Earth's life-sustaining oceans harbor diverse bacterial communities that display varying composition across time and space. While particular patterns of variation have been linked to a range of factors, unifying rules are lacking, preventing the prediction of future changes. Here, analyzing the distribution of fast- and slowgrowing bacteria in ocean datasets spanning seasons, latitude, and depth, we show that higher seawater temperatures universally favor slower-growing taxa, in agreement with theoretical predictions of how temperaturedependent growth rates differentially modulate the impact of mortality on species abundances. Changes in bacterial community structure promoted by temperature are independent of variations in nutrients along spatial and temporal gradients. Our results help explain why slow growers dominate at the ocean surface, during summer, and near the tropics and provide a framework to understand how bacterial communities will change in a warmer world.

Background: Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings: Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance: For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.

Ecological significance of trees growing in urban and peri-urban settings is likely to increase in future land-use regimes, calling for better understanding of their role as potential reservoirs or stepping stones for associated biodiversity. We studied the diversity of fungal endophytes in woody tissues of asymptomatic even aged pedunculate oak trees, growing as amenity trees in a peri-urban setting. The trees were classified into three groups according to their phenotypic vitality (high, medium, and low). Endophytes were cultured on potato dextrose media from surface sterilized twigs and DNA sequencing was performed to reveal the taxonomic identity of the morphotypes. In xylem tissues, the frequency and diversity of endophytes was highest in oak trees showing reduced vitality. This difference was not found for bark samples, in which the endophyte infections were more frequent and communities more diverse than in xylem. In general, most taxa were shared across the samples with few morphotypes being recovered in unique samples. Leaf phenolic profiles were found to accurately classify the trees according to their phenotypic vitality. Our results confirm that xylem is more selective substrate for endophytes than bark and that endophyte assemblages in xylem are correlated to the degree of host vitality. Thus, high vitality of trees may be associated with reduced habitat quality to wood-associated endophytes.

Cluster 5 picocyanobacteria significantly contribute to primary productivity in aquatic ecosystems. Estuarine populations are highly diverse and consist of many co-occurring strains, but their physiology remains largely understudied. In this study, we characterized 17 novel estuarine picocyanobacterial strains. Phylogenetic analysis of the 16S rRNA and pigment genes (cpcBandcpeBA) uncovered multiple estuarine and freshwater-related clusters and pigment types. Assays with five representative strains (three phycocyanin rich and two phycoerythrin rich) under temperature (10–30°C), light(10–190 μmol  photons  m^-2s^-1), and salinity (2–14  PSU) gradients revealed distinct growth optima and tolerance, indicating that genetic variability was accompanied by physiological diversity. Adaptability to environmental conditions was associated with differential pigment content and photosynthetic performance. Amplicon sequence variants at a coastal and an offshore station linked population dynamics with phylogenetic clusters, supporting that strains isolated in this study represent key ecotypes within the Baltic Sea picocyanobacterial community. The functional diversity found within strains with the same pigment type suggests that understanding estuarine picocyanobacterial ecology requires analysis beyond the phycocyanin and phycoerythrin divide. This new knowledge of the environmental preferences in estuarine picocyanobacteria is important for understanding and evaluating productivity in current and future ecosystems.

Cyanobacteria are globally widespread photosynthetic prokaryotes and are major contributors to global biogeochemical cycles. One of the most critical processes determining cyanobacterial eco-physiology is cellular death. Evidence supports the existence of controlled cellular demise in cyanobacteria, and various forms of cell death have been described as a response to biotic and abiotic stresses. However, cell death research in this phylogenetic group is a relatively young field and understanding of the underlying mechanisms and molecular machinery underpinning this fundamental process remains largely elusive. Furthermore, no systematic classification of modes of cell death has yet been established for cyanobacteria. In this work, we analyzed the state of knowledge in the field of cyanobacterial cell death. Based on that, we propose unified criterion for the definition of accidental, regulated, and programmed forms of cell death in cyanobacteria based on molecular, biochemical, and morphologic aspects following the directions of Nomenclature Committee on Cell Death (NCCD). With this, we aim to provide a guide to standardize the nomenclature related to this topic in a precise and consistent manner, which will facilitate further ecological, evolutionary and applied research in the field of cyanobacterial cell death

Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 mu M and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803.

Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene bla(OXA-48). Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat to human health and are increasingly being isolated from nonclinical settings. OXA-48-producing Escherichia coli sequence type 38 (ST38) is the most frequently reported CRE type in wild birds and has been detected in gulls or storks in North America, Europe, Asia, and Africa. The epidemiology and evolution of CRE in wildlife and human niches, however, remains unclear. We compared wild bird origin E. coli ST38 genome sequences generated by our research group and publicly available genomic data derived from other hosts and environments to (i) understand the frequency of intercontinental dispersal of E. coli ST38 clones isolated from wild birds, (ii) more thoroughly measure the genomic relatedness of carbapenem-resistant isolates from gulls sampled in Turkey and Alaska, USA, using long-read whole-genome sequencing and assess the spatial dissemination of this clone among different hosts, and (iii) determine whether ST38 isolates from humans, environmental water, and wild birds have different core or accessory genomes (e.g., antimicrobial resistance genes, virulence genes, plasmids) which might elucidate bacterial or gene exchange among niches. Our results suggest that E. coli ST38 strains, including those resistant to carbapenems, are exchanged between humans and wild birds, rather than separately maintained populations within each niche. Furthermore, despite close genetic similarity among OXA-48-producing E. coli ST38 clones from gulls in Alaska and Turkey, intercontinental dispersal of ST38 clones among wild birds is uncommon. Interventions to mitigate the dissemination of antimicrobial resistance throughout the environment (e.g., as exemplified by the acquisition of carbapenem resistance by birds) may be warranted.IMPORTANCE Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene bla(OXA-48). This is the most frequently reported carbapenem-resistant clone in wild birds, though it was unclear if it circulated within wild bird populations or was exchanged among other niches. The results from this study suggest that E. coli ST38 strains, including those resistant to carbapenems, are frequently exchanged among wild birds, humans, and the environment. Carbapenem-resistant E. coli ST38 clones in wild birds are likely acquired from the local environment and do not constitute an independent dissemination pathway within wild bird populations. Management actions aimed at preventing the environmental dissemination and acquisition of antimicrobial resistance by wild birds may be warranted.

Objectives: A variety of methods have been developed to detect antimicrobial resistance (AMR) in differ-ent environments to better understand the evolution and dissemination of this public health threat. Com-parisons of results generated using different AMR detection methods, such as quantitative PCR (qPCR) and whole-genome sequencing (WGS), are often imperfect, and few studies have analysed samples in parallel to evaluate differences. In this study, we compared bacterial culture and WGS to a culture-independent commercially available qPCR assay to evaluate the concordance between methods and the utility of each in answering research questions regarding the presence and epidemiology of AMR in wild bird habitats.Methods: We first assessed AMR gene detection using qPCR in 45 bacterial isolates from which we had existing WGS data. We then analysed 52 wild bird faecal samples and 9 spatiotemporally collected water samples using culture-independent qPCR and WGS of phenotypically resistant indicator bacterial isolates.Results: Overall concordance was strong between qPCR and WGS of bacterial isolates, although concor-dance differed among antibiotic classes. Analysis of wild bird faecal and water samples revealed that more samples were determined to be positive for AMR via qPCR than via culture and WGS of bacterial isolates, although qPCR did not detect AMR genes in two samples from which phenotypically resistant isolates were found.Conclusions: Both qPCR and culture followed by sequencing may be effective approaches for characteris-ing AMR genes harboured by wild birds, although data streams produced using these different tools may have advantages and disadvantages that should be considered given the application and sample matrix.Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Anthropogenic inputs into the environment may serve as sources of antimicrobial resistant bacteria and alter the ecology and population dynamics of synanthropic wild animals by providing supplemental forage. In this study, we used a combination of phenotypic and genomic approaches to characterize antimicrobial resistant indicator bacteria, animal telemetry to describe host movement patterns, and a novel modeling approach to combine information from these diverse data streams to investigate the acquisition and long-distance dispersal of antimicrobial resistant bacteria by landfill-foraging gulls. Our results provide evidence that gulls acquire antimicrobial resistant bacteria from anthropogenic sources, which they may subsequently disperse across and between continents via migratory movements. Furthermore, we introduce a flexible modeling framework to estimate the relative dispersal risk of antimicrobial resistant bacteria in western North America and adjacent areas within East Asia, which may be adapted to provide information on the risk of dissemination of other organisms and pathogens maintained by wildlife through space and time. Published by Elsevier B.V.

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenicallyassociated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

Bacteria in the sea are constantly exposed to environmental challenges (e.g. variations in nutrient concentrations, temperature and light conditions), and therefore appropriate gene expression response strategies to cope with them efficiently are evolved. This thesis investigates some interconnected questions regarding such adaptive strategies employed by marine bacteria.

The recently discovered ability of bacteria to use the membrane protein proteorhodopsin (PR) to harvest light energy for cell metabolism were investigated in Vibrio sp. AND4 and Dokdonia sp. MED134. PR phototrophy in AND4 promoted survival during starvation, the molecular basis for which were the upregulation of the PR gene by nutrient limitation rather than light. MED134, in contrast, uses PR phototrophy to grow better, and we discovered that the light-stimulated growth was stronger in seawater with the single carbon compound alanine compared to a mixture of complex organic matter. Thus, differences between bacteria in PR gene expression regulation in response to light, nutrients or organic matter quality critically determine the ecological role of PR phototrophy in the sea.

Current observations that membrane transporters (including PR) are highly expressed in seawater inspired a comparative analysis of transporter distributions in marine bacteria. Totally, 192 transporter families were found in 290 genome-sequenced strains. Consistent differences, but also similarities, in the number of transporters were found between major bacterial groups. Interestingly, sodium transporters were found to be more abundant in PR-containing SAR11. These findings suggest that bacteria have inherently distinctive potentials to adapt to resource variations in the sea.

To examine links between transcriptional responses and growth of bacteria under controlled environmental settings, a mesocosm phytoplankton bloom experiment was performed. Transcriptional analysis of the microbial community (i.e. metatranscriptomics) revealed 2800 categories of functional genes (SEED functions), of which around 10% were overrepresented in either the bloom mesocosms or the controls. Importantly, these functions indicated potential metabolic mechanisms (e.g. TonB mediated nutrient transport) by which bacteria took advantage of the bloom conditions.

