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Microbial Interactions in the Respiratory Tract

Murphy, Timothy F. MD*†; Bakaletz, Lauren O. MD, PhD‡§; Smeesters, Pierre R. MD, PhD

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The Pediatric Infectious Disease Journal: October 2009 - Volume 28 - Issue 10 - p S121-S126
doi: 10.1097/INF.0b013e3181b6d7ec
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Abstract

Upper respiratory tract infections (URTIs) are highly prevalent pediatric diseases associated with significant morbidity and socioeconomic cost. Because the nasopharynx lies between the nose, sinuses, ears, larynx, and the lower respiratory tract, resident pathogens of the nasopharynx can be the source for both upper and lower respiratory tract infections.1,2 This is particularly true of middle ear infections, as the nasopharynx is linked with the middle ear via the Eustachian tube. The nasopharynx is also a major source of secretions containing bacteria that can easily spread between individuals and these may subsequently become pathogenic in the new host. In this respect, the issue of nasopharyngeal carriage is critical, because it plays an important role in both the development of disease and the spread of pathogens. The nasopharyngeal flora becomes established during the first year of life.3–5 A broad variety of microorganisms, including commensal bacteria as well as potential pathogens such as S. pneumoniae, H.influenzae (predominantly nontypeable strains), and M. catarrhalis, colonize the nasopharynx. In most cases these organisms are carried without causing clinical symptoms. However, when homeostatic conditions of the host are altered, microorganisms may invade adjacent sites and/or invade the bloodstream, causing disease.

URTIs are caused by the synergistic and antagonistic associations of upper respiratory tract viruses and bacterial pathogens. The role of these agents has been studied for many years. This article reviews the interactions between these microorganisms in the pathogenesis of URTIs.

OTITIS MEDIA

In the United States (US) as in other developed countries, otitis media (OM) is the most common reason for children to visit doctors and emergency rooms and also the most common reason for prescribing antibiotics.6 Importantly, it is estimated that about 10% of children are otitis-prone7,8; however, it is difficult, to predict which children will become otitis-prone. This has important implications for vaccine strategies for preventing OM.

Figure 1 shows a compilation of 8 different studies involving tympanocentesis and culture of middle ear fluid from 1990–20079–16: the 3 predominant pathogens are S. pneumoniae, non-typeable H. influenzae (NTHi), and M. catarrhalis, which are members of the commensal flora of the nasopharynx. The microbial ecology is evolving and undoubtedly will continue to change.17

F1-2
FIGURE 1.:
The 3 predominant pathogens in otitis media: S. pneumoniae, NTHi and M. catarrhalis (from 8 different studies involving tympanocentesis and culture of middle ear fluid from 1990–2007).9–16

Streptococcus pneumoniae

S. pneumoniae (or pneumococcus), is a bacterium that commonly resides in the upper respiratory tract of children and adults. Asymptomatic nasopharyngeal carriage of pneumococci is widely prevalent among young children and is an important factor in the development and transmission of pneumococcal disease. Up to 54% of children will carry pneumococci in their nasopharynx by the time they are 1-year-old.2 Nasopharyngeal carriage may occur in up to 60% of healthy preschool children and up to 30% of older children and adults.18

S. pneumoniae can evade host defenses in normal and impaired hosts and spread to the upper or lower respiratory tract. This results in infections such as acute otitis media (AOM), pneumonia, and sinusitis. Or, it may invade the bloodstream causing invasive diseases.19 In addition, nasopharyngeal secretions also facilitate the transmission of these potentially pathogenic bacteria between individuals.

