SECTION B

Lung Injury—

Bronchopulmonary

Dysplasia

6

Prenatal and Postnatal Microbial Colonization and Respiratory

Outcome in Preterm Infants

Rose Marie Viscardi, MD

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Introduction

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Antenatal Infection and Pulmonary Outcomes

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Postnatal Microbial Colonization and Adverse Pulmonary Outcomes

Introduction

Bronchopulmonary dysplasia (BPD) was first described more than 40 years ago as a progression of characteristic chest radiographic findings that correlated with patho- logic changes consisting of acute and chronic lung inflammation, fibrosis, and bron- chial smooth muscle hypertrophy in premature, ventilator-dependent infants.^1,2 Although BPD remains one of the major morbidities of preterm birth, improvements in perinatal care such as antenatal steroids, exogenous surfactant, lung-protective ventilator strategies, and alternatives to mechanical ventilation have limited the disease to the most immature infants.³ Compared with the lung histology observed in the ventilated preterm lung during the pre–exogenous surfactant era, the “new” BPD is characterized by more uniform inflation, fewer but larger alveoli, and less fulminant, but persistent inflammation.⁴ Currently the incidence of “new” BPD is 30% in infants born at or before 28 weeks of gestation, but only 3% in infants born after 28 weeks.⁵ Long-term sequelae include prolonged dependence on supplemental oxygen, reactive airway disease, risk for pulmonary infections, and neurodevelopmental delays.

There is accumulating evidence that the central event in BPD pathogenesis is the interruption of normal developmental signaling during early stages of lung development by lung injury and subsequent dysregulated inflammatory response, a complex process that is often initiated in utero by intrauterine infection and aug- mented postnatally by exposure to hyperoxia, volutrauma, and post-natal infection.³ The objectives of this chapter are to describe (1) the consequences of fetal lung exposure to infection/inflammation in utero—and experimental evidence that pro- vides insight into potential mechanisms for how this exposure alters lung develop- mental signaling—and of interactions with postnatal injurious stimuli; (2) the effects of postnatal infections in the preterm lung such as ventilator-associated pneumonia that may exacerbate lung injury, prolong hospitalization, and increase mortality; and (3) potential effects of these perinatally acquired infections on long-term pulmonary outcomes. The discussion focuses on the epidemiologic and experimental evidence that the genital mycoplasma species Ureaplasma parvum and Ureaplasma urealyticum contribute to neonatal lung injury and may affect long-term pulmonary outcomes in infants whose mothers were infected.

Antenatal Infection and Pulmonary Outcomes

Epidemiologic studies indicate that intrauterine infection is the leading cause of very early preterm birth.⁶ The frequency of positive culture results in amniotic fluid and placental tissues along with histologic and biochemical evidence of intrauterine

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inflammation among women with spontaneous preterm labor and premature preterm rupture of the membranes at less than 30 weeks of gestation is 70% to 80% and is inversely related to gestational age (Fig. 6-1).^5,7 The organisms most commonly isolated from amniotic fluid⁸ and placentas^9,10 are the genital mycoplasmas U.

parvum, U. urealyticum, and Mycoplasma hominis, and those less frequently isolated are gram-positive organisms (Streptococcus spp., Staphylococcus spp.), gram-negative organisms (Escherichia. coli, Pseudomonas spp.), anaerobes (e.g. Bacteroides spp.), and organisms associated with bacterial vaginosis. Analysis of amniotic fluid from women

Figure 6-1 Distribution of maternal and fetal stages of histologic chorioamnionitis by gesta- tional age. A, Maternal stages of histological chorioamnionitis are as follows: stage 1: poly- morphonuclear neutrophils/leukocytes (PMNs) in subchorionic plate fibrin and/or membranous chorionic trophoblast layer (gray); stage 2: PMNs in chorionic plate or chorionic connective tissue and/or amnion (pink); and stage 3: necrotizing chorioamnionitis with degenerated neu- trophils, thickened eosinophilic membrane, and amnionic epithelial degeneration (red).

