The impact of cyanobacteria on growth and death of opportunistic pathogenic bacteria

Climate change may cause increased microbial growth in water sources and more knowledge is required on how this may affect the hygienic water quality, i.e., whether increased occurrence of cyanobacteria and algae may stimulate the growth rate of opportunistic pathogenic bacteria. Laboratory experiments were performed to investigate if the presence of the cyanobacteria Anabanea lemmermannii and Microcystis aeruginosa affected the survival and growth rate of the opportunistic pathogenic bacteria Aeromonas hydrophila and Pseudomonas aeruginosa, and the faecal indicators Escherichia coli and coliforms. Cyanobacteria were cultured in bottles containing the nutrient-poor medium 02. Sewage, A. hydrophila or P. aeruginosa was added to cyanobacterial cultures and the bacterial growth and survival was followed. E. coli and coliforms from sewage died within few days and the decay rate was not affected by the presence of cyanobacteria. The presence of Anabaena stimulated the growth rate of P. aeruginosa, but had no effect on the growth rate of A. hydrophila. Microcystis had no effect on the growth rate of P. aeruginosa and an inhibiting effect on the growth rate of A. hydrophila.     

Microbiological quality of groundwater sources used by rural communities in Limpopo Province, South Africa.

A survey of the microbiological quality of water from 194 boreholes (97 privately owned and 97 communal boreholes) in the rural Thitale-Hlanganani area of the Limpopo Province, South Africa was carried out between August 2002 and August 2003. Very little information on the microbiological quality of privately-owned boreholes in rural communities is available raising concerns about the safety of these groundwater supplies. In this study, levels of total coliforms, thermotolerant (faecal) coliforms, faecal enterococci, Clostridium perfringens (vegetative cells and spores) and somatic coliphages were determined for community and privately-owned borehole water. The average counts for total coliforms, faecal coliforms, faecal enterococci and Clostridium perfringens exceeded the South African recommended guideline limits of 0-10 counts.100 ml(-1) for total coliforms and 0 counts. 100 ml(-1) for faecal coliforms, faecal enterococci and Clostridium perfringens respectively. Comparisons between the levels of indicator bacteria present in private and communal boreholes during dry seasons indicated a statistical difference for faecal enterococci bacteria (p = 0.005) and Clostridium perfringens (p = 0.08). Comparisons between the levels of indicator bacteria present in private and communal boreholes during rainy seasons indicated statistical differences between total coliforms (p = 0.05), faecal coliforms (p = 0.03) and Clostridium perfringens (p = 0.009) bacteria. No significant differences in the presence of somatic coliphages in both private and communal borehole water were see. The results indicated the need for environmental impact assessment studies to monitor the microbiological quality of groundwater sources in rural communities.     

Biodegradation of estrogen conjugates by bacteria isolated from river sediments

The objective of this study was to investigate the ability of E. coli in river sediments to degrade estrogen conjugates. Biodegradation experiments on glucuronide estrogens (E1-GLU, E2-GLU and E3-GLU) using E. coli, non-E. coli bacteria as well as sediment crude extracts were carried out in batch mode. A pure identified E. coli strain (KCTC 2571) was used for comparison of enzyme activity. The results showed that the degradation rate of estrogen conjugates by KCTC 2571 and E. coli isolated from sediments followed a similar trend. Fecal bacteria showed a high ability to deconjugate glucuronided estrogens. Approximately 50% of glucuronide moieties were cleaved within 4 h of contact time in experiments using pure E. coli. The degradation rate was slower in experiments using crude extracts of sediments, and conjugated estrogens were not completely degraded even after 12 h of reaction. These results provide a clear understanding of the fate and behavior of estrogen by bacteria in the environment.     

Experience with the antibiotic resistance analysis and DNA fingerprinting in tracking faecal pollution at two lake beaches.

Posting or closing of swimming beaches because of faecal contamination is a widespread problem reported in many locations. In a risk-based approach to this problem, the risk to swimmers' health is assessed by field monitoring of indicator bacteria and the associated risks are managed by source controls and other remedial measures. In risk assessment, great advances have been made in recent years with the introduction of microbial source tracking (MST) techniques. Two such techniques, antibiotic resistance analysis and DNA fingerprinting, were applied in a study of causes of faecal contamination at two lake beaches in Toronto, Ontario. Both methods identified bird faeces as the dominant sources of E. coli. Coping with this type of pollution presents a major environmental challenge.     

Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.     

Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques.

Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because OPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination.     

Microbiological water quality in a large in-building, water recycling facility.

