The influence of combined treatment of municipal wastewater and landfill leachate on the spread of antibiotic resistance in the environment - A preliminary case study.

Environmentally-friendly management of landfill leachate (LL) poses a challenge, and LL is usually co-treated with municipal wastewater in wastewater treatment plants (WWTPs). The extent to which the co-treatment of LL and municipal wastewater influences the spread of antibiotic resistance (AR) in the environment has not been examined to date. Two WWTPs with similar wastewater composition and technology were studied. Landfill leachate was co-treated with wastewater in one of the studied WWTPs. Landfill leachate, untreated and treated wastewater from both WWTPs, and river water sampled upstream and downstream from the wastewater discharge point were analyzed. Physicochemical parameters, microbial diversity, and antibiotic resistance genes (ARGs) abundance were investigated to determine the impact of LL co-treatment on chemical and microbiological contamination in the environment. Landfill leachate increased pollutant concentrations in untreated wastewater and river water. Cotreatment of LL and wastewater could affect the abundance and diversity of microbial communities and the interactions between microbial species. Co-treatment also decreased the stability of microbial co-occurrence networks in the examined samples. The mexF gene was identified as a potential marker of environmental pollution with LL. This is the first study to explore the impact of LL on the occurrence of AR determinants in wastewater and rivers receiving effluents.

Chlorine disinfection modifies the microbiome, resistome and mobilome of hospital wastewater - A nanopore long-read metagenomic approach.

The aim of the present study was to analyze changes in the microbiome, resistome, and mobilome of hospital wastewater (HWW) induced by disinfection with chlorine compounds. Changes in bacterial communities and specific antibiotic resistance genes (ARGs) in HWW were determined with the use of a nanopore long-read metagenomic approach. The main hosts of ARGs in HWW were identified, and the mobility of resistance mechanisms was analyzed. Special attention was paid to the prevalence of critical-priority pathogens in the HWW microbiome, which pose the greatest threat to human health. The results of this study indicate that chlorine disinfection of HWW can induce significant changes in the structure of the total bacterial population and antibiotic resistant bacteria (ARB) communities, and that it can modify the resistome and mobilome of HWW. Disinfection favored the selection of ARGs, decreased their prevalence in HWW, while increasing their diversity. The mobility of the HWW resistome increased after disinfection. Disinfection led to the emergence of new drug resistance mechanisms in previously sensitive bacterial taxa. In conclusion, this study demonstrated that HWW disinfected with low (sublethal) concentrations of free chlorine significantly contributes to the mobility and transfer of drug resistance mechanisms (including critical mechanisms) between bacteria (including pathogens).

The Impact of Chlorine Disinfection of Hospital Wastewater on Clonal Similarity and ESBL-Production in Selected Bacteria of the Family Enterobacteriaceae.

Hospitals are regarded as ecological niches of antibiotic-resistant bacteria (ARB). ARB can spread outside the hospital environment via hospital wastewater (HWW). Therefore, HWW is often disinfected in local stations to minimize that risk. Chlorine-based treatment is the most popular method of HWW disinfection around the world, however, recent research has suggested that it can contribute to the spread of antimicrobial resistance (AMR). The aim of this study is to determine the impact of HWW disinfection on the clonal similarity of Enterobacteriaceae species and their ability to produce extended-spectrum beta-lactamases (ESBLs). The study was conducted in a hospital with a local chlorine-based disinfection station. Samples of wastewater before disinfection and samples of disinfected wastewater, collected in four research seasons, were analyzed. Bacteria potentially belonging to the Enterobacteriaceae family were isolated from HWW. The Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was used to generate DNA fingerprints of all bacterial isolates. The isolates were phenotypically tested for the production of ESBLs. Antibiotic resistance genes (blaSHV, blaTEM, and blaOXA, blaCTX-M-1-group, blaCTX-M-2-group, blaCTX-9-group and blaCTX-M-8/25-group) were detected by PCR in strains with confirmed phenotypic ability to produce ESBLs. The ESBL+ isolates were identified by the sequencing of 16S rDNA. In the present study, the same bacterial clones were isolated from HWW before and after disinfection and HWW was sampled in different seasons. Genetic and phenotypic variations were observed in bacterial clones. ESBL+ strains were isolated significantly more often from disinfected than from non-disinfected HWW. The blaOXA gene was significantly more prevalent in isolates from disinfected than non-disinfected HWW. Enterobacter hormaechei and Klebsiella pneumoniae were the dominant species in ESBL+ strains isolated from both sampling sites. The results of this study indicate that chlorine-based disinfection promotes the survival of ESBL-producing bacteria and/or the transmission of genetic determinants of antimicrobial resistance. As a result, chlorination increases the proportion of ESBL-producing Enterobacteriaceae in disinfected wastewater. Consequently, chlorine-based disinfection practices may pose a risk to the environment and public health by accelerating the spread of antimicrobial resistance.

Metagenomics analysis of probable transmission of determinants of antibiotic resistance from wastewater to the environment - A case study.

