Hospital wastewater as source of human pathogenic bacteria: A phenotypic and genomic analysis of international high-risk clone VIM-2-producing Pseudomonas aeruginosa ST235/O11.

Pseudomonas aeruginosa belong to the special pathogen group capable of causing serious infections, with high mortality rates. The aim of this study was to describe the antibiotic resistance and genomic characteristics of Pseudomonas aeruginosa belonging to international high-risk clone ST235 (GPAE0131 isolate), obtained from hospital wastewater. P. aeruginosa GPAE0131 was isolated from ward tertiary hospital in Brazil and the antibiotic resistance profile was determined by the disc-diffusion method. Genomic characteristics related to antibiotic resistance and virulence factors were evaluated by genomic DNA sequencing on the Illumina MiSeq platform and bioinformatic analysis. GPAE0131 isolate showed resistance to piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, levofloxacin and tobramycin. Resistome comprehend of resistance genes to ß-lactams (blaVIM-2, blaOXA-4, blaOXA-488, blaPDC-35), aminoglycosides (aph(3')-IIb, aac(6')-IIc, aac(6')-Ib9, aadA1), fosfomycin (fosA), chloramphenicol (catB7) and sulfonamides (sul1). Genome comparisons evidence insertion of blaVIM-2 and blaOXA-4 genes. GPAE0131 isolate was predicted to be pathogenic to humans and several virulence factors were found, including encoding gene for ExoU and exotoxin A. All of these features into a pathogenic international high-risk clone (ST235), classified as critical priority, stands out as public health concern due to the widespread dispersal of human pathogens through wastewater. It is suggested that mitigating measures be implemented, such as the treatment of hospital sewage and the addition of tertiary treatment, to prevent the escape of pathogens at this level into the environment.

Spatiotemporal distribution, interactions and toxic effect of microorganisms and ARGs/MGEs from the bioreaction tank in hospital sewage treatment facility.

Antibiotic resistance genes (ARGs) can be emitted from wastewater to ambient air and impose unignorable inhalable hazards, which could be exacerbated in antibiotic-concentrated hospital sewage. However, whether the ARG-carrying pathogens are more likely to infect cells remains largely unknown. Here, this study investigated and analyzed the spatiotemporal distribution, interaction, and toxicity of airborne microorganisms and their hosting ARGs in a hospital sewage treatment facility. The average concentration of ARGs/MGEs in sewage of bioreaction tank (BRT-W) was 2.27 × 104 gene copies/L. In the air of bioreaction tank (BRT-A), the average concentration of ARGs/MGEs was 15.86 gene copies/m3. In the four seasons, the ARGs concentration of sewage gradually decreased over time; The concentration of ARGs in the air first decreased and then increased. In spring, the concentration of ARGs/MGEs (qacedelta1-01) in BRT-W was highest (1.05 × 105 gene copies/L); The concentration of ARGs/MGEs (strB) in BRT-A in winter was higher than other seasons (26.18 gene copies/m3). Different from the past, this study also paid attention to the pathogenic potential of ARGs/MGEs in the air. The results of cell experiments showed that the cytotoxicity of drug-resistant Escherichia coli could reach Grade V. This suggested that the longer the drug-resistant E. coli were exposed to cells, the greater the cytotoxicity. Moreover, the cytotoxicity of bacteria increased with the increase in exposure time. In spring, the toxic effect of ARGs/MGEs in sewage of BRT-W was highest. Traceability analysis proved that BRT-W was an essential source of microorganisms and ARGs/MGEs in BRT-A. Furthermore, the combined risk of people exposed to the air of BRT in spring was higher than that in other seasons.

Novel insights into genetic characteristics of blaGES-encoding plasmids from hospital sewage.

