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Article

Performance of a Pilot-Scale Continuous Flow Ozone-Based Hospital Wastewater Treatment System

by
Takashi Azuma
1,
Miwa Katagiri
2,
Naobumi Sasaki
3,
Makoto Kuroda
3,* and
Manabu Watanabe
2,*
1
Department of Pharmaceutical Sciences, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Japan
2
Department of Surgery, Toho University Ohashi Medical Center, Tokyo 153-8515, Japan
3
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
*
Authors to whom correspondence should be addressed.
Antibiotics 2023, 12(5), 932; https://doi.org/10.3390/antibiotics12050932
Submission received: 9 March 2023 / Revised: 16 May 2023 / Accepted: 17 May 2023 / Published: 19 May 2023
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Abstract

:
Antimicrobial resistance (AMR) is becoming a global concern. Recently, research has emerged to evaluate the human and environmental health implications of wastewater from medical facilities and to identify acceptable wastewater treatment methods. In this study, a disinfection wastewater treatment system using an ozone-based continuous flow system was installed in a general hospital located in Japan. The effectiveness of antimicrobial-resistant bacteria (ARB) and antimicrobials in mitigating the environmental impact of hospital wastewater was evaluated. Metagenomic analysis was conducted to characterize the microorganisms in the wastewater before and after treatment. The results demonstrated that ozone treatment enables effective inactivation of general gut bacteria, including Bacteroides, Prevotella, Escherichia coli, Klebsiella, DNA molecules, and ARGs, as well as antimicrobials. Azithromycin and doxycycline removal rates were >99% immediately after treatment, and levofloxacin and vancomycin removal rates remained between 90% and 97% for approximately one month. Clarithromycin was more readily removed than the other antimicrobials (81–91%), and no clear removal trend was observed for ampicillin. Our findings provide a better understanding of the environmental management of hospital wastewater and enhance the effectiveness of disinfection wastewater treatment systems at medical facilities for mitigating the discharge of pollutants into aquatic environments.

1. Introduction

In recent years, antimicrobial resistance (AMR) has become dangerously close to our daily lives, raising concerns about sustainable human development [1,2,3]. A significant issue with AMR is that it not only poses a direct health risk of infecting people through hospital- and community-acquired infections, but also an indirect risk of infecting humans via the environment [4,5]. O’Neill Commission, under the request of the UK government, estimated that if effective measures are not taken to combat the prevalence of AMR, annual global deaths from AMR will increase from 0.7 million in 2014 to 10 million by 2050, surpassing cancer-caused deaths, whereas the economic loss to global GDP will be $100 trillion [6]. According to the latest report in 2022 published in The Lancet, an internationally renowned medical journal, annual deaths attributable to AMR are anticipated to nearly double in five years to 1.27 million in 2019 [7]. The World Health Organization (WHO) has proposed the “One Health” approach as a comprehensive measure for humans-animals-environment and has called for the formulation of national action plans in each country [8]. In Japan, action plans have been established and measures are progressing [2,9].
Previous research has indicated the impact of AMR originating from wastewater entering the environment, despite the fact that the origins of AMR detected in aquatic environments are diverse [3,10,11]. Social interest in the risk management of wastewater originating from medical facilities, such as hospitals, has been rapidly growing [12,13,14]. Previous studies have reported antimicrobial-resistant bacteria (ARB) as a problem in clinical practice, suggesting that hospital wastewater may serve as a reservoir for AMR [15,16,17,18,19]. In addition to ARB, residual antimicrobials can be detected in hospital effluents [12,20,21]. The pollution load of hospital wastewater in aquatic environments ranges from several tens to 71%, with rates varying by country [22,23,24,25,26].
Human environmental risks from AMR environmental contamination include the health impacts associated with direct inoculation exposure to viable bacteria surviving in the environment [27,28,29]. Furthermore, it is important to consider the indirect effects of the propagation of antimicrobial resistance genes (ARGs) on E. coli, which are widely distributed in the environment, by encouraging the development of new AMR, even though the direct health effects of ARGs are considered minor, as is the case with viable bacteria [30,31,32]. Moreover, antimicrobials detected in the aquatic environment could be a contributing factor to the unexpected emergence of AMR from the environment, in addition to the toxic effects on the river environment [33,34,35]. Under these circumstances, assessing the actual situation of ARB and antimicrobials in hospital wastewater and their environmental risk, as well as seeking initiatives to develop treatments that can reduce or eliminate such risks, would contribute to protecting human health as well as improving the quality of medical care [36,37,38]. Furthermore, ARB is considered an important issue in ensuring the safety of the water environment and watershed preservation to secure healthy drinking and irrigation water [39,40].
With the recent remarkable development of science and technology, wastewater treatment systems that are effective in treating hospital wastewater have been developed [41,42,43,44,45]. Among them, ozone treatment has been the focus of research in recent years because it has strong sterilization and pollutant removal potential, including chemical-free deodorizing and residue-free wastewater after treatment [46,47,48]. However, the efficacy of wastewater systems based on ozone treatment for hospital wastewater has been primarily evaluated in small-scale (several hundred milliliters to several L) test systems in laboratories [49,50], with minimal research done on the actual hospital wastewater scale [51,52]. Our research group installed an ozone-based hospital wastewater treatment system in a hospital facility located in an urban Japanese region. A medium-scale batch-type treatment tank (effective volume of 1 m3) was installed in the hospital to evaluate the inactivation effect of ozone treatment on ARB and antimicrobials. Both ARB and residual antimicrobials were reduced to ≤1% after 20 min of treatment [21]. If the wastewater treatment of a hospital on a practical scale is proven to be a feasible solution to environmental AMR concerns, it may be conceived as an effective mechanism against AMR, thus contributing to the One Health initiative [32,53,54]. Furthermore, the results can potentially contribute to both the public interest in regional security and the safety of the local population [55,56,57].
As part of the efforts to implement a hospital wastewater treatment system in society, a pioneering trial has begun to verify the effectiveness of measures utilized to reduce the environmental burden of the hospital wastewater treatment system by conducting continuous treatment of the entire hospital wastewater before discharging it into the public sewer system. Therefore, in the present study, a continuous treatment system that can effectively treat hospital wastewater without interfering with hospital operations was developed. To achieve more effective treatment, we additionally tested an ultraviolet light-emitting diode (UV-LED) [58,59], which has recently been shown to be effective in disinfecting pathogenic microorganisms, including SARS-CoV2 [60,61,62], and is rapidly becoming more widely used. The disinfection process, which uses ozone plus an ozone catalyst, has previously been reported for wastewater treatment plants [63,64,65]. In cases such as hospital wastewater where it is virtually impossible to install large-scale treatment facilities or secure a new hospital site for wastewater treatment, the evaluation of the effectiveness of direct disinfection treatment for untreated raw hospital wastewater is a new challenge of great social interest in terms of hospitals and the environment [66,67]. The effect of applying disinfection wastewater treatment systems to hospital wastewater to reduce the burden of AMR in the environment was determined by clarifying the inactivation effect on ARB, ARGs, and antimicrobials, and analyzing the characteristics of microorganisms in wastewater before and after treatment using metagenomic analysis.

2. Materials and Methods

2.1. Hospital Wastewater Treatment Using Ozone Treatment Based on the Continuous Flow System

An ozone inactivation system for bacteria and antimicrobials present in hospital wastewater based on a continuous flow system was installed at the University Hospital, Ohashi Medical Center (BN; 35.652578° N, 139.683959° E), with a capacity of 319 beds, in Toho University, Tokyo, Japan, as previously reported [21]. Various wastewaters generated as a result of hospital activities were stored in two underground wastewater tanks (influent) with a total volume of 22.5 m3. The annual daily inflow of the wastewater tanks was 50 m3/day, which was approximately equal to the volume of the wastewater tanks. The supernatant was pumped directly into the public sewage system at 2 m3 per discharge with an average frequency of 25 discharges per day (50 m3/day as outflow). First, hospital wastewater from one of the two storage tanks was introduced into wastewater treatment tank 1 (ozone) at a flow rate of 20 L/min, with an effective volume of 1 m3 for ozone treatment. The ozone-treated wastewater was then flowed into wastewater treatment tank 2 (UV-LED) with an effective volume of 1 m3, which was connected to a UV disinfection unit for further inactivation owing to overflow inflow at a flow rate of 20 L/min. Finally, the treated wastewater was returned to the original storage tank on one side using a return pump (flow rate 150 L/min) for circulation to the original storage tanks (influent). The appearance and configuration of the ozone treatment system used in this study are shown in Figure 1, and a three-dimensional (3D) view of the equipment is shown in Figure S1.
Ozone was generated using an ozone generator (Ozonia® TOGC45X, Suez Environment, Paris, France) equipped with a Pressure Swing Adsorption (PSA) oxygen generator. The hospital wastewater in the wastewater treatment tank was circulated using a circulation pump (32LPS5.75E, Ebara Corporation, Tokyo, Japan) at a flow rate of 5 L/min, and ozone gas was fine-bubbled through fine-bubble generating nozzles (YJ-9, For EARTH Co., Ltd., Tokyo, Japan) (microbubbles with diameters between 1 μm and 100 μm, using mode 30–50 μm, and ultrafine bubbles with diameters less than 1 μm in the 50–200 nm [68,69]) and introduced into the wastewater treatment tank. The ozone treatment was performed at an ozone generation rate of 27.5 g/h. UV irradiation was performed using a UV-LED system (DWM13-S06-XX-K, NIKKISO Co., Ltd., Tokyo, Japan) with a peak emission of 280 nm and an intensity of 43 mW/cm2. A portion (1 L) of the solution in this tank was collected 0, 1, 4, 6, 8, 15, and 29 days after the start of the experiment. Basic water quality parameters (biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solids (SS), and total nitrogen (TN)) during treatment in this investigation, along with DNA concentration and total reads of metagenomic DNA sequences, are shown in Table S1. Sodium thiosulfate was immediately added to mitigate the effects of residual ozone on the samples [70,71]. Samples were stored at 4 °C in the dark and processed for 12 h.

