Journal of Applied
Microbiology
Volume 101 Page 977 - November 2006
doi:10.1111/j.1365-2672.2006.03018.x
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| Volume 101 Issue
5
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| ORIGINAL ARTICLE
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| Evaluation of
novel fluorogenic substrates for the detection of glycosidases in Escherichia
coli and enterococci
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| J.D. Perry1,
A.L. James2,
K.A. Morris1,
M. Oliver1,
K.F. Chilvers1,
R.H. Reed2
and F.K. Gould1
|
Aims: Enzyme substrates based
on 4-methylumbelliferone are widely used for the detection of Escherichia
coli and enterococci in water, by detection of β-glucuronidase
and β-glucosidase activity respectively. This study aimed to synthesize
and evaluate novel umbelliferone-based substrates with improved sensitivity
for these two enzymes.
Methods and Results: A novel β-glucuronide
derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized
and compared with 4-methylumbelliferyl-β-d-glucuronide (MUG) using
42 strains of E. coli in a modified membrane lauryl sulfate broth.
Over 7 h of incubation, the fluorescence generated from the hydrolysis
of CMUG by E. coli was over twice that from MUG, and all of the
38 glucuronidase-positive strains generated a higher fluorescence with
CMUG compared with MUG. Neither substrate caused inhibition of bacterial
growth in any of the tested strains. Four β-glucosidase substrates
were also synthesized and evaluated in comparison with 4-methylumbelliferyl-β-d-glucoside
(MU-GLU) using 42 strains of enterococci in glucose azide broth. The four
substrates comprised β-glucoside derivatives of umbelliferone-3-carboxylic
acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl,
ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found
to be superior to MU-GLU for the detection of enterococci, especially after
18 h of incubation, while umbelliferone-3-carboxylic acid-β-d-glucoside
was inferior. However, the variability in detectable β-glucosidase
activity among the different strains of enterococci in short-term assays
using the three carboxylate esters (7 h incubation) may compromise
their use for rapid detection and enumeration of these faecal indicator
bacteria.
Conclusions: The β-glucuronidase
substrate CMUG appears to be a more promising detection system than the
various β-glucosidase substrates tested.
Significance and Impact of the Study: The
novel substrate CMUG showed enhanced sensitivity for the detection of β-glucuronidase-producing
bacteria such as E. coli, with a clear potential for application
in rapid assays for the detection of this indicator organism in natural
water and other environmental samples. |
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Journal of Applied
Microbiology
Volume 101 Page 1015 - November 2006
doi:10.1111/j.1365-2672.2006.03011.x
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| Volume 101 Issue
5
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| ORIGINAL ARTICLE
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| F+RNA
coliphage typing for microbial source tracking in surface waters
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| J. Stewart-Pullaro1,2,
J.W. Daugomah1,
D.E. Chestnut3,
D.A. Graves3,
M.D. Sobsey2
and G.I. Scott1
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Aims: The utility of coliphages
to detect and track faecal pollution was evaluated using South Carolina
surface waters that exceeded State faecal coliform standards.
Methods and Results: Coliphages
were isolated from 117 surface water samples by single agar layer (SAL)
and enrichment presence/absence (EP/A) methods. Confirmed F+RNA
coliphages were typed for microbial source tracking using a library-independent
approach. Concentrations of somatic coliphages using 37 and 44·5°C incubation
temperatures were found to be significantly different and the higher temperature
may be more specific for faecal contamination. The EP/A technique detected
coliphages infecting Escherichia coli Famp in 38 (66%) of the 58
surface water samples negative for F+ coliphages by the SAL
method. However, coliphages isolated by EP/A were found to be less representative
of coliphage diversity within a sample. Among the 2939 coliphage isolates
tested from surface water and known source samples, 813 (28%) were found
to be F+RNA. The majority (94%) of surface water F+RNA
coliphage isolates typed as group I. Group II and/or III viruses were identified
from 14 surface water stations, the majority of which were downstream of
wastewater discharges. These sites were likely contaminated by human-source
faecal pollution.