Introduction: The prevalence of Guiana extended-spectrum (GES)-type carbapenemase producers is increasing worldwide, and hospital water environments are considered as potential reservoirs. However, the genetic features underlying this resistance are not yet fully understood. This study aimed to characterize blaGES-encoding plasmids from a single-hospital sewage sample in Japan. Methods: Carbapenemase producers were screened using carbapenemase-selective agar and polymerase chain reaction. Whole-genome sequencing analyzes were performed on the carbapenemase-producing isolates. Results: Eleven gram-negative bacteria (four Enterobacter spp., three Klebsiella spp., three Aeromonas spp., and one Serratia spp.) with blaGES-24 (n = 6), blaGES-6 (n = 4), and blaGES-5 (n = 1) were isolated from the sewage sample. Five blaGES-24 and a blaGES-5 were localized in IncP-6 plasmids, whereas three blaGES-6 plasmids were localized in IncC plasmids with IncF-like regions. The remaining blaGES-6 and blaGES-24 were, respectively, localized on IncFIB-containing plasmids with IncF-like regions and a plasmid with an IncW-like replication protein. The IncP-6 and IncW-like plasmids had a close genetic relationship with plasmids from Japan, whereas the IncC/IncF-like and IncFIB/IncF-like plasmids were closely related to those from the United States and Europe. All blaGES genes were located on the class 1 integron cassette of the Tn3 transposon-related region, and the IncC/IncF-like plasmid carried two copies of the integron cassette. Eight of the eleven blaGES-encoding plasmids contained toxin-antitoxin system genes. Discussion: The findings on the plasmids and the novel genetic content from a single wastewater sample extend our understanding regarding the diversity of resistance and the associated spread of blaGES, suggesting their high adaptability to hospital effluents. These findings highlight the need for the continuous monitoring of environmental GES-type carbapenemase producers to control their dissemination.

Identification of TMexCD-TOprJ-producing carbapenem-resistant Gram-negative bacteria from hospital sewage.

Carbapenems and tigecycline are crucial antimicrobials for the treatment of gram-negative bacteria infections. Recently, a novel resistance-nodulation-division (RND) efflux pump gene cluster, tmexCD-toprJ, which confers resistance to tigecycline, has been discovered in animals and clinical isolates. It was reported that hospital sewage could act as a reservoir for gram-negative bacteria with high antimicrobial resistance genes. In this study, we analyzed 84 isolates of carbapenem-resistant gram-negative bacteria (CR-GNB) from hospital sewage, and identified five isolates of TMexCD-ToprJ-producing CR-GNB, including one Raoultella ornithinolytica isolate and four Pseudomonas spp. isolates. All these five isolates carried at least one carbapenem resistance gene and were resistant to multiple antibiotics. Multiple tmexCD-toprJ clusters were detected, including tmexC2D2-toprJ2, tmexC3D3-toprJ3, tmexC3.2D3.3-toprJ1b and tmexC3.2D3-toprJ1b. Among these clusters, the genetic construct of tmexC3.2D3-toprJ1b showed 2-fold higher minimum inhibitory concentration (MIC) of tigecycline than other three variants. In addition, it was found that the tmexCD-toprJ gene cluster was originated from Pseudomonas spp. and mainly located on Tn6855 variants inserted in the same umuC-like genes on chromosomes and plasmids. This unit co-localized with blaIMP or blaVIM on IncHI5-, IncpJBCL41- and IncpSTY-type plasmids in the five isolates of TMCR-GNB. The IncHI5- and IncpSTY-type plasmids had the ability to conjugal transfer to E. coli J53 and P. aeruginosa PAO1, highlighting the potential risk of transfer of tmexCD-toprJ from Pseudomonas spp. to Enterobacterales. Importantly, genomic analysis showed that similar tmexCD-toprJ-harboring IncHI5 plasmids were also detected in human samples, suggesting transmission between environmental and human sectors. The emergence of TMCR-GNB from hospital sewage underscores the need for ongoing surveillance of antimicrobial resistance genes, particularly the novel resistance genes such as the tmexCD-toprJ gene clusters in the wastewater environment.

Emergence of Aeromonas veronii strain co-harboring blaKPC-2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b cluster from hospital sewage in China.

