Poultry manure is a reservoir for antibiotics and antibiotic resistance genes and composting is an effective biological treatment for manure. This study explored the effect of using two methods of adding a complex microbial agent to the composting of laying-hen manure on doxycycline degradation and tetracycline resistance genes elimination. The results showed that incorporating a complex microbial agent at 0.8% (w/w) on the 0ᵗʰ and 11th day (group MT2) effectively degraded doxycycline with a final degradation rate of 46.83 ± 0.55%. The half-life of doxycycline in this group was 21.90 ± 0.00 days and was significantly lower than that of group MT1 (1.6% (w/w) complex microbial agent added on the 0ᵗʰ day) and group DT (compost without complex microbial agent). But there was no significant difference in the final degradation rate of doxycycline between group DT and group MT1. The addictive with the complex microbial agent changed the microbial community structure. Bacteroidetes, Firmicutes and Proteobacteria were the dominant phyla during composting. Aerococcus, Desemzia, Facklamia, Lactobacillus, Streptococcus, and Trichococcus were the bacteria related to the degradation of doxycycline. Moreover, the incorporation of a complex microbial agent could decrease the risk on spreading tetracycline resistance genes. The single addition promoted the elimination of tetM, whose possible hosts were Enterococcus, Lactobacillus, Staphylococcus, and Trichococcus. Adding the complex microbial agent twice promoted the elimination of tetX, which was related to the low abundance of Chryseobacterium, Flavobacterium and Neptunomonas in group MT2. Redundancy analysis showed that the bacterial community, residual doxycycline and physiochemical properties have a potential effect on the variation in tetracycline resistance genes levels. Overall, adding the complex microbial agent twice is an effective measure to degrade doxycycline.

The dissemination of antibiotic resistance (AR) has attracted global attention because of the increasing antibiotic treatment failure it has caused. Through natural transformation, a live bacterium takes up extracellular DNA (exDNA), which facilitates AR dissemination. However, recovery of exDNA from water samples is challenging. In this study, we validated a consecutive ultrafiltration-based protocol to simultaneously recover intracellular DNA (inDNA), dissolved exDNA (Dis_exDNA, dissolved in the bulk water), and adsorbed exDNA (Ads_exDNA, adsorbed to the surfaces of suspended particles). Using hollow fiber ultrafiltration (HFUF), all DNA fractions were concentrated from environmental water samples, after which Dis_exDNA (supernatant) was separated from inDNA and Ads_exDNA (pellets) using centrifugation. Ads_exDNA was washed off from the pellets with proteinase K and sodium phosphate buffer. Dis_exDNA and Ads_exDNA were further concentrated using centrifugal ultrafiltration, from which silica binding was performed. inDNA was extracted from washed pellets with a commercial kit. For inDNA, HFUF showed recovery efficiencies of 96.5 ± 18.5% and 88.0 ± 2.0% for total cells and cultured Escherichia coli, respectively (n = 3). To represent all possible DNA fragments in water environment, exDNA with different lengths (10.0, 4.0, 1.0, and 0.5 kbp) were spiked to test the recovery efficiencies for Dis_exDNA. The whole process achieved 62.2%–62.9% recovery for 10 and 4 kbp exDNA, and 38.8%–44.5% recovery for 1.0 and 0.5 kbp exDNA. Proteinase K treatment enhanced the recovery of Ads_exDNA by 4.0–10.7 times. The protocol was applied to water samples from an urban river in Tokyo, Japan. The abundance of AR genes (ARGs) in inDNA, Dis_exDNA, and Ads_exDNA increased downstream of wastewater treatment plants. ARGs in Ads_exDNA and Dis_exDNA accounted for 1.8%–26.7% and 0.03%–20.9%, respectively, of the total DNA, implying that Ads_exDNA and Dis_exDNA are nonnegligible potential pools for the horizontal transfer of ARGs.

