Name: a mouse model for the transition of streptococcus pneumoniae from colonizer to pathogen upon viral co-infection recapitulates age-exacerbated illness
Uploaded: 2022-09-28T14:15:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness at JoVE.

We would like to thank Nick Lenhard for the critical reading and editing of this manuscript. We would also like to thank Andrew Camilli and Anthony Campagnari for the bacterial strains and Bruce Davidson for the viral strains. This work was supported by the National Institute of Health Grant (R21AG071268-01) to J.L. and the National Institute of Health Grants (R21AI145370-01A1), (R01AG068568-01A1), (R21AG071268-01) to E.N.B.G.

The authors have no conflicts of interest to disclose.

Most of the existing S. pneumoniae/IAV co-infection experimental studies rely on bacterial delivery into the lungs of mice pre-infected with IAV. These models have helped identify changes in the pulmonary environment and systemic immune response that render the host susceptible to secondary bacterial infection15,16,17,32,33,34,35,36,37. However, these models have failed to mimic the transition of S. pneumoniae from an asymptomatic colonizer to a pathogen capable of causing serious lung and systemic infections. Further, these models are not suitable for studying the host factors and host-pathogen interactions in the upper respiratory tract that contribute to susceptibility to infection. A prior model for the movement of pneumococci from the nasopharynx to the lung after IAV infection relied on bacterial infection of the nasopharynx followed by viral infection. However, it failed to reproduce the severe signs of disease observed in human patients21. The modified murine infection model described here recapitulates the transition of S. pneumoniae from asymptomatic carriage to a pathogen that causes severe clinical disease.
A critical step of this model is establishing S. pneumoniae infection in the nasopharynx. Streptococcus pneumoniae form biofilms and colonize the nasopharynx at different efficiencies21,38. To establish consistent infection, at least 5 &#215; 106 CFU of the biofilm-grown bacterial&#160;strains tested so far are required23. It is recommended that any new bacterial strain be tested for stable infection of the nasopharynx prior to viral infection. For viral co-infection, previous studies have found that intranasal infection with IAV is required for the dispersion of the bacteria from the nasopharynx21,22,23. In those prior studies, 500 PFU of IAV for intranasal delivery were used, while in this study, 200 PFU were sufficient to increase bacterial numbers in the nasopharynx. IAV infection is not limited to the upper airways and can spread to the lungs39,40, which is key for rendering the pulmonary environment more permissive for bacterial infection15,16,41. The delivery of IAV to the lungs can be achieved by either intranasal delivery or intratracheal installation of anesthetized mice. Prior work with BALB/cByJ mice found that intranasal delivery results in viral pneumonia21; however, access of the inoculum to the lungs following intranasal inoculation is more restricted in C57BL/6 mice. In C57BL/6 mice, intratracheal installation is required for consistent delivery of the virus23. In this model, prior bacterial colonization accelerates the presentation of disease symptoms after viral infection23. As viral infection can itself cause disease symptoms with potential variation in kinetics, it is recommended to first test a range of doses for any new viral strain tested and choose a dose that reveals accelerated kinetics in co-infected hosts.
The lungs provide another critical readout for disease evaluation in this model. For the assessment of pathogen burden and immune cell influx, a lung from the same mouse can be used. However, as infection and inflammation severity can differ between lobes, it is recommended to not take different lobes of the same lung for the various assessments. Rather, all the lobes can be minced into small pieces, mixed well together, and then parsed out equally for the different assessments. Similarly, the nasopharynx can be used for the enumeration of bacterial CFU or viral PFU and immune response. However, the number of cells obtained from the washes and tissue is too low to perform flow cytometry without pooling the samples from mice within the same group. Alternatively, inflammation in the nasopharynx can be assessed histologically23.
A critical feature of this model is that it recapitulates the clinical disease seen in patients. In humans, secondary pneumococcal pneumonia following IAV infection often results in obvious signs of disease, including cough, dyspnea, fever, and muscle aches that can lead to hospitalizations, respiratory failure, and even death8,15,42,43. This model recapitulates the severe signs of clinical disease observed in humans in terms of difficulty in breathing (reflected in the breathing score) and overall malaise (reflected in posture and movement scores) displayed by the mice, as well as death in some of the healthy young controls. The exacerbated disease symptoms in co-infected mice are likely a result of both bacterial dissemination to the lungs and impaired viral clearance in mice with pneumococcal carriage23. A limitation of the model is that the incidence of clinical disease and bacterial dissemination from the nasopharynx varies between mice and is influenced by bacterial strain, host age, and genotype21,22,23. Reflecting this, for invasive strains, the progression from localized infection (with no detectable bacteremia) to death can occur within 24 h. Therefore, for a true assessment of systemic spread, bacteremia should be followed over shorter intervals (every 6-12 h). Similarly, the disease score can change rapidly, particularly in the first 72 h following co-infection. Therefore, to closely track the disease symptoms, it is advisable to monitor mice three times per day for days 1-3 post IAV infection.
In summary, this model replicates the movement of S. pneumoniae from an asymptomatic colonizer of the nasopharynx to a pathogen capable of causing pulmonary and systemic disease upon IAV infection. In this model, IAV triggers the transition of S. pneumoniae via modifying the bacterial behavior in the nasopharynx, increasing bacterial spread to the lung, and altering antibacterial immunity23. Similarly, bacterial carriage blunts the antiviral immune responses and impairs IAV clearance from the lungs23. This renders this model ideal for parsing out changes in immune responses in single versus polymicrobial infections. Additionally, the course of disease following co-infection is, in part, dependent on the strain of pneumococci present in the nasopharynx. Therefore, the model is suited to dissecting the bacterial factors required for asymptomatic colonization versus pathogenic transition of S. pneumoniae. Lastly, this model reproduces the susceptibility of aging to co-infections, and although this was not tested here, it can be easily used to assess the impact of host background on the disease course. In conclusion, separating carriage and disease into distinct steps provides the opportunity to analyze the genetic variants of both the pathogens and the host, allowing the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. Moving forward, this model can be used to tailor treatment options for vulnerable hosts.

