quantitative determination of e. coli, and fecal coliforms in water using a
DESCRIPTION
Quantitative determination of E. coli, and fecal coliforms in water using aTRANSCRIPT
This article was downloaded by: [University of Stellenbosch]On: 10 May 2013, At: 10:55Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UK
Journal of EnvironmentalScience and Health,Part A: Toxic/HazardousSubstances andEnvironmentalEngineeringPublication details, including instructionsfor authors and subscription information:http://www.tandfonline.com/loi/lesa20
Quantitativedetermination of E.coli, and fecal coliformsin water using achromogenic mediumJ.L. Alonso a , A. Soriano b , I. Amoros a &M.A. Ferrus ca Instituto de Hidrología y Medio Natural,Universidad Politécnica, Camino de Vera 14,Valencia, 46022b Gamaser S.L., c/ Pedrapiquers 4, Valencia,46014c Departamento de Biotecnología,Universidad Politecnica, SpainPublished 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 achromogenic medium, Journal of Environmental Science and Health, Part A:Toxic/Hazardous Substances and Environmental Engineering, 33:6, 1229-1248
To link to this article: http://dx.doi.org/10.1080/10934529809376785
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private studypurposes. Any substantial or systematic reproduction, redistribution,reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden.
The publisher does not give any warranty express or implied ormake any representation that the contents will be complete oraccurate or up to date. The accuracy of any instructions, formulae,and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions,claims, proceedings, demand, or costs or damages whatsoever orhowsoever caused arising directly or indirectly in connection with orarising out of the use of this material.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
J. ENVIRON. SCI. HEALTH, A33(6), 1229-1248 (1998)
QUANTITATIVE DETERMINATION OF E. COLI AND FECAL
COLIFORMS IN WATER USING A CHROMOGENIC MEDIUM
Key Words: Kcoli, β -galactosidase, β-glucuronidase, water, fecal coliforms
J.L. Alonso1 A. Soriano2 I. Amoros1 and M.A. Ferrus3
1Instituto de Hidrología y Medio Natural, Universidad Politécnica, Camino de Vera 14,46022 Valencia.
2 Gamaser S.L., c/ Pedrapiquers 4, 46014 Valencia.3Departamento de Biotecnología 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 β-
1229
Copyright © 1998 by Marcel Dekker, Inc. www.dekker.com
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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-37°C plus 22h-44.5°, and 44.5°C) as determined by variance
analysis. In our study K coli represented, on average 70.5-92.5% of the fecal coliform
population. A high incidence of false negative KEC (19.5%) and E. coli (29.6%)
colonies was detected at 44.5°C. 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 37°C, 29 KEC strains that failed to produce β-
galactosidase at 44.5°C were able to produce the enzyme at 37°C. In our opinion the
physiological condition of the fecal coliform isolates could be responsible for the non-
expression of P-galactosidase and P-glucuronidase activities at 44.5°C.
