Consideration of within-patient diversity highlights transmission pathways and antimicrobial resistance gene variability in vancomycin-resistant Enterococcus faecium.

BACKGROUND: WGS is increasingly being applied to healthcare-associated vancomycin-resistant Enterococcus faecium (VREfm) outbreaks. Within-patient diversity could complicate transmission resolution if single colonies are sequenced from identified cases. OBJECTIVES: Determine the impact of within-patient diversity on transmission resolution of VREfm. MATERIALS AND METHODS: Fourteen colonies were collected from VREfm positive rectal screens, single colonies were collected from clinical samples and Illumina WGS was performed. Two isolates were selected for Oxford Nanopore sequencing and hybrid genome assembly to generate lineage-specific reference genomes. Mapping to closely related references was used to identify genetic variations and closely related genomes. A transmission network was inferred for the entire genome set using Phyloscanner. RESULTS AND DISCUSSION: In total, 229 isolates from 11 patients were sequenced. Carriage of two or three sequence types was detected in 27% of patients. Presence of antimicrobial resistance genes and plasmids was variable within genomes from the same patient and sequence type. We identified two dominant sequence types (ST80 and ST1424), with two putative transmission clusters of two patients within ST80, and a single cluster of six patients within ST1424. We found transmission resolution was impaired using fewer than 14 colonies. CONCLUSIONS: Patients can carry multiple sequence types of VREfm, and even within related lineages the presence of mobile genetic elements and antimicrobial resistance genes can vary. VREfm within-patient diversity could be considered in future to aid accurate resolution of transmission networks.

Assessment of Risks of Dioxins for Aryl Hydrocarbon Receptor-Mediated Effects in Polar Bear (Ursus maritimus) by in Vitro and in Silico Approaches.

Polar bear (Ursus maritimus) populations accumulate dioxins and related compounds (DRCs) at levels that are of health concern. The toxicities of DRCs are primarily mediated via aryl hydrocarbon receptor (AHR) signaling pathway. To evaluate the sensitivity and responses to DRCs in polar bears, we assessed the activation potencies of polar bear-specific AHR (pbAHR) by DRCs through in vitro and in silico approaches. In vitro assays showed that the pbAHR was as sensitive to DRCs as C3H/lpr mouse AHR, which is well-known to be highly sensitive to DRCs. Comparison of pbAHR transactivation potencies indicated that TCDF, 2,3,4,7,8-PeCDF, and BaP exhibited high induction equivalency factors (IEFs). Considering the accumulation levels of DRCs in polar bears, PCB126 was found to be the most active inducer of pbAHR. The in vitro transactivation potencies of ligands of pbAHR showed a significant relationship with in silico ligand docking energies in a pbAHR homology model. The protein ligand interaction fingerprint (PLIF) analysis showed different interaction patterns depending on the ligands. Several amino acids which are highly conserved among mammals may be involved in species-specific responses via backbone interactions with neighboring amino acid residues which are specific to pbAHR. We document high susceptibility of polar bears to DRCs, through a mechanistic approach, for the first time.

Interleukin-17 Is Required for Control of Chronic Lung Infection Caused by Pseudomonas aeruginosa.

Chronic pulmonary infection with Pseudomonas aeruginosa is a feature of cystic fibrosis (CF) and other chronic lung diseases. Cytokines of the interleukin-17 (IL-17) family have been proposed as important in the host response to P. aeruginosa infection through their role in augmenting antibacterial immune responses, although their proinflammatory effect may contribute to lung damage that occurs as a result of chronic infection. We set out to explore the role of IL-17 in the host response to chronic P. aeruginosa infection. We used a murine model of chronic pulmonary infection with CF-related strains of P. aeruginosa We demonstrate that IL-17 cytokine signaling is essential for mouse survival and prevention of chronic infection at 2 weeks postinoculation using two different P. aeruginosa strains. Following infection, there was a marked expansion of cells within mediastinal lymph nodes, comprised mainly of innate lymphoid cells (ILCs); ∼90% of IL-17-producing (IL-17+) cells had markers consistent with group 3 ILCs. A smaller percentage of IL-17+ cells had markers consistent with a B1 phenotype. In lung homogenates harvested 14 days following infection, there was a significant expansion of IL-17+ cells; about 50% of these were CD3+, split equally between CD4+ Th17 cells and γδ T cells, while the CD3- IL-17+ cells were almost exclusively group 3 ILCs. Further experiments with B cell-deficient mice showed that B cell production of IL-17 or natural antibodies did not provide any defense against chronic P. aeruginosa infection. Thus, IL-17 rather than antibody is a key element in host defense against chronic pulmonary infection with P. aeruginosa.

