New Estimation of Antibiotic Resistance Genes in Sediment Along the Haihe River and Bohai Bay in China: A Comparison Between Single and Successive DNA Extraction Methods

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.

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Prevalence And Characterization Of Plasmid-Mediated Quinolone Resistance In Various Aquatic Sources

10.32920/ryerson.14652291.v1 ◽

2021 ◽

Author(s):

Farhan Yusuf ◽

Kimberley Gilbride

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Quinolone Resistance ◽

Resistant Bacteria ◽

Rrna Gene ◽

Environmental Isolates ◽

Topoisomerase Iv ◽

Phenotypic Resistance ◽

Ciprofloxacin Resistance

Bacterial isolates found in aquatic ecosystems often carry antibiotic resistance genes (ARGs). These ARGs are often found on plasmids and transposons, which allows them to be proliferate throughout bacterial communities via horizontal gene transfer (HGT) causing dissemination of multidrug resistance. The increase in antibiotic resistance has raised concerns about the ability to continue to use these drugs to fight infectious diseases. Novel synthetic antibiotics like ciprofloxacin that are not naturally found in the environment were developed to prevent resistances. However, ciprofloxacin resistance has occurred through chromosomal gene mutations of type 2 topoisomerases or by the acquisition of plasmid-mediated quinolone resistances (PMQR). A particular PMQR, qnr genes, encoding for pentapeptide repeat proteins that confer low levels of quinolone resistance and protect DNA gyrase and topoisomerase IV from antibacterial activity. These qnr genes have been identified globally in both clinical and environmental isolates. The aim of this study was to determine the prevalence of ciprofloxacin-resistant bacteria in aquatic environments in the Greater Toronto Area and the potential dissemination of ciprofloxacin resistance. With the selective pressure of ciprofloxacin, we hypothesize that ciprofloxacin-resistant bacteria (CipR) in the environment may carry PMQR mechanisms while the sensitive population (CipS) would not carry PMQR genes. Isolates were tested for resistance to an additional 12 different antibiotics and identified using Sanger sequencing PCR products of the 16S rRNA gene. To determine which genes are responsible for ciprofloxacin resistance, multiplex PCR of associated qnr genes, qnrA, qnrB, and qnrS, was carried out on 202 environmental isolates. Our data demonstrate a similar prevalence of qnr genes was found in CipR (19%) and CipS (14%) populations suggesting that the presence of these genes was not necessarily correlated with the phenotypic resistance to the antibiotic. Furthermore, ciprofloxacinresistant bacteria were found in all locations at similar frequencies suggesting that resistance genes are widespread and could possibly arise through HGT events. Overall, determining the underlying cause and prevalence of ciprofloxacin resistance could help re-establish the effectiveness of these antimicrobial compounds.

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Degradation of antibiotic resistance genes and mobile gene elements in dairy manure anerobic digestion

PLoS ONE ◽

10.1371/journal.pone.0254836 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0254836

Author(s):

Yi Wang ◽

Pramod K. Pandey ◽

Sundaram Kuppu ◽

Richard Pereira ◽

Sharif Aly ◽

...

Keyword(s):

Antibiotic Resistance ◽

Anaerobic Digestion ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Dairy Manure ◽

Health Concern ◽

Animal Origin ◽

Rrna Gene ◽

Transposase Gene ◽

Integrase Gene

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

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Analysis of Antibiotic Resistance Genes, Environmental Factors, and Microbial Community From Aquaculture Farms in Five Provinces, China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.679805 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xu Cheng ◽

Yitong Lu ◽

Yanzhen Song ◽

Ruifang Zhang ◽

Xinyan ShangGuan ◽

...

Keyword(s):

