Genomic surveillance of multidrug-resistant Escherichia coli and Klebsiella in clinical and wastewater isolates from a paediatric hospital in Lima, Peru

IntroductionWastewater surveillance has recently emerged as a promising method for AMR surveillance, but few studies have directly compared wastewater surveillance samples with clinical isolates and the clinical significance of wastewater surveillance for AMR bacteria is still unclear.MethodsWe carried out monthly surveillance of hospital wastewater and surrounding community water at a tertiary hospital in Chonburi, Thailand from March to December 2024 and compared our findings with clinical isolates collected at the same hospital during the same period. For each wastewater sample, we isolated ESKAPEE pathogens, identified species by mass spectrometry, performed antimicrobial susceptibility testing (AST), followed by PCR testing of AMR genes and whole genome sequencing (WGS) on a subset of wastewater isolates and compared our results to clinical isolates.ResultsWe obtained 2,735 AMR isolates from untreated hospital wastewater, of which 1,550 were ESKAPEE pathogens including: Klebsiella pneumoniae (35.8%), Enterobacter spp. (397, 25.6%), Escherichia coli (24.9%), Enterococcus faecium (10.1%), Acinetobacter baumannii (2.7%), and Pseudomonas aeruginosa (0.8%). Based on AST data, we found that 85% K. pneumoniae isolates, 43% A. baumannii isolates, and 98% E. coli isolates, 62% Enterobacter spp. isolates, and 26% E. faecium isolates were classified as multi-drug resistant. We carried out hierarchical clustering of the AST data for a subset of 416 wastewater isolates along with 743 clinical isolates and found significant overlap in AST profiles of wastewater and clinical isolates. Using the clustering data, we selected a subset of 52 wastewater isolates with similar AST profiles to clinical isolates for WGS and identified 19 wastewater isolates that were highly genetically related (≤10 different alleles by cgMLST) to a clinical isolate, including 9 isolates with zero different alleles from closely related clinical isolates.ConclusionOur results show that surveillance of untreated hospital wastewater is capable of identifying ESKAPEE that share similar drug resistance profiles, AMR genes, and clonal lineages found in the clinical isolates over the same time period. To our knowledge, this is one of the first studies to demonstrate a high level of genetic-relatedness between hospital wastewater and clinical isolates and demonstrate the clinical relevance of bacterial wastewater surveillance for MDR ESKAPEE pathogens.

Virulence and antimicrobial resistance genes are enriched in the plasmidome of clinical Escherichia coli isolates compared with wastewater isolates from western Kenya.

Cefiderocol-resistant Aeromonas with expanded Resistomes in German hospital wastewater: Phenotypic and genomic evidence from the environment-clinical Interface.

Abstract. Endraputra PN, Kuntaman K, Wasito EB, Shirakawa T, Raharjo D, Setyarini W. 2021. Profile variation of bla genes among non-lactose fermenting Gram negative bacilli between clinical and environmental isolates of Dr. Soetomo Hospital, Surabaya, Indonesia. Biodiversitas 22: 5047-5054. Carbapenem-resistant non-fermenter Gram-negative bacilli are notorious opportunistic pathogens in hospitalized patients and hospital environments. This study explored the carbapenemase gene among non-fermenter Gram-negative bacilli from hospital wastewater and clinical isolates in Dr. Soetomo Hospital, Surabaya, Indonesia. All samples were screened on MacConkey agar with meropenem 2 µg/ml and gene detected by Multiplex PCR. All samples were screened on MacConkey agar with meropenem 2 µg/ml and gene detected by Multiplex PCR. A total of 121 isolates consisted of 76 clinical (41 carbapenem-resistant Acinetobacter baumannii and 35 carbapenem-resistant Pseudomonas aeruginosa), 45 environmental isolates (6 carbapenem-resistant Pseudomonas aeruginosa and 32 carbapenem-resistant Pseudomonas spp.), and 7 screening samples (all CRPAs). Clinical isolates carbapenemase genes were identified, blaOXA-23-like 21 (28%), blaOXA-24-like 30 (39%), blaNDM-1 1 (1%), and blaIMP-1 6 (8%) while environmental isolates were blaOXA-23-like 5 (13%), blaOXA-24-like 4 (11%), blaOXA-48-like 2 (5%), blaNDM-1 13 (34%), blaVIM 12 (32%), and blaIMP-1 4 (11%). Rectal swab screening specimens presented blaOXA-23-like 3 (43%), blaOXA-24-like 3 (43%), and blaNDM-1 1 (14%). The carbapenemase gene pattern was different between clinical and environmental isolates. The blaOXA-23-like and blaOXA-24-like were most prevalent among in both clinical and wastewater, while blaVIM was mostly in wastewater. The presence of carbapenem-resistant non-fermenter Gram-negative bacilli carrying carbapenemase genes in hospital effluents indicated that the community river was seeded with an antimicrobial resistance gene.