The Effects of Smoking on Human Pharynx Microbiota Composition and Stability.

The oral microbiota is essential to the health of the host, yet little is known about how it responds to disturbances. We examined the oropharyngeal microbiota of 30 individuals over 40 weeks. As the oropharynx is an important gateway to pathogens, and as smoking is associated with increased incidence and severity of respiratory infections, we compared the microbiota of smokers and nonsmokers to shed light on its potential for facilitating infections. We hypothesized that decreased species diversity, decreased community stability, or increased differences in community structure could facilitate invading pathogens. We found that smoking is associated with reduced alpha diversity, greater differences in community structure, and increased environmental filtering. The effects of short-term perturbations (antibiotic use and participants exhibiting cold symptoms) were also investigated. Antibiotic use had a negative effect on alpha diversity, irrespective of smoking status, and both antibiotic use and cold symptoms were associated with highly unique bacterial communities. A stability analysis of models built from the data indicated that there were no differences in local or global stability in the microbial communities of smokers, compared to nonsmokers, and that their microbiota are equally resistant to species invasions. Results from these models suggest that smoker microbiota are perturbed but characterized by alternative stable states that are as stable and invasion-resistant as are the microbiota of nonsmokers. Smoking is unlikely to increase the risk of infectious disease through the altered composition and ecological function of the microbiota; this is more likely due to the effects of smoking on the local and systemic immune system. IMPORTANCE Smoking is associated with an increased risk of respiratory infections. Hypothetically, the altered community diversity of smokers' pharyngeal microbiota, together with changes in their ecological stability properties, could facilitate their invasion by pathogens. To address this question, we analyzed longitudinal microbiota data of baseline healthy individuals who were either smokers or nonsmokers. While the results indicate reduced biodiversity and increased species turnover in the smokers' pharyngeal microbiota, their ecological stability properties were not different from those of the microbiota of nonsmokers, implying, in ecological terms, that the smokers' microbial communities are not less resistant to invasions. Therefore, the study suggests that the increased propensity of respiratory infections that is seen in smokers is more likely associated with changes in the local and systemic immune system than with ecological changes in the microbial communities.

Regional contamination versus regional dietary differences: understanding geographic variation in brominated and chlorinated contaminant levels in polar bears.

The relative contribution of regional contamination versus dietary differences to geographic variation in polar bear (Ursus maritimus) contaminant levels is unknown. Dietary variation between Alaska, Canada, East Greenland, and Svalbard subpopulations was assessed by muscle nitrogen and carbon stable isotope (δ(15)N, δ(13)C) and adipose fatty acid (FA) signatures relative to their main prey (ringed seals). Western and southern Hudson Bay signatures were characterized by depleted δ(15)N and δ(13)C, lower proportions of C(20) and C(22) monounsaturated FAs and higher proportions of C(18) and longer chain polyunsaturated FAs. East Greenland and Svalbard signatures were reversed relative to Hudson Bay. Alaskan and Canadian Arctic signatures were intermediate. Between-subpopulation dietary differences predominated over interannual, seasonal, sex, or age variation. Among various brominated and chlorinated contaminants, diet signatures significantly explained variation in adipose levels of polybrominated diphenyl ether (PBDE) flame retardants (14-15%) and legacy PCBs (18-21%). However, dietary influence was contaminant class-specific, since only low or nonsignificant proportions of variation in organochlorine pesticide (e.g., chlordane) levels were explained by diet. Hudson Bay diet signatures were associated with lower PCB and PBDE levels, whereas East Greenland and Svalbard signatures were associated with higher levels. Understanding diet/food web factors is important to accurately interpret contaminant trends, particularly in a changing Arctic.

Spatial and temporal trends of selected trace elements in liver tissue from polar bears (Ursus maritimus) from Alaska, Canada and Greenland.

