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Applied and Environmental Microbiology, August 1999, p. 3746-3749, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparison and Recovery of Escherichia coli and
Thermotolerant Coliforms in Water with a Chromogenic Medium
Incubated at 41 and 44.5°C
Jose L.
Alonso,1,*
Adela
Soriano,2
Oscar
Carbajo,3
Inmaculada
Amoros,1 and
Hemda
Garelick3
Instituto de Hidrologia y Medio Natural,
Universidad Politecnica, 46022 Valencia,1
Gamaser S.L., 46014 Valencia,2
Spain, and School of Health Biological and Environmental
Sciences, Middlesex University, London N11 2NQ, United
Kingdom3
Received 10 February 1999/Accepted 19 May 1999
 |
ABSTRACT |
This study compared the performance of a commercial chromogenic
medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate
(CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA)
for enumeration of Escherichia coli and non-E.
coli thermotolerant coliforms (KEC). To establish that we could
recover the maximum KEC and E. coli population, we compared
two incubation temperature regimens, 41 and 44.5°C. Statistical
analysis by the Fisher test of data did not demonstrate any
statistically significant differences (P = 0.05) in
the enumeration of E. coli for the different media (CECC
and CECCP) and incubation temperatures. Variance analysis of data
performed on KEC counts showed significant differences (P = 0.01) between KEC counts at 41 and 44.5°C on
both CECC and CECCP. Analysis of variance demonstrated statistically
significant differences (P = 0.05) in the enumeration
of total thermotolerant coliforms (TTCs) on CECC and CECCP compared
with mLSA. Target colonies were confirmed to be E. coli at
a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4%
when using CECCP incubated at 41°C. The results of this study showed
that CECCP agar incubated at 41°C is efficient for the simultaneous
enumeration of E. coli and KEC from river and marine waters.
| |
TEXT |
The significance of various coliform
organisms in water has been and remains an extensively studied subject.
Since fecal coliforms are not defined taxonomically, Escherichia
coli is the only member species for which standardized data
exists. According to Leclerc et al. (16), the coliform
species of fecal origin and their isolation frequency in human feces
(15) are as follows: E. coli, 100%;
Citrobacter diversus, 70%; Citrobacter
amalonaticus, 70%; Citrobacter freundii, 70%;
Klebsiella pneumoniae, 49%; Klebsiella oxytoca,
49%; Enterobacter cloacae, 9%; and Enterobacter
aerogenes, 9%. The following species will probably be of nonfecal
origin (16): Klebsiella trevisamii,
Enterobacter agglomerans, E. gergoviae, E. sakazakii, Hafnia alvei, Serratia
marcescens, S. liquefaciens, S. marinorubra,
and S. odorifera. Unfortunately, the specificity of fecal
coliforms as indicators of fecal pollution varies considerably depending on the environmental conditions and the presence of industrial effluent (3). Some authors (7, 19)
have suggested that the term "fecal coliforms" should be excluded
from microbiology. "Thermotolerant coliforms" (TTC) is considered
to be a more appropriate description of these organisms (4).
The acronym KEC is introduced in this study to describe the
-galactosidase positive thermotolerant coliforms other than E. coli. On the other hand, E. coli is generally considered a more reliable sanitary indicator (8). Therefore a direct enumeration of E. coli is needed to monitor fecal
contamination in surface waters (20).
Conventional procedures for verification of fecal coliforms as E. coli when fecal contamination is questionable are very laborious and time-consuming (11). The use of media containing
chromogenic and fluorogenic substrates for the enzymes
-galactosidase (LAC) and -glucuronidase (GUD) for simultaneous
detection of coliforms and E. coli is increasing
(23). One such medium, CHROMagarECC, developed by CHROMagar,
simultaneously detects coliforms and E. coli.
Addition of pyruvate to media improves the recovery of injured bacteria
through its action in degrading peroxides and promoting cell recovery
(18). Incubation temperature should also be considered in an
effort to recover the maximum number of fecal coliform strains. Some
authors (17) have indicated that the optimal temperature for
incubation of all fecal coliform strains is 41°C.
