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Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

A large number of antibiotic-resistant P. aeruginosa isolates are continuously discharged into natural water basins mainly through sewage. However, the environmental reservoirs of antibiotic resistance factors are poorly understood. In this study, the antibiotic resistance patterns of 245 isolates from various aquatic sites in Greece were analysed. Twenty-three isolates with resistance patterns cefotaxime-aztreonam-ceftazidime, cefotaxime-aztreonam-meropenem, cefotaxime-ceftazidime-meropenem, cefotaxime-ceftazidime-aztreonam-meropenem and cefotaxime-ceftazidime-cefepime-aztreonam-meropenem were screened phenotypically for the presence of extended spectrum β-lactamases (ESBLs), while 77 isolates with various resistant phenotypes were screened for the presence of class 1 and class 2 integrase genes. The aztreonam-resistant isolates and ESBL producers were the main resistant phenotypes in all habitats tested. In 13/77 isolates class 1 integron was detected, while all tested isolates were negative for the presence of the class 2 integrase gene. CTX-M group 9 β-lactamase was present in a small number of isolates (three isolates) highlighting the emergence of ESBL genes in aquatic environments. As a conclusion, it seems that Greek water bodies could serve as a potential reservoir of resistant P. aeruginosa isolates posing threats to human and animal health.

Water and swab samples were collected from 13 hospitals and analyzed for Legionella counts. Legionella was detected in eight out of 13 hospitals and in 22 of 130 water and swab-collected samples. A total of 72.7% of the strains were L. pneumophila ser. 1, 22.7% were L. pneumophila ser. 2-14, and 4.5% did not belong to any of these groups. AFLP typing of the L. pneumophila ser. 1 strains generated two distinguishable AFLP types. There was no significant correlation to the sample type with Legionella recovery. Legionella isolation was more likely to occur in the cooling towers than the water system. Water temperatures of 30-40 degrees C seem to favor Legionella growth. Of the 265 serum samples taken from the medical and technical staff for the control of IgG titre, 89.4% were negative, 7.2% were positive, and for 3.4% the result was doubtful. No association between IgG titre and maximum observed level of Legionella occurrence was detected.

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.