a numerical taxonomic study of fluorescent pseudomonas strains isolated from natural mineral waters

Journal of Applied Bacteriology 1995, 78, 71-81

A numerical taxonomic study of fluorescent Pseudomonas strains isolated from natural mineral waters

M. Elomari, L. Coroler, D. lzard and H. Leclerc Service de bacteriologie, Faculte de Medecine, Lille, France

4927105194: received 20 May 1994, revised 5 September 1994 and accepted 7 September 1994

M. E L O M A R I , L. C O R O L E R , D . I Z A R D A N D H. L E C L E R C . 1995. Forty-six strains offluorescent pseudomonads, isolated from natural mineral waters, together with 12 strains from clinical material and 44 reference strains, were phenotypically classified by 28 1 characteristics. T h e data were processed by the Dice similarity coefficient and unweighted pair group algorithm with arithmetic averages. Eight clusters were defined a t the 62% similarity level. Clusters I, I1 and IV were further divided into nine subclusters. Virtually all t h e mineral water strains fall into three groups : Ib (eight strains), I I a (14 strains) and V (16 strains). Subclusters Ib and I I a included natural mineral water strains only. Cluster V contained 13 mineral water strains and three culture collection strains of Pseudomonas~uorescens biovar 111. DNA/DNA hybridization studies are needed to define the taxonomic status of these three groups within the genus Pseudomonas.

INTRODUCTION

Natural mineral waters are characterized by their bacterial flora and their chemical and physical composition (Schwaller and Schmidt-Lorenz 1981). These properties are indicators of natural and original qualities of the water. There have been few published studies on the bacterial flora of mineral waters (Guillot and Leclerc 1993). At the point of emergence, natural mineral waters always contain bacteria which are capable of growth on an appropriate culture medium. Identification of these organisms poses real problems (Quevedo-Sarmiento et al . 1986; Gonzalez et al. 1987; Manaia et al. 1990; Morais and Da Costa 1990). They are Gram-negative heterotrophic bacteria, which obtain their cell carbon by oxidative assimilation of organic compounds. The majority are fluorescent pseudomonads. Recent studies on pseudomonads have partially clarified their taxonomy (Palleroni 1992) and have led to the revival of some genera such as Comamonas, the creation of new genera, such as Hydrogenophaga and Acidovorax, and to the description of a new family, Comamonadaceae. Identifica- tion of pseudomonads to species level, however, continues to be a difficult task, especially of those found in drinking water. Specific taxonomic studies, comparable to those done on Enterobacteriaceae isolated from drinking water (Leclerc et al. 1983), are required.

There is no question that comprehensive phenotypic analysis is very useful for pseudomonads at the species level

Correspondence to : H . Leclerc, Service de barthriologte, Faculth de Mhdectne, I place de eerdun, 59045 Ldle Cedex, France.

and will continue to be so (Palleroni 1992). The description of the species Pseudomonas lundensis and Ps. fragi by Molin and Ternstrom (1982, 1986) and Molin et al. (1986), among organisms isolated from meat, demonstrates the power of numerical analysis based on data from extensive phenotypic screening.

In the present work, a numerical phenotypic analysis was conducted on fluorescent Pseudomonas strains isolated from natural mineral waters.

MATERIALS AND METHODS

Bacterial strains

The 100 strains studied included 46 wild strains isolated from natural mineral water and identified as fluorescent pseudomonads according to the criteria of Palleroni (1984) and 54 type, collection and wild strains selected for control purposes and representative of the fluorescent pseudomonads (Ps. aeruginosa, Ps. JEzlorescens biovars I-V, Ps. chlororaphis, Ps. aureofaciens, Ps. marginalis, Ps. putida biovars A and B, Ps. lundensis, Ps. syringae, Ps. cichorii, Ps. tolaasii and Ps. asplenii). Details of the strains, including reference numbers and origins (when known) are given in Table 1.

