WO2020264239A1 - Methods of diagnosing and predicting chronic lung and bowel disease in pre-term infants - Google Patents

Abstract

The invention relates to methods for diagnosing or assessing the risk of developing chronic lung and bowel disease in pre-term infants by measuring the nitrate reductase activity in oral microbiome samples from the infant. The invention further relates to methods for guiding the care of pre-term infants based on measuring the nitrate reductase activity in oral microbiome samples from the infant.

Description

METHODS OF DIAGNOSING AND PREDICTING CHRONIC LUNG

AND BOWEL DISEASE IN PRE-TERM INFANTS

STATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/867,405, filed June 27, 2019, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention relates to methods for diagnosing or assessing the risk of developing chronic lung and bowel disease in pre-term infants, as well as guiding the care of pre-term infants, by measuring the nitrate reductase activity in oral microbiome samples from the infant.

BACKGROUND OF THE INVENTION

[0003] Infants born prematurely are at risk for different co-morbidities including gastrointestinal and pulmonary disease. Up to 12% of infants born prematurely will develop necrotizing enterocolitis, a devastating gastrointestinal disease with mortality rates as high a 30% for extremely premature infants. Up to 20% of preterm infants will develop severe chronic lung disease, some of which will also develop pulmonary hypertension, which increases an infant’s risk for death multifold. Currently no reliable biochemical predictive tools are available for necrotizing enterocolitis and chronic lung disease associated with pulmonary hypertension in preterm infants.

[0004] The present invention overcomes previous shortcomings in the art by providing new tools for diagnosing and predicting risk for developing chronic lung disease and bowel disease in preterm infants.

SUMMARY OF THE INVENTION

[0005] One aspect of the invention provides a method of diagnosing bronchopulmonary dysplasia (BPD) or determining the risk of developing BPD in an infant that is born at a gestational age of about 28 weeks or less ( e.g ., a preterm infant or newborn), comprising: measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth (e.g., within about one minute to about 96 hours after birth) and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional oral microbiome samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD or determining the infant to be at risk of developing BPD when no spike ( e.g ., no increase) in NR activity is observed in the sample taken from the infant at about 29 weeks PMA relative to NR activity before and/or after 29 weeks PMA.

[0006] A second aspect of the invention provides a method of diagnosing pulmonary hypertension (PH) or determining the risk of developing PH in an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having PH or determining the infant to be at risk of developing PH when no spike in NR activity is observed in the sample taken from the infant at about 29 weeks PMA relative to NR activity before and/or after 29 weeks PMA.

[0007] An additional aspect of the invention provides a method of diagnosing necrotizing enterocolitis (NEC) or determining the risk of developing NEC in an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken at birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having NEC or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA relative to NR activity before and/or after 29 weeks PMA.

[0008] A further aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD or

determining the infant to be at risk of developing BPD when no spike (e.g., no increase) in NR activity is observed at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA); and guiding the care of the infant that is diagnosed to have or to be at risk of developing BPD by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering oxygen and/or nitric oxide, (b) administering at least one phosphodiesterase inhibitor ( e.g ., sildenafil), (c) administering a composition comprising a bronchodilator, a steroid, and/or a diuretic, (d) administering a composition comprising at least one probiotic bacterial species/strain, and/or (e) prolongation of pharmacologic exposure to caffeine.

[0009] An additional aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having PH or determining the infant to be at risk of developing PH when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed to have PH or to be at risk of developing PH by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering nitric oxide and/or oxygen, (b) administering a composition comprising at least one phosphodiesterase inhibitor (e.g., sildenafil), (c) administering a composition comprising at least one endothelin receptor antagonist (e.g, bosentan), (d) administering a composition comprising at least one NO precursor (e.g, L-citrulline) and/or (e) administering a composition comprising at least one prostacyclin (e.g, epoprostanol).

[0010] A further aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having necrotizing enterocolitis (NEC) or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed to be at risk of developing NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) replacing oral feeding with parenteral feeding, (b) removing air and fluid from the stomach and intestine, (c) administering a composition comprising intravenous fluids, d) administering a composition comprising at least one antibiotic, (e) administering a composition comprising at least one probiotic bacterial species/strain, and/or (f) administering a composition comprising L-arginine. [0011] Further provided are kits comprising one or more reagents for use in assessing the risk of developing BPD, PH, and/or NEC in an infant that is born at a gestational age of about 28 weeks or less and/or for analysing the microbes in an oral microbiome sample taken from an infant that is born at a gestational age of about 28 weeks or less.

[0012] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1C provide representative tracings of nitrate dependent nitrite formation. Fig. 1A, nitrite formation from control samples and fresh tongue swabs; (-) indicates isolate to which no nitrate has been added, (+) indicates isolate following nitrate addition. Fig. IB, summary of data from all samples. Fig. 1C, nitrite formation following 18 hour incubation from tongue swab and air swab (control).

[0014] FIGS. 2A-2C provide longitudinal nitrate reductase activity in preterm infants (n=28) as a measure of nitrite formation following addition of nitrate to saliva samples. Fig. 2A shows oral nitrate reductase activity increases at 29 weeks PMA in preterm infants (p < O.Olcompared to birth and 27 weeks PMA, and p < 0.05 relative to 34 weeks PMA. Data analyzed by paired t-test. Fig. 2B provides colony forming units (CFU) following 18 hour culture of swabs (collected at indicated times) did not change over time (p = 0.22). Fig. 2C shows nitrate reductase activity normalized to CFU.

[0015] FIG. 3 shows percent relative abundance by microbial genus and species. There were statistically more Rothia and Veillonella at 34 weeks PMA compared to all other time points (p <0.03). Analysis performed with 1-way ANOVA with Tukey multiple comparisons test.

[0016] FIG. 4 provides Spearman correlation metrics demonstrating associations between individual bacteria and NR activity. The microbial abundance of the NR producing bacteria Veillonella and Rothia and the NR-negative bacteria Streptococcus were positively correlated with adjusted NR activity.

[0017] FIG. 5 shows change in oral nitrate reductase activity as measured between PMA 28 and 29 weeks for pre-term infants that developed BPD with PH, BPD only, or neither after 34 weeks PMA. P-value was determined by t-test between“no BPD nor PH” and“BPD with PH”. N values indicate the number of patients per group. DETAILED DESCRIPTION

[0018] The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0020] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

[0021] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0022] As used in the description of the invention and the appended claims, the singular forms“a,”“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. [0023] Also as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative (“or”).

[0024] The term“about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example,“about X” where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein. In reference to the age of a preterm infant,“about” means ±1 to 5 days, e.g .,“about 28 weeks” means 28 weeks ± 1-5 days,“about 29 weeks PMA” means 29 weeks PMA± 1-5 days, “about 30 weeks” means 30 weeks ± 1-5 days, and the like.

[0025] As used herein, phrases such as“between X and Y” and“between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as“between about X and Y” mean“between about X and about Y” and phrases such as“from about X to Y” mean “from about X to about Y.”

[0026] The term“comprise,”“comprises” and“comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0027] As used herein, the transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term“consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to“comprising.”

[0028] As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and“enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.

