Você está na página 1de 35

An ISO/TR 13843 Method Performance Validation for

the Enterlolert*-DW and Quanti-Tray* System for the


Quantitative Detection of Enterococci in Drinking Water

APRIL 2010

One IDEXX Drive • Westbrook, Maine 04092 USA


idexx.com
© 2011 IDEXX Laboratories, Inc. All rights reserved. • 8980-01

*Enterolert, Define Substrate Technology, DST and Quanti-Tray are trademarks or registered trademarks
of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.
The IDEXX Privacy Policy is available at www.idexx.com.
1. Introduction
The Enterolert*-DW test, based on Defined Substrate Technology* (DST*), is designed to detect
enterococci in drinking water samples. As enterococci grow in the liquefied medium, the organisms’
enzymes metabolize a substrate specific for β-glucosidase, changing the medium’s color from blue
to green and confirming the presence of enterococci in water samples.
The Enterolert-DW and Quanti-Tray* system (Enterolert-DW/Quanti-Tray) is used for the detection
and enumeration of enterococci in water samples. A blister pack of Enterolert-DW reagent is added
to a sterile bottle containing the water sample. After the powder reagent is added, the vessel is
shaken to dissolve the powder. The dissolved sample/medium mixture is added to a Quanti-Tray
or Quanti-Tray 2000, and the tray is sealed with the Quanti-Tray sealer. The tray is incubated at
41 ± 0.5ºC for 24 hours. The quantification of confirmed enterococci in the sample is determined
by counting the number of green colored wells on the Quanti-Tray device using the Most Probable
Number (MPN) statistics.

2. Scope of application of Enterolert-DW/Quanti-Tray system


The Enterolert-DW/Quanti-Tray system is intended for the analysis of drinking water samples. It
is designed to be comparable in performance to ISO 7899-2:2000 Water Quality – Detection and
enumeration of intestinal enterococci – Part 2: Membrane filtration method (ISO 7899-2) and The
Microbiology of Drinking Water (2006) – Part 5: The isolation and enumeration of enterococci by
membrane filtration (MoDW 2006, Part 5) in drinking water applications. The Enterolert-DW/Quanti-
Tray system provides quantitative, confirmed results for the detection of enterococci in drinking
water samples within 24 hours

3. Target organism identification (ISO/TR 13843 sections 10.2.1 and 9.2)


The Enterolert-DW/Quanti-Tray system detects enterococci based on their β-glucosidase activity
using the Defined Substrate Technology. When enterococci are present in the test sample, the
medium changes color from blue to green. The Quanti-Tray system provides quantitative results
based on the MPN statistics.

3.1 Pure culture challenge (ISO/TR 13843 section 10.2.1)


Typical reactions of the Enterolert-DW/Quanti-Tray system were characterized by challenging the
medium using pure cultures of reference strains from the American Type Culture Collection (ATCC).
The target enterococcus bacteria, as well as nontarget Gram-negative and Gram-positive bacteria,
were tested. Typical results for these reference strains are listed in Table 3.1. These strains are
used by IDEXX for routine quality control of the Enterolert-DW/Quanti-Tray system.

IDEXX Laboratories, Inc. 1


Table 3.1 Reference strains of enterococcus bacteria and nontarget Gram-negative and Gram-positive
bacteria used for confirming typical positive and negative reactions in the Enterolert-DW/Quanti-
Tray system

2BU Bacterium 1BU Strain 3BU Typical reaction in Enterolert-DW


Enterococcus faecium ATCC 35667 Green
Enterococcus faecalis ATCC 33186 Green
Enterococcus hirae ATCC 8043 Green
Serratia marcescens** ATCC 43862 Blue (no color change)
Staphylococcus aureus*** ATCC 6538 Blue (no color change)
6 4
** Up to 10 CFU/100 mL *** Up to 10 CFU/100 mL

3.2 Sensitivity, specificity, false positive/negative rates, efficiency and selectivity studies
(ISO/TR 13843 section 9.2)
In terms of microbiological methods these characteristics are defined by ISO/TR 13843 as:
a) number of presumptive positives found positive (true positives)
b) number of presumptive negatives found positive (false negatives)
c) number of presumptive positives found negative (false positives)
d) number of presumptive negatives found negative (true negatives)

1) sensitivity = a/(a + b), the fraction of the total positives correctly assigned in the presumptive count
2) specificity = d/(c + d), the fraction of the total negatives correctly assigned in the presumptive count
3) false positive rate = c/(a + c), the fraction of the observed positives wrongly assigned
4) false negative rate = b/(b + d), the fraction of the observed negatives wrongly assigned
5) Efficiency (E) = (a + d)/n
6) Selectivity (F) = lg [(a + c)/n]

For the Enterolert-DW/Quanti-Tray system, these characteristics relate to the number of wells which
were positive for enterococci that actually contained target bacteria and the number of negative
wells that were truly negative for the target bacteria.
The assessment of sensitivity, specificity and selectivity of the Enterolert-DW/Quanti-Tray system
was achieved through the identification of bacterial isolates from positive and negative wells of
tested samples. The samples were prepared by seeding drinking water samples with either
chlorinated or nonchlorinated sewage effluent samples.
In the present validation, false positives and false negatives of the Enterolert-DW/Quanti-Tray
system were determined by the selective isolation and bacterial identification from 163 positive wells
and 186 negative wells selected at random from samples collected in 49 locations in the U.S., U.K.,
Italy, Belgium and Spain. The VITEK II biochemical identification system (bioMérieux, Inc. Durham,
NC, U.S.A.) was used. Results are detailed in Appendix I and Appendix II, respectively.
The performance characteristics of the Enterolert-DW/Quanti-Tray system, as defined by ISO/TR
13843, are listed in Table 3.2.1

IDEXX Laboratories, Inc. 2


Table 3.2.1 Enterolert-DW/Quanti-Tray system sensitivity, specificity, false positive/negative rates,
efficiency and selectivity

Characteristic Results
Sensitivity 92%
Specificity 98%
False-positive rate 2%
False-negative rate 7%
Efficiency 95%
Selectivity -0.3

Bacterial isolates from four (4) of 163 positive wells were identified as nonenterococcus species and
thirteen (13) isolates from the 186 negative wells were actually found to be enterococcus species.
The bacterial species giving rise to false-positive and false-negative results in this validation are
detailed in Tables 3.2.2 and 3.2.3 respectively.

