We evaluated two clinically relevant pneumococcal strains in vitro and in a chinchilla model of disease. We hypothesized that FG2319A would outcompete MIB0210215B/C in the chinchilla model as it does in vitro. However, our data indicate that both 19A and 15B/C strains of ST199 have similar pathogenic potential in experimental OM. The observation that serotypes previously classified as having lower invasive potential (i.e. 15B/C) have similar capacity to produce experimental OM as serotypes classified as having greater disease potential (i.e. 19A) has implications for the effectiveness of PCV7, PHiD-CV, and PCV13 in preventing otitis media and possibly non-bacteremic pneumonia.
Both capsule type and genetic background contribute to disease potential and presentation [10
]. Current methods of prevention and control focus on the polysaccharide capsule, an important pneumococcal virulence factor. Surprisingly, PCV7 has not reduced nasopharyngeal carriage of S. pneumoniae
but has resulted in complete replacement, with virtually the same prevalence of pneumococcal carriage, of non-vaccine serotypes [17
]. With over 90 different serotypes expressed and high levels of genomic plasticity, researchers cannot accurately predict which pneumococcal serotypes are likely to produce mucosal and/or invasive disease and which will be important to include in future vaccines.
Second generation vaccines in development include serotypes 1, 3, 5, 6A, 7F, and 19A. Serotypes 3, 6A, and 19A are common middle ear pathogens [27
]. Recent studies identified capsule type 19A as an important expanding serotype [21
] and inclusion of 19F capsular polysaccharide in the currently licensed vaccine formulation, PCV7, has not provided cross-protection against serotype 19A-associated disease [23
]. Although serogroups 1, 5, and 7 are common invasive pneumococcal disease pathogens worldwide [58
], these are rarely associated with OM [27
]. Therefore PHiD-CV may provide only minimal improvement over the PCV7 in preventing pneumococcal OM.
Minor differences between serotype 19A strains and serotype 15B/C strains may explain why serotype 19A has expanded more quickly than serotype 15B/C over the last decade. These differences include a shorter generation time observed in serotype 19A. Shouval et al. reported a slight increase in the odds of finding serotype 19A in disease compared to carriage (versus no different than 1.0 in serotype 15B/C) [9
]. Epidemiological data suggest that serogroup 15 strains, are increasing in carriage and disease [21
]. Between 2001 and 2007, Huang et al. observed a statistically significant increase in 19A colonization and a 50% increase, though not significant, in 15B/C colonization [31
]. Casey et al. recovered both serotype 19A and 15B/C from the middle ear space of children with and without acute OM [59
]. The most common serotypes recovered were 19A, 11, 15, and 23B from non-acute OM visits and 19A, 15, and 6C from acute OM visit [59
]. Hicks et al. found statistically significant increases in the rates of invasive pneumococcal disease due to 19A and 15 in young children and older adults after the introduction of PCV7 in the United States [32
]. Gonzalez et al. reported a significant increase in invasive pneumococcal disease caused by serogroup 15, with ST199 being the predominant clone [28
appeared more robust than MIB0210215B/C
only in vitro
. In the experimental OM model, we show that FG23, which expresses a 19A capsule, does not have a significant advantage over a serotype 15B/C strain, MIB02102, in its ability to colonize or cause disease. This is consistent with observations that 19A and 15B/C are two of the most prevalent serotypes found in the nasopharynx of children [31
]. As serotype 19A isolates are recovered more frequently than serotype 15B/C from children with invasive pneumococcal disease, yet colonization appears similar, tissue-specific genetic requirements beyond capsule are potentially responsible for differences in colonization, mucosal infection, and bloodstream invasion.
Weinberger et al. [61
] proposed that the metabolic costs of specific polysaccharide capsules could be used to predict the emergence of individual pneumococcal serotypes. These authors postulate that there is a greater metabolic cost to produce capsule in serotypes with more carbons and high-energy bonds per polysaccharide repeat unit. Thus, serotypes with low production costs are increasing in prevalence because they can produce more polysaccharide capsule and are consequently better able to evade neutrophil-mediated killing. Data indicate that serotype 15B/C has more carbons and high-energy bonds per repeat unit than serotype 19A [61
]. The shorter generation time of FG2319A
found here supports this “cost of capsule” model. Serotype 19A has 7 high energy bonds and 20 carbons, whereas serotype 15B/C has 11.4 high energy bonds and 15B and 15C have 33.4 and 32 carbons, respectively. Weinberger et al. did not directly examine the degree of encapsulation and neutrophil-mediated killing of serotype 15 strains [61
]. Based on their results, we would also expect that FG2319A
would persist longer in carriage and would be a better competitor in the disease model because it can produce more capsule for the same energy cost and thus should be able to evade destruction by the immune system. However, our chinchilla model did not identify significant differences between the two serotypes when inoculated in monoculture or in co-culture. Although a small number of animals were studied for duration of colonization, we utilized a non-parametric test appropriate for the small number and expect any differences in duration of colonization would be slight.