This thesis combines analyses of model organisms with community analysis and highlights the possibilities to identify important mechanisms that underlie the ecological success of different bacteria in the marine environment. 

Picocyanobacteria (< 2 mu m in diameter) are significant contributors to total phytoplankton biomass. Due to the high diversity within this group, their seasonal dynamics and relationship with environmental parameters, especially in brackish waters, are largely unknown. In this study, the abundance and community composition of phycoerythrin rich picocyanobacteria (PE-SYN) and phycocyanin rich picocyanobacteria (PC-SYN) were monitored at a coastal (K-station) and at an offshore station (LMO; similar to 10 km from land) in the Baltic Sea over three years (2018-2020). Cell abundances of picocyanobacteria correlated positively to temperature and negatively to nitrate (NO3) concentration. While PE-SYN abundance correlated to the presence of nitrogen fixers, PC-SYN abundance was linked to stratification/shallow waters. The picocyanobacterial targeted amplicon sequencing revealed an unprecedented diversity of 2169 picocyanobacterial amplicons sequence variants (ASVs). A unique assemblage of distinct picocyanobacterial clades across seasons was identified. Clade A/B dominated the picocyanobacterial community, except during summer when low NO3, high phosphate (PO4) concentrations and warm temperatures promoted S5.2 dominance. This study, providing multiyear data, links picocyanobacterial populations to environmental parameters. The difference in the response of the two functional groups and clades underscore the need for further high-resolution studies to understand their role in the ecosystem.

Background: Prokaryotes dominate the biosphere and regulate biogeochemical processes essential to all life. Yet, our knowledge about their biology is for the most part limited to the minority that has been successfully cultured. Molecular techniques now allow for obtaining genome sequences of uncultivated prokaryotic taxa, facilitating in-depth analyses that may ultimately improve our understanding of these key organisms. Results: We compared results from two culture-independent strategies for recovering bacterial genomes: single-amplified genomes and metagenome-assembled genomes. Single-amplified genomes were obtained from samples collected at an offshore station in the Baltic Sea Proper and compared to previously obtained metagenome-assembled genomes from a time series at the same station. Among 16 single-amplified genomes analyzed, seven were found to match metagenome-assembled genomes, affiliated with a diverse set of taxa. Notably, genome pairs between the two approaches were nearly identical (average 99.51% sequence identity; range 98.77-99.84%) across overlapping regions (30-80% of each genome). Within matching pairs, the single-amplified genomes were consistently smaller and less complete, whereas the genetic functional profiles were maintained. For the metagenome-assembled genomes, only on average 3.6% of the bases were estimated to be missing from the genomes due to wrongly binned contigs. Conclusions: The strong agreement between the single-amplified and metagenome-assembled genomes emphasizes that both methods generate accurate genome information from uncultivated bacteria. Importantly, this implies that the research questions and the available resources are allowed to determine the selection of genomics approach for microbiome studies.

Data from several years of monthly samplings are combined with a 1-year detailed study of carbon flux through bacteria at a NW Mediterranean coastal site to delineate the bacterial role in carbon use and to assess whether environmental factors or bacterial assemblage composition affected the in situ rates of bacterial carbon processing. Leucine (Leu) uptake rates [as an estimate of bacterial heterotrophic production (BHP)] showed high interannual variability but, on average, lower values were found in winter (around 50 pM Leu(-1) h(-1)) as compared to summer (around 150 pM Leu(-1) h(-1)). Leu-to-carbon conversion factors ranged from 0.9 to 3.6 kgC mol Leu(-1), with generally higher values in winter. Leu uptake was only weakly correlated to temperature, and over a full-year cycle (in 2003), Leu uptake peaked concomitantly with winter chlorophyll a (Chl a) maxima, and in periods of high ectoenzyme activities in spring and summer. This suggests that both low molecular weight dissolved organic matter (DOM) released by phytoplankton, and high molecular weight DOM in periods of low Chl a, can enhance BHP. Bacterial respiration (BR, range 7-48 mu g C l(-1) d(-1)) was not correlated to BHP or temperature, but was significantly correlated to DOC concentration. Total bacterial carbon demand (BHP plus BR) was only met by dissolved organic carbon produced by phytoplankton during the winter period. We measured bacterial growth efficiencies by the short-term and the long-term methods and they ranged from 3 to 42%, increasing during the phytoplankton blooms in winter (during the Chl a peaks), and in spring. Changes in bacterioplankton assemblage structure (as depicted by denaturing gradient gel electrophoresis fingerprinting) were not coupled to changes in ecosystem functioning, at least in bacterial carbon use.

