Hosts Of Antibiotic Resistance Genes Research Articles

Impact of Combined Pollution of Ciprofloxacin and Copper on the Diversity of Archaeal Communities and Antibiotic-Resistance Genes

This study aimed to explore the response of archaeal communities and antibiotic-resistance genes (ARGs) to ciprofloxacin (CIP, 0.05–40 mg/L) and copper (Cu, 3 mg/L) combined pollution during stress- and post-effect periods in an activated sludge system. With the increase in the CIP concentration, the diversity of archaea decreased, but the richness increased under the stress of 10 mg/L CIP. Under stress and post effects, the change in unknown archaeal community structure was more significant than that of the known archaea. The relative abundance of unknown archaea was significantly reduced with the increase in CIP concentration. Meanwhile, there were certain archaea that belonged to abundant and rare taxa with different resistance and recovery characteristics. Among them, Methanosaeta (49.15–83.66%), Methanoculleus (0.11–0.45%), and Nitrososphaera (0.03–0.36%) were the typical resistant archaea to combined pollution. And the resistance of the abundant taxa to combined pollution was significantly higher than that of the rare taxa. Symbiotic and competitive relationships were observed between the known and the unknown archaea. The interactions of abundant known taxa were mainly symbiotic relationships. While the rare unknown taxa were mainly competitive relationships in the post-effect period. Rare archaea showed an important ecological niche under the stress-effect. Some archaea displayed positive correlation with ARGs and played important roles as potential hosts of ARGs during stress- and post-periods. Methanospirillum, Methanosphaerula, Nitrososphaera and some rare unknown archaea also significantly co-occurred with a large number of ARGs. Overall, this study points out the importance of interactions among known and unknown archaeal communities and ARGs in a wastewater treatment system under the stress of antibiotics and heavy metal combined pollution.

High-throughput profiling of antibiotic resistance genes in the Yellow River of Henan Province, China

Profiling antibiotic resistance genes (ARGs) in the Yellow River of China’s Henan Province is essential for understanding the health risks of antibiotic resistance. The profiling of ARGs was investigated using high-throughput qPCR from water samples in seven representative regions of the Yellow River. The absolute and relative abundances of ARGs and moble genetic elements (MGEs) were higher in summer than in winter (ANOVA, p < 0.001). The diversity and abundance of ARGs were higher in the Yellow River samples from PY and KF than the other sites. Temperature (r = 0.470 ~ 0.805, p < 0.05) and precipitation (r = 0.492 ~ 0.815, p < 0.05) positively influenced the ARGs, while pH had a negative effect (r = − 0.462 ~ − 0.849, p < 0.05). Network analysis indicated that the pathogenic bacteria Rahnella, Bacillus, and Shewanella were the possible hub hosts of ARGs, and tnpA1 was the potential MGE hub. These findings provide insights into the factors influencing ARG dynamics and the complex interaction among the MGEs, pathogenic bacteria and environmental parameters in enriching ARGs in the Yellow River of Henan Province.

Differential drivers on the occurrence of antibiotic resistance genes in agricultural soils and crops: Evidence from the different fertilization regimes

Organic fertilizer application caused bacterial resistance contamination in farming systems has been widely documented, and long-term fertilization will exacerbate the migration of antibiotic resistance genes (ARGs) to crops and humans. However, it remains unclear whether a combined pattern of chemical and organic fertilizer application can arrest the high prevalence of ARGs in soil-crop. Here we investigated the ARGs occurrence under different fertilization regimes, and explored the mechanisms by which micro-ecological shifts and geochemical factors in modulating the ARGs fate in soil and crop. The results showed that the soil ARGs abundance was highest under 100% organic fertilizer, while the highest ARGs abundance in crops was observed at 100% chemical fertilizer. Application of organic fertilizers with more than 50% ration intensified the soil accumulation and migration of tetA, sul1, sul2 and macA genes. And, multidrug_transporter, macA and sul1 were co-shared in soil and crop, where ARGs potential hosts differed complete in the two, suggesting that these ARGs may be transferred across media by horizontal transfer. Procrustes analysis revealed that soil microbial community was significantly correlated with ARG hosts, and soil microbial evolutionary pathway was congruent with antibiotic resistance, suggesting that fertilizers affect soil ARGs abundance mainly by altering soil microbial composition and their ecological evolutionary trajectories. While, no significant correlation was observed between endophytes and crop ARG host. Structural equations demonstrated that soil nutrients and crop growth characteristics contributed largely to the prevalence of crop ARGs. This outcome will provide new insights into the high prevalence of ARGs in soil and crop, and offer fertilizer recommendations for effectively curbing antibiotic-resistance contamination in farming systems.

