Pathophysiology of Neonatal Acute Bacterial Meningitis

Bacterial Meningitis

Meningitis is a life-threatening infection of the central nervous system (CNS) that affects the pia mater, the arachnoid, and the subarachnoid space. The microorganisms can activate the host immune response, and both inflammatory mediators and microorganisms can break down the blood-brain barrier, allowing the influx of fluid and solutes into the brain, triggering the brain edema. This infection is a significant cause of morbidity and mortality worldwide, primarily in neonates, in whom it is associated with long-term neurologic sequelae in up to 50% of the survivors. In Europe and North America, Streptococcus agalactiae (group B Streptococcus ) is the most common etiologic agent of neonatal meningitis, followed by Escherichia coli . The remaining agents include Listeria monocytogenes, Streptococcus pneumoniae , and Neisseria meningitidis .

The multiplication of bacteria within the subarachnoid space occurs concomitantly with the release of bacterial products, such as peptidoglycan, lipoteichoic acid (a constituent of the cell wall of gram-positive bacteria; e.g., S. agalactiae , S. pneumoniae , and Listeria spp.), lipopolysaccharide (LPS, a constituent of the cell wall of gram-negative bacteria; e.g., E. coli ), lipooligosaccharide (a constituent of the cell wall of N. meningitidis ), flagellum (motility, e.g. , E. coli and Listeria spp . ), pilus (mediating the attachment of the bacteria on cells, e.g., S. agalactiae , E. coli, and N. meningitidis ), DNA, and cell wall fragments. These bacterial compounds are all denoted as pathogen-associated molecular patterns (PAMPs). ^, These PAMPs are recognized by pattern-recognition receptors (PRRs) and non-PRRs, which are essential constituents of the immune system. ^, PRRs include several families, including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), and intracellular DNA-sensing molecules. The non-PRRs receptors include receptors for advanced glycation end products (RAGEs), triggering receptors expressed on myeloid cells (TREMs), and G-protein-coupled receptors (GPCRs). The sensing of PAMPs by immune receptors triggers a cascade of signaling pathways that activate several transcription factors and promote the production of proinflammatory mediators. The inflammatory mediators include cytokines, chemokines, and antimicrobial peptides, which are necessary to eliminate the invading pathogens.

Endogenous molecules that are released from stressed or injured cells during meningitis infection also trigger the innate immune system. These molecules are recognized by PRRs and non-PRRs and are known as damage-associated molecular patterns (DAMPs) . ^, Among the DAMPs identified, there are cellular proteins and nucleic acid–related molecules, such as the heat shock protein (HSP), the high mobility group box-1 (HMGB-1) protein, members of the S-100 family, cytochrome-c, nucleic acids, and adenosine triphosphate. ^{, ,} Thus the development of brain damage during bacterial meningitis results from the combined effects of both the pathogen and the innate immune response. The recognition of PAMPs and DAMPs by immune receptors result in amplification of the proinflammatory response and can lead to long-term cognitive impairment in survivors of neonatal meningitis. ^,

Colonization

S. agalactiae is a gram-positive diplococcus, and it is categorized according to capsular antigens such as Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX. ^, Although all of the serotypes can cause infections, in North America, serotypes Ia, Ib, II, III, and V are most frequently associated with invasive disease. However, in other studies, the most frequent serotype associated with invasive disease was type III, demonstrating that the dominant serotypes vary regionally and differ by invasive and colonizing isolates. This microorganism is considered part of the healthy microflora in the vagina in up to 10% to 30% of healthy women, and it is a risk factor for early-onset meningitis due to S. agalactiae . ^, The adherence of the microorganism to epithelial cells has shown to be an essential factor in the colonization of the mucous membranes of both the human rectum and vagina. The pathogens can also attach to the placental membranes, respiratory tract epithelium, and blood-brain barrier endothelium.

The human placenta does not present a microbiome and also does not have evidence for the presence of bacteria in complicated and uncomplicated pregnancies. The neonatal infection occurs when the infant is exposed during the birth process to a large number of pathogens that can be transmitted through the vagina to ruptured amniotic membranes, or due to contact of the infant during passage through the birth canal. In addition, S. agalactiae can be aspirated into the fetal lungs and transferred haematogenously into the CNS. ^, The colonization of S. agalactiae is associated with their ability to bind to extracellular matrix proteins, predominantly to human fibrinogen. Fibrinogen is a glycoprotein present in the bloodstream and on the surface of the cells and tissues. S. agalactiae interactions with fibrinogen are linked with the expression of the cell wall–anchored LP X TG protein fibrinogen-binding protein-A (FbsA) and the secreted protein FbsB, which promote invasion of epithelial cells. The LP X TG glycoproteins including Srr1 and Srr2also contribute to fibrinogen binding. In addition, Srr1 and FbsC can mediate the microorganism invasion of brain vascular endothelial cells and the translocation through the blood-brain barrier. ^, Another critical pathogenic mechanism for S. agalactiae is the production of the β-hemolysin. This toxin is cytolytic for brain endothelial cells and causes a disruption of the blood-brain barrier. This inflammatory mechanism also induces the production of interleukin (IL)-8 and the neutrophil receptor intercellular adhesion molecule-1 (ICAM-1), thereby promoting neutrophil migration across microvascular endothelial cells, suggesting that this toxin is crucial to the invasion of S. agalactiae into the CNS.

