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The polar regions are the fastest warming places on earth. Accelerated glacial melting causes increased supply of nutrients such as metal oxides (i.e., iron and manganese oxides) into the surrounding environment, such as the marine sediments of Potter Cove, King George Island/Isla 25 de Mayo (West Antarctic Peninsula). Microbial manganese oxide reduction and the associated microbial communities are poorly understood in Antarctic sediments. Here, we investigated this process by geochemical measurements of in situ sediment pore water and by slurry incubation experiments which were accompanied by 16S rRNA sequencing. Members of the genus Desulfuromusa were the main responder to manganese oxide and acetate amendment in the incubations. Other organisms identified in relation to manganese and/or acetate utilization included Desulfuromonas, Sva1033 (family of Desulfuromonadales) and unclassified Arcobacteraceae. Our data show that distinct members of Desulfuromonadales are most active in organotrophic manganese reduction, thus providing strong evidence of their relevance in manganese reduction in permanently cold Antarctic sediments.

IntroductionDiagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements.MethodsThis study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing.ResultsGenome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5’-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination.ConclusionThis study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

The threat of multidrug-resistant organisms (MDROs) and antimicrobial resistance (AMR) are real and increasing every day. They affect not only healthcare systems but also communities, causing economic and public health concerns. Governments must take action to tackle AMR and prevent the spread of MDROs and regional hubs have a critical role to play in achieving this outcome. Furthermore, bacteria have no borders, consequently, cooperation networks should be extended between countries as a crucial strategy for achieving the success of infection control. Euregions, which are a specific form of cooperation between local authorities of two or more bordering European countries, can help solve common problems and improve the lives of people living on both sides of the border. Regional collaboration strategies can enhance infection control and build resilience against antimicrobial resistance. This review identifies risk factors and the correct approaches to infection prevention and control, including education and awareness programs for healthcare professionals, appropriate prescribing practices, and infection prevention control measures. These measures can help reduce the incidence of antimicrobial resistance in the region and save lives. It is therefore essential to take concrete actions and foster the creation of more effective regional and cross-border centers to ensure the success of infection control policies and the management of healthcare-associated infections. This work sheds light on the issue of MDRO infections within healthcare settings, while also acknowledging the crucial role of the One Health concept in understanding the broader context of these infections. By recognizing the interdependence of human and animal health and the environment, we can take constructive steps toward mitigating the risks of these infections and promoting better health outcomes for all.

ABSTRACT In this study, we determined the presence of virulence factors in nonoutbreak, high-risk clones and other isolates belonging to less common sequence types associated with the spread of OXA-48-producing Klebsiella pneumoniae clinical isolates from The Netherlands (n = 61) and Spain (n = 53). Most isolates shared a chromosomally encoded core of virulence factors, including the enterobactin gene cluster, fimbrial fim and mrk gene clusters, and urea metabolism genes (ureAD). We observed a high diversity of K-Locus and K/O loci combinations, KL17 and KL24 (both 16%), and the O1/O2v1 locus (51%) being the most prevalent in our study. The most prevalent accessory virulence factor was the yersiniabactin gene cluster (66.7%). We found seven yersiniabactin lineages—ybt 9, ybt 10, ybt 13, ybt 14, ybt 16, ybt 17, and ybt 27—which were chromosomally embedded in seven integrative conjugative elements (ICEKp): ICEKp3, ICEKp4, ICEKp2, ICEKp5, ICEKp12, ICEKp10, and ICEKp22, respectively. Multidrug-resistant lineages—ST11, ST101, and ST405—were associated with ybt 10/ICEKp4, ybt 9/ICEKp3, and ybt 27/ICEKp22, respectively. The fimbrial adhesin kpi operon (kpiABCDEFG) was predominant among ST14, ST15, and ST405 isolates, as well as the ferric uptake system kfuABC, which was also predominant among ST101 isolates. No convergence of hypervirulence and resistance was observed in this collection of OXA-48-producing K. pneumoniae clinical isolates. Nevertheless, two isolates, ST133 and ST792, were positive for the genotoxin colibactin gene cluster (ICEKp10). In this study, the integrative conjugative element, ICEKp, was the major vehicle for yersiniabactin and colibactin gene clusters spreading. IMPORTANCE Convergence of multidrug resistance and hypervirulence in Klebsiella pneumoniae isolates has been reported mostly related to sporadic cases or small outbreaks. Nevertheless, little is known about the real prevalence of carbapenem-resistant hypervirulent K. pneumoniae since these two phenomena are often separately studied. In this study, we gathered information on the virulent content of nonoutbreak, high-risk clones (i.e., ST11, ST15, and ST405) and other less common STs associated with the spread of OXA-48-producing K. pneumoniae clinical isolates. The study of virulence content in nonoutbreak isolates can help us to expand information on the genomic landscape of virulence factors in K. pneumoniae population by identifying virulence markers and their mechanisms of spread. Surveillance should focus not only on antimicrobial resistance but also on virulence characteristics to avoid the spread of multidrug and (hyper)virulent K. pneumoniae that may cause untreatable and more severe infections.

