사람 및 가축 유래 분변 미생물 군집과 항생제 내성 유전자 간 상관 관계에 대한 연구

정지원,반다리 아프라지타,운노 타쯔야 한국환경농학회 2022 한국환경농학회지 Vol.41 No.4

BACKGROUND: Antibiotics used in animal husbandry for disease prevention and treatment have resulted in the rapid progression of antibiotic resistant bacteria which can be introduced into the environment through livestock feces/manure, disseminating antibiotic resistant genes (ARGs). In this study, fecal samples were collected from the livestock farms located in Jeju Island to investigate the relationship between microbial communities and ARGs. METHODS AND RESULTS: Illumina MiSeq sequencing was applied to characterize microbial communities within each fecal sample. Using quantitative PCR (qPCR), ten ARGs encoding tetracycline resistance (tetB, tetM), sulfonamide resistance (sul1, sul2), fluoroquinolone resistance (qnrD, qnrS), fluoroquinolone and aminoglycoside resistance (aac(6')-Ib), beta-lactam resistance (blaTEM, blaCTX-M), macrolide resistance (ermC), a class 1 integronsintegrase gene (intI1), and a class 2 integrons-integrase gene (intI2) were quantified. The results showed that Firmicutes and Bacteroidetes were dominant in human, cow, horse, and pig groups, while Firmicutes and Actinobacteria were dominant in chicken group. Among ARGs, tetM was detected with the highest number of copies, followed by sul1 and sul2. Most of the genera belonging to Firmicutes showed positive correlations with ARGs and integron genes. There were 97, 34, 31, 25, and 22 genera in chicken, cow, pig, human, and horse respectively which showed positive correlations with ARGs and integron genes. In network analysis, we identified diversity of microbial communities which correlated with ARGs and integron genes. CONCLUSION(S): In this study, antibiotic resistance patterns in human and livestock fecal samples were identified. The abundance of ARGs and integron genes detected in the samples were associated with the amount of antibiotics commonly used for human and livestocks. We found diverse microbial communities associated with antibiotics resistance genes in different hosts, suggesting that antibiotics resistance can disseminate across environments through various routes. Identifying the routes of ARG dissemination in the environment would be the first step to overcome the challenge of antibiotic resistance in the future.

Characterization of antibiotic resistance genes in representative organic solid wastes: Food waste-recycling wastewater, manure, and sewage sludge

Lee, Jangwoo,Shin, Seung Gu,Jang, Hyun Min,Kim, Young Beom,Lee, Joonyeob,Kim, Young Mo Elsevier 2017 Science of the Total Environment Vol.579 No.-

<P><B>Abstract</B></P> <P>In this research, the distribution of antibiotic resistance genes (ARGs) was characterized in representative organic solid waste (OSW) in Korea: food waste-recycling wastewater (FRW), manure, and sewage sludge. The amounts of total ARG (gene copies/16S rRNA gene copies) was greatest in manure followed by sewage sludge and FRW. Interestingly, there were significantly different patterns in the diversity and mechanisms of ARGs. For example, a significant proportion of ARGs were tetracycline resistant genes in all the OSW (40.4–78.2%). β-lactam antibiotics resistant genes were higher in the FRW samples than in other types of OSW but sulfonamides resistant genes represented the greatest proportion in sludge. Regarding the characteristics of antibiotic resistance mechanisms, there was a relatively higher proportion of the ribosomal protection mechanism to tetracycline observed in the FRW and manure samples. However, tetracycline resistant genes with direct interaction were relatively higher in the sewage sludge samples. <I>sul</I>1 was the dominant subtype in all the OSW types and detection of <I>erm</I>B was observed although there was no <I>erm</I>C detected in sewage sludge. There were significant correlations between the occurrences of ARG subtypes: <I>tet</I>B and <I>tet</I>G in all OSW (<I>P</I> <0.01); <I>tet</I>E and <I>tet</I>Q only in sludge (<I>P</I> <0.01). The Class 1 integron-integrase gene (<I>intI</I>1) was significantly correlated with total ARGs only in manure and sludge (<I>P</I> <0.05), revealing potential horizontal gene transfer in these OSW.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Total relative amount of ARGs were highest in the manure, followed by sludge, and FRW. </LI> <LI> Diversity and mechanisms of ARGs were significantly different between OSWs. </LI> <LI> Sulfonamides resistant genes represented the greatest proportion in sludge. </LI> <LI> The ribosomal protection mechanism to tetracycline was dominant in FRW and manure. </LI> <LI> Total ARGs and <I>intI</I>1 were positively correlated in manure and sludge, but not in FRW. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Prevalence of antibiotic resistance genes from effluent of coastal aquaculture, South Korea