Spatial trends and comparative changes in time of selected trace elements were studied in liver tissue from polar bears from ten different subpopulation locations in Alaska, Canadian Arctic and East Greenland. For nine of the trace elements (As, Cd, Cu, Hg, Mn, Pb, Rb, Se and Zn) spatial trends were investigated in 136 specimens sampled during 2005-2008 from bears from these ten subpopulations. Concentrations of Hg, Se and As were highest in the (northern and southern) Beaufort Sea area and lowest in (western and southern) Hudson Bay area and Chukchi/Bering Sea. In contrast, concentrations of Cd showed an increasing trend from east to west. Minor or no spatial trends were observed for Cu, Mn, Rb and Zn. Spatial trends were in agreement with previous studies, possibly explained by natural phenomena. To assess temporal changes of Cd, Hg, Se and Zn concentrations during the last decades, we compared our results to previously published data. These time comparisons suggested recent Hg increase in East Greenland polar bears. This may be related to Hg emissions and/or climate-induced changes in Hg cycles or changes in the polar bear food web related to global warming. Also, Hg:Se molar ratio has increased in East Greenland polar bears, which suggests there may be an increased risk for Hg(2+)-mediated toxicity. Since the underlying reasons for spatial trends or changes in time of trace elements in the Arctic are still largely unknown, future studies should focus on the role of changing climate and trace metal emissions on geographical and temporal trends of trace elements.

Genomic Analysis of Global Staphylococcus argenteus Strains Reveals Distinct Lineages With Differing Virulence and Antibiotic Resistance Gene Content.

Infections due to Staphylococcus argenteus have been increasingly reported worldwide and the microbe cannot be distinguished from Staphylococcus aureus by standard methods. Its complement of virulence determinants and antibiotic resistance genes remain unclear, and how far these are distinct from those produced by S. aureus remains undetermined. In order to address these uncertainties, we have collected 132 publicly available sequences from fourteen different countries, including the United Kingdom, between 2005 and 2018 to study the global genetic structure of the population. We have compared the genomes for antibiotic resistance genes, virulence determinants and mobile genetic elements such as phages, pathogenicity islands and presence of plasmid groups between different clades. 20% (n = 26) isolates were methicillin resistant harboring a mecA gene and 88% were penicillin resistant, harboring the blaZ gene. ST2250 was identified as the most frequent strain, but ST1223, which was the second largest group, contained a marginally larger number of virulence genes compared to the other STs. Novel S. argenteus pathogenicity islands were identified in our isolates harboring tsst-1, seb, sec3, ear, selk, selq toxin genes, as well as chromosomal clusters of enterotoxin and superantigen-like genes. Strain-specific type I modification systems were widespread which would limit interstrain transfer of genetic material. In addition, ST2250 possessed a CRISPR/Cas system, lacking in most other STs. S. argenteus possesses important genetic differences from S. aureus, as well as between different STs, with the potential to produce distinct clinical manifestations.

A Longitudinal Study of the Human Oropharynx Microbiota Over Time Reveals a Common Core and Significant Variations With Self-Reported Disease.

Our understanding of human microbial communities, in particular in regard to diseases is advancing, yet the basic understanding of the microbiome in healthy subjects over time remains limited. The oropharynx is a key target for colonization by several important human pathogens. To understand how the oropharyngeal microbiome might limit infections, and how intercurrent infections might be associated with its composition, we characterized the oropharyngeal microbiome of 18 healthy adults, sampled weekly over a 40-weeks using culture-independent molecular techniques. We detected nine phyla, 202 genera and 1438 assignments on OTU level, dominated by Firmicutes, Bacteroidetes, and Proteobacteria on phylum level. Individual microbiomes of participants were characterized by levels of high alpha diversity (mean = 204.55 OTUs, sd = 35.64), evenness (19.83, sd = 9.74) and high temporal stability (mean Pearson's correlation between samples of 0.52, sd = 0.060), with greater differences in microbiome community composition between than within individuals. Significant changes in community composition were associated with disease states, suggesting that it is possible to detect specific changes in OTU abundance and community composition during illness. We defined the common core microbiota by varying occurrence and abundance thresholds showing that individual core microbiomes share a substantial number of OTUs across participants, chiefly Streptococci and Veillonella. Our results provide insights into the microbial communities that characterize the healthy human oropharynx, community structure and variability, and provide new approaches to define individual and shared cores. The wider implications of this result include the potential for modeling the general dynamics of oropharynx microbiota both in health and in response to antimicrobial treatments or probiotics.

Origin, maintenance and spread of antibiotic resistance genes within plasmids and chromosomes of bloodstream isolates of Escherichia coli.

