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Journal of Environmental
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Quantitative
determination of E.
coli, and fecal coliforms
in water using a
chromogenic medium
a

J.L. Alonso , A. Soriano , I. Amoros &

M.A. Ferrus

Instituto de Hidrologa y Medio Natural ,

Universidad Politcnica , Camino de Vera
14, Valencia, 46022
b

Gamaser S.L. , c/ Pedrapiquers 4,

Valencia, 46014
c

Departamento de Biotecnologa ,
Universidad Politecnica , Spain
Published online: 15 Dec 2008.

To cite this article: J.L. Alonso , A. Soriano , I. Amoros & M.A. Ferrus (1998)
Quantitative determination of E. coli, and fecal coliforms in water using a
chromogenic medium, Journal of Environmental Science and Health, Part A:
Toxic/Hazardous Substances and Environmental Engineering, 33:6, 1229-1248,
DOI: 10.1080/10934529809376785
To link to this article: http://dx.doi.org/10.1080/10934529809376785

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J. ENVIRON. SCI. HEALTH, A33(6), 1229-1248 (1998)

QUANTITATIVE DETERMINATION OF E. COLI AND FECAL

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COLIFORMS IN WATER USING A CHROMOGENIC MEDIUM

Key Words: Kcoli, -galactosidase, -glucuronidase, water, fecal coliforms

J.L. Alonso A. Soriano I. Amoros and M.A. Ferrus

1

Instituto de Hidrologa y Medio Natural, Universidad Politcnica, Camino de Vera 14,

46022 Valencia.
2
Gamaser S.L., c/ Pedrapiquers 4, 46014 Valencia.
3
Departamento de Biotecnologa Universidad Politecnica
Spain

ABSTRACT

A new medium, Chromocult Coliform Agar (CC agar) developed by E.

Merck AG (Darmstadt, Germany) was compared with the Standard

Methods

membrane filtration fecal coliform (mFC) medium for fecal coliform detection and
enumeration. In the CC agar, non-E. coli fecal coliforms (Klebsiella, Enterobacter and
Citrobacter) (KEC) were identified by the production of a salmon to red colour from
p-galactosidase (LAC) cleavage of the substrate Salmon-GAL, while E. coli colonies
were detected by the blue colour, produced by the cleavage of X-glucuronide by -

1229
Copyright 1998 by Marcel Dekker, Inc.

www.dekker.com

1230

ALONSO ET AL.

glucuronidase (GUS). Statistically, there was no significant differences between fecal

coliform counts obtained with the two media (CC agar and mFC agar) and two
incubation procedures (2h-37C plus 22h-44.5, and 44.5C) as determined by variance
analysis. In our study K coli represented, on average 70.5-92.5% of the fecal coliform

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population. A high incidence of false negative KEC (19.5%) and E. coli (29.6%)
colonies was detected at 44.5C. Two K coli GUS negative phenotype upon
reinoculation into CC agar were GUS+. A total of 31 KEC LAC colonies were
streaked onto CC agar and incubated at 37C, 29 KEC strains that failed to produce galactosidase at 44.5C were able to produce the enzyme at 37C. In our opinion the
physiological condition of the fecal coliform isolates could be responsible for the nonexpression of P-galactosidase and P-glucuronidase activities at 44.5C.

INTRODUCTION
The detection and enumeration of indicator organisms are of primary
importance for the monitoring of sanitary and microbiological quality of water. Fecal
coliforms have been long used as indicators of fecal contamination in water and food.
The term fecal coliform include all coliforms that can ferment lactose at 44.5C, trying
to separate the non ubiquitous coliforms from those of true fecal origin (Dockins and
McFeters, 1978). The presence of. coli directly relates to fecal contamination with its
implied threat of the presence of enteric disease agents (Rice et al., 1990). The other
members of the fecal coliform group (Klebsiella, Enterobacter and Citrobacter) may
be isolated in feces, but their presence does not always suggest fecal contamination