Despite the high abundance of Archaea in the global ocean, their metabolism and biogeochemical roles remain largely unresolved. We investigated the population dynamics and metabolic activity of Thaumarchaeota in polar environments, where these microorganisms are particularly abundant and exhibit seasonal growth. Thaumarchaeota were more abundant in deep Arctic and Antarctic waters and grew throughout the winter at surface and deeper Arctic halocline waters. However, in situ single-cell activity measurements revealed a low activity of this group in the uptake of both leucine and bicarbonate (<5% Thaumarchaeota cells active), which is inconsistent with known heterotrophic and autotrophic thaumarchaeal lifestyles. These results suggested the existence of alternative sources of carbon and energy. Our analysis of an environmental metagenome from the Arctic winter revealed that Thaumarchaeota had pathways for ammonia oxidation and, unexpectedly, an abundance of genes involved in urea transport and degradation. Quantitative PCR analysis confirmed that most polar Thaumarchaeota had the potential to oxidize ammonia, and a large fraction of them had urease genes, enabling the use of urea to fuel nitrification. Thaumarchaeota from Arctic deep waters had a higher abundance of urease genes than those near the surface suggesting genetic differences between closely related archaeal populations. In situ measurements of urea uptake and concentration in Arctic waters showed that small-sized prokaryotes incorporated the carbon from urea, and the availability of urea was often higher than that of ammonium. Therefore, the degradation of urea may be a relevant pathway for Thaumarchaeota and other microorganisms exposed to the low-energy conditions of dark polar waters.

Heterotrophic microflagellates isolated from the Baltic Sea and grown under laboratoryconditions were shown to release dissolved free amino acids (DFAA) when grazing bacteria. Flagellatesreleased 3H-amino acids when fed 3H-leucine-labelled bacteria, and concentrations of aminoacids increased in the experimental medium. Serine showed a strong positive correlation withflagellate feeding. Aspartic acid, glutamic acid and ornithine also increased more than other aminoacids. During consumption of bacteria, the flagellates released 13% of the ingested nitrogen asammonia, and 30 % of the ingested phosphorus as phosphate. In a field experiment off Scripps Pier, wemeasured bacterial production, flagellate abundance, and concentration of DFAA over a 28 h period.The concentration of DFAA showed a covariation with the flagellate numbers. Results from our fieldand laboratory experiments suggest that flagellates may be a source of DFAA in the sea. 

Variation in traits causes bacterial populations to respond in contrasting ways to environmental drivers. Learning about this will help us understand the ecology of individual populations in complex ecosystems. We used 454 pyrosequencing of the hypervariable region V6 of the 16S rRNA gene to study seasonal dynamics in Baltic Sea bacterioplankton communities, and link community and population changes to biological and chemical factors. Surface samples were collected from May to October 2003 and in May 2004 at the Landsort Deep in the central Baltic Sea Proper. The analysis rendered, on average, 20 200 sequence reads for each of the eight samples analyzed, providing the first detailed description of Baltic Sea bacterial communities. Community composition varied dramatically over time, supporting the idea of strong temporal shifts in bacterioplankton assemblages, and clustered according to season (including two May samples from consecutive years), suggesting repeatable seasonal succession. Overall, community change was most highly correlated with change in phosphorus concentration and temperature. Individual bacterial populations were also identified that tightly co-varied with different Cyanobacteria populations. Comparing the abundance profiles of operational taxonomic units at different phylogenetic distances revealed a weak but significant negative correlation between abundance profile similarity and genetic distance, potentially reflecting habitat filtering of evolutionarily conserved functional traits in the studied bacterioplankton.

Cercopithifilaria species are tick-transmitted filarial parasites of mammals. In Europe, three Cercopithifilaria spp. are known to parasitize dogs, all occurring mainly in the Mediterranean countries. In Romania, Cercopithifilaria bainae has been reported in a single dog in eastern Romania but the occurrence in other parts of the country is not known. To further elucidate the geographic distribution of Cercopithifilaria spp. infection, 544 ticks were collected from dogs in several locations across Romania. The presence of Cercopithifilaria spp. was investigated with real-time PCR. A single Dermacentor reticulatus female tick was found to be infected with Cercopithifilaria bainae. The finding in the present study is geographically separated from the previous finding in Romania by 800 km, as well as by the Carpathian mountain range. Hence, C. bainae is more geographically widespread in Romania than previously recognized. However, the single detection does suggest that infection is rather uncommon in Romanian dogs. Nevertheless, further studies on Cercopithifilaria spp. distribution and prevalence are needed.

Romania has a highly diverse tick fauna. Consequently, a high diversity of tick-transmitted pathogens might be a potential threat to humans. However, only a limited number of tick species regularly infest humans, and pathogens present in such species are therefore of particular interest from a medical perspective. In this study, 297 ticks were collected from humans during 2013 and 2014. Ixodes ricinus was the predominant tick species, accounting for 272 specimens or 91.6% of the ticks in the study. Nevertheless, other tick species were also found to infest humans: Dermacentor marginatus constituted 7% of the ticks found on humans (21/297), Haemaphysalis punctata 1% (3/297), and Haemaphysalis concinna 0.3% (1/297). Ticks were tested by PCR for a wide range of tick-borne pathogens. In total, 11.8% of the ticks carried human pathogenic bacteria, while no viral or protozoan pathogens were detected. The most frequently detected pathogen was Rickettsia spp., occurring in 5.4% of the ticks (16/297) and comprising three species: Rickettsia (R.) raoultii, R. monacensis, and R. helvetica. Borrelia s.l. occurred in 3% (9/297) of the ticks. "Candidatus Neoehrlichia mikurensis" occurred in 1.7% (5/297) and Anaplasma phagocytophilum in 1.3% (4/297). Anaplasma bovis was detected in an H. punctata and Borrelia miyamotoi in an I. ricinus. These results point to the need for further studies on the medical importance of tick-borne pathogens in Romania.