[0029] As used herein, the term“spike in NR activity” refers to an increase in NR activity at about 29 weeks PMA as compared to a time point prior or after 29 weeks PMA (e.g, at about 28 weeks PMA or earlier than 29 weeks PMA, or about 30 weeks PMA or later than 29 weeks PMA) of at least about 5% (e.g, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 175, 200

250, 300, 350, 400, 450, 500%, or more and any range or value therein,) ( e.g ., about 5% to about 100%, about 5% to about 150%, about 5% to about 300%, about 15% to about 100%, about 15% to about 150%, about 15% to about 200%, about 15% to about 300%, about 15% to about 500%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 300%, about 50% to about 500%, and the like.

[0030] As used herein, the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” and“decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0031] As used herein,“gestational age” as applied to the age of an infant refers to the time elapsed between the first day of the last normal menstrual period of the infant’s mother and the day of delivery.

[0032] “Chronological age” (or“postnatal” age) is the time elapsed after birth. It is usually described in days, weeks, months, and/or years.

[0033] As used herein,“postmenstrual age” or“PM A” refers to the perinatal period beginning after the day of birth. It is the time el apsed between the first day of the last menstrual period of the mother and birth of the infant (gestational age) plus the time elapsed after birth (chronological age) and is usually provided in number of weeks. As an example, a preterm infant born at a gestational age of 30 weeks who is at a chronological age of 8 weeks would have a PM A of 38 weeks.

[0034] As used herein,“an infant that is a gestational age of about 28 weeks or less” is a preterm infant or preterm newborn having a gestational age of about 21 to about 28 weeks.

An infant for which this invention is useful may also be described as having a birth weight of less than about 2500 grams (low birth weight), less than about 1500 grams (very low birth weight), or less than about 1000 grams (extremely low birth weight).

[0035] As used herein, the term“oral microbiome” refers to the microorganisms that reside in the oral cavity. In some embodiments, the oral microbiome refers the microorganisms that reside on the tongue and/or at the posterior dorsum of the tongue.

[0036] The term“bronchopulmonary dysplasia” or“BPD” is a form of chronic lung disease that affects newborns (mostly premature) and infants requiring supplemental oxygen and/or positive pressure at 36 weeks PMA. This disease is most commonly seen in premature infants who required mechanical ventilation and oxygen therapy for acute respiratory distress.

[0037] The term“pulmonary hypertension” or“PH” is high blood pressure in the arteries of the lungs (the pulmonary arteries) and affects the right side of the heart. The disease can result in hypoxemia secondary to right-to-left intracardiac shunting of blood.

[0038] The term“necrotizing enterocolitis” or“NEC” refers to a disease process of the gastrointestinal (GI) tract and is characterized by ischemic necrosis of the intestinal (large and small) mucosa, which is associated with severe inflammation, invasion of enteric gas forming organisms, and dissection of gas into the bowel wall and portal venous system

[0039] The terms“increased risk” and“decreased risk” as used herein define the level of risk that a subject ( e.g ., an infant that is a gestational age of about 28 weeks or less) has of developing BPD, PH and/or NEC, as compared to a control subject (e.g., a subject that exhibits an increase in nitrate reductase activity at about 29 weeks postmenstrual age).

[0040] By the terms“treat,”“treating,” or“treatment,” it is intended that the severity of the subject’s condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.

[0041] As used herein, the terms“prevent,”“prevents,” or“prevention” and“inhibit,” “inhibits,” or“inhibition” (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.

[0042] “Diagnosing,”“assessing the risk,”“predicting the risk” or“determining the risk,” as used herein means providing an indication that a subject may be afflicted with or at risk of developing a disease, e.g., a disease such as BDP, PH or NEC, and includes other terms such as screening for a disease, providing a risk assessment for disease, determining

responsiveness to treatment, etc. It will be appreciated that no such technique is perfect and that such diagnosis, assessment, prognosis, prediction, or the like may be confirmed by other procedures such as physical examination, imaging, histological examination of tissue samples, etc. The term“prognosing” or“responsiveness” as used herein includes providing an assessment or indication of disease in response to treatment (such as administration of probiotics, antibiotics, oxygen and/or nitric oxide, a phosphodiester inhibitor, a

bronchodilator, a steroid, a diuretic, and the like, and combinations thereof) after initial diagnosis, as an indication of the efficacy of the treatment, risk of the disease returning, severity of disease following treatment, or the like. [0043] An“effective,”“prophylactically effective,” or“therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an“effective,”“prophylactically effective,” or“therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the effects need not be complete or curative, as long as some benefit is provided to the subject. For example, an“effective,”“prophylactically effective,” or “therapeutically effective” amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. As appropriate, an“effective,”“prophylactically effective,” or“therapeutically effective” amount in any individual case for any particular agent, compound or composition can be determined by one of skill in the art by reference to the pertinent texts and literature such as Remington, The Science and Practice of Pharmacy (latest edition).

[0044] Many distinct microbial ecosystems have been described throughout the human body. Associations between these microbiota and human disease have focused efforts into understanding mechanisms by which these organisms may influence disease susceptibility, progression, and severity. The microbiota over the tongue dorsum are particularly unique in that several facultative anaerobes residing in the crypts of the tongue dorsum express an enzyme, nitrate reductase (NR) (Duncan et al, Nat. Med. 1(6): 546 (1995)), not found in the human genome. NR containing bacteria catalyze the reduction of nitrate to nitrite, which is a substrate for nitric oxide (NO) via protonation within the acidic gastric environment or further electron reduction through a number of hypoxia sensitive enzymes and proteins in the blood and tissues (Weitzberg et al, Annu. Rev. Nutr. 33 : 129 (2013)). Accumulating evidence suggests that reduction of nitrate to nitrite and NO is an important and parallel pathway, with nitric oxide synthase, to control systemic NO-bioavailability.

[0045] NR significantly impacts systemic, gastrointestinal, and pulmonary vasculature homeostasis. Several studies have demonstrated that nitrate administration significantly lowers systolic and diastolic blood pressures by levels comparable to a single

antihypertensive agent (Larsen et al., N. Engl. J. Med. 355(26):2792 (2006); Kapil et al, Hypertension 56(2):274 (2010); Webb et al., Hypertension 51(3):784 (2008)). Conversely, decreased oral and plasma nitrite and subsequent NO bioavailability following the use of antibacterial mouthwash is associated with increased blood pressure and loss of NO- dependent signaling (Kapil et al, Free Radic. Biol. Med. 55:93 (2013); Govoni el al, Nitric Oxide 19(4): 333 (2008)). Animal models have demonstrated a gastroprotective role of nitrite through its ability to increase mucosal blood flow and mucosal thickness of the intestines (Bjorne et al, J. Clin. Invest. 113(1): 106 (2004)); these benefits are lost after antiseptic mouthwash application (Petersson et al, Free Radic. Biol. Med. 46(8): 1068 (2009)). Similarly, nitrate conversion to nitrite may also serve to limit ischemia-reperfusion injury, endothelial dysfunction, and provide host defense via antimicrobial effects on gut pathogens (Dykhuizen et al, Antimicrob. Agents Chemother. 40(6): 1422 (1996); Xia et al, Chin. Med. J. (Engl.) 119(22): 1904 (2006); Fite et al, Antimicrob. Agents Chemother.