QUANTITATIVE DETERMINATION OF E. COLI

1231

(Covert et al., 1992). The abbreviation "KEC" will be used in this study for the
designation of non-E. coli fecal coliforms (Klebsiella, Enterobacter and Citrobacter).
A major limitation of current membrane filtration methods used for counting
fecal coliforms is the enumeration of microorganisms which are not exclusively of fecal

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origin, thereby giving a false indication of the sanitary quality of the water
(Augoustinos et al., 1993). Identification of coli in the past has been laborious, and
only recently methods have been developed that detect E. coli rapidly with accuracy
and specificity (Alonso et al., 1996; Shadix et al., 1993). The identification of coliforms
based on detection of P-galactosidase activity (Manafi et al., 1991) is a significant
departure from methods that utilize the bacterial end products of lactose fermentation
(APHA, 1995).
A new chromogenic medium, Chromocult Coliform agar (CC agar), has been
developed by E. Merck AG (Darmstadt, Germany), to detect coliforms and K coli
simultaneously. A combination of two chromogenic glycosides is used for the detection
of p-galactosidase (LAC) and P-glucuronidase (GUS). The Salmon-GAL substrate
causes a salmon to red colour of the KEC coliform colonies (LAC+ GUS") and the
substrate X-glucuronide is used for the identification of P-glucuronidase. E. coli
cleaves both Salmon-GAL and X-glucuronide, so that positive colonies take on a dark
blue to violet colour (LAC+ GUS*).
In this study, the CC agar was compared with the M-FC medium recommended
in Standard Methods (1995) for the enumeration of fecal coliforms by the membrane
filtration technique.

1232

ALONSO ET AL.
MATERIAL AND METHODS

Sampling
A total of 40 water samples were collected from 6 different environmental
sources in the Valencia area. The water samples were as follows: 6 samples from the

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Turia river near Valencia drinking water treatment plant (site TR); 11 samples from
two well water supplies (site PI, 6 samples, and site P2, 5 samples); 8 samples from a
heavily polluted stream (site AP); 8 samples of seawater (salinity 21%o) (Malvarrosa
beach) influenced by sewage discharge (site M l ) and 7 samples of seawater from a
point located 200 m south of the previously mentioned sewage discharge (salinity
34%o) (site M2). All samples were collected in sterile glass bottles, refrigerated and
assayed within 24 h after collection. Samples from sites TR, AP, Ml and M2 were
preassayed within 2 h to estimate bacterial density. Several dilutions of these samples
were filtered to estimate the number of KEC and K coli present in collected waters.
After 22 h incubation, the most appropriate dilution was chosen and samples were
definitively analyzed.
Bacterial Strains
The 32 reference strains from the Coleccin Espaola de Cultivos Tipo (CECT)
and 6 Salmonella strains from the Instituto de Hidrologa y Medio Natural (IHMN)
stock culture collection used in this study are listed in Table 1. All strains, except
Enterococcus strains, were grown and maintained on nutrient agar (Merck). The
Enterococcus strains were grown and maintained on brain heart agar (Merck).
Incubation Temperature Effect
Pure-culture studies were conducted with reference and IHMN strains.
Bacteria were resuspended in 5 ml of phosphate buffer (APHA, 1995). A loopfiil of the

QUANTITATIVE DETERMINATION OF E. COLI

1233

TABLE 1
Growth Conditions at 37, 41 and 44.5C of Different
Bacteria on Chromocult Coliform Agar

