Frequencies of detected ARGs Environmental spread of antibiotic resistance genes (ARGs) in aquatic systems with different levels of microbial contamination Perri V.1, Luprano M.L.1, Visino F.2, Masciopinto C.2, Tandoi V. 1, Levantesi C1 1) Water Research Institute (IRSA) CNR, via Salaria km 29.300, 00015 Monterotondo Scalo, Roma (Italy) 2) Water Research Institute (IRSA) CNR, Via Francesco De Blasio 5, 70132 Bari (Italy) Introduction Antibiotic resistance represents a significant global health problem due to the continuous increase of resistant bacteria frequencies in the clinical setting that reduce the capacity to control and treat infectious diseases. Water environments, is considered a significant reservoir of Antibiotic Resistance Genes (ARGs) which confer resistance to bacteria and could potentially be transferred from environmental bacteria to human pathogens through horizontal gene transfer processes (Rizzo et al., 2013; Marti et al., 2014). Of particular concern, for the recent alarming worldwide increase of β-lactam resistance in Enterobacteriaceae causes of nosocomial infections, is the spread of resistance genes encoding for beta- lactamase and for Extended Spectrum Beta Lactamases that confers resistance also to the third-generation cephalosporin antibiotic. Recently the presence of beta lactamase in water environment in Italy was shown confirming that these contaminants are present in the environment and might represent a risk for human health in our country . Due to the wide variety of ARGs, the definition of appropriate indicators of ARGs presence in the environments is necessary. sul1 and intI1, encoding respectively for resistance to sulfonamides and class 1 integrons integrase, commonly associated to mobile elements and thus more prone to horizontal transfer have been proposed as ARGs marker (Berendonk et al 2015). At present, limited data are available on the presence and diversity of ARGs in the water environment in Italy, however more attention should be directed to understanding the ARGs contamination level in our country and consequently for the definition of suitable monitoring strategies necessary for the assessment of the risk related to the spread of ARGs. Objectives of this work: To investigate the presence of ARGs encoding resistance to 5 different class of antibiotics (ermB, tetO, vanA, mecA, ampC, blaSHV, blaTEM, blaCTX-M and blaCTX-M1 genes) in Italian water environment characterized by different levels of contamination and anthropic impact (municipal wastewater, contaminated surface water and contaminated groundwater). To evaluate to two marker genes of anthropogenic pollution and ARGs presence (sul1 and intI1 genes) and assess the relationships between ARGs and water pollution levels, namely total bacteria (16SrDNA) and fecal bacteria ( E. coli uidA) contamination. Figure 1. Different anthropogenic activities that result in the dissemination of antibiotic resistance genes (ARGs) in aquatic environments (Marti et al., 2014) Water samples Methods Totally 34 water samples comprising wastewater (10) , contaminated surface (12) and impacted ground waters (12) collected in Ostuni site Apulia region were analysed : WASTEWATER: municipal wastewater samples coming from two different sites located in the south of Italy. SURFACE WATER: samples were collected from Lama D’Antelmi channel, a small channel (Fig1-A) heavily contaminated Situated in the south-east of Italy (Ostuni, Puglia) which collect local run-offs and effluents from the Ostuni WWTP (white in fig.1-B). Samples were collected from January to July 2015. GROUNDWATER: aquifer is located in the same area of contaminated channel (Ostuni, Puglia). The groundwater is affected by a natural phenomenon of seawater intrusion and contamination of the soil. Samples were collected from January to July 2015 (Fig. 1-C). PCR and quantitative PCR (qPCR) DNA estraction Target gene amplification by PCR/ qPCR Figure 1. A) Lama D’Antelmi channel (sampling sites Ponte Fontanelle) B) Research area of channel’s and groundwater’s sampling sites (in yellow are showed 3 sampling points of Ostuni groundwater; in red 3 sampling points of Lama D’Antelmi channel) A B Detection Target genes: PCR: ermB, tetO, vanA, mecA, ampC, blaSHV, blaTEM, blaCTX-M and blaCTX-M1 Q-PCR: E. coli uidA, 16S rDNA, sul1 and intI1 Results and discussions Water microbiological quality ARGs PCR screening 6 of the 9 analysed genes were detected: ermB and the b lactamases ampC, blaSHV, and