Phenotypic characterization

Forty-five conventional tests were performed as described by Gavini et al . (1989). The assimilation of 49 carbo-

72 M. ELOMARI ET A L .

Table 1 List of organisms studied and their classification in phenotypic analysis

Cluster Sub-cluster Culture collection or other reference no. Name as received Isolated from

I Ia ATCC 17815 DSM 50275 DSM 50276

NCPPB 667T ( = ATCC 108MT) DSM 50106 ( = ATCC 17826)

CFML 92-132

Ps. jluorescens biovar I1 Ps. marginalis Ps. marginalis

Ps. marginalis Ps. jluorescens biovar I1

Lettuce Plant Soft rot of potato tuber Mineral water Endive Sea water

Ib CFML 92-143 CFML 92-135 CFML 92-133 CFML 92-140 CFML 92-134 CFML 92-138 CFML 92-123 CFML 92-124

Ic ATCC 15916 ATCC 17563 CIP 56-90 NCPPB 2192T (=ATCC 33618T

= PDDCC 2290T) CIP 73-25 (=ATCC 17397) ATCC 1352ST ATCC 17518 ATCC 14150

Id CFML 92-127 CFML 92-146 NCPPB 1616 ( = PDDCC 2839)

CFML 92-141

Inc ATCC 17482 ATCC 17816

I1 IIa CFML 92-103 CFML 92-104 CFML 92-108 CFML 92-1 13 CFML 92-1 12 CFML 92-1 1 1 CFML 92-102 CFML 92-122 CFML 92-120 CFML 92-1 15 CFML 92-1 16 CFML 92-119 CFML 92-126 CFML 92-125

IIb ATCC 17573 DSM 50148 (=ATCC 17533) DSM 50415 ATCC 12983

Ps. jluorescens biovar V Ps. jluorescens biovar I Ps. jluorescens biovar I Ps. tolaasii

Ps. jluorescens biovar I Ps. jluorescens biovar I Ps. jluorescens biovar V Ps. jluorescens biovar V

Ps. tolaasii

Ps. jiuorescens biovar I1 Ps. jluorescens biovar I I

Ps. jfuorescens biovar V Ps. jluorescens biovar V Ps. jluorescens biovar IV

Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water

Sea water Sputum Egg Cultivated mushroom

Agaricus bisporus Tap water Pre-filter tanks Soil ?

Mineral water Mineral water Cultivated mushroom

Agaricus bisporus Mineral water

Naphtalene-enriched medium Flower

Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water

Polluted sea water Soil ?

Ps. fluorescens biovar IV Soil

TAXONOMIC STUDY O F FLUORESCENT PSEUDOMONAS 73

Table 1 (continued)


111 CCUG 18758 CCUG 18757T (=CCM 573T)

IV IVa CFML 90-50 CFML 90-20 CFML 90-44 CFML 90-87

DSM 50208 (= ATCC 17485) CIP 52-191T (=ATCC 12633T

CFML 90-96

=ATCC 23647T) CIP 63-23 (= CCEB 520) CFML 90-22 CFML 90-60

V

VI

VII

IVb ATCC 17484 LMG 1246 (=ATCC 17430

CCUG 1317 =NCIB 10534)

IVC CFML 90-23

ATCC 17414

CCEB 518T ( = ATCC 13985T) ATCC 17415 DSM 50083T ( = ATCC 9446T) ATCC 9447 ATCC 17386

CFML 90-81

CFML 92-130

CFML 92-105 CFML 92-106 CFML 92-101 CFML 92-131 CFML 92-139 CFML 92-144 CFML 92-145 ATCC 17559 CFML 92-135 CFML 92-142 CFML 92-137 ATCC 17571 CFML 92-114 CFML 92-1 18 ATCC 17400 CFML 92-107

CFML 92-109 CFML 92-1 17 CFML 92-121

CIP 76-1 10 (= ATCC 27853) CIP 63-52T (=ATCC 1014ST) ATCC 15692

Ps. lundensis Ps. lundensis

Ps. putida Ps. putida Ps. putida Ps. juorescens Ps. juorescens Ps. putida biovar A Ps. putida biovar A

Ps. putida Ps. putida Ps. putida

Ps. putida biovar B Ps. putida biovar B

Ps. putida biovar B

Ps. putida Ps. juorescens Ps. ch lororaphis

Ps. aureofaciens Ps. aureofaciens Ps. chlororaphis Ps. chlororaphis Ps. Juorescens biovar V

Ps. juorescens biovar 111



Ps. aeruginosa Ps. aeruginosa Ps. aeruginosa

Beef meat Beef meat

Tracheal aspirate Antiseptic solution Stool Spinal fluid Spinal fluid Soil Soil

? Urine Biological fluid

Naphtalene-enriched media ?