2.2. Viable Bacterial Counting of Wastewater Samples

To determine the efficacy of ozone treatment in inactivating potential β-lactam-resistant bacteria, an aliquot (100 µL) of influent or treated wastewater sample was 10-fold serially diluted with phosphate-buffered saline, followed by spreading on non-selective media BTB (Bromothymol Blue, lactose agar; Drigalski Agar, Modified) agar and CHROMagar ESBL plates (bioMérieux S.A., Marcy-l’Étoile, France) for extended-spectrum β-lactamases (ESBL) producing bacteria. Colony-forming units per mL (CFU/mL) were determined at the appropriate dilution for each treatment time point.

2.3. Metagenomic DNA-Seq Analysis of Wastewater Samples

To collect organisms larger than bacteria, ozone-treated wastewater samples were passed through TPP Rapid Filtermax Vacuum Filtration systems (TPP, Trasadingen, Switzerland) in 100 mL bottles fitted with 49 cm2, 0.2 µm polyethersulfone membranes. The membranes were removed from the bottles and stored at −30 °C until DNA extraction. One-fourth of the collected membrane, corresponding to 25 mL of influent or treated water, was cut into small pieces and placed in ZR-96 BashingBead Lysis Tubes (0.1 and 0.5 mm; Zymo Inc., Irvine, CA, USA). Bacterial lysis buffer (800 µL; Roche, Basel, Switzerland) was added to the bead tube, which was then frozen at −30 °C and thawed at 23 °C. The tube was subjected to bead-beating (1500 rpm for 10 min) using a GenoGrinder 2010 homogenizer. After brief centrifugation (8000× g for 3 min), 400 µL of the supernatant was collected. The DNA in the supernatant was purified using a Roche MagNa Pure Compact instrument (DNA_Bacteria_v3 protocol; elution:50 µL). DNA concentration and purity were measured using a Qubit DNA HS kit (Thermo Fisher Scientific, Waltham, MA, USA).
Metagenomic DNA-seq libraries were prepared using the QIAseq FX DNA Library Kit (Qiagen, Hilden, Germany), followed by short-read sequencing using the NextSeq 500 platform (2 × 150-mer paired-end) (Illumina, San Diego, CA, USA). Adapter and low-quality sequences were trimmed using Sickle version 1.33 (https://github.com/najoshi/sickle) considering the following parameters: average quality threshold “−q 20” and minimum length threshold “−l 40.” Metagenomic DNA-seq analysis was performed using clean reads for homology searches without de novo assembly for all subsequent analyses. Detailed scripts and databases are described below.
Taxonomic classification of every read from the metagenomic analysis was performed using mega-BLAST (e-value threshold, 1E−20; identity threshold, 95%) against the NCBI nt database using MePIC2 [72] and was subsequently analyzed using MEGAN 6 [73]. Statistical analysis by ozone and subsequent UV-LED treatments was analyzed using two-way repeated ANOVA (R-Studio 2022.12.0+353).
Resistome analysis using metagenomic DNA-seq reads was performed using ARGs-OAP v3.2.1 against the implemented ARG database [74,75].
All raw read sequence files are available from the DRA/SRA database (Table S2).

2.4. Analytical Procedures for Antimicrobials

A total of six antimicrobials grouped into five classes, β-lactams (ampicillin), new quinolones (levofloxacin), macrolides (azithromycin and clarithromycin), tetracyclines (doxycycline), and glycopeptide (vancomycin) (>98%), were examined in the present investigation on the basis of a previous report on their concentrations and detection frequencies in hospital effluent, wastewater, and river water, both in Japan and around the world [25,76], as well as on the basis of antimicrobial use in clinical sites in Japan [77,78].
The concentrations of the target antimicrobials in the wastewater were analyzed as previously described [21,25]. Briefly, 10 mL of wastewater was filtered through a glass fiber filter (GF/B, 1 μm pore size, Whatman, Maidstone, UK), and the solutions were subjected to OASIS HLB solid-phase extraction cartridges (Waters Corp., Milford, MA, USA) at a flow rate of 1 mL/min. The adsorbed antimicrobials were eluted with 3 mL acetone and 3 mL methanol and then evaporated mildly to dryness under a gentle stream of N2 gas at 37 °C. The residue was solubilized in 200 μL of a 90:10 (v/v) mixture of 0.1% formic acid solution in methanol, and 10 μL of this solution was analyzed using an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC) system coupled to a tandem quadrupole mass spectrometer (TQD, Waters Corp.). Quantification was performed by subtracting the blank data from the corresponding data yielded by the spiked sample solutions to account for matrix effects and losses during sample extraction [79,80]. The recovery rates of antimicrobials in the wastewater influent ranged from 77% to 108% (Table S6), and the limits of detection (LODs) and limits of quantification (LOQs) were calculated as the concentrations at signal-to-noise ratios of 3 and 10, respectively [81,82].

3. Results

3.1. Proportion of Bacteria in Hospital Wastewater after Ozone Treatment

Hospital wastewater was treated with ozone followed by UV-LED irradiation in a continuous-flow pilot plant (Figure 1) for 0, 1, 4, 6, 8, 15, and 29 days after starting continuous treatment. The visible brown color of the wastewater disappeared on day 1, and continuous treatment kept the treated water clear until the end of the treatment on day 29 (Figure 2A). Furthermore, the general water quality parameters (BOD, COD, SS, and TN) did not decrease or differ after the treatment (Table S1).
Although it did not exhibit significant inactivation of the general parameters apart from the visible color, the treated water collected from the ozone or UV-LED tanks showed >90% inactivation of viable bacteria on BTB agar after 1 d of treatment (Figure 2B,C), and it maintained the level of reduced viable bacteria until day 29 (Figure 2C). In addition, the original storage tank (influent) had a reduced CFU of viable bacteria because the original storage tank (influent) received treated water (20 L/min) after ozone/UV-LED treatment from the bypass route (Figure 1). Compared with the bacterial CFU on BTB agar, the potential ESBL-producing Enterobacteriaceae on CHROMagar ESBL demonstrated a rather low susceptibility to ozone/UV-LED treatment, but finally underwent >90% inactivation on day 29 (Figure 2D).
To characterize the actual susceptibility of bacterial genera to ozone, membrane-trapped bacteria in the treated sample (corresponding to the 25 mL water sample) were subjected to genomic DNA extraction (Table S1), and metagenomic DNA-seq analysis was performed (summarized in Table S3). Notable fecal bacteria, Bacteroides and Prevotella, showed 90% less detection following ozone treatment compared with the original storage on day 1 (Figure 3). In addition, the most ubiquitous ESBL producers, Escherichia and Klebsiella, showed >80% less detection from the original storage on day 1 following treatment (Figure 3). On the other hand, the environmental bacteria Acinetobacter and Pseudomonas, which include potential nosocomial pathogens, exhibited increasing CFU after ozone/UV-LED treatment; however, there were low amounts of inputs from the influent tank for these two genera (Figure 3). Further taxonomic classification at the species level using the MEGAN 6 software suggested that possible bacterial isolates, which could be genetically similar to Acinetobacter sp. WCHAc010034, and Pseudomonas spp. LTGT-11-2Z significantly increased growth over 100-fold from day 0 onwards (Table S4). Acinetobacter sp. WCHAc010034 was isolated from hospital sewage in China (BioSample SAMN05356835) and Pseudomonas sp. LTGT-11-2Z was isolated from the roots of Alhagi sparsifolia Shap. in the Taklamakan Desert, China (BioSample: SAMN10219285), suggesting that neither were identified as clinical specimens; however, their pathogenicity remains to be investigated.

3.2. Resistome Analysis in Hospital Wastewater Subjected to Ozone Treatment

In addition to the bacterial taxonomic analysis, ARG resistome analysis was performed using metagenomic DNA-seq reads. The top 12 most abundant ARGs with a combined display of specific numerical values and a composite display of colored bars are shown in Figure 4 (all the results obtained for other ARGs are summarized in Table S5). Class 1 integrons (sul1 and qacEdelta), β-lactamase GES variants (blaGES-15, blaGES--14, and blaGES--5), aminoglycoside acetyl transferase (aac(6′)-31), and tetracycline resistance (tet(39) and tet(36)) were mainly detected in sewage samples. Most of these were significantly inactivated to less than 10% of the sequencing reads on day 1, consistent with the results of the CFU (Figure 2) and metagenomic analyses (Figure 3). No increase in the ARGs was observed during the experiment (Table S5). Additional statistical analysis showed that the effect of ozone and subsequent UV treatment on the wastewater was significant (adjusted p-value < 0.05, two-way repeated ANOVA and pairwise t-test) among Bacteroides, Prevotella, Pseudomonas, Bifidobacterium, and Ruminococcus families, and partial effects on the other bacterial families were also suggested (Figure S2).

