Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.
Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.
Secondary bacterial pneumonia following influenza infection is a leading cause of death in the United States and an active area of research1,2. Despite numerous studies using murine models of secondary bacterial pneumonia, inconsistencies regarding pathogen instillation and interrogation remain3,4,5. In addition, while many previous efforts have focused on the immunomodulatory effects of influenza that lead to an increased susceptibly to secondary bacterial infection, more recent data suggests virulence regulation of the bacterial pathogen is an equal contributor towards the establishment of disease6,7,8,9. These new data necessitate a more precise method to explore secondary bacterial pneumonia in murine models of co-infection that facilitate investigation into the pathogen response.
Unique to influenza co-infection models, host organisms are intentionally immunocompromised by a primary influenza infection prior to the administration of the secondary bacterial agent. In order to best replicate the disease pathogenesis observed in human hosts, it is imperative that the pathogen load of both primary and secondary agents be controlled so as to observe the individual and combinatorial effects of each infectious agent. Most commonly, respiratory infections in mice have been established through an intranasal administration3,4,5,6,10. Inasmuch as this route is noted for being technically simple and can be appropriate in some single-agent infection applications, it is unsuitable for co-infection models, as instillation procedures, dose volumes, and efficacy are highly variable within published literature3,4,5,6.
To gain a more complete understanding of secondary bacterial pneumonia pathogenesis, contributions of both the host and the pathogen must be considered. To that end, we have developed a straightforward and reproducible approach for recovery of viable bacteria and pathogen RNA from infected lungs. This method uses a simplified, non-invasive intratracheal instillation procedure followed by subsequent isolation of bacterial RNA. The intratracheal instillation procedure described herein is similar to previously described methods and is not limited to pathogen delivery11,12,13. Use of this particular procedure benefits from being low-cost and does not require the use of specialized equipment such as cannulas, guide wires, or fiber optic cable; furthermore, because this procedure is non-invasive it insures minimal stress on murine subjects, minimizes an inflammatory response from the inoculation mechanics, and provides an efficient delivery route for the infection of multiple subjects. Briefly, isoflurane anesthetized mice are suspended from the incisors. Forceps are used to gently grasp the tongue followed by insertion of a pre-loaded bent, blunt-tipped, 21-gauge needle into the trachea and delivery of pathogen load. Validation of this procedure is demonstrated by visual confirmation of dye equally distributed into the pulmonary compartment and recovery of bacterial load. We then demonstrate how to recover viable Staphylococcus aureus (S. aureus) from infected lungs and describe a reproducible method to isolate high quality pathogen RNA.
All methods conform to the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at Montana State University.
1. Intratracheal Instillation
2. Excision of Infected Lungs
3. Pathogen Recovery and Analysis
Figure 1 utilizes a 0.1% weight/volume Coomassie brilliant blue solution to demonstrate that an intratracheal instillation delivers the inoculum directly and evenly within the lower respiratory tract. Figure 2 shows that the bacteria (S. aureus) CFUs recovered directly from homogenized lung tissue. Figure 3 demonstrates the use of this system for precise delivery and recovery of inoculum in the lower respiratory tract by plotting the input and recovery CFUs from individual mice. Figure 4 shows the qRT-PCR amplification curve of the bacterial housekeeping gene gyrB to demonstrate that bacterial RNA can be extracted directly from infected lung tissue with minimal DNA contamination. Figure 5 shows the construction of a standard curve using qRT-PCR amplification of the influenza A virus M-segment to demonstrate viral RNA can be extracted directly from infected lung tissue.
Figure 1: Intratracheal instillation enables even distribution into the lower respiratory tract. (A, B) Uninfected lungs were excised from a healthy mouse following intratracheal instillation of sterile PBS and photographed from (A) dorsal and (B) ventral perspectives. (C, D) 50 µL of a 0.1% Coomassie brilliant blue solution were administered to an anesthetized mouse via intratracheal administration. (C) Dorsal. (D) Ventral. Please click here to view a larger version of this figure.
Figure 2: Representative recovery of bacterial CFUs from infected lung homogenate. Lungs were excised one day post-challenge with S. aureus. Following homogenation, 100 µL of the lung slurry was serially diluted through 10-6. To enumerate CFUs recovered, 10 µL drops were plated from the 10-5 and 10-6 dilutions onto tryptic soy agar (TSA) and incubated at 37 °C with 5% C02 overnight. Please click here to view a larger version of this figure.
Figure 3: Precise delivery and recovery of the pathogen inoculum following intratracheal administration. Mice were divided into two groups containing three mice per group. Mice were subjected to intratracheal instillation of S. aureus at 1 x 108 (low) and 2 x 108 (high) CFU/mL. One hour post-infection, mice were euthanized and the lungs were excised to demonstrate the precision of the instillation and recovery. Bacterial inoculum and bacteria recovered from the lung homogenate were plated on TSA (tryptic soy agar). No significant differences were reported between bacterial input and recovery. Please click here to view a larger version of this figure.
Figure 4: Representative bacterial RNA recovery and purity. Six-hours following intratracheal instillation with 1 x 108 CFU/50 µL of S. aureus, mice were euthanized. Lungs were excised and homogenized followed by resuspension of the lung slurry in buffer RLT-ß-mercaptoethanol. RNA was purified as described in step 3.914. qRT-PCR was used to detect transcripts of the bacterial housekeeping gene gyrB. A control containing no reverse transcriptase (nRT) was included to demonstrate the purity of the RNA recovered. At a threshold of 0.1, gyrB transcripts were detected at an average cycle of 21.1, 20.3, and 20.5. nRT controls were not detected until cycle averages 35.9, 35.5, and 35.0. n = 3 biological replicates, containing 3 technical replicates/biological replicate. Please click here to view a larger version of this figure.
