Pathoadaptive Alteration of Salmonella Biofilm Formation in Response to the Gallbladder Environment

Abstract

Typhoid fever, a human-specific disease, is primarily caused by the pathogen Salmonella enterica serovar Typhi. It is estimated that 3 to 5% of people infected with typhoid fever become chronic carriers. Studies have demonstrated that a mechanism of chronic carriage involves biofilm formation on gallstone surfaces. In the course of a previous study using a chronic carriage mouse model, a Salmonella enterica serovar Typhimurium isolate was recovered from a mouse gallstone that exhibited a 2-fold increase in biofilm formation over the wild type. In order to identify the gene(s) responsible for the phenotype, the genomic sequences of this isolate and others were determined and compared. These sequences identified single nucleotide polymorphisms (SNPs) in 14 genes. Mutations in the most promising candidates, envZ and rcsB, were created, but neither showed increased biofilm-forming ability separately or in combination. The hyperbiofilm isolate did, however, present variations in cellular appendages observable using different techniques and a preferential binding to cholesterol. The isolate was also examined for systemic virulence and the ability to colonize the gallbladder/gallstones in a mouse model of chronic infection, demonstrating a systemic virulence defect and decreased gallbladder/gallstone colonization. Finally, to determine if the appearance of hyperbiofilm isolates could be replicated in vitro and if this was a common event, wild-type Salmonella spp. were grown long term in vitro under gallbladder-mimicking conditions, resulting in a high proportion of isolates that replicated the hyperbiofilm phenotype of the original isolate. Thus, Salmonella spp. acquire random mutations under the gallbladder/gallbladder-simulating conditions that may aid persistence but negatively affect systemic virulence. IMPORTANCE Chronic carriers are the main reservoirs for the spread of typhoid fever in regions of endemicity. Salmonella Typhi forms biofilms on gallstones in order to persist. A strain with enhanced biofilm-forming ability was recovered after a nine-month chronic-carriage mouse study. After sequencing this strain and recreating some of the mutations, we could not duplicate the phenotype. The isolate did show a difference in flagella, a preference to bind to cholesterol, and a systemic virulence defect. Finally, gallbladder conditions were simulated in vitro. After 60 days, there was a 4.5-fold increase in hyperbiofilm isolates when a gallstone was present. These results indicate that Salmonella spp. can undergo genetic changes that improve persistence in gallbladder albeit at the cost of decreased virulence. RESULTSSalmonella spp. isolated from GS at 9 months postinfection show increased biofilm formation. S. Typhimurium strains isolated from various organs following a long-term infection study (10) displayed different biofilm formation abilities ( Fig. 1a). Strains obtained from feces and organs including the liver and pancreas showed a diminished biofilm-forming capacity. Interestingly, isolates from the GB showed...

Parallel evolution leading to impaired biofilm formation in invasive Salmonella strains

“…Typhi can form biofilms on human gallstones [57,58], a process which has been simulated with S . Typhimurium in a mouse model of chronic carriage; however, the nature of the extracellular matrix in such biofilms is still under investigation [37,59,60]. Our analysis highlighted the presence 4 premature stop codons in the bcsC of the S .…”

Bacterial Biofilm and its Role in the Pathogenesis of Disease

“…Antibiotics 2020, 9,59 6 of 27 the chronic gall bladder infection. The host immune response changes from an early proinflammatory response to a later anti-inflammatory response [76] whereas S. Typhi adapts to the gall bladder environment by increasing the biofilm forming ability and the capacity for persistence and simultaneously reducing the ability to cause acute infections [77,78]. It has also been shown that S. Paratyphi A can persist in the gallbladder [79] and accumulating evidence indicate a role for nontyphoidal Salmonellae in persistent infections.…”

Microbe-Driven Genotoxicity in Gastrointestinal Carcinogenesis

“…Although the majority of bacteria ingested are killed by stomach acid, bile or intestinal defensins, if the dose is high enough (only about 1000–10,000 colony forming units) surviving Salmonella can colonize the gut lumen or even invade the mucosa and proliferate within epithelial cells [ 148 ]. Importantly, from the intestine passing through the liver Salmonella can enter the gallbladder [ 149 , 150 ].…”