We thank the Central Animal Facility, IISc for supplying us with mice for research. This study was funded by grants to DpN from the Department of Biotechnology and Science and Engineering Research Board, Government of India. The infrastructural support from the DBT-IISc program and DST-FIST grants are greatly acknowledged. We thank all previous and current members of the DpN lab for their support.

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&#160;Typhimurium&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
<ol>
	<li>Add 100 &#181;L of Salmonella Typhimurium NCTC 12023 glycerol stock to 3 mL of Luria Bertani (LB) broth. Incubate the culture at 160 rpm at 37 &#176;C overnight.</li>
	<li>Streak 50 &#181;L of the overnight grown culture in LB broth onto a Salmonella Shigella (SS) agar plate and incubate at 37 &#176;C for ~12 h. Store the SS agar plate with the bacterial colonies at 4 &#176;C for several days before the in vivo infection experiment.</li>
	<li>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 &#176;C overnight.</li>
	<li>Add 0.1 mL of the bacterial culture to 50 mL of LB broth and incubate at 37 &#176;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.</li>
	<li>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.</li>
	<li>Centrifuge the tubes at 7,750 &#215; g for 15 min. Discard the supernatant and wash the pellet with 1 mL of 1x PBS 2x. Centrifuge the tubes at 7,750 &#215; g for 15 min.</li>
	<li>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 &#215; 108 colony-forming units (CFU)/mL.</li>
	<li>Prepare a bacterial cell suspension of 1 &#215; 106 CFU/mL by diluting this stock solution. 
	&#8203;CAUTION: Optimize the CFU corresponding to OD under specific laboratory conditions to determine the CFU for OD 1.0 before initiating the experiments.</li>
</ol>
2. Mice and infections
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Inject 0.5 mL of 1 &#215; 106 CFU/mL bacterial suspension intraperitoneally with the help of a 1 mL syringe. Therefore, each mouse receives 5 &#215; 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.</li>
	<li>Put the mice back in the cages as assigned.</li>
	<li>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 &#176;C and acute distress at 96 h as a humane endpoint. 
	&#8203;NOTE: In this model, some mice may start dying after 12 h of Salmonella injection. Therefore, plan experiments properly involving multiple time points.</li>
</ol>
3. CFU assessment of organs
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge the tubes at 200 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Collect the supernatant into fresh microfuge tubes and prepare dilutions of 1 &#215; 10&#8722;1 and 10&#8722;2 in a 96-well plate.</li>
	<li>Spread 50 &#181;L of the diluent onto fresh SS agar plates and incubate the plates at 37 &#176;C for 12 h.</li>
	<li>Count the number of colonies that appear in each condition and normalize the data with the organ weight using Equation (1): 
	CFU/mg = <img alt="Equation 1" src="/files/ftp_upload/63695/63695eq01.jpg" style="margin: auto;" /> (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 &#181;L. 
	For example, if 100 colonies are found in an SS agar plate, where 50 &#181;L of a 1 &#215; 10-1 dilution of a homogenized organ weighing 10 mg is spread, then 
	CFU/mg = <img alt="Equation 2" src="/files/ftp_upload/63695/63695eq02.jpg" style="margin: auto;" /></li>
</ol>
4. Flow cytometric analysis of various immune cell populations in peritoneal exudate
<ol>
	<li>Collect the peritoneal cells as described previously by Ray and Dittel6.</li>
	<li>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.</li>
	<li>Spin the cells down at 200 &#215; g at 4 &#176;C for 10 min, discard the supernatant, and wash the cells 1x with 1x cold PBS. Centrifuge the cells at 200 &#215; g at 4 &#176;C for 10 min.</li>
	<li>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.</li>
	<li>Centrifuge the cells at 200 &#215; g at 4 &#176;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.</li>
	<li>Incubate ~0.2 million cells in 200 &#181;L 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 &#181;L of blocking buffer without antibody.</li>
	<li>Incubate the samples on ice for 45 min with intermittent tapping every 15 min.</li>
	<li>Centrifuge the cells at 200 &#215; g for 10 min at 4 &#176;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.&#160;However, it is best to acquire data from freshly stained samples in a flow cytometer.</li>
	<li>Resuspend the cells in 200 &#181;L of FACS staining buffer (2% FBS in PBS). Acquire the data in a flow cytometer.</li>
</ol>

