The diversity of the antimicrobial resistome of lake Tanganyika increases with the water depth.

The presence of antimicrobial resistance genes (ARGs) in the microbiome of freshwater communities is a consequence of thousands of years of evolution but also of the pressure exerted by anthropogenic activities, with potential negative impact on environmental and human health. In this study, we investigated the distribution of ARGs in Lake Tanganyika (LT)'s water column to define the resistome of this ancient lake. Additionally, we compared the resistome of LT with that of Lake Baikal (LB), the oldest known lake with different environmental characteristics and a lower anthropogenic pollution than LT. We found that richness and abundance of several antimicrobial resistance classes were higher in the deep water layers in both lakes. LT Kigoma region, known for its higher anthropogenic pollution, showed a greater richness and number of ARG positive MAGs compared to Mahale. Our results provide a comprehensive understanding of the antimicrobial resistome of LT and underscore its importance as reservoir of antimicrobial resistance. In particular, the deepest water layers of LT are the main repository of diverse ARGs, mirroring what was observed in LB and in other aquatic ecosystems. These findings suggest that the deep waters might play a crucial role in the preservation of ARGs in aquatic ecosystems.

Nutrient optimization in bioleaching: are we overdosing?

The general trend in biomining (i.e., bioleaching and biooxidation) is the use of media with high concentrations of the nutrients (nitrogen as ammonium, phosphorous as phosphate, and K), which are considered to be essential for microbial growth. The depletion of any of the nutrients would affect negatively the bioleaching (and biooxidation) capacity of the microorganisms, so the formulation of the different media ensures that there is a surplus of nutrients. However, some of these nutrients (e.g., phosphate, K) may be already present in the ore and are made available to the microorganisms when the ore is exposed to the low-pH media used during bioleaching. The effect of phosphate addition (109 mg/L) and depletion on the bioleaching of low-grade sulfidic ore alongside the determination of ammonium (i.e., 25 mg/L, 50 mg/L, 109 mg/L, 409 mg/L, and 874 g/L) requirements were studied. The results of the experiments presented showed that the addition of phosphate did not have any effect on the bioleaching of the low-grade sulfidic ore while the addition of ammonium was necessary to obtain higher redox potentials (>650 mV vs. Ag/AgCl) and higher metal (Co, Cu, Ni, and Zn) dissolutions. Temperature was the factor that shaped the microbial communities, at 30°C, the microbial community at the end of all the experiments was dominated by Acidithiobacillus sp. as well as at 42°C, except when nutrients were not added and Sulfobacillus sp. was the dominant microorganism. At 55°C, DNA recovery was unsuccessful, and at 60°C, the microbial communities were dominated by Sulfolobus sp. In conclusion, the amount of nutrients in bioleaching could be reduced significantly to achieve the redox potentials and metal dissolution desired in bioleaching without affecting the microbial communities and bioleaching efficiencies.

Treated wastewater: A hotspot for multidrug- and colistin-resistant Klebsiella pneumoniae.

Wastewater treatment plants are hotspots for the release of antimicrobial resistant pathogenic bacteria into aquatic ecosystems, significantly contributing to the cycle of antimicrobial resistance. Special attention should be paid to antimicrobial resistant ESKAPE bacteria, which have been identified as high-priority targets for control measures. Among them, Klebsiella pneumoniae is particularly noteworthy. In this study, we collected wastewater samples from the inlet, sedimentation tank, and effluent water of a wastewater treatment plant in June, July, October, and November of 2018. We detected and characterized 42 K. pneumoniae strains using whole genome sequencing (15 from the inlet, 8 from the sedimentation tank, and 19 from the effluent). Additionally, the strains were tested for their antimicrobial resistance phenotype. Using whole genome sequencing no distinct patterns were observed in terms of their genetic profiles. All strains were resistant to tetracycline, meanwhile 60%, 47%, and 37.5% of strains isolated from the inlet, sedimentation tank, and effluent, respectively, were multidrug resistant. Some of the multidrug resistant isolates were also resistant to colistin, and nearly all tested positive for the eptB and arnT genes, which are associated with polymyxin resistance. Various antimicrobial resistance genes were linked to mobile genetic elements, and they did not correlate with detected virulence groups or defense systems. Overall, our results, although not quantitative, highlight that multidrug resistant K. pneumoniae strains, including those resistant to colistin and genetically unrelated, being discharged into aquatic ecosystems from wastewater treatment plants. This suggests the necessity of monitoring aimed at genetically characterizing these pathogenic bacteria.

Parity in bacterial communities and resistomes: Microplastic and natural organic particles in the Tyrrhenian Sea.