3.3. Removal of Antimicrobials by Ozone Treatment

All six targeted antimicrobials were detected in the hospital wastewater before treatment. The detected concentrations of the antimicrobials ranged from 746 ng/L to a maximum of 37.9 μg/L, and the order of the detected concentrations was different for each compound. The detected concentration of each antimicrobial at the start of treatment was 17.9 μg/L ± 17.6 μg/L for ampicillin, 13.6 μg/L ± 6.1 μg/L for levofloxacin, 1.8 μg/L ± 1.5 μg/L for azithromycin, 1.4 μg/L ± 136 ng/L for clarithromycin, 1.1 μg/L ± 95 ng/L for doxycycline, and 11.0 μg/L ± 0.8 μg/L for vancomycin. LC–MS/MS parameters and validations of each antimicrobial and the validation of the recovery rates of antimicrobials in the wastewater and the limits of detection (LODs) and limits of quantification (LOQs) are summarized in Tables S6 and S7. Residual antimicrobials detected in hospital wastewater are thought to originate from antimicrobials used to treat diseases in clinical settings [83]. These values were largely similar to those reported in a survey conducted in an urban hospital located in a different region of Japan (734 ng/L to a maximum of 13.4 µg/L) [84], and largely consistent with those previously reported in other countries [12,85,86]. The time course of the antimicrobial concentrations in hospital wastewater during treatment is summarized in Figure 5, and the detailed concentrations are shown in Table S8.
Ozone treatment was effective in removing all the targeted antimicrobials. More than 99% of azithromycin and doxycycline was removed immediately after treatment and was not detected in the ozone treatment tank throughout the experiment. The removal rates for levofloxacin and vancomycin remained between 90% and 97% during treatment. Clarithromycin was more readily removed than the other antimicrobials (81% to 91%), and no clear removal trend was observed for ampicillin. In the wastewater treatment tank where UV-LED irradiation followed ozonation, ampicillin and levofloxacin additionally decreased from their levels in the ozone treatment tank by an average of 15% and 53%, respectively. However, no notable changes in the concentration were observed for the other compounds. Finally, the concentration of antimicrobials before discharge into the public sewage system decreased during continuous-type treatment. The average removal rates of antimicrobials in the present study were 71 ± 24% for levofloxacin, 82 ± 16% for azithromycin, 88 ± 10% for doxycycline, and 44 ± 42% for vancomycin. However, no clear removal trends for ampicillin and clarithromycin were observed during the experiment, which was largely consistent with the removal trend of antimicrobials in the ozone treatment tank.

4. Discussion

The detection of ARB and antimicrobials in hospital wastewater throughout the period of this treatment test suggests that a certain amount of these pollutants is released from every hospital into the public sewage system, and that it is vital to implement social countermeasures on a large scale. The original storage tank (influent) showed an increase/decrease in the detected concentrations of both ARB and antimicrobials, which was associated with the continuous inflow of untreated raw hospital wastewater into the original tank.
General gut bacteria, including Bacteroides, Prevotella, E. coli, and Klebsiella, were significantly inactivated by ozone treatment (Figure 3). However, environmental bacteria, such as Acinetobacter and Pseudomonas, exhibited notable persistence to ozone, as in a previous batch treatment trial [21]. Based on the AMR issue, the persistence of Acinetobacter and Pseudomonas could play a major role in AMR reservoirs, although ozone treatment might act efficiently. Such persistence remains to be characterized in future studies to achieve a sustainable treatment process under continuous hospital operation. This study demonstrated that ozone treatment enabled the effective inactivation of viable bacteria (Figure 3) and DNA molecules, including ARGs (Figure 4); however, the general water quality parameters did not differ (BOD, COD, SS, and TN) (Table S1). This finding strongly suggests that such parameters may not be essential criteria for reducing hospital-associated AMR factors because ozone causes partial damage to viable organisms and DNA molecules, leading to dead bacteria and damaged DNA molecules that no longer function.
The characteristics of the components most likely to be removed (>90% removal after 10 min treatment) were consistent for the antimicrobials and for ampicillin and clarithromycin, which were not adequately removed by the sequential treatment in this study; the removal rate was 96–100% for these antimicrobials at 40 min after treatment. These data suggest that it is essential to upgrade the treatment system and conduct demonstration tests and evaluations at an actual plant scale when developing from a batch (small-scale) system to the actual treatment of hospital wastewater.
The results of the present investigation show that ozone treatment removes antimicrobials, and the treatment time required for removal differs for each compound, supporting the results reported in previous evaluations of ozonation of pharmaceuticals in environmental water [87,88]. Antimicrobials, which tended to remain in the treated water compared to other antimicrobials, such as clarithromycin and ampicillin, could possibly be removed by increasing the ozone injection volume and prolonged treatment (high ozone exposure volume) [89,90]. Additionally, it would be effective to combine ozone treatment with other treatments for environmental pollutants that are difficult to treat effectively with ozone treatment alone. The fact that the UV-LED treatment in this study further improved the removal rates of ampicillin and levofloxacin [91,92], which are known to be easily degraded by light irradiation in the UV region, will prove useful when examining the effectiveness of the treatment for a wide spectrum of environmental pollutants in wastewater. Some antimicrobials, including β-lactam antimicrobials such as ampicillin, are attenuated in water within a few hours [93,94]. Clarithromycin is highly persistent in the environment as it is not susceptible to attenuation in the aquatic environment through photolysis or biodegradation [95,96]. Levofloxacin is considered the primary antimicrobial agent that causes clinical problems with ARB [97]. Previous studies have reported that ozone and/or UV treatment reduces ecotoxicological effects to approximately 1/10–1/20,000 compared to untreated compounds [47,98,99,100]. On the other hand, some researchers pointed out that the toxicity increases approximately 2–100 fold in some cases [99,100]. It is known that differences in susceptibility to these toxic effects occur in different target species, and that under conditions where multiple compounds coexist, weakening or strengthening effects would occur compared to exposure to a single substance [101,102]. Whole effluent toxicity (WET), as recommended by the US Environmental Protection Agency (EPA), which evaluates the toxic effects of a target water body as a whole using species at different trophic levels, would be a comprehensive approach to address these issues [103,104,105]. In addition, the strong oxidizing action of ozone or hydroxyl radicals can decrease the formation of transformation products by providing sufficient processing time and by acting in combination with catalysts, such as UV and hydrogen peroxide [46,106,107]. The results demonstrated that when antimicrobials with an environmental impact were effectively removed before flowing into the environment and kept at a low level, they are notable as an effective measure to reduce the environmental impact caused by AMR.
It is possible to reduce or eliminate the inflow of ARB and antimicrobials as environmental pollutants into the aquatic environment through the advancement of wastewater treatment systems [3,65,108]. In addition, it is important to conduct further optimization and refinement tests to maximize the synergistic effect of ozone treatment with an ozone catalyst, which is often applied to secondary-treated water after biological treatment in the wastewater treatment area [44,47,109]. However, a practical issue persists in that the cost of these advanced treatments increases as treatment systems become sophisticated and/or multiple treatments are combined [13,110]. Research that considers the cost aspects of actual implementation should be conducted in the future. The operating costs of this disinfectant system were approximately US $300 for one month using the maximum electronic power of the system, which could be an affordable cost for AMR disinfectants. The legalization of the required reduction levels in conjunction with research on environmental risk assessment for AMR discharge into the aquatic environment, as well as the promotion of the development of new mitigation strategies for dealing with AMR from an environmental perspective, needs to be emphasized. In addition, it will be challenging to deepen social understanding and support hospital incentives. Further development and return to society in both academia and industry are required.
As studies have revealed the antimicrobial-resistant nature of hospital wastewater and the significant impact of the loads discharged into the environment, more attention is being paid to how hospital wastewater should be treated [12,56]. However, research on environmental management and mitigation control of ARB and antimicrobials in hospital wastewater is still limited worldwide, owing to the general difficulty in researching hospital wastewater [111,112,113]. To the best of our knowledge, this is the first report on the effectiveness of a pilot-scale continuous wastewater treatment system based on ozonation for the inactivation of ARB and antimicrobials in the entire effluent generated by a hospital.

5. Limitations

The limitations of this study are as follows: The first is the optimization of wastewater treatment systems. An ideal wastewater treatment system involves the continuous direct discharge of treated water into a public sewage system. However, there are still issues that have not been fully covered in the present investigation in terms of practical aspects, such as technology, funding, and the need to reconstruct the entire hospital facility to continue treating the entire hospital wastewater while maintaining a balance with the constant inflow of untreated wastewater [114,115]. Another issue is the technical restrictions in maintaining the effect of UV light from UV-LED for a long period because raw hospital wastewater contains multiple solid organic substances. Further improvements in these aspects of the processing equipment need to be examined in the future.
Second, the inactivation effects of ARB and antimicrobials were investigated. Neither ARB nor antimicrobials were completely inactivated during the trial of hospital wastewater treatment. Under these conditions, microorganisms remain viable in the treated hospital wastewater. It is essential to elucidate the potential of these microorganisms to form biofilms in treatment tanks [116] and to evaluate the pathogenicity and environmental impact of microorganisms that require more ozone than other microorganisms [66,117].
Finally, the treatment effectiveness for the basic general water quality parameters was noted. For the hospital wastewater treatment in this study, the treatment system was found to be capable of inactivating ARB and antimicrobials, which have become new environmental pollutants of concern in recent years, although some improvements, including water quality, have not yet been achieved. Improving the treatment to include these water quality items can be achieved by increasing the amount of ozone injected, but in some respects, this treatment strategy is not practical or energy-efficient and it would be more effective in combination with other treatments [118,119,120]. These issues can be improved via biodegradation, as it is known that ozone treatment alone makes it difficult to convert a persistent substance into a biodegradable substance [47,49]. Our results support the need for further conclusive research considering experimental, technical, and regional customs, bias, and unknown factors.

6. Conclusions

In the present study, an ozonation-based continuous-flow disinfection wastewater treatment system was implemented in a core hospital located in the center of Japan, and its effectiveness in mitigating the environmental impact of AMR associated with hospital wastewater was evaluated. The results showed that both ARB and antimicrobials that would have an impact on the environment were effectively removed and maintained at a low level during treatment, which would be an effective countermeasure to mitigate the environmental impact caused by AMR. These findings are significant for implementing feasible and effective countermeasures to address AMR in the environment. The overall results facilitate a comprehensive understanding of the AMR risk posed by hospital wastewater and provide insights for devising strategies to eliminate or mitigate the burden of ARB and flow of antimicrobials into aquatic environments. Our findings could help enhance the effectiveness of introducing wastewater treatment systems, not only in wastewater treatment plants, but also in medical facilities, to reduce the discharge of pollutants into rivers, thereby contributing to environmental and human health safety.

