Ofélia Godinho

Background/Objectives: It is of the utmost importance to study environmental bacteria, as these microorganisms remain poorly characterized regarding their diversity, antimicrobial resistance, and impact on the global ecosystem. This knowledge gap is particularly pronounced for marine bacteria. In this study, we aimed to isolate bacteria from different marine samples and to gain insights into the environmental bacterial resistome, an aspect that remains largely neglected. Methods: Bacteria were isolated from several marine sources using two different culture media, and their identification was based on 16S rRNA gene analysis. Whole-genome sequencing was performed for selected isolates belonging to novel taxa. Antimicrobial susceptibility to seven antibiotics was evaluated using the disk diffusion method. Results: A total of 171 bacterial isolates belonging to the phyla Pseudomonadota, Bacteroidota, Planctomycetota, Actinomycetota, and Bacillota were obtained from diverse marine samples. The most abundant group belonged to the class Alphaproteobacteria. Thirty isolates represented novel taxa, comprising 16 new species and one new genus. Despite the challenges associated with determining antibiotic resistance profiles in environmental bacteria, only one isolate (1.8%) was pan-susceptible, whereas 54 (98.2%) showed resistance to at least one of the tested antibiotics. Moreover, 33 isolates exhibited a multidrug-resistant phenotype. Genome analysis of four novel taxa revealed the presence of an incomplete AdeFGH efflux pump. Conclusions: This study highlights the high bacterial diversity in marine environments, the striking prevalence of antibiotic resistance, and the major methodological challenges in studying environmental bacteria. Importantly, it emphasizes the relevance of culturomics-based approaches for uncovering hidden microbial diversity and characterizing environmental resistomes.

Background/Objectives: It is of upmost importance to study environmental bacteria, as these microorganisms remain poorly characterized regarding their diversity, antimicrobial resistance, and impact on the global ecosystem. This knowledge gap is particularly pronounced for marine bacteria. In this study, we aimed to isolate marine bacteria from different sources and to gain insights into the environmental bacterial resistome, an aspect that remains largely neglected. Methods: Bacteria were isolated from several marine sources using two different culture media, and their identification was based on 16S rRNA gene analysis. Whole-genome sequencing was performed for selected isolates belonging to novel taxa. Antimicrobial susceptibility to seven antibiotics was evaluated using the disk diffusion method. Results: A total of 171 bacterial isolates belonging to the phyla Pseudomonadota, Bacteroidota, Planctomycetota, Actinomycetota, and Bacillota were obtained from diverse marine samples. The most abundant group belonged to the class Alphaproteobacteria. Thirty isolates represented novel taxa, comprising 16 new species and one new genus. Despite the challenges associated with determining antibiotic resistance profiles in environmental bacteria, only one isolate (1.8%) was pan-susceptible, whereas 54 (98.2%) showed resistance to at least one of the tested antibiotics. Moreover, 33 isolates exhibited a multidrug-resistant phenotype. Genome analysis of four novel taxa revealed the presence of an incomplete AdeFGH efflux pump. Conclusions: This study highlights the high bacterial diversity in marine environments, the striking prevalence of antibiotic resistance, and the major methodological challenges in studying environmental bacteria. Importantly, it emphasizes the relevance of culturomics-based approaches for uncovering hidden microbial diversity and characterizing environmental resistomes.

One hundred years after planctomycetes were discovered and 50 years since the first isolate was successfully cultured, this bacterial phylum remains enigmatic in many ways. In the last few decades, a significant effort to characterize new isolates has resulted in >150 described species, allowing a more comprehensive analysis of their features. However, metagenomic studies reveal that a diverse group of planctomycetes has yet to be cultured and characterized, and that many biological surprises are yet to be revealed. This is the case for the recently discovered phagotrophic Candidatus Uabimicrobium, which challenges our understanding of the distinction between prokaryotes and eukaryotes. The unique biology of planctomycete cells, such as their ability to divide without the FtsZ protein, their complex structure and characteristic morphology, their relatively large genomes containing many genes with unknown function, and their variable metabolic capabilities, imposes significant barriers for researchers. Although ubiquitous, the precise ecological roles of planctomycetes in various environments are still not fully understood. However, their distinctive metabolism opens the door to a large number of potential biotechnological applications, which are beginning to be unveiled. In this article, we first review the historical milestones in planctomycetes research and describe the pioneers of the field. We then describe the controversies and their resolutions, we highlight the past discoveries and current interrogations related to planctomycetes, and discuss the ongoing challenges that hinder a comprehensive understanding of their biology. We end up with directions for exploring the biology and ecological roles of these fascinating organisms.