This study was designed to evaluate CHROMagarECC (CECC), modified by
the addition of sodium pyruvate (CECCP), for the simultaneous enumeration of E. coli and KEC by the membrane filtration
technique in river and marine waters. To help maximize the recovery of
both E. coli and KEC populations, we incubated the bacteria
at 41 and 44.5°C.
Sampling.
A total of 50 water samples were collected from
different environmental sources in the area of Valencia, Spain. The
water samples were as follows: 7 samples from Turia River (site TR) near the Valencia drinking-water treatment plant, 3 samples from the
Jucar River (site JR), 20 samples of seawater from Malvarrosa beach
(sites M1 and M2), and 20 samples of seawater from Alboraya beach
(sites A1 and A2). All samples were collected in sterile glass bottles,
refrigerated, and assayed within 2 h after collection.
Medium comparison.
All membrane filtration analyses were
carried out in duplicate, and bacterial concentrations were reported as
the mean of these replicates. Specific incubation and enumeration
procedures for each test medium were as follows. The water samples were
diluted, and duplicates of each dilution were filtered through sterile 0.45-µm-pore-size membranes (Whatman, Maidstone, England). One membrane of each set of duplicates was placed on a pre-treated layer of
CECC agar (CHROMagar Microbiology, Paris, France) in a 47-mm-diameter
petri dish. The second membrane of each duplicate pair was placed on
CECC supplemented with 0.05% (wt/vol) sodium pyruvate (Sigma Chemical
Co., St. Louis, Mo.) (CECCP agar). A set of duplicate membranes placed
on CECC and CECCP media was incubated at 41°C, and a second set of
membranes was incubated at 44.5°C. All blue colonies
(LAC+ GUD+) were counted as E. coli,
and all red colonies (LAC+ GUD
) were counted
as KEC. The number of TTCs was calculated as the sum of blue and red
colonies. For comparison, duplicates of each dilution were processed by
a standard membrane filtration method for fecal coliforms
(24). The membranes were placed onto membrane filtration
lauryl sulfate-based medium (mLSB; Oxoid, UNIPATH Ltd., Basingstoke,
England) solidified by adding 1.2% agar (mLSA). The membranes placed
on mLSA were incubated in a water bath at 44.5°C for 24 h, and
duplicate membranes were incubated for 30°C for 4 h
(resuscitation) followed by 44.5°C for 20 h. Fecal coliforms, if
present, appeared as yellow colonies.
Biochemical identification.
A total of 521 colonies from the
most appropriate dilutions of CECCP agar incubated at 41°C were
identified. This medium and the 41°C incubation temperature was
selected for the specificity study because these conditions gave the
best overall recovery of E. coli, KEC, and TTC in our
initial experiments. E. coli colonies (blue), KEC colonies
(red), and nontarget colonies (white) were tested for cytochrome
oxidase activity. E. coli colonies were identified by using
the Microbact 12E system (Medvet Sxcience Pty Ltd., Adelaide,
Australia). KEC and nontarget colonies were identified by using the API
20E system (BioMérieux, Marcy l'Etoile, France).
Statistical analysis.
Results were analyzed by linear
regression to verify the linearity of the relationship between E. coli and KEC isolated on CECCP agar incubated at 41°C. To
examine the medium performance (CECCP agar incubated at 41°C) over a
range of sample types and concentrations, the samples were grouped by
sample site; by E. coli, KEC, and TTC counts; and by
incubation temperatures. Overall significant effects and interaction
effects were tested by two-way analysis of variance ANOVA. Each
procedure category was further separated by one-way ANOVA. When the
P value of the F-test was less than 0.05, a multiple-range
test was used to find which means were significantly different. All
E. coli, KEC, and TTC counts were converted to
log10 values for statistical analysis. All statistical methods were performed with Statgraphics Plus 2.1 software.
Enumeration of E. coli, KEC, and TTC.