Soil

Tracheal aspirate Urine ? Mineral water Mass river clay in kerosene Soil Plate contaminant ? Tryptophan-enriched water

Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Mineral water Plant Mineral water Mineral water Mineral water Polluted sea water Mineral water Mineral water

Mineral water Egg

Mineral water Mineral water Mineral water

Blood culture ? Infected wound

74 M . E L O M A R I E T A L .

Table 1 (continued)


VIII

NC

CFML 92-128 CFML 92-129

CFML 90-78 ATCC 23835T DSM 50259T CFML 92-1 10 CFBP 1392' (=ATCC 19310T) ATCC 17397

Mineral water Mineral water

Ps. putida Placenta Ps. asplenii Plant Ps. cichorii Endive

Ps. syringae Plant Ps. Juorescens biovar I

Mineral water

Tap water

ATCC, American Type Culture Collection, Rockville, MD, USA ; CCEB, Culture Collection of Entomogenous Bacteria, Department of Insect Pathology, Institute of Entomology, Prague, Czechoslovakia; CCM, Czechoslovak Collection of Microorganism, J.E. Purkyne, University, Bmo, Czechoslovakia; CCUG, Culture Collection, University of Goteborg, Goteborg, Sweden ; CIP, Collection of Institut Pasteur, Paris, France ; CFML, Collection de la faculti de Midecine, Lille, France ; DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany; NCIB, National Collection of Industrial Bacteria, Aberdeen, Scotland, UK; NCPPB, National Collection of Plant Pathogenic Bacteria, Ministry of Agriculture, Fisheries and Food, Plant Pathology Laboratory, Harpenden, Hertford- shire, UK; PDDCC, Plant Diseases Division Culture Collection, Auckland, New Zealand; Inc, unclustered strains of cluster I ; NC, strains not belonging to any cluster.

hydrates, 49 organic acids and 49 amino acids (Gavini el al. 1989) was studied with the API 50-CH, API SO-A0 and API 50-AA systems (BioMirieux), respectively. Enzymatic tests were performed on experimental enzymatic strips (89 tests). The enzymatic activities tested included 59 pepti- dase, 10 esterase, and 20 oxidase activities (Gavini et al. 1991). These enzymatic activities were also studied with the API systems which were incubated at 30°C for 4 h. The tests were scored as positive according to the manufac- turer's recommendations. In all, 28 1 characters were deter- mined. Eighty of these, which were either positive or negative for all the strains, were not included in the numerical analysis, which was performed as described by Gavini et al. (1989).

Test reproduciblllty

All the tests were repeated on 10% of strains. The average probability of error of 2.5%, as calculated by the formula of Sneath and Johnson (1972), would not produce serious dis- tortion of taxonomic structure.

RESULTS AND DISCUSSION

At a similarity level of 62%, the numerical analysis yielded eight main clusters, designated I-VIII (Fig. 1 and Table 1) . Clusters I, I1 and IV could be subdivided at higher levels of similarity into nine subclusters: Ia-Id, IIa, I Ib and IVa-IVc. Six strains did not cluster: Ps. asplenii ATCC 2383ST, Ps. cichorii DSM 50259T, Ps. syringae CFBP 1392', Ps. Juorescens biovar I ATCC 17397 and the wild strains CFML 90-78 and 92-110 isolated from a clini-

cal specimen and mineral water, respectively. Phenotypic characteristics for differentiating clusters and subclusters are shown in Tables 2 and 3.

The following clusters or subclusters contained one type strain only and their phenotypic characteristics were in good agreement with published data (Stanier et al. 1966; Champion et al. 1980; Palleroni 1984; Barrett et al. 1986): subcluster Ia (Ps. marginalis NCPPB 667'), including strains mostly of plant origin; cluster I11 (Ps. lundensis CCUG 18757') contained two strains from beef meat; subcluster IVa (Ps. putida biovar A CIP 52-191T) contained reference strains of mainly clinical origin, as did cluster VII (Ps. aeruginosa CIP 63-52T).

