SUMMARYObservations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7ϫ10 -4 and 1.7ϫ10 -3 ·mol·l -1 , doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a ʻsignaling centerʼ for mosquito aging.

BackgroundIn this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.Methodology/Principal FindingsCompared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001.Conclusions/SignificanceAlthough further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ⌬tonB ⌬hcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain. Burkholderia mallei, the causative agent of glanders, is a Gramnegative, obligate mammalian pathogen. Glanders is primarily a disease of solipeds, with rare cases occurring among humans (1, 2). Naturally acquired human cases occur in areas of endemicity, particularly among those exposed to infected solipeds (2-4). Additionally, cases have been reported among laboratory workers (5, 6). The World Organization for Animal Health coordinates ongoing efforts toward worldwide eradication; however, regional endemicity still exists in Africa, Asia, the Middle East, and South America (2). Recent equid outbreaks in the Middle East and Asia (7-9) set the stage for possible glanders reintroduction into disease-free regions; as a result, glanders has been classified as a reemerging disease.Glanders is a debilitating and often fatal disease transmitted via cutaneous and respiratory routes. Disease course and severity are route dependent, with respiratory cases being the most severe (3). Respiratory infection is characterized by rapid onset of symptoms, including fever, lymphadenopathy, pulmonary abscesses, pneumonia, disseminated organ infection, and, ultimately, septicemia (3, 5, 10). Because of the high incidence of septicemia following respiratory infection, fatality rates in human respiratory cases have been estimated at 90% without treatment and 40% with aggressive antibiotic therapy (11).The use of B. mallei as a biothreat agent has been documented in different world military conflicts (10,12,13). Its amenability to aerosolization, low infectious dose, high case fatality rate, and high-level antibiotic resistance make B. mallei a top candidate for malevolent use (1, 10). Because of its perceived public health threat, the Department of Health and Human Services has categorized B. mallei as a tier 1 select agent. The lack of effective treatments against these bacteria highlights the need for an effective vaccine. Numerous vaccine strategies have been tested; however, to date there are n...

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Purpose: The rapid emergence of multidrug-resistant (MDR) bacteria and the lack of new therapies to eliminate them poses a major threat to global health. With the alarming rise in antimicrobial resistance (AMR), focus has now shifted to the use of the polymyxin class of antibiotics as the last line of defense for treatment of Gram-negative infections. Unfortunately, the growing resistance of bacteria against polymyxins is threatening the treatment of MDR infections, necessitating the need for novel strategies. The objective of this study was to determine if combination of polymyxin (polymyxin B or colistin) with a nonantibiotic small molecule AR-12, a celecoxib derivative that is devoid of cyclooxygenase 2 (COX-2) inhibitory activities, can be an effective strategy against polymyxin-resistant MDR bacteria. Methods: Growth inhibition studies, time-kill assays and permeability assays were conducted to investigate the effect of AR-12 on the antibacterial activity of polymyxins. Results: Growth studies were performed on a panel of polymyxin-resistant MDR strains using the combination of AR-12 with either colistin or polymyxin B. The combination treatment had no effect on strains that have inherent polymyxin resistance; however, AR-12 was effective in lowering the minimal inhibitory concentration (MIC) of polymyxins by 4–60-fold in several strains that had acquired polymyxin resistance. Time-kill assays using the combination of AR-12 and colistin with select MDR strains suggest rapid killing and bactericidal activity, while the permeability assays using fluorescently labeled dansylated polymyxin and 1-N-phenylnaphthylamine (NPN) in these MDR strains suggest that AR-12 can potentiate the antibacterial activity of polymyxins by possibly altering the bacterial outer membrane via modification of lipopolysaccharide and thereby improving the uptake of polymyxins. Conclusion: Our studies indicate that the combination of AR-12 and polymyxin is effective in targeting select Gram-negative bacteria that have acquired polymyxin resistance. Further understanding of the mechanism of action of AR-12 will provide new avenues for developing narrow-spectrum antibacterials to target select Gram-negative MDR bacteria. Importantly, our studies show that the use of nonantibiotic small molecules in combination with polymyxins is an attractive strategy to counter the growing resistance of bacteria to polymyxins.

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Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infections with a number of bacterial pathogens. Among them, the Burkholderia cepacia complex (Bcc) bacteria, consisting of nine related species, have emerged as problematic CF pathogens due to their antibiotic resistance, incidence of nosocomial infection, and person-to-person transmission. Bcc organisms present the clinical microbiologist with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that reliably distinguish among these organisms. The complex taxonomy of the Bcc species colonizing the CF respiratory tract makes accurate identification problematic. Despite the clinical implications of Bcc identification, a clinical laboratory differentiation of species within the Bcc is lacking. Additionally, no commercial assays are available to further identify the Bcc species. In the current study, secretory proteins present in the cultured supernatants of Burkholderia cenocepacia and Burkholderia multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis. In total, 341 protein spots were detected, of which 23 were unique to each species, demonstrating that potential diagnostic candidates between these two members of the Bcc exist.The Burkholderia cepacia complex (Bcc) consists of nine genetically distinct but phenotypically similar Gram-negative bacilli distinguished by their role as important agents of pulmonary disease in cystic fibrosis (CF) patients (12, 16). Associated with increased morbidity and mortality, Bcc bacteria make eradication very difficult due to their ability to establish infection and maintain chronic colonization in the CF lung (6,9,18,22). Furthermore, these organisms are capable of person-to-person spread, presenting the threat of nosocomial acquired infection. Highly variable and unpredictable, clinical outcomes of Bcc infection range from asymptomatic carriage to bacteremic disease and death (12). This variation is speculated to be the result of genomovar heterogeneity and differential expression of a virulence factor(s) during Bcc infection. Although all species of the Bcc have been recovered from CF patients, Burkholderia cenocepacia and Burkholderia multivorans are the most prevalent, accounting for 85% of pulmonary infections (16).Establishing accurate methods of Bcc identification is paramount for the implementation of infection control measures and appropriate treatment of infected and/or exposed CF patients. Furthermore, species identification within the Bcc is vital not only for surgical and clinical management of CF patients but also for the elucidation of the group's epidemiology. Bcc organisms present clinical microbiologists with a diagnostic dilemma due to the l...

Our data demonstrate that previous germination-induction experiments performed in suspension can be reproduced when Bacillus spores are deposited onto reference surfaces materials. Our proof of concept experiment illustrated that a germination pretreatment step significantly improves conventional secondary decontamination strategies and remediation plans.