48(2):655 (2004); Jadert et al, Redox Biol. 2:73 (2014)). The prevention of right ventricular hypertrophy through nitrate supplementation in mice supports the possible role of enterosalivary nitrate reduction in attenuating pulmonary vascular resistance in mice (Baliga et al, Circulation 125(23):2922 (2012)) [14] with growing evidence to suggest nitrate based therapeutics for modification of pulmonary vascular disease (Koch et al, Free Radic. Biol. Med. 105:48 (2017)).

[0046] Angiogenesis and vascular resistance play important roles in the development of certain co-morbidities of prematurity in infants. In characterizing NEC, a devastating gastrointestinal illness in preterm infants, a high intestinal vascular resistance pattern in the superior mesenteric artery has been demonstrated in infants that develop NEC (Murdoch et al, Pediatrics 118(5): 1999 (2006)) as well as low splanchnic tissue oxygenation in animal models of NEC (Zamora et al, PLoS One 10(6): e0125437 (2015)). PH, a multifactorial disease that develops in around 20% of extremely preterm infants (Bhat et al, Pediatrics 129(3):e682 (2012)) with a mortality risk up to 48% (Khemani et al, Pediatrics 120(6): 1260 (2007)), is influenced by pulmonary vascular growth and function in the developing neonate. A commonality between both NEC and PH is the involvement of small vessels. Given the role of vascular resistance and angiogenesis in disease development in preterm infants and the possible attenuating role of NR mediated NO production, the present inventors have evaluated NR activity in preterm infants and associations with covariates including co morbidities, exposures, and nutrition, and unexpectedly determined that NR activity from the oral microbiome of preterm infants ( e.g. , those born at a gestational age of about 28 weeks or less) may be used for diagnosing and predicting the risk of developing BPD, PH and/or NEC in these infants. [0047] In some embodiments, the present invention provides a method of diagnosing BPD and/or determining the risk of developing BPD in an infant that is bom at a gestational age of about 28 weeks or less, the method comprising: measuring NR activity ( e.g ., nM nitrite formed/10³ CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g., about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD and/or determining the infant to be at risk of developing BPD when no spike (e.g, no increase) in NR activity is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).

[0048] In some embodiments, a method of diagnosing PH and/or determining the risk of developing PH in an infant that is a gestational age of about 28 weeks or less is provided, the method comprising: measuring NR activity (nM nitrite formed/10³ CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having PH and/or determining the infant to be at risk of developing PH when no spike in NR activity is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).

[0049] In some embodiments, a method of diagnosing NEC and/or determining the risk of developing NEC in an infant that is a gestational age of about 28 weeks or less is provided, the method comprising: measuring NR activity (nM nitrite formed/10³ CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken at birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having NEC and/or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).

[0050] In some embodiments, the present invention provides a method of diagnosing BPD, PH, and/or NEC and/or determining the risk of developing BPD, PH or NEC in an infant that is born at a gestational age of about 28 weeks or less, the method comprising: quantifying the bacterial species ( e.g ., the number of viable bacterial counts, and/or measuring the DNA associated with the bacteria) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g., about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD, PH and/or NEC and/or

determining the infant to be at risk of developing BPD, PH and/or NEC when no spike (e.g, no increase) in the amount of specific bacterial species is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).

[0051] Another aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD and/or determining the infant to be at risk of developing BPD when no spike (e.g, no increase) in NR activity is observed at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA); and guiding the care of the infant that is diagnosed as having BPD and/or to be at risk of developing BPD by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering a therapeutically effective amount of oxygen and/or nitric oxide, (b) administering a therapeutically effective amount of a phosphodiesterase inhibitor, (c) administering a therapeutically effective amount of a composition comprising a

bronchodilator, a steroid, and/or a diuretic, (d) administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (e) prolongation of pharmacologic exposure to caffeine, thereby guiding the care of the infant.

[0052] In some embodiments, a phosphodiesterase inhibitor that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, sildenafil, prostacyclin, iloprost and/or treprostinil.

[0053] In some embodiments, a bronchodilator that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, albuterol. [0054] In some embodiments, a steroid that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, hydrocortisone, dexamethasone, and/or prednisone.

[0055] In some embodiments, a diuretic that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, furesomide and/or diuril.

[0056] In some embodiments, a probiotic bacterial strain or species that may be

administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella,

Lactobacillus, Hemophilus, Staphylococcus , Streptoccocus, Enter occocus and/or Prevolella. In some embodiments, the probiotic bacterial strain or species can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia and/or Veillonella. A therapeutically effective amount of a probiotic bacterium or composition of probiotic bacteria can be for example a daily dose of probiotic bacterium or composition of probiotic bacteria of about 10⁴ to about 10¹² CFU, for example 10⁵ to 10¹⁰, or 10⁶ to 10⁸, or 10⁷ to 10⁹,or 10⁸ to 10¹⁰ total CFUs of bacteria may be used. In some embodiments, a daily dose of one or each bacteria may be around 10⁸ or 10⁹ total CFUs, e.g., 10⁷ to 10¹⁰ or 10⁸ to 10¹⁰ or 10⁸ to 10⁹. Such doses can be in the form of CFU/g or CFU/unit-dosage form.

[0057] In some embodiments, the present invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having PH and/or determining the infant to be at risk of developing PH when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed as having PH and/or to be at risk of developing PH by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering a therapeutically effective amount of nitric oxide and/or oxygen, (b) administering a therapeutically effective amount of a composition comprising at least one phosphodiesterase inhibitor, (c) administering a therapeutically effective amount of a composition comprising at least one endothelin receptor antagonist, (d) administering a therapeutically effective amount of a composition comprising at least one NO precursor and/or (e) administering a

therapeutically effective amount of a composition comprising at least one prostacyclin. [0058] In some embodiments, a phosphodiesterase inhibitor that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, sildenafil.

[0059] In some embodiments, an endothelin receptor antagonist that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, bosentan.

[0060] In some embodiments, a nitric oxide precursor that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, L-arginine and/or nitrate.

[0061] In some embodiments, a prostacyclin that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, epoprostanol, iloprost, and/or treprostinil.

[0062] Further provided is a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, the method comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks ( e.g ., about 30, 31, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having NEC and/or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed as having NEC and/or to be at risk of developing NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) replacing oral feeding with parenteral feeding, (b) removing air and fluid from the stomach and intestine, (c)

administering a therapeutically effective amount of a composition comprising intravenous fluids, (d) administering a therapeutically effective amount of a composition comprising at least one antibiotic, (e) administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (f) administering a therapeutically effective amount of a composition comprising L-arginine.

[0063] In some embodiments, an antibiotic that may be administered to the infant diagnosed with or at risk of developing NEC includes, but is not limited to, vancomycin, tobramycin, flagyl, zosyn, and/or fluconazole.