B, Fetal vasculitis stages are as follows: stage 1: PMNs in the wall of chorionic plate vessels or umbilical vein (gray); stage 2: PMNS in one or both umbilical arteries (pink); and stage 3:

PMNs in concentric band(s) around one or more umbilical vessels accompanied by cellular debris, or eosinophilic precipitate, and necrotizing funisitis (red).¹⁶ (From Viscardi RM, Muhu- muza CK, Rodriguez A, et al. Inflammatory markers in intrauterine and fetal blood and CSF compartments are associated with adverse pulmonary and neurologic outcomes in preterm infants. Pediatr Res. 2004;55:1009-1017.)

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6 with preterm labor and intact membranes using advanced molecular techniques combined with culture demonstrated a greater prevalence (15%) and diversity (18 taxa) of microbes than culture or species-specific polymerase chain reaction (PCR) studies.⁸ The microbes included a related group of fastidious bacteria, such as Sneathia sanguinegens, Leptotrichia amnionii, and an unassigned, uncultivated, and previously uncharacterized bacterium. Using culture-independent approaches such as analysis of the bacterial 16S ribosomal RNA gene^11,12 and denaturing gel electro- phoresis (DGGE)¹³ to analyze gastric and tracheal aspirates (TAs) to identify perina- tally acquired microbes in preterm infants, other studies have identified at least 22 microbial species other than the Mycoplasma species, confirming the diversity of organisms potentially involved in intrauterine infection and it sequelae.

Histologic Chorioamnionitis and Pulmonary Outcomes

Chorioamnionitis has been defined by histologic criteria (presence of polymorpho- nuclear cells in choriodecidual space, fetal membranes, and/or cord), microbiologic criteria (positive results of culture or molecular detection methods), and/or bio- chemical criteria (elevated amniotic fluid cytokine and chemokine levels) as evidence of infection/inflammation in the intrauterine compartment.¹⁴ Clinical chorioamnio- nitis defined by maternal fever, maternal and fetal tachycardia, uterine tenderness, foul-smelling vaginal discharge, and leukocytosis is present in less than 40% of cases of histologic chorioamnionitis,⁵ indicating that the majority of intrauterine infections are subclinical.¹⁵

Standardization of placental pathology review has provided a framework for characterizing the stage or extent of polymorphonuclear cell infiltration and grading for severity.¹⁶ In response to microbial invasion, maternal polymorphonuclear cells progress from infiltration of the subchorionic plate fibrin and/or membranous cho- rionic trophoblast layer (stage 1) to infiltration of the chorionic plate or chorionic connective tissue and/or amnion (stage 2) to necrotizing chorioamnionitis with degenerated neutrophils, thickened eosinophilic membrane, and amnionic epithelial degeneration (stage 3). A grade of inflammation is assigned to describe the number of neutrophils or presence of microabcesses.¹⁶ It has been proposed that the presence of advanced maternal stage of necrotizing amnionitis is indicative of prolonged infection/inflammation.^16-18

The fetal inflammatory response syndrome (FIRS) has been defined biochemi- cally by elevated umbilical cord concentrations of cytokines (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor-α [TNFα]).^5,19 However, this response also involves a broader inflammatory response, including upregulation of chemokines (IL-8, macrophage inflammatory protein-1β [MIP-1β], and RANTES [regulated upon acti- vation, normal T cell expressed and secreted]), adhesion molecules (intracellular adhesion molecule-1 [ICAM-1], ICAM-3, and E-selectin), matrix metalloproteinases (MMP-1 and MMP-9), an angiogenic factor (vascular endothelial growth factor [VEGF]), and an acute-phase protein (C-reactive protein [CRP]), in venous blood in the first few days of life.¹⁸ Histologically, the response is defined by fetal vasculitis/

funisitis characterized by polymorphonuclear infiltration of the chorionic vessels or umbilical cord.¹⁴ Stages of fetal vasculitis describe the progression from neutrophils in the wall of chorionic plate vessels or umbilical vein (stage 1) to one or both umbilical arteries (stage 2) to concentric bands around one or more umbilical vessel accompanied by cellular debris, or eosinophilic precipitate (stage 3, necrotizing funisitis).¹⁶ Intensity of the inflammation is graded according to the number of neutrophils present.