Viral Communities Suppress the Earthworm Gut Antibiotic Resistome by Lysing Bacteria on a National Scale.

Earthworms are critical in regulating soil processes and act as filters for antibiotic resistance genes (ARGs). Yet, the geographic patterns and main drivers of earthworm gut ARGs remain largely unknown. We collected 52 earthworm and soil samples from arable and forest ecosystems along a 3000 km transect across China, analyzing the diversity and abundance of ARGs using shotgun metagenomics. Earthworm guts harbored a lower diversity and abundance of ARGs compared to soil, resulting in a stronger distance-decay rate of ARGs in the gut. Greater deterministic assembly processes of ARGs were found in the gut than in soil. The earthworm gut had a lower frequency of co-occurrence patterns between ARGs and mobile genetic elements (MGEs) in forest than in arable systems. Viral diversity was higher in the gut compared to soil and was negatively correlated with bacterial diversity. Bacteria such as Streptomyces and Pseudomonas were potential hosts of both viruses and ARGs. Viruses had negative effects on the diversity and abundance of ARGs, likely due to the lysis on ARG-bearing bacteria. These findings provide new insights into the variations of ARGs in the earthworm gut and highlight the vital role of viruses in the regulation of ARGs in the soil ecosystem.

Efficient elimination of antibiotic resistome in livestock manure by semi-permeable membrane covered hyperthermophilic composting

Livestock manure is a hotspot for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), and an important contributor to antibiotic resistance in non-clinical settings. This study investigated the effectiveness and potential mechanisms of a novel composting technology, semi-permeable membrane covered hyperthermophilic composting (smHTC), in removal of ARGs and MGEs in chicken manure. Results showed that smHTC was more efficient in removal of ARGs and MGEs (92% and 93%) compared to conventional thermophilic composting (cTC) (76% and 92%). The efficient removal in smHTC is attributed to direct or indirect negative effects caused by the high temperature, including reducing the involvement of bio-available heavy metals (HMs) in co-selection processes of antibiotic resistance, decreasing the bacterial abundance and diversity, suppressing the horizontal gene transfer and killing potential ARGs hosts. Overall, smHTC can efficiently remove the resistome in livestock manure, reducing the risk to crops and humans from ARGs residues in compost products.

A comparison of antibiotic resistance genes and mobile genetic elements in wild and captive Himalayan vultures.

As the most widely distributed scavenger birds on the Qinghai-Tibetan Plateau, Himalayan vultures (Gyps himalayensis) feed on the carcasses of various wild and domestic animals, facing the dual selection pressure of pathogens and antibiotics and are suitable biological sentinel species for monitoring antibiotic resistance genes (ARGs). This study used metagenomic sequencing to comparatively investigate the ARGs and mobile genetic elements (MGEs) of wild and captive Himalayan vultures. Overall, the resistome of Himalayan vultures contained 414 ARG subtypes resistant to 20 ARG types, with abundances ranging from 0.01 to 1,493.60 ppm. The most abundant resistance type was beta-lactam (175 subtypes), followed by multidrug resistance genes with 68 subtypes. Decreases in the abundance of macrolide-lincosamide-streptogramin (MLS) resistance genes were observed in the wild group compared with the zoo group. A total of 75 genera (five phyla) of bacteria were predicted to be the hosts of ARGs in Himalayan vultures, and the clinical (102 ARGs) and high-risk ARGs (35 Rank I and 56 Rank II ARGs) were also analyzed. Among these ARGs, twenty-two clinical ARGs, nine Rank I ARG subtypes, sixteen Rank II ARG subtypes were found to differ significantly between the two groups. Five types of MGEs (128 subtypes) were found in Himalayan vultures. Plasmids (62 subtypes) and transposases (44 subtypes) were found to be the main MGE types. Efflux pump and antibiotic deactivation were the main resistance mechanisms of ARGs in Himalayan vultures. Decreases in the abundance of cellular protection were identified in wild Himalayan vultures compared with the captive Himalayan vultures. Procrustes analysis and the co-occurrence networks analysis revealed different patterns of correlations among gut microbes, ARGs, and MGEs in wild and captive Himalayan vultures. This study is the first step in describing the characterization of the ARGs in the gut of Himalayan vultures and highlights the need to pay more attention to scavenging birds.