The sea-level rise during the Holocene (11–0 ky BP) and its resulting sedimentation and biogeochemical processes may control microbial life in Arctic sediments. To gain further insight into this interaction, we investigated a sediment core (up to 10.7 m below the seafloor) from the Chuckchi Shelf of the western Arctic Ocean using metabarcoding-based sequencing and qPCR to characterize archaeal and bacterial 16S rRNA gene composition and abundance, respectively. We found that Arctic Holocene sediments harbor local microbial communities, reflecting geochemical and paleoclimate separations. The composition of bacterial communities was more diverse than that of archaeal communities, and specifically distinct at the boundary layer of the sulfate–methane transition zone. Enriched cyanobacterial sequences in the Arctic middle Holocene (8–7 ky BP) methanogenic sediments remarkably suggest past cyanobacterial blooms. Bacterial communities were phylogenetically influenced by interactions between dispersal limitation and environmental selection governing community assembly under past oceanographic changes. The relative influence of stochastic and deterministic processes on the bacterial assemblage was primarily determined by dispersal limitation. We have summarized our findings in a conceptual model that revealed how changes in paleoclimate phases cause shifts in ecological succession and the assembly process. In this ecological model, dispersal limitation is an important driving force for progressive succession for bacterial community assembly processes on a geological timescale in the western Arctic Ocean. This enabled a better understanding of the ecological processes that drive the assembly of communities in Holocene sedimentary habitats affected by sea-level rise, such as in the shallow western Arctic shelves.

Objectives: Urinary tract infections (UTIs) caused by multidrug-resistant Escherichia coli have become a major medical concern. Old antibiotics such as fosfomycin have become an alternative therapeutic option due to their effectiveness and, as a result, fosfomycin is now used as a first-line drug for the treatment of UTIs in many countries. Despite low resistance rates, fosfomycin heteroresistance, defined as a phenomenon where subpopulations of bacteria are resistant to high antibiotic concentrations whereas most of the bacteria are susceptible, is an underestimated problem. Methods: The frequency of heteroresistance in E. coli isolated from hospitalized patients in Brazil and its effect on susceptibility of E. coli in biofilms was studied and the isolates were molecularly characterized to reveal the mechanisms behind their fosfomycin heteroresistance using whole-genome sequencing. Results: A higher frequency of fosfomycin heteroresistance compared with other studies was found. In biofilms, most heteroresistant isolates were less sensitive to fosfomycin than control isolates and showed overexpression of metabolic genes thereby increasing their survival rate. Molecular characterization showed that some resistant subpopulations derived from heteroresistant isolates had a defect in their fosfomycin uptake system caused by mutations in transporter and regulatory genes, whereas others overexpressed the murA gene. None to minor effects on bacterial fitness were observed. Oxidative stress protection, virulence and metabolic genes were differentially expressed in resistant subpopulations and heteroresistant isolates. Conclusion: Frequent detection of heteroresistance in UTIs may play a role in the failure of antibiotic treatments and should therefore be more carefully diagnosed.

Acidobacteria occur in a large variety of ecosystems worldwide and are particularly abundant and highly diverse in soils. In spite of their diversity, only few species have been characterized to date which makes Acidobacteria one of the most poorly understood phyla among the domain Bacteria. We used a culture-independent niche modeling approach to elucidate ecological adaptations and their evolution for 4,154 operational taxonomic units (OTUs) of Acidobacteria across 150 different, comprehensively characterized grassland soils in Germany. Using the relative abundances of their 16S rRNA gene transcripts, the responses of active OTUs along gradients of 41 environmental variables were modeled using hierarchical logistic regression (HOF), which allowed to determine values for optimum activity for each variable (niche optima). By linking 16S rRNA transcripts to the phylogeny of full 16S rRNA gene sequences, we could trace the evolution of the different ecological adaptations during the diversification of Acidobacteria. This approach revealed a pronounced ecological diversification even among acidobacterial sister clades. Although the evolution of habitat adaptation was mainly cladogenic, it was disrupted by recurrent events of convergent evolution that resulted in frequent habitat switching within individual clades. Our findings indicate that the high diversity of soil acidobacterial communities is largely sustained by differential habitat adaptation even at the level of closely related species. A comparison of niche optima of individual OTUs with the phenotypic properties of their cultivated representatives showed that our niche modeling approach (1) correctly predicts those physiological properties that have been determined for cultivated species of Acidobacteria but (2) also provides ample information on ecological adaptations that cannot be inferred from standard taxonomic descriptions of bacterial isolates. These novel information on specific adaptations of not-yet-cultivated Acidobacteria can therefore guide future cultivation trials and likely will increase their cultivation success.

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis.Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content.Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster’s closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes.Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients.

Anaerobic methane oxidizing archaea (ANME) mediate anaerobic oxidation of methane (AOM) in marine sediments and are therefore important for controlling atmospheric methane concentrations in the water column and ultimately the atmosphere. Numerous previous studies have revealed that AOM is coupled to the reduction of different electron acceptors such as sulfate, nitrate/nitrite or Fe(III)/Mn(IV). However, the influence of electron acceptor availability on the in situ ANME community composition in sediments remains largely unknown. Here, we investigated the electron acceptor availability and compared the microbial in situ communities of three methane-rich locations offshore the sub-Antarctic island South Georgia, by Illumina sequencing and qPCR of mcrA genes. The methanic zone (MZ) sediments of Royal Trough and Church Trough comprised high sulfide concentrations of up to 4 and 19 mM, respectively. In contrast, those of the Cumberland Bay fjord accounted for relatively high concentrations of dissolved iron (up to 186 μM). Whereas the ANME community in the sulfidic sites Church Trough and Royal Trough mainly comprised members of the ANME-1 clade, the order-level clade “ANME-1-related” (Lever and Teske, 2015) was most abundant in the iron-rich site in Cumberland Bay fjord, indicating that the availability of electron acceptors has a strong selective effect on the ANME community. This study shows that potential electron acceptors for methane oxidation may serve as environmental filters to select for the ANME community composition and adds to a better understanding of the global importance of AOM.

