Culture Methods

Analytical limits of four -glucuronidase and -galactosidase-based commercial
culture methods used to detect Escherichia coli and total coliforms

Andre F. Maheux
a,b
, Vicky Hupp
a,b
, Maurice Boissinot
a,b
, Franois J. Picard
a,b
, Luc Bissonnette
a,b
,
Jean-Luc T. Bernier
a,b
, Michel G. Bergeron
a,b,
a
Centre de Recherche en Infectiologie de l'Universit Laval, Centre Hospitalier Universitaire de Qubec (Pavillon CHUL), Qubec (Qubec), Canada
b
Dpartement de Biologie Mdicale, Facult de Mdecine, Universit Laval, Qubec (Qubec), Canada
a b s t r a c t a r t i c l e i n f o
Article history:
Received 14 July 2008
Received in revised form 4 August 2008
Accepted 5 August 2008
Available online 8 August 2008
Keywords:
E. coli
MI agar
Colilert
Chromocult Coliform agar ES
Readycult Coliforms 100
Total coliforms
Colilert
(Colilert), Readycult
Coliforms 100 (Readycult), Chromocult
Coliform agar ES (Chromocult), and

MI agar (MI) are -galactosidase and -glucuronidase-based commercial culture methods used to assess
water quality. Their analytical performance, in terms of their respective ability to detect different strains of
Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their
ability to detect -glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of
different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to
detect -galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and
environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected -
glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4
methods detected -galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total
coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to
detect -glucuronidase production and MI the weakest to detect -galactosidase production. Furthermore,
the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they
may not be appropriate for identication of presumptive E. coli strains.
2008 Elsevier B.V. All rights reserved.
1. Introduction
The multiple-tube fermentation and membrane lter techniques
are classical reference methods used for water quality monitoring
(APHA et al., 1998) that have been associated with their own
limitations. The multiple-tube fermentation method provides results
only after 3 to 4 days and the interference by a high number of non-
coliform bacteria have been shown to alter the efciency of the
analysis (Evans et al., 1981; Means and Olson, 1981; Seidler et al.,
1981). For the membrane lter technique, the most widely used
medium for drinking water analysis are m-Endo and mFC media in
United States and Canada (APHAet al., 1998) and Tergitol-TTC medium
in Europe (AFNOR, 1990). However, since these media lack specicity,
coliform conrmation is required (APHA et al., 1998) which delays the
results by 2 to 3 days. Also, the presence of a high number of
background heterotrophic bacteria was shown to decrease coliform
recovery (Burlingame et al., 1984; Clark, 1980). The inherent limita-
tions of these two methods make them unable to provide, within
hours, useful public health information.
To diminish background effects of heterotrophic bacteria and
circumvent the need for a conrmation stage required by both
multiple-tube fermentation and membrane lter techniques, methods
based on the enzymatic properties of coliforms (-galactosidase for
total coliforms and -glucuronidase enzymes for Escherichia coli
detection) have been developed. These enzymes have been chosen
because conventional coliformmonitoring is based on detection of the
presence of -galactosidase and because the gene encoding the -
glucuronidase enzyme (uidA) was found to be specic (Brenner et al.,
1972) and present in more than 97% of E. coli isolates (Lupo and
Halpern, 1970; Martins et al., 1993).
Colilert
(Colilert, IDEXX Laboratories, Westbrook, ME, USA),