Subcluster IVc contained two type strains (Ps. chlororaphis CCEB 518' and Ps. aureofaciens DSM 50083T) which have been shown to be members of the same geno- species (Champion et al. 1980). Subcluster Ic also included two type strains (Ps. Juorescens biovar I ATCC 1352!jT and Ps. tolaasii NCPPB 2192'). The fluorescent species Ps. tolaasii is described in section V of Bergey's Manual of Sys- tematic Bacteriology (Palleroni 1984). This section includes a number of Pseudomonas species whose natural relationships with well-characterized species of the genus are largely unknown. Subcluster I Ib contained collection strains designated biovars IV (DSM 50415 and ATCC 12983) and V (ATCC 17573 and DSM 50148) of Ps. fruo- rescens, whereas subcluster IVb contained only collection strains of Ps. putida biovar B (ATCC 17484, L M G 1246 and CCUG 1317).

Strains isolated from mineral water grouped into clusters V, VI and VIII and subclusters Ib, Id and IIa. Of these clusters VI and VIII and subcluster Id contained relatively

TAXONOMIC STUDY OF FLUORESCENT PSEUDOMONAS 75

0 10 20 30 40 50 60 70 80 90 100 1 1 1 1 1 1 I Ps. fluorescens biovar II:

I b

Inc Ps fluorescens biovar II: Ps fluorescens biovar I Ps fluorascens biovar P

Ps fluorescens biovar I Ic Ps fOl0OSll

Id Ps toloosir

~~ 10

Ps. fluorescens biovar ISC Ilb Ps. fluorescens biovar P

mo Ps putiduT biovarA

E b Ps puttdo biovar B

Ps fluorescens b i o v a r x ~ - mc Ps. chioromphisT

Ps auraofociens’

I

I

It

m

Fig. 1 Phenotypic dendrogram based on unweighted pair group average linkage. SD, Dice similarity index. The cut-off level from clusters is 62%

PS. oeruqinoso’ Ps. fluorescens biovar I

few strains (three, two and four, respectively) and conse- quently their characteristics are not discussed here. I t should be noted that, although subcluster Id also contained Ps. toluusii NCPPB 1616, the taxonomic status of the strain is not clear. The other three groups contained most of the mineral water strains. Cluster V contained 13 wild strains isolated from mineral water and three of Ps. fluorestens biovar I11 (ATCC 17559, ATCC 17571 and ATCC 17400). Subclusters Ib (eight strains) and IIa (14 strains) contained only mineral water isolates.

The test results which differentiate the clusters and subclusters containing the numerically significant mineral water strains from those containing other strains are shown

in Table 4. All of the strains isolated from mineral water grew on malonate and inositol, whereas most of the other strains did not. Most of the mineral water strains reduced nitrate and utilized L-tyrosine, while strains of other origins were characterized by the presence of L-alanyl-L- phenylalanyl-L-prolyl-L-alanine arylamidase and esterase C12. Phenotypic tests useful for differentiating between clusters that included strains from natural mineral waters are shown in Table 5. All the mineral water strains of subcluster Ib grew on L-tryptophan, itaconate, trigonelline, 5-keto-gluconate and mesaconate but none of the mineral water strains of subcluster IIa was able to use these compounds. The mineral water strains of subcluster I b and

Tabl

e 2

Phen

otyp

ic ch

arac

teri

stic

s of c

lust

ers

Clu

ster

s (no

. of

stra

ins)

I (n =

28)

I1

(n

= 1

8)

I11 (n =

2)

IV (n =

22)

V

(n =

16)

V

I (n

= 3

) V

II (n =

3)

VII

I (n =

2)

