Impact of Fish Farming on Antibiotic Resistome and Mobile Elements in Baltic Sea Sediment

Antibiotic resistance has become a serious threat to the efficacy of antibiotics used in human and veterinary medicine. Understanding the abundance and prevalence of antibiotic resistance genes (ARGs) in the environmental resistome is important for maintaining the efficacy of antibiotics and predicting a risk of the ARGs spreading in the environment and moving into previously non-resistant bacteria, including human pathogens. Fish farms are an environmental reservoir of ARGs due to the treatment of fish with antibiotics that also are important for human medicine. The two main topics of this thesis are (1) determining the abundance and diversity of ARGs and mobile elements in sediments impacted by fish farming and (2) investigating the major source of ARGs in the farm sediments in the Northern Baltic Sea. In addition, correlations between ARGs and mobile elements were examined to estimate the potential risk of ARG mobilization in the environment. This study employed a high-throughput qPCR array, which permits quantifying hundreds of ARGs and genes associated with mobile elements in the environmental resistome in a single experiment. Fish farming impacts the composition of ARGs in sediments below fish farms in the Northern Baltic Sea. However, the impact is local and mostly limited to enrichment of ARGs associated with antibiotics used at the farms. In the current conditions, the risk of ARG spread from the farm sediments to the surrounding sediments is low in the Northern Baltic Sea. However, the enriched ARGs persist in the farm sediments during the 6-year observations even when the selection pressure of the antibiotics is negligible. Moreover, significant correlations between mobile elements and ARGs may imply the persistence of certain ARGs in the fish farming environments and their potential for mobilizing the ARGs to other bacteria including pathogens. The persistence of ARGs at the farm facilities is a threat to the efficacy of the antibiotics against fish diseases, potentially leading to fish production losses. We provide indirect evidence suggesting that certain ARGs are being constantly introduced by feces of the farmed fish into the sediments below the fish farms. Further studies could focus on investigating the development of ARGs in juvenile fish before they are introduced into the Baltic Sea open-cage farms. We conclude that a high throughput qPCR array is a powerful tool that provides unprecedented insights into the ARG composition in the environmental resistome associated with fish farming.Bakteerien vastustuskyky antibiooteille eli antibioottiresistenssi uhkaa heikentää ihmisten ja eläinten lääkinnässä käytettävien antibioottien tehoa. Bakteerien vastustuskyky aiheutuu antibioottiresistenssigeeneistä, joita bakteerit kantavat perimässään. Antibioottiresistenssigeenien määrien seuraaminen on keskeistä antibioottien tehon säilyttämiseksi sekä riskien ymmärtämiseksi. Kalankasvattamoilla käytetään antibiootteja kalojen sairauksien hoitamiseksi, minkä seurauksena kasvattamoiden ympäristössä havaitaan resistenssigeenejä keskimääräistä enemmän. Tämän väitöskirjan kaksi aihetta ovat (1) antibioottiresistenssigeenien sekä liikkuvien geneettisten elementtien määrien ja monimuotoisuuden selvittäminen Itämerellä sijaitsevien kalankasvatuslaitosten pohjasedimenteissä sekä (2) resistenssigeenien alkuperän selvittäminen. Tutkimuksissa käytettiin uudenlaista qPCR-pohjaista analyysimenetelmää, joka mahdollistaa satojen resistenssigeenien ja liikkuvien geneettisten elementtien samanaikaisen mittaamisen näytteistä. Kalankasvatus vaikuttaa paikallisesti antibioottiresistenssigeenien määriin ja monimuotoisuuteen kalankasvattamoiden pohjasedimenteissä Itämerellä. Tulostemme perusteella resistenssigeenien leviäminen kasvattamoiden lähiympäristöön on melko vähäistä. Toisaalta resistenssigeenit säilyvät sedimenteissä kuuden vuoden seurantajakson ajan, vaikka antibioottien aiheuttama valintapaine on ollut heikko. Tämä on uhka kalankasvatuksessa käytettävien antibioottien teholle ja saattaa johtaa kasvaneisiin kustannuksiin tuotannon laskun vuoksi. Tutkimus antaa epäsuoraa näyttöä siitä, että tietyt resistenssigeenit saapuvat kasvattamoille kasvatettavan kalan ulosteen mukana. Jatkotutkimuksissa olisi syytä mitata nuorten kalojen kantamia antibioottiresistenssigeenejä ennen kalojen saapumista kasvattamoille