Conclusions: The results suggest
that faecal contamination in surface waters can be detected and source
identifications aided by coliphage analyses.
Significance and Impact of the Study: This
study supports the premise that coliphage typing can provide useful, but
not absolute, information to distinguish human from animal sources of faecal
pollution. Furthermore, the comparison of coliphage isolation methods detailed
in this study should provide valuable information to those wishing to incorporate
coliphage detection into water quality assessments. |
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Journal of Applied
Microbiology
Volume 101 Page 1123 - November 2006
doi:10.1111/j.1365-2672.2006.02997.x
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| Volume 101 Issue
5
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| ORIGINAL ARTICLE
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| Human astroviruses
in raw sewage samples in Hungary
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| E. Meleg1,2,
F. Jakab1,2,
B. Kocsis2,
K. Banyai1,2,
B. Melegh3
and G. Szucs1,2
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Aims: Routine procedures for monitoring
viruses in water samples have not been drawn up for the water-microbiology
screening panel. Enteric viruses, including astroviruses, are able to persist
under environmental conditions and may cause public health problems by
contaminating natural and drinking water resources. The aim of this study
was to detect human astroviruses (HAstVs) from raw wastewater samples.
Methods and Results: To obtain
data on whether human astroviruses are shed in the environment, 35 raw
sewage samples from 22 sewage plants in different regions of Baranya County,
Hungary were tested for astrovirus using a polyethylene glycol method for
concentration and a guanidinium thiocyanate–silica procedure for extraction
of viral RNA. Reverse transcription-polymerase chain reaction (RT-PCR)
with HAstV-specific primer pairs was used for amplification and the specificity
of amplicons was confirmed by nucleotide sequencing and phylogenetic analysis.
Among the 35 raw sewage samples, 15 (43%) contained HAstV and by sequence
analysis, 10 genotype HAstV-1 and one genotype HAstV-2 were identified.
Conclusions: The high detection
rate of astroviruses we encountered in this study provide convincing evidence
that HAstVs circulate at a relatively high frequency in the Hungarian population.
No correlation between the standard indicators of faecal pollution and
the presence of HAstVs was found.
Significance and Impact of the Study: Our
study is the first report on detection of HAstV in sewage in Hungary and
suggests that HAstV might be potent indicators of viral pollution in environmental
specimens. |
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Journal of Applied
Microbiology
Volume 101 Page 1188 - November 2006
doi:10.1111/j.1365-2672.2006.02984.x
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| Volume 101 Issue
5
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| ORIGINAL ARTICLE
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| Campylobacter
jejuni inactivation in New Zealand soils
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| C.M. Ross and
A.M. Donnison
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Aim: The study was undertaken
to determine the inactivation rate of Campylobacter jejuni in New
Zealand soils.
Methods and Results: Farm dairy
effluent (FDE) inoculated at c. 105 ml 1
with C. jejuni was applied to intact soil cores at a rate of
2 l m 2.
Four soils were used: Hamilton (granular); Taupo (pumice); Horotiu and
Waihou (allophanic). After FDE application cores were incubated at 10°C
for up to 32 days. For all four soils all the FDE remained within
the cores and at least 99% of C. jejuni were retained in the
top 5 cm. Campylobacter jejuni had declined to the limit of
detection (two C. jejuni 100 g 1)
by 25 days in Hamilton and Taupo soils and by 32 days in Waihou
soil. In contrast, in Horotiu soil the decline was only three orders of
magnitude after 32 days. Simulated heavy rainfall was applied 4 and
11 days after FDE application and only about 1% of the applied C. jejuni
were recovered in leachates.
Conclusions: This study demonstrated
that at least 99% of applied C. jejuni were retained in the
top 5 cm of four soils where they survived for at least 25 days
at 10°C.
Significance and Impact of the Study: Soil
retention of C. jejuni is efficient at FDE application rates
that prevent drainage losses. The low infectious dose of C. jejuni
and its ability to survive up to 25 days have implications for stock
management on dairy farms. |
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