Antimicrobial resistance may develop in nature including in hospital wastewater through horizontal genetic transfer. Few studies were conducted on the antimicrobial resistance genes in hospital wastewater and wastewater isolates in Indonesia. The prevalence and abundance of beta-lactam resistance genes in hospital wastewater and Enterobacterales wastewater isolates were investigated. Twelve wastewater samples were collected from an influent wastewater treatment plant. Escherichia coli and Klebsiella pneumoniae were isolated from the wastewater samples by culture-based methods. DNA was extracted from wastewater samples and the isolates. Nineteen beta-lactam resistance genes were tested by a high throughput qRT-PCR method. blaGES and blaTEM were the most abundant genes detected in hospital wastewater and Escherichia coli, respectively (p < 0.001). The relative abundance of blaCMY_2, blaCTX-M5, blaCTX-M8, blaGES, blaNDM, and blaSHV11 in Klebsiella pneumoniae was higher than in the wastewater and Escherichia coli (p < 0.001; p = 0.006; p = 0.012; p < 0.001; p = 0.005; p < 0.001). Klebsiella pneumoniae might be associated with resistance to piperacillin/tazobactam, ceftriaxone, and cefepime (p < 0.001; p = 0.001; p < 0.001). In conclusion, ESBL genes showed higher abundance than carbapenemase genes in hospital wastewater samples. The ESBL-producing bacteria that were predominantly found in hospital wastewater may originate from clinical specimens. The culture-independent antibiotic resistance monitoring system might be developed as an early warning system for the increasing beta-lactam resistance level in clinical settings.

Background: Hospital wastewater is a major source of antimicrobial resistance (AMR) outflow into the environment. This study uses metagenomics to study how hospital clinical activity impacts antimicrobial resistance genes (ARGs) abundances in hospital wastewater.Methods: Sewage was collected over a 24-h period from multiple wastewater collection points (CPs) representing different specialties within a tertiary hospital site and simultaneously from community sewage works. High throughput shotgun sequencing was performed using Illumina HiSeq4000. ARG abundances were correlated to hospital antimicrobial usage (AMU), data on clinical activity and resistance prevalence in clinical isolates.Results: Microbiota and ARG composition varied between CPs and overall ARG abundance was higher in hospital wastewater than in community influent. ARG and microbiota compositions were correlated (Procrustes analysis, p=0.014). Total antimicrobial usage was not associated with higher ARG abundance in wastewater. However, there was a small positive association between resistance genes and antimicrobial usage matched to ARG phenotype (IRR 1.11, CI 1.06–1.16, p<0.001). Furthermore, analyzing carbapenem and vancomycin resistance separately indicated that counts of ARGs to these antimicrobials were positively associated with their increased usage [carbapenem rate ratio (RR) 1.91, 95% CI 1.01–3.72, p=0.07, and vancomycin RR 10.25, CI 2.32–49.10, p<0.01]. Overall, ARG abundance within hospital wastewater did not reflect resistance patterns in clinical isolates from concurrent hospital inpatients. However, for clinical isolates of the family Enterococcaceae and Staphylococcaceae, there was a positive relationship with wastewater ARG abundance [odds ratio (OR) 1.62, CI 1.33–2.00, p<0.001, and OR 1.65, CI 1.21–2.30, p=0.006 respectively].Conclusion: We found that the relationship between hospital wastewater ARGs and antimicrobial usage or clinical isolate resistance varies by specific antimicrobial and bacterial family studied. One explanation, we consider is that relationships observed from multiple departments within a single hospital site will be detectable only for ARGs against parenteral antimicrobials uniquely used in the hospital setting. Our work highlights that using metagenomics to identify the full range of ARGs in hospital wastewater is a useful surveillance tool to monitor hospital ARG carriage and outflow and guide environmental policy on AMR.

