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Faecal indicator bacteria have traditionally been used in the detection of faecal pollution in water, but due to concerns about the lack of reliability of these indicators, alternative methods have been developed. One of which is the detection of sterols present in human and animal excreta via GC-MS analysis of water in this study. The Szűcs method was used to detect six target sterols (coprostanol, cholesterol, dehydrocholesterol, stigmasterol, β-sitosterol, and stigmastanol) in environmental water samples. An initial study was done by analysing raw sewage and effluent (human faecal sterol biomarkers) and water samples were spiked with excreta from cattle, chickens, horses, pigs, and sheep to determine faecal sterol fingerprints. The method was evaluated for quantitation and differences between the water samples from each species. Following liquid-liquid extraction, silylation and derivatization, samples were analysed by GC-MS. Standard curve assays were linear up to 160ng and the limit for quantification was 20ng. The human faecal sterol biomarker was coprostanol, while herbivore profiles were dominated by terrestrial sterol biomarkers (stigmasterol and stigmastanol). Sterol fingerprints and differences in concentrations of sterols between various animals and between animals and humans occurred, providing the opportunity to determine whether faecal pollution was from humans or from animals. The method proved sensitive enough to evaluate faecal contamination in environmental water. Groundwater was collected from bore-holes and surface water samples were collected from the Baberspan Inland Lake. Physico-chemical parameters analysed indicated that pH for surface water samples was above 6.9. The total dissolved solids (TDS) in groundwater indicated that the water was not suitable for human consumption, but could be used for livestock watering. Surface water electrical conductivity (EC) and inorganic nitrates was too high to be used for irrigational purposes. Nitrates in groundwater were too high to be consumed by humans. In groundwater, the total coliform target water quality range (TWQR) was exceeded at 53% of sites analysed and faecal coliform TWQR were exceeded at 77% sites. Surface water samples complied with TWQR with regards to faecal coliforms for full contact recreational activities and livestock watering. The TWQR for E. coli, with regards to full contact recreational activities, was within a safe range for surface water. Faecal streptococci were found in 85% of groundwater sampling sites. And surface water faecal streptococci counts exceeded the TWQR for full contact recreational activities. There is no TWQR for faecal sterols in water, but concentrations of cholesterol and coprostanol was found at three of the groundwater sites analysed. This indicates faecal contamination from possible animal and human origin. Surface water samples analysed showed that the Harts River water is clean and free of faecal sterols, while the water analysed from the inflow, hotel and outflow, cholesterol eluted, which showed faecal contamination, possibly from animals. Faecal sterol markers could be detected in groundwater and surface water, adding an extra dimension to determining the quality of water systems. An optimization and sensitivity study of the method was done on waste water treatment plant (WWTP) raw sewage and effluent. The WWTP sample analysed form Potchefstroom and Carletonville WWTP yielded all six target sterols in the raw sewage water samples, but no sterols eluted in the effluent samples. The raw sewage water sample taken from the Fochville WWTP yielded all six target sterols as well, however, the effluent yielded an unknown compound as well as cholesterol. An alternative study was done where the effluent sample volume was increased. By increasing the volume of water, one can possibly increase the amount (“load”) of sterols extracted and analysed, resulting in a higher abundance of target sterols. By using the target qualifier ions of the six target sterols, and the GC-TOF/MS software, the target sterols could still be qualitatively determined. Optimal volume for raw sewage is 300 ml water sample as this is enough to yield all 6 target sterols. For optimum water quality monitoring via faecal sterol analysis of effluent and other environmental samples, at least 1L sample volume needs to be collected and analysed. The methods described here can be applied to the analysis of environmental water samples. The technical advantages also make it suitable for routine environmental monitoring of faecal pollution. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Faecal indicator bacteria have traditionally been used in the detection of faecal pollution in water, but due to concerns about the lack of reliability of these indicators, alternative methods have been developed. One of which is the detection of sterols present in human and animal excreta via GC-MS analysis of water in this study. The Szűcs method was used to detect six target sterols (coprostanol, cholesterol, dehydrocholesterol, stigmasterol, β-sitosterol, and stigmastanol) in environmental water samples. An initial study was done by analysing raw sewage and effluent (human faecal sterol biomarkers) and water samples were spiked with excreta from cattle, chickens, horses, pigs, and sheep to determine faecal sterol fingerprints. The method was evaluated for quantitation and differences between the water samples from each species. Following liquid-liquid extraction, silylation and derivatization, samples were analysed by GC-MS. Standard curve assays were linear up to 160ng and the limit for quantification was 20ng. The human faecal sterol biomarker was coprostanol, while herbivore profiles were dominated by terrestrial sterol biomarkers (stigmasterol and stigmastanol). Sterol fingerprints and differences in concentrations of sterols between various animals and between animals and humans occurred, providing the opportunity to determine whether faecal pollution was from humans or from animals. The method proved sensitive enough to evaluate faecal contamination in environmental water. Groundwater was collected from bore-holes and surface water samples were collected from the Baberspan Inland Lake. Physico-chemical parameters analysed indicated that pH for surface water samples was above 6.9. The total dissolved solids (TDS) in groundwater indicated that the water was not suitable for human consumption, but could be used for livestock watering. Surface water electrical conductivity (EC) and inorganic nitrates was too high to be used for irrigational purposes. Nitrates in groundwater were too high to be consumed by humans. In groundwater, the total coliform target water quality range (TWQR) was exceeded at 53% of sites analysed and faecal coliform TWQR were exceeded at 77% sites. Surface water samples complied with TWQR with regards to faecal coliforms for full contact recreational activities and livestock watering. The TWQR for E. coli, with regards to full contact recreational activities, was within a safe range for surface water. Faecal streptococci were found in 85% of groundwater sampling sites. And surface water faecal streptococci counts exceeded the TWQR for full contact recreational activities. There is no TWQR for faecal sterols in water, but concentrations of cholesterol and coprostanol was found at three of the groundwater sites analysed. This indicates faecal contamination from possible animal and human origin. Surface water samples analysed showed that the Harts River water is clean and free of faecal sterols, while the water analysed from the inflow, hotel and outflow, cholesterol eluted, which showed faecal contamination, possibly from animals. Faecal sterol markers could be detected in groundwater and surface water, adding an extra dimension to determining the quality of water systems. An optimization and sensitivity study of the method was done on waste water treatment plant (WWTP) raw sewage and effluent. The WWTP sample analysed form Potchefstroom and Carletonville WWTP yielded all six target sterols in the raw sewage water samples, but no sterols eluted in the effluent samples. The raw sewage water sample taken from the Fochville WWTP yielded all six target sterols as well, however, the effluent yielded an unknown compound as well as cholesterol. An alternative study was done where the effluent sample volume was increased. By increasing the volume of water, one can possibly increase the amount (“load”) of sterols extracted and analysed, resulting in a higher abundance of target sterols. By using the target qualifier ions of the six target sterols, and the GC-TOF/MS software, the target sterols could still be qualitatively determined. Optimal volume for raw sewage is 300 ml water sample as this is enough to yield all 6 target sterols. For optimum water quality monitoring via faecal sterol analysis of effluent and other environmental samples, at least 1L sample volume needs to be collected and analysed. The methods described here can be applied to the analysis of environmental water samples. The technical advantages also make it suitable for routine environmental monitoring of faecal pollution. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Η διατήρηση της μικροβιολογικής ποιότητας του υδάτινου περιβάλλοντος είναι υψίστης σημασίας δεδομένων των κινδύνων που ενέχονται για τη δημόσια υγεία. Η αξιολόγηση της μικροβιολογικής ποιότητας των υδάτων πραγματοποιείται με την ανίχνευση της κοπρανώδους μόλυνσης και με τον έλεγχο της παρουσίας και συγκέντρωσης συγκεκριμένων μικροοργανισμών – δεικτών, όπως είναι η Escherichia coli. Ωστόσο, η απλή ανίχνευση κοπρανώδους μόλυνσης δεν επαρκεί για την υπόδειξη τρόπων εξυγίανσης και αντιμετώπισης του εκάστοτε προβλήματος. Οι δύο κύριες ομάδες στις οποίες διακρίνεται η κοπρανώδης μόλυνση είναι η ανθρώπινη και η ζωική, οι οποίες υποδηλώνουν πιθανή παρουσία διαφορετικών κάθε φορά παθογόνων μικροοργανισμών για τον άνθρωπο. Έτσι, προκειμένου να οριοθετηθεί ο κίνδυνος για τη δημόσια υγεία και να καθοριστούν μέτρα αντιμετώπισης της μόλυνσης ενδείκνυται ο προσδιορισμός της ανθρώπινης ή ζωικής προέλευσης της κοπρανώδους μόλυνσης. Στην παρούσα μελέτη αναπτύχθηκαν, εφαρμόστηκαν και αξιολογήθηκαν οι μέθοδοι: α)Έλεγχος πολλαπλής ανθεκτικότητας σε αντιβιοτικά (Multiple Antibiotic Resistance – MAR – φαινοτυπική μέθοδος) και β) PCR με τυχαία ενισχυμένα τμήματα πολυμορφικού DNA - Random Amplified Polymorphic DNA-PCR (RAPD-PCR – γονοτυπική μέθοδος), ως τεχνικές προσδιορισμού και διάκρισης προέλευσης μικροοργανισμών. Κατά το πρώτο στάδιο καθορίστηκαν οι παράμετροι των μεθόδων για το διαχωρισμό στελεχών E. coli γνωστής προέλευσης (60 στελέχη απομονωμένα από ζωικά κόπρανα και 68 στελέχη από ανθρώπινα). Για το διαχωρισμό και κατηγοριοποίηση των στελεχών εφαρμόστηκαν η Ιεραρχική Ανάλυση Κατά Συστάδες και η Διαχωριστική Ανάλυση. Με τη MAR ανάλυση τα στελέχη E. coli εμφάνισαν διαφορετικούς συνδυασμούς ανθεκτικότητας και διαχωρίστηκαν βάσει της προέλευσής τους με μέσο ποσοστό σωστής ταξινόμησης (ARCC) 99,2%. Με την RAPD-PCR χρησιμοποιήθηκαν δύο εκκινητές ξεχωριστά (1254 & 1290) και τα 128 στελέχη E. coli γνωστής προέλευσης διαχωρίστηκαν σε ανθρώπινης και ζωικής πηγής με ARCC 98,4% και με τους δύο εκκινητές. Η διακριτική ικανότητα της RAPD-PCR με τους δύο εκκινητές ήταν D1254=0,97 & D1290=0,90. Επιπλέον, η αξιολόγηση της επαναληψιμότητας της RAPD-PCR και με τους δύο εκκινητές έδωσε ικανοποιητικά αποτελέσματα με την εμφάνιση ίδιων ηλεκτροφορητικών εικόνων για τα ίδια βακτηριακά στελέχη. Στη συνέχεια οι επιλεγμένες τεχνικές εφαρμόστηκαν για την ταξινόμηση και κατηγοριοποίηση στελεχών E. coli άγνωστης προέλευσης εκτιμώντας την ανθρώπινη ή ζωική πηγή τους βάσει του μοντέλου διαχωρισμού των E. coli γνωστής προέλευσης. Οι E. coli άγνωστης προέλευσης (234 στελέχη) απομονώθηκαν από δείγματα πόσιμου νερού δικτύου από 11 περιοχές και δείγματα μη επεξεργασμένων λυμάτων από τις εισόδους τεσσάρων σταθμών βιολογικού καθαρισμού (ΚΕΡΕΦΥΤ – Νομός Αττικής, ΨΥΤΤΑΛΕΙΑ – Νομός Αττικής, ΡΙΟ – Νομός Αχαΐας και ΠΑΤΡΑ - Νομός Αχαΐας). Τα 234 στελέχη με τη MAR ανάλυση ταξινομήθηκαν ως ανθρώπινα και ζωικά σε ποσοστά 46,6% και 53,4% αντίστοιχα. Τα αποτελέσματα ταξινόμησης ήταν διαφορετικά με τη μέθοδο RAPD-PCR. Με τον εκκινητή 1254 τα άγνωστα στελέχη προσδιορίστηκαν ως ανθρώπινα κατά το 64,9% και ως ζωικά κατά το 35,1%. Αντίστοιχα, με τον εκκινητή 1290 τα ποσοστά ήταν 60,3% ανθρώπινα και 39,7% ζωικά. Τα στελέχη του πόσιμου νερού που προέρχονταν από τους σταθμούς δειγματοληψίας που ήταν αστικά κέντρα χαρακτηρίστηκαν εξ ολοκλήρου ως ανθρώπινης προέλευσης. Αντίθετα, στις περιοχές δειγματοληψίας με ανεπτυγμένη κτηνοτροφία βρέθηκαν και στελέχη ζωικής προέλευσης, γεγονός που υποδηλώνει την είσοδο στο δίκτυο κοπρανώδους υλικού προερχόμενου από ζώα των συγκεκριμένων περιοχών, τα οποία ενδεχομένως να έχουν άμεση πρόσβαση στις πηγές και γεωτρήσεις. Όσον αφορά στο χαρακτηρισμό των E. coli που καταλήγουν στους αναφερόμενους βιολογικούς καθαρισμούς, η πλειοψηφία ανίχνευσης ανθρωπίνων στελεχών δηλώνει την πιθανή παρουσία στα ακατέργαστα λύματα πολλών ανθρωπίνων εντερικών παθογόνων σημαντικών για τη δημόσια υγεία. Δεδομένου ότι τα τελευταία χρόνια οι ερευνητές έχουν αποδυθεί σε μια προσπάθεια επαναχρησιμοποίησης επεξεργασμένων λυμάτων επισημαίνεται η ανάγκη επεξεργασίας τους σε διάφορα στάδια για τη διασφάλιση της δημόσιας υγείας. Παρατηρήθηκε συμφωνία αποτελεσμάτων με τη χρήση των δύο εκκινητών καθώς η διαφορά στα ποσοστά δεν ήταν στατιστικά σημαντική (P>0,05). Συγκρίνοντας τα αποτελέσματα που ελήφθησαν με τις δύο μεθόδους, τη φαινοτυπική (MAR ανάλυση) και τη γονοτυπική (RAPD-PCR), υπήρξε στατιστικά σημαντική διαφορά (P<0,05), με συνέπεια να τίθεται θέμα επιλογής της πιο ενδεδειγμένης μεθόδου τυποποίησης και διάκρισης περιβαλλοντικών μικροοργανισμών. H παρούσα μελέτη αναδεικνύει την RAPD-PCR ως μια γονοτυπική μέθοδο με ικανοποιητική διακριτική ικανότητα, ευαισθησία, επαναληψιμότητα υπό αυστηρά καθορισμένες συνθήκες και χαμηλού κόστους. Η ευκολία εφαρμογής για την τυποποίηση μεγάλου αριθμού βακτηριακών στελεχών, χωρίς την απαίτηση γνώσης της νουκλεοτιδικής αλληλουχίας του γενετικού