Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic E. coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters

Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamics

The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

BACKGROUND: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. METHODOLOGY/PRINCIPAL FINDINGS: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. CONCLUSIONS/SIGNIFICANCE: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health

On the track for an efficient detection of Escherichia coli in water : A review on PCR-based methods

Ensuring water safety is an ongoing challenge to public health providers. Assessing the presence of fecal contamination indicators in water is essential to protect public health from diseases caused by waterborne pathogens. For this purpose, the bacteria Escherichia coli has been used as the most reliable indicator of fecal contamination in water. The methods currently in use for monitoring the microbiological safety of water are based on culturing the microorganisms. However, these methods are not the desirable solution to prevent outbreaks as they provide the results with a considerable delay, lacking on specificity and sensitivity. Moreover, viable but non-culturable microorganisms, which may be present as a result of environmental stress or water treatment processes, are not detected by culture-based methods and, thus, may result in false-negative assessments of E. coli in water samples. These limitations may place public health at significant risk, leading to substantial monetary losses in health care and, additionally, in costs related with a reduced productivity in the area affected by the outbreak, and in costs supported by the water quality control departments involved. Molecular methods, particularly polymerase chain reaction-based methods, have been studied as an alternative technology to overcome the current limitations, as they offer the possibility to reduce the assay time, to improve the detection sensitivity and specificity, and to identify multiple targets and pathogens, including new or emerging strains. The variety of techniques and applications available for PCR-based methods has increased considerably and the costs involved have been substantially reduced, which together have contributed to the potential standardization of these techniques. However, they still require further refinement in order to be standardized and applied to the variety of environmental waters and their specific characteristics. The PCR-based methods under development for monitoring the presence of E. coli in water are here discussed. Special emphasis is given to methodologies that avoid pre-enrichment during the water sample preparation process so that the assay time is reduced and the required legislated sensitivity is achieved. The advantages and limitations of these methods are also reviewed, contributing to a more comprehensive overview toward a more conscious research in identifying E. coli in water.Diana Mendes (SFRH/BDE/33752/2009) was recipient of a fellowship from the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) and Frilabo, Lda. The authors thank Tatiana Aguiar (Centre of Biological Engineering) for English proofreading, the financial support from the Project "Desenvolvimento de um kit de detecao e quantificacao de E. coli e bacterias coliformes em aguas", Ref. 2009/5787, Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER, the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "Biolnd-Biotechnology and Bioengineering for improved Industrial and processes", REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER

Methodological approaches for studying the microbial ecology of drinking water distribution systems

The study of the microbial ecology of drinking water distribution systems (DWDS) has traditionally been based on culturing organisms from bulk water samples. The development and application of molecular methods has supplied new tools for examining the microbial diversity and activity of environmental samples, yielding new insights into the microbial community and its diversity within these engineered ecosystems. In this review, the currently available methods and emerging approaches for characterising microbial communities, including both planktonic and biofilm ways of life, are critically evaluated. The study of biofilms is considered particularly important as it plays a critical role in the processes and interactions occurring at the pipe wall and bulk water interface. The advantages, limitations and usefulness of methods that can be used to detect and assess microbial abundance, community composition and function are discussed in a DWDS context. This review will assist hydraulic engineers and microbial ecologists in choosing the most appropriate tools to assess drinking water microbiology and related aspects

A somatic coliphage threshold approach to improve the management of activated sludge wastewater treatment plant effluents in resource-limited regions

Versión aceptada para publicaciónEffective wastewater management is crucial to ensure the safety of water reuse projects and 29 effluent discharge into surface waters. Multiple studies have demonstrated that municipal 30 wastewater treatment with conventional activated sludge processes is inefficient for the removal 31 of the wide spectrum of viruses in sewage. In this study, a well-accepted statistical approach was 32 used to investigate the relationship between viral indicators and human enteric viruses during 33 wastewater treatment in a resource-limited region. Influent and effluent samples from five urban 34 wastewater treatment plants (WWTP) in Costa Rica were analyzed for somatic coliphage and 35 human enterovirus, hepatitis A virus, norovirus genotype I and II, and rotavirus. All WWTP 36 provide primary treatment followed by conventional activated sludge treatment prior to 37 discharge into surface waters that are indirectly used for agricultural irrigation. The results 38 revealed a statistically significant relationship between the detection of at least one of the five 39 human enteric viruses and somatic coliphage. Multiple logistic regression and Receiver Operating Characteristic curve analysis identified a threshold of 3.0 ×103 40 (3.5-log10) somatic 41 coliphage plaque forming unit per 100 mL, which corresponded to an increased likelihood of encountering enteric viruses above the limit of detection (>1.83×102 42 virus target/100 mL). 43 Additionally, quantitative microbial risk assessment was executed for famers indirectly reusing 44 WWTP effluent that met the proposed threshold. The resulting estimated median cumulative 45 annual disease burden complied with World Health Organization recommendations. Future 46 studies are needed to validate the proposed threshold for use in Costa Rica and other regions.Universidad de Costa Rica/[]/UCR/Costa RicaNational Science Foundation/[OCE-1566562]/NSF/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí