(~) INSTITUTPASTEUR/ELSEVIER Paris 1991

Res. Microb:oi 1991, 142, 95-i01

Haemolysin occurrence among Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria strains isolated from different aquatic ecosystems P. Monfort and B. Baleux

Laboratoire d'Hydrobiologie Marine et Continentale/URA CNRS 1355, Universitd de Montpellier I.', Sciences et Techniques du Languedoc, 34095 Montpellier Cedex 02 (France)

SUMMARY

A total of 909 Aeromonas spp. isolates from different aquatic ecosystems w e r e tested for haemolysin production by both sheep and horse blood agar-plate assays and by rabbit erythrocytes in broth assay. A comparison of these different methods was undertaken in order to appreciate their capacity to evaluate the haemolytic activity of Aeromonas spp. isolated from aquatic ecosystems. The haemolytic activity was associated particularly with A. hydrophila and A. sobria (about 950/bof strains), whereas oA. caviae did not produce haemolysin (about 95% of strains). A method suitable for use in routine diagnostic microbiology laboratories is proposed for quantifying both groups of A. hydrophila/A, sobria and A. ccviae in environmental water. Key-words : Haemolysin, Aeromonas hydmphila, Aeromonas caviae, Aeromonas sobria, Water ; Ecosystem, Enumeration.

INTRODUCTION

Motile A e r o m o n a s species (A. hydrophila, A . caviae and A . sobria) are widely distributed in aquatic environments (Hazen et al., 1978) and are recognized as primary pathogens of fish, reptiles and amphibia (Hazen et aL, 1978a ; Hird et al., 1981). At one time, A e r o m o n a s spp. were thought to cause diseases only in immunocompromised humans. Thus, they are usually considered to be a secondary or opportunistic pathogen (Von Graevenitz and Mensch, 1968 ; Picard et aL, 1984). Recent attention has focused on Submitted May 20, 1990, accepted November26, 1990.

these microorganisms owing to their inc: :='asing association with infections in pai .'nts without known immunological abnorma! .'ies. A e r o m o n a s spp. have been implicat~l in many infections of wounds exposed d: ring aquatic activity (Beaune et aL, 1978 ; J~,-:el.~l~et al., 1979 ; Delbecke et aL, 1985 ; The:r non et al., 1987). Reports from many parts ~:( the world suggest that A e r o m o n a s spp. are c :usative agents of acute diarrhoeal disease in: m=a~ following ingestion of water (Cumberba: :h et al., 1979 ; Pitarangsi et al., 1982 ; Bm :e er al., 1984 ; Figura et al., 1986 ; Kirov , ' aL, 1986; M6graud, 1986). Therefore, :hesc

96

P. M O N F O R T A N D B. B A L E U X

organisms can be considered as significant enteric pathogens (Janda and Duffey, 1988). This suggestion is supported by the finding of various exotoxins including enterotoxin, haemolysin and cytotoxic proteins (Cumberbatch et al., 1979 ; Turnbull et al., 1984 ; Janda, 1985 ; Watson et al., 1985).

erythrocyte haemolysis in culture filtrates or on blood agar plates (Brenden and Janda, 1987). The haemolysin test is suitable for use without facilities for enterotoxin testing such as suckling mouse or ileal loop assays. The present study was undertaken to assess the haemolytic activity of environmental Aeromonas spp. strains from different aquatic ecosystems and to compare different methods of haemolysin testing.