Antimicrobial resistance is presently one of the leading causes of death worldwide. The surveillance of different environments, namely, wastewater treatment plants (WWTPs), as hotspots of antibiotic-resistant bacteria, has become crucial under the One Health approach. This study aimed to characterize, phenotypically and genotypically, antibiotic-resistant bacteria along a WWTP receiving domestic and industrial sewage. Four sampling sites, representing distinct treatment points of the WWTP, were selected for sampling bacterial isolation in selective media supplemented, or not, with antibiotics, and subsequent antimicrobial susceptibility testing. Antibiotic resistance encoding genes were screened by molecular methods. A total of 50 bacterial isolates were obtained, 50% of which were affiliated with the genus Enterococcus. The antimicrobial susceptibility testing revealed antibiotic phenotypic resistance in isolates obtained from all the four treatment points of the wastewater samples, with resistance to tetracycline (32.5%) and ampicillin (25%) being the most common. Three isolates were found to be multidrug resistant and were affiliated with the genera Citrobacter, Shigella and Klebsiella. Molecular screening revealed the presence of tet(M), bla_TEM, bla_SHV and bla_CTX-M, as well as class 1 integrons carrying dfrA25, ANT(3″)-IIa and aadA6 genes. This study highlights the relevance of bacterial isolation and their antimicrobial susceptibility evaluation in WWTP systems since antibiotic-resistant strains were found from the raw influent to the final effluent discharged into the environment, denoting the need for surveillance and containment measures.

Background: Planctomycetota isolation in pure culture is still challenging with most of the reported data coming from molecular-based methods. Here, we intended to isolate Planctomycetota from the filter-feeder Pacific oyster Magallana gigas, extending the search to a not yet explored natural reservoir and to characterize their antimicrobial resistance phenotype. Methods: Oyster samples from different supermarkets and from a farm producer were subject to isolation in selective medium. Inoculation was performed from the shell biofilm and after an enrichment of the edible content. Results: Planctomycetota isolates (n = 65) were only obtained from the shell biofilm with four different species identified: Rhodopirellula baltica (n = 62), Rhodopirellula rubra (n = 1), Rhodopirellula heiligendammensis (n = 1) and Gimesia chilikensis (n = 1). This study reports the first association of Planctomycetota members with oysters and the first description of R. heiligendammensis in Portugal. Moreover, R. rubra, originally identified in Portugal, was isolated from oysters of French origin. Antibiotic susceptibility testing, conducted in strains belonging to two species never assayed before revealed multidrug resistance phenotypes with bacteria showing resistance to several classes of clinically relevant antibiotics (e.g., β-lactams and aminoglycosides). Conclusion: The ecological role and impact of Planctomycetota on oyster holobiont and, ultimately, in public health, under the One Health concept, is discussed.

A bacterial strain was isolated from a brackish water sample of Tagus river, Alcochete, Portugal and was designated TO1_6^T. It forms light pink colonies on M13 medium supplemented with N-acetylglucosamine. Cells are pear-shaped to spherical, form rosettes and divide by budding. Strain TO1_6^T presents a mesophilic and neutrophilic profile, with optimum growth at 20 to 25 °C and pH 7.0 to 7.5, and vitamin supplementation is not required to promote its growth. The genome of the novel isolate is 7.77 Mbp in size and has a DNA G + C content of 56.3%. Based on its 16S rRNA gene sequence, this strain is affiliated with the phylum Planctomycetota. Further taxonomic characterization using additional phylogenetic markers, namely rpoB gene sequence (encoding the β-subunit of the DNA-dependent RNA polymerase), as well as Percentage of conserved proteins, average nucleotide identity and average amino acid identity, suggest the affiliation of strain TO1_6^T to the genus Stieleria, a recently described taxon in the family Pirellulaceae, order Pirellulales and class Planctomycetia. Based on the genotypic, phylogenetic and physiological characterization, we here describe a new species represented by the type strain TO1_6^T (= CECT 30432^T, = LMG 32465^T), for which the name Stieleria tagensis sp. nov. is proposed.

Bacteria from the distinctive Planctomycetes phylum are well spread around the globe; they are capable of colonizing many habitats, including marine, freshwater, terrestrial, and even extreme habitats such as hydrothermal vents and hot springs. They can also be found living in association with other organisms, such as macroalgae, plants, and invertebrates. While ubiquitous, only a small fraction of the known diversity includes axenic cultures. In this study, we aimed to apply conventional techniques to isolate, in diverse culture media, planctomycetes from two beaches of the Portuguese north-coast by using sediments, red, green, and brown macroalgae, the shell of the mussel Mytilus edulis, an anemone belonging to the species Actinia equina, and seawater as sources. With this approach, thirty-seven isolates closely related to seven species from the families Planctomycetaceae and Pirellulaceae (class Planctomycetia) were brought into pure culture. Moreover, we applied an iChip inspired in-situ culturing technique to successfully retrieve planctomycetes from marine sediments, which resulted in the isolation of three additional strains, two affiliated to the species Novipirellula caenicola and one to a putative novel Rubinisphaera. This work enlarges the number of isolated planctomycetal strains and shows the adequacy of a novel methodology for planctomycetes isolation.