Extraintestinal pathogenic E. coli (ExPEC) is the most frequent etiological agent of urinary tract infections (UTIs). Particular evolutionary successful lineages are associated with severe UTIs and higher incidences of multidrug resistance. Most of the resistance genes are acquired by horizontal transfer of plasmids and other mobile genetic elements (MGEs), and this process has been associated with the successful dissemination of particular lineages. Here, we identified the presence of MGEs and their role in virulence and resistance profiles of isolates obtained from the urine of hospitalized patients in Brazil. Isolates belonging to the successful evolutionary lineages of sequence type (ST) 131, ST405, and ST648 were found to be multidrug-resistant, while those belonging to ST69 and ST73 were often not. Among the ST131, ST405, and ST648 isolates with a resistant phenotype, a high number of mainly IncFII plasmids was identified. The plasmids contained resistance cassettes, and these were also found within phage-related sequences and the chromosome of the isolates. The resistance cassettes were found to harbor several resistance genes, including blaCTX-M-15. In addition, in ST131 isolates, diverse pathogenicity islands similar to those found in highly virulent ST73 isolates were detected. Also, a new genomic island associated with several virulence genes was identified in ST69 and ST131 isolates. In addition, several other MGEs present in the ST131 reference strain EC958 were identified in our isolates, most of them exclusively in ST131 isolates. In contrast, genomic islands present in this reference strain were only partially present or completely absent in our ST131 isolates. Of all isolates studied, ST73 and ST131 isolates had the most similar virulence profile. Overall, no clear association was found between the presence of specific MGEs and virulence profiles. Furthermore, the interplay between virulence and resistance by acquiring MGEs seemed to be lineage dependent. Although the acquisition of IncF plasmids, specific PAIs, GIs, and other MGEs seemed to be involved in the success of some lineages, it cannot explain the success of different lineages, also indicating other (host) factors are involved in this process. Nevertheless, the detection, identification, and surveillance of lineage-specific MGEs may be useful to monitor (new) emerging clones.

Spatial and temporal processes shaping microbial communities are inseparably linked but rarely studied together. By Illumina 16S rRNA sequencing, we monitored soil bacteria in 360 stations on a 100 square meter plot distributed across six intra-annual samplings in a rarely managed, temperate grassland. Using a multi-tiered approach, we tested the extent to which stochastic or deterministic processes influenced the composition of local communities. A combination of phylogenetic turnover analysis and null modeling demonstrated that either homogenization by unlimited stochastic dispersal or scenarios, in which neither stochastic processes nor deterministic forces dominated, explained local assembly processes. Thus, the majority of all sampled communities (82%) was rather homogeneous with no significant changes in abundance-weighted composition. However, we detected strong and uniform taxonomic shifts within just nine samples in early summer. Thus, community snapshots sampled from single points in time or space do not necessarily reflect a representative community state. The potential for change despite the overall homogeneity was further demonstrated when the focus shifted to the rare biosphere. Rare OTU turnover, rather than nestedness, characterized abundance-independent β-diversity. Accordingly, boosted generalized additive models encompassing spatial, temporal and environmental variables revealed strong and highly diverse effects of space on OTU abundance, even within the same genus. This pure spatial effect increased with decreasing OTU abundance and frequency, whereas soil moisture – the most important environmental variable – had an opposite effect by impacting abundant OTUs more than the rare ones. These results indicate that – despite considerable oscillation in space and time – the abundant and resident OTUs provide a community backbone that supports much higher β-diversity of a dynamic rare biosphere. Our findings reveal complex interactions among space, time, and environmental filters within bacterial communities in a long-established temperate grassland.

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur’s BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0–10 and 0–14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and

Background: Virtually no studies on the dynamics of the intestinal microbiota in patients admitted to the intensive care unit (ICU) are published, despite the increasingly recognized important role of microbiota on human physiology. Critical care patients undergo treatments that are known to influence the microbiota. However, dynamics and extent of such changes are not yet fully understood. To address this topic, we analyzed the microbiota before, during and after planned major cardio surgery that, for the first time, allowed us to follow the microbial dynamics of critical care patients. In this prospective, observational, longitudinal, single center study, we analyzed the fecal microbiota using 16S rRNA gene sequencing.Results: Samples of 97 patients admitted between April 2015 and November 2016 were included. In 32 patients, data of all three time points (before, during and after admission) were available for analysis. We found a large intra-individual variation in composition of gut microbiota. During admission, a significant change in microbial composition occurred in most patients, with a significant increase in pathobionts combined with a decrease in strictly anaerobic gut bacteria, typically beneficial for health. A lower bacterial diversity during admission was associated with longer hospitalization. In most patients analyzed at all three time points, the change in microbiota during hospital stay reverted to the original composition post-discharge.Conclusions: Our study shows that, even with a short ICU stay, patients present a significant change in microbial composition shortly after admission. The unique longitudinal setup of this study displayed a restoration of the microbiota in most patients to baseline composition post-discharge, which demonstrated its great restorative capacity. A relative decrease in benign or even beneficial bacteria and increase of pathobionts shifts the microbial balance in the gut, which could have clinical relevance. In future studies, the microbiota of ICU patients should be considered a good target for optimisation.