The enteropathogenic potential of Aeromonas spp. is notably mediated by the cytotoxic enterotoxin as well as other extracellular substances (CT-cross-reactive factor, proteases, etc.) (Asao et aL, 1984; Cumberbatch et aL, 1979; Janda, 1985) and it has been shown that enterotoxin production is significantly correlated with haemolysin production (Cumberbatch et al., 1979; Burke et aL, 1983). Cytotoxin production and other virulence factors are preferentially related to A. hydrophila and A . sobria (Turnbull et aL, 1984, Barer et al., 1986). Both A . hydrophila and A . sobria are the major a e r o m o n a d enteric pathogens (Janda, 1985; Megraud, 1986). However, A . caviae, which never possesses virulence factors, was frequently found in faeces of patients with symptoms o f enteric infection (Megraud, 1986 ; Moyer, I987) and it is not clear whether this bacterium is part of the transitory flora or whether it can be enteropathogenic (Megraud, 1986). Cumberbatch et aL (1979) suggested that the cytotoxic and haemolytic activities may be either the expressions of the same molecule or subject to the same genetic control. The works o f Asao et al. (1984) support the notion that Aeromonas haemolysin is a cytotoxic enterotoxin. Results of different studies have revealed a significant correlation between haemolysin production, enterotoxin production and diarrhoeal disease (Cumberbatch et aL, 1979; Burke et aL, 1983, Brauer et aL, 1985). The enterotoxin can be detected by animal assays in culture filtrates (Burke et aL, 1981 ; Turnbull et aL, 1984) and the haemolysin by

After the sampling from PXA agar, each Aeromonas colony was cultured onto nutrient agar (bioM&ieux) and 50 ml of heart infusion broth (HIB, Difco Laboratories) in 100-ml Erlenmeyer flasks were inoculated with the Aeromonas strain to be tested and incubated at 37°C in a water bath shaking at 100 rpm for 20 to 24 h. Cell-free prepa-

ADH = arginine dihydrolase. HB = h o r s e blood. HT = haemolysin titre.

0 D C = ornithine decarboxylase. SB =sheep blood. WD = without dilution.

MATERIALS AND M E T H O D S

Strains

A group of 909 Aeromonas isolates from different aquatic ecosystems were studied (table I): 184 strains were recovered from a sewage treatment pond and 178 from receiving brackish water of this sewage treatment system ; 64 strains were recovered from a sewage treatment plant (activated sludge) and 483 from different surface waters. The Aeromonas isolations were made on PXA agar (Rogol et al., 1979; Monfort and Baleux, 1988). All isolates were initially identified as Aeromonas genus by the following standard tests: motility (+), Gram ( - ) , cytochrome oxidase (+), D-glucose fermentation (+) (37°C, 24h), arginine dihydrolase (+) (30°C, 24 to 48 h), ornithine decarboxylase ( - ) (30°C, 24 to 48 h), ONPG test (+) (37°C, 2 h), sensitivity to O/129 ( - ) (Diagnostics Pasteur) (30°C, 24 h). The species was determined using the following screening tests (30°C, 24 to 48 h) (Popoff, 1984): esculin hydrolysis, L-arabinose utilization, fermentation of salicin, acetoin from glucose (VosgesProskauer), gas from glucose, H2S from cysteine. Bacterial preparations :

HAEMOL YSIN OCCURRENCE AMONG AQUATIC AEROMONAS

97

Table I. Distribution of the 909 Aeromonas spp. strains isolated from different aquatic ecosystems. Origin Sewage treatment pond (M~ze) Receiving brackish water (Thau lagoon) Sewage treatment system (Montpellier) Lez river Vis river H6rault river Salagou lake Total

A . hydrophila

A . caviae

A . sobria

Aeromonas sp.

9

83

79

13

39

1

136

2

1 15 9 5 15

52 36 69 42 1

2 69 93 49 62

9 5 6 2 5

93

284

490

42

Aeromonas sp. = unidentifiedAeromonas strains by Popoff's scheme(1984).

rations were made by filtration of the cultured broth through a membrane filter (Millipore 0.45/~m). The preparations were stored at 4°C and tested on the day of preparation (Burke et aL, 1983).

tion was assayed four times. The trays were read with a spectrophotometer (Multiskan, Titertek) after and incubation at 37°C for I h followed by another incubation at 4°C for 2 h (Wretlind et al., 1973). The haemolysin titre (HT) was expressed as the log2 of the reciprocal of the greatest dilution showing 50°7o haemolysis of the erythrocytes (Burke et aL, 1983). Haemolytic activity, as interpreted, is shown in table II.

