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The literature data on the microbiological water quality of the surface and groundwaters in Montenegro is very scarce. Therefore, this chapter aims to provide an insight in the microbiological water quality of rivers in Montenegro by compiling the data obtained in period 2009–2018 in national monitoring program and the data collected within the Montenegro survey in 2019 with an emphasis on the hotspots of faecal pollution and possible sources of pollution. Despite the high risk that poor implementation of wastewater treatment might represent for Montenegro, the obtained dataset indicates that overall microbiological water quality of rivers in Montenegro is quite good. About 80% of the investigated sites have bathing water quality according to Montenegrin legislation. Only four sites were identified as hotspots of faecal pollution where the human source of pollution prevails.

High-Throughput Sequencing technologies are providing unprecedented inventories of microbial communities in aquatic samples, offering an invaluable tool to estimate the impact of anthropogenic pressure on marine communities. In this case study, the Mediterranean touristic site of Aci Castello (Italy) was investigated by High-Throughput Sequencing of 16S and 18S rRNA genes. The sampling area falls within a Marine Protected Area and, notwithstanding, features an untreated urban wastewater discharge. Seawater samples were collected close to the wastewater output (COL) and at a second station about 400 m further off (PAN), before and after a summer increase in population. Prokaryotic communities clustered according to stations, rather than to seasons. While PAN showed a typical, not impacted, marine microbial composition, COL was consistently enriched in Epsilonproteobacteria and Firmicutes. Protist communities showed a peculiar clustering, as COL at springtime stood alone and was dominated by Ciliophora, while the other samples were enriched in Dinophyta. Analysis of alternative, detectable by High-Throughput Sequencing, microbial indicators, including both faecal- and sewage-associated, allowed uncovering the different sources of pollution in coastal and anthropogenically impacted marine ecosystems, underpinning the relevance of High-Throughput Sequencing-based screening as rapid and precise method for water quality management.

Microbial pollution of recreational waters poses a significant public health risk which, unless mitigated, will continue to increase with population growth. Water managers must implement strategies to accurately discriminate and source human from animal faecal contamination in complex urbanised environments. Our case-study used a new combination of chemical (i.e. ammonia) and microbial (i.e. Escherichia coli, Bacteroides spp.) faecal monitoring tools in a targeted multi-tiered approach to quickly identify pollution hot-spots and track high-risk subterranean stormwater drains in real-time. We successfully located three point sources of human faecal pollution (both episodic and constant pollution streams) within 11 catchments in a total monitoring time of four months. Alternative approaches for obtaining such fine-scale accuracy are typically labour intensive and require expensive equipment.

Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of faecal pollution sources in environmental waters. They rely on the association of certain fecal microorganisms with a particular host, that ranges from human [1] to agricultural animals [2,3] to pets [4] and wild animals such as gulls [5]. MST is used to appraise recreational water quality and to correlate with human health risk.

Microorganisms are the ideal indicators of the organic pollution of the surface waters because of their ability to promptly respond to environmental changes. Due to poor economic situation and lack of law implementation, most wastewaters in Serbia are released directly into a surface water recipient without any, or a limited, treatment. This practice has created numerous highly polluted surface waters in the region. Therefore, we conducted seasonal monitoring of the bacteriological quality of the Nišava River located in southeastern Serbia in order to assess the extent of its organic and faecal pollution. A total number of heterotrophs, Escherichia coli and intestinal enterococci counts were determined by standard cultivation methods in samples from five locations along the river. In most instances, bacteriological quality of Nišava water belonged to the classes of low or moderate organic and faecal pollution. The samples taken downstream of the city of Niš were the most polluted. Intestinal enterococci and E. coli counts indicated a moderate faecal contamination. Since bacterial counts can reveal a presence of faecal pollution and therefore presence of potentially pathogenic bacteria, a proper microbiological monitoring of surface waters used as a source of drinking water, like the Nišava River itself, is critically important.

Bottled water can be contaminated by coliforms and/or Escherichia coli (E. coli). These bacteria are considered as indicators of faecal pollution, and their detection in bottled water indicates the potential contamination by pathogenic enteric microorganisms. In recent decades, different methods were developed for the detection of coliforms and E. coli in drinking water and in bottled water including mineral water. Since 1976, the Italian regulation has defined microbiological methods to evaluate microbiological characteristics of mineral waters. Three different methods for the detection of coliforms and E. coli in bottled water were compared in this study: the Italian reference method, according to the “Italian Ministerial Rule,” the ISO 9308–1:2002 method, and a new rapid method. The results have demonstrated that the ISO method 9308–1:2002 and the new rapid method are as sensitive and specific as Italian reference method, and that both could be used to evaluate the contamination level of coliform and E. coli in drinking water and in bottled water including mineral water.