Enterobacter aerogenes
Enterobacter cloacae
Enterobacter sakazakii
Enterobacter gergoviae
Klebsiella pneumoniae
Klebsiella oxytoca
Klebsiella ozaenae
Citrobacter diversus
Citrobacter amalonaticus
Citrobacter freundii
Escherichia coli
Hafnia alvei
Serratia odorfera
Serratia marcescens
Serratia rubiadea
Cedecea davisae
Kluyvera ascorbata
Shigella flexneri
Shigella boydii
Shigella sonnei
Aeromonas hydrofila
Aeromonas caviae
Aeromonas media
A eromonas jandaei
Aeromonas schubertii
Aeromonas trota
Aeromonas eucrenophila
A. veronii bv. veronii
Vibrio cholerae
Pseudomonas aeruginosa
Enterococcus faecalis
Enterococcusfaecium
Salmonella derby
S. bredeney (4 strains)
Salmonella london

No.'
684
194
858
857
140
860
851
856
863
401
678
157
867

159
868
842
861
585
583
413
398
838
4232
4228
4241
4255
4224
4257
557
108
184
410
IHMN
IHMN
IHMN

37C
Gb
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Cc
r
r
r
r
r
r
r
r
r
r
b
r
r
r
r
c
r
c
t
b
r
r
r
r
c
r
r
r
r
c

c
t
t

41C
G ~ ~~c
+
r
+
r
+
r
r
+
r
+
+
r
+
r
+
r
c
+
r
+
b
+
r
+
r
+
+
r
+
r
+
c
t
+
+
b
+
r
+
r
+
r
+
r
+
r
+
r
+
r
+
r
+
c
+
c
+
t
+
t
t-i

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Test Strain

44.5C
G
C
+
r
+
r
+
r
+
c
+
r
+
r
+
c
+
c
+
r
+
b
+
r
+
r
+
r
+
r
+
r
+
c
+
t
+
b
+
+
+
+
+

c
t
t

* No. of reference strain from the CECT

'G: Growth; +=Good; +=Weak; -=None.
b
C: Colour; r=Salmon to Red; b=Dark Blue to Violet; t=Light Blue to Turquoise;
c=Colourless.

1234

ALONSO ET AL.

phosphate buffer culture was streaked onto CC agar plates and incubated at three
different incubation temperatures (37C, 41C and 44.5C). After growth was
observed, the P-galactosidase and p-glucuronidase activities of 32 reference strains and
6 Salmonella strains were tested.

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Microbiological Analysis
Samples were decimal diluted or concentrated according to the expected
bacterial density as above described. Duplicates of each sample dilution were filtered
through sterile 0.45 urn pore size membranes (Whatman) using the standard membrane
filtration technique. The membranes were placed onto a pre-prepared layer of CC agar
in a 47-mm petri-dish. These were then incubated at 44.5C in a water bath for 24 h.
All salmon to red colonies (LAC+ GUS") were counted as presumptive KEC coliforms,
and all blue to violet colonies (LAC* GUS*) were counted as presumptive K coli. For
comparison, the second duplicate membrane of each pair was processed by a standard
method for fecal coliforms. The membranes were layered onto M-FC agar (Merck) and
incubated at 44.5C in a water bath for 24 h. All blue colonies were counted as fecal
coliforms (APHA, 1995). Rosolic acid from M-FC medium was eliminated as
suggested by Presswood and Strong (1978). These authors observed that eliminating
rosolic acid from M-FC medium improves the M-FC procedure by allowing higher
fecal coliform colony recoveries.
In the modified method, the membranes were placed on CC agar and M-FC
agar, and were incubated at 37C for 2 h before incubation at 44.5C in a water bath
for 22-24 h. Rose et al. (1975) suggested the need for a repair phase prior to incubation
at the elevated temperature.