Petroleum-based microplastic particles (MPs) are carriers of antimicrobial resistance genes (ARGs) in aquatic environments, influencing the selection and spread of antimicrobial resistance. This research characterized MP and natural organic particle (NOP) bacterial communities and resistomes in the Tyrrhenian Sea, a region impacted by plastic pollution and climate change. MP and NOP bacterial communities were similar but different from the free-living planktonic communities. Likewise, MP and NOP ARG abundances were similar but different (higher) from the planktonic communities. MP and NOP metagenome-assembled genomes contained ARGs associated with mobile genetic elements and exhibited co-occurrence with metal resistance genes. Overall, these findings show that MPs and NOPs harbor potential pathogenic and antimicrobial resistant bacteria, which can aid in the spread of antimicrobial resistance. Further, petroleum-based MPs do not represent novel ecological niches for allochthonous bacteria; rather, they synergize with NOPs, collectively facilitating the spread of antimicrobial resistance in marine ecosystems.

Anthropogenic pollution may enhance natural transformation in water, favouring the spread of antibiotic resistance genes.

Aquatic ecosystems are crucial in the antimicrobial resistance cycle. While intracellular DNA has been extensively studied to understand human activity's impact on antimicrobial resistance gene (ARG) dissemination, extracellular DNA is frequently overlooked. This study examines the effect of anthropogenic water pollution on microbial community diversity, the resistome, and ARG dissemination. We analyzed intracellular and extracellular DNA from wastewater treatment plant effluents and lake surface water by shotgun sequencing. We also conducted experiments to evaluate anthropogenic pollution's effect on transforming extracellular DNA (using Gfp-plasmids carrying ARGs) within a natural microbial community. Chemical analysis showed treated wastewater had higher anthropogenic pollution-related parameters than lake water. The richness of microbial community, antimicrobial resistome, and high-risk ARGs was greater in treated wastewaters than in lake waters both for intracellular and extracellular DNA. Except for the high-risk ARGs, richness was significantly higher in intracellular than in extracellular DNA. Several ARGs were associated with mobile genetic elements and located on plasmids. Furthermore, Gfp-plasmid transformation within a natural microbial community was enhanced by anthropogenic pollution levels. Our findings underscore anthropogenic pollution's pivotal role in shaping microbial communities and their antimicrobial resistome. Additionally, it may facilitate ARG dissemination through extracellular DNA plasmid uptake.

Teaching transposon classification as a means to crowd source the curation of repeat annotation - a tardigrade perspective.

BACKGROUND: The advancement of sequencing technologies results in the rapid release of hundreds of new genome assemblies a year providing unprecedented resources for the study of genome evolution. Within this context, the significance of in-depth analyses of repetitive elements, transposable elements (TEs) in particular, is increasingly recognized in understanding genome evolution. Despite the plethora of available bioinformatic tools for identifying and annotating TEs, the phylogenetic distance of the target species from a curated and classified database of repetitive element sequences constrains any automated annotation effort. Moreover, manual curation of raw repeat libraries is deemed essential due to the frequent incompleteness of automatically generated consensus sequences. RESULTS: Here, we present an example of a crowd-sourcing effort aimed at curating and annotating TE libraries of two non-model species built around a collaborative, peer-reviewed teaching process. Manual curation and classification are time-consuming processes that offer limited short-term academic rewards and are typically confined to a few research groups where methods are taught through hands-on experience. Crowd-sourcing efforts could therefore offer a significant opportunity to bridge the gap between learning the methods of curation effectively and empowering the scientific community with high-quality, reusable repeat libraries. CONCLUSIONS: The collaborative manual curation of TEs from two tardigrade species, for which there were no TE libraries available, resulted in the successful characterization of hundreds of new and diverse TEs in a reasonable time frame. Our crowd-sourcing setting can be used as a teaching reference guide for similar projects: A hidden treasure awaits discovery within non-model organisms.

Extracellular DNA includes an important fraction of high-risk antibiotic resistance genes in treated wastewaters.

Wastewater treatment plants are among the main hotspots for the release of antibiotic resistance genes (ARGs) into the environment. ARGs in treated wastewater can be found in the intracellular DNA (iDNA) and in the extracellular DNA (eDNA). In this study, we investigated the fate and the distribution (either in eDNA or in iDNA) of ARGs in the treated wastewaters pre and post-disinfection by shotgun metagenomics. The richness of the intracellular resistome was found to be higher than the extracellular one. However, the latter included different high risk ARGs. About 11% of the recovered metagenome assembled genomes (MAGs) from the extracted DNA was positive for at least one ARG and, among them, several were positive for more ARGs. The high-risk ARG bacA was the most frequently detected gene among the MAGs. The disinfection demonstrated to be an important driver of the composition of the antibiotic resistomes. Our results demonstrated that eDNA represents an important fraction of the overall ARGs, including a number of high-risk ARGs, which reach the environment with treated wastewater effluents. The studied disinfections only marginally affect the whole antibiotic resistome but cause important shifts from intracellular to extracellular DNA, potentially threating human health.