Jang, Hyun Min,Kim, Young Beom,Choi, Sangki,Lee, Yunho,Shin, Seung Gu,Unno, Tatsuya,Kim, Young Mo Elsevier 2018 Environmental pollution Vol.233 No.-

<P><B>Abstract</B></P> <P>The wide use of antibiotics in aquaculture for prophylactic and therapeutic purposes can potentially lead to the prevalence of antibiotic resistance genes (ARGs). This study reports for the first time the profile of ARGs from effluents of coastal aquaculture located in South Jeolla province and Jeju Island, South Korea. Using quantitative PCR (qPCR), twenty-two ARGs encoding tetracycline resistance (<I>tetA</I>, <I>tetB</I>, <I>tetD</I>, <I>tetE</I>, <I>tetG</I>, <I>tetH</I>, <I>tetM</I>, <I>tetQ</I>, <I>tetX</I>, <I>tetZ</I>, <I>tetBP</I>), sulfonamide resistance (<I>sul1</I>, <I>sul2</I>), quinolone resistance (<I>qnrD</I>, <I>qnrS</I>, <I>aac(6′)-Ib-cr</I>), β-lactams resistance (<I>bla</I> <SUB>TEM</SUB>, <I>bla</I> <SUB>CTX</SUB>, <I>bla</I> <SUB>SHV</SUB>), macrolide resistance (<I>ermC</I>), florfenicol resistance (<I>floR</I>) and multidrug resistance (<I>oqxA</I>) and a class 1 integrons-integrase gene (<I>intI1</I>) were quantified. In addition, Illumina Miseq sequencing was applied to investigate microbial community differences across fish farm effluents. Results from qPCR showed that the total number of detected ARGs ranged from 4.24 × 10<SUP>−3</SUP> to 1.46 × 10<SUP>−2</SUP> copies/16S rRNA gene. Among them, <I>tetB</I> and <I>tetD</I> were predominant, accounting for 74.8%–98.0% of the total ARGs. Furthermore, <I>intI1</I> gene showed positive correlation with <I>tetB</I>, <I>tetD</I>, <I>tetE</I>, <I>tetH</I>, <I>tetX</I>, <I>tetZ tetQ</I> and <I>sul1</I>. Microbial community analysis revealed potential host bacteria for ARGs and <I>intI1</I>. Two genera, <I>Vibrio</I> and <I>Marinomonas</I> belonging to <I>Gammaproteobacteria</I>, showed significant correlation with <I>tetB</I> and <I>tetD</I>, the most dominant ARGs in all samples. Also, operational taxonomic units (OTUs)-based network analysis revealed that ten OTUs, classified into the phyla <I>Proteobacteria</I>, <I>Cyanobacteria/Chloroplast</I>, <I>Bacteroidetes</I>, <I> <U>Verrucomicrobia</U> </I> and an unclassified phylum, were potential hosts of tetracycline resistance genes (i.e., <I>tetA</I>, <I>tetG</I>, <I>tetH</I>, <I>tetM</I>, <I>tetQ</I> and <I>tetZ</I>). Further systematic monitoring of ARGs is warranted for risk assessment and management of antibacterial resistance from fish farm effluents.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We found prevalence of various ARGs from effluent of coastal aquaculture. </LI> <LI> Among the targeted ARGs, both <I>tet</I>B and <I>tet</I>D were predominant. </LI> <LI> <I>intI</I>1 had positive correlation with tetracycline and sulfonamide resistance genes. </LI> <LI> Network analysis revealed potential host bacteria for ARGs and <I>intI</I>1. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

MinION 메타지놈 시퀀싱을 이용한 사람과 가축 간 항생제 내성 유전자 전파에 대한 연구

정지원 ( Jiwon Jeong ),운노타쯔야 ( Tatsuya Unno ) 한국환경농학회 2023 한국환경농학회 학술대회집 Vol.2023 No.0

