Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman’s rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.

BackgroundAn ancestral trait of eukaryotic cells is the presence of mitochondria as an essential element for function and survival. Proper functioning of mitochondria depends on the import of nearly all proteins that is performed by complexes located in both mitochondrial membranes. The complexes have been proposed to contain subunits formed by proteins common to all eukaryotes and additional subunits regarded as lineage specific. Since Amoebozoa is poorly sampled for the complexes we investigated the outer membrane complexes, namely TOM, TOB/SAM and ERMES complexes, using available genome and transcriptome sequences, including transcriptomes assembled by us.ResultsThe results indicate differences in the organization of the Amoebozoa TOM, TOB/SAM and ERMES complexes, with the TOM complex appearing to be the most diverse. This is reflected by differences in the number of involved subunits and in similarities to the cognate proteins of representatives from different supergroups of eukaryotes.ConclusionsThe obtained results clearly demonstrate structural variability/diversity of these complexes in the Amoebozoa lineage and the reduction of their complexity as compared with the same complexes of model organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2402-2) contains supplementary material, which is available to authorized users.

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

BackgroundThe existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences.ResultsWhen compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones.ConclusionThe results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-4383-1) contains supplementary material, which is available to authorized users.

The global prevalence of infections caused by ESBL-producing Enterobacterales (ESBL-E) is increasing and for Escherichia coli observations indicate that this is partly driven by community-onset cases. The ESBL-E population structure in the community is scarcely described and data on risk factors for carriage are conflicting. Here, we report the prevalence and population structure of fecal ESBL-producing E. coli and Klebsiella pneumoniae (ESBL-Ec/Kp) in a general adult population, examine risk factors, and compare carriage isolates with contemporary clinical isolates. Fecal samples obtained from 4999 participants (54% women) ≥40 years in the seventh survey of the population-based Tromsø Study, Norway (2015-2016) were screened for ESBL-Ec/Kp. In addition, we included 118 ESBL-Ec clinical isolates from the Norwegian surveillance program in 2014. All ESBL-producing isolates were whole-genome sequenced. Risk factors associated with carriage were analyzed using multivariable logistic regression. ESBL-Ec gastrointestinal carriage prevalence was 3.3% (95%CI 2.8-3.9%, no sex difference) and 0.08% (0.02-0.20%) for ESBL-Kp. For ESBL-Ec, travel to Asia was the only independent risk factor (AOR 3.47, 95%CI 2.18-5.51). E. coli ST131 was most prevalent in both collections. However, the ST131 proportion was significantly lower in carriage (24%) vs. clinical isolates (58%, p<0.001). Carriage isolates were genetically more diverse with a higher proportion of phylogroup A (26% vs. 5%, p<0.001), indicating that ESBL gene acquisition occurs in a variety of E. coli lineages colonizing the gut. STs commonly related to extra-intestinal infections were more frequent in the clinical isolates also carrying a higher prevalence of antimicrobial resistance, which could indicate clone associated pathogenicity.

