Somatic Coliphages as an Operational Tool to Assess Loss of Bathing Water Quality after Heavy Rain Events.

Rapid population growth and coastal development has led to increased fecal contamination of coastal surface waters worldwide, enhancing the potential risk of waterborne human pathogens in bathing areas. More frequent heavy rainfall events, attributed to global warming, have further exacerbated the problem by causing sometimes sewer overflows into recreational waters. As traditional bacterial indicators have limited accuracy for predicting health risks associated with waterborne viruses, the additional use of viral indicators such as coliphages is recommended. In this study, we compared the behavior of bacterial and viral indicators of water quality at 10 Barcelona beaches during three bathing seasons, in dry conditions, and after four rainstorms that caused specific pollution events due to rain runoff with combined sewer overflows (CSO). Levels of all target indicators increased after the rainstorms, but compared to Escherichia coli and intestinal enterococci, somatic coliphages exhibited a slower decline and higher environmental persistence following a rain event. Daily continuous sampling carried out during the days following a rainstorm allowed not only the determination of the decay kinetics of each target indicator but also the day when the water quality recovered the values established in the current European regulation in approximately 2 -3 days after each CSO. These observations indicate that the combined use of bacterial and viral indicators can enhance the surveillance of microbial quality of bathing waters. Moreover, coliphages can swiftly provide insights into transient fecal pollution linked to rainfall episodes, thanks to available analytical techniques that enable same-day recommendations. The management of urban wastewater and recreational water regulations should consistently employ microbial indicators to address rainwater runoff or sewer overflows resulting from heavy rainfall.

Modelling the seasonal impacts of a wastewater treatment plant on water quality in a Mediterranean stream using microbial indicators.

Faecal pollution modelling is a valuable tool to evaluate and improve water management strategies, especially in a context of water scarcity. The reduction dynamics of five faecal indicator organisms (E. coli, spores of sulphite-reducing clostridia, somatic coliphages, GA17 bacteriophages and a human-specific Bifidobacterium molecular marker) were assessed in an intermittent Mediterranean stream affected by a wastewater treatment plant (WWTP). Using Bayesian inverse modelling, the decay rates of each indicator were correlated with two environmental drivers (temperature and streamflow downstream of the WWTP) and the generated model was used to evaluate the self-depuration distance (SDD) of the stream. A consistent increase of 1-2 log10 in the concentration of all indicators was detected after the discharge of the WWTP effluent. The decay rates showed seasonal variation, reaching a maximum in the dry season, when SDDs were also shorter and the stream had a higher capacity to self-depurate. High seasonality was observed for all faecal indicators except for the spores of sulphite-reducing clostridia. The maximum SDD ranged from 3 km for the spores of sulphite-reducing clostridia during the dry season and 15 km for the human-specific Bifidobacterium molecular marker during the wet season. The SDD provides a single standardized metric that integrates and compares different contamination indicators. It could be extended to other Mediterranean drainage basins and has the potential to integrate changes in land use and catchment water balance, a feature that will be especially useful in the transient climate conditions expected in the coming years.

Dynamics of crAssphage as a human source tracking marker in potentially faecally polluted environments.

Recent studies have shown that crAssphage is abundant in human faecal samples worldwide. It has thus been postulated as a potential microbial source tracking (MST) marker to detect human faecal pollution in water. However, an effective implementation of crAssphage in water management strategies will depend on an understanding of its environmental dynamics. In this work, the abundance and temporal distribution of crAssphage was analysed in the effluent of wastewater treatment plants using different sewage treatments, and in two rivers (water and sediments) that differ in pollution impact and flow regime. Additionally, the influence of environmental conditions (temperature and rainfall) on the removal of the marker was studied along a river section, and natural inactivation was assessed by a mesocosms approach. Molecular and culture-based tools were used to compare crAssphage abundance and dynamics with those of bacteria and bacteriophages currently applied as global indicators (E. coli, somatic coliphages, Bacteroides GA17 bacteriophages, and the human-associated MST markers HF183 and HMBif). CrAssphage concentrations in sewage effluent and river samples were similar to those of HF183 and HMBif and higher than other general and/or culture-based indicators (by 2-3 orders of magnitude). Measurement of crAssphage abundance revealed no temporal variability in the effluent, although rainfall events affected the dynamics, possibly through the mobilisation of sediments, where the marker was detected in high concentrations, and an increase in diffuse and point pollution. Another factor affecting crAssphage inactivation was temperature. Its persistence was longer compared with other bacterial markers analysed by qPCR but lower than culturable markers. The results of this study support the use of crAssphage as a human source tracking marker of faecal pollution in water, since it has similar abundances to other molecular human MST markers, yet with a longer persistence in the environment. Nevertheless, its use in combination with infectious bacteriophages is probably advisable.

