Name: a mouse model for the transition of streptococcus pneumoniae from colonizer to pathogen upon viral co-infection recapitulates age-exacerbated illness
Uploaded: 2022-09-28T14:15:00.000+00:00
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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strain</td></tr><tr><td>Uracil</td><td>Fisher</td><td>AC157300250</td><td>Mix II stock</td></tr><tr><td>Worthington Biochemical Corporation&amp;nbsp;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 ...

a-mouse-model-for-transition-streptococcus-pneumoniae-from-colonizer

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.3K Views.  University at Buffalo School of Medicine. Deze methode beschrijft een nieuw muismodel voor het bestuderen van de overgang van pneumokokken van een asymptomatische kolonisator naar een ziekteverwekkende ziekteverwekker tijdens virale infectie. Het bootst beter na wat er bij mensen gebeurt. Dit model kan een belangrijk hulpmiddel zijn voor het identificeren van potentiële therapeutische doelen tegen de secundaire pneumokokkenpneumonie bij gevoelige gastheren.Dit model reproduceert de gevoeligheid van veroudering. Daarom kan het...

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

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

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

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Medicine

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

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

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