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 44.5°C, 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
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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 Ml) 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 Colección Española de Cultivos Tipo (CECT)
and 6 Salmonella strains from the Instituto de Hidrología 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
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
QUANTITATIVE DETERMINATION OF E. COLI
TABLE 1Growth Conditions at 37°, 41° and 44.5°C of Different
Bacteria on Chromocult Coliform Agar
1233
Test Strain
Enterobacter aerogenesEnterobacter cloacaeEnterobacter sakazakiiEnterobacter gergoviaeKlebsiella pneumoniaeKlebsiella oxytocaKlebsiella ozaenaeCitrobacter diversusCitrobacter amalonaticusCitrobacter freundiiEscherichia coliHafnia alveiSerratia odoríferaSerratia marcescensSerratia rubiadeaCedecea davisaeKluyvera ascorbataShigella flexneriShigella boydiiShigella sonneiAeromonas hydrofilaAeromonas caviaeAeromonas mediaA eromonas jandaeiAeromonas schubertiiAeromonas trotaAeromonas eucrenophilaA. veronii bv. veroniiVibrio choleraePseudomonas aeruginosaEnterococcus faecalisEnterococcusfaeciumSalmonella derbyS. bredeney (4 strains)Salmonella london
No.'684194858857140860851856863401678157867159868842861585583413398838
423242284241425542244257
557108184410
IHMNIHMNIHMN
37°CGb 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
41G ~++++++++
++++++-
++++++++-+++++--
+++
°C~~c
rrrrrrrrcrbrr
t-i
r-rctbrrrr-rrrrc--ctt
44.5°CG C+ r+ r+ r-
+ c+ r+ r+ c+ c+ r+ b+ r+ r+ r+ r-
+ r+ c+ t+ b--
+ r-----+ c--
+ c+ t+ t
* No. of reference strain from the CECT'G: Growth; +=Good; +=Weak; -=None.bC: Colour; r=Salmon to Red; b=Dark Blue to Violet; t=Light Blue to Turquoise;c=Colourless.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1234 ALONSO ET AL.
phosphate buffer culture was streaked onto CC agar plates and incubated at three
different incubation temperatures (37°C, 41°C and 44.5°C). After growth was
observed, the P-galactosidase and p-glucuronidase activities of 32 reference strains and
6 Salmonella strains were tested.
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 44.5°C 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 44.5°C 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 37°C for 2 h before incubation at 44.5°C 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.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1236 ALONSO ET AL.
TABLE 2Non-£ 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*
Samplingsource
TR:EC-CCAb
KEC-CCAC
EKEC-CCAd
FC-mFCPI:EC-CCAKEC-CCAEKEC-CCAFC-mFCAP:EC-CCAKEC-CCAEKEC-CCAFC-mFC
Ml:EC-CCAKEC-CCAEKEC-CCAFC-mFC
M2:EC-CCAKEC-CCAEKEC-CCAFC-mFC
Mean
2.151.972.392.45
1.950.811.501.68
6.725.966.796.73
5.354.515.415.36
3.072.533.183.13
2h37°-44.5°CSD
1.511.631.561.67
0.620.250.800.71
0.200.140.190.19
1.241.121.221.25
0.930.830.910.98
Min
0.700.300.850.85
1.110.480.480.60
6.465.786.566.52
3.382.703.463.36
2.081.542.202.08
Max
4.084.204.454.61
2.581.082.592.62
7.006.237.057.03
6.725.706.766.71
4.483.704.544.62
Mean
2.872.642.372.41
1.790.601.611.61
6.715.826.766.73
5.324.395.375.36
3.002.403.113.07
44.5SD
1.411.481.571.58
0.550.260.720.77
0.190.160.180.19
1.221.291.231.23
1.020.850.981.01
°CMin
1.581.000.781.04
1.180.300.480.48
6.515.606.576.52
3.382.303.413.38
1.941.402.051.81
Max
4.264.204.534.48
2.500.952.502.56
6.996.117.037.04
6.696.046.786.68
4.573.784.634.58
"Data are reported as log values per 100 ml. The results are expressed asarithmetic 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.dEKEC-CCA = E. coli and non-Ecoli fecal coliforms recovered on CC agar.TC-mFC = Fecal coliforms recovered on mFC agar.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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, Ml 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-37°C plus 22h-44.5°C, and 44.5°C)
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
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1238 ALONSO ET AL.