Background Environmental monitoring of enterobacteria in hospital wastewater could be a useful tool to understand the composition of the microbiota in patients, their visits, and healthcare personnel, but it also may be useful for monitoring antimicrobial resistance among healthcare-associated infections in hospitalized patients. The aim of this study was to characterize and describe the phenotypic and genotypic antimicrobial resistance of Escherichia coli and Klebsiella sp. strains from wastewater and from clinical isolates in a tertiary care children’s hospital in Lima, Perú. Methods We systematically collected 70 isolates of Escherichia coli and Klebsiella sp. from wastewater and 24 isolates of the same enterobacteria from blood and urine cultures at Instituto Nacional de Salud del Niño San Borja (INSN-SB) in Lima from December 2018 to May 2019. Susceptibility profiles were evaluated following CLSI criteria. We used the Jarlier method for the detection of extended-spectrum beta-lactamases (ESBL). For detection of AmpC beta-lactamases, we used discs of cefoxitin, ceftazidime, and ceftriaxone combined with cloxacillin and cefotaxime. For the detection of carbapenemase production, we used EDTA and phenylboronic acid inhibitors between meropenem and imipenem disks. We performed genomic sequencing for the detection of resistance genes and to perform a phylogenetic cluster analysis. Results We collected a total of 94 isolates (70 from wastewater and 24 from clinical samples). Among the total isolates, 19 (20.2%) were ESBL producers. The frequency of ESBL producers in wastewater was 7.1% (5/24), whereas the frequency of ESBL producers in clinical samples was 58.3% (14/70). The most frequent resistance genes were tet (variant 34 and A) and blaTEM-1. The frequency of tet(34) and tet(A) were 10% and 7%, respectively, in isolates from wastewater, whereas the frequency of these genes were 0% and 6%, respectively, in clinical isolates. The gene blaCTX-M-15 was present in isolates from wastewater (1%) and clinical samples (2%). Phylogenetic cluster analysis found no similarities between isolates from wastewater and to those from clinical samples, suggesting that the population of these enterobacteria were different in wastewater compared with clinical samples. Conclusions Enterobacteria from hospital wastewater may not reflect the profile of infections caused by these microorganisms. However, they may reflect the microbiological microbiota among patients, their visits, and hospital healthcare personnel. Further studies that compare the phenotypic and genotypic characteristics among isolates from wastewater and the enteric microbiota from these individuals would be necessary to assess this hypothesis.

Characterisation, dissemination and persistence of gentamicin resistant Escherichia coli from a Danish university hospital to the waste water environment

Are antibiotic-resistant Pseudomonas aeruginosa isolated from hospitalised patients recovered in the hospital effluents?