During mechanical-biological treatment, wastewater droplets reach the air with bioaerosols and pose a health threat to wastewater treatment plant (WWTP) employees and nearby residents. Microbiological pollutants and antimicrobial resistance determinants are discharged to water bodies with treated wastewater (TWW), which poses a potential global epidemiological risk. In the present study, the taxonomic composition of microorganisms was analyzed, and the resistome profile and mobility of genes were determined by metagenomic next-generation sequencing in samples of untreated wastewater (UWW), wastewater collected from an activated sludge (AS) bioreactor, TWW, river water collected upstream and downstream from the wastewater discharge point, and in upper respiratory tract swabs collected from WWTP employees. Wastewater and the emitted bioaerosols near WWTP's facilities presumably contributed to the transmission of microorganisms, in particular bacteria of the phylum Actinobacteria and the associated antibiotic resistance genes (ARGs) (including ermB, ant(2″)-I, tetM, penA and cfxA2) to the upper respiratory tract of WWTP employees. The discharged wastewater increased the taxonomic diversity of microorganisms and the concentrations of various ARGs (including bacA, emrE, sul1, sul2 and tetQ) in river water. This study fills in the knowledge gap on the health risks faced by WWTP employees. The study has shown that microbiological pollutants and antimicrobial resistance determinants are also in huge quantities discharged to rivers with TWW, posing a potential global epidemiological threat.

Characterization of carbapenem resistance in environmental samples and Acinetobacter spp. isolates from wastewater and river water in Poland.

The aim of this study was to analyze the prevalence of carbapenem resistance genes in Acinetobacter spp. isolated from wastewater in a municipal WWTP and to determine their spread from treated wastewater to river water with the use of conventional and molecular microbiology methods (qualitative and quantitative PCR and metagenomic analysis). Samples of untreated and treated wastewater and samples of river water obtained upstream and downstream from the wastewater discharge point were collected in 3 seasons (February, June, and September) of 2019. Acinetobacter spp. isolates were obtained by the culture method on the CHROMagar™ Acinetobacter medium. Additionally, environmental DNA was extracted from the samples for metagenomic and qPCR analyses. The presence of beta-lactam resistance genes (Ambler class B and D), insertion sequence ISAba1, and class I, II, and III integron-integrase genes was determined, and the bacterial taxonomic structure and wastewater and river samples was analyzed. Out of the 301 isolates obtained on the CHROMagar™ Acinetobacter medium, 258 belonged to the genus Acinetobacter, including 21 isolates that were identified as Acinetobacter baumannii. The highest number of Acinetobacter spp. and A. baumannii isolates were obtained from wastewater and river water samples collected in June and September. The ISAba1/blaOXA-51 complex was identified in 13 isolates, which confirms the occurrence of carbapenem-resistance isolates in the analyzed samples. The number of Acinetobacter isolates carrying antibiotic resistance genes (ARGs) increased in river water samples collected downstream from the wastewater discharge point (48 out of 258 isolates - 18.6%) compared to river water samples collected upstream from the wastewater discharge point (34 out of 258 isolates - 13.2%), which suggests that WWTP is a source of pollution in the natural environment. The conducted research provides evidence that bacteria of the genus Acinetobacter may spread alarming beta-lactam resistance in the environment and, therefore, pose a serious epidemiological threat.

Structure of the manure resistome and the associated mobilome for assessing the risk of antimicrobial resistance transmission to crops.

In this study, the impact of bovine and poultry manure on the quantitative and qualitative composition of antibiotic resistance genes (ARGs) and the environmental mobilome associated with antimicrobial resistance in soil and crops was determined with the use of next generation sequencing methods. The aim of the study was to perform a metagenomic analysis of manure to estimate the risk of the transmission of ARGs and bacterial drug resistance carriers to fertilized soil and crops. The total copy number of ARGs was nearly four times higher in poultry manure (555 ppm) than in bovine manure (140 ppm), and this relationship was also noted in fertilized soil. Poultry manure induced a much greater increase in the concentrations of ARGs in the soil environment (196.4 ppm) than bovine manure (137.8 ppm) immediately after supplementation. The application of poultry manure led to the highest increase in the abundance of genes encoding resistance to tetracyclines (9%), aminoglycosides (3.5%), sulfonamides (3%), bacitracin (2%), chloramphenicol (2%), and macrolide-lincosamide-streptogramin antibiotics (1%). Heavy metals were stronger promoters of antibiotic resistance in the environment than antibiotics. Antibiotics exerted a greater influence on maintaining the diversity of ARGs than on increasing their abundance in soil. Large quantities of insertion sequences (IS), including those associated with the mobility of ARGs in the population of ESKAPEE pathogens, are introduced to soil with manure. These IS remain stable for up to several months, which indicates that manure, in particular poultry manure, significantly increases the risk of rapid ARG transfer to the environment. Manure also largely contributes to an increase in the diversity of the resistome and mobilome in the metagenome of bacteria isolated from crops. Bacteria of the phylum Proteobacteria appear to play a major role in the transmission of multiple ARGs in crops grown for human and animal consumption.

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.

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.