Routine wastewater-based monitoring of antibiotic resistance in two Finnish hospitals : focus on carbapenem resistance genes and genes associated with bacteria causing hospital-acquired infections

Background: Wastewater-based monitoring represents a useful tool for antibiotic resist-ance surveillance. Aim: To investigate the prevalence and abundance of antibiotic resistance genes (ARGs) in hospital wastewater over time. Methods: Wastewater from two hospitals in Finland (HUS1 and HUS2) was monitored weekly for nine weeks (weeks 25-33) in summer 2020. A high-throughput real-time poly-merization chain reaction (HT-qPCR) system was used to detect and quantify 216 ARGs and genes associated with mobile genetic elements (MGEs), integrons, and bacteria causing hospital-acquired infections (HAIs), as well as the 16S rRNA gene. Data from HT-qPCR were analysed and visualized using a novel digital platform, ResistApp. Eight carbapenem resistance genes (blaGES, blaKPC, blaVIM, blaNDM, blaCMY, blaMOX, blaOXA48, and blaOXA51) and three genes associated with bacteria causing HAIs (Acinetobacter bau-mannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were studied. Findings: There was a significantly higher number of ARGs at both hospitals in weeks 27-30 (174-191 genes) compared to other sampling weeks (151-171 genes). Our analyses also indicated that the two hospitals, which used different amounts of antibiotics, had significantly different resistance gene profiles. Carbapenem resistance genes were more prevalent and abundant in HUS1 than HUS2. Across both hospitals, blaGES and blaVIM were the most prevalent and abundant. There was also a strong positive association between blaKPC and K. pneumoniae in HUS1 wastewater. Conclusion: Routine wastewater-based monitoring using ResistApp can provide valuable information on the prevalence and abundance of ARGs in hospitals. This helps hospitals understand the spread of antibiotic resistance in hospitals and identify potential areas for intervention. (c) 2021 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer reviewe

Profiles of environmental antibiotic resistomes in the urban aquatic recipients of Sweden using high-throughput quantitative PCR analysis

Antibiotic resistance in aquatic ecosystems presents an environmental health issue worldwide. Urban recipient water quality is susceptible to effluent discharges with antibiotic resistance contaminants and needs to be protected, particularly for those as sources of drinking water production. Knowledge on aquatic resistome profiles in downstream of wastewater treatment plants allows a better understanding of the extent to which antibiotic resistance contaminants emerge and spread in recipient waters, but such information remains very limited in Sweden. The key objective of this study was to determine the resistome profiles of numerous antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and other genes in urban recipient water systems connected to Sweden's major drinking water reservoir. This was achieved through analysis of surface water samples for 296 genes using high-throughput quantitative PCR arrays. A total of 167 genes were detected in at least one of the samples, including 150 ARGs conferring resistance to 11 classes of antibiotics, 7 integrase MGEs and 9 other genes. There was a spatial difference in the resistome profiles with the greatest average relative abundance of resistance genes observed in the water body of Vasteras followed by Uppsala, Stockholm and Eskilstuna, as similar to the general pattern of the antibiotic sales for these regions. ARGs against beta-lactams and sulfonamides showed the highest average relative abundance in the studied water bodies, while vancomycin resistance genes were only found in the Uppsala water environment. Generally, the recipient water bodies were detected with higher numbers of genes and greater relative abundances as compared to the upstream sites. Anthropogenic pollution, i.e., wastewater discharge, in the recipient water was also reflected by the finding of intI, sul1 and crAssphage. Overall, this study provided the first quantitative assessment of aquatic environmental resistomes in Sweden, highlighting the widespread of antibiotic resistance contaminants in urban recipient waters.Peer reviewe

