ABSTRACT
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.
INTRODUCTION
Transmission 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 with viral respiratory infection are thought to be a consequence of both more-frequent transmission and a higher burden of colonizing bacteria (8–10). The altered dynamics of bacterial carriage during influenza outbreaks is associated with higher rates of pneumococcal disease, particularly among vulnerable populations (11). Horizontal transmission from colonized hosts has been modeled by McCullers in ferrets and is enhanced by influenza infection (11). Intralitter pneumococcal transmission has also been demonstrated among infant mice, a more tractable model host, but requires influenza coinfection (12, 13). Our group used this model to show that increased pneumococcal acquisition by initially uncolonized “contact” pups correlates with increased mucin production and bacterial shedding by colonized “index” pups with influenza A virus (IAV) coinfection (7). These responses among index pups were chiefly driven by the mucosal inflammatory response to IAV, which recapitulates the effects of respiratory viruses in children (6, 12). Although most pneumococcal transmission in the human population may be seasonal, it is not generally associated with influenza coinfection (14). It is therefore unclear whether the more mild inflammatory response that characterizes pneumococcal monoinfection is sufficient to allow levels of shedding that could promote transmission (7, 15). Here, we describe the adaptation of the infant mouse model to enable the study of pneumococcal shedding and transmission without the confounding effects of viral coinfection.
MATERIALS AND METHODS
Ethics statement.This study was conducted according to the guidelines outlined by National Science Foundation Animal Welfare Requirements and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. The New York University Medical Center (NYUMC) IACUC oversees the welfare, well-being, and proper care and use of all vertebrate animals used for research and educational purposes at the NYU Langone Medical Center and School of Medicine. The NYUMC IACUC Assurance Number is A3435-01. The approved-protocol numbers for this project are 150216-01 and 150520-01.
Bacterial strain construction and growth conditions.Strains used in this study are listed in Table 1. Pneumococcal strains were grown statically in tryptic soy (TS) broth (Becton Dickinson, Franklin Lakes, NJ) to mid-exponential phase at 37°C. When the bacterial culture reached the desired optical density at 620 nm (OD620), cells were washed and diluted in sterile phosphate-buffered saline (PBS) for inoculation. Ten-fold serial dilutions were plated on selective media in triplicate for quantitative culture. TS agar plates were supplemented with either 5% sheep blood or catalase (Worthington Biochemical Corporation, NJ) (6,300 U/plate) and were incubated overnight at 37°C with 5% CO2. Bacteria were stored in 20% glycerol at −80°C.
Strains used in this study
P1542 (type 4) and P1547 (type 6A) were made streptomycin resistant (Smr) by transformation with a PCR product made using the rpsL-forward (5′-GATAAGGACAGAACCAGTTCC-3′) and rpsL-reverse (5′GCATCCTATCTTACCAACGG-3′) primers amplifying regions 1,000 bp upstream and downstream of the rpsL gene. The template for the PCR was genomic DNA, obtained using a MasterPure DNA purification kit (Illumina), from strain P1397 (type 23F) (16), which contains a point mutation in rpsL. Resistant strains were selected on 5% sheep blood agar plates containing streptomycin (200 μg/ml). To construct P2422, a capsule-deficient mutant, strain P2406 was transformed with genomic DNA from P2407, containing a previously described insertion of the “sweet Janus” cassette in the cps locus, with selection on TS agar plates supplemented with 5% blood and kanamycin (kan) (TS-kan) (500 μg/ml) (20). Single colonies were replated on TS-kan, and unencapsulated colonies were selected based on their “rough” phenotype. To construct a cps+ corrected mutant (P2438), P2422 was transformed with genomic DNA from P2406 with selection on TS agar plates supplemented with streptomycin (200 μg/ml) and 10% (wt/vol) sucrose. Both the capsule-deficient mutant and cps+ strains were confirmed by an immunoblot assay using an anti-type 4 capsular polysaccharide monoclonal antibody (MAb) kindly provided by M. Nahm (University of Alabama at Birmingham). TIGR4 lacking its pilus (P2454) was constructed as follows: genomic DNA isolated from strain AC2306 (TIGR4, ΔrlrA), kindly provided by A. Camilli (Tufts University), was used to transform strain P2406 (TIGR4 Smr), and the resulting transformants were selected on TS agar plates supplemented with chloramphenicol (4 μg/ml).
