Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADHdehydrogenasesisrefractorytotheearlyNADPHoxidasedependent antimicrobial activity of IFN␥-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO ؊ ) and hydrogen peroxide (H 2 O 2 ). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H 2 O 2 . NADH antagonizes the hydroxyl radical (OH ⅐ ) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.Oxidative stress engendered by the sustained synthesis of NO mediates cytotoxicity against a variety of eukaryotic and prokaryotic cells (1-3). Because of its unpaired electron, NO directly reacts with metal prosthetic groups of cytochromes in the electron transport chain (ETC) 2 and [Fe-S] clusters of dehydratases (4, 5). Alternatively, reactive nitrogen species (RNS) generated through the interaction of NO with O 2 and superoxide (O 2 . ) indirectly mediate cytotoxicity of this diatomic radical.

By remodeling the phagosomal membrane, the type III secretion system encoded within the Salmonella pathogenicity island-2 (SPI2) helps Salmonella thrive within professional phagocytes. We report here that nitric oxide (NO) generated by IFNγ-activated macrophages abrogates the intracellular survival advantage associated with a functional SPI2 type III secretion system. NO congeners inhibit overall expression of SPI2 effectors encoded both inside and outside the SPI2 gene cluster, reflecting a reduced transcript level of the sensor kinase SsrA that governs overall SPI2 transcription. Down-regulation of SPI2 expression in IFNγ-treated macrophages does not seem to be the result of global NO cytotoxicity, because transcription of the housekeeping rpoD sigma factor remains unchanged, whereas the expression of the hmpA-encoded, NO-metabolizing flavohemoprotein is stimulated. Because of the reduced SPI2 expression, Salmonella-containing vacuoles interact more efficiently with compartments of the late endosomal/lysosomal system in NO-producing, IFNγ-treated macrophages. These findings demonstrate that inhibition of intracellular SPI2 transcription by NO promotes the interaction of Salmonella phagosomes with the degradative compartments required for enhanced antimicrobial activity. Transcriptional repression of a type III secretion system that blocks phagolysosome biogenesis represents a novel mechanism by which NO mediates resistance of IFNγ-activated phagocytes to an intracellular pathogen.

Our investigations have identified a mechanism by which exogenous production of nitric oxide (NO) induces resistance of Gram-positive and -negative bacteria to aminoglycosides. An NO donor was found to protect Salmonella spp. against structurally diverse classes of aminoglycosides of the 4,6-disubstituted 2-deoxystreptamine group. Likewise, NO generated enzymatically by inducible NO synthase of gamma interferon-primed macrophages protected intracellular Salmonella against the cytotoxicity of gentamicin. NO levels that elicited protection against aminoglycosides repressed Salmonella respiratory activity. NO nitrosylated terminal quinol cytochrome oxidases, without exerting long-lasting inhibition of NADH dehydrogenases of the electron transport chain. The NO-mediated repression of respiratory activity blocked both energy-dependent phases I and II of aminoglycoside uptake but not the initial electrostatic interaction of the drug with the bacterial cell envelope. As seen in Salmonella, the NO-dependent inhibition of the electron transport chain also afforded aminoglycoside resistance to the clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Together, these findings provide evidence for a model in which repression of aerobic respiration by NO fluxes associated with host inflammatory responses can reduce drug uptake, thus promoting resistance to several members of the aminoglycoside family in phylogenetically diverse bacteria.

