Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Summary

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Abstract

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.

This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.

Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Introduction

Sepsis is defined as a dysregulated systemic inflammatory and immune response to microbial invasion or tissue damage, leading to organ injury distant from the site of infection or damage. Septic shock is a subset of sepsis characterized by hypotension persisting during volume resuscitation, with a substantially increased risk of mortality¹. The general public has become more aware of this disorder during the COVID-19 pandemic. Despite its high associated mortality, comprehensive epidemiological data on the global burden of sepsis is lacking because of the complexity of its diagnosis. In 2017, there were 48.9 million sepsis incidences and 11 million deaths worldwide, accounting for 19.7% of all global deaths². Further, a study on the extended prevalence of infection and related sepsis in intensive care unit patients found that 62% of the positive isolates from patients were Gram-negative organisms³.

Initially, the investigations on sepsis focused on delineating microbial pathogenesis. However, understanding the "danger hypothesis", which dictates how the host distinguishes self and non-self, led to the tilting of the balance of sepsis research toward understanding the host response to an invading pathogen. The widely used mice models of sepsis include the lipopolysaccharide (LPS)-induced endotoxemia model, polymicrobial sepsis models, cecal ligation and puncture (CLP) and colon ascendens stent peritonitis (CASP), and monobacterial infection models⁴.

We have standardized a mouse model system by inducing peritoneal sepsis using Salmonella Typhimurium. This model is advantageous over others because Salmonella Typhimurium is an intracellular pathogen that mimics the clinically relevant condition of Gram-negative sepsis. The outcome of peritonitis sepsis in this model is systemic, with 100% mortality within 96 h post infection. Therefore, this model is instrumental in studying the inflammatory and lethal host responses. In this model, sepsis is induced by intraperitoneally injecting 0.5 million colony-forming units (CFU) of Salmonella Typhimurium into an 8-10-week-old C57BL/6 mouse. Systemic infection can be confirmed by assessing organ bacterial burden ~16 h post infection. This article demonstrates Salmonella Typhimurium-induced peritonitis sepsis in mice, characterizes the resulting alterations in peritoneal cell composition, and quantifies bacterial burden in different organs.

Protocol

All experiments using Salmonella Typhimurium were conducted in Bio Safety Level 2 (BSL-2) facilities. Care must be taken to use proper personal protective equipment (PPE), ensure safety, and follow standard BSL-2 biohazard disposal methods. All mice experiments were conducted following guidelines stated by the Institutional Animal Ethics Committee, IISc. Mice were bred and maintained at the Central Animal Facility of IISc (Registration number: 48/1999/CPCSEA, dated 1/3/1999), approved by the Ministry of Environment and Forest, Government of India. The experimental protocols were approved by the Committee for Purpose and Control and Supervision of Experiments on Animals with the approved permit number CAF/Ethics/797/2020.

BSL2 definition: A BSL2 rating represents that the biohazardous agents pose a moderate threat to the environment and laboratory staff⁵.

1. Culture preparation of Salmonella Typhimurium         

1. Add 100 µL of Salmonella Typhimurium NCTC 12023 glycerol stock to 3 mL of Luria Bertani (LB) broth. Incubate the culture at 160 rpm at 37 °C overnight.
2. Streak 50 µL of the overnight grown culture in LB broth onto a Salmonella Shigella (SS) agar plate and incubate at 37 °C for ~12 h. Store the SS agar plate with the bacterial colonies at 4 °C for several days before the in vivo infection experiment.
3. Pick a single colony from the streaked SS agar plate using a microtip. Eject the microtip into 3 mL of LB broth and culture at 160 rpm at 37 °C overnight.
4. Add 0.1 mL of the bacterial culture to 50 mL of LB broth and incubate at 37 °C in a shaker incubator at 160 rpm for 3-4 h to reach the logarithmic phase of growth. Dilute the culture by a factor of 2 using LB broth.
   NOTE: During the logarithmic phase, bacterial cells are at their best health and are actively dividing.
5. Measure the optical density (OD) of the culture at 600 nm wavelength of light in a spectrophotometer or microplate reader. Once the OD reaches 1.0, make two aliquots of 1 mL of culture in 1.5 mL microfuge tubes.
6. Centrifuge the tubes at 7,750 × g for 15 min. Discard the supernatant and wash the pellet with 1 mL of 1x PBS 2x. Centrifuge the tubes at 7,750 × g for 15 min.
7. Resuspend the pellet in 0.5 mL of 1x PBS in two different 1.5 mL microfuge tubes. Combine the suspensions from both the tubes into one 1.5 mL tube now containing ~2 × 10⁸ colony-forming units (CFU)/mL.
8. Prepare a bacterial cell suspension of 1 × 10⁶ CFU/mL by diluting this stock solution.
   ​CAUTION: Optimize the CFU corresponding to OD under specific laboratory conditions to determine the CFU for OD 1.0 before initiating the experiments.