The global prevalence of infections caused by extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) is increasing, and for Escherichia coli , observations indicate that this is partly driven by community-onset cases. The ESBL-E population structure in the community is scarcely described, and data on risk factors for carriage are conflicting. Here, we report the prevalence and population structure of fecal ESBL-producing E. coli and Klebsiella pneumoniae (ESBL-Ec/Kp) in a general adult population, examine risk factors, and compare carriage isolates with contemporary clinical isolates. Fecal samples obtained from 4,999 participants (54% women) ≥40 years in the seventh survey of the population-based Tromsø Study, Norway (2015, 2016), were screened for ESBL-Ec/Kp. In addition, we included 118 ESBL-Ec clinical isolates from the Norwegian surveillance program in 2014. All isolates were whole-genome sequenced. Risk factors associated with carriage were analyzed using multivariable logistic regression. ESBL-Ec gastrointestinal carriage prevalence was 3.3% [95% confidence interval (CI) 2.8%–3.9%, no sex difference] and 0.08% (0.02%–0.20%) for ESBL-Kp. For ESBL-Ec, travel to Asia was the only independent risk factor (adjusted odds ratio 3.46, 95% CI 2.18–5.49). E. coli ST131 was most prevalent in both collections. However, the ST131 proportion was significantly lower in carriage (24%) versus clinical isolates (58%, P < 0.001). Carriage isolates were genetically more diverse with a higher proportion of phylogroup A (26%) than clinical isolates (5%, P < 0.001), indicating that ESBL gene acquisition occurs in a variety of E. coli lineages colonizing the gut. STs commonly related to extraintestinal infections were more frequent in clinical isolates also carrying a higher prevalence of antimicrobial resistance, which could indicate clone-associated pathogenicity. IMPORTANCE ESBL-Ec and ESBL-Kp are major pathogens in the global burden of antimicrobial resistance. However, there is a gap in knowledge concerning the bacterial population structure of human ESBL-Ec/Kp carriage isolates in the community. We have examined ESBL-Ec/Kp isolates from a population-based study and compared these to contemporary clinical isolates. The large genetic diversity of carriage isolates indicates frequent ESBL gene acquisition, while those causing invasive infections are more clone dependent and associated with a higher prevalence of antibiotic resistance. The knowledge of factors associated with ESBL carriage helps to identify patients at risk to combat the spread of resistant bacteria within the healthcare system. Particularly, previous travel to Asia stands out as a major risk factor for carriage and should be considered in selecting empirical antibiotic treatment in critically ill patients.

The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times.

concentration of FFAs than BTC-6, due to the increased sensitivity of these cells. Mitochondrial function was monitored by measurements of the mitochondrial oxygen consumption (high-resolution respirometry), ATP content (PerkinElmer), mitochondrial membrane potential [flow cytometry (BD)], activity of the caspase-9 (R&D Systems) and ROS generation (DCFH-DA). In preadipocyte cell line: palmitic acid impaired mitochondrial function that was manifested by a decreased intracellular ATP content and an increased ROS generation. Arachidonic acid increased ROS generation and caspase-9 activity related with the mitochondrial apoptosis pathway. Eicosapentaenoic acid decreased mitochondrial respiration and ATP content. In BTC6 line increased the amount of early apoptotic cells after incubation with AA and EPA was observed. There was also a tendency to increased caspase-9 activity after incubation with FFAs. Arachidonic acid slightly increased mitochondrial respiration in beta cells. In conclusion, free fatty acids affect mitochondrial function of selected adipose and pancreatic cell lines which is interesting in the light of a recent data, indicating that regulation of mitochondrial activity may be the target pathway for treating obesity.Systemic energy metabolism requires coordination of different organs to maintain homeostasis and overcome physiological and stress challenges. Skeletal muscle is a major metabolic organ that responds to energy shortage. Here, we show that nutrient deprivation of cultured muscle cells (C2C12) changes their bioenergetic profile. Differentiated myotubes were deprived of glucose and lglutamine for 30 min up to 24 h. Plate-based respirometry showed that the lack of glycolysis was first compensated by an increased mitochondrial ATP turnover. In the sequelae of nutrient deprivation, mitochondrial respiration and ATP production were progressively compromised until reaching levels below reliable detection after 24 h. After 8 h of nutrient deprivation, respiration linked to ATP synthesis was decreased to 36% of non-fasted controls. Fibroblast Growth Factor 21 (FGF21), previously shown to be induced upon a reduced mitochondrial energy output in muscle mediating endocrine crosstalk between organs, increased mRNA levels by 13-fold and the secretion of FGF21 protein by 5-fold. Under these conditions, we assessed the protein secretome unbiased using mass spectrometric analysis. In the complex secretome, we found the highest released protein to be FGF21, confirming the suitability of our analysis to discover novel players mediating fasting induced systemic crosstalk. We identified a total of 1498 proteins; 93 of these were significantly induced by starvation, of which 21 were previously published to be secreted and thus represent potential myokines. While FGF21 regulates carbohydrate and lipid metabolism, other novel proteins may display similar effects to maintain systemic homeostasis and will be explored in vivo, offering novel therapeutics to treat metabolic diseases.