Integrated site-specific quantification of faecal bacteria and detection of DNA markers in faecal contamination source tracking as a microbial risk tracking tool in urban Lake ecosystems

The presence of feacal-derived pathogens in water is responsible for several infectious diseases and deaths worldwide. As a solution, sources of fecal pollution in waters must be accurately assessed, properly determined and strictly controlled. However, the exercise has remained challenging due to the existing overlapping characteristics by different members of faecal coliform bacteria and the inadequacy of information pertaining to the contribution of seasonality and weather condition on tracking the possible sources of pollution. There are continued ef forts to improve the Faecal Contamination Source Tracking(FCST) techniques such as Microbial Source Tracking(MST). This study aimed to make contribution to MST by evaluating the efficacy of combining site specific quantification of faecal contamination indicator bacteria and detection of DNA markers while accounting for seasonality and weather conditions' eff ects in tracking the major sources of faecal contamination in a freshwater system(Donghu Lake, China). The results showed that the use of cyd gene in addition to lacZ and uidA genes differentiates E. coli from other closely related faecal bacteria. The use of selective media increases the pollution source tracking accuracy. BSA addition boosts PCR detection and increases FCST efficiency. Seasonality and weather variability also influence the detection limit for DNA markers.

Sanitary Status of 44 Hog Manures in Brittany： Comparison of the Effectiveness of Manure Treatments Based on the Levels of Indicator Bacteria and Two Pathogenic Bacteria

The hygienic performance of simple storage and biological treatment of manure was evaluated using effluents collected from 44 hog farms across Brittany, France. Analyses were carried out on raw manure, on the sludge stored after biological treatment and on the liquid phase stored in a lagoon after sludge settling or sludge dewatering. The effect of the treatments was evaluated on E. coli, enterococci, Clostridium perfringens, Salmonella and Listeria monocytogenes. Sequential feeding of manure maintained a high level of enteric bacteria in storage tanks regardless the duration of storage, and biological treatment only slightly reduced the concentration of enteric bacteria （average reduction in raw manure and sludge 〈 2.2 log10）. Salmonella and L. monocytogenes were identified in raw manure （50% and 18.2% of samples, respectively）, in sludge （14.8% and 11.1%）, and in lagoons （8.0% and 24%）, suggesting that there is still a sanitary risk after biological treatment of manure. Salmonella Derby and L. monocytogenes serotype 4b each accounted for 50% of the serotypes identified in the samples. There was no correlation between the presence of the two pathogenic bacteria and the size of the herd or the duration of manure storage. However, environmental conditions in the lagoons appeared to favor the presence ofL. monocytogenes and hence a risk of disseminating this pathogen in the environment.

Method of Improving the Bacteriological Monitoring Quality in Water

Due to lack of quantitative standard reference material, bacteriological monitoring has the characteristics of strong professionality and heavy workload. It is necessary to conduct quality control in sampling preparation, sample collection technology, sample preservation and transportation, laboratory equipment, supply quality and monitoring personnel requirements in order to improve the quality of monitoring data and ensure the representativeness, accuracy, precision, comparability and integrity of the monitoring results.

Metagenomic Shotgun Sequencing Provides Prevalence Data for Pathogens, and Source-Tracking Indices Useful in Public Health Risk Assessment of Environmental Waters

State-approved membrane filtration (MF) techniques for water quality assessments were contrasted with metagenomic shotgun sequencing (MSS) protocols to evaluate their efficacy in providing precise health-risk indices for surface waters. Samples from a freshwater receiving pond (ABI-1002) and two upstream storm water ditches (ABI-1003) and (ABI-1004) yielded alarmingly high Fecal coliform MF densities of 220, >2000 and >2000 CFU/100ml respectively. The indicator, Enterococcus bacteria exceeded allowable limits in all but the equipment control (ABI-1001). Using MSS, the relative numerical abundance of pathogenic bacteria, virulence and antibiotic resistance genes revealed the status and potential pollution sources of each ditch. High levels of Shigella sp. (0 (ABI-1001), 4945 (ABI-1002), 55,008 (ABI-1003), and 2221 (ABI-1004) genomic reads/100ml) correlated with virulence genes and antibiotic resistance genes found in fecal samples for ABI1003 and not ABI1004. Traditional culture methods (TCM) showed possible fecal contamination in two of the four samples, and no contamination in the others. MSS clearly distinguished between fecal and environmental bacteria contamination sources, and pinpointed actual risks from pathogens. Our data underscore the potential utility of MSS in precision risk assessment for public and biodiversity health and tracking of environmental microbiomes shifts by field managers and policy makers.