2. Mice and infections

1. House 8-10-week-old male C57BL/6 mice weighing ~20 g in the clean-air room of the animal facility for several days for acclimation.
2. On the day of infection, hold the mouse with one hand, wipe the abdominal skin with 70% ethanol, and spread the hind legs for better accessibility of the abdominal wall.
3. Inject 0.5 mL of 1 × 10⁶ CFU/mL bacterial suspension intraperitoneally with the help of a 1 mL syringe. Therefore, each mouse receives 5 × 10⁵ CFU. Control, uninfected mice receive 0.5 mL of PBS alone. Post infection, plate the culture to check the actual CFU injected, which may vary from 0.2-0.8 million CFU/0.5 mL.
4. Put the mice back in the cages as assigned.
5. Sacrifice the mice using CO₂ asphyxiation ~12-18 h post infection for the best response. Usually, all infected mice die within 96 h. Under some experimental interventions, some mice may survive. Euthanize these mice after 96 h. Also, euthanize any mouse with a body temperature below 33.2 °C and acute distress at 96 h as a humane endpoint.
   ​NOTE: In this model, some mice may start dying after 12 h of Salmonella injection. Therefore, plan experiments properly involving multiple time points.

3. CFU assessment of organs

1. Sacrifice the infected mouse by CO₂ asphyxiation, and wipe the abdomen with a piece of cotton dipped in 70% ethanol. Cut open the abdominal skin. Refer to the article by Ray and Dittel for a video protocol on how to collect peritoneal lavage fluid⁶. Cut open the peritoneal cavity and collect the organs of interest in microfuge tubes. Additionally, as the blood in mice with sepsis gets coagulated fast and the amounts are low, collect it quickly after sacrificing.
   NOTE: This video demonstrates the enumeration of organ CFU from the liver as the liver undergoes extensive histopathological damage in this model of sepsis.
2. Cut a small piece of the liver and place it in a microfuge tube.
   NOTE: This can be stored on ice for a maximum of 2-3 h before proceeding to the next step.
3. Weigh and transfer the pieces to microcentrifuge tubes. Preferably, cut pieces weighing ~10-15 mg for proper homogenization. Use entire organs in the case of smaller ones such as the mesenteric lymph nodes (MLNs) or thymus.
4. Add 0.5 mL of 1x PBS to the tube and homogenize the organs using a hand homogenizer. Make sure that the organs are completely homogenized. Make up the volume to 1 mL by adding 0.5 mL of 1x PBS.
5. Centrifuge the tubes at 200 × g for 5 min at 4 °C.
6. Collect the supernatant into fresh microfuge tubes and prepare dilutions of 1 × 10⁻¹ and 10⁻² in a 96-well plate.
7. Spread 50 µL of the diluent onto fresh SS agar plates and incubate the plates at 37 °C for 12 h.
8. Count the number of colonies that appear in each condition and normalize the data with the organ weight using Equation (1):
   CFU/mg = (1)
   NOTE: The number 20 is used in the formula to convert the colonies per plate to CFU/mL. This number is arrived at by dividing 1 mL by the amount of a given volume of the culture plated-in this case, 50 µL.
   For example, if 100 colonies are found in an SS agar plate, where 50 µL of a 1 × 10^-1 dilution of a homogenized organ weighing 10 mg is spread, then
   CFU/mg =

4. Flow cytometric analysis of various immune cell populations in peritoneal exudate

1. Collect the peritoneal cells as described previously by Ray and Dittel⁶.
2. Resuspend the cell pellet from peritoneal lavage fluid in 1 mL of RPMI supplemented with 10% fetal bovine serum (FBS). Enumerate the total cell numbers in the peritoneal lavage using a hemocytometer. Adjust the cell number so that every tube receives 0.2-0.5 million cells.
   NOTE: Peritoneal lavage in mice with sepsis may contain RBCs, which probably appear due to hemorrhage. Be careful to exclude RBCs while counting peritoneal cells. In the brightfield microscope, RBCs appear much smaller than the immune cells. These appear as flat disks or doughnuts, round, with an indentation in the center, but not hollow. A step to lyse RBCs may be added⁷.
3. Spin the cells down at 200 × g at 4 °C for 10 min, discard the supernatant, and wash the cells 1x with 1x cold PBS. Centrifuge the cells at 200 × g at 4 °C for 10 min.
4. Block the Fc receptors on PECs using FcR blocker (1:400 dilution), prepared in blocking buffer consisting of 5% FBS and 0.02% sodium azide in PBS. Incubate on ice for 15 min.
5. Centrifuge the cells at 200 × g at 4 °C for 10 min. Discard the supernatant. Dilute the fluorochrome-conjugated antibodies of interest in blocking buffer. Use a 1:500 dilution of anti-mouse LY6G to stain the neutrophils.
   NOTE: Other immune cell populations can also be detected using, for example, anti-mouse B220 for B cells, anti-mouse CD3 for T cells, and anti-mouse F4/80 for macrophages.
6. Incubate ~0.2 million cells in 200 µL of the diluted solutions of antibodies in separate tubes. As a negative control, set aside one tube in each fluorochrome type for the unstained control to incubate the cells with 200 µL of blocking buffer without antibody.
7. Incubate the samples on ice for 45 min with intermittent tapping every 15 min.
8. Centrifuge the cells at 200 × g for 10 min at 4 °C. Discard the supernatant. Fix the cells with 4% paraformaldehyde for ~15 min at room temperature if they need to be be stored for several days. However, it is best to acquire data from freshly stained samples in a flow cytometer.
9. Resuspend the cells in 200 µL of FACS staining buffer (2% FBS in PBS). Acquire the data in a flow cytometer.

