Towards the definition of an antibiotic resistome signature in wastewater and downstream environments

ABSTRACTIn many low- and middle-income countries, antibiotic-resistant bacteria spread in the environment due to inadequate treatment of wastewater and the poorly regulated use of antibiotics in agri- and aquaculture. Here, we characterized the abundance and diversity of antibiotic-resistant bacteria and antibiotic resistance genes in surface waters and sediments in Bangladesh through quantitative culture of extended-spectrum beta-lactamase (ESBL)-producing coliforms and shotgun metagenomics. Samples were collected from highly urbanized settings (n = 7), rural ponds with a history of aquaculture-related antibiotic use (n = 11), and rural ponds with no history of antibiotic use (n = 6). ESBL-producing coliforms were found to be more prevalent in urban samples than in rural samples. Shotgun sequencing showed that sediment samples were dominated by the phylum Proteobacteria (on average, 73.8% of assigned reads), while in the water samples, Cyanobacteria were the predominant phylum (on average, 60.9% of assigned reads). Antibiotic resistance genes were detected in all samples, but their abundance varied 1,525-fold between sites, with the highest levels of antibiotic resistance genes being present in urban surface water samples. The abundance of antibiotic resistance genes was significantly correlated (R2 = 0.73; P = 8.9 × 10−15) with the abundance of bacteria originating from the human gut, which suggests that the release of untreated sewage is a driver for the spread of environmental antibiotic resistance genes in Bangladesh, particularly in highly urbanized settings.IMPORTANCE Low- and middle-income countries (LMICs) have higher burdens of multidrug-resistant infections than high-income countries, and there is thus an urgent need to elucidate the drivers of the spread of antibiotic-resistant bacteria in LMICs. Here, we study the diversity and abundance of antibiotic resistance genes in surface water and sediments from rural and urban settings in Bangladesh. We found that urban surface waters are particularly rich in antibiotic resistance genes, with a higher number of them associated with plasmids, indicating that they are more likely to spread horizontally. The abundance of antibiotic resistance genes was strongly correlated with the abundance of bacteria that originate from the human gut, suggesting that uncontrolled release of human waste is a major driver for the spread of antibiotic resistance in the urban environment. Improvements in sanitation in LMICs may thus be a key intervention to reduce the dissemination of antibiotic-resistant bacteria.

Animal manures are commonly used to enhance soil fertility, but there are growing concerns over the impact of this practice on the development and dissemination of antibiotic resistance. The aim of this field study was to determine the effect of annual dairy manure applications on the occurrence and abundance of antibiotic resistance genes (ARGs) in an agricultural soil during four years of crop production. Treatments included (i) control (no fertilizer or manure), (ii) inorganic fertilizer and (iii) dairy manure at three application rates. Quantitative PCR was used to determine absolute (per g dry soil) and relative (per 16S rRNA gene) abundances of ARGs in DNA extracted from soils. Six ARGs and one class 1 integron were targeted. This study found that (i) manure application increases ARG abundances above background soil levels; (ii) the higher the manure application rate, the higher the ARG abundance in soil; (iii) the amount of manure applied is more important than reoccurring annual applications of the same amount of manure; (iv) absolute abundance and occurrence of ARGs decreases with increasing soil depth, but relative abundances remained constant. This study demonstrated that dairy manure applications to soil significantly increase the abundance of clinically relevant ARGs when compared to control and inorganic fertilized plots.

Abundance, diversity, and host assignment of total, intracellular, and extracellular antibiotic resistance genes in riverbed sediments

The dissemination of antibiotic resistance genes to the environment is an important factor causing increased prevalence of resistant pathogens. Manure is an important fertilizer, but it contains diverse resistance genes. Therefore, its application to fields may lead to increased abundance of resistance genes in the environment. Farming environments exposed to animal manure have not been studied extensively in countries with comparably low antibiotic use, such as Finland. The effects of manure storage and application to fields on the abundance of resistance genes were studied on two dairy cattle farms and two swine farms in southern Finland. Samples were taken from farms during the 2013 cropping season. Copy numbers of carbapenem (), sulfonamide (), and tetracycline () resistance genes were measured with quantitative polymerase chain reaction, and the data were analyzed using linear mixed models. The relative abundance of antibiotic resistance genes increased about fourfold in soil after manure application. Carbapenemase encoding was detected on all of the studied farms, which indicated that the gene is dispersed in the farm environment. The relative abundance of antibiotic resistance genes increased in stored manure compared with fresh manure roughly fivefold. This study shows that antibiotic resistance genes are disseminated on Finnish production animal farms. The spreading of resistance genes in farm-associated environments could possibly be limited by experimenting with new manure handling methods that could reduce the abundance of the genes in manure used for land application.

