Bruce D. McCollister

Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration

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 NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNγ-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 (H2O2). 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 H2O2. NADH antagonizes the hydroxyl radical (OH·) 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 Salmonellas 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.

Epidemiology of Healthcare‐Associated Bloodstream Infection Caused by USA300 Strains of Methicillin‐Resistant Staphylococcus aureus in 3 Affiliated Hospitals

OBJECTIVE To describe the epidemiology of bloodstream infection caused by USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA), which are traditionally associated with cases of community-acquired infection, in the healthcare setting. DESIGN Retrospective cohort study. SETTING Three academically affiliated hospitals in Denver, Colorado. METHODS Review of cases of S. aureus bloodstream infection during the period from 2003 through 2007. Polymerase chain reaction was used to identify MRSA USA300 isolates. RESULTS A total of 330 cases of MRSA bloodstream infection occurred during the study period, of which 286 (87%) were healthcare-associated. The rates of methicillin resistance among the S. aureus isolates recovered did not vary during the study period and were similar among the 3 hospitals. However, the percentages of cases of healthcare-associated MRSA bloodstream infection due to USA300 strains varied substantially among the 3 hospitals: 62%, 19%, and 36% (P<.001) for community-onset cases and 33%, 3%, and 33% (P=.005) for hospital-onset cases, in hospitals A, B, and C, respectively. In addition, the number of cases of healthcare-associated MRSA bloodstream infection caused by USA300 strains increased during the study period at 2 of the 3 hospitals. At each hospital, USA300 strains were most common among cases of community-associated infection and were least common among cases of hospital-onset infection. Admission to hospital A (a safety-net hospital), injection drug use, and human immunodeficiency virus infection were independent risk factors for healthcare-associated MRSA bloodstream infection due to USA300 strains. CONCLUSIONS The prevalence of USA300 strains among cases of healthcare-associated MRSA bloodstream infection varied dramatically among geographically clustered hospitals. USA300 strains are replacing traditional healthcare-related strains of MRSA in some healthcare settings. Our data suggest that the prevalence of USA300 strains in the community is the dominant factor affecting the prevalence of this strain type in the healthcare setting.

Nitric Oxide Protects Bacteria from Aminoglycosides by Blocking the Energy-Dependent Phases of Drug Uptake

ABSTRACT 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.

Constitutive Acid Sphingomyelinase Enhances Early and Late Macrophage Killing of Salmonella enterica Serovar Typhimurium

ABSTRACT 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 ΔspiC::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.

Redox sensor SsrB Cys203 enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection

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 Cys203 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 Cys203 with reactive nitrogen species (RNS). These findings identify the two-component response regulator SsrB Cys203 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 NRAMPR, C3H/HeN murine model of acute oral infection. Given the widespread occurrence of the wild-type allele in Salmonella enterica, these findings indicate that SsrB Cys203 increases Salmonella virulence by serving as a redox sensor of NO resulting from the host immune response to oral infection.

Hydrogen-Peroxide-Enhanced Nonthermal Plasma Effluent for Biomedical Applications

A novel nonthermal plasma dielectric-barrier discharge (DBD) system for decontamination, sterilization, and medical applications has been developed. The discharge is physically removed from the disinfection zone, and plasma-induced free radicals are delivered through an air stream. The physical distance between the discharge and the treatment surface can be up to 3 m, making the technology robust and flexible for applications in the medical clinic. The bactericidal properties of the free-radical effluent are enhanced by hydrogen peroxide additives. We report a 6-log reduction in Staphylococcus aureus and Pseudomonas aeruginosa bacteria strains in under 1 min of exposure in vitro and inactivation of Bacillus atrophaes spores and Escherichia coli biofilms. The concentration of hydrogen peroxide additives is seen to be a key variable in inactivation efficacy, suggesting that active species in our experiment may be different than in other DBD configurations. Precise chemical concentration measurements using direct frequency comb spectroscopy show presence of ozone ( O3), hydrogen peroxide (H2O2), nitrous oxide (N2O), and nitrogen dioxide (NO2). In vivo multiple exposures of mouse skin to the plasma effluent do not yield any adverse effects.

Antibiotic prescribing practices in a multicenter cohort of patients hospitalized for acute bacterial skin and skin structure infection.

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 2 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 greater than 10 days. RESULTS A total of 533 cases were included: 320 with nonpurulent 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.

Systemic CD8 T-Cell Memory Response to a Salmonella Pathogenicity Island 2 Effector Is Restricted to Salmonella enterica Encountered in the Gastrointestinal Mucosa