Typ

e st

rain

s

Ps. p

utid

a A

Ps

. Jlu

ores

cens

A

Ps. c

hlor

orap

his

Ps m

argi

nalis

Ps

. lud

ensi

s Ps

. aureofaciens

Ps. a

eruginosa

Con

vent

iona

l te

sts

Mot

ility

C

ultu

re a

t 4°C

C

ultu

re a

t 41

°C

Nitr

ate

redu

ctio

n T

TC

(10%

) T

ribu

tyri

n G

elat

inas

e T

wee

n-es

tera

se

Lec

ithin

ase

Car

bon sources

Gly

cero

l Er

ythr

itol

L-ar

abin

ose

Rib

ose

wx

ylos

e A

doni

tol

Gal

acto

se

pman

nose

In

osito

l So

rbito

l N

-ace

tyl-g

luco

sam

ine

Xyl

itol

Buty

rate

M

alon

ate

Adi

pate

Su

bera

te

Aze

late

Se

baca

te

Lev

ulin

ate

I tac

onat

e M

esac

onat

e A

coni

tate

59

100 3 66

24

48

17

14

31

100 87

90

100 69

41

100

100 97

100 90

31

31

72

24 0 0 0 21

93

90

83

22

100 0 78 0 39 0 6 44

100 0

100

100 83 0

100

100 83

67

84 0 39

89 0 0 0 0 0 0 0

100

100

100 0 0

100 0 0 0 0

100 0

100 0 0 0 0

100 50 0 0 0 50 0 0 0 0 0 0 0 0 0

59

100 5 23

77

77 5 32

36

100 14

60

95

28 0 32

82

27

14

41 0 91

64

14 5 5 5 23

50

45

91

19

100 0 94 6 62.5

12

.5

50

75

100 94 0

100 6

100 87.5

10

0 10

0 0 10

0 81 0 94

100

100

100

100 12.5

87

-5

87.5

19

33

100 0 67 0 0 33 0 0

100 67

100

100

100 0

100 67

100

100

100 0

100

100 0 0 0 0 0 67

67

100

100

0 0

100

100

0 10

0 0

100

0 10

0 0

66

100

100

100

66

0

100

0 0

0 0

0 66

0

33

0 0

0 0

0 0

0 0

0 0

0 10

0 0

33

0 66

10

0 66

100

100

0 66

0

66

0 66

0

100

100

100

0 10

0 0

0 0

Tabl

e 2

(con

tinue

d)

Clu

ster

s (n

o. o

f stra

ins)

Ben

zoat

e p-

Hyd

roxy

-ben

zoat

e L-

man

dela

te

L-try

ptop

han

L-l y

sine

D

L-5-

amin

o-va

lera

te

2-A

min

o-be

nzoa

te

Sper

min

e H

ista

min

e

I (n =

28)

I1

(M =

18)

I1

1 (n

= 2

) IV

(n =

22)

V

(n =

16)

V

I (n =

3)

VII

(n =

3)

VII

I (n

= 2

)

Typ

e st

rain

s

Ps. p

utid

a A

Ps. f

ruor

esce

ns A

Ps. c

hlor

orap

his

Ps m

argi

nalis

Ps. l

unde

nsis

Ps. a

ureo

faci

ens

Ps. a

erug

inos

a

14

56

50

82

94

0 10

0 10

0 93

10

0 0

100

31

100

100

100

0 6

0 5

0 0

100

0 79

0

0 45

94

10

0 66

0

14

39

50

86

12.5

0

100

100

76

100

100

95

37.5

10

0 10

0 0

52

10

0 45

75

10

0 66

50

28

83

0

100

75

0 10

0 10

0 10

22

0

100

6 0

66

100

Enz

ymat

ic te

sts

L-se

r y I-t

yro

sine

ar

ylam

idas

e 86

a-

L-gl

utam

yl-a

-L-

hist

idin

e ar

ylam

idas

e 34

Es

tera

se C

18

10

100

100

95

100

0 66

0

67

100

82

6 0

32

44

0 66

0

31

0 10

0 10

0 ~~

All

the

stra

ins

in t

he s

tudy

wer

e ox

idas

e an

d ca

tala

se-p

ositi

ve,

with

gro

wth

at 0

and

0.8

% N

aCl;

hydr

olys

ed ty

rosi

ne;