[0064] In some embodiments, a probiotic bacterial strain or species that may be

administered to an infant diagnosed with or at risk of developing NEC can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella,

Lactobacillus, Hemophilus, Staphylococcus , Streptoccocus, Enter occocus and/or Prevotella. In some embodiments, the probiotic bacterial strain or species can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia and/or Veillonella. A therapeutically effective amount of a probiotic bacterium or composition of probiotic bacteria can be for example a daily dose of probiotic bacterium or composition of probiotic bacteria of about 10⁴ to about 10¹² CFU, for example 10⁵ to 10¹⁰, or 10⁶ to 10⁸, or 10⁷ to 10⁹,or 10⁸ to 10¹⁰ total CFUs of bacteria. In some embodiments, a daily dose of one or each bacteria may be around 10⁸ or 10⁹ total CFUs, e.g., 10⁷ to 10¹⁰ or 10⁸ to 10¹⁰ or 10⁸ to 10⁹. Such doses can be in the form of CFU/g or CFU/unit-dosage form.

[0065] In some embodiments, a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: quantifying the bacterial species (e.g, the number of viable bacterial counts, and/or measuring the DNA associated with the bacteria in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD, PH and/or NEC and/or determining the infant to be at risk of developing BPD, PH and/or NEC when no spike (e.g, no increase) in the amount of specific bacterial species is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA); and guiding the care of the infant that is diagnosed as having BPD, PH and/or NEC and/or determined to be at risk of developing BPD, PH and/or NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as: for BPD: (a) administering a therapeutically effective amount of oxygen and/or nitric oxide, (b) administering a therapeutically effective amount of a phosphodiesterase inhibitor, (c) administering a therapeutically effective amount of a composition comprising a bronchodilator, a steroid, and/or a diuretic, (d) administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (e) prolongation of pharmacologic exposure to caffeine, thereby guiding the care of the infant; for PH: (a) administering a therapeutically effective amount of nitric oxide and/or oxygen, (b) administering a therapeutically effective amount of a composition comprising at least one phosphodiesterase inhibitor, (c) administering a therapeutically effective amount of a composition comprising at least one endothelin receptor antagonist, (d) administering a therapeutically effective amount of a composition comprising at least one NO precursor and/or (e) administering a therapeutically effective amount of a composition comprising at least one prostacyclin; and/or for NEC: (a) replacing oral feeding with parenteral feeding, (b) removing air and fluid from the stomach and intestine, (c) administering a therapeutically effective amount of a composition comprising intravenous fluids, (d) administering a therapeutically effective amount of a composition comprising at least one antibiotic, (e) administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (f) administering a therapeutically effective amount of a composition comprising L-arginine.

[0066] Further provided is a method for treating an infant that is a gestational age of about 28 weeks or less for BPD, PH and/or NEC, the method comprising diagnosing the infant as having or at increased risk of having BPD, PH and/or NEC by the methods of the invention and treating the infant for BPD, PH and/or NEC.

[0067] In some embodiments, an oral microbiome sample is taken from the mouth of the infant, optionally the tongue of the infant. In some embodiments, the oral microbiome sample may be taken from the posterior dorsum of the tongue of the infant. An oral microbiome sample may be obtained from the infant in any acceptable manner. In some embodiments, an oral microbiome sample may be obtained by a swab, tongue scraper, suction bulb, and/or cotton pad.

[0068] In some embodiments, a first oral microbiome sample taken after birth may be taken/obtained from the infant within one minute to about 72 hours, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 54,

60, 66, 72 hrs, or any range or value therein, after birth. Thus, a range of time for which a first oral microbiome sample may be obtained from the infant may be about 5, 10, 15, 30, 45, 60, 90, 120, 180 min to about 10, 15, 24, 36, 48, 60, or 72 hours after birth, and any range or value therein. In some embodiments, a first oral microbiome sample may be obtained at about 78, 84, 90, or 96 hours after birth. In some embodiments, a first oral microbiome sample may be obtained from the infant from about 24 hours after birth to about 72 hours after birth, optionally at about 45 hours to about 70 hours after birth.

[0069] In some embodiments, the at least two additional oral microbiome samples are two samples, a second sample taken at about 29 weeks PMA and a third sample taken at about 30 weeks PMA, at about 31 weeks PMA, at about 32 weeks PMA, at about 33 weeks PMA, or at about 34 weeks PMA. Of course, the gestational age of the infant will determine the timing and number of samples taken from the infant. Thus, for example, an infant having a gestational age of about 28 weeks, will have a first oral microbiome sample taken at birth ( e.g ., about 1 minute to about 96 hrs after birth) and then a second oral microbiome sample taken at about 29 weeks PMA and at least one further oral microbiome sample taken at about 30, 31, 32, 33, and/or 34 weeks PMA. As a further example, an infant having a gestational age of about 25 weeks, will have a first oral microbiome sample taken at birth and then at least a second sample taken at about 29 weeks PMA (and possibly a sample taken at about 26, 27 and/or 28 weeks), and one or more oral microbiome samples taken at about 30, 31, 32, 33, and/or 34 weeks PMA. Thus, the at least two additional oral microbiome samples may be obtained at weekly intervals following the first oral microbiome sample obtained after birth until the infant reaches about 30 to 34 weeks PMA. In some embodiments, following the first oral microbiome sample taken after birth, the at least two additional oral microbiome samples may be taken periodically about every 2 days to about every 14 days ( e.g ., about every 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 days). In some embodiments, when taking a sample at about 29 weeks PMA, a sample may be across a series of consecutive days or every other day for ± 1-5 days from the 29 week PMA time point (e.g., samples may be taken every other day or every day for a set series of days that are ± 1-5 days from the 29 week PMA time point, e.g., if October 23^rd is the actual date of the 29 week PMA for a subject, then samples may be taken on October 18^th (-5), 19^th (-4), 20^th (-3), 21^st (-2), 22^nd (-1), 23^rd (0), 24^th (±1), 25^th (±2), 26^th (±3), 27^th (±4), and/or 28^th (+5)) or any subset thereof (e.g., October 18^th (-5), 19^th (-4), 20^th (-3), 21^st (-2), 22^nd (-1), 23^rd (0); 21^st (-2), 22^nd (-1), 23^rd (0), 24^th (±1), 25^th (±2); 22^nd (- 1), 23^rd (0), 24^th (±1), and the like.

[0070] In some embodiments, the at least two additional oral microbiome samples are taken about 1 min to about 4 hours post-feeding (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75,

80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,

230, 240 min post feeding, and any range or value therein). In some embodiments, the at least two additional oral microbiome samples are taken about 2 hours post-feeding.

[0071] In some embodiments, the method further comprises culturing the oral microbiome samples for about 5 min to about 36 hours (e.g, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,

220, 230, 240, 250, 275, 300, 350, 400, 450, 500, 550, 600 min, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, and any range or value therein) prior to determining (measuring) the NR activity. In some embodiments, the method may further comprise culturing the oral microbiome samples for about 6 hours to 30 hours prior to determining (measuring) the NR activity. In some embodiments, the method may further comprise culturing the oral microbiome samples for about 12 hours to 24 hours prior to determining (measuring) the NR activity. In some embodiments, the method may further comprise culturing the oral microbiome samples, for about 18 hours prior to determining (measuring) the NR activity.

