Enterobacteriaceae of the “Coliform Group” in Drinking Water: Identification and Worldwide Distribution

System. App!. Microbio!. 6, 312-318 (1985)

Enterobacteriaceae of the "Coliform Group" in Drinking Water: Identification and Worldwide Distribution FRAN<;OISE GA VINI, HENRI LECLERC, and DAVID A. A. MOSSEL Institut National de la Sante et de la Recherche Mcdicale Unite 146, 59651 Villeneuve-d' Ascq-Cedex, France

Received May 5, 1985

Summary A total of 1017 strains of Bgalactosidase positive Enterobacteriaceae isolated from drinking water by 23 laboratories, were identified. Saprophytic Enterobacteriaceae constituted 51 % of the identified strains irrespective of the type of water, its source or geographical origin. A significant correlation was observed between the numbers of faecal streptococci and faecal Enterobacteriaceae in the water samples examined. On the other hand, no relationship was apparent between the presence of faecal or non-faecal Enterobacteriaceae, and presence or absence of sulphite-reducing Clostridium sp., or the total bacterial colony count at 20°C and 3rc. The frequency distribution patterns of the enterobacteria were not related to water treatment.

Key words: Enterobacteriaceae - Coliform - Drinking water

Introduction Taxonomic studies performed in recent years on ~ galactosidase positive Enterobacteriaceae have resulted in (i) a precise phenotypic definition of the species belonging to the coli-aerogenes and faecal coliform groups, Esche-

richia coli, Citrobacter (reundii, C. diversus, C. amalonaticus, Klebsiella pneumoniae, K. oxytoca, K. mobilis, Enterobacter cloacae, and (ii) the differentiation of species of environmental saprophytic enterobacteria, Serratia (onticola (Gavini et al., 1979), Rahnella aquatilis (Izard et al., 1979), Enterobacter intermedium (Izard et al., 1980), E. amnigenus (Izard et al., 1981b), Buttiauxella agrestis (Ferragut et al., 1981), Klebsiella trevisanii (Ferragut et al., 1983), Klebsiella terrigena (Izard et al., 1981a). To the latter group, the species Enterobacter gergoviae (Brenner et al., 1980) and E. sakazakii (Farmer et al., 1980) were added, and also some other species which are isolated more rarely. These observations prompted a new approach to the definition of the "coliform group" (Leclerc et al., 1983). An investigation was performed on Enterobacteriaceae isolated from drinking water in Northern France by Oger et al. (1981). In order to interpret the significance of the presence of the different species noted in the study of Oger et al. (1981) and to identify Enterobacteriaceae of importance in assessing the safety and quality of drinking water,

a study was carried out on 1017 isolates from drtnking waters from different areas of the world. Relationships between identification, water source and water treatment, and also the correlation between the occurence of Enterobacteriaceae and other faecal indicator bacteria, sulphite-reducing Clostridium sp. and mesophilic colony counts at 20°C and 37°C were studied.

Materials and Methods Strains

All strains studied were B galactosidase positive Enterobacteriaceae. They were sent to us by the 23 laboratories listed in Table 1. The strains were isolated from drinking water samples which had been analyzed according to the prevailing standards in each country; in France, those issued by the Ministry of Public Health (Ruling of March, 15, and July 15, 1980). For each strain, information was provided on (i) the source of isolation (spring, well, drilling, river, lake) and the water type (catchwork, pumping station, tank, water tower, pipe waters); (ii) the treatment applied (if any) e. g. chlorination, ozonization or other; (iii) the other bacteriological parameters of the sample (faecal streptococci, mesophilic colony counts at 20°C and 37°C, and presence or absence of sulphite-reducing Clostridium sp.)