Antibiotic Resistance ◽

Environmental Factors ◽

Bacterial Community ◽

Correlation Analysis ◽

Resistance Genes ◽

Environmental Risk ◽

Antibiotic Resistance Genes ◽

Community Diversity ◽

Sediment Samples ◽

Positive Correlation

The excessive use of antibiotics speeds up the dissemination and aggregation of antibiotic resistance genes (ARGs) in the environment. The ARGs have been regarded as a contaminant of serious environmental threats on a global scale. The constant increase in aquaculture production has led to extensive use of antibiotics as a means to prevent and treat bacterial infections; there is a universal concern about the environmental risk of ARGs in the aquaculture environment. In this study, a survey was conducted to evaluate the abundance and distributions of 10 ARGs, bacterial community, and environmental factors in sediment samples from aquatic farms distributed in Anhui (AP1, AP2, and AP3), Fujian (FP1, FP2, and FP3), Guangxi (GP1, GP2, and GP3), Hainan (HP1, HP2, and HP3), and Shaanxi (SP1, SP2, and SP3) Province in China. The results showed that the relative abundance of total ARGs was higher in AP1, AP2, AP3, FP3, GP3, HP1, HP2, and HP3 than that in FP1, FP2, GP1, GP2, SP1, SP2, and SP3. The sul1 and tetW genes of all sediment samples had the highest abundance. The class 1 integron (intl1) was detected in all samples, and the result of Pearson correlation analysis showed that the intl1 has a positive correlation with the sul1, sul2, sul3, blaOXA, qnrS, tetM, tetQ, and tetW genes. Correlation analysis of the bacterial community diversity and environmental factors showed that the Ca2+ concentration has a negative correlation with richness and diversity of the bacterial community in these samples. Of the identified bacterial community, Proteobacteria, Firmicutes, Chloroflexi, and Bacteroidota were the predominant phyla in these samples. Redundancy analysis showed that environmental factors (TN, TP, Cl–, and Ca2+) have a positive correlation with the bacterial community (AP1, GP1, GP2, GP3, SP1, SP2, and SP3), and the abundance of ARGs (sul1, tetW, qnrS, and intl1) has a positive correlation with the bacterial community (AP2, AP3, HP1, HP2, and HP3). Based on the network analysis, the ARGs (sul1, sul2, blaCMY, blaOXA, qnrS, tetW, tetQ, tetM, and intl1) were found to co-occur with bacterial taxa from the phyla Chloroflexi, Euryarchaeota, Firmicutes, Halobacterota, and Proteobacteria. In conclusion, this study provides an important reference for understanding the environmental risk associated with aquaculture activities in China.

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Distribution of Antibiotic Resistance Genes in Three Different Natural Water Bodies-A Lake, River and Sea

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17020552 ◽

2020 ◽

Vol 17 (2) ◽

pp. 552 ◽

Cited By ~ 4

Author(s):

Sicong Su ◽

Chenyu Li ◽

Jiping Yang ◽

Qunying Xu ◽

Zhigang Qiu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Natural Water ◽

Poyang Lake ◽

Antibiotic Resistance Genes ◽

Water Bodies ◽

Human Beings ◽

Absolute Abundance ◽

Haihe River ◽

Natural Water Bodies

Currently, due to abuse in the use of human antibiotics and the weak regulatory control that the authorities have over sewage discharge and manure management, antibiotic resistance genes (ARGs) have become a new type of environmental pollutant. Three different natural water bodies (Poyang Lake, Haihe River and Qingdao No.1 Bathing Beach seawater) were sampled during the same periods to conduct a longitudinal comparison of distribution. The distribution and expression of 11 ARGs in 20 species were studied, and the correlations between the expression and the distribution of time and space of the ARGs in different water bodies were also analyzed. With the exception of ermA, blaNDM-1 and vanA, which were not detected in seawater, the other ARGs could be detected in all three water bodies. Tetracycline resistance genes (tetC, tetM and tetQ) in the seawater and Haihe River had even reached 100%, and sulfa ARGs (sul1 and sul2) in the seawater and Poyang Lake, as well as sul2 and sul3 in the Haihe River, had also reached 100%. The ARG pollution in Haihe River was much more serious, since 14 and 17 of 20 ARG species were significantly higher compared with seawater and Poyang Lake, respectively. Some ARGs also had a high absolute abundance. The absolute abundance of macrolide resistance genes (ermB) in seawater was as high as 8.61 × 107 copies/L, and the anti-tuberculosis resistant genes (rpoB and katG) in the Haihe River Basin were highly abundant at 1.32 × 106 copies/L and 1.06 × 107 copies/L, respectively. This indicates that ARGs have gradually become more diverse and extensive in natural water bodies. The results of a redundancy analysis (RDA) of the three water bodies showed that although each water body is affected by different factors in space and time, overall, the presence of AGRs is closely related to the production and life of human beings and the migration of animals.

