Hidden Resistome: Enrichment Reveals the Presence of Clinically Relevant Antibiotic Resistance Determinants in Treated Wastewater-Irrigated Soils.

Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring γ-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.

Changes in Antibiotic Resistance Gene Levels in Soil after Irrigation with Treated Wastewater: A Comparison between Heterogeneous Photocatalysis and Chlorination.

Wastewater (WW) reuse is expected to be increasingly indispensable in future water management to mitigate water scarcity. However, this increases the risk of antibiotic resistance (AR) dissemination via irrigation. Herein, a conventional (chlorination) and an advanced oxidation process (heterogeneous photocatalysis (HPC)) were used to disinfect urban WW to the same target of Escherichia coli <10 CFU/100 mL and used to irrigate lettuce plants (Lactuca sativa) set up in four groups, each receiving one of four water types, secondary WW (positive control), fresh water (negative control), chlorinated WW, and HPC WW. Four genes were monitored in water and soil, 16S rRNA as an indicator of total bacterial load, intI1 as a gene commonly associated with anthropogenic activity and AR, and two AR genes blaOXA-10 and qnrS. Irrigation with secondary WW resulted in higher dry soil levels of intI1 (from 1.4 × 104 copies/g before irrigation to 3.3 × 105 copies/g after). HPC-treated wastewater showed higher copy numbers of intI1 in the irrigated soil than chlorination, but the opposite was true for blaOXA-10. The results indicate that the current treatment is insufficient to prevent dissemination of AR markers and that HPC does not offer a clear advantage over chlorination.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-ß-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-ß-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Making waves: The NORMAN antibiotic resistant bacteria and resistance genes database (NORMAN ARB&ARG)-An invitation for collaboration to tackle antibiotic resistance.

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Antibiotic resistance and class 1 integron gene dynamics along effluent, reclaimed wastewater irrigated soil, crop continua: elucidating potential risks and ecological constraints.

Reuse of municipal wastewater is a growing global trend, but currently there is lack of consensus regarding the potential dissemination of antibiotic resistance elements by treated wastewater irrigation. We tracked intI1, a proxy for anthropogenic pollution, and an assemblage of antibiotic resistance genes associated with mobile elements and/or wastewater (blaGES, blaOXA2, blaOXA10, blaTEM, blaCTX-M-32 and qnrS) in treated wastewater effluents, effluent stabilization reservoirs, and along irrigation water-soil-crop continua in experimental lysimeters and large-scale commercial fields. While several of the targeted antibiotic resistance genes were profuse in effluents, there was almost no correlation between gene abundance in irrigation water and those detected in soil, and no evidence of systematic gene transfer to irrigated soil or crops. In contrast, soil intI1 abundance correlated strongly to irrigation water levels in lysimeters and sandy field soils, but this was not the case for clay-rich soils or for most of the analyzed crops, suggesting that intI1 may not always be a reliable marker for tracking the impact of treated wastewater irrigation. We hypothesize that "ecological boundaries" expedited by biotic and abiotic factors constrain dissemination of antibiotic resistance elements, and assert that a more holistic perception of these factors is crucial for understanding and managing antibiotic resistance dissemination.

Enhanced Bacterial Fitness Under Residual Fluoroquinolone Concentrations Is Associated With Increased Gene Expression in Wastewater-Derived qnr Plasmid-Harboring Strains.

Plasmids harboring qnr genes confer resistance to low fluoroquinolone concentrations. These genes are of significant clinical, evolutionary and environmental importance, since they are widely distributed in a diverse array of natural and clinical environments. We previously extracted and sequenced a large (∼185 Kbp) qnrB-harboring plasmid, and several small (∼8 Kbp) qnrS-harboring plasmids, from Klebsiella pneumoniae isolates from municipal wastewater biosolids, and hypothesized that these plasmids provide host bacteria a selective advantage in wastewater treatment plants (WWTPs) that often contain residual concentrations of fluoroquinolones. The objectives of this study were therefore to determine the effect of residual fluoroquinolone concentrations on the growth kinetics of qnr plasmid-harboring bacteria; and on the copy number of qnr plasmids and expression of qnr genes. Electrotransformants harboring either one of the two types of plasmids could grow at ciprofloxacin concentrations exceeding 0.5 µg ml-1, but growth was significantly decreased at concentrations higher than 0.1 µg ml-1. In contrast, plasmid-free strains failed to grow even at 0.05 µg ml-1. No differences were observed in plasmid copy number under the tested ciprofloxacin concentrations, but qnr expression increased incrementally from 0 to 0.4 µg ml-1, suggesting that the transcription of this gene is regulated by antibiotic concentration. This study reveals that wastewater-derived qnr plasmids confer a selective advantage in the presence of residual fluoroquinolone concentrations and provides a mechanistic explanation for this phenomenon.