Aims We aimed at quantifying bacteriophages in raw and treated wastewaters of human and animal origin in Tunisia to assess their usefulness for tracking the origin of faecal pollution and in the follow-up of effectiveness of water treatments process. Methods and Results The concentrations of bacteriophages in wastewater samples were determined by double layer agar technique. Somatic coliphages and F-specific RNA bacteriophages were present in all types of samples in high concentrations. The values of Escherichia coli were variable depending on geographical location. On the other hand, bacteriophages infecting strain GA17 were detected preferably when human faecal contamination was occurred. Conclusions Bacteriophages appear as a feasible and widely applicable manner to detect faecal contamination in Tunisia. On the other hand, phages infecting GA17 could be good markers for tracking the origin of faecal pollution in the area studied. Significance and Impact of the Study The reuse of treated wastewaters can be a solution to meet the needs of water in the geographical area of study. Bacteriophages seem to predict differently the presence of faecal contamination in water than bacterial indicators. Consequently, they can be a valuable additional tool to improve water resources management for minimizing health risks.

Faecal contamination of water has both anthropogenic and zoogenic origins that can shade various point and nonpoint/diffuse sources of pollution. Due to the dual origin and number of sources of faecal contamination, there are immense challenges in the implementation of effective measures to protect water bodies from pollution that poses threats to human and environmental health. The main health threats refer to infections, illnesses and deaths caused by enteric pathogenic microbes, in particular those responsible for waterborne zoonoses. To detect and identify the origins and sources of faecal pollution simultaneously, various methods and indicators have been compiled into a comprehensive measuring toolbox. Molecular diagnostics using genetic markers derived from Bacteroidales 16S rRNA gene sequences are quite prevalent in the current methodological implementation for the identification of faecal contamination sources in water. For instance, a culture- and library-independent microbial source tracking toolbox combining micro- and molecular biology tests run as a three-step procedure has been implemented in Norway. Outcomes from the Norwegian studies have identified two general trends in dominance of contributors to faecal water contamination in urban environments. Firstly, there is a tendency of higher contributions from anthropogenic sources during the cold season. Secondly, the identification of the dominance of zoogenic sources in faecal water contamination during warm periods of the year.

Laboratory-scale experiments on the survival of Escherichia coli in raw, undiluted, freshly collected, source-separated yellowwater were performed. Concentrations of E. coli and its survival at different temperature regimes and storage times were measured in yellowwater originally cross-contaminated with faeces and yellowwater purposely contaminated (deliberately spiked) with faecal material. The temperature regimes of cold (4°C), mild (10°C) and warm (22°C) were the limited factors, whereas the storage time of the contaminated yellowwater was unlimited and lasted until the E. coli concentrations reached the limit of detection of

Background information: The post-2015 Sustainable Development Goals for sanitation call for universal access to adequate and equitable sanitation and an end to open defaecation by 2030. In Isiolo County, a semi-arid region lying in the northern part of Kenya, poor sanitation and water shortage remain a major problem facing the rural communities. Objective: The overall aim of the study was to assess the relationship between sanitation practices and the bacteriological quality of drinking water sources. The study also assessed the risk factors contributing to open defaecation in the rural environments of the study area. Methods: A cross-sectional study of 150 households was conducted to assess the faecal disposal practices in open defaecation free (ODF) and open defaecation not free (ODNF) areas. Sanitary surveys and bacteriological analyses were conducted for selected community water sources to identify faecal pollution sources, contamination pathways, and contributory factors. Analysis of data was performed using SPSS (descriptive and inferential statistics at α = .05 level of significance). Results: Open defaecation habit was reported in 51% of the study households in ODNF villages and in 17% households in ODF villages. Higher mean colony counts were recorded for water samples from ODNF areas 2.0, 7.8, 5.3, and 7.0 (×103) colony-forming units (CFUs)/100 mL compared with those of ODF 1.8, 6.4, 3.5, and 6.1 (×103) areas for Escherichia coli, faecal streptococci, Salmonella typhi, and total coliform, respectively. Correlation tests revealed a significant relationship between sanitary surveys and contamination of water sources ( P = .002). Conclusions: The water sources exhibited high levels of contamination with microbial pathogens attributed to poor sanitation. Practising safe faecal disposal in particular is recommended as this will considerably reverse the situation and thus lead to improved human health.