Identification and Distribution of "Coliform" Bacteria in Drinking Water Table 1. Laboratories collaborating in the study Laboratoire Departemental de I'Eau Laboratoire de Controle des Eaux de la Ville de Paris Laboratoire d'Hygiene de la Ville de Nancy Laboratoire Departemental d'Hygiene Laboratoire Municipal Laboratoire Regional de Controle des Eaux Laboratoire Departemental et Regional de Biologie et d'Hygiene Laboratoire d'Hygiene et d'Epidemiologie Provinciaal Instituut voor Hygiene Service des Eaux et du Gaz Laboratorio Medico Provinciale d'Igiene-Profilassi Laboratorio Provinciale d'IgieneProfilassi Laboratorio d'Igiene-Profilassi Laboratorio Municipal Depart. de Hydrologia Thames Water Authority New River Head Laboratory Institut Pasteur Unifer Societe Marocaine de Distribution Institut Pasteur, Laboratoire de Controle des Eaux et des Denrees Alimentaires Manager Environmental Biology Universidad de Concepcion, Departamento de Microbiologia Ministere de l'Environnement Ontari9 Ministry of Health, Public Health Laboratory Laboratoire d'Hygiene de la Ville de Paris

Toulouse (France) Paris (France) Nancy (France) La Rochelle (France) Le Havre (France) Montpellier (France) Caen (France)

Bruxelles (Belgium) Anvers (Belgium) Neuchatel (Switzerland) Milan (Italy) Venice (Italy) Novara (Italy) Barcelone (Spain) London (G.B.) Alger (Algeria) Casablanca (Morocco ) Tunis (Tunisia) Sao Paulo (Brazil) Concepcion (Chile) Quebec (Canada) Ontario (Canada) Paris (France)

Identification All enterobacteria were identified by a computer program which used a multitest system performed on microtitre plates. This system, based on 24 tests, allowed the identification of 29 species (Gavini and Leclerc, 1982). The identification program was developed on a microcomputer, ISTC 5000, and calculated according to Bayes' theorem with equal prior probabilities for the species (Willcox et aI., 1980). An unknown strain was identified at an identification level greater than or equal to 0.990. When a lower value was obtained, further tests were performed and the identification attempted again.

Results

Isolation frequency of the different enterobacterial species: correlation with source and type of water The identified species are listed in Table 2 in order of decreasing frequency. Waters from pumping stations were grouped as "catchwork" (CA), while water tower, tank and pipe waters were grouped as "distribution system"

313

(DS). The number of strains isolated from pumping stations, water tower, tanks and piges was very low. On the whole, C. freundii was found to be the predominant species (18 % of the total strains). It was the most prevalent in all waters, except DS waters from spring, well and river sources. In addition to significant numbers of C. freundii, Serratia fonticola occured in 30 % of DS waters from wells, Rahnella aquatiUs, in 28 % of DS waters from springs, and Enterobacter agglomerans in 27 % of DS waters from rivers. It should be noted that a high proportion of these strains were isolated in a particular laboratory. Thus, 40 % of R. aquatilis strains were sent in by the Toulouse laboratory, 52 % of S. fonticola by the Quebec laboratory and 43 % of E. agglomerans by the Paris laboratory. Thirty eight percent of the enterobacteria isolated were considered to be of faecal origin and 51 % of non-faecal origin; 10 % of the strains were not identified (Table 3). The number of non-faecal exceeded that of faecal Enterobacteriaceae in spring waters (CA and DS waters), even when the high number of R. aquatilis received from the Toulouse laboratory was neglected. A high level of saprophytic enterobacteria was also observed in DS water from wells; 53 % of these saprophytic enterobacteria were identified as S. fonticola, which were all received from the Quebec laboratory. A higher incidence of faecal enterobacteria was observed in well waters (CA waters), drillings (DS and CA waters), and in DS waters the water source of which was not known.