Introduction: The raise of multi-drug resistant bacteria involving carbapenem, colistin, or tigecycline resistance constitutes a threat to public health, which partly results from the transmission of corresponding mobile resistance genes, such as blaKPC and blaNDM for carbapenem, mcr for colistin, and tmexCD-toprJ gene cluster for tigecycline. Herein, we described the emergence of an Aeromonas veronii strain HD6454 co-harboring blaKPC-2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster from hospital sewage. Methods: Whole genome sequencing (WGS) was used to determine the genome sequence of HD6454, and the detailed genomic analysis of genetic elements or regions carrying key antimicrobial resistance genes (ARGs) from HD6454 were performed. Cloning experiment was conducted to confirm the function of key ARGs in mediating antimicrobial resistance. Conjugation experiment was conducted to determine the mobility of the plasmid. Results: The results showed that this strain belonged to a novel sequence type (ST) variant ST1016, and carried 18 important ARGs. Among them, the blaKPC-2 was carried by non-self-transmissible IncP-6 plasmid, while tmexC3.2-tmexD3.3-toprJ1b gene cluster and mcr-3.17 were carried by integrative and mobilizable element (IME) or IME-related region in chromosome. The mcr-3.17, mcr-3.6, and mcr-3-like3 genes were further inferred to originate from IMEs of Aeromonas species. Additionally, for the first time, the mcr-3.17 was confirmed to confer low-level resistance to colistin under inducible expression, while tmexC3.2-tmexD3.3-toprJ1b gene cluster was confirmed to confer low-level resistance to tigecycline. Discussion: This is the first report of a strain co-harboring blaKPC-2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster. Although the resistance and/or mobility of these ARGs are limited in this strain, the emergence of this multiple important ARGs-carrying strain deserves further attention.

Seasonal analysis of commonly prescribed antibiotics in Istanbul city.

Antibiotics are among the most common medicine groups since they are used to treat infectious diseases, as nutritional supplements in livestock breeding, and for preservation in the food industry. Turkey is among the highest antibiotic consumers in the world. In the present study, the most popular 14 antibiotics available in Turkey were monitored in one hospital sewage and two urban wastewater treatment plant influents and effluents seasonally in Istanbul province, the largest metropolitan center in Turkey. The present research aimed to develop a robust analytical method to determine 14 antibiotics, including six chemical groups, in environmental matrices which are considered significant antibiotic pollution sources, namely hospital sewage and urban wastewater. Solid-phase extraction (SPE) and UPLC-MS/MS analysis parameters included optimized column temperature, eluent, mobile phase, and flow rate. Three SPE cartridges were employed in recovery studies. The antibiotic recovery rates varied between 40 and 100%, and all analytes were identified within 3 min with UPLC-MS/MS under optimal conditions. It was determined that method detection limits (MDLs) varied between 0.07 and 2.72 µg/L for the antibiotics. In all seasons, the highest beta-lactam group antibiotic concentrations were identified in hospital sewage. The season with the greatest variety of antibiotics in urban wastewater was spring. Clarithromycin and ciprofloxacin were the antibiotics determined at the highest concentration in the influent and effluent of the wastewater treatment plant in all seasons. This study showed that the most widely used beta-lactam group antibiotics were found in high amounts in hospital sewage wastewater but in low concentrations in the treatment plants, and hence, it is seen that the degradability of beta-lactam group antibiotics was high. The presence of clarithromycin, ciprofloxacin, lincomycin, levofloxacin, and trimethoprim antibiotics in hospital sewage in higher amounts and also in inlet and outlet of wastewater treatment plants proves that those are resistant antibiotics.

Coexistence of tmexCD3-toprJ1b tigecycline resistance genes with two novel bla VIM-2-carrying and bla OXA-10-carrying transposons in a Pseudomononas asiatica plasmid.