Cilj: Osiguranje zdravstveno ispravne vode osnovna je potreba svakog čovjeka. S obzirom na dugogodišnje mikrobiološko onečišćenje u vodoopskrbnoj mreži Gorskog kotara, analizirano je petogodišnje razdoblje. Cilj ovog rada bio je pratiti utjecaj promjena zakonske regulative i sanitarno-tehničkih uvjeta – ugradnje većeg broja automatskih klorinatora na broj ispitanih uzoraka iz vodoopskrbnog sustava, kao i na rezultate ispitivanja parametara zdravstvene ispravnosti vode za ljudsku potrošnju. U radu je uspoređen profil onečišćenja vode za ljudsku potrošnju u Gorskom kotaru i ostalim dijelovima županije. Metode: Statistički su obrađeni i interpretirani rezultati županijskog monitoringa zdravstvene ispravnosti vode za ljudsku potrošnju na području Gorskog kotara u razdoblju od 2011. do 2015. (broj ispitanih uzoraka N = 7.047). Rezultati: Stupanjem na snagu Pravilnika o parametrima sukladnosti i metodama analize vode za ljudsku potrošnju (NN 125/13, 141/13 i 128/15) broj uzoraka na temelju kojih se prati stanje vode u razvodnoj mreži Gorskog kotara je šesterostruko smanjen, ali je broj ispitanih pokazatelja povećan. Ugradnja automatskih klorinatora (2012. g. i 2013. g.) rezultirala je dvostrukim smanjenjem broja nesukladnih uzoraka. Zaključci: Primjena Pravilnika (NN 125/13, 141/13 i 128/15) rezultirala je značajnim smanjenjem broja analiziranih uzoraka u pojedinim vodoopskrbnim sustavima, što je procjenu njihovog stanja uvelike otežalo. Ugradnjom automatskih klorinatora broj nesukladnih uzoraka u Gorskom kotaru upola je smanjen. Glavni razlog zdravstvene neispravnosti vode za ljudsku potrošnju u vodovodima Gorskog kotara je fekalno onečišćenje vode, dok u ostalim dijelovima županije najčešće od zadanih kriterija odstupaju mutnoća vode, povećani broj kolonija (UBB) i nusprodukti klorinacije., Aim: Ensuring the safety of potable water is a necessity of every human being. Taking into account long-term microbiological contamination of water supply network of Gorski Kotar, the results of 5-year County monitoring program of potable water safety in that region have been analysed. The aim of this study was to monitor the impact of amendment of legal standards as well as sanitary and technical requirements – the insertion of higher number of chlorinators on the number of examined samples deriving from the water supply system as well as the influence on the results of testing of the health parameters of water intended for human consumption. This study compared the contamination profile of water for human consumption in Gorski Kotar and other regions of the Primorje-Gorski Kotar County. Methods: This study presents the elaborated and interpreted results of the County monitoring program of potable water safety in the region of Gorski Kotar during the 5-year period (2011–2015; the number examined samples N = 7,047). Results: Application of the Regulations on the parameters of assessment and methods for the analysis of water for human consumption (Official Gazette 125/2013, 141/2013/, 128/2015) provides six times lesser number of samples for the monitoring of water supply system network in Gorski Kotar, but the number of analysed parameters was increased. Installation of automatic chlorinators (2012 and 2013) resulted with twice smaller number of non-compliant samples of drinking water. Conclusions: Application of the the Regulations (Official Gazette 125/2013, 141/2013, 128/2015) resulted with the significant reduction of samples for certain water supply systems making the objective assessment of their condition much harder. The installation of automatic chlorinators reduced the number of non-compliant water samples in Gorski Kotar by half. The main reason for non-complying samples of water for human consumption in the water supply systems of Gorski Kotar is faecal contamination, while for the other parts of the County major deviation from stipulated criteria are turbidity, increased number of colonies (Heterotrophic Plate Count – HPC) and chlorination by-products.

BACKGROUND: Coliforms and other bacterial indicators are known to be present in Community water samples causing faecal pollution of water. There is also a growing body of evidence demonstrating that the aquatic environmental microbes are drug resistant. OBJECTIVE: The objective of this study was to evaluate the microbial quality of the water from tap, well and pond in urban households of Kochi, Kerala state. MATERIALS AND METHODS: A total of 100 water samples (46 from well water, 45 from tap water and 9 from lakes and ponds) were analysed by the multiple fermentation tube method to determine the presumptive coliform count or the most probable number (MPN) of coliforms, and the isolates were identified using standard procedures, followed by susceptibility testing. RESULTS: Eighty samples were positive for growth (76 grew coliforms, four samples grew non-pathogens). A total of 105 coliform isolates were grown in culture. The MPN numbers were noted to be >100 for over 50% of all the samples (n = 56), thus making the water unsatisfactory for drinking purposes. The highest number of organism isolated was Klebsiella pneumonia (n = 55), followed by Enterobacter spp (n = 34) and Escherichia coli (n = 16). Well water had the highest proportion of all three of these organisms. Up to 26%–40% resistance was seen to amoxicillin–clavulanic acid and 5.4%–5.8% of resistance was noted to third-generation cephalosporins, while two isolates were extended-spectrum beta-lactamase-producing Enterobacteriaceae (CTX-M and TEM types). CONCLUSION: The city municipal water authorities need to adopt more aggressive treatment/disinfection practices to combat high coliform contamination. The bacteriological quality of well water at the source needs to be monitored. Meanwhile, continued adequate home purification is suggested for drinking water. The city water does not pose a threat of antibiotic resistance for now. Environmental sampling should be given equal priority as clinical sampling in the coming days.

El presente estudio fue realizado con el objetivo de evaluar el uso de los colifagos somáticos como indicadores de contaminación fecal en aguas recreacionales marinas. Los colifagos somáticos fueron cuantificados en 140 muestras provenientes de 5 playas del circuito de la “Costa Verde”, Lima, y correlacionados con los indicadores de contaminación fecal convencionales (Coliformes fecales, E. coli y Enterococos). Los colifagos fueron determinados por el método de “capa simple” previa aplicación de la técnica modificada de “filtración de la muestra” y utilizando a E. coli ATCC 13706 como cepa hospedero. Los Coliformes fecales (CF), E. coli (EC) y Enterococos (EN) fueron cuantificados por el método del Número Más Probable (NMP). Para efectos de análisis estadístico se consideró niveles de contaminación fecal en función de valores arbitrarios de 1000 CF/100 mL (“alta contaminación”). Las playas evaluadas presentaron niveles de contaminación fecal de