ABSTRACT To better understand the evolution of a systemic memory response to a mucosal pathogen, we monitored antigen-specific OT1 CD8 T-cell responses to a fusion of the SspH2 protein and the peptide SIINFEKL stably expressed from the chromosome of Salmonella enterica and loaded into the class I pathway of antigen presentation of professional phagocytes through the Salmonella pathogenicity island 2 type III secretion system (TTSS). This strategy has revealed that effector memory CD8 T cells with low levels of CD62L expression (CD62Llow) are maintained in systemic sites months after vaccination in response to low-grade infections with Salmonella. However, the CD8 T-cell pool eventually declines. Low numbers of central memory cells surviving after prolonged resting from an antigen encounter can nevertheless reconstitute the systemic effector memory pool in a route-specific recall response to cognate antigens encountered in the gut. Accordingly, populations of CD62Lhigh interleukin-7 receptor-positive progenitor central memory cells grafted into naïve mice expand in response to orally administered Salmonella expressing the chromosomal translational fusion of sspH2 and the sequence encoding the SIINFEKL peptide but fail to proliferate following systemic stimulation. Moreover, populations of systemic memory CD8 T cells restricted to Salmonella in oral vaccines selectively expand in response to cognate antigens presented by cells isolated from mesenteric lymph nodes (MLN). Together, these findings have revealed the imprinting of systemic CD8 central memory T-cell recall responses against enteropathogens by MLN. MLN restriction represents a novel mechanism by which systemic CD8 T-cell immunity is confined to periods of high risk for extraintestinal dissemination.

Microbiology and Initial Antibiotic Therapy for Injection Drug Users and Non–Injection Drug Users with Cutaneous Abscesses in the Era of Community‐associated Methicillin‐resistant Staphylococcus aureus

OBJECTIVES The incidence of cutaneous abscesses has increased markedly since the emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Injection drug use is a risk factor for abscesses and may affect the microbiology and treatment of these infections. In a cohort of patients hospitalized with cutaneous abscesses in the era of CA-MRSA, the objectives were to compare the microbiology of abscesses between injection drug users and non-injection drug users and evaluate antibiotic therapy started in the emergency department (ED) in relation to microbiologic findings and national guideline treatment recommendations. METHODS This was a secondary analysis of two published retrospective cohorts of patients requiring hospitalization for acute bacterial skin infections between January 1, 2007, and May 31, 2012, in seven academic and community hospitals in Colorado. In the subgroup of patients with cutaneous abscesses, microbiologic findings and the antibiotic regimens started in the ED were compared between injection drug users and non-injection drug users. Antibiotic regimens involving multiple agents, lack of activity against MRSA, or an agent with broad Gram-negative activity were classified as discordant with Infectious Diseases Society of America (IDSA) guideline treatment recommendations. RESULTS Of 323 patients with cutaneous abscesses, 104 (32%) occurred in injection drug users. Among the 235 cases where at least one microorganism was identified by culture, S. aureus was identified less commonly among injection drug users compared with non-injection drug users (55% vs. 75%, p = 0.003), with similar patterns observed for MRSA (33% vs. 47%, p = 0.054) and methicillin-susceptible S. aureus (17% vs. 26%, p = 0.11). In contrast to S. aureus, streptococcal species (53% vs. 25%, p < 0.001) and anaerobic organisms (29% vs. 10%, p < 0.001) were identified more commonly among injection drug users. Of 88 injection drug users and 186 non-injection drug users for whom antibiotics were started in the ED, the antibiotic regimens were discordant with IDSA guideline recommendations in 47 (53%) and 101 (54%), respectively (p = 0.89). In cases where MRSA was ultimately identified, the antibiotic regimen started in the ED lacked activity against this pathogen in 14% of cases. CONCLUSIONS Compared with non-injection drug users, cutaneous abscesses in injection drug users were less likely to involve S. aureus, including MRSA, and more likely to involve streptococci and anaerobes; however, MRSA was common in both groups. Antibiotic regimens started in the ED were discordant with national guidelines in over half of cases and often lacked activity against MRSA when this pathogen was present.

Comparison of the microbiology and antibiotic treatment among diabetic and nondiabetic patients hospitalized for cellulitis or cutaneous abscess

BACKGROUND Among diabetics, complicated skin infections may involve gram-negative pathogens; however, the microbiology of cellulitis and cutaneous abscess is not well established. OBJECTIVE To compare the microbiology and prescribing patterns between diabetics and nondiabetics hospitalized for cellulitis or abscess. DESIGN Secondary analysis of 2 published retrospective cohorts. SETTING/PATIENTS Adults hospitalized for cellulitis or abscess, excluding infected ulcers or deep tissue infections, at 7 academic and community facilities. METHODS Microbiological findings and antibiotic use were compared among diabetics and nondiabetics. Multivariable logistic regression was performed to identify factors associated with exposure to broad gram-negative therapy, defined as receipt of at least 2 calendar days of β-lactamase inhibitors, second- to fifth-generation cephalosporins, fluoroquinolones, carbapenems, tigecycline, aminoglycosides, or colistin. RESULTS Of 770 total patients with cellulitis or abscess, 167 (22%) had diabetes mellitus. Among the 38% of cases with a positive culture, an aerobic gram-positive organism was isolated in 90% of diabetics and 92% of nondiabetics (P = 0.59); aerobic gram-negative organisms were isolated in 7% and 12%, respectively (P = 0.28). Overall, diabetics were more likely than nondiabetics to be exposed to broad gram-negative therapy (54% vs 44% of cases, P = 0.02). By logistic regression, diabetes mellitus was independently associated with exposure to broad gram-negative therapy (odds ratio: 1.66, 95% confidence interval: 1.15-2.40). CONCLUSION In cases of cellulitis or abscess associated with a positive culture, gram-negative pathogens were not more common among diabetics compared with nondiabetics. However, diabetics were overall more likely to be exposed to broad gram-negative therapy suggesting this prescribing practice may not be not warranted.