Pathogens, faecal indicators and human-specific microbial source-tracking markers in sewage.

The objective of this review is to assess the current state of knowledge of pathogens, general faecal indicators and human-specific microbial source tracking markers in sewage. Most of the microbes present in sewage are from the microbiota of the human gut, including pathogens. Bacteria and viruses are the most abundant groups of microbes in the human gut microbiota. Most reports on this topic show that raw sewage microbiological profiles reflect the human gut microbiota. Human and animal faeces share many commensal microbes as well as pathogens. Faecal-orally transmitted pathogens constitute a serious public health problem that can be minimized through sanitation. Assessing both the sanitation processes and the contribution of sewage to the faecal contamination of water bodies requires knowledge of the content of pathogens in sewage, microbes indicating general faecal contamination and microbes that are only present in human faecal remains, which are known as the human-specific microbial source-tracking (MST) markers. Detection of pathogens would be the ideal option for managing sanitation and determining the microbiological quality of waters contaminated by sewage; but at present, this is neither practical nor feasible in routine testing. Traditionally, faecal indicator bacteria have been used as surrogate indicators of general faecal residues. However, in many water management circumstances, it becomes necessary to detect both the origin of faecal contamination, for which MST is paramount, and live micro-organisms, for which molecular methods are not suitable. The presence and concentrations of pathogens, general faecal indicators and human-specific MST markers most frequently reported in different areas of the world are summarized in this review.

Assessment of the decay rates of microbial source tracking molecular markers and faecal indicator bacteria from different sources.

AIMS: Evaluate the T90 and compare the decay of different faecal indicator bacteria (FIB) and molecular microbial source tracking (MST) markers of human and animal sources during summer and winter. METHODS AND RESULTS: The persistence of Escherichia coli and enterococci and several MST molecular markers targeting host-specific Bifidobacterium and Bacteroidales species (BifHM, BifCW, BifPL, HF183/BFD, Rum2Bac and Pig2Bac) was assessed at the same time using mesocosms. Dialysis bags filled with diluted wastewater from different sources were kept in an outdoor water tank and monitored regularly to assess the inactivation rates. The T90 values of E. coli by culture methods ranged from 1·52 to 5·69 days in summer and 2·06 to 6·19 days in winter, whereas with qPCR 2·29-4·23 days in summer and 4·17-8·09 days in winter. T90 values for enterocci ranged from 1·15 to 3·10 days in summer and from 3·01 to 5·46 days in winter. Significant differences were observed between faecal sources for both markers. For the MST makers similar T90 values were obtained in summer (1·05-1·91 days), whereas higher variability was observed in winter (2·90-6·12 days). CONCLUSIONS: Different decay rates were observed for the FIB from the different sources, especially for E. coli in ruminant samples. A higher variability among T90 values of the different MST markers in winter was observed, whereas similar T90 values were detected in summer highlighting the stronger effect of environmental parameters during this season. SIGNIFICANCE AND IMPACT OF THE STUDY: The diverse inactivation rates observed in bacteria from different faecal sources have implications when these rates are used to model faecal pollution in water. The use of FIBT90 of different sources is essential to develop reliable predictive models. Since different inactivation of E. coli regarding the source of pollution has been observed, the source of the pollution has to be considered for modelling approaches.

Bluephage: A rapid method for the detection of somatic coliphages used as indicators of fecal pollution in water.

