiment, the subject is a newborn.

[0021] In one embodiment, the subject is a child. In one embodiment, the age of the child is less than 12 months.

[0022] In one embodiment, the subject is an adult.

[0023] As used herein, the term "biological sample" is used in its broadest sense herein. A biological sample is generally obtained from a subject. The sample can be any sample of biological tissue or fluid that can be used to assay the biomarkers of the present invention. Often, the sample is a "clinical sample", i.e., a sample derived from a patient. Such samples can include body fluids that may or may not contain cells, such as blood (e.g., whole blood, serum, or plasma), synovial fluid, saliva, tissue, or samples from fine needle biopsies, as well as archival samples with known diagnostic, treatment, and / or prognostic histories, but are not limited thereto. Also, biological samples can include tissue sections such as frozen sections taken for histological purposes. The term "biological sample" also encompasses any substance obtained by processing a biological sample. The resulting substance includes, but is not limited to, cells isolated from the sample (or their progeny), or proteins, DNA, or RNA extracted from the sample.

[0024] In one embodiment, the biological sample is a bronchoalveolar lavage fluid (BAL), or sputum, or a protected specimen brush (from bronchoscopic sampling) or a throat swab. In one embodiment, the biological sample is a sample of spontaneous or induced sputum.

[0025] As used herein, the term "predict" refers to the likelihood or probability that a subject will manifest an event. Preferably, the event is, herein, bronchopulmonary colonization / infection by Pseudomonas aeruginosa.

[0026] As used herein, the term "risk" refers to the likelihood that an event, such as the occurrence of Pseudomonas aeruginosa lung colonization / infection, will occur over a specific period of time, and can mean the "absolute" or "relative" risk of a subject. The absolute risk can be measured with reference to the actual post-measurement findings for an appropriate time cohort or to the index values obtained from a statistically valid historical cohort followed over an appropriate period of time. The relative risk refers to the ratio of the absolute risk of a patient compared to either the absolute risk of a low-risk cohort or the average population risk, which can vary depending on how the clinical risk factors are evaluated.

[0027] In accordance with the method of the present invention, the abundance of bacteria of the genus Porphyromonas is measured. Any method for measuring abundance known to those skilled in the art can be used. Examples of these methods include, but are not limited to, direct counting by microscopy, electronic counting chambers, indirect viable cell counting, culture-based techniques, or molecular methods.

[0028] In one embodiment, the abundance of Porphyromonas bacteria is measured by any conventional method well-known in the art and typically by using molecular methods. In one embodiment, the abundance of Porphyromonas bacteria is measured using 16S rRNA deep sequencing. In one embodiment, the abundance of Porphyromonas bacteria is measured using an abundance table generated by next-generation sequencing of the 16S rRNA gene of all bacteria in a given biological sample using qPCR technology. Nucleic acids can be extracted from the sample by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C.). U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected target nucleic acid sequence. Primers useful in the present invention include oligonucleotides that can act as a starting point for nucleic acid synthesis within the target nucleic acid sequence. qPCR involves the use of a thermostable polymerase. The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when exposed to high temperatures for the time required to denature the double-stranded template nucleic acid. Generally, synthesis begins at the 3' end of each primer and proceeds in the 5'→3' direction along the template strand.Thermostable polymerases are isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, if the enzyme is replenished, non-thermostable polymerases can also be used in PCR assays. Typically, the polymerase is Taq polymerase (i.e., Thermus aquaticus polymerase). The primers are combined with the PCR reagents under reaction conditions that induce primer extension. The newly synthesized strands form double-stranded molecules that can be used in subsequent reaction steps. The steps of strand separation, annealing, and extension may be repeated the number of times necessary to produce the desired amount of amplification product corresponding to the target nucleic acid sequence molecule. The rate-limiting factors in the reaction are the amounts of primer, thermostable enzyme, and nucleotide triphosphates present during the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. When used in detection, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps are required to amplify a sufficient amount of the target sequence for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or even 100 times.

[0029] 16S deep sequencing technology is well described in the current state of the art, for example, in Shendure and Ji, "Next-generation DNA sequencing", Nature Biotechnology, 26(10):1135-1145 (2008).