TABLE3Regression and Correlation Parameters from Data Obtained Using Chromocult
Coliform Agar (CC Agar)
Samplesite
TR
PI
AP
Ml
M2
Parameters*
EC37-KEC37EC44-KEC44
EC37-KEC37EC44-KEC44
EC37-KEC37EC44-KEC44
EC37-KEC37EC44-KEC44
EC37-KEC37EC44-KEC44
R
0.990.98
0.690.64
0.680.40
0.990.99
0.990.98
P
<0.01<0.01
NSb
NS
NSNS
<0.01<0.01
<0.01<0.01
Intercept(a)
0.3400.405
0.5711.729
0.9773.981
0.4191.185
0.2570.170
Slope
(b)
0.9160.936
1.7010.108
0.9630.468
1.0930.941
1.1120.182
aEC37-FC37=£sc/;OT'c/7/ij coli and non-£ coli fecal coliforms (Klebsiella,Enterobacier and Citrobacter) (KEC) recovered on CC agar (2h 37°-44.5°C). EC44-FC44=£'. coli and non-£. coli fecal coliforms recovered on CC agar (44.5°C).^ 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 37°C, 41°C and 44.5°C 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 44.5°C. The growth of Aeromonas
reference strains was inhibited at 44.5°C, except in the case of Aeromonas jandaei.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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 false-
negative 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 44.5°C, were incubated on CC agar at 37°C to
determine whether the expression of GUS formation was temperature dependent. Both
E. coli strains showed GUS production at 37°C. Alonso et al. (1996) found that false
negative K coli GUS' colonies occurred less frequently at 35°C than at 44.5°C. 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
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1240 ALONSO ET AL.
TABLE 4
Number of & coli and Non-E coli Fecal Coliforms Isolates Grown on CC AgarIdentified on the Basis of IMVIC, Cytochrome Oxidase, Catalase and TSI Agar
Reactions
Phenotype
LAC GUS"APC
MlM2TRPIP2
TotalLAC+ GUS+
APMlM2TRPIP2
TotalLACGUS*
APMlM2TRPIP2
TotalLAC GUS-
APMlM2TRPIP2
Total
IsolatesNo.
373142413111
193
242948485211
212
290320
16
172315364629166
E.No.
14102519181
87
242748465210
207
280320
15
0215109
coli(%)
38325946589
45
10093
10096
1009198
10089
0100100
094
097
14205
KECNo.
2320171781
86
0202015
0100001
1720121462
71
(%)
6268414226
945
0704092
01100006
100878039137
43
Noncoliformb
No.
0000358
0000000
0000000
012
12221047
(%)
0000
10464
0000000
0000000
04
1333483428
Notidentified
No.
010524
12
0000000
0000000
0005
171739
(%)
030
126
366
0000000
0000000
000
14375924
"KEC: Klebsiella, Enterobacter and Citrobacter.bOxidase +: Pseudomonas spp., Vibrio spp., Aeromonas spp.'Sampling sites.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
TABLE 5Identification of Colonies Picked from CC Agar Using the API 20E System
I
O
m
3§oTI
LAC+GUS" No. LAC+GUS* No. LACGUS4* No. LACGUS- No.
Enterobacter cloacaeKlebsiella oxytocaK. PneuntoniaeCitrobacterfreundiiC. AmalonaticusEscherichia coli
422616
£ co//C.freundii
81
Total
E co//
21
Pseudomonas spp.P. fluorescensAcinetobacter spp.Flavobacterium spp.
Proteus spp.Salmonella typhiCitrobacterfreundiiC. amalonaticusKlebsiella oxytocaK. pneumoniaeEnterobacter cloacaeE agglomeransE sakazakiiEscherichia coli
41111181423114
33
aLAC+ GUS': salmon to red colonies.•"LAC* GUS+: dark-blue to violet colonies.lAC" GUS+: light-blue to turquoise colonies.dLAC GUS': colourless colonies.D
ownl
oade
d by
[U
nive
rsity
of
Stel
lenb
osch
] at
10:
55 1
0 M
ay 2
013
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
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, C.
amalonaticus, Escherichia vulneris, and Hqfnia alvet), Aeromonas sp. and
Acinetobacter sp. (Heizmann, 1988; Kámpfer 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"
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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 37°C, 29 KEC
strains that failed to produce P-galactosidase at 44.5°C were able to produce the
enzyme at 37°C. Dockins and McFeters (1978) observed that optimal activity of 0-
galactosidase enzyme in freshly sonic extracts fecal coliforms typically occurred at
30+2°C, and the activity decreased rapidly as the temperature increased above 35 to
38°C. At 44.5°C 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 44.5°C incubation temperature by 1 or 2°C resulted in
significantly faster rate of ONPG hydrolysis. Munro et al. (1987) observed that P-
galactosidase 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 44.5°C.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
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
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 44.5°C 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.