The Resistome of Farmed Fish Feces Contributes to the Enrichment of Antibiotic Resistance Genes in Sediments below Baltic Sea Fish Farms

Our previous studies showed that particular antibiotic resistance genes (ARGs) were enriched locally in sediments below fish farms in the Northern Baltic Sea, Finland, even when the selection pressure from antibiotics was negligible. We assumed that a constant influx of farmed fish feces could be the plausible source of the ARGs enriched in the farm sediments. In the present study, we analyzed the composition of the antibiotic resistome from the intestinal contents of 20 fish from the Baltic Sea farms. We used a high-throughput method, WaferGen qPCR array with 364 primer sets to detect and quantify ARGs, mobile genetic elements (MGE), and the 16S rRNA gene. Despite a considerably wide selection of qPCR primer sets, only 28 genes were detected in the intestinal contents. The detected genes were ARGs encoding resistance to sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(32), tetM, tetO, tetW], aminoglycoside (aadA1, aadA2), chloramphenicol (catA1), and efflux-pumps resistance genes (emrB, matA, mefA, msrA). The detected genes also included class 1 integron-associated genes (intI1, qacE?1) and transposases (tnpA). Importantly, most of the detected genes were the same genes enriched in the farm sediments. This preliminary study suggests that feces from farmed fish contribute to the ARG enrichment in farm sediments despite the lack of contemporaneous antibiotic treatments at the farms. We observed that the intestinal contents of individual farmed fish had their own resistome compositions. Our result also showed that the total relative abundances of transposases and tet genes were significantly correlated (p = 0.001, R-2 = 0.71). In addition, we analyzed the mucosal skin and gill filament resistomes of the farmed fish but only one multidrug-efflux resistance gene (emrB) was detected. To our knowledge, this is the first study reporting the resistome of farmed fish using a culture-independent method. Determining the possible sources of ARGs, especially mobilized ARGs, is essential for controlling the occurrence and spread of ARGs at fish farming facilities and for lowering the risk of ARG spread from the farms to surrounding environments.Peer reviewe

Antibiotic Resistomes and Microbiomes in the Surface Water along the Code River in Indonesia Reflect Drainage Basin Anthropogenic Activities

Water and sanitation are important factors in the emergence of antimicrobial resistance in low-and middle-income countries. Drug residues, metals, and various wastes foster the spread of antibiotic resistance genes (ARGs) with the help of mobile genetic elements (MGEs), and therefore, rivers receiving contaminants and enfluents from multiple sources are of special interest. We followed both the microbiome and resistome of the Code River in Indonesia from its pristine origin at the Merapi volcano through rural and then city areas to the coast of the Indian Ocean. We used a SmartChip quantitative PCR with 382 primer pairs for profiling the resistome and MGEs and 16S rRNA gene amplicon sequencing to analyze the bacterial communities. The community structure explained the resistome composition in rural areas, while the city sampling sites had lower bacterial diversity and more ARGs, which correlated with MGEs, suggesting increased mobility potential in response to pressures from human activities. Importantly, the vast majority of ARGs and MGEs were no longer detectable in marine waters at the ocean entrance. Our work provides information on the impact of different influents on river health as well as sheds light on how land use contributes to the river resistome and microbiome.Peer reviewe

Sulphonamide and Trimethoprim Resistance Genes Persist in Sediments at Baltic Sea Aquaculture Farms but Are Not Detected in the Surrounding Environment

Peer reviewe

Antibiotics and antibiotic resistance genes in global lakes:A review and meta-analysis

