Abstract
The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner.
Highlights
Nasopharyngeal colonization with the respiratory pathogen Streptococcus pneumoniae is a prerequisite for the development of pneumococcal disease
To determine if agglutination can be measured by flow cytometry, we compared agglutinated with non-agglutinated pneumococci
We found that a large proportion of the bacteria incubated with ST4-specific serum had increased on both the FSC and SSC axis, as compared to the bacteria incubated with serotype 14 (ST14)-specific serum (Fig 1A & 1B)
Summary
Nasopharyngeal colonization with the respiratory pathogen Streptococcus pneumoniae is a prerequisite for the development of pneumococcal disease. Following dissemination of bacteria to the ear, lung, bloodstream or brain, otitis media, pneumonia, sepsis or meningitis may develop, respectively. Several mucosal defense mechanisms, such as antibody-mediated opsonization and opsonophagocytosis by phagocytes, have been suggested to be important in the reduction or complete prevention of colonization [1,2]. Roche et al (2015) showed that the presence of agglutinating antibodies on the mucosal surface plays an important role in the prevention of pneumococcal colonization in a mouse model of colonization and transmission [3]. The agglutinating properties of antibodies raised against novel vaccine candidates.
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