The flux of methane, a potent greenhouse gas, from the seabed is largely controlled by anaerobic oxidation of methane (AOM) coupled to sulfate reduction (S-AOM) in the sulfate methane transition (SMT). S-AOM is estimated to oxidize 90% of the methane produced in marine sediments and is mediated by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria. An additional methane sink, i.e., iron oxide coupled AOM (Fe-AOM), has been suggested to be active in the methanic zone of marine sediments. Geochemical signatures below the SMT such as high dissolved iron, low to undetectable sulfate and high methane concentrations, together with the presence of iron oxides are taken as prerequisites for this process. So far, Fe-AOM has neither been proven in marine sediments nor have the governing key microorganisms been identified. Here, using a multidisciplinary approach, we show that Fe-AOM occurs in iron oxide-rich methanic sediments of the Helgoland Mud Area (North Sea). When sulfate reduction was inhibited, different iron oxides facilitated AOM in long-term sediment slurry incubations but manganese oxide did not. Especially magnetite triggered substantial Fe-AOM activity and caused an enrichment of ANME-2a archaea. Methane oxidation rates of 0.095 ± 0.03 nmol cm–3 d–1 attributable to Fe-AOM were obtained in short-term radiotracer experiments. The decoupling of AOM from sulfate reduction in the methanic zone further corroborated that AOM was iron oxide-driven below the SMT. Thus, our findings prove that Fe-AOM occurs in methanic marine sediments containing mineral-bound ferric iron and is a previously overlooked but likely important component in the global methane budget. This process has the potential to sustain microbial life in the deep biosphere.

ABSTRACT In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage. IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2. An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.

Soil protists are increasingly appreciated as essential components of soil foodwebs; however, there is a dearth of information on the factors structuring their communities. Here we investigate the importance of different biotic and abiotic factors as key drivers of spatial and seasonal distribution of protistan communities. We conducted an intensive survey of a 10 m2 grassland plot in Germany, focusing on a major group of protists, the Cercozoa. From 177 soil samples, collected from April to November, we obtained 694 Operational Taxonomy Units representing >6 million Illumina reads. All major cercozoan taxonomic and functional groups were present, dominated by the small flagellates of the Glissomonadida. We found evidence of environmental selection structuring the cercozoan communities both spatially and seasonally. Spatial analyses indicated that communities were correlated within a range of 3.5 m. Seasonal variations in the abundance of bacterivores and bacteria, followed by that of omnivores suggested a dynamic prey-predator succession. The most influential edaphic properties were moisture and clay content, which differentially affected each functional group. Our study is based on an intense sampling of protists at a small scale, thus providing a detailed description of the biodiversity of different taxa/functional groups and the ecological processes involved in shaping their distribution.

Rapid and reliable identification of bacterial pathogens directly from patient samples is required for optimizing antimicrobial therapy. Although Sanger sequencing of the 16S ribosomal RNA (rRNA) gene is used as a molecular method, species identification and discrimination is not always achievable for bacteria as their 16S rRNA genes have sometimes high sequence homology. Recently, next generation sequencing (NGS) of the 16S–23S rRNA encoding region has been proposed for reliable identification of pathogens directly from patient samples. However, data analysis is laborious and time-consuming and a database for the complete 16S–23S rRNA encoding region is not available. Therefore, a better, faster, and stronger approach is needed for NGS data analysis of the 16S–23S rRNA encoding region. We compared speed and diagnostic accuracy of different data analysis approaches: de novo assembly followed by Basic Local Alignment Search Tool (BLAST), operational taxonomic unit (OTU) clustering, or mapping using an in-house developed 16S–23S rRNA encoding region database for the identification of bacterial species. De novo assembly followed by BLAST using the in-house database was superior to the other methods, resulting in the shortest turnaround time (2 h and 5 min), approximately 2 h less than OTU clustering and 4.5 h less than mapping, and a sensitivity of 80%. Mapping was the slowest and most laborious data analysis approach with a sensitivity of 60%, whereas OTU clustering was the least laborious approach with 70% sensitivity. Although the in-house database requires more sequence entries to improve the sensitivity, the combination of de novo assembly and BLAST currently appears to be the optimal approach for data analysis.

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

Objectives: Daptomycin (DAP) resistance in Staphylococcus aureus is uncommon but there are increasing reports of the emergence of resistance during DAP therapy. Most clinical DAP-resistant S. aureus isolates investigated carried mutations in the mprF gene. The aim of this study was to identify mutations between a clinical pair of methicillin-susceptible S. aureus (MSSA) isolates (DAP-susceptible and DAP-resistant). Additionally, the activity of genes previously associated with DAP resistance was assessed.Materials and Methods: Two MSSA isolates from patient with left-sided endocarditis were analyzed by whole genome sequencing (WGS) and reverse transcription-quantitative real-time PCR (RT-qPCR). The first isolate, DAP-susceptible, was obtained before initiation of treatment and the second isolate, DAP-resistant, was recovered after 4 weeks of DAP therapy.Results: Comparison of complete genomes of DAP-susceptible and its DAP-resistant variant identified two non-synonymous and one synonymous mutations. The non-synonymous mutations consisted of a S829L substitution in mprF and a T331I substitution in vraS. The RT-qPCR experiments revealed an increased expression of vraS, dltA, mprF, and sceD genes in DAP-resistant variant. Strikingly, the expression of dltA and mprF genes was significantly downregulated by DAP.Conclusion: The mprF and vraS genes were previously associated with DAP resistance, however, none of the mutations described in this study had been previously identified and linked to DAP resistance. Moreover, we provide a new insight into the DAP action on S. aureus, in which the expression of key genes in DAP resistance is decreased by the antibiotic.