Representative Results

A detailed characterization of the host immune response using this particular model is shown in previous publications^8,9. A few representative results of the described protocol are depicted in this section. This model aims to induce systemic infection of S. Typhimurium by intraperitoneal injection of the bacterial culture to induce sepsis. To confirm the infection, the lysates of the liver and spleen from septic mice were spread on SS agar plates, and the number of colonies was counted. In Figure 1, the images of SS agar plates indicate the organ CFU burden in the liver and spleen of septic mice. The homogenized organ lysates were spread at a dilution of 1 × 10⁻¹ and incubated at 37 °C. The black pigmented S. Typhimurium colonies appeared after ~12 h of incubation at 37 °C. Furthermore, upon infection of mice, the cultures from blood and peritoneal lavage are reported to be positive for bacterial cells⁸. These results indicated that the intraperitoneally injected bacterial cells disseminated systemically and colonized the internal organs. A part of the plate is shown as a zoomed-in inset to highlight the colonies. The pathogen successfully disseminated systemically and colonized the internal organs.

Figure 2 shows the sera isolated from healthy and septic mice. The volume of collectible blood through cardiac puncture from septic mice is usually 100-200 µL, which is lower than the amount from healthy mice, where ~500 µL of blood can be obtained. As a result, the serum volume isolated from septic mouse blood is low. This phenomenon happens because of the increased coagulation of the blood in septic mice. Moreover, the sera from septic mice show distinct red coloration, indicating the occurrence of extensive hemolysis^8,9.

As shown in Figure 3, flow cytometry-based immunophenotyping of peritoneal exudate cells was performed to assess the infiltration of neutrophils in the peritoneal cavity of healthy and septic mice. As neutrophils express LY6G protein on the cell surface, FITC-tagged anti-LY6G antibody was used to stain the neutrophil cell population. These images represent data from one healthy and two infected mice. After data acquisition, the cells were gated to include only singlets in an FSC-A versus SSC-A plot. Then, the histogram plots and dot-plot were drawn. Here, the septic mice showed increased infiltration of neutrophils in the peritoneal cavity.

Figure 1: Visualization of organ CFU post infection. Organs were homogenized as described in the protocol. The lysate was spread on SS agar plates using a spreader. Images of plates spread with a 1 × 10^-1 dilution of homogenized lysates of (A) liver and (B) spleen. A part of the plate is shown as a zoomed inset to highlight the colonies. Inset magnification = 13x (area). Please click here to view a larger version of this figure.

Figure 2: Enhanced hemolysis in septic mice. The blood was collected in microcentrifuge tubes and left undisturbed at room temperature for 30 min. The clot was removed by centrifuging the tubes at 2,000 × g for 10 min in a refrigerated centrifuge. Please click here to view a larger version of this figure.

Figure 3: Flow cytometric profiling of neutrophil population in peritoneal lavage fluid. Peritoneal lavage fluid was collected 16 h post infection. The peritoneal cells were stained with FITC-tagged anti-LY6G antibody. Septic mice showed increased infiltration of neutrophils in the peritoneal cavity. Abbreviations: FITC = fluorescein isothiocyanate; LY6G = lymphocyte antigen 6 complex locus G6D; SSC-A = side-scatter area. Please click here to view a larger version of this figure.

Discussion

This article describes a method of inducing a severe form of bacterial sepsis by intraperitoneal injection of Salmonella Typhimurium. This model is advantageous over others as Salmonella Typhimurium is an intracellular pathogen and, hence, highly pathogenic, mimicking the clinically relevant condition of Gram-negative sepsis. The outcome of peritonitis sepsis in this model is systemic, with 100% mortality within 96 h post infection. Therefore, this model is instrumental in studying the inflammatory and lethal host responses⁸ and the effects of therapeutic intervention in attenuating sepsis.