Introduction: Tigecycline and carbapenems are considered the last line of defense against microbial infections. The co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomononas asiatica was not investigated. Methods: P. asiatica A28 was isolated from hospital sewage. Antibiotic susceptibility testing showed resistance to carbapenem and tigecycline. WGS was performed to analyze the antimicrobial resistance genes and genetic characteristics. Plasmid transfer by conjugation was investigated. Plasmid fitness costs were evaluated in Pseudomonas aeruginosa transconjugants including a Galleria mellonella infection model. Results: Meropenem and tigecycline resistant P. asiatica A28 carries a 199, 972 bp long plasmid PLA28.4 which harbors seven resistance genes. Sequence analysis showed that the 7113 bp transposon Tn7389 is made up of a class I integron without a 5'CS terminal and a complete tni module flanked by a pair of 25bp insertion repeats. Additionally, the Tn7493 transposon, 20.24 kp long, with a complete 38-bp Tn1403 IR and an incomplete 30-bp Tn1403 IR, is made up of partial skeleton of Tn1403, a class I integron harboring bla OXA-10, and a Tn5563a transposon. Moreover, one tnfxB3-tmexC3.2-tmexD3b-toprJ1b cluster was found in the plasmid and another one in the the chromosome. Furthermore, plasmid PLA28.4 could be conjugated to P. aeruginosa PAO1, with high fitness cost. Discussion: A multidrug-resistant plasmid carrying tmexCD3-toprJ1b and two novel transposons carrying bla VIM-2 and bla OXA-10 -resistant genes was found in hospital sewage, increasing the risk of transmission of antibiotic-resistant genes. These finding highlight the necessary of controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Phage-based therapy against biofilm producers in gram-negative ESKAPE pathogens.

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5-1 µg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

Sewage from a secondary hospital in Ribeirão Preto, southeastern Brazil: a source of multidrug-resistant Enterobacteriaceae.

Antimicrobial resistance is one of the severe threats to global health. Hospital sewage can serve as a reservoir for multi-resistant bacteria and promote the spread of antimicrobial resistance. This study aimed to investigate the antimicrobial susceptibility and the pathogenic potential of Enterobacteriaceae isolated from the sewage of a secondary hospital in Ribeirão Preto, a city in southeastern Brazil. The strains were isolated by membrane filtration and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The antimicrobial susceptibility profile was performed by disk diffusion. Polymerase chain reaction (PCR) assays were used to detect virulence genes among the strains. Twenty-eight isolates were obtained, with Klebsiella pneumoniae being the predominant species (71.4%, n = 20). All isolates were classified as multidrug-resistant, including four isolates that were non-susceptible to at least 50% of the tested antibiotics. All isolates were also non-susceptible to cefuroxime and sulfonamides antibiotics; however, they were susceptible to norfloxacin, ofloxacin, amikacin, gentamicin, netilmicin, ertapenem, cefazolin, cefaclor, and cefotetan. The virulence genes ycfM, fimH, mrkD, kfu, and entB were detected in several isolates. Our study showed that even in a secondary hospital, without the routine of major surgeries and intensive care admissions, the hospital sewage can harbor a high percentage of multidrug-resistant bacteria with pathogenic potential. This leads to the worrying risk of public health and environmental contamination.

Multidrug-resistant and potentially pathogenic Enterobacteriaceae found in a tertiary hospital sewage in southeastern Brazil.

Hospital sewage is considered an environment with the potential to favor the spread and increase of multidrug-resistant bacteria (MDR). The increase in antimicrobial resistance is one of the greatest global threats today. Therefore, this study aimed to evaluate the profile of antimicrobial susceptibility and virulence factors in Enterobacteriaceae isolated from the sewage of a tertiary hospital located in southeastern Brazil. For bacterial isolation, membrane filtering, serial dilution, and spread-plate techniques were used. The bacterial isolates were identified using the MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) technique. Antimicrobial susceptibility profile was performed by disk-diffusion test. Virulence genes were screened by Polymerase Chain Reaction (PCR) and the hypermucoviscosity phenotype by string test. In total, 13 enterobacteria distributed in three species were identified (Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii) and 76.9% (n = 10) were classified as MDR. Two K. pneumoniae demonstrated the hypermucoviscosity phenotype. The virulence genes ycfM and entB were detected in all K. pneumoniae isolates (other genes found were fimH, mrkD, and kfu). The results indicated that the sewage from the analyzed hospital receives MDR bacteria and has the potential to contaminate and spread through the environment.

Detection of E. coli IncX1 Plasmid-Mediated mcr-5.1 Gene in an Indian Hospital Sewage Water Using Shotgun Metagenomic Sequencing: A First Report.

