Metagenomic survey and whole genome sequencing of antimicrobial resistant bacteria in the sewage of a Japanese hospital

Background The dissemination of antimicrobial-resistant bacteria (ARB) and the transfer of antimicrobial resistance genes (ARGs) are a threat to public health. Antibiotics are indispensable therapeutic agents essential for the treatment of infectious diseases; however, inappropriate use of antibiotics leads to the emergence of ARB. It is established that hospitals are closely involved in the spread of antimicrobial resistance (AMR), which impedes antibiotic treatment and subsequently increases mortality. In addition, excrement of patients or healthy carriers of ARB are discharged from the hospital sewage through the wastewater treatment plant (WWTP) into the rivers, causing an AMR burden on the environment. Method Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of the extended spectrum β-lactamase (ESBL)-producing organisms (EPOs). A comparative genome analysis was also performed between EPO isolates from sewage and clinical isolates. Results Metagenomic analysis showed that the hospital sewage tanks had bacterial ora corresponding to the human gut. During the study period, the hospital was relocated to a newly constructed building with new sewage tanks; however, the presence of ARB/ARGs in the new hospital sewage tanks became markedly equivalent to that of the old hospital within one month. The ESBL bla CTX−M and carbapenemase bla IMP genes were not much detected in the original hospital sewage samples by metagenome analysis, but selection on CHROMagar ESBL increased the sensitivity to detect those β-lactamase genes. Comparative genome analysis between sewage and clinical EPO isolates revealed partial similarity; however, most EPO isolates exhibited a notable difference ( ≥ 50) in single nucleotide variations based on core-genome phylogeny. This result suggests that only some of the sewage EPO isolates were originated from the clinical patient. Therapeutic agents in the hospital sewage were analyzed and the concentration of levooxacin and clarithromycin was 0.0325 and 0.0135 µg/mL, respectively. Conclusions Whole genome analysis between sewage and clinical isolates suggested that healthy or asymptomatic carriers may be involved in the of hospital sewage. Moreover, the hospital hotspot the


Introduction
The rapid dissemination of antimicrobial-resistant bacteria (ARB) has become one of the major public health concerns in the world. In case novel antibiotics against ARB are not developed, the annual number of deaths due to bacterial infections by ARB is predicted to increase to 10 million by 2050 [1]. In addition to nosocomial ARB infections, other environmental sources of ARB should also be investigated. The Global Action Plan on Antimicrobial Resistance drafted by World Health Organization [2] states that there is a need to understand the impact of human activities on the environment, particularly the spread and transfer of antimicrobial resistance genes (ARG) and strains. The Nippon National Action Plan on Antimicrobial Resistance One Health Report 2018 in Japan addressed that 12.4 − 27.5% of tested invasive Escherichia coli isolates were resistant to 3rd generation cephalosporins and 0.1% were resistant to carbapenems. Additionally, 5.7 − 9.4% of tested invasive Klebsiella pneumoniae isolates were resistant to 3rd generation cephalosporins and 0.3 − 0.5% were resistant to carbapenems [3]. The isolation rates of these resistant strains are lower in Japan compared to other countries, but are certainly increasing. Therefore, antimicrobial resistance (AMR) should be well monitored in the β-lactam-resistant Enterobacteriaceae family of bacteria.
In particular, hospital wastewater is contaminated by ARB and residual antibiotics present in the excrement of patients, visitors, and medical personnel, and is considered to be a hot spot for the growth and propagation of ARB [5].
Although it is not yet clear whether the hospital wastewater-related ARB disseminate into the water bodies, ARBs are observed to exhibit deleterious effects on human health. In the clinical setting, resistance to carbapenems in Enterobacteriaceae-related infections [39] is a serious concern, owing to limited treatment options [40,41] and high mortality rates [42]. The presence of CPOs in hospital sewage [5,13,21,22], urban sewage [43], and coastal water environments [31,44] has also increased. Therefore, active surveillance and management are necessary to avoid undesirable dissemination of ARB from hospital sewage to WWTP. Similar to the general sewage system, hospital sewage is also discharged into a public WWTP and released into rivers after primary puri cation, activated sludge process, and occasional chlorine disinfection. In recent years, technically and economically viable management strategies for ARB, ARG, and residual antibiotics discharged from hospitals have been studied in some countries [45][46][47][48][49][50][51]; however, there is a lack of similar reports from Japan. The aim of this study is to illustrate the contamination of hospital sewage with ARB containing resistant genes of clinical origin, and the effect of hospital relocation on the state of ARB/ARGs in the hospital sewage using comprehensive metagenomic sequencing. In addition, we compared the whole genome sequence of extended spectrum β-lactamase (ESBL)-producing organisms (EPOs) from hospital sewage and clinical isolates.