The polysaccharide capsule of S. pneumoniae is known to act as a critical virulence factor protecting the organism from clearance by host defenses.20 Specific pneumococcal capsular polysaccharides influence immunogenicity and determine serotypes. At least 91 capsular pneumococcal serotypes have been identified.21 These capsular types differ in chemistry, prevalence, extent of drug resistance, and virulence.22–24

Haemophilusinfluenzae

Type b Versus Nontypeable Strains

H. influenzae has 6 different capsular serotypes, a through f, in addition to nonencapsulated or nontypeable strains. The 2 most important human pathogens are the serotype b strains that have a capsule and the nontypeable strains which are nonencapsulated. The pathogenesis of infection for type b strains is hematogenous spread,25 whereas NTHi proceeds through mucosal infection or local spread.26 The clinical manifestations are reflected by these differences in pathogenesis: type b strains cause invasive disease, in particular meningitis, epiglottitis, cellulitis, and pneumonia, while nontypeable strains cause mucosal infections such as OM (the predominant clinical manifestation in children), sinusitis, conjunctivitis and less likely pneumonia. Type b strains are essentially a clonal population, whereas nontypeable strains show enormous genetic heterogeneity among strains.27,28 It was initially thought that nontypeable strains were type b strains that had lost their capsule; however, it has been shown genetically that these are a different population of organisms. Lastly, there is a highly effective vaccine for type b strains, whereas there is no such vaccine yet available for NTHi. However, recent, exciting progress has been made on the development of vaccines for nontypeable strains.29

NTHi: Patterns of Nasopharyngeal Colonization

The pathogenesis of OM involves movement of bacterial pathogens from the nasopharynx to the middle ear.2,30,31 Therefore, events in the nasopharynx and nasopharyngeal colonization are keys to understanding OM pathogenesis. When a child acquires a strain of NTHi, many things can happen: the strain can be cleared with or without disease; some children are colonized for long periods of time by one strain, whereas others acquire and clear sequential strains from the nasopharynx. An important observation by Faden et al in the 1980s and 1990s was that colonization by NTHi in the first months of life was associated with recurrent OM.32 OM-prone children are colonized at a higher rate than non-OM-prone children. A recent study from the University of Michigan that used molecular typing of strains observed frequent transmission of strains among children in daycare centers.33

Another recent study in Galveston Texas examined nasopharyngeal colonization at the time of OM, comparing pre- and post-pneumococcal vaccine periods in fully immunized children.34 The study showed that the relative distribution of colonizing pathogens changed with the widespread use of the pneumococcal conjugate vaccine (PCV).

Clinical Manifestations of NTHi-Induced Diseases in Children

OM is an important and a common clinical manifestation of NTHi infection in children. NTHi also causes sinusitis35; an etiological diagnosis of sinusitis requires an invasive procedure to access the sinus. NTHi and pneumococcus are the predominant pathogens in bacterial conjunctivitis.36 The importance of NTHi as a cause of pneumonia in children remains to be elucidated, despite evidence in adults. Interestingly, NTHi causes respiratory tract infections early in the course of cystic fibrosis, before children become colonized with mucoid strains of Pseudomonas.37

Moraxella catarrhalis

There are many reasons why M. catarrhalis has been overlooked as a pathogen, including the successive modifications of the name. Previously this pathogen was called “Neisseria catarrhalis.” In a landmark study, published in the Lancet in 1953, regarding the bacteriology of chronic bronchitis, pneumococcus, and H. influenzae were stated to be the most important pathogens in chronic obstructive pulmonary disease (COPD).38 Although, M. catarrhalis was present in sputum samples more often than pneumococcus or H. influenzae, the authors stated that M. catarrhalis was an “organism whose pathogenic propensities are known to be slight or nonexistent.” Thus, the organism was ignored in COPD for the next 3 or 4 decades, as a result of contemporary thinking which was reflected in this statement.

M. catarrhalis is an important pathogen both in children and in adults. There is variability in epidemiology among geographic sites. In children with OM, M. catarrhalis is present in the middle ear fluid, a normally sterile site. In adults, M. catarrhalis is also an important cause of exacerbations of COPD, resulting in an estimated 2 to 4 million exacerbations per year in the US.39

Microbial Interactions

Bacterial pathogens are generally studied individually, although in their natural environment they often coexist or compete with multiple microbial species. Similarly, diagnosis of infections often proceeds via an approach which assumes a single-agent etiology. However, mixed infections are probably frequent and complex interactions occur between the different infectious microorganisms living in the same ecological niche.40

Direct effects of one microbe on another occur within a species and effects of microbes on each other can also occur through the host. One bacterial species may affect the disease caused by another. Direct effects of one organism may target the pathogenic factors of another. Highly evolved relationships exist between microbes that live in the upper respiratory tract. It will be important to understand these relationships, especially in settings when flora is manipulated, with the possibility of affecting other pathogens.