Readycult
Coliforms 100 (Readycult; Merk KGaA, Darmstadt, Ger-

many), Chromocult
Coliform agar ES (Chromocult; Merk KGaA,

Darmstadt, Germany), and MI agar (MI; BD, Franklin Lakes, NJ, USA)
are four commercial test methods based on the determination of -
galactosidase and -glucuronidase enzyme activities which are used
to detect, within 24 h, total coliforms and E. coli in water samples.
These tests are easy to use, require no additional conrmatory step,
and provide a more rapid estimate of indicators of bacteriological
contamination of water as compared to classical techniques (Brenner
Journal of Microbiological Methods 75 (2008) 506514
Corresponding author. Centre de Recherche en Infectiologie de l'Universit Laval,
Centre Hospitalier Universitaire de Qubec (Pavillon CHUL), 2705 Laurier Blvd. Suite RC-
709, Qubec (Qubec), Canada G1V 4G2. Tel.: +1 418 654 2705; fax: +1 418 654 2715.
E-mail address: Michel.G.Bergeron@crchul.ulaval.ca (M.G. Bergeron).
0167-7012/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2008.08.001
Contents lists available at ScienceDirect
Journal of Microbiological Methods
j our nal homepage: www. el sevi er. com/ l ocat e/ j mi cmet h
Table 1
Ability of Colilert, Readycult Coliforms 100, Chromocult Coliform agar ES and MI agar test methods to detect Escherichia coli and Shigella sp. strains
Strains (origin) No.
reference
Serotype Test methods
Colilert Readycult Coliforms 100 Chromocult Coliform agar ES MI agar
Escherichia coli (n=74)
Escherichia coli (clinical) ATCC 11775 O1:K1:H7 + + + +
Escherichia coli (clinical) ATCC 23511 O16:K1(L):NM + + + +
Escherichia coli (clinical) ATCC 35401 O78:H11 + + + +
Escherichia coli (clinical) ATCC 43886 O25:K98:NM + + +
Escherichia coli (clinical) ATCC 43890 O157:H7
Escherichia coli (clinical) ATCC 43896 O78:K80:H12 + + + +
Escherichia coli (clinical) CCRI-1191 N/A + + + +
Escherichia coli (clinical) CCRI-2099 N/A + + +
Escherichia coli (clinical) CCRI-2109 N/A +
Escherichia coli (clinical) CCRI-2166 N/A
Escherichia coli (clinical) CCRI-8831 O157:H7
Escherichia coli (clinical) CCRI-8832 O157:H7
Escherichia coli (clinical) CCRI-8833 O103:H2 + + +
Escherichia coli (clinical) CCRI-8834 O103:H2 + + +
Escherichia coli (clinical) CCRI-8835 O111:H- + + +
Escherichia coli (clinical) CCRI-8836 O111:H- + + +
Escherichia coli (clinical) CCRI-8837 O26:NM
Escherichia coli (clinical) CCRI-8838 O26:NM + + + +
Escherichia coli (clinical) CCRI-8839 O145:NM + +
Escherichia coli (clinical) CCRI-8840 O145:NM + + + +
Escherichia coli (environmental) CCRI-14813 N/A + + + +
Escherichia coli (environmental) CCRI-14871 N/A + +
Escherichia coli (environmental) CCRI-16537 N/A + + +
Escherichia coli (clinical) LSPQ 2086 O8:H9 + + + +
Escherichia coli (clinical) LSPQ 2092 O18:NM + + + +
Escherichia coli (clinical) LSPQ 2096 O26:NM + + + +
Escherichia coli (clinical) LSPQ 2113 O111:NM +
Escherichia coli (clinical) LSPQ 2115 O128:H8 + + + +
Escherichia coli (clinical) LSPQ 2117 O113:H21 + +
Escherichia coli (clinical) LSPQ 2118 O117:H4 + + +
Escherichia coli (clinical) LSPQ 2125 O128:NM + + +
Escherichia coli (clinical) LSPQ 2127 O157:H7
Escherichia coli (clinical) LSPQ 3760 O157:H7 +
Escherichia coli (clinical) LSPQ 3761 O157:H7 +
(continued on next page) (continued on next page)
507 A.F. Maheux et al. / Journal of Microbiological Methods 75 (2008) 506514
et al., 1993, 1996b; Edberg et al., 1988; Horman and Hanninen, 2006;
Pitkanen et al., 2007).
Different collections of strains were also tested with each
commercial -galactosidase and -glucuronisade-based test methods
to establish their ability to recover total coliforms and E. coli strains.
All of these methods were found to be at least as efcient as classical
reference methods (Landre et al., 1998; Rice et al., 1990, 1991, 1993).
However, the expression of the -glucuronidase enzyme was found to
be variable depending on the medium and technique used (Chang et
al., 1989; Feng and Lampel, 1994; Shadix and Rice, 1991). Furthermore,
there is no study comparing the analytical limits of these methods
using pure cultures of bacteria, in order to determine their respective
analytical ability to detect E. coli strains and total coliforms.
In this study, we compared four commercial -galactosidase and
-glucuronidase-based test methods (Colilert, Readycult, Chromocult
agar, and MI agar) using a collection of bacteria representing strains of
different geographical origins and serotypes obtained in both fecal
and environmental settings. More specically, we have determined
their ability to detect target E. coli and total coliform strains, as well as
non-coliform strains (specicity testing).
2. Materials and methods
2.1. Bacterial strains
The ability of the Colilert
(Colilert), Chromocult
coliforms agar
ES (Chromocult), Readycult
Coliform 100 (Readycult), and MI agar