Table 3.2.2 False-positive bacterial identification using VITEK II

Occurrences
U

False-positive ID (out of 163 positives)


Lactococus raffinolactis 1

Elizabethkingia meningoseptica 3

Table 3.2.3 False-negative bacterial identification using VITEK II

Occurrences
False-negative ID (out of 186 negatives)
Enterococcus hirae 2

Enterococcus faecium 4

Enterococcus avium 5

Enterococcus faecalis 1

Enterococcus casseliflavus 1

The outcomes of this validation indicate that the Enterolert-DW/Quanti-Tray system is sensitive and
specific (92% and 98%, respectively). The method is also selective, having a value of -0.3, which is
considerably better than the guidance value of -1 suggested by ISO/TR 13843 for colony-count
methods. With a calculated efficiency value of 95%, the data suggest that the method is highly
efficient. The identification data indicate that the Enterolert-DW/Quanti-Tray system recovers a
broad range of enterococcus species.

4. Counting uncertainty (ISO 13843 section 10.2.1 and Annex B)


Repeatability and reproducibility are two estimates of the reliability of a method that can be achieved
analytically. Repeatability (r) and reproducibility (R) are defined as follows:
Repeatability: Closeness of the agreement between the results of successive measurements of
the same measure carried out under the same conditions of measurement
Reproducibility: Closeness of the agreement between the results of measurements of the same
measure carried out under changed conditions of measurement

IDEXX Laboratories, Inc. 3


When applied to counting of microbiological colonies on an agar plate, or positive reactions in an
MPN test, the analyst is the ‘condition’ that either remains the same or is changed. Thus, in this
context, repeatability is the agreement in counts obtained by repeated counting events by the same
analyst, and reproducibility is the agreement between subsequent counts obtained by two or more
analysts.
ISO/TR 13843 Annex B provides guidance on the assessment of counting repeatability and
reproducibility using the relative standard deviations (RSDc) of repeated counts. It also
recommends that, when using pure cultures, RSDc should ideally be < 0.02 (i.e., not more than 2%
deviation).
Counting uncertainty studies were conducted by IDEXX analysts, and the resulting data are
presented in Appendix III. The counting was performed such that each analyst was unaware of
other analysts’ counts, and the resulting MPN values were recorded as whole integers. The
calculated RSDs for MPN values for the Enterolert-DW/Quanti-Tray system, inoculated with
Enterococcus faecalis ATCC 19433, are listed in Table 4.1.

Table 4.1 Enterolert-DW repeatability and reproducibility

U Relative standard deviation


U Counting uncertainty (RSDc)
Repeatability (r) 0.006

Reproducibility (R) 0.005

The calculated values for typical enterococci are much less than the guidance value of < 0.02
(ISO/TR 13843 section B1), indicating that reliable MPN counting of enterococci can be achieved
with the Enterolert-DW/Quanti-Tray method.

5. Robustness (ISO/TR 13843 sections 10.2.2 and B.4)


The aspect of robustness that is relevant to the Enterolert-DW/Quanti-Tray method is the incubation
period of 24 hours with a valid result up to 28 hours, as described in the method. The results of
triplicate counts of suspensions of reference cultures of E. faecium, E. faecalis, E. durans, E. hirae,
E. mundtii and E. gallinarum analysed using the same batch of Enterolert-DW at both the 24-hour
and 28-hour incubation periods are presented in Appendix IV.
There was no change in the counts observed for 6 of the 7 type strains validated. The E. gallinarum
ATCC 49573 counts increased by one positive well during the 24- to 28-hour read window. This
strain of E. gallinarum ATCC 49573 appears to show a relatively slower growth rate or lower
enzyme activity compared to the other bacterial strains tested.
The data suggest that the recommended incubation period of a minimum of 24 hours and up to 28
hours is robust. However, for some strains of enterococci that either grow slowly or have reduced β-
glucosidase activity, there may be a marginal increase in counts at 28 hours of incubation,
compared to those at 24 hours of incubation.
An increase in counts over a prescribed incubation period (e.g., 40–48 hours for typical membrane
filtration methods, such as ISO 7899-2 or MoDW 2006, part 5) is a well recognized phenomenon,
particularly with environmental enterococci. However, provided that the increase in counts after an
extended incubation is not excessive, for practical and operational considerations, counts from the
Enterolert-DW/Quanti-Tray system at either 24 or 28 hours incubation can be considered
acceptable.

6. Upper working limits (ISO/TR 13843 sections 10.2.4 and 6.3.3)


The Enterolert-DW/Quanti-Tray system is an MPN-based method with an arbitrary counting range of
0 to 201 for Quanti-Tray and 0 to 2,420 for Quanti-Tray/2000, determined by the number of wells in
the Quanti-Tray. According to ISO/TR 13843, section 6.3.3, no upper limit can be set for MPN

IDEXX Laboratories, Inc. 4


methods for practical and statistical reasons, “because precision does not depend in a simple way
on the number of particles introduced in the detection set.”

7. Precision (ISO/TR 13843 sections 10.2.5 and 9.5.5)


The precision of an MPN method is described by the 95% confidence intervals (CIs) calculated for
each MPN value (ISO/TR 13843, section 9.5.5). The CIs for counts from the Enterolert-DW/Quanti-
Tray system, calculated using the program available online from the U.S. Food and Drug
Administration (FDA) Bacteriological Analytical Manual (BAM) (at
http://www.cfsan.fda.gov/~ebam/bam-a2.html#excl), are given in Appendix V.
The theoretical repeatability of each MPN value can be derived from the MPN and its CIs. Relative
uncertainty values for each MPN value (log10SD, calculated from the MPN 95% CI values) and
repeatability (relative standard uncertainty), given as coefficients of variance (CV%), are also
presented in Appendix VI. These Poisson values can be used as minimum precision values that can
be combined with any appropriate data a laboratory may have (e.g., on variation in decanting the
test volume or counting uncertainty) to derive an overall estimate of repeatability for each MPN
value.