Bacteriophages limitedly contribute to the antimicrobial resistome of microbial communities in wastewater treatment plants.

Bacteriophages are known as players in the transmission of antimicrobial resistance genes (ARGs) by horizontal gene transfer. In this study, we characterized the bacteriophage community and the associated ARGs to estimate the potential for phages to spread ARGs in aquatic ecosystems analyzing the intra- and extracellular DNA isolated from two wastewater treatment plants (WWTPs) by shotgun metagenomics. We compared the phage antimicrobial resistome with the bacterial resistome and investigated the effect of the final disinfection treatment on the phage community and its resistome. Phage community was mainly composed by Siphoviridae and other members of the order Caudovirales. The final disinfection only marginally affected the composition of the phage community, and it was not possible to measure its effect on the antimicrobial resistome. Indeed, only three phage metagenome-assembled genomes (pMAGs) annotated as Siphoviridae, Padoviridae, and Myoviridae were positive for putative ARGs. Among the detected ARGs, i.e., dfrB6, rpoB mutants, and EF-Tu mutants, the first one was not annotated in the bacterial MAGs. Overall, these results demonstrate that bacteriophages limitedly contribute to the whole antimicrobial resistome. However, in order to obtain a comprehensive understanding of the antimicrobial resistome within a microbial community, the role of bacteriophages needs to be investigated. IMPORTANCE WWTPs are considered hotspots for the spread of ARGs by horizontal gene transfer. In this study, we evaluated the phage composition and the associated antimicrobial resistome by shotgun metagenomics of samples collected before and after the final disinfection treatment. Only a few bacteriophages carried ARGs. However, since one of the detected genes was not found in the bacterial metagenome-assembled genomes, it is necessary to investigate the phage community in order to gain a comprehensive overview of the antimicrobial resistome. This investigation could help assess the potential threats to human health.

Anthropogenic pollution drives the bacterial resistome in a complex freshwater ecosystem.

Aquatic ecosystems in anthropogenically impacted areas are important reservoirs of antibiotic resistance genes (ARGs) of allochthonous origin. However, the dynamics of the different ARGs within the bacterial communities of lakes and rivers, as well as the factors that drive their selection, are not completely understood. In this study, we analysed the fate of the bacterial resistome (total content of ARGs and of metal resistance genes, MRGs) for a period of six months (summer-winter) in a continuum lake-river-lake system (Lake Varese, River Bardello, Lake Maggiore) in Northern Italy, by shotgun metagenomics. The metagenomic data were then compared with chemical, physical and microbiological data, to infer the role of anthropogenic pressure in the different sampling stations. ARGs and MRGs were more abundant and diverse in the River Bardello, characterised by the highest anthropogenic pollution. The date of sampling influenced ARGs and MRGs, with higher abundances in summer (August) than in fall or in winter, when the impact of the treated wastewater discharge in the river was limited by a higher water flow from Lake Varese. ARG and MRG abundances were significantly correlated and they co-occurred in the main network analysis modules with potential pathogenic bacteria. Different levels of anthropogenic impact selectively promoted specific ARGs while others, generally abundant in waters, were not affected by anthropogenic pressure. Reducing the level of anthropogenic pressure resulted in a rapid decrease of most ARGs. From our results, the role of anthropogenic pressure in promoting the spread of specific antibiotic resistances and of potential pathogens in aquatic ecosystem becomes clear. Finally we highlight the strict correlation between ARGs and MRGs suggesting their potential co-selection in stressed aquatic bacterial communities.

Metagenome Analysis Reveals a Response of the Antibiotic Resistome to Mars-like Extraterrestrial Conditions.

The spread of antibiotic resistance is becoming a serious global health concern. Numerous studies have been done to investigate the dynamics of antibiotic resistance genes (ARGs) in both indoor and outdoor environments. Nonetheless, few studies are available about the dynamics of the antibiotic resistome (total content of ARGs in the microbial cultures or communities) under stress in outer space environments. In this study, we aimed to experimentally investigate the dynamics of ARGs and metal resistance genes (MRGs) in Kombucha Mutualistic Community (KMC) samples exposed to Mars-like conditions simulated during the BIOMEX experiment outside the International Space Station with analysis of the metagenomics data previously produced. Thus, we compared them with those of the respective non-exposed KMC samples. The antibiotic resistome responded to the Mars-like conditions by enriching its diversity with ARGs after exposure, which were not found in non-exposed samples (i.e., tet and van genes against tetracycline and vancomycin, respectively). Furthermore, ARGs and MRGs were correlated; therefore, their co-selection could be assumed as a mechanism for maintaining antibiotic resistance in Mars-like environments. Overall, these results highlight the high plasticity of the antibiotic resistome in response to extraterrestrial conditions and in the absence of anthropogenic stresses.