The use of Antibiotics in livestock industry can result in the spread of antibiotic resistance among pathogenic bacteria, leading to an increase in the frequency of antibiotic resistant bacteria. Since livestock farms are exposed to the natural environment, they can be a route for transmission of antibiotic resistant genes (ARGs) from livestock feces to the surrounding environment. Additionally, livestock feces are commonly used as fertilizer for various crops through composting process. Advancements in metagenome sequencing and data processing have enabled the analysis of the genomes of microbial communities in different environments. Long-read based MinION sequencing (ONT; Oxford Nanopore Technology, UK) can locate mobile genetic elements (MGEs), such as integrons and transposons that help in the mobility of ARGs. This makes it suitable for comprehensive research on the patterns of ARG propagation. In this study, we used MinION sequencing to analyze the dissemination pattern of ARGs with MGEs in the feces of both human and livestock animals. Livestock feces were collected from four pig farms, five cow farms, five chicken farms and five horse riding grounds on Jeju Island. Five healthy adults donated human feces. All fecal samples were homogenized, freeze-dried, and stored at -80 ℃ until DNA extraction. A total of 72 DNA samples were extracted using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, Germany), and prepared as a library for MinION sequencing, following the manufacturer's instructions. After sequencing, the fastq files were trimmed using Porechop to remove sequencing adapters. ARG-containing reads and bacteria that have these reads were identified using ARGpore2, based on BLASTP. The number of ARG-containing reads were normalized by the number of total nanopore reads. Using the EPI2ME software from Oxford Nanopore Technology, we classified the microbial communities of each sample. Collectively, we found 7, 16, 8, 13, and 2 types of antibiotic classes in humans, pigs, cows, chickens, and horses, respectively. Resistance to aminoglycoside and tetracycline was observed in all samples. In addition, we identified the genetic organization from nanopore reads containing a clinically relevant gene among the detected ARGs. Analyzing these reads will not only allow us to understand the relationship between ARGs and MGEs but also provide the basis for future studies aiming to find, remove, or deactivate ARGs.

Exploring the antibiotic resistome in activated sludge and anaerobic digestion sludge in an urban wastewater treatment plant via metagenomic analysis

유근제,Hyunji Yoo,Jangho Lee,Eun Joo Choi,Joonhong Park 한국미생물학회 2020 The journal of microbiology Vol.58 No.2

Antibiotic resistance genes (ARGs) are emerging contaminants that pose a potential threat to human health worldwide. Urban wastewater treatment plants (WWTPs) are a main source of both antibiotic-resistant bacteria and ARGs released into the environment. Nevertheless, the propagation of ARGs and their underlying mechanisms and the dynamics of mobile genetic elements (MGEs) in WWTPs have rarely been investigated in South Korea. In this study, shotgun metagenomic analysis was used to identify comprehensive ARGs and their mechanisms, bacterial communities, and MGEs from 4 configurations with 2 activated sludge (AS) and 2 anaerobic digestion sludge (ADS) samples. A total of 181 ARG subtypes belonging to 22 ARG types were broadly detected, and the ARG abundances in the AS samples were 1.3–2.0 orders of magnitude higher than in the ADS samples. Multidrug and bacitracin resistance genes were the predominant ARG types in AS samples, followed by ARGs against sulfonamide, tetracycline, and β-lactam. However, the composition of ARG types in ADS samples was significantly changed. The abundance of multidrug and β-lactam resistance genes was drastically reduced in the ADS samples. The resistance genes of MLS were the predominant, followed by ARGs against sulfonamide and tetracycline in the ADS samples. In addition, plasmids were the dominant MGEs in the AS samples, while integrons (intI1) were the dominant MGEs in the ADS samples. These results provide valuable information regarding the prevalence of ARG types and MGEs and the difference patterns between the AS and ADS systems.