8. Relative recoveries (ISO/TR 13843 section 10.2.6 and ISO 17994)


The relative recovery of enterococcus spp. by the Enterolert-DW/Quanti-Tray system was compared
to that of the reference methods of ISO 7899-2 and MoDW 2006, part 5, following the requirements
of ISO 17994:2004 Water Quality – Criteria for establishing equivalence between microbiological
methods (ISO 17994).
A total of 403 drinking water samples from geographically diverse locations within the United
Kingdom, Italy, Spain, Belgium and the U.S. were used in this validation. Split samples were used in
each of the three methods under comparison. Among those, 198 samples were chlorinated and 205
were not chlorinated.
Paired confirmed count data for the two methods (either the Enterolert-DW/Quanti-Tray system and
ISO 7899-2 or the Enterolert-DW/Quanti-Tray system and MoDW part 5) were transformed using
natural logarithms (ln), and the relative difference in counts for each pair (xi) was calculated using
the formula [ln(ai) – ln(bi)] x 100% (where ai and bi are the paired confirmed counts for the trial and
reference methods respectively).
Note: If one of the counts is zero, then 1 is added to both counts prior to log-transformation.
The mean relative difference and the standard deviation were calculated from the data. Evaluation
of equivalence was based upon the mean relative difference and the expanded uncertainty (U)
(calculated from the standard deviation of the mean value [U = 2s/√n]), from which the lower (xL)
and higher (xH) limits of the confidence interval are calculated. The outcomes of the studies are
listed in Tables 8.1 and 8.2.
The analysis showed that the Enterolert-DW/Quanti-Tray method gives overall higher recoveries in
comparison with ISO 7899-2. This result was largely driven by the relative differences obtained
using chlorinated sample matrices. For the nonchlorinated samples, the validation indicated
“Methods not different” between the two methods.
The overall conclusion was “Methods not different” when comparing the Enterolert-DW/Quanti-Tray
system to MoDW 2006, Part 5, with results from the Enterolert-DW/Quanti-Tray system being higher
in the chlorinated samples and showing no difference in the nonchlorinated samples.
The observed differences in recoveries between the Enterolert-DW/Quanti-Tray system and the two
standard methods were most likely driven by the performance degradation of the standard methods
when chlorinated samples were used. This is further supported by the low false-positive rate (2%)
and high specificity (98%) determined for the Enterolert-DW/Quanti-Tray system. Since it was a
split-sample validation, the results indicated that the standard methods were unable to detect some

IDEXX Laboratories, Inc. 5


target organisms from the same water sample. The relative recoveries for each method are
summarized in Appendix VII.
During this validation, it was observed that there were significant differences in relative recovery
between the two standard methods, ISO 79-899-2 and MoDW 2006, Part 5. The results are
summarized in Table 8.3.

Table 8.1 Enterolert-DW/Quanti-Tray system vs. ISO 7899-2

Expanded Expanded
Drinking water Mean relative uncertainty LO uncertainty HI
sample matrix difference (%RD) (%XL) (%XH) Conclusion

Enterolert-DW
All matrices 18 11 26
higher

Chlorinated Enterolert-DW
31 19 43
matrix higher
Nonchlorinated Methods no
6 -3 13
matrix difference

Table 8.2 Enterolert-DW/Quanti-Tray system vs. MoDW 2006, Part 5

Expanded Expanded
Drinking water Mean relative uncertainty LO uncertainty HI
sample matrix difference (%RD) (%XL) (%XH) Conclusion
All matrices 3 -4 10 Methods no
difference

Chlorinated 13 3 26 Enterolert-DW
matrix higher
Nonchlorinated -8 -15 0 Inconclusive
matrix

IDEXX Laboratories, Inc. 6


Table 8.3 ISO 7899-2 vs. MoDW 2006, Part 5

Expanded Expanded
Drinking water Mean relative uncertainty LO uncertainty HI
sample matrix difference (%RD) (%XL) (%XH) Conclusion
All matrices -15 -14 -16 Methods
significantly
different
Chlorinated matrix -17 -15 -19 Methods
significantly
different
Nonchlorinated -13 -12 -15 Methods
matrix significantly
different

9. References
 ISO 7899-2:2000 Water Quality – Detection and enumeration of intestinal enterococci - Part
2: Membrane filtration method. Geneva: International Organization for Standardization
 The Microbiology of Drinking Water (2006) - Part 5 - The isolation and enumeration of
enterococci by membrane filtration. Nottingham: Environment Agency
 The Microbiology of Drinking Water (2002) - Part 3 - Practices and procedures for
laboratories. Nottingham: Environment Agency
 ISO/TR 13843:2000(E) Water Quality – Guidance on validation of microbiological methods.
Geneva: International Organization for Standardization
 ISO 17994:2004 Water Quality – Criteria for establishing equivalence between
microbiological methods. Geneva: International Organization for Standardization

IDEXX Laboratories, Inc. 7


Appendix I: Identification of isolates from positive Enterolert-DW/Quanti-Tray wells
U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
1 + E. gallinarum 99%
2 + E. faecium 97%
3 + E. faecium 99%
4 + E. gallinarum 99%
5 + E. gallinarum 99%
6 + E. faecium 99%
7 + E. faecalis 99%
8 + E. faecium 99%
9 + E. faecium 96%
10 + E. gallinarum 99%
11 + E. gallinarum 99%
12 + E. faecium 97%
13 + E. hirae 99%
14 + E. faecalis 99%
15 + E. faecium 98%
16 + E. gallinarum 99%
17 + E. faecium 98%
18 + E. gallinarum 93%
19 + E. faecalis 99%
20 + E. avium 92%
21 + E. faecalis 99%
22 + E. durans/hirae Low Dis
23 + E. faecalis 99%
24 + E. seriolicida 99%
25 + E. faecalis 99%
26 + E. seriolicida 99%
27 + E. seriolicida 95%
28 + E. gallinarum 98%
E. seriolicida/L. lactis
29 + ssp lactis Low Dis
30 + E. faecium 99%
31 + Lactococus raffinolactis 90%
32 + E. gallinarum 99%
33 + E. seriolicida 99%
34 + E. faecium 99%
35 + E. faecium 99%
36 + E. faecium 99%
37 + E. gallinarum 99%
38 + E. faecium 99%
39 + E. faecium 99%

