Insights into the role of the respiratory tract microbiome in defense against bacterial pneumonia

Abstract

The respiratory tract microbiome (RTM) is a microbial ecosystem inhabiting different niches throughout the airway. A critical role for the RTM in dictating lung infection outcomes is underlined by recent efforts to identify community members benefiting respiratory tract health. Obligate anaerobes common in the oropharynx and lung such as Prevotella and Veillonella are associated with improved pneumonia outcomes and activate several immune defense pathways in the lower airway. Colonizers of the nasal cavity, including Corynebacterium and Dolosigranulum, directly impact the growth and virulence of lung pathogens, aligning with robust clinical correlations between their upper airway abundance and reduced respiratory tract infection risk. Here, we highlight recent work identifying respiratory tract bacteria that promote airway health and resilience against disease, with a focus on lung infections and the underlying mechanisms driving RTM protective benefits.

Graphical Abstract

Introduction

The primary site of microbial colonization in the respiratory tract is the upper airway, which comprises the nasal cavity, nasopharynx, and oropharynx. In the lower airway, microbial density diminishes with passage from the large airways of the trachea through the bronchial tree, terminating in the alveoli [1,2]. Bacteria in the oropharynx are the dominant source of lung microbiome ‘seeding’, which occurs throughout life due to frequent aspiration [3,4]. All prevalent bacterial pneumonia pathogens, including Streptococcus pneumoniae (the pneumococcus), Staphylococcus aureus, Haemophilus influenzae, and others originate from the nasal cavity and nasopharynx of the upper airway, where they colonize asymptomatically. Accordingly, the dynamics between opportunistic pathogens, or ‘pathobionts’, and other members of the RTM throughout the upper and lower airway influence both pathogen colonization and lung infection risk.

Several internal cues shape an individual’s RTM, including age, infection history, and co-morbidities such as chronic lung disease [2,5]. While the RTM is maintained in a state of equilibrium during health, respiratory tract infections are associated with dysbiosis, signaled by a decline in microbial diversity [6–9]. Pathogens are more resistant to antibiotic therapy, which unintentionally depletes non-pathogenic, or commensal, RTM bacteria [10,11] and extended antibiotic therapy increases infection risk [12]. Altered infection profiles following RTM disruption suggests a beneficial role for commensals which are otherwise abundant in health.

In this review, we focus our discussion on clinical associations paired with experimental evidence identifying bacterial species with potentially protective effects that negatively correlate with opportunistic bacterial pathogens (Figure 1). However, none of these species exist in isolation, and we are likely just scratching the surface of the ecological dynamics influencing bacterial pneumonia. Further, our understanding of how other RTM members impact bacterial pneumonia, including viruses and fungi as well as the understudied protozoa and archaea, is far from complete. Significant alterations in the lung virome were reported in patients with bacterial pneumonia [13,14], whereas the lung mycobiome appeared to be relatively stable [15]. As reviewed elsewhere, lower fungal diversity correlates with reduced respiratory function in the context of chronic lung diseases, suggesting a beneficial impact [16]. Transient exposures to respiratory tract viruses may improve anti-viral immune defense against viral pneumonia, though the implications for bacterial pneumonia are less clear [17]. In the following sections, we review current knowledge regarding the potential protective functions of the bacterial component of the RTM in the context of bacterial pneumonia.

Figure 1.

Cooperative and inhibitory dynamics between beneficial bacteria within the RTM and lung pathogens. Arrows highlight positive and negative correlations (dotted lines) and interactions for which experimental evidence demonstrates inhibitory effects on the indicated respiratory tract pathogen (solid lines). Numbers correspond to citation(s) supporting the relationship. Rothia species (spp) include R. aeria and R. similmucilaginosa. Corynebacterium pseudodiphth. = C. pseudodiphtheriticum.