The use of somatic coliphages as indicators of fecal and viral pollution in water and food has great potential due to the reliability, reproducibility, speed and cost effectiveness of methods for their detection. Indeed, several countries already use this approach in their water management policies. Although standardized protocols for somatic coliphage detection are available, user-friendly commercial kits would facilitate their routine implementation in laboratories. The new method presented here allows detection of up to 1 somatic coliphage in under 3.5 h, well within one working day. The method is based on a modified Escherichia coli strain with knocked-out uidB and uidC genes, which encode the transport of glucuronic acid inside cells, and overexpressing uidA, which encodes the enzyme ß-glucuronidase. The enzyme accumulated in the bacterial cells only has contact with its substrate after cell lysis, such as that caused by phages, since the strain cannot internalize the substrate. When the enzyme is released into the medium, which contains a chromogen analogous to glucuronic acid, it produces a change of color from yellow to dark blue. This microbiological method for the determination of fecal pollution via the detection of culturable microorganisms can be applied to diverse sample types and volumes for qualitative (presence/absence) and quantitative analysis and is the fastest reported to date.

Effect of hygienization treatment on the recovery and/or regrowth of microbial indicators in sewage sludge.

AIMS: Escherichia coli (EC) is the primary indicator micro-organism in regulations for sewage sludge reuse. The aim of this work was to assess the ability of EC to enter and recover from a viable-but-not-culturable state (VBNC) after sludge hygienization treatments. METHODS AND RESULTS: The persistence of EC, somatic coliphages (SOMCPH), spores of sulphite-reducing clostridia (SRC) and Salmonella spp. was assessed in digested sludge after different pasteurization treatments and storage conditions. Pasteurization at 55°C produced EC-injured cells that were resuscitated during the first 24 h. Different sludge treatments altered the inactivation kinetics of EC, while SOMCPH and SRC did not resuscitate and showed lower die-off than EC. No regrowth was observed in stored sludge for up to 60 days. CONCLUSIONS: EC monitoring by culturable methods is not by itself a suitable method for assessing the hygienization achieved in sludge as EC can enter into VBNC from which it can recover during the first hours of storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulations should indicate the time when monitoring of EC should be performed to avoid the period when EC can resuscitate from VBNC or add alternative microbial indicators, such as SOMCPH, which do not have a VBNC state.

Hygienization performances of innovative sludge treatment solutions to assure safe land spreading.

The present research aims at the evaluation of the hygienization performances of innovative sludge treatment processes applied for the separated treatment of secondary sludge. Namely, two digestion pretreatments (sonication and thermal hydrolysis) and two sequential biological processes (mesophilic/thermophilic and anaerobic/aerobic digestion) were compared to the mesophilic (MAD) and thermophilic anaerobic digestion (TAD). Microbial indicators (Escherichia coli, somatic coliphages and Clostridium perfringens spores) and pathogens (Salmonella and enteroviruses), which show different resistances to treatment processes, were monitored in untreated and treated sludge. Overall, microbial load in secondary sludge was shown to be similar or lower than previously reported in literature for mixed sludge. Notably, the anaerobic/aerobic digestion process increased the removal of E. coli and somatic coliphages compared to the simple MAD and always achieved the hygienization requirement (2-log-unit removal of E. coli) proposed by EU Commission in the 3rd Working Document on sludge (April 2000) for the use of treated sludges in agriculture with restriction on their application. The microbial quality limits for the unrestricted use of sludge in agriculture (no Salmonella in 50 g wet weight (WW) and E. coli <500 CFU/g) were always met when thermal digestion or pretreatment was applied; however, the required removal level (6-log-unit removal of E. coli) could not be assessed due to the low level of this microorganism in raw sludge. Observed levels of indicator removal showed a higher resistance of viral particles to thermal treatment compared with bacterial cells and confirmed the suitability of somatic coliphages as indicators in thermal treatment processes.

EU policy on sewage sludge utilization and perspectives on new approaches of sludge management.