prod

uced

lev

an fr

om s

ucro

se a

nd u

tiliz

ed s

ucci

nate

, D

L-la

ctat

e, L-

mal

ate,

pyru

vate

, L-a

lani

ne a

nd L

-glu

tam

ate

as so

le c

arbo

n so

urce

s. A

ll st

rain

s po

sses

sed

the

follo

win

g en

zym

e ac

tiviti

es; e

ster

ase-

C4;

est

eras

e-C

5 ; e

ster

ase-

C6;

es

tera

se-C

8 ; L

-ala

nine

ary

lam

idas

e an

d B

-ala

nine

; B

-xyl

osid

ase,

lysi

ne a

nd o

rnith

ine

deca

rbox

ylas

e; in

dole

and

ace

toin

e pr

oduc

tion ; p

oly-

B-h

ydro

xybu

tyra

te a

ccum

ulat

ion;

gr

owth

on

war

abin

ose,

~-x

ylos

e, B

-met

hyl-w

xylo

side

, so

rbos

e, d

ulci

tol,

a-m

ethy

l-wm

anno

side

, a-

met

hyl-D

-glu

cosi

de,

amyg

dalin

e, a

rbut

ine,

aes

culin

e, s

alic

in, c

ello

bios

e,

mal

tose

, la

ctos

e, m

elez

itose

, mel

ibio

se, r

aftin

ose,

sta

rch,

gly

coge

n, g

entib

iose

, w

tura

nose

, ~-

lyxo

se, w

taga

tose

, ox

alat

e, p

htal

ate,

iso

phta

late

, L-

norle

ucin

e, -

trypt

opha

n,

3am

inob

enzo

ate,

+am

inob

enzo

ate,

ure

a, a

ceta

min

e an

d et

hyla

min

e as

sole

car

bon

sour

ce.

Non

e of

the

stra

ins

poss

esse

d th

e fo

llow

ing

enzy

me

activ

ities

: a-

wga

lact

osid

ase;

/h-

gala

ctos

idas

e ;

phos

pho-

8-m

acto

sida

se;

a-L-

arab

inos

idas

e; a

-wgl

ucos

idas

e;

/3-~

ga

lact

uron

oh y d

rola

se ;

B-D

-glu

curo

nida

se ;

m-m

alto

sidas

e ; B

-mal

tosi

dase

; N

-ace

t y I-

a-D

-glu

cosa

min

idas

e ; a

-L-f

ucos

idas

e ; B

-wfu

cosi

dase

; 8

-L-f

ucos

idas

e ; /3

-wla

ctos

idas

e ;

a-w

man

nosi

dase

; 8-

wm

anno

sida

se a

nd B

-wxy

losi

dase

. All

of th

e st

rain

s w

ere

nega

tive

for

the

follo

win

g te

sts:

gro

wth

in th

e pr

esen

ce o

f a c

once

ntra

tion

of s

odiu

m c

hlor

ide

of 7%

w/v

; /3

-gal

acto

sida

se; D

Nas

e an

d hy

drol

ysis

of s

tarc

h.

TT

C, 2,3,5-Triphenyltetrazolium c

hlor

ide.

Tab

le 3

Phe

noty

pic

char

acte

ristic

s of

sub

clus

ters

Subc

lust

ers

(no.

of

stra

ins)

Ib (

n =

8)

Ic (n =

8)

Id (n =

4)

IIa (n

= 1

4)

IIb

(n =

4)

IVa (n

= 1

0)

IVb

(n =

3)

IVc (n

= 9

) Ia

(n =

6)

~~

Typ

e st

rain

s

Ps. t

olaa

sit

Ps. a

ureo

faci

ens

Ps. m

argz

nabs

Ps

. Juo

resc

ens

A

Ps. p

utid

a A

Ps

. chl

oror

aphi

s

Con

vent

iona

l te

sts

Mot

ility

G

row

th a

t N

aCl

3%

TT

C (1

0%)