Supplementary Materials

The following supporting information was downloaded from https://www.mdpi.com/article/10.3390/antibiotics12050932/s1. Figure S1. 3D view of the equipment of the continuous-flow pilot-scale ozone treatment system implemented in a hospital facility; Figure S2. Two-way repeated ANOVA of each bacterial family based on the effect of ozone and subsequent UV treatment on the wastewater (adjusted p-value < 0.05, two-way repeated ANOVA and pairwise t-test). Table S1. Summary of water quality parameters during hospital wastewater treatment with ozone; Table S2. All the raw read sequence files were obtained from the DRA/SRA database; able S3. Counts of sequencing reads were detected for each bacterial genus using metagenomic DNA-seq analysis; Table S4. Counts of sequencing reads for Acinetobacter or Pseudomonas species detected by metagenomic DNA-seq analysis; Table S5. Counts of sequencing reads for each antimicrobial resistance gene (ARG) detected using metagenomic DNA-seq analysis; Table S6. LC-MS/MS parameters and validation of each antimicrobial; Table S7. Validation of the method characteristics for the analysis of antimicrobials in wastewater; Table S8. Concentration of targeted antimicrobials in hospital wastewater during continuous-flow treatment.

Author Contributions

M.K. (Miwa Katagiri), M.K. (Makoto Kuroda) and M.W. collected water samples. Conceptualization: T.A., M.K. (Makoto Kuroda) and M.W.; investigation: T.A., M.K. (Miwa Katagiri), N.S. and M.K. (Makoto Kuroda); methodology: T.A., N.S., M.K. (Makoto Kuroda) and M.W.; formal analysis: T.A., M.K. (Miwa Katagiri) N.S., M.K. (Makoto Kuroda) and M.W.; writing—original draft: T.A., M.K. (Makoto Kuroda) and M.W.; writing—review and editing: T.A., M.K. (Miwa Katagiri), N.S., M.K. (Makoto Kuroda) and M.W.; supervision: T.A., M.K. (Makoto Kuroda) and M.W.; funding acquisition: T.A., M.K. (Makoto Kuroda) and M.W.; project administration: M.K. (Makoto Kuroda) and M.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Research Program on Emerging and Reemerging Infectious Diseases of the Japan Agency for Medical Research and Development (grant number: JP22fk0108131). This work was also supported by a grant for research on emerging and reemerging infectious diseases and immunization (H30 Shinkogyosei-Ippan-002 and 21HA1002) from the Ministry of Health, Labor and Welfare, Japan and research grants and scholarships (20H02289) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All raw read sequence files are available from the DRA/SRA database (accession numbers DRR439511–DRR439531 [see Table S2]).