BlaTEM. Accordingly, macrolide and betalactam antibiotics are still commonly used in Italy in veterinary and human medicines. Notably ARGs of clinical relevance were most frequently detected in aquatic environment. ErmB the most common (56%) is frequently found in resistant S. pneumoniae infection in Italy. Beta lactamases, namely BlaSHV (44%) and blaTEM (29%) are known to confer resistance to Enterobacteriaceae causes of nosocomial infections. ESBL genes (BlaCTX-M and bla CTX-M1) conferring resistance to the third generation cephalosporin, were also detected. PCR screening showed that ARGs are present in various water ecosystems of Italy. in: 100% of wastewater samples, in 92% of channel and also in groundwater samples (16%). ARGs frequency was higher in more contaminated water, and surprisingly often, is greater in the channel than in the wastewater. PCR screening of ARGs: images showed ARGs frequency in total samples (big pie chart) and in each aquatic system (little pie chart) Frequencies of detected ARGs Average concentration of 16S rDNA, E.coli uidA, sul1, intI1 gene in the monitored water The monitored surface and groundwater were highly impacted by anthropogenic pollution . As expected the higher contamination level was observed in wastewater and channel water, showing similar levels of 16SrDNA and E.coli uid genes. Faecal contamination was also observed, although at lower level, in 41% of the groundwater samples. sul1 and int1 were shown to be very common in water with different contamination levels, indicating that the resistance to Sulphonamides and genetic elements, likely involved in ARGs gene transfer, are widely distributed in the microbial community in the water environment. Sul 1 and int1 were more concentrated in wastewater and channel water confirming that the these genes are indicators of anthropogenic pollution Differently from E.coli uid gene Sul 1 and int1 were also present and abundant in 100% and 83 % of the analyzed ground water samples. Relationships between ARGs and contamination indicators Frequency of beta-lactamase in E. coli positive and in negative samples With the exception of one sample all samples negative for E.coli by qPCR are also negative for beta-lactamase supporting the use of this parameter for the monitoring of beta lactamases in impacted water environments. 60 % of positive samples to the qPCR quantification of E. coli are positive to blaSHV (A), 40% to blaTEM (B), while 10% is positive to beta-lactamase CTX-M (C). Frequency of detection of b-lactamase in positive and negative samples for E.coli, was estimated. Level of sul1, int1, 16SrDNA and E.coli in ARGs positive and negative samples Conclusions ARGs, comprising clinically relevant genes detected in resistant nosocomial infections and that represent a risk for human health, were found in Italy in the waters environment underling, consequently, the importance of considering these pollutants in water treatment and managing strategies. The higher frequencies of ARGs observed in more impacted waters, confirm that WWTPs discharges and agricultural run-off are common sources these contaminants. sul1 and intI1 were shown to be widely spread in water environments microbial community indicating that genetic elements likely involved ARGs transfer are commonly present in these populations. Results confirm that the definition of suitable ARGs monitoring parameter is still necessary: E. coli, seems a good indicator of Beta-lactamase presence but might fail to show the presence of ARGs diffused in gram positive bacteria, while sul 1 and int1 are good indicators of anthropogenic pollution might provide an overestimate of the real ARGs pollution level. However, these results should be confirmed in a wider set of data.   ARGs are present in more impacted waters and all the analyzed indicators were significantly more abundant ( t test p< 0,05) in ARGs positive in respect to ARGs negative samples sul1 and int1 were always present in ARGs positive samples; however both genes were also frequently detected at high level in ARGs negative samples. E. coli was only rarely detected by PCR in ARGs negative samples (33%) which, in agreement with the high frequencies of beta lactamases in our samples, indicate a link between ARGs presence and water fecal contamination. However, E.coli was not present in one of the ARGs positive samples were ermB was detected suggesting that E.coli might not be a suitable indicator of the presence of gram positive ARGs.