(MI) test methods to detect E. coli strains was veried using 74 strains
of E. coli (Table 1). Eight (8) E. coli strains were obtained from the
American Type Culture Collection (ATCC; Manassas, VA): ATCC 11775,
ATCC 43886, ATCC 23511, ATCC 35401, ATCC 43890, ATCC 43894, ATCC
43895, and ATCC 43896. Clinical isolates of E. coli (n=38) were
obtained from Huashan Hospital (Shanghai, China; n=1), Hpital
Ambroise Par (Boulogne, France; n=1), Institut fr Hygiene und
Mikrobiologie der Universitt Wrzburg (Wrzburg, Germany;
n=10), Laboratoire de Sant Publique du Qubec (Sainte-Anne de
Bellevue, Qubec, Canada; n=12), National Institute of Public Health
(Warsaw, Poland; n=1), South African Institute for Medical Research
(Johannesburg, South Africa; n=2), Microbiology Laboratory of the
Centre Hospitalier de l'Universit Laval (Qubec, Qubec, Canada;
n=5), University of Edinburgh (Edinburgh, Scotland; n=5), and
Wyeth-Ayerst Research (Pearl River, NY; n=1). Environmental isolates
of E. coli (n=28) were obtained from various sources and isolated by
different methods including Colilert, MI, Chromocult, mFC agar, and
modied mTEC agar. These environmental strains were isolated from
(i) drinking water samples obtained from the Service d'Analyse
Environmentale Bodycote (Qubec, Qubec, Canada; n=9), (ii) water
samples from Bermuda (n=5), (iii) river water samples across Canada,
were obtained from the Centre d'Expertise en Analyse Environne-
mentale du Qubec (Qubec, Qubec, Canada; n=14). The analytical
limits of the four culture methods were also assessed by using 8 Shi-
gella strains representing four nomen species (Table 1).
The ability of the four methods to detect total coliform strains was
also veried by using 33 reference and environmental non-E. coli total
coliform strains (Table 2) consisting of Citrobacter spp. (n=12), Enter-
obacter spp. (n=4), non-E. coli Escherichia spp. (n=4), Hafnia spp.
(n=2), Klebsiella spp. (n=5), Pantoae spp. (n=1), Raoultella spp. (n=3),
Serratia spp. (n=1), and Yersinia spp. (n=1).
The specicity of the four methods was demonstrated by using a
battery of clinical and environmental strains consisting of 52 Gram-
positive bacterial species and 37 non-total coliforms Gram-negative
bacterial species (Table 3).
The identity of all reference, clinical and environmental strains
used in this study was conrmed using a MicroScan Autoscan-4
system(Siemens Healthcare Diagnostic Inc., Newark, DE, USA) or with
a Vitek 32 (bioMrieux SA, Marcy l'toile, France). Bacterial strains
were grown from frozen stocks, kept at 80 C in brain heart infusion
(BHI) medium (BD, Franklin Lakes, NJ, USA) containing 10% glycerol,
and cultured on sheep blood agar. Three passages were performed
prior to analysis of each strain with each culture method.
2.2. Bacterial cell suspension preparation
Each bacteria described in Tables 13 was grown to logarithmic
phase (0.50.6 OD
600
) andadjusted to a 0.5 McFarlandstandard, before
being serially diluted ten-fold in phosphate-buffered saline (PBS;
137 mMNaCl, 6.4 mMNa
2
HPO
4
, 2.7 mMKCl, 0.88 mMKH
2
PO
4
, pH7.4).
For each bacterial strains, an aliquot of the 10
5
dilution was spiked in
spring water (Labrador, Anjou, Qubec, Canada) ltered on a 0.22 m
pore size membrane lter (Millipore Corporation, Billerica, MN, USA)
to produce a suspension having approximately 100 colony forming
unit (CFU)/100 mL of water. Bacterial count was veried by ltering
100 mL of each spiked water sample through a GN-6 membrane lter
(47 mm diameter, 0.45 m pore size) with a standard platform
manifold (Millipore Corporation, Billerica, MA, USA). Then, the lter
was incubated on sheep blood agar plates for 242 h at 35.00.5 C
prior to the determination of colony counts. Tests to conrm the
sterility of lter membranes and buffer used for rinsing the ltration
apparatus were also performed.
2.3. Membrane ltration methods
For membrane ltration methods, two 100 mL volumes of each
spiked water samples were ltered on GN-6 membrane lters with a
standard platform manifold. One lter was incubated on Chromocult
agar plates (Merck KGaA, Darmstadt, Germany) while the other lter
was incubated on MI agar plates (BD) for 242 h at 35.00.5 C.
Subsequently, colonycount and color were determinedfor Chromocult
Table 1 (continued)
reference
Serotype Test methods
Escherichia coli (clinical) LSPQ 3762 O157:H7
All E. coli strains 38/74 (51.4%) 60/74 (81.1%) 59/74 (79.7%) 59/74 (79.7%)
All E. coli non-O157:H7 strains 38/65 (58.5%) 58/65 (89.2%) 59/65 (90.8%) 59/65 (90.8%)
All environmental strains 22/28 (78.6%) 28/28 (100%) 27/28 (96.4%) 28/28 (100%)
Shigella sp. (n=8)
Shigella boydii ATCC 9207 N/A + + +
Shigella dysenteriae ATCC 11835 type 1
Shigella dysenteriae CCRI-8843 N/A
Shigella dysenteriae CCRI-8844 N/A
Shigella exneri CCRI-2198 N/A
Shigella exneri ATCC 12022 type 2b
Shigella sonnei CCRI-2196 N/A +
Shigella sonnei ATCC 29930 N/A +
All Shigella strains 0/8 (0%) 1/8 (12.5%) 1/8 (12.5%) 3/8 (37.5%)
Table 2
Ability of Colilert, Readycult Coliforms 100, Chromocult Coliform agar ES and MI agar test methods to detect total coliforms
reference
Test methods
E. coli (n=74)
Escherichia coli (clinical) ATCC 11775 + + + +
Escherichia coli (clinical) ATCC 43890 + + +
Escherichia coli (clinical) CCRI-1191 + + + +
Escherichia coli (clinical) CCRI-2108 + + +
Escherichia coli (clinical) CCRI-2109 +
Escherichia coli (clinical) CCRI-8852 + +
Escherichia coli (environmental) CCRI-14813 + + + +
Escherichia coli (clinical) LSPQ 2086 + + + +
Escherichia coli (clinical) LSPQ 2113 +
Escherichia coli (clinical) LSPQ 2117 + +
Escherichia coli (clinical) LSPQ 2127 + + +
(continued on next page) (continued on next page)
and MI, while uorescence under UV light (=365 nm) was measured
for MI. Each preparation of Chromocult and MI plates was tested for
performance using pure cultures of target and non-target microorgan-
isms, as recommended by the USEPA microbiology methods manual.
Tests to conrm the sterility of the lter membranes and buffer used
for rinsing the ltration apparatus were also performed.
2.4. Liquid culture methods
For liquid culture methods, preparation, validation, storage, and
handling steps were all performed according to the manufacturer's
instructions. For the Colilert method, one snap pack containing the
Colilert reagent (IDEXX Laboratories Canada Corp., Toronto, Ontario,
Canada) was dissolved in 100 mL of spiked water sample. The solution
was then added to a Quanti-Tray
, sealed, and incubated at 35.0

