Dataset Information

Efflux Pump Overexpression Contributes to Tigecycline Heteroresistance in Salmonella enterica serovar Typhimurium.

ABSTRACT: 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-?-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/?p52 were 0.5, 1, and 1 ?g/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/?p52 strain. Phe-Arg-?-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.

SUBMITTER: Chen Y

PROVIDER: S-EPMC5313504 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Efflux Pump Overexpression Contributes to Tigecycline Heteroresistance in Salmonella enterica serovar Typhimurium.

Chen Yi Y Hu Daxing D Zhang Qijing Q Liao Xiao-Ping XP Liu Ya-Hong YH Sun Jian J

Frontiers in cellular and infection microbiology 20170217

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 analys ...[more]

PMID: 28261566

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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:BackgroundBacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.ResultsA small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).ConclusionsSmall RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

Project description:AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.IMPORTANCE Antibiotic resistance is a major public health concern. In Gram-negative bacteria, overexpression of the AcrAB-TolC multidrug efflux system confers resistance to clinically useful drugs. Here, we show that loss of AcrB efflux function causes loss of virulence in Salmonella enterica serovar Typhimurium. This is due to the reduction of bacterial factors necessary for infection, which is likely to be caused by the retention of noxious molecules inside the bacterium. We also show that, in contrast to loss of AcrB protein, loss of efflux does not induce overexpression of other efflux pumps from the same family. This indicates that there are differences between loss of efflux protein and loss of efflux that make gene deletion mutants unsuitable for studying the biological function of membrane transporters. Understanding the biological role of AcrB will help to assess the risks of targeting efflux pumps as a strategy to combat antibiotic resistance.

Dataset Information

Efflux Pump Overexpression Contributes to Tigecycline Heteroresistance in Salmonella enterica serovar Typhimurium.

Publications

Efflux Pump Overexpression Contributes to Tigecycline Heteroresistance in <i>Salmonella enterica</i> serovar Typhimurium.

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