Ixodes (Ixodes) apronophorus is a neglected tick species and its geographical distribution, host associations, and role as a disease vector are not well known. We collected I. apronophorus from several locations in Romania. Morphological identification of ticks was confirmed by analysis of 16S rDNA and 12S rDNA gene sequences. We report new host associations of I. apronophorus, which was collected from dogs, foxes, and a hare-all new hosts for this tick species in Romania. Furthermore, we report for the first time occurrence of Ehrlichia sp. HF in I. apronophorus. Ehrlichia sp. HF was identified by sequencing a part of the 16S rDNA gene and was found in 16% (3/19) of the tested ticks. Ehrlichia sp. HF has not been previously reported in Eastern Europe and seems to have a much larger geographic distribution than previously known. Currently, it is unknown whether I. apronophorus is a competent vector for Ehrlichia sp. HF, or if the findings in this study represent infection in the hosts, namely dogs and fox.

Background: Ticks are transmitting a wide range of bacterial pathogens that cause substantial morbidity and mortality in domestic animals. The full pathogen burden transmitted by tick vectors is incompletely studied in many geographical areas, and extensive studies are required to fully understand the diversity and distribution of pathogens transmitted by ticks. Results: We sampled 824 ticks of 11 species collected in 19 counties in Romania. Ticks were collected mainly from dogs, but also from other domestic and wild animals, and were subjected to molecular screening for pathogens. Rickettsia spp. was the most commonly detected pathogen, occurring in 10.6% (87/824) of ticks. Several species were detected: Rickettsia helvetica, R. raoultii, R. massiliae, R. monacensis, R. slovaca and R. aeschlimannii. A single occurrence of the zoonotic bacterium Bartonella vinsonii berkhoffii was detected in a tick collected from a dog. Anaplasma phagocytophilum occurred in four samples, and sequences similar to Anaplasma marginale/ovis were abundant in ticks from ruminants. In addition, molecular screening showed that ticks from dogs were carrying an Ehrlichia species identical to the HF strain as well as the enigmatic zoonotic pathogen "Candidatus Neoehrlichia mikurensis". An organism similar to E. chaffeensis or E. muris was detected in an Ixodes ricinus collected from a fox. Conclusions: We describe an abundant diversity of bacterial tick-borne pathogens in ticks collected from animal hosts in Romania, both on the level of species and genotypes/strains within these species. Several findings were novel for Romania, including Bartonella vinsonii subsp. berkhoffii that causes bacteremia and endocarditis in dogs. "Candidatus Neoehrlichia mikurensis" was detected in a tick collected from a dog. Previously, a single case of infection in a dog was diagnosed in Germany. The results warrant further studies on the consequences of tick-borne pathogens in domestic animals in Romania.

Babesia spp., Theileria spp., and Hepatozoon spp. are tick-transmitted apicomplexan parasites that cause several important diseases in animals. To increase current knowledge about the diversity of tick-transmitted pathogens in Romania, we investigated the occurrence of Babesia spp., Theileria spp., and Hepatozoon spp. in a wide range of tick species infesting animal hosts. We collected 852 ticks from 10 different animal species from 20 counties in Romania. The assessment was based on detection of parasite DNA by PCR. Five different apicomplexan parasite species were detected; among them three different species of Babesia: B. canis, B. microti, and B. ovis. Hepatozoon canis was the most frequently detected parasite, found predominately in Ixodes ricinus ticks collected from domestic dogs. It was also detected in I. ricinus collected from goat, fox, and cat. Furthermore, H. canis was found in Haemaphysalis punctata and Haemaphysalis concinna ticks. In addition, Theileria buffeli was detected in Rhipicephalus bursa ticks collected from cattle.

Babesiosis is a severe disease in cattle worldwide. In Europe, the main causative agent of bovine babesiosis is Babesia divergens. In some areas, this species is reported to have declined or even disappeared, and its etiological role overtaken by other piroplasmid species. Moreover, co-infection with other tick-transmitted pathogens can be expected to complicate diagnosis in cattle. Hence, molecular identification of the causative agent of babesiosis should be a priority. Therefore, samples from 71 domestic cattle, 39 with clinical signs of babesiosis and 32 without, from southern Sweden were screened for Babesia spp. and Anaplasma spp. using molecular methods Babesia divergens was detected in 38 of the samples, and Anaplasma phagocytophilum in 17. Co-infections with both pathogens were frequent, occurring in 18% of the animals with a B. divergens infection. The possibility of co-infection should be considered in diagnosis and treatment of bovine babesiosis.

Bacteria in the bloodstream may cause sepsis, therefore early and correct treatment is important. Antibiotic resistance is an increasing problem and antimicrobial susceptibility testing (AST) is essential for the patients survival. In sepsis with diagnosis based on blood culture, at least two days are required for results from AST.