0.5 C for 24 h prior to the identication of samples presenting a
yellow color and uorescence under UV light (=365 nm). All
reactions remaining negative after 24 h of incubation were incubated
at 35 C for an additional 4 h prior to a second analysis evaluation of
the reaction color and uorescence signal. Positive and negative
controls, using pure cultures of target and non-target microorganisms,
were performed as recommended by the manufacturer.
For the Readycult method, one snap pack containing the Readycult
reagent (Merck, Darmstadt, Germany) was dissolvedin100 mL of spiked
water and incubated at 35.00.5 Cfor 242 h prior to the identication
of samples presenting a green color and uorescence under UV light
(=365nm). Positive andnegative controls, using pure cultures of target
and non-target microorganisms, were also performed as recommended
by the manufacturer.
3. Results
3.1. Detection of E. coli strains
Seventy four (74) E. coli strains of different serotypes isolated from
fecal and environmental settings as well as from different geographic
origins were used to demonstrate the ability of the four culture
methods to detect various E. coli strains (Table 1). For conrmatory
purposes, all strains that have presented negative results were tested a
second time with a different lot of kit/media.
Fifty-nine (59) of the 74 E. coli strains tested (79.7%) yielded a -
glucuronidase-positive signal with both MI and Chromocult methods
(Table 1). Fluorescence was observed for 60 of these strains (81.1%)
tested with Readycult. Of the 74 E. coli strains tested, 55 (74.3%) were
detected by all three methods, whereas 9 (12.2%) were undetectable by
the three methods. In addition, discordant results between these three
methods were observed for 10 strains (13.5%). The Colilert method gave
the lowest percentage of detection since only 38 of the 74 E. coli strains
(51.4%) showed uorescence after 24 h of incubation at 35 C. However,
in two cases, Colilert detected a strain that was tested negative by one or
two of the other methods. In the rst case, the E. coli strain detected by
Colilert, Readycult, and Chromocult methods was not detected by MI
and in the second, the strain detected by Colilert and Chromocult was
not detected by both Readycult and MI methods. Chromocult, MI, and
Table 2 (continued)
reference
Test methods
All E. coli strains 70/74 (94.6%) 71/74 (95.9%) 72/74 (97.3%) 64/74 (86.5%)
All environmental strains 28/28 (100%) 28/28 (100%) 28/28 (100%) 28/28 (100%)
Non-E. coli total coliforms (n=33)
Citrobacter amalonaticus (clinical) ATCC 25405 + +
Citrobacter braakii (clinical) ATCC 43162 + + +
Citrobacter farmeri (clinical) ATCC 51112 + +
Citrobacter freundii (clinical) ATCC 8090 + + + +
Citrobacter freundii (environmental) CCRI-14856 + + +
Citrobacter gillenii (clinical) ATCC 51117 + + + +
Citrobacter koseri (clinical) ATCC 27156
Citrobacter koseri (clinical) ATCC 27028 + +
Citrobacter murliniae (clinical) ATCC 51641 + + +
Citrobacter sedlakii (clinical) ATCC 51115 + + + +
Citrobacter werkmanii (clinical) ATCC 51114 + + + +
Citrobacter youngae (food) ATCC 29935 + + +
Enterobacter aerogenes (clinical) ATCC 13048 + + +
Enterobacter cloacae (clinical) ATCC 13047 +
Enterobacter cloacae (environmental) CCRI-17108 + + +
Enterobacter sakazakii (environmental) CCRI-17037 + + +
Escherichia blattae (animal) ATCC 29907
Escherichia fergusonii (clinical) ATCC 35469 +
Escherichia hermannii (clinical) ATCC 33650 + + +
Escherichia vulneris (clinical) ATCC 33821 + + +
Hafnia alvei (clinical) ATCC 13337
Hafnia alvei (environmental) CCRI-16651 +
Klebsiella oxytoca (clinical) ATCC 13182 + +
Klebsiella pneumoniae (clinical) ATCC 27799 + +
Klebsiella pneumoniae (environmental) CCRI-17014 + + +
Pantoea agglomerans (clinical) ATCC 27155 + +
Raoultella ornithinolytica (clinical) ATCC 31898 + + + +
Raoultella planticola (environmental) ATCC 33531 + +
Raoultella terrigena (environmental) ATCC 33257
Serratia marcescens (environmental) ATCC 13880
Yersinia enterocolytica (clinical) ATCC 9610
All non-E. coli total coliforms 20/33 (60.6%) 19/33 (57.6%) 19/33 (57.6%) 15/33 (45.5%)
All environmental non-E. coli total coliforms 6/7 (85.7%) 6/7 (85.7%) 7/7 (100%) 0/7 (0%)
All total coliforms 90/107 (84.1%) 90/107 (84.1%) 91/107 (85.0%) 79/107 (73.8%)
All environmental total coliforms 34/35 (97.1%) 34/35 (97.1%) 35/35 (100%) 28/35 (80.0%)
Readycult tests yielded a positive signal for all other strains found
positive by Colilert. Overall, discordant results between Colilert and, at
least one of the three other methods, were observed for 29 of the 74
strains panel (39.2%). Anadditional 4 E. coli strains showed positive with
