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 mil....

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 Environm.......

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 .......

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