Current knowledge about the spread of pathogens in aquatic environments is scarce probably because bacteria, viruses, algae and their toxins tend to occur at low concentrations in water, making them very difficult to measure directly. The purpose of this study was the development and validation of tools to detect pathogens in freshwater systems close to an urban area. In order to evaluate anthropogenic impacts on water microbiological quality, a phylogenetic microarray was developed in the context of the EU project µAQUA to detect simultaneously numerous pathogens and applied to samples from two different locations close to an urban area located upstream and downstream of Rome in the Tiber River. Furthermore, human enteric viruses were also detected. Fifty liters of water were collected and concentrated using a hollow-fiber ultrafiltration approach. The resultant concentrate was further size-fractionated through a series of decreasing pore size filters. RNA was extracted from pooled filters and hybridized to the newly designed microarray to detect pathogenic bacteria, protozoa and toxic cyanobacteria. Diatoms as indicators of the water quality status, were also included in the microarray to evaluate water quality. The microarray results gave positive signals for bacteria, diatoms, cyanobacteria and protozoa. Cross validation of the microarray was performed using standard microbiological methods for the bacteria. The presence of oral-fecal transmitted human enteric-viruses were detected using q-PCR. Significant concentrations of Salmonella, Clostridium, Campylobacter and Staphylococcus as well as Hepatitis E Virus (HEV), noroviruses GI (NoGGI) and GII (NoGII) and human adenovirus 41 (ADV 41) were found in the Mezzocammino site, whereas lower concentrations of other bacteria and only the ADV41 virus was recovered at the Castel Giubileo site. This study revealed that the pollution level in the Tiber River was considerably higher downstream rather than upstream of Rome and the downstream location was contaminated by emerging and re-emerging pathogens.

A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.

Microbiological indicators are commonly used in the assessment of public health risks associated with fecal contamination of freshwater ecosystems. Sediments are a reservoir of microorganisms, and can thus provide information on past pollution events, not obtainable through the testing of surface water. Moreover, pathogens present in sediment may represent future threats to human health. Clostridium perfringens, a typical colonizer of sediments, has been suggested as an alternative indicator of fecal pollution. In order to be suitable for such purpose, the microorganism should be widely distributed in contaminated environments. The objective of this study was thus to determine the composition of the anaerobic community in sediment samples of the lower Tiber basin, in central Italy, through a combined approach involving granulometric analysis of sediment samples, as well as a microbiological and molecular (16S rRNA) analysis of strains.

Around the globe, escalation in rare opportunistic microbial infections is alarming as they are heading steadily towards 'superbug' status. In aquatic ecosystems, plastic fosters multidrug-resistant pathogenic bacteria and plays a significant role in trafficking antibiotic-resistant genes. In this study, we focused on a multidrug-resistant bacterial strain isolated from microbial communities found on plastic substrates of a volcanic lake in central Italy. Extended-spectrum beta-lactamase-producing strains were isolated from both raw water and plastic substrates for a comparative investigation using microbiological and molecular methods, and antibiotic susceptibility profiling was performed against a panel of ten antibiotics. Molecular identification and Basic Local Alignment Search Tool analysis confirmed an almost identical sequencing pattern of two isolated strains and their homology with Morganella morganii. Antibiotic susceptibility tests revealed their resistance to almost all tested antibiotics. Class 1 integron-associated gene (intI1) and seven antibiotic resistance genes were detected in both strains, confirming their superbug status. To our knowledge, this is the first study on the characterization of extended-spectrum beta-lactamase-producing M. morganii isolated from the biofilm of plastic substrates, depicting the potential toxicity of plastic in harbouring and dispersing virulent, multidrug-resistant, opportunistic human pathogens.

Antibiotic-resistant bacteria (ARB) adhesion onto plastic substrates is a potential threat to environmental and human health. This current research investigates the prevalence of two relevant human pathogens, Staphylococcus spp. and Klebsiella spp., and their sophisticated equipment of antibiotic-resistant genes (ARGs), retrieved from plastic substrates submerged into an inland water body. The results of microbiological analysis on selective and chromogenic media revealed the presence of colonies with distinctive phenotypes, which were identified using biochemical and molecular methods. 16S rDNA sequencing and BLAST analysis confirmed the presence of Klebsiella spp., while in the case of Staphylococcus spp., 63.6% of strains were found to be members of Lysinibacillus spp., and the remaining 36.3% were identified as Exiguobacterium acetylicum. The Kirby–Bauer disc diffusion assay was performed to test the susceptibility of the isolates to nine commercially available antibiotics, while the genotypic resistant profile was determined for two genes of class 1 integrons and eighteen ARGs belonging to different classes of antibiotics. All isolated bacteria displayed a high prevalence of resistance against all tested antibiotics. These findings provide insights into the emerging risks linked to colonization by potential human opportunistic pathogens on plastic waste commonly found in aquatic ecosystems.

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.