Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Groundwater is essential for human activities and is sometimes referred to as a non-renewable resource in the same way as oil and gas. During the past century, precious groundwater reserves worldwide have been threatened by anthropogenic release of chemical compounds. Among these, chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) belong to the most common class of groundwater contaminants. In oxic conditions, PCE and TCE are recalcitrant to any form of degradation and constitute a long-term source of groundwater contamination. It has been reported that CEs can be degraded biologically in anoxic conditions, serving as electron acceptors of an anaerobic respiratory process called organohalide respiration (OHR). In this process, PCE is sequentially reduced to TCE, cis-dichloroethene (cDCE), vinyl chloride (VC), and finally to harmless ethene (Eth). Engineering strategies based on reductive dechlorination, such as monitored natural attenuation (MNA), have been designed for the bioremediation of aquifers contaminated with CEs. However, stalling of the sequence of dechlorination has often been observed, resulting from incomplete or impeded biodegradation of the highly toxic daughter molecules (cDCE and VC) and leading to their accumulation in situ. It was shown in laboratory experiments that OHR of CEs is more efficient in mixed cultures and that organohalide-respiring bacteria (OHRB) live in association with other microorganisms in microbial consortia. In the aquifer ecosystem, OHRB rely on complex interactions between members of bacterial communities for their electron donors and carbon supplies. On the other hand, they are in competition for these resources with other terminal electron-accepting processes (TEAPs). These complex interactions together with the intrinsic structural heterogeneity of aquifers make it difficult to understand the reasons for lower CEs accumulation, to predict the fate of OHR, and to design bioremediation strategies. This thesis aimed at elucidating the reasons for lower CEs accumulation in situ. An ecological approach considering the aquifer ecosystem in its whole complexity was developed and applied to the specific cases of two contaminated sites showing accumulation of lower CEs. The proposed methodology relies on the precise depiction of both aquifer habitat and microbial communities interacting therein. However, the potential impact of the first technical step, namely the impact of the pumping parameters on groundwater samples used for the description of the bacterial communities, was not known at the start of this thesis work. Results of investigations addressing this topic showed that parameters related to the tubing characteristics did not impact the apparent bacterial community structures (BCS) in a laboratory experiment. However, the study revealed a significant impact of the pumping flow rate on apparent BCS extracted from the aquifer. Terminal-restriction fragment length polymorphism (T-RFLP), coupled with an appropriate groundwater sampling strategy, enabled an accurate profiling of the BCS. However no indication was provided concerning the identities of the community members. A bioinformatics tool called PyroTRF-ID was developed to overcome this obstacle. The software enabled affiliating terminal-restriction fragments (T-RFs) to precise phylotypes by coupling T-RFLP and pyrosequencing data. An additional function enables in silico assessment of restriction enzymes for designing appropriate T-RFLP procedures. The developed methodology was applied all along the thesis work. Optimized tools and procedures were used to investigate the reasons for the accumulation of VC, and to a lesser extent cDCE, in the first aquifer showing relatively homogeneous lithological composition and slow groundwater fluxes. Statistical findings indicated that VC reduction was outcompeted by Fe(III) reduction in some sections of the aquifer. T-RFs showing significant correlations with VC reduction variables were identified by sequencing and with PyroTRF-ID. Results showed sequences closely affiliated to uncultured bacteria of the "Lahn Cluster" (LC) within the class Dehalococcoidetes, previously reported as PCE-to-cDCE dechlorinating microorganisms, and to the genus "Dehalococcoides". According to present knowledge, only members of this genus are able to reduce cDCE and VC. A major T-RF negatively correlated with the LC T-RF affiliated to the genus Rhodoferax, containing iron-reducing bacteria (IRB). Furthermore, indications were obtained of a mutualistic interaction between IRB and sulfate-reducing bacteria, potentially playing an important role by reducing the Fe(III) contents locally. The importance of the aquifer hydrogeological structure and functioning was exemplified in the study of the second aquifer ecosystem, where discrepancies in the fate of OHR were observed. In the immediate vicinity of the source zone, complete OHR was occurring, with production of Eth, whereas cDCE was accumulating further downstream, in apparent similar redox conditions. The profiling and metagenomic techniques revealed drastically different bacterial communities in the zones displaying different OHR fate. The source zone displayed bacterial populations typically found in highly reduced environments and involved in OHR, such as Dehalococcoides sp. In contrast, the downstream zone showing an accumulation of cDCE displayed bacterial populations typically found in more oxidized environments, such as aerobic bacteria, nitrifiers, and denitrifiers. In this zone, the conditions were probably slightly oxidized periodically by the combination of aquifer recharge, a specific lithological configuration, and lower organic matter content. Finally, and based on these considerations, a methodology for the investigation of the reasons for lower CEs accumulation in contaminated aquifers is proposed and discussed. This methodology follows on a multidisciplinary ecological approach for the description of the functioning of the aquifer ecosystem, and enables formulating scenarii of the reasons for lower CEs accumulation

Linking bacterial population dynamics and nutrient removal in the granular sludge biofilm ecosystem engineered for wastewater treatment