Repeated hydrogen peroxide dosing briefly reduces cyanobacterial blooms and microcystin while increasing fecal bacteria indicators in a eutrophic pond

This study explored the effects of H_(2)O_(2)on Cyanobacteria and non-target microbes using fluorometry,microscopy,flow cytometry,and high throughput DNA sequencing of the 16S rRNA gene during a series of mesocosm and whole-ecosystem experiments in a eutrophic pond in NY,USA.The addition of H_(2)O_(2)(8 mg/L)significantly reduced Cyanobacteria concentrations during a majority of experiments(66%;6 of 9)and significantly increased eukaryotic green and unicellular brown algae in 78%and 45%of experiments,respectively.While heterotrophic bacteria declined significantly following H_(2)O_(2)addition in all experiments,bacteria indicative of potential fecal contamination(Escherichia coli,Enterococcus,fecal coliform bacteria)consistently and significantly increased in response to H_(2)O_(2),evidencing a form of‘pollution swapping’.H_(2)O_(2)more effectively reduced Cyanobacteria in enclosed mesocosms compared to whole-ecosystem applications.Ten whole-pond H_(2)O_(2)applications over a twoyear period temporarily reduced cyanobacterial levels but never reduced concentrations below bloom thresholds and populations always rebounded in two weeks or less.The bacterial phyla of Cyanobacteria,Actinobacteria,and Planctomycetes were the most negatively impacted by H_(2)O_(2).Microcystis was always reduced by H_(2)O_(2),as was the toxin microcystin,but Microcystis remained dominant even after repeated H_(2)O_(2)treatments.Although H_(2)O_(2)favored the growth of eukaryotic algae over potentially harmful Cyanobacteria,the inability of H_(2)O_(2)to end cyanobacterial blooms in this eutrophic waterbody suggests it is a non-ideal mitigation approach in high biomass ecosystems and should be used judiciously due to potential negative impacts on non-target organisms and promotion of bacteria indicative of fecal contamination.

Comparison of two methods for detection of fecal indicator bacteria used in water quality monitoring of the Three Gorges Reservoir

Scientifically sound methods to rapidly measure fecal indicator bacteria are important to ensure safe water for drinking and recreational purposes.A total of 200 water samples obtained from the Three Gorges Reservoir during three successive one-year study periods（October 2009 to September 2012） were analyzed using multiple-tube fermentation（MTF）and most probable numbers combined with polymerase chain reaction（MPN-PCR）.The MPN-PCR method was found to be significantly more sensitive than the MTF method for detecting Escherichia coli and Enterococcus spp.,and of equal sensitivity for detecting total coliforms when all surface water samples were grouped together.The two analytical methods had a strong,significant relationship,but MPN-PCR took only 12-18 hr,compared with the 3-8 days needed using the MTF method.Bacterial concentrations varied per sampling site but were significantly lower in the mainstream of the Yangtze River than those in the backwater areas of tributaries.The water quality of 85.8% of water samples from the mainstream was suitable for use as a centralized potable water source,while the water quality of 52.5% of water samples from the backwater areas was unsuitable for recreational activities.Relationships between fecal indicator bacteria showed significant correlation（r = 0.636-0.909,p 〈 0.01,n = 200）,while a weak but significant correlation was found between fecal indicators and water turbidity,water temperature,daily inflow,and total dissolved solids（r = 0.237-0.532,p 〈 0.05,n = 200）.The study indicated that MPN-PCR is a rapid and easily performed deoxyribonucleic acid（DNA）-based method for quantitative detection of viable total coliforms,E.coli,and Enterococcus spp.in surface water.

Evaluation of the relationship between two different methods for enumeration fecal indicator bacteria: Colony-forming unit and most probable number

Most probable number （MPN） and colony-forming unit （CFU） estimates of fecal indicator bacteria （FIB） concentration are common measures of water quality in aquatic environments. Thus, FIB intensively monitored in Yeongsan Watershed in an attempt to compare two different methods and to develop a statistical model to convert from CFU to MPN estimates or vice versa. As a result, the significant difference was found in the MPN and CFU estimates. The enumerated Escherichia coli concentrations in MPN are greater than those in CFU, except for the measurement in winter. Especially in fall, E. coli concentrations in MPN are one order of magnitude greater than that in CFU. Contrarily, enterococci bacteria in MPN are lower than those in CFU. However, in general, a strongly positive relationship are found between MPN and CFU estimates. Therefore, the statistical models were developed, and showed the reasonable converting FIB concentrations from CFU estimates to MPN estimates. We expect this study will provide preliminary information towards future research on whether different analysis methods may result in different water quality standard violation frequencies for the same water sample.