Multiple epidemiologic studies have addressed the association of histologic chorioamnionitis with and without cord involvement with gestation-independent effects on neonatal pulmonary outcomes (summarized in a 2010 review¹⁴). In 1996,Watterberg and colleagues²⁰ observed that histologic chorioamnionitis was associated with a reduced risk for respiratory distress syndrome (RDS), but an increased risk for bronchopulmonary dysplasia (BPD) in a small cohort of mechani- cally ventilated preterm infants with birth weight less than 2000 g who were not exposed to antenatal steroids or exogenous surfactant. Subsequent studies over the

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past 15 years have found an association of chorioamnionitis with a reduced effect^21-24 or no effect^25-27 on RDS risk and an increased effect,^5,25,27 a decreased effect,²⁸ or no effect22,26,29,30 on BPD risk.

Limitations of many of the studies include single-center cohorts with varying gestational age ranges, racial/ethnic distributions, and inclusion criteria, and sample sizes inadequately powered to analyze possible confounding or risk modification of gestational age and other important variables. For instance, Redline and associates²⁹ observed in a retrospective study that histologic chorioamnionitis tended to be higher in white infants and lower in African-American infants with BPD at 36 weeks postmenstrual age, suggesting that racial differences in response to chorioamnionitis may explain, in part, differences in study outcomes. Interpretation of these studies is also complicated by the potential selection bias of center-specific placental review criteria and nonstandardized criteria for chorioamnionitis diagnosis. A major chal- lenge is the lack of a true “normal” comparison group, because many preterm pla- centas without chorioamnionitis have other lesions that may affect outcomes.¹⁴

It has been proposed that the timing, duration of exposure, and severity of chorioamnionitis affects neonatal pulmonary outcomes. In a cohort of 276 preterm infants born before 33 weeks of gestation at our institution, histologic chorioamnio- nitis was associated with 3.6-fold higher risk for BPD in infants born at 28 weeks gestation or earlier, but not in infants of 29 to 32 weeks gestation. Maternal stage of chorioamnionitis was significantly correlated with BPD severity⁵ as defined by National Institutes of Health (NIH) consensus criteria.³ Forty percent of the infants in whom moderate or severe BPD developed were exposed to longstanding or nec- rotizing amnionitis (stage 3) compared with 34% of infants in whom mild BPD developed and 19% infants without BPD (Fig. 6-2). Fetal vasculitis was not associ- ated with BPD in this study. Subacute chorioamnionitis, a pathologic diagnosis distinct from acute chorioamnionitis, is characterized by mixed degenerative poly- morphonuclear neutrophils/leukocytes (PMNs) and mononuclear cells in the chori- onic plate with greatest severity involving the amnion.³¹ In a study comparing 90 singleton placentas with stage 3 acute and subacute chorioamnionitis at 23 to32 weeks of gestation with gestational age– and birth weight–matched controls without chorioamnionitis, the presence of amniotic necrosis was independently associated

Figure 6-2 Relationship of severity of bronchopulmonary dysplasia (BPD) and maternal stage of chorioamnionitis. Percentages of study infants with mild, moderate-to-severe, and no BPD according to maternal involvement with chorioamnionitis by stages. Maternal stages are as described in Figure 6-1. BPD severity is defined according to National Institutes of Health (NIH) criteria.³(From Viscardi RM, Muhumuza CK, Rodriguez A, et al. Inflammatory markers in intrauterine and fetal blood and CSF compartments are associated with adverse pulmonary and neurologic outcomes in preterm infants. Pediatr Res. 2004;55:1009-1017.)

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6 with BPD,³¹ suggesting that longstanding or chronic inflammation contributes to lung injury in utero.

