Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp.

Abstract

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected—24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012–2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

New Molecular Tools: Application of the Μaqua Phylochip and Concomitant Fish Probes to Study Freshwater Pathogens From Samples Taken Along the Tiber River, Italy

“…Fifty litres of river water were filtered through a polysulfone hollow-fibre ultrafilter module (HF80S, Fresenius Medical Care, Bad Homberg, Germany) by the MICROCOKIT team (JRC and CNR-IRSA). Filtration and cell concentration process is described in detail in [7]- [9]. Thirty mls of the concentrate were preserved in Tri-reagent (Sigma, France) at a ratio of 1:4 concentrate:Tri-reagent to ensure sufficient concentrate dilution for optimal RNA extraction [3], [10], [11].…”

“…Validation was performed by four µAQUA partners using either traditional cell counting regimes (cyanobacterial) to plate counts (bacteria) to magnetic beds/antibodies (protozoa) with good correlations between both methods. Results for three of the monitoring sites are published (the Tiber River above and below Rome, Italy [7], rivers near Paris, France [8], one lake in Bulgaria (9]). The µAQUA microarray was transferred into the EU project, MICROCOKIT (www.microcokit.eu), who sampled the entire length of the Tiber River in spring and autumn for two years, concentrating on four sites with pollution distinct types: pristine, agricultural, industrial and anthropogenic.…”

Microarray (phylochip) analysis of freshwater pathogens at several sites along the Northern German coast transecting both estuarine and freshwaters

“…The μAQUA microarray was field tested in six countries. Microarray results for one of the monitoring sites, the Tiber River in Italy, have been published (Marcheggiani et al 2015) as has the PCR and rtPCR results from a Bulgarian site (Panaiotov et al 2015), and in this paper, we present microarray results for bacteria, cyanobacterial and protozoa for selected sites in Germany monitored regularly by Marilim for 3 years from 2012 until 2014.…”

Scientific Symposium “Small Solution for Big Water-Related Problems: Innovative Microarrays and Small Sensors to Cope with Water Quality and Food Security”

“…Finally, Panaiotov et al . [ 33 ] evaluated the water quality of the Black Sea and the Dam of Iskar by applying molecular tests for the detection of viruses, bacteria and protozoa during the years 2012–2014. The approach used microscopic and PCR analyses for key-pathogens.…”