6 research outputs found
Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR
We
evaluated the ability of chip-based digital PCR (dPCR) to quantify
enterococci, the fecal indicator recommended by the United States
Environmental Protection Agency (USEPA) for water-quality monitoring.
dPCR uses Poisson statistics to estimate the number of DNA fragments
in a sample with a specific sequence. Underestimation may occur when
a gene is redundantly encoded in the genome and multiple copies of
that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted
using two commercial DNA extraction kits, we confirmed that dPCR could
discern individual copies of the redundant 23s rRNA gene in the enterococcal
genome. dPCR quantification was accurate when compared to the nominal
concentration inferred from fluorometer measurements (linear regression
slope = 0.98, intercept = 0.03, <i>R</i><sup>2</sup> = 0.99,
and <i>p</i> value <0.0001). dPCR quantification was
also consistent with quantitative PCR (qPCR) measurements as well
as cell counts for BioBall reference standard and 24 environmental
water samples. qPCR and dPCR quantification of enterococci in the
24 environmental samples were significantly correlated (linear regression
slope =1.08, <i>R</i><sup>2</sup> of 0.96, and <i>p</i> value <0.0001); the group mean of the qPCR measurements was 0.19
log units higher than that of the dPCR measurements. At environmentally
relevant concentrations, dPCR quantification was more precise (i.e.,
had narrower 95% confidence intervals than qPCR quantification). We
observed that humic acid caused a similar level of inhibition in both
dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than
qPCR. Inhibition of dPCR was partially relieved when the number of
thermal cycles was increased. Based on these results, we conclude
that dPCR is a viable option for enumerating enterococci in ambient
water
Application of Phylogenetic Microarray Analysis to Discriminate Sources of Fecal Pollution
Conventional methods for fecal source tracking typically
use single
biomarkers to systematically identify or exclude sources. High-throughput
DNA sequence analysis can potentially identify all sources of microbial
contaminants in a single test by measuring the total diversity of
fecal microbial communities. In this study, we used phylogenetic microarray
analysis to determine the comprehensive suite of bacteria that define
major sources of fecal contamination in coastal California. Fecal
wastes were collected from 42 different populations of humans, birds,
cows, horses, elk, and pinnipeds. We characterized bacterial community
composition using a DNA microarray that probes for 16S rRNA genes
of 59 316 different bacterial taxa. Cluster analysis revealed
strong differences in community composition among fecal wastes from
human, birds, pinnipeds, and grazers. Actinobacteria, Bacilli, and
many Gammaproteobacteria taxa discriminated birds from mammalian sources.
Diverse families within the Clostridia and Bacteroidetes taxa discriminated
human wastes, grazers, and pinnipeds from each other. We found 1058
different bacterial taxa that were unique to either human, grazing
mammal, or bird fecal wastes. These OTUs can serve as specific identifier
taxa for these sources in environmental waters. Two field tests in
marine waters demonstrate the capacity of phylogenetic microarray
analysis to track multiple sources with one test
Decay of Coliphages in Sewage-Contaminated Freshwater: Uncertainty and Seasonal Effects
Understanding the
fate of enteric viruses in water is vital for
protection of water quality. However, the decay of enteric viruses
is not well characterized, and its uncertainty has not been examined
yet. In this study, the decay of coliphages, an indicator for enteric
viruses, was investigated in situ under both sunlit and shaded conditions
as well as in summer and winter. The decay rates of coliphages and
their uncertainties were analyzed using a Bayesian approach. The results
from the summer experiments revealed that the decay rates of somatic
coliphages were significantly higher in sunlight (1.29 ± 0.06
day<sup>–1</sup>) than in shade (0.96 ± 0.04 day<sup>–1</sup>), but the decay rates of male-specific (F+) coliphages were not
significantly different between sunlight (1.09 ± 0.09 day<sup>–1</sup>) and shaded treatments (1.11 ± 0.08 day<sup>–1</sup>). The decay rates of both F+ coliphages (0.25 ±
0.02 day<sup>–1</sup>) and somatic coliphages (0.12 ±
0.01 day<sup>–1</sup>) in winter were considerably lower than
those in summer. Temperature and chlorophyll a (chla) concentration
varied significantly (<i>p</i> < 0.001) between the two
seasons, suggesting that these parameters might be important contributors
to the seasonal variation of coliphage decay. Additionally, the Bayesian
approach provided full distributions of decay rates and reduced the
uncertainty, offering useful information for comparing decay rates
under different conditions
A Bottom-Up Approach to Dual Shape-Memory Effects
This
study demonstrates how to successfully bridge the gap between
nanoscale shape-memory function and macroscale motion using a bottom-up
approach. This was achieved by first fabricating a photoswitchable
surface-molecular-imprinted layer-by-layer (LbL) film capable of memorizing
the shape and size of template molecules when illuminated. This photoswitch
was built on the fundamental supramolecular interaction between an
α-cyclodextrin-modified template acting as a photosocket and
an azobenzene-modified polyÂ(acrylic acid) photoplug. Corresponding
patterns applied by cover-printing and wet photolithography were used
to illustrate the stability of the binding sites; a simple and clean
method was developed for removing the template–dye by UV irradiation.
