Poultry manure-derived microorganisms as a reservoir and source of antibiotic resistance genes transferred to soil autochthonous microorganisms.

Animal husbandry is increasing yearly due to the growing demand for meat and livestock products, among other reasons. To meet these demands, prophylactic antibiotics are used in the livestock industry (i.e., poultry farming) to promote health and stimulate animal growth. However, antibiotics are not fully metabolized by animals, and they are evacuated to the environment with excreta. Animal manure is used as fertilizer to reduce the volume of waste generated in the livestock sector. However, manure often contains microorganisms harboring antibiotic resistance genes (ARGs). Then, the microbiome of manure applicate to the soil may contribute to the spread of antibiotic resistance in the environment, including autochthonous soil-dwelling microorganisms. The present study was conducted during the crops growing season in Poland (May to September 2019) to determine the influence of poultry manure as well as poultry manure supplemented with selected antibiotics on the diversity of the soil microbiome in treatments that had not been previously fertilized with manure and the ability of antibiotic-resistant bacteria to transfer ARGs to other soil bacteria. Antibiotic concentrations were elevated at the beginning of the study and decreased over time. Poultry manure induced significant changes in the structure of microbial communities in soil; the diversity of the soil microbiome decreased, and the abundance of bacterial genera Bradyrhizobium, Streptomyces, and Pseudomonas, which are characteristic of the analyzed manure, increased. Over time, soil microbial diversity was restored to the state observed before the application of manure. Genes conferring resistance to multiple drugs as well as genes encoding resistance to bacitracin and aminoglycosides were the most frequently identified ARGs in the analyzed bacteria, including on mobile genetic elements. Multidrug resistance was observed in 17 bacterial taxa, whereas ARGs were identified in 32 bacterial taxa identified in the soil microbiome. The results of the study conclude that the application of poultry manure supplemented with antibiotics initially affects soil microbiome and resistome diversity but finally, the soil shows resilience and returns to its original state after time, with most antibiotic resistance genes disappearing. This phenomenon is of great importance in sustainable soil health after manure application.

The challenges in the identification of Escherichia coli from environmental samples and their genetic characterization.

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.

Determination of antimicrobial agents and their transformation products in an agricultural water-soil system modified with manure.

Manure fertilization is the primary source of veterinary antimicrobials in the water-soil system. The research gap is the fate of antimicrobials after their release into the environment. This study aimed to provide a detailed and multi-faceted examination of fertilized cultivated fields using two types of manure (poultry and bovine) enriched with selected antimicrobials. The research focused on assessing the mobility and stability of antimicrobials in the water-soil system. Additionally, transformation products of antimicrobials in the environment were identified. The extraction (solid-phase extraction and/or solid-liquid extraction) and LC-MS/MS analysis procedures were developed to determine 14 antimicrobials in the soil and pore water samples. Ten out of fourteen antimicrobials were detected in manure-amended soil and pore water samples. The highest concentration in the soil was 109.1 ng g-1 (doxycycline), while in pore water, it was 186.6 ng L-1 (ciprofloxacin). Sixteen transformation products of antimicrobials were identified in the soil and soil-related pore water. The same transformation products were detected in both soil and soil pore water extracts, with significantly higher signal intensities observed in soil extracts than in water. Transformation products were formed in oxidation, carbonylation, and ring-opening reactions.

Solar-light driven photodegradation of antimicrobials, their transformation by-products and antibiotic resistance determinants in treated wastewater.

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.

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.