[0030] 16S deep sequencing technology, also known as "next-generation DNA sequencing" ("NGS"), "high-throughput sequencing", "ultra-parallel sequencing", and "deep sequencing", refers to a method of sequencing multiple nucleic acids in parallel. See, for example, Bentley et al, Nature 2008, 456:53-59. The major commercially available platforms produced by Roche / 454 (Margulies et al, 2005a), Illumina / Solexa (Bentley et al, 2008), Life / APG (SOLiD) (McKernan et al, 2009), and Pacific Biosciences (Eid et al, 2009) can be used for deep sequencing. For example, in the 454 method, the DNA to be sequenced can be fragmented and the DNA segments can be PCR amplified using primers that supply adapters or contain adapters. The adapter is a 25-mer nucleotide necessary for binding to DNA capture beads and for annealing of emulsion PCR amplification primers and sequencing primers. The DNA fragments are made single-stranded and bound to DNA capture beads such that only one DNA fragment binds to one bead. Next, the DNA-containing beads are emulsified in a water-in-oil mixture to obtain microreactors each containing only one bead. Within the microreactors, the fragments are PCR amplified to obtain millions of copies per bead. After PCR, the emulsion is broken and the beads are loaded onto a picotiter plate. Each well of the picotiter plate can accommodate only one bead. Sequencing enzymes are added to the wells and nucleotides are flowed through the wells in a defined order. As a result of nucleotide incorporation, pyrophosphate is released, which catalyzes the reaction to produce a chemiluminescent signal. This signal is recorded by a CCD camera and the signal is translated into a DNA sequence using software. In the Illumina method (Bentley (2008)), fragments supplied with single-stranded adapters are bound to an optically transparent surface and subjected to "bridge amplification".As a result of this procedure, millions of clusters are obtained, each containing a copy of a unique DNA fragment. DNA polymerase, primers, and four types of labeled reversible terminator nucleotides are added, and the surface is imaged by laser fluorescence to determine the position and nature of the labels. The protecting groups are then removed, and the process is repeated for several cycles. The SOLiD process (Shendure (2005)) is similar to 454 sequencing in that DNA fragments are amplified on the surface of beads. Sequencing involves cycles of ligation and detection of labeled probes. Several other technologies for high-throughput sequencing are currently under development. Examples of such technologies are The Helicos system (Harris (2008)), Complete Genomics (Drmanac (2010)), and Pacific Biosciences (Lundquist (2008)). Since this is a very rapidly evolving technical field, the applicability of high-throughput sequencing methods to the present invention will be apparent to those skilled in the art.

[0031] A further object of the present invention is a method for preventing Pseudomonas aeruginosa lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - predicting the risk of expression of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; - administering to the subject a therapeutically effective amount of a Pseudomonas aeruginosa-specific antibiotic if the subject is concluded to be at high risk of expressing Pseudomonas aeruginosa lung colonization / infection. The invention relates to a method comprising the above.

[0032] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is an aminoglycoside (gentamicin, amikacin, tobramycin, excluding kanamycin).

[0033] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is a quinolone (ciprofloxacin, levofloxacin, excluding moxifloxacin).

[0034] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is a cephalosporin (ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole, excluding cefuroxime, cefotaxime, or ceftriaxone).

[0035] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is ceftazidime-avibactam or ceftolozane-tazobactam.

[0036] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is an anti-Pseudomonas aeruginosa penicillin: carboxypenicillin (carbenicillin and ticarcillin) and ureidopenicillin (mezlocillin, azlocillin, and piperacillin).

[0037] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is a carbapenem (meropenem, imipenem, doripenem, excluding ertapenem).

[0038] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is polymyxin (polymyxin B and colistin).

[0039] In one embodiment, the Pseudomonas aeruginosa-specific antibiotic is a monobactam (aztreonam).

[0040] In a preferred embodiment, the Pseudomonas aeruginosa-specific antibiotics are oral ciprofloxacin and inhaled colistin.

[0041] In a preferred embodiment, the Pseudomonas aeruginosa-specific antibiotic is inhaled tobramycin.

[0042] A further object of the present invention is a method for preventing Pseudomonas aeruginosa lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - predicting the risk of development of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; and - administering a therapeutically effective amount of a Porphyromonas probiotic to the subject if the subject is concluded to be at high risk of developing Pseudomonas aeruginosa lung colonization / infection. Relates to a method comprising.

[0043] As used herein, the term "prevention" refers to a reduction in the risk of acquiring or manifesting a given medical condition.

[0044] As used herein, the term "probiotics" refers to living microorganisms that confer a health benefit on a subject when administered in an appropriate therapeutic amount. The health benefit is the result of the production of nutrients and / or cofactors by the probiotics, competition between the probiotics and pathogens, and / or stimulation of the immune response in the subject by the probiotics.

[0045] The term "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance that exists outside the body to a subject by, for example, mucosal, intradermal, intravenous, subcutaneous, intramuscular, intra-articular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, the administration of the substance is typically performed after the occurrence of the disease or its symptoms. When preventing a disease or its symptoms, the administration of the substance is typically performed before the occurrence of the disease or its symptoms.

[0046] "A therapeutically effective amount" means an amount of a Pseudomonas aeruginosa - specific antibiotic sufficient for use in a method of preventing Pseudomonas aeruginosa lung colonization / infection at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound being used; the duration of the treatment; and like factors well - known in the medical arts. For example, it is well within the skill of the art to start with a dosage of the compound at a level lower than that required to achieve the desired treatment effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary widely from 0.01 to 1,000 mg per day per adult. Typically, the compositions contain the active ingredient in amounts of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg to adjust the dosage to the symptoms of the subject being treated. The medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is usually supplied at dosage levels of from 0.0002 mg / kg (body weight) to about 20 mg / kg (body weight) per day, particularly at dosage levels of from about 0.001 mg / kg (body weight) to 7 mg / kg (body weight) per day.

[0047] Another object of the present invention is a method of adjusting patient monitoring, comprising - predicting the risk of manifestation of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; - increasing the frequency of examinations when the subject is concluded to be at high risk of manifesting Pseudomonas aeruginosa lung colonization / infection and related to a method comprising.

[0048] For example, if it is concluded that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection, screening may be performed monthly or every 15 days, although standard patient monitoring includes screening every three months.

[0049] To confirm the prediction of the risk of developing Pseudomonas aeruginosa lung colonization / infection in a subject with cystic fibrosis obtained using the method of the present invention, it is possible to detect Pseudomonas aeruginosa by qPCR (Hery-Arnaud et al., CMI 2017: qPCR provides a window of opportunity of 8 months).

[0050] Another object of the present invention is a method for stratifying a subject with cystic fibrosis, comprising: - determining the bacterial species with a high abundance in the airway of the subject; - when the bacterial species with a high abundance in the airway of the subject is Streptococcus or Haemophilus, concluding that it is a favorable cystic fibrosis progression, or when the bacterial species with a high abundance in the airway of the subject is Staphylococcus, concluding that it is an unfavorable cystic fibrosis progression; and relates to a method comprising the above.

[0051] Another object of the present invention is a method for predicting the risk of developing Pseudomonas aeruginosa lung colonization / infection in a subject with cystic fibrosis (CF), comprising: - detecting the presence or absence of rhinovirus in the airway of the subject; - when the presence of rhinovirus is detected, concluding that the subject has a low risk of developing Pseudomonas aeruginosa lung colonization / infection, or when the absence of rhinovirus is detected, concluding that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection; and relates to a method comprising the above.

[0052] Another object of the present invention is a method for predicting the risk of manifestation of Pseudomonas aeruginosa lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - determining the bacterial species with a high abundance in the airway of the subject; - when the bacterial species with a high abundance in the airway of the subject is from the genus Streptococcus or Haemophilus, concluding that the subject has a low risk of manifestation of Pseudomonas aeruginosa lung colonization / infection, or when the bacterial species with a high abundance in the airway of the subject is from the genus Staphylococcus, concluding that the subject has a high risk of manifestation of Pseudomonas aeruginosa lung colonization / infection; and relates to a method comprising the above.

[0053] The present invention will be further described by the following drawings and examples. However, these examples and drawings should not be construed as limiting the scope of the present invention by any means.