Hospital setting
The study was conducted at the Ohashi Medical Center in Toho University, located at Jonan-area suburb of Tokyo, Japan. The Ohashi Medical Center (35.652573, 139.685833) was opened in 1973 with a single East building and expanded to Central, Administration, and West buildings to increase patient capacity. Eventually, the number of beds in this medical center was 430 (Fig. 1). The hospital included the following departments in the West building (BW); Cardiology, Respiratory, Internal medicine, and Neurology wards, Intensive Care Unit, examination (dialysis, MRI, endoscopy, and radiography) and operating rooms; Administration building (BA); outpatient clinics of Surgery and Pediatric departments, and administrative staff room; Central building (BC); laboratories, Cardiovascular surgery, Orthopedics, Gynecology, and emergency outpatient clinics and inpatient wards of Gastrointestinal medicine, Surgery and Orthopedics, and dining rooms; East building (BE); outpatient clinics of Internal medicine, Plastic surgery, Ophthalmology, Otorhinolaryngology, Dermatology, and Urology and inpatient wards of Cardiology, Neurosurgery, Ophthalmology, and private wards.
As part of a renovation plan, a new hospital building (BN) (35.652578, 139.683959) with 319 beds was constructed approximately 50 m away from the old hospital (35.652573, 139.685833) and was inaugurated on June 20, 2018. Since May 2018, we introduced a policy of restricted hospitalization of seriously ill patients, and transferred outpatients to neighboring hospitals to reduce the number of patients at the time of relocation. We stopped emergency services from June 6 to June 20 and outpatients from June 16 to June 19, and transferred the hospitalized patients to the BN on June 16. We started accepting outpatients and hospitalized patients on June 20. The BN attends to 1000 outpatients per day and has a staff count of 2000 employees.
In both the old and new hospitals, stool and urine were stored in the underground sewage tanks without mixing with other drainage and were pumped to the sewage system several times a day (Fig. 1). In the old hospital, each building had respective sewage tanks (STW, STA, STC, and STE as shown in Fig. 1); however, the new hospital had two connected storage tanks of 22.5 m 3 collecting all the sewage (STN shown in Fig. 1). It was impossible to quantify daily in ow and out ow of sewage tanks as there is no system for regular measurement. The sewage discharged from the tanks was sent to the wastewater treatment plants (WWTP) and treated using ltering, and microbiological and biochemical mechanisms.
After treatments, the e uent was discharged into the nearby river.

Collection of water samples
In the period from May 8, 2018 to Jun 12, 2018, sewage water samples were collected from four separate sewage tanks of the old hospital (STW, STA, STC, and STE) once a week. In the period from Jun 6 to July 17, sewage samples were collected from the sewage tank of the new hospital (STN) once a week. At 9 a.m., 20 ml of sewage sample was collected in sterile bottles from the manhole of the sewage tank and processed for analysis within 2 h.