Twee

n es

tera

se

Leci

thin

ase

Elas

tase

Carbon sources

Gxy

lose

R

ham

nose

In

osito

l su

cros

e Tr

ehal

ose

Inul

in

Xyl

itol

5-K

eto-

gluc

onat

e B

utyr

ate

Isov

aler

ate

Mal

onat

e A

dipa

te

Gly

cola

te

Levu

linat

e 2-

Cet

oglu

tara

te

Citr

acon

ate

Mes

acon

ate

Aco

nita

te

L-ph

enyl

alan

ine

L-ty

rosi

ne

t-tr

ypto

phan

Tr

igon

ellin

C

itrul

ine

DL-

2-am

ino-

benz

oate

B

ut yl

amin

e A

myl

amin

e Sp

erm

ine

Tryp

tam

ine

71

71

14 0 29

14

71

14

100 43

86 0 0 71 0 14

86

14

14 0 14

57

86

57

86

71

57

71

57

43 0 0 0 0

25

75 0 0 0 75

100 0

100

100 87.5

0 0

100

100 0

100 50

62.5

75

37

.5

0 10

0 10

0 10

0

100

100 25

12.5

0 0 0 0 87

-5

100

100 50

37.5

37

.5

12.5

50

12.5

10

0 75

100 0 75

37.5

0 0 37.5

0 0 0 37.5

50

87

.5

100 62.5

50

87

.5

25

50

100 0 12.5

50

12

.5

0 10

0 25

25

100 0 75

25

100 0

100 0 75

25 0 0 75

50 0 0 50

75

100

100 50

50

75

25

75

75

25 0

100 0

7 75

86

25

0

0 0

25

50

25

36

0

93

50

0 0

93

50

21

75

86

100

57

0 0

0 0

0 43

25

57

50

10

0 50

0

0 14

0

0 0

21

75

36

75

0 0

100

100

100

100

100

0 0

0 0

50

21

0 7

0 29

0

43

0 93

50

7

0

50

33

100

0 90

0 0

33

0 33

20

0

10

67

10

0 0

0 0

0 0

0 0

0 0

0 0

0 90

10

0 10

0 10

0 80

33

10

0

30

0 0

0 50

33

0

66

30

0 10

0 10

0 50

10

0 0

67

0 10

0 10

0 10

0 30

0

10

100

50

100

50

100

100

100

0 10

0

78

100 89

78

78

11

33 0 67

78

78 0 0 56

89

100 56

22 0 56 0 11

78

78

100 89

78

11

44

67

78

78

100 0

Tabl

e 3

(con

tinue

d)

Subc

lust

ers

(no.

of

stra

ins)

~

~ ~~

~

~~

~ ~

Ia (n =

6)

Ib (n =

8)

IC (n

= 8

) Id

(n =

4)

IIa (n

= 1

4)

IIb

(n =

4)

IVa (n =

10)

IV

b (n

= 3

) IV

c (n

= 9

)