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
QUANTITATIVE DETERMINATION OF E. COLI 1245
TABLEÓPercentage of Thermotolerant, ThermosensUive and índole Negative K coli
(LAC* GUS*) Strains Recovered in CC Agar
Samplingsites
APMlM2TRPIP2
No. ofstrains
243053465511
Thermo-tolerant*
No.
24285142519
%
1009396919382
Thermo-sensitiveb
No.
02243
12
%
074955
Indol-No.
023219
%
076424
'Thermotolerant: gas formed from lactose a 144.5°CkThermosensitive: gas not formed from lactose at 44.5°C
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
44.5°C incubation temperature to 41°C may reverse the expression of P-galactosidase
and P-glucuronidase activities of some metabolically injured fecal coliforms.
REFERENCES
Alonso, J.L., Amoros, I., Chong, S. and Garelick, H., J. Microbiol. Methods, 25, 309-315(1996).
APHA, "Standard Methods for the Examination of Water and Wastewater, 19thedition" American Public Health Association, New York (1995), 9, pp. 1-117.
Augoustinos, M.T., Grabow, N.A. and Kfir, R., Wat. Sci. Tech., 27, 267-270 (1993).
Bej, A.K., McCarty, S.C. and Atlas, R.M., Appl. Environ. Microbiol., 57, 2429-2432(1991).
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1246 ALONSO ET AL.
Brenner, K.P., Rankin, C.C, Roybal, Y.R. and Stelma, Jr.G., Appl. Environ.Microbiol., 59, 3534-3544 (1993).
Brodsky, M.H., ASM News, 63, 345-346 (1997).
Caplenas, N.R. and Kanarek, M.S., Am. J. Publ. Hlth., 74, 1273-1275 (1984).
Charriere, G., Mossel, D.A.A., Beaudeau, P. and Leclerc, H., J. Appl. Bacteriol., 76,336-344 (1992).
Ciebin, B.W., Brodsky, M.H., Eddington, R, Horsnell, G., Choney, A., Palmateer, G.,Ley, A., Joshi, R. and Shears, G, Appl. Environ. Microbiol., 61, 3940-3942 (1995).
Clark, D.L., Milner, B.B., Stewart, M.H., Wolfe, R.L. and Olson, B.H., Appl. Environ.Microbiol., 57, 1528-1534(1991).
Cleuziat, P. and Robert-Baudoy, J., FEMS Microbiol. Lett., 72, 315-322 (1990).
Covert, T.C., Rice, E.W., Johnson, S.A, Berman, D., Johnson, C.H. and Mason, P.J.,J. A. W. W. A., 84, 98-104 (1992).
Coyne, M.S. and Schuler, J.C, J. Environ. Qual, 23, 126-129 (1994).
Dockins, W.S. and McFeters, G.A., Appl. Environ. Microbiol., 36, 341-348 (1978).
Dufour, A.P., "Bacterial Indicators/Health Hazards Associated with Water" Ed. A.W.Hoadley and B.J. Dutka, American Society for Testmg Materials, Philadelphia (1977),pp. 48-58.
Edberg, S.C., Allen, M.J., Smith, D.B., and the National Collaborative Study, Appl.Environ. Microbiol., 54, 1595-1601 (1988).
Evison, L.M., Wat. Sci. Tech., 20, 309-315 (1988).
Feng, P., Lum, R. and Chang, G.W, Appl. Environ. Microbiol, 57, 320-323 (1991).