Brief Description of Drawings

[0054]

Figure 1

Figure 2A

Figure 2B

Figure 2C

[0055] Example: Materials and Methods Patient cohort, inclusion criteria, and global data. In this study, 34 CF patients (17 females and 17 males) with a median age of 13.8 years ([IQR: 7.8 - 31 years]) were included and followed for a median of 8 months [IQR: 1 - 23 months]. CF patients were classified as "free" and "never" infected based on the Lee criteria that define the patient's Pseudomonas aeruginosa infection status at the first sampling. Other clinical and biological data collected at each sampling, including age, gender, CFTR mutations, clinical status according to the four stages defined by Price et al. (baseline, exacerbation, treatment, or recovery) (DOI: 10.1186 / 2049-2618-1-27), antibiotic treatment, BMI, Lee status (DOI: 10.1016 / S1569-1993(02)00141-8), and quality of sputum samples (score established according to cytological parameters, epithelial cells, and white blood cells), were collected. Analyses of the bacterial and viral communities in the lungs were performed on spontaneous sputum samples collected at two time points: at enrollment (time point 0) and at a median of 8 months later (Figure 1). At the end of the follow-up, the patients were divided into two groups. Group 1 included patients who remained Pseudomonas aeruginosa-free, while patients in Group 2 became Pseudomonas aeruginosa-positive under culture during the follow-up (Figure 1).

[0056] Nucleic acid extraction from sputum samples. To assess the bacterial composition, total DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Courtaboeuf, France). After treatment with 25 μL of proteinase K (10 mg / mL) at 56°C for 4 hours, viral RNA and DNA were extracted from the same sputum samples using the NUCLISENS® easyMAG™ automated extractor (bioMerieux, Marcy l’Etoile, France).

[0057] Characterization of the bacterial microbiota. Barcoded high-throughput 454 pyrosequencing was performed on the amplified V3 and V4 hypervariable regions of the 16S rRNA gene (Bioproject PRJNA 297396).

[0058] Screening for respiratory viruses. The RespiFinder® SMART 22 FAST kit (PathoFinder, Maastricht, The Netherlands) was used in the GeneAmp® PCR System 9700 (Applied Biosystems, Courtaboeuf, France) to simultaneously detect 18 human respiratory viruses. Furthermore, specific qPCR was performed to discriminate between both HRV and HEV.

[0059] Bioinformatics and statistical analysis. Sequences were analyzed using the standard UPARSE pipeline. For all statistical analyses, the significance threshold was set at 0.05. The false discovery rate (FDR) was calculated to correct for multiple hypothesis testing.

[0060] Results Overall composition of the airway microbiota in cystic fibrosis. Bacterial community. Five dominant phyla were observed, namely Firmicutes (43.11%), Proteobacteria (32.18%), Bacteroidetes (13.31%), Actinobacteria (7.66%), and Fusobacteria (3.62%), and 11 dominant genera (relative abundance ≥ 1%), namely Streptococcus (22.73%), Haemophilus (14.80%), Staphylococcus (10.66%), Neisseria (10.19%), Prevotella (7.57%), Rothia (7.01%), Porphyromonas (5.33%), Veillonella (4.14%), Fusobacterium (2.98%), Granulicatella (1.74%), and Pseudomonas (1.26%). Only one OTU assigned to the Streptococcus mitis group was shared among all samples.

[0061] Viral community. Respiratory viruses were detected in 29.2% (n = 19) of sputum samples. Coinfection (≥ 2 viruses) was observed in 4.6% (n = 3) of sputum samples. HRV / HEV was the most dominant virus as it was detected in 24.6% of the samples (HRV, n = 7; HEV, n = 9). Adenovirus, parainfluenza 2, coronavirus 229E, and NL63 were detected in 4.6% (n = 3), 3.1% (n = 2), 1.5% (n = 1), and 1.5% (n = 1), respectively. There was no significant correlation between the presence of rhinovirus and / or non-rhinovirus and lung exacerbation.