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Conjugative multi-resistant plasmids in Haihe River and their impacts on the abundance and spatial distribution of antibiotic resistance genes

Water Research ◽

10.1016/j.watres.2016.12.046 ◽

2017 ◽

Vol 111 ◽

pp. 81-91 ◽

Cited By ~ 53

Author(s):

Bingjun Dang ◽

Daqing Mao ◽

Yan Xu ◽

Yi Luo

Keyword(s):

Antibiotic Resistance ◽

Spatial Distribution ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Haihe River

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Occurrence and distribution of antibiotic resistance genes in the coastal area of the Bohai Bay, China

Marine Pollution Bulletin ◽

10.1016/j.marpolbul.2016.03.064 ◽

2016 ◽

Vol 107 (1) ◽

pp. 245-250 ◽

Cited By ~ 40

Author(s):

Zhi-Guang Niu ◽

Kai Zhang ◽

Ying Zhang

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Coastal Area ◽

Antibiotic Resistance Genes ◽

Bohai Bay

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Study of Occurrence Characteristics of Antibiotic Resistance Genes in Pig manure during Anaerobic Digestion and Digestate Fertilization

E3S Web of Conferences ◽

10.1051/e3sconf/202123301130 ◽

2021 ◽

Vol 233 ◽

pp. 01130

Author(s):

PAN Xin-rong ◽

CHEN Lei ◽

YU Heng ◽

ZUO Jian-e

Keyword(s):

Antibiotic Resistance ◽

Anaerobic Digestion ◽

Relative Abundance ◽

Resistance Genes ◽

Poultry Manure ◽

Antibiotic Resistance Genes ◽

Pig Manure ◽

Rrna Gene ◽

Occurrence Characteristics ◽

Biogas Residue

Antibiotic resistance genes (ARGs) existing in livestock and poultry manure have the risk to spread and proliferate. This might endanger people’s health. The common treatment of livestock and poultry manure is anaerobic digestion. But the change of ARGs during anaerobic digestion require further study, and the effect of digestate fertilization to the antibiotic resistance of cropland soil is still unclear. This study investigated the pig manure, biogas liquid, biogas residue, and cropland soils fertilized with and without digestate. The results showed that, the relative abundance of ARGs in biogas residue was much higher than other samples. The average relative abundance was 1.46×10-1 copy ratio (copy of ARG/copy of 16S rRNA gene), and the total relative abundance was 3.07 copy ratio. There were 21 ARGs detected in the 5 samples. 11 of them were shared by the 5 samples. The main ARGs were aminoglycoside, chloramphenicol, sulfonamide, tetracycline, and multidrug. Aminoglycoside had the highest relative abundance, and the total relative abundance in all samples was 1.18 copy ratio. Anaerobic digestion increased the total relative abundance of ARGs in pig manure from 1.14×10-1 to 1.70×10-1 copy ratio. Fertilization of digestate increased the total relative abundance of AGRs in soil from 3.27×10-1 to 7.29×10-1 copy ratio.

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Antibiotic resistance genes and class 1 integron: Evidence of fecal pollution as a major driver for their abundance in water and sediments impacted by metal contamination and wastewater in the Andean region of Bolivia

10.1101/2020.03.23.003350 ◽

2020 ◽

Author(s):

Jorge Agramont ◽

Sergio Gutierrez-Cortez ◽

Enrique Joffré ◽

Åsa Sjöling ◽

Carla Calderon Toledo

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Fecal Pollution ◽

Mining Activities ◽

Class 1 Integron ◽

Sediment Samples ◽

Water And Sediment ◽

Class 1 ◽

Water And Sediment Samples

AbstractWater and sediment samples affected by mining activities were collected from three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake directly impacted by acid mine drainage, and the Uru-Uru lake located close to Oruro city and highly polluted by mining activities and human wastewater discharges. Physicochemical parameters, including metal compositions, were analyzed in water and sediment samples. Antibiotic resistance genes (ARGs), were screened for, and verified by quantitative PCR together with the mobile element class 1 integron (intl1) as well as crAssphage, a marker of human fecal pollution. The gene intl1 showed a positive correlation with sul1, sul2, tetA and blaOXA-2. CrAssphage was only detected in Uru-Uru lake and its tributaries and significantly higher abundance of ARGs were found in these sites. Multivariate analysis showed that crAssphage abundance, electrical conductivity and pH were positively correlated with higher levels of intl1 and ARGs. Taken together our results suggest that fecal pollution is the major driver of higher ARGs and intl1 in wastewater and mining contaminated environments.

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