Although histologic chorioamnionitis may decrease the risk for RDS because of maturational effects in the fetal lung, it may also contribute to an increase in sus- ceptibility to augmented postnatal lung injury. In a case-control study, Van Marter and coworkers³² observed that histologic chorioamnionitis was associated with a decreased rate of BPD in infants undergoing ventilation for less than 1 week but an increased rate of BPD in infants undergoing prolonged ventilation or with postnatal sepsis. Furthermore, infants exposed to histologic chorioamnionitis with fetal vas- culitis in another study had impaired responses to exogenous administration of surfactant, which contributed to both prolonged mechanical ventilation after surfac- tant administration and the development of BPD.³³ Peripheral blood leukocytosis (leukocyte count > 30,000/mm³) in the first 2 days of life in chorioamnionitis- exposed infants has been found to increase the risk for BPD 4.6-fold but to decrease the risk for death.³⁴ Antenatal exposure to histologic chorioamnionitis may be an important response modifier of other postnatal interventions. Although there was no overall benefit of low-dose prophylactic hydrocortisone therapy in preterm infants in a randomized trial, the subgroup of chorioamnionitis-exposed infants in the treatment arm experienced significantly decreased mortality and improved sur- vival without BPD in comparison with placebo-treated infants.³⁵

Role of Infection-Mediated Cytokine Signaling in Bronchopulmonary Dysplasia

Clinical studies of biomarkers in intrauterine and pulmonary compartments and experimental animal and in vitro studies have demonstrated that the host inflam- matory response to intrauterine microbial invasion is the link between antenatal infection and altered lung development. Amniotic fluid concentrations of proinflam- matory cytokines IL-1β, IL-6, TNFα, and IL-8 were higher in pregnancies produc- ing infants in whom BPD developed than in pregnancies producing infants without BPD.³⁶ The fetal inflammatory response characterized by placental vasculitis and/or elevated cord serum IL-6 concentrations was an independent risk factor for BPD.^19,37 Increased concentrations of IL-6 and IL-1β have been detected in TAs of preterm infants on the first day of life in association with prolonged rupture of the mem- branes³⁸ and histologic chorioamnionitis,^20,39 respectively. A series of longitudinal studies comparing the temporal changes in inflammatory mediators and their inhib- itors in TAs from preterm infants with and without lung disease have shown that there is an imbalance in the levels of pro- and anti-inflammatory cytokines during the first week of life in infants in whom BPD develops.^40-43 The increase in expres- sion of pulmonary proinflammatory cytokines, chemokines, adhesion molecules, proteases, and angiogenic factors in concert with a decreased capacity to downregu- late this response in infants who experience BPD suggests that persistent endoge- nous generation of these factors might contribute to chronic lung injury and inflammation. Bose and coworkers⁴⁴ summarized the contribution of different aspects of the inflammatory response to BPD in an extensive review of TA biomarker studies.

Chronic Inflammation in the Immature Lung Alters Developmental Signaling and Fibrosis

Transforming growth factor-β1 (TGF-β1) is involved in lung morphogenesis, repair of lung injury, airway remodeling, lung fibrosis, and BPD.⁴⁵ TGF-β was detected at sites of lung injury in association with myofibroblast proliferation in lungs of infants dying with RDS, implicating TGF-β in the preterm lung response to injury.⁴⁶ TGF-β1 is elevated in TAs of infants who progress to BPD.⁴⁷ Overexpression of TGF-β1 in the lungs of newborn transgenic mice produces a phenotype similar to human BPD, with arrest of lung sacculation, epithelial differentiation, and vascular development.^48-50 TGF-β1–expressing adenoviral vectors were found to have similar effects in the newborn rodent lung.^45,51 Intra-amniotic endotoxin inoculation in pregnant sheep

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induced fetal lung TGF-β1 messenger RNA (mRNA) and protein expression and phosphorylation of protein Smad2, indicating TGF-β1 signaling.⁵² These studies show that excessive TGF-β1 signaling during lung development contributes to the arrest of both alveolarization and fibrosis, both hallmarks of BPD.

