C. Bernasconi*, G. Volponi and L. Bonadonna** *European Commission, Joint Research Center, Institute for Environment and Sustainability, Via E. Fermi 1, 21020 Ispra (Varese), Italy (E-mail:
[email protected] ) **Istituto Superiore di Sanita’, Rome, Italy Abstract The European Drinking Water Directive defines reference methods for the enumeration of microbiological parameters in drinking water. The method to be used for Escherichia coli and coliforms is the membrane filtration technique on Lactose TTC agar with Tergitol 7. Many technical drawbacks of the procedure, as well as its limitations regarding the recent taxonomy of coliforms, make it necessary to evaluate alternative methods. Two alternative assays, a chromogenic media (m-ColiBlu24w) and a defined substrate technology-DST test (Colilert 18/Quanty Traye) were compared with the ISO standard with attention to the phenotypic characteristic of the isolates. Results showed that the ISO method failed to detect an important percentage of coliforms and E. coli while m-ColiBlu24w and Colilert 18 provided results in a shorter time allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps. Keywords Coliforms; E. coli; microbiological methods; water
Introduction
Coliform bacteria and Escherichia coli are recommended as hygienic indicator organisms. The EU Directive (EC, 1998) specified the reference method for their enumeration in drinking waters (ISO 9308-1) based on membrane filtration and growth on Lactose TTC agar with Tergitol 7 (ISO, 2000). Readability of results and time to obtain the response are the main shortcomings of the method. Based on the traditional coliform definition (oxidase-negative, facultative anaerobic rod-shaped bacteria that ferment lactose to produce acid and gas within 48 h at 35 8C (Breed and Norton, 1937)), coliforms weakly reduce TTC giving rise to yellow-orange colonies. However, from our experience, other organisms can often be present in water. Serratia, Shigella, Proteus, Yersinia and Hafnia belonging to the Enterobacteriaceae but not all species are able to ferment lactose. Furthermore, some E. coli strains neither ferment lactose nor produce indole (Leclerc et al., 2001). Advances in molecular methods and typing have recently redesigned coliform taxonomy: the group, including E. coli, possesses b-galactosidase which hydrolyses orthonitrophenil-b-galactopyranoside (ONPG) to the yellow-coloured o-nitrophenol; unlike other coliforms, E. coli also possesses b-glucuronidase which catalyses the hydrolysis of the 4-methyl-umbelliferyl-b-D -glucuronide (MUG). New substrate and methods have been designed on the basis of the new coliform taxonomic scheme. Generally, during the process of substrate utilisation, a chromogen or fluorochrome is released from the substrate indicating the presence of the target microorganism (Bonadonna, 2003). For environmental monitoring the use of chromogenic media is increasing. They allow a better differentiation and readability of the analysis results with the additional advantage of providing response in a shorter time compared with other methods (Edberg and Kontnick, 1986; Sartory and Howard, 1992; Frampton and Restaino, 1993). doi: 10.2166/wst.2006.460
Water Science & Technology Vol 54 No 3 pp 141–145 Q IWA Publishing 2006
Comparison of three different media for the detection of E. coli and coliforms in water
141
The present study compared m-ColiBlu24w (Millipore, MA, USA) and Colilert 18/Quanti-Traye (IDEXX, ME, USA) to the ISO standard for the enumeration of coliforms and E. coli in different water samples, taking into account practical aspects such as readability of results and user friendliness. Materials and methods C. Bernasconi et al.