Aims: In this study, faecal indicator bacteria (FIB) namely Escherichia coli and Enterococcus spp. were seeded into slurries of possum faeces and placed on the roof and in the gutter of a roof-captured rainwater (RCR) system. The persistence of FIB in these circumstances was determined under ambient climatic conditions. FIB persistence was also determined under in situ conditions in tank water using diffusion chambers. Methods and Results: The numbers of surviving FIB at different time intervals were enumerated using culture-based methods. Both FIB were rapidly inactivated on the roof under sunlight conditions (T= 2 h) compared with shade conditions (T= 9-53 h). Significant differences were observed between sunlight and shade conditions on the roof for both T values of E. coli (P < 0·001) and Enterococcus spp. (P < 0·001). E. coli showed biphasic inactivation patterns under both clean and unclean gutter conditions. Enterococcus spp., however, showed rapid inactivation (T = 2 h for the clean gutter and T = 6 h for the unclean gutter) compared with E. coli (T = 22 h for the clean gutter and T = 20 h for the unclean gutter). Significant differences were also observed between the T values of E. coli and Enterococcus spp. for both clean (P < 0·001) and unclean (P < 0·001) gutters. Both E. coli and Enterococcus spp. showed nonlinear biphasic inactivation in tank water. Significant difference was observed between the T value of E. coli inactivation compared with Enterococcus spp. (P < 0·001) in the tank water. Conclusions: In this study, FIB were observed to survive longer (T = 9-53 h) on the roof under shade conditions compared with sunlight conditions (T = 2 h). If there is a rainfall event within two to three days after the deposition of faecal maters on the roof, it is highly likely that FIB would be transported to the tank water. When introduced into the tank, a relatively slow inactivation process may take place (T = 38-72 h). Significance and Impact of the Study: The presence of FIB in water indicates faecal pollution and potential presence of enteric pathogens. Therefore, the information on the resilience of FIB, as obtained in this study, can be used for indirect assessment of health risks associated with using roof-captured rainwater for potable and nonpotable purposes.

Fish pond manuring is often used in fish farming for intensification of fish production by balancing the ratio between carbon and other nutrients. However, the using of manure, classified as hazardous organic matter originating from animal faeces, poses a risk to the water environment.The aim of this study was to evaluate the effect of semi-liquid pig manure application on faecal pollution and related potential health risk of fish pond utilization.The evaluation of faecal pollution was made during 6 years; the microbiological monitoring was completed with the water chemistry data. Our results showed that a better situation was in ponds, processes of which can be regulated by changes in nutrient contents, i.e., by controlled manure application. The absolute counts of microbial indicators were not significantly different in the control non-manured and manured fish ponds, but sporadically detected high faecal indicators counts in summer can represent a potential health risk. According to our results, the water quality of fish ponds is influenced due to manuring but the immediate impact of manuring in fish ponds and recipients in our study proved neither in deterioration of microbiological quality and health risk enhancement nor in the water quality assessed by chemical analyses.

Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers.

Viral indicators were used to evaluate the effectiveness of water treatment processes and to determine the source of faecal pollution with a view to promote water source management. Water samples were collected from three critical points of the Temba, Klipdrift and Wallmansthal water treatment plants (raw water, settled water and filtered water). The viral indicator concentrations and selected physicochemical parameters were measured using standard methods. Random water samples were subjected to real-time polymerase chain reaction analyses. Subsequent to filtration, the removal efficiency of the three Waterworks was recorded as follows: 93.2, 96.1 and 95.7 % for somatic coliphages, respectively; and 69.4 and 68.4 % FRNA coliphages for Temba and Klipdrift. There was a notable 50 % reappearance of FRNA coliphages in filtered water samples from Wallmansthal. Positive correlations were observed between the physicochemical parameters and somatic coliphages, with the exception of the pH and the temperature in Wallmansthal. Both turbidity and temperature indicated positive correlations with FRNA counts from Temba and Klipdrift. Negative correlations were observed between all the physicochemical parameters and FRNA counts from Wallmansthal. The real-time polymerase chain reaction analyses revealed the persistence of GA genotype FRNA (Group II, which is of human faecal origin) in all the water samples. The MS2 genotype (Group I) and Qβ genotype (Group III) FRNA, which are of animal origin, were detected only in the raw water from Temba. Efforts should be focused on the protection of water sources and the optimisation of treatment processes in order to prevent viral persistence during water treatment.

