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Abstract coliresides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. colican persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil. Background Escherichia coli is a Gram-negative, facultative anaerobe, belonging to the Enterobacteriaceae family, which inhabits the intestinal tract of humans, warm-blooded animals and reptiles (Berg, 1996; Gordon and Cowling, 2003). It can be transferred through water and sediments via faeces and is used as an indicator of faecal contamination in drinking and recreational water. The use of E. coli as a faecal indicator is based, at least in part, on the assumption that it exists transiently outside of the host gastrointestinal tract (Ishii and Sadowsky, 2008) and does not survive for a long time in the external environment. However, several studies have isolated E. coli from various natural environments such as municipal wastewater, freshwater, beach water, beach sand and soils (Jiménez et al., 1989; Brennan et al., 2010; Chiang et al., 2011; Byappanahalli et al., 2012; Zhi et al., 2016). The capacity of these E. coli strains to survive for long periods of time and grow in the external environment raises questions about the validity of its continued use as indicator of water quality (Brennan et al., 2010). To understand the genetic mechanism underlying the survival and persistence of E. coli in soil, we developed a soil survival assay to evaluate the role of the different genetic factors on soil survival. We investigated the role of the general stress response regulator, RpoS, in the survival of long-term soil persistent E. coli in soil. The ability of the rpoS mutant (COB583ΔrpoS) to survive in soil was compared with the wildtype strain (COB583) and RpoS was demonstrated to be important for the survival and long-term persistence of E. coli in soil (Somorin et al., 2016). Here, we present the detailed protocol for the soil survival assay and the recovery of E. coli from soil. Abdos Product Used Sterile micropipette tips (100-1,000 µl) (Abdos Labtech, catalog number: P10102 ) Sterile micropipette tips (2-200 µl) (Abdos Labtech, catalog number: P10101 ) Microcentrifuge tube (1.5 ml) (Abdos Labtech, catalog number: P10202 ) Centrifuge tube (15 ml graduated) (Abdos Labtech, catalog number: P10402 ) 90 mm Petri dishes (Abdos Labtech, catalog number: P10901 ) Author : Yinka Somorin, Conor O'Byrne Read More

Abstract Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below. Background Group A Streptococcus (GAS), or Streptococcus pyogenes , is a human-specific Gram-positive bacterial pathogen. It remains among the top ten pathogens causing several diseases with mild to life-threatening clinical symptoms (Bisno and Stevens, 1996;Efstratiou and Lamagni, 2016). The clinical presentation of these diseases includes pharyngitis, impetigo, erysipelas, tonsilitis, scarlet fever, necrotizing fasciitis, rheumatic fever, glomerulonephritis, septicemia, and others. More than 220 different GAS serotypes are circulating in the human population worldwide. M1T1 is the dominant serotype spread globally and causing diseases in the population of economically well-equipped countries (Aziz and Kotb, 2008; Nelson et al., 2016; Lynskey et al., 2019). GAS classification is based on variation in the N-terminal amino acid sequence of the surface M-protein. Antibiotic regimens are the only definitive strategy to cure GAS infections, because of the unavailability of any licensed universal vaccine on the market. Recent reports on the development of antibiotic resistance in GAS are worrisome and have motivated us to develop a new alternative therapeutic approach to cure its infections. Invasive GAS infections, like sepsis, bacteremic pneumonia, and necrotizing fasciitis (NF), spread in sterile sites (Metzgar and Zampolli, 2011). NF begins as a small lesion with the appearance of mild erythema, which rapidly progresses with inflammation in the subcutaneous tissue, and destructs skin and soft tissue over large body areas (Walker et al., 2014). Antibiotic administration, surgical debridement of infected tissues, and supportive care are the main line of treatment for invasive GAS diseases (Stevens and Bryant, 2017). Unfortunately, the mortality rate from NF ranges from 23 to 35% in resource-rich settings, despite quick medical interventions (Davies et al., 1996; Carapetis et al., 2005;Allen and Moore, 2010;Olsen and Musser, 2010; Cole et al., 2011;Ralph and Carapetis, 2013). Therefore, alternative therapeutic approaches are urgently needed to combat invasive soft-tissue GAS infections. Our recent studies showed that GAS causes endoplasmic reticulum stress (ER stress) and triggers the unfolded protein response (UPR) in the host cells upon infection. This results in enhanced asparagine synthesis and release, which is utilized by GAS to increase virulence and rate of proliferation. Furthermore, the PERK-eIF2α-ATF4 branch of UPR was exclusively triggered during GAS infection (Anand et al., 2021). When we treated GAS-infected mice with PERK pathway inhibitors (GSK2656157 and ISRIB), the bacteria were cleared faster than in untreated infected mice. We discuss the method of treatment using PERK pathway inhibitors in a murine model of human soft tissue infection. Abdos Product Used Sterile 2 mL tubes (Abdos Labtech Private Limited, catalog number: P10203) Author Aparna Anand, Abhinay Sharma, Miriam Ravins, Atul Kumar Johri,  Boaz Tirosh, Emanuel Hanski Read More