Upper Respiratory Tract Colonization

S. pneumoniae, H. influenzae, M. catarrhalis, and Staphylococcus aureus typically asymptomatically colonize the nasopharynx of young children. There is substantial interest in how the human microflora may be contributing to disease and to the maintenance of health. Antibiotics and vaccines modify the flora by removing organisms that are part of the commensal flora.2 These organisms are in a complex yet balanced relationship with each other and thereby manipulation of one may trigger effects on other components of the flora. Because multiple species simultaneously colonize the same niche, the survival of an organism may depend on its ability to compete with coinhabitants of the niche. Moreover, host factors appear to have an influence on the result of this competition.

Interbacterial Competition Between Streptococcus pneumoniae and Haemophilusinfluenzae

In vivo and in vitro studies on S. pneumoniae and H. influenzae competition show interesting and sometimes contradictory results. Using a multivariate longitudinal model, Jacoby et al described a positive association between S. pneumoniae and H. influenzae colonization in Aboriginal and non-Aboriginal children in Australia41; whereas, Pettigrew et al showed that S. pneumoniae colonization is negatively associated with colonization by H. influenzae on 968 swabs collected from 212 American children.42 It has to be underscored that the Australian study included healthy children, whereas the American study included children who had an URTI.

Culture supernatant from S. pneumoniae inhibits the growth of H. influenzae in vitro, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. This selective effect seems to be caused by hydrogen peroxide production by S. pneumoniae under aerobic growth conditions.43 Similarly, S. pneumoniae secretes a neuraminidase that desialylates the H. influenzae lipopolysaccharide. This desialylation probably increases the bactericidal effect of complement on H. influenzae.44

Interestingly, a mouse model of mucosal colonization demonstrates different competitive relationships between these 2 bacteria. When given individually, both bacterial species occupy a similar microenvironment within the nasopharynx. However, when given simultaneously, rapid clearance of S. pneumoniae is observed. This competitive effect is eliminated if neutrophil-like cells or complement are depleted from the host mice.45 Moreover, the host nucleotide-binding oligomerization domain-1 (Nod1) plays a central role in this enhanced opsonophagocytic killing of S. pneumoniae.46 These studies demonstrate that the host innate immune response may influence the competition between species and therefore impacts the composition of the colonizing flora.

Viral Potentiation of Bacterial Infections of the Respiratory Tract

It has been postulated for a long time that viral infections of the respiratory tract predispose it to bacterial superinfections, notably by the disruption of the respiratory mucosal epithelium.47 Similarly, NTHi and S. pneumoniae are more frequently recovered from patients with viral infections than from those without, suggesting a link between viral infection and bacterial colonization.48,49 New experimental data provide additional interesting elements. Several studies have demonstrated, for example, a major increase in the development of experimental OM in chinchillas coinoculated with influenza A virus and S. pneumoniae.50,51 Influenza and parainfluenza viruses possess neuraminidase activity. It has been suggested that neuraminidase activity promotes S. pneumoniae colonization by exposing host cell receptors otherwise covered by sialic acid.44,52 Globally, respiratory viruses promote bacterial adhesion to respiratory epithelial cells.47,52 As adhesion is the first step toward colonization and infection, this viral “priming” has a significant impact on disease. A recent study also demonstrated that influenza A virus infection predisposes mice to fatal septicemia following superinfection with S. pneumoniae serotype 3.53 The molecular mechanism responsible for these observations is not fully understood but high levels of neutrophil activator granulocyte colony-stimulating factor (G-CSF) have been found in superinfected animals, suggesting that G-CSF is a major contributor to synergistic exacerbation of disease leading to fatal septicemia. These data suggest that, as for bacterial interaction, viral-bacterial interactions are driven, at least in part, by host-linked immunologic factors.