Blood stream invasion by Escherichia coli is the commonest cause of bacteremia in the UK and elsewhere with an attributable mortality of about 15-20 %; antibiotic resistance to multiple agents is common in this microbe and is associated with worse outcomes. Genes conferring antimicrobial resistance, and their frequent location on horizontally transferred genetic elements is well-recognised, but the origin of these determinants, and their ability to be maintained and spread within clinically-relevant bacterial populations is unclear. Here, we set out to examine the distribution of antimicrobial resistance genes in chromosomes and plasmids of 16 bloodstream isolates of E. coli from patients within Scotland, and how these genes are maintained and spread. Using a combination of short and long-read whole genome sequencing methods, we were able to assemble complete sequences of 44 plasmids, with 16 Inc group F and 20 col plasmids; antibiotic resistance genes located almost exclusively within the F group. blaCTX-M15 genes had re-arranged in some strains into the chromosome alone (five strains), while others contained plasmid copies alone (two strains). Integrons containing multiple antibiotic genes were widespread in plasmids, notably many with a dfrA7 gene encoding resistance to trimethoprim, thus linking trimethoprim resistance to the other antibiotic resistance genes within the plasmids. This will allow even narrow spectrum antibiotics such as trimethoprim to act as a selective agent for plasmids containing antibiotic resistance genes mediating much broader resistance, including blaCTX-M15. To our knowledge, this is the first analysis to provide complete sequence data of chromosomes and plasmids in a collection of pathogenic human bloodstream isolates of E. coli. Our findings reveal the interplay between plasmids and integrative and conjugative elements in the maintenance and spread of antibiotic resistance genes within pathogenic E. coli.

Optic flow cues help explain altitude control over sea in freely flying gulls.

For studies of how birds control their altitude, seabirds are of particular interest because they forage offshore where the visual environment can be simply modelled by a flat world textured by waves then generating only ventral visual cues. This study suggests that optic flow, i.e. the rate at which the sea moves across the eye's retina, can explain gulls' altitude control over seas. In particular, a new flight model that includes both energy and optical invariants helps explain the gulls' trajectories during offshore takeoff and cruising flight. A linear mixed model applied to 352 flights from 16 individual lesser black backed gulls (Larus fuscus) revealed a statistically significant optic flow set-point of ca 25° s-1. Thereafter, an optic flow-based flight model was applied to 18 offshore takeoff flights from nine individual gulls. By introducing an upper limit in climb rate on the elevation dynamics, coupled with an optic flow set-point, the predicted altitude gives an optimized fit factor value of 63% on average (30-83% in range) with respect to the GPS data. We conclude that the optic flow regulation principle helps gulls to adjust their altitude over sea without having to directly measure their current altitude.

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.

The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling.

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.

Genetic analysis of invasive Escherichia coli in Scotland reveals determinants of healthcare-associated versus community-acquired infections.

Bacteraemia caused by Escherichia coli is a growing problem with a significant mortality. The factors that influence the acquisition and outcome of these infections are not clear. Here, we have linked detailed genetic data from the whole-genome sequencing of 162 bacteraemic isolates collected in Scotland, UK, in 2013-2015, with clinical data in order to delineate bacterial and host factors that influence the acquisition in hospital or the community, outcome and antibiotic resistance. We identified four major sequence types (STs) in these isolates: ST131, ST69, ST73 and ST95. Nearly 50 % of the bacteraemic isolates had a urinary origin. ST69 was genetically distinct from the other STs, with significantly less sharing of accessory genes and with a distinct plasmid population. Virulence genes were widespread and diversely distributed between the dominant STs. ST131 was significantly associated with hospital-associated infections (HAIs), and ST69 with those from the community. However, there was no association of ST with outcome, although patients with HAI had a higher immediate mortality compared to those with community-associated infections (CAIs). Genome-wide association studies revealed genes involved in antibiotic persistence as significantly associated with HAIs and those encoding elements of a type VI secretion system with CAIs. Antibiotic resistance was common, and there were networks of correlated resistance genes and phenotypic antibiotic resistance. This study has revealed the complex interactions between the genotype of E. coli and its ability to cause bacteraemia, and some of the determinants influencing hospital or community acquisition. In part, these are shaped by antibiotic usage, but strain-specific factors are also important.

Land or sea? Foraging area choice during breeding by an omnivorous gull.