IDEXX Laboratories, Inc. 8


U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
40 + E. faecium 98%
41 + E. hirae 95%
42 + E. hirae 99%
43 + E. faecium 98%
44 + E. hirae 95%
45 + E. hirae 99%
46 + E. gallinarum 99%
47 + E. faecium 99%
48 + E. faecium 99%
49 + E. faecium 99%
50 + E. hirae 99%
51 + E. durans/faecium Low Dis
52 + E. faecalis 99%
53 + E. gallinarum 93%
54 + E. durans 98%
55 + E. faecium 96%
56 + E. faecium 99%
57 + E. faecalis 99%
58 + E. hirae 99%
59 + E. faecium 92%
60 + E. hirae 99%
61 + E. faecium 99%
62 + E. faecium 99%
63 + E. faecium 94%
64 + E. faecium 99%
65 + E. durans 97%
66 + E. faecium 98%
67 + E. faecalis 99%
68 + E. hirae 95%
69 + E. faecium 99%
70 + E. faecalis 99%
71 + E. faecium 96%
72 + E. casseliflavus 89%
73 + E. faecalis 99%
74 + E. casseliflavus 99%
75 + E. seriolicida 99%
76 + E. seriolicida / lactis Low Dis
77 + E. durans/faecium/hirae Low Dis
78 + E. casseliflavus 97%
79 + E. gallinarum 99%
80 + E. casseliflavus 89%
81 + E. durans/hirae Low Dis

IDEXX Laboratories, Inc. 9


U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
82 + E. gallinarum 98%
83 + E. gallinarum 99%
84 + E. hirae 95%
85 + E. faecalis 99%
86 + E. seriolicida /lactis Low Dis
87 + E. faecium 95%
88 + E. seriolicida /lactis Low Dis
89 + E. faecium 98%
90 + E. avium 95%
91 + E. gallinarum 99%
92 + E. faecium 93%
93 + E. gallinarum 99%
94 + E. gallinarum 99%
95 + E. gallinarum 99%
96 + E. faecium 98%
97 + E. faecium 99%
98 + E. faecium 99%
99 + E. faecalis 99%
100 + E. gallinarum 99%
101 + E. seriolicida /lactis Low Dis
102 + E. faecium 96%
103 + E. faecium 99%
104 + E. faecium 92%
105 + E. gallinarum 99%
106 + E. faecium 99%
107 + E. durans/gallinarum/hirae Low Dis
108 + E. gallinarum 99%
109 + E. faecalis 99%
110 + E. faecium 98%
Elizabethkingia
111 + meningoseptica 95%
112 + E. gallinarum 99%
Elizabethkingia
113 + meningoseptica 99%
114 + E. faecium 98%
115 + E. casseliflavus 99%
116 + E. faecium 99%
117 + E. casseliflavus/gallinarum Low Dis
118 + E. faecium 99%
119 + E. casseliflavus 99%
120 + E. gallinarum 99%
121 + E. faecium/gallinarum Low Dis

IDEXX Laboratories, Inc. 10


U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
122 + E. gallinarum 98%
123 + E. hirae 99%
124 + E. raffinosus 91%
125 + E. faecium 99%
126 + E. faecalis 85%
127 + E. faecalis 99%
Elizabethkingia
128 + meningoseptica 99%
129 + E. hirae 99%
130 + E. durans 95%
131 + E. casseliflavus 93%
132 + E. seriolicida / lactis Low Dis
133 + E. faecium 86%
134 + E. casseliflavus/gallinarum Low Dis
135 + E. gallinarum 98%
136 + E. casseliflavus/gallinarum Low Dis
137 + E. faecium 99%
138 + E. hirae 99%
139 + E. faecium 98%
140 + E. gallinarum 99%
141 + E. faecium 98%
142 + E. gallinarum 99%
143 + E. faecium 99%
144 + E. faecium 99%
145 + E. gallinarum 98%
146 + E. durans/faecium Low Dis
147 + E. faecium 99%
148 + E. faecium 99%
149 + E. durans 85%
150 + E. gallinarum 99%
151 + E. faecium/gallinarum Low Dis
152 + E. faecium 99%
153 + E. faecium 94%
154 + E. faecium 99%
155 + E. faecalis 99%
156 + E. hirae 91%
157 + E. durans 94%
158 + E. faecium 99%
159 + E. faecium 98%
160 + E. durans 98%
161 + E. faecalis 99%
162 + E. durans 92%

IDEXX Laboratories, Inc. 11


U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
163 + E. faecalis 99%
164 + E. durans/faecium Low Dis
165 + E. faecium 99%
166 + E. faecium 99%
167 + E. faecium 98%
168 + E. gallinarum 99%
169 + E. faecalis 99%
170 + E. casseliflavus/gallinarum Low Dis
171 + E. durans 98%
172 + E. faecium 99%
173 + E. hirae 99%
174 + E. faecium 99%
175 + E. gallinarum 99%
176 + E. faecium 98%
177 + E. hirae 95%
178 + E. avium 90%
179 + E. gallinarum 98%
180 + E. faecalis 99%
181 + E. gallinarum 99%
182 + E. faecium 99%
183 + E. hirae 99%
184 + E. faecium 95%
185 + E. gallinarum 98%
186 + E. gallinarum 99%
187 + E. gallinarum 99%
188 + E. faecium 93%
189 + E. faecium 99%
190 + E. faecium 93%
191 + E. faecium 99%
192 + E. gallinarum 99%
193 + E. gallinarum 99%
194 + E. faecium 99%
195 + E. faecalis 99%
196 + E. faecium 99%
197 + E. gallinarum 99%
198 + E. faecium 98%
199 + E. faecium 98%
200 + E. durans/faecium Low Dis
201 + E. faecium 91%
202 + E. faecium 99%
203 + E. faecalis 99%
204 + E. faecalis 99%