These data further confirm that serotype is not the sole factor in determining prevalence in carriage or virulence potential. Other factors contribute to virulence in disease models and in patients with pneumococcal disease, including pneumococcal genetic background, doubling time, pathogenicity islands, prevalence in the population, and host factors [62
]. There is evidence that genetic background, beyond clonal complex and serotype, influence pneumococcal disease potential [10
] S. pneumoniae
is a highly plastic species with a high level of genetic diversity. Accessory genes exist within the pneumococcal supragenome, which may cluster into regions [66
]. Accessory regions vary not only between, but also within clonal types. These accessory regions may also encode redundant functions, i.e. phosphotransferase sugar transport systems and ATP-binding cassette transport systems [66
]. This redundancy suggests that a single gene or gene profile will not necessarily identify strains that are more or less pathogenic. A more complete understanding of the pneumococcal supragenome, including accessory regions, serotype, and clonal complex, when combined with epidemiological and clinical data will help elucidate disease potential.
Although the chinchilla model does not use a genetically pure line of chinchillas, this is outweighed by its usefulness in closely mirroring middle ear disease in young children. An advantage to this model is that it allows nasopharyngeal colonization to be established before infection, further improving upon previous chinchilla models of experimental OM which used direct middle ear inoculation [12
]. Following colonization, barotrauma negative pressure in the middle ear cavity, allowing bacteria into the middle ear space and leading to infection. Eustachian tube dysfunction in humans is often cause by a viral upper respiratory tract infection. Influenza, RSV, coronavirus, and adenovirus are associated with otitis media [67
] but may differentially impact Eustachian tube function [68
]. Rather than choosing one particular virus, we used barotrauma to create the negative pressure, which enabled us to reproduce the Eustachian tube dysfunction in a consistent, simplified manner. Day of barotrauma may affect the outcome of experimental OM. In the literature challenge to the middle ear via barotrauma or bacterial inoculation varies from 48 hours to 7 days [15
]. We selected day 5 for barotrauma to allow bacterial colonization to be established before infection and also to reflect peak incidence of acute OM following upper respiratory tract infection in children which occurs on days 3 and 5 [73
We developed a culture-independent assay to measure mixed culture strain composition. Generally, competition studies rely on spontaneous mutations or selectable markers in laboratory-altered strains for differentiation. However, these markers may introduce artificial fitness costs not found in the parent strain [38
]. By using viable count data and Quellung reaction serotyping data, we showed the qPCR assay is selectively and accurately amplifying the S. pneumoniae
strains in monoculture, and in mixed cultures in vitro
and in the chinchilla model of disease. To our knowledge, this is the first qPCR assay to take advantage of innate genetic divergences for differentiation between strains of the same species in order to measure in vitro
growth characteristics and in vivo
capacity. Traditionally, serotype assignment is completed on a limited number of colony forming units. Serotyping several hundred colonies per sample per animal is inefficient, costly and impractical. The qPCR assay uses the larger sample and provides definitive, quantitative results. The ability to assess capacity to colonize and to cause experimental OM in the disease model will assist in the search for virulence factors in S. pneumoniae
strains. In addition, the assay may be adapted for use with other bacterial species.
As with any assay, there are limitations to our method for assessing growth characteristics and infection potential. If the bacterial load in the chinchilla model is low, there is a limit of detection in qPCR. Also, it is possible to overload the qPCR with too much template [75
]. The simplest solutions are to decrease the volume of template added or to dilute the template. We found a 5-fold dilution was sufficient when saturation was a problem. qPCR does not provide a method for differentiating between metabolically active and inactive bacteria. We cannot definitely state whether the viable count data are yielding 100% of the live bacteria present in the sample or whether the qPCR assay is overestimating absolute bacterial counts by detecting DNA from dead bacteria. Conversely, researchers have demonstrated the presence of viable bacteria in culture-negative middle ear samples [76
]. Therefore, the assay provides a culture-independent method, which may be more accurate than culture methods. Post et al. have shown that after three days, DNA from dead bacteria in the chinchilla middle ear is no longer amplifiable [77
]. In addition, the qPCR analyses are dependent upon plasmid-based standard curve copy number calculations, which could contribute to either underestimation or overestimation of viable count. In this project, data from viable count and qPCR analyses were comparable. These limitations regarding detection of viable bacteria must be taken into consideration when evaluating absolute viable counts. This may be less of a problem here, when comparing strain relative ratios.
Despite these limitations, we believe our qPCR assay, when designed properly, provides a convenient, accurate, novel method for comparing closely related clinical isolates. The assay was designed to be easily adapted for use with other pneumococcal strains as well as other bacterial species with genomic plasticity, such as H. influenzae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus
]. The assay's ability to compare closely related strains of pneumococci without laboratory-based manipulations makes it a useful tool for evaluating virulence potential in the search for putative virulence factors among clinical isolates.
Data reported here, along with the observations that serogroup 15 is increasing in nasopharyngeal carriage, otitis media, and invasive disease all suggest that serotype 15B/C may soon become an important cause of pneumococcal disease [29
]. Shouval et al. describe that the likelihood of finding serotype 15B/C strains in invasive disease or acute OM following colonization is equal to finding it in carriage [9
]. Therefore, as the prevalence of 15B/C carriage increases, serotype 15B/C strains may become responsible for more disease. Continually adding serotypes to the current vaccines is not a sustainable approach to prevention and control of pneumococcal disease. As both serotype 15B/C and 19A were demonstrated to be virulent in our experimental OM model, further evaluation of non-vaccine serotypes to determine their capacity to colonize and produce acute OM is needed to understand the potential impact of next generation capsular vaccines. Further research is needed to identify other non-capsule virulence factors and to develop alternative strategies for prevention and control.