The Thames Water recycling plant at the Millennium Dome, London, reclaimed three sources of water: greywater from the washbasins, rainwater from the Dome roof and groundwater from a borehole on site. These were pre-treated separately, and the mixed stream filtered using ultrafiltration and reverse osmosis membranes. Monitoring for indicator microorganisms was undertaken throughout the plant and in the reclaimed water distribution system, as well as ad-hoc monitoring for the presence of pathogens in the raw waters. Treatment to the level of ultrafiltration was more than adequate to produce a water quality meeting existing worldwide reclaimed water guidelines for toilet flushing. Owing to the excellent quality of the water leaving the plant, no significant microbiological growth was observed in the reclaimed water distribution system during the year. The raw greywater exhibited a higher faecal bacterial load than the rainwater and groundwater, as predicted from more human contact (i.e. hand washing). Environmental strains of Legionella were observed in the three raw greywater samples analysed for pathogens, as was Cryptosporidium, Giardia and faecal enterococci. The rainwater had relatively high levels of faecal bacteria, probably of avian origin. Giardia was detected in one rainwater sample confirming the potential for this water source to contain pathogens.     

Antimicrobial resistance of fecal indicators in disinfected wastewater

The main objective of the study was to assess the potential of three systems (UV irradiation, ozonation, and micro/ultrafiltration) operated in a pilot scale in removal of antimicrobial-resistant fecal bacteria from secondary effluent of the local wastewater treatment plant (700,000 population equivalent). The effectiveness of the processes was analysed using the removal ratio of fecal indicators (Escherichia coli and Enterococcus spp.). The susceptibility of fecal indicators to antimicrobial agents important in human therapy was examined. Resistance to nitrofurantoin and erythromycin was common among enterococci and followed by resistance to fluoroquinolones and tetracycline. Resistance to high-level aminoglycosides and glycopeptides was also observed. E. coli isolates were most frequently resistant to penicillins and tetracycline. The extended-spectrum beta-lactamase-producing E. coli was detected once, after ozonation. Substantial attention should be paid to the E. coli and enterococci resistant to three or more chemical classes of antimicrobials (MAR), which in general constituted up to 15 and 49% of the tested isolates, respectively. Although the applied methods were effective in elimination of fecal indicators (removal efficiency up to 99.99%), special attention has to be paid to the application of sufficient disinfection and operation conditions to avoid selection of antimicrobial resistant bacteria.     

Helsinge sludge reed bed system: reduction of pathogenic microorganisms.

Reduction of pathogenic bacteria: Salmonella, enterococci and Escherichia coli was investigated using the sludge reed bed system. The system at Helsinge was established in 1996 and has a capacity of 630 tonnes dry solids per year and consists of 10 basins. Since 2000 the individual basins have been subjected to an average area-specific loading rate of 46-56 kg dry solids/m2/year. The total sludge residue height in April 2006 was approximately 1.40 m. The sludge (approximately 0.5-0.8% dry solids), with which the individual basins were loaded, contained a large number of bacteria. Salmonella, enterococci and E. coli were found in the sludge in the following quantities: 10-300/100 g (wet weight), 7,000-25,000 CFU/g (wet weight) and 800,000-10,000,000 CFU/100 g (wet weight), respectively. The analysis of the reduction in pathogens in the sludge residue through a period of 3-4 months after the last loading indicated that the pathogen content was reduced down to < 2/100 g (Salmonella), < 10 CFU/g (enterococci) and < 200 number/100 g (E. coli). For enterococci and E. coli the reduction was approximately log 5 and log 6-7, respectively. In the same period the sludge residue achieved a dry solids content of approximately 20-35%.     

Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish.

Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be considered.     

Removal improvement of bacteria (Escherichia coli and enterococci) in maturation ponds using baffles

Korba wastewater treatment plant is a conventional activated sludge followed by three maturation ponds (MP1, MP2, MP3) in series acting as a tertiary treatment. The first study of wastewater treatment plants showed that the effluent concentration of Escherichia coli and enterococci at the outlet of the (MP3) varies between 10(3) and 10(4)CFU/100 ml. After the hydrodynamic study conducted by Rhodamine WT which showed short-circuiting in the MP1, two baffles were introduced in the first maturation pond (MP1) to improve the hydrodynamic and the sanitary performances. The second hydraulic study showed that the dispersion number 'd' was reduced from 1.45 to 0.43 by this engineering intervention and the Peclet number was raised from 0.69 to 2.32. The hydraulic retention time was increased by 14 h. Because of well-designed baffles, the removal efficiency of E. coli and enterococci was raised between 0.2 and 0.7 log units for the first maturation pond.     

Isolation and characterization of thermophilic bacteria capable of lysing microbial cells in activated sludge.