Release of antibiotics into the environment, which often occurs downstream of wastewater treatment plants, poses a human health threat due to the potential development of bacterial antibiotic resistance. In this study, laboratory experiments were conducted to evaluate the performance of ball milled biochar on the removal of two sulfonamide antibiotics, sulfamethoxazole (SMX) and sulfapyridine (SPY) from water and wastewater. Aqueous batch sorption experiment using both pristine and ball milled biochar derived from bagasse (BG), bamboo (BB) and hickory chips (HC), made at three pyrolysis temperatures (300, 450, 600 °C), showed that ball milling greatly enhanced the SMX and SPY adsorption. The 450 °C ball milled HC biochar and BB biochar exhibited the best removal efficiency for SMX (83.3%) and SPY (89.6%), respectively. A range of functional groups were produced by ball milling, leading to the conclusion that the adsorption of sulfonamides on the biochars was controlled by multiple mechanisms including hydrophobic interaction, π–π interaction, hydrogen bonding, and electrostatic interaction. Due to the importance of electrostatic interaction, SMX and SPY adsorption was pH dependent. In laboratory water solutions, the Langmuir maximum adsorption capacities of SMX and SPY reached 100.3 mg/g and 57.9 mg/g, respectively. When tested in real wastewater solution, the 450 °C ball milled biochar still performed well, especially in the removal of SPY. The maximum adsorption capacities of SMX and SPY in wastewater were 25.7 mg/g and 58.6 mg/g, respectively. Thus, ball milled biochar has great potential for SMX and SPY removal from aqueous solutions including wastewater.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) have become a critical global public health issue in this century. There is increasing evidence for the presence and transmission of ARGs by air transmission. In this research, ARGs and ARB in air conditioner filter dust (AC dust) and urine samples from 55 kindergarten children in 17 kindergartens and nearby 10 soil samples in Hong Kong were analyzed. The results showed the presence of 16 ARG subtypes and the mobile genetic element (MGE) intI1 in AC dust, and 12 ARG subtypes in the soil samples. ARGs presenting resistance to sulfonamide (6.9 × 10⁻³–0.17) (expressed as relative abundance of the 16 S rRNA genes) were most abundant followed by macrolides (1.8 × 10⁻³–3.3 × 10⁻²), sul1, sul2 (sulfonamide), ermF (macrolides) and intI1 genes in AC dust in 17 kindergartens. For soil samples, 12 ARG subtypes and the intI1 were detected, and the genes providing resistance to sulfonamide (1.6 × 10⁻³–2.7 × 10⁻¹) were the most abundant ARGs in the 10 soil samples, followed by tetracycline (ND–1.4 × 10⁻²). Multi-resistant bacteria with sul1, sul2, intI1, or tetQ were detected in all AC dust samples and some urine samples. Based on bacterial genera and ARG co-occurrence network analysis and Hong Kong’s special geographical location and cultural environment, there might be two origins for the ARGs detected in the kindergartens: β-lactam/macrolide ARGs mainly derived from human medicine use and tetracycline/sulfonamide ARGs mainly from other areas, as well as IntI1 may play a role in the spread of ARGs in Hong Kong. The widely detection of ARGs in AC dust in kindergartens in Hong Kong highlights the need for the improvement of management measures.

Wastewater effluents released in surface water provides suitable nutrient rich environment for the growth and proliferation of antibiotic resistant bacteria (ARB) and genes (ARG). Consequently, bacterial resistance has highly evolved over the recent years and diversified that each antibiotic class is inhibited by a distinct mechanism. In the present study, the prevalence of Multidrug resistant (MDR), extended spectrum β-lactamases (ESBL) and plasmid mediated Amp-C producing strains was analyzed in 28 surface water samples collected near domestic effluent discharge sites in river Ganga located across 11 different geographical indices of Uttar Pradesh, India. A total of 243 bacterial strains with different phenotypes were isolated. Among 243 isolates, 206 (84.77%) exhibited MDR trait displaying maximum resistance towards β-lactams (P = 78.19%; AMX = 72.84%), glycopeptides (VAN = 32.92%; TEI = 79.42%), cephalosporins (CF = 67.90%; CFX = 38.27%), and lincosamides (CD = 78.18%) followed by sulfonamide, macrolide and tetracycline. ESBL production was confirmed in 126 (51.85%) isolates that harbored the genes: blaTEM (95.24%), blaSHV (22.22%), blaOXA (11.90%) and blaCTX-M group (14.28%). The presence of plasmid mediated AmpC was detected only in 6.17% of isolates. The existence of such pathogenic strains in the open environment generates an urgent need for incorporating stringent measures to reduce the antibiotic consumption and hence its release.