Colilert upon an extended incubation for an additional 4 h at 35 C
thereby increasing its level of detection from 51.4 to 56.8%.
When only non-O157:H7 E. coli strains were considered, 59 of 65
(90.8%) werefoundtobepositivewithbothMI andChromocult methods
while 58 (89.2%) were positive with Readycult. Colilert presented the
lowest level of detectionby yielding a uorescent positive signal for only
38 of the 65 non-O157:H7 E. coli strains (58.5%) tested. Finally, when
only the 28 environmental E. coli strains were considered, 22 (78.6%)
were detectable with Colilert while 27 (96.4%) were detectable with
Chromocult and 28 (100%) with both MI and Readycult.
3.2. Detection of Shigella strains
Eight (8) Shigella spp. strains were used to demonstrate the ability
of the four tests to detect Shigella species (Table 1). S. exneri and S.
dysenteriae strains were undetected by all 4 methods, while the S.
sonnei strains were -galactosidase-positive with the four methods
and also -glucuronidase-positive based on the MI test. S. boydii was
detected as -galactosidase-positive by both Chromocult and MI tests
and as glucuronidase-positive by Readycult, Chromocult, and MI test
methods.
3.3. Detection of total coliform strains
The ability of the four methods to detect -galactosidase produc-
tion by total coliform strains was veried with the 74 E. coli strains
(Table 1) as well as with the 33 clinical and environmental non-E. coli
total coliform strains (Table 2). Colilert, Readycult and Chromocult
showed comparable detection rates of respectively 20 (60.6%), 19
(57.6%), and 19 (57.6%) of the 33 non-E. coli total coliform strains
tested, whereas the MI test method gave the lowest percentage by
detecting only 15 (45.5%) of these strains. Only 4 strains (12.1%) were
detected by the four test methods whereas 6 (18.2%) were undetect-
able by all methods. Thus, discordant results were observed for 23
Table 3
Strains used for the specicity analysis
Gram-positive bacteria (n=52) Gram-negative bacteria (n=37)
Abiotrophia defectiva ATCC 49176 Acinetobacter baumanii ATCC 19606
Enterococcus avium ATCC 14025 Acinetobacter haemolyticus ATCC 17906
Enterococcus casseliavus ATCC 25788 Aeromonas caviae CCUG 44411
Enterococcus cecorum ATCC 43198 Aeromonas hydrophila ATCC 7966
Enterococcus columbae ATCC 51263 Burkholderia cepacia ATCC 25416
Enterococcus dispar ATCC 51266 Haemophilus haemolyticus ATCC 33390
Enterococcus durans ATCC 19432 Haemophilus inuenzae ATCC 9007
Enterococcus faecalis ATCC 19433 Haemophilus parahaemolyticus ATCC 10014
Enterococcus faecium ATCC 19434 Haemophilus parainuenzae ATCC 7901
Enterococcus avescens ATCC 49996 Legionella pneumophila ATCC 33156
Enterococcus gallinarum LSPQ 3364 Moraxella atlantae ATCC 29525
Enterococcus hirae ATCC 8043 Moraxella catarrhalis ATCC 25238
Enterococcus mundtii ATCC 43186 Neisseria caviae ATCC 14659
Enterococcus pseudoavium ATCC 49372 Neisseria elongata ATCC 25295
Enterococcus rafnosus ATCC 49427 Neisseria gonorrhoeae ATCC 35201
Enterococcus ratti ATCC 700914 Neisseria meningitidis ATCC 13077
Enterococcus saccharolyticus ATCC 43076 Neisseria mucosa ATCC 19696
Enterococcus solitarius ATCC 49428 Pasteurella aerogenes ATCC 27883
Enterococcus sulfureus ATCC 49903 Photorhabdus luminescens ATCC 43948
Gemella haemolysans ATCC 10379 Proteus mirabilis ATCC 25933
Granulicatella adiacens ATCC 49175 Proteus vulgaris ATCC 29513
Lactobacillus acidophilus ATCC 4356 Providencia alcalifaciens ATCC 9886
Leifsonia aquaticus ATCC 14665 Providencia rettgeri ATCC 9250
Listeria grayi ATCC 19120 Providencia rustigianii ATCC 33673
Listeria innocua ATCC 33090 Providencia stuartii ATCC 43664
Listeria ivanovii ATCC 19119 Pseudomonas aeruginosa ATCC 35554
Listeria monocytogenes ATCC 15313 Pseudomonas uorescens ATCC 13525
Listeria seeligeri ATCC 35967 Pseudomonas stutzeri ATCC 17588
Micrococcus luteus ATCC 9341 Salmonella cholerasuis ATCC 7001
Staphylococcus aureus ATCC 25923 Salmonella indica ATCC 43976
Staphylococcus capitis ATCC 27840 Salmonella typhimurium ATCC 14028
Staphylococcus epidermidis ATCC 14990 Stenotrophomonas maltophilia ATCC 13637
Staphylococcus haemolyticus ATCC 29970 Vibrio alginolyticus CCRI-14794
Staphylococcus hominis ATCC 27844 Vibrio cholerae ATCC 25870
Staphylococcus lugdunensis ATCC 43809 Vibrio uvialis CCRI-14795
Staphylococcus saprophyticus ATCC 15305 Vibrio parahaemolyticus ATCC 17802
Staphylococcus simulans ATCC 27848 Vibrio vulnicus ATCC 27562
Staphylococcus warneri ATCC 27836
Streptococcus agalactiae ATCC 13813
Streptococcus anginosus ATCC 33397
Streptococcus bovis ATCC 33317
Streptococcus constellatus ATCC 27823
