RESUMO
Escherichia coli bacteria are an essential indicator in evaluations of environmental pollution, which is why they must be correctly identified. This study aimed to determine the applicability of various methods for identifying E. coli strains in environmental samples. Bacterial strains preliminary selected on mFc and Chromocult media as E. coli were identified using MALDI Biotyper techniques, based on the presence of genes characteristic of E. coli (uidA, uspA, yaiO), as well as by 16S rRNA gene sequencing. The virulence and antibiotic resistance genes pattern of bacterial strains were also analyzed to investigate the prevalence of factors that may indicate adaptation to unsupportive environmental conditions and could have any significance in further identification of E. coli. Of the strains that had been initially identified as E. coli with culture-based methods, 36-81% were classified as E. coli with the use of selected techniques. The value of Cohen's kappa revealed the highest degree of agreement between the results of 16S rRNA gene sequencing, the results obtained in the MALDI Biotyper system, and the results of the analysis based on the presence of the yaiO gene. The results of this study could help in the selection of more accurate and reliable methods which can be used in a preliminary screening and more precise identification of E. coli isolated from environmental samples.
Assuntos
Bactérias , Escherichia coli , RNA Ribossômico 16S/genética , Bactérias/genética , VirulênciaRESUMO
This study aimed to assess the possibility of using solar light-driven photolysis and TiO2-based photocatalysis to remove (1) antibiotic residues, (2) their transformation products (TPs), (3) antibiotic resistance determinants, and (4) genes identifying the indicator bacteria in a treated wastewater (secondary effluent). 16 antimicrobials belonging to the different classes and 45 their transformation by-products were selected for the study. The most susceptible to photochemical decomposition was tetracycline, which was completely removed in the photocatalysis process and in more than 80% in the solar light-driven photolysis. 83.8% removal (on average) was observed using photolysis and 89.9% using photocatalysis in the case of the tested genes, among which the genes sul1, uidA, and intI1 showed the highest degree of removal by both methods. The study revealed that applied methods promisingly remove the tested antibiotics, their TPs and genes even in such a complex matrix including treated wastewater and photocatalysis process had a higher removal efficiency of antibiotics, TPs and genes tested. Moreover, the high percentage removal of the intI1 gene (>93%) indicates the possibilities of use of the solar light-driven photolysis and TiO2-based photocatalysis in minimizing the antibiotic resistance genes transfer by mobile genetic elements.
Assuntos
Águas Residuárias , Poluentes Químicos da Água , Antibacterianos/farmacologia , Catálise , Resistência Microbiana a Medicamentos/genética , Fotólise , Titânio/química , Águas Residuárias/química , Poluentes Químicos da Água/análiseRESUMO
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(2â³)-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.
Assuntos
Anti-Infecciosos , Poluentes Ambientais , Antibacterianos/análise , Antibacterianos/farmacologia , Anti-Infecciosos/análise , Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Poluentes Ambientais/análise , Genes Bacterianos , Metagenômica , Águas Residuárias/análise , Água/análiseRESUMO
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.
Assuntos
Acinetobacter baumannii , Águas Residuárias , Antibacterianos/análise , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Testes de Sensibilidade Microbiana , Polônia , Águas Residuárias/análise , Água/análise , beta-Lactamases/genéticaRESUMO
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.
Assuntos
Antibacterianos , Esterco , Animais , Antibacterianos/farmacologia , Bovinos , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Humanos , Solo , Microbiologia do SoloRESUMO
The aim of the work was to develop a new HPLC-MS/MS method that allows for the simultaneous detection of antimicrobials agents (targeted analysis) and their transformation products (non-targeted analysis), which enabled the elucidation of their transformation pathways in the environment. Targeted analysis was performed for 16 selected antimicrobials agents (AMs) in wastewater collected at different stages of the treatment process and river water from sections before and after wastewater discharge. The samples were collected in the Lyna sewage treatment plant (Olsztyn, Poland) in three measuring periods at different seasons. Analytes were selected from tetracyclines, fluoroquinolones, ß-lactams, macrolides, glycopeptides, lincosamides and synthetic antibiotics. As a part of the targeted analysis, 13 AMs were detected in wastewater samples, and 7 of them in river water samples. However, their presence and concentrations were closely related to the type of the sample and the season in which the sample was taken. The highest concentrations of AMs were detected in samples collected in September (max. 1643.7 ng L-1 TRI), while the lowest AMs concentrations were found in samples collected in June (max. 136.1 ng L-1 CLR). The total content of AMs in untreated wastewater was in the range of 1.42-1644 ng L-1, while in the river water was for upstream 1.22-48.73 ng L-1 and for downstream 2.24-149 ng L-1. In the non-target analysis, 33 degradation products of the selected AMs were identified, and the transformation pathways of their degradation were speculated. In the course of the research, it was found that as a result of the processes taking place in wastewater treatment plant, the parent substances are transformed into a number of stable transformation products. Transformation products resulted from hydroxylation, ring opening, oxidation, methylation or demethylation, carboxylation, or cleavage of the CN bond of the parent AMs.
