Host Defense Mechanisms Against Bacteria

Introduction

Globally, infections cause an estimated 1 million neonatal deaths annually, representing over 40% of all neonatal deaths. ^, Overwhelming host response to a microbial infection, or neonatal sepsis, is defined as infection in the first 28 days of life; for preterm infants, this period includes up to 4 weeks after the expected due date. This is further subdivided into early-onset neonatal sepsis (EOS), with an onset during the first 72 hours of age, and late-onset neonatal sepsis (LOS), where incidence peaks in the second to third week of postnatal life but includes events up to 1 month of age.

It is difficult to clinically differentiate between serious bacterial infections (SBI) versus viral, fungal, or other causes without concomitant microbial identification. ^, Unfortunately, the incidence of culture-positivity in cases of clinically suspected bacterial sepsis is on average only 10% in the United States; this rate is substantially higher in prematurely born and/or very-low- birth-weight (VLBW) infants. Available data suggest that in several populations around the world the rate of SBI in newborns may be orders of magnitude higher than in the United States. However, few high-quality studies exist that examined culture-proven SBI in resource-restricted regions of the world, although this is where most neonatal deaths occur. In two studies, one from South Africa and another one from south Asia, pathogen detection succeeded in no more than approximately one-fourth of all cases of clinically suspected SBI (also known as possible SBI or pSBI), despite cutting-edge study design and methodology (including culture and molecular testing). ^, The fact that approximately three-fourths of all cases had no pathogen identified likely indicates that some pathogens were missed, but also that not all pSBI are related to a pathogen; it may be that some are driven by the host for yet unknown reasons. Among infectious pathogens identified, bacteria were the leading cause of neonatal infectious deaths. For example, a meta-analysis of studies between 2008 and 2018 highlighted Staphylococcus aureus (SA), Klebsiella , and Escherichia coli spp. as the dominant causes of culture-proven SBI in neonates (<28 days) in sub-Saharan Africa. Ureaplasma spp. and Group B Streptococcus (GBS) were most frequently identified among pSBI cases in South Africa. Ureaplasma spp. were also the most commonly identified pathogens in cases of pSBI in 60-day-old or younger infants in south Asia.

Despite the development of potent antimicrobial agents, the mortality rate associated with neonatal bacterial sepsis remains very high, especially in preterm infants. In the pre-antibiotic era the case fatality rate of neonatal sepsis exceeded 80%; with the introduction of antibiotics and advances in perinatal care, the case fatality rate has dropped to under 20%. However, this rate is still far higher than in the pediatric or young adult age groups. Beyond mortality, neonatal sepsis also causes significant immediate and long-term morbidity in those that survive. In particular, the risks for central nervous system injury leading to cerebral palsy, abnormal neurodevelopment, visual impairment, and poor growth are significantly elevated with each episode of sepsis. Lastly, neonatal sepsis also causes significant strain on the health care system. In North America alone, it is estimated that each episode of bacterial sepsis prolongs the duration of a neonate’s hospital stay by about 2 weeks, resulting in an incremental cost of USD $25,000 per episode.

The high prevalence of specific species associated with infection in early life, such as Ureaplasma spp . and several gram-negative bacteria, suggests that there are particular virulence factors peculiar to these pathogens, or to age-specific host responses to those microorganisms, which are centrally involved in the high morbidity and mortality of newborn bacterial sepsis. In this chapter, we will review virulence factors known to be involved for the most important bacterial pathogens in early life and what is known about the age-specific host response to bacterial infections, with a strong focus on those aspects presumed to be of relevance for protection from bacterial infection in early life.

Bacterial Factors Contributing to Infection in Early Life

As causative agents of chorioamnionitis, preterm delivery, and neonatal sepsis, Ureaplasma species ( Ureaplasma spp.) are a major contributor to maternal-fetal morbidity worldwide. Ureaplasma spp. are frequent commensals of the female lower genital tract; colonization rates are influenced by multiple factors, including ethnicity and age. Clinical outcome of infection with Ureaplasma spp. is highly variable across human populations and also bacterial species (genotype- and even serovar)-dependent. ^, In vitro studies showed that some Ureaplasma spp. bind host cells and can actively suppress innate immune pathways, while in vivo (animal) infection demonstrated that Ureaplasma spp. can induce inflammatory responses in fetal immune cells. ^, Several virulence factors for Ureaplasma spp. have been identified, including the multiple band antigen (MBA). The N-terminal domain of the MBA is conserved among the 14 serovars of Ureaplasma ; antigenic size variation has, however, been reported for the C-terminal region of MBA protein and is hypothesized to be involved in immune evasion, allowing chronic infection during pregnancy. Variation of the size of the MBA protein also appears associated with distinct cord blood innate immune responses. While MBA may be an important contributor to Ureaplasma spp. pathogenicity, much remains to be learned about how and why Ureaplasma spp. are so frequently linked to pSBI of the newborn, especially in low-resource settings.

Klebsiella pneumoniae has emerged has an important cause of morbidity and mortality in neonates. Drug-resistant strains of Klebsiella that have been associated with outbreaks in neonatal intensive care units around the globe are especially problematic. K. pneumoniae has developed multiple immune evasion strategies, including inhibition of complement activation, dampening of inflammatory response, and apoptosis of macrophages, which often appears to involve the capsular polysaccharide. While K. pneumoniae is now considered as an “urgent threat to human health,” surprisingly little is known about this important bacterial pathogen, not only in regards to its role in early life infections but overall.