A functional fusion of nanoimprints and macroscopic materials was
subsequently established by applying LbL coating technology to polyÂ(d,l-lactic acid) (PDLLA) modified to have a shape-memory
effect. Macroscopic changes in shape were found to cause deformation
of recognition cavities in terms of their shape and size, thereby
enabling us to visualize the effect of the specific adsorption behavior
toward template–dye on a patterned PDLLA sheet. The rapid swelling
and surface erosion of PDLLA also revealed that an increase in the
number of deposited layers can significantly affect the interfacial
properties of both the substrate and LbL film. It is believed that
such novel designs and methods should prove useful for the development
of multifunctional biomaterials
Solar Inactivation of Enterococci and <i>Escherichia coli</i> in Natural Waters: Effects of Water Absorbance and Depth
The decay of sewage-sourced <i>Escherichia coli</i> and
enterococci was measured at multiple depths in a freshwater marsh,
a brackish water lagoon, and a marine site, all located in California.
The marine site had very clear water, while the waters from the marsh
and lagoon contained colored dissolved organic matter that not only
blocked light but also produced reactive oxygen species. First order
decay rate constants of both enterococci and <i>E. coli</i> were between 1 and 2 d<sup>–1</sup> under low light conditions
and as high as 6 d<sup>–1</sup> under high light conditions.
First order decay rate constants were well correlated to the daily
average UVB light intensity corrected for light screening incorporating
water absorbance and depth, suggesting endogenous photoinactivation
is a major pathway for bacterial decay. Additional laboratory experiments
demonstrated the presence of colored dissolved organic matter in marsh
water enhanced photoinactivation of a laboratory strain of <i>Enterococcus faecalis</i>, but depressed photoinactivation of
sewage-sourced enterococci and <i>E. coli</i> after correcting
for UVB light screening, suggesting that although the exogenous indirect
photoinactivation mechanism may be active against <i>Ent. faecalis,</i> it is not for the sewage-source organisms. A simple linear regression
model based on UVB light intensity appears to be a useful tool for
predicting inactivation rate constants in natural waters of any depth
and absorbance
Interlaboratory Comparison of Real-Time PCR Protocols for Quantification of General Fecal Indicator Bacteria
The application of quantitative real-time PCR (qPCR)
technologies
for the rapid identification of fecal bacteria in environmental waters
is being considered for use as a national water quality metric in
the United States. The transition from research tool to a standardized
protocol requires information on the reproducibility and sources of
variation associated with qPCR methodology across laboratories. This
study examines interlaboratory variability in the measurement of enterococci
and <i>Bacteroidales</i> concentrations from standardized,
spiked, and environmental sources of DNA using the Entero1a and GenBac3
qPCR methods, respectively. Comparisons are based on data generated
from eight different research facilities. Special attention was placed
on the influence of the DNA isolation step and effect of simplex and
multiplex amplification approaches on interlaboratory variability.
Results suggest that a crude lysate is sufficient for DNA isolation
unless environmental samples contain substances that can inhibit qPCR
amplification. No appreciable difference was observed between simplex
and multiplex amplification approaches. Overall, interlaboratory variability
levels remained low (<10% coefficient of variation) regardless
of qPCR protocol