υλικού την καθιστούν ιδιαίτερα προσιτή σε εργαστήρια μοριακής μικροβιολογίας, ως τεχνική διάκρισης προέλευσης της κοπρανώδους μόλυνσης στο υδάτινο περιβάλλον. / Maintenance of the microbiological quality and safety of water systems is imperative, as their faecal contamination may exact high risks to human health as well as result in significant economic losses. The microbiological quality of water systems is evaluated by detecting their faecal pollution and especially specific faecal indicators such as Escherichia coli. Simple detection of faecal pollution is not sufficient in order to apply appropriate management plans to remedy the problem and to prevent any further contamination. Human faecal material is generally perceived as constituting a grater human health risk than animal faecal material, considering that it is more likely to contain human-specific enteric pathogens. Thus, it would be desirable to determine the source of the faecal material, especially for the assessment of risk for public health and for the development of monitoring plans. In the present study the development and assessment of Multiple Antibiotic Resistance Analysis (MAR – phenotypic method) and Randomly Amplified Polymorphic DNA-PCR Analysis (RAPD-PCR – genotypic method) were established as microbial source tracking methods. Firstly, parameters of the two selected methods were determined for the discrimination of E. coli isolates of known source (60 isolates from animal faecal material & 68 isolates from human faecal material). Hierarchical Cluster Analysis and Discriminant Analysis were applied for the classification of the isolates. With MAR analysis E. coli isolates developed different resistance profiles and were discriminated according to their source with an average rate of correct classification (ARCC) of 85.2%. With RAPD-PCR analysis two different 10-nt primers of arbitrary sequence were used (1254 & 1290) and the 128 E. coli isolates of known origin were classified as human and animal with the following ARCC: ARCC1254= 87.5% & ARCC1290= 81.3%. The discriminatory power of RAPD-PCR with the two selected primers was D1254=0.97 & D1290=0.90. Furthermore, the assessment of reproducibility of RAPD-PCR analysis provided satisfactory results with both primers, as RAPD profiles were identical for the same bacterial isolates. The assessment of specificity of the method resulted in the discrimination among RAPD profiles of E. coli isolates and other reference bacteria. The selected methods were applied for the classification and the source tracking of E. coli isolates, derived from tap water and raw sewage samples. In total 234 E. coli strains were isolated from tap water from 11 areas and raw sewage samples from four treatment plants (KEREFYT – prefecture of Attiki, PSITALIA - prefecture of Attiki, RIO - prefecture of Achaia and PATRA - prefecture of Achaia). With MAR analysis the 234 isolates were classified as human and animal in percentages of 46.6% & 53.4%, respectively. Classification results were different with RAPD-PCR analysis. With primer 1254 the classification was: 64.9% of human origin and 35.1% of animal origin and with primer 1290 the classification was: 60.3% of human origin and 39.7% of animal origin. Isolates derived from tap water of urban areas were classified in total as of human origin. On the contrary, in areas with many farm breeders many isolates were classified as of animal origin, indicating presence of faecal material in the water systems derived animal activities. As far as E. coli isolates from raw sewage samples are concerned, the majority of them were classified as of human source, indicating the possible presence of other human enteric pathogens as well. Taking into account the fact that there has been an effort in order to reuse treated sewage, it seems necessary a multi-stage process to renovate wastewater before it re-enters a body of water. There was an agreement of results of classification obtained form the use of the two different primers as the percentages did vary statistically (P>0.05). Comparing results obtained from the two selected methods, the difference was statistically significant (P<0.05), raising a question of the appropriate method for the typing and discrimination of environmental microorganisms. The present study demonstrates RAPD analysis as a simple, cost effective genotypic method with satisfactory discriminatory power, sensitivity and reproducibility. It can be applied for the analysis of a large number of bacterial isolates without the prior knowledge of nucleotide sequence of DNA to be necessary. Finally, it may fulfil environmental for the determination of origin of faecal pollution protecting water resources and public health.