This paper presents the current sewage sludge legislation in Europe and expected developments regarding the coming directives on the application of the "End-of-waste" criteria and on fertilizers. Discussion on sludge production and processing is also included. The Directive 86/278 has regulated the use in agriculture of residual sludge from domestic and urban wastewater. After 1986, this directive was transposed in the different member state legislation and currently the national limit values on heavy metals, some organic micropollutants and pathogens are placed in a rather wide range. This seems the inevitable consequence of different attitudes towards sludge management practices in the member states. The discussion by the European Joint Research Center (JRC) in Seville regarding application of end-of-waste criteria for compost and digestate has produced a final document (IPTS 2014) where sludge was excluded from the organic wastes admitted for producing an end-of-waste compost. Sludge processing in Europe seems addressed to different goals: sludge minimization, full stabilization and hygienization by thermal hydrolysis processes before anaerobic digestion, and on-site incineration by fluidized bed furnace. Thermophilic anaerobic digestion was applied with success on the Prague WWTP with a preliminary lysimeter centrifugation. Coming techniques, like wet oxidation and pyrolysis, are applied only on very few plants.

Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms.

AIMS: To assess the diversity in production of acylated homoserine lactones (AHLs) among Vibrio spp and related species. METHODS AND RESULTS: A total of 106 isolates, with representatives of 28 Vibrio spp and related species, were investigated for the production of AHLs. For this, a rapid method for the screening of AHLs was developed based on the use of bacterial biosensors using a double-layer microplate assay. At least one bacterial biosensor was activated in 20 species, Agrobacterium tumefaciens being the most frequently activated biosensor. One isolate of Vibrio anguillarum, Vibrio rotiferianus and Vibrio metschnikovii activated the Chromobacterium violaceum biosensor, which is not common among the Vibrionaceae family. For those species with more than one isolate, the biosensor activation profile was the same except for two species, V. anguillarum and V. metschnikovii, which varied among the different isolates. CONCLUSIONS: AHL production was observed in the majority of the studied species, with a diverse biosensor activation profile. SIGNIFICANCE AND IMPACT OF THE STUDY: The high diversity in AHL production is in consistence with the high diversity in ecological niches of the Vibrionaceae family. The absence of AHL detection in eight species warrants further work on their quorum-sensing systems.

Persistence and diversity of faecal coliform and enterococci populations in faecally polluted waters.

AIM: To assess the persistence and diversity of faecal bacterial populations (faecal coliforms and enterococci) that have recently been included in microbial source tracking (MST) predictive models. METHODS AND RESULTS: The analysed bacterial populations included members of the enterococci group (ENT) [Enterococcus faecium (FM), Enterococcus faecalis (FS) and Enterococcus hirae (HIR)] and the faecal coliform group (FC) [diverse Escherichia coli phenotypes (ECP) and cellobiose-negative faecal coliforms (CNFC)]. The inactivation of these distinct groups was monitored over time on-site in river by biochemical fingerprinting, and diversity indices were calculated. Among the different analysed species belonging to the ENT group, HIR persisted longer and was able to replicate in the environment at a higher rate. On the other hand, ECP and NCFC showed a similar persistence throughout the different seasons. The diversity index (Di) for FC increased substantially in the summer after 96 h to a maximum value of 0·96. On the other hand, the Di for ENT diminished over the same period to a value of 0·86, suggesting a different persistence for the different species integrating this group. CONCLUSIONS: The persistence of ECP, CNFC, FM and FS in the aquatic environment is high, particularly for the members of the FC and in the summer season. On the contrary, HIR is able to replicate in the environment at a high rate even in winter, and therefore, its inclusion in MST predictive models is discouraged. SIGNIFICANCE AND IMPACT OF THE STUDY: ECP, CNFC, FMFS and HIR have been proposed as additional variables in MST predictive models. However, the different persistence of HIR compared with the other variables should be taken into account for the development of such models.

Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality.

