80 Lipinska BS, Sharma.s,.Georgopour-o^s c. Sequence anarysis ancr reguration of the htrA gene of Escherichia coi:'a o32-indepena.nt r".tt'unitr. oi'tr.ut-incllcible transcription. Nucleic Acids Res lggg; 16: 1b053_67. 8l Johnson K, charles.I, Dougan G, et ar. The rore of stress-response protein in ^^ Salmotrclla ryphitnuriutn virulence. Mol Micrcbiol l99l;5: +Of _i]tt 3::::^l?:.*\Id..l_K, Lourenco RV, et ar. Inflammaiory: r",,.iion and airway

BACKGROUND:Much evidence of HIV-exposed uninfected (HEU) infant infectious morbidity predates availability of maternal combination antiretroviral therapy and does not control for universal risk factors (preterm birth, low birth weight, suboptimal breastfeeding and poverty).METHODS:This prospective cohort study identified HIV-infected and HIV-uninfected mothers and their newborns from South African community midwife unit. The primary outcome, infectious cause hospitalization or death before 6 months of age, was compared between HEU and HIV-unexposed (HU) infants and classified for type and severity using validated study-specific case definitions. Adjusted odds ratios (aORs) were calculated by logistic regression including stratified analyses conditioned on breastfeeding.RESULTS:One hundred and seventy-six (94 HEU and 82 HU) mother-infant pairs were analyzed. HIV-infected mothers were older (median, 27.8 vs. 24.7 years; P < 0.01) and HU infants more often breastfed (81/82 vs. 35/94; P < 0.001). Groups were similar for maternal education, antenatal course, household characteristics, birth weight, gestational age and immunizations. The primary outcome occurred in 17 (18%) HEU and 10 (12%) HU infants [aOR, 1.45; 95% confidence interval (CI): 0.44-4.55]. In stratified analysis restricted to breastfed infants, the aOR for hospitalization due to very severe infection or death was 4.2 (95% CI: 1.00-19.2; P = 0.05) for HEU infants. Hospitalization for diarrhea was more common in HEU than HU infants [8/94 (8.5%) vs. 1/82 (1.2%); P = 0.04].CONCLUSION:The difference between HEU and HU infants in the probability of infectious cause hospitalization or death in the first 6 months of life was not significant. However, among breastfed infants, severe infectious morbidity occurred more often in HEU than HU infants.

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.

Purpose: Serratia marcescens is a common pathogen in the hospital environment and colonizes patients. It is a serious cause of nosocomial outbreaks of invasive infections. There is limited experience using polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) method for typing of S. marcescens isolates for timely investigation of an outbreak. We sought to evaluate the performance of PCR-RAPD method using different primer sets compared to a reference method in the context of a neonatal intensive care unit (NICU) outbreak of invasive S. marcescens. Methods & Materials: Stored isolates of S. marcescens and cluster-related patient isolates over 2 months, were sub-cultured and genomic DNA was extracted. Blinded PCR amplification of each isolate DNA was performed using 4 primer sets, 2 of which were previously published primers for fingerprinting S. marcescens and 2 RAPD primers which were tested for the first time (272 P. aeruginosa and 270 B. cepacia primer sets). The PCR products were run on agarose gels, and the band patterns were compared visually and with the aid of computer analysis. Then the results were compared to those obtained from the traditional typing methods of pulse field gel electrophoresis (PFGE). Results: Overall, 4 blood isolates, 4 non-invasive samples, a control S. marcescens and a control Pseudomonas aeruginosa were included. All the invasive outbreak isolates (4 blood cultures from 3 different patients) were found to harbor identical S. marcesens strains using PFGE and RAPD typing methods, and they clustered separately from non-invasive isolates. Of the 4 primer sets used, the 272 P. aeruginosa primer gave the highest discrimination and the most number of bands allowing for accurate clustering of for the S. marcesens isolates. Results for RAPD were available within 24 hours from testing initiation and were replicable. Conclusion: In a small invasive S. marcescens outbreak in the NICU, typing by PCR-RAPD using the 272 P. aeruginosa primer set performed well compared to the reference method.. A particular attraction of this method is that it is economic and provides rapid results to inform infection control measures in real time. The use of PCR-RAPD could be a reasonable option for outbreak investigations of S. marcescens.

Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 μg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 μg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 μg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 μg/ml for B. cepacia and B. dolosa (24 h) and ≥100 μg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 μg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 μg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF.

Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient's lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle.IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient's lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.

At a Glance Commentary: Our data provide evidence for the new Cystic Fibrosis Foundation guideline that advises people with CF to wear a surgical mask in healthcare

Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease-dependent cytokine degradation. In subacute pulmonary infections, lasR mutant-infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

Burkholderia cenocepacia and other members of the Burkholderia cepacia complex (BCC) are highly multidrug-resistant bacteria that cause severe pulmonary infections in patients with cystic fibrosis. A screen of 2686 compounds derived from marine organisms identified molecules that could synergise with polymyxin B (PMB) to inhibit the growth of B. cenocepacia. At 1 μg/mL, five compounds synergised with PMB and inhibited the growth of B. cenocepacia by ≥70% compared with growth in PMB alone. Follow-up testing revealed that one compound from the screen, the aminocoumarin antibiotic novobiocin, synergised with PMB and colistin against tobramycin-resistant clinical isolates of B. cenocepacia and Burkholderia multivorans. In parallel, we show that novobiocin sensitivity is common among BCC species and that these bacteria are even more susceptible to an alternative aminocoumarin, clorobiocin, which also had an additive effect with PMB against B. cenocepacia. These studies support using aminocoumarin antibiotics to treat BCC infections and show that synergisers can be found to increase the efficacy of antimicrobial peptides and polymyxins against BCC bacteria.