Acknowledgments

We would like to thank Hiroki Furuya (For EARTH Co., Ltd. (fine bubble generator and technical data provided by YJ nozzle developer Takashi Yamamoto)) and Takashi Ichinose (To-rei Co., Ltd.) for technical support with the ozone treatment system and the facility staff at Toho University Ohashi Medical Center. We would like to thank Rina Tanaka and Risa Someno for their help with whole-genome sequencing of wastewater samples.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Li, S.; Zhang, C.; Li, F.; Hua, T.; Zhou, Q.; Ho, S.H. Technologies towards antibiotic resistance genes (ARGs) removal from aquatic environment: A critical review. J. Hazard. Mater. 2021, 411, 125148. [Google Scholar] [CrossRef] [PubMed]
  2. Baba, H.; Nishiyama, M.; Watanabe, T.; Kanamori, H. Review of antimicrobial resistance in wastewater in Japan: Current challenges and future perspectives. Antibiotics 2022, 11, 849. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, Y.; Han, Y.; Li, L.; Liu, J.; Yan, X. Distribution, sources, and potential risks of antibiotic resistance genes in wastewater treatment plant: A review. Environ. Pollut. 2022, 310, 119870. [Google Scholar] [CrossRef] [PubMed]
  4. World Health Organization (WHO). Antimicrobial Resistance: Global Report on Surveillance; World Health Organization: Geneva, Switzerland, 2014; pp. 1–232. [Google Scholar]
  5. US Department of Health and Human Services. Centers for Disease Control and Prevention (CDC). Antibiotic Resistance Threats in the United States. 2019; p. 1. Available online: https://www.cdc.gov/drugresistance/biggest-threats.html (accessed on 18 February 2023).
  6. Jim, O.N. Antimicrobial resistance: Tackling a crisis for the health and wealth of nations. Rev. Antimicrob. Resist. 2014, 1–16. [Google Scholar]
  7. Murray, C.J.L.; Ikuta, K.S.; Sharara, F.; Swetschinski, L.; Robles Aguilar, G.; Gray, A.; Han, C.; Bisignano, C.; Rao, P.; Wool, E.; et al. Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. Lancet 2022, 399, 629–655. [Google Scholar] [CrossRef]
  8. World Health Organization (WHO). Global Action Plan on Antimicrobial Resistance; World Health Organization: Geneva, Switzerland, 2015; pp. 1–19. [Google Scholar]
  9. National Action Plan on Antimicrobial Resistance (AMR) (2016–2020); The Government of Japan: Tokyo, Japan, 2016; pp. 1–69.
  10. Pazda, M.; Kumirska, J.; Stepnowski, P.; Mulkiewicz, E. Antibiotic resistance genes identified in wastewater treatment plant systems–A review. Sci. Total Environ. 2019, 697, 134023. [Google Scholar] [CrossRef]
  11. Ezeuko, A.S.; Ojemaye, M.O.; Okoh, O.O.; Okoh, A.I. Technological advancement for eliminating antibiotic resistance genes from wastewater: A review of their mechanisms and progress. J. Environ. Chem. Eng. 2021, 9, 106183. [Google Scholar] [CrossRef]
  12. Cai, M.; Wang, Z.; Gu, H.; Dong, H.; Zhang, X.; Cui, N.; Zhou, L.; Chen, G.; Zou, G. Occurrence and temporal variation of antibiotics and antibiotic resistance genes in hospital inpatient department wastewater: Impacts of daily schedule of inpatients and wastewater treatment process. Chemosphere 2022, 292, 133405. [Google Scholar] [CrossRef]
  13. Pariente, M.I.; Segura, Y.; Álvarez-Torrellas, S.; Casas, J.A.; de Pedro, Z.M.; Diaz, E.; García, J.; López-Muñoz, M.J.; Marugán, J.; Mohedano, A.F.; et al. Critical review of technologies for the on-site treatment of hospital wastewater: From conventional to combined advanced processes. J. Environ. Manag. 2022, 320, 115769. [Google Scholar] [CrossRef]
  14. Ulvi, A.; Aydın, S.; Aydın, M.E. Fate of selected pharmaceuticals in hospital and municipal wastewater effluent: Occurrence, removal, and environmental risk assessment. Environ. Sci. Pollut. Res. 2022, 29, 75609–75625. [Google Scholar] [CrossRef]
  15. Cahill, N.; O’Connor, L.; Mahon, B.; Varley, Á.; McGrath, E.; Ryan, P.; Cormican, M.; Brehony, C.; Jolley, K.A.; Maiden, M.C.; et al. Hospital effluent: A reservoir for carbapenemase-producing Enterobacterales? Sci. Total Environ. 2019, 672, 618–624. [Google Scholar] [CrossRef] [PubMed]
  16. Al Salah, D.M.M.; Ngweme, G.N.; Laffite, A.; Otamonga, J.-P.; Mulaji, C.; Poté, J. Hospital wastewaters: A reservoir and source of clinically relevant bacteria and antibiotic resistant genes dissemination in urban river under tropical conditions. Ecotoxicol. Environ. Saf. 2020, 200, 110767. [Google Scholar] [CrossRef] [PubMed]
  17. Gwenzi, W.; Musiyiwa, K.; Mangori, L. Sources, behaviour and health risks of antimicrobial resistance genes in wastewaters: A hotspot reservoir. J. Environ. Chem. Eng. 2020, 8, 102220. [Google Scholar] [CrossRef]
  18. Sekizuka, T.; Itokawa, K.; Tanaka, R.; Hashino, M.; Yatsu, K.; Kuroda, M. Metagenomic analysis of urban wastewater treatment plant effluents in tokyo. Infect. Drug Resist. 2022, 15, 4763–4777. [Google Scholar] [CrossRef] [PubMed]
  19. Sekizuka, T.; Tanaka, R.; Hashino, M.; Yatsu, K.; Kuroda, M. Comprehensive genome and plasmidome analysis of antimicrobial resistant bacteria in wastewater treatment plant effluent of Tokyo. Antibiotics 2022, 11, 1283. [Google Scholar] [CrossRef] [PubMed]
  20. Yao, S.; Ye, J.; Yang, Q.; Hu, Y.; Zhang, T.; Jiang, L.; Munezero, S.; Lin, K.; Cui, C. Occurrence and removal of antibiotics, antibiotic resistance genes, and bacterial communities in hospital wastewater. Environ. Sci. Pollut. Res. 2021, 28, 57321–57333. [Google Scholar] [CrossRef]
  21. Azuma, T.; Katagiri, M.; Sekizuka, T.; Kuroda, M.; Watanabe, M. Inactivation of bacteria and residual antimicrobials in hospital wastewater by ozone treatment. Antibiotics 2022, 11, 862. [Google Scholar] [CrossRef]
  22. Weissbrodt, D.; Kovalova, L.; Ort, C.; Pazhepurackel, V.; Moser, R.; Hollender, J.; Siegrist, H.; McArdell, C.S. Mass flows of X-ray contrast media and cytostatics in hospital wastewater. Environ. Sci. Technol. 2009, 43, 4810–4817. [Google Scholar] [CrossRef]
  23. Santos, L.H.M.L.M.; Gros, M.; Rodriguez-Mozaz, S.; Delerue-Matos, C.; Pena, A.; Barceló, D.; Montenegro, M.C.B.S.M. Contribution of hospital effluents to the load of pharmaceuticals in urban wastewaters: Identification of ecologically relevant pharmaceuticals. Sci. Total Environ. 2013, 461–462, 302–316. [Google Scholar] [CrossRef]
  24. Aydin, S.; Aydin, M.E.; Ulvi, A.; Kilic, H. Antibiotics in hospital effluents: Occurrence, contribution to urban wastewater, removal in a wastewater treatment plant, and environmental risk assessment. Environ. Sci. Pollut. Res. 2019, 26, 544–558. [Google Scholar] [CrossRef]
  25. Azuma, T.; Otomo, K.; Kunitou, M.; Shimizu, M.; Hosomaru, K.; Mikata, S.; Ishida, M.; Hisamatsu, K.; Yunoki, A.; Mino, Y.; et al. ; et al. Environmental fate of pharmaceutical compounds and antimicrobial-resistant bacteria in hospital effluents, and contributions to pollutant loads in the surface waters in Japan. Sci. Total Environ. 2019, 657, 476–484. [Google Scholar] [CrossRef] [PubMed]
  26. Afsa, S.; Hamden, K.; Lara Martin, P.A.; Mansour, H.B. Occurrence of 40 pharmaceutically active compounds in hospital and urban wastewaters and their contribution to mahdia coastal seawater contamination. Environ. Sci. Pollut. Res. 2020, 27, 1941–1955. [Google Scholar] [CrossRef] [PubMed]
  27. Harbarth, S.; Balkhy, H.H.; Goossens, H.; Jarlier, V.; Kluytmans, J.; Laxminarayan, R.; Saam, M.; Van Belkum, A.; Pittet, D. Antimicrobial resistance: One world, one fight! Antimicrob. Resist. Infect. Control 2015, 4, 49. [Google Scholar] [CrossRef]
  28. Noman, E.; Al-Gheethi, A.; Radin Mohamed, R.M.S.; Talip, B.; Al-Sahari, M.; Al-Shaibani, M. Quantitative microbiological risk assessment of complex microbial community in prawn farm wastewater and applicability of nanoparticles and probiotics for eliminating of antibiotic-resistant bacteria. J. Hazard. Mater. 2021, 419, 126418. [Google Scholar] [CrossRef] [PubMed]
  29. Schoen, M.E.; Jahne, M.A.; Garland, J.; Ramirez, L.; Lopatkin, A.J.; Hamilton, K.A. Quantitative microbial risk assessment of antimicrobial resistant and susceptible Staphylococcus aureus in reclaimed wastewaters. Environ. Sci. Technol. 2021, 55, 15246–15255. [Google Scholar] [CrossRef] [PubMed]
  30. Zainab, S.M.; Junaid, M.; Xu, N.; Malik, R.N. Antibiotics and antibiotic resistant genes (ATGs) in groundwater: A global review on dissemination, sources, interactions, environmental and human health risks. Water Res. 2020, 187, 116455. [Google Scholar] [CrossRef]
  31. Anand, U.; Reddy, B.; Singh, V.K.; Singh, A.K.; Kesari, K.K.; Tripathi, P.; Kumar, P.; Tripathi, V.; Simal-Gandara, J. Potential environmental and human health risks caused by antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs) and emerging contaminants (ECS) from municipal solid waste (MSW) landfill. Antibiotics 2021, 10, 374. [Google Scholar] [CrossRef]
  32. Booton, R.D.; Meeyai, A.; Alhusein, N.; Buller, H.; Feil, E.; Lambert, H.; Mongkolsuk, S.; Pitchforth, E.; Reyher, K.K.; Sakcamduang, W.; et al. ; et al. One health drivers of antibacterial resistance: Quantifying the relative impacts of human, animal and environmental use and transmission. One Health 2021, 12, 100220. [Google Scholar] [CrossRef]
  33. González-Plaza, J.J.; Blau, K.; Milaković, M.; Jurina, T.; Smalla, K.; Udiković-Kolić, N. Antibiotic-manufacturing sites are hot-spots for the release and spread of antibiotic resistance genes and mobile genetic elements in receiving aquatic environments. Environ. Int. 2019, 130, 104735. [Google Scholar] [CrossRef]
  34. Menz, J.; Olsson, O.; Kümmerer, K. Antibiotic residues in livestock manure: Does the eu risk assessment sufficiently protect against microbial toxicity and selection of resistant bacteria in the environment? J. Hazard. Mater. 2019, 379, 120807. [Google Scholar] [CrossRef]