The 10 river
water samples tested had E. coli counts ranging from 10 to
2.9 × 103 CFU/100 ml, with an arithmetic mean of
3.3 × 102 CFU/100 ml (CECCP incubated at 41°C). The
40 marine samples analyzed had E. coli counts ranging from 5 to 7.2 × 103 CFU/100 ml with an arithmetic mean of
1.2 × 103 CFU/100 ml. Analysis of variance of the
E. coli data demonstrated no difference between temperatures
and media, with the F test yielding a P value of 0.994 for
river samples and a P value of 0.666 for marine samples. It
should, however, be noted that CECCP incubated at 41°C exhibited the
highest E. coli counts. Ho and Tam (14) found
that the performance of CHROMagar Liquid ECC was comparable to mLSB
plus urea but the chromogenic medium was superior to the conventional
medium in sensitivity and specificity. Sartory (24) reported
that incorporation of sodium pyruvate into mLSA resulted in significant
improvements on membrane filtration recovery of E. coli from
chlorinated drinking-water samples.
Positive correlations (P = 0.01) between the
concentration of E. coli and KEC were found at sites TR
(r = 0.99), M1 (r = 0.96), M2
(r = 0.88), A1 (r = 0.97), and A2
(r = 0.98). At these sites, E. coli
represented on average 22.0 to 35.2% of the TTC population. At site
JR, there was no correlation (r = 
0.15) between
E. coli and KEC. The significant correlation coefficients
obtained between E. coli and KEC at sites TR, M1, M2, A1,
and A2 confirm the fecal origin of KEC at these sites, although KEC at
site JR were probably of nonfecal origin. In our study, E. coli represented a range of 8.8 to 35.2% of the TTC population.
Bordalo (5), studying different types of water ranging from
polluted seawater to unpolluted freshwater, found that E. coli made up 82% of the TTC. However, in this study, E. coli level was compared with TTC counts in samples incubated at
44.5°C. This temperature inhibited the growth of all the coliforms of
nonfecal origin as well as a large number of fecal coliforms
(16). A temperature of 41°C is near the optimum temperature of fecal coliform growth (16, 17).
The 10 river water samples had KEC counts ranging from 10 to 5.1 × 103 CFU/100 ml. The 40 marine water samples analyzed had
KEC counts ranging from 5 to 1.9 × 104 CFU/100 ml.
KEC counts on CECC were compared with KEC counts on CECCP incubated at
both incubation temperatures (41 and 44.5°C). Data from the ANOVA
performed on KEC counts showed significant differences
(P = 0.01) between KEC counts at 41 and 44.5°C both on CECC and on CECCP, with the F test yielding a P value of
0.00001 for river and marine samples. The mean KEC counts obtained with CECCP at 41°C was 6.2 times the mean KEC counts at 44.5°C.
Geldreich (13) suggested that an effort should be made to
optimize the recovery of coliforms while excluding environmental
strains of no significance by investigating incubation temperatures
between 39 and 42°C.
Counts of TTC on CECC and CECCP were compared with counts on mLSA. A
summary of the TTC counts by medium and incubation temperatures
is
given in Table
1. The concentration of
TTC in the marine water
samples was higher; the range of TTC for marine
samples was 10
to 2.6 × 10
4 CFU/100 ml, whereas the
range of TTC for the river samples was
70 to 7.9 × 10
3 CFU/100 ml. The differences between TTC counts obtained
with
the two chromogenic media incubated at 41°C and mLSA at two
incubation
temperatures (4 h at 30°C plus 20 h at 44.5°C, and
incubation
at only 44.5°C) were statistically different as determined
by
ANOVA and the multiple-range test. The use of CECCP and incubation
at 41°C resulted in significantly greater detection of TTC in
river
and marine water samples compared with the use of mLSA in
two
incubation procedures.
Characterization of E. coli, KEC, and nontarget
isolates.
The identities of the three types of colonies
(LAC+ GUD, LAC+ GUD+,
and LAC GUD) on CECCP agar incubated at
41°C are shown in Table 2 (river samples) and Table 3 (marine samples).