E. coli colonizes the digestive tract of humans and animals, and it is the most common gram-negative microorganism that causes neonatal meningitis. In 80% of gram-negative neonatal meningitis cases, E. coli with K1 capsular polysaccharides are isolated in the cerebrospinal fluid (CSF). E. coli K1 colonizes the gastrointestinal mucosa and translocates from the lumen of the small intestine or colon into the systemic circulation before entering into the CNS across the blood-brain barrier. In an experimental meningitis model, the oral administration of E. coli K1 resulted in stable and persistent gastrointestinal colonization in newborn rats. Using the same experimental model in a different study, E. coli K1 colonized the gut and then crossed the gastrointestinal barrier. With the microorganism present in the blood circulation and brain tissue, the neonatal rats developed a lethal systemic infection.

L. monocytogenes is a facultative anaerobic, intracellular gram-positive bacillus that is motile by the use of its peritrichous flagella. This microorganism is a foodborne pathogen that can grow at low temperatures and cause meningitis, sepsis, and meningoencephalitis in neonates, pregnant women, and immunocompromised patients. Maternal bacteremia can lead to fetal intrauterine infection; however, L. monocytogenes can also gain access to the neonate via oral exposure during the passage through the birth canal. An elevated concentration of this pathogen was found in the lung and gastrointestinal tract, suggesting that this infection can also be acquired in utero via the inhalation and ingestion of infected amniotic fluid and via the hematogenous route cross the blood-brain barrier. In a preclinical model, 2 days after the oral inoculation with L. monocytogenes into pregnant guinea pigs, the pathogen was isolated from the placenta, fetal liver, and fetal brain.

S. pneumoniae is a gram-positive lancet-shaped diplococcus arranged in pairs. The human nasopharynx is the main reservoir of S. pneumoniae, which usually leads to asymptomatic colonization. This microorganism is transferred between people by coughing and sneezing. ^, Pneumococcus colonizes the human nasopharynx through the degradation of mucus by several enzymes, such as neuraminidase A, β-galactosidase A, β-acetylglucosaminidase, and neuraminidase B. Moreover, this microorganism is capable of expressing more than 90 serotypes based on differences in their capsular polysaccharides. The expression of a polysaccharide capsule is required for the full pathogenicity of S. pneumoniae because the capsule can repulse the sialic acid residues of mucus, thereby decreasing the probability of pneumococcal immobilization. Another virulence factor is a pore-forming toxin known shown as pneumolysin . This enzyme can induce pores in cholesterol-rich membranes and decrease cell ciliary beating to further activate the host immune response. ^, The pneumolysin can activate TLR 4 and NLRP3 (a PRR) and increase the proinflammatory response.

N. meningitidis is a gram-negative diplococcus that colonizes the nasopharynx of up to 35% of healthy individuals. ^, This pathogen presents 13 different capsular polysaccharide structures; however, only A, B, C, W-135, Y, and X cause the most life-threatening disease. This microorganism may be acquired through the inhalation of respiratory droplets. N. meningitidis colonizes the nonciliated mucosal epithelial cells of the upper respiratory tract, where it can enter cells briefly before migrating back to the apical surfaces of the cells for the propagation to a new host. The pathogen uses the polysaccharide capsules, pili, and outer membrane adhesion to develop an interface between the host and meningococcus. Thus, after the microorganism gains access to the bloodstream, it can multiply and disseminate into various tissues, thereby causing sepsis, purpura fulminans, and meningitis.

Central Nervous System Bacterial Invasions

Mechanisms Implicated in Microbial Traversal of Blood-Brain Barrier

The passage of microorganisms across the blood-brain barrier is a vital step in the development of meningitis. The pathogens can cross the blood-brain barrier via the following different mechanisms: transcellular traversal, paracellular traversal, or Trojan-horse (microorganism inside of phagocytes). ^, Transcellular traversal occurs when the microorganism penetrates the cells without any traces or intracellular tight-junction disruption. In paracellular traversal, the microorganism penetrates between cells with or without evidence of tight-junction disruption, and by Trojan-horse, the microorganism crosses endothelial cells transmigrating into phagocytes.