Microorganisms can use crystalline iron minerals for iron reduction linked to organic matter degradation or as conduits for direct interspecies electron transfer (mDIET) to syntrophic partners, e.g., methanogens. The environmental conditions that lead either to reduction or conduit use are so far unknown. We investigated microbial community shifts and interactions with crystalline iron minerals (hematite and magnetite) in methanic ferruginous marine sediment incubations during organic matter (glucose) degradation at varying temperatures. Iron reduction rates increased with decreasing temperature from 30°C to 4°C. Both hematite and magnetite facilitated iron reduction at 4°C, demonstrating that microorganisms in the methanic zone of marine sediments can reduce crystalline iron oxides under psychrophilic conditions. Methanogenesis occurred, however, at higher rates with increasing temperature. At 30°C, both hematite and magnetite accelerated methanogenesis onset and maximum process rates. At lower temperatures (10°C and 4°C), hematite could still facilitate methanogenesis but magnetite served more as an electron acceptor for iron reduction than as a conduit. Different temperatures selected for different key microorganisms: at 30°C, members of genus Orenia, Halobacteroidaceae, at 10°C, Photobacterium and the order Clostridiales, and at 4°C Photobacterium and Psychromonas were enriched. Members of the order Desulfuromonadales harboring known dissimilatory iron reducers were also enriched at all temperatures. Our results show that crystalline iron oxides predominant in some natural environments can facilitate electron transfer between microbial communities at psychrophilic temperatures. Furthermore, temperature has a critical role in determining the pathway of crystalline iron oxide utilization in marine sediment shifting from conduction at 30°C to predominantly iron reduction at lower temperatures.

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications.

Urinary tract infections (UTIs) are often caused by Escherichia coli. Their increasing resistance to broad-spectrum antibiotics challenges the treatment of UTIs. Whereas, E. coli ST131 is often multidrug resistant (MDR), ST69 remains susceptible to antibiotics such as cephalosporins. Both STs are commonly linked to community and nosocomial infections. E. coli phylogenetic groups B2 and D are associated with virulence and resistance profiles making them more pathogenic. Little is known about the population structure of E. coli isolates obtained from urine samples of hospitalized patients in Brazil. Therefore, we characterized E. coli isolated from urine samples of patients hospitalized at the university and three private hospitals in Rio de Janeiro, using whole genome sequencing. A high prevalence of E. coli ST131 and ST69 was found, but other lineages, namely ST73, ST648, ST405, and ST10 were also detected. Interestingly, isolates could be divided into two groups based on their antibiotic susceptibility. Isolates belonging to ST131, ST648, and ST405 showed a high resistance rate to all antibiotic classes tested, whereas isolates belonging to ST10, ST73, ST69 were in general susceptible to the antibiotics tested. Additionally, most ST69 isolates, normally resistant to aminoglycosides, were susceptible to this antibiotic in our population. The majority of ST131 isolates were ESBL-producing and belonged to serotype O25:H4 and the H30-R subclone. Previous studies showed that this subclone is often associated with more complicated UTIs, most likely due to their high resistance rate to different antibiotic classes. Sequenced isolates could be classified into five phylogenetic groups of which B2, D, and F showed higher resistance rates than groups A and B1. No significant difference for the predicted virulence genes scores was found for isolates belonging to ST131, ST648, ST405, and ST69. In contrast, the phylogenetic groups B2, D and F showed a higher predictive virulence score compared to phylogenetic groups A and B1. In conclusion, despite the diversity of E. coli isolates causing UTIs, clonal groups O25:H4-B2-ST131 H30-R, O1:H6-B2-ST648, and O102:H6-D-ST405 were the most prevalent. The emergence of highly virulent and MDR E. coli in Brazil is of high concern and requires more attention from the health authorities.

The human intestinal microbiota is a homeostatic ecosystem with a remarkable impact on human health and the disruption of this equilibrium leads to an increased susceptibility to infection by numerous pathogens. In this study, we used shotgun metagenomic sequencing and two different bioinformatic approaches, based on mapping of the reads onto databases and on the reconstruction of putative draft genomes, to investigate possible changes in the composition of the intestinal microbiota in samples from patients with Shiga Toxin-producing E. coli (STEC) infection compared to healthy and healed controls, collected during an outbreak caused by a STEC O26:H11 infection. Both the bioinformatic procedures used, produced similar result with a good resolution of the taxonomic profiles of the specimens. The stool samples collected from the STEC infected patients showed a lower abundance of the members of Bifidobacteriales and Clostridiales orders in comparison to controls where those microorganisms predominated. These differences seemed to correlate with the STEC infection although a flexion in the relative abundance of the Bifidobacterium genus, part of the Bifidobacteriales order, was observed also in samples from Crohn's disease patients, displaying a STEC-unrelated dysbiosis. The metagenomics also allowed to identify in the STEC positive samples, all the virulence traits present in the genomes of the STEC O26 that caused the outbreak as assessed through isolation of the epidemic strain and whole genome sequencing. The results shown represent a first evidence of the changes occurring in the intestinal microbiota of children in the course of STEC infection and indicate that metagenomics may be a promising tool for the culture-independent clinical diagnosis of the infection.