Western blot analysis.Loss of the pilus in strain TIGR4 was confirmed by Western blotting. Strains were grown as described above to an OD620 of ∼1.0, collected by centrifugation, and resuspended in loading buffer. Samples were boiled for 10 min and loaded onto a Bolt 4–12% Bis-Tris Plus gel (Invitrogen). Proteins were transferred to a nitrocellulose membrane using a Novex iBlot system (Life Technologies). After blocking with 1% bovine serum albumin (BSA) in 1× PBS, the following primary and secondary antibodies were used: polyclonal rabbit antisera to RrgB (pilus subunit), kindly provided by R. Malley (Boston Children's Hospital), at a concentration of 1:200,000, and goat anti-rabbit IgG alkaline phosphatase antibody (Sigma A3687), at a 1:4,000 concentration. Signal was visualized with 1-Step NBT/BCIP (Nitro Blue Tetrazolium/5-bromo-4-chloro-3-indolylphosphate) substrate for alkaline phosphatase (Thermo Scientific).
Colonization and shedding in infant mice.Wild-type (WT) C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred and maintained in a conventional animal facility. The pups were delivered in our facility and maintained with their dam for the course of the experiment. Pups infected intranasally (i.n.) with infectious agents were monitored throughout the course of the experiments and appeared healthy and gained weight at a rate similar to that seen with uninfected animals.
Four-day-old pups were inoculated i.n. without anesthesia with ∼2,000 CFU of S. pneumoniae suspended in 3 μl of PBS. Briefly, the bacterial suspension was placed on the nares with a blunt pipette tip and the pup was allowed to inhale the inoculum, at which point it was returned to its dam. To quantify pneumococcal shedding from the upper respiratory tract, the nasal secretions of infected pups were cultured by gently tapping the nares (20 taps/pup) of the pup onto a TS agar plate supplemented with streptomycin (200 μg/ml) to prevent the growth of contaminants (7). When shedding was quantified for a strain not resistant to streptomycin, TS agar plates supplemented with neomycin (10 to 20 μg/ml) were used. The sample was then evenly spread across the plate using a sterile swab and incubated overnight at 37°C with 5% CO2.
At age 9 days, pups were euthanized by CO2 asphyxiation followed by cardiac puncture, the upper respiratory tract was subjected to lavage with 200 μl of sterile PBS from a needle inserted into the trachea, and fluid was collected from the nares. Ten-fold serial dilutions of the collected fluid were plated as described above. The limit of detection was 33 CFU/ml unless otherwise noted.
In other experiments to determine the effect of IAV on pneumococcal shedding, at age 9 days, pups were inoculated i.n. with the IAV/HKx31 strain (2 × 104 50% tissue culture infective doses [TCID50]) suspended in 3 μl of PBS. IAV/HKx31 (H3N2) was grown in the allantoic fluid of 10-day-old embryonated chicken eggs (B&E Eggs) and stored at −80°C. Viral concentrations for infection were determined by titration in Madin-Darby canine kidney cells, as described previously (21).
Transmission in infant mice.The transmission model described in this study is based on a modification of a previously described study (7). Briefly, for IAV coinfection transmission experiments, 1 in 3 pups in a litter were randomly selected as index mice and at age 4 days were infected with TIGR4SmR as described above. The index mice were then returned to presence of the dam and the other uninfected pups (contact mice). At 8 days of age, all pups were inoculated i.n. with the influenza A virus/HKx31 strain as described above. To detect bacterial transmission from the index pups to the contact pups, all pups were euthanized at age 14 days, and nasal lavage samples were collected and serial dilutions plated on selective medium. For experiments performed with the type 6A isolate (P2432), pups were euthanized at age 12 days, because at later time points the pups developed septic infection. Transmission experiments performed with pneumococcal monoinfection were carried out as described above with bacterial inoculation of mice at age 4 days and no treatment at age 8 days. In transmission experiments performed with a 1:1 ratio of index mice to contact mice, the same protocol was followed with half of the litter designated index mice and inoculated at age 4 days.