The microbial traversal of the blood-brain barrier occurs via an interaction between the microorganisms and host receptors. S. agalactiae binds to the microvascular endothelial cells via the laminin-binding protein (LmB), FbsA, pilus tip adhesion (Pil-A), and invasion protein regulator (IagA) through lipoteichoic acid anchoring. Pil-A binds to collagen, which promotes the S. agalactiae interaction with the α₂β₁ integrin. Experimental meningitis in Pil-A–deficient mutant mice exhibited delayed mortality, a decrease in leukocyte infiltration, and bacterial dissemination into the CNS. The FbsA binds with the Fn-integrin; the antifibronectin antibody that blocks fibronectin binding to integrins reduced the invasion of the wild-type S. agalactiae .

E. coli K1 interacts with CD48 on brain microvascular endothelial cells through a type 1 fimbrial adhesion (FimHa bacterial adhesin), an outer-membrane protein A (OmpA) through N -acetylglucosamine (GlcNAc) or glucose-regulated protein-96 (Gp96), and cytotoxic-necrotizing factor 1 (CNF1) through the laminin receptor (LR). CNF1 is a bacterial virulence factor that is primarily related to E. coli strains that cause meningitis. This toxin contributes to the E. coli K1 invasion of brain endothelial cells in vitro and the traversal of the blood-brain barrier in a neonatal experimental meningitis model. Furthermore, an isogenic mutant lacking CNF1 was less invasive in brain endothelial cells and less able to penetrate the brain in vivo. In vitro, a double-knockout mutant with deleted OmpA and CNF1 genes was less invasive in human brain microvascular endothelial cells.

L. monocytogenes can cause life-threatening meningitis, and during this process, the pathogen crosses the blood-brain barrier by paracellular, transcellular, and intracellular mechanisms or within infected phagocytes. ^, The intracellular L. monocytogenes escapes from the phagosome by disrupting the phagosome membrane secreting the phosphatidylinositol-specific phospholipase C (Pic)-A and PIcB phospholipases, and the toxin listeriolysin O. Ultimately, the bacteria are released and multiply in the cytoplasm and disseminate into adjacent cells. Another mechanism of invasion for this microorganism involves the functions of its surface proteins, internalin (Inl)-A and Inl-B. These proteins bind with the receptor for the globular head of the complement component C1q (gC1qR) or vascular endothelial (VE)-cadherin on endothelial cells. ^, The deletion of either or both of the proteins leads to decreased invasion, suggesting an interdependency of Inl-A and Inl-B during the invasion of choroid plexus epithelial cells.

S. pneumoniae adheres to the blood-brain barrier endothelium before causing meningitis. This adhesion occurs when the pneumococcal surface protein C (PspC) interacts with the laminin receptor or the polymeric immunoglobulin receptor (pIgR) on brain endothelial cells. ^, S. pneumoniae adhesion is associated with pIgR on brain endothelial cells, and blocking the pIgR reduces the pneumococcal adhesion on endothelial cells. Another interaction is between the cell-wall phosphorylcholine and the platelet-activating-factor receptor (PAFr) on endothelial cells. The PAFr knockout mice showed an impaired ability to support bacterial translocation from the blood to the brain. S. pneumoniae may penetrate the CSF intracellularly by disrupting tight intraepithelial junctions or by the transcellular mechanism. ^,

The N. meningitidis interaction involves numerous microbial structures and proteins, such as type IV pili, the CD147 receptor, and the β2-adrenoceptor (β2AR) on endothelial cells. CD147 is a receptor for the type IV pilus that facilitates the adhesion of meningococci on endothelial cells and in the brain. The pilin subunits PilE and PilIV activate β2AR, thereby promoting endothelial cell signaling and facilitating the passage of the microorganisms through the blood-brain barrier. ^, This interaction between meningococcal ligands and the cellular host receptor is essential for N. meningitidis adhesion to human endothelial cells. Interfering with this interaction could prevent meningococcus-induced vascular dysfunctions ( Fig. 168.1 ).

Recognition of Bacterial Infection by Innate Immune Sensors

Toll-Like Receptor-Sensing Bacteria

The innate immune system detects pathogens via several receptor families. TLRs recognize molecular motifs that are expressed by pathogens or endogenous ligands released by tissue insult. TLRs trigger the immune response during meningitis, with TLR signaling representing a pivotal role in innate immune function. The TLR receptors are organized into two broad categories: one group of receptors is expressed at the cell surface for extracellular ligand recognition (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10), and the other group is localized in intracellular endosomal compartments to recognize pathogen nucleic acids (TLR3, TLR7, TLR8, and TLR9) (see Fig. 168.1 ).