Figure 5: Representative viral RNA recovery. Six days following intratracheal instillation with 100 PFU/50 µL of influenza A/PR/8/1934(H1N1), mice were euthanized. Lungs were excised and homogenized, and 200 µL of the homogenized slurry was collected and passed through a 70 µm cell strainer prior to viral RNA purification. Purified RNA was serially diluted (10-1-10-4) followed by amplification of influenza A M-segment. (A) Amplification plot of influenza A RNA recovered from an infected lung and diluted 10-1–10-4. (B) Standard curve of influenza A M-segment. Threshold = 0.2, R2 = 0.994, slope = -3.46. Please click here to view a larger version of this figure.
Use of this model provides a highly efficient and reproducible method to study secondary bacterial infections. The ability to tightly control the delivery of the pathogen inoculum enables more precise observations of the individual and combinatorial effects of each pathogen. Inefficiencies in the more common intranasal instillation route have likely contributed to the discrepancies in dose volumes and concentrations present in the literature. It is reasonable that the lack of a precise murine system to study secondary bacterial pneumonia has delayed findings identifying bacterial specific responses that contribute to the severity of pulmonary co-infections. Developing a reproducible model to study virulence expression during secondary bacterial infections could lead to the identification of vaccine or drug targets to ameliorate these infections.
The intratracheal instillation step is critical to successfully establish a lower respiratory tract infection and any down-stream analysis of the pathogens. When learning this technique, it may be helpful to practice using a dye (as described in the methods) prior to administering infectious material. Using a dye allows for the direct visualization of the inoculum into the respiratory tract. A common mistake that can occur is insertion of the blunt-needle into the esophagus rather than the trachea. This will result in delivery of the inoculum into the stomach rather than the lungs. To correct this mistake, angle the needle further away from the body and pass it down into the trachea. Once mastered, this procedure is very efficient and can be used to conduct experiments with large numbers of mice. Working in batches to anesthetize mice, the intratracheal instillation can be completed in approximately 30 seconds per mouse. In addition, the excision of the lungs can be completed in 2 to 3 minutes per mouse.
Recovery of viable and pure bacterial RNA from infected tissues is critical for transcript analysis. RNases are ubiquitous and can quickly ruin an experiment15. Some methods include using RNase inhibitors; however, we have found that freezing the sample at -80 °C in RLT-ß-mercaptoethanol or immediately processing the sample for RNA isolation using all RNase free tubes and reagents are effective at reducing RNase contamination. Additionally, we recommend that a maximum of six samples be purified at one time. Including more than six samples can result in prolonged latencies between protocol steps that can culminate in RNA degradation. Once purified, care should also be taken to avoid any unnecessary freeze-thaw cycles. Thus, if multiple analyses will be done on one sample, aliquoting purified RNA for storage at -80 °C is recommended.
In addition to the techniques reviewed herein, this method can be supplemented by performing bronchial alveolar lavage prior to the excision and homogenation of the lungs16. This can be accomplished by lavage of the entire lower respiratory tract or by using suture thread to restrict one branching arm of the bronchial tree followed by lavage through the remaining branch. Often this leads to a reduction in the recovery of the pathogen load but provides a sample whereupon information such as lactate dehydrogenase activity, cellular population identity, and cytokine profiles can be obtained16. Together these data can form a more complete understanding of the host-pathogen interactions occurring during secondary bacterial pneumonia.
While the methods discussed have been within the context of secondary bacterial pneumonia, they are suitable to be extended to any murine model of lower respiratory infection; specifically, those that would benefit from tightly controlled delivery and recovery of the installed inoculum. Furthermore, like many other infection routes, the intratracheal instillation can be utilized in non-infectious applications, such as the administration of therapeutics and environmental compounds12.
The authors have nothing to disclose.
The authors would to thank Nicole Meissner, M.D./Ph.D., Montana State University, for her help in establishing the intratracheal instillation method. This work was supported by the U.S. National Institutes of Health (Grants NIH-1R56AI135039-01A1, GM110732, R21AI128295, U54GM115371), as well as funds from the Montana University System Research Initiative (51040-MUSRI2015-03) and Montana State University Agriculture Experiment Station.
Lysing Matrix B | MP Biomedicals | 6911100 | Referred to in text as "0.1 mm silica beads" |
21-gauge blunt needle | SAI | B21-150 | 1.5" is recommended. |
RNase-Free DNase Set | Qiagen | 79254 | DNase used in the accompanying text. |
FastPrep-24 Classic Instrument | MP Biomedicals | 116004500 | FastPrep FP120 is no longer available. Referred to in text as "Bead Beater" |
TaqMan AIV-Matrix Reagents | Applied Biosystems | 4405543 | Influenza A M-segment qRT-PCR kit. |
Intubation Stand | Kent Scientific | ETI-MES-01 | Referred to in text as "intubation platform." Intubation platform used in the accompanying video was made in house. |
RNeasy Mini Kit | Qiagen | 74106 | RNA purification kit; contains RNeasy columns, Buffer RLT, Buffer RW1, and Buffer RPE |
QIAamp Viral RNA Mini Kit | Qiagen | 52904 | Viral RNA purification kit. |
Tissue Grinders | Thermo Fisher Scientific | 02-542-08 | |
2-Mercaptoethanol (β-Mercaptoethanol) | Calbiochem | UN2966 |