Dataset Information

Cooperation of the multidrug efflux pump and lipopolysaccharides in the intrinsic antibiotic resistance of Salmonella enterica serovar Typhimurium.

ABSTRACT:

Objectives

In Gram-negative bacteria, drug susceptibility is associated with multidrug efflux systems and an outer membrane (OM) barrier. Previous studies revealed that Salmonella enterica serovar Typhimurium has 10 functional drug efflux pumps. Among them, AcrB is a major factor to maintain the intrinsic drug resistance in this organism. The lipopolysaccharide (LPS) content of OM is also important for resistance to lipophilic drugs; however, the interplay between the multidrug efflux pump and LPS in the intrinsic antibiotic resistance of Salmonella remains to be studied in detail. The aim of this study was to investigate the relationship between AcrB and LPS in the intrinsic drug resistance of this organism.

Methods

The genes encoding LPS core biosynthetic proteins and AcrB were disrupted from the wild-type S. enterica strain ATCC 14028s. The plasmid carrying acrB was transformed into these mutants and then the drug susceptibilities of the mutants and transformants were determined.

Results

Our results showed that the levels of Salmonella intrinsic antibiotic resistance were decreased when the length and branches of core oligosaccharide were lost. Furthermore, the deletion of acrB reduced multidrug resistance of all LPS mutants and AcrB production from the plasmid complemented this phenotype. However, AcrB production could not completely compensate for LPS function in intrinsic resistance.

Conclusions

Both pump inactivation and shortened LPS enhanced drug susceptibility, although the maximum susceptibility was achieved when the two were combined. Hence, these results indicated that the multidrug efflux system and OM barrier are both essential for maintaining intrinsic antibiotic resistance in Salmonella.

SUBMITTER: Yamasaki S

PROVIDER: S-EPMC3625434 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Publications

Cooperation of the multidrug efflux pump and lipopolysaccharides in the intrinsic antibiotic resistance of Salmonella enterica serovar Typhimurium.

Yamasaki Seiji S Nagasawa Saya S Fukushima Aiko A Hayashi-Nishino Mitsuko M Nishino Kunihiko K

The Journal of antimicrobial chemotherapy 20130201 5

<h4>Objectives</h4>In Gram-negative bacteria, drug susceptibility is associated with multidrug efflux systems and an outer membrane (OM) barrier. Previous studies revealed that Salmonella enterica serovar Typhimurium has 10 functional drug efflux pumps. Among them, AcrB is a major factor to maintain the intrinsic drug resistance in this organism. The lipopolysaccharide (LPS) content of OM is also important for resistance to lipophilic drugs; however, the interplay between the multidrug efflux pu ...[more]

PMID: 23378414

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Project description:UnlabelledMultidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump in Salmonella, has previously been linked to virulence in mice. The molecular mechanism of this link between macAB and infection is unclear. We demonstrate that macAB plays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection. macAB is induced upon exposure to H2O2 and is critical for survival of Salmonella enterica serovar Typhimurium in the presence of peroxide. Furthermore, we determined that macAB is required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages. macAB mutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay, macAB mutants appear to be unable to induce protection against exogenous H2O2 in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue the macAB mutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection of Salmonella against oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2 compound secreted by Salmonella cells through a MacAB-dependent mechanism.ImportanceThe ABC-type multidrug efflux pump MacAB is known to be required for Salmonella enterica serovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killing in vitro and for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect of macAB mutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.

Project description:Bacterial heteroresistance has been identified in several combinations of bacteria and antibiotics, and it complicated the therapeutic strategies. Tigecycline is being used as one of the optimal options for the treatment of infections caused by multidrug-resistant Salmonella. This study investigated whether heterorresistance to tigecycline exists in a Salmonella enterica serovar Typhimurium strain harboring the oqxAB-bearing IncHI2 plasmid pHXY0908. MIC and population analyses were performed to evaluate population-wide susceptibility to tigecycline. The effects of efflux pumps on MIC levels were assessed using the efflux pump inhibitor Phe-Arg-β-naphthylamide, measuring intracellular tigecycline accumulation as well as mRNA levels of regulatory and efflux pump genes. DNA sequencing of regulatory regions were performed and plasmid curing from a resistant strain provided an appropriate control. Results showed that MICs of a parental strain with and without pHXY0908 as well as a plasmid-cured strain 14028/Δp52 were 0.5, 1, and 1 μg/mL, respectively. Population analysis profiling (PAP) illustrated that only the pHXY0908-containg strain was heteroresistant to tigecycline. A fraction of colonies exhibited stable profiles with 4- to 8-fold increases in MIC. The frequencies of emergence of these isolates were higher in the plasmid-containing strain pHXY0908 than either the parental or the 14028/Δp52 strain. Phe-Arg-β-naphthylamide addition restored tigecycline susceptibility of these isolates and intracellular tigecycline accumulation was reduced. Heteroresistant isolates of the strain containing pHXY0908 also had elevated expression of acrB, ramA, and oqxB. DNA sequencing identified numerous mutations in RamR that have been shown to lead to ramA overexpression. In conclusions, heteroresistance to tigecycline in Salmonella enterica serovar Typhimurium was manifested in a plasmid-bearing strain. Our results suggest that this phenotype was associated with overexpression of the AcrAB-TolC and OqxAB efflux pumps.

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Dataset Information

Cooperation of the multidrug efflux pump and lipopolysaccharides in the intrinsic antibiotic resistance of Salmonella enterica serovar Typhimurium.

Objectives

Methods

Results

Conclusions

Publications

Cooperation of the multidrug efflux pump and lipopolysaccharides in the intrinsic antibiotic resistance of Salmonella enterica serovar Typhimurium.

Similar Datasets

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