Gut Microbiota and Other Factors Associated With Increased Regulatory T Cells in Hiv-exposed Uninfected Infants.

HIV-exposed uninfected infants (HEU) have higher infectious morbidity than HIV-unexposed infants (HUU). HEU have multiple immune defects of unknown origin. We hypothesized that HEU have higher regulatory T cells (Treg) than HUU, which may dampen their immune defenses against pathogens. We compared 25 Treg subsets between HEU and HUU and sought the factors that may affect Treg frequencies. At birth, 3 Treg subsets, including CD4 + FOXP3 + and CD4 + FOXP3 + CD25+, had higher frequencies in 123 HEU than 117 HUU and 3 subsets were higher in HUU. At 28 and 62 weeks of life, 5 Treg subsets were higher in HEU, and none were higher in HUU. The frequencies of the discrepant Treg subsets correlated at birth with differential abundances of bacterial taxas in maternal gut microbiome and at subsequent visits in infant gut microbiomes. In vitro, bacterial taxa most abundant in HEU expanded Treg subsets with higher frequencies in HEU, recapitulating the in vivo observations. Other factors that correlated with increased Treg were low maternal CD4 + T cells in HEU at birth and male sex in HUU at 28 weeks. We conclude that maternal and infant gut dysbiosis are central to the Treg increase in HEU and may be targeted by mitigating interventions.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 µM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

Aging-Associated Augmentation of Gut Microbiome Virulence Capability Drives Sepsis Severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition but also an overabundance of genomic virulence factors that have functional consequence on host immune evasion. IMPORTANCE Older adults suffer more frequent and worse outcomes from sepsis, a critical illness secondary to infection. The reasons underlying this unique susceptibility are incompletely understood. Prior work in this area has focused on how the immune response changes with age. The current study, however, focuses instead on alterations in the community of bacteria that humans live with within their gut (i.e., the gut microbiome). The central concept of this paper is that the bacteria in our gut evolve along with the host and "age," making them more efficient at causing sepsis.

Aging-associated augmentation of gut microbiome virulence capability drives sepsis severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host, but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition, but an overabundance of genomic virulence factors that have functional consequence on host immune evasion. One Sentence Summary: The severity of sepsis in the aged host is in part mediated by longevity-associated increases in gut microbial virulence.

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium.

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.

Miliary Histoplasmosis in a Patient with Rheumatoid Arthritis.

Miliary histoplasmosis is a rare presentation that may mimic miliary tuberculosis. We report a case of miliary histoplasmosis in a 52-year-old male who was being treated with hydroxychloroquine, methotrexate, and sulfasalazine for his rheumatoid arthritis and presented to the emergency department with shortness of breath and fevers. Computed tomography (CT) chest revealed miliary pulmonary nodules. Urine Histoplasma antigen and serum Histoplasma antigen were negative; however, Coccidioides immitis complement immunofixation assay and Coccidioides IgM were positive. The patient was initiated on treatment for pulmonary coccidioidomycosis and immunosuppression was held. However, a few days later, Histoplasma capsulatum was isolated from cultures from bronchoscopy. This case highlights the difficulty in diagnosing histoplasmosis in immunocompromised patients and the importance of having a broad differential diagnosis for miliary pulmonary nodules. Tissue culture and histopathology remain the gold standard for the diagnosis of histoplasmosis. Further research needs to be conducted to determine the optimal duration of histoplasmosis treatment in immunocompromised patients.

Cryptococcosis and cryptococcal meningitis: New predictors and clinical outcomes at a United States academic medical centre.

As the diagnosis of cryptococcosis is challenging in low-prevalence settings, uncovering predictive factors can improve early diagnosis and timely treatment. The aim of the study was to relate clinical outcomes to predictive variables for the presence of cryptococcosis. A retrospective case-control study matched by collection date, age and gender at a 1:2 ratio (55 cases and 112 controls) was performed in case patients diagnosed with Cryptococcus infection at the University of Colorado Hospital between 2000 and 2017 (n = 167). A bivariate and a forward, stepwise multivariable logistic regression model were performed to identify predictors of cryptococcosis infection. In an adjusted multivariable model, cryptococcal infection was significantly associated with the presence of respiratory symptoms, hyponatremia, lung disease or corticosteroids. Additionally, cryptococcal meningitis was associated with headaches, corticosteroids or increased CSF protein. Conversely, a reduced risk of cryptococcosis was associated with hypertension or peripheral monocytosis. Cryptococcal meningitis leads to subsequent hearing impairment (16% vs 4% (control), P = .013), muscle weakness (40% vs 20%, P = .021), cognitive deficits (33% vs 6%, P = .0001) or any adverse outcome (84% vs 29%, P = .0001). We uncovered novel clinical predictors for the presence of cryptococcal infection or cryptococcal meningitis. This study in patients at a low-prevalence US medical centre underscores the importance of early diagnosis in this population.