BACKGROUND: Generalist predators may vary their diet and use of habitat according to both internal state (e.g. breeding stage) and external (e.g. weather) factors. Lesser black-backed gulls Larus fuscus (Linnaeus 1758) are dietary generalists, foraging in both terrestrial and marine habitats during breeding. We investigate what affects the gulls' propensity to forage at sea or on land. We assess the importance of terrestrial foraging to gulls in the Baltic Sea (sub. sp. L. f. fuscus), looking especially at their use of agricultural fields. RESULTS: Through the GPS tracking of 19 individuals across 3 years we tracked 1038 foraging trips and found that 21.2 % of foraging trips were predominantly terrestrial, 9.0 % were a mix of terrestrial and marine, and 68.5 % were exclusively marine. Terrestrial trips were (1) more frequent when departing around sunrise, whereas marine trips occurred throughout the day. Additionally, trips with mostly land-based foraging decreased as the breeding season progressed, suggesting dietary switching coincident with the onset of chick provisioning. (2) During cloudy and cold conditions terrestrial foraging trips were more likely. (3) We found no differences between sexes in their land-based foraging strategy. (4) Gull individuals showed great variation in foraging strategy. Using observations of agricultural fields, carried out for one year, we found that (5) gulls preferentially foraged on fields with short vegetation, and there was a positive association with occurrence of waders and other species of gulls. (6) The availability and use of these preferred fields decreased through the breeding period. CONCLUSIONS: This study found high prevalence of terrestrial foraging during early breeding as well as support for dietary switching early in the breeding season. The overall tendency for marine or terrestrial foraging was consistent within individuals, with gull identity accounting for much of the variation observed in foraging trips. Our results suggest that anthropogenic terrestrial food sources may play a role in the low breeding success of these gulls through either variation in quantity and/or quality. Finally, our study demonstrates the potential of combining data from GPS-tracking of individual animals with the 'ground-truthing' of habitat visited to elucidate the otherwise nebulous behavior of a generalist predator.

Efficacy of species-specific protein antibiotics in a murine model of acute Pseudomonas aeruginosa lung infection.

Protein antibiotics, known as bacteriocins, are widely produced by bacteria for intraspecies competition. The potency and targeted action of bacteriocins suggests that they could be developed into clinically useful antibiotics against highly drug resistant Gram-negative pathogens for which there are few therapeutic options. Here we show that Pseudomonas aeruginosa specific bacteriocins, known as pyocins, show strong efficacy in a murine model of P. aeruginosa lung infection, with the concentration of pyocin S5 required to afford protection from a lethal infection at least 100-fold lower than the most commonly used inhaled antibiotic tobramycin. Additionally, pyocins are stable in the lung, poorly immunogenic at high concentrations and efficacy is maintained in the presence of pyocin specific antibodies after repeated pyocin administration. Bacteriocin encoding genes are frequently found in microbial genomes and could therefore offer a ready supply of highly targeted and potent antibiotics active against problematic Gram-negative pathogens.

IL-22 exacerbates weight loss in a murine model of chronic pulmonary Pseudomonas aeruginosa infection.

BACKGROUND: Interleukin (IL)-22 is a critical mediator of mucosal immunity and tissue regeneration, protecting against a number of respiratory pathogens. Whether IL-22 confers protection against chronic Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) is unknown. METHODS: Explanted CF lungs were examined for IL-22 production and immune-localization. A murine model of persistent pulmonary PA infection was used to examine production of IL-22 following infective challenge. The role of IL-22 was examined using IL-22 knockout (KO) animals. RESULTS: IL-22 is produced within the adult CF lung and localizes to the airway epithelium. IL-22 is produced by murine pulmonary lymph node cells following lung infection. The absence of IL-22 resulted in no significant difference in acute mortality, bacterial burden, chronic infection rates, histological changes or neutrophilic inflammation in the chronic PA infection model. However, IL-22 KO animals lost less weight following infection. CONCLUSION: IL-22 is produced in the CF lung and in response to PA infection yet is dispensable in protection against chronic pulmonary P. aeruginosa infection in a murine model. However, we identified a novel role for the cytokine in promoting infection-related weight-loss, a significant prognostic factor in the CF population.

Chlorinated hydrocarbon contaminants and metabolites in polar bears (Ursus maritimus) from Alaska, Canada, East Greenland, and Svalbard: 1996-2002.