To find an efficient biological method to solubilize waste activated sludge (WAS) from the biological wastewater treatment process, several strains of thermophilic bacteria capable of solubilizing WAS were isolated from sewage sludge compost. The culture supernatants of the isolates were able to lyse vegetable bacterial cells and the lytic activity mainly came from the exoenzyme produced by the isolates. The culture supernatants of the different isolates showed different lysis characteristics. The factors affecting bacterial cell lysis were investigated using E. coli as a model bacterium. The E. coli cells were lysed easily at higher temperature (60 degrees C or 70 degrees C) while little lytic activity by the supernatants of isolates was observed at lower temperature (50 degrees C). The level of pH also had great influence on the lysis of E. coli cells. The E. coli cells in the early stationary growth phase were easier to lyse than those in the late stationary growth phase or death phase.     

Microbiological quality of reclaimed water used for golf courses' irrigation.

Microbial quality of reclaimed water used for irrigation in two golf courses located in the southern Iberian Peninsula (Spain and Portugal) was evaluated. Bacterial indicators for faecal pollution (total and faecal coliforms, Escherichia coli and enterococci) were tested by membrane filtration using appropriate selective media. In addition, somatic E. coli bacteriophages, enteric viruses (entero-, hepatitis A and rota-) and Legionella pneumophila were also analysed. The results obtained showed that all wastewater treatment processes reduced adequately the number of indicator microorganisms although a significant correlation between pathogenic and indicator microorganisms tested was not found. L. pneumophila was detected by PCR but not confirmed by culture. Survival experiments of pathogenic microorganisms in aerosols and irrigated turf are conducted to determine the health hazards for the golf practice and to propose a microbial standard for wastewater used for irrigation of golf courses.     

Comparison of total and faecal coliforms as faecal indicator in eutrophicated surface water.

The aim of this study was to evaluate the use of total coliforms (TC) and faecal coliforms (FC) using a membrane filtration method for precise monitoring of faecal pollution in Korean surface water. The samples were collected in Korea from both main rivers and their tributaries. Presumptive TC * FC were enumerated. The ratios of presumptive FC to TC were not constant, but varied widely, and TC were difficult to enumerate because of overgrowth by background colonies. For FC this was not the case. Seven hundred and three purified strains of presumptive TC * FC and their background colonies were biotyped using API 20E. Among 272 presumptive TC, non-faecal related species, Aeromonas hydrophila dominated (34.6%) and E. coli accounted for only 5.1%. In contrast, E. coli made up 89% of the 209 presumptive FC. Furthermore, of 164 background colonies on Endo Agar LES, 54.9% was A. hydrophila, while background colonies on m-FC Agar were few (58 strains), and despite their atypical colony appearance, most of them were biotyped as enteric bacteria. These results reveal that the detection of FC rather than TC using m-FC Agar is more appropriate for faecal pollution monitoring in eutrophicated surface water located in a temperate region.     

Comparative stability and growth requirements of S. aureus and faecal indicator bacteria in seawater.

The fate (stability, multiplication) of S. aureus, E. coli and E. faecalis was determined in three classes of recreational waters (seawater, estuarine, stream) supplemented with nutrients in the form of sewage and peptone. In the absence of sunlight (24 +/- 2 degrees C), all bacteria in all water samples did not multiply and were slowly (days) inactivated. When 50% sewage was added to all water samples, E. coli and E. faecalis multiplied but S. aureus did not. When peptone (0.05%, 0.5%) was the added nutrient, the three bacteria multiplied. In the presence of sunlight (15-27 degrees C), S. aureus was inactivated rapidly (hours) in all water samples. These results show that when their nutritional requirements are met, S. aureus, E. coli and E. faecalis can multiply in the high salinity conditions of seawater. However, under environmental conditions, sunlight is an effective natural bactericidal agent.     

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.     

The association of E. coli and soil particles in overland flow.

The removal of E. coli from overland flow under saturation-excess runoff conditions was investigated in experimental field plots that were 1 m wide and 5 m long. Variation in the attenuation of bacteria and distance transported was quantified under contrasting flow conditions. In addition, the impact of soil tillage upon microbial attenuation was examined by comparing results derived from grassed plots (intact) with those subject to tillage with the soil left bare (cultivated). For intact plots subjected to a flow of 2 L/min, 27% of the E. coli in the flow was removed after 5 m with removal following a logarithmic function with respect to distance. For the higher flow rates of 6 L/min and 20 L/min, no attenuation trend was observed over this distance. E. coli removal during flow across the cultivated plots was significantly greater compared to the intact plots. This was attributed to a greater infiltration rate in the cultivated plots (due to the tillage) which promoted a greater volume of flow to pass through the soil matrix, providing the opportunity for filtration and adsorption of microbes. Logarithmic trends with respect to distance were observed for all flow rates tested on the cultivated plots (2, 6 and 20 L/min). Total removal after 5 m at a flow rate of 2 L/min was 41% and again removal efficiency decreased as the flow rate increased. Analysis of the transported state of the E. coli revealed that the bacteria were being transported predominantly in particles less than 20 microm in diameter and were not attached to large (dense) soil particles. The limited removal (< 50%) of bacteria from overland flow under saturation-excess runoff conditions in these experiments appeared, therefore, to be primarily due to a lack of settling or deposition. Instead, most bacteria remained entrained within the overland flow down the length of the plots.     

