Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

New Endohyphal Relationships between Mucoromycota and Burkholderiaceae Representatives.

Mucoromycota representatives are known to harbor two types of endohyphal bacteria (EHB)-Burkholderia-related endobacteria (BRE) and Mycoplasma-related endobacteria (MRE). While both BRE and MRE occur in fungi representing all subphyla of Mucoromycota, their distribution is not well studied. Therefore, it is difficult to resolve the evolutionary history of these associations in favor of one of the following two alternative hypotheses explaining their origin: "early invasion" and "late invasion." Our main goal was to fill this knowledge gap by surveying Mucoromycota fungi for the presence of EHB. We screened 196 fungal strains from 16 genera using a PCR-based approach to detect bacterial 16S rRNA genes, complemented with fluorescence in situ hybridization (FISH) imaging to confirm the presence of bacteria within the hyphae. We detected Burkholderiaceae in ca. 20% of fungal strains. Some of these bacteria clustered phylogenetically with previously described BRE clades, whereas others grouped with free-living Paraburkholderia Importantly, the latter were detected in Umbelopsidales, which previously were not known to harbor endobacteria. Our results suggest that this group of EHB is recruited from the environment, supporting the late invasion scenario. This pattern complements the early invasion scenario apparent in the BRE clade of EHB.IMPORTANCE Bacteria living within fungal hyphae present an example of one of the most intimate relationships between fungi and bacteria. Even though there are several well-described examples of such partnerships, their prevalence within the fungal kingdom remains unknown. Our study focused on early divergent terrestrial fungi in the phylum Mucoromycota. We found that ca. 20% of the strains tested harbored bacteria from the family Burkholderiaceae Not only did we confirm the presence of bacteria from previously described endosymbiont clades, we also identified a new group of endohyphal Burkholderiaceae representing the genus Paraburkholderia We established that more than half of the screened Umbelopsis strains were positive for bacteria from this new group. We also determined that, while previously described BRE codiverged with their fungal hosts, Paraburkholderia symbionts did not.

Revealing antimicrobial resistance in stormwater with MinION.

Discharge of urban stormwater containing organic matter, heavy metals and sometime human feces, to the natural aquatic reservoirs without any treatment is not only an environmental problem. It can lead to prevalence of antibiotic resistant bacteria in stormwater systems and transmission of antibiotic resistance genes to the environment. We performed antibiotic resistome identification and virus detection in stormwater samples from Stockholm, using publicly available metagenomic sequencing MinION data. A MinION platform offers low-cost, precise environmental metagenomics analysis. 37 groups of antibiotic resistant bacteria (ARB), 11 resistance types with 26 resistance mechanisms - antibiotic resistance genes (ARGs) giving tolerance to the aminoglycoside, beta-lactams, fosmidomycin, MLS, multidrug and vancomycin were identified using ARGpore pipeline. The majority of the identified bacteria species were related to the natural environment such as soil and were not dangerous to human. Alarmingly, human pathogenic bacteria carrying resistance to antibiotics currently used against them (Bordetella resistant to macrolides and multidrug resistant Propionibacterium avidum) were also found in the samples. Most abundant viruses identified belonged to Caudovirales and Herpesvirales and they were not carrying ARGs. Unlike the virome, resistome and ARB were not unique for stormwater sampling points. This results underline the need for extensive monitoring of the microbial community structure in the urban stormwater systems to assess antimicrobial resistance spread.

The morphology and metabolic potential of the Chloroflexi in full-scale activated sludge wastewater treatment plants.

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in the operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of Danish wastewater treatment plants (WWTPs) with nutrient removal were screened to identify numerically important Chloroflexi genera. Fluorescence in situ hybridization probes were designed for their in situ characterization. All abundant Chloroflexi phylotypes were putatively identified as facultative anaerobic chemoorganotrophs involved in sugar fermentation. They were all filamentous but differed in their morphology and spatial arrangement. 'Candidatus Villigracilis' was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of 'Candidatus Amarolinea' was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important systems.

Factors affecting population of filamentous bacteria in wastewater treatment plants with nutrients removal.

Filamentous population in activated sludge and key operational parameters of full-scale municipal wastewater treatment plants (WWTPs) with bulking problems representative for Poland were investigated with quantitative fluorescence in situ hybridization. Statistical analyses revealed few relationships between operational parameters and biovolume of filamentous bacteria. Sludge age was not only positively correlated with abundance of Chloroflexi (parametric correlation and principal component analysis (PCA)), but also differentiated Microthrix population (analysis of variance (ANOVA)). Phylum Chloroflexi and pH presented a negative relation during the study (PCA). ANOVA showed that pH of influent and sludge volume index (SVI) differentiated abundance of types 0803 and 1851 of Chloroflexi and candidate division TM7. SVI increased along with higher abundance of Microthrix (positive parametric and non-parametric correlations and positive relation in PCA). Biovolumes of morphotypes 0803 and 1851 of Chloroflexi were differentiated by organic matter in influent, also by nutrients in the case of Chloroflexi type 1851. Chemical and biological oxygen demands (COD and BOD5, respectively) were negatively correlated with Microthrix. COD also differentiated the abundance of Haliscomenobacter hydrossis. Results of the study can be used to prevent WWTPs from excessive proliferation of filamentous bacteria and operational problems caused by them--bulking and foaming of activated sludge.