Dataset Information

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.

ABSTRACT: The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies.

SUBMITTER: VanCleave TT

PROVIDER: S-EPMC5732358 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Impact of Gentamicin Concentration and Exposure Time on Intracellular <i>Yersinia pestis</i>.

VanCleave Tiva T TT Pulsifer Amanda R AR Connor Michael G MG Warawa Jonathan M JM Lawrenz Matthew B MB

Frontiers in cellular and infection microbiology 20171211

The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens <i>in vitro</i> ...[more]

PMID: 29312891

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Project description:Plague is a flea-borne rodent-associated zoonotic disease caused by Yersinia pestis The disease is characterized by epizootics with high rodent mortalities, punctuated by interepizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between Y. pestis and the ubiquitous soil free-living amoeba (FLA) Acanthamoeba castellanii to assess if the bacterium can survive within soil amoebae and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoebae. The results demonstrate that during coculture with amoebae, representative Y. pestis strains of epidemic biovars Medievalis, Orientalis, and Antiqua are phagocytized and able to survive within amoebae for at least 5 days. Key Y. pestis determinants of the intracellular interaction of Y. pestis and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the Y. pestis-amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in the intracellular survival of Y. pestis in macrophages, a PhoP mutant is unable to survive when cocultured with amoebae. Additionally, induction of the T3SS blocks phagocytic uptake of Y. pestis by amoebae, similar to that which occurs during macrophage infection. Electron microscopy revealed that in A. castellanii, Y. pestis resides intact within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of Y. pestis within FLA suggests that protozoa may serve as a protective soil reservoir for Y. pestisIMPORTANCEYersinia pestis is a reemerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly, causing high rodent mortality, and an interepizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of Y. pestis in the context of its persistence in the environment and its reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here, we demonstrate prolonged survival and subversion of intracellular digestion of Y. pestis within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for Y. pestis, which may help explain the recrudescence of plague epizootics.

Project description:BACKGROUND: Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches. CONCLUSIONS/SIGNIFICANCE: We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.

Dataset Information

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.

Publications

Impact of Gentamicin Concentration and Exposure Time on Intracellular <i>Yersinia pestis</i>.

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