Hospital sanitary facilities on wards with high antibiotic exposure play an important role in maintaining a reservoir of resistant pathogens, even over many years.

BACKGROUND: Hospitals with their high antimicrobial selection pressure represent the presumably most important reservoir of multidrug-resistant human pathogens. Antibiotics administered in the course of treatment are excreted and discharged into the wastewater system. Not only in patients, but also in the sewers, antimicrobial substances exert selection pressure on existing bacteria and promote the emergence and dissemination of multidrug-resistant clones. In previous studies, two main clusters were identified in all sections of the hospital wastewater network that was investigated, one K. pneumoniae ST147 cluster encoding NDM- and OXA-48 carbapenemases and one VIM-encoding P. aeruginosa ST823 cluster. In the current study, we investigated if NDM- and OXA-48-encoding K. pneumoniae and VIM-encoding P. aeruginosa isolates recovered between 2014 and 2021 from oncological patients belonged to those same clusters. METHODS: The 32 isolates were re-cultured, whole-genome sequenced, phenotypically tested for their antimicrobial susceptibility, and analyzed for clonality and resistance genes in silico. RESULTS: Among these strains, 25 belonged to the two clusters that had been predominant in the wastewater, while two others belonged to a sequence-type less prominently detected in the drains of the patient rooms. CONCLUSION: Patients constantly exposed to antibiotics can, in interaction with their persistently antibiotic-exposed sanitary facilities, form a niche that might be supportive for the emergence, the development, the dissemination, and the maintenance of certain nosocomial pathogen populations in the hospital, due to antibiotic-induced selection pressure. Technical and infection control solutions might help preventing transmission of microorganisms from the wastewater system to the patient and vice versa, particularly concerning the shower and toilet drainage. However, a major driving force might also be antibiotic induced selection pressure and parallel antimicrobial stewardship efforts could be essential.

Antibiotic concentrations in raw hospital wastewater surpass minimal selective and minimum inhibitory concentrations of resistant Acinetobacter baylyi strains.

Antibiotics are essential for modern medicine, they are employed frequently in hospitals and, therefore, present in hospital wastewater. Even in concentrations, that are lower than the minimum inhibitory concentrations (MICs) of susceptible bacteria, antibiotics may exert an influence and select resistant bacteria, if they exceed the MSCs (minimal selective concentrations) of resistant strains. Here, we compare the MSCs of fluorescently labelled Acinetobacter baylyi strains harboring spontaneous resistance mutations or a resistance plasmid with antibiotic concentrations determined in hospital wastewater. Low MSCs in the µg/L range were measured for the quinolone ciprofloxacin (17 µg/L) and for the carbapenem meropenem (30 µg/L). A 24 h continuous analysis of hospital wastewater showed daily fluctuations of the concentrations of these antibiotics with distinctive peaks at 7-8 p.m. and 5-6 a.m. The meropenem concentrations were always above the MSC and MIC values of A. baylyi. In addition, the ciprofloxacin concentrations were in the range of the lowest MSC for about half the time. These results explain the abundance of strains with meropenem and ciprofloxacin resistance in hospital wastewater and drains.

Metagenomic Insights Into the Changes of Antibiotic Resistance and Pathogenicity Factor Pools Upon Thermophilic Composting of Human Excreta.

In times of climate change, practicing a form of sustainable, climate-resilient and productive agriculture is of primordial importance. Compost could be one form of sustainable fertilizer, which is increasing humus, water holding capacity, and nutrient contents of soils. It could thereby strengthen agriculture toward the adverse effects of climate change, especially when additionally combined with biochar. To get access to sufficient amounts of suitable materials for composting, resources, which are currently treated as waste, such as human excreta, could be a promising option. However, the safety of the produced compost regarding human pathogens, pharmaceuticals (like antibiotics) and related resistance genes must be considered. In this context, we have investigated the effect of 140- and 154-days of thermophilic composting on the hygienization of human excreta and saw dust from dry toilets together with straw and green cuttings with and without addition of biochar. Compost samples were taken at the beginning and end of the composting process and metagenomic analysis was conducted to assess the fate of antibiotic resistance genes (ARGs) and pathogenicity factors of the microbial community over composting. Potential ARGs conferring resistance to major classes of antibiotics, such as beta-lactam antibiotics, vancomycin, the MLSB group, aminoglycosides, tetracyclines and quinolones were detected in all samples. However, relative abundance of ARGs decreased from the beginning to the end of composting. This trend was also found for genes encoding type III, type IV, and type VI secretion systems, that are involved in pathogenicity, protein effector transport into eukaryotic cells and horizontal gene transfer between bacteria, respectively. The results suggest that the occurrence of potentially pathogenic microorganisms harboring ARGs declines during thermophilic composting. Nevertheless, ARG levels did not decline below the detection limit of quantitative PCR (qPCR). Thresholds for the usage of compost regarding acceptable resistance gene levels are yet to be evaluated and defined.

The Role of ß-Glycosylated Wall Teichoic Acids in the Reduction of Vancomycin Susceptibility in Vancomycin-Intermediate Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 µg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 µg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 µg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly ß-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of ß-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, ß-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus. IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the ß-conformation. By experimentally increasing the proportion of ß-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the ß-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.