DNA-seq analysis
Metagenomic analysis for whole organisms in original hospital sewage: First, 5 ml of sewage was centrifuged at 5,000 x g for 5 min and the resultant cell pellet was vortexed with a remaining 500 µl of sewage water. Next, the cell suspension was mixed with 500 µl phenol/chloroform/isoamylalcohol (PCI) in a microcentrifuge tube with 2 ml of beads. Cell breaking was performed by GenoGrinder 2010 by shaking at 1,500 rpm for 5 min. The PCI mixture was centrifuged at 8,000 rpm for 5 min, followed by DNA puri cation using a Gel DNA Recovery Kit, Zymoclean-96 (ZYMO RESEARCH, Irvine, CA,USA). A metagenome DNA-seq library was prepared using the QIAseq FX DNA library prep kit (Qiagen: Venlo, Netherlands), followed by sequencing using NextSeq 500 (Illumina) with NextSeq 500 mid output kit v2.5 (300 cycle).
Metagenomic analysis for EPOs on CHROMagar-ESBL: Two microliter of sewage sample was diluted with 100 µl phosphate buffered saline (PBS), plated on CHROMagar-ESBL (bioMérieux, Marcy-l'Etoile, France) for selection of EPOs, and incubated at 36°C overnight. Cultured colonies were harvested and mixed with 1000 µl of PBS. Next, 100 µl of the bacterial suspension from CHROMagar-ESBL was transferred to a 2 ml ZR BashingBead™ Lysis tube, and mixed with 500 µl PCI and 500 µl elution buffer (10 mM Tris pH 8.0). The tube was shaken by GenoGrinder 2010 at 1,500 rpm for 5 min. A metagenome DNA-seq library was prepared using the QIAseq FX DNA library prep kit (Qiagen: Venlo, Netherlands), followed by sequencing using NextSeq 500.

EPO isolates in hospital sewage
To selectively grow EPO isolates from sewage samples, 50 µl of sewage from the glycerol stocks prepared on May 22 and July 17 were diluted with 1000 µl PBS, followed by plating on CHROMagar-ESBL, and incubation at 37℃ overnight. Subsequently, 80 colonies were selected as potential EPOs and streaked on fresh CHROMagar-ESBL plates cultured at 37℃ overnight to obtain pure cultures. ESBL producing bacteria were cultured in tryptic soy broth, and the cell suspension was transferred to 2 ml ZR BashingBead™ Lysis tubes, and mixed with 500 µl PCI. The tube was shaken by GenoGrinder 2010 at 1,500 rpm for 5 min. The PCI extract was centrifuged at 8,000 rpm for 5 min, followed by DNA puri cation using a Gel DNA Recovery Kit, Zymoclean-96 (ZYMO RESEARCH). A DNA-seq library was constructed by a QIAseq FX, followed by sequencing using NextSeq 500.

EPO clinical isolates
All 20 EPO clinical isolates obtained between May 8 and July 17, 2018 were subjected to whole-genome sequencing as described above, and comparative genomics. Among the 20 clinical isolates, 12 isolates were obtained from outpatients and 11 isolates were obtained from urological patients. The samples were from urine, sputum, and central venous catheter (75%, 10%, and 10%, respectively). Some specimens were obtained from the same patient through subsequent diagnosis. The ethical committee in Toho University Ohashi Medical Center waived the need for written consent regarding the research into bacterial isolates (Investigation No. pH). The personal data related to the clinical information were anonymized, and our procedure does not require a written consent from patients suffering from bacterial infections. Antibiotic susceptibility of samples cultured in the microbiology laboratory within the same period were determined using breakpoints standardized by the Clinical and Laboratory Standards Institute (CLSI). Screening (broth microdilution method) and con rmatory tests (the disk diffusion method) on the EPOs were conducted according to CLSI recommendations. (CLSI Performance Standards for antimicrobial disk susceptibility tests; Approved standard-13th edition CLSI document M02. Wayne, PA: Clinical and Laboratory Standards Institute; 2018).

Bioinformatics
To characterize microbial ora in the sewage samples, the sequencing reads were analyzed by MePIC2 [52] and Krona [53] and MEGAN v6 software [54]. To characterize isolates of bacterial species and identify antimicrobial resistance genes, sequencing reads were analyzed by multi locus sequence typing (MLST) [55] and ResFinder [56], respectively.

Principal Coordinate Analysis (PCoA)
The sequenced reads were assigned to a taxonomic hierarchy using MEGAN v6 software based on a megaBLAST nucleic acid homology search.

Core genome single nucleotide variation phylogenetic analysis
To compare the genotype of 37 E. coli strains isolated from patients and hospital sewage, the Illumina short reads, excluding low quality and adapter sequences, were aligned using BWA-MEM [57] against the complete chromosome sequences of E. coli STN0717-11, followed by extraction of single nucleotide variants (SNVs) using VarScan v2.3.4 [58]. The prophage and repeat regions were predicted by PHASTER [59] and MUMmer 3 [60], respectively, and the detected SNVs in these regions were excluded. Regions of recombination in the chromosome were predicted using Gubbins v. 2.3.4 [61], followed by masking SNVs in the recombination regions. A maximum likelihood phylogenetic tree was constructed from SNV sites in the core genome region using FastTree2. De novo assembly was performed using SKESA v.2.3.0 [62] with short reads of each strain, followed by analyzing sequence type, putative serotype, and AMR gene prediction using pubMLST (https://pubmlst.org/escherichia/), SeroTypeFinder [63], and Bacterial Antimicrobial Resistance Reference Gene Database (BioProject ID: PRJNA313047), respectively.