Obligate anaerobes are associated with improved lung infection outcomes

In a healthy state, obligate anaerobes including Prevotella and Veillonella are among the most abundant genera in the lungs [3,18,19]. Conversely, airway anaerobes are diminished during pneumonia [20–23], indicating a potential role in respiratory tract health maintenance. The notion that anaerobic bacteria in the RTM have beneficial effects is reinforced by clinical data demonstrating the impact of antibiotics with anaerobic coverage in critically ill patients. Prescription of anti-anaerobic antibiotics for patients with suspected aspiration pneumonia remains widespread despite current recommendations [24–26]. In patients hospitalized with pneumonia, low microbiome diversity and fewer obligate anaerobes correlated with reduced survival [27]. In this study, both anti-anaerobic antibiotics and chronic oxygen therapy were associated with depletion of obligate anaerobes [27]. A separate analysis of 3,032 patients on mechanical ventilation similarly noted reduced overall survival and a significant increase in the combined metric of mortality and ventilator associated pneumonia in patients treated with anti-anaerobic antibiotics [28]. As with the prior study, anti-anaerobic coverage was associated with an enrichment in facultative anaerobes, most notably Enterobacteriaceae [28]. In other reports, reduced obligate anaerobes corresponded with increased ventilator associated pneumonia [29] and enrichment of Streptococcus, another facultative anaerobe, in patients with aspiration pneumonia [30]. The link between anti-anaerobic antibiotic coverage and increased patient mortality was confirmed and expanded in a recent retrospective analysis of 15,908 patients in the Netherlands [31]. While it is difficult to discriminate between importance for the RTM versus the gut microbiome, which also supports respiratory tract health, these findings are consistent with a positive relationship between airway anaerobe abundance and resilience against pneumonia.

Mechanisms driving the beneficial effects of obligate anaerobes in the respiratory tract

Analysis of differential RTM clustering in the healthy lung has provided clues regarding the identity of impactful obligate anaerobes [32,33]. Segal et al. described an RTM pneumotype enriched in Prevotella and Veillonella associated with increased neutrophils and lymphocytes in the lower airway, indicative of subclinical inflammation [32]. Expanding these observations, Wu et al. demonstrated that lung installation with an oral commensal compilation including Prevotella melaningogenica and Veillonella parvula increased protection against secondary challenge with S. pneumoniae for up to two weeks in mice [34]. S. pneumoniae clearance was accompanied by an early increase in pro-inflammatory signaling pathways followed by a Th17 response propagated primarily by V. parvula [34]. In our investigations with P. melaninogenica, we found that enhanced clearance of S. pneumoniae at 24 hours post-exposure involved increased killing of S. pneumoniae by lung neutrophils [35]. Improved neutrophil-mediated clearance required the innate immune receptor TLR2 and pro-inflammatory cytokine TNFα, with inflammation restrained by co-induction of the anti-inflammatory cytokine IL-10 [35]. Notably, immune pathways including TLR signaling and IL-10 were elevated in the lungs of people with the Prevotella and Veillonella pneumotype [33] and TNFα and IL-10 positively correlated with Prevotella abundance and improved lung function in lung transplant patients [36]. Together, these studies suggest that subclinical lung inflammation induced by exposure to oral anaerobes may provide a protective benefit against pneumococcal pneumonia (Figure 2).

Figure 2.

Mechanisms for enhanced immune protection against pneumococcal lung infection following aspiration of obligate oral anaerobes into the lungs. Clinical studies indicate that an increased presence of oral anaerobes correlates with reduced mortality risk in patients with pneumonia (left box). Interactions between P. melaninogenica and V. parvula with the immune system are highlighted. AM = alveolar macrophage; Th17 = T helper 17 cell.

It remains unclear how oral anaerobes influence lung physiology in the setting of chronic lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). In both cases, Prevotella abundance is inversely correlated with disease severity [37,38], and in people with CF, Prevotella is more common in the absence of CF lung pathogens [39,40]. Prevotella and other obligate anaerobes isolated from the CF airway reduced NFκB activation and secretion of the neutrophil chemokine IL-8 from human epithelial cells exposed to lipopolysaccharide (LPS), suggesting that they restrain inflammation [41]. Similarly, alveolar macrophage responsiveness to the innate immune receptor TLR4, which detects bacterial LPS, was lower in cells from healthy individuals with the Prevotella and Veillonella enriched pneumotype [33]. In animal models, endogenous Prevotella were reduced in the context of chronic lung inflammation induced by LPS and elastase [42], but enriched along with Bacteroides following bleomycin-induced fibrosis [43]. In the bleomycin fibrosis model, membrane vesicles from P. melaninogenica and Bacteroides stercoris increased lung immunopathology [43], suggesting a detrimental impact for oral anaerobes in some settings.