[0072] In some embodiments, the culturing is under aerobic conditions. In some embodiments, the conditions for culturing include nutrient rich conditions.

[0073] Following culturing, the samples are then tested for NR activity. Any method of measuring NR activity may be used. For example, after establishing baseline nitrite levels in the sample ( e.g ., cultured sample). The sample (or portion thereof) may be incubated at about 37°C for about 10 min), nitrate may be added to a portion of the sample (e.g., about 10 mΐ of 10 mM nitrate to a 90 mΐ sample) and the sample mixed (e.g, by vortex). Nitrite formation may be measure using any method known in the art (e.g, triiodide chemiluminescence as previously described (Pelletier et al, Free Radic Biol Med. 41(4):541 (2006)). In some embodiments, nitrite may be measured by Griess reaction by visible spectroscopy or after HPLC purification. In some embodiments, nitrite test strips may be used.

[0074] The present invention further comprises a kit or kits to carry out the methods of this invention. A kit of this invention can comprise reagents, buffers, and apparatus for mixing, measuring, sorting, labeling, etc., as well as instructions and the like as would be appropriate for measuring NR activity and/or for analyzing the microbes present in an oral microbiome sample from an infant.

[0075] In some embodiments, the invention provides a kit for assessing the risk of developing BPD, PH, and/or NEC in an infant, comprising one or more reagents for measuring NR activity in the cultured samples, with optional instructions for the use thereof.

[0076] In some embodiments, the invention provides a kit for microbial analysis of a microbiome sample taken from an infant that is born at a gestational age of about 28 weeks or less, comprising one or more reagents for identifying the microbes present in the oral microbiome sample, with optional instructions for the use thereof.

[0077] In some embodiments, a kit of the invention may comprise three or more

implements for taking oral microbiome samples from an infant (e.g., from the posterior dorsum of the tongue of the infant) and/or one or more reagents for culturing the oral microbiome samples.

[0078] In some embodiments, the kit of the invention may comprise markers for identifying at least one of a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella, Lactobacillus, Hemophilus, Staphylococcus, Streptoccocus, Enter occocus and/or Prevolella. [0079] The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.

EXAMPLES

Example 1.

[0080] Institutional Review Board approval at the University of Alabama at Birmingham was obtained prior to study initiation. Infants were recruited from the UAB Regional Neonatal Intensive Care Unit (RNICU). Preterm infants enrolled were < 28 6/7 weeks gestation at birth. Initial samples for oral microbiome and NR activity were taken within 72 hours of birth. Weekly samples were then obtained 2 hours post feeding until infants reached 34 weeks post-menstrual age (PMA).

Salivary Nitrate Reductase Activity

[0081] Samples were taken from the posterior dorsum of each infant’s tongue using sterile wood Fisherbrand cotton-tipped applicators (Cat. No. 23-400-115) by rotating the applicator 360° over the right posterior tongue dorsum. All samples were collected by the same researcher to minimize inter-sampling variability. Samples were then inserted immediately in 1.5 ml of Brain Heart Infusion Broth (Anaerobe Systems, Morgan Hill, CA) and placed on ice. NR activity was measured two times after sample collection: within 2 hours of collection and after 18 hour incubation under aerobic conditions in BHI at 37°C. Two 90 pL aliquots (one for control and one to which nitrate was later added) were incubated in a water bath at 37°C for 10 minutes prior to analysis and baseline nitrite levels measured. Ten pL of 10 mM nitrate was then added (ImM final nitrate concentration), samples vortexed (~3 seconds), and nitrite formation sequentially measured over 20 minutes using triiodide chemiluminescence as previously described (Pelletier et al, Free Radic. Biol. Med. 41(4):541 (2006)). These nitrite levels were then compared to the sample controls to which no nitrate was added.

Standard curves generated on the day of sampling from a nitrite solution of known concentration were then used for final nitrite calculations. One way ANOVA was performed to discern differences in NR activity over time. Mann-Whitney U testing was used to determine the influence of other co-variates with NR activity.

Oral microbiome analysis [0082] Weekly samples were taken for microbiome analysis using Fisherfmest Dry

Transport Swabs (Fisher Healthcare, Houston, Texas). At the same time points used for NR sampling, the applicator was rotated 360° over the left posterior tongue dorsum, immersed in 1 ml sterile normal saline, and placed in a -80°C freezer until further analysis. DNA was then extracted using a QIAGEN QIAamp DNA Stool Mini Kit (QIAGEN, Germany).

Microbiome analysis using 16S rRNA sequencing was performed at four time points via methods previously described (Lai et al, Sci. Rep. 6:31023 (2016)): birth, 27 PMA, 29 weeks PMA, and at > 34 weeks PMA. To determine bacterial numbers, colony forming unit (CFU) assays were performed on each sample used to measure NR activity. Ten pL of sample were serially diluted in normal saline and plated in triplicate on Tryptic Soy Blood Agar plates (Anaerobe Systems), incubated for 24 hours, and CFUs averaged between the three plated samples.

[0083] The top fifty most abundant microbiota represented across all samples were reported. To determine the variations in oral microbiota over the time sampled, the percent microbial abundance of both NR producing and non-producing microbiota were analyzed at each sampling time point with 1-way ANOVA analysis to detect differences at each PMA. Spearman correlation metrics were used to determine associations between the microbial abundance of individual bacteria and NR activity.

Nitrate Reductase Activity in yre-term infants

[0084] NR activity was measured in 190 samples taken from 28 preterm infants between birth and 34 weeks PMA. For samples taken at birth, the mean time of sample collection was 49 ± 18 h, with a median of 48.5 h of life. The average gestational age of the preterm cohort was 25.6 ± 1.6 weeks with an average birth weight of 728 g ± 218 g (other demographic data are displayed in Table 1).

[0085] No infants in the cohort developed NEC Stage II or higher. In the first 7 preterm infants enrolled, a total of 23 samples were collected (from birth to 21d PMA). No NR activity, assessed by nitrate-dependent formation of nitrite, was detectable in these samples. FIG. 1A shows representative traces for nitrite formation measured by triiodide mediated reduction to NO, and FIG. IB summary data from all samples. However, significant NR activity was observed if tongue swabs were first cultured for 18 hours, and then nitrate- dependent formation of nitrite assessed (FIG. 1A). No NR activity was evident in swabs exposed to air only (FIG. 1C), suggesting that NR expressing bacteria are present on the newborn tongue, but at low levels. Table 1: Demographic Data from Enrolled Preterm Infants (mean ± SD)

[0086] For all subsequent samples, oral swabs were cultured for 18 h and then NR activity measured. NR activity remained constant over the 10 week observation period, except for a significant increase in activity at 29 weeks PMA (FIG. 2A), with more activity compared to infants at 24, 27, and 28 weeks PMA by one-way ANOVA. No differences in the number of bacteria were observed (FIG. 2B). FIG. 2C plots NR activity normalized to CFU. A significant spike in NR activity at 29 weeks PMA was still evident, with significantly more activity at every PMA less than 29 weeks except 28 weeks PMA. Preterm infants born via spontaneous vaginal delivery had higher NR activity at birth compared to those born via cesarean section. Samples taken after 48 hour from delivery had higher NR activity compared to samples taken within 48 hours after delivery. Exposure to antenatal antibiotics did not result in statistically different NR activity at birth in preterm infants (p=0.12).