E. coli has long been recognized as one of the leading bacterial agents for EOS and LOS, as well as neonatal meningitis. ^, E. coli contains several virulence factors, promoting translocation through the amniotic membrane and subsequent invasion of the fetal and newborn blood-brain barrier, which are likely to be responsible for its high prevalence in cases of newborn sepsis and meningitis. The majority of E. coli strains causing meningitis belong to a specific capsular serotype (K1) that possesses type 1 pili and the outer membrane protein OmpA, promoting adhesion and penetration across endothelial layers. In vitro, OmpA can also suppress dendritic cell (DC) maturation and function, dampen pro-inflammatory cytokine production, and increased production of anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. Further emphasizing OmpA’s importance in neonatal virulence is the finding that different portions of the extracellular loops of OmpA allow invasion and subsequent survival inside of the very host cells that should eliminate this pathogen, such as neutrophils. Despite its long history as a neonatal pathogen, the precise molecular host-bacterial interactions that give E. coli this infamously prominent role in early life infections are barely understood.

Among the gram-positive bacteria, Streptococcus agalactiae , or GBS remains one of the important invasive pathogens for newborns, despite antibiotic prophylaxis regimens implemented in many parts of the world. In North America and Europe, where GBS is a commensal of the human intestinal and vaginal tract in 15% to 30% of healthy adults, every 10th neonate acquires GBS vertically during passage through the birth canal or shortly thereafter. Yet, 99% of colonized infants will never develop invasive GBS disease. Some of the underlying mechanisms that lead to disease are beginning to emerge. For example, the ability of type III GBS in particular to adhere to the neonatal epithelium facilitates colonization and predominance in early-onset neonatal sepsis. Crossing the mucosal barrier and the blood-brain barrier seems to be mechanistically linked, as GBS serotype III is a particularly frequent isolate in neonatal meningitis. The high-level neurotropism is at least partially due to expression of the adhesion molecule hypervirulent GBS adhesin (HvgA). HvgA efficiently supports bacterial adhesion and transfer through to the intestinal wall and later across the blood-brain barrier, specifically the vascular endothelium of the choroid plexus. Expression levels of HvgA and other GBS virulence factors, such as pili and toxins, are regulated by the upstream two-component control system CovR/S. This in turn is modulated by acidic pH and high glucose levels, which the microbe encounters during the passage through the intestine. After invasion, GBS has the ability to subvert innate immunity by different mechanisms. The GBS enzyme glyceraldehyde-3-phosphate-dehydrogenase induces the production of IL-10 and thereby decreases the recruitment of neutrophils and limits their bactericidal activity. GBS capsular polysaccharides, allowing the identification of 10 unique serotypes (Ia, Ib, II to IX), have a terminal sialic acid residue. This SIA residue binds to host inhibitory sialic acid binding immunoglobulin (Ig)-like lectins (SIGLECs 5, 9 and 14) and thereby dampens phagocytosis, oxidative burst, and platelet-mediated antimicrobial killing. ^, On the host side, sensing of GBS nucleic acids and lipopeptides by both Toll-like receptors (TLRs) and the inflammasome appears to be critical for host resistance against GBS; these host functions display age-dependent changes in function (see below).

Staphylococcus spp. and particularly S. epidermidis are other leading causes of sepsis in neonates (reviewed by Marchant and colleagues and Power Coombs and colleagues ^, ). Newborns are often colonized via horizontal rather than vertical transfer. S. epidermidis produces a biofilm that favors its persistence on medical devices. Epidemic clones that display significant antibiotic resistance preferentially produce extracellular polymers, such as polysaccharide intercellular adhesion (PIA), that are part of the biofilm. PIA modulates host innate immune responses by different mechanisms, including inhibition of phagocytosis. Staphylococcus spp. also evade clearance by the immune system by using exoenzymes such as protease and endopeptidase, and in part by generating adenosine. Adenosine is an endogenous purine metabolite that acts via cognate seven-transmembrane receptors to induce immunomodulatory intracellular cyclic adenosine monophosphate (cAMP). cAMP enhances production of IL-6, which impairs neutrophil function while inhibiting production of tumor necrosis factor (TNF)α, which is important for neutrophil activation. Neonatal mononuclear cells are particularly sensitive to the effects of adenosine.

Protein toxins are another group of important virulence factors contributing to neonatal sepsis (reviewed in Sonnen and Henneke ). Most of the major pathogens responsible for neonatal sepsis, namely GBS, E. coli, and S. aureus , secrete toxins of different molecular natures, but each is key for defining the disease. These pore-forming exotoxins are expressed as soluble monomers prior to engagement of the target cell membrane with subsequent formation of an aqueous membrane pore. Membrane pore formation allows penetration of epithelial barriers as well as evasion of the immune system. In the process, pore formation contributes to inflammation and hence to the manifold manifestations of sepsis. S. epidermidis produces phenol-soluble modulin toxins that participate in biofilm formation and induction of pro-inflammatory responses that may be associated with necrotizing enterocolitis.

Age-Dependent Aspects of Host Defense Contributing to Bacterial Infection Early in Life

Immune-mediated protection, in evolution as well as ontogeny, starts with a focus on primitive host defenses of single-cells, designated cell-autonomous immunity. This is followed by increasingly complex interactions, such as biochemical coordination in collections of cells via nutritional immunity, to increasingly specialized cells that provide barrier function and innate immunity. Only at the last stages of evolution and ontogeny is adaptive immunity, with its highly specialized tissues, readily identifiable. During fetal and early neonatal life, the host protective immune system undergoes profoundly rapid developmental changes; these changes occur in adaptation to specific functional demands as well as changes encoded in the host genome. The early life immune system is thus not simply stuck in a fixed state of “immaturity” but has in fact been shaped over millennia of human phylogeny to assure our survival as a species; yet it remains highly responsive to the rapidly changing demands of each individual’s ontogeny. The increased risk for bacterial infection then must arise from this interphase between phylogenetically selected survival programs and extraordinary demands during ontogeny.