Tracking human faecal contamination in tropical reservoirs in Puerto Rico

Using a combination of chemical and microbiological (culture-dependent and -independent) approaches, sources of human faecal contamination were identified in two water reservoirs in Puerto Rico – Guajataca and La Plata. Fluorescence from optical brighteners (OB) – commonly found in laundry detergents – was used as an indicator of contamination from septic systems and other household discharges. Traditional indicators of faecal contamination (e.g. Escherichia coli; faecal enterococci) were enumerated, and human faecal contamination was confirmed through detection of Bifidobacterium adolescentis utilizing polymerase chain reaction (PCR)-based analyses. For Guajataca Reservoir, four of 19 sampling sites (21%) were positive for the presence of B. adolescentis under baseflow conditions. The OB fluorescence data suggested that the most likely source for three of these sites was residential, whereas the source of contamination at the remaining site, although of human origin, was probably non-residential. B. adolescentis was present in 83% (19 of 23) of the sampling sites in La Plata. The La Plata sources were more difficult to identify because samples were taken under stormflow conditions, although the presence of OB fluorescence suggested a residential origin in a number of instances. OB fluorescence and traditional bacterial indicators of faecal contamination produced a number of false positive and negative findings for both reservoirs, pointing to the importance of understanding the limitations of these tools for tropical freshwater systems. The results of this study should be useful in developing a weight-of-evidence approach for the identification of potential sources and extent of human faecal contamination in similar tropical reservoirs, a necessary step in the development of management plans to reduce or eliminate these sources.     

E. coli transport in soil columns: implications for reuse of treated wastewater in irrigation.

Reuse of treated wastewater in irrigation is gaining recognition as a vital element in the water resources management plan of developing countries, especially those situated in arid and semi-arid regions. An understanding of the transport of residual pollutants from treated wastewater, such as bacteria, in soil as a result of irrigation is critical to assessing health risks and the possible contamination of limited groundwater resources. In this work, retention of E. coli is evaluated for a soil that is irrigated by treated wastewater for growth of non-food crops near Egypt's Red Sea coast. In particular, the effects of soil organic fraction (SOF) and hydraulic loading rate (HLR) were investigated in laboratory soil columns. The matrix of experiments included three HLRs and three SOFs. The retention of bacteria by adsorption was observed at HLRs of 5 and 13 cm/h, with the magnitude of the adsorption increasing proportionally to the SOF. The impact of SOF was greater for the lower HLR. At the lowest HLR investigated (5 cm/h), filtration was also observed for the two higher SOFs (0.674 and 2.04 per cent). At a high HLR (66 cm/h) simulating flood irrigation, retention of bacteria was minimal regardless of the SOF. Since the bacterial solution is applied to a dry soil column to simulate field conditions, E. coli breakthrough after two pore volumes of throughput (vs. one) provided a meaningful comparison of bacterial retention as a function of HLR and SOF.     

Global Warming Effects on Faecal Coliform Bacterium Watershed Impairments in Portugal

Impairment of surface water quality by faecal coliform bacteria is an issue of great importance across the globe. A water quality model, Hydrological Simulation Program FORTRAN, was used to predict the impacts of farming and climate change on faecal coliform loads and concentrations in streams of the Lis River watershed, in the Leiria region, Portugal. The calibrated faecal coliform model simulated well the patterns and range of observed faecal coliform concentrations. The accuracy of the model was evaluated by the per cent bias coefficient and the coefficient of determination. The results indicate a general deterioration of the water quality regarding faecal contamination in Lis River. Maximum daily loads were calculated for each of the impaired streams; an average of 77% reduction in the current faecal coliform load from the watershed is necessary to achieve the established water quality goals by the Council Directive 75/440/EEC ( 1975 ). Climate change scenarios (increments on temperature and precipitation) were assumed to predict the behaviour of faecal coliform bacteria in the watershed. The simulated results showed that an increase of 1°C in air daily temperature results in an increase of water temperature of 1.1°C and a 1.5% decrease on faecal coliform bacteria in stream concentration. The combined effect of air temperature (+1°C) and precipitation (+7%) increment leads to an increase of ~2% in bacteria inflow to the basin. Copyright © 2014 John Wiley & Sons, Ltd.