QUANTITATIVE DETERMINATION OF E. COLI

1235

A total number of 587 colonies from the most appropriate dilution of CC agar
were submitted to qualitative analysis. For each sample site, salmon to red colonies
(LAC+ GUSO, dark blue to violet colonies (LAC* GUS4), light blue to turquoise (LAC"
GUS*) and colourless colonies (LAC" GUS") were randomly picked and subcultured on

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nutrient agar (Merck). Purified cultures were further identified by the following cultural
characteristics: indole production, growth on Simmons' citrate agar (Merck), methyl
red and Voges-Proskauer reactions, gas production in EC broth (Merck), reaction on
triple sugar iron agar (TSI) (Merck), and possesion of cytochrome oxidase and
catalase. A total number of 66 isolates were further identified using the API 20E
system (bioMerieux).
Statistical Analysis
Bacterial counts were logarithmically transformed prior to statistical treatment.
Results were analyzed by linear regression to verify the linearity of the relationship
between E. coli and KEC coliforms obtained with CC agar. To examine the medium
performance (CC agar) over a range of sample types and concentrations, the samples
were grouped by sample site, by E. coli and KEC coliform counts on CC agar, by fecal
coliform counts on mFC agar, and by incubation temperatures. A unifactorial variance
analysis was performed on the means of the data. All statistics were obtained using
Statgraphics software.

RESULTS AND DISCUSSION

E. coli and KEC counts on CC agar, and fecal coliform counts on mFC agar, at
two incubation procedures are compared in Table 2. In this study E coli was isolated

1236

ALONSO ET AL.

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TABLE 2
Non- coli Fecal Coliforms {Klebsiella spp., Enterobacter spp. and Citrobacter
spp.) (KEC) and Escherichia coli Recovered on Chromocult Coliform Agar
(CC agar), and Fecal Coliforms Recovered on MFC Agar*
Sampling
source

Mean

2h37-44.5C
SD
Min

Max

Mean

44.5 C
SD
Min

Max

TR:
EC-CCAb
KEC-CCAC
EKEC-CCAd
FC-mFC

2.15
1.97
2.39
2.45

1.51
1.63
1.56
1.67

0.70
0.30
0.85
0.85

4.08
4.20
4.45
4.61

2.87
2.64
2.37
2.41

1.41
1.48
1.57
1.58

1.58
1.00
0.78
1.04

4.26
4.20
4.53
4.48

1.95
0.81
1.50
1.68

0.62
0.25
0.80
0.71

1.11
0.48
0.48
0.60

2.58
1.08
2.59
2.62

1.79
0.60
1.61
1.61

0.55
0.26
0.72
0.77

1.18
0.30
0.48
0.48

2.50
0.95
2.50
2.56

6.72
5.96
6.79
6.73

0.20
0.14
0.19
0.19

6.46
5.78
6.56
6.52

7.00
6.23
7.05
7.03

6.71
5.82
6.76
6.73

0.19
0.16
0.18
0.19

6.51
5.60
6.57
6.52

6.99
6.11
7.03
7.04

5.35
4.51
5.41
5.36

1.24
1.12
1.22
1.25

3.38
2.70
3.46
3.36

6.72
5.70
6.76
6.71

5.32
4.39
5.37
5.36

1.22
1.29
1.23
1.23

3.38
2.30
3.41
3.38

6.69
6.04
6.78
6.68

3.07
2.53
3.18
3.13

0.93
0.83
0.91
0.98

2.08
1.54
2.20
2.08

4.48
3.70
4.54
4.62

3.00
2.40
3.11
3.07

1.02
0.85
0.98
1.01

1.94
1.40
2.05
1.81

4.57
3.78
4.63
4.58

PI:
EC-CCA
KEC-CCA
EKEC-CCA
FC-mFC
AP:
EC-CCA
KEC-CCA
EKEC-CCA
FC-mFC
Ml:
EC-CCA
KEC-CCA
EKEC-CCA
FC-mFC

M2:
EC-CCA
KEC-CCA
EKEC-CCA
FC-mFC

"Data are reported as log values per 100 ml. The results are expressed as
arithmetic mean (Mean), standard deviation (SD), minimum (Min),
and maximum (Max).
""EC-CCA = Escherichia coli (LAC* GUS*) recovered on CC agar.
"KEC-CCA = Non- coli fecal coliforms (LAC+ GUS") recovered on CC agar.
d
EKEC-CCA = E. coli and non-Ecoli fecal coliforms recovered on CC agar.
TC-mFC = Fecal coliforms recovered on mFC agar.