Typ

e st

rain

s

Ps. t

alaa

sii

Ps. a

ureo

faci

ens

Ps. c

hlor

orap

his

Ps. m

argi

nalis

Ps

. Juo

resc

ens

A

Ps. p

utid

a A

Enz

ymat

ic te

sts

L-h

ydro

xypr

olin

e ar

ylam

idas

e L-

aspa

rtate

ar

y lam

idas

e L-

isol

euci

ne

ar y l

amid

ase

L-al

any

l-L-p

heny

lala

n yl

-L-

prol

yl-L

-ala

nine

ar

ylam

idas

e a-

L-gl

utam

yl-a

-L-

glut

amic

-ar y

lam

idas

e GI

ycy

l-~-a

rgin

ine

aryl

amid

ase

~-hi

stid

yl-~

-leu

cyl-

L-

hist

idin

e ar

ylam

idas

e L-

histi

dyl-L

-ser

ine

aryl

amid

ase

L-ph

enyl

alan

y I-L

-pro

line

aryl

amid

ase

L-Va

l y 1-1

-t yr

osy I

-L-s

erin

e ar

ylam

idas

e L-

hist

id yl

-L-p

heny

lal

anin

e ar

ylam

idas

e L-

1 ys

yl-L

-ser

ine

ar yl

amid

ase

Este

rase

-C12

Es

tera

se-C

14

Este

rase

-Clb

8-

w lu

cosi

dase

100 86

43

71 0

100 29

86

57

57

100 86

57

14 0 86

100 25

50

25 0 75 0 25 0 0 0 0 62.5

0 0 0

100 87.5

50

87.5

37.5

100 37.5

75

50

62.5

100 75

100 50

25 0

50

100 0 75

25

100 0 75

75

75

75 0

100 0 0 0

57

43

29

29 0

100 0 50

86

36

86

21 7 0 0 0

50

75

100 75

75

100 25

75

25

50

100 75

100 50 0 0

70

50

20

100 0

100 20

0

100 20

100 60

10

0 0 0 0

0 0 0 67

33

100 0 0 67

0

100

100 33 0 0 0

89

100 89

89

56

100

100 78

78

67

100 67

100 78

44

11

?TC

, 2,3

,5-T

riph

enyl

tetr

azol

ium

chlo

ride.

80 M. ELOMARI ET A L .

Table 4 Phenotypic characteristics for differentiating mineral water pscudomonad strains from other strains

Phenotypic tests A B (% A) - ($4 B)

1.-alanyl-L-phen y lalan yl- L-pro1 yl-r .-alanine arylamidase

Motility i.-lysyl-r .-serine-4-

Growth on TTC Nitrate reduction Denitrilication Inositol L-pro1 yl-L-arginine

Lsterase-C 12 Malonate Histamine Isovalerate L-tyrosine

tncthoxy arylamidase

arylamidase

20* 87 67

11 77 66 11 70 59

3 60 57 91 40 51 66 15 51 97 49 48 32 79 47

40 a7 47 100 55 45 14 55 41 23 64 41 83 42 40

* "h of positive strains. A, Natural mineral water strains from groups Ib, IIa and V; B, strains of other origins; TTC, 2,3,5-triphenyltetrazolium chloride.

cluster V showed clear phenotypic differences : all strains of subcluster Ib used L-arabinose, trigonellin, sucrose and sorbitol, whereas strains of cluster V did not. Conversely all strains of cluster V were able to grow on adonitol, L-arabitol, suberate, azelate and sebacate, whereas mineral water strains of subcluster Ib failed to do so. The mineral water strains of subcluster IIa and cluster V showed obvious phenotypic differences. All strains of subcluster IIa utilized 1.-arabinose but none was able to utilize erythritol, L-arabitol, suberate, azelate, sebacate, L-tryptophan and adonitol. In contrast, all of the strains of cluster V utilized all of these compounds except L-arabinose. Tests for assimilation of xylitol, L-arabitol, adonitol, suberate, azelate and sebacate were most helpful in differentiation among subclusters of strains originating from mineral water. On the basis of these results it is possible to allocate a strain isolated from mineral water to one of the three groups.

In conclusion, three numerically important phenotypic clusters containing almost only the strains of fluorescent pseudomonads isolated from natural mineral water have been characterized phenotypically. Further studies (in par- ticular DNA/DNA hybridization) are needed in order to determine the taxonomic status of these groups, their

1.-arahinose Adonitol Sorbitol Xylitol L-arabitol Suberate Azelate Sebacate Aconitate 0-x yli t ol Trigonelline 5-Keto-gluconate Spermine Sucrose lsobutyrate Benzoate L-serine L-lysyl-L-serine-4-

Levulinate Erytritol I taconate Amylamine L-tr yptophan r .-ornithine Mesaconate

methoxy arylamidase

Table 5 Phenotypic characteristics for

including strains of mineral water from each other Phenotypic tests Subcluster IIa Subcluster Ib Cluster v differentiating groups of pseudomonads

1 00* 100 0 0

71.5 0 0 0 0 0

100 93 0 0

93 21.5 7

50 100

86 0 0 0

43 0

28.5 0

0 100

0 0 0 0 0

100 100 100 100

0 100 100

0 25

0 75 75

100 0

100 100 100

100 0

81 100 1 00 100 100 18 6 0 6

75 0

12.5 94

100

82 12.5 93 87.5 0

94 100 87.5

~

* of positive strains.

TAXONOMIC STUDY OF FLUORESCENT PSEUDOMONAS 81

relationships to the genus Pseudomonas and their ecological significance.

REFERENCES

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a numerical taxonomic study of fluorescent pseudomonas strains isolated from natural mineral waters

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