Summary Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through faeces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent faecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extraintestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a faecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics revealed the diversity and complexity of E. coli strains in various environments, which are affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments with regard to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2017 Bacteriophages are studied in an effort to establish the viral safety of water as well as gather information as to what source of faecal pollution is dominant in a water system. The aim of this study was to determine bacteriophage levels and associated characteristics in selected temperate water systems. The methods used in this study were the double-agar–layer plaque assay, for enumeration of somatic coliphage and F-RNA phage levels. Transmission Electron Microscopy was used for characterisation and identification of somatic coliphages. RT-PCR was employed for identification of F-RNA phages. Physical parameter levels of the water systems were measured on site by multi-meter probes. Chemical parameter levels of the water systems were measured in the laboratory using a spectrophotometer. Six water systems in the North West Province of South Africa was studied. These water systems were: Mooi River, Harts River, Barberspan, Crocodile River, Marico River, and Schoonspruit River. Somatic coliphages were found at 34 of the 37 sites that were sampled in this study. The two sites that had no somatic coliphage detected in their water were both in the Crocodile River. The Delarey site in the Harts River was not analysed for phage levels. The highest somatic coliphage level detected in this study was 23 000.00 ± 989.95 pfp/100 mL in the Schoonspruit River. The highest F-RNA phage level recorded during the period of this study was 4 270.00 ± 11.84 pfp/100 mL in the Barberspan water system. The Marico River was the system least affected by environmental and species variables. Barberspan was the water system most severely impacted by F-RNA phage pollution sources. TEM images of plaques showed 3 different morphologies which could indicate a possible link between virion size of somatic coliphages and plaque morphology. However, definite statements regarding this is premature and requires further investigation. Human faecal pollution is entering Barberspan near the hotel sampling site as well as near the outflow of Barberspan into Leeupan sampling site. The physico-chemical parameter levels of the six water systems studied were all indicative of temperate water systems. Considering the bacteriophage and physicochemical parameter levels it is evident that all six water systems studied were being impacted by pollution from domestic and/or agricultural sources Masters

MSc (Microbiology), North-West University, Potchefstroom Campus, 2017 Clostridium are ubiquitous in nature and common inhabitants of the gastrointestinal track of humans and animals. Some are pathogenic or toxin producers. These pathogenic Clostridium species can be introduced into surface water systems through various sources, such as effluent from wastewater treatment plants (WWTP) and surface runoff from agricultural areas. In a South African context, little information is available on this subject. Therefore, this study aimed to characterize Clostridium species isolated from surface water and aquatic sediment in selected river systems across the North West Province in South Africa. To achieve this aim, this study had two main objectives. The first objective focused on determining the prevalence of Clostridium species in surface water of the Schoonspruit, Crocodile and Groot Marico Rivers and evaluate its potential as an indicator of faecal pollution, along with the possible health risks associated with these species. The presence of sulphite-reducing Clostridium (SRC) species were confirmed in all three surface water systems using the Fung double tube method. The high levels of SRC were correlated with those of other faecal indicator organisms (FIO). WWTP alongside the rivers were identified as one of the major contributors of SRC species and FIO in these surface water systems. These findings supported the potential of SRC species as a possible surrogate faecal indicator. However, limitations of SRC species as FIO were noticed in this study. Furthermore, the results showed that the physico-chemical parameters such as temperature, dissolved oxygen, chemical oxygen demand, nitrates, phosphates and sulphates present in the water had a great effect in the Clostridium spp. levels during the warm-rainy season. This was possibly due to non-point source pollution such as surface runoff which promoted eutrophication in parts of these river systems. The second objective of the study was to investigate antibiotic resistance in Clostridium species isolated from both surface water and aquatic sediment and the presence of antibiotic resistance gene in these isolates. A total of 67 Clostridium isolates obtained from the Schoonspruit and Crocodile Rivers showed resistance against Ampicillin, Tetracycline or Clindamycin. No antibiotic resistant isolates were obtained from the Groot Marico River. The minimum inhibitory concentration (MIC) of 6 antibiotics were determined using the recommended agar dilution method. MIC values of Ampicillin (AMP) ranged from 0.25-2 μg/ml, 0.5 to >256 μg/ml for Tetracycline (TE), 0.25 to >256 μg/ml for Clindamycin (DA), 0.5-16 μg/ml for Amoxicillin (AMX), 0.5-32 μg/ml for Chloramphenicol (C) and 0.5-64 μg/ml for Metronidazole (MTZ). Using these MIC values, resistance profile could be generated for each antibiotic resistant Clostridium isolate. These results revealed that Antibiotics such as Amoxicillin and Chloramphenicol were the most effective in inhibiting the growth of antibiotic resistant Clostridium species. Whereas the majority of the isolates showed resistance against Ampicillin and Tetracycline. None of the antibiotics tested for in this study were 100% effective against the Clostridium isolates. Furthermore, ten different multi-antibiotic resistant (MAR) phenotypes were also observed across these isolates. The most prevalent one being AMP-TE-DA-MTZ-C-AMX. All the isolates that presented this phenotype were obtained from aquatic sediment, suggesting that aquatic sediment may be a reservoir for antibiotic resistance and MAR Clostridium species. Additionally, the presence of several antibiotic resistance genes was also screened for using PCR. One of the genes encoding for macrolide-lincosamide-streptogramin (MLS) (ermF), and β-lactam (blaTEM) resistance were not found to be present in any Clindamycin and Ampicillin resistant isolates, respectively. However, several Clindamycin resistant Clostridium isolates were found to harbour the ermB gene, which also encodes for MLS resistance. Two genes encoding for efflux mechanisms against Tetracycline (tetK and tetL) were found in the genomes of some of the Tetracycline resistant isolates. Using both Gram and endospore staining, alongside DNA sequencing, 7 Clostridium species were identified throughout both studies, which included Clostridium bifermentans, C. perfringens, C. sordellii, C. baratii, C. ghonii, C. lituseburense and C. dakarense. Several of these Clostridium species are known pathogens and have been associated with severe gastrointestinal diseases, botulism and necrotising gas-gangrene in both humans and animals. To conclude, the data generated revealed the presence of potentially pathogenic Clostridium species in both surface water and sediment. The presence of antibiotic resistant genes in environmental Clostridium species are also a cause for concern. The expression of these genes could contribute to MAR in these potential pathogenic bacteria. Furthermore, these results highlighted the necessity to screen for other antibiotic resistant pathogens in the aquatic environment and to further investigate the potential sources. Additionally, it is recommended that SRC species should be used as an additional indicator of faecal pollution in surface water systems. Lastly, all these findings indicate that the surface water systems in the North West Province are exposed to various pollutants such as antibiotics and faecal contaminants from runoff and WWTP. This is cause for concern, considering that many rural and informal communities are directly dependent on these water sources and as a result affecting the health of its users, particularly the immune-compromised individuals and livestock. Masters

Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2016/2017 [Resumen] En el Ayuntamiento de Ribadeo (Lugo) se estimó la carga microbiológica presente en el agua de cuatro fuentes (fuente del Valín, Sta. Cruz, Ove y fuente de Cedofeita), un río (río de Esteiro) y un pozo para determinar si cumplían los parámetros microbiológicos establecidos en el Real Decreto 140/2003 referidos a la calidad del agua de consumo humano. Los microorganismos analizados fueron los indicadores de contaminación fecal (grupo coliforme, enterococos y Clostridium perfringens), los microorganismos totales cultivables a 22ºC y 37ºC y Pseudomonas aeruginosa. Los resultados obtenidos mostraron una baja abundancia de microorganismos cultivables y ausencia total del resto de indicadores en las cuatro fuentes y el pozo, mientras que la muestra de río presentó contaminación de origen fecal, siendo así sus aguas una posible fuente de transmisión de microorganismos patógenos. [Resumo] No Concello de Ribadeo (Lugo) estimouse a carga microbiolóxica presente na auga de catro fontes (fonte do Valín, Sta. Cruz, Ove e fonte de Cedofeita), un río e un pozo para determinar se cumprían os parámetros microbiolóxicos establecidos no Real Decreto 140/2003 referidos á calidade da auga de consumo humano. Os microorganismos analizados foron os indicadores de contaminación fecal (grupo coliforme, enterococos e Clostridium perfringens), os microorganismos cultivables a 22ºC e 37ºC e Pseudomonas aeruginosa. Os resultados obtidos amosaron unha baixa abundancia de microorganismos cultivables e ausencia total do resto nas catro fontes e no pozo, mentres que no río supéranse amplamente tódolos parámetros analizados, sendo así as súas augas unha posible fonte de transmisión de microorganismos patóxenos. [Abstract] Microbiological analyses of water from different points in Ribadeo village were carried out: four fountains (Fuente del Valín, fuente de Sta. Cruz, fuente de Ove y fuente de Cedofeita), one well and one river (río de Esteiro). Microbiological parameters related to water quality were estimated taking into account the normative published in the Real Decreto 140/2003 referred to potability of water. The microorganisms analysed were the indicators of faecal pollution (“coliform group”, enterococci and Clostridium perfrigens), the cultivable microorganisms at a 22 and 37ºC and Pseudomonas aeruginosa. The results obtained show a low concentrations of cultivable microorganisms and a complete absence of the remaining indicators in the four fountains and the well, while in the river all parameters analysed are substantially exceeded. Therefore, the river waters could be a potential source of transmission of pathogenic microorganisms.