Using CaCO3 armor to alleviate PFOA-induced stress on microorganisms in porous aquatic environments

Perfluorooctanoic acid (PFOA), even at trace levels, can cause considerable microbial responses in porous aquatic environments. We propose the use of CaCO3 to reduce PFOA migration through the formation of Ca-PFOA bonding, which impede the intracellular penetration of PFOA and thus alleviate PFOA-induced damage to microbes. In this work, a porous environment was simulated by sand columns (100 mm in height), and a thin layer of CaCO3 was placed on the top of the columns. Upon the dissolution of CaCO3, Ca2+ reduced the zeta potential of the effluent as the influent containing 2.4 μM PFOA passed through the CaCO3-containing system. This promoted the bonding of Ca-PFOA, resulting in more than a twofold increase in the blocking efficiency of PFOA during the initial 13 days. A calcite calcium armor (50–100 nm) formed on the cell surface effectively reduced the oxidative damage caused by PFOA: intracellular reactive oxygen species were reduced by 39.3 %, and bioactivity (in adenosine triphosphate) increased by 454.5 %. The alleviation of PFOA-induced stress reduced the synthesis of extracellular polymerizing substances by 67.1 %, but microbial growth remained healthy. CaCO3 introduction stimulated quorum sensing through the generation of C6-HSL to up-regulate the carbon fixation pathway. Under PFOA-induced stress, CaCO3 introduction resulted in a microbial community that was similar to that in the absence of PFOA; additionally, the dominance of denitrifying bacteria as hosts of antibiotic resistance genes (ARGs) decreased, resulting in minimal or no impact on water purification efficiency. Additionally, microbial-induced carbonate precipitation functional microorganisms were enriched as Ca2+ alleviated PFOA-induced stress. In conclusion, CaCO3 effectively alleviated PFOA-induced stress and reduced the environmental effects of PFOA on the porous aquatic environment. Microbial enhancement strategy provides a new perspective for PFOA pollution remediation.

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.

Parachlorometaxylenol stress caused multidrug-type antibiotic resistance genes proliferation via simultaneously reshaping microbial community and interfering metabolic traits during wastewater treatment process

Despite biological wastewater treatment processes (e.g., sequencing batch reactors (SBR)) being able to reduce the dissemination of antibiotic resistance genes (ARGs), the variation of ARGs under exogenous pollutant stress is an open question. This work investigated the impacts of para-chloro-meta-xylenol (PCMX, typical antibacterial contaminants) on ARGs spread in long-term SBR operation. Although the SBR process inherently decreased ARGs abundance, the presence of PCMX substantially amplified both the prevalence (mainly multidrug) and abundance of total ARGs (1.17-fold of the control). Further analysis demonstrated that PCMX disintegrated sludge structures as well as increased membrane permeability, facilitating the release of mobile genetic elements and subsequent horizontal transfer of ARGs. In addition, PCMX selectively enriched potential ARG hosts, notably Nitrospira and Candidatus Accumulibacter, which predominantly served as multidrug ARG hosts. Concurrently, the self-adaptive functions of ARGs hosts in the PCMX-exposed SBR system were activated via quorum sensing, two-component regulatory system, ATP-binding cassette transporters, and bacterial secretion system. The upregulation of these metabolic pathways also contributed to the dissemination of ARGs.

