Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota

One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from environmental and human-associated bacteria. Yet, it is poorly understood to what extent and by what mechanisms these so-called reservoirs contribute to emerging resistance. Therefore, the work described in this thesis focussed on generating novel insights into different niches as sources of resistance, with a particular focus on the human gut microbiota as well as on microbial communities associated with marine sponges, especially because the latter have been described as one of the richest sources of bioactive secondary metabolites, including a broad range of antimicrobials. Cultivation-based methods were complemented with culture-independent approaches in order to study bacterial taxa that are not readily cultivated. Using metatranscriptomics it was found that clinically relevant antibiotic resistance genes are expressed in a broad range of environmental niches including human, mouse and pig gut microbiota, sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment. The diversity of resistance gene transcripts differed greatly per niche indicating that the environment contains a rich reservoir of functional resistance that could be accessible by pathogens. Even though resistance gene expression might be linked to the presence of natural antibiotics, we did not detect expression of the corresponding secondary metabolite biosynthesis clusters. Thirty-one antibiotic-resistant bacteria, amongst which three belonging to potentially novel Flavobacteriaceae spp., were isolated from the Mediterranean sponges Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis. Isolates were identified in a high throughput manner by double-barcoded 16S rRNA gene amplicon sequencing. Furthermore, analysis of sponge tissue-derived bacterial biomass growing on agar media showed that many novel bacterial taxa can still be isolated by conventional cultivation methods. Genomic DNA from the 31 antibiotic resistant bacteria was interrogated with respect to the presence of active resistance genes by functional metagenomics. In addition, we also screened metagenomic libraries prepared from DNA directly isolated from sponge tissue in order to circumvent the need for cultivation. In total, 37 unique resistance genes were identified, and the predicted gene products of 15 of these shared blaPSV-1), which was classified into a new β-lactamase family, was found to be exclusive to the marine specific genus Pseudovibrio. These findings raised questions as to the functional roles of these genes in sponges, but more importantly, the functionality of these genes in E. coli shows that they can potentially be harnessed by phylogenetically distinct bacteria in other environments, including human pathogens. As such, it is a wake-up call as to the significance of marine resistance reservoirs. Pseudovibrio, a genus of α-Proteobacteria, was studied in more detail by comparative genomics as it comprises bacteria that potentially play a role as sponge symbionts and marine hubs of antibiotics resistance. Based on gene content, members of the genus Pseudovibrio were found to cluster by sponge sampling location indicating geographic speciation. Furthermore, Pseudovibrio spp. isolated from sponges near the Spanish coast clustered by sponge, suggesting host-specific colonization or adaptation. Strong support for Pseudovibrio spp. forming symbiotic relations with sponges came from the presence of a plethora of (predicted) conserved symbiosis-related functions in their genomes. A final study aimed to isolate novel antibiotic resistant reservoir species from the human gut microbiota using a targeted approach. Faecal samples from hospitalized patients that received Selective Digestive Decontamination (SDD), a prophylactic treatment with a cocktail of different antibiotics (tobramycin, polymyxin E, amphotericin B and cefotaxime), were inoculated anaerobically on agar media, after which bacterial biomass was analysed by 16S rRNA gene amplicon sequencing. Six novel taxa were identified that, based on their growth on media supplemented with the SDD antibiotics, could serve as clinically relevant reservoirs of antibiotic resistance. For one of these six taxa a member was obtained in pure culture by targeted isolation. The abundance of antibiotic resistant uncultivated taxa in the human gut microbiota warrants further research as to their potential roles in resistance dissemination. In conclusion, this thesis provides deeper insights into different environmental niches as reservoirs of antibiotic resistance. The results can serve to prime and inspire future research.</p

Sponge Microbiota are a Reservoir of Functional Antibiotic Resistance Genes

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n=6), gentamicin (n=1), amikacin (n=7), trimethoprim (n=17), chloramphenicol (n=1), rifampicin (n=2) and ampicillin (n=3). Fifteen of 37 inserts harboured resistance genes that shared <90% amino acid identity with known gene products, whereas on 13 inserts no resistance gene could be identified with high confidence, in which case we predicted resistance to be mainly mediated by antibiotic efflux. One marine-specific ampicillin-resistance-conferring β-lactamase was identified in the genus Pseudovibrio with 41% global amino acid identity to the closest β-lactamase with demonstrated functionality, and subsequently classified into a new family termed PSV. Taken together, our results show that sponge microbiota host diverse and novel resistance genes that may be harnessed by phylogenetically distinct bacteria

Comparative genomics highlights symbiotic capacities and high metabolic flexibility of the marine genus Pseudovibrio

