Detection of carbapenemase-producing, hypervirulent Klebsiella spp. in wastewater and their potential transmission to river water and WWTP employees.

Wastewater treatment plants (WWTPs) release drug-resistant microorganisms to water bodies (with effluents), and WWTP employees are exposed to bioaerosol emissions from the processed wastewater. Bacteria of the genus Klebsiella, in particular carbapenemase-producing (CP), hyper-virulent (Hvr) strains of Klebsiella pneumoniae, play a special role in this process. Klebsiella spp. strains isolated from wastewater, river water and the upper respiratory tract of WWTP employees were analyzed in this study. The isolated strains were identified as K. pneumoniae (K. pn) or K. non-pneumoniae (K. npn). The prevalence of nine types of genes encoding resistance to beta-lactams, nine genes encoding virulence factors and K1/K2 capsular serotypes, three genes encoding multi drug effluent pump systems, and the class 1 integron-integrase gene was determined by PCR. A total of 284 Klebsiella spp. isolates were obtained in the study: 270 environmental strains and 14 strains from the upper respiratory tract. Among environmental isolates 90.7% (245/270) harbored beta-lactam resistance genes, 17.4% (47/270) were classified as CP strains, 11.1% (30/270) were classified as Hvr strains, and 1.9% (5/270) were classified as CP-Hvr strains. CP-Hvr strains were also isolated from WWTP employees. Genes encoding ß-lactamases (including carbapenemases), complete efflux pump systems and the K1 serotype were identified more frequently in K. pn strains. In turn, K. npn strains were characterized by a higher prevalence of blaSHV and intI1 genes and K2 serotype gene. The strains isolated from wastewater and river water also differed in the abundance of drug resistance and virulence genes. The results of the study indicate that CP-Hvr K. pn strains are possibly transmitted from wastewater via bioareosol to the upper respiratory tract of WWTP employees. blaGES-type carbapenemases significantly contributed to the spread of drug resistance in the environment.

Industrialization as a source of heavy metals and antibiotics which can enhance the antibiotic resistance in wastewater, sewage sludge and river water.

The spread of antibiotic resistance is closely related with selective pressure in the environment. Wastewater from industrialized regions is characterized by higher concentrations of these pollutants than sewage from less industrialized areas. The aim of this study was to compare the concentrations of contaminants such as antibiotics and heavy metals (HMs), and to evaluate their impact on the spread of genes encoding resistance to antimicrobial drugs in samples of wastewater, sewage sludge and river water in two regions with different levels of industrialization. The factors exerting selective pressure, which significantly contributed to the occurrence of the examined antibiotic resistance genes (ARGs), were identified. The concentrations of selected gene copy numbers conferring resistance to four groups of antibiotics as well as class 1 and 2 integron-integrase genes were determined in the analyzed samples. The concentrations of six HMs and antibiotics corresponding to genes mediated resistance from 3 classes were determined. Based on network analysis, only some of the analyzed antibiotics correlated with ARGs, while HM levels were correlated with ARG concentrations, which can confirm the important role of HMs in promoting drug resistance. The samples from a wastewater treatment plant (WWTP) located an industrialized region were characterized by higher HM contamination and a higher number of significant correlations between the analyzed variables than the samples collected from a WWTP located in a less industrialized region. These results indicated that treated wastewater released into the natural environment can pose a continuous threat to human health by transferring ARGs, antibiotics and HMs to the environment. These findings shed light on the impact of industrialization on antibiotic resistance dissemination.

Microbial and chemical pollutants on the manure-crops pathway in the perspective of "One Health" holistic approach.

This study determined the impact of poultry and bovine manure fertilization on the content of antibiotics, heavy metals (HMs), and the quantitative and qualitative composition of integrase and antibiotic resistance genes in soil, groundwater, and crops cultivated on manure-amended plots. Antibiotic concentration levels were analyzed using the HPLC-MS/MS, heavy metal concentration level were measured by HGAAS and ICP-OES, while the integrase genes and ARGs were quantified using Real-Time PCR (qPCR) method. Manure, soil, and crops samples contained the highest concentration of Zn (104-105 ng gdm-1) and Cu (103-105 ng gdm-1) of all HMs tested. Manure-supplemented soil was characterised by a high concentration of doxycycline and enrofloxacin. A high abundance of integrase genes was noted in samples of manure (109-1010 copies gdm-1) and soil (107-108 copies gdm-1). Among all the analyzed genes, sul1, sul2, blaTEM, and integrase genes were the most common. Results of the study demonstrate the selective character of ARGs transfer from poultry and bovine manure to plants. The only gene to occur in all studied environmental compartments was sul1 (from 102 - groundwater to 1011 - poultry manure). It was also found that animal manure may cause an increase in the HMs concentration in soil and their accumulation in crops, which may influence the health of humans and animals consuming crops grown on manure-amended soil. The high abundance of integrase genes and ARGs and their reciprocal correlations with HMs pose a serious risk of the rapid spread of antibiotic resistance in the environment. Moreover, unusual dependencies between integrase genes and selected ARGs indicate the possibility of changes in the mobility nature of genetic elements.