Streptococcus pneumoniae (pneumococcus) are Gram-positive bacteria that asymptomatically reside in the nasopharynx of most healthy individuals1,2. Promoted by factors that are not completely defined, pneumococci can transition from benign colonizers of the nasopharynx to pathogens that spread to other organs resulting in serious infections, including otitis media, pneumonia, and bacteremia3. Pneumococcal disease presentation is, in part, dependent on strain-specific differences, including the serotype, which is based on the composition of capsular polysaccharides. There have been over 100 serotypes characterized so far, and some are associated with more invasive infections4,5. Several other factors increase the risk of pneumococcal disease. One such factor is viral infection, where the risk of pneumococcal pneumonia is increased 100-fold by IAV6,7. Historically, S. pneumoniae is one of the most common causes of secondary bacterial pneumonia following influenza and is associated with worse outcomes8. Another major risk factor is advanced age. In fact, S. pneumoniae is the leading cause of community-acquired bacterial pneumonia in elderly individuals above 65 years old9,10. Elderly individuals account for the majority (&#62;75%) of deaths due to pneumonia and influenza, indicating that the two risk factors-aging and IAV infection-synergistically worsen disease susceptibility11,12,13,14. However, the mechanisms by which viral infection prompts the transition of pneumococci from asymptomatic colonizer to invasive pathogen and how this is shaped by host factors remain poorly defined. This is largely due to the absence of a small animal model that recapitulates the transition from asymptomatic pneumococcal colonization to critical clinical disease.
Co-infection studies have classically been modeled in mice inoculated with pneumococci directly into the lungs 7 days following influenza infection15,16. This reproduces the susceptibility to secondary bacterial pneumonia and is ideal for studying how antiviral immune responses impair antibacterial defenses17. However, longitudinal studies in humans have demonstrated that pneumococcal carriage in the nasopharynx, where the bacteria can form asymptomatic biofilms18, is uniformly associated with invasive diseases19,20. Bacterial isolates from infections of the middle ear, lung, and blood are genetically identical to those found in the nasopharynx20. Thus, to study the transition from asymptomatic carriage to invasive disease following IAV infection, a model was established in which mice were intranasally administered biofilm-grown pneumococci followed by IAV infection of the nasopharynx21,22. Viral infection of the upper airway led to changes in the host environment that led to the dispersal of pneumococci from biofilms and their spread to the lower airways21. These dispersed bacteria had upregulated expression of virulence factors important for infection, converting them from colonizers to pathogens21. These observations highlight the complex interaction between the virus, host, and bacteria and demonstrate that the changes to the host triggered by viral infection have a direct impact on the pneumococcal behavior, which, in turn, alters the course of bacterial infection. However, this model fails to recapitulate the severe signs of illness observed in humans, likely because the virus is limited to the nasal cavity, and the systemic effects of viral infection on host immunity and lung damage are not recapitulated.
We recently established a model that incorporates the complex interaction between the host and pathogens but also more closely mimics the disease severity observed in humans23. In this model, mice are first infected intranasally with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This resulted in bacterial dissemination to the lungs, pulmonary inflammation, and illness that progressed to lethality in a fraction of young mice23. This previous study demonstrated that both viral and bacterial infection altered host defense: viral infection promoted bacterial dissemination, and prior bacterial colonization impaired the ability of the host to control pulmonary IAV levels23. Examining the immune response revealed that IAV infection diminished the antibacterial activity of neutrophils, while bacterial colonization blunted the type I interferon response critical to antiviral defense23. Importantly, this model reproduced the susceptibility of aging. Compared to young mice, old mice displayed signs of disease earlier, showed more severe clinical illness, and succumbed to infection more frequently23. The work presented in this manuscript shows that the degree of disease is also dependent on the bacterial strain, because invasive pneumococcal strains display more efficient dissemination upon IAV infection, show more overt signs of pulmonary inflammation, and result in accelerated rates of disease compared to non-invasive strains. Thus, this S. pneumoniae/IAV co-infection model permits the detailed examination of both pathogen and host factors and is well-suited for studying immune responses to polymicrobial infections at the different phases of disease progression.