The mean confirmation rate of target blue colonies was 91.3% in river
water samples and 92% in marine water samples. Other
investigators
(
6) have shown a good correlation between GUD
detection and
E. coli when using chromogenic media with indoxyl
derivatives. The
E. coli false-positive rate was 8.7% (9 of
104
colonies) in river samples and 8.0% (7 of 88 colonies) in marine
samples. In our study, GUD activity was found in some strains
of
Citrobacter freundii,
Enterobacter agglomerans,
and
Klebsiella pneumoniae. The GUD
colonies
which were confirmed to be
E. coli were found at rates
of
2.5 and 3.1% for river and marine waters, respectively. Four
LAC
GUD
E. coli strains at
41°C were further cultured in CECCP at 37°C
for 24 h to
determine whether the expression of LAC and GUD was
temperature
dependent. The four
E. coli strains showed LAC production
but not GUD production at 37°C (LAC
+ GUD
).
Schets and Havelaar (
25) found that although the GUD
reaction
was specific for
E. coli with
4-methylumbelliferyl-
-
D-glucuronide
(MUG) substrate, an
average of 14% of these strains were GUD negative
at 44°C; of these
strains, 24% showed GUD activity at 37°C. Alonso
et al.
(
2) found that false-negative
E. coli colonies
occurred
less frequently at 37°C than at 44.5°C. However, other
authors
(
10) found more false-negative colonies at the lower
incubation
temperature of 35 to 37°C. Olson et al. (
21)
suggested that
injury, impermeability, lack of gene expression, or
nonutilization
of the GUD substrate may all account for the
GUD
phenotype in
E. coli.
The criteria for the identification of KEC of likely fecal origin used
for the evaluation of CECCP in this study is based
on the work by
Leclerc et al. (
16). The percentage of KEC of
likely fecal
origin isolated on CECCP agar incubated at 41°C was
higher in marine
water than in river water. Of the 125 LAC
+
GUD
colonies, 90 (72.0%) were confirmed as KEC of likely
fecal origin
(21
E. agglomerans strains, 2
E. sakazakii strains, 2
Hafnia alvei strains, 1
Serratia liquefaciens strain, and 1
S. marcescens
strain
were not included) in river waters, and of the 58 LAC
+ GUD
colonies, 48 (82.7%) were confirmed
as KEC coliforms of likely
fecal origin (3
E. agglomerans
strains and 7
E. sakazakii strains
were not included) in
marine waters. The dominating KEC species
of likely fecal origin was
Citrobacter freundii in river (19.2%)
and marine (33.9%)
water samples. A total of 9 LAC
+ GUD
+ colonies
and 4 LAC
GUD
colonies in river water
samples and 7 LAC
+ GUD
+ colonies in marine
water samples were KEC coliforms, resulting
in a false-negative rate of
7.2% (13 of 181 colonies) in river
water samples and 4.5% (7 of 157 colonies) in marine water samples.
None of the
Aeromonas or
Vibrio strains showed LAC activity at
41°C. It is evident
that this incubation temperature is useful
for the elimination of
background interference due to
Aeromonas and helps to avoid
other LAC
+ noncoliform bacteria belonging to the genus
Vibrio. The main
sources of false-positive results in other
types of coliform chromogenic
media are
Vibrio and
Aeromonas sp. (
1,
22).
In summary, the results of this study showed that CECCP incubated at
41°C is efficient for the simultaneous enumeration of
E. coli and KEC from river and marine
waters.
| |
ACKNOWLEDGMENTS |
We thank CHROMagar Microbiology for providing us with CHROMagarECC medium.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Hidrologia y Medio Natural, Universidad Politecnica, Camino de Vera
s/n, 46022 Valencia, Spain. Phone: 34963877090. Fax: 34963877009. E-mail: jalonso{at}ihdr.upv.es.
| |
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Applied and Environmental Microbiology, August 1999, p. 3746-3749, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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