Assuntos
Anti-Infecciosos , Corrida , Poluentes Químicos da Água , Antibacterianos , Espectrometria de Massas em Tandem , Águas Residuárias , Poluentes Químicos da Água/análiseRESUMO
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.
Assuntos
Klebsiella , Águas Residuárias , Antibacterianos , Proteínas de Bactérias/genética , Klebsiella/genética , Testes de Sensibilidade Microbiana , Rios , Água , beta-Lactamases/genéticaRESUMO
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.
Assuntos
Antibacterianos/análise , Água Doce/análise , Metais Pesados/análise , Esgotos/análise , Águas Residuárias/análise , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Água Doce/microbiologia , Desenvolvimento Industrial , Integrases/genética , Integrons/genética , Limite de Detecção , Esgotos/microbiologia , Eliminação de Resíduos Líquidos , Águas Residuárias/microbiologiaRESUMO
Conventional mechanical and biological wastewater treatment is unable to completely eliminate all pollutants, which can therefore enter surface water bodies together with treated wastewater. In addition, bioaerosols produced during wastewater treatment can pose a threat to the health of the wastewater treatment plant staff. In order to control the impact of a wastewater treatment plant (WWTP) on the surrounding environment, including its employees, samples of wastewater and water from a river which received treated wastewater were analysed in terms of their content of antibiotics and heavy metals, levels of selected physiochemical parameters, concentrations of antibiotic-resistance genes (ARGs) and genes of integrases. Furthermore, a quantitative analysis of ARGs in the metagenomic DNA from nasal and throat swabs collected from the WWPT employees was made. Both untreated and treated wastewater samples were dominated by genes of resistance to sulphonamides (sul1, sul2), MLS group of drugs (ermF, ermB) and beta-lactams (blaOXA). A significant increase in the quantities of ARGs and concentrations of antibiotics was observed in the river following the discharge of treated wastewater in comparison to their amounts in the river water upstream from the point of discharge. Moreover, a higher concentration of ARGs was detected in the DNA from swabs obtained from the wastewater treatment plant employees than from ones collected from the control group. Many statistically significant (p < 0.05) correlations between the concentration of the gene of resistance to heavy metals cnrA versus ARGs, and between the ARGs content and the concentrations of heavy metals in both wastewater and river water samples were observed. The study has demonstrated that the mechanical and biological methods of wastewater treatment are not efficient and may affect the transmission of hazardous pollutants to the aquatic environment and to the atmospheric air. It has been shown that an activated sludge bioreactor can be a potential source of the presence of multi-drug resistant microorganisms in the air, which is a health risk to persons working in WWTPs. It has also been found that an environment polluted with heavy metals is where co-selection of antibiotic resistance may occur, in the development of which integrase genes play an essential role.
Assuntos
Antibacterianos , Purificação da Água , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos/genética , Humanos , Integrases/farmacologia , Águas Residuárias/análiseRESUMO
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.
Assuntos
Poluentes Ambientais , Esterco , Animais , Antibacterianos , Bovinos , Genes Bacterianos , Humanos , Solo , Microbiologia do Solo , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Agricultura , Esgotos/química , Poluentes do Solo/análise , Eliminação de Resíduos Líquidos/métodos , Animais , Antibacterianos , Produtos Agrícolas , Resistência Microbiana a Medicamentos/genética , Poluentes Ambientais , Humanos , Metais Pesados/análise , Solo/químicaRESUMO
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.