Routine AST-method in Swedish microbiological laboratories is the disc diffusion method standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In disc diffusion, bacteria are grown on agar plates and inhibition zones around paper disks with antibiotics are used to measure the antibiotic effect. The method requires bacterial colonies as source material and takes 16–20 hours. EUCAST has further developed the method for Rapid Antimicrobial Susceptibility Testing (RAST). RAST is performed directly from positive blood culture bottles and inhibition zones are measured at 4, 6 and 8 hours. The method uses different breakpoints for categorization at the different timepoints. The purpose of the study was to evaluate RAST for Staphylococcus aureus and Streptococcus pneumoniae by examining bacterial isolates inoculated into blood culture bottles. Zone diameters were measured, and results were compared with those of standardized disk diffusion. For S.aureus, at 4 hours 102/184 (55%) readings were categorizable, at 6 hours 164/184 (89%) and at 8 hours 174/184 (95%). A total of 7 error categorizations occurred, 5 of which were underestimation of clindamycin resistance at 4 hours. For S.pneumoniae at 4 hours, 74/80 (92%) readings were categorizable, at 6 hours 73/80 (91%) and at 8 hours 77/80 (96%). Resistance was overestimated at 21 zone measurements at 4 hours.. RAST showed good results for S.aureus after 6 hours. For S.pneumoniae, further studies are required but RAST may work after 6 hours for penicillin resistance assessment.

A novel Gram-staining-negative, chemoorganotrophic, moderately halophilic, strictly aerobic bacterium, strain MED121(T), was isolated from a seawater sample collected at the Blanes Bay Microbial Observatory in the north-western Mediterranean Sea. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to Marinomonas dokdonensis and other Marinomonas species (96.3 and 93.3-95.7% sequence similarities, respectively), within the family Oceanospirillaceae. Strain MED121(T) was included into a whole-genome sequencing study and, subsequently, it was characterized using a polyphasic taxonomic approach. It was found to be oxidase and catalase positive, its cells are cocci to short rods, it does not ferment carbohydrates and does not reduce nitrate to nitrite or gas and it requires at least 2.5% (w/v) marine salts and tolerates up to 7% (w/v) salts. Its major cellular fatty acids in order of abundance are C-16:1 omega 7c/C-16:1 omega 6c,C-18:1 omega 7c(1), C-16:0 and C-10:0 3-OH. Its genome had an approximate length of 5.1 million bases and a DNA G+C content equal to 40.9 mol%. Analysis of the annotated genes reveals the capacity for the synthesis of ubiquinone 8 (O8) and the polar lipids phosphatidylglycerol and phosphatidylethanolannine, in agreement with other members of the genus. All the data collected supported the creation of a novel species to accommodate this bacterium, for which the name Marinomonas blandensis sp. nov. is proposed. The type strain is MED121(T) (=CECT 7076(T)=LMG 29722(T)).

Antibiotic use and its implications have been discussed extensively in the past decades. This situation has global consequences when antibiotic resistance becomes widespread in the intestinal bacterial flora of stationary and migratory birds. This study investigated the incidence of fecal bacteria and general antibiotic resistance, with special focus on extended spectrum beta-lactamase (ESBL) isolates, in two species of seabirds at remote Easter Island. We identified 11 species of bacteria from masked booby (Sula dactylatra) and Christmas shearwater (Puffinus nativitatis); five species of gram-negative bacilli, four species of Streptococcus (Enterococcus), and 2 species of Staphylococcus. In addition, 6 types of bacteria were determined barely to the genus level. General antibiotic susceptibility was measured in the 30 isolated Enterobacteriaceae to 11 antibiotics used in human and veterinary medicine. The 10 isolates that showed a phenotypic ESBL profile were verified by clavulanic acid inhibition in double mixture discs with cefpodoxime, and two ESBL strains were found, one strain in masked booby and one strain in Christmas shearwater. The two bacteria harboring the ESBL type were identified as Serratia odorifera biotype 1, which has zoonotic importance. Despite minimal human presence in the masked booby and Christmas shearwater habitats, and the extreme geographic isolation of Easter Island, we found several multiresistant bacteria and even two isolates with ESBL phenotypes. The finding of ESBLs has animal and public health significance and is of potential concern, especially because the investigation was limited in size and indicated that antibiotic-resistant bacteria now are distributed globally.

Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response, and programmed cell death (PCD) in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to ~4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (<0.8 μm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e., sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.

Campylobacter jejuni is the primary cause of bacterial gastroenteritis worldwide, infecting humans mostly through consumption of contaminated poultry. C. jejuni is common in the gut of wild birds, and shows distinct strain-specific association to particular bird species. This contrasts with farm animals, in which several genotypes co-exist. It is unclear if the barriers restricting transmission between host species of such specialist strains are related to environmental factors such as contact between host species, bacterial survival in the environment, etc., or rather to strain specific adaptation to the intestinal environment of specific hosts. We compared colonization dynamics in vivo between two host-specific C. jejuni from a song thrush (ST-1304 complex) and a mallard (ST-995), and a generalist strain from chicken (ST-21 complex) in a wild host, the mallard (Anas platyrhynchos). In 18-days infection experiments, the song thrush strain showed only weak colonization and was cleared from all birds after 10 days, whereas both mallard and chicken strains remained stable. When the chicken strain was given 4 days prior to co-infection of the same birds with a mallard strain, it was rapidly outcompeted by the latter. In contrast, when the mallard strain was given 4 days prior to co-infection with the chicken strain, the mallard strain remained and expansion of the chicken strain was delayed. Our results suggest strain-specific differences in the ability of C. jejuni to colonize mallards, likely associated with host origin. This difference might explain observed host association patterns in C. jejuni from wild birds.