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			<td>D39</td>
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			<td>D-Alanine</td>
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			<td>D-Calcium pantothenate</td>
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			<td>Dextrose</td>
			<td>Fisher Chemical</td>
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			<td>EF3030</td>
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			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
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			<td>Falcon&#160;Round-Bottom Polypropylene Test Tubes With Cap</td>
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			<td>Falcon&#160;Round-Bottom Polystyrene Test Tubes (5 mL)</td>
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			<td>Gibco&#160;Penicillin-Streptomycin (10,000 U/mL)</td>
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			<td>L-Cysteine hydrochloride monohydrate</td>
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			<td>L-Cystine</td>
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			<td>Fisher</td>
			<td>AC156211000</td>
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			<td>LIFE TECHNOLOGIES LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
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			<td>Amino acid stock</td>
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			<td>Low endotoxin BSA&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A1470-10G</td>
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		<tr>
			<td>L-Proline</td>
			<td>Fisher</td>
			<td>AAA1019922</td>
			<td>Amino acid stock</td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Fisher</td>
			<td>AC132660250</td>
			<td>Amino acid stock</td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Fisher</td>
			<td>AC138930250</td>
			<td>Amino acid stock</td>
		</tr>
		<tr>
			<td>L-Tryptophan</td>
			<td>Fisher</td>
			<td>AAA1023014</td>
			<td>Amino acid stock</td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Fisher</td>
			<td>AAA1272014</td>
			<td>Amino acid stock</td>
		</tr>
		<tr>
			<td>Ly6C BV 605</td>
			<td>BD Bioscience</td>
			<td>BDB563011</td>
			<td>Clone: AL-21 DF 1:300</td>
		</tr>
		<tr>
			<td>Ly6G AF 488&#160;</td>
			<td>Biolegend</td>
			<td>NC1102120</td>
			<td>Clone: IA8, DF 1:300</td>
		</tr>
		<tr>
			<td>Madin-Darby Canine Kidney (MDCK) cells&#160;</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>CCL-34</td>
			<td>MDCK cell line for PFU analuysis&#160;</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate 7-Hydrate</td>
			<td>Fisher</td>
			<td>60-019-68</td>
			<td>CDM starter stock</td>
		</tr>
		<tr>
			<td>Manganese Sulfate</td>
			<td>Fisher</td>
			<td>M113-500</td>
			<td>Mix I stock</td>
		</tr>
		<tr>
			<td>MilQ water</td>
			<td></td>
			<td></td>
			<td>Ultra-pure water</td>
		</tr>
		<tr>
			<td>Mouse Fc Block</td>
			<td>BD Bioscience</td>
			<td>BDB553142</td>
			<td>Clone: 2.4G2 DF 1:100</td>
		</tr>
		<tr>
			<td>MWI VETERINARY&#160;PURALUBE VET OINTMENT</td>
			<td>Fisher</td>
			<td>NC1886507</td>
			<td>Eye lubricant for infection</td>
		</tr>
		<tr>
			<td>NCI-H292 mucoepidermoid carcinoma cell line&#160;</td>
			<td>ATCC</td>
			<td>CRL-1848</td>
			<td>H292 lung epithelial cell line for biofilm growth</td>
		</tr>
		<tr>
			<td>Niacinamide</td>
			<td>Fisher</td>
			<td>18-604-792</td>
			<td>Vitamin stock</td>
		</tr>
		<tr>
			<td>NK 1.1 AF 700</td>
			<td>BD Bioscience</td>
			<td>50-112-4692</td>
			<td>Clone: PK136 DF 1:200</td>
		</tr>