Intensive nutrient removal from wastewater in anaerobic-aerobic systems using granular sludge should rely on optimal balances at biofilm and microbial ecology levels. This study targets the impacts of reactor characteristics and fluctuations in operation conditions on nutrient removal and bacterial community structures by means of microbial and numerical ecology methods. The dynamics of both predominant and accompanying populations were investigated with high resolution on temporal and phylogenetic scales in two reactors operated during 5 months with synthetic wastewater. Multivariate analyses highlighted significant correlations from process to microbial scales in the first reactor, whereas nitrification and phosphorus removal might have been affected by oxygen mass transfer limitations with no impact at population level in the second system. The bacterial community continuum of the first reactor was composed of two major antagonistic Accumulibacter-Nitrosomonas-Nitrospira and Competibacter-Cytophaga-Intrasporangiaceae clusters that prevailed under conditions leading to efficient P- (> 95%) and N-removal (> 65%) and altered P- (< 90%) and N-removal (< 60%), respectively. A third cluster independent of performances was dominated by Xanthomonadaceae affiliates that were on average more abundant at 25 °C (31 ± 5%) than at 20 °C (22 ± 4%). Starting from the physiological traits of the numerous phylotypes identified, a conceptual model is proposed as a base for functional analysis in the granular sludge microbiome and for future investigations with complex real wastewate

OpenGenomeBrowser: a versatile, dataset-independent and scalable web platform for genome data management and comparative genomics.

BACKGROUND As the amount of genomic data continues to grow, there is an increasing need for systematic ways to organize, explore, compare, analyze and share this data. Despite this, there is a lack of suitable platforms to meet this need. RESULTS OpenGenomeBrowser is a self-hostable, open-source platform to manage access to genomic data and drastically simplifying comparative genomics analyses. It enables users to interactively generate phylogenetic trees, compare gene loci, browse biochemical pathways, perform gene trait matching, create dot plots, execute BLAST searches, and access the data. It features a flexible user management system, and its modular folder structure enables the organization of genomic data and metadata, and to automate analyses. We tested OpenGenomeBrowser with bacterial, archaeal and yeast genomes. We provide a docker container to make installation and hosting simple. The source code, documentation, tutorials for OpenGenomeBrowser are available at opengenomebrowser.github.io and a demo server is freely accessible at opengenomebrowser.bioinformatics.unibe.ch . CONCLUSIONS To our knowledge, OpenGenomeBrowser is the first self-hostable, database-independent comparative genome browser. It drastically simplifies commonly used bioinformatics workflows and enables convenient as well as fast data exploration

Linking bacterial population dynamics and nutrient removal in the granular sludge biofilm ecosystem engineered for wastewater treatment

Intensive nutrient removal from wastewater in anaerobic–aerobic systems using granular sludge should rely on optimal balances at biofilm and microbial ecology levels. This study targets the impacts of reactor characteristics and fluctuations in operation conditions on nutrient removal and bacterial community structures by means of microbial and numerical ecology methods. The dynamics of both predominant and accompanying populations were investigated with high resolution on temporal and phylogenetic scales in two reactors operated during 5 months with synthetic wastewater. Multivariate analyses highlighted significant correlations from process to microbial scales in the first reactor, whereas nitrification and phosphorus removal might have been affected by oxygen mass transfer limitations with no impact at population level in the second system. The bacterial community continuum of the first reactor was composed of two major antagonistic Accumulibacter-Nitrosomonas-Nitrospira and Competibacter-Cytophaga-Intrasporangiaceae clusters that prevailed under conditions leading to efficient P- (> 95%) and N-removal (> 65%) and altered P- (< 90%) and N-removal (< 60%), respectively. A third cluster independent of performances was dominated by Xanthomonadaceae affiliates that were on average more abundant at 25 °C (31 ± 5%) than at 20 °C (22 ± 4%). Starting from the physiological traits of the numerous phylotypes identified, a conceptual model is proposed as a base for functional analysis in the granular sludge microbiome and for future investigations with complex real wastewater

Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either ‘sensitive’ or ‘resistant’ based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains’ genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing

Fn14•Trail effectively inhibits hepatocellular carcinoma growth.

BACKGROUND: New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL\u27s capacity to inhibit HCC growth was tested. RESULTS: Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL\u27s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice. CONCLUSIONS: In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein\u27s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies

Inflammatory Activation of Astrocytes Facilitates Melanoma Brain Tropism via the CXCL10-CXCR3 Signaling Axis

Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis

Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either &lsquo;sensitive&rsquo; or &lsquo;resistant&rsquo; based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains&rsquo; genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing

BACTERIAL COMMUNITIES IN A CONTAMINATED AQUIFER: INFLUENCE OF ENVIRONMENTAL VARIABLES

Numerous industries have been using chlorinated ethenes (CEs), mainly tetrachlorethene and trichloroethene, as non-flammable solvents since the beginning of the 20th century. Massive usage, along with careless handling and storage, made CEs one of the most abundant classes of aquifer contaminants. Porous aquifers are dynamic ecosystems showing complex interactions between physical, chemical and biotic components. These environments are inherently extremely heterogeneous in their structure and composition in terms of lithology, grain size distribution and chemical nature, and thus provide a large variety of living conditions. A possible approach for studying reductive dehalogenation processes in such environments consists in the analysis of the correlations between bacterial communities and environmental variables. Among the latter, the grain size distribution is expected to have a major impact on the structuring and functioning of the communities. In this study, this hypothesis was tested on samples issued from a CE-contaminated aquifer in which the dehalorespiration process was effectively occurring. Previous studies showed that this aquifer was semi-confined and had a slow groundwater flow. The bacterial communities were analyzed at different locations on this site using T-RFLP profiling, together with the analysis of multiple environmental factors. The analysis of the correlations between environmental factors and T-RFLP profiles surprisingly revealed that grain size distribution had only a limited influence. This unexpected result led us to hypothesize that the possible impact of the grain size distribution was decreased due to the slow groundwater flow. Diffusion phenomena would then be the principal mechanism of mass transfer of nutrients to specialized bacterial micro-niches