Streptococcus cristatus ATCC 51100
Streptococcus gordonii ATCC 33399
Streptococcus intermedius ATCC 27335
Streptococcus mutans ATCC 25175
Streptococcus parasanguis ATCC 15912
Streptococcus pneumoniae ATCC 6303
Streptococcus pyogenes ATCC 19615
Streptococcus salivarius ATCC 7073
Streptococcus sanguinis ATCC 10556
Streptococcus suis ATCC 43765
(69.7%) of the 33 non-E. coli total coliform strains tested (Table 2).
Again, when considering all total coliform strains, including all E. coli
strains, MI presented the lowest percentage of detection with only 80
(74.8%) of the 107 strains tested whereas Colilert, Readycult, and
Chromocult detected -galactosidase production in respectively 90
(84.1%), 90, and 91 (85.0%) of all total coliform strains tested.
Interestingly, we observed that with environmental non-E. coli
total coliform strains only, 6 of the 7 (85.7%) total coliform strains
tested positive for -galactosidase production with both Colilert and
Readycult, while all were positive with Chromocult. MI did not detect
any of these strains. However, when only the 28 environmental E. coli
strains were considered, all were positive with MI as well as with the 3
other methods.
3.4. Specicity
Specicity (i.e. the ability to detect only -galactosidase and -
glucuronidase production by the target species of the four test
methods) was veried by testing 89 non-total coliform strains
representing 52 species of Gram-positive and 37 species of Gram-
negative bacteria that are frequently encountered in fecal or
environmental settings (Table 3). Eighty-six (86) of these 89 (96.6%)
bacterial species were undetectable with the 4 test methods. Provi-
dencia alcalifaciens and Providencia rustigianii yielded a -galactosi-
dase-positive signal while Salmonella indica yielded positive results
for both -galactosidase and -glucuronidase with Readycult, Chro-
mocult and MI test methods.
4. Discussion
By using a panel of 74 E. coli strains of different serotypes isolated
fromfecal and environmental settings, we have determined the ability
of the test methods to detect -glucuronidase production. To our
knowledge, this is the rst report on the comparison of these test
methods, using pure cultures. We found that Chromocult, MI and
Readycult present a similar percentage of detection varying from 79.9
to 81.1%, whereas Colilert detected only 51.4% of the E. coli strains. As
opposed to Rice et al. (1990) who observed -glucuronidase
production for 99.5% of the E. coli strains tested after 28 h, an
additional 4 h of incubation for uorescent-negative Colilert strains
allowed only a slight improvement in the detection of -glucuroni-
dase production (increased to 56.8%). The lowest level of detection
obtained by Colilert, as compared to the 3 other tests, is not explained
by the presence of E. coli O157:H7 strains, that have been reported to
be uniformly -glucuronidase-negative (Krishnan et al., 1987; Ratnam
et al., 1988). Indeed, when these strains were excluded in the data set,
Chromocult, MI and Readycult methods still showed a similar
percentage of detection varying from 89.2 to 90.8%. Whereas the
detection level with Colilert was 58.8%.
The ability to detect E. coli was also veried with environmental
isolates only. Surprisingly, and contrary to what have been published
previously by Rice et al. (1993) and Shadix et al. (1993) who detected -
glucuronidase production for more than 95% of the E. coli strains tested,
Colilert showed the lowest detection level of environmental E. coli
strains (i.e. 78.6%), whereas almost all E. coli strains were detected with
Chromocult, MI andReadycult (96.4to100%). Suchdiscrepant results are
not totally unexpected since studies previously realized by Doyle et al.
(1955), Feng and Lampel (1994), Chang et al. (1989), and Shadix and Rice
(1991) showed that the percentage of -glucuronidase-negative E. coli
strains of a given nature source population will be variable and that the
compositionof the media will inuence the rate of detection. The results
of this study, obtained by comparing a collection of E. coli strains, show
that Colilert systematically detected approximately 20% less E. coli
strains than the 3 other culture methods, even if they are based on the
same enzymatic principles. A similar lower percentage of E. coli
detection using minimal media o-nitrophenyl--D-galactopyranoside
(MMO)-MUG preparations, such as Colilert, was also observed by
Martins et al. (1993). The dened substrate technology of Colilert is
based on the choice and the amount of ingredients for providing a strict
requirement for E. coli specic growth. Our results and those of Martins