[0062] Effect of gender. The estimated and observed richness and diversity of bacterial species were significantly greater in male patients (observed species: 54.07 ± 18.4; H’: 3.4 ± 1.04) than in female patients (observed species: 43.78 ± 16.4; H’: 2.8 ± 0.95). For both male and female cohorts, this difference was not explained by patient age (t-test, p-value > 0.5). Furthermore, the composition of the CF lung microbiota varied by gender, as revealed by Unifrac weighted analysis (adonis test; p < 0.05). The relative abundances (RAs) of the genera Selenomonas, Leptotrichia, Parvimonas, and Atopobium were significantly more important in the male group (Mann-Whitney test, p-value < 0.05), and the M / F ratios were 3.3, 3.1, 15.8, and 6.2, respectively, while Bifidobacterium was only seen in the male group (low abundance, RA < 0.1%). Furthermore, Parvimonas was significantly more abundant in male patients who remained non-infected during follow-up (Mann-Whitney test, p-value = 0.01). Samples were classified with an error rate of 0.24 + / - 0.08, which is twice as high as the baseline error rate of random guessing (0.49).

[0063] Study of biomarkers associated with a low risk of initial Pseudomonas aeruginosa colonization. In patients in group 2, bacterial diversity was similar to that before Pseudomonas aeruginosa colonization. Also, in patients in group 1, diversity did not change over time.

[0064] The important genus Porphyromonas. As expected, the RA of the genus Pseudomonas was significantly (p-value = 1.79e-10) in samples from group 1 -7) There were more (Figure 2a). Interestingly, in the patients of Group 1 who remained non-infected with Pseudomonas aeruginosa during follow-up observation, the relative abundance (RA) of Porphyromonas at T0 was significantly higher than that in the patients of Group 2 (Mann-Whitney test, p-value < 0.001) (Figure 2b). The average RA was 252.9 reads in Group 1 and 130.7 reads in Group 2 (ratio 1.9). At T0, the patients in Group 1 who would remain non-infected with Pseudomonas aeruginosa had three times more abundance of Porphyromonas compared to the other three groups (T1 of Group 1, T0 and T1 of Group 2). Furthermore, these results were well supported by the observation of random forest, which showed an important relationship between the RA of Pseudomonas and Porphyromonas (Figure 2c). Interestingly, Porphyromonas was present in a larger amount in the male group that remained non-infected with Pseudomonas aeruginosa, indicating an important potential role of this genus.

[0065] The CF airway microbiota was clustered into pulmotypes.

[0066] The three pneumotype drivers are Streptococcus, Haemophilus, and Staphylococcus. Cohort samples were clustered into three pneumotypes dominated by either Streptococcus (pneumotype A), Haemophilus (pneumotype B), or Staphylococcus (pneumotype C). This clustering was consistent with the PCA results and was confirmed using the Kruskal–Wallis test (p-value = 0.001). The pneumotypes were driven by differences in the RA of these three dominant genera and other co-existing genera. Indeed, for pneumotype A, Streptococcus (30.0%), followed by Neisseria (13.3%), Rothia (9.2%), Prevotella (8.5%), and Porphyromonas (6.9%); for pneumotype B, Haemophilus (62.0%), Streptococcus (9.5%), and Aggregatibacter (5.0%); and for pneumotype C, Staphylococcus (54.6%), Streptococcus (13.5%), and Neisseria (6.7%) were dominant. Overall, 69.2% (n = 45), 15.4% (n = 10), and 15.4% (n = 10) of sputum samples were assigned to pneumotypes A, B, and C, respectively. It is worth mentioning that the cytological score had no effect on the clustering (Anosim test using Bray Curtis distance, p-value < 0.05; R 2 = 0.073).

[0067] The pneumotypes showed different bacterial community structures (Adonis test using Bray Curtis distance, p-value = 0.001), except for H’ when H’ of pneumotype A (H’: 3.39 ± 0.83) was significantly higher (p-value = 0.012) than that of pneumotype C (H’: 2.4 ± 1.18), but the alpha diversity was not different.

[0068] Other parameters (e.g., patient age, BETR classification, CFTR mutation, BMI) were tested to find relationships between pneumotypes, but none reached significance (data not shown).

[0069] The inventors also observed the variability within and between important pneumotypes. During long-term follow-up, changes in pneumotype were observed in five CF patients. In fact, the pneumotypes of patients 023 and 226 changed from pneumotype A to pneumotype C, while the reverse change was observed for patient 065. Patient 253 changed from pneumotype A to B, in contrast to patient 076.