QUANTITATIVE DETERMINATION OF E. COLI

1237

from all of the six zones analyzed but at different densities (Table 2). The data of site
P2 were not reported because of low number of samplings with positive results. The
highest levels of E. coli were detected at sites AP and Ml, with densities up to 10s
CFU/100 ml. These zones also showed high numbers of KEC coliforms. Table 3

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summarizes the values of the correlation coefficients (r) and the confidence levels (P)
obtained between the concentrations of K coli and KEC. At site P2, the presence of E.
coli (1 CFU/100 ml) was detected only in four samples and it was not included in the
statistical analysis. Positive correlations (P<0.01) were found at sites TR, M l and M2.
There was no correlation at sites PI and AP. Counts of E. coli and KEC on CC agar
were compared with fecal coliform counts on mFC agar. Statistically, there was no
significant differences between coliform counts obtained with the two media (CC agar
and mFC agar) and two incubation procedures (2h-37C plus 22h-44.5C, and 44.5C)
as determined by variance analysis. ANOVA on the K coli data at two incubation
procedures of CC agar indicated no significant differences among incubation
procedures. KEC coliforms represented, on average 7.9-29.5% of the fecal coliform
population. Figueras et al. (1994) demonstrated the low specificity of mFC medium for
the enumeration and detection of fecal coliforms from seawater, on the basis of the high
incidence of false positive colonies (thermotolerant non-fecal coliforms). Many authors
(Caplenas and Kanarek, 1984; Charriere et al., 1992; Dufour, 1977; Evison, 1988)
consider that the adjective "fecal" is not properly applied and questioned the usefulness
of fecal coliforms other than E. coli as fecal indicators. We agree with other authors
(Brodsky, 1997; Mossel, 1997) that in order to provide more comparative results, the

ALONSO ET AL.

1238

TABLE3
Regression and Correlation Parameters from Data Obtained Using Chromocult
Coliform Agar (CC Agar)

Intercept
(a)

Slope
(b)

0.99
0.98

<0.01
<0.01

0.340
0.405

0.916
0.936

EC37-KEC37
EC44-KEC44

0.69
0.64

NS b
NS

0.571
1.729

1.701
0.108

AP

EC37-KEC37
EC44-KEC44

0.68
0.40

NS
NS

0.977
3.981

0.963
0.468

Ml

EC37-KEC37
EC44-KEC44

0.99
0.99

<0.01
<0.01

0.419
1.185

1.093
0.941

M2

EC37-KEC37
EC44-KEC44

0.99
0.98

<0.01
<0.01

0.257
0.170

1.112
0.182

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Sample
site

Parameters*

TR

EC37-KEC37
EC44-KEC44

PI

EC37-FC37=sc/;OT'c/7/ij coli and non- coli fecal coliforms (Klebsiella,

Enterobacier and Citrobacter) (KEC) recovered on CC agar (2h 37-44.5C). EC44FC44='. coli and non-. coli fecal coliforms recovered on CC agar (44.5C).
^ 5 = ^ 1 significant.

term fecal coliform should be revised and replaced with the more definitive fecal index
organism Escherichia coli.
The p-galactosidase and P-glucuronidase activities of 32 reference strains and 6
Salmonella strains at 37C, 41C and 44.5C are shown in Table 1. The ability to
produce p-galactosidase of Klebsiella pneumoniae, Citrobacter diversus and C.
amalonaticus strains on CC agar was inhibited at 44.5C. The growth of Aeromonas
reference strains was inhibited at 44.5C, except in the case of Aeromonas jandaei.