et al. (1993) suggest that such minimal media could be lacking some
essential nutrients. Thus, the low recovery of some E. coli strains by
minimal medium may not be totally attributed to the strain itself but
may also be inuenced by the composition of the medium.
In this study, S. dysenteriae and S. exneri strains were not detected
by any of the four methods. For Readycult and Colilert, S. boydii and S.
sonnei are recognized as total coliforms, whereas MI yielded an E. coli-
phenotype result for these two species. These results are in
accordance with those published by McDaniels et al. (1996), where
positive signal for -glucuronidase were observed within members of
the genus Shigella.
The ability of the four test methods to detect -galactosidase
production was veried by using, in addition to the 74 E. coli strains,
33 reference and environmental non-E. coli total coliform strains
found in fecal and environmental settings of different geographic
origins. For the non-E. coli total coliforms, Chromocult, Colilert and
Readycult showed a similar percentage of detection ranging from
57.6 to 60.6% whereas MI detected only 45.5% of the strains tested. It
is well known in environmental microbiology that the total
coliforms group, based on phenotypic characteristics, is not well
dened. In accordance with Olstadt et al. (2007), the results of our
study show that, in addition to this fact, there is a total lack of
correlation between test methods based on the same enzymatic
principle to recognize a strain as non-E. coli total coliform. Indeed,
our results showed that there is no correlation between the 4
methods tested either within the same genera or within the same
species. Furthermore, when only environmental non-E. coli total
coliforms are considered, Chromocult achieved to detect all strains,
while Colilert and Readycult detected 85.7% of them (7/8). MI was
unable to detect any of these environmental isolates. When all total
coliforms strains are considered, Colilert, Chromocult, and Readycult
showed similar percentages of detection ranging from 94.6 to 97.3%
of E. coli strains, whereas MI detected 86.5% of these strains.
Interestingly, the four methods detected -galactosidase production
in all environmental E. coli strains. However, studies using a higher
number of environmental strains are required to extend this
observation. Finally, no correlation was observed between -
galactosidase production results obtained by the Microscan identi-
cation test and the results obtained by the four other methods
tested. Indeed, the orthonitrophenyl--D-galactopyranoside (ONPG)
test performed on the Microscan system yielded -galactosidase-
positive signal for 94.4% of total coliformstrains tested. These results
suggest that automated phenotypic identication tests may also
have difculties to induce production of -galactosidase. Thus,
identication methods, solely relying on the activity of a single
enzyme, are subject to a lack of robustness and may lead to
misinterpretations since enzymatic activity can be transient and
highly regulated by environmental factors.
By comparing the -glucuronidase and the -galactosidase
production detection between environmental and clinical E. coli
strains, one could be tempted to conclude that these 4 methods are
more efcient to detect environmental E. coli strains than clinical E.
coli strains. However, we must keep in mind that the environmental
strains used in this study were isolated from recommended media
used for the recovery of E. coli in water. Thus, environmental E. coli
strains that cannot be detected by those media could not have been
included in the strain collection used for in this study. This bias limits
the interpretation of the difference between the rate of detection of
environmental E. coli strains and strains isolated by clinical
techniques.
The specicity was veried by testing 89 non-total coliform strains
frequently encountered in fecal or environmental settings. Only P.
alcalifaciens, P. rustigianii, and S. indica yielded positive results. It was
previously reported that species of Providencia yielded false-positive
results with Colilert especially in a marine environment (Pisciotta et
al., 2002). Furthermore, some strains of Salmonella species are known
to express -galactosidase and -glucuronidase enzyme (Feng and
Hartman, 1982; Kaluzewski and Tomczuk, 1995). Globally, all four tests
methods studied here presented a low level of false-positive results
based on testing of a bacterial strains collection representing 52 Gram-
positive and 37 Gram-negative species. These results are in accor-