[0070] Association between pneumotype and CF characteristics. The inventors observed that the ratio of the genus Porphyromonas was very high in some samples belonging to pneumotype A, with an average RA of 6.9% per sample (on average, 2.5% per sample for pneumotype B and 3.8% per sample for pneumotype C), but these trends did not reach significance. Interestingly, the viral community did not have a significant effect on pneumotype clustering (Kruskal-Wallis test, p-value > 0.1). However, according to the g-test of independence, the presence of rhinovirus was correlated with two OUTs: the presence of Haemophilus influenzae and the absence of Pseudomonas aeruginosa (p-value < 0.05). Interestingly, 90% (n = 9 out of 10 samples) of the samples belonging to pneumotype C were in a "non-infected" state according to the Lee criteria. No association with the Lee criteria was observed for the two other pneumotypes. The inventors also observed that the prevalence of the genus Leptotrichia, a male biomarker, was significantly lower in pneumotype C compared to pneumotypes A and B (Kruskal-Wallis test, p-value < 0.01) (see SI results for details).

[0071] Discussion This cohort study was to find early biomarkers of Pseudomonas aeruginosa colonization in CF. The inventors hypothesized that the microbiota could be more or less permissive to Pseudomonas aeruginosa in the CF airway. To evaluate this hypothesis, the inventors examined the characteristics of these microbiota, including bacteria and viruses, during the early stages of Pseudomonas aeruginosa colonization. Surprisingly, instead of pathogenic symbionts, the inventors identified characteristics of CF lungs with a low risk of Pseudomonas aeruginosa infection and three pulmotypes potentially correlated with the progression of lung disease.

[0072] At the stage of chronic Pseudomonas aeruginosa infection, low bacterial diversity was positively correlated with the progression of CF and the presence of P. aeruginosa. In this cohort, which consisted mostly of CF children who were not chronically infected with P. aeruginosa, the alpha diversity index did not decrease over the 8-month follow-up period even after acquisition of P. aeruginosa. Therefore, other biomarkers were explored. The relative abundance (RA) of Porphyromonas was significantly higher in patients of group 1 (patients who remained uninfected with P. aeruginosa) than in those of group 2 (p value < 0.001). Conversely, patients with Porphyromonas RA below the threshold of 5.33% showed a 3.7-fold higher risk of P. aeruginosa acquisition. Bacteria of the genus Porphyromonas are anaerobic symbionts of the oral microbiota and are thought to be part of the core microbiota of the CF lung. In a previous study by our team aimed at characterizing the effect of the CFTR potentiator (ivacaftor) on the microbiota of the CF airway (Hery-Arnaud G (2015) Impact of the CFTR-potentiator ivacaftor on airway in cystic fibrosis patients carrying a G551D mutation. PLoS One 10(4):e0124124), it was demonstrated that the RA of Porphyromonas increased persistently after the start of treatment, which was positively correlated with the percentage of predicted forced expiratory volume in one second (FEV-1). Taken together, these results suggest that Porphyromonas could be a favorable prognostic biomarker in CF. This finding also raises questions about the relationship between this bacterium and the P. aeruginosa-negative phenotype. Even if one bacterial genus cannot fully explain the stability of CF progression in this way, in vitro experiments must be conducted to study the interaction between Porphyromonas and P. aeruginosa in order to confirm the possible favorable role of this anaerobic bacterium in the CF airway.

