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.

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.

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.

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.

Detection, enumeration and isolation of strains carrying the stx2 gene from urban sewage.

Verotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks and reported to carry the shiga toxin genes. Shiga toxins, stx1 and stx2, are important virulence factors of these strains. These genes have been linked to bacteriophages and consequently are susceptible to lateral transmission. To better understand the ecology of these genes a study of the presence of the shiga toxin 2 gene (stx2) among coliform bacteria present in sewage samples was carried out. A procedure based on colony hybridisation was developed for the isolation of enterobacteria carrying this gene. Colony growth on Chromocult agar was transferred to a membrane and hybridised with a gene specific probe. The procedure allowed detection of about one colony carrying the gene among around 1,000 faecal coliform colonies. The numbers of bacteria carrying the gene in sewage were also estimated by PCR indicating that the numbers of bacteria carrying the stx2 gene were about 1/1,000 faecal coliforms. The detected numbers by both methods were similar. Positive colony hybridisation was detected in four sewage origins. Fifty-two colonies showing positive signal were isolated from the Chromocult agar plates, confirmed to be stx2 positive by PCR and phenotypically characterised. Results of the characterisation showed certain diversity among the isolates even in isolates from the same sample. Most of these isolates would not have been isolated with the methods regularly used for the isolation of E. coli 0157:H7 strains. The method will allow study of the numbers and characteristics of bacteria carrying the stx2 gene in different water environments and isolate them in order to determine their role in the spread of the gene.