Molecular methods based on nucleic acid recognition and amplification are valuable tools to complement and support water management decisions. At present, these decisions are mostly supported by the principle of end-point monitoring for indicators and a small number of selected measured by traditional methods. Nucleic acid methods show enormous potential for identifying isolates from conventional culture methods, providing data on cultivable and noncultivable micro-organisms, informing on the presence of pathogens in waters, determining the causes of waterborne outbreaks, and, in some cases, detecting emerging pathogens. However, some features of water microbiology affect the performance of nucleic acid-based molecular techniques and thus challenge their suitability for routine water quality control. These features include the variable composition of target water samples, the generally low numbers of target micro-organisms, the variable water quality required for different uses and the physiological status or condition of such micro-organisms. The standardization of these molecular techniques is also an important challenge for its routine use in terms of accuracy (trueness and precision) and robustness (reproducibility and reliability during normal usage). Most of national and international water regulations recommend the application of standard methods, and any new technique must be validated respect to established methods and procedures. Moreover, molecular methods show a high cost-effectiveness value that limits its practicability on some microbial water analyses. However, new molecular techniques could contribute with new information or at least to supplement the limitation of traditional culture-based methods. Undoubtedly, challenges for these nucleic acid-based methods need to be identified and solved to improve their feasibility for routine microbial water monitoring.

The persistence of bifidobacteria populations in a river measured by molecular and culture techniques.

AIMS: To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR). METHODS AND RESULTS: Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T(90) values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB. CONCLUSIONS: Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T(90) when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST).

Resistance of faecal coliforms and enterococci populations in sludge and biosolids to different hygienisation treatments.

The composition of the most abundant facultative anaerobic bacteria populations [faecal coliforms (FC) and enterococci (ENT)] in sludge can be modified after different treatments. These involve the disposal or reuse of sludge and include: anaerobic digesters, incineration, composting, pasteurization and lime treatments. In this study, three treatment types (mesophilic anaerobic digestion, composting and pasteurization) were compared in terms of their ability to reduce both bacterial populations. The diversity and any changes in composition of main phenotypic groups for both populations were also analyzed. Mesophilic anaerobic digestion (MAD) was carried out at 35 degrees C for 20 days. Digested sludge was then dehydrated by centrifugation at 2,500 rpm. Composting (COM) was performed at 55 degrees C with windrow phases. Pasteurization was assayed at 60 degrees C for 90 min (P60), at 80 degrees C for 60 min (P80). A 1-1.5 log unit reduction was observed for FC, and 1 log unit reduction was noted for ENT by MAD treatment. In composting, this reduction proved higher for FC than for ENT (6 log and 3-4 log units, respectively). Optimal pasteurization was obtained at 80 degrees C for 60 min, resulting in a 5 log unit reduction for FC and a 2 log unit reduction for ENT. High diversity indices (Di) for both bacterial populations were detected both before and after implementation of the different treatments. Analyses of the population's similarity provided that FC were diverse both before and after COM, P60 and P80 treatments. However, no differences were observed on the composition of ENT populations after the different treatments assayed.

Comparison of Vibrio spp. populations found in seawater, in exhibition aquaria, in fish intestine and in fish feed.

AIMS: Vibrio populations in the seawater supply and the water of seven exhibition aquaria that simulate various Mediterranean and tropical ecosystems were compared. The similarity of Vibrio populations in the intestine of various fish species, feed and water was examined. Resistance to the antibiotics used in fish health management was analysed for the dominant Vibrio isolates. METHODS AND RESULTS: Samples were collected for 1 year from seven exhibition tanks reproducing different ecosystems. The diversity and population similarity among vibrios were determined using a miniaturized biochemical phenotyping method. Similar Vibrio populations were found in the water supply and in the water of the Mediterranean ecosystems. However, different Vibrio populations were found in the water of tanks with tropical ecosystems. Vibrio populations in the water seemed to have a greater effect on the composition of intestinal Vibrio populations than those in feed. No resistance to antibiotics was observed, indicating their appropriate use for health management. CONCLUSIONS: Water characteristics have a greater impact on the composition of Vibrio populations in aquaria and fish intestinal microbiota than bacteria in feed. SIGNIFICANCE AND IMPACT OF THE STUDY: The microbiological monitoring water could provide valuable information for managing the health of exhibition aquaria.

Identification of Vibrio spp. with a set of dichotomous keys.