Fate of antibiotic resistance genes and metal resistance genes during thermophilic aerobic digestion of sewage sludge

Jang, Hyun Min,Lee, Jangwoo,Kim, Young Beom,Jeon, Jong Hun,Shin, Jingyeong,Park, Mee-Rye,Kim, Young Mo Elsevier 2018 Bioresource technology Vol.249 No.-

<P><B>Abstract</B></P> <P>This study examines the fate of twenty-three representative antibiotic resistance genes (ARGs) encoding tetracyclines, sulfonamides, quinolones, β-lactam antibiotics, macrolides, florfenicol and multidrug resistance during thermophilic aerobic digestion (TAD) of sewage sludge. The bacterial community, class 1 integrons (<I>intI</I>1) and four metal resistance genes (MRGs) were also quantified to determine the key drivers of changes in ARGs during TAD. At the end of digestion, significant decreases in the quantities of ARGs, MRGs and <I>intI</I>1 as well as 16S rRNA genes were observed. Partial redundancy analysis (RDA) showed that shifts in temperature were the key factors affecting a decrease in ARGs. Shifts in temperature led to decreased amounts of ARGs by reducing resistome and bacterial diversity, rather than by lowering horizontal transfer potential via <I>intI</I>1 or co-resistance via MRGs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Changes in ARGs, MRGs and <I>intI</I>1 were investigated during TAD of sewage sludge. </LI> <LI> Significant decreases in ARGs, MRGs and <I>intI</I>1 genes were observed in TAD. </LI> <LI> Shifts in temperature were responsible for changes in ARGs. </LI> <LI> A decline in bacterial diversity resulted in decrease in resistome during TAD. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Removal of antibiotics from wastewater and its problematic effects on microbial communities by bioelectrochemical Technology: Current knowledge and future perspectives

Mahdi Hassan,Guangcan Zhu,Yong-ze LU,Ali Hamoud AL-Falahi,Yuan LU,Shan Huang,Ziren Wan 대한환경공학회 2021 Environmental Engineering Research Vol.26 No.1

In this review, antibiotics are considered an emerging pollutant that has drawn worldwide attention in recent years. Therefore, the effective removal of antibiotic contaminants has become a hot issue in the field of environmental research. Most antibiotics applied to humans eventually enter municipal Wastewater Treatment Plants (WWTPs), because there are no appropriate commercially available pretreatment techniques. However, increasing anthropogenic activities, the high demand for animal-protein in developing countries as a nutritional alternative, and the extensive usage of antibiotics are mainly responsible for the persistence of antibiotic pollutants. One of the serious concerns regarding the presence of antibiotics in water and their potential role in exacerbating the emergence of antibiotics-resistance bacteria (ARB) and antibiotics-resistance genes (ARGs). In recent years, bioelectrochemical technologies are found promising for suppressing antibiotic contaminants through microbial metabolism and electrochemical redox reactions. Therefore, this review provides up-to-date insight research on bioelectrochemical systems (BESs), which improves the removal of the antibiotic in an efficient way. The focus of this review has been on the environmental sources of antibiotics, their health effects and possible degradation pathways, bacterial-antibiotics resistance mechanisms, and treatment of antibiotic-contained water using BES technology.

Prevalence of multidrug-resistant (MDR) Escherichia coli in untreated effluents from a wastewater treatment plant (WWTP) in Dhaka, Bangladesh

Md Naeem Hossain,Asim kumar Roy,Humayra Habib,Zenat Zebin Hossain,Humaira Akhter,Anowara Begum 한국미생물학회 2022 미생물학회지 Vol.58 No.3

The widespread use of antibiotics in human and animal husbandry results in the potential release of antibiotics into surrounding environments and leads to the emergence of antibiotic-resistant bacteria and antibiotic resistance genes. With this concern, we aimed to study the molecular mechanisms associated with multidrug resistance (MDR) and biofilm formation capacity in Escherichia coli isolates from Dhaka City’s wastewater treatment plant. A total of 37 antimicrobialresistant E. coli isolates were analyzed for the presence of genes associated with antibiotic resistance and biofilm formation using several phenotypic and polymerase chain reaction (PCR) assays and curing plasmids through the SDS method. Among the 37 isolates, most prevalent gene detected was qnrS (32.43%), followed by blaTEM (29.73%), blaCTX-M-15 (24.32%), dfrA17 (21.62%), sul2 (8.1%), qnrB (5.41%), sul1 (5.41%), and dfrA1 (2.70%). Twelve E. coli isolates (32.43%) possessed only class 1 integrons. A comparison of results prior to and after plasmid curing revealed most E. coli had plasmid-mediated antibiotic resistance. Among the 37 isolates, nine strains showed weak biofilm production and one showed strong biofilm production. All of the isolates were curli producers; fimH and csgA genes were present in 72.9% and 64.9% of the isolates, respectively. The dynamic antibiotic resistance diversity revealed in this study may pose concern regarding the potential development of drug-resistant bacteria and their dissemination into the surrounding environment via WWTPs.

