You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Single Cell Bottlenecks in the Pathogenesis of Streptococcus pneumoniae}
Article:
Abstract:
Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A( IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion.
Introduction:
The pathogenesis of microbial diseases generally involves multiple stages( entry, establishment, invasion, exit) that often begin with the colonization of host surfaces. For organisms without an environmental reservoir, their continued success requires proliferation within their obligate host and transmission to new susceptible hosts. The induction of disease, which usually results from a combination of impaired host defense and microbial virulence attributes, may benefit the organism if it increases proliferation and/or transmission. An example of an organism with a predominantly commensal lifestyle that also is a leading cause of disease is Streptococcus pneumoniae( the pneumococcus)[1]. Pneumococci serially and sequentially colonize the mucosal surface of the human nasopharynx asymptomatically beginning in early childhood( the carrier state). Transmission occurs from carriers to non-carriers and is most frequent in settings of close contact, such as among siblings or in daycare centers, and results from direct or indirect exposure to respiratory secretions[2][3]. Disease occurs when the organism transits to normally sterile sites within the respiratory tract to the middle ear cavity or lungs. The organism may also gain access to the bloodstream from the nasopharynx or sites of localized disease to cause systemic infection. Because of high rates of carriage and these complications, the pneumococcus is a leading cause of otitis media, pneumonia, and sepsis[1][4]. An additional consideration is that recent upper respiratory viral infection, particularly with influenza, increases rates of carriage and is a major risk factor for all pneumococcal diseases[5][6][7]. Pneumococcal infection has been partially controlled through immunization. The most well established effect of vaccine-induced immunity in adults is protection of the individual from pneumococcal bacteremia[8][9]. More recently, widespread immunization of children, which blocks the acquisition of colonization, has led to lower rates of transmission within the community and protection of unvaccinated populations( ‘herd immunity’)[10][11]. Together these clinical observations suggest that it is possible to impact pneumococcal disease at discrete steps in its pathogenesis. In order to better understand the mechanisms responsible for protection, we sought to identify the steps during infection where pneumococci must pass through a population bottleneck( s) –a sharp reduction in the size of the population due to environmental constraints[12][13]. A bottleneck would occur, for instance, if only a fraction of infecting organisms are able to pass through a host barrier or evade a local host defense. Alternatively, a bottleneck could result from microbial competition or a requirement for increased fitness via genetic adaptation( within-host evolution) to meet different challenges in the host. Tight bottlenecks would be attractive ‘weak points’ for intervention strategies since there would be relatively few organisms to target. Also, by targeting the stages when the bacterial population size is most restricted, there is less opportunity for selection among the phenotypically and genetically diverse population that characterizes the pneumococcus. Since there are no tractable experimental models to study steps beyond colonization in the natural host, we examined these in an animal model. Our study employed an infant mouse model that recapitulates many of the key features of pneumococcal pathogenesis. These include increased susceptibility early in life, occurrence of localized disease in normally sterile sites within the respiratory tract( otitis media) and invasive infection( bacteremia) following colonization, and close contact leading to increased host-to-host transmission.
Discussion:
This report describes an animal model that allows for the study of many of the consequences of bacterial colonization including localized and systemic disease and host-to-host transmission. We utilized this model to examine the effects of a clinically important viral co-infection. We found that IAV increases the burden of colonizing pneumococci consistent with prior observations in infant mice[18]. A similar effect had been shown in adult mice and was attributed to the increased availability of the nutrient sialic acid in secretions stimulated by IAV[16]. The greatly increased bacterial shedding and transmission in the setting of IAV described in the current report was out of proportion to the relatively modest difference in colonization density and could be attributable to the effect of the virus on secretions enhancing bacterial egress from the host. The higher rate of otitis media in the setting of IAV is consistent with prior reports and could be due to either an increased number of colonizing organisms or eustachian tube dysfunction[19][20]. The use of the infant mice avoided the ‘limited effective population’ that has been proposed in colonization of adult mice and that could hamper the analysis of population bottlenecks[21]. In this regard, we established that following a low inoculum dose all infant mice were colonized by all three of the strains and that each of three strains was shed every day post-challenge. This enabled a comprehensive analysis of bottlenecks for events following colonization. A limitation of our study using infant mice was the inability to obtain multiple samples at different times during infection from the same animal. Thus, we could only assess population bottlenecks at a single time point. Our study suggests that the host response rather than microbial factors are the main determinants of the size of these population bottlenecks. In the case of bacteremia, the population bottleneck in passage from the mucosal surface to the bloodstream was tight, but even more restricted in the setting of IAV co-infection. This increased barrier could be due to the mucosal inflammatory response that accompanies IAV infection. The effect on IAV on restricting the size of the bottleneck in bacteremia added further support that this bottleneck exists at the mucosal interface since IAV infection does not cause viremia. It is interesting that the onset of sepsis/bacteremia was delayed up to eight days after colonization was established and did not occur until about twelve days age regardless of the duration of colonization. This age-related invasive infection in infant mice differs from congenic adult mice, which become septic within 2–4 days following IN challenge with the same pneumococcal isolate[22]. The overall