Whole-Genome Sequencing Identifies In Vivo Acquisition of a blaCTX-M-27-Carrying IncFII Transmissible Plasmid as the Cause of Ceftriaxone Treatment Failure for an Invasive Salmonella enterica Serovar Typhimurium Infection.

We report a case of ceftriaxone treatment failure for bacteremia caused by Salmonella enterica subsp. enterica serovar Typhimurium, due to the in vivo acquisition of a blaCTX-M-27-encoding IncFII group transmissible plasmid. The original ß-lactamase-susceptible isolate ST882S was replaced by the resistant isolate ST931R during ceftriaxone treatment. After relapse, treatment was changed to ciprofloxacin, and the patient recovered. Isolate ST931R could transfer resistance to Escherichia coli at 37°C. We used whole-genome sequencing of ST882S and ST931R, the E. coli transconjugant, and isolated plasmid DNA to unequivocally show that ST882S and ST931R had identical chromosomes, both having 206 identical single-nucleotide polymorphisms (SNPs) versus S Typhimurium 14028s. We assembled a complete circular genome for ST931R, to which ST882S reads mapped with no SNPs. ST882S and ST931R were isogenic except for the presence of three additional plasmids in ST931R. ST931R and the E. coli transconjugant were ceftriaxone resistant due to the presence of a 60.5-kb IS26-flanked, blaCTX-M-27-encoding IncFII plasmid. Compared to 14082s, ST931R has almost identical Gifsy-1, Gifsy-2, and ST64B prophages, lacks Gifsy-3, and instead carries a unique Fels-2 prophage related to that found in LT2. ST882S and ST931R both had a 94-kb virulence plasmid showing >99% identity with pSLT14028s and a cryptic 3,904-bp replicon; ST931R also has cryptic 93-kb IncI1 and 62-kb IncI2 group plasmids. To the best of our knowledge, in vivo acquisition of extended-spectrum ß-lactamase resistance by S Typhimurium and blaCTX-M-27 genes in U.S. isolates of Salmonella have not previously been reported.

Risk Factors for Cryptococcal Meningitis: A Single United States Center Experience.

Cryptococcal meningitis carries a high mortality. Further understanding of immune suppression factors associated with neuroinvasive infection will improve risk stratification and enhance early diagnosis and treatment with antifungal therapy. The aim of the study was to corroborate established or find novel clinical predictors for cryptococcal meningitis. We performed a matched case-control study of Cryptococcus infection in immunocompromised patients with or without cryptococcal meningitis. Data of all patients with a diagnosis of cryptococcal disease were collected at University of Colorado Hospital between 2000 and 2015 (n = 51). Thirty patients were diagnosed with cryptococcal meningitis. We built a logistic regression model for risk factors associated with cryptococcal meningitis. The single-predictor univariate model found that a positive blood culture, positive serum cryptococcal antigen, current malignancy, and headaches were significantly associated with cryptococcal meningitis (p = 0.02). In the adjusted multivariate model, central nervous system disease was significantly associated with a diagnosis of HIV infection (OR 24.45, 95 % CI 1.62-350.37; p = 0.022) and a positive serum cryptococcal antigen test (OR 42.92, 95 % CI 3.26-555.55; p = 0.0055). In patients with HIV infection or a positive serum cryptococcal antigen, the pretest probability of neuroinvasive Cryptococcus infection is increased and an aggressive diagnostic evaluation should be conducted to exclude infection and consider empiric therapy.

Risk factors and influence of carbapenem exposure on the development of carbapenem resistant Pseudomonas aeruginosa bloodstream infections and infections at sterile sites.

