Analysing carbapenemases in hospital wastewater: Insights from intracellular and extracellular DNA using qPCR and digital PCR.

The widespread dissemination of carbapenem-resistant bacteria in wastewater systems, particularly from clinical sources, poses a significant public health risk. This study assessed the concentrations and distributions of extracellular DNA (exDNA) and intracellular DNA (iDNA) harboring carbapenemase genes in wastewater from six tertiary care hospitals in Germany. We collected a total of 36 samples, comprising six biological replicates from each hospital, and analysed them using quantitative real-time PCR (qPCR) and digital PCR (dPCR). The analysis targeted seven carbapenemase genes: blaNDM, blaKPC, blaIMP, blaVIM, blaOXA-23-like, blaOXA-48-like, and blaOXA-58-like across both DNA fractions. Our results revealed significant variability in the concentrations of exDNA and iDNA across the sampling sites, with iDNA typically present at higher concentrations. Using NanoDrop One spectrophotometry and the Qubit dsDNA kit, exDNA concentrations ranged from 2.7 to 7.7 ng/mL, while Qubit recorded lower values between 1.1 and 4.0 ng/mL. Conversely, iDNA concentrations were markedly higher, spanning from 42.3 to 191.7 ng/mL with NanoDrop and 12.0 to 46.5 ng/mL with Qubit, highlighting the variability between DNA types and quantification methods. Despite its lower concentrations, exDNA comprised up to 18.2 % of total DNA, highlighting its potential role in the horizontal transfer of antimicrobial resistance genes (ARGs). The study detected target ARGs in both DNA fractions at all sites, with notable differences in their concentrations; iDNA consistently exhibited higher levels of ARGs, with the highest concentrations reaching 10.57 ± 0.20 log gene copies per liter (GC/L) for blaVIM in iDNA and 6.96 ± 0.72 log GC/L for blaIMP in exDNA. dPCR demonstrated greater sensitivity than qPCR, especially effective for detecting low-abundance targets like blaOXA-23-like in the exDNA fraction. Additionally, qPCR's susceptibility to inhibition and contamination emphasizes the superior robustness of dPCR. This research highlights the need for improved monitoring and the implementation of advanced treatment technologies to mitigate ARG dissemination in wastewater.

High Genetic Diversity in Third-Generation Cephalosporin-Resistant Escherichia coli in Wastewater Systems of Schleswig-Holstein.

The spread of multidrug-resistant bacteria from humans or livestock is a critical issue. However, the epidemiology of resistant pathogens across wastewater pathways is poorly understood. Therefore, we performed a detailed comparison of third-generation cephalosporin-resistant Escherichia coli (3GCREC) from wastewater treatment plants (WWTPs) to analyze dissemination pathways. A total of 172 3GCREC isolated from four WWTPs were characterized via whole genome sequencing. Clonal relatedness was determined using multi-locus sequence typing (MLST) and core genome MLST. Resistance genotypes and plasmid replicons were determined. A total of 68 MLST sequence types were observed with 28 closely related clusters. Resistance genes to eight antibiotic classes were detected. In fluoroquinolone-resistant isolates, resistance was associated with three-or-more point mutations in target genes. Typing revealed high genetic diversity with only a few clonal lineages present in all WWTPs. The distribution paths of individual lines could only be traced in exceptional cases with a lack of enrichment of certain lineages. Varying resistance genes and plasmids, as well as fluoroquinolone resistance-associated point mutations in individual isolates, further corroborated the high diversity of 3GCREC in WWTPs. In total, we observed high diversity of 3GCREC inside the tested WWTPs with proof of resistant strains being released into the environment even after treatment processes.

Phenotypic Variation in Clinical S. aureus Isolates Did Not Affect Disinfection Efficacy Using Short-Term UV-C Radiation.

Pigmentation, catalase activity and biofilm formation are virulence factors that cause resistance of Staphylococcus aureus to environmental stress factors including disinfectants. In recent years, automatic UV-C room disinfection gained greater importance in enhanced disinfection procedures to improve disinfection success in hospitals. In this study, we evaluated the effect of naturally occurring variations in the expression of virulence factors in clinical S. aureus isolates on tolerance against UV-C radiation. Quantification of staphyloxanthin expression, catalase activity and biofilm formation for nine genetically different clinical S. aureus isolates as well as reference strain S. aureus ATCC 6538 were performed using methanol extraction, a visual approach assay and a biofilm assay, respectively. Log10 reduction values (LRV) were determined after irradiation of artificially contaminated ceramic tiles with 50 and 22 mJ/cm2 UV-C using a commercial UV-C disinfection robot. A wide variety of virulence factor expression was observed, indicating differential regulation of global regulatory networks. However, no direct correlation with the strength of expression with UV-C tolerance was observed for either staphyloxanthin expression, catalase activity or biofilm formation. All isolates were effectively reduced with LRVs of 4.75 to 5.94. UV-C disinfection seems therefore effective against a wide spectrum of S. aureus strains independent of occurring variations in the expression of the investigated virulence factors. Due to only minor differences, the results of frequently used reference strains seem to be representative also for clinical isolates in S. aureus.