Antimicrobial resistance in hospital wastewater in Scotland: a cross-sectional metagenomics study

Hospital wastewater is a reservoir of antimicrobial resistance (AMR), yet the genetic diversity and resistance mechanisms of environmental Escherichia coli in such settings remain underexplored. This study aimed to investigate the genomic characteristics, resistance profiles, and virulence potential of E. coli isolates recovered from a hospital wastewater in Jakarta, Indonesia. Six Escherichia coli isolates from hospital wastewater were sequenced using short-read next-generation sequencing (NGS). Raw reads were quality-checked and assembled with SPAdes, with five genomes retained for downstream analysis. Antimicrobial resistance (AMR) genes were identified using Staramr and the CARD database, while virulence factors were predicted using Abricate against the Virulence Factors Database (VFDB). Plasmid replicons were detected with PlasmidFinder. Phylogroup assignment followed the Clermont typing method, and phylogenetic analysis was conducted using a neighbor-joining tree based on core genome MLST (cgMLST) generated with chewBBACA v3.3.10. Multilocus sequence typing (MLST) revealed five distinct sequence types (ST744, ST156, ST1196, ST38, and ST10) across three phylogroups (A, B1, and D). A total of 57 AMR genes were detected, including blaCTX-M-15, blaCMY-2, and blaOXA-1 along with plasmid-mediated and chromosomal mutations conferring resistance to fluoroquinolones, aminoglycosides, and tetracyclines. E. coli from hospital wastewater in Jakarta exhibited high genomic diversity, multidrug resistance, and variable virulence profiles. The findings support the role of untreated hospital effluents as a hotspot for AMR emergence and horizontal gene transfer. This showed the need for routine environmental surveillance to mitigate the public health risks associated with environmental reservoirs of resistant pathogens.

Summary Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial‐resistant (AMR) Aeromonas strains (A. hydrophila GSH8‐2 and A. caviae GSH8M‐1) carrying the carabapenemase bla KPC‐2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8‐2 and GSH8M‐1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired bla KPC‐2‐positive IncP‐6‐relative plasmids (pGSH8‐2 and pGSH8M‐1‐2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to bla KPC‐2, pGSH8M‐1‐2 carries an IS26‐mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Metagenomic studies have made it possible to deepen the analysis of the abundance of bacterial populations that carry resistance and virulence determinants in the wastewater environment. In this study, a longitudinal collection of samples of community and hospital wastewater from August 2021 to September 2022 was obtained. Shotgun metagenomic sequencing and bioinformatic analysis were performed to characterize the bacterial abundance, antimicrobial resistance genes (ARGs), plasmids, and virulence factor genes (VFGs) contained in the wastewater. The microbial composition of the community and hospital wastewater showed that the most abundant bacterial phyla detected in all samples were: Proteobacteria, Bacteroides, Firmicutes, Campylobacterota, and Actinobacteria. Seasonal differences in the relative abundances of species, ARGs, plasmids, and VFGs were observed. In this study, a total of 270 ARGs were detected, and it was found that the absolute abundance of ARGs only showed a 39% reduction in the treated wastewater. Furthermore, the ARGs detected in this study were found to encode resistance to antibiotics of the last choice. Our results showed that plasmids carrying resistance genes were more abundant in raw wastewater, and 60% more abundant in hospital wastewater compared to community wastewater. Several of the VFGs detected in this study encode for adhesion, motility, and biofilm formation, which likely allows bacteria to remain and persist in the wastewater environment and survive WWTP treatment systems, thus managing to escape into the environment via treated wastewater.