Colistin is used against a multitude of multidrug-resistant and extremely drug-resistant Gram-negative bacterial infections. The emergence of colistin resistance is highly concerning as it may lead to the failure of this last-resort antibiotic. Since the identification of first mobile colistin resistance (mcr) genes, several variants of mcr genes have been reported, but still there are limited studies detecting mcr genes in hospital sewage water. The prevalence of mcr in the hospital environment is extremely hazardous putting health care workers, patients, and visitors at a higher risk of exposure. It may lead to a multidrug-resistant bacterial infection outbreak. In this study, we report mcr-5.1 gene in an Indian hospital sewage water using shotgun metagenomics, as a first report. The mcr-5.1 gene in the metagenome has been explored using RGI, ABRicate, NCBI database, CARD, and Resfinder. This mcr-5.1 gene harbored by Escherichia coli is a plasmid-mediated gene carried by an IncX1 plasmid pSGMCR103. The bioinformatics analysis revealed the genetic environment of mcr-5.1 gene, which consisted of mobile element protein, ChrB domain protein, putative major facilitator superfamily type transporter, and a hypothetical protein.

blaKPC-24-Harboring Aeromonas veronii from the Hospital Sewage Samples in China.

KPC-24, different from KPC-2 by a single amino acid alteration at codon 6 (R6P), was initially discovered in Klebsiella pneumoniae in Chile. Here, we reported KPC-24-producing Aeromonas veronii isolates from hospital sewage in China. The blaKPC-24 was cloned and the MICs were tested against ß-lactams antimicrobial agents. KPC-24 exhibited a ß-lactam susceptibility profile similar to that of KPC-2. Whole-genome sequencing and analysis revealed that blaKPC-24 was located within a Tn6296-related region on an IncP-6 plasmid. IMPORTANCE Our study described a variant of K. pneumoniae carbapenemase (KPC), KPC-24, from two A. veronii strains isolated from hospital sewage, in which antibiotics, biocides, pharmaceuticals, and heavy metals may supply an appropriate condition for the evolution of carbapenemases. Some variants exhibited stronger hydrolysis activity to antibiotics and gave rise to a major public health concern. More seriously, Aeromonas species are prevalent in aquatic environments and, thus, may act as a suitable vector for antibiotics-resistance genes and foster the transmission of resistance. We should attach importance to surveying the evolution and transmission of antibiotics-resistance genes.

blaKPC-2-Encoding IncP-6 Plasmids in Citrobacter freundii and Klebsiella variicola Strains from Hospital Sewage in Japan.

Klebsiella pneumoniae carbapenemase (KPC) producers are an emerging threat to global health, and the hospital water environment is considered an important reservoir of these life-threatening bacteria. We characterized plasmids of KPC-2-producing Citrobacter freundii and Klebsiella variicola isolates recovered from hospital sewage in Japan. Antimicrobial susceptibility testing, whole-genome sequencing analysis, bacterial conjugation, and transformation experiments were performed for both KPC-2 producers. The blaKPC-2 gene was located on the Tn3 transposon-related region from an IncP-6 replicon plasmid that could not be transferred via conjugation. Compared to the blaKPC-2-encoding plasmid of the C. freundii isolate, alignment analysis of plasmids with blaKPC-2 showed that the blaKPC-2-encoding plasmid of the K. variicola isolate was a novel IncP-6/IncF-like hybrid plasmid containing a 75,218-bp insertion sequence composed of IncF-like plasmid conjugative transfer proteins. Carbapenem-resistant transformants harboring blaKPC-2 were obtained for both isolates. However, no IncF-like insertion region was found in the K. variicola donor plasmid of the transformant, suggesting that this IncF-like region is not readily functional for plasmid conjugative transfer and is maintained depending on the host cells. The findings on the KPC-2 producers and novel genetic content emphasize the key role of hospital sewage as a potential reservoir of pathogens and its linked dissemination of blaKPC-2 through the hospital water environment. Our results indicate that continuous monitoring for environmental emergence of antimicrobial-resistant bacteria might be needed to control the spread of these infectious bacteria. Moreover, it will help elucidate both the evolution and transmission pathways of these bacteria harboring antimicrobial resistance. IMPORTANCE Antimicrobial resistance is a significant problem for global health, and the hospital environment has been recognized as a reservoir of antimicrobial resistance. Here, we provide insight into the genomic features of blaKPC-2-harboring isolates of Citrobacter freundii and Klebsiella variicola obtained from hospital sewage in Japan. The findings of carbapenem-resistant bacteria containing this novel genetic context emphasize that hospital sewage could act as a potential reservoir of pathogens and cause the subsequent spread of blaKPC-2 via horizontal gene transfer in the hospital water environment. This indicates that serial monitoring for environmental bacteria possessing antimicrobial resistance may help us control the spread of infection and also lead to elucidating the evolution and transmission pathways of these bacteria.