Effect of Mn-HA on ARGs and MRGs in nitrogen-culturing sludge

The simultaneous escalation in ARGs (antibiotic resistance genes) and MRGs (metal resistance genes) further complicates the intricate network of factors contributing to the proliferation of microbial resistance. Manganese, which has been reported to affect the resistance of bacteria to antibiotics and metals, plays a vital role in microbial nitrogen metabolism. Moreover, nitrifying and denitrifying populations are potential hosts for ARGs. In this study, manganese was introduced in its prevalent organic chelated form in the environment (Manganese humus chelates, Mn-HA) to a N metabolism sludge to explore the effect of manganese on MRGs and ARGs dissemination. Metagenomics results revealed that manganese availability enhances nitrogen metabolism, while a decrease in ARGs was noted which may be attributed to the inhibition of horizontal gene transfer (HGT), reflected in the reduced integrase -encoded gene int. Population analysis revealed that nitrifier and denitrifier genus harbor MRGs and ARGs, indicating that nitrifier and denitrifier are hosts of MRGs and ARGs. This raises the question of whether the prevalence of ARGs is always increased in metal-contained environments.

Metagenomic insights into effects of carbon/nitrogen ratio on microbial community and antibiotic resistance in moving bed biofilm reactor

This study investigated the effects of carbon/nitrogen (C/N) ratio on microbial community in moving bed biofilm reactor (MBBR) using metagenomic analysis, and the dynamic changes of relevant antibiotic resistance genes (ARGs) were also analyzed. The results showed that under low C/N ratio, MBBR exhibited average removal rates of 98.41 % for ammonia nitrogen and 75.79 % for total nitrogen. Metagenomic analysis showed low C/N ratio altered the structure of biofilm and water microbiota, resulting in the detachment of bacteria such as Actinobacteria from biofilm into water. Furthermore, sulfamethazine (SMZ)-resistant bacteria and related ARGs were released into water under low C/N ratio, which lead to the increase of SMZ resistance rate to 90%. Moreover, most dominant genera are potential hosts for both nitrogen cycle related genes and ARGs. Specifically, Nitrosomonas that carried gene sul2 might be released from biofilm into water. These findings implied the risks of antibiotic resistance dissemination in MBBR under low C/N ratio.

Longitudinal Analysis of Urban Stormwater Microbiome and Resistome from Watersheds with and without Green Infrastructure using Long-Read Sequencing

Since stormwater conveys a variety of contaminants into water bodies, green infrastructure (GI) is increasingly being adopted as an on-site treatment solution in addition to controlling peak flows. The purpose of this study was to identify differences in microbial water quality of stormwater in watersheds retrofitted with GI vs. those without GI. Considering stormwater is recently recognized as a contributor to the antibiotic resistance (AR) threat, another goal of this study was to characterize changes in the microbiome and collection of AR genes (resistome) of urban stormwater with season, rainfall characteristics, and fecal contamination. MinION long-read sequencing was used to analyze stormwater microbiome and resistome from watersheds with and without GI in Columbus, Ohio, United States, over 18 months. We characterized fecal contamination in stormwater via culturing Escherichia coli and with molecular microbial source tracking (MST) to identify sources of fecal contamination. Overall, season and storm event (rainfall) characteristics had the strongest relationships with changes in the stormwater microbiome and resistome. We found no significant differences in microbial water quality or the microbiome of stormwater in watersheds with and without GI implemented. However, there were differences between the communities of microorganisms hosting antibiotic resistance genes (ARGs) in stormwater from watersheds with and without GI, indicating the potential sensitivity of AR bacteria to treatment. Stormwater was contaminated with high concentrations of human-associated fecal bacterial genes, and the ARG host bacterial community had considerable similarities to human feces/wastewater. We also identified 15 potential pathogens hosting ARGs in these stormwater resistome, including vancomycin-resistant Enterococcus faecium (VRE) and multidrug-resistant Pseudomonas aeruginosa. In summary, urban stormwater is highly contaminated and has a great potential to spread AR and microbial hazards to nearby environments. This study presents the most comprehensive analysis of stormwater microbiome and resistome to date, which is crucial to understanding the potential microbial risk from this matrix. This information can be used to guide future public health policy, stormwater reuse programs, and urban runoff treatment initiatives.