IDEXX Laboratories, Inc. 12


U Sample ID U Enterolert-DW reactions U VITEK ID U ID confidence
205 + E. faecium 97%
206 + E. gallinarum 99%
207 + E. faecium 99%
208 + E. faecium 98%
209 + E. gallinarum 99%
210 + E. faecalis 99%
211 + E. faecium 99%
212 + E. casseliflavus/E. gallinarum Low Dis
213 + E. faecalis 95%
214 + E. faecium 99%
215 + E. durans 93%
216 + E. faecium 98%
217 + E. durans 99%
218 + E. faecalis 99%
219 + E. faecium 97%
220 + E. seriolicida /lactis Low Dis
221 + E. faecium 98%
222 + E. faecium 99%
223 + E. faecalis 99%
224 + E. faecium 97%
225 + E. faecalis 99%
226 + E. faecalis 99%
227 + E. faecalis 99%
228 + E. faecium 99%
229 + E. faecium 98%

IDEXX Laboratories, Inc. 13


Appendix II: Identification of isolates from negative Enterolert-DW/Quanti-Tray wells
Enterolert-DW
Sample ID VITEK ID ID confidence
U

reaction
U U U

1 - None NA
2 - None NA
3 - E. hirae 99%
4 - E. hirae 95%
5 - None NA
6 - None NA
7 - None NA
8 - None NA
9 - None NA
10 - None NA
11 - None NA
12 - None NA
13 - None NA
14 - None NA
15 - E. faecium 99%
16 - E. faecium 98%
17 - None NA
18 - None NA
19 - None NA
20 - None NA
21 - None NA
22 - None NA
23 - None NA
24 - None NA
25 - None NA
26 - None NA
27 - None NA
28 - None NA
29 - None NA
30 - None NA
31 - None NA
32 - E. faecium 99%
33 - None
34 - None
35 - E. avium 99%
36 - E. avium 99%
37 - None NA
38 - None NA
39 - None NA
40 - None NA

IDEXX Laboratories, Inc. 14


Enterolert-DW
Sample ID VITEK ID ID confidence
U

reaction
U U U

41 - None NA
42 - None NA
43 - None NA
44 - None NA
45 - None NA
46 - None NA
47 - None NA
48 - None NA
49 - None NA
50 - None NA
51 - None NA
52 - None NA
53 - None NA
54 - None NA
55 - None NA
56 - None NA
57 - None NA
58 - E. avium 99%
59 - None NA
60 - None NA
61 - None NA
62 - None NA
63 - None NA
64 - None NA
65 - None NA
66 - None NA
67 - None NA
68 - None NA
69 - None NA
70 - None NA
71 - None NA
72 - None NA
73 - None NA
74 - None NA
75 - None NA
76 - None NA
77 - None NA
78 - None NA
79 - None NA
80 - None NA
81 - None NA

IDEXX Laboratories, Inc. 15


Enterolert-DW
Sample ID VITEK ID ID confidence
U

reaction
U U U

82 - None NA
83 - None NA
84 - None NA
85 - None NA
86 - None NA
87 - None NA
88 - None NA
89 - E. avium 99%
90 - None NA
91 - None NA
92 - None NA
93 - None NA
94 - None NA
95 - E. faecalis 99%
96 - None NA
97 - None NA
98 - None NA
99 - None NA
100 - None NA
101 - None NA
102 - None NA
103 - None NA
104 - None NA
105 - E. casseliflavus 99%
106 - None NA
107 - None NA
108 - None NA
109 - None NA
110 - None NA
111 - None NA
112 - None NA
113 - None NA
114 - None NA
115 - None NA
116 - None NA
117 - None NA
118 - None NA
119 - None NA
120 - None NA
121 - None NA
122 - None NA

IDEXX Laboratories, Inc. 16


Enterolert-DW
Sample ID VITEK ID ID confidence
U

reaction
U U U

123 - E. avium 99%


124 - None NA
125 - None NA
126 - None NA
127 - None NA
128 - None NA
129 - None NA
130 - None NA
131 - E. faecium 98%
132 - None NA
133 - None NA
134 - None NA
135 - None NA
136 - None NA
137 - None NA
138 - None NA
139 - None NA
140 - None NA
141 - None NA
142 - None NA
143 - None NA
144 - None NA
145 - None NA
146 - None NA
147 - None NA
148 - None NA
149 - None NA
150 - None NA
151 - None NA
152 - None NA
153 - None NA
154 - None NA
155 - None NA
156 - None NA
157 - None NA
158 - None NA
159 - None NA
160 - None NA
161 - None NA
162 - None NA
163 - None NA

IDEXX Laboratories, Inc. 17


Enterolert-DW
Sample ID VITEK ID ID confidence
U

reaction
U U U

164 - None NA
165 - None NA
166 - None NA
167 - None NA
168 - None NA
169 - None NA
170 - None NA
171 - None NA
172 - None NA
173 - None NA
174 - None NA
175 - None NA
176 - None NA
177 - None NA
178 - None NA
179 - None NA
180 - None NA
181 - None NA
182 - None NA
183 - None NA
184 - None NA
185 - None NA
186 - None NA