Objectives This study investigates the frequency and characteristics of carbapenemase-producing Escherichia coli/Klebsiella pneumoniae (CPE/K) and extended-spectrum cephalosporinase-producing E. coli/K. pneumoniae (ESCE/K) in healthy humans and livestock in rural Cambodia. Additionally, household practices as risk factors for faecal carriage of ESCE/K are identified. Methods Faecal samples were obtained from 307 humans and 285 livestock including large ruminants, pigs and poultry living in 100 households in rural Cambodia in 2011. Each household was interviewed, and multilevel logistic model determined associations between household practices/meat consumption and faecal carriage of ESCE/K. CPE and ESCE/K were detected and further screened for colistin resistance genes. Results CPE/K isolates harbouring bla(OXA-48 )were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: bla(CTX-M-15), bla(CTX-M-14), bla(CTX-M-27), bla(CTX-M-55), bla(SHV-2), bla(SHV-12), bla(SHV-28); AmpC genes: bla(CMY-2), bla(CMY-42,) bla(DHA-1); and colistin resistance genes: mcr-1-like and mcr-3-like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K. Conclusions Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans.

Infections due to multidrug resistant Gram-negative bacteria are a major problem in large parts of the world. Due to lack of treatment options, a previously banned antibiotic, colistin, has been reintroduced. Unfortunately, susceptibility testing of colistin is problematic and currently the only recommended method is broth microdilution (BMD). Colistin is a positively charged molecule that binds to lipid A at the negatively charged outer cell membrane, which leads to cell disruption. Several mutations causing colistin resistance have been identified, most of them cause alterations of lipid A, resulting in impaired colistin outer cell membrane interaction.

The aim of this study was to evaluate four potential screening methods for colistin resistance and compare the results with BMD (reference method). The four screening methods were broth disk elution, disk diffusion and the commercially available tests Superpolymyxin and Rapid polymyxin NP.

A collection of 57 whole genome sequenced Enterobacterales consisting of 28 Escherichia coli and 29 Klebsiella pneumoniae were included in the study. Reference results were obtained by BMD, where the estimated minimum inhibitory concentration (MIC) values were used for classification of the isolates as sensitive (MIC ≤ 2 mg/l) or resistant (MIC > 2 mg/l).

Sensitivity and specificity were calculated for the methods. The highest sensitivity (100 %) was noted for Superpolymyxin and Rapid polymyxin NP, and the lowest specificity (76 %) for broth disk elution. Considering the results, prevalence, durability, time and cost efficiency, it was concluded that Rapid polymyxin NP is the best alternative as a screening method of evaluated methods.

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10(4) CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.

We present a novel and simple technique for storing live Acanthamoeba for long periods of time. The amoebae are maintained at refrigerator temperatures in a peptone-yeast extract-glucose (PYG) medium normally used for cultivation. Using this method, we obtained survival rates of at least 4 years for Acanthamoeba polyphaga and 3 years for Acanthamoeba castellanii and Acanthamoeba rhysodes. Advantages of this storage method are: (1) it is quick and simple, (2) inexpensive, (3) does not require encystment before storage, (4) resuscitation of cysts can be achieved within a week of culture in PYG medium at 27A degrees C, and does not require co-culture with bacteria or any special equipment.

Background: Although Campylobacter jejuni is a frequent cause of bacterial gastroenteritis, one of the enigmas is how thisfragile organism can survive the transit through the acid milieu of the stomach. C. jejuni is very sensitive to low pH, but cansurvive in moderately acid environment for short periods of time. We have previously shown that C. jejuni can colonize andeven replicate in different species of amoebas, thereby gaining protection from adverse environments.

Objectives: We evaluated the effects of hydrochloric acid (HCl) on C. jejuni at various pH and time intervals, to study whetherco-cultivation with amoeba influenced C.jejuni acid tolerance. The setup was chosen to mimic the acidified milieu of the humangastrointestinal tract.

Methods: Cultures of C. jejuni (CCUG 11284) were co-cultured with Acanthamoeba polyphaga in either PBS or tap wateracidified with HCl to pH 1, 2, 3 and 4. We also evaluated different treatments effect on campylobacter survival, by exposingsome bacterial samples to an acid shock and some to a slower acidification process.

Results and conclusions: We show that C. jejuni can withstand pH below the normal range of survival, when co-cultured withA. polyphaga. C. jejuni co-cultured with amoebae survived acidified conditions at pH 3 for 20 hours and pH 2 for approximately5 hours. We also found a pH increase during the experiment, which correlated with campylobacter survival. These results pointto an unknown mechanism for C.jejuni to survive at low pH levels. This could be in the form of excretion of pH-increasingsubstances and simultaneous chemotaxic orientation towards a protective host. Our results could give one possible explanationto C. jejuni survival through the low pH of the gastrointestinal tract.

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.

Microorganisms that have a pH optimum for growth of less than pH 3 are termed "acidophiles". To grow at low pH, acidophiles must maintain a pH gradient of several pH units across the cellular membrane while producing ATP by the influx of protons through the F(0)F(1) ATPase. Recent advances in the biochemical analysis of acidophiles coupled to sequencing of several genomes have shed new insights into acidophile pH homeostatic mechanisms. Acidophiles seem to share distinctive structural and functional characteristics including a reversed membrane potential, highly impermeable cell membranes and a predominance of secondary transporters. Also, once protons enter the cytoplasm, methods are required to alleviate effects of a lowered internal pH. This review highlights recent insights regarding how acidophiles are able to survive and grow in these extreme conditions.