Improving antibiotic resistance removal from piggery wastewater by involving zero-valent iron within an integrated anoxic-aerobic biofilm reactor

This study introduces zero-valent iron (ZVI) to enhance the removal of antibiotic resistance in piggery wastewater. Two integrated anoxic-aerobic biofilm reactors (IAOBRs) were operated simultaneously for 120 days, with one serving as a control reactor and the other as experimental reactor supplemented with ZVI. The experimental reactor demonstrated superior antibiotic removal efficiencies (89.8 ± 4.5 %), eliminating 99.7 ± 0.7 % of sulfonamides, 91.6 ± 4.5 % of tetracyclines and 81.1 ± 8.4 % of quinolones. The antibiotic resistance bacteria, antibiotic resistance genes (ARGs) and mobile genetic elements removal efficiency reached 0.69–1.74, 2.06 and 2.15 logs, respectively. In comparison to the control reactor, the activated sludge in the experimental reactor also experienced a reduction in total antibiotic concentrations by 57.58 ± 19.99 %. The enhanced performance of the iron-mediated process in antibiotic resistance removal is attributed to the augmentation of enzymatic activity, biodegradation ability, and enrichment of specific microorganisms. The introduction of ZVI also modified the microbial community composition and decreased the potential hosts of ARGs. The proposed iron-mediated process greatly enhances antibiotic resistance removal from wastewater and mitigates antibiotic accumulation in activated sludge, offering new insights into wastewater biological treatment for eliminating antibiotic resistance.

Suppressing Ralstonia solanacearum and Bacterial Antibiotic Resistance Genes in Tomato Rhizosphere Soil through Companion Planting with Basil or Cilantro

The effects of companion planting on soil antibiotic resistance genes (ARGs) and associated microbial composition have remained largely unclear until now. In this study, we assessed the changes in the soil microbiome and ARGs frequencies for tomato growing soils that were companion planted with basil (TB) or cilantro (TC) using a metagenome approach. The abundance of the phytopathogen Ralstonia solanacearum was significantly lower in the TC or TB treatments compared to the tomato monoculture soils (TT). A significant enrichment of Pseudomonas and Aquabacterium and a depletion of Nocardioides and Streptomyces were observed in the TC treatment. Interestingly, both TC and TB companion planting reduced the absolute abundance and the number of subtypes of ARGs. The TC soil showed the lowest numbers of unique ARG subtypes, especially the ARGs resistant to vancomycin and rifamycin, as well as those associated with multidrug resistance. Furthermore, network analysis further revealed that Nocardioides and Streptomyces were potential hosts of ARGs, whereas Flavobacterium negatively correlated with mdtG, suggesting a suppressive effect in reducing ARGs. Together, our results suggest that the companion planting of tomatoes with basil or cilantro can reduce the risk of ARG accumulation, making it a feasible farming management tool to promote soil and plant health in sustainable agriculture.

