Metagenomic investigation of antibiotic resistance genes and resistant bacteria contamination in pharmaceutical plant sites in China.

Pharmaceutical plant sites play a significant role in the dissemination of antibiotic resistance genes (ARGs) into the environment. It is imperative to comprehensively monitor of ARGs across various environmental media at these sites. This study focused on three pharmaceutical plants, two located in North China and one in South China. Through metagenomic approaches, we examined the composition, mobility potential, and bacterial hosts of ARGs in diverse media such as process water, groundwater, topsoil, soil cores, and pharmaceutical fermentation residues across diverse environmental matrices, including topsoil, soil cores, process water, groundwater, and pharmaceutical fermentation residues. We identified a wide array of ARGs, comprising 21 types and 740 subtypes, with process water exhibiting the highest abundance and diversity. Treatment processes varied in their efficacy in eliminating ARGs, and the clinically relevant ARGs should also be considered when evaluating wastewater treatment plant efficiency. Geographical distinctions in groundwater ARG distribution between northern and southern regions were observed. Soil samples from the three sites showed minimal impact from pharmaceutical activity, with vancomycin-resistance genes being the most prevalent. High levels of ARGs in pharmaceutical fermentation residues underscore the necessity for improved waste management practices. Metagenomic assembly revealed that plasmid-mediated ARGs were more abundant than chromosome-mediated ARGs. Metagenome-assembled genomes (MAGs) analysis identified 166 MAGs, with 62 harboring multiple ARGs. Certain bacteria tended to carry specific types of ARGs, revealing distinct host-resistance associations. This study enhances our understanding of ARG dissemination across different environmental media within pharmaceutical plants and underscores the importance of implementing strict regulations for effluent and residue discharge to control ARG spread.

Linking bacterial life strategies with the distribution pattern of antibiotic resistance genes in soil aggregates after straw addition.

Straw addition markedly affects the soil aggregates and microbial community structure. However, its influence on the profile of antibiotic resistance genes (ARGs), which are likely associated with changes in bacterial life strategies, remains unclear. To clarify this issue, a soil microcosm experiment was incubated under aerobic (WS) or anaerobic (AnWS) conditions after straw addition, and metagenomic sequencing was used to characterise ARGs and bacterial communities in soil aggregates. The results showed that straw addition shifted the bacterial life strategies from K- to r-strategists in all aggregates, and the aerobic and anaerobic conditions stimulated the growth of aerobic and anaerobic r-strategist bacteria, respectively. The WS decreased the relative abundances of dominant ARGs such as QnrS5, whereas the AnWS increased their abundance. After straw addition, the macroaggregates consistently exhibited a higher number of significantly altered bacteria and ARGs than the silt+clay fractions. Network analysis revealed that the WS increased the number of aerobic r-strategist bacterial nodes and fostered more interactions between r-and K-strategist bacteria, thus promoting ARGs prevalence, whereas AnWS exhibited an opposite trend. These findings provide a new perspective for understanding the fate of ARGs and their controlling factors in soil ecosystems after straw addition. ENVIRONMENTAL IMPLICATIONS: Straw soil amendment has been recommended to mitigate soil fertility degradation, improve soil structure, and ultimately increase crop yields. However, our findings highlight the importance of the elevated prevalence of ARGs associated with r-strategist bacteria in macroaggregates following the addition of organic matter, particularly fresh substrates. In addition, when assessing the environmental risk posed by ARGs in soil that receives crop straw, it is essential to account for the soil moisture content. This is because the species of r-strategist bacteria that thrive under aerobic and anaerobic conditions play a dominant role in the dissemination and accumulation of ARG.

Tidal flat aquaculture pollution governs sedimentary antibiotic resistance gene profiles but not bacterial community based on metagenomic data.

Coastal tidal flats are intersection zones between terrestrial and marine environments and are considered repositories of pollutants from anthropogenic activities (e.g., fishery and aquaculture). Specifically, the prevalence of antibiotics and antibiotic resistance genes (ARGs) in coastal aquaculture environments pose critical threats to estuarine ecosystems. However, the contribution of aquaculture to the occurrence and abundance of ARGs and community assemblies has not been fully explored in tidal flat zones. Thus, we investigated ARGs profiles, ARG-carrying host bacteria, and their associate microbial community in the Dongtai and Sheyang tidal flat aquaculture regions of Jiangsu, China using metagenomic assembly methods. The antibiotic concentrations in the sediment samples ranged from nd to 35.50 ng/g dw, and the antibiotic pollution in the Dongtai tidal flat was more severe than in the Sheyang tidal flats. Metagenomic assembly indicated that a total of 247 ARG subtypes associated with ARG 33 types were characterized across all samples and their abundance in the Dongtai region exceeded that in the Sheyang region. Meanwhile, 21 bacteria in the tidal flat aquaculture were identified as ARG-carrying pathogens, including Escherichia coli, Vibrio fluvialis, and Staphylococcus aureus. Using neutral and null modeling analysis to determine the community ecological processes, the results revealed bacterial and ARG communities were generally dominated by stochastic and deterministic processes, respectively. The above results suggested that aquaculture pollution was contributed to shape ARG profiles in tidal flats. The observed deterministic processes affecting the ARG community in tidal flat aquaculture also provides an effective foundation to control the risks of environmental antibiotic resistance through reducing aquaculture antibiotic usage.