Pseudovibrio is a marine bacterial genus members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesised that Pseudovibrio spp. form mutualistic relationships with their hosts. Here, we studied Pseudovibrio phylogeny and genetic adaptations that may play a role in host colonization by comparative genomics of 31 Pseudovibrio strains, including 25 sponge isolates. All genomes were highly similar in terms of encoded core metabolic pathways, albeit with substantial differences in overall gene content. Based on gene composition, Pseudovibrio spp. clustered by geographic region, indicating geographic speciation. Furthermore, the fact that isolates from the Mediterranean Sea clustered by sponge species suggested host-specific adaptation or colonization. Genome analyses suggest that Pseudovibrio hongkongensis UST20140214-015BT is only distantly related to other Pseudovibrio spp., thereby challenging its status as typical Pseudovibrio member. All Pseudovibrio genomes were found to encode numerous proteins with SEL1 and tetratricopeptide repeats, which have been suggested to play a role in host colonization. For evasion of the host immune system, Pseudovibrio spp. may depend on type III, IV and VI secretion systems that can inject effector molecules into eukaryotic cells. Furthermore, Pseudovibrio genomes carry on average seven secondary metabolite biosynthesis clusters, reinforcing the role of Pseudovibrio spp. as potential producers of novel bioactive compounds. Tropodithietic acid, bacteriocin and terpene biosynthesis clusters were highly conserved within the genus, suggesting an essential role in survival e.g. through growth inhibition of bacterial competitors. Taken together, these results support the hypothesis that Pseudovibrio spp. have mutualistic relations with sponges

Biotechnological Potential of Bacteria Isolated from the Sea Cucumber Holothuria leucospilota and Stichopus vastus from Lampung, Indonesia

In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS1) as well as product/daughter ions (MS2) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential

A review of machine learning applications for the proton MR spectroscopy workflow

This literature review presents a comprehensive overview of machine learning (ML) applications in proton MR spectroscopy (MRS). As the use of ML techniques in MRS continues to grow, this review aims to provide the MRS community with a structured overview of the state-of-the-art methods. Specifically, we examine and summarize studies published between 2017 and 2023 from major journals in the MR field. We categorize these studies based on a typical MRS workflow, including data acquisition, processing, analysis, and artificial data generation. Our review reveals that ML in MRS is still in its early stages, with a primary focus on processing and analysis techniques, and less attention given to data acquisition. We also found that many studies use similar model architectures, with little comparison to alternative architectures. Additionally, the generation of artificial data is a crucial topic, with no consistent method for its generation. Furthermore, many studies demonstrate that artificial data suffers from generalization issues when tested on in vivo data. We also conclude that risks related to ML models should be addressed, particularly for clinical applications. Therefore, output uncertainty measures and model biases are critical to investigate. Nonetheless, the rapid development of ML in MRS and the promising results from the reviewed studies justify further research in this field.</p

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included beta-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.Peer reviewe

Repeated Quantum Error Detection in a Surface Code

The realization of quantum error correction is an essential ingredient for reaching the full potential of fault-tolerant universal quantum computation. Using a range of different schemes, logical qubits can be redundantly encoded in a set of physical qubits. One such scalable approach is based on the surface code. Here we experimentally implement its smallest viable instance, capable of repeatedly detecting any single error using seven superconducting qubits, four data qubits and three ancilla qubits. Using high-fidelity ancilla-based stabilizer measurements we initialize the cardinal states of the encoded logical qubit with an average logical fidelity of 96.1%. We then repeatedly check for errors using the stabilizer readout and observe that the logical quantum state is preserved with a lifetime and coherence time longer than those of any of the constituent qubits when no errors are detected. Our demonstration of error detection with its resulting enhancement of the conditioned logical qubit coherence times in a 7-qubit surface code is an important step indicating a promising route towards the realization of quantum error correction in the surface code.Comment: 12 pages, 11 figure

Mono- and multispecies biofilms from a crustose coralline alga induce settlement in the scleractinian coral Leptastrea purpurea

Microorganisms have been reported to induce settlement in various marine invertebrate larvae but their specificity of inductive capacities for the settlement of coral larvae remains poorly understood. In this study, we isolated 56 microbial strains from the crustose coralline alga (CCA) Hydrolithon reinboldii using five different media either with or without additional antibiotics and/or spiked CCA extract. We tested the isolates for their potential to induce settlement behavior in larvae of the brooding scleractinian coral Leptastrea purpurea. From these 56 CCA-associated microbial strains, we identified six bacterial classes and 18 families. The culturable bacterial community associated with H. reinboldii was dominated by Gammaproteobacteria, Actinobacteria, and Alphaproteobacteria while the Illumina MiSeq analysis showed that the culture-independent bacterial community was dominated by Gammaproteobacteria, Alphaproteobacteria, and Flavobacteria. Furthermore, we found no correlation between inductive settlement capacities and phylogenetic relationships. Instead, settlement behavior of L. purpurea larvae was induced by specific isolated species. Strains #1792 (Pseudovibrio denitrificans), #1678 (Acinetobacter pittii), #1633 (Pseudoalteromonas phenolica), #1772 (Marine bacterium LMG1), #1721 (Microbulbifer variabilis), and #1783 (Pseudoalteromonas rubra) induced settlement behavior in coral larvae at mostly high and significant levels (>= 40%) but the remaining isolates strongly varied in their activity. Multispecies biofilms consisting of four strains (#1792, #1678, #1633, and #1721) were observed to synergistically increase settlement behavior levels (> 90%); however, the addition of #1772 to the multispecies biofilms negatively affected coral larvae and resulted in a total loss of inducing activity. The findings provide new insights into the role of bacteria in the settlement process of scleractinian corals and may help to identify the true nature of bacteria-derived morphogenic cues