Statistical analysis.All of the statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Differences were determined using the Mann-Whitney U test (comparing two groups) or the Kruskal-Wallis test with Dunn's postanalysis (comparing multiple groups).
RESULTS
Shedding of pneumococcal strain TIGR4.Because of the critical role in transmission for bacterial exit from the colonized host, we screened for factors affecting pneumococcal shedding by infant mice in the absence of IAV coinfection. Infant C57BL/6 mice were colonized by i.n. inoculation of ∼2,000 CFU of strain TIGR4SmR (type 4) in PBS (3 μl) and the pups returned to the dam. Shedding was monitored daily by gently tapping the content of the nares of each pup (20 taps/pup) onto selective media as previously described (7). When pups were inoculated at age 4 days, shedding typically peaked at age 6 to 7 days and diminished significantly by age 9 days (Fig. 1A). At this point, the pups were euthanized and nasal lavage samples cultured to assess colonization density.
Quantification of bacterial shedding from the nares of pups infected with Streptococcus pneumoniae either alone or in a coinfection with influenza A virus. (Ai) Schematic representation of the inoculation, shedding, and nasal lavage schedule. Four-day-old pups were intranasally (i.n.) inoculated with ∼2,000 CFU of TIGR4SmR. Daily shedding results were collected and quantified on the days shown. The cross indicates the day of euthanization. (Aii) Shedding values for each day postinoculation, with median values shown and each symbol representing the CFU observed from a single mouse on that day. Data represent results of multiple independent experiments. The panel-wide horizontal line represents the 300-CFU threshold level described in Results. (Bi) Schematic representation of the inoculation, shedding, and nasal lavage schedule with influenza A virus coinfection. Four-day-old pups were inoculated i.n. with ∼2,000 CFU of TIGR4SmR and infected i.n. with the influenza A virus/HKx31 strain (2 × 104 TCID50) at 9 days of age, with daily shedding results collected and quantified on the days shown. (Bii) Shedding values for each day postinoculation after i.n. infection with influenza A virus. (C) Pattern of shedding from three individual pups during pneumococcal monoinfection, with determinations performed at least twice per day, with daily means for each mouse connected by a line. ***, P < 0.001.
In the previously described model, IAV was typically administered to pups aged 8 to 9 days (7). Coinfection with the HKx31 strain of IAV of pups previously colonized with TIGR4SmR increased the numbers of shed pneumococci, which peaked at about age 11 days (Fig. 1B). Almost all shedding events from age 11 days to age 13 days exceeded 300 CFU/20 taps—a level that was considered a significant threshold because average shedding above this level in our prior study was associated with pup-to-pup transmission (7). Although we observed less shedding without IAV coinfection, a substantial portion of TIGR4SmR shedding events in pups from 5 to 9 days of age were above this threshold level.
The quantification of shedding during pneumococcal monoinfection was characterized by marked pup-to-pup variation (results for three representative pups are shown in Fig. 1C). Additionally, when individual pups were tested several times in a single day, the level of shedding varied by as much as ∼8-fold in some pups, whereas shedding values were relatively constant for other pups in the same litter. Due to this variable and hectic pattern, daily shedding values were pooled and compared over a 5-day observation period in all subsequent experiments. According to the data combined in this manner, the mean level of shedding varied from pup to pup within a litter by less than ∼30%.
Pneumococcal shedding is age dependent.We focused on infant mice because transmission has not been observed among adults, an effect that could be due to the higher dose required to colonize adult mice (22). To further explore the contribution of age at the time of inoculation, mice were infected at age 4, 8, or 12 days and shedding was monitored over the next 5 days (Fig. 2A). There was a steady decrease in overall shedding with increasing age at the time of infection. This decline in shedding with increasing age could have been caused by the lower density of colonizing pneumococci in older pups (Fig. 2B).
Shedding and colonization density decline with increasing age of pups at the time of infection. (A) Pups were challenged i.n. with TIGR4SmR at age 4, 8, or 12 days, and daily shedding values are shown for all 5 days of the experiment. Each symbol represents CFU from 20 taps from a single mouse on 1 day, with the median value shown. (B) The colonization density for each pup in the experiment described for panel A was determined at the age indicated after shedding was measured over 5 days postinoculation, with the median value shown. *, P < 0.05; ***, P < 0.001. L.O.D, limit of detection.