[0087] Subgroup analyses for associations between NR activity and presence or absence of comorbidities were also conducted using NR activity adjusted for CFU. All associations were analyzed using two-way ANOVA with Fisher’s LSD test for multiple comparisons. There was a trend for decreased NR activity between 29 and 34 weeks PMA in infants that developed bronchopulmonary dysplasia (BPD) (n=14) defined as need for oxygen supplementation at 36 weeks PMA compared to infants that did not develop BPD (n=9), though non-significant (p=0.12 using two way ANOVA). When infants with both BPD and pulmonary hypertension (n=6) were compared to those infants that had neither BPD nor pulmonary hypertension (n=9), there was significantly decreased adjusted NR activity at 29, 30, and 32 weeks PMA via Fisher’s LSD test for multiple comparisons (p=0.0047). Infants that received multiple antibiotic courses (defined as > 3 antibiotic courses and/or > 7 days of broad spectrum antibiotic therapy) had less NR activity at 34 weeks PMA compared to infants with fewer courses or days of therapy (p=0.015). Infants exclusively maternal breast milk fed had statistically more NR activity at 28 weeks PMA than formula fed infants.

Oral Microbiome abundance and composition

[0088] FIGS. 2A-2C indicate an increase in NR activity at a PMA of 29 weeks (FIGS. 2A, 2C) that was not associated with altered bacterial count (FIG. 2B), suggesting an increase in specific activity and/or changes in bacterial composition. To further explore this and better characterize which bacteria colonize the distal tongue in premature infants, microbiome analysis was performed on samples collected at birth, 27, 29 and 34 weeks PMA, the time points over which NR activity increased then decreased (FIG. 2A). FIG. 3 shows the relative abundance of bacterial species at each PMA tested.

[0089] One hundred one isolates were analyzed. Firmicutes, Proteobacteria, and

Bacteroides were the most abundant phyla on the tongue dorsum over the time period sampled representing 58%, 22.5%, and 7% of all samples respectively (FIG. 3). Based on previous studies (Kanady et al, Nitric Oxide 27(4): 193 (2012); Doel et al, Eur. ./. Oral Sci. 113(1): 14 (2005)) or the ENSEMBL database, NR expressing bacteria identified in the cohort included Actinomyces, Rothia, Veillonella, Lactobacillus, Hemophilus , and P re vote l la, all of which were present from birth. Rothia were significantly more abundant at 34 weeks PMA compared to all other time points (p=0.01) as was the abundance of Veillonella (p=0.0003) and Actinomyces (p=0.04) (FIG. 3). Based on Spearman correlation metrics, the NR producing bacteria Veillonella and Rothia as well as the NR- bacteria Streptococcus were positively correlated with NR activity (FIG. 4). Upon investigation of trends in

Streptococcus over the PMAs analyzed, significantly more bacteria were present at 29 and 34 weeks PMA compared to infants at birth (p=0.0044).

[0090] Given the associated trends in covariates including form of enteral nutrition, BPD, antibiotic exposure, and presence or absence of pulmonary hypertension, analysis of trends in the bacteria Veillonella, Rothia, and Streptococcus were conducted. [0091] The alpha biodiversity of oral microbiota were higher at birth (4.41) compared to all other time points of 27 weeks PMA (2.35), 29 weeks PMA (2.59), and > 34 w PMA (2.60) (p<0.05). The number of colony forming units did not statistically differ during the period during which infants were sampled (FIG. 2B).

Discussion

[0092] Formation of NO in mammals is mediated by nitric oxide synthase dependent and independent mechanisms. The latter involves nitrate-reduction to nitrite by commensal oral nitrate-reducing bacteria. The nitrite formed provides substrate for various NR systems that mediate NO-signaling by hypoxia and pH-dependent mechanisms (Benjamin et al, Nature 368(6471):502 (1994)). However, little is known regarding the role of the oral microbiome in mediating nitrate-reduction and contributing to NO-homeostasis mechanisms in the newborn and specifically premature infants. Nitric oxide is a primary vasodilator of the intestinal vasculature (Reber et al, Am. ./. Physiol. Gastrointest. Liver Physiol. 280(1):G43 (2001)). The importance of endothelial nitric oxide synthase (eNOS), an enzyme responsible for local NO production within tissue vasculature, has been explored within mice models demonstrating NEC pathology in knockout mice as well as gastrointestinal protection in pig models exposed to L-arginine supplementation, a substrate for endothelial NO production (Yazji et ah, Proc. Natl. Acad. Sci. USA 110(23):9451 (2013); De Plaen, Clin. Perinatol. 40(1): 109 (2013)). A recent study of NEC in an eNOS knockout mice model not only demonstrated the importance of eNOS in gastrointestinal pathology, but also in regulating inflammatory mediators in the lung (Drucker et al, J. Pediatr. Surg. 53(6):1208 (2018)). Multiple randomized controlled trials substantiate inhaled NO as a life-saving therapy in full term infants with persistent pulmonary hypertension with a potential for benefit in preterm infants with developing lung disease (Barrington et al., Cochrane Database Syst. Rev.

ECD000399 (2017); Barrington et al., Cochrane Database Syst. Rev. ECD000509 (2017)).

[0093] Given the importance of eNOS on gastrointestinal perfusion and mounting evidence for its role in conjunction with NO in pulmonary pathology, other pathways for NO generation warrant further evaluation. Data from this prospective cohort study are the first to longitudinally evaluate NR production in ELGANs. Our results are consistent with previous conclusions in older preterm infants with an average gestational age of 30 weeks at birth wherein NR activity remained undetectable until two weeks of age, and then when activity became detectable, it remained low, about 10% that of adults (Kanady et al, Nitric Oxide 27(4): 193 (2012)). Oral NR activity in this cohort was low and only detectable after bacterial amplification. This underscores the low abundance of NR bacteria in the oral cavity from preterm infants and is similar to our prior studies investigating oral NR activity in adult mice (Ahmed et al, Nitric Oxide 66:62 (2017)). We note that in mice, which have similar low NR activities, a nitrate- and oral microbiome dependent signaling has been demonstrated (Ahmed et al, Nitric Oxide 66:62 (2017)).

[0094] Moreover, the oral NR activity measured here may not directly relate to any nitrate- dependent activation of NO-signaling cascades, and how much nitrite is needed to elicit NO- dependent effects in the newborn is also unknown. Interestingly, inhaled NO therapy for persistent pulmonary hypertension of the newborn, previously thought to have limited effect systemically, doubles serum nitrite levels and increases nitrate levels four fold (Ibrahim et al, J Pediatr. 160(2):245 (2012)). While these levels still remain low, they have been demonstrated to still influence systolic blood pressure in adults (Webb et al, Hypertension 51(3):784 (2008)). The physiologic relevance of NR activity may simply be negligible at birth prior to oral microbiome establishment and serum nitrate and nitrite levels may initially better reflect maternally derived levels. Thus, while the oral NR activity is low, our data suggests that it is present and increases from birth in premature babies.