ROLE OF BACTERIAL BIOFILMS IN OTITIS MEDIA

Definition and Characteristics

A biofilm is a highly-organized, multicellular community that is encased in a polymeric matrix, in close association with a surface. This definition has been the source of some debate in literature based on whether or not the polymeric matrix is comprised of a unique product produced by the bacteria.54 However, this definition is evolving and the biofilm matrix is highly dependent not only on the organism that produces the biofilm, but also on the environment in which the biofilm is produced. The definition, nonetheless, refers to the preferred state of bacterial growth in nature. Bacteria growing within a biofilm, as opposed to their planktonic or free-floating counterparts, have a reduced growth rate and a distinct transcriptome.55,56 They reduce their growth rate to optimize conditions for residence in a nutrient-limited environment. They also exchange genetic material at an increased frequency.

Bacteria growing in a biofilm have substantially increased resistance not only to effectors of innate and acquired immunity, but also to the action of antibiotics.57 For example, antibiotics that target the cell wall are not effective against bacteria that have slowed their metabolism and are no longer dividing. This is an effective mechanism to resist antibiotics. Diseases with a biofilm component would require novel methods for prevention, diagnosis and treatment, owing to these characteristics.

The Biofilm Paradigm and Otitis Media

The biofilm paradigm was recently put forth because OM is difficult to treat with antibiotics; it is often chronic and recurrent in nature. Moreover, effusions recovered from OM middle ears are often bacteriologically sterile. However, although bacteria cannot be cultured from these effusions, they are also often PCR-positive for bacterial DNA. Initially, this paradigm that biofilms were present in the middle ear was not uniformly or even enthusiastically embraced. One of the first points that convinced many that this concept was worth pursuing was the demonstration by Rayner et al58 that, in addition to bacterial DNA, there was also bacterial messenger RNA present in middle ear fluids. Messenger RNA from bacteria has a very short half-life, and the presence of this message suggested metabolically active bacteria present within those fluids, despite the inability to culture them. To date, all 3 major otopathogens—S. pneumoniae, NTHi, and M. catarrhalis—have been shown to form biofilms both in vitro and in vivo.59–63

Direct detection of a bacterial biofilm in the middle ears of children, however, was the missing link required by many to be convinced that biofilms were indeed a component or part of the disease course of OM. Recently, Hall-Stoodley et al64 showed the presence of bacterial biofilms in association with middle-ear mucosa samples recovered from children with chronic and recurrent OM.

Relevance of Biofilms in Otitis Media

Current data support the role of biofilms in recurrent and chronic OM. It would be counterintuitive however, to not consider the possibility that biofilms also contribute to AOM because bacteria take only minutes to begin building a biofilm in a favorable environment. This is relevant to the therapeutic management of OM because the susceptibility profiles are distinctly different between planktonically grown or broth-grown micro-organisms compared with those that are resident within a biofilm. Bacteria in a biofilm are up to 1000 times more resistant to the action of antibiotics than those that are growing planktonically.57,65,66

Biofilms in the middle ear may be of mixed microbial etiology, suggesting that a unique therapeutic regimen may be required. Methods are being developed to consider the mechanical or enzymatic dispersal of biofilms as a way to treat and eradicate the biofilm from the middle ear space. Some challenges to this approach are expected in the case of OM, concerning the frequency and method of delivery necessary for efficacy. Nonetheless, this is an area of active investigation. It has been suggested that the practice of “watchful waiting” and of ventilation tube insertion itself may favor the biofilm phenotype in the tubotympanum and thus this approach may also favor the development of tube-associated otorrhea,67–69 whereas others have suggested that tube-associated aeration of the middle ear favors disruption of the bacterial biofilms.