AIMS: To define a binary biochemical key for the identification of all recognized Vibrio spp. METHODS AND RESULTS: A matrix of phenotypical results was developed based on the previous taxonomical studies and the first description manuscripts. A unification of results from various sources was also performed to integrate different taxonomical studies within the same data matrix. Established criteria for selecting the optimal set of tests yielded the highest discrimination, as well as the lowest number of tests. An initial identification key was defined using arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase tests, as well as defining eight different clusters. This key leads each cluster to a secondary key for species identification. Most of Vibrio spp. presented an identification threshold of 100%. CONCLUSIONS: A new set of biochemical keys has been determined provides a scheme for the rapid identification of clinical and environmental species of Vibrio. No more than 14 are needed for even the most complicated identifications. This newly defined set of keys updates and improves similar findings published in previous studies. SIGNIFICANCE AND IMPACT OF THE STUDY: These biochemical keys are designed for use in routine applications, particularly in environmental and clinical studies involving a high number of isolates.

Characterization of bacterial coliform occurrences in different zones of a drinking water distribution system.

AIMS: To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. METHODS AND RESULTS: Conventional and standardized protocols for identifying enterobacterial populations were applied. Additional tests to confirm isolates were included. Analyses of diversity and population similarity were performed using the Phene Plate System, a miniaturized biochemical phenotyping method. Isolates were identified by the API 20E system in tandem with biochemical phenotyping. A total of 16 576 samples were taken from the water distribution system, with 1416 isolates analysed. A low number of coliform occurrences were observed (2%). Escherichia coli was not detected in either water origin or in Zone 2 samples; however, in Zones 1 and 3 a low number of cases of E. coli were recorded. The percentages of E. coli depended on the identification criteria. Eight biochemical profiles for coliform populations were defined according to the results of the confirmative tests. There was a high diversity among these populations in the three zones studied, although no significant variations in their composition (associated with occurrences in the different zones) were observed. Klebsiella oxytoca was the most commonly detected species irrespective of zone, although seven other enterobacterial genera were also found. CONCLUSIONS: Analysis of the enzymatic activity of beta-glucuronidase or application of the criteria established in the norm ISO 9308-1, in tandem with thermotolerance was needed to evaluate the occurrence of E. coli in the distribution systems. Detected occurrences of bacterial coliforms could be associated with re-growth patterns for specific sampling points in the distribution system. Seasonal differences, independent of the studied zones, were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical phenotyping of bacterial coliforms was shown to be a useful method on the characterization of occurrences in water distribution systems.

Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments.

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.

Combined use of an immunomagnetic separation method and immunoblotting for the enumeration and isolation of Escherichia coli O157 in wastewaters.

AIMS: The detection of Escherichia coli O157:H7 in environmental samples is a human concern. The high persistence of this serotype in the environment suggests that contaminated animal wastewater could act as a potential reservoir. Nevertheless, the high levels of background microflora and cell damage because of environmental stress hamper the isolation of this pathogen without using enrichment methods. This study develops a method for the detection of E. coli and investigates its prevalence in animal and human wastewaters. METHODS AND RESULTS: Incubation of the sample for 1 h 30 min at 37 degrees C in peptone water supplemented with vancomycin and cefsulodin, enhanced the recovery of bacteria whilst ensuring that no growth occurred. Subsequently, a combination of immunomagnetic separation, cefixime-tellurite-sorbitol MacConkey (CT-SMAC) plating and immunoblotting with specific O157 antibodies allowed the detection, enumeration and isolation of E. coli O157 strains in human, swine and cattle wastewaters, which presented values of 0.2, 0.4, and 1.0 log10 ml(-1) units, respectively. Some of the isolates carried genes coding for Shiga toxins, intimin and enterohemolysin. CONCLUSIONS: Escherichia coli O157 is commonly present in animal and human wastewaters. The developed method reduced the high rate of false positives reported for other technical approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The confirmation of serotype by specific immunomethods is necessary to prevent false-positive detection and incorrect enumeration.

Multiplex PCR with 16S rRNA gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution.

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.