Variation in shedding by different pneumococcal strains.We next considered strain-to-strain variation in pneumococcal shedding. Four additional unrelated isolates of different serotypes were compared for shedding over a 5-day period following inoculation of 4-day-old pups with ∼2,000 CFU. Two (types 2 and 19F) of the four strains tested shed significantly less than TIGR4, whereas the type 6A and 23F isolates showed equivalent levels of shedding (Fig. 3A). However, the proportion of high-shedding events (>300 CFU/20 taps) was significantly greater for the type 4 isolate (TIGR4) than for the type 6A (P = 0.01), type 19F (P = 0.01), and type 23F (P = 0.0065) strains as assessed by Fisher's exact test. Although shedding results differed among strains, colonization density levels at the time of sacrifice at age 9 days were equivalent, except for the type 2 strain (D39), which colonized poorly (Fig. 3B). Our findings confirmed that low colonization density is associated with reduced shedding but also suggested that strain-specific factors that do not impact colonization affect shedding.
Differences among pneumococcal strains in the levels of shedding and colonization. (A) Pups were challenged i.n. at age 4 days with an isolate of the type indicated below, and daily shedding values are shown for all 5 days of the experiment. Each symbol represents CFU obtained from a single mouse on 1 day, with the median value shown. The panel-wide horizontal line represents the 300-CFU threshold level described in Results. (B) The colonization density for each pup tested as described above was determined at age 9 days, after shedding was measured over 5 days postinoculation, with the median value shown. **, P < 0.01; ***, P < 0.001.
Pneumococcal factors contributing to shedding and colonization.To examine the contribution of the major cell surface feature and virulence determinant of the pneumococcus, we tested shedding of a capsule-deficient mutant of strain TIGR4SmR. This was generated using an insertion mutation in the cps locus. To confirm the role of this locus, a corrected mutant that restored expression of the type 4 capsule was also constructed. The capsule-deficient mutant shed significantly less than the parent wild-type (WT) strain (Fig. 4A). This effect was cps specific, as shedding was restored to WT levels when the cps locus was reintroduced (cps+). However, colonization of the capsule-deficient mutant was significantly lower than that of both the encapsulated parent strain and the corrected mutant strain (Fig. 4B). These findings demonstrate the importance of capsule in shedding but suggest that the major role of capsule in shedding is that of its effect on colonization density as previously reported in adult mice (23). This observation, together with data from strain D39, supported the conclusion that high colonization density is a requirement for shedding to exceed threshold levels.
The effect of pneumococcal surface factors on shedding and colonization. (A) Pups were challenged i.n. with TIGR4SmR (WT), with a capsule-deficient mutant, or with an encapsulated corrected mutant (cps+) at age 4 days, and daily shedding values are shown for all 5 days of the experiment. Each symbol represents CFU obtained from a single mouse on 1 day, with the median value shown. (B) The colonization density for each pup tested as described above was determined after shedding was measured over 5 days postinoculation, with the median value shown. (C) Western blot using antibody to the RgrB subunit of pneumococcal pilus. (D) Pups were challenged i.n. with the TIGR4SmR (WT) strain or a pilus islet deletion mutant (ΔrlrA strain) at age 4 days, and daily shedding values are shown for all 5 days of the experiment. (E) The colonization density of each pup tested as described for panel D was determined after 5 days postinoculation, with median values shown. **, P < 0.01; ***, P < 0.001.
Since shedding was highest for strain TIGR4, we investigated the role of the pneumococcal pilus, a surface adhesion expressed by this strain but present in the minority of other pneumococcal isolates (24). Expression of the RrgB pilin subunit revealed by Western blotting was used as a marker of piliation. Of the isolates tested for shedding, only TIGR4 expressed the RrgB pilin subunit (Fig. 4C). There was, however, no significant difference in shedding or colonization between a pilus-locus deletion mutant of TIGR4 and the parent strain, making it unlikely that the pneumococcal pilus contributes to the phenotypes tested in our model (Fig. 4D and E).