Haemolysin assays

Agar-plate assays. - - Haemolytic activity was determined by a zone of haemolysis around colonies on blood agar plates (bioM6rieux) containing 507o (v/v) either sheep blood (SB) or horse blood (HB) after 18 to 24 h of incubation at 37°C (Janda et aL, 1984 ; Brenden and Janda, 1987). Broth assay. - - Volumes (I00/~1) of serial dilutions (1/10, 1/20, etc.) of cell-free preparation in phosphate-buffered saline (pH 7.4) were added to equal volumes of a 1°70 suspension of rabbit erythrocytes (bioM6rieux) in microtitre trays (Linbro, Flow Laboratories). Each cell-free prepara-

RESULTS

The distribution o f the different A e r o monas species f r o m d i f f e r e n t aquatic ecosystems is shown in table I ; 867 o f 909 A e r o m o n a s strains were identified as A.

Table II. P.cadings of erythrocytes haemolysis in microtitre trays. Reading Haemolysis Haemolysis Haemolysis Haemolysis Haemolysis Haemolysis Haemolysis

negative WD at 1/I0 at 1/20 at 1/40 at 1/80 at 1/160

HT n

WD 3.32 4.32 5.32 6.32 7.32

Interpretation Non-haemolytic strain Slight-haemolytic strain Medium-haemolytic strain High-haemolytic strain High-haemolytic strain High-haemolytic strain High-haemolytic strain

WD= without dilution The haemolysintitre (HT) is expressed as the log2 of the reciprocal of the greatest dilution showing 50% haemolysisof the erythrocytes.

P. MONFORT AND B. BALEUX

98

hydrophila, A. caviae or A . sobria according to the classification o f P o p o f f (1984) ; 42 o f 909 (4.6%) Aeromonas strains could not be identified with this classification.

w h e n t e s t e d on b l o o d agar ( S B - , H B - ) produced haemolysin against rabbit erythrocytes in broth assay (HT+), whereas the conserve situation occurred only a m o n g 0.3% o f Aeromonas strains (SB-, HB +, H T - ) .

Table III shows that 94.2% o f Aeromonas strains have the same response by agar-plate assays and by broth assay : 59°70 o f all isolates were f o u n d to be haemolysin-positive with the three tests (SB +, H B +, H T ÷) and 35.2°70 were n o n - h a e m o l y t i c ( S B - , H B - , H T - ) . These results show a slight difference in the sensitivity to Aeromonas haemolysin o f the three erythrocyte species used. M o r e important was the difference due to the negative response o f 4.6% o f all isolates on sheep blood agar when the response was positive on horse blood agar (SB-, HB ÷) : 1.2070 o f Aeromonas strains which were non-haemolytic

Table

The Aeromonas species are classified into four groups according to the haemolysin titre ( H T ) : non-haemolytic ( H T - ) , slight-haemolytic (HT = WD), medium-haemolytic ( H T = 3 . 3 2 ) and high-haemolytic (HT~>4.32) (as shown in tables II and III). A. caviae is an occasionally haemolytic species (93.7% o f A. caviae strains are n o n - h a e m o l y t i c ) . Conversely, A . hydrophila and A. sobria are haem o l y t i c species (respectively 9 5 . 7 % a n d 95.3%). The haemolysin titre shows that about half o f A. hydrophila and A . sobria strains are s l i g h t - h a e m o l y t i c (respectively

IlL Haemolysin assays on blood agar plates and in microtitre trays of 909 Aeromonas strains.

Species

A. hydrophila

No

SB

HB

HT

No.

%

93

~

~

4 12 37 20 13 5 2 266

4.3 12.9 40 21.5 13.9 5.3 2.1 93.7 3.8 0.4 1.4 0.7 4.3 0.4 4.5 39.6 23.7 13 9.8 4.5 0.2 69 2.5

+ + + + + m

+ + + + + _

~ WD WD 3.32 4.32 5.32 6.32 _

--

m +

WD WD

11 1

+ + --

+ + m

3.32 5.32 _

4 2 21

+

A. caviae

A. sobria

284

490

+ +

Aeromonas sp.

42

+ + + + + + ~ + + +

~

2

+ + + + + + --

WD WD 3.32 4.32 5.32 6.32 7.32 --

+ +

~ WD

1 4

+ + +

WD 3.32 4.32

5 1 2

SB=sheep blood ; HB=horse blood, HT=haemolysintitre ; WD=without dilution.