Deciphering the pathogenic risks of microplastics as emerging particulate organic matter in aquatic ecosystem

Microplastics are accumulating rapidly in aquatic ecosystems, providing habitats for pathogens and vectors for antibiotic resistance genes (ARGs), potentially increasing pathogenic risks. However, few studies have considered microplastics as particulate organic matter (POM) to elucidate their pathogenic risks and underlying mechanisms. Here, we performed microcosm experiments with microplastics and natural POM (leaves, algae, soil), thoroughly investigating their distinct effects on the community compositions, functional profiles, opportunistic pathogens, and ARGs in Particle-Associated (PA) and Free-Living (FL) bacterial communities. We found that both microplastics and leaves have comparable impacts on microbial community structures and functions, enriching opportunistic pathogens and ARGs, which may pose potential environmental risks. These effects are likely driven by their influences on water properties, including dissolved organic carbon, nitrate, DO, and pH. However, microplastics uniquely promoted pathogens as keystone species and further amplified their capacity as hosts for ARGs, potentially posing a higher pathogenic risk than natural POM. Our research also emphasized the importance of considering both PA and FL bacteria when assessing microplastic impacts, as they exhibited different responses. Overall, our study elucidates the role and underlying mechanism of microplastics as an emerging POM in intensifying pathogenic risks of aquatic ecosystems in comparison with conventional natural POM.

Deciphering the reduction of antibiotic resistance genes (ARGs) during medium-chain fatty acids production from waste activated sludge: Driven by inhibition of ARGs transmission and shift of microbial community

Medium-chain fatty acids (MCFAs) production from waste activated sludge (WAS) by chain extension (CE) is a promising technology. However, the effects and mechanisms of CE process on the fate of antibiotic resistance genes (ARGs) remain unclear. In this study, the results showed that the removal efficiency of ARGs was 81.15 % in CE process, suggesting its efficacy in reducing environmental risks. Further, the observed decrease in mobile genetic elements (MGEs) indicated that CE process restricted the horizontal gene transfer (HGT). Complementing this, the increase in soluble organic matters and extracellular 16 S rDNA confirmed that MCFAs production caused bacterial damage. Decreased intracellular ARGs and increased extracellular ARGs further revealed that MCFAs production impaired ARGs hosts, thereby limiting the vertical gene transfer (VGT) of ARGs. Shift of microbial community combined with co-occurrence network analysis demonstrated that functional bacteria without host potential for ARGs were enriched, but potential ARGs and MGEs hosts decreased, showing the role of functional bacterial phylogeny and selection pressure of MCFAs in reducing ARGs. Finally, partial least squares path model was used to systematic verify the mechanism of ARGs removal in CE process, which was attributed to the inhibition of ARGs transmission (HGT and VGT) and shift of microbial community.

Mitigated dissemination of antibiotic resistance genes by nanoscale zero-valent iron and iron oxides during anaerobic digestion: Roles of microbial succession and regulation

Nanoscale zero-valent iron (ZVI) and the oxides have been documented as an effective approach for mitigating the dissemination of antibiotic resistance genes (ARGs) during anaerobic digestion (AD). However, the mechanism of ARGs dissemination mitigated by nanoscale ZVI and iron oxides remain unclear. Here, we investigated the influencing mechanisms of nanoscale ZVI and iron oxides on ARGs dissemination during AD. qPCR results indicated that nanoscale ZVI and iron oxides significantly declined the total ARGs abundances, and the strongest inhibiting effect was observed by 10 g/L nanoscale ZVI. Mantel test showed ARGs distribution was positively correlated with physiochemical properties, integrons and microbial community, among which microbial community primarily contributed to ARGs dissemination (39.74%). Furthermore, redundancy and null model analyses suggested the dominant and potential ARGs host was Fastidiosipila, and homogeneous selection in the determinism factors was the largest factor for driving Fastidiosipila variation, confirming the inhibition of Fastidiosipila was primary reason for mitigating ARGs dissemination by nanoscale ZVI and iron oxides. These results were related to the inhibition of ARGs transfer related functions. This work provides novel evidence for mitigating ARGs dissemination through regulating microbial succession and regulation induced by ZVI and iron oxides.

Bacterial hosts of clinically significant beta-lactamase genes in Croatian wastewaters.