Correlation with faecal streptococci, sulphite-reducing Clostridium spp and colony counts Faecal streptococci. Occurrence of faecal streptococci in the same water samples is based only on the information provided on 692 enterobacterial strains. In general there was a significant correlation (Chi-square test, P < 0.05) between the presence of non-faecal Enterobacteriaceae and the absence of faecal streptococci in 100 ml of water (Table 4). The distribution of Enterobacteriaceae species was studied in waters contaminated and uncontaminated by faecal streptococci (Table 5). In such contaminated waters a predominance of the faecal enterobacterial species, E. coli (8 %), K. pneumoniae (4 %), K. mobilis (2 %), was noted, whereas in the uncontaminated waters the saprophytic species, E. agglomerans (8 %), K. trevisanii (4 %), R. aquatilis (10 %), E. amnigenus (7 %), B. agrestis (3 %) dominated. A similar percentage of isolates of C. freundii (18 %), S. fonticola (10 %), K. oxytoca (3 %), H. alvei (2 %) was found in waters with or without detectable faecal streptococci. Sulphite-reducing Clostridium sp., colony counts at 20°C and 3rc. Information on sulphite-reducing Clostridium sp. was provided for 491 enterobacterial isolates from the same waters, while information on colony counts at 20 and 37°C was given for 328 and 468 enterobacterial isolates respectively.

314

F. Gavini, H. Leclerc, and D. A. A. Mosse!

Water source

Spring

Lake

River

Drilling

Well

Water type

CA

DS

CA

DS

CA

DS

CA

DS

CA

DS

SpecieslNumber of strains (Total 1017)

119

186

163

178

61

57

12

226

12

3

15

25

38

29

29

18

13

12

12

6

5

2

12

12

2

53

0

0

3

5

53

4

9

0

0

o

0

8

16

5

5

2

13

5

0

7

4

29

2

4

o

26

2

o

8

o o

e. freundii

E. agglomerans b S. fonticola b

R. aquatilis b E. cloacae

K. trevisaniib K.oxytoca E. amnigenus b K. terrigena b E. coli

184 18%" 113 11% 86 8% 73 7% 68 7% 63 6% 50 5% 46 5% 44 4% 36 4%

3

28 62

o 31

3

4

7

4

10

7

12

5

8

11

8

6

4

2

o

4

10

6

10

0

3

6

o

Table 2. Frequency of distribution of enterobacterial species (number of strains) according to source and type of water

0

o

o

3

o

2

o

o

o

o o

o

o

o

K. pneumoniae (2%); B. agrestis b (2%); H. alvei (2%); S.liquefaciens b (2%); E. sakazakiib (1 %); E. intermedium b (1 %); K. mobilis (1 %); S. plymuthica b (1 %); S. marcescens b (1 %); Y. intermedia b (1 %); Y. enterocolitica b (1 %); Y. frederiksenii b (1 %); unidentified (10%). Percentage calculated from the total number of strains (1017) Non-faecal strains CA: catchwork water DS: distribution system water

a

b

On the basis of the Chi-square test, no relationship was observed between the presence of non-faecal Enterobacteriaceae and the absence of sulphite-reducing Clostridium sp., the colony count at 20°C (less than 100 bacteria per ml) or the colony count at 37°C (less than 10 bacteria per ml) (Table 4). As depicted in Table 5 and 6, the distribution of Enterobacteriaceae species was generally very heterogeneous. A higher incidence of faecal isolates (identified as E. coli

Water source

Spring

Well

and C. (reundii) was observed in waters which did not contain sulphite-reducing Clostridium sp. Non-faecal enterobacteria such as K. trevisanii, K. terrigena or B. agrestis were isolated more frequently from waters with colony counts at 20°C exceeding 102/ml than from less contaminated waters. Similar distributions were observed with the species E. agglomerans, S. (onticola, K. trevisanii which were isolated more frequently from water samples with colony counts at 37°C exceeding 10/ml.