In transgenic mice, overexpression of IL-1β, TNFα, IL,-6 or IL-11 was found to inhibit alveolarization, indicating that prolonged exposure of the preterm lung to a proinflammatory environment may contribute to abnormal alveolar septation.^3,53,54 Bry and coworkers⁵³ developed a bitransgenic mouse model in which the expression of mature human IL-1β is conditionally expressed in airway epithelial cells in the fetal and neonatal lung. In this model, IL-1β expression increased on E14.5, was maximal by E16.5 (pseudoglandular period), and decreased postnatally. Postnatal growth was impaired and mortality was higher in the IL-1β–expressing newborn mice. The lungs of these newborn mice demonstrated many features of the BPD phenotype, including disrupted alveolar septation and capillary development, and disordered deposition of α-smooth muscle actin and elastin in alveolar septa of distal air spaces.⁵³

The effects of prolonged exposure to proinflammatory cytokines on alveolariza- tion may be mediated by up-regulation of TGF-β1. Transient overexpression of TNFα⁵⁵ or IL-1β⁵⁶ in rat lung by adenoviral gene transfer produces lung fibrosis due to stimulation of TGF-β1, and induction of myofibroblasts. Absence of the β6 inte- grin subunit, an activator of TGF-β1, improves alveolar development in transgenic IL-1β–expressing newborn mice, implicating TGF-β signaling in the pathogenesis of BPD.⁵⁷ TNFα, IL-1β, and TGF-β1 are elevated in TAs of infants who progress to BPD.40,41,47,58,59

Taken collectively, these data indicate that prolonged exposure of the develop- ing lung to proinflammatory, profibrotic factors may contribute to BPD by disrupting normal developmental signaling. The next section reviews the human and experi- mental evidence that the low-virulence pathogens U. parvum and U. urealyticum contribute to preterm birth and lung injury, and augment a dysregulated inflamma- tory response by stimulating the proinflammatory, profibrotic signaling pathways.

Role of Genital Mycoplasmas in Intrauterine Infection and Neonatal Lung Injury

The Mollicute class comprises at least 200 species; humans are the primary hosts for at least 17 of them. The organisms reside primarily in association with the mucosal surfaces of the urogenital and respiratory tracts. The four urogenital species belong to two different phylogenetic groups within the Mollicute class. M. hominis belongs to the Hominis group, whereas the Ureaplasma spp. and Mycoplasma genitalium belong to the Pneumoniae group.⁶⁰ Ureaplasma consists of 2 species and 14 serovars.

U. parvum contains serovars 1, 3, 6, and 14, and U. urealyticum contains the remain- ing serovars.⁶¹ The Mycoplasma species are the smallest self-replicating, free-living organisms. The M. genitalium genome is the smallest, with 580 kilo–base pairs (kbp), the U. parvum serovar 3 genome is the second smallest, with 751 kbp, and the newly sequenced M. hominis genome is 665 kbp.^60,62 Owing to their small genome size, the genital Mycoplasma species have limited biosynthetic capacities, requiring a parasitic relationship with a host. These species all lack cell walls and share 247 core coding sequences⁶⁰ but have distinct energy-generating pathways and pathogenic roles in human disease. M. genitalium utilizes glycolysis, but the Ureaplasma spp. and M.

hominis hydrolyze urea and arginine, respectively, to generate adenosine triphosphate (ATP).^60,61 M. genitalium is associated with male urethritis and cervicitis but is not known to be a pathogen in infants. M. hominis is associated with pyelonephritis, bacterial vaginosis, pelvic inflammatory disease, and postpartum endometritis but has not been consistently associated with histologic chorioamnionitis or BPD.^13,61 It is much less commonly isolated as a single organism from amniotic fluid, chorio- amnion, and neonatal tracheal and gastric aspirates than the Ureaplasma species.^13,61 The following section focuses on the evidence implicating the Ureaplasma species in neonatal lung disease.

