poster - International Conference of Agricultural Engineering

on the post-emission side, the monitoring of effluent discharges from WWTP in the aquatic
environment (Kummerer, 2009a; Jury et al., 2010).
This paper describes a research project to assess the impact of hospital, urban, rural and
agricultural activities in the spread of ARB in the water supply and drainage in urban and
agricultural systems. This project addresses two key areas: i) impacts of livestock on water
quality for domestic supply, ii) impacts of diverse sources as livestock or hospitals on water
and soil quality in agriculture, particularly to irrigation and livestock supply. The research
team already developed successfully ARB research in other matrixes, such as: ready-to-eat
foods (Amador et al., 2011), hospital samples (Fernandes & Prudêncio, 2010). Additionally,
the enterprise (Águas Mondego e Bairrada, S.A.) that supplies water to Coimbra integrates
this team, having the know-how to provide infield the means to access and collect water
samples and characterise the regional water networks. The results achieved encourage
developing new application to water research.
2. Methodology
The field and laboratorial work is idealized to be executed in four main steps (Fig. 1). At first,
water samples collection in Coimbra Region, Portugal, follows a defined sampling
methodology in relation to: (i) the sites selection in the water network of distribution and
drainage, upstream and downstream the location of some human activities, namely hospital,
WWTP and livestock farm, to enable evaluating the effect of each activity in the spread of
ARB; (ii) the definition of a sampling schedule throughout the year, to monitor the putative
seasonal contribution of each activity to ARB dissemination and (iii) the edapho-climatic and
hydraulic characterisation of sampling sites.
Sampling is followed by the microbiological analysis of water samples, which includes: (i) the
determination of their microbial charge, through quantitative methods and (ii) the screening of
Enterobacteriaceae, through differential and selective media. After bacteria isolation and
biochemical identification, the patterning the antibiotic susceptibility of the isolates is carried
out through the Minimal Inhibitory Concentration (MIC) tests by the disk diffusion method on
Mueller Hinton agar with antibiotic disks, according to the Clinical Laboratory Standards
Institute (NCCLS, 2005). The antibiotic selection involves the main antibiotic classes used in
Portugal, namely β-lactams, Quinolones, Chloramphenicol, Sulphonamides, Tetracycline,
Aminoglycosides, Glycopeptides, Macrolides, Lyncosamide.
Afterwards, the isolated antibiotic-resistant bacteria are molecularly characterised aiming: (i)
their identification and (ii) the detection and identification of the antibiotic-resistance genes
that they harbour. Briefly, the total and/or plasmidic bacterial DNA is extracted and the
resistance genes detected with specific PCR primers and identified by sequencing of PCR
amplicons.
Next, the antibiotic-resistant bacteria are used to perform horizontal transference assays with
the liquid mating method (Amador et al., 2011), to assess their ability to transmit the
antibiotic-resistance genes among bacteria.
Finally, the last step of the project is carried out through the integration of all data gathered in
the previously described steps, to assess the impact of human activities (hospital, WWTP
and livestock farm) in the spread of ARB into water.
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