Emergence of Metallo-ß-Lactamases and OXA-48 Carbapenemase Producing Gram-Negative Bacteria in Hospital Wastewater in Algeria: A Potential Dissemination Pathway Into the Environment.

Antibiotic-resistant bacteria can leave hospitals and therefore contaminate the environment and, most likely, humans and animals, through different routes, among which wastewater discharge is of great importance. This study aims to assess the possible role of hospital sewage as reservoir and dissemination pathway of carbapenem-resistant Gram-negative bacilli (GNB). Carbapenem-resistant GNB were selectively isolated from wastewater collected from a public hospital in Batna, Algeria. Species identification was carried out using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry, and antibiotic susceptibility was evaluated by the disc diffusion method. ß-Lactamase production was investigated phenotypically using the double-disk synergy assay and the modified CarbaNP test, then the molecular mechanisms of ß-lactam-resistance were studied by PCR and sequencing. Ten Enterobacteriaceae and 14 glucose-nonfermenting GNB isolates were obtained. All Enterobacteriaceae isolates were positive for OXA-48 and TEM-1D ß-lactamases, where seven of them coproduced an extended-spectrum ß-lactamase. VIM-2 carbapenemase was detected in six glucose-nonfermenting GNB isolates. However, three Pseudomonas aeruginosa, one Comamonas jiangduensis and one Acinetobacter baumannii isolates were positive for VIM-4 variant. In addition, NDM-1 enzyme was detected in four A. baumannii isolates. Our findings highlight the potential impact of hospital wastewater in the spread of drug resistance mechanisms outside of hospitals.

Identification of a Novel blaNDM Variant, blaNDM-33, in an Escherichia coli Isolate from Hospital Wastewater in China.

Since the discovery of NDM-1 and the worldwide reporting of different variants have raised alarms concerning global health, the problem of carbapenem-resistant Enterobacterales (CRE) has become increasingly serious. Therefore, research on the hydrolytic activity and molecular structure of NDM variants is beneficial to the development of antibacterial drugs. NDM has been evolving into variants that possess different hydrolysis activities toward ß-lactam antibiotics. Here, we characterized a novel blaNDM variant, named blaNDM-33, identified from a multidrug-resistant Escherichia coli strain from hospital sewage. NDM-33 differed from NDM-5 with a single-amino-acid substitution (A72T). blaNDM-5 was located in the Tn125-related blaNDM-33 region from an IncX3-type plasmid, pHD6415-NDM, that can be transferred horizontally. The genetic construct of blaNDM-33 showed higher MICs of carbapenems than a blaNDM-5 construct. Enzyme kinetics showed that NDM-33 had higher enzymatic activity for meropenem and cefazolin than NDM-5. The emergence of this novel NDM variant could pose a threat to public health because of its transferability and enhanced carbapenem activity. IMPORTANCE Our study described a novel NDM-33 variant from an E. coli strain isolated from hospital sewage, where it was associated with human disease and antibiotic exposure. Importantly, hospital sewage was increasingly considered to be related to CRE hosts. Pathogens were transmitted from reservoirs through direct and indirect contact, ingestion, and inhalation of contaminated water or aerosols. In addition, under the selective pressure of antibiotics, NDM variants will become the main strain in the hospital water system and evolve into high virulence and high resistance. The monitoring of NDM mutants is of great significance for preventing and controlling the evolution of superbugs.

