SUMMARY Host-to-host transmission is a critical step for infection. Here we studied transmission of the opportunistic pathogen Streptococcus pneumoniae in an infant mouse model. Transmission from nasally colonized pups required high levels of bacterial shedding in nasal secretions and was temporally correlated with, and dependent upon, the acute inflammatory response. Pneumolysin, a pore-forming cytotoxin and major virulence determinant, was both necessary and sufficient to promote inflammation, which increased shedding and allowed for intralitter transmission. Direct contact between pups was not required for transmission indicating the importance of an environmental reservoir. An additional in vivo effect of pneumolysin was to enhance bacterial survival outside of the host. Our findings provide experimental evidence of a microbial strategy for transit to new hosts and explain why an organism expresses a toxin that damages the host upon which it depends.

One of the least understood aspects of the bacterium Streptococcus pneumoniae (pneumococcus) is its transmission from host to host, the critical first step in both the carrier state and the disease state. To date, transmission models have depended on influenza A virus coinfection, which greatly enhances pneumococcal shedding to levels that allow acquisition by a new host. Here, we describe an infant mouse model that can be utilized to study pneumococcal colonization, shedding, and transmission during bacterial monoinfection. Using this model, we demonstrated that the level of bacterial shedding is highest in pups infected intranasally at age 4 days and peaks over the first 4 days postchallenge. Shedding results differed among isolates of five different pneumococcal types. Colonization density was found to be a major factor in the level of pneumococcal shedding and required expression of capsule. Transmission within a litter occurred when there was a high ratio of colonized "index" pups to uncolonized "contact" pups. Transmission was observed for each of the well-colonizing pneumococcal isolates, with the rate of transmission proportional to the level of shedding. This model can be used to examine bacterial and host factors that contribute to pneumococcal transmission without the effects of viral coinfection.T ransmission from one infected individual to another is necessary for most agents of infection. For Streptococcus pneumoniae (the pneumococcus), a leading opportunistic Gram-positive human pathogen, transmission occurs primarily from hosts colonized on the mucosal surfaces of their upper respiratory tract (the carrier state; 1). Bacterial acquisition and colonization of the mucosal surfaces of the nasopharynx by pneumococci are dynamic, transient processes which are generally asymptomatic and occur most commonly during early childhood. Under certain circumstances, however, pneumococci gain access to normally sterile environments within the host, resulting in diseases such as otitis media, pneumonia, sepsis, and meningitis. Acute respiratory infections associated with the pneumococcus represent a significant public health burden, with an estimated 14.5 million cases of serious pneumococcal disease worldwide (2). None of its many disease states, however, is thought to promote pneumococcal contagion (3).While bacterial and host factors contributing to the carrier state have been studied in the natural host and modeled in animals, there is relatively little mechanistic understanding of transmission (4). Person-to-person spread is thought to require close contact, such as within families or day care centers, either directly from nasal secretions or possibly via contact with contaminated surfaces (fomites; 3, 5). Therefore, one of the key steps in pneumococcal contagion involves the exit or shedding of the organism from the respiratory tract of a colonized individual.A factor known to enhance pneumococcal shedding is a concurrent or recent viral respiratory infection (6, 7). Increased rates of carriage associated ...

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.

An important yet poorly understood facet in the life cycle of a successful pathogen is the host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae (Kpn), a gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat as Kpn has become multi-drug resistant. Epidemiological studies suggest that Kpn host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Herein, we describe a murine model that can be utilized to study mucosal (oropharynx and gastrointestinal [GI]) colonization, shedding within feces, and transmission of Kpn through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we show that Kpn can asymptomatically colonize the GI tract of immunocompetent mice, and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent Kpn isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, Kpn carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient super-shedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors towards Kpn dissemination.

The capsular polysaccharide (CPS) of Streptococcus pneumoniae is characterized by its diversity, as it has over 95 known serotypes, and the variation in its thickness as it surrounds an organism. While within-host effects of CPS have been studied in detail, there is no information about its contribution to host-to-host transmission. In this study, we used an infant mouse model of intralitter transmission, together with isogenic capsule switch and cps promoter switch constructs, to explore the effects of CPS type and amount. The determining factor in the transmission rate in this model is the number of pneumococci shed in nasal secretions by colonized hosts. Two of seven capsule switch constructs showed reduced shedding. These constructs were unimpaired in colonization and expressed capsules similar in size to those of the wild-type strain. A cps promoter switch mutant expressing ~50% of wild-type amounts of CPS also displayed reduced shedding without a defect in colonization. Since shedding from the mucosal surface may require escape from mucus entrapment, a mucin-binding assay was used to compare capsule switch and cps promoter switch mutants. The CPS type or amount constructs that shed poorly were bound more robustly by immobilized mucin. These capsule switch and cps promoter switch constructs with increased mucin-binding affinity and reduced shedding also had lower rates of pup-to-pup transmission. Our results demonstrate that CPS type and amount affect transmission dynamics and may contribute to the marked differences in prevalence among pneumococcal types.

This study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that (i) differences in viral shedding of various IAV strains are dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins, (ii) host age plays a key role in the efficiency of IAV transmission, (iii) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV, and (iv) expression of sialidases by colonizing S. pneumoniae antagonizes transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion and its potential for optimizing vaccines and therapeutics that target disease spread.

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism’s transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM−/− host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism’s capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

According to the pre-recruitment hypothesis, Escherichia coli SoxS activates transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase that scan the chromosome for class I and class II SoxS-dependent promoters. Previously, we showed that the a subunit's C-terminal domain plays a role in activating both classes of promoter by making proteinprotein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the pre-recruitment hypothesis. Here, we identified seven single alanine substitutions of region 4 of the σ 70 subunit (σ 70 R4) of RNA polymerase that reduce SoxS activation of class II promoters. With genetic epistasis tests between these σ 70 R4 mutants and positive control mutants of SoxS we identified ten pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays we showed that SoxS and σ 70 R4 can interact in solution, i.e., "off-DNA". The interaction requires amino acids of the class I/II but not the class II positive control surface of SoxS and five amino acids of σ 70 R4 that reduce activation in E. coli also reduce the SoxS-σ 70 R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system, i.e., off-DNA. Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts σ 70 R4 both off-DNA as well as on-DNA is consistent with the pre-recruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNA polymerase.