Shallow-water hydrothermal systems represent extreme environments with unique biogeochemistry and high biological productivity, at which autotrophic microorganisms use both light and chemical energy for the production of biomass. Microbial communities of these ecosystems are metabolically diverse and possess the capacity to transform a large range of chemical compounds. Yet, little is known about their diversity or factors shaping their structure or how they compare to coastal sediments not impacted by hydrothermalism. To this end, we have used automated ribosomal intergenic spacer analysis (ARISA) and high-throughput Illumina sequencing combined with porewater geochemical analysis to investigate microbial communities along geochemical gradients in two shallow-water hydrothermal systems off the island of Dominica (Lesser Antilles). At both sites, venting of hydrothermal fluids substantially altered the porewater geochemistry by enriching it with silica, iron and dissolved inorganic carbon, resulting in island-like habitats with distinct biogeochemistry. The magnitude of fluid flow and difference in sediment grain size, which impedes mixing of the fluids with seawater, were correlated with the observed differences in the porewater geochemistry between the two sites. Concomitantly, individual sites harbored microbial communities with a significantly different community structure. These differences could be statistically linked to variations in the porewater geochemistry and the hydrothermal fluids. The two shallow-water hydrothermal systems of Dominica harbored bacterial communities with high taxonomical and metabolic diversity, predominated by heterotrophic microorganisms associated with the Gammaproteobacterial genera Pseudomonas and Pseudoalteromonas, indicating the importance of heterotrophic processes. Overall, this study shows that shallow-water hydrothermal systems contribute substantially to the biogeochemical heterogeneity and bacterial diversity of coastal sediments.

Highly resistant microorganisms (HRMOs) may evade screening strategies used in routine diagnostics. Bacteria that have evolved to evade diagnostic tests may have a selective advantage in the nosocomial environment. Evasion of resistance detection can result from the following mechanisms: low-level expression of resistance genes not resulting in detectable resistance, slow growing variants, mimicry of wild-type-resistance, and resistance mechanisms that are only detected if induced by antibiotic pressure. We reviewed reports on hospital outbreaks in the Netherlands over the past 5 years. Remarkably, many outbreaks including major nation-wide outbreaks were caused by microorganisms able to evade resistance detection by diagnostic screening tests. We describe various examples of diagnostic evasion by several HRMOs and discuss this in a broad and international perspective. The epidemiology of hospital-associated bacteria may strongly be affected by diagnostic screening strategies. This may result in an increasing reservoir of resistance genes in hospital populations that is unnoticed. The resistance elements may horizontally transfer to hosts with systems for high-level expression, resulting in a clinically significant resistance problem. We advise to communicate the identification of HRMOs that evade diagnostics within national and regional networks. Such signaling networks may prevent inter-hospital outbreaks, and allow collaborative development of adapted diagnostic tests.

Objectives: To reveal the prevalence and epidemiology of extended-spectrum β-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- and carbapenemase (CP) producing Enterobacteriaceae and vancomycin-resistant enterococci (VRE) across the Northern Dutch–German border region.Methods: A point-prevalence study on ESBL/pAmpC/CP producing Enterobacteriaceae and VRE was carried out in hospitalized patients in the Northern Netherlands (n = 445, 2012–2013) and Germany (n = 242, 2012). Healthy individuals from the Dutch community (n = 400, 2010–2012) were also screened. In addition, a genome-wide gene-by-gene approach was applied to study the epidemiology of ESBL-Escherichia coli and VRE.Results: A total of 34 isolates from 27 patients (6.1%) admitted to Dutch hospitals were ESBL/pAmpC positive and 29 ESBL-E. coli, three pAmpC-E. coli, one ESBL-Enterobacter cloacae, and one pAmpC-Proteus mirabilis were found. In the German hospital, 18 isolates (16 E. coli and 2 Klebsiella pneumoniae) from 17 patients (7.7%) were ESBL positive. In isolates from the hospitalized patients CTX-M-15 was the most frequently detected ESBL-gene. In the Dutch community, 11 individuals (2.75%) were ESBL/pAmpC positive: 10 ESBL-E. coli (CTX-M-1 being the most prevalent gene) and one pAmpC E. coli. Six Dutch (1.3%) and four German (3.9%) hospitalized patients were colonized with VRE. Genetic relatedness by core genome multi-locus sequence typing (cgMLST) was found between two ESBL-E. coli isolates from Dutch and German cross-border hospitals and between VRE isolates from different hospitals within the same region.Conclusion: The prevalence of ESBL/pAmpC-Enterobacteriaceae was similar in hospitalized patients across the Dutch–German border region, whereas VRE prevalence was slightly higher on the German side. The overall prevalence of the studied pathogens was lower in the community than in hospitals in the Northern Netherlands. Cross-border transmission of ESBL-E. coli and VRE seems unlikely based on cgMLST analysis, however continuous monitoring is necessary to control their spread and stay informed about their epidemiology.

Objectives:Staphylococcus aureus infections in burn patients can lead to serious complications and death. The frequency of S. aureus infection is high in low- and middle-income countries presumably due to limited resources, misuse of antibiotics and poor infection control. The objective of the present study was to apply population genomics to precisely define, for the first time, the transmission of antibiotic resistant S. aureus in a resource-limited setting in sub-Saharan Africa.Methods:Staphylococcus aureus surveillance was performed amongst burn patients and healthcare workers during a 7-months survey within the burn unit of the Korle Bu Teaching Hospital in Ghana.Results: Sixty-six S. aureus isolates (59 colonizing and 7 clinical) were obtained from 31 patients and 10 healthcare workers. Twenty-one of these isolates were ST250-IV methicillin-resistant S. aureus (MRSA). Notably, 25 (81%) of the 31 patients carried or were infected with S. aureus within 24 h of admission. Genome comparisons revealed six distinct S. aureus clones circulating in the burn unit, and demonstrated multiple transmission events between patients and healthcare workers. Further, the collected S. aureus isolates exhibited a wide range of genotypic resistances to antibiotics, including trimethoprim (21%), aminoglycosides (33%), oxacillin (33%), chloramphenicol (50%), tetracycline (59%) and fluoroquinolones (100%).Conclusion: Population genomics uncovered multiple transmission events of S. aureus, especially MRSA, within the investigated burn unit. Our findings highlight lapses in infection control and prevention, and underscore the great importance of active surveillance to protect burn victims against multi-drug resistant pathogens in resource-limited settings.