Monitoring of hospital sewage shows both promise and limitations as an early-warning system for carbapenemase-producing Enterobacterales in a low-prevalence setting.

Carbapenemase-producing Enterobacterales (CPE) constitute a significant threat to healthcare systems. Continuous surveillance is important for the management and early warning of these bacteria. Sewage monitoring has been suggested as a possible resource-efficient complement to traditional clinical surveillance. It should not least be suitable for rare forms of resistance since a single sewage sample contains bacteria from a large number of individuals. Here, the value of sewage monitoring in early warning of CPE was assessed at the Sahlgrenska University Hospital in Gothenburg, Sweden, a setting with low prevalence of CPE. Twenty composite hospital sewage samples were collected during a two-year period. Carbapenemase genes in the complex samples were analyzed by quantitative PCR and the CPE loads were assessed through cultures on CPE-selective agar followed by species determination as well as phenotypic and genotypic tests targeting carbapenemases of presumed CPE. The findings were related to CPE detected in hospitalized patients. A subset of CPE isolates from sewage and patients were subjected to whole genome sequencing. For three of the investigated carbapenemase genes, blaNDM, blaOXA-48-like and blaKPC, there was concordance between gene levels and abundance of corresponding CPE in sewage. For the other two analyzed genes, blaVIM and blaIMP, there was no such concordance, most likely due to the presence of those genes in non-Enterobacterales populating the sewage samples. In line with the detection of OXA-48-like- and NDM-producing CPE in sewage, these were also the most commonly detected CPE in patients. NDM-producing CPE were detected on a single occasion in sewage and isolated strains were shown to match strains detected in a patient. A marked peak in CPE producing OXA-48-like enzymes was observed in sewage during a few months. When levels started to increase there were no known cases of such CPE at the hospital but soon after a few cases were detected in samples from patients. The OXA-48-like-producing CPE from sewage and patients represented different strains, but they carried similar blaOXA-48-like-harbouring mobile genetic elements. In conclusion, sewage analyses show both promise and limitations as a complement to traditional clinical resistance surveillance for early warning of rare forms of resistance. Further evaluation and careful interpretation are needed to fully assess the value of such a sewage monitoring system.

Thermal plasma activation and UV/H2O2 oxidative degradation of pharmaceutical residues.

The aquatic environment becomes increasingly contaminated by anthropogenic pollutants such as pharmaceutical residues. Due to poor biodegradation and continuous discharge of persistent compounds in sewage water samples, pharmaceutical residues might end up in surface waters when not removed. To minimize this pollution, onsite wastewater treatment techniques might complement conventional waste water treatment plants (WWTPs). Advanced oxidation processes are useful techniques, since reactive oxygen species (ROS) are used for the degradation of unwanted medicine residues. In this paper we have studied the advanced oxidation in a controlled laboratory setting using thermal plasma and UV/H2O2 treatment. Five different matrices, Milli-Q water, tap water, synthetic urine, diluted urine and synthetic sewage water were spiked with 14 pharmaceuticals with a concentration of 5 µg/L. All compounds were reduced or completely decomposed by both 150 W thermal plasma and UV/H2O2 treatment. Additionally, also hospital sewage water was tested. First the concentrations of 10 pharmaceutical residues were determined by liquid chromatography mass spectrometry (LC-MS/MS). The pharmaceutical concentration ranged from 0.08 up to 2400 µg/L. With the application of 150 W thermal plasma or UV/H2O2, it was found that overall pharmaceutical degradation in hospital sewage water were nearly equivalent to the results obtained in the synthetic sewage water. However, based on the chemical abatement kinetics it was demonstrated that the degree of degradation decreases with increasing matrix complexity. Since reactive oxygen and nitrogen species (RONS) are continuously produced, thermal plasma treatment has the advantage over UV/H2O2 treatment.

Comprehensive Genomic Survey of Antimicrobial-Resistance Bacteria in the Sewage Tank Replacement with Hospital Relocation.