Measurement of concentrations of chemical compounds
Forty drug components in the sewage samples were analyzed using solid-phase extraction (SPE) and ultra-performance liquid chromatography -tandem mass spectrometry (LC-MS/MS) based on a previously described method [64] with minor modi cations. Brie y, the sample was ltered by a polyethersulfone membrane (0.22 µm pore size, Merck) and 100 ml of the ltrate was spiked with 1 g/l ascorbic acid, 1 g/l EDTA, and a surrogate standard mixture, and then concentrated by SPE cartridge (Oasis HLB cartridges, 200 mg/6 cc, Waters, Japan). The analytes concentrated on the cartridge were extracted with 6 ml of methanol before being measured by LC-MS/MS and quanti ed by the alternative surrogate method [64].

Results And Discussion
Hospital setting The Ohashi Medical Center in Toho University is a general hospital opened in 1973. Prior to relocation, the hospital consisted of four buildings: West building (BW), Administration building (BA), Central building (BC), and East building (BE) and had a total of 430 beds, and accepted an average of 850 outpatients per day ( Fig. 1). In the BW, there were intensive care wards (ICU, cardiovascular surgery, cardiovascular internal medicine, respiratory, and internal medicine), but no outpatient departments. In the BA, there were several outpatient departments, but the toilets were for only healthcare workers. In the BC, patients with digestive diseases, urinary diseases, and bone fracture were mainly accommodated, and the number of beds was the largest among the old hospital buildings. The BE included the highest number of outpatient departments with no intensive care wards.
The hospital relocated to the new building (BN) on June 20 th , 2018. In the BN, all outpatient departments and wards were integrated into one building. The sewage from patients was not mixed with other wastewater sources (laboratory and general wastewater) in the wastewater systems of the old and new hospital (Fig. 1).
The average number of hospitalized patients in May was 246, and the number of hospitalized patients in BC, BE, and BW building started to decrease in June. Consequently, 66 patients were transferred to BN on June 16 (Fig. 2). After the new hospital was opened on June 20, we started outpatient examinations and accepted a new hospitalized patient, and the number of inpatients gradually increased and exceeded 250 on July 2.

