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  1. Miller, Samuel I. (Ed.)
    ABSTRACT Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses of two rodents: Peromyscus leucopus , the white-footed deermouse and reservoir for the agents of Lyme disease and other zoonoses, and the house mouse Mus musculus . Four hours after injection with LPS or saline, blood, spleen, and liver samples were collected and subjected to transcriptome sequencing (RNA-seq), metabolomics, and specific reverse transcriptase quantitative PCR (RT-qPCR). Differential expression analysis was at the gene, pathway, and network levels. LPS-treated deermice showed signs of sickness similar to those of exposed mice and had similar increases in corticosterone levels and expression of interleukin 6 (IL-6), tumor necrosis factor, IL-1β, and C-reactive protein. By network analysis, the M. musculus response to LPS was characterized as cytokine associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in the expression levels of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 responses in deermice. Analysis of metabolites in plasma and RNA in organs revealed species differences in tryptophan metabolism. Two genes in particular signified the different phenotypes of deermice and mice: the Slpi and Ibsp genes. Key RNA-seq findings for P. leucopus were replicated in older animals, in a systemic bacterial infection, and with cultivated fibroblasts. The findings indicate that P. leucopus possesses several adaptive traits to moderate inflammation in its balancing of infection resistance and tolerance. IMPORTANCE Animals that are natural carriers of pathogens that cause human diseases commonly manifest little or no sickness as a consequence of infection. Examples include the deermouse, Peromyscus leucopus , which is a reservoir for Lyme disease and several other disease agents in North America, and some types of bats, which are carriers of viruses with pathogenicity for humans. Mechanisms of this phenomenon of infection tolerance and entailed trade-off costs are poorly understood. Using a single injection of lipopolysaccharide (LPS) endotoxin as a proxy for infection, we found that deermice differed from the mouse ( Mus musculus ) in responses to LPS in several diverse pathways, including innate immunity, oxidative stress, and metabolism. Features distinguishing the deermice cumulatively would moderate downstream ill effects of LPS. Insights gained from the P. leucopus model in the laboratory have implications for studying infection tolerance in other important reservoir species, including bats and other types of wildlife. 
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  2. Miller, Samuel I. (Ed.)
    ABSTRACT The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii , the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis. IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii , which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities. 
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