Abundance of Class 1 IntegronIntegrase and Sulfon

Authors: Ryszard Koczura Joanna Mokracka Agata Taraszewska Natalia Łopacinska

Publish Date: 2016/09/06

Volume: 72, Issue: 4, Pages: 909-916

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Abstract

In this study we determined the presence of class 1 integronintegrase gene in culturable heterotrophic bacteria isolated from river water and sediment sampled upstream and downstream of a wastewater treatment plant effluent discharge Moreover we quantified intI1 and sulfonamide resistance genes sul1 and sul2 in the water and sediment using qPCR There was no correlation between the results from water and sediment samples which suggests integroncontaining bacteria are differentially retained in these two environmental compartments The discharge of treated wastewater significantly increased the frequency of intI1 among culturable bacteria and the gene copy number in river water and increased the number of sul1 genes in the sediment We also observed seasonal differences in the frequency of the class 1 integronintegrase gene among culturable heterotrophs as well as intI1 copy number in water but not in sediment The results suggest that the abundance of class 1 integrons in aquatic habitat depends on anthropogenic pressure and environmental factorsSurface waters act as important recipients reservoirs and vectors of biotic contaminants like antibioticresistant bacteria ARBs and antibiotic resistance genes ARGs in the environment 1 2 3 Antibioticresistant bacteria can enter the environment through for example wastewater treatment plant effluents and agricultural runoff Agricultural and wastewater input of antibiotics biocides and heavy metals into surface waters imposes selective pressure enabling the maintenance and amplification of ARBs and enhancing lateral transfer of ARGs in the environment 4 5Class 1 integrons are genetic assembly elements involved in capture and spread of ARGs They are characterized by a 5′conserved region that consists of an integrase gene intI attachment site attC and a promoter PC that directs transcription of the incorporated genes The 3′conserved region contains qacΔE1 and sul1 genes responsible for resistance to quaternary ammonium compounds and sulfonamides respectively 6 7 Class 1 integrons capture gene cassettes that determine resistance to antimicrobials code for transport proteins esterases phosphatases transposases and also proteins of unknown function Over 130 different resistance gene cassettes have been described in the variable regions of class 1 integrons so far These integrons are widely distributed among clinical strains and in environmental isolates their presence can be associated with increased frequency of virulence genes 8 Class 1 resistance integrons are located on mobile elements like transposons and plasmids and thus are involved in spread of antibiotic resistance genes in bacteria by lateral gene transfer 9 10Integrons have been detected in the genomes of cultivable heterotrophs isolated from surface waters with different anthropogenic pressure wastewater and ground waters 3 11 12 However culturebased methods do not take into account nonculturable microorganisms which constitute the vast majority of environmental microorganisms 13 The prevalence of integrons is proposed to serve as a marker of antibiotic resistance level 14 and anthropogenic pollution in the environment 15 Investigations into the levels of intI1 and sul genes along a gradient of anthropogenic impact not only can give insights into the spread and proliferation of ARGs in surface water but also can identify critical point sources of ARGs in river water 15The aim of this study was to evaluate the copy number and abundance of class 1 integronintegrase and sulfonamide resistance genes in river water and sediment at three sites with different anthropogenic pressure We tested hypotheses that the copy number and/or relative abundance of class 1 integronintegrase and sulfonamide resistance genes in river habitat a are affected by anthropogenic pressure and b show seasonal fluctuations Additionally we set up another hypothesis that water and sediment microbiomes may differ in the abundance of the aforementioned genesWater and sediment samples were taken monthly from January to December 2014 from the Warta River Altogether there were 12 sampling events 3 for each season—January February and March in winter April May and June in spring July August and September in summer and October November and December in autumn The Warta River is 808km long and flows in centralwestern Poland There were three sampling sites localized ca 15 km away from each other site no 1 referred to as “upstream” in Rogalinek GPS coordinates 522488 168948 under relatively low anthropogenic pressure localized upstream of a municipal water intake site no 2 referred to as “city” in Poznań GPS 523814 169362 a city with a population of 550000 and several hospitals without their own wastewater treatment facilities and site no 3 referred to as “downstream” near Czerwonak GPS 524946 169734 5 km downstream of the outlet of a municipal wastewater treatment plant serving Poznań and the nearby areas The wastewater treatment plant WWTP is capable of taking up 200000 m3 of sewage per day During sewage treatment the chemical oxygen demand is reduced from 1158 to 36 mg O2 L−1 biological oxygen demand from 483 to 35 mg O2 L−1 total suspended solids from 555 to 5 mg L−1 total nitrogen from 88 to 10 mg N L−1 and total phosphorus from 15 to 06 mg P L−1 The water samples were collected to sterile containers from the riverbed The sediment samples were taken 10 cm below the bottom surface The samples were transported to laboratory at 4 °C and processed within 3 hPhysicochemical parameters of water namely temperature pH total dissolved solids TDS salinity conductivity and rugged dissolved oxygen RDO concentration were measured in situ with the use of a portable multiparameter meter OrionStar A329 Thermo Scientific Biological oxygen demand BOD was measured with OxiTop Control system WTW

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