		<tr>
			<td>Oxyrase For Broth 50Ml Bottle 1/Pk</td>
			<td>Fisher</td>
			<td>50-200-5299</td>
			<td>To remove oxygen from liquid cultures</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% in PBS</td>
			<td>Thermoscientific</td>
			<td>J19932-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Pivetal Isoflurane&#160;</td>
			<td>Patterson Veterinary</td>
			<td>07-893-8440</td>
			<td>Isoflurane for anesthesia during infection&#160;</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Chemical</td>
			<td>P288-500</td>
			<td>Starter stock</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Chemical</td>
			<td>P285-500</td>
			<td>Starter stock</td>
		</tr>
		<tr>
			<td>Pyridoxal hydrochloride&#160;</td>
			<td>Fisher</td>
			<td>AC352710250</td>
			<td>Vitamin stock</td>
		</tr>
		<tr>
			<td>Pyridoxamine dihydrochloride</td>
			<td>Fisher</td>
			<td>AAJ6267906</td>
			<td>Mix I stock</td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Fisher</td>
			<td>AC132350250</td>
			<td>Vitamin stock</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>VWR</td>
			<td>0530-500G</td>
			<td>Starter stock</td>
		</tr>
		<tr>
			<td>Sodium Azide&#160;</td>
			<td>Fisher Bioreagents</td>
			<td>BP922I-500</td>
			<td>For FACS buffer</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Chemical</td>
			<td>S233-500</td>
			<td>Starter stock and final supplement to CDM</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Fisher Chemical</td>
			<td>S374-500</td>
			<td>Starter stock</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Fisher Chemical</td>
			<td>S369-500</td>
			<td>Starter stock</td>
		</tr>
		<tr>
			<td>TCR APC&#160;</td>
			<td>BD Bioscience</td>
			<td>50-112-8889</td>
			<td>Clone: GL-3 DF 1:200</td>
		</tr>
		<tr>
			<td>TCR&#946; APC-Cy7</td>
			<td>BD Pharmigen</td>
			<td>BDB560656</td>
			<td>Clone: H57-597 DF 1:200</td>
		</tr>
		<tr>
			<td>Thermo Scientific Blood Agar with Gentamicin</td>
			<td>Fisher</td>
			<td>R01227</td>
			<td>Blood agar plates with the antibiotic gentamicin&#160;</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Trypsin, TPCK Treated</td>
			<td>Fisher</td>
			<td>PI20233</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine hydrochloride</td>
			<td>Fisher</td>
			<td>AC148991000</td>
			<td>Vitamin stock</td>
		</tr>
		<tr>
			<td>TIGR4</td>
			<td>ATCC</td>
			<td>BAA-334</td>
			<td>Streptococcus pneumoniae strain</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Fisher</td>
			<td>AC157300250</td>
			<td>Mix II stock</td>
		</tr>
		<tr>
			<td>Worthington Biochemical Corporation&#160;Collagenase, Type 2, 1 g</td>
			<td>Fisher</td>
			<td>NC9693955</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model for the transition of streptococcus pneumoniae from colonizer to pathogen upon viral co-infection recapitulates age-exacerbated illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

Watch this Scientific Journal Video about A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness at JoVE.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

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Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

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Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

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Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

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Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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