River

Drilling

Lake

Water type

CA

DS

CA

DS

CA

DS

CA

DS

CA

DS

SpecieslNumber of strains (Total 10 17) F.e. 389 38%' N.F.e. 522 51% N.id.e. 105 10%

119

186

163

178

61

57

12

226

12

3

37 61% 20 33% 4 7%

31 54% 17 30% 9 16%

3 25% 9 75% 0

2 98 43% 17% 112 8 49% 67% 2 16 7% 17%

50 28 24%b 27% 69 118 58% 63% 18 22 18% 10%

76 63 47% 35% 67 100 41% 56% 15 20 12% 8%

F. e.: Faecal coliforms; N. F. e.: Non faecal coliforms; N. id. e. : Non identified coliforms. Percentage of strains calculated from the total number of strains (1017). b Percentage of strains calculated from the total number of strains from each water type.

a

1 33% 2 67% 0

Table 3. Frequency of distribution of faecal and non-faecal Enterobacteriaceae from each source and type of water

Identification and Distribution of "Coliform" Bacteria in Drinking Water Table 4. Relationships between the distribution of faecal and non-faecal Enterobacteriaceae and the presence or absence of faecal streptococci, sulphite-reducing Clostridium sp. and colony counts

STR

STR

CLO

+ Number of 163' strains F.C 67 N.F.C 72 N.id.C. 24

CLO

+

315

M.B.20°C MB 20°C MB 20°C

M. B. 37°C MB 37°C MB 37°C

+

+

529

222

269

93

235

202

266

181 288 60

90 114 18

111 125 33

40 42 11

76 133 26

79

96

96 145 26

27

SIR + or -: presence or absence of faecal streptococci in 100 ml of water; CLO + or -: presence or absence of sulphite-reducing Clostridium sp. in 20 ml of water; M. B. 20°C + or -: colony count at 20 ° greater or less than 100 in 1 ml of water; M.B. 37°C + or -: colony count at 37° greater or less than 10 in 1 ml of water. Number of enterobacterial strains for which the information on STR, CLO, M. B. 20°C or M. B. 37°C was known. F. C: Faecal coliforms; N. F. c.: Non-faecal coliforms; N. id. C: Non identified coliforms.

a

Table 5. Relationships between the distribution of enterobacterial species and the presence or absence of faecal streptococci or sulphitereducing Clostridium sp.

Species

Total strains

STR

STR

+ 163

C. freundii

E. agglomerans S. fonticola R. aquatilis E. cloacae K. trevisanii K.oxytoca E. amnigenus K. terrigena E.coli K. pneumoniae B. agrestis H. alvei S.liquefaciens E. sakazakii E. intermedium K. mobilis S. plymuthica S. marcescens Y. intermedia Y. enterocolitica Y. frederiksenii non identified