Pneumococcal transmission without influenza A virus coinfection.Our previous infant coinfection study linked high pneumococcal shedding to the transmission of the type 23F isolate (7). These conditions used a ratio of colonized index (donor) pups to uncolonized contact (recipient) pups of 1:3. Transmission was assessed in nasal lavage samples obtained from contact pups at age 14 days to allow time after peak shedding for stable colonization to be established. In the current study, we first recapitulated this result using TIGR4SmR with IAV coinfection and observed a transmission rate of 100% with the same inoculation protocol (Fig. 5A). Next, we examined the results to determine whether transmission occurs without IAV coinfection under these same conditions, but no transmission events were detected (Fig. 5B).
Pneumococcal transmission with and without the influence of influenza A virus (IAV) coinfection. (Ai) Schematic representation of the schedule used to determine pneumococcal transmission with IAV coinfection. Strain TIGR4SmR (∼2,000 CFU) was administered i.n. to index mice only on day 4 of life, and then the HKx31 strain of IAV (2 × 104 TCID50) was given i.n. to both index mice and contact mice on day 8 of life. The cross indicates the day of euthanization. (Aii) Pups were inoculated and euthanized following the schedule described, with a 1:3 ratio of index mice to contact mice. Results show the colonization density in nasal lavage samples for individual pups, with median values indicated. (Bi) Schematic representation of the inoculation and nasal lavage schedule used to determine transmission of strain TIGR4SmR without IAV. (Bii) Pups were inoculated and euthanized following the schedule as described above with two different ratios of index mice to contact mice as indicated. Results show the colonization density in nasal lavage samples for individual pups, with median values indicated. *, P < 0.05.
Since the shedding of TIGR4SmR was severalfold lower in the monoinfection model than in an IAV coinfection model, we postulated that a higher ratio of index pups to contact pups would increase the overall number of shed pneumococci/litter and facilitate transmission. With a 1:1 ratio of index pups to contact pups, we observed transmission of TIGR4SmR, albeit at a rate that was lower than that seen with the coinfection model (29%) (Fig. 5B). We then determined whether we could use this ratio (1:1) to observe transmission without IAV coinfection with other isolates that shed at different levels (types 6A, 19F, and 23F) but colonize robustly (Fig. 6A). Transmission was observed at a 1:1 ratio of index mice to contact mice for each isolate tested. Rates of transmission among the strains paralleled the level of shedding and proportion of high shedding values (>300 CFU/20 taps) (compare Fig. 3A to Fig. 6A). All the strains colonized index mice equally well at the time of sacrifice of the mice at age 14 days, with the exception of the 6A isolate (Fig. 6B). The period of contact between the index mice and the contact mice was also extended to age 21 days (age at weaning) to determine if the rate of transmission would increase with more time. However, there was no change in the transmission rate despite extending the contact period (data not shown).
Transmission of pneumococcal isolates of other serotypes during monoinfection. (A) Transmission from index mice to contact mice with a 1:1 ratio of index mice to contact mice for isolates of the types indicated according to the schedule shown in Fig. 5Bi. (B) Comparison of the colonization density levels measured at the time of sacrifice for the index mice used in transmission experiments performed with isolates of different types, with median values indicated. **, P < 0.01.
In summary, we have described a tractable model and suitable conditions, including the commonly used strain TIGR4, for studying pneumococcal transmission in the absence of viral coinfection.
DISCUSSION
Our understanding of the biology of transmission for infectious agents has been limited by a lack of model systems and particularly of tractable animal models. In this report, we describe the use of infant mice to study intralitter transmission of S. pneumoniae. This model of pneumococcal monoinfection was developed by modification of a previously described protocol that depended on concurrent IAV infection (7, 13). The ability to study transmission without viral coinfection will allow a thorough examination of pneumococcal factors and of host responses to pneumococcal colonization that affect transmission.
An important determining factor for pneumococcal transmission is the burden of exiting or shed organisms in secretions of the colonized host. We used a quantitative shedding assay to screen for bacterial and host factors that could affect transmission to optimize this model. The shedding assay is limited, however, by considerable animal-to-animal and temporal variability requiring multiple sampling events. Using this approach, we found that daily shedding was maximal over the first 4 days after colonization was established—an observation that correlates with the peak of the acute inflammatory response to pneumococcal colonization (15). In a prior report, we described how the inflammatory response to IAV in the airway mucosa drives shedding, and the results described above suggest that this may also be the case for inflammation in response to pneumococcal monoinfection (7).