A suite of chlorinated hydrocarbon contaminants (CHCs) including organochlorine pesticides (OCPs) and by-products, polychlorinated biphenyls (PCBs), and methyl sulfone (MeSO2) PCB and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) metabolites were determined in adipose tissue of 107 adult and sub-adult polar bears, almost exclusively females, sampled between 1996 and 2002 from populations spanning Arctic and Subarctic regions of Alaska, Canada, East Greenland, and Svalbard. The East Greenland and Svalbard populations of polar bears were distinguished by higher proportions of dichlorodiphenyldichloroethane (DDT)-related compounds, nonachlors, oxychlordane, and higher-chlorinated and persistent PCB congeners (hepta- to nona-chlorinated). Conversely, Alaska, the westernmost population of the North American Arctic, was characterized by higher proportions of relatively volatile compounds such as hexachlorocyclohexanes (HCHs) and pentachlorobenzene (PnCBz), lower-chlorinated PCB congeners (tri- to penta-chlorinated), and lower proportions of oxychlordane. Geometric mean (GM) with 95% confidence limits (CL) SigmaHCH concentrations were highest in Alaska male polar bear fat samples (GM 593; CL 363-909 ng g-1 lipid weight), SigmaDDT concentration were highest in East Greenland female samples (GM 309; CL 249-490 ng g-1 l.w.), and Sigma42PCB (GM 5972; CL 4637-9129 ng g-1 l.w.) and SigmaMeSO2-PCB (GM 198; CL 162-279 ng g-1 l.w.) concentrations were highest in female samples collected from Svalbard. The distribution of Sigma-chlordane-related compounds (SigmaCHL), SigmaCBz, mirex, and dieldrin was relatively uniform among the populations of polar bears investigated. The present 1996-2002 data of female polar bear fat samples was compared to spatial assessments of female polar bear fat samples collected between 1989 and 1993 from comparable populations. The two-point temporal comparisons showed a general decrease for age-adjusted mean concentrations of SigmaCHL, p,p'-DDE, Sigma42PCB, SigmaMeSO2-PCB and 3-MeSO2-p,p'-DDE over a period of approximately 10 years. However, concentrations of dieldrin were comparatively unchanged. Comparisons of present 2001-2002 concentrations in fat of female polar bears from Western Hudson Bay showed great consistency with temporal trends (1991-1999) previously reported for the same region, i.e. the apparent non-decreasing trend of SigmaCHL, beta-HCH, SigmaHCH and dieldrin, and the apparent declining trend for SigmaPCB. However, present concentrations of alpha-HCH and SigmaCBz were elevated, and SigmaDDT was notably lower in Western Hudson Bay samples compared to the last measurements in fat samples collected in 1999, which was not in accord with reported temporal trends for this region. As a result of their relatively high degree of contamination, East Greenland and Svalbard polar bears are at higher health risk of contaminant exposure among Arctic and Subarctic populations. In addition to continued biomonitoring, further research on health and population status is needed to evaluate the impact from chronic exposure of polar bear populations to CHCs and their metabolites.

Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis.

Pseudomonas aeruginosa is the most common pathogen that colonizes the lungs of patients with cystic fibrosis. Isolates from sputum are typically all derived from the same strain of bacterium but show extensive phenotypic heterogeneity. One of these variants is the so-called small colony variant, which also shows increased ability to form a biofilm and is frequently resistant to multiple antibiotics. The presence of small colony variants in the sputum of patients with cystic fibrosis is associated with a worse clinical condition. The underlying mechanism responsible for generation of the small colony phenotype remains unclear, but a final common pathway would appear to be elevation of intracellular levels of cyclic di-GMP. This phenotypic variant is thus not just a laboratory curiosity, but a significant bacterial adaptation that favors survival within the lung of patients with cystic fibrosis and contributes to the pulmonary damage caused by P. aeruginosa.

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy.

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.

Caspase-1 cleavage of the TLR adaptor TRIF inhibits autophagy and ß-interferon production during Pseudomonas aeruginosa infection.

Bacterial infection can trigger autophagy and inflammasome activation, but the effects of inflammasome activation on autophagy are unknown. We examined this in the context of Pseudomonas aeruginosa macrophage infection, which triggers NLRC4 inflammasome activation. P. aeruginosa induced autophagy via TLR4 and its adaptor TRIF. NLRC4 and caspase-1 activation following infection attenuated autophagy. Caspase-1 directly cleaved TRIF to diminish TRIF-mediated signaling, resulting in inhibition of autophagy and in reduced type I interferon production. Expression of a caspase-1 resistant TRIF mutant enhanced autophagy and type I interferon production following infection. Preventing TRIF cleavage by caspase-1 in an in vivo model of P. aeruginosa infection resulted in enhanced bacterial autophagy, attenuated IL-1ß production, and increased bacterial clearance. Additionally, TRIF cleavage by caspase-1 diminished NLRP3 inflammasome activation. Thus, caspase-1 mediated TRIF cleavage is a key event in controlling autophagy, type I interferon production, and inflammasome activation with important functional consequences.