Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.     

Correlation between bacterial indicators and bacteriophages in sewage and sludge

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

Occurrence and densities of bacteriophages proposed as indicators and bacterial indicators in river waters from Europe and South America

AIMS: To evaluate the feasibility of bacteriophages as a complementary tool for water quality assessment in surface waters from different parts of the globe. METHODS AND RESULTS: Faecal coliform bacteria, enterococci, spores of sulphite-reducing clostridia, somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis were determined by standardized methods in raw sewage and in 392 samples of river water from 22 sampling sites in 10 rivers in Argentina, Colombia, France and Spain, which represent very different climatic and socio-economic conditions. The results showed that the indicators studied maintained the same relative densities in the raw sewage from the different areas. Classifying the river water samples according to the content of faecal coliform bacteria, it can be observed that the relative densities of the different bacterial indicators and bacteriophages changed according to the concentration of faecal coliform bacteria. There was a relative increase in the densities of all groups of bacteriophages and sulphite-reducing clostridia with respect to faecal coliforms and enterococci in the samples with low counts of faecal coliform bacteria. CONCLUSIONS: The numbers of bacterial indicators and bacteriophages were similar in the different geographical areas studied. Once released in rivers, the persistence of the different micro-organisms differed significantly. Bacteriophages and spores of sulphite-reducing clostridia persisted longer than faecal coliforms and enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages in river water samples provide additional information to that provided by bacteria about the fate of faecal micro-organisms in river water. The easy, fast and cheap methods for phage determination are feasible both in industrialized and developing countries.     

Occurrence and levels of indicators and selected pathogens in different sludges and biosolids

AIMS: Determine the occurrence and levels of pathogens and indicators in raw and treated sludges and compare their persistence after two different treatments. METHODS AND RESULTS: Helminth ova, Cryptosporidium spp., Salmonella spp., enteroviruses, and bacterial and viral indicators were determined in raw sludges and biosolids produced after mesophilic and thermophilic treatments. Except Salmonella, all of the parameters were quantified. Helminth ova were found at very low concentrations even in raw sludges. Viable Cryptosporidium oocysts were still present in most samples of treated sludges. Faecal coliforms, spores of sulphite-reducing clostridia (SSRC), and somatic coliphages were the only indicators with values above their detection limits in most of the samples. CONCLUSIONS: Pathogens were still detected in some treated sludge samples. SSRC were the most resistant micro-organisms to treatments and hence may be an indicator for the reduction of protozoan oocysts. Somatic coliphages constitute an alternative as viral indicators due to their detection in sludges before and after treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the persistence of some pathogens after sludge treatments, additional indicators are needed. SSRC and somatic coliphages are good candidates. Easy and inexpensive methods for the determination of these indicators are feasible both in industrialized and developing countries.     

Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination

AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Abstract Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

Occurrence and removal of parasites, enteric bacteria and faecal contamination indicators in wastewater natural reclamation systems in Tenerife-Canary Islands, Spain

Two wastewater natural reclamation systems (WWNRS) have been compared regarding their efficiencies on faecal bacteria removal and the persistence of enteric pathogens. These WWNRS are constituted of a combination of anaerobic treatment, small sub-surface flow constructed wetland refilled of volcanic ashes and a final pond as water reservoir. Faecal coliforms, enterococci, Escherichia coli, Clostridium perfringens, somatic coliphages, Salmonella sp., Campylobacter sp., Cryptosporidium sp., Giardia sp. and helminth eggs were analyzed in constructed wetlands inlet and outlet and storage pond effluent. Low numbers of protozoan positive samples (4.54% in Albergue de Bolico for both protozoa, and 19.05% in Carrizal Alto for Giardia sp.) and absence of helminth eggs were found. Both systems demonstrated efficient reduction of faecal contamination indicators in the wastewaters (removal rates values of 2 log₁₀). The natural systems for wastewater treatment used to be efficient in Salmonella abatement, this fact was confirmed in the reported systems, since enterobacteriaceae were found in only one of the effluents. Campylobacter species associated with the access of animals to storage ponds were detected in the reclaimed water.     

An examination of the bacteriophages and bacteria of the Namib desert

Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth's terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55 approximately 65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94 approximately 98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and 8-Proteobacteria groups were also observed.     

Survival kinetics of faecal bacterial indicators in spent broiler litter composting

Aims:  The objective of this study was to deduce and analyse equations that best describe the behaviour of faecal bacterial indicators and two decomposition parameters during broiler litter composting. Mathematical models were fitted and the order of rate equations were identified.
Methods and Results:  The Levenburg-Marquardt algorithm was used to fit nonlinear mathematical models to total coliforms (TC), faecal coliforms (FC), Escherichia coli (EC), faecal enteroccocci (FE), organic-C and volatile solids reduction, VS_Red, by the least squares procedure. The rate equations showed that TC, FC and EC reductions were expressed by second-order decay kinetics. FE reduction followed first-order decay. Temperature dependency of decomposition rate was effectively verified by applying empirically derived rate equations.
Conclusions:  The governing mathematical models critically compare the inactivation kinetics of faecal indicators. TC, FC and EC were rapidly destroyed while FE was more resistant. Temperature elevation, organic-C and VS_Red dynamics provide an accurate understanding of composting-induced decomposition of the broiler litter.
Significance and Impact of the Study:  The conservative performance of FE with respect to the other indicators has been established. Hence, FE presents better opportunities to encompass the totality of the composting process in terms of attainment of hygiene efficacy compared with EC.     

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans)

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.     

Occurrence of airborne enteric bacteria in Mexico city

Summary An investigation of microbial air quality in the area of the National Autonomous University of Mexico, located in the southern part of Mexico City, was conducted for one year. Ambient outdoor concentrations and size distribution of airborne bacteria were measured, 130 samples were taken at noon, using an Andersen 6 stage sampler, located 2 m above ground level. Concentration ranges and colony-forming units per cubic meter of air (CFU m^–3) found, were as follows:14 to 12999 for total bacteria, No growth (NG) to 55 for coliform bacteria, NG to 11 for fecal coliform and NG to 10 for fecal Streptococci.Bacteria associated with the potentially respirable fraction (0.65 to 4.7 µm) averaged 37% and 9% for total bacteria and coliform bacteria respectively. In 23% of the samples, coliform bacteria were recovered, with higher incidences during dry season. The most common of these were:Escherichia coli (15%), followed bySerratia (13%) andEnterobacter (10%),The total bacteria correlated significatively (p<0.05) with the following parameters: particulate matter smaller than 10 µm (PM₁₀) (r=0.40), total suspended particulates (TSP) (r=0.26), daily variation of temperature (r=0.18), and vapor pressure (r=–0.16). These relationships indicate that fecal soil pollution could affect air quality with potential health risks.     

红壤地下水厌氧氨氧化菌分布特征及环境响应

【目的】进一步了解红壤区地下水中厌氧氨氧化菌群落结构分布规律及其生态功能差异化机制。【方法】以酸性红壤地下水为研究对象,结合理化指标和微生物高通量测序技术,研究吉安市地下水中微生物群落结构和厌氧氨氧化菌的分布规律及其影响因子。【结果】11个地下水研究点中,氮含量超标点位有3个,超标率达27.3%;仅有2个站点的水质符合《生活饮用水卫生标准》(GB 5749—2022)要求,超标率达81.8%。地下水中厌氧氨氧化菌广泛存在,11个样品的厌氧氨氧化细菌hzsB基因拷贝数在3.67×10⁴-6.62×10⁸ copies/g,与其他生态系统相比,红壤地下水厌氧氨氧化菌功能基因拷贝数更高,说明地下水环境特性更适合厌氧氨氧化细菌生长代谢。每个点位均检测出厌氧氨氧化菌的4个属：CandidatusBrocadia为优势菌属,平均丰度值为62.47%,Candidatus Scalindua平均丰度值为17.44%,Candidatus Jettenia平均丰度值为14.41%,Candidatus Kuenenia平均丰度值为5.67%。相关性分析表明,厌氧氨氧化菌基因丰度与氨氮、锰、氯化物相关性显著,可见地下水中氨氮、锰、氯化物含量的升高可能会增强厌氧氨氧化菌的活性及丰度。【结论】本研究结果突出了厌氧氨氧化菌对红壤地下水元素地球化学生物循环的重要价值,可为我国红壤区地下水污染的生物防治技术的研究提供基础资料。     

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s- triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 μg L⁻¹ and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum . PCR detection of the genes encoding s -triazine-degrading enzymes indicated the presence of the atz A and atz B genes in A. incenata and the atz B and atz C genes in J. lividum . The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.     

Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges

A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1.1 x 10(5) pfu g-1) and the lowest being that of Bacteroides fragilis phages (110 pfu g-1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others.     

Occurrence of coliphages in urban stormwater and their fate in stormwater management systems

AIMS: To investigate the occurrence of coliphages in, and their removal from, urban stormwater. METHODS AND RESULTS: Inflow and outflow concentrations of somatic and f-specific RNA coliphages to two stormwater treatment systems were determined on 21 occasions over a period of 5 months. Somatic coliphages were detected in 19 (90%) of the constructed wetland inlet samples, 13 (62%) of the pond inlet samples, and less frequently at the outlets of the two systems. F-specific RNA coliphages were detected at the inlets but only occasionally at the pond outlet. Somatic coliphages were found to attach preferentially to particles <5 microm in size and persisted in the sediments of the two systems. CONCLUSIONS: Treatment systems providing conditions that are conducive to the settlement of fine particles may effectively remove sediment-bound coliphages and, therefore, possibly enteric viruses from stormwater. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid the design of systems for effective removal of viral contaminants from urban stormwater.     

Molecular detection of bacterial indicators in cosmetic/pharmaceuticals and raw materials

PCR assays were compared with standard microbiological methods for rapid detection of the United States Pharmacopoeia (USP) bacterial indicators in artificially contaminated samples of raw materials and cosmetic/pharmaceutical products. DNA primers containing the specific sequences of the uidA gene of the β-glucuronidase enzyme for Escherichia coli, the membrane lipoprotein gene oprL for Pseudomonas aeruginosa, and the 16S ribosomal gene for Staphylococcus aureus were used for detection in the PCR reaction. Contaminated samples were incubated for 24 h at 35°C. After incubation in broth media with and without 4% Tween 20, samples were streaked on selective growth media. After 5–6 days, all microbial indicators were morphologically and biochemically identified using standard methods while detection and identification by the PCR-based assays was completed within 27–30 h. Rapid PCR detection of E. coli, S. aureus, and P. aeruginosa will allow a faster quality evaluation and release of raw materials and cosmetic/pharmaceutical products sensitive to microbial contamination. Received 21 June 1998/ Accepted in revised form 11 January 1999