Metagenomic analysis of tank ora in each building
To elucidate the differential microbial ora in the hospital sewage tank (tank ora), metagenome DNAseq analysis of sewage samples was conducted. The dominant bacteria in the sewage were classi ed according to the metagenomic data Supplemental Data Set S1), wherein the most common genus was Aeromonas, followed by Citrobacter and Bacteroidetes (Fig. 3a). In addition, the proportion of genera varied depending on the building; the largest genus comprised of Citrobacter in STA, Aeromonas in STC, Bacteroides in STE, Acinetobacter and Citrobacter in STW, and variable genera in STN. Notably, less diversity was observed in STA (Fig. 3b) tanks because the toilet users in BA were limited to staff members, and the amount of excretion was also small.
In our hospital's sewage tank, bacteria originating from human gut ora were expected to be predominant as only stool and urine excreted from the toilet are stored. Composition of bacterial ora in hospital sewage has been reported to comprise of human gut ora Bacteroides, Faecalibacterium, Bi dobacterium, Blautia, Roseburia, and Ruminococcus, in addition to Klebsiella, Aeromonas, Enterobacter, Prevotella, and Comamonas [5]. In the gut ora of healthy Japanese adults, the anaerobes Bi dobacterium, Blautia, Bacteroides, Faecalibacterium, Eubacterium, Ruminococcus, and Collinsella are predominant [66]. However, hospital sewage is mainly in uenced by the patients' gut ora, which is different from healthy individuals. Thus, ora in each tank at the old hospital (STA, STW, STC, and STE) exhibited different bacterial compositions depending on the characteristics of the patients present in each building (Fig. 1).
Since June 5th, only construction company and hospital staff visited the BN, and the STN was composed of a few genera such as Citrobacter, Aeromonas, Escherichia, and Klebsiella. Thereafter, Arcobacter, Citrobacter, Aeromonas, Bacteroides, and Acinetobacter comprised the main constituents, and the variety of genera increased with time. In the STN, the composition of bacteria was partially consistent with that in the STC, STE, and STW. It appears that all bacterial species detected in the old sewage tanks (STA, STC, STE and STW) were integrated into STN. For instance, Aeromonas originated from STC and Citrobacter and Acinetobacter originated from STW. In the STN, Comamonas and Arcobacter were signi cantly detected after the transfer of inpatients. Comamonas is a genus of aerobic proteobacteria, generally considered as environmental bacteria with less pathogenicity. It is reported that one of the Comamonas species, C. testosterone, aerobically degrades testosterone when cultured in testosteronecontaining media [67]. In the STN, the enrichment of Comamonas in sewage suggests the possibility of its role in decontamination of medical compounds. Arcobacter spp. are gram-negative rod-shaped bacteria of the family Campylobacteraceae. A. butzleri, A. cryaerophilus, and A. skirrowii have been implicated in severe acute gastrointestinal infections and bacteremia after exposure to shell sh and/or ingestion of contaminated water, or inadequately cooked meat/shell sh [International Commission on Microbial Speci cation for Foods (ICMSF, 2002)]. In the present study, there were no instances of infectious enteritis due to Arcobacter in the hospital; however, the causative bacteria of infectious enteritis are not easily identi able, except in limited conditions. Arcobacter is detected in WWTP in several countries [68][69][70][71], and the presence of antimicrobial resistant Arcobacter has also been documented [70,72]. E. coli and Klebsiella were not abundant, but were consistently detected, in all sewage tanks of buildings. It is unclear whether the difference in the bacterial composition of each tank re ects characteristics of each building (department, diseases, drugs administered). Unlike the sewage system in the old hospital (STA, STC, STE, and STW), the new hospital has a single sewage tank (STN). Thus, the bacterial composition of STN looks similar to that of STW, suggesting that the ora in STW was most in uenced by STN.
Similarity and diversity of bacterial population amongst tanks was analyzed using principal co-ordinates analysis (PCoA) based on bacterial genus level (Fig. 4). A PCoA used a total of 25 sewage samples of each tank excluding STA, and showed that the STN and STW groups were closely plotted, while the STE and STC groups were largely separated. The BW and BN contain rooms where seriously ill patients are treated, such as intensive care unit (ICU), coronary care unit (CCU), and respiratory disease wards (Fig. 1). According to PCoA analysis, the distribution of ora in STN after the transfer of patients was similar to that of STW, indicated by the presence of Aeromonas, Citrobacter, and Comamonas (Fig. 4). The distribution of tank ora can be strongly in uenced by the wards for patients who were prescribed strong doses of antibiotics and other therapeutic agents. Furthermore, tank ora can be instantly affected by excrement of hospital patient/staff/visitor because the bacterial composition of STN was comparable to that of STW within one month after the relocation.
In this study, the old hospital sewage tanks (STA, STC, STE, and STW) were separately installed in each building; thus, a marked difference was observed among tanks. In contrast, it was di cult to identify the relationship between the ora and speci c departments in the new hospital sewage tank, STN, because of a single tank in the building. For the purpose of monitoring department-speci c ARB, it may be bene cial to install department-speci c sewage tanks.

Analysis of β-lactamase genes in ora of tanks
The metagenome next generation sequence (NGS) reads corresponding to β-lactamase genes were identi ed in original hospital sewage samples (Fig. 5a) and EPOs from each tank were selected on CHROMagar ESBL (Fig. 5b). In the original hospital sewage samples, various β-lactamase genes such as bla GES , bla OXA, and bla CMY were detected in ora of STC, STW, and STN, while almost no β-lactamase genes were detected in ora of STA and STE. This may be due to the limited sensitivity of metagenomic detection in this study. The bla IMP gene was detected from STC and STW samples, and the bla CTX-M gene was detected from STW and STN samples (Fig. 5a). CHROMagar ESBL selection facilitated the detection of EPOs as β-lactamase genes were detected in STA and STE using this technique. Moreover, bla IMP and bla CTX-M genes were present in ora of all sewage tanks (Fig. 5b).

