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Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network.

Condell O, Power KA, Händler K, Finn S, Sheridan A, Sergeant K, Renaut J, Burgess CM, Hinton JC, Nally JE, Fanning S - Front Microbiol (2014)

Bottom Line: Typhimurium compared with its isogenic sensitive progenitor.The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur.In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism.

View Article: PubMed Central - PubMed

Affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland ; European Program for Public Health Microbiology Training, European Centre for Disease Prevention and Control Stockholm, Sweden.

ABSTRACT
Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24(CHX)) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.

No MeSH data available.


Related in: MedlinePlus

Expression of differentially expressed genes and/or proteins associated with anaerobic metabolism/energy production in (A) the reference isolate ST24WT following chlorhexidine exposure and (B) the mutant isolate ST24CHX compared to its isogenic ST24WT without chlorhexidine exposure. Enzyme names, descriptions, gene identifiers and fold-change values are given in Table 2, Tables S1, S4. Color blocks without a white diagonal line represent gene fold change values (Table S1), color blocks containing a white diagonal line represent protein fold change values (Table 2, Table S4).
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Figure 5: Expression of differentially expressed genes and/or proteins associated with anaerobic metabolism/energy production in (A) the reference isolate ST24WT following chlorhexidine exposure and (B) the mutant isolate ST24CHX compared to its isogenic ST24WT without chlorhexidine exposure. Enzyme names, descriptions, gene identifiers and fold-change values are given in Table 2, Tables S1, S4. Color blocks without a white diagonal line represent gene fold change values (Table S1), color blocks containing a white diagonal line represent protein fold change values (Table 2, Table S4).

Mentions: When analyzed using the transcriptomic platform, genes associated with central metabolism and energy production in ST24WT exhibited altered expression. Enzymes associated with glycolysis and the TCA cycle were down-regulated, whilst those of other alternative pathways, such as mixed acid fermentation, fatty acid biosynthesis and glyceroplipid metabolism were up-regulated (Figure 5A). The expression of formate dehydrogenase was increased, and this enzyme functions in the conversion of formate to CO2 in the absence of alternative electron acceptors. In addition, acetyl-CoA carboxylase, an enzyme that converts acetyl-CoA to malonyl-CoA, a regulatory step for the inititation of fatty acid biosynthesis, was also up-regulated. Genes associated with anaerobic glycerolipid metabolism, such as glyB, were up-regulated in ST24WT. Interestingly, genes encoded by the pdu operon, which is associated with the catabolism of 1,2-propanediol and linked with the formation of polyhedral bodies- large proteinaceous structures of unknown function (Bobik et al., 1999; Havemann et al., 2002), were up-regulated. This metabolic pathway is known to be involved with anoxic metabolism in Salmonella (Bobik et al., 1999; Havemann et al., 2002), and it has previously been proposed that chlorhexidine blocks oxygen utilization in bacteria (Barrett-Bee et al., 1994). ST24WT appeared to up-regulate alternative metabolic pathways for anoxic energy production to mitigate this inhibitory action of chlorhexidine (Figure 5A).


Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network.

Condell O, Power KA, Händler K, Finn S, Sheridan A, Sergeant K, Renaut J, Burgess CM, Hinton JC, Nally JE, Fanning S - Front Microbiol (2014)

Expression of differentially expressed genes and/or proteins associated with anaerobic metabolism/energy production in (A) the reference isolate ST24WT following chlorhexidine exposure and (B) the mutant isolate ST24CHX compared to its isogenic ST24WT without chlorhexidine exposure. Enzyme names, descriptions, gene identifiers and fold-change values are given in Table 2, Tables S1, S4. Color blocks without a white diagonal line represent gene fold change values (Table S1), color blocks containing a white diagonal line represent protein fold change values (Table 2, Table S4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117984&req=5

Figure 5: Expression of differentially expressed genes and/or proteins associated with anaerobic metabolism/energy production in (A) the reference isolate ST24WT following chlorhexidine exposure and (B) the mutant isolate ST24CHX compared to its isogenic ST24WT without chlorhexidine exposure. Enzyme names, descriptions, gene identifiers and fold-change values are given in Table 2, Tables S1, S4. Color blocks without a white diagonal line represent gene fold change values (Table S1), color blocks containing a white diagonal line represent protein fold change values (Table 2, Table S4).
Mentions: When analyzed using the transcriptomic platform, genes associated with central metabolism and energy production in ST24WT exhibited altered expression. Enzymes associated with glycolysis and the TCA cycle were down-regulated, whilst those of other alternative pathways, such as mixed acid fermentation, fatty acid biosynthesis and glyceroplipid metabolism were up-regulated (Figure 5A). The expression of formate dehydrogenase was increased, and this enzyme functions in the conversion of formate to CO2 in the absence of alternative electron acceptors. In addition, acetyl-CoA carboxylase, an enzyme that converts acetyl-CoA to malonyl-CoA, a regulatory step for the inititation of fatty acid biosynthesis, was also up-regulated. Genes associated with anaerobic glycerolipid metabolism, such as glyB, were up-regulated in ST24WT. Interestingly, genes encoded by the pdu operon, which is associated with the catabolism of 1,2-propanediol and linked with the formation of polyhedral bodies- large proteinaceous structures of unknown function (Bobik et al., 1999; Havemann et al., 2002), were up-regulated. This metabolic pathway is known to be involved with anoxic metabolism in Salmonella (Bobik et al., 1999; Havemann et al., 2002), and it has previously been proposed that chlorhexidine blocks oxygen utilization in bacteria (Barrett-Bee et al., 1994). ST24WT appeared to up-regulate alternative metabolic pathways for anoxic energy production to mitigate this inhibitory action of chlorhexidine (Figure 5A).

Bottom Line: Typhimurium compared with its isogenic sensitive progenitor.The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur.In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism.

View Article: PubMed Central - PubMed

Affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland ; European Program for Public Health Microbiology Training, European Centre for Disease Prevention and Control Stockholm, Sweden.

ABSTRACT
Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24(CHX)) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.

No MeSH data available.


Related in: MedlinePlus