(1) 122 49 71 58 34 28 22 43 40 36 13 18 17 17 6 7 4 5 5 5 6 1 84

(2) 29 6 17 7 6 5 5 8 13 13 7 3 4 6 2 2 3 0 1 0 1 0 24

(3) 18% 4% 10% 4% 4% 3% 3% 5% 8% 8% 4% 2% 2% 4% 1% 1% 2% 1% 1% 15%

Total strains

+ 222

529 (2) 93 43 54 51 28 23 17 35 27 23 6 15 13 11 4 5 1 5 4 5 5 1 60

(3) 18% 8% 10% 10% 5% 4% 3% 7% 5% 4% 1% 3% 2% 2% 1% 1% 1% 1% 1% 1% 1% 1% 11%

CLO

CLO

(1) 82 84 14 28 44 33 30 23 19 26 11 10 4 2 11 5 4 4 2 1 2 0 51

(2) 32 61 2 4 24 23 21 6 5 5 6 1 1 1 9 0 1 1 0 0 1 0 18

(3) 14% 27% 1% 2% 11% 10% 9% 3% 2% 2% 3% 1% 1% 1% 4% 0 1% 1%

1% 8%

269 (2) 50 23 12 24 20 10 9 17 14 21 5 9 3 1 2 5 3 3 2 1 1 0 33

(3) 19% 9% 4% 9% 7% 4% 3% 6% 5% 8% 2% 3% 1% 1% 1% 2% 1% 1% 1% 1% 1% 12%

(1) Number of strains of each enterobacterial species, for which information on the presence or absence of "faecal streptococci" (STR) or "sulphite-reducing Clostridium sp." (CLO) was known. (2 ) Number of strains of each enterobacterial species related with the presence or absence of faecal streptococci, or of sulphite- reducing Clostridium sp. (3) Percentage of strains of each enterobacterial species calculated from the total number of strains isolated in the presence or the absence of faecal streptococci or of sulphite-reducing Clostridium sp.

316

F. Gavini, H. Leclerc, and D . A. A. Mosse!

Species

M.B.20 oe

Total strains

M . B. 20 °C

(2) 18

(1)

Non identified

55 37 17 33 19

8 2 4 2 6 6 6 6 6 4 2 3 1 0 3 1 3 0 0 1 0

11 10 20 16 20 5 9 5 3 4 7 2 7 2 4 4 1 37

11

(3 ) 19 % 9% 2% 4% 2% 6% 5% 5% 5% 5% 4% 2% 3% 1% 0 3% 1% 3% 0 0 1% 0 12%

(2) 37 29 15 29 17 5 4 14 10 l4 1 7 2 2 4 4 1 4 2 4 3 1 26

M.B.37°e

+ 202

235

93

C. freundii E. agglomerans S. fonticola R. aquatilis E. cloacae K. trevisanii K. oxytoca E. amnigenus K. terrigena E. coli K. pneumoniae B. agrestis H. alvei S.liquefaciens E. sakazakii E. intermedium K.mobilis S. plymuthica S. marcescens Y. intermedia Y. enterocolitica Y. frederiksenii

Total strains

+

M . B. 3r e

(1)

(3) 16% 12 % 6% 12% 7% 2% 2% 6% 4% 6% 1% 3% 1'Yo 1% 2% 2% 1% 2% 1% 2% 1% 1% 11%

109 32 19 37 24 20 15 35 30 14 7 22 5

8 4 11 1 7 4 5 5 1 52

(2 ) 45 18 10 3 10 12 8 20 8 7 4 9 4 5 2 2 1 3 1 1 2 0 27

(3) 22% 9% 5% 1% 5% 6% 4% 10% 4% 3% 2% 4% 2% 2% 1% 1% 1% 1% 1% 1% 1% 0 13 %

266

(2) 64

14 9 34 14 8 7 15 22 7 3 13 1 3 2 9 0 4 3 4 3 1 25

Table 6. Relationships between the distribution of enterobacterial species and colony counts at 20 °C and 37"C

(3 ) 24% 5% 3% 13% 5% 3% 3% 6% 8% 3% 1% 5% 1% 1% 1% 3% 0 2% 1% 2% 1% 1% 9%

(1) Number of strains of each species, for which the information colony counts at 20 °C or at 37°C was known . (2) Number of strains of each species related with the colony counts at 20 °C (greater or less than 100 in 1 ml of water) or at 37"e (greater o r less than 10 in 1 ml of water). (3) Percentage of strains of each species calculated from the total number of identified strains related to colony counts at 20 °C or at 37°e.

Correlation with water treatment

Most enterobacterial isolates (60 %) originated from untreated waters. Chlorinated and ozonized waters accounted respectively for 27 % and 13 % of the strains. Saprophytic enterobacterial species occurred more frequently in untreated waters than in chlorinated waters though the proportion was not statistically significant (Chi-square test). This effect was much less clear when the distribution frequency of individual species was studied

Table 7. Distribution of faecal and non-faecal Enterobacteriaceae in untreated, chlorinated and ozonized waters

Number of strains F.e. N.F.e. N. id.e.