Genome comparison between sewage and clinical isolates
Although metagenomic analysis showed the number of each bacterial species and ARG, it is impossible to deduce which bacterial strain carries a particular ARG. Thus, whole genome sequencing was performed for 78 EPO isolates from STW0522 and STN0717, and 20 EPO clinical isolates (May 8, 2018 to July 17, 2018) for comparison of genomic sequences of EPOs from sewage tanks and clinical sources. In the STW0522 sample, bacteria such as E. coli, Klebsiella, Enterobacter, Citrobacter, and Achromobacter were detected, and in the STN0717 sample, E. coli, Klebsiella, Enterobacter, and Citrobacter were detected (Table 1). Clinical isolates included only E. coli and the Klebsiella spp., wherein E. coli displayed sequence variations compared to sewage isolates ( Table 2).
Thus far, the healthy carriage rate of EPOs has been rising worldwide. In Japan, detection rate of EPO was reported at 12.2% in healthy adult volunteers [78] and at 15.6% in healthy food handlers [79]. Additionally, it is reported that 92.9% of EPOs were bla CTX-M gene positive [80]. The CTX-M genes (bla CTX-M-14 , bla CTX-M-27 , bla CTX-M-15 and bla CTX-M-2 in descending order in size) are mainly identi ed in Japan and its reported sequence is similar to that observed in EPOs in the hospital sewage and clinical isolates in this study (Table 3). Prominent global trends in CTX-M epidemiology indicate that the reduced occurrence of bla CTX-M-2 and emergence of bla CTX-M-27 [75], suggesting that healthy carriers could be at risk for nosocomial infections by EPOs.
In the sewage samples, isolates possessed various β-lactamase genes; bla SHV-12 , bla CTX-M-14 , and bla CTX-M-62, in Enterobacter kobei; bla SHV-12 in Enterobacter cloacae; bla CTX-M-9 in Enterobacter homaechei; bla IMP-11 in Pseudomonas monteilii, and bla SHV-12 in Raoultella planticola (Supplemental Data Set S2). Many of the potential EPOs harboring ARGs in the sewage tanks were different from the clinical isolates. It is not clear whether these EPOs were excreted by healthy carriers or were transformed by acquiring the ARGs in the sewage tank.

Concentration of residual antimicrobial agents in hospital sewage tanks
There is a possibility that sewage tanks provide an environment conducive to the development of novel drug resistant bacteria. Sewage tanks are reservoirs of ARGs and transfer of genes between bacteria may occur under the selective pressure of antimicrobial agents and antiseptics. Indeed, concentration of chemical contaminants in the tank (Table 4) showed that the most predominant antimicrobial agent was levo oxacin (32,500 ng/l) and clarithromycin (13500 ng/l), which may potentially exhibit inhibitory action in the sewage tank environment, although their concentrations were below MIC breakpoints.
In hospital wastewater, cipro oxacin concentration was reported to be 22,000 -179,000 ng/L in Germany [91], 19,110 -428,00 ng/L in Vietnam, and clarithromycin was reported to be up to 960 ng/L in Portugal [92] and 760 -72,800 ng/l in Singapore [27]. The present study detected an antibiotic concentration similar to the above reports. Although β-lactam antibiotics were not measured in this study, they are known to be almost undetectable in environmental samples [93,94]. This is because unstable lactam rings increase susceptibility of β-lactam antibiotics to hydrolysis immediately after excretion [94,95]. Hospital sewage tanks containing a large amount of residual antibiotics [96] may change the composition of tank ora and further promote the development of AMR by high selective pressure on bacteria [20,97,98]. Antibiotics exert selective pressure in favor of the growth resistant bacteria, even at very low concentrations [99][100][101]. Moreover, there are some reports that concentration of antibiotics in hospital sewage are often higher than the reported no-effect or minimum concentrations for resistance selection, suggesting that the selection of ARB can occur in this environment [97,102]. We presume that selective pressure of antibiotics exists in our hospital sewage tanks; however, this will be veri ed in future studies.
Horizontal gene transfer is a cause of great concern as it is one of the most important mechanisms of the spread of antibiotic resistance in the environment [103,104]. It is suggested that nutrient-rich environments such as wastewater offer optimal conditions for horizontal gene transfer, involving the transconjugation of AMR plasmids encoding ARGs [105] and transformation of non-pathogenic bacteria into reservoirs of ARGs. It is known that a microbial gut ora composed of a spectrum of bacteria functions as a reservoir for ARGs and horizontal plasmid transfer between bacteria is common [103]. This is plausible as sewage tanks consists of an accumulation of excrement and acquisition of resistance may occur frequently.
In Sweden, carbapenemase-producing Klebsiella was also detected from clinical specimens, in Svartån River and the rivers downstream of it [11]. In Japan, KPC-2 producing Klebsiella [16,17] and NDM-5coproducing Escherichia coli [17] were detected from the e uent of urban wastewater treatment plants. Effective actions should be taken including advanced wastewater treatment processes such as ozone and UV treatment[47, 106,107], and ultra ltration [108] to accelerate the removal of ARB in WWTP.
However, even the above methods do not ensure a complete removal of resistant bacteria, therefore, treatment processes may be introduced prior to the release of hospital sewage into the main sewage to reduce ARB and residual drug concentrations. In Japan, there are a few reports of contamination of sewage with ARB and presence of ARGs in microbial ora of hospital sewage tanks [109,110].
Nevertheless, this study is the rst comprehensive description of AMR in a hospital setting using metagenomic and whole genome analysis.

