Dried Saliva Spots: A Robust Method for Detecting Streptococcus pneumoniae Carriage by PCR.

The earliest studies in the late 19th century on Streptococcus pneumoniae (S. pneumoniae) carriage used saliva as the primary specimen. However, interest in saliva declined after the sensitive mouse inoculation method was replaced by conventional culture, which made isolation of pneumococci from the highly polymicrobial oral cavity virtually impossible. Here, we tested the feasibility of using dried saliva spots (DSS) for studies on pneumococcal carriage. Saliva samples from children and pneumococcus-spiked saliva samples from healthy adults were applied to paper, dried, and stored, with and without desiccant, at temperatures ranging from -20 to 37 °C for up to 35 days. DNA extracted from DSS was tested with quantitative-PCR (qPCR) specifically for S. pneumoniae. When processed immediately after drying, the quantity of pneumococcal DNA detected in spiked DSS from adults matched the levels in freshly spiked raw saliva. Furthermore, pneumococcal DNA was stable in DSS stored with desiccant for up to one month over a broad range of temperatures. There were no differences in the results when spiking saliva with varied pneumococcal strains. The collection of saliva can be a particularly useful in surveillance studies conducted in remote settings, as it does not require trained personnel, and DSS are resilient to various transportation conditions.

Carriage of Streptococcus pneumoniae in aged adults with influenza-like-illness.

Incidence of pneumococcal disease is disproportionally high in infants and elderly. Nasopharyngeal colonisation by Streptococcus pneumoniae is considered a prerequisite for disease but unlike in children, carriage in elderly is rarely detected. Here, we tested for S. pneumoniae in nasopharyngeal and saliva samples collected from community-dwelling elderly with influenza-like-illness (ILI). Trans-nasal nasopharyngeal, trans-oral nasopharyngeal and saliva samples (n = 270 per sample type) were collected during winter/spring 2011/2012 from 135 persons aged 60-89 at onset of ILI and 7-9 weeks later following recovery. After samples were tested for pneumococci by conventional culture, all plate growth was collected. DNA extracted from plate harvests was tested by quantitative-PCRs (qPCR) specific for S. pneumoniae and serotypes included in the 13-valent pneumococcal conjugated vaccine (PCV13). Pneumococci were cultured from 14 of 135 (10%) elderly with none of the sampled niches showing superiority in carriage detection. With 76/270 (28%) saliva, 31/270 (11%) trans-oral and 13/270 (5%) trans-nasal samples positive by qPCR, saliva was superior to nasopharyngeal swabs (p<0.001) in qPCR-based carriage detection. Overall, from all methods used in the study, 65 of 135 (48%) elderly carried pneumococci at least once and 26 (19%) at both study time points. The difference between carriage prevalence at ILI (n = 49 or 36%) versus recovery (n = 42 or 31%) was not significant (p = 0.38). At least 23 of 91 (25%) carriage events in 19 of 65 (29%) carriers were associated with PCV13-serotypes. We detected a large reservoir of pneumococci in saliva of elderly, with PCV13-serotype distribution closely resembling the contemporary carriage of serotypes reported in the Netherlands for PCV-vaccinated infants.

Respiratory microbiota dynamics following Streptococcus pneumoniae acquisition in young and elderly mice.

The upper respiratory tract (URT) is a distinct microbial niche of low-density bacterial communities and, also, a portal of entry for many potential pathogens, including Streptococcus pneumoniae. Thus far, animal models have been used to study the dynamics of and interactions between limited numbers of different species in the URT. Here, we applied a deep sequencing approach to explore, for the first time, the impact of S. pneumoniae acquisition on URT microbiota in a mouse model, as well as potential age-dependent effects. Young-adult and elderly mice were inoculated intranasally with S. pneumoniae, and nasal lavage samples were collected for up to 28 days postcolonization. Bacterial DNA extracted from lavage samples was subjected to barcoded pyrosequencing of the V5-to-V7 hypervariable region of the small-subunit rRNA gene. We observed highly diverse microbial profiles, with the presence overall of 15 phyla and approximately 645 operational taxonomic units (OTUs). We noted differences in the composition of microbiota between young and elderly mice, with a significantly higher abundance of Bacteroidetes in the young mice. The introduction of S. pneumoniae into the URT led to a temporary dominance of pneumococci in the microbiota of all mice, accompanied by a significant decrease in microbial diversity. As mice gradually cleared the colonization, the diversity returned to baseline levels. Diversification was accompanied by an early expansion of Bacteroidetes, Staphylococcus spp., and Lachnospiraceae. Moreover, the Bacteroidetes expansion was significantly greater in young-adult than in elderly mice. In conclusion, we observed differences in URT microbiota composition between naive young-adult and elderly mice that were associated with differences in pneumococcal clearance over time.