Gram-positive bacteria in the upper airway impede lung pathogen survival

In the nasal cavity and nasopharynx, the Gram-positive bacteria Corynebacterium and Dolosigranulum are consistently linked with reduced pneumococcal colonization and infection risk in both children and adults [44–47]. Depletion of Corynebacterium and Dolosigranulum following antibiotic therapy also corresponds with otitis media (ear infection) recurrence [48–50]. For S. pneumoniae, the negative relationship between colonization and Corynebacterium abundance was replicated in vitro, as spent media from Corynebacterium accolens cultures inhibited S. pneumoniae growth through the lipase LipS1 [51]. Using a murine model, we found that C. accolens expression of LipS1 contributed to improved defense against S. pneumoniae lung infection [52]. In addition to S. pneumoniae, Corynebacterium abundance negatively correlates with S. aureus [5,53]. In vitro, C. pseudodiphtheriticum, but not C. accolens, inhibited S. aureus growth, suggesting a species-dependent phenotype [54]. C. pseudodiphtheriticum also attenuated S. aureus virulence, as downregulation of the Agr quorum sensing system was required for survival in the presence of C. pseudodiphtheriticum [55]. While C. accolens hasn’t been shown to directly inhibit growth, it can reduce S. aureus biofilm biomass [56], corresponding with less S. aureus-induced barrier damage [57]. Another species, C. propinquum, produces siderophores which inhibit growth of coagulase-negative staphylococci, though S. aureus was largely resistant [58]. Collectively, these studies identify a direct impact for several Corynebacterium species against pathogen growth (Figure 3).

Figure 3.

Beneficial bacteria in the RTM alter pathogen growth and virulence through diverse mechanisms. Mechanisms for Corynebacterium and within-genus inhibition of respiratory tract pathogens through increased opsonophagocytic killing (cross-reactive antibodies), direct killing (bacteriocins, epithelial cell secretion of AMPs, LipS1 cleavage of host triacyglycerols resulting in production of toxic FFAs), reduced expression of virulence genes (Agr quorum sensing system), reduced biofilms (Esp), and nutritional immunity (Hpl sequestration of heme). Abs = antibodies; FFAs = free fatty acids; AMPs = antimicrobial peptides; NTHi = non-typeable H. influenzae.

Corynebacterium is frequently co-identified with Dolosigranulum, and, as such, they are often examined in tandem [49,59,60]. D. pigrum, the only known species of Dolosigranulum, can synergize with Corynebacterium to inhibit growth of S. aureus and S. pneumoniae in vitro [61]. However, D. pigrum in isolation had a selective impact on S. aureus growth, and reduced mortality of S. aureus infected larvae [44,61]. While the factors contributing to D. pigrum-mediated inhibition remain elusive, community modeling of D. pigrum and S. aureus metabolic interactions identified reactions catalyzed by D. pigrum which may contribute to S. aureus growth restriction [62]. Enzymatic activity is a shared theme among Gram-positive commensals with inhibitory activity against respiratory tract pathogens, as in addition to Corynebacterium lipases and candidate D. pigrum enzymes, select species of the Gram-positive aerobe Rothia express a peptidoglycan endopeptidase that inhibited growth of the pathogen Moraxella catarrhalis [63]. D. pigrum also suppressed proinflammatory cytokine secretion from airway epithelial cells exposed to S. aureus [44]. In addition, D. pigrum improved clearance of S. pneumoniae from the lungs of infant mice [64,65]. The same group proposed that modulation of respiratory tract epithelial cells by the D. pigrum may impact COVID-19, as D. pigrum reduced SARS-CoV-2 replication in a lung epithelial cell line [66]. Together, these studies highlight diverse beneficial effects of Corynebacterium and D. pigrum, mediated by direct pathogen inhibition with potential contributions from host immune modulation.

Close relatives of lung pathogens: the case for species-specific analysis of the RTM

Accumulating evidence implicates beneficial roles for close relatives of the major bacterial respiratory tract pathogens, which may be overlooked using traditional 16S rRNA microbiome sequencing. One example is the commensal Streptococcus mitis, which was more susceptible than S. pneumoniae to complement-mediated killing [67], but shares enough homology to elicit cross-reactive antibodies that improved opsonophagocytic killing of S. pneumoniae and increased pneumococcal clearance from the lungs of mice [68–70]. Another commensal Streptococcus species, Streptococcus salivarius, produces bacteriocins that inhibited S. pneumoniae growth and reduced pneumococcal adherence to respiratory tract epithelial cells [71,72]. S. salivarius also decreased the burdens of P. aeruginosa in a rat infection model and inhibited growth of M. catarrhalis and S. aureus in vitro, indicating the potential for a broad impact beyond S. pneumoniae [73,74]. Non-pneumococcal streptococci are not universally beneficial, as S. mitis can donate penicillin-binding protein gene fragments to S. pneumoniae, increasing resistance to β-lactam antibiotics [75]. Regardless, these findings suggest that pneumococcal acquisition and persistence is affected by prior exposures and species interactions within the Streptococcus genus.