QUANTITATIVE DETERMINATION OF E. COLI

1239

Salmonella bredeney (4 strains) and S. london showed P-glucuronidase activity at the

three temperatures tested.
The identities of the four types of colonies (LAC+ GUS\ LAC+ GUS+, LAC"
GUS* and LAC GUS") on CC agar are shown in Table 4. The identity of 66 isolates

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was verified with the API 20E system (Table 5). The KEC LAC+ GUD' species
identified were Klebsiella oxytoca (2 strains), K. pneumoniae (2 strains), Enierobader
cloacae (4 strains), Citrobacterfreundii (6 strains) and C. amalonaticus (1 strain).
Of the 212 blue colonies (LAC+ GUS4) 207 (98%) were confirmed as E. coli,
giving a false positive rate of 2% (5 of 212 colonies). A total of 9 LAC GUS' colonies,
15 LAC GUS+ colonies and 87 LAC+ GUS' were E. coli, resulting in a false negative
rate of 29.6% (111 of 375 colonies). Covert et al. (1992) reported that the falsenegative rates with natural populations of E coli ranged from 18.6% with the
Coliquik test (CL) to 23.4% with the Colilert test (CL) (these enzyme detection tests
contains the fluorogenic substrate 4-methylumbelliferyl-P-D-glucuronide, MUG).
Ciebin et al. (1995) encountered a lower incidence of P-glucuronidase-negative E. Coli
isolates with river (9.8 and 9.3%) and lake (7.8 and 8.8%) samples with FC-BCIG and
TEC-BCIG media (m-FC and m-TEC media supplemented with the chromogenic
substrate 5-bromo-6-chloro-3-indolyl-P-D-gIucuronide, BCIG), respectively. Two E.
coli, GUS negative phenotype at 44.5C, were incubated on CC agar at 37C to
determine whether the expression of GUS formation was temperature dependent. Both
E. coli strains showed GUS production at 37C. Alonso et al. (1996) found that false
negative K coli GUS' colonies occurred less frequently at 35C than at 44.5C. Several
authors (Clark et al., 1991; Covert et al., 1992; Palmer et al., 1995) showed that some
MUG negative Ecoli isolates regained the MUG phenotype upon further culture. One
mechanism that could cause GUS negative phenotype would be failure of the permease

ALONSO ET AL.

1240

TABLE 4
Number of & coli and Non-E coli Fecal Coliforms Isolates Grown on CC Agar
Identified on the Basis of IMVIC, Cytochrome Oxidase, Catalase and TSI Agar
Reactions
Phenotype

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Isolates
No.

E. coli
No. (%)

KEC
No.
(%)

Non
coliformb
No.
(%)

LAC GUS"
23
APC
37
14
38
62
0
20
10
32
68
0
Ml
31
17
M2
42
25
59
41
0
17
41
46
42
0
TR
19
8
PI
31
18
58
26
3
11
1
1
P2
9
9
5
86
Total
193
87
45
45
8
LAC+ GUS+
AP
24
24
100
0
0
0
Ml
29
0
27
93
2
7
M2
48
48
100
0
0
0
TR
48
46
0
96
2
4
PI
52
52
100
0
0
0
11
1
P2
10
91
9
0
Total
212
207
98
5
0
2
LACGUS*
AP
2
2
100
0
0
0
1
11
Ml
9
8
89
0
M2
0
0
0
0
0
0
TR
3
3 100
0
0
0
PI
2
2 100
0
0
0
P2
0
0
0
0
0
0
1
16
15
94
6
Total
0
LAC GUSAP
17
0
0
17
100
0
Ml
23
9
20
2
87
1
M2
15
1
7
12
80
2
TR
36
5
14
14
39
12
1
PI
46
2
6
13
22
P2
29
0
0
2
7
10
166
9
5
71
Total
43
47
"KEC: Klebsiella, Enterobacter and Citrobacter.
b
Oxidase +: Pseudomonas spp., Vibrio spp., Aeromonas spp.
'Sampling sites.

