Dataset Information

Genetic Variants and Phenotypic Characteristics of Salmonella Typhimurium-Resistant Mutants after Exposure to Carvacrol.

ABSTRACT: The emergence of antimicrobial resistance has raised questions about the safety of essential oils and their individual constituents as food preservatives and as disinfection agents. Further research is required to understand how and under what conditions stable genotypic resistance might occur in food pathogens. Evolution experiments on Salmonella Typhimurium cyclically exposed to sublethal and lethal doses of carvacrol permitted the isolation of SeSCar and SeLCar strains, respectively. Both evolved strains showed a significant increase in carvacrol resistance, assessed by minimum inhibitory and bactericidal concentrations, the study of growth kinetics in the presence of carvacrol, and the evaluation of survival under lethal conditions. Moreover, antibiotic susceptibility tests revealed a development of SeLCar resistance to a wide range of antibiotics. Whole genome sequencing allowed the identification of single nucleotide variations in transcriptional regulators of oxidative stress-response: yfhP in SeSCar and soxR in SeLCar, which could be responsible for the increased resistance by improving the response to carvacrol and preventing its accumulation inside the cell. This study demonstrates the emergence of S. Typhimurium-resistant mutants against carvacrol, which might pose a risk to food safety and should therefore be considered in the design of food preservation strategies, or of cleaning and disinfection treatments.

SUBMITTER: Berdejo D

PROVIDER: S-EPMC7356045 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Genetic Variants and Phenotypic Characteristics of <i>Salmonella</i> Typhimurium-Resistant Mutants after Exposure to Carvacrol.

Berdejo Daniel D Merino Natalia N Pagán Elisa E García-Gonzalo Diego D Pagán Rafael R

Microorganisms 20200622 6

The emergence of antimicrobial resistance has raised questions about the safety of essential oils and their individual constituents as food preservatives and as disinfection agents. Further research is required to understand how and under what conditions stable genotypic resistance might occur in food pathogens. Evolution experiments on <i>Salmonella</i> Typhimurium cyclically exposed to sublethal and lethal doses of carvacrol permitted the isolation of SeSCar and SeLCar strains, respectively. B ...[more]

PMID: 32580471

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Project description:Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

Project description:BackgroundSmall colony variants (SCVs), constituting a slow-growing subpopulation of bacteria that facilitates persistence in lethal environmental conditions, are able to revert to the phenotype of rapid growth for further proliferation and transmission. Salmonella enterica serotype Typhimurium is one of the most important foodborne pathogens. This study investigated the genetic mechanisms how SCVs induced by streptomycin reverted to the fast-growing phenotype and the phenotypic changes of SCVs among their complete life cycle in S. Typhimurium.MethodsSalmonella Typhimurium SCVs were obtained by streptomycin treatment and their revertants were collected in the absence of antibiotics. The fitness, antimicrobial susceptibility, biofilm formation, and the biofilm-related genes expression were analyzed in comparison to their wild type strain, and the whole genome sequencing was performed to identify the genetic changes in the life cycle of S. Typhimurium SCVs.ResultsSmall colony variants were characterized by an increased antimicrobial resistance to streptomycin (64-fold), imipenem (twofold), and gentamicin (fourfold). A significant increase in biofilm production with higher expression of csgB was observed in SCVs (P < 0.01). The genetic alterations of all SCVs occurred in ubiE gene (coenzyme Q8 and menaquinone synthesis) with frameshift mutations. However, all fast-growing revertants again lost the trait of increased biofilm production (P > 0.05), in which two modes of the genetic changes for reversing to the rapidly growing form were observed: four revertants harbored a secondary mutation in ubiE, which reinstated most of the amino acid sequence of the ubiE, and other four revertants harbored a mutation in prfB.ConclusionsSalmonella Typhimurium could switch to the phenotype of SCVs under the treatment of streptomycin by a mutation in ubiE, partially combined with increased production of biofilm, and these SCVs could escape from growth restriction by a compensatory mutation in prfB or a new mutation in ubiE. These findings may contribute to establishing phenotype-directed treatments against SCVs of S. Typhimurium.

Project description:Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Project description:Salmonella enterica serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10(-4) per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10(-6) per cell per generation. Two highly virulent S. Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the Salmonella pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.

Dataset Information

Genetic Variants and Phenotypic Characteristics of Salmonella Typhimurium-Resistant Mutants after Exposure to Carvacrol.

Publications

Genetic Variants and Phenotypic Characteristics of <i>Salmonella</i> Typhimurium-Resistant Mutants after Exposure to Carvacrol.

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