항생제 내성 제어를 위한 소독 기법간의 비교 ; 염소, 오존 및 전자빔

오준식,김성표 대한상하수도학회 2013 상하수도학회지 Vol.27 No.6

Recently, a number of countries are now considering the reuse of effluents from wastewater treatment for various water applications. To improve the reuse of wastewater effluent, the development of appropriate micro-pollutant removal technology is necessary. Although several researche have been studied for removing micro-pollutants in water, little study has been conducted for the removal of emerging contaminant such as antibiotic resistant genes (ARGs) by disinfection processes. Therefore, the aim of this study is to compare the capacity of disinfection technologies such as chlorination, ozone, and electron beam, for removing antibiotic resistant bacteria (ARB) and ARGs. Based on this study, better ARG removal can be achieved by ozonation and electron beam. Relatively, high CT values of chlorination or ozonation are needed to remove ARB and ARG compared to conventional pathogens.

Quantitative and qualitative changes in antibiotic resistance genes after passing through treatment processes in municipal wastewater treatment plants

Lee, Jangwoo,Jeon, Jong Hun,Shin, Jingyeong,Jang, Hyun Min,Kim, Sungpyo,Song, Myoung Seok,Kim, Young Mo Elsevier 2017 The Science of the total environment Vol.605 No.-

<P><B>Abstract</B></P> <P>In this study, quantitative and qualitative changes in antibiotics resistance genes (ARGs) were investigated in two municipal wastewater treatment plants (WWTPs) treating pretreated livestock or industrial wastewater as well as municipal sewage. Total eight ARGs (<I>tet</I>X, <I>tet</I>M, <I>tet</I>A, <I>sul</I>1, <I>sul</I>2, <I>erm</I>B, <I>qnr</I>D, and <I>bla</I> <SUB>TEM</SUB>) were quantified, and their relative abundance was assessed by ARGs copies/16S rRNA gene copies. The fate of ARGs was observed to be different between two WWTPs: <I>sul</I>, <I>qnr</I>D, and <I>bla</I> <SUB>TEM</SUB> were proliferated during the treatment processes only in the WWTP1 which received pretreated livestock wastewater. Furthermore, dynamic shifts in patterns of ARGs occurrence were observed during biological, secondary sedimentation and coagulation processes. During biological treatment in both WWTPs, relative abundance of <I>tet</I> and <I>erm</I>B changed: <I>tet</I> increased significantly by 211.6–357.6%, while <I>erm</I>B decreased by 70.4–92.0%. Little variation was observed in <I>sul</I>, <I>qnr</I>D and <I>bla</I> <SUB>TEM</SUB>. Subsequently, the relative abundance of <I>tet</I> decreased during the secondary sedimentation and coagulation in both WWTPs: <I>tet</I> decreased by 56.0–86.3% during sedimentation and by 48.2–75.7% during coagulation, respectively. During the final treatment, different responses of antibiotic resistance bacteria (ARB) and ARGs to ultraviolet (UV) disinfection were found: removal efficiencies of ARB were observed in the range of 34–75%, while obvious reduction in ARGs was not observed at the UV dose of 27mJ/cm<SUP>2</SUP>. Although ARGs underwent various treatment processes, considerable levels of ARGs remained at discharge amounting to 4.2×10<SUP>18</SUP> copies/day from WWTP1 and 5.4×10<SUP>16</SUP> copies/day from WWTP2, respectively.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Quantitative and qualitative changes in ARGs were investigated in WWTPs. </LI> <LI> Changes were unique for each ARG community while undergoing treatment processes. </LI> <LI> Variation in ARGs was largest during biological and post-physiochemical processes. </LI> <LI> ARGs showed limited response to UV disinfection. </LI> <LI> ARB were reduced by UV disinfection. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>