Due to reclaimed water, irrigation can cause human health and environmental risks. Soil amendments are applied to reveal the abundance of pathogens and antibiotic resistance genes in rhizosphere soil irrigated by reclaimed water and to better understand the effects of environmental factors on the rhizosphere soil bacterial composition, which has guiding significance for the reasonable use of soil amendments. In this study, the effects of biochar, bioorganic fertilizer, humic acid, loosening soil essence, and corn vinasse on bacterial community diversity and certain gene abundances in rhizosphere soil under drip irrigation with reclaimed water were studied using high-throughput assays and quantitative PCR. The results showed that biochar significantly increased pH, organic matter, and total nitrogen contents in the rhizosphere soil. The corn vinasse significantly decreased soil pH and increased the contents of total nitrogen and total phosphorus but significantly increased the soil EC value (P<0.05). The effects of the five soil amendments on the α-diversity of rhizosphere bacteria were not significantly different. The bacterial community structure and diversity of rhizosphere bacteria were similar at different taxonomic levels, but their relative abundance was different. α-Proteobacteria, γ-Proteobacteria, Bacteroidia, Actinobacteria, Acidimicrobiia, and Anaerolineae were the dominant bacteria in all treatments. The dominant genera consisted of Pseudomonas, Sphingobium, Sphingomonas, Cellvibrio, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Flavobacterium, and Algoriphagus (relative abundance>1%). Correlation analysis of environmental factors showed that the composition of the rhizosphere bacterial community was strongly correlated with pH, EC, total nitrogen, and total phosphorus content. The abundances of pathogenic bacteria and antibiotic resistance genes were 103-107 copies·g-1 and 104-108 copies·g-1, respectively. There were significant differences in the detection levels of pathogens and antibiotic resistance genes. Bioorganic fertilizer, loosening soil essence, and corn vinasse significantly increased the abundances of some antibiotic resistance genes, whereas humic acid and corn vinasse significantly decreased the abundances of Pseudomonas syringae, Ralstonia solanacearum, and total coliforms (P<0.05). A significant correlation was found between pathogens (Arcobacter, Bacillus cereus, Pantoea agglomerans, and Fecal bacteroidetes) and antibiotic resistance genes (tetA, tetB, tetO, tetQ, sul1, ermB, and ermC). In conclusion, while monitoring pathogens and antibiotic resistance genes in the agricultural environment under reclaimed water irrigation, attention should be paid to the rational application of soil amendments to avoid exacerbating the spread of biological contamination.

Effects of industrial effluents containing moderate levels of antibiotic mixtures on the abundance of antibiotic resistance genes and bacterial community composition in exposed creek sediments

Metagenomic and viromic analysis reveal the anthropogenic impacts on the plasmid and phage borne transferable resistome in soil

Antibiotic resistance is a global threat to public health, with antibiotic resistance genes (ARGs) being one of the emerging contaminants; furthermore, animal manure is an important reservoir of biocide resistance genes (BRGs) and metal resistance genes (MRGs). However, few studies have reported differences in the abundance and diversity of BRGs and MRGs between different types of animal manure and the changes in BRGs and MRGs before and after composting. This study employed a metagenomics-based approach to investigate ARGs, BRGs, MRGs, and mobile genetic elements (MGEs) of yak and cattle manure before and after composting under grazing and intensive feeding patterns. The total abundances of ARGs, clinical ARGs, BRGs, MRGs, and MGEs were lower in the manure of grazing livestock than in the manure of the intensively fed group. After composting, the total abundances of ARGs, clinical ARGs, and MGEs in intensively fed livestock manure decreased, whereas those of ARGs, clinical ARGs, MRGs, and MGEs increased in grazing livestock manure. The synergy between MGEs mediated horizontal gene transfer and vertical gene transmission via host bacteria proliferation, which was the main driver that altered the abundance and diversity of ARGs, BRGs, and MRGs in livestock manure and compost. Additionally, tetQ, IS91, mdtF, and fabK were potential indicators for estimating the total abundance of clinical ARGs, BRGs, MRGs, and MGEs in livestock manure and compost. These findings suggest that grazing livestock manure can be directly discharged into the fields, whereas intensively fed livestock manure should be composted before returning to the field. IMPORTANCE The recent increase in the prevalence of antibiotic resistance genes (ARGs), biocide resistance genes (BRGs), and metal resistance genes (MRGs) in livestock manure poses risks to human health. Composting is known to be a promising technology for reducing the abundance of resistance genes. This study investigated the differences and changes in the abundances of ARGs, BRGs, and MRGs between yak and cattle manure under grazing and intensive feeding patterns before and after composting. The results indicate that the feeding pattern significantly affected the abundances of resistance genes in livestock manure. Manure in intensive farming should be composted before being discharged into the field, while grazing livestock manure is not suitable for composting due to an increased number of resistance genes.