[0073] Beyond pathogens, host factors shape the composition of the airway microbiota in the context of mucus-microbe-host crosstalk. The inventors have found that gender influences the composition of the CF airway microbiota. Gender differences in the mean lifespan of CF patients have been examined over the years in the United States and European countries (Jain R (2014) Gender differences in outcomes of patients with cystic fibrosis. J Women's Health 23(12):1012-1020.). Despite significant improvements in healthcare and treatment, women with CF still have worse outcomes, higher mortality, earlier colonization with Pseudomonas aeruginosa, and a higher risk of conversion of the non-mucoid to the mucoid form of Pseudomonas aeruginosa than men. Similarly, gender differences have been reported in other lung diseases. Gender differences in the immune response may be involved in the gender differences in such lung diseases. Also, sex hormones may affect lung function; for example, estrogen has been shown to induce the mucoid phenotype of Pseudomonas aeruginosa (McElvaney NG (2012) Effect of estrogen on Pseudomonas mucoidy and exacerbations in cystic fibrosis. NEJM 366(21):1978-1986.). However, the fact that gender differences manifest before puberty and after menopause suggests that hormones may not fully explain them. Given that respiratory infections are major contributors to morbidity and mortality, a hypothesis has recently been proposed that the respiratory state is the most important factor in the gender differences in CF. Therefore, this study provides a missing link between gender differences and susceptibility, which may be relevant to a wide range of pathogens, not just Pseudomonas aeruginosa. Also, this is the first time that microbiome research has supported epidemiological studies focusing on gender differences in CF.The present inventors have identified an overabundance in men of the genera Selenomonas, Leptotrichia, Atopobium, Parvimonas, and Bifidobacterium, which are obligate anaerobes and have already been associated with health status (Martinez FJ, for the COMET investigators (2014) Lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the COMET study. Lancet Respir Med 2:548-556.). The present inventors can hypothesize that the genera associated with these men and the genus Porphyromonas may contribute as a protective barrier against Pseudomonas aeruginosa, particularly in male CF patients. In vitro and in vivo analyses are necessary to confirm this hypothesis. The genus Leptotrichia was found to be dominant in healthy oral communities. This study observed that the RA of Actinobacteria (the phylum of the genus Atopobium) has a negative correlation with Pseudomonas aeruginosa, suggesting a direct effect of the resident microbiota on bacterial pathogens through competition for similar ecological niches and behaviors. In men, the abundance of the genus Bifidobacterium was found to be low (RA < 0.1%), and it was not present at all in women. Other studies have demonstrated that a decrease in the genus Bifidobacterium is associated with the asthmatic phenotype. Also, these four bacterial genera are usually described as bacteria that inhabit the intestine and are found in patients with chronic obstructive pulmonary disease. Recently, it has been found that intestinal colonization precedes airway colonization, and the gut-lung relationship in CF has been clarified, namely that the composition of the gut microbiota in young CF is directly involved in the progression of lung disease.

[0074] Samples were strongly clustered into three pulmotypes, with the dominant one being Streptococcus (pulmotype A), and the other two being dominated by either Haemophilus (pulmotype B) or Staphylococcus (pulmotype C). In previous studies pooling pediatric and adult cohorts, two ecotypes represented by either Pseudomonas or Streptococcus were detected (Dalpke AH (2015) Comparison of microbiomes from different niches of upper and lower airways in children and adolescents with cystic fibrosis. PLoS One 10(1):e0116029.). Here, pulmotype A was characterized by the highest Shannon diversity. The dominance of Streptococcus has already been recognized to be correlated with high bacterial diversity, increased respiratory function, and the first months of life in CF patients. The early establishment of the Streptococcus group in the airways is also considered a favorable biomarker in CF progression, having a direct and positive effect in the acquisition model of CF pathogens, more specifically Pseudomonas. Therefore, species of the genus Streptococcus can be defined as keystone species whose early and persistent colonization is a favorable characteristic in CF. Furthermore, the Streptococcus salivarius group was positively correlated with an increase in FEV-1. Interestingly, it was also observed that Porphyromonas was significantly dominant in samples belonging to pulmotype A.

[0075] The genus Haemophilus was the main contributor to pulmotype B. Haemophilus influenzae and Pseudomonas aeruginosa are under strong interspecies competition for colonization of the CF airway microbiota. Despite the fact that the majority of the cohorts tested consisted of patients negative for P. aeruginosa, the inventors actually observed that the genus Haemophilus had a strong influence on the structure of the CF airway microbiota. Furthermore, the genus Haemophilus was present in higher amounts in rhinovirus-positive samples, which in turn harbored low-RA P. aeruginosa. Based on this finding, the inventors can hypothesize that pulmotype B appears after pulmotype A following the initiation of antibiotic therapy or a perturbation of the microbiota such as a viral infection.