We found that the level of pneumococcal shedding is highest when mice are infected at a younger age, is proportional to the level of bacterial colonization, and varies significantly among pneumococcal isolates. Other than differences in colonization density, it is unclear why there is strain-to-strain variation. Pneumococci must adhere to the host to establish stable carriage but must also detach at a sufficient frequency to allow exit and transmission to a new host. Among the strains tested, shedding was the highest for TIGR4, so we examined and excluded the potential for its pilus adhesin to mediate its increased shedding. Similarly, pilus islet-1-positive strains were no more likely to be transmitted in a longitudinal carriage study (25). A number of other pneumococcal adhesins have been described, and the distributions of these differ considerably between strains (26). Among the factors that could affect levels of shedding are differences in capsular polysaccharide composition. The amounts and types of capsule impact adherence to host cells as well as interactions with, and escape from, mucus in the nasopharyngeal niche (23). The expression of capsular polysaccharide was shown to be critical to attainment of significant levels of shedding, but this could be due to the effect of capsule on colonization density. Because of the marked variation in capsule amount and chemical composition, it would be interesting to explore whether these affect the proportion of colonizing pneumococci that are shed and transmitted.
Although the median level of shedding was ∼100 CFU/20 taps for TIGR4 during monoinfection, daily values of >2,000 CFU/20 taps were occasionally observed. This observation raised the possibility that transmission required variant “supershedder” bacteria or pups that acted as “superspreaders” (27). However, when a putative high-shedding (>2,000 CFU/20 taps) strain was retested in another litter it did not display significantly increased shedding (data not shown). Moreover, when a putative superspreader pup was followed, it was no more likely to continue to shed at a higher level than its littermates (data not shown). Therefore, we were unable to show that either supershedder bacteria or superspreader mice contribute to transmission in our model.
We found that transmission during pneumococcal monoinfection was undetectable when the protocol described for IAV coinfection was followed (7). By using TIGR4 and modifying the ratio of donor index mice to recipient contact mice, we were able to achieve a reliable rate of transmission. We observed that, with pneumococcal monoinfection using a ratio of one index pup to one contact pup, the transmission rate was as high as ∼29%. This higher ratio could increase the chances that a contact pup would have sufficient physical interaction with a colonized index pup or with fomites harboring sufficient numbers of the organism. Our ongoing studies suggest that there is a tight population bottleneck in transmission between index pups and contact pups in the setting of IAV coinfection (unpublished data). More index pups/cage could make it more likely that this bottleneck would be overcome in a situation in which there was less shedding/pup in the absence of IAV coinfection. A caveat concerning the model is that once a contact pup becomes colonized it may be a source of further transmission, and, therefore, the transmission rates that we report here could be overestimates. That said, in an average litter with about eight pups, using a 1:1 ratio of index mice to contact mice, we estimate there would be on average one transmission event per litter.
Thus, we have described a useful model to study pneumococcal transmission without the potentially confounding effects of viral coinfection. This model recapitulates many of the key features of pneumococcal transmission in the natural host in the absence of viral upper respiratory infection. These include an increased rate in early childhood, strain-to-strain differences, and the requirement for close contact (15). The transmission rate among colonized children has not been characterized in detail and may be higher or lower than the rates determined in this report using infant mice.
Epidemiologic studies performed following the introduction of widespread pneumococcal immunization in early childhood have shown that colonized children are the major source of the pathogen for older populations (28). The most vulnerable groups, including the elderly, still suffer from unacceptably high rates of disease because coverage by the vaccine is incomplete (29). Since the majority of vaccine efficacy is now known to be a consequence of decreased spread from immunized individuals, an understanding of the biology of pneumococcal transmission may foster alternative and potentially better means of prevention (30).
FOOTNOTES
- Received 16 May 2016.
- Returned for modification 15 June 2016.
- Accepted 5 July 2016.
- Accepted manuscript posted online 11 July 2016.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