Samples
Simulated samples were prepared by spiking drinking water with chlorine-injured organisms derived from surface water according to DWI procedures (DWI, 2000). The same volumes of the same samples (presumptive levels of 1– 50 target organisms/100 mL) were processed simultaneously and in parallel with the three methods. ISO 9308-1 (Lactose-TTC)
Analysis was performed according to ISO 9308-1 (ISO, 2000). Samples were filtered and the membrane was incubated at 36 ^ 2 8C for 21 ^ 3 h. Confirmation steps (oxidase activity and indole production from tryptophane) were necessary in order to discriminate coliforms and E. coli. Oxidase (OX)-negative colonies were considered coliforms whilst those that were oxidase-negative and indole-positive were counted as E. coli. Results were available within 72 h. Colilert 18/Quanti-Traye (Colilert 18)
Colilert 18 was used according to the manufacture’s instructions. It is based on the most probable number (MPN) technique and is an enzyme-based, semi-automatic assay reduced to multi-wells. It can analyse coliforms and E. coli in one phase with incubation of 18 h at 36 ^ 1 8C. Yellow-coloured wells were considered total coliforms whereas E. coli-positive wells were yellow and fluorescent under UV (365 nm). M-ColiBlu24 (CB24)
The method uses the traditional membrane filtration technique and a chromogenic substrate. Samples were filtered and incubated at 36 ^ 2 8C for 22 ^ 2 h. Blue colonies were considered E. coli whereas coliforms appeared as red colonies. Confirmation tests
The isolated colonies from all three media were considered as presumptive and subjected to confirmatory tests. Following incubation, presumptive colonies were counted and isolated, in a percentage of 70– 100%, for further confirmation. m-ColiBlu24 and TTClactose isolates were sub-cultured onto plate count agar (PCA) medium and those from Colilert 18 were plated on MacConkey agar. Presumptive coliform isolates were analysed for cytochrome oxidase (negative), the ability to ferment lactose (LAC) (positive) and the hydrolysis of ortho-nitrophenyl-b-D -galactopyranoside (ONPG) (positive). Presumptive E. coli isolates were analysed for the same biochemical reactions (OX negative, LAC positive, ONPG hydrolysis positive) and indole production (positive). In the case of absence of lactose fermenting colonies, a well isolated negative colony was isolated and characterised. Some colonies, negative for lactose fermentation but positive for ONPG reaction or with an unusual biochemical profile, were identified at species level with the VITEK System (Biome´rieux, France). Results and discussion 142
The number of coliforms isolated with each method and subjected to the OX test is reported in Table 1.
Table 1 Number of presumptive coliforms isolated with each method and subjected to the OX test
No presumptive OX positive (%)
ISO 9308-1
Colilert 18
m-ColiBlu24
Totals
103 54 (53)
188 19 (10)
224 18 (8)
515 91 (18)
C. Bernasconi et al.
In this first screening, 54 colonies (52%) from Lactose TTC were OX positive and thus not belonging to the coliform group. From the same samples analysed using the Colilert 18 and the CB24, 19 (10%) and 18 (8%), respectively, had the same characteristic. Among the OX-positive colonies, 22 (24%) were identified. Plesiomonas shigelloides and Aeromonas hydrophila were the predominant isolated species (27%). Results obtained from OX-negative colonies, combining in pairs the biochemical characteristics LAC and ONPG, are reported in Table 2. Different yields were obtained on the colonies isolated with the three methods. From the same sample, each method was able to sustain the growth of fractions of bacterial populations, even if those belonging to the group of coliforms, were at least in part, dissimilar. Particularly, Lactose TTC agar did not recover some typical coliforms (LAC and ONPG positive) in comparison with the other methods. Furthermore, when compared with the Lactose-TTC, Colilert 18 and CB24 were able to detect a higher number of coliforms unable to ferment lactose (and thus atypical on the base of the traditional coliform definition). From colonies with an atypical biochemical profile in at least one of the confirmation tests, 32 were also biochemically identified. Results of the identification tests are reported in Table 3. Citrobacter freundii complex, Enterobacter cloacae and Serratia marcescens were the predominant species (13%). Other species of coliforms showed atypical profiles; most of them (41%) were LAC negative vs. those (25%) showing a negative reaction to ONPG. The verification of ONPG hydrolysis therefore seemed, based on obtained data, to be the more appropriate test for the identification of the target microorganisms with respect to the more traditional lactose fermentation. In Table 4 the number of E. coli isolated with each method and subjected to the OX test is reported. In this first screening, 17 colonies (21%) from Lactose TTC were OX positive. From the same samples analysed using Colilert 18 and CB24, five (3%) and four (3%), respectively, had the same characteristic. All the OX-negative colonies were subcultivated at 44 8C. Different yields were obtained on the colonies isolated with the three methods: 34 (41%), 168 (97%) and 164 (98%) colonies from Lactose TTC, Colilert 18 and CB24, respectively, were able to grow at this temperature. Results obtained by grouping in pairs the biochemical characteristics LAC and ONPG for E. coli, are reported in Table 5. Also in this case, the lowest recovery of the typical target organism was found when Lactose TTC was used. From colonies with an atypical biochemical profile in at least one of the confirmation tests, 30 isolates were also biochemically identified (Table 6). Klebsiella pneumonia/oxytoca Table 2 Results obtained from OX-negative colonies, combining in pairs the biochemical characteristics LAC and ONPG Method
ISO 9308-1 Colilert 18 m-ColiBlu24 Totals
LAC/ONPG
Totals
1 / 1 (%)
1 /2 (%)
2 /1(%)
2/2 (%)
34 132 188 354
13 (13) 12 (7) 0 25 (5)
0 19 (10) 18 (8) 37 (7)
2 (1) 6 (3) 0 8 (1)
(33) (70) (84) (69)
Note: The percentage was calculated on the total number of presumptive coliforms
49 169 206 424 143
Table 3 Number of species identified with the corresponding biochemical characteristics lactose (LAC) and ortho-nitrophenil-b-galactopyranoside (ONPG)
