Microbial abundance and community composition in biofilms on in-pipe sensors in a drinking water distribution system.

Collecting biofilm samples from drinking water distribution systems (DWDSs) is challenging due to limited access to the pipes during regular operations. We report here the analysis of microbial communities in biofilm and water samples collected from sensors installed in a DWDS where monochloramine is used as a residual disinfectant. A total of 52 biofilm samples and 14 bulk water samples were collected from 17 pipe sections representing different water ages. Prokaryotic genome copies (bacterial and archaeal 16S rRNA genes, Mycobacterium spp., ammonia-oxidizing bacteria (AOB), and cyanobacteria) were quantified with droplet digital PCR, which revealed the abundance of these genes in both biofilm and water samples. Prokaryotic 16S rRNA gene sequencing analysis was carried out for a subset of the samples (12 samples from four sites). Mycobacterium and AOB species were dominant in the DWDS sections with low water age and sufficient residual monochloramine, whereas Nitrospira species (nitrite-oxidizing bacteria) dominated in the sections with higher water age and depleted monochloramine level, suggesting the occurrence of nitrification in the studied DWDS. The present study provides novel information on the abundance and identity of prokaryotes in biofilms and water in a full-scale operational DWDS.

Variably improved microbial source tracking with digital droplet PCR.

This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85-1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74-100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ±â€¯2.8 for B. theta and 18.7 ±â€¯10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.

Bacteroidales markers for microbial source tracking in Southeast Asia.

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively.

Increasing capacity for environmental engineering in Salta, Argentina.

BACKGROUND: The Fogarty International Center (FIC) of the United States National Institutes of Health includes the International Training and Research in Environmental and Occupational Health (ITREOH) Program. The "International Training Program in Environmental Toxicology and Public Health" Center, funded in 2002 is based at the University of California, Davis, and is part of the ITREOH group of Centers. It has major efforts focused at the public universities in Montevideo, Uruguay, and Salta, Argentina. RESULTS: Training and research efforts in Salta begun in 2005 in the College of Engineering. A donated used real-time PCR machine was the starting point and the initial FIC support was instrumental to face other problems including physical space, research projects and grants, trainees, training, networking, and distractions/opportunities in order to develop local capacities in Environmental Engineering using modern methodology. After 6 years of successful work, the Salta center has become a reference Center in the field, and is still growing and consolidating. CONCLUSIONS: This program has had a significant impact locally and regionally. The model used in Argentina could be easily adapted to other fields or types of projects in Argentina and in other developing countries.

Diarrhea and parasitosis in Salta, Argentina.

BACKGROUND: Salta city is the capital of the province with the same name located in the northwest of Argentina. Its great growth over the last decade was not organized and the population expanded to occupy places where water and sanitation were not yet available. Although the Arenales River, crossing the city, receives the impact of point and non-point source pollution, the water is used for many purposes, including domestic in the poorest areas, industrial, and recreational with children as the main users. According to the World Health Organization, an estimated 24% of the global disease burden and 23% of all deaths can be attributed to environmental factors. In particular, an estimated 94% of the diarrheal burden of disease is attributable to environment, and is associated with risk factors such as unsafe drinking water and poor sanitation and hygiene. Chronic diarrhea can be caused by an infection or other etiologies; however, most of the times the etiological agent is not identified. METHODOLOGY: All the cases of diarrhea and parasitosis reported during 2005 in four public health centers of the city of Salta were classified by gender and age, analyzed, and represented geographically to show areas of higher morbidity rates, which were probably related to environmental factors. RESULTS: Water, poor sanitation, and pollution are candidate risk factors. Diarrhea cases showed seasonality, with the highest incidence during late spring and summer, while parasitosis was persistent throughout the year. CONCLUSION: Our spatial analysis permitted us to detect the regions of higher incidence of diarrhea and parasitosis during 2005 in the area of study.