The human pathogenic bacteria (HPB) in animal feces may disseminate to agricultural soils with their land application as organic fertilizer. However, the knowledge about the impacts of different sources and rates of animal manures on the temporal changes of soil HPB remains limited, which hamper our ability to estimate the potential risks of their land application. Here, we constructed an HPB database including 565 bacterial strains. By blasting the 16 S rRNA gene sequences against the database we explored the occurrence and fate of HPB in soil microcosms treated with two rates of swine, poultry or cattle manures. A total of 30 HPB were detected in all of manure and soil samples. Poultry manure at the high level obviously improved the abundance of soil HPB. The application of swine manure could introduce concomitant HPB into the soils. Of which, Pseudomonas syringae pv. syringae B728a and Escherichia coli APEC O78 may deserve more attention because of their survival for a few days in manured soils and being possible hosts of diverse antibiotic resistance genes (ARGs) as revealed by co-occurrence pattern. Bayesian source tracking analysis showed that the HPB derived from swine manure had a higher contribution to soil pathogenic communities than those from poultry or cattle manures in early days of incubation. Mantel test together with variation partitioning analysis suggested that bacterial community and soil physicochemical properties were the dominant factors determining the profile of HPB and contributed 64.7% of the total variations. Overall, our results provided experimental evidence that application of animal manures could facilitate the potential dissemination of HPB in soil environment, which should arouse sufficient attention in agriculture practice and management to avoid the threat to human health.

The use of the phenicol antibiotic florfenicol in livestock can select for the optrA gene, which also confers resistance to the critically important oxazolidinone antibiotic linezolid. However, the occurrence and dissemination of florfenicol and linezolid cross-resistance genes in anaerobic treatment systems for livestock waste are unknown. Herein, the phenotypes and genotypes (optrA, fexA, fexB, and cfr) of florfenicol and linezolid cross-resistance were investigated in 339 enterococci strains isolated from lab- and full-scale mesophilic anaerobic digestion systems treating swine waste. It was found that optrA, fexA, and fexB were frequently detected in isolated enterococci in both systems by PCR screening, whereas cfr was not detected. The most abundant gene was optrA, which was detected in 73.5% (n = 50) and 38.9% (n = 23) of enterococci isolates in the full-scale influent and effluent, respectively. Most strains carried more than two resistance genes, and the average percentage of co-occurrence of optrA/fexA was 16.6%. Based on minimum inhibitory concentrations of the enterococci strain phenotypes, 85.7%, 77.5%, and 77.5% of strains in influent were resistant to chloramphenicol, florfenicol, and linezolid, respectively, while 56.3%, 65.2%, and 13% in the effluent isolates were found, respectively, which was consistent with the genotype results. The phenotypes and genotypes of florfenicol and linezolid resistance were relative stable in the enterococci isolated from the influent and effluent in lab-scale anaerobic digestion system. The findings signify the enterococci isolates harboring the optrA gene remained in effluents of both full- and lab-scale swine waste anaerobic digestion system; hence, effective management strategies should be implemented to prevent the discharge of antibiotic resistance from the livestock waste treatment systems.

Antibiotic resistance surveillance data is lacking in many parts of the world, limiting effective therapy and management of resistance development. Analysis of urban wastewater, which contains bacteria from thousands of individuals, opens up possibilities to generate informative surveillance data in a standardized and resource-efficient way. Here, we evaluate the relationship between antibiotic resistance prevalence in E. coli from wastewater and clinical samples by studying countries with different resistance situations as assessed by traditional clinical surveillance. Composite, influent wastewater samples were collected over 24 h from treatment plants serving major cities in ten European countries. Using a broth screening method, resistance to six antibiotic classes was analyzed for 2507 E. coli isolates (n = 247–252 per country). Resistance prevalence in wastewater E. coli was compared to that in clinical E. coli reported by the European Antibiotic Resistance Surveillance Network. Resistance prevalence was lower in wastewater than clinical E. coli but followed similar geographic trends. Significant relationships were found for resistance to aminopenicillins (R² = 0.72, p = 0.0019) and fluoroquinolones (R² = 0.62, p = 0.0072), but not for aminoglycosides (R² = 0.13, p = 0.31) and third-generation cephalosporins (R² = 0.00, p = 0.99) where regression analyses were based on considerably fewer resistant isolates. When all four antibiotic classes were taken into account, the relationship was strong (R² = 0.85, p < 0.0001). Carbapenem resistance was rare in both wastewater and clinical isolates. Wastewater monitoring shows promise as method for generating surveillance data reflecting the clinical prevalence of antibiotic resistant bacteria. Such data may become especially valuable in regions where clinical surveillance is currently limited.

Lincomycin mycelial residues (LMRs) are one kind of byproduct of the pharmaceutical industry. Hydrothermal treatment has been used to dispose of them and land application is an attractive way to reuse the treated LMRs. However, the safe dose for soil amendment remains unclear. In this study, a lab-scale incubation experiment was conducted to investigate the influence of the amendment dosage on lincomycin resistance genes and soil bacterial communities via quantitative PCR and 16S rRNA sequencing. The results showed that introduced lincomycin degraded quickly in soil and became undetectable after 50 days. Degradation rate of the high amendment amount (100 mg kg−1) was almost 4 times faster than that of low amendment amount (10 mg kg−1). Moreover, the introduced LMRs induced the increase of lincomycin resistance genes after incubation for 8 days, and two genes (lmrA and lnuB) showed a dosage-related increase. For example, the abundance of gene lmrA was 17.78, 74.13 and 128.82 copies g−1 soil for lincomycin concentration of 10, 50 and 100 mg kg−1, respectively. However, the abundance of lincomycin resistance genes recovered to the control level as the incubation period extended to 50 days, indicating a low persistence in soil. In addition, LMRs application markedly shifted the bacterial composition and significant difference was found between control soil, 10 mg kg−1 and 50 mg kg−1 lincomycin amended soil. Actually, several genera bacteria were significantly related to the elevation of lincomycin resistance genes. These results provided a comprehensive understanding of the effects of lincomycin dosage on the fate of resistance genes and microbial communities in LMRs applied soil.

Growing evidence suggests that microplastics can adsorb antibiotics and may consequently exacerbate effects on the health of exposed organisms. Our current understanding of the combined effects of microplastics and antibiotics on antibiotic resistance genes (ARGs) in soil invertebrates is limited. This study aimed to investigate changes in the microbiome and ARGs in Enchytraeus crypticus following exposure to a soil environment that contained both microplastics and antibiotics. Tetracycline (TC), polyamide (PA) and polyvinyl chloride (PVC) were used to construct microcosms of polluted soil environments (TC, PA, PVC, PA+TC, PVC+TC). The differences in microbiomes and ARGs were determined by bacterial 16S rRNA gene amplicon sequencing and high throughput quantitative PCR. The results show that compared with the Control or microplastics alone treatments, TC was significantly accumulated in E. crypticus when exposed to TC alone or in combination with microplastics (P < 0.05), but there were no significant differences about TC accumulation between TC, PA+TC, and PVC+TC treated E. crypticus (P > 0.05). Microplastics and TC significantly disturbed the microbial community, and decreased the microbial alpha diversity of E. crypticus (P < 0.05). However, there were no significant differences between TC, microplastics and their combined exposure treatments, and no toxic synergies on the diversity of E. crypticus microbiome between tetracycline and microplastics in soil environment. All the treatments increased the diversity of ARGs in E. crypticus (39–49 ARGs vs. 25 ARGs of control). In particular, treatments combining PVC and TC or PA and TC exposure resulted in greater ARGs abundance than the treatments when E. crypticus was exposed to PVC, PA or TC alone. These results add to our understanding of the combined effects of microplastics and antibiotics on the ARGs and microbiome of soil invertebrates.