Long time persistence and evolution of carbapenemase-producing Enterobacterales in the wastewater of a tertiary care hospital in Germany.

BACKGROUND: Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. METHODS: In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). RESULTS: Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum ß-lactamases decreased over time. CONCLUSIONS: This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future.

DNA methylation-based age estimation for adults and minors: considering sex-specific differences and non-linear correlations.

DNA methylation patterns change during human lifetime; thus, they can be used to estimate an individual's age. It is known, however, that correlation between DNA methylation and aging might not be linear and that the sex might influence the methylation status. In this study, we conducted a comparative evaluation of linear and several non-linear regressions, as well as sex-specific versus unisex models. Buccal swab samples from 230 donors aged 1 to 88 years were analyzed using a minisequencing multiplex array. Samples were divided into a training set (n = 161) and a validation set (n = 69). The training set was used for a sequential replacement regression and a simultaneous 10-fold cross-validation. The resulting model was improved by including a cut-off of 20 years, dividing the younger individuals with non-linear from the older individuals with linear dependence between age and methylation status. Sex-specific models were developed and improved prediction accuracy in females but not in males, which might be explained by a small sample set. We finally established a non-linear, unisex model combining the markers EDARADD, KLF14, ELOVL2, FHL2, C1orf132, and TRIM59. While age- and sex-adjustments did not generally improve the performance of our model, we discuss how other models and large cohorts might benefit from such adjustments. Our model showed a cross-validated MAD and RMSE of 4.680 and 6.436 years in the training set and of 4.695 and 6.602 years in the validation set, respectively. We briefly explain how to apply the model for age prediction.

Movement building responses to COVID-19: lessons from the JASS mobilisation fund.

This article draws on the work of Just Associates (JASS), a feminist movement support organisation that strengthens the leadership and organising capacity of community-based women networks in Southern Africa, Southeast Asia, and Mesoamerica, to transform the structures that perpetuate inequality and violence. We analyse qualitative interviews and surveys drawn from recipients of the JASS mobilisation fund (JMF), an innovative financial crisis support mechanism for feminist movements. We argue that localisation strategies deployed by women's networks supported by the JMF in response to COVID-19, challenge dominant humanitarian responses that de-centre feminist movements, local knowledge, and expertise. By accounting for local knowledge generated from long histories of movement building, building collective power, and challenging racialised and gendered responses to humanitarian crises, women's collectives and networks supported through the JMF developed contextually relevant responses that challenge patriarchal structural barriers heightened by COVID-19.

High burden and diversity of carbapenemase-producing Enterobacterales observed in wastewater of a tertiary care hospital in Germany.

Hospitals are one of the main reservoirs of multi-resistant Enterobacterales (MRE). As MRE are resistant to the most frequently used antibiotics, therapy for patients with MRE infections is challenging. It has been previously described that MRE from hospital wastewater can pass into municipal wastewater and even surface water. In this study, we investigated the diversity and epidemiology of MRE in the wastewater of a large tertiary care hospital. Wastewater samples were collected for a four-day period and tested for the presence of Enterobacterales resistant to 3rd gen. cephalosporins. Representative isolates were further characterized by whole genome sequencing. In 120 ß-glucuronidase-producing isolates, 68 Escherichia coli and, interestingly, also 52 Citrobacter freundii were identified. In 120 ß-glucosidase-producing isolates 45 Serratia marcescens, 34 Klebsiella oxytoca, 32 Enterobacter cloacae and 9 Klebsiella pneumoniae were observed. For all species various MLST sequence types and different clusters of resistance genes were determined, showing a great diversity within the different Enterobacterales, further corroborated by clonal analysis performed by cgMLST. The most prominent clone was wastewater associated E. coli ST635, which accounted for 47.1% of all E. coli isolates. Interestingly, 45.6% of E. coli, 88.5% of C. freundii, 95.6% of S. marcescens, 91.2% of K. oxytoca, 96.9% of E. cloacae and 88.9% of K. pneumoniae isolates carried a carbapenemase gene, indicating a high burden with carbapenemase-producing Enterobacterales. Comparison with clinical isolates from the same hospital displayed few clonal matches. One wastewater isolate of K. pneumoniae was identified to be closely related compared to a clone that had been introduced into the hospital during an outbreak four years earlier. One E. coli isolate was identified as identical to an isolate from a patient, with inpatient stay during the sampling period. The data obtained in this study highlight the problem of antibiotic resistance of Enterobacterales in hospital wastewater. In particular, the clustered occurrence of carbapenemase genes is of great concern and underscores the problem of increasingly scarce antibiotic options against these bacteria.