Immunosenescence and pneumococcal disease: an imbalance in host-pathogen interactions.

Respiratory infections are among the most important causes of morbidity and mortality from infectious diseases worldwide. The most common causative bacterium, Streptococcus pneumoniae, frequently colonises the upper respiratory tract, where it resides mostly asymptomatically. Occasionally, however, S pneumoniae can cause severe disease such as pneumonia. Local host immunity is essential to control colonising pathogens by preventing overgrowth, spread, and invasion. However, age-related immune deficits in elderly people, known as immunosenescence, might contribute to increased disease burden. We review present knowledge about immunosenescence in the respiratory tract against Gram-positive bacteria, particularly S pneumoniae. We discuss the possible underdetection of pneumococcal colonisation in elderly people, and suggest changes to present surveillance methods to improve understanding of the relation between colonisation and disease. We conclude that present knowledge about alteration of host-pathogen interactions by immunosenescence in the respiratory tract is insufficient, and that research is needed to enable improved measures for prevention.

Impaired innate mucosal immunity in aged mice permits prolonged Streptococcus pneumoniae colonization.

Streptococcus pneumoniae is a frequent asymptomatic colonizer of the nasopharyngeal niche and only occasionally progresses toward infection. The burden of pneumococcal disease is particularly high in the elderly, and the mechanisms behind this increased susceptibility are poorly understood. Here we used a mouse model of pneumococcal carriage to study immunosenescence in the upper respiratory tract (URT). Nasal mucosa-associated lymphoid tissue (NALT) showed increased expression of Toll-like receptor 1, interleukin-1ß, NLRp3 inflammasome, and CCL2 in naive elderly compared to young animals. This suggests an increased proinflammatory expression profile in the NALT of aged mice at baseline. Simultaneously, we observed a more tolerogenic profile in respiratory epithelia of naive elderly compared to young adult mice with upregulation of the NF-κß pathway inhibitor peroxisome proliferator-activated receptor gamma (PPARγ). After nasal instillation of pneumococci, pneumococcal colonization was prolonged in elderly mice compared to in young adults. The delay in clearance was associated with absent or delayed upregulation of a proinflammatory mediator(s) in the NALT, diminished influx of macrophages into the URT niche, and absent downregulation of PPARγ in respiratory epithelium, accompanied by diminished expression of cathelicidin (CRAMP) at the site of colonization. These findings suggest that unresponsiveness to pneumococcal challenge due to altered mucosal immune regulation is the key to increased susceptibility to disease in the elderly.

Dry-powder pulmonary insufflation in the mouse for application to vaccine or drug studies.

Pulmonary delivery of substances in small animal models is often useful for experimental testing of various vaccine and drug candidates. One of the most challenging elements to such protocols is the efficient disposition of test materials in the lungs of mice. Herein we detail a means to deliver dry powders of an inhalant live-attenuated Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine against Mycobacterium tuberculosis to the lungs of mice. The direct delivery methodology is quick, safe, and allows for repeated pulmonary insufflation of substances if boosting is desired. This model system could be easily adapted for use with other dry-powder vaccine and drug candidates.

Mycobacterium tuberculosis Rv2224c modulates innate immune responses.

Tuberculosis remains a major global health problem that kills up to 2 million people annually. Central to the success of Mycobacterium tuberculosis (Mtb) as a pathogen is its ability to evade host immunity and to establish a chronic infection. Although its primary intracellular niche is within macrophages, the underlying molecular mechanisms are poorly understood. Here we show that Rv2224c, a cell envelope-associated predicted protease, is critical for Mtb virulence. Disruption of Rv2224c led to prolonged survival of infected mice and highly reduced lung pathology. Absence of Rv2224c enhanced host innate immune responses, compromised the intracellular survival of Mtb in macrophages, and increased its susceptibility to lysozyme. We provide insights into the molecular basis for Rv2224c function by showing that Rv2224c activity promotes processing and extracellular release of the Mtb protein, GroEL2. Inhibition of Rv2224c and its targets offers opportunities for therapeutic interventions and immune-modulatory strategies.