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 ±â€¯0.20 log gene copies per liter (GC/L) for blaVIM in iDNA and 6.96 ±â€¯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.

Do asymptomatic STEC-long-term carriers need to be isolated or decolonized? New evidence from a community case study and concepts in favor of an individualized strategy.

Asymptomatic long-term carriers of Shigatoxin producing Escherichia coli (STEC) are regarded as potential source of STEC-transmission. The prevention of outbreaks via onward spread of STEC is a public health priority. Accordingly, health authorities are imposing far-reaching restrictions on asymptomatic STEC carriers in many countries. Various STEC strains may cause severe hemorrhagic colitis complicated by life-threatening hemolytic uremic syndrome (HUS), while many endemic strains have never been associated with HUS. Even though antibiotics are generally discouraged in acute diarrheal STEC infection, decolonization with short-course azithromycin appears effective and safe in long-term shedders of various pathogenic strains. However, most endemic STEC-strains have a low pathogenicity and would most likely neither warrant antibiotic decolonization therapy nor justify social exclusion policies. A risk-adapted individualized strategy might strongly attenuate the socio-economic burden and has recently been proposed by national health authorities in some European countries. This, however, mandates clarification of strain-specific pathogenicity, of the risk of human-to-human infection as well as scientific evidence of social restrictions. Moreover, placebo-controlled prospective interventions on efficacy and safety of, e.g., azithromycin for decolonization in asymptomatic long-term STEC-carriers are reasonable. In the present community case study, we report new observations in long-term shedding of various STEC strains and review the current evidence in favor of risk-adjusted concepts.

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.