UT

eL

OZ

643'

184 88 83 13

160

230 330 83

66 85 9

UT: untreated waters; eL: chlorinated waters; OZ: ozonized waters a Number of strains in each type of water (untreated, chlorinated or ozonized waters)

(Table 8). An exception was S. (onticola, which was isolated infrequently from chlorinated waters (1 % of the population) but was the most frequently found species (13 %) in untreated waters; C. (reundii was the next most frequent. No relation was noted between the origin of strains (faecal or non-faecal) and their frequency in chlorinated and ozonized together and untreated waters, nor between their origin and their frequency in ozonized alone and untreated waters. One species, E. agglomerans, was the most prevalent in ozonized waters, accounting for 33 % of the strains isolated from these waters. These E. agglomerans strains accounted for 53 % of the strains identified as belonging to this species. They all originated from river water (DS waters).

Discussion The results of this investigation corroborate those of a previous study, performed on strains isolated from waters in the North of France (Oger et aI., 1981). In that survey 59 % of the enterobacteria isolated were identified as sap-

317

Identification and Distribution of "Coliform" Bacteria in Drinking Water Table 8. Distribution of enterobacterial species in treated and untreated waters

Species

c. freundii E. agglomerans S. fonticola R. aquatilis E. cloacae K. trevisanii K.oxytoca E. amnigenus K. terrigena E.coli K. pneumoniae B. agrestis H.alvei S.liquefaciens E.sakazakii E. intermedium K.mobilis S. plymuthica S. marcescens Y. intermedia Y. enterocolitica Y. frederiksenii Non identified

Treated waters OZ 160

CL 184 (1) 37 15 2 14 15 12 14 13 13 14 4 2 2 2 5 2 2 2 0 1 0 0 13

(2) 20% 8% 1% 8% 8% 7% 8% 7% 7% 8% 2% 1% 1% 1% 3% 1% 1% 1% 1%

7%

(1) 20 53 0 0 23 21 17 3 3 2 4 0 0 0 5 0 0 0 0 0 0 0 9

(2) 12% 33%

14% 13% 11% 2% 2% 1% 3%

3%

6%

Untreated waters 643

Total 344 (1) 57 68 2 14 38 33 31 16 16 16 8 2 2 2 10 2 2 2 0 1 0 0 22

(2) 17% 20 % 1% 4% 11% 10% 9% 5% 5% 5% 2% 1% 1% 1% 3% 1% 1% 1% 1%

6%

(1)

125 41 86 57 28 26 16 33 26 20 16 22

18 16 4 7 7 6 8 5 5 1 83

(2) 19% 6% 13% 9% 4% 4% 2% 5% 4% 3% 2% 3% 3% 2% 1% 1% 1% 1% 1% 1% 1% 1% 13%

CL: chlorinated waters; OZ: ozonized waters. (1) Number of identified strains isolated in treated waters (chlorinated or ozonized or in total treated waters) or in untreated waters. (2) Percentage of strains of each species calculated from the total strains isolated in chlorinated or ozonized or total treated waters, or in untreated waters.

rophytic species, compared with 51 % in this study; 34 % were of faecal origin and 7 % non identifiable, compared with 38 % and 10 % in this study. More than 700 strains, 75 % of the bacteria studied, belonged to only 10 species. The most common species was C. freundii followed by E. agglomerans. These species, together with K. pneumoniae and E. coli, also predominated in the drinking waters from a rural area investigated by Lamka et al. (1980). Clark et al. (1982) found that in municipal drinking waters E. cloacae was the most frequent member of the Enterobacteriaceae, while in raw waters from lake and river sources E. coli predominated. The strains identified in this investigation as E. amnigenus, E. intermedium and R. aquatilis would have been grouped as E. cloacae by Clark et al. (1982), i. e. 20 % of the isolates or a percentage exceeding that of E. coli. The relationship between the presence or absence of faecal streptococci and of faecal or non-faecal Enterobacteriaceae is very significant (Table 4). It confirms the validity of using the count of group D streptococci as an indicator of faecal contamination rather than the incidence of sulphite-reducing Clostridium sp. or the mesophilic colony count. The mesophilic bacterial count is known to be of importance especially in treatment surveillance, but it