Rainfall increases the abundance of antibiotic resistance genes within a riverine microbial community

Occurrence and temporal variation of antibiotic resistance genes (ARGs) in shrimp aquaculture: ARGs dissemination from farming source to reared organisms

Research on the distribution of antibiotic resistance genes (ARGs) in urban sewage treatment systems is extensive, but there is still insufficient research on their abundance in industrial wastewater recycling systems. In this study, a printing and dyeing wastewater (PDWW) recycling system was constructed, and 16S rDNA and high-throughput sequencing technology was used to analyze the microbial communities and ARG abundance during the treatment process. A total of 52 ARGs in nine categories were detected, of which the relative abundance of β-lactam resistance genes was the highest. During the treatment cycle, the concentration of aromatic pollutants increased with an increase in the number of cycles, while the abundance of β-lactam resistance genes increased first, decreased, and then increased (reaching 61.85% on the 100th day). At the same time, the abundance of Firmicutes, Actinobacteria, and Cyanobacteria related ARGs decreased significantly (by 84.66%, 64.38%, and 85.15%, respectively). More than 21 kinds of ARGs were significantly affected by the enrichment by the aromatic pollutants. Among them, 6 kinds of ARGs were significantly positively correlated with changes in the concentrations of the aromatic pollutants (P<0.01), while 6 were significantly negatively correlate (P<0.01). These results show that the abundance of ARGs was affected by the microbial communities and the aromatic pollutants, which increased at first, decreased, and then increased during the PPDW recycling process. This study reveals the effects of the enrichment of aromatic contaminants and changes in microbial communities on ARGs during PPDW recycling, and provides theoretical guidance for the recycling of PDWW to reduce environmental pollution associated with ARGs.

ABSTRACTThe reversibility of antibiotic resistance is theoretically attractive due to the prospect of restoring the clinical potency of antibiotics. It is important to find out the factors that affect the reversibility of antibiotic resistance. Here, an mcr-1-positive multidrug-resistant (MDR) environmental Escherichia coli isolate was successively passaged under four antibiotic-free culture conditions. The relative abundances of multiple antibiotic resistance genes (ARGs) kept decreasing during the successive passages. The linear correlations between abundances of ARGs on the same MDR plasmid reflected that the decay of antibiotic resistance during the passage was mainly due to the elimination of the MDR plasmid (pMCR_W5-6). Colistin-susceptible strains were isolated at the end of the passage. The whole-genome sequencing of two susceptible isolates detected the elimination of the MDR plasmid and deletion of the mcr-1 gene. Deletions of DNA fragments from chromosome and plasmid were closely related to a variety of insertion sequences (ISs). The results of coculture of resistant and susceptible strains at different antibiotic concentrations indicated that the high fitness cost led to the poor stability of mobile ARGs. Strict control of the use of antibiotics can at least reverse the severe antibiotic resistance caused by mobile ARGs of high fitness cost. IMPORTANCE The dissemination of bacterial antibiotic resistance is a serious threat to human health. The development of new antibiotics faces both economic and technological challenges. The reversibility of antibiotic resistance has become an important issue causing wide concern due to the prospect of restoring the clinical potency of antibiotics. Our study suggests that the high mobility of ARGs of high fitness cost may just reflect their poor stability. Therefore, strict control of the use of antibiotics can at least reverse the severe antibiotic resistance caused by mobile ARGs of high fitness cost. This study brings hope for the possibility of curbing the dissemination of antibiotic resistance.

Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what alters those is a first step in understanding our environmental resistome. Toward this goal, 62 samples were collected over 3 years from soils near the 30-year old Gondwana Research Station and for 4 years before and during development of the new Jang Bogo Research Station, both at Terra Nova Bay in Antarctica. These sites reflect limited and more extensive human impact, respectively. A qPCR array with 384 primer sets targeting antibiotic resistance genes and mobile genetic elements (MGEs) was used to detect and quantify these genes. A total of 73 ARGs and MGEs encompassing eight major antibiotic resistance gene categories were detected, but most at very low levels. Antarctic soil appeared to be a common reservoir for seven ARGs since they were present in most samples (42%-88%). If the seven widespread genes were removed, there was a correlation between the relative abundance of MGEs and ARGs, more typical of contaminated sites. There was a relationship between ARG content and distance from both research stations, with a significant effect at the Jang Bogo Station especially when excluding the seven widespread genes; however, the relative abundance of ARGs did not increase over the 4 year period. Silt, clay, total organic carbon, and SiO2 were the top edaphic factors that correlated with ARG abundance. Overall, this study identifies that human activity and certain soil characteristics correlate with antibiotic resistance genes in these oligotrophic Antarctic soils and provides a baseline of ARGs and MGEs for future comparisons.

Impact of chicken litter pre-application treatment on the abundance, field persistence, and transfer of antibiotic resistant bacteria and antibiotic resistance genes to vegetables

Fungicides enhanced the abundance of antibiotic resistance genes in greenhouse soil