BACKGROUND: Excrement containing antimicrobial-resistant bacteria (ARB) is discharged from the hospital sewage through wastewater treatment plants (WWTP) into rivers, increasing the antimicrobial resistance (AMR) burden on the environment. PURPOSE: We illustrate the contamination of hospital sewage tanks with ARB harboring antimicrobial resistance genes (ARGs) using comprehensive metagenomic sequencing. During the study period, we moved to a new hospital building constructed for renovation. Therefore, we investigated the difference in bacterial flora in the sewage tanks for each building with different departments, and the change in bacterial flora over time in new sewage tanks. Furthermore, we performed a comparative genome analysis of extended spectrum ß-lactamase (ESBL)-producing organisms (EPOs) from hospital sewage and clinical samples. Residual antibiotics in the sewage tank were also measured. METHODS: Metagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of EPOs. RESULTS: The bacterial composition of new sewage tanks was comparable with that of old tanks within 1 month after relocation and was instantly affected by excrement. The bacterial composition of sewage tanks in the old and new buildings, containing rooms where seriously ill patients were treated, was similar. Selection on CHROMagar ESBL allowed detection of EPOs harboring bla CTX-M and carbapenemase genes in all sewage tanks. One of the sewage Escherichia coli strain comprising ST393 harboring bla CTX-M-27 corresponded to the clinical isolates based on core genome analysis. Moreover, the levels of levofloxacin and clarithromycin in the hospital sewage were 0.0325 and 0.0135 µg/mL, respectively. CONCLUSION: Hospital sewage was contaminated with many ARB species, ARGs and residual antibiotics, which can cause a burden on WWTP sewage treatment. The bacterial flora in the sewage tank was rapidly affected, especially by the ward with seriously ill patients. AMR monitoring of hospital sewage may help detect carriers prior to nosocomial ARB-associated outbreaks and control the outbreaks.

Complete genome sequencing and comparative plasmid analysis of KPC-2-producing Klebsiella pneumoniae isolated from hospital sewage water in Japan.

OBJECTIVES: The Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. METHODS: A KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. RESULTS: Klebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. CONCLUSION: pKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Detection of New Delhi metallo-beta-lactamase enzyme gene bla NDM-1 associated with the Int-1 gene in Gram-negative bacteria collected from the effluent treatment plant of a tuberculosis care hospital in Delhi, India.

BACKGROUND: Organisms possessing the bla NDM-1 gene (responsible for carbapenem resistance) with a class-1 integron can acquire many other antibiotic resistance genes from the community sewage pool and become multidrug-resistant superbugs. In this regard, hospital sewage, which contains a large quantity of residual antibiotics, metals and disinfectants, is being recognized as a significant cause of antimicrobial resistance (AMR) origination and spread across the major centres of the world and is thus routinely investigated as a marker for tracing the origin of drug resistance. Therefore, in this study, an attempt has been made to identify and characterize the carbapenem-resistant microbes associated with integron genes amongst the organisms isolated from the effluent treatment plant (ETP) installed in a tertiary respiratory care hospital in Delhi, India. METHODS: One hundred and thirty-eight organisms belonging to Escherichia , Klebsiella , Pseudomonas and Acinetobacter spp. were collected from the incoming and outgoing sewage lines of the ETP. Carbapenem sensitivity and characterization was performed by the imipenem and imipenem-EDTA disc diffusion method. Later DNA extraction and PCR steps were performed for the Int-1 and bla NDM-1 genes. RESULTS: Of the 138 organisms, 86 (62.3 %) were imipenem-resistant (P<0.05). One hundred and twenty-four (89.9 %) organisms had one or both of the genes. Overall, the bla NDM-1 gene (genotypic resistance) was present in 71 % (98/138) of organisms. 53.6 % (74/138) organisms were double gene-positive (bla NDM-1 + Int-1), of which 40 were producing the metallo-beta-lactamase enzyme, making up almost 28.9 % (40/138) of the collected organisms. CONCLUSION: The current study strengthens the hypothesis that Carbapenem resistant organisms are in a high-circulation burden through the human gut and hospital ETPs are providing an environment for resistance origination and amplification.