should not be considered as an indicator of faecal contamination. The relationship also confirms the value of determining the numbers of the ~ galactosidase positive Enterobacteriaceae during water analysis. If the observed incidence of S. fonticola strains is neglected, the distribution of faecal and non-faecal Enterobacteriaeceae does not appear to be related to water treatment. Nearly all the S. fonticola strains were isolated from untreated waters, i. e. wells and springs and originated from the Quebec laboratory. Their presence in these waters cannot easily be explained. This investigation confirms the high incidence of saprophytic bacteria, whatever the source, or geographic origin of drinking water. Apart from E. coli strains, accepted as faecal indicator bacteria and commonly sought during water analysis, other faecal coliforms accounted for 34 % of the isolated Enterobacteriaceae, non-faecal strains for 55 %. An improved insight into the ecology of these two types of bacteria should allow a more rational interpretation of the results of examinations of water and food than does the present policy of monitoring which is based on the isolation and enumeration of poorly defined faecal enterobacteria.

318

F. Gavini, H. Leclerc, and D. A. A. Mosse!

Acknowledgements. We thank the laboratories collaborating in this study and C Caron for her technical assistance. - This study was financially supported by 1.N.S.E.R.M. (Institut National de la Sante et de la Recherche Medicaie, France)

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Gavini, F., Leclerc, H.: Quality assurance of a multiple well system for the mechanical identification of Enterobacteriaceae strains. Arch. Lebensmitt.-Hyg. 33,137-176 (1982) Izard, D., Ferragut, C, Gavini, F., Kersters, K., De Ley, J. Leclerc, H.: Klebsiella terrigena, a new species from soil and water. Int. J. system. Bact. 31, 116-127 (1981a) Izard, D., Gavini, F., Leclerc, H.: Polynucleotide sequence relatedness and genome size among Enterobacter intermedium sp. nov. and the species Enterobacter cloacae and Klebsiella pneumoniae. Zbl. Bakt. Hyg., I. Abt. Orig. C 1, 51-60 (1980)

Izard, D., Gavini, F., Trinel, P. A., Leclerc, H.: Rahnella aquatilis, nouveau membre de la famille des Enterobacteriaceae. Ann. Microbiol. (Inst. Pasteur) 130, 163-167 (1979) Izard, D., Gavini, F., Trinel, P. A., Leclerc, H.: Deoxyribonucleic acid relatedness between Enterobacter cloacae and Enterobacfer amnigenus sp. nov. Int. J. system. Bact. 31, 35-42 (1981b) Lamka, K. G., Lechevalier, M. W., Seidler, R. ].: Bacterial contamination of drinking water supplies in a modern rural neighborhood. Appl. Environ. Microbiol. 39, 734-738 (1980) Leclerc, H., Gavini, F., Izard, D., Trinel, P. A.: Les coliformes: my the et realite. Colloque INSERM: Les bacilles a Gram negatif d'interet medical et en sante pubJique, Lille France 1983 Oger, C, Gavini, F., Delattre, ]. M., Leclerc, H.: A propos des coliformes et de la colimetrie des eaux d'alimentation. Ann. Microbiol. (lnst. Pasteur) 132 A, 183-189 (1981) Willcox, W. R., The late, Lapage, S. P., Holmes, B.: A review of numerical methods in bacterial identification. Antonie v. Leeuwenhoek 46, 233-299 (1980)

Dr. Fram;oise Gavini, INSERM U. 146, Domaine du CERTIA, 369, rue J. Guesde, F-59651 Villeneuve d'Ascq-Cedex, France