Staphylococcus epidermidis improves protection against S. aureus by several mechanisms, including the secretion of a serine protease called Esp that was reported to disrupt S. aureus biofilms and correlated with reduced S. aureus carriage in humans [76]. S. epidermidis also induced secretion of antimicrobial peptides from respiratory tract epithelial cells, allowing it to outcompete S. aureus and M. catarrhalis in a murine infection model [77]. Additionally, S. epidermidis activated epithelial expression of IFN-λ, which improved protection against influenza A virus infection in mice [78]. Unlike S. aureus, S. epidermidis doesn’t impede neutrophil recruitment [79], consistent with a less virulent profile. However, select clones of S. epidermidis expressing an S. aureus-type cell wall teichoic acid increased infection fatality in mice and facilitated DNA exchange with S. aureus [80], providing a mechanistic explanation for the rise of multidrug resistant S. epidermidis clones [81]. These findings suggest that analyses beyond the species level would provide a more comprehensive understanding of the implications of Staphylococcus carriage.

In a final example, the commensal Haemophilus haemolyticus produces a unique heme-binding protein called haemophilin (Hpl) which inhibited growth and adherence of the pathogen non-typeable H. influenzae (NTHi) to lung epithelial cells [82–84]. This effect was accompanied by increased epithelial cell secretion of the pro-inflammatory cytokine IL-6 and neutrophil chemokine IL-8 in response to H. haemolyticus, as compared with NTHi, which suppressed IL-6 and IL-8 secretion [85]. Clinically, carriage of Hpl+ H. haemolyticus isolates correlated with reduced NTHi colonization [86]. Together, these findings emphasize the importance of looking beyond the genus level to inform the impact of RTM composition on lung infection risk (Figure 3).

Therapeutic applications

Identifying the protective mechanisms of beneficial airway bacteria provides insights for the development of new RTM-based therapeutics, including respiratory tract directed probiotics. Several live bacterial oral and nasal sprays have demonstrated efficacy against recurrent pharyngitis and pathogen colonization in pre-clinical trials in young children and adults, indicating proof of concept for this approach [87–89]. Probiotics targeting the respiratory tract present novel opportunities in contrast to gut probiotics, which must contend with survival through the acidic stomach and establish colonization in the most microbe-dense part of the body. However, respiratory tract probiotics face similar challenges, including barriers to uptake and conflicting reports regarding efficacy [90–92]. In the pre-clinical trials with probiotic sprays, subjects were treated daily for a period of weeks to several months. Reduced treatment frequency will likely require improved RTM incorporation.

Another focal point for RTM therapeutic applications lies in vaccine development. Bacterium-like particles prepared from C. pseudodiphtheriticum were investigated as a vaccine adjuvant, with improved efficacy of the pneumococcal conjugate vaccine reported in mice [93]. OM-85, which contains lysates from opportunistic respiratory tract pathogens, reduced infection frequency and antibiotic use in children with recurrent respiratory tract infections in randomized, placebo-controlled clinical trials [94,95]. Similarly, oral administration of mixed bacterial lysates from both opportunistic pathogens and uncharacterized species of Streptococcus, Staphylococcus, Corynebacterium, and Pseudomonas was associated with reduced frequency and severity of respiratory tract infections [96]. While promising, more placebo-controlled clinical trials will be critical for evaluating the therapeutic potential of different respiratory tract bacterial compilations.

Outlook

In conclusion, studies highlighted in this review implicate the RTM in maintaining respiratory tract health through direct and indirect control of prominent bacterial pathogens. Efforts to capitalize on the protective benefits of RTM community members will rely on further identification and characterization of the critical species mediating pathogen inhibition. In addition, understanding the mechanisms by which different RTM species engage with the host immune defense against bacterial pneumonia may open new avenues to immunobiotic and immunotherapy approaches for respiratory tract infections.

Highlights.

• Obligate anaerobes in the lung improve protection against pneumonia

• Gram-positive bacteria in the nasal cavity correlate with reduced infection risk

• Within-genus relatives restrain pathogen virulence and growth