In this model system, the cellular composition of the peritoneal cavity changes dramatically to combat the inoculated pathogen. A large number of LY6G⁺ neutrophils infiltrate the peritoneal cavity, with a simultaneous decrease in F4/80⁺ macrophages, resulting in the macrophage disappearance reaction (MDR)^8,10. Therefore, this model is useful in studying the kinetic changes in immune cell compositions and functions during sepsis, which is still a vastly unexplored area. Salmonella Typhimurium systemically infects, resides in, and proliferates in the organs of mice. The serum proinflammatory cytokines such as TNF-α, IL-6, and IFN-γ also increase⁸. A cascade of proinflammatory responses also leads to hemolysis and coagulation of the blood, which becomes visible upon dissection of the infected mice^10,11. The interplay between the host response to this bacterial burden and the damaging effects of the bacteria leads to disfigured tissue architecture, loss of functions, and tissue necrosis, especially in the liver.

Moreover, previous reports from the laboratory have shown that such effects are modulated by the upregulated expression of nitric oxide synthase 2 (NOS2), which leads to increased reactive oxygen species (ROS) and higher levels of proinflammatory cytokines, leading to an enhanced inflammatory response⁸. This mouse sepsis model is advantageous over others because the induction of sepsis is faster with more lethal effects. It does not require technical expertise to perform the surgery on anesthetized mice, as is the case with the current gold-standard CLP-sepsis mouse model. In the CLP-sepsis model, only a subset of mice develops sepsis, whereas all mice develop sepsis in the model described here.

Moreover, compared to the LPS-induced endotoxemia sepsis model, this model is more clinically relevant for Gram-negative monobacterial sepsis. The model is also useful in understanding the increases in intracellular ROS, proinflammatory cytokine levels, hemolysis, and blood coagulation, all of which happen during sepsis. It is reported that the use of the nitric oxide donor drug DETA-NONOate provides a survival benefit in Nos2 knockout mice with sepsis using this model⁸. This suggests that this model can be used to identify novel drugs to treat sepsis. Also, this model can be quickly adopted by any laboratory equipped with BSL-2 facilities.

While using this model, precautions must be taken at the level of bacterial cell health and the CFU number to obtain optimal results. One should always use freshly streaked Salmonella colonies from SS agar plates. It is also recommended to optimize the process of culture preparation by finding the OD value in the spectrophotometer corresponding to the mentioned CFU.

The disadvantage of using this model is that the effect of sepsis in mice is intensely lethal within 96 h post infection. This limits the study of host immune responses at late time points. It can be overcome by using a more attenuated bacterial strain of Salmonella Typhimurium.

There are differences between human patients suffering from sepsis and mice models attempting to mimic human sepsis. For example, patients take longer to develop sepsis and are put on supportive interventions and therapeutics. However, sepsis in mouse models develops much faster, and supportive interventions are not done⁴. However, there are some similarities between sepsis in humans and mice, with patients with sepsis showing higher levels of proinflammatory cytokines, blood coagulation, and hemolysis in clinical settings. All these are mimicked in this mouse model of sepsis. Therefore, some readouts of this model are relevant to clinical scenarios, although a direct correlation may not be advisable.

Materials

Name	Company	Catalog Number	Comments
Consumables
1 mL Sterile Syringe with 26 G needle	Beckton Dickinson, Singapore	303060
1.5 mL Microcentrifuge Tube	Tarsons, USA	500010
10 mL Sterile Syringe with 21 G needle	Beckton Dickinson, Spain	307758
50 mL Conical Flask	Tarsons, USA	441150
50 mL Graduated Centrifuge Tube	Tarsons, USA	546041
50 mL Graduated Centrifuge Tube	Tarsons, USA	546021
Cell spreader	VWR, USA	VWRU60828-680
Dulbecco’s Phosphate Buffered Saline	HiMedia, Mumbai, India	TS1006
Ethanol	Merck	100983
FcR blocker	BD Biosciences	553142
Fetal Bovine Serum	Gibco	10270-106
FITC Rat anti-mouse Ly6G (Clone 1A8)	BD Pharmingen	551460
Glycerol	Sigma-Aldrich	G9012
Hand based Homogenizer	-	-
Hemocytometer (Neubauer counting chamber)	Rohem, India	I.S. 10269
Luria Bertani Broth	HiMedia, Mumbai, India	M1245
Paraformaldehyde	Sigma-Aldrich	158127
Petriplates	Tarsons, USA	460091
RPMI	Himedia, Mumbai, India	AT060-10X1L
Salmonella-Shigella Agar	HiMedia, Mumbai, India	M108
Sodium azide	Sigma-Aldrich	S2002
Equipments
Centrifuge	Kubota
Flow cytometer	BD FACSverse
Incubator	N-biotek
Spectrophotometer	Shimadzu
Weighing machine	Sartorius