Conclusions
In many countries including Japan, hospital wastewater is discharged into sewage without treatment, and ARB are detected from the drained rivers, and soil and water environment. Therefore, the frequency of ARGs in bacteria increases and consequently, there is an AMR burden on the environment. The dissemination of ARB/ARGs in the environment can increase the risk of infectious diseases [18]; however, there are very few direct suggestive data about their epidemiological effects [111]. Our study reveals the presence of ARB/ARGs in the hospital sewage tank and suggests that every hospital patient/staff/visitor can be a potential carrier. Furthermore, the hospital sewage tank is a hot spot where various bacterial species may acquire ARGs. Monitoring of ARB/ARGs in hospital sewage is expected to identify the presence of carriers, and control nosocomial outbreaks and dissemination of ARB/ARGs in the community.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable

Availability of data and materials
The metagenomic short-read sequences for DNA-seq were deposited in the DNA Data Bank of Japan (BioProject PRJDB9036; BioSample SAMD00195117-SAMD00195180; DRA accession DRA009310) (Supplemental Data Set S1). The NGS data for whole genome sequencing were deposited in the DNA Data Bank of Japan (BioProject PRJDB9036; BioSample SAMD00195843-SAMD00196018; DRA accession DRA009309). All complete sequences in this study are available from the DDBJ/ENA/GenBank database (accession numbers AP022380-AP022556), as shown in the supplementary data les (Supplemental Data Set S2).

Competing interests
The authors declare that they have no competing interests.         PCoA plot using detected NGS read counts by metagenome DNA-Seq. PCoA was performed according to Bray Curtis distance (the average linkage). The genera, Acinetobacter, Citrobacter, and Comamonas were common present in STW and STN samples. Most severely ill inpatients were treated in the BW and BN, thus their excretion may have a major impact on the bacterial content of the sewage tanks.  Core genome phylogeny using single-nucleotide variations (SNVs) of ESBL-producing E. coli isolates.
Core genome phylogeny was constructed using ESBL-producing E. coli isolates; 20 clinical isolates (THOnumber, orange highlighted), one sewage isolate from STW0522 (brown highlighted), and 20 sewage isolates from STN0717 (blue highlighted). Complete genome sequence of STN0717-11 was used as a genome reference and 39.48% of the genome sequence was used as core-genome regions among all tested strains. Few clinical isolates were obtained from same patient ( §, patient No.8; ¶, patient No.9; †, patient No.7 in supplement Data Set S1). Heatmap of pairwise differences of core genome SNVs are shown using a color gradient with pink and red. The lower half part indicates core genome SNVs among all strains, and the upper half part shows core genome SNVs between indicated two strains. THO-008 and -

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. DataSetS1Metagenome.xlsx DataSetS2Isolates.xlsx