<ol>
	<li>Hotchkiss, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+and+septic+shock.">Sepsis and septic shock.</a> Nature Reviews Disease Primers. 2 (1), 1-21 (2016).</li><li>Rudd, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional,+and+national+sepsis+incidence+and+mortality,+1990-2017:+Analysis+for+the+Global+Burden+of+Disease+Study.">regional, and national sepsis incidence and mortality, 1990-2017: Analysis for the Global Burden of Disease Study.</a> The Lancet. 395 (10219), 200-211 (2020).</li><li>Vincent, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+study+of+the+prevalence+and+outcomes+of+infection+in+intensive+care+units.">International study of the prevalence and outcomes of infection in intensive care units.</a> JAMA. 302 (21), 2323-2329 (2009).</li><li>Lewis, A. J., Seymour, C. W., Rosengart, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+murine+models+of+sepsis.">Current murine models of sepsis.</a> Surgical Infections. 17 (4), 385-393 (2016).</li><li>Ta, L., Gosa, L., Nathanson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+and+biohazards:+Understanding+biosafety+levels+and+meeting+safety+requirements+of+a+biobank.">Biosafety and biohazards: Understanding biosafety levels and meeting safety requirements of a biobank.</a> Biobanking. 1897, 213-225 (2019).</li><li>Ray, A., Dittel, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Peritoneal+Cavity+Cells.">Isolation of Mouse Peritoneal Cavity Cells.</a> Journal of Visualized Experiments: JoVE. (35), e1488 (2010).</li><li>Liu, X., Quan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+isolation+from+mouse+femur+bone+marrow.">Immune cell isolation from mouse femur bone marrow.</a> Bio-protocol. 5 (20), 1631 (2015).</li><li>Yadav, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+2+enhances+the+survival+of+mice+during+Salmonella+Typhimurium+infection-induced+sepsis+by+increasing+reactive+oxygen+species,+inflammatory+cytokines+and+recruitment+of+neutrophils+to+the+peritoneal+cavity.">Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity.</a> Free Radical Biology &amp; Medicine. 116, 73-87 (2018).</li><li>Verma, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+hemoglobin+is+a+marker+of+systemic+inflammation+in+mouse+models+of+sepsis:+A+Raman+spectroscopic+study.">Cell-free hemoglobin is a marker of systemic inflammation in mouse models of sepsis: A Raman spectroscopic study.</a> Analyst. 146 (12), 4022-4032 (2021).</li><li>Cassado, A. D. A., Lima, M. R. D., Bortoluci, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+mouse+peritoneal+macrophages:+Heterogeneity,+development,+and+function.">Revisiting mouse peritoneal macrophages: Heterogeneity, development, and function.</a> Frontiers in Immunology. 6, 225 (2015).</li><li>Yadav, S., Verma, T., Chattopadhyay, A., Nandi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+pathophysiology+of+sepsis,+an+inflammatory+disorder:+Key+roles+of+oxidative+and+nitrosative+stress.">Factors affecting the pathophysiology of sepsis, an inflammatory disorder: Key roles of oxidative and nitrosative stress.</a> Indian Journal of Inflammation Research. 3 (1), 2 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

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-&#945;, IL-6, and IFN-&#947; also increase8. A cascade of proinflammatory responses also leads to hemolysis and coagulation of the blood, which becomes visible upon dissection of the infected mice10,11. 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.

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 &#34;danger hypothesis&#34;, 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.

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

characterizing salmonella typhimurium-induced septic peritonitis in mice

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.

A detailed characterization of the host immune response using this particular model is shown in previous publications8,9. 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 &#215; 10&#8722;1 and incubated at 37 &#176;C.&#160;The black pigmented S. Typhimurium colonies appeared after ~12 h of incubation at 37 &#176;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 &#181;L, which is lower than the amount from healthy mice, where ~500 &#181;L 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 hemolysis8,9.
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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63695/63695fig01.jpg" /> 
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 &#215; 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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63695/63695fig02.jpg" /> 
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 &#215; g for 10 min in a refrigerated centrifuge. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63695/63695fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63695/63695fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice at JoVE.com

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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 ...

characterizing-salmonella-typhimurium-induced-septic-peritonitis

Nanyang Technological University

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JoVE Journal

Immunology and Infection

5.2K Views.  Indian Institute of Science. Sepsis is een ontregelde immuunrespons van de gastheer op microbiële invasie of weefselbeschadiging, wat leidt tot orgaanbeschadiging op een plaats die ver verwijderd is van die van infectie of schade. Het grote publiek is zich meer bewust geworden van deze aandoening, tijdens de COVID-19-pandemie. In 2017 waren er 48,9 miljoen sepsis-incidenten en 11 miljoen sterfgevallen wereldwijd, goed voor bijna 20% van alle wereldwijde sterfgevallen.Zoals te verwachten is, zijn de meeste gevalle...

Video: Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Medicine

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 ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Biology

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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