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A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

ABSTRACT: Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5?mM H2O2 (H2O2H). We identified two sets of overlapping genes required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for H2O2 resistance included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), and H2O2 scavenging enzymes. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. On the contrary, a subset of conditionally essential proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated by H2O2. The identified genes will deepen our understanding on S. Typhimurium survival mechanisms in macrophages, and can be exploited to develop new antimicrobial drugs.

SUBMITTER: Karash S

PROVIDER: S-EPMC5719062 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

Karash Sardar S Liyanage Rohana R Qassab Abdullah A Lay Jackson O JO Kwon Young Min YM

Scientific reports 20171206 1

Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2</s ...[more]

PMID: 29213059

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Project description:Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

Project description:BACKGROUND: Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. RESULTS: The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. CONCLUSIONS: The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the insertion and deletion of DNA stretches have shaped their evolutionary histories.

Dataset Information

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

Publications

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

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