6 Are There Ureaplasma Species- or Serovar-Specific

Virulence Factors?

U. parvum is more commonly isolated from clinical vaginal,⁶³ amniotic fluid,⁶⁴ and infant respiratory specimens^12,65,66 and is the predominant species detected by PCR in newborn serum and/or cerebrospinal fluid (CSF) samples.⁶⁷ It has been proposed that some serovars have greater association with adverse pregnancy outcomes than others.^63,65,68 Abele-Horn and colleagues reported a higher rate of BPD in U. urealyti- cum respiratory tract colonized infants. In contrast, Katz and associates^63,69 observed no difference in prevalence of either species detected by PCR between infants with and without BPD. In a prospective study of respiratory secretions in infants born at less than 33 weeks of gestation, the distribution of Ureaplasma species and serovars was determined by real-time PCR using species- and serovar-specific primers/

probes.^66,70 U. parvum was much more common (63%) than U. urealyticum (33%).

Serovars 3 and 6 alone and in combination accounted for 96% of U. parvum isolates.

U. urealyticum isolates were commonly a mixture of multiple serovars, serovar 11 either alone or combined with other serovars (59%) being the most common serovar.

No individual species/serovars or serovar mixtures were associated with moderate to severe BPD. This finding supports the contention that Ureaplasma virulence is independent of species and serovar with regard to neonatal lung disease which nevertheless must be confirmed.

Previously proposed ureaplasmal virulence factors include immunoglobulin A (IgA) protease, urease, phospholipases A and C, and production of hydrogen per- oxide.⁶² These factors may allow the organism to evade mucosal immune defenses by degrading IgA and injuring mucosal cells through the local generation of ammonia, membrane phospholipid degradation and prostaglandin synthesis, and membrane peroxidation, respectively. Although functionally active IgA protease and phospho- lipase A and C have been found in Ureaplasma spp., the genes that code for these proteins have not been identified in the U. parvum serovar 3 genome.⁶² The urea- plasmal enzymes may have unique gene sequences compared with analogous genes in other species.

The ureaplasmal MB antigen, which contains both serovar-specific and cross- reactive epitopes, is the predominant antigen recognized during ureaplasmal infec- tions in humans. It exhibits highly variable size in vitro,⁷¹ in clinical isolates in vivo,⁷² and in an experimental ovine intra-amniotic infection model,⁷³ suggesting that antigen size variation may be another mechanism through which the organism evades host defenses.⁶¹ Garcia-Castillo and coworkers⁷⁴ demonstrated that Urea- plasma isolates from patients with urethritis or chronic prostatitis formed biofilms in vitro, suggesting another means to evade the host immune response and alter antibiotic susceptibility.

Genetic diversity among ten clinical U. parvum isolates obtained from vaginal swabs was analyzed in one study by comparative genomic DNA hybridization to a DNA macroarray.⁷⁵ Although at least 538 genes (92%) were common to all the iso- lates, strain-specific genes mostly with unknown function represented 8% of the genome. One hypervariable plasticity region identified in this analysis had genetic features consistent with a putative pathogenicity island. Further genetic studies of the ureaplasmal genome are likely to identify virulence factors that may be novel therapeutic targets.

Potential Role of Ureaplasma Species in Preterm Birth and Intrauterine Inflammation

Because Ureaplasma is a commensal in the adult female genital tract, it has been regarded as being of low virulence. However, it has been associated with multiple obstetrical complications, including infertility, stillbirth, and preterm delivery.⁶¹ Ureaplasma spp. are the organisms most commonly isolated from amniotic fluid obtained from women who present with preterm-onset labor (POL) with intact membranes,^76,77 preterm premature rupture of membranes (pPROM),⁷⁸ and short cervix associated with microbial invasion of the amniotic cavity,⁷⁹ as well as from infected placentas.⁷⁸ The prevalence of infected amniotic fluid with cultivated