Not
identified
No.
(%)

0
0
0
0
10
46
4

0
1
0
5
2
4
12

0
3
0
12
6
36
6

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0
4
13
33
48
34
28

0
0
0
5
17
17
39

0
0
0
14
37
59
24

Downloaded by [Southern Illinois University] at 21:05 18 July 2015

TABLE 5
Identification of Colonies Picked from CC Agar Using the API 20E System

LAC + GUS"

No.

LAC + GUS*

No.

LACGUS 4 *

No.

LACGUS-

No.
O

Enterobacter cloacae
Klebsiella oxytoca
K. Pneuntoniae
Citrobacterfreundii
C. Amalonaticus
Escherichia coli

Total
a

4
2
2
6
1
6

co//

C.freundii

21

LAC+ GUS': salmon to red colonies.

"LAC* GUS+: dark-blue to violet colonies.
l A C " GUS+: light-blue to turquoise colonies.
d
LAC GUS': colourless colonies.

8
1

E co//

Pseudomonas spp.
P. fluorescens
Acinetobacter spp.
Flavobacterium spp.
Proteus spp.
Salmonella typhi
Citrobacterfreundii
C. amalonaticus
Klebsiella oxytoca
K. pneumoniae
Enterobacter cloacae
E agglomerans
E sakazakii
Escherichia coli

4
1
1
1
1
1
8
1
4
2
3
1
1
4
33

o
TI

1242

ALONSO ET AL.

to transport the glucuronide substrate across the cell membrane (Coyne and Schuler,
1994). Some authors (Bej et al., 1991; Cleuziat and Robert-Baudoy, 1990; Feng et al.,
1991; Flicker and Flicker, 1994; Green et al., 1991; Martins et al., 1993;
Venkateswaran et al., 1996) observed that part of the genetic sequences of the uidA

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gene, which encodes for the GUS enzyme, was present in most if not all E coli
isolates, regardless of the GUS phenotype. Frampton and Restaino (1993) indicated
that the following factors may influence the GUS assay substantially, whichever GUS
detection system is used: strain differences in response to particular substrates and
substrate concentration; effects of carbohydrate content and selective agents in the
medium; incubation time and temperature; pH changes; ionic strength effects; and
possible interference by large numbers of competing bacteria or substances in the
sample itself. We have isolated one strain of Citrobacter freundii LAC+ GUS+.
Although P-glucuronidase activity has been reported in some strains of coliforms
(Enterobacter agglomerans, E. cloacae, E. amnigenus, Citrobacter freundii,
amalonaticus,

Escherichia vulneris, and Hqfnia alvet), Aeromonas

C.

sp. and

Acinetobacter sp. (Heizmann, 1988; Kmpfer et al., 1991; Perez et al., 1986; Sartory y
Howard, 1992; Watkins et al., 1988), their occurrence appears to be very infrequent
(Sartory and Howard, 1992). The reason for the production of p-glucuronidase by
these strains is not known, but other investigators (Brenner et al., 1993) have suggested
that the reaction may be plasmid mediated.
The specificity of the medium for KEC coliforms was low. Of the 193 salmon to
red colonies (LAC+ GUS") 86 (45%) were confirmed as KEC coliforms, giving a false
positive rate of 55% (127 of 193 colonies). A total of 71 LAC" GUS" colonies, 1 LAC"

QUANTITATIVE DETERMINATION OF E. COLI

1243

GUS+ colony and 5 LAC+ GUS+ colonies were KEC coliforms, resulting in a false
negative rate of 19.5% (77 of 394 colonies). A high incidence of false negative (LAC)
KEC colonies was detected. Because enzyme activities are subject to the physiological
status of the bacteria, a variable fraction of the coliform bacteria may be stressed when