Escherichia coli (E. coli) O157 that do not produce Shiga toxin and do not possess flagellar antigen H7 are of diverse H serotypes. In this study, the antibiotic resistance properties, genotype of a set of virulence associated genes and the phylogenetic background of E. coli O157:non-H7 groups were compared. Whole genome sequencing was performed on fourteen O157:non-H7 isolates collected in the STEC-ID-net study. The genomes were compared with E. coli O157 genomes and a typical Enteropathogenic E. coli (tEPEC) genome downloaded from NCBI. Twenty-six (86%) of the analyzed genomes had the intimin encoding gene eae but of different types mostly correlating with their H types, e.g., H16, H26, H39 and H45 carried intimin type ε, β, κ and α, respectively. They belonged to several E. coli phylogenetic groups, i.e., to phylogenetic group A, B1, B2 and D. Seven (50%) of our collected O157:non-H7 isolates were resistant to two or more antibiotics. Several mobile genetic elements, such as plasmids, insertion elements and pathogenicity islands, carrying a set of virulence and resistance genes were found in the E. coli O157:non-H7 isolates. Core genome phylogenetic analysis showed that O157:non-H7 isolates probably evolved from different phylogenetic lineages and were distantly related to the E. coli O157:H7 lineage. We hypothesize that independent acquisition of mobile genetic elements by isolates of different lineages have contributed to the different molecular features of the O157:non-H7 strains. Although distantly related to the STEC O157, E. coli O157:non-H7 isolates from multiple genetic background could be considered as pathogen of concern for their diverse virulence and antibiotic resistance properties.

ABSTRACT The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains.Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model.

Plants shape distinct, species-specific microbiomes in their rhizospheres. A main premise for evaluating microbial communities associated with root-soil compartments is their successful separation into the rhizosphere (soil-root interface), the rhizoplane (root surface), and the endosphere (inside roots). We evaluated different approaches (washing, sonication, bleaching) regarding their efficiency to separate microbial cells associated with different root compartments of soil-grown rice using fluorescence microscopy and community fingerprinting of 16S rRNA genes. Vigorous washing detached 45% of the rhizoplane population compared to untreated roots. Additional sonication reduced rhizoplane-attached microorganisms by up to 78% but caused various degrees of root tissue destruction at all sonication intensities tested. Treatment with sodium hypochlorite almost completely (98%) removed rhizoplane-associated microbial cells. Community fingerprinting revealed that microbial communities obtained from untreated, washed, and sonicated roots were not statistically distinguishable. Hypochlorite-treated roots harbored communities significantly different from all other samples, likely representing true endospheric populations. Applying these procedures to other root samples (bean, clover) revealed that treatment efficiencies were strongly affected by root morphological parameters such as root hair density and rigidity of epidermis. Our findings suggest that a careful evaluation of separation strategies prior to molecular community analysis is indispensable, especially when endophytes are the subject of interest.

Interrelated successive transformation steps of nitrification are performed by distinct microbial groups – the ammonia-oxidizers, comprising ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizers such as Nitrobacter and Nitrospira, which are the dominant genera in the investigated soils. Hence, not only their presence and activity in the investigated habitat is required for nitrification, but also their temporal and spatial interactions. To demonstrate the interdependence of both groups and to address factors promoting putative niche differentiation within each group, temporal and spatial changes in nitrifying organisms were monitored in an unfertilized grassland site over an entire vegetation period at the plot scale of 10 m². Nitrifying organisms were assessed by measuring the abundance of marker genes (amoA for AOA and AOB, nxrA for Nitrobacter, 16S rRNA gene for Nitrospira) selected for the respective sub-processes. A positive correlation between numerically dominant AOA and Nitrospira, and their co-occurrence at the same spatial scale in August and October, suggests that the nitrification process is predominantly performed by these groups and is restricted to a limited timeframe. Amongst nitrite-oxidizers, niche differentiation was evident in observed seasonally varying patterns of co-occurrence and spatial separation. While their distributions were most likely driven by substrate concentrations, oxygen availability may also have played a role under substrate-limited conditions. Phylogenetic analysis revealed temporal shifts in Nitrospira community composition with an increasing relative abundance of OTU03 assigned to sublineage V from August onwards, indicating its important role in nitrite oxidation.

In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in Germany. In 2013 E. coli O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis. All E. coli O104:H4 isolates tested were positive for stx2a, aatA and terD but were negative for escV. All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies.

Extended-spectrum ß-lactamase producing Klebsiella pneumoniae have emerged as one of the major nosocomial pathogens. Between July and September 2012, a CTX-M-15 producing K. pneumoniae caused an outbreak in a university hospital in the Netherlands. The outbreak isolates were characterized and assigned to a novel sequence type (ST1427). An epidemiological link between affected patients was supported by patient contact tracing and whole-genome phylogenetic analysis. Genetic diversity was detected among multiple isolates obtained from different body sites of the index patient, which may relate to antibiotic treatment and/or host adaptation. Environmental contamination caused by the outbreak clone was found in the patient rooms even on medical equipment. The novel clone was not closely related to any known endemic/epidemic clone, but carried a set of a plasmid-borne resistance genes (blaCTX-M-15, blaTEM-1, blaOXA-1, aac(6')-Ib-cr, qnrB1, tetA(A), aac(3)-II). Analysis of its virulence factors revealed a previously uncharacterized capsular biosynthesis region and two uncharacterized fimbriae gene clusters, and suggested that the new clone was not hypervirulent. To our knowledge, this is the first outbreak report of K. pneumoniae ST1427, and our study could be of help to understand the features of this newly emerging clone.