Lakes are an important source of freshwater, containing nearly 90% of the liquid surface fresh water worldwide. Long retention times in lakes mean pollutants from discharges slowly circulate around the lakes and may lead to high ecological risk for ecosystem and human health. In recent decades, antibiotics and antibiotic resistance genes (ARGs) have been regarded as emerging pollutants. The occurrence and distribution of antibiotics and ARGs in global freshwater lakes are summarized to show the pollution level of antibiotics and ARGs and to identify some of the potential risks to ecosystem and human health. Fifty-seven antibiotics were reported at least once in the studied lakes. Our meta-analysis shows that sulfamethoxazole, sulfamerazine, sulfameter, tetracycline, oxytetracycline, erythromycin, and roxithromycin were found at high concentrations in both lake water and lake sediment. There is no significant difference in the concentration of sulfonamides in lake water from China and that from other countries worldwide; however, there was a significant difference in quinolones. Erythromycin had the lowest predicted hazardous concentration for 5% of the species (HC5) and the highest ecological risk in lakes. There was no significant difference in the concentration of sulfonamide resistance genes (sul1 and sul2) in lake water and river water. There is surprisingly limited research on the role of aquatic biota in propagation of ARGs in freshwater lakes. As an environment that is susceptible to cumulative build-up of pollutants, lakes provide an important environment to study the fate of antibiotics and transport of ARGs with a broad range of niches including bacterial community, aquatic plants and animals

Occurrence of sul and tet(M) genes in bacterial community in Japanese marine aquaculture environment throughout the year : Profile comparison with Taiwanese and Finnish aquaculture waters

The use of antibiotics in aquaculture causes selection pressure for antibiotic-resistant bacteria (ARB). Antibiotic resistance genes (ARGs) may persist in ARB and the environment for long time even after stopping drug administration. Here we show monthly differences in the occurrences of genes conferring resistance to sulfonamides (i.e. sul1, sul2, sul3), and tetracyclines (tet(M)) in Japanese aquaculture seawater accompanied by records of drug administration. sul2 was found to persist throughout the year, whereas the occurrences of sul1, sul3, and tet(M) changed month-to-month. sul3 and tet(M) were detected in natural bacterial assemblages in May and July, but not in colony-forming bacteria, thus suggesting that the sul3 was harbored by the non-culturable fraction of the bacterial community. Comparison of results from Taiwanese, Japanese, and Finnish aquaculture waters reveals that the profile of sul genes and tet(M) in Taiwan resembles that in Japan, but is distinct from that in Finland. To our knowledge, this work represents the first report to use the same method to compare the dynamics of sul genes and tet(M) in aquaculture seawater in different countries. (C) 2019 Elsevier B.V. All rights reserved.Peer reviewe

The qPCR Assay for Detecting The Presence and Relative Abundance of Pseudomonas aerugionosa and Antibiotic Resistance Gene aadA2 in Hospital Wastewater of National Reference Hospital Dr. Cipto Mangunkusumo (RSCM)

Antimicrobial resistance is one of the top 10 global health threats. The hospital wastewater (HWW) potentially becomes the reservoir and dissemination of antibiotic resistance gene (ARG) and bacterial pathogens. In Indonesia, the protocol to monitor the ARGs form HWW has not been established. This study aimed to detect the presence and find the relative abundance of P. aeruginosa and aadA2 genes from Dr. RSUPN. Cipto Mangungkusumo (RSCM) inlet and outlet wastewater through qPCR assay. The primers used were supported by Resistomap. The study revealed that the qPCR assay was able to detect the Ct value of P. aeruginosa and aadA2. The aadA2 gene was found in all waste water samples, meanwhile P. aeruginosa was only found in some of inlet samples. aadA2 had the highest relative abundance and this gene’s mobility uses plasmids and integrons that potentially enhance the acquired antimicrobial resistance (AMR) mechanism. This study implicated that qPCR assay was capable to detect pathogenic bacteria and ARG, and ARG could be released to the environment even though the wastewater samples have been proceeded in wastewater treatment plants (WWTP). The qPCR assay can be used as the method to monitor the AMR status in a hospital and the spreading potency to the environment using the HWW