  35. Hossain, A.; Habibullah-Al-Mamun, M.; Nagano, I.; Masunaga, S.; Kitazawa, D.; Matsuda, H. Antibiotics, antibiotic-resistant bacteria, and resistance genes in aquaculture: Risks, current concern, and future thinking. Environ. Sci. Pollut. Res. 2022, 29, 11054–11075. [Google Scholar] [CrossRef] [PubMed]
  36. He, H.; Zhou, P.; Shimabuku, K.K.; Fang, X.; Li, S.; Lee, Y.; Dodd, M.C. Degradation and deactivation of bacterial antibiotic resistance genes during exposure to free chlorine, monochloramine, chlorine dioxide, ozone, ultraviolet light, and hydroxyl radical. Environ. Sci. Technol. 2019, 53, 2013–2026. [Google Scholar] [CrossRef] [PubMed]
  37. Khan, N.A. Hospital Wastewater Treatment: Global Scenario and Case Studies; IWA Publishing: London, UK, 2022. [Google Scholar]
  38. Parida, V.K.; Sikarwar, D.; Majumder, A.; Gupta, A.K. An assessment of hospital wastewater and biomedical waste generation, existing legislations, risk assessment, treatment processes, and scenario during COVID-19. J. Environ. Manag. 2022, 308, 114609. [Google Scholar] [CrossRef] [PubMed]
  39. Anthony, E.T.; Ojemaye, M.O.; Okoh, O.O.; Okoh, A.I. A critical review on the occurrence of resistomes in the environment and their removal from wastewater using apposite treatment technologies: Limitations, successes and future improvement. Environ. Pollut. 2020, 263, 113791. [Google Scholar] [CrossRef]
  40. Mahaney, A.P.; Franklin, R.B. Persistence of wastewater-associated antibiotic resistant bacteria in river microcosms. Sci. Total Environ. 2022, 819, 153099. [Google Scholar] [CrossRef]
  41. Ahmed, S.; Khan, F.S.A.; Mubarak, N.M.; Khalid, M.; Tan, Y.H.; Mazari, S.A.; Karri, R.R.; Abdullah, E.C. Emerging pollutants and their removal using visible-light responsive photocatalysis– A comprehensive review. J. Environ. Chem. Eng. 2021, 9, 106643. [Google Scholar] [CrossRef]
  42. Ao, X.; Eloranta, J.; Huang, C.H.; Santoro, D.; Sun, W.; Lu, Z.; Li, C. Peracetic acid-based advanced oxidation processes for decontamination and disinfection of water: A review. Water Res. 2021, 188, 116479. [Google Scholar] [CrossRef]
  43. Divyapriya, G.; Singh, S.; Martínez-Huitle, C.A.; Scaria, J.; Karim, A.V.; Nidheesh, P.V. Treatment of real wastewater by photoelectrochemical methods: An overview. Chemosphere 2021, 276, 130188. [Google Scholar] [CrossRef]
  44. Asghar, A.; Lutze, H.V.; Tuerk, J.; Schmidt, T.C. Influence of water matrix on the degradation of organic micropollutants by ozone based processes: A review on oxidant scavenging mechanism. J. Hazard. Mater. 2022, 429, 128189. [Google Scholar] [CrossRef]
  45. Zhou, S.; Marcelino, K.R.; Wongkiew, S.; Sun, L.; Guo, W.; Khanal, S.K.; Lu, H. Untapped potential: Applying microbubble and nanobubble technology in water and wastewater treatment and ecological restoration. ACS ES&T Eng. 2022, 2, 1558–1573. [Google Scholar]
  46. Loeb, B.L. Forty years of advances in ozone technology. A review of ozone: Science & engineering. Ozone Sci. Eng. 2018, 40, 3–20. [Google Scholar]
  47. Rekhate, C.V.; Srivastava, J.K. Recent advances in ozone-based advanced oxidation processes for treatment of wastewater- a review. Chem. Eng. J. Adv. 2020, 3, 100031. [Google Scholar] [CrossRef]
  48. Lim, S.; Shi, J.L.; von Gunten, U.; McCurry, D.L. Ozonation of organic compounds in water and wastewater: A critical review. Water Res. 2022, 213, 118053. [Google Scholar] [CrossRef]
  49. Aleksić, S.; Žgajnar Gotvajn, A.; Premzl, K.; Kolar, M.; Turk, S.Š. Ozonation of amoxicillin and ciprofloxacin in model hospital wastewater to increase biotreatability. Antibiotics 2021, 10, 1407. [Google Scholar] [CrossRef] [PubMed]
  50. Azuma, T.; Hayashi, T. Disinfection of antibiotic-resistant bacteria in sewage and hospital effluent by ozonation. Ozone Sci. Eng. 2021, 43, 413–426. [Google Scholar] [CrossRef]
  51. Czekalski, N.; Imminger, S.; Salhi, E.; Veljkovic, M.; Kleffel, K.; Drissner, D.; Hammes, F.; Bürgmann, H.; von Gunten, U. Inactivation of antibiotic resistant bacteria and resistance genes by ozone: From laboratory experiments to full-scale wastewater treatment. Environ. Sci. Technol. 2016, 50, 11862–11871. [Google Scholar] [CrossRef]
  52. Basturk, I.; Varank, G.; Murat-Hocaoglu, S.; Yazici-Guvenc, S.; Oktem-Olgun, E.E.; Canli, O. Characterization and treatment of medical laboratory wastewater by ozonation: Optimization of toxicity removal by central composite design. Ozone Sci. Eng. 2021, 43, 213–227. [Google Scholar] [CrossRef]
  53. Walsh, T.R. A one-health approach to antimicrobial resistance. Nat. Microbiol. 2018, 3, 854–855. [Google Scholar] [CrossRef]
  54. Miłobedzka, A.; Ferreira, C.; Vaz-Moreira, I.; Calderón-Franco, D.; Gorecki, A.; Purkrtova, S.; Jan, B.; Dziewit, L.; Singleton, C.M.; Nielsen, P.H.; et al. ; et al. Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health Cycle. J. Hazard. Mater. 2022, 424, 127407. [Google Scholar] [CrossRef]
  55. Hernando-Amado, S.; Coque, T.M.; Baquero, F.; Martínez, J.L. Antibiotic resistance: Moving from individual health norms to social norms in one health and global health. Front. Microbiol. 2020, 11, 01914. [Google Scholar] [CrossRef]
  56. Lépesová, K.; Olejníková, P.; Mackuľak, T.; Cverenkárová, K.; Krahulcová, M.; Bírošová, L. Hospital wastewater—Important source of multidrug resistant coliform bacteria with ESBL-production. Int. J. Environ. Res. Public Health 2020, 17, 7827. [Google Scholar] [CrossRef] [PubMed]
  57. Qiu, D.; Ke, M.; Zhang, Q.; Zhang, F.; Lu, T.; Sun, L.; Qian, H. Response of microbial antibiotic resistance to pesticides: An emerging health threat. Sci. Total Environ. 2022, 850, 158057. [Google Scholar] [CrossRef] [PubMed]
  58. Keshavarzfathy, M.; Malayeri, A.H.; Mohseni, M.; Taghipour, F. Uv-led fluence determination by numerical method for microbial inactivation studies. J.Photochem. Photobiol. A Chem. 2020, 392, 112406. [Google Scholar] [CrossRef]
  59. Shimoda, H.; Matsuda, J.; Iwasaki, T.; Hayasaka, D. Efficacy of 265-nm ultraviolet light in inactivating infectious SARS-CoV-2. J. Photochem. Photobiol. 2021, 7, 100050. [Google Scholar] [CrossRef]
  60. Biancullo, F.; Moreira, N.F.F.; Ribeiro, A.R.; Manaia, C.M.; Faria, J.L.; Nunes, O.C.; Castro-Silva, S.M.; Silva, A.M.T. Heterogeneous photocatalysis using UVA-LEDs for the removal of antibiotics and antibiotic resistant bacteria from urban wastewater treatment plant effluents. Chem. Eng. J. 2019, 367, 304–313. [Google Scholar] [CrossRef]
  61. Gerchman, Y.; Mamane, H.; Friedman, N.; Mandelboim, M. Uv-led disinfection of coronavirus: Wavelength effect. J. Photochem. Photobiol. B Biol. 2020, 212, 112044. [Google Scholar] [CrossRef]
  62. Wan, Q.; Cao, R.; Wen, G.; Xu, X.; Xia, Y.; Wu, G.; Li, Y.; Wang, J.; Xu, H.; Lin, Y.; et al. Efficacy of UV-LED based advanced disinfection processes in the inactivation of waterborne fungal spores: Kinetics, photoreactivation, mechanism and energy requirements. Sci. Total Environ. 2022, 803, 150107. [Google Scholar] [CrossRef]
  63. Dong, C.; Fang, W.; Yi, Q.; Zhang, J. A comprehensive review on reactive oxygen species (ROS) in advanced oxidation processes (AOPs). Chemosphere 2022, 308, 136205. [Google Scholar] [CrossRef]
  64. Lozano, I.; Pérez-Guzmán, C.J.; Mora, A.; Mahlknecht, J.; Aguilar, C.L.; Cervantes-Avilés, P. Pharmaceuticals and personal care products in water streams: Occurrence, detection, and removal by electrochemical advanced oxidation processes. Sci. Total Environ. 2022, 827, 154348. [Google Scholar] [CrossRef]
  65. Saravanan, A.; Deivayanai, V.C.; Kumar, P.S.; Rangasamy, G.; Hemavathy, R.V.; Harshana, T.; Gayathri, N.; Alagumalai, K. A detailed review on advanced oxidation process in treatment of wastewater: Mechanism, challenges and future outlook. Chemosphere 2022, 308, 136524. [Google Scholar] [CrossRef]
  66. Yuan, T.; Pian, Y. Hospital wastewater as hotspots for pathogenic microorganisms spread into aquatic environment: A review. Front. Environ. Sci. 2023, 10, 1734. [Google Scholar] [CrossRef]
  67. Zhu, L.; Yuan, L.; Shuai, X.Y.; Lin, Z.J.; Sun, Y.J.; Zhou, Z.C.; Meng, L.X.; Ju, F.; Chen, H. Deciphering basic and key traits of antibiotic resistome in influent and effluent of hospital wastewater treatment systems. Water Res. 2023, 231, 119614. [Google Scholar] [CrossRef] [PubMed]
  68. Yamada, S.; Amano, T.; Minagawa, H. A study for distribution of microbubbles and effects of oxygen supplying into water. Trans. Jpn. Soc. Mech. Eng. B 2005, 71, 1301–1306. [Google Scholar] [CrossRef]
  69. Hashimoto, K.; Kubota, N.; Okuda, T.; Nakai, S.; Nishijima, W.; Motoshige, H. Reduction of ozone dosage by using ozone in ultrafine bubbles to reduce sludge volume. Chemosphere 2021, 274, 129922. [Google Scholar] [CrossRef]
  70. Zheng, J.; Su, C.; Zhou, J.; Xu, L.; Qian, Y.; Chen, H. Effects and mechanisms of ultraviolet, chlorination, and ozone disinfection on antibiotic resistance genes in secondary effluents of municipal wastewater treatment plants. Chem. Eng. J. 2017, 317, 309–316. [Google Scholar] [CrossRef]
  71. Dunkin, N.; Weng, S.; Coulter, C.G.; Jacangelo, J.G.; Schwab, K.J. Impacts of virus processing on human norovirus gi and gii persistence during disinfection of municipal secondary wastewater effluent. Water Res. 2018, 134, 1–12. [Google Scholar] [CrossRef]
  72. Takeuchi, F.; Sekizuka, T.; Yamashita, A.; Ogasawara, Y.; Mizuta, K.; Kuroda, M. Mepic, metagenomic pathogen identification for clinical specimens. Jpn. J. Infect. Dis. 2014, 67, 62–65. [Google Scholar] [CrossRef]