We present the case of a 3-year-old boy who was diagnosed with cerebral abscesses due to Aspergillus nidulans infection on day 28 of induction chemotherapy for acute lymphoblastic leukemia. He responded well to treatment with voriconazole and caspofungin, making a full recovery. There are very few cases of invasive aspergillosis reported in children during induction chemotherapy for acute leukemia and A. nidulans is rare in the absence of chronic granulomatous disease.

Rationale: We have been collecting Burkholderia species bacteria from patients with cystic fibrosis (CF) for the last 30 years. During this time, our understanding of their multispecies taxonomy and infection control has evolved substantially.Objectives: To evaluate the long-term (30 year) epidemiology and clinical outcome of Burkholderia infection in CF, and fully define the risks associated with infection by each species.Methods: Isolates from Burkholderia-positive patients (n=107) were speciated and typed annually for each infected patient. Microbiological and clinical data were evaluated by thorough review of patient charts, and statistical analyses performed to define significant epidemiological factors.Measurements and Main Results: Before 1995, the majority of new Burkholderia infections were caused by epidemic clones of Burkholderia cenocepacia. After implementation of new infection control measures in 1995, Burkholderia multivorans became the most prevalent species. Survival analysis showed that patients with CF infected with B. cenocepacia had a significantly worse outcome than those with B. multivorans, and a novel finding was that, after Burkholderia infection, the prognosis for females was significantly worse than for males.Conclusions: B. multivorans and B. cenocepacia have been the predominant Burkholderia species infecting people with CF in Vancouver. The implementation of infection control measures were successful in preventing new acquisition of epidemic strains of B. cenocepacia, leaving nonclonal B. multivorans as the most prevalent species. Historically, survival after infection with B. cenocepacia has been significantly worse than B. multivorans infection, and, of new significance, we show that females tend toward worse clinical outcomes.

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.

ABSTRACT Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans . The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1- N -phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.

Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with an increased risk for infection. We showed previously that whole-blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors compared with whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, a significant reduction in the intracellular cytokine production on the single-cell level was only detected in South African children’s monocytes, conventional dendritic cells, and plasmacytoid dendritic cells. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children compared with children from the other continents. Together, our data identify differences in cell composition, as well as profoundly lower functional responses of innate cells, in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored.

ABSTRACT Burkholderia cepacia complex and Stenotrophomonas maltophilia infections are associated with poor clinical outcomes in persons with cystic fibrosis (CF). The MIC 50 based on planktonic growth and the biofilm concentration at which 50% of the isolates tested are inhibited (BIC 50 ) of tobramycin were measured for 180 B. cepacia complex and 101 S. maltophilia CF isolates and were 100 μg/ml for both species. New inhalation devices that deliver high tobramycin levels to the lung may be able to exceed these MICs.

BACKGROUND:Susceptibility to infection as well as response to vaccination varies among populations. To date, the underlying mechanisms responsible for these clinical observations have not been fully delineated. Because innate immunity instructs adaptive immunity, we hypothesized that differences between populations in innate immune responses may represent a mechanistic link to variation in susceptibility to infection or response to vaccination.OBJECTIVE:Determine whether differences in innate immune responses exist among infants from different continents of the world.METHODS:We determined the innate cytokine response following pattern recognition receptor (PRR) stimulation of whole blood from 2-year-old infants across 4 continents (Africa, North America, South America, and Europe).RESULTS:We found that despite the many possible genetic and environmental exposure differences in infants across 4 continents, innate cytokine responses were similar for infants from North America, South America, and Europe. However, cells from South African infants secreted significantly lower levels of cytokines than did cells from infants from the 3 other sites, and did so following stimulation of extracellular and endosomal but not cytosolic PRRs.CONCLUSIONS:Substantial differences in innate cytokine responses to PRR stimulation exist among different populations of infants that could not have been predicted. Delineating the underlying mechanism(s) for these differences will not only aid in improving vaccine-mediated protection but possibly also provide clues for the susceptibility to infection in different regions of the world.

Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

Background:Early in life, HIV-exposed uninfected (HEU) infants are at an increased risk of morbidity and mortality from infectious disease compared with HIV-unexposed (UE) infants. To improve our understanding of the mechanisms underlying their increased risk, we contrasted innate immune development between HEU and UE infants in a developing world setting, where early life infectious disease risk is exceptionally high.Methods:A prospective longitudinal cohort of HEU and UE newborns was established, and the most detailed characterization to date of HEU infant immune development was performed. Single-cell cytokine production was analyzed by flow cytometry after stimulation of whole blood with pathogen-associated molecular patterns (PAMPs).Results:Monocyte, classical dendritic cell, and plasmacytoid dendritic cell composition was similar between HEU and UE infants throughout the first year of life. However, HEU mononuclear cells mounted an enhanced pro-inflammatory response to PAMP stimulation, both in quantity of cytokine produced per cell and in proportion of responder cells. Significant differences in cytokine production were detected on the single-cell level in a PAMP-specific pattern, but only at 2 and 6 weeks of age; all differences normalized by 12 months of age.Conclusions:This time course of innate immune deviation early in life corresponds to the clinical window of vulnerability to infections in HEU infants and may be at least partially responsible for their increased morbidity and mortality from infectious disease.

BACKGROUND:Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.FINDINGS:Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [3H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro.CONCLUSIONS:These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.