C. Bernasconi et al.
LAC fermentation
ONPG hydrolysis
Identified species
Number (%)
þ þ þ 2 2 þ 2 2 2 þ 2 2 2 2 2 2 2 2 þ
2 2 2 þ þ 2 þ þ þ 2 þ þ þ þ þ 2 2 þ 2
Citrobacter farneri Citrobacter freundii complex Citrobacter braaki Enterobacter aerogenes Enterobacter amnigenus Enterobacter intermedius Enterobacter cloacae Escherichia coli Escherichia hermannii Klebsiella pneumoniae Klebsiella pneumoniae Klebsiella ornithinolytica Leclercia adecarboxylata Morganella morganii Pantoea agglomerans Providencia retgeri Providencia stuarti Serratia marcescens Yersinia intermedia
1 (3) 4 (13) 1 (3) 1 (3) 3 (9) 1 (3) 4 (13) 1 (3) 1 (3) 2 (6) 1 (3) 1 (3) 1 (3) 2 (6) 1 (3) 1 (3) 1 (3) 4 (13) 1 (3)
Table 4 Number of presumptive E. coli isolated with each method and subjected to OX test ISO 9308-1
No presumptive OX positive (%)
Colilert 18
82 17 (21)
m-ColiBlu24
173 5 (3)
168 4 (3)
Totals
423 26 (6)
Table 5 Results obtained from OX-negative colonies, E. coli presumptive, combining in pairs the biochemical characteristics LAC and IND Method
ISO 9308-1 Colilert 18 m-ColiBlu24 Totals
LAC/IND 1/1(%)
1 /2 (%)
2/1(%)
2/2 (%)
Totals
20 (23) 128 (74) 148 (88) 296 (70)
9 17 14 40
0 4 (2) 1 (0.6) 5 (1)
5 (1) 19 (11) 1 (0.7) 25 (6)
34 168 164 366
(10) (10) (8) (9)
Note: The percentage was calculated on the total number of presumptive E. coli Table 6 Number of species identified with the corresponding biochemical characteristics lactose (LAC) and indole (IND)
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LAC fermentation
IND production
Identified species
þ þ 2 2 2 2 þ þ
2 2 þ þ þ þ 2 2
Citrobacter farneri Citrobacter freundii Citrobacter freundii Citrobacter koseri Enterobacter cloacae Escherichia coli Escherichia coli Klebsiella pneumoniae
Number (%)
6 (20) 3 (10) 2 (7) 1 (3) 4 (13) 2 (7) 3 (10) 9 (30)
(30%) and Citrobacter farmeri (20%) were the prevalent species. Among the colonies with typical reactions for LAC (þ) and IND (þ), 30 confirmed E. coli were also identified: 73% confirmed as E. coli, C. farmeri (17%), K. pneumoniae/oxytoca (7%) and C. freundii (3%) were also isolated.
Under the specific test conditions and for the analysed water samples, the reference method allowed a large variety of interfering bacteria to grow. This was, presumably, due to both the composition of the growth substrate and to the non-selective incubation temperature. In fact, the calculated confirmation rate for coliforms was low (27%) for Lactose TTC against 85% for Colilert 18 and 73% for CB24. A similar confirmation rate was observed for E. coli: Lactose TTC 32%, Colilert 87% and CB24 94%.
Two different systems (Colilert 18/Quanti Tray and m-ColiBlu24) for the detection and enumeration of coliforms and E. coli in water samples were compared with ISO 9308-1 (Lactose-TTC). This study demonstrated that the presence of background flora affected the counts on Lactose TTC. Other authors have reached similar conclusions (Schets et al., 2002; Bonadonna et al., 2005). Interfering organisms, cultivated as orange to brown colonies on the medium, made it extremely difficult to select and read results. As a consequence, most of the presumptive isolates failed to be confirmed. On the other hand, results from Colilert 18 and m-ColiBlu24 were easier to read. Colilert 18 and m-ColiBlu24 were more sensitive than the ISO reference method and were also able to detect coliforms not fermenting lactose. In addition, the two alternative methods had the advantage of requiring no confirmatory steps. For coliform analysis, Colilert 18 had the highest confirmation rate (85%) whereas for E. coli, presumptive target organisms were confirmed at 94% with m-ColiBlu24. Moreover, Colilert 18 provided results faster than the other methods (18 h). The use of procedures that provide quicker results are of fundamental importance when an environmental intervention decision is needed, especially when there is a potential public health risk.
C. Bernasconi et al.
Conclusions
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