In low- and middle-income countries, living in homes with soil floors and animal cohabitation may expose children to fecal organisms, increasing the risk of enteric and antimicrobial-resistant infections. Our objective was to understand whether cow cohabitation in homes with soil floors in rural Bangladesh contributed to the presence and diversity of potential pathogens and antimicrobial resistance genes (ARGs) in the home. In 10 randomly selected households in rural Sirajganj District, we sampled floor soil and cow dung, which is commonly used as sealant in soil floors. We extracted DNA and performed shotgun metagenomic sequencing to explore potential pathogens and ARGs in each sample type. We detected 7 potential pathogens in soil only, 38 pathogens in cow dung only, and 182 pathogens in both soil and cow dung. Cow dung exhibited modestly higher potential pathogen genus richness compared to soil floors (Wilcoxon signed-rank test, P = 0.002). Using Bray-Curtis dissimilarity, potential pathogen species community composition differed between floors and cow dung (permutational multivariate analysis of variance, P < 0.001). All soil floor and cow dung samples contained ARGs; detected ARGs confer resistance to antibiotic classes including sulfonamides, rifamycin, aminoglycosides, lincosamides, and tetracycline. Paired floor and cow dung samples shared ARGs against rifamycin and glycopeptides, but otherwise, there was little overlap in resistomes between sample types. Our findings contribute to the growing literature on household soil and domestic animals as potentially important contributors to disease transmission and as reservoirs of antimicrobial resistance in low-income country settings.IMPORTANCEIn low-income countries, inadequate housing materials and animal cohabitation can lead to fecal contamination of rural homes. Contaminated soil floors are difficult to clean and may harbor organisms causing illness and antibiotic resistance, especially in young children, who frequently ingest soil. We sequenced soil floor and cow dung samples from households in Sirajganj district, Bangladesh, and identified potential pathogens and antibiotic resistance genes. We detected 182 potential pathogens in both soil and cow dung; organisms present in both sample types at the highest relative abundances were Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Pseudomonas aeruginosa. Antibiotic resistance genes were found in all samples. In cow dung, the most common genes conferred resistance to the antibiotics lincosamide, rifamycin, cephamycin, tetracycline, and multiple antibiotics. In soil floors, the most common genes conferred resistance to rifamycin, sulfonamides, and multiple antibiotics. Household soil and cow dung may be important reservoirs of pathogens and antimicrobial resistance in low-income country settings with high levels of animal cohabitation compared to settings with finished household floors and minimal animal cohabitation.

This longitudinal study tests correlations between antimicrobial agents (AA) and corresponding antimicrobial resistance genes (ARGs) generated by a community of >100 k people inhabiting one city (Bath) over a 13 month randomised monitoring programme of community wastewater. Several AAs experienced seasonal fluctuations, such as the macrolides erythromycin and clarithromycin that were found in higher loads in winter, whilst other AA levels, including sulfamethoxazole and sulfapyridine, stayed consistent over the study period. Interestingly, and as opposed to AAs, ARGs prevalence was found to be less variable, which indicates that fluctuations in AA usage might either not directly affect ARG levels or this process spans beyond the 13-month monitoring period. However, it is important to note that weekly positive correlations between individual associated AAs and ARGs were observed where seasonal variability in AA use was reported: ermB and macrolides CLR-clarithromycin and dmCLR-N-desmethyl clarithromycin, aSPY- N-acetyl sulfapyridine and sul1, and OFX-ofloxacin and qnrS. Furthermore, ARG loads normalised to 16S rRNA (gene load per microorganism) were positively correlated to the ARG loads normalised to the human population (gene load per capita), which indicates that the abundance of microorganisms is proportional to the size of human population and that the community size, and not AA levels, is a major driver of ARG levels in wastewater. Comparison of hospital and community wastewater showed higher number of AAs and their metabolites, their frequency of occurrence and concentrations in hospital wastewater. Examples include: LZD-linezolid (used only in severe bacterial infections) and AMX-amoxicillin (widely used, also in community but with very low wastewater stability) that were found only in hospital wastewater. CIP-ciprofloxacin, SMX-sulfamethoxazole, TMP-trimethoprim, MTZ-metronidazole and macrolides were found at much higher concentrations in hospital wastewater while TET-tetracycline and OTC-oxytetracycline, as well as antiretrovirals, had an opposite trend. In contrast, comparable concentrations of resistant genes were observed in both community and hospital wastewater. This supports the hypothesis that AMR levels are more of an endemic nature, developing over time in individual communities. Both hospital and community wastewater had AAs that exceeded PNEC values (e.g. CLR-clarithromycin, CIP-ciprofloxacin). In general, though, hospital effluents had a greater number of quantifiable AAs exceeding PNECs (e.g. SMX-sulfamethoxazole, ERY-erythromycin, TMP-trimethoprim). Hospitals are therefore an important consideration in AMR surveillance as could be high risk areas for AMR.