  73. Huson, D.H.; Beier, S.; Flade, I.; Górska, A.; El-Hadidi, M.; Mitra, S.; Ruscheweyh, H.-J.; Tappu, R. Megan community edition—Interactive exploration and analysis of large-scale microbiome sequencing data. PLoS Comput. Biol. 2016, 12, e1004957. [Google Scholar] [CrossRef]
  74. Yin, X.; Jiang, X.T.; Chai, B.; Li, L.; Yang, Y.; Cole, J.R.; Tiedje, J.M.; Zhang, T. Args-oap v2.0 with an expanded sarg database and hidden markov models for enhancement characterization and quantification of antibiotic resistance genes in environmental metagenomes. Bioinformatics 2018, 34, 2263–2270. [Google Scholar] [CrossRef]
  75. Yin, X.; Zheng, X.; Li, L.; Zhang, A.N.; Jiang, X.T.; Zhang, T. ARGs-OAP v3.0: Antibiotic-resistance gene database curation and analysis pipeline optimization. Engineering 2022, in press. [Google Scholar] [CrossRef]
  76. Tran, N.H.; Reinhard, M.; Gin, K.Y.H. Occurrence and fate of emerging contaminants in municipal wastewater treatment plants from different geographical regions-A review. Water Res. 2018, 133, 182–207. [Google Scholar] [CrossRef] [PubMed]
  77. Ministry of Health Labour and Welfare (Japan). Ministry of Health Labour and Welfare, Japan. Japan Nosocomial Infections Surveillance (JANIS), Nosocomial Infections Surveillance for Drug-Resistant Bacteria. Available online: https://janis.mhlw.go.jp/english/index.asp (accessed on 24 March 2023).
  78. Ministry of Health Labour and Welfare (Japan). Annual Report on Statistics of Production by Pharmaceutical Industry in 2021. Available online: https://www.mhlw.go.jp/topics/yakuji/2021/nenpo/index.html (accessed on 24 March 2023).
  79. Prasse, C.; Schlüsener, M.P.; Schulz, R.; Ternes, T.A. Antiviral drugs in wastewater and surface waters: A new pharmaceutical class of environmental relevance? Environ. Sci. Technol. 2010, 44, 1728–1735. [Google Scholar] [CrossRef]
  80. Azuma, T.; Ishiuchi, H.; Inoyama, T.; Teranishi, Y.; Yamaoka, M.; Sato, T.; Mino, Y. Occurrence and fate of selected anticancer, antimicrobial, and psychotropic pharmaceuticals in an urban river in a subcatchment of the Yodo River basin, Japan. Environ. Sci. Pollut. Res. 2015, 22, 18676–18686. [Google Scholar] [CrossRef] [PubMed]
  81. Petrović, M.; Škrbić, B.; Živančev, J.; Ferrando-Climent, L.; Barcelo, D. Determination of 81 pharmaceutical drugs by high performance liquid chromatography coupled to mass spectrometry with hybrid triple quadrupole–linear ion trap in different types of water in Serbia. Sci. Total Environ. 2014, 468–469, 415–428. [Google Scholar] [CrossRef] [PubMed]
  82. Schlüsener, M.P.; Hardenbicker, P.; Nilson, E.; Schulz, M.; Viergutz, C.; Ternes, T.A. Occurrence of venlafaxine, other antidepressants and selected metabolites in the rhine catchment in the face of climate change. Environ. Pollut. 2015, 196, 247–256. [Google Scholar] [CrossRef] [PubMed]
  83. Chaturvedi, P.; Shukla, P.; Giri, B.S.; Chowdhary, P.; Chandra, R.; Gupta, P.; Pandey, A. Prevalence and hazardous impact of pharmaceutical and personal care products and antibiotics in environment: A review on emerging contaminants. Environ. Res. 2021, 194, 110664. [Google Scholar] [CrossRef]
  84. Azuma, T.; Arima, N.; Tsukada, A.; Hirami, S.; Matsuoka, R.; Moriwake, R.; Ishiuchi, H.; Inoyama, T.; Teranishi, Y.; Yamaoka, M.; et al. ; et al. Detection of pharmaceuticals and phytochemicals together with their metabolites in hospital effluents in Japan, and their contribution to sewage treatment plant influents. Sci. Total Environ. 2016, 548–549, 189–197. [Google Scholar] [CrossRef]
  85. Oliveira, T.S.; Murphy, M.; Mendola, N.; Wong, V.; Carlson, D.; Waring, L. Characterization of pharmaceuticals and personal care products in hospital effluent and waste water influent/effluent by direct-injection LC-MS-MS. Sci. Total Environ. 2015, 518–519, 459–478. [Google Scholar] [CrossRef]
  86. Singh, R.R.; Angeles, L.F.; Butryn, D.M.; Metch, J.W.; Garner, E.; Vikesland, P.J.; Aga, D.S. Towards a harmonized method for the global reconnaissance of multi-class antimicrobials and other pharmaceuticals in wastewater and receiving surface waters. Environ. Int. 2019, 124, 361–369. [Google Scholar] [CrossRef]
  87. Kharel, S.; Stapf, M.; Miehe, U.; Ekblad, M.; Cimbritz, M.; Falås, P.; Nilsson, J.; Sehlén, R.; Bester, K. Ozone dose dependent formation and removal of ozonation products of pharmaceuticals in pilot and full-scale municipal wastewater treatment plants. Sci. Total Environ. 2020, 731, 139064. [Google Scholar] [CrossRef]
  88. Issaka, E.; Amu-Darko, J.N.-O.; Yakubu, S.; Fapohunda, F.O.; Ali, N.; Bilal, M. Advanced catalytic ozonation for degradation of pharmaceutical pollutants―A review. Chemosphere 2022, 289, 133208. [Google Scholar] [CrossRef]
  89. Iakovides, I.C.; Michael-Kordatou, I.; Moreira, N.F.F.; Ribeiro, A.R.; Fernandes, T.; Pereira, M.F.R.; Nunes, O.C.; Manaia, C.M.; Silva, A.M.T.; Fatta-Kassinos, D. Continuous ozonation of urban wastewater: Removal of antibiotics, antibiotic-resistant Escherichia coli and antibiotic resistance genes and phytotoxicity. Water Res. 2019, 159, 333–347. [Google Scholar] [CrossRef] [PubMed]
  90. Chávez, A.M.; Beltrán, F.J.; López, J.; Javier Rivas, F.; Álvarez, P.M. On the importance of reactions in the proximity of the gas–water interface: Application to direct ozone reactions of antibiotics in water. Chem. Eng. J. 2023, 458, 141408. [Google Scholar] [CrossRef]
  91. Ge, L.; Na, G.; Zhang, S.; Li, K.; Zhang, P.; Ren, H.; Yao, Z. New insights into the aquatic photochemistry of fluoroquinolone antibiotics: Direct photodegradation, hydroxyl-radical oxidation, and antibacterial activity changes. Sci. Total Environ. 2015, 527–528, 12–17. [Google Scholar] [CrossRef] [PubMed]
  92. Liu, X.; Lv, K.; Deng, C.; Yu, Z.; Shi, J.; Johnson, A.C. Persistence and migration of tetracycline, sulfonamide, fluoroquinolone, and macrolide antibiotics in streams using a simulated hydrodynamic system. Environ. Pollut. 2019, 252, 1532–1538. [Google Scholar] [CrossRef]
  93. Lima, L.M.; Silva, B.N.M.d.; Barbosa, G.; Barreiro, E.J. β-lactam antibiotics: An overview from a medicinal chemistry perspective. Eur. J. Med. Chem. 2020, 208, 112829. [Google Scholar] [CrossRef]
  94. Robles-Jimenez, L.E.; Aranda-Aguirre, E.; Castelan-Ortega, O.A.; Shettino-Bermudez, B.S.; Ortiz-Salinas, R.; Miranda, M.; Li, X.; Angeles-Hernandez, J.C.; Vargas-Bello-Pérez, E.; Gonzalez-Ronquillo, M. Worldwide traceability of antibiotic residues from livestock in wastewater and soil: A systematic review. Animals 2022, 12, 60. [Google Scholar] [CrossRef]
  95. Vione, D.; Feitosa-Felizzola, J.; Minero, C.; Chiron, S. Phototransformation of selected human-used macrolides in surface water: Kinetics, model predictions and degradation pathways. Water Res. 2009, 43, 1959–1967. [Google Scholar] [CrossRef]
  96. Carvalho, I.T.; Santos, L. Antibiotics in the aquatic environments: A review of the european scenario. Environ. Int. 2016, 94, 736–757. [Google Scholar] [CrossRef]
  97. Izadi, E.; Afshan, G.; Patel, R.P.; Rao, V.M.; Liew, K.B.; Meor Mohd Affandi, M.M.R.; Kifli, N.; Suleiman, A.; Lee, K.S.; Sarker, M.M.R.; et al. ; et al. Levofloxacin: Insights into antibiotic resistance and product quality. Front. Pharmacol. 2019, 10, 00881. [Google Scholar] [CrossRef]
  98. Norte, T.H.d.O.; Marcelino, R.B.P.; Medeiros, F.H.A.; Moreira, R.P.L.; Amorim, C.C.; Lago, R.M. Ozone oxidation of β-lactam antibiotic molecules and toxicity decrease in aqueous solution and industrial wastewaters heavily contaminated. Ozone Sci. Eng. 2018, 40, 385–391. [Google Scholar] [CrossRef]
  99. Tufail, A.; Price, W.E.; Mohseni, M.; Pramanik, B.K.; Hai, F.I. A critical review of advanced oxidation processes for emerging trace organic contaminant degradation: Mechanisms, factors, degradation products, and effluent toxicity. J. Water Proc. Eng. 2021, 40, 101778. [Google Scholar] [CrossRef]
  100. Zilberman, A.; Gozlan, I.; Avisar, D. Pharmaceutical transformation products formed by ozonation—Does degradation occur? Molecules 2023, 28, 1227. [Google Scholar] [CrossRef] [PubMed]
  101. Wang, H.; Xi, H.; Xu, L.; Jin, M.; Zhao, W.; Liu, H. Ecotoxicological effects, environmental fate and risks of pharmaceutical and personal care products in the water environment: A review. Sci. Total Environ. 2021, 788, 147819. [Google Scholar] [CrossRef]
  102. Li, J.; Li, W.; Liu, K.; Guo, Y.; Ding, C.; Han, J.; Li, P. Global review of macrolide antibiotics in the aquatic environment: Sources, occurrence, fate, ecotoxicity, and risk assessment. J. Hazard. Mater. 2022, 439, 129628. [Google Scholar] [CrossRef]
  103. Magdeburg, A.; Stalter, D.; Oehlmann, J. Whole effluent toxicity assessment at a wastewater treatment plant upgraded with a full-scale post-ozonation using aquatic key species. Chemosphere 2012, 88, 1008–1014. [Google Scholar] [CrossRef]
  104. Díaz-Garduño, B.; Pintado-Herrera, M.G.; Biel-Maeso, M.; Rueda-Márquez, J.J.; Lara-Martín, P.A.; Perales, J.A.; Manzano, M.A.; Garrido-Pérez, C.; Martín-Díaz, M.L. Environmental risk assessment of effluents as a whole emerging contaminant: Efficiency of alternative tertiary treatments for wastewater depuration. Water Res. 2017, 119, 136–149. [Google Scholar] [CrossRef]
  105. Tamura, I.; Yasuda, Y.; Kagota, K.; Yoneda, S.; Nakada, N.; Kumar, V.; Kameda, Y.; Kimura, K.; Tatarazako, N.; Yamamoto, H. Contribution of pharmaceuticals and personal care products (PPCPs) to whole toxicity of water samples collected in effluent-dominated urban streams. Ecotoxicol. Environ. Safe. 2017, 144, 338–350. [Google Scholar] [CrossRef]