[0095] Interestingly, a statistically significant increase in NR activity was noted at 29 weeks PMA. This observation was consistent across the cohort of 28 preterm infants. Changes in the microbial abundance of NR producing bacteria remains the most plausible mechanism from the data herein for these changes in NR activity. Variables previously noted to influence initial microbiota include delivery mode (Dominguez-Bello et al, Proc. Natl Acad. Sci. USA 107(26): 11971 (2010)), the diversity and progression of breast milk flora (Cabrera- Rubio et al, Am. J. Clin. Nutr. 96(3):544 (2012)) vs formula (Patel et al, PLoS One

10(7):e0114664 (2015)), and maternal intrapartum antibiotic exposure (Keski-Nisula et al, Acta Paediatr. 102(5):480 (2013)). The oral cavity is colonized within hours (Nelson-Filho et al., Braz. Dent. J. 2013. 24(4):415 (2013)) and, while initially similar, divergence between oral and intestinal microbiota later occurs at around 15 days of life (Costello et al, MBio 4(6):e00782 (2013)). Delivery mode influenced the NR activity in this cohort as did the time of first isolate collection and whether an infant was breast milk or formula fed. The bacteria in this cohort most associated with NR production based on Spearman analysis were

Veillonella and Rothia. Previous studies have also reported these bacteria to be NR producers in the adult mouth (Doel et al, Eur. J. Oral Sci. 113(1): 14 (2005)). Streptococcus, which was also noted to be associated with NR activity, does not produce NR. However, the relative abundance of Streptococcus has been shown to be as high as 89% in isolates with high NR activity compared to 72% in isolates with low NR activity (Hyde et al, PLoS One 9(3):e88645 (2014)), which may support its role as a commensal organism.

[0096] The 29 week PMA time point at which activity significant increased coincides with a critical window where NEC occurrence peaks (Yee et al, Pediatrics 129(2):e298 (2012)). Several studies have investigated how changes in endogenous NO metabolism may be linked with NEC. In a recent study of extremely preterm infants, the serum nitrate and nitrite levels increased up until the twentieth day of life (Pun et al, Pediatr. Res. 2016. 79(3):432 (2016)). Urinary nitrite levels from the same study, while initially similar in infants that did and did not develop NEC, increased following NEC development possibly from excessive NO production from inducible NOS (iNOS) (Petrosyan et al, Pediatr. Surg. Int. 25(4):309 (2009)). The spike in NR activity observed at 29 weeks may be a plausible mechanism for the rise in serum nitrite, as increased NR activity would lead to more conversion of salivary nitrate to nitrite. A randomized trial in preterm infants showed decreased NEC risk in infants receiving L-arginine (Polycarpou et al, J. Parenter. Enteral Nutr. 37(5):617 (2013)); an important finding given that preterm infants are deficient in arginine (Wu et al, J. Nutr. Biochem. 15(8):442 (2004)). Additionally, decreased endothelial nitric oxide synthase function has been found in resected tissue from infants with NEC (Nowicki et al, J. Pediatr. 150(1):40 (2007)). Since disruptions in the balance of active intestinal vasoconstriction and vasodilation, mediated in part by lower nitric oxide bioavailability, have been implicated in NEC pathogenesis (Nankervis et al, Semin. Perinatol. 32(2): 83 (2008)), the increased oral NR activity observed at 29 weeks may have physiological significance in providing an additional pathway for NO generation at this crucial time in development. Our study was not powered to observe associations between NR activity and NEC. None of the patients in this study developed NEC, and all demonstrated increased NR activity at 29 weeks leading us to hypothesize that a failure to increase oral NR activity at this time may be associated and/or contribute to NEC development.

[0097] Considering this increase in NR activity was even more pronounced in infants receiving maternal breast milk compared to formula fed infants, this rise in NR activity is of particular interest as NEC occurs significantly more frequently in formula fed infants compared to human milk fed infants (Sisk et al, J. Perinatol. 27(7):428 (2007); Meinzen- Derr et al, J. Perinatol. 29(1):57 (2009); Lucas et al, Lancet 336(8730): 1519 (1990)). The mechanism by which infants fed human milk are protected from necrotizing enterocolitis are multifactorial and include components including nitrate, human milk oligosaccharides, and lactoferrin. Some have reported higher amounts of nitrate in human and mouse milk compared to formula (Yazji et al, Proc. Natl. Acad. Sci. USA 110(23):9451 (2013)), however, the amount of nutritional nitrate and/or nitrite are likely variable based on whether milk is fresh, frozen, mature, or colostrum (Jones et al, J. Parenter. Enteral Nutr. 38(7):856 (2014)). Upregulation of eNOS via certain oligosaccharides found in human milk has also been a recent reported means by which gastrointestinal perfusion is restored in a NEC mouse model (Good et al, Br. J. Nutr. 116(7): 1175 (2016)). The microbial contribution of maternal human milk compared to other forms of nutrition such as donor breast milk (which requires pasteurization), formula, and parenteral forms of nutrition may be yet another benefit. These microbiota are of particular importance for NR activity, as it is microbial production of NR allowing bioactivation of nutritional and salivary nitrate.

[0098] The relative increase in NR activity occurring around 29 weeks PMA may also have physiologic implications for intestinal growth, handling of enteral nutrition, and potentially gastrointestinal pathology. The gastrointestinal tract of the preterm infant has immature motility and an incomplete ability to enzymatically digest and absorb food substrates

(Commare et al, Nutr. Clin. Pract. 22(2): 159 (2007)). Small feedings initiated after birth promote intestinal maturation after which feedings are slowly advanced to balance maturational benefit with concerns for inciting pathological changes such as necrotizing enterocolitis. Increased mesenteric blood flow has been associated with better feeding tolerance presumably due to better vascular support of this increased enteral milk burden (Fang et al, Arch. Dis. Child Fetal Neonatal Ed. 85(1):F42 (2001)). The role of NO in mesenteric blood flow and preventing ischemic injury in NEC is supported by pre-clinical and clinical studies previously cited.

[0099] The relationship between NR derived NO and development of BPD has not been previously described in humans despite a growing body of evidence suggesting its possible role as a therapeutic target (Koch et al, Free Radic. Biol. Med. 105:48 (2017)). In this cohort of ELGANs, NR activity was significantly lower in infants that developed BPD compared to those infants that did not develop BPD (FIG. 5). The development of BPD in preterm infants is the result of impaired development of pulmonary microvasculature due to many factors including prenatal environment, genetic predisposition, and postnatal exposures (Mourani et al., Clin. Perinatol. 2015. 42(4):839 (2015)). Inhaled NO has the largest amount of safety data and is approved for use in term infants with persistent pulmonary hypertension of the newborn. Nitric oxide has been well described as a mediator of pulmonary vascular homeostasis. NO leads to smooth muscle relaxation in pulmonary vasculature via activation of cyclic guanosine monophosphate. In pulmonary hypertension there is an incomplete response to signals normally responsible for vasodilatation as well as reduced NO signaling (Tonelli et al, Pulm. Circ. 3(1):20 (2013)). In eNOS knockout mice, restoration of the gene through an adenoviral vector leads to improved pulmonary arterial pressure (Champion et al, Proc. Natl. Acad. Sci. USA 99(20): 13248 (2002)) and exposure to L-arginine has also been noted to cause vasodilatation in the setting of pulmonary hypertension in a rat model (Goret et al., Eur. J. Pharmacol. 581(1-2): 148 (2008)).