VACCINE FOR NTHi

The medical and public health burden of invasive pneumococcal infections have driven the development and implementation of widespread use of PCV-7 in many countries.70 Although the need to prevent invasive pneumococcal disease has been apparent, there is also an urgent need for vaccines to prevent infections caused by NTHi and M. catarrhalis based largely on the magnitude of the worldwide burden of OM and its consequences.29

Developing a vaccine for NTHi and M. catarrhalis necessitates a different strategy from the pneumococcal vaccines, because both organisms are nonencapsulated. The Hib vaccine is based on capsules, as are the pneumococcal vaccines. Therefore, an approach is to find surface-exposed proteins that are conserved among strains.29 An ideal vaccine antigen should be (1) present on the bacterial surface, (2) conserved among strains, ie, a similar sequence from strain to strain, (3) expressed when the organism is in the human upper respiratory tract, and (4) induce a protective immune response. There are a variety of antigens in various stages of development and Table 1 summarizes 6 promising antigens. There is a need to test these proteins in clinical trials to determine whether or not they will prevent OM and other infections caused by NTHi.

T1-2
TABLE 1:
Vaccine for NTHi. Six Promising Antigens in Various Stages of Development

The children who are OM-prone will particularly benefit from vaccines for NTHi. It is difficult to predict who will be otitis-prone, so it is likely that universal vaccination for OM will be necessary. A recent prospective study by Prymula et al71 evaluated a vaccine formulation that consisted of protein D, which is a conserved surface-exposed outer membrane protein of NTHi, conjugated to 11 different pneumococcal polysaccharides. A total of 4968 infants were randomly allocated to 2 groups. Approximately half received the test vaccine and half received a hepatitis A vaccine as the placebo control. Clinical diagnosis followed by tympanocentesis and middle ear fluid culture demonstrated more episodes of AOM in the control group compared with the experimental group. Importantly, the efficacy for the vaccine was calculated to be 57.6% for pneumococcal vaccine serotypes and 35.3% for NTHi. Although there is a need to improve efficacy against NTHi, this is the first clinical trial showing that it is possible to prevent OM caused by this otopathogen.

As novel methods to prevent OM and other diseases of the airway (sinusitis, adenotonsillitis, exacerbations of COPD, bronchitis) are developed and tested, it will be important to consider that biofilm formation is likely important in the pathogenesis of these infections. One approach may be to develop vaccines designed to prevent the movement of bacteria from the nasopharynx into the middle ear and thereby prevent the initiation of biofilm formation in this anatomic niche.

CONCLUSION

Synergistic interactions occur between viruses and bacteria in the nasopharynx. Nasopharyngeal colonization and movement of bacterial pathogens from the nasopharynx to the middle ear are important in the pathogenesis of OM, a disease that is the most common reason for prescribing antibiotics in children. Reducing the carrier state, therefore, may have an impact on the subsequent burden of disease and confer herd protection. However, altering colonization by one pathogen may have consequences for other microbes including other potential pathogens in this niche.

OM includes a biofilm component as part of the disease course. Understanding the physiology and the biochemistry of these biofilms should contribute to the development of novel methods to treat and preferably prevent this disease. Due to the highly resistant nature of biofilms, preventing the first episode of AOM is likely to be an important part of the preventative strategy. One should consider the possible contribution of biofilms to OM in situations of treatment failure, chronicity, and recurrence. Given the potential for mixed microbial etiology of OM, developing a vaccine that targets 2 or more of the bacterial candidate otopathogens would seem to be a rational and promising approach. Vaccines that show a clinically meaningful impact on OM may have a similar impact on sinusitis and conjunctivitis, considering the continuous nature of the mucosa. Further studies are required to quantify such expanded benefits, particularly on immunization against NTHi, a bacterium that is increasingly being recognized as a significant pathogen in respiratory tract infections.

ACKNOWLEDGMENTS

The authors thank Dr. Armine Najand (Medical Education Global Solutions, France) for medical writing and Mr. Yann Colardelle (Medical Education Global Solutions, France) for manuscript coordination. GlaxoSmithKline Biologicals provided editorial assistance and sponsored this supplement.

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Keywords:

upper respiratory tract; bacterial interactions; otitis media; pathogenesis; nontypeable Haemophilus influenzae; Streptococcus pneumoniae; Moraxella catarrhalis; nasopharyngeal flora; serotype replacement; biofilm; polymicrobial disease; vaccine; heptavalent pneumococcal conjugate vaccine

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