IDEXX Laboratories, Inc. 18


Appendix III: Counting Uncertainty for the Enterolert-DW/Quanti-Tray System
Enterolert-DW / Quanti-Tray in MPN/100mL Repeatability Reproducibility
Analyst A Analyst A Analyst B Analyst C
Sample count 1 count 2 count count Mean SD RSD sq Mean SD RSD sq
1 16 16 16 16 16.00 0.0000 0.0000 16.00 0.0000 0.0000
2 16 16 16 16 16.00 0.0000 0.0000 16.00 0.0000 0.0000
3 21 21 21 21 21.00 0.0000 0.0000 21.00 0.0000 0.0000
4 21 21 21 21 21.00 0.0000 0.0000 21.00 0.0000 0.0000
5 22 22 22 22 22.00 0.0000 0.0000 22.00 0.0000 0.0000
6 27 27 27 27 27.00 0.0000 0.0000 27.00 0.0000 0.0000
7 27 27 27 27 27.00 0.0000 0.0000 27.00 0.0000 0.0000
8 36 36 36 36 36.00 0.0000 0.0000 36.00 0.0000 0.0000
9 25 25 25 25 25.00 0.0000 0.0000 25.00 0.0000 0.0000
10 34 34 34 34 34.00 0.0000 0.0000 34.00 0.0000 0.0000
11 38 38 38 38 38.00 0.0000 0.0000 38.00 0.0000 0.0000
12 31 31 31 31 31.00 0.0000 0.0000 31.00 0.0000 0.0000
13 32 32 32 32 32.00 0.0000 0.0000 32.00 0.0000 0.0000
14 31 31 31 31 31.00 0.0000 0.0000 31.00 0.0000 0.0000
15 27 27 27 27 27.00 0.0000 0.0000 27.00 0.0000 0.0000
16 45 45 45 45 45.00 0.0000 0.0000 45.00 0.0000 0.0000
17 45 45 45 45 45.00 0.0000 0.0000 45.00 0.0000 0.0000
18 45 45 45 45 45.00 0.0000 0.0000 45.00 0.0000 0.0000
19 41 43 41 43 42.00 1.4142 0.0011 41.67 1.1547 0.0008
20 56 56 56 56 56.00 0.0000 0.0000 56.00 0.0000 0.0000
21 50 50 50 50 50.00 0.0000 0.0000 50.00 0.0000 0.0000
22 50 50 50 50 50.00 0.0000 0.0000 50.00 0.0000 0.0000
23 50 50 50 50 50.00 0.0000 0.0000 50.00 0.0000 0.0000
24 56 56 56 56 56.00 0.0000 0.0000 56.00 0.0000 0.0000
25 70 70 70 70 70.00 0.0000 0.0000 70.00 0.0000 0.0000
26 70 70 70 70 70.00 0.0000 0.0000 70.00 0.0000 0.0000
27 59 59 59 59 59.00 0.0000 0.0000 59.00 0.0000 0.0000
28 56 56 56 56 56.00 0.0000 0.0000 56.00 0.0000 0.0000
29 62 62 62 62 62.00 0.0000 0.0000 62.00 0.0000 0.0000
30 70 70 70 70 70.00 0.0000 0.0000 70.00 0.0000 0.0000

Sum Sum
RSD 0.0011 RSD 0.0008
RSDc 0.0061 RSDc 0.0051

IDEXX Laboratories, Inc. 19


Appendix IV: Robustness assessment for Enterolert-DW using typed strains
Number of positive wells
Rep 24 hr 28 hr % Change
1 15 15 0%
E. faecium ATCC 35667 2 10 10 0%
3 10 10 0%
1 23 23 0%
E. faecalis ATCC 33186 2 21 21 0%
3 21 21 0%
1 13 13 0%
E. faecalis ATCC 19433 2 17 17 0%
3 13 13 0%
1 19 19 0%
E. durans ATCC 11576 2 18 18 0%
3 30 30 0%
1 23 23 0%
E. hirae ATCC 8043 2 19 19 0%
3 24 24 0%
1 11 11 0%
E. mundtii ATCC 43187 2 19 19 0%
3 17 17 0%
1 25 26 4%
E. gallinarum ATCC 49573 2 27 28 4%
3 37 38 3%

IDEXX Laboratories, Inc. 20


Appendix V: MPN Table
95% Confidence Limits
# positive wells MPN/100 ml Lower Upper
0 <1.0 0.0 3.7
1 1.0 0.3 5.6
2 2.0 0.6 7.3
3 3.1 1.1 9.0
4 4.2 1.7 10.7
5 5.3 2.3 12.3
6 6.4 3.0 13.9
7 7.5 3.7 15.5
8 8.7 4.5 17.1
9 9.9 5.3 18.8
10 11.1 6.1 20.5
11 12.4 7.0 22.1
12 13.7 7.9 23.9
13 15.0 8.8 25.7
14 16.4 9.8 27.5
15 17.8 10.8 29.4
16 19.2 11.9 31.3
17 20.7 13.0 33.3
18 22.2 14.1 35.2
19 23.8 15.3 37.3
20 25.4 16.5 39.4
21 27.1 17.7 41.6
22 28.8 19.0 43.9
23 30.6 20.4 46.3
24 32.4 21.8 48.7
25 34.4 23.3 51.2
26 36.4 24.7 53.9
27 38.4 26.4 56.6
28 40.6 28.0 59.5
29 42.9 29.7 62.5
30 45.3 31.5 65.6
31 47.8 33.4 69.0
32 50.4 35.4 72.5
33 53.1 37.5 76.2
34 56.0 39.7 80.1
35 59.1 42.0 84.4
36 62.4 44.6 88.8
37 65.9 47.2 93.7
38 69.7 50.0 99.0
39 73.8 53.1 104.8
40 78.2 56.4 111.2
41 83.1 59.9 118.3
42 88.5 63.9 126.2

IDEXX Laboratories, Inc. 21


95% Confidence Limits
# positive wells MPN/100 ml Lower Upper
43 94.5 68.2 135.4
44 101.3 73.1 146.0
45 109.1 78.6 158.7
46 118.4 85.0 174.5
47 129.8 92.7 195.0
48 144.5 102.3 224.1
49 165.2 115.2 272.2
50 200.5 135.8 387.6
51 > 200.5 146.1 infinite