Inhalable Antibiotic Resistome from Wastewater Treatment Plants to Urban Areas: Bacterial Hosts, Dissemination Risks, and Source Contributions.

Antibiotic resistance genes (ARGs) are commonly detected in the atmosphere, but questions remain regarding their sources and relative contributions, bacterial hosts, and corresponding human health risks. Here, we conducted a qPCR- and metagenomics-based investigation of inhalable fine particulate matter (PM2.5) at a large wastewater treatment plant (WWTP) and in the ambient air of Hong Kong, together with an in-depth analysis of published data of other potential sources in the area. PM2.5 was observed with increasing enrichment of total ARGs along the coastal-urban-WWTP gradient and clinically relevant ARGs commonly identified in urban and WWTP sites, illustrating anthropogenic impacts on the atmospheric accumulation of ARGs. With certain kinds of putative antibiotic-resistant pathogens detected in urban and WWTP PM2.5, a comparable proportion of ARGs that co-occurred with MGEs was found between the atmosphere and WWTP matrices. Despite similar emission rates of bacteria and ARGs within each WWTP matrix, about 11-13% of the bacteria and >57% of the relevant ARGs in urban and WWTP PM2.5 were attributable to WWTPs. Our study highlights the importance of WWTPs in disseminating bacteria and ARGs to the ambient air from a quantitative perspective and, thus, the need to control potential sources of inhalation exposure to protect the health of urban populations.

Long-read sequencing revealed cooccurrence, host range, and potential mobility of antibiotic resistome in cow feces.

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and ß-lactam-resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum ß-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

Distribution patterns of antibiotic resistance genes and their bacterial hosts in pig farm wastewater treatment systems and soil fertilized with pig manure.

Vast reservoirs of antibiotic resistance genes (ARG) are discharged into the environment via pig manure. We used metagenomic analysis to follow the distribution and shifts of ARGs and their bacterial hosts along wastewater treatment in three large pig farms. The predominating ARGs potentially encoded resistance to tetracycline (28.13%), aminoglycosides (23.64%), macrolide-lincosamide-streptogramin (MLS) (12.17%), sulfonamides (11.53%), multidrug (8.74%) and chloramphenicol (6.18%). The total relative ARG abundance increased along the treatment pathway prior to anaerobic digestion that had a similar degradative capacity for different ARGs and these ARGs were reduced by about 25% after digestion, but ARGs enriched erratically in manured soils. Distinctive ARG distribution patterns were found according to the three sample locations; feces, soil and wastewater and the differences were primarily due to the tetracycline ARGs (feces > wastewater > soil), sulfonamide ARGs (soil > wastewater > feces) and MLS ARGs (feces > wastewater > soil). Metagenomic assembly-based host analyses indicated the Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were primary ARG carriers. The Streptococcaceae increased the abundance of multidrug, MLS and aminoglycoside ARGs in feces; Moraxellaceae were the primary contributors to the high abundance of multidrug ARGs in wastewater; the Comamonadaceae led to the higher abundance of bacA in wastewater and soil than feces. We found a high level of heterogeneity for both ARGs and ARG-hosts in the wastewater treatment system and in the agricultural soils for these pig farms.

Metagenomic insights into the profile of antibiotic resistomes in a large drinking water reservoir.

Reservoirs play a vital role in the control and management of surface water resources. However, the long water residence time in the reservoir potentially increases the storage and accumulation of antibiotic resistant genes (ARGs). The full profiles and potential health risks of antibiotic resistomes in reservoirs are largely unknown. In this study, we investigated the antibiotic resistomes of water and sediment during different seasons in the Danjiangkou Reservoir, which is one of the largest reservoirs in China, using a metagenomic sequencing approach. A total of 436 ARG subtypes belonging to 20 ARG types were detected from 24 water and 18 sediment samples, with an average abundance of 0.138 copies/cell. The overall ARG abundance in the sediment was higher than that in the water, and bacitracin and vancomycin resistance genes were the predominant ARG types in the water and sediment, respectively. The overall ARG abundance in the dry season was higher than that in the wet season, and a significant difference in ARG subtype compositions was observed in water, but not in the sediment, between the different seasons. The potential horizontal gene transfer frequency in the water was higher than that in the sediment, and the ARGs in water mainly came from the sediment upstream of the reservoir. The metagenomic assembly identified 14 contigs as ARG-carrying pathogens including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and 3 of 14 carried virulence factors. Overall, the potential public health risks posed by resistomes in the water of the Danjiangkou Reservoir were higher in the dry season than in the wet season. Based on these results, strategies including sediment control and pathogen monitoring are suggested for water safety management in drinking water reservoirs.