'Ferroplasma acidarmanus' strain Fer1 is an extremely acidophilic archaeon involved in the genesis of acid mine drainage, and was isolated from copper-contaminated mine solutions at Iron Mountain, CA, USA. Here, the initial proteomic and molecular investigation of Cu(2+) resistance in this archaeon is presented. Analysis of Cu(2+) toxicity via batch growth experiments and inhibition of oxygen uptake in the presence of ferrous iron demonstrated that Fer1 can grow and respire in the presence of 20 g Cu(2+) l(-1). The Fer1 copper resistance (cop) loci [originally detected by Ettema, T. J. G., Huynen, M. A., de Vos, W. M. & van der Oost, J. Trends Biochem Sci 28, 170-173 (2003)] include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB). Transcription analyses demonstrated that copZ and copB are co-transcribed, and transcript levels were increased significantly in response to exposure to high levels of Cu(2+), suggesting that the transport system is operating for copper efflux. Proteomic analysis of Fer1 cells exposed to Cu(2+) revealed the induction of stress proteins associated with protein folding and DNA repair (including RadA, thermosome and DnaK homologues), suggesting that 'Ferroplasma acidarmanus' Fer1 uses multiple mechanisms for resistance to high levels of copper.

'Ferroplasma acidarmanus' Fer1 is an arsenic-hypertolerant acidophilic archaeon isolated from the Iron Mountain mine, California; a site characterized by heavy metals contamination. The presence of up to 10 g arsenate per litre [As(V); 133 mM] did not significantly reduce growth yields, whereas between 5 and 10 g arsenite per litre [As(III); 67-133 mM] significantly reduced the yield. Previous bioinformatic analysis indicates that 'F. acidarmanus' Fer1 has only two predicted genes involved in arsenic resistance and lacks a recognizable gene for an arsenate reductase. Biochemical analysis suggests that 'F. acidarmanus' Fer1 does not reduce arsenate indicating that 'F. acidarmanus' Fer1 has an alternative resistance mechanism to arsenate other than reduction to arsenite and efflux. Primer extension analysis of the putative ars transcriptional regulator (arsR) and efflux pump (arsB) demonstrated that these genes are co-transcribed, and expressed in response to arsenite, but not arsenate. Two-dimensional polyacrylamide gel electrophoresis analysis of 'F. acidarmanus' Fer1 cells exposed to arsenite revealed enhanced expression of proteins associated with protein refolding, including the thermosome Group II HSP60 family chaperonin and HSP70 DnaK type heat shock proteins. This report represents the first molecular and proteomic study of arsenic resistance in an acidophilic archaeon.

'Ferroplasma acidarmanus' Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the 'F. acidarmanus' Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in 'F. acidarmanus' Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.

To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates-assumed to be related to autotrophic metabolisms-were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.

Bacterioplankton communities are made up of a small set of abundant taxa and a large number of low-abundant organisms (i.e. 'rare biosphere'). Despite the critical role played by bacteria in marine ecosystems, it remains unknown how this large diversity of organisms are affected by human-induced perturbations, or what controls the responsiveness of rare compared to abundant bacteria. We studied the response of a Mediterranean bacterioplankton community to two anthropogenic perturbations (i.e. nutrient enrichment and/or acidification) in two mesocosm experiments (in winter and summer). Nutrient enrichment increased the relative abundance of some operational taxonomic units (OTUs), e.g. Polaribacter, Tenacibaculum, Rhodobacteraceae and caused a relative decrease in others (e.g. Croceibacter). Interestingly, a synergistic effect of acidification and nutrient enrichment was observed on specific OTUs (e.g. SAR86). We analyzed the OTUs that became abundant at the end of the experiments and whether they belonged to the rare (<0.1% of relative abundance), the common (0.1-1.0% of relative abundance) or the abundant (>1% relative abundance) fractions. Most of the abundant OTUs at the end of the experiments were abundant, or at least common, in the original community of both experiments, suggesting that ecosystem alterations do not necessarily call for rare members to grow.

Despite important ecological roles posited for virocells (i.e., cells infected with viruses), studying individual cells in situ is technically challenging. We introduce here a novel correlative microscopic approach to study the ecophysiology of virocells. By conducting concerted virusFISH, 16S rRNA FISH, and scanning electron microscopy interrogations of uncultivated archaea, we linked morphologies of various altiarchaeal cells to corresponding phylogenetic signals and indigenous virus infections. While uninfected cells exhibited moderate separation between fluorescence signals of ribosomes and DNA, virocells displayed complete cellular segregation of chromosomal DNA from viral DNA, the latter co-localizing with host ribosome signals. A similar spatial separation was observed in dividing cells, with viral signals congregating near ribosomes at the septum. These observations suggest that replication of these uncultivated viruses occurs alongside host ribosomes, which are used to generate the required proteins for virion assembly. Heavily infected cells sometimes displayed virus-like particles attached to their surface, which agree with virus structures in cells observed via transmission electron microscopy. Consequently, this approach is the first to link genomes of uncultivated viruses to their respective structures and host cells. Our findings shed new light on the complex ecophysiology of archaeal virocells in deep subsurface biofilms and provide a solid framework for future in situ studies of virocells.