TLR2 recognizes bacterial compounds, such as lipoproteins, peptidoglycan, and lipoteichoic acid. ^, In vitro, L. mono-cytogenes cell wall components were recognized by TLR2 with the help of CD14 and TLR6 ; the lack of TLR2 was associated with a diminished bacterial clearing and impaired host resistance to S. agalactiae infection. LPS is the significant component of the outer membrane of gram-negative bacteria, such as E. coli , which is recognized by TLR4; however, HMGB1, HSP, hyaluronic acid, fibronectin, and fibrinogen are also recognized by TLR4. ^, In a preclinical study, the prestimulation of TLR4 increased the phagocytosis of E. coli strains by murine microglial cells. Similarly, brain abscess caused by E. coli in a twin pair of neonatal patients was associated with a TLR4 gene mutation. The important exotoxin pneumolysin produced by S. pneumoniae is recognized by TLR4 and confers resistance to pneumococcal infection. Gram-positive and gram-negative bacteria can also have a flagellin protein, which is a component of the flagella, and TLR5 recognizes this protein. TLR9 is a receptor for oligodeoxy nucleotides that contain unmethylated CpG motifs (CpG-ODN) ^, and recognizes bacterial cytosine-guanine (CpG)-containing DNA, mitochondrial DNA (mtDNA), and DAMPs.

TLR signaling is initiated by the dimerization and recruitment of adaptor proteins. TLR1, TLR2, and TLR6 signal via myeloid differentiation factor 88 (MyD88) and the Toll/IL-1 receptor (TIR)-containing adaptor protein (TIRAP). TLR3 recruits the TIR domain–containing adaptor protein inducing interferon-β (TRIF). TLR5, TLR7, and TLR9 recruit only the MyD88 adaptor. TLR4 can signal through four of the adaptor molecules. TLR4 uses the TIRAP to bind MyD88 and the TRIF-related adaptor molecule (TRAM) to bind TRIF to stimulate the production of proinflammatory mediators. ^, E. coli , S. pneumoniae, and N. meningitidis are sensed by TLR4, thereby initiating a potent immune response through the four adaptors. Based on this feature, N. meningitides , S. pneumoniae , and E. coli can rapidly lead to a fatal septic shock. ^, After the recruitment by TLRs, the MyD88 adaptor molecule associates with IL-1 receptor-associated kinase-4 (IRAK-4), which is a serine/threonine kinase that phosphorylates IRAK-1 and IRAK-2. Subsequently, IRAK interacts with the receptor-associated factor (TRAF) family and provides a link to the TAK1/TAB1/TAB2/TAB3 complex. TAK1 phosphorylates the IKK complex, which in turn phosphorylates IKQ, resulting in the release and nuclear translocation of the transcription factor, nuclear factor-κB (NF-κB). NF-κB is a transcriptional activator of various genes involved in the pathogenesis of meningitis, such as tumor necrosis factor-alpha (TNF-α), IL-1β–inducible nitric oxide synthase (iNOS), and ICAMs. ^, TAK1 activation is also associated with the mitogen-activated protein kinase (MAPK) pathway. In addition, MAPKK-3/6, MAPKK-4/7, and MEK-1/2 induce the activation of p38, c-jun N-terminal kinase (JNK), and extracellular signal-regulated (ERK-1/2), respectively. These cascades lead to the translocation of protein-1 (AP-1) to the nucleus, thus promoting the transcription of inflammatory cytokines ( Fig. 168.2 ).

Among patients with a MyD88 deficiency or an IRAK-4 deficiency, 68% presented with invasive pneumococcal disease, and 45% presented with meningitis. ^, IRAK-4 and MyD88 deficiencies predispose patients to recurrent life-threatening bacterial infections in infancy and early childhood, resulting in minimal clinical features despite invasive bacterial infection. ^, According to the aforementioned studies in humans, in experimental pneumococcal meningitis, MyD88-deficient mice presented with a decrease of pleocytosis, proinflammatory mediators, cytokines, and chemokines in the CNS. Nevertheless, MyD88 deficiency was associated with severe bacteremia. Similarly, in E. coli K1 neonatal meningitis, MyD88-deficient mice were unable to control meningitis, demonstrating that MyD88 has a critical role in early host defense. In mice, vulnerability to neurologic morbidity changes intensely during the first few weeks of life. This may elucidate why the neonatal brain is particularly sensitive to infection and why infection during this time can lead to long-term neurologic sequelae.