IDEXX Laboratories, Inc. 22


Appendix VI: 95% percent confidence intervals and theoretical repeatability for each MPN
value for the 51 well Enterolert-DW/Quanti-Tray system
95% CI Theoretical repeatability
# positive MPN per 100 ml Repeatability Relative standard
wells sample Lower Upper SD (Log10 SD) uncertainty (CV%)
0 <1.0 0.0 3.7 - -
1 1.0 0.3 5.6 0.4343 100.0
2 2.0 0.6 7.3 0.3071 70.7
3 3.1 1.1 9.0 0.2508 57.7
4 4.2 1.7 10.7 0.2172 50.0
5 5.3 2.3 12.3 0.1943 44.7
6 6.4 3.0 13.9 0.1774 40.9
7 7.5 3.7 15.5 0.1643 37.8
8 8.7 4.5 17.1 0.1537 35.4
9 9.9 5.3 18.8 0.1450 33.4
10 11.1 6.1 20.5 0.1376 31.7
11 12.4 7.0 22.1 0.1313 30.2
12 13.7 7.9 23.9 0.1257 29.0
13 15.0 8.8 25.7 0.1209 27.8
14 16.4 9.8 27.5 0.1166 26.8
15 17.8 10.8 29.4 0.1127 26.0
16 19.2 11.9 31.3 0.1092 25.1
17 20.7 13.0 33.3 0.1061 24.4
18 22.2 14.1 35.2 0.1032 23.8
19 23.8 15.3 37.3 0.1005 23.1
20 25.4 16.5 39.4 0.0981 22.6
21 27.1 17.7 41.6 0.0959 22.1
22 28.8 19.0 43.9 0.0938 21.6
23 30.6 20.4 46.3 0.0919 21.2
24 32.4 21.8 48.7 0.0902 20.8
25 34.4 23.3 51.2 0.0885 20.4
26 36.4 24.7 53.9 0.0870 20.0
27 38.4 26.4 56.6 0.0856 19.7
28 40.6 28.0 59.5 0.0843 19.4
29 42.9 29.7 62.5 0.0830 19.1
30 45.3 31.5 65.6 0.0819 18.9
31 47.8 33.4 69.0 0.0809 18.6
32 50.4 35.4 72.5 0.0799 18.4
33 53.1 37.5 76.2 0.0791 18.2
34 56.0 39.7 80.1 0.0783 18.0
35 59.1 42.0 84.4 0.0776 17.9

IDEXX Laboratories, Inc.


95% CI Theoretical repeatability
# positive MPN per 100 ml Repeatability Relative standard
wells sample Lower Upper SD (Log10 SD) uncertainty (CV%)
36 62.4 44.6 88.8 0.0770 17.7
37 65.9 47.2 93.7 0.0765 17.6
38 69.7 50.0 99.0 0.0761 17.5
39 73.8 53.1 104.8 0.0758 17.4
40 78.2 56.4 111.2 0.0756 17.4
41 83.1 59.9 118.3 0.0756 17.4
42 88.5 63.9 126.2 0.0757 17.4
43 94.5 68.2 135.4 0.0761 17.5
44 101.3 73.1 146.0 0.0768 17.7
45 109.1 78.6 158.7 0.0778 17.9
46 118.4 85.0 174.5 0.0794 18.3
47 129.8 92.7 195.0 0.0819 18.9
48 144.5 102.3 224.1 0.0859 19.8
49 165.2 115.2 272.2 0.0929 21.4
50 200.5 135.8 387.6 0.1094 25.2
51 > 200.5 146.1 infiniti - -

IDEXX Laboratories, Inc.


Appendix VII: Enterolert-DW vs. ISO 7899-2 and MoDW 2006, part 5, relative recovery results
Chlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
1 23 80 95
2 69 70 86
3 7 7 11
4 54 78 87
5 79 96 106
6 116 90 100
7 10 6 10
8 52 79 95
9 68 61 72
10 39 48 59
11 47 43 52
12 9 10 13
13 58 53 60
14 57 58 70
15 18 2 2
16 45 48 54
17 109 29 43
18 64 43 53
19 53 82 87
20 70 73 78
21 13 62 72
22 83 67 71
23 56 22 25
24 124 75 89
25 34 26 29
26 62 63 73
27 119 66 72
28 74 77 78
29 16 4 7
30 96 37 50
31 183 172 181
32 101 60 68
33 123 129 153
34 56 31 38
35 201 203 208
36 173 141 153
37 62 154 167
38 41 26 33
39 0 0 0
40 12 3 8

IDEXX Laboratories, Inc. 25


Chlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
41 95 85 116
42 13 3 7
43 68 74 95
44 9 2 3
45 201 156 161
46 142 79 114
47 13 139 149
48 5 4 6
49 21 0 0
50 74 17 18
51 165 114 130
52 74 17 29
53 118 55 72
54 45 3 6
55 201 123 124
56 159 97 103
57 39 79 103
58 40 5 6
59 24 6 7
60 25 19 25
61 51 22 25
62 35 16 18
63 35 16 18
64 59 56 57
65 49 28 31
66 14 5 5
67 50 40 44
68 61 45 49
69 28 19 20
70 63 50 54
71 56 57 63
72 30 48 52
73 56 54 55
74 40 95 102
75 56 45 50
76 16 9 12
77 64 84 90
78 65 104 105
79 27 6 8
80 35 31 39
81 70 22 28

IDEXX Laboratories, Inc. 26


Chlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
82 15 17 22
83 78 46 48
84 49 72 87
85 144 44 53
86 5 2 3
87 63 72 81
88 58 87 96
89 66 36 37
90 55 12 14
91 8 0 0
92 1 0 0
93 78 13 15
94 57 27 35
95 75 48 53
96 123 22 34
97 24 0 0
98 74 5 7
99 43 48 50
100 59 29 35
101 16 16 18
102 8 1 1
103 66 43 46
104 38 45 46
105 38 52 54
106 53 38 41
107 37 22 24
108 44 39 41
109 91 42 51
110 91 48 51
111 48 19 21
112 5 3 5
113 95 41 51
114 86 34 45
115 106 41 56
116 68 39 47
117 55 30 32
118 78 40 48
119 165 123 132
120 165 103 112
121 106 109 118
122 119 105 119