Cilj: Osiguranje zdravstveno ispravne vode osnovna je potreba svakog čovjeka. S obzirom na dugogodišnje mikrobiološko onečišćenje u vodoopskrbnoj mreži Gorskog kotara, analizirano je petogodišnje razdoblje. Cilj ovog rada bio je pratiti utjecaj promjena zakonske regulative i sanitarno-tehničkih uvjeta – ugradnje većeg broja automatskih klorinatora na broj ispitanih uzoraka iz vodoopskrbnog sustava, kao i na rezultate ispitivanja parametara zdravstvene ispravnosti vode za ljudsku potrošnju. U radu je uspoređen profil onečišćenja vode za ljudsku potrošnju u Gorskom kotaru i ostalim dijelovima županije. Metode: Statistički su obrađeni i interpretirani rezultati županijskog monitoringa zdravstvene ispravnosti vode za ljudsku potrošnju na području Gorskog kotara u razdoblju od 2011. do 2015. (broj ispitanih uzoraka N = 7.047). Rezultati: Stupanjem na snagu Pravilnika o parametrima sukladnosti i metodama analize vode za ljudsku potrošnju (NN 125/13, 141/13 i 128/15) broj uzoraka na temelju kojih se prati stanje vode u razvodnoj mreži Gorskog kotara je šesterostruko smanjen, ali je broj ispitanih pokazatelja povećan. Ugradnja automatskih klorinatora (2012. g. i 2013. g.) rezultirala je dvostrukim smanjenjem broja nesukladnih uzoraka. Zaključci: Primjena Pravilnika (NN 125/13, 141/13 i 128/15) rezultirala je značajnim smanjenjem broja analiziranih uzoraka u pojedinim vodoopskrbnim sustavima, što je procjenu njihovog stanja uvelike otežalo. Ugradnjom automatskih klorinatora broj nesukladnih uzoraka u Gorskom kotaru upola je smanjen. Glavni razlog zdravstvene neispravnosti vode za ljudsku potrošnju u vodovodima Gorskog kotara je fekalno onečišćenje vode, dok u ostalim dijelovima županije najčešće od zadanih kriterija odstupaju mutnoća vode, povećani broj kolonija (UBB) i nusprodukti klorinacije., Aim: Ensuring the safety of potable water is a necessity of every human being. Taking into account long-term microbiological contamination of water supply network of Gorski Kotar, the results of 5-year County monitoring program of potable water safety in that region have been analysed. The aim of this study was to monitor the impact of amendment of legal standards as well as sanitary and technical requirements – the insertion of higher number of chlorinators on the number of examined samples deriving from the water supply system as well as the influence on the results of testing of the health parameters of water intended for human consumption. This study compared the contamination profile of water for human consumption in Gorski Kotar and other regions of the Primorje-Gorski Kotar County. Methods: This study presents the elaborated and interpreted results of the County monitoring program of potable water safety in the region of Gorski Kotar during the 5-year period (2011–2015; the number examined samples N = 7,047). Results: Application of the Regulations on the parameters of assessment and methods for the analysis of water for human consumption (Official Gazette 125/2013, 141/2013/, 128/2015) provides six times lesser number of samples for the monitoring of water supply system network in Gorski Kotar, but the number of analysed parameters was increased. Installation of automatic chlorinators (2012 and 2013) resulted with twice smaller number of non-compliant samples of drinking water. Conclusions: Application of the the Regulations (Official Gazette 125/2013, 141/2013, 128/2015) resulted with the significant reduction of samples for certain water supply systems making the objective assessment of their condition much harder. The installation of automatic chlorinators reduced the number of non-compliant water samples in Gorski Kotar by half. The main reason for non-complying samples of water for human consumption in the water supply systems of Gorski Kotar is faecal contamination, while for the other parts of the County major deviation from stipulated criteria are turbidity, increased number of colonies (Heterotrophic Plate Count – HPC) and chlorination by-products.

Aims To identify faecal pollution along the southeastern Florida coast and determine the performance of a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) method for pepper mild mottle virus (PMMoV). Methods and Results In 2014, bimonthly surface water samples were collected from inlets, exposed to runoff and septic seepage, and coastal sites, exposed to ocean outfalls. Analysis of culturable enterococci and a suite of microbial source tracking (MST) markers (BacHum, CowM2, DogBact, HF183, HPyV, PMMoV) revealed faecal pollution, primarily of human origin, at all sites. Since PMMoV was detected more frequently than other MST markers, the process limits of quantification (undiluted to 10-2 dilution) and detection (10-2 dilution) for the RT-qPCR method were determined by seeding untreated wastewater into the coastal waters. Simulated quantitative microbial risk assessment, employing human norovirus as a reference pathogen, calculated a 0.286 median risk of gastrointestinal illness associated with the PMMoV limit of detection. Conclusions All sites met the U.S. EPA recreational water criteria, despite detection of domestic astewater associated MST markers. PMMoV correlated only with human-associated MST markers. Significance and Impact of Study This study demonstrated that PMMoV is an important domestic wastewater-associated marker that should be included in the MST toolbox; therefore, future studies should thoroughly investigate the health risks associated with its detection and quantification in environmental waters. This article is protected by copyright. All rights reserved.

There is currently great interest in monitoring the microbiological quality of bathing waters and ensuring the safety of users. The European Bathing Water Directive 2006/7/EC which came into place in 2011 sets strict new standards for the classification of bathing waters. Ability to rapidly test microbiological quality of bathing waters is a powerful tool for meeting water quality standards and guidelines, risk assessment and management systems. Real-time and on-line monitoring are key factors for consideration in current method developments for continuous indicator organism assessment to meet early warning requirements and water safety plans. The aim of this work is to address the need for tests capable of rapid on-site and in-situ assessment of microbiological water quality. To achieve an active management of bathing areas and to reduce the risk associated with the presence of faecal pollution, such tests are essential. In the 1st part of this thesis, commercial in-situ sensors are used to collect continuous high frequency water quality data for a period of 7 months in Dublin Port. These data are used to identify pollution hot-spots at the site and coupled with data collected from discrete samples it is shown that continuous monitoring can be used as a decision support tool. In the 2nd part, the thesis looks at developing a rationale for monitoring water for biological contamination and follows by establishing a proposed sensor technology based on a bacterial marker enzyme and fluorescence optical detection. A novel protocol for the recovery and detection of faecal pollution indicator bacteria, E. coli, using β-glucuronidase (GUS) activity is developed. The developed protocol involves two main steps: sample preparation and GUS activity measurement and has a time–toresult of less than 75 min.