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changes in irradiation, salinity, temperature, and nutrient concentration of the

environment occur (Pommepuy et al., 1992). Fecal coliform bacteria comprise several
bacterial species and their response to environmental factors may not be the same for
each species (Pommepuy et al., 1996). In treated drinking water injured coliforms can
comprise between 50 and >90% of coliforms present (McFeters, 1989). A total of 31
LAC GUS' colonies were streaked onto CC agar and incubated at 37C, 29 KEC
strains that failed to produce P-galactosidase at 44.5C were able to produce the
enzyme at 37C. Dockins and McFeters (1978) observed that optimal activity of 0galactosidase enzyme in freshly sonic extracts fecal coliforms typically occurred at
30+2C, and the activity decreased rapidly as the temperature increased above 35 to
38C. At 44.5C fecal P-galactosidase activity was 25 to 50% of the optimal
temperature (Dockins and McFeters, 1978). This decrease in p-galactosidase activity in
fecal coliforms has been indirectly observed by Warren et al. (Warren et al., 1976) who
found that lowering the 44.5C incubation temperature by 1 or 2C resulted in
significantly faster rate of ONPG hydrolysis. Munro et al. (1987) observed that Pgalactosidase activity of coli starved cells disappeared gradually with time. The
physiological condition of KEC isolates could be responsible for the non-expression of
enzyme activity at 44.5C.

1244

ALONSO ET AL.
When LAC+ GUS', LAC+ GUS+ and LAC GUS* colonies were considered as

fecal coliforms (included E. coli), more than 95% (401 of 421 colonies) of the
identified colonies belonged to the fecal coliform group, giving a false positive rate of
4.8% (20 of 421 colonies). Nevertheless, LAC GUS' colonies represented 48.1% (80

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of 166 colonies) of the identified coliform group.

Results of the study indicated that 94% (205 of 219 colonies) of the E. coli
LAC+ GUS* strains produced gas in the EC medium (Table 6). Thermotolerant E. coli
was the most frequently isolated in the 6 environmental conditions, as expected.
However, the percentage was variably ranging from 82% (P2) to 100% (AP). A total
of 219 E. coli strains (LAC+ GUS*) were verified in EC broth and 12 (5%) gas
negative strains were encountered. In EC broth, K coli must transport lactose through
the cell membrane, transform the substrate to glucose, metabolize glucose through the
glycolytic cycle to pyruvate, and then convert pyruvate to the desired end product,
either acid or gas (Edberg et al., 1988). Because lactose fermentation at 44.5C is
determined by a complex of different enzymes, a number of anomalous results may
occur, such as false negative gas production (Edberg et al., 1988; Gtammanco et al.,
1992). Leclerc et al. (1977) observed that the activity of formic hydrogen lyase, which
is needed for gas production from lactose, is quite often reduced and sometimes
entirely suppressed under conditions that do not favour survival of coliforms in water.
Munro et al. (1987) suggested that the disappearance of P-galactosidase activity in
non-salt adapted E coli cells starved in seawater could have implications for their
enumeration by standard cultural methods, all of which being grounded on the
acidification and fermentation of lactose.

QUANTITATIVE DETERMINATION OF E. COLI

1245

TABLE
Percentage of Thermotolerant, ThermosensUive and ndole Negative K coli
(LAC* GUS*) Strains Recovered in CC Agar

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Sampling
sites

AP
Ml
M2
TR
PI
P2

No. of
strains

24
30
53
46
55
11

Thermotolerant*

Thermosensitiveb

Indol-

No.

No.

No.

24
28
51
42
51
9

100
93
96
91
93
82

0
2
2
4
3
12

0
7
4
9
5
5

0
2
3
2
1
9

0
7
6
4
2
4

'Thermotolerant: gas formed from lactose a 144.5C

Thermosensitive: gas not formed from lactose at 44.5C

The data obtained suggested that specificity of CC agar for fecal coliforms was
related to the incubation temperature and we are of the opinion that lowering the
44.5C incubation temperature to 41C may reverse the expression of P-galactosidase
and P-glucuronidase activities of some metabolically injured fecal coliforms.

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Received: December 22, 1997