The role of microorganisms in the cycling of sedimentary organic carbon is a crucial one. To better understand relationships between molecular composition of a potentially bioavailable fraction of organic matter and microbial populations, bacterial and archaeal communities were characterized using pyrosequencing-based 16S rRNA gene analysis in surface (top 30 cm) and subsurface sediments (30-530 cm) of the Helgoland mud area, North Sea. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) was used to characterize a potentially bioavailable organic matter fraction (hot-water extractable organic matter, WE-OM). Algal polymer-associated microbial populations such as members of the Gammaproteobacteria, Bacteroidetes and Verrucomicrobia were dominant in surface sediments while members of the Chloroflexi (Dehalococcoidales and candidate order GIF9) and Miscellaneous Crenarchaeota Groups (MCG), both of which are linked to degradation of more recalcitrant, aromatic compounds and detrital proteins, were dominant in subsurface sediments. Microbial populations dominant in subsurface sediments (Chloroflexi, members of MCG, and Thermoplasmata) showed strong correlations to total organic carbon content. Changes of WE-OM with sediment depth reveal molecular transformations from oxygen-rich (high Oxygen to Carbon (O/C), low Hydrogen to Carbon (H/C) ratios) aromatic compounds and highly unsaturated compounds towards compounds with lower O/C and higher H/C ratios. The observed molecular changes were most pronounced in organic compounds containing only CHO atoms. Our data thus, highlights classes of sedimentary organic compounds that may serve as microbial energy sources in methanic marine subsurface environments.

The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx1a or stx2a subtypes. Interestingly, stx2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-β, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx2a.

Background: Antimicrobial resistance rates are increasing. This is, among others, caused by incorrect or inappropriate use of antimicrobials. To target this, a multidisciplinary Antimicrobial Stewardship-Team (A-Team) was implemented at the University Medical Center Groningen on a urology ward. Goal of this study is to evaluate the clinical effects of the case-audits done by this team, looking at length of stay (LOS) and antimicrobial use.Methods: Automatic e-mail alerts were sent after 48 hours of consecutive antimicrobial use triggering the case-audits, consisting of an A-Team member visiting the ward, discussing the patient’s therapy with the bed-side physician and together deciding on further treatment based on available diagnostics and guidelines. Clinical effects of the audits were evaluated through an Interrupted Time Series analysis and a retrospective historic cohort. Results: A significant systemic reduction of antimicrobial consumption for all patients on the ward, both with and without case-audits was observed. Furthermore, LOS for patients with case-audits who were admitted primarily due to infections decreased to 6.20 days (95% CI: 5.59-6.81) compared to the historic cohort (7.57 days; 95% CI: 6.92-8.21) (p=0.012). Antimicrobial consumption decreased for these patients from 8.17 DDD/patient (95% CI: 7.10-9.24) to 5.93 DDD/patient (95% CI: 5.02-6.83) (p=0.008). For patients with severe underlying diseases (e.g. cancer) these outcome measures remained unchanged.Conclusions: The evaluation showed a considerable positive impact. Antibiotic use of the whole ward was reduced, transcending the intervened patients. Furthermore, LOS and mean antimicrobial consumption for a subgroup was reduced, thereby improving patient care and potentially lowering resistance rates.

Iron reduction in subseafloor sulfate-depleted and methane-rich marine sediments is currently a subject of interest in subsurface geomicrobiology. While iron reduction and microorganisms involved have been well studied in marine surface sediments, little is known about microorganisms responsible for iron reduction in deep methanic sediments. Here, we used quantitative PCR (Q-PCR)-based 16S rRNA gene copy numbers and pyrosequencing-based relative abundances of bacteria and archaea to investigate covariance between distinct microbial populations and specific geochemical profiles in the top 5 m of sediment cores from the Helgoland mud area, North Sea. We found that gene copy numbers of bacteria and archaea were specifically higher around the peak of dissolved iron in the methanic zone (250-350 cm). The higher copy numbers at these depths were also reflected by the relative sequence abundances of members of the candidate division JS1, methanogenic and Methanohalobium/ANME-3 related archaea. The distribution of these populations was strongly correlated to the profile of pore-water Fe2+ while that of Desulfobacteraceae corresponded to the pore-water sulfate profile. Furthermore, specific JS1 populations also strongly co-varied with the distribution of Methanosaetaceae in the methanic zone. Our data suggest that the interplay among JS1 bacteria, methanogenic archaea and Methanohalobium/ANME-3-related archaea may be important for iron reduction and methane cycling in deep methanic sediments of the Helgoland mud area and perhaps in other methane-rich depositional environments.

Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet a limited number of isolates makes it difficult to understand physiology and ecological impact of the microorganisms involved. Here, two-staged cultivation was implemented to selectively enrich and isolate crystalline iron(III) reducers in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by two-year successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. The Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) media in order to stimulate proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. These isolates had 94.8–98.1% sequence similarities of 16S rRNA genes to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. The results demonstrate the successful enrichment and isolation of novel iron(III) reducers that were able to thrive by reducing highly crystallized ferric irons.