[0076] Haemophilus influenzae and Staphylococcus aureus are considered the most dominant pathogens in the pediatric CF population and are gradually replaced by P. aeruginosa later. Interestingly, the last pulmotype defined by cluster analysis is driven by the genus Staphylococcus and can be considered pathogenic given that its driver is a pathogen that has been well characterized. In the early stages of CF, high-RA S. aureus was shown to be positively associated with increased airway inflammation. Furthermore, S. aureus colonization is thought to be a risk factor for early P. aeruginosa colonization. Ninety percent of pulmotype C samples corresponded to patients in a more advanced state of lung infection than patients in a "non-infected" state with respect to P. aeruginosa, in other words, patients who "had never been infected".

[0077] Finally, the inventors investigated the relationship between pulmotypes and CF progression. During long-term follow-up, changes in pulmotypes were observed in only five CF patients. These findings indicated the relative stability of the CF airway microbiota over time in childhood. For patient 023, who changed from pulmotype A to pulmotype C, a dramatic loss of FEV-1 (from 76.2% to 60.5% in just three months; data not shown) was observed. In further long-term studies, it would be interesting to understand the long-term evolution of the microbiome and the associated pulmotypes in order to accurately define their dynamics over the entire disease history. Such studies could evaluate whether changes in pulmotypes (e.g., from A to C) are harmful to respiratory function and whether they can correlate with the clinical status. The inventors hypothesized that the pulmotype "Streptococcus" represents the founding pulmotype of a stable CF lung microbiome; its boundaries are rarely delimited in terms of species composition and it has a high diversity synonymous with a healthy respiratory state. Since it may be inappropriate to speak of a "healthy lung" in relation to CF, the inventors propose to define the change from pulmotype A to another pulmotype as the main microbial dysbiosis of the CF lung. The pulmotypes "Haemophilus", "Staphylococcus" (and in adults "Pseudomonas") correspond to entry points in a disrupted CF ecosystem and are thus defined as pathogens.

[0078] Conclusion This study demonstrated the great importance of microbiota data in the management of CF patients. The concept of pulmotypes is a way to simplify the complexity of the CF airway microbiota, which is already defined as polymicrobial and spatially heterogeneous. Using this concept, the inventors were able to identify characteristics that may be useful in predicting CF progression. Identification of bacteria of interest opens the possibility of using the bacteria as prognostic biomarkers to identify new treatment options. To validate these findings and to address the question of causality, further cohort studies are needed. The effect of the input microbiota on CF progression in childhood also needs to be investigated. In the not-too-distant future, studies of both biochemical and microbiological properties will constitute a new approach to understanding CF microbiology.

[0079] References: Throughout this application, various references are provided that describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure.

Claims

1. A method for predicting the risk of Pseudomonas aeruginosa lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - measuring the abundance of bacteria of the genus Porphyromonas in a biological sample obtained from the subject; and - when the abundance of bacteria of the genus Porphyromonas is measured to be high, concluding that the subject has a low risk of developing Pseudomonas aeruginosa lung colonization / infection, or when the abundance of bacteria of the genus Porphyromonas is measured to be low, concluding that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection. A method comprising the above steps.

2. The method according to claim 1, wherein the subject is a child.

3. The method according to claim 1, wherein the biological sample is bronchoalveolar lavage fluid (BAL) or sputum.

4. The method according to claim 1, wherein the abundance of bacteria of the genus Porphyromonas is measured using 16S rRNA deep sequencing.

5. A method for preventing Pseudomonas aeruginosa lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - predicting the risk of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; and - when it is concluded that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection, administering a therapeutically effective amount of a Pseudomonas aeruginosa-specific antibiotic to the subject. A method comprising the above steps.

6. A method for preventing Pseudomonas aeruginosa (Pa) lung colonization / infection in a subject suffering from cystic fibrosis (CF), comprising: - predicting the risk of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; and - when it is concluded that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection, administering a therapeutically effective amount of Porphyromonas probiotics to the subject. A method comprising the above steps.

7. A method for adjusting patient monitoring, comprising: - predicting the risk of Pseudomonas aeruginosa lung colonization / infection by using the method of the present invention; and - when it is concluded that the subject has a high risk of developing Pseudomonas aeruginosa lung colonization / infection, increasing the frequency of examinations. A method comprising the above steps.

8. The method according to any one of claims 1 to 7, wherein the abundance is an absolute abundance or a relative abundance.

Images