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Objective Hospitalizations for acute bacterial skin and skin structure infection (ABSSSI) are common. Optimizing antibiotic use for ABSSSIs requires an understanding of current management. The objective of this study was to evaluate antibiotic prescribing practices and factors affecting prescribing in a diverse group of hospitals. Design Multicenter, retrospective cohort study Setting Seven community and academic hospitals Methods Children and adults hospitalized between June 2010 and May 2012 for cellulitis, wound infection, or cutaneous abscess were eligible. The primary endpoint was a composite of two prescribing practices representing potentially avoidable antibiotic exposure: 1) use of antibiotics with a broad spectrum of activity against gram-negative bacteria; or 2) treatment duration >10 days. Results 533 cases were included: 320 with non-purulent cellulitis, 44 with wound infection or purulent cellulitis, and 169 with abscess. Of 492 cases with complete prescribing data, the primary endpoint occurred in 394 (80%) cases and varied significantly across hospitals (64 – 97%, p<.001). By logistic regression, independent predictors of the primary endpoint included wound infection or purulent cellulitis (odds ratio [OR] 5.12, 95% confidence interval [CI] 1.46 – 17.88), head or neck involvement (OR 2.83, 95%CI 1.17 – 6.82), adult cases (OR 2.20, 95%CI 1.18 – 4.11), and admission to a community hospital (OR 1.90, 95%CI 1.05 – 3.44). Conclusions Among patients hospitalized for ABSSSI, use of antibiotics with broad gram-negative activity or treatment courses longer than 10 days were common. There may be substantial opportunity to reduce antibiotic exposure through shorter courses of therapy targeting gram-positive bacteria.

We show herein that the Salmonella pathogenicity island 2 (SPI2) response regulator SsrB undergoes S-nitrosylation upon exposure of Salmonella to acidified nitrite, a signal encountered by this enteropathogen in phagosomes of macrophages. Mutational analysis has identified Cys 203 in the C-terminal dimerization domain of SsrB as the redox-active residue responding to nitric oxide (NO) congeners generated in the acidification of nitrite. Peroxynitrite and products of the autooxidation of NO in the presence of oxygen, but not hydrogen peroxide, inhibit the DNA-binding capacity of SsrB, demonstrating the selectivity of the reaction of Cys 203 with reactive nitrogen species (RNS). These findings identify the twocomponent response regulator SsrB Cys 203 as a thiol-based redox sensor. A C203S substitution protects SsrB against the attack of RNS while preserving its DNA-binding capacity. When exposed to SPI2-inducing conditions, Salmonella expressing the wild-type ssrB allele or the ssrB C203S variant sustain transcription of the sifA, sspH2, and srfJ effector genes. Nonetheless, compared with the strain expressing a redox-resistant SsrB C203S variant, wild-type Salmonella bearing the NO-responsive allele exhibit increased fitness when exposed to RNS in an NRAMP R , C3H/HeN murine model of acute oral infection. Given the widespread occurrence of the wildtype allele in Salmonella enterica, these findings indicate that SsrB Cys 203 increases Salmonella virulence by serving as a redox sensor of NO resulting from the host immune response to oral infection.macrophages | pathogenesis | two-component regulatory system

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

We have identified acid sphingomyelinase (ASM) as an important player in the early and late anti-Salmonella activity of macrophages. A functional ASM participated in the killing activity of macrophages against wild-type Salmonella enterica serovar Typhimurium. The role of ASM in early macrophage killing of Salmonella appears to be linked to an active NADPH phagocyte oxidase enzymatic complex, since the flavoprotein inhibitor diphenyleneiodonium not only blocked a productive respiratory burst but also abrogated the survival advantage of Salmonella in macrophages lacking ASM. Lack of ASM activity also increased the intracellular survival of an isogenic ⌬spiC::FRT Salmonella strain deficient in a translocator and effector of the Salmonella pathogenicity island 2 (SPI2) type III secretion system, suggesting that the antimicrobial activity associated with ASM is manifested regardless of the SPI2 status of the bacteria. Constitutively expressed ASM is responsible for the role that this lipid-metabolizing hydrolase plays in the innate host defense of macrophages against Salmonella. Accordingly, the ASM activity and intracellular concentration and composition of ceramide, gangliosides, and neutral sphingolipids did not increase upon Salmonella infection. Salmonella triggered, nonetheless, a significant increase in the secreted fraction of ASM. Collectively, these findings have elucidated a novel role for constitutive ASM in the anti-Salmonella activity of murine macrophages.