Figueras, M.J, Polo, F , Inza, I. and Guarro, J., Lett. Appl. Microbiol, 19, 446-450(1994).
Frampton, E.W. and Restaino, L., J. Appl. Bacteriol, 74: 223-233.
Fricker, E.J. and Fricker, C.R., Lett. Appl. Microbiol, 19, 44-46 (1994).
Giammanco, G, Pignato, S. and Biondi, M , Zbl. Hyg, 193, 99-105 (1992).
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
QUANTITATIVE DETERMINATION OF E. COLI 1247
Green, D.H., Lewis, G.D., Rodtong, S. and Loutit, M.W., J. Microbiol. Methods, 13,207-214 (1991).
Heizmann, W , Döller, P.C, Gütbrod, B. and Werner H., J. Clin. Microbiol., 26, 2682-2684 (1988).
Kampfer, P , Rauhoff, D. and Dott, W., J. Clin. Microbiol, 29, 2877-2879 (1991).
Leclerc, H., Mossel, D.A.A, Trinel, P.A. and Gavini, F. "Bacterial Indicators/HealthHazard Associated with Water" Ed. A.W. Hoadley and B.J. Dutka, American Societyfor Testing Materials, Philadelphia (1977), pp. 22-36.
Manafi, M , Kneifel, W. and Bascomb, S., Microbiol. Rev, 55, 335-348 (1991).
Martins, M.T., Rivera, I.G., Clark, D.L, Stewart, M.H, Wolfe, R.L. and Olsen, B.H,Appl. Environ. Microbiol, 59, 2271-2276 (1993).
McFeters, G.A, "Injured index and pathogenic bacteria: occurrence and detection infoods, water and feeds" Ed. B. Ray, CRC Press, Boca Raton (1989), pp. 179-210.
Mossel, D.A.A, ASM News, 63, 175 (1997).
Munro, P.M, Gauthier, M.J. and Laumond, F.M, Appl. Environ. Microbiol, 53,1476-1481 (1987).
Palmer, C.J, Tsai, Y , Lang, A.L. and Sangermano, L.R, Appl. Environ. Microbiol,59, 786-790(1995).
Perez, J.L, Berrocal, C.I. and Berrocal, L , J. Appl. Bacteriol, 61, 541-545 (1986).
Pommepuy, M , Guillard, J.F, Duprey, E , Derrien, A , Le Guyader, F. and Cormier,M , Wat. Sci. Tech, 25, 93-103 (1992).
Pommepuy, M , Fiksdal, L , Gourmelon, M , Melikechi, H , Caprais, M.P., Cormier,M. and Colwell, R.R., J. Appl. Bacteriol, 81, 174-180 (1996).
Presswood, W.G. and Strong D.K, Appl. Environ. Microbiol, 36, 90-94 (1978).
Rice, E.W, Allen, M.J. and Edberg, S.C., Appl. Environ. Microbiol, 56, 1203-1205(1990).
Rose, R.E, Geldreich, E.E. and Litsky, W, Appl. Microbiol, 29, 532-536 (1975).
Sartory, D.P. and Howard, L , Lett. Appl. Microbiol, 15, 273-276 (1992).
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3
1248 ALONSO ET AL.
Shadix, L.C., Dunnigan, ME. and Rice, E.W, Can. J. Microbiol, 39, 1066-1070(1993).
Venkateswaran, K., Murakoshi, A. and Satake, M., Appl. Environ. Microbiol., 62,2236-2243 (1996).
Warren, L.S., Benoit, R.E. and Jessee J.A., Appl. Environ. Microbiol., 35, 136-141(1976).
Watkins, W.D., Rippey, S.R., Clavet, C.R., Kelley-Reitz, D. J. and Burkhardt, W.,Appl. Environ. Microbiol., 54, 1874-1875 (1988).
Received: December 22, 1997
Dow
nloa
ded
by [
Uni
vers
ity o
f St
elle
nbos
ch]
at 1
0:55
10
May
201
3