Sanitary inspection of wells was performed according to World Health Organization (WHO) procedures using risk-of-contamination (ROC) scoring in the peri-urban tropical lowlands of Dar es Salaam, Tanzania. The ROC was assessed for its capacity to predict bacterial faecal pollution in the investigated well water. The analysis was based on a selection of wells representing environments with low to high presumptive faecal pollution risk and a multi-parametric data set of bacterial indicators, generating a comprehensive picture of the level and characteristics of faecal pollution (such as vegetative Escherichia coli cells, Clostridium perfringens spores and human-associated sorbitol fermenting Bifidobacteria). ROC scoring demonstrated a remarkable ability to predict bacterial faecal pollution levels in the investigated well water (e.g. 87% of E. coli concentration variations were predicted by ROC scoring). Physicochemical characteristics of the wells were not reflected by the ROC scores. Our results indicate that ROC scoring is a useful tool for supporting health-related well water management in urban and suburban areas of tropical, developing countries. The outcome of this study is discussed in the context of previously published results, and future directions are suggested.

To follow faecal pollution steroid compounds have been analysed in 106 sediment samples from the Siak River, E Sumatra, Indonesia. Coprostanol was detected in 40 of these. Contents ranged from 50 to 10,530 ng/g d.w. with a mean of 878 (TOC-normalised: range 7.4-393.0, mean 44.1 μg/g TOC). Total contents and the coprostanol/cholesterol ratio argue for a major contribution from untreated sewage which is also evident from field observations. The distribution along the river indicates the quantitatively dominant source to be the city of Pekanbaru with an estimated population of 1.5 million. Coprostanol contents decrease downstream indicating ongoing degradation either during transport or in the surface sediment. However, additional sources of coprostanol become evident further downstream. On the other hand, the 5β/(5β + 5α)-cholestan-3β-ol ratio versus cholesterol and a ternary plot using C27 sterols suggest that plant sources also contribute to the sedimentary coprostanol due to its formation by bacteria in suboxic/anoxic sediments.

Data mining is a set of computer-assisted techniques designed to automatically mine large volumes of integrated data for new, hidden or unexpected information, or patterns. Two artificial neural networks (ANN) models were developed for prediction of Cryptosporidium oocysts and Giardia cysts respectively using multiple water quality parameters as input. These neural models were feed forward networks, trained by back propagation algorithm. Eight water quality parameters were used to predict Cryptosporidium peak concentration and seven parameters were used to model Giardia concentration in Kano River, Nigeria. The ANN models correctly predicted oocysts and cysts concentration with accuracy of 90% and 92% respectively. The neural network model gave excellent results.

Das von Theodor Escherich vor 125 Jahren entdeckte E. coli-Bakterium hat wie kein anderes die Entwicklung der molekularbiologischen Forschung und der medizinischen und industriellen Biotechnologie beeinflusst. Vor allem die Eigenschaften des K12-Stammes im Hinblick auf Apathogenitat, Kultivierbarkeit und Transformierbarkeit haben E. coli zum “Haustier” der Genetiker und Molekularbiologen gemacht. Die Leichtigkeit, mit der gentechnisch veranderte E. coli hergestellt werden konnen, liesen dieses Bakterium zum beliebten Produktionsorganismus in der modernen Biotechnologie zur Erzeugung von Medikamenten und Feinchemikalien werden. Als physiologischer Darmbewohner von Menschen und Tieren wird E. coli als Indikatororganismus fur fakale Verunreinigungen von Grund- und Trinkwasser verwendet. Neben seiner mikrookologischen Rolle im Magen-Darm-Trakt kommt ihm in Form von pathogenen Stammen auch eine Bedeutung als Erreger von Durchfallerkrankungen zu. The history of colibacteria The E. coli bacterium discovered by Theodor Escherich 125 years ago has influenced the development of molecular-biological research and medicinal and industrial biotechnology like no other bacterium. In particular, the characteristics of the K12-strain with respect to apathogenicity, culturability and transformability made E. coli the “workhorse” of geneticists and molecular biologists. The easiness with which genetically modified E. coli can be made let this bacterium become a popular production organism of modern biotechnology for the making of drugs and fine chemicals. As a physiological inhabitant of the intestine of humans and animals, E. coli is used as an indicator organism of faecal pollution of ground and drinking water. Alongside its micro-ecological role in the gastrointestinal tract, the E. coli bacterium, in terms of pathogenic strains, also has significance as a causative agent of diarrhoeal diseases.

Faecal pollution of river water can lead to health problems because of the presence of infectious microorganisms. These may be derived from human sewage or animal sources. Water safety or quality is best described by a combination of sanitary inspection and microbial water quality assessment. This approach provides data on possible sources of pollution in Arieş river water, as well as numerical information on the actual level of faecal pollution according with the analzyed bacteriological parameters.

We have developed a method for the detection of viruses in environmental samples that we have called enterophages, that specifically infect Enterococcus faecalis. This method has allowed us to determine the prevalence and to study the ecology of this group of phages. The enterophages replicate at 37 degrees C, and at 41 degrees C. The presence of NaN(3) in the media inhibits the growth of background microbiota and allows an accurate, specific and rapid detection of these viruses. Enterophages were present in raw domestic sewage at lower concentrations (average 1.8 x 10(2) PFU/100 mL) than those of coliphages (average 1.7 x 10(5) PFU/100 mL). Phages were characterised by transmission electron microscopy showing icosahedral capsids, some with non-contractile tails as well as icosahedral non-tailed capsids. Different isolates had capsid sizes ranging from 20 nm to about 75 nm in diameter. These data describe a new group of phages that may serve as alternate indicators of human faecal pollution, especially in recreational waters. The ecology of these enterophages indicates that these may be strictly of human origin.