dance with studies claiming that a further systematic identication
and conrmation for these techniques are not necessary for water
samples monitoring (Edberg et al., 1988; Rompre et al., 2002), except
for marine water monitoring in the case of Colilert (Landre et al., 1998;
Pisciotta et al., 2002).
This study examined the relative performance of the Colilert, MI
agar, Chromocult agar and Readycult test methods by comparing their
respective analytical limits, using a collection of strains consisting of E.
coli, Shigella, total coliforms, and other non-target bacteria. These
results do not necessarily correlate with their individual efciency to
detect E. coli and total coliforms in natural water samples. This is
explained by the fact that environmental samples present a hetero-
genic population of bacteria and the chance that a contaminated water
sample only contains strains that are undetectable by one of these
methods is quite low. Indeed, the majority of published studies
comparing these methods, using natural water samples, showed a
similar efciency between them and classical reference methods
(APHA et al., 2005; Bernasconi et al., 2006; Brenner et al., 1993, 1996a,
b; Buckalew et al., 2006; Clark and el-Shaarawi, 1993; Colquhoun
et al., 1995; Cowburn et al., 1994; Eckner, 1998; Edberg et al., 1988,
1990; Fricker and Fricker, 1996; Fricker et al., 1997; Horman and
Hanninen, 2006; Macy et al., 2005; Niemela et al., 2003; Schets et al.,
2002). However, it cannot be excluded that a lower rate of detection of
E. coli could be observed with Colilert when natural water samples
analysed present low diversity of strains or when the water samples
only contain low levels of bacterial contamination. Indeed, this may
provide an explanation for the unexpected level of false-negative
results observed with Colilert method by Clark et al. (1991), Hall and
Moyer (1989), Lewis and Mak (1989), and Pitkanen et al. (2007), when
it was compared to the standard membrane ltration fecal coliform
(mFC) and thermotolerant E. coli (mTEC) test methods.
Finally, classical microbiology techniques for water quality mon-
itoring, such as mFC and Tergitol-TTC media, require strain conrma-
tion. Thus, to conrm the identity of their E. coli presumptive strains,
laboratories using classical techniques could be tempted to use one of
the four commercial methods tested in this study. The relatively high
level of false-negative results reached by the four methods suggests
that they should not be used to conrm the identity of presumptive E.
coli strains. Tests asserting multiple phenotypes are recommended for
species identication. Future alternatives may include molecular tests
for conserved species-specic genetic targets (Martinez et al., 2006).
Acknowledgements
We thank ve Brub and Marie-Claude Hlie for technical
assistance. We also thank Drs Louise Ct, director of the Microbiology
Laboratory of CHUL (Centre Hospitalier Universitaire de Qubec),
Philippe Cantin (Centre d'Expertise en Analyse Environnementale du
Qubec), Pierre Simard and Lynda Rodrigue (Bodycote Canada), Pierre
Harbec (Laboratoire de Sant Publique du Qubec), Wang Fu (Huashan
Hospital), Helge Karch (Institut fr Hygiene und Mikrobiologie der
Universitat), Jordy Vila (Servei de Microbiologia, Centre de Diagnstic
Biomdic, Universitat de Barcelona), Nicolas Chamoine (Hpital
Ambroise Par), Patricia Bradford (Wyeth-Ayerst Research), Sebastian
G. B. Amyes (University of Edinburgh), Marek Gniadkowski (National
Institute of Public Health), and Mignon du Plessis (South African
Institute for Medical Research) for providing E. coliShigella strains.
This research was supported by grants PA-15586 from the
Canadian Institutes of Health Research (CIHR) and FCI-5251 from the
Canadian Foundation for Innovation (CFI). Andre Maheux, Vicky
Hupp, and Jean-Luc Bernier received a scholarship from Nasivvik
(Center for Inuit Health and Changing Environment; Canadian
Institutes for Health Research).
Disclosure of interests: The authors of this study do not have any links
with companies manufacturing or commercializing water testing
products.
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Analytical limits of four -glucuronidase and -galactosidase-based commercial

culture methods used to detect Escherichia coli and total coliforms

Coliforms 100 (Readycult), Chromocult

Coliform agar ES (Chromocult), and

(Colilert, IDEXX Laboratories, Westbrook, ME, USA),

Coliforms 100 (Readycult; Merk KGaA, Darmstadt, Ger-

Coliform agar ES (Chromocult; Merk KGaA,

Coliform 100 (Readycult), and MI agar

, sealed, and incubated at 35.0

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