Targeting Salmonella Typhimurium Invasion and Intracellular Survival Using Pyrogallol.

Biruk Tesfaye Birhanu,Seung-Chun Park,Eon-Bee Lee,Seung-Jin Lee

doi:10.3389/fmicb.2021.631426

Biruk Tesfaye Birhanu, Seung-Chun Park + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2021.631426

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Abstract

Salmonella enterica serovar Typhimurium, an intracellular pathogen, evades the host immune response mechanisms to cause gastroenteritis in animals and humans. After invading the host cells, the bacteria proliferate in Salmonella-containing vacuole (SCV) and escapes from antimicrobial therapy. Moreover, Salmonella Typhimurium develops resistance to various antimicrobials including, fluoroquinolones. Treating intracellular bacteria and combating drug resistance is essential to limit the infection rate. One way of overcoming these challenges is through combination therapy. In this study, Pyrogallol (PG), a polyphenol, is combined with marbofloxacin (MAR) to investigate its effect on Salmonella Typhimurium invasion and intracellular survival inhibition. The Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PG against Salmonella Typhimurium were 128 and 256 μg/mL, respectively. The lowest fractional inhibitory concentration (FIC) index for a combination of PG and MAR was 0.5. The gentamycin protection assay revealed that PG (30 μg/mL) alone and in combination with sub-MIC of MAR inhibited 72.75 and 76.18% of the invading bacteria in Caco-2 cells, respectively. Besides, the intracellular survival of Salmonella Typhimurium was reduced by 7.69 and 74.36% in treatment with PG alone and combined with sub-MIC of MAR, respectively, which was visualized by the confocal microscopy. PG has also shown to increase the intracellular accumulation of fluoroquinolone by 15.2 and 34.9% at 30 and 100 μg/mL concentration, respectively. Quantitative real-time PCR demonstrated PG suppressed the genetic expression of hilA, invF, sipB, and acrA by 14.6, 15.4, 13.6, and 36%, respectively. However, the downregulation of hilA, invF, sipB, and acrA increased to 80, 74.6, 78, and 70.1%, in combination with sub-MIC of MAR, respectively. Similarly, PG combined with MAR inhibited the expression of sdiA, srgE, and rck genes by 78.6, 62.8, and 61.8%, respectively. In conclusion, PG has shown antimicrobial activity against Salmonella Typhimurium alone and in combination with MAR. It also inhibited invasion and intracellular survival of the bacteria through downregulation of quorum sensing, invading virulence, and efflux pump genes. Hence, PG could be a potential antimicrobial candidate which could limit the intracellular survival and replication of Salmonella Typhimurium.

Highlights

Salmonella enterica subsp. enterica serovar Typhimurium, an intracellular Gram-negative bacterium, is a non-typhoidal Salmonella serotype known to cause diarrhea and intestinal inflammation in humans and animals (Laupland et al, 2010)
In this study, we discovered the effect of PG alone or in combination with MAR in the invasion and intracellular survival of Salmonella Typhimurium isolated from pigs
We have indicated the antibacterial activity of PG against two field strains of Salmonella Typhimurium isolated from a clinical infection of pigs and ATCC control strains

Summary

Introduction

Salmonella enterica subsp. enterica serovar Typhimurium, an intracellular Gram-negative bacterium, is a non-typhoidal Salmonella serotype known to cause diarrhea and intestinal inflammation in humans and animals (Laupland et al, 2010). The bacteria invade specialized epithelial cells called M cells. It triggers membrane ruffling and actin rearrangement which leads to bacterial internalization upon injecting its effector proteins (Patel and Galán, 2005). The T3SS-1 genes plays a significant role to invade the intestinal epithelial cells and mediate bacterial entry and their persistence in the host cells (Thiennimitr et al, 2012). Successful adherence and invasion of the host cell are essential to cause infections. Salmonella Typhimurium invades epithelial cells either through cytoskeletal rearrangement known as the “Trigger” mechanism or receptor-mediated entry “Zipper” mechanism (Velge et al, 2012). Identifying natural compounds with a higher penetration capacity has significant importance (Liu et al, 2020)

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