Sewage sludge in agriculture - the effects of selected chemical pollutants and emerging genetic resistance determinants on the quality of soil and crops - a review.

In line with sustainable development principles and in order to combat climate change, which contributes to progressive soil depletion, various solutions are being sought to use treated sewage sludge as a soil amendment to improve soil quality and enrich arable soils with adequate amounts of biogenic compounds. This review article focuses on the effects of the agricultural use of biosolids on the environment. The article reviews the existing knowledge on selected emerging contaminants in treated sewage sludge and describes the impact of these pollutants on the environment and living organisms based on 183 publications selected from over 16,000 papers on related topics published over the last ten years. This study deals not only with chemical contaminants but also genetic determinants of resistance to these compounds. Current research has questioned the agricultural use of biosolids due to the presence of mutual interactions between antibiotics, heavy metals, the genetic determinants of resistance (antibiotic resistance genes - ARGs and heavy metal resistance genes - HMRGs) and non-steroidal anti-inflammatory drugs as well as the risks associated with their transfer to the environment. This study emphasizes the need for more extensive legal regulations that account for other pollutants of environmental concern (PEC), particularly in countries where sewage sludge is applied in agriculture most extensively. Future research should focus on more effective methods of eliminating PEC from sewage sludge, especially from the sludge that is used to fertilize agricultural land, because even small amounts of these micropollutants can have serious implications for the health and life of humans and animals.

The prevalence of drug-resistant and virulent Staphylococcus spp. in a municipal wastewater treatment plant and their spread in the environment.

Drug-resistant and pathogenic Staphylococcus spp. strains can reach surface waters and air with wastewater evacuated to the environment. These strains increase the environmental pool of genetic determinants conferring antibiotic resistance and virulence, and constitute a health risk for the employees of wastewater treatment plants (WWTP) who come into daily contact with bioaerosols. The aim of this study was to identify the genetic determinants of drug resistance and virulence in Staphylococcus spp. strains isolated from untreated (UWW) and treated wastewater (TWW), an activated sludge (AS) bioreactor, river water collected upstream and downstream from the wastewater discharge point (URW and DRW), and WWTP employees. All isolates were analysed for the presence of the rpoB gene, and were subjected to clonal analysis by ERIC fingerprinting. As a result, 249 of the 455 analysed isolates were selected for PCR. The presence of the gene encoding nuclease activity in S. aureus (nuc), the methicillin resistance gene (mecA), vancomycin resistance gene (vanA), antiseptic resistance gene (qacA/B) and virulence genes (sasX, pvl, tst1, hla, sec) was determined. The prevalence of nuc, mecA, vanA and qacA/B genes in wastewater and river water was determined by quantitative PCR (qPCR). In the group of strains isolated from wastewater and water samples, 63% were identified as S. aureus, and 20% of the strains carried the vanA gene. The hla virulence gene was present in 80% of the isolates, and the pvl gene was detected in 27% of the isolates. In the group of strains isolated from the employees, 82% were identified as S. aureus, and the presence of vanA and mecA genes was confirmed in 14% and 16% of the isolates, respectively. The most prevalent virulence gene was hla (74%), whereas pvl was observed in 43% of the isolates. The quantitative analysis revealed the highest concentrations of the studied genes in UWW samples, at 2.56x104 gene copies/ml for nuc, 1.18x103 gene copies /ml for mecA, 8.28x105 gene copies /ml for vanA and 3.83x105 gene copies /ml for qacA/B. Some of analysed genes were identified in the isolates from both URW and DRW samples, as well as in genomic DNA of these samples. These results indicate that wastewater is not effectively treated in the analysed WWTP, which could contribute to the dissemination of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) to the environment. An analysis of the genetic relatedness of selected isolates revealed clusters of strains originating from UWW samples, AS samples and the employees. These observations suggest that ARGs and ARB are transmitted by wastewater bioaerosols to the upper respiratory tract mucosa of the plant's employees, thus increasing their exposure to infectious factors.