inflammatory milieu of the respiratory mucosa is elevated in infant compared to young adult mice[16]. This more inflamed state could tighten the bottleneck and result in a more delayed onset of bacterial invasion in infants. An alternative explanation is that additional events are required during infant infection and raises the possibility of a reservoir for the organism outside the upper respiratory tract from which seeding of the bloodstream occurs. This reservoir did not appear to reside in the lower respiratory tract. A reservoir was also proposed by Moxon and Murphy, who demonstrated single cell bottlenecks in bacteremic infection of infant rats nasally challenged with Haemophilus influenzae type b following an eclipse phase[23]. We previously described how TLR- or TGFβ-signaling in polarized epithelial cells and in vivo opens tight junctions between cells and facilitates bacterial invasion[24]. It would be interesting to test whether such signaling has the effect of loosening the size of the bottleneck in bacteremia. Lastly, our data showing the lack of tight bottleneck following IP challenge with a low inoculum contrasts with those of Gerlini et al, who described a tight bottleneck in pneumococcal bacteremia of adult mice following intravenous challenge with a high inoculum( 3×105 CFU) with a different bacterial strain and in a different mouse line[25]. Of note, their challenge with ~103 more pneumococci appears to overwhelm splenic clearance. The tight bottleneck we observed following invasion from the mucosal surface, where pneumococcal bacteremia originates, suggests that a large bolus of pneumococci may not access the bloodstream in this manner. Also, the Gerlini et al study followed bacteremia with multiple blood cultures over 3 days; whereas our use of infant mice precluded multiple sampling and all pups had to be sacrificed within hours of acquiring invasive infection due to the rapid onset of sepsis. In the case of transmission, the identification of a tight bottleneck explains the requirement for IAV co-infection. This was attributed to mucosal inflammation and increased secretions in the setting of IAV facilitating bacterial exit and transit to a new host. The use of mice lacking TLR2-signaling needed to control responses to IAV that showed increased bacterial shedding and a looser bottleneck confirmed these effects of viral infection[14][26]. This correlation between the number of shed bacteria and the incidence of transmission provides an explanation why IAV co-infection is required to raise transmission events to a detectable level. The effect of IAV on nasal secretions could be explained by its stimulation of TLR3-signaling since IN administration of the analog of viral dsRNA, poly-ICLC, was sufficient to increase bacterial shedding in the absence of viral infection. Other pathways involved in innate responses to IAV may also contribute to these effects on the host. In contrast, intranasal administration of bacterial PAMPs were ineffective in stimulating an inflammatory response sufficient to enhance bacterial shedding. These findings suggest there may be specificity for signaling pathways triggered by viral infection. We propose that in this model without IAV the number of shed bacteria would be generally insufficient to surpass the threshold required to transit the tight bottleneck. A further consideration is that the higher inoculum required for establishing colonization in adult mice is unlikely to pass through this tight bottleneck explaining the lack of transmission beyond the infant period[16]. The demonstration of a single cell bottleneck in transmission provides context for understanding some its requirements. There did not appear to be a requirement for multiple organisms to enter a new host or for genetic adaptation to establish colonization upon entry into a new host. Rather our findings suggest that events prior to entry are critical. In particular, since transmission rates correlate with the density of shed organisms, it appears that there must be a sufficient number of bacteria exiting their niche on the mucosal surface for a single organism/chain to succeed in reaching the nasal mucosa of a new, susceptible host. This level of shedding might have to be actively induced by environmental factors in the host as demonstrated by the requirement for IAV co-infection in this model system. Shed organisms must also survive for a sufficient period to encounter a new host. Bacterial constituents that allow for survival between hosts are poorly understood, although a prior report documented that this species is highly resistant to desiccation[27]. The small number of pneumococci able to succeed in being transmitted is likely why additional transmission events were not observed in pups where colonization was already established, since the minimum colonizing dose is greater than two-logs higher in this setting. A tight bottleneck in transmission also explains why close contact between individuals, and in particular with young children, is typically required for pneumococcal ‘contagion’. We are currently determining whether there are specific bacterial factors that actively induce shedding and enhance transmission. The plasticity of the pneumococcus is the major challenge to prevention of disease through immunization and control by antibiotics. Here we demonstrate two key steps in its pathogenesis, transmission and bacteremia following colonization, where its population size is highly restricted and, therefore, its ability to take advantage of its marked genetic diversity and adapt by genetic exchange is minimized.
Many discrete steps are involved in the progression of infectious diseases . Bottlenecks represent key points where the population size/genetic diversity is at a minimum and the pathogen is most vulnerable to intervention strategies . Our study used an infant mouse model for a comprehensive analysis of bottlenecks in infection by the major pathogen Streptococcus pneumoniae . In our model , we also considered influenza A virus , a clinically important and common co-infection . The main findings reveal i ) a single cell bottleneck during host-to-host transmission and ii ) the bottleneck in transmission occurs during events between bacterial exit from one host and establishment in another host . We manipulated innate immune responses involved in viral control and inflammation to show that viral co-infection allows the bottleneck in transmission to be overcome by increasing bacterial exit . Finally , we demonstrate that a specific host response stimulated by influenza A is sufficient to recapitulate effects of viral co-infection . Thus , our study identifies key vulnerable stages during S . pneumoniae infection and provides mechanistic understanding for how viral infection promotes bacterial contagion .