IDEXX Laboratories, Inc. 27


Chlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
123 159 121 133
124 31 24 31
125 137 110 124
126 106 103 117
127 96 90 94
128 114 79 86
129 98 76 93
130 70 73 85
131 53 73 82
132 61 71 77
133 11 9 13
134 137 70 80
135 98 67 77
136 70 65 78
137 44 70 76
138 37 22 28
139 49 25 30
140 44 36 37
141 37 28 34
142 37 29 35
143 21 4 5
144 50 22 27
145 42 24 33
146 31 22 30
147 26 20 24
148 17 46 52
149 31 43 55
150 23 48 54
151 17 61 70
152 48 53 61
153 31 25 32
154 22 42 48
155 48 36 44
156 63 41 51
157 50 42 49
158 116 98 103
159 130 80 86
160 137 82 86
161 106 82 85
162 94 78 81
163 47 31 38

IDEXX Laboratories, Inc. 28


Chlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
164 39 44 51
165 130 49 56
166 159 69 78
167 103 52 57
168 12 41 44
169 28 36 43
170 19 41 45
171 21 34 40
172 27 33 34
173 5 0 0
174 5 7 8
175 11 2 2
176 37 18 22
177 22 12 17
178 58 51 58
179 48 59 68
180 6 30 36
181 9 29 36
182 7 25 31
183 10 16 22
184 13 23 29
185 5 23 32
186 2 0 0
187 32 38 49
188 29 33 40
189 26 40 49
190 26 44 49
191 33 37 39
192 29 37 39
193 1 3 4
194 159 122 127
195 155 131 138
196 64 29 40
197 65 42 48
198 22 74 91

IDEXX Laboratories, Inc. 29


Nonchlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
1 15 21 23
2 19 20 25
3 8 21 22
4 20 18 27
5 17 21 27
6 23 29 35
7 15 17 18
8 17 19 23
9 23 20 24
10 24 23 25
11 21 24 29
12 21 20 26
13 17 25 29
14 29 20 23
15 20 22 29
16 201 22 25
17 21 21 25
18 17 18 23
19 21 23 33
20 29 27 30
21 25 29 32
22 17 21 22
23 35 29 32
24 41 21 25
25 34 29 31
26 25 20 22
27 28 26 30
28 44 21 25
29 25 21 23
30 31 53 54
31 39 50 51
32 35 50 51
33 34 51 52
34 30 51 52
35 42 51 54
36 36 51 54
37 35 48 49
38 17 64 65
39 32 57 58
40 76 53 55
41 72 44 44
42 72 49 53
43 61 51 57
44 44 42 51
45 59 45 50
46 48 51 70
47 63 49 63
48 33 58 65

IDEXX Laboratories, Inc. 30


Nonchlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
49 23 47 52
50 28 29 30
51 23 35 37
52 30 44 44
53 26 38 41
54 28 35 40
55 34 34 37
56 47 32 35
57 35 37 39
58 19 35 37
59 33 40 41
60 41 28 30
61 42 37 40
62 52 34 37
63 45 34 38
64 34 36 40
65 49 33 36
66 33 35 37
67 21 21 23
68 39 32 38
69 44 25 32
70 32 23 25
71 21 26 29
72 19 19 22
73 19 23 28
74 18 21 26
75 19 22 29
76 23 21 26
77 17 16 17
78 23 17 24
79 25 29 32
80 33 26 32
81 30 34 36
82 38 39 41
83 32 39 47
84 35 31 38
85 34 33 40
86 47 32 37
87 21 29 34
88 37 40 48
89 51 37 42
90 41 35 37
91 32 36 38
92 32 28 30
93 10 17 18
94 31 28 31
95 36 30 36
96 38 33 39
97 118 34 36

IDEXX Laboratories, Inc. 31


Nonchlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
98 201 29 30
99 29 30 31
100 65 31 33
101 165 35 37
102 35 26 26
103 52 20 22
104 173 37 38
105 44 38 39
106 44 36 39
107 183 36 40
108 144 37 39
109 183 31 33
110 42 19 24
111 53 14 15
112 44 18 26
113 4 11 14
114 32 15 20
115 47 15 18
116 50 13 19
117 67 12 19
118 29 18 22
119 49 21 25
120 4 3 4
121 4 4 5
122 5 6 6
123 5 4 4
124 2 3 4
125 1 2 2
126 2 4 4
127 2 4 5
128 5 4 5
129 2 3 3
130 40 39 46
131 43 31 37
132 40 27 34
133 38 32 37
134 30 28 36
135 24 31 35
136 33 29 43
137 38 36 46
138 35 39 44
139 41 31 39
140 12 10 12
141 11 8 14
142 15 15 17
143 12 14 16
144 15 9 10
145 11 15 16
146 11 9 10

IDEXX Laboratories, Inc. 32


Nonchlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
147 14 13 15
148 11 11 13
149 15 12 13
150 5 35 42
151 22 33 40
152 21 30 36
153 15 35 40
154 44 34 39
155 36 29 35
156 25 32 37
157 28 30 33
158 35 30 34
159 32 28 32
160 26 41 45
161 34 28 32
162 31 30 35
163 29 30 32
164 21 36 39
165 25 26 31
166 21 38 39
167 24 23 28
168 34 28 30
169 24 35 38
170 35 32 42
171 50 25 35
172 32 33 40
173 35 36 44
174 38 37 41
175 29 33 41
176 27 28 35
177 34 28 35
178 40 33 41
179 26 35 45
180 28 38 42
181 28 33 38
182 25 38 41
183 29 32 36
184 33 30 30
185 22 33 39
186 33 60 61
187 31 43 51
188 31 50 55
189 35 48 52
190 37 41 46
191 30 44 48
192 26 46 47
193 29 36 40
194 37 41 46
195 29 37 42

IDEXX Laboratories, Inc. 33


Nonchlorinated Sample Matrix
Sample Enterolert-DW ISO 7899-2 MoDW 2006, part 5
number (MPN/100mL) (CFU/100mL) (CFU/100mL)
196 31 36 42
197 27 39 44
198 30 34 39
199 22 36 43
200 37 40 43
201 43 38 41
202 36 38 40
203 35 39 41
204 15 25 28
205 17 25 26

IDEXX Laboratories, Inc. 34