  106. Ike, I.A.; Karanfil, T.; Cho, J.; Hur, J. Oxidation byproducts from the degradation of dissolved organic matter by advanced oxidation processes—A critical review. Water Res. 2019, 164, 114929. [Google Scholar] [CrossRef]
  107. Tufail, A.; Price, W.E.; Hai, F.I. A critical review on advanced oxidation processes for the removal of trace organic contaminants: A voyage from individual to integrated processes. Chemosphere 2020, 260, 127460. [Google Scholar] [CrossRef]
  108. Castellano-Hinojosa, A.; Gallardo-Altamirano, M.J.; González-López, J.; González-Martínez, A. Anticancer drugs in wastewater and natural environments: A review on their occurrence, environmental persistence, treatment, and ecological risks. J. Hazard. Mater. 2023, 447, 130818. [Google Scholar] [CrossRef] [PubMed]
  109. Tripathi, S.; Pathak, V.; Tripathi, D.M.; Tripathi, B.D. Application of ozone based treatments of secondary effluents. Bioresour. Technol. 2011, 102, 2481–2486. [Google Scholar] [CrossRef] [PubMed]
  110. Foroughi, M.; Khiadani, M.; Kakhki, S.; Kholghi, V.; Naderi, K.; Yektay, S. Effect of ozonation-based disinfection methods on the removal of antibiotic resistant bacteria and resistance genes (ARB/ARGs) in water and wastewater treatment: A systematic review. Sci. Total Environ. 2022, 811, 151404. [Google Scholar] [CrossRef] [PubMed]
  111. Zhang, L.; Ma, X.; Luo, L.; Hu, N.; Duan, J.; Tang, Z.; Zhong, R.; Li, Y. The prevalence and characterization of extended-spectrum β-lactamase- and carbapenemase-producing bacteria from hospital sewage, treated effluents and receiving rivers. Int. J. Environ. Res. Public Health 2020, 17, 1183. [Google Scholar] [CrossRef] [PubMed]
  112. Khan, N.A.; Vambol, V.; Vambol, S.; Bolibrukh, B.; Sillanpaa, M.; Changani, F.; Esrafili, A.; Yousefi, M. Hospital effluent guidelines and legislation scenario around the globe: A critical review. J. Environ. Chem. Eng. 2021, 9, 105874. [Google Scholar] [CrossRef]
  113. Verlicchi, P. Trends, new insights and perspectives in the treatment of hospital effluents. Curr. Opin. Environ. Sci. Health 2021, 19, 100217. [Google Scholar] [CrossRef]
  114. Ajala, O.J.; Tijani, J.O.; Salau, R.B.; Abdulkareem, A.S.; Aremu, O.S. A review of emerging micro-pollutants in hospital wastewater: Environmental fate and remediation options. Results Eng. 2022, 16, 100671. [Google Scholar] [CrossRef]
  115. Bian, J.; Wang, H.; Ding, H.; Song, Y.; Zhang, X.; Tang, X.; Zhong, Y.; Zhao, C. Unveiling the dynamics of antibiotic resistome, bacterial communities, and metals from the feces of patients in a typical hospital wastewater treatment system. Sci. Total Environ. 2023, 858, 159907. [Google Scholar] [CrossRef]
  116. Perveen, S.; Pablos, C.; Reynolds, K.; Stanley, S.; Marugán, J. Growth and prevalence of antibiotic-resistant bacteria in microplastic biofilm from wastewater treatment plant effluents. Sci. Total Environ. 2023, 856, 159024. [Google Scholar] [CrossRef]
  117. Korichi, W.; Ibrahimi, M.; Loqman, S.; Ouhdouch, Y.; Younes, K.; Lemée, L. Assessment of actinobacteria use in the elimination of multidrug-resistant bacteria of Ibn Tofail hospital wastewater (Marrakesh, Morocco): A chemometric data analysis approach. Environ. Sci. Pollut. Res. 2021, 28, 26840–26848. [Google Scholar] [CrossRef]
  118. Kovalova, L.; Siegrist, H.; von Gunten, U.; Eugster, J.; Hagenbuch, M.; Wittmer, A.; Moser, R.; McArdell, C.S. Elimination of micropollutants during post-treatment of hospital wastewater with powdered activated carbon, ozone, and UV. Environ. Sci. Technol. 2013, 47, 7899–7908. [Google Scholar] [CrossRef] [PubMed]
  119. Hiller, C.X.; Hübner, U.; Fajnorova, S.; Schwartz, T.; Drewes, J.E. Antibiotic microbial resistance (AMR) removal efficiencies by conventional and advanced wastewater treatment processes: A review. Sci. Total Environ. 2019, 685, 596–608. [Google Scholar] [CrossRef] [PubMed]
  120. Mousazadeh, M.; Kabdaşlı, I.; Khademi, S.; Sandoval, M.A.; Moussavi, S.P.; Malekdar, F.; Gilhotra, V.; Hashemi, M.; Dehghani, M.H. A critical review on the existing wastewater treatment methods in the COVID-19 era: What is the potential of advanced oxidation processes in combatting viral especially SARS-CoV-2? J. Water Proc. Eng. 2022, 49, 103077. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic representation of pilot-scale continuous-flow ozone-based treatment system implemented in a hospital facility. The picture depicts the appearance of the hospital wastewater disinfection treatment system equipped with the ozone treatment system tested in this study. The technical specifications of the equipment used in the system are described in detail below. A 3D view of the equipment is shown in Figure S1.
Figure 1. Schematic representation of pilot-scale continuous-flow ozone-based treatment system implemented in a hospital facility. The picture depicts the appearance of the hospital wastewater disinfection treatment system equipped with the ozone treatment system tested in this study. The technical specifications of the equipment used in the system are described in detail below. A 3D view of the equipment is shown in Figure S1.
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Figure 2. Characterization of treated wastewater by bacterial viability. (A) Visual image of the collected waste or treated wastewater after ozone or UV-LED treatment. (B) Isolation of bacteria from treated wastewater samples on BTB agar and CHROMagar ESBL. An aliquot (100 µL) of influent, ozone-, or UV-treated wastewater samples was spread on the agar plate at a 10-fold dilution. (C,D) Colony forming units per milliliter (CFU/mL) were determined at the appropriate dilution for each treatment time point.
Figure 2. Characterization of treated wastewater by bacterial viability. (A) Visual image of the collected waste or treated wastewater after ozone or UV-LED treatment. (B) Isolation of bacteria from treated wastewater samples on BTB agar and CHROMagar ESBL. An aliquot (100 µL) of influent, ozone-, or UV-treated wastewater samples was spread on the agar plate at a 10-fold dilution. (C,D) Colony forming units per milliliter (CFU/mL) were determined at the appropriate dilution for each treatment time point.
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Figure 3. Metagenomic DNA-seq analysis of bacteria trapped on a 0.2 µm filter after ozone-UV-LED treatment. Notable bacterial genera (Bacteroides, Prevotella, Escherichia, Klebsiella, Acinetobacter, and Pseudomonas) were highlighted to show the metagenomic sequencing read counts detected by megablast search and subsequent taxonomic classification using the MEGAN 6 application. The results obtained for each bacterial genus are summarized in Table S3.
Figure 3. Metagenomic DNA-seq analysis of bacteria trapped on a 0.2 µm filter after ozone-UV-LED treatment. Notable bacterial genera (Bacteroides, Prevotella, Escherichia, Klebsiella, Acinetobacter, and Pseudomonas) were highlighted to show the metagenomic sequencing read counts detected by megablast search and subsequent taxonomic classification using the MEGAN 6 application. The results obtained for each bacterial genus are summarized in Table S3.
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Figure 4. Metagenomic DNA-seq analysis of antimicrobial resistance genes (ARGs) after ozone-UV-LED treatment. Notable top 12 ARGs were selected to show the sequencing reads corresponding to the targeted ARGs using the ARGs_OAP program. DNA conc. (ng/µL) and Metagenomic DNA-seq (total reads) are shown in Table S1, and all obtained results for other ARGs are summarized in Table S5.
Figure 4. Metagenomic DNA-seq analysis of antimicrobial resistance genes (ARGs) after ozone-UV-LED treatment. Notable top 12 ARGs were selected to show the sequencing reads corresponding to the targeted ARGs using the ARGs_OAP program. DNA conc. (ng/µL) and Metagenomic DNA-seq (total reads) are shown in Table S1, and all obtained results for other ARGs are summarized in Table S5.
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Figure 5. Time course of antimicrobial concentrations in hospital wastewater during ozone treatment. Removal of antimicrobials over time during treatment of hospital wastewater. (A): original storage tank (influent), (B): wastewater treatment tank 1 (ozone), and (C): wastewater treatment tank 2 (UV-LED). A summary of antimicrobial concentrations at each treatment time is shown in Table S8.
Figure 5. Time course of antimicrobial concentrations in hospital wastewater during ozone treatment. Removal of antimicrobials over time during treatment of hospital wastewater. (A): original storage tank (influent), (B): wastewater treatment tank 1 (ozone), and (C): wastewater treatment tank 2 (UV-LED). A summary of antimicrobial concentrations at each treatment time is shown in Table S8.
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Azuma, T.; Katagiri, M.; Sasaki, N.; Kuroda, M.; Watanabe, M. Performance of a Pilot-Scale Continuous Flow Ozone-Based Hospital Wastewater Treatment System. Antibiotics 2023, 12, 932. https://doi.org/10.3390/antibiotics12050932

AMA Style

Azuma T, Katagiri M, Sasaki N, Kuroda M, Watanabe M. Performance of a Pilot-Scale Continuous Flow Ozone-Based Hospital Wastewater Treatment System. Antibiotics. 2023; 12(5):932. https://doi.org/10.3390/antibiotics12050932

Chicago/Turabian Style

Azuma, Takashi, Miwa Katagiri, Naobumi Sasaki, Makoto Kuroda, and Manabu Watanabe. 2023. "Performance of a Pilot-Scale Continuous Flow Ozone-Based Hospital Wastewater Treatment System" Antibiotics 12, no. 5: 932. https://doi.org/10.3390/antibiotics12050932

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