[0100] The oral microbiota of the extremely preterm infant has not been well described.

Our data show that oral bacteria are present from birth. While it is broadly assumed that colonization with commensal bacteria occurs after birth, recent studies show that organs systems including the lung contain commensal bacteria at birth (Lai et al, Sci. Rep. 6:31023 (2016)). To better characterize the oral microbiome in extremely premature infants, and to gain insights into potential mechanisms and associations underlying NR activity changes, we performed microbiome analysis. The most abundant phyla detected were similar to the most abundant bacteria in the adult distal tongue oral microbiome including Firmicutes,

Proteobacteria, and Bacteroides (Ahn et al, PLoS One 6(7):e22788 (2011)).

[0101] In this cohort of extremely preterm infants, the oral microbiota of the tongue dorsum demonstrated that NR reducing bacteria are present at birth, but increase in abundance with infants’ post-menstrual age. The presence of nitrate reducing species from birth has also been reported in moderately preterm infants (Kanady et al, Nitric Oxide 27(4): 193 (2012)). The specific NR producing bacteria identified are consistent with those previously reported (Doel et al, Eur. J. Oral Sci. 113(1): 14 (2005)), including Veillonella, Actinomyces, Rothia, and Staphylococcus. Although bacteria capable of nitrate reduction do increase in abundance at later PMAs, there was not a discrete increase at the time of peak NR activity observed at 29 weeks PMA. Those bacteria that were statistically more abundant, Rothia and Veillonella , were the same bacteria found to be positively correlated with NR activity based on Spearman correlation analysis.

[0102] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method of diagnosing bronchopulmonary dysplasia (BPD) or determining the risk of developing BPD in an infant that is born at a gestational age of about 28 weeks or less, comprising:

measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA; and

diagnosing the infant as having BPD or determining the infant to be at risk of developing BPD when no spike ( e.g ., no increase) in NR activity is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).

2. A method of diagnosing pulmonary hypertension (PH) or determining the risk of developing PH in an infant that is a gestational age of about 28 weeks or less, comprising: measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA; and

diagnosing the infant as having PH or determining the infant to be at risk of developing PH when no spike in NR activity is observed in the sample taken from the infant at about 29 weeks PMA.

3. A method of diagnosing necrotizing enterocolitis (NEC) or determining the risk of developing NEC in an infant that is a gestational age of about 28 weeks or less, comprising: measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken at birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having NEC or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA.

4. A method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising:

measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA;

diagnosing the infant to have bronchopulmonary dysplasia (BPD) or determining the infant to be at risk of developing BPD when no spike ( e.g ., no increase) in NR activity is observed at about 29 weeks PMA; and

guiding the care of the infant that is diagnosed to be at risk of developing BPD by providing one or more suitable therapeutic and/or prophylactic treatments, such as:

(a) administering oxygen and/or nitric oxide,

(b) administering a pulmonary vasodilator,

(c) administering a composition comprising a bronchodilator, a steroid, and/or a diuretic,

(d) administering a composition comprising at least one probiotic bacterial species/strain, and/or

(e) prolongation of pharmacologic exposure to caffeine.

5. A method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising:

measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA;

diagnosing the infant to have pulmonary hypertension (PH) or determining the infant to be at risk of developing PH when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed to be at risk of developing PH by providing one or more suitable therapeutic and/or prophylactic treatments, such as:

(a) administering nitric oxide and/or oxygen,

(b) administering a composition comprising at least one phosphodiesterase inhibitor,

(c) administering a composition comprising at least one endothelin receptor antagonist,

(d) administering a composition comprising at least one NO precursor, and/or

(e) administering a composition comprising at least one prostacyclin.

6. A method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising:

measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional samples is taken at about 29 weeks PMA;

diagnosing the infant to have necrotizing enterocolitis (NEC) or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA; and

guiding the care of the infant that is diagnosed to be at risk of developing NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as:

(a) replacing oral feeding with parenteral feeding,

(b) removing air and fluid from the stomach and intestine,

(c) administering a composition comprising intravenous fluids,

(d) administering a composition comprising at least one antibiotic,

(e) administering a composition comprising at least one probiotic bacterial species/strain, and/or

(f) administering a composition comprising L-arginine.

7. The method of any one of the preceding claims, wherein the oral microbiome sample is taken from tongue of the infant.

8. The method of any one of the preceding claims, wherein the oral microbiome sample is taken from the posterior dorsum of the tongue of the infant.

9. The method of any one of the preceding claims, wherein the first oral microbiome sample is taken at about 96 hour or less after birth, optionally between about 1 minute to about 72 hours after birth.

10. The method of any one of the preceding claims, wherein the first oral microbiome sample is taken at about 24 hours to about 72 hours after birth, optionally at about 45 hours to about 70 hours after birth.

11. The method of any one of the preceding claims, wherein the at least two additional oral microbiome samples are two samples, a second sample taken at about 29 weeks PMA and a third sample taken at about 30 weeks PMA, at about 31 weeks PMA, at about 32 weeks PMA, at about 33 weeks PMA, or at about 34 weeks PMA.

12. The method of any one of claims 1 to 10, wherein the at least two additional oral microbiome samples are taken at weekly intervals following the first oral microbiome sample taken after birth until the infant reaches about 30 to 34 weeks PMA.

13. The method of any one of claims 1 to 10, wherein the at least two additional oral microbiome samples are taken periodically following the first oral microbiome sample taken after birth about every 2 days to about every 14 days.

14. The method of any of the preceding claims wherein the at least two additional oral microbiome samples are taken post-feeding.

15. The method of claim 14, wherein the at least two additional oral microbiome samples are taken about 2 hours post-feeding.

16. The method of any one of the preceding claims, further comprising culturing the oral microbiome samples for about 5 min to about 36 hours prior to determining (measuring) the NR activity.

17. The method of claim 4 or claim 6, wherein the at least one probiotic bacterial strain/species is a species/strain of the genus Actinomyces, Rothia, Veillonella, Lactobacillus, Hemophilus, Staphylococcus , Streptoccocus, Enter occocus and/or Prevotella.

18. The method of claim 4 or claim 6, wherein the at least one probiotic bacterial strain/species is a species/strain of the genus Actinomyces, Rothia and/or Veillonella.

19. A kit for assessing the risk of developing bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), and/or necrotizing enterocolitis (NEC) in an infant that is born at a gestational age of about 28 weeks or less, comprising one or more reagents for measuring nitrate reductase (NR) activity in the cultured samples, with optional instructions for the use thereof.

20. A kit for microbial analysis of a microbiome sample taken from an infant that is bom at a gestational age of about 28 weeks or less, comprising one or more reagents for identifying the microbes present in the microbiome sample, with optional instructions for the use thereof.

21. The kit of claim 19 or claim 20, further comprising three or more implements for taking oral microbiome samples from an infant and/or one or more reagents for culturing the oral microbiome samples.