BACKGROUND: Patients with Pseudomonas aeruginosa infections from blood or sterile sites were evaluated to determine risk factors associated with carbapenem resistance (CRPA) compared to carbapenem sensitivity (CSPA) as well as prior carbapenem use and the development of resistance. FINDINGS: Retrospective chart review of 80 patients hospitalized with a documented P. aeruginosa infection during 2010-2011. Stored isolates were retested with both Kirby-Bauer disk diffusion and E-tests. Clinical characteristic of patients in the CRPA (N = 21) and the CSPA (N = 59) groups were similar. Hospital acquired (HA) infections were more common in the CRPA group compared to the CSPA group (71 vs 44 %, p = 0.04) and CRPA patients were more likely to have a Foley catheter at the time of infection (71 vs 37 %, p = 0.01). There was more carbapenem use in the CRPA group prior to onset of infection (59 vs 22 %, OR 5.1, 95 % CI 1.3-20.8, p = 0.01). Length of stay was significantly longer in the CRPA group (mean 44 days) compared to the CSPA group (mean 23 days), p = 0.02. Mortality between the two groups was similar and there were no differences between groups for death attributable to Pseudomonas. CONCLUSIONS: Patients with CRPA were more likely to have HA infections and to have a multidrug resistant profile. Other identifiable risks included a Foley catheter in place at the time of infection and exposure to a carbapenem prior to infection. Prompt removal of devices and judicious use of antibiotics may be interventions that can impact the development of this kind of infections.

Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection.

Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff. Methods. Escherichia coli was incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015. In vitro techniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff. Results. Mean bacterial colony formation in PBS was greater for E. coli incubated in the presence of GTM (1.48 × 10(7) CFU/mL) versus without (9.95 × 10(5) CFU/mL) following 20-hour incubation (p = 0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) with Enterococcus faecalis. Conclusion. In vitro experiments support a facilitating role of GTM on colony formation of E. coli in PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence of E. faecalis, but results were inconclusive. Further studies are recommended.

Failure of outpatient antibiotics among patients hospitalized for acute bacterial skin infections: What is the clinical relevance?

BACKGROUND: Infectious Diseases Society of America guidelines recommend that patients hospitalized for acute bacterial skin infections after failure of outpatient antibiotic therapy be managed as "severe" infections; however, the clinical relevance of apparent failure of outpatient therapy is not clear. METHODS: This was a secondary analysis of a multicenter, retrospective cohort of adults and children hospitalized for cellulitis, abscess, or wound infection. We compared clinical features, laboratory and microbiology findings, antibiotic treatment, and outcomes among patients who received outpatient antibiotics prior to admission and those who did not. RESULTS: Of 533 patients, 179 (34%) received outpatient antibiotics prior to admission. Compared with those who did not, patients who received antibiotics prior to admission less frequently had fever (18% vs 26%, P=.04) and leukocytosis (33% vs 51%, P<.001). In the 202 cases where a microorganism was identified, Staphylococcus aureus was more common among those who received antibiotics prior to admission (75% vs 58%, P=.02), particularly methicillin-resistant S aureus (41% vs 27%, P=.049), whereas aerobic gram-negative bacilli were less common (3% vs 13%, P=.03). After hospitalization, clinical failure occurred with similar frequency between the 2 groups (12% vs 11%, P=.73). CONCLUSIONS: Patients hospitalized with skin infections after apparently failing outpatient therapy had clinical features suggestive of less severe infection and similar outcomes compared with patients who did not receive antibiotics prior to admission. Our results suggest that inpatient treatment for patients not responding to outpatient therapy should focus on methicillin-resistant S aureus, not gram-negative pathogens.

Highly Bactericidal Polyurethane Effective Against Both Normal and Drug-Resistant Bacteria: Potential Use as an Air Filter Coating.

The battle against the prevalence of hospital-acquired infections has underscored the importance of identifying and maintaining the cleanliness of possible infection transmission sources in the patient's environment. One of the most crucial lines of defense for mitigating the spread of pathogens in a healthcare facility is the removal of microorganisms from the environment by air filtration systems. After removing the pathogenic microorganisms, the filters used in these systems can serve as reservoirs for the pathogens and pose a risk for secondary infection. This threat, combined with the ever-growing prevalence of drug-resistant bacterial strains, substantiates the need for an effective bactericidal air filter. To this end, a broad-spectrum bactericidal polyurethane incorporating immobilized quaternary ammonium groups was developed for use as an air filter coating. In this study, the bactericidal activity of the polymer coating on high-efficiency particulate air (HEPA) filter samples was quantified against eight bacterial strains commonly responsible for nosocomial infection-including drug-resistant strains, and confirmed when applied as a filter coating in conditions mimicking those of its intended application. The coated HEPA filter samples exhibited high bactericidal activity against all eight strains, and the polyurethane was concluded to be an effective coating in rendering HEPA filters bactericidal.

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 ß-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.

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.

Nitric oxide protects bacteria from aminoglycosides by blocking the energy-dependent phases of drug uptake.

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.

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 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 two-component 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 wild-type 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.

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.