Wastewater treatment plants (WWTPs) provide a suitable environment for the interaction of antibiotic resistant bacteria and antibiotic-resistance genes (ARGs) from human, animal, and environmental sources. The aim was to study the influent and effluent of two WWTPs in Croatia to identify bacterial hosts of clinically important beta-lactamase genes (blaTEM, blaVIM, blaOXA-48-like) and observe how their composition changes during the treatment process. A culture-independent epicPCR (Emulsion, Paired isolation and Concatenation Polymerase Chain Reaction) was used to identify the ARG hosts, and 16S rRNA amplicon sequencing to study the entire bacterial community. Different wastewater sources contributed to the significant differences in bacterial composition of the wastewater between the two WWTPs studied. A total of 167 genera were detected by epicPCR, with the Arcobacter genus, in which all ARGs studied were present, dominating in both WWTPs. In addition, the clinically important genera Acinetobacter and Aeromonas contained all ARGs examined. The blaOXA-48-like gene had the highest number of hosts, followed by blaVIM, while blaTEM had the narrowest host range. Based on 16S rRNA gene sequencing, ARG hosts were detected in both abundant and rare taxa. The number of hosts carrying investigated ARGs was reduced by wastewater treatment. EpicPCR provided valuable insights into the bacterial hosts of horizontally transmissible beta-lactamase genes in Croatian wastewater.

Effect of Mineral Fertilizers and Pesticides Application on Bacterial Community and Antibiotic-Resistance Genes Distribution in Agricultural Soils

Soils are a hotspot for the emergence and spread of antibiotic resistance. The effects of agrochemical treatments on the bacterial community of agricultural soils and the content of antibiotic-resistance genes (ARGs) were studied. Treatments included the following: control, mineral fertilizers (NPKs), pesticides, and the combined treatment of soils under soya (Glycine max), sunflower (Helianthus annuus L.), and wheat (Triticum aestivum). Bacterial community taxonomic composition was studied using 16S rRNA gene sequencing. The content of 10 ARGs and 3 integron genes (intI1, intI2, intI3) was determined using quantitative real-time PCR. The results showed that the treatments had little effect on the taxonomic composition and diversity of the soil bacterial community. The most significant factors determining differences in the microbial community were sampling time and soil physico-chemical parameters. A significant role of the bacterial community in ARG distribution in soils was demonstrated. Representatives of the Pseudomonas, Bacillus, Sphingomonas, Arthrobacter genera, and the Nocardioidaceae and Micrococcaceae families were likely ARG hosts. The presence of integron genes of all three classes was detected, the most numerous being intI3. This work provides important information on the role of agricultural soils in ARG transfer, and the findings may be useful for sustainable and safe agricultural development.

Diverse bacterial hosts and potential risk of antibiotic resistomes in ship ballast water revealed by metagenomic binning

Ship ballast water promoting the long-range migration of antibiotic resistance genes (ARGs) has raised a great concern. This study attempted to reveal ARGs profile in ballast water and decipher their hosts and potential risk using metagenomic approaches. In total, 710 subtypes across 26 ARG types were identified among the ballast water samples from 13 ships of 11 countries and regions, and multidrug resistance genes were the most dominant ARGs. The composition of ARGs were obviously different across samples, and only 5% of the ARG subtypes were shared by all samples. Procrustes analysis showed the bacterial community contributed more than the mobile genetic elements (MGEs) in shaping the antibiotic resistome. Further, 79 metagenome-assembled genomes (46 genera belong to four phyla) were identified as ARG hosts, with predominantly affiliated with the Proteobacteria. Notably, potential human pathogens (Alcaligenes, Mycolicibacterium, Rhodococcus and Pseudomonas) were also recognized as the ARG hosts. Above 30% of the ARGs hosts contained the MGEs simultaneously, supporting a pronounced horizontal gene transfer capability. A total of 43 subtypes (six percent of overall ARGs) of ARGs were assessed with high-risk, of which 23 subtypes belonged to risk Rank I (including rsmA, ugd, etc.) and 20 subtypes to the risk Rank II (including aac(6)-I, sul1, etc.). In addition, antibiotic resistance risk index indicated the risk of ARGs in ballast water from choke points of maritime trade routes was significantly higher than that from other regions. Overall, this study offers insights for risk evaluation and management of antibiotic resistance in ballast water.