22 194 116 64 48 22 I 29

9

12 2.5 5

HAEMOL YSIN OCCURRENCE AtvlONG AQUATIC AEROMONAS

52.9% and 44.1%), about 20% of these two species are medium-haemolytic (respectively 21.507o and 23.7070) and about 20070 are highhaemolytic (respectively 21.3070 and 27.5070). The X2 test shows that the two populations of A . hydrophila and A . sobria are not significantly different through these four descriptive factors. If haemolysin assays were used to discriminate A. hydrophila/A, sobria group from A . caviae, 94.8070 would have been correctly classified by broth assay (HT), and 96.3070 by horse blood agar plate assay (HB).

DISCUSSION

The results of bacterial identification show that 42 out of 909 (4.6070) Aeromonas strains are not identifiable according to the classification of Popoff (1984). These strains could belong to the three new species recently described i.e. : A.media (Allen et aL, 1983), A. veronii (Hickman-Brenner et aL, 1987) and A . sc,~uberii (Hickman-Brenner et aL, 1988). The characteristics of these species are different from the motile species of the Aeromonas genus described in the last edition of Bergey's Manual of Determinative Bacteriology (1984). Thus A. media is non-motile and produces a diffusible brown non-fluorecent pigment (Alien et aL, 1983). A . veronii does not produce an arginine dilydrolase ( A D H - ) but produces an ornithine decarboxylase (ODC ÷) (Hickman-Brenner et aL, 1987). Mannitol is not fermented and indole is not produced by A . schubertii (Hickman-Brenner et aL, 1988). Non-motile strains producing a brown pigment were not found in our study. The A D H - and ODC ÷ characteristic may enable a distinction between A. veronii and other motile species. However, Kuijper et al. (1989) showed, in DNA hybridization studies, that some Aeromonas strains A D H ÷ and O D C may belong to the species veronii. The Aeromonas strains were not tested for mannitol fermentation and indole production because such tests are not useful for differentiating the Aeromonas genus from other genera or

99

for differentiating between Aeromonas species according to the classification of Popoff (1984). Furthermore, the A . veronii and A. schubertii strains were isolated essentially from clinical samples and their taxonomic characteristics are still subject to discussion. Therefore, it is not possible by phenotypic analysis to tell whether a strain with an A . sobria biochemical pattern belongs to A . sobria (hybridization group 7) or to A . veronii (hybridization group 8 or 10) (Kuijper et aL, 1989). In the following discussion, we prefer to use Popoff's classification of Aeromonas species (1984) and to consider the unidentified Aeromonas strains as Aeromonas spp. The use of three different erythrocytes to haemolysin assays shows that sheep erythrocytes are less sensitive to Aeromonas haemolysin than horse and rabbit erythrocytes. Brenden and Janda (1987) also find that sheep erythrocytes are relatively insensitive in detection of haemolytic activity of Aeromonas spp. from clinical sources. Furthermore in our study, 98.5°/0 of Aeromonas isolates had the same response by both horse blood agar assay and broth assay with rabbit er~hrocytes. These two methods are equally efficient in detecting the Aeromonas spp. haemolysin. However, broth assay allows quantification of haemolytic activity by a haemolysin titre whereas plate assay is easier to implement. Also, horse blood agar assay is a good routine test to assess the haemolytic activity of Aeromonas environmental strains. The results of this study show that about 95°/0 of A . hydrophila and A . sobria strains produce haemolysin, whereas about 95°70 of A. caviae strains do not produce haemolysin (about 95070 of strains). Janda et aL (1984) have found similar results among 121 Aeromonas spp. strains of clinical sources : 55 of 58 (95070)A. hydrophila, 28 of 33 (93070)A. sobria and 6 of 30 (20°70) A. caviae were haemolytic. Similar results have been found by Barer et aL (1986) among 95 Aeromonas strains, about 70070 of which were from clinical sources and about 30°70 from chlorinated

100

P. M O N F O R T A N D B. B A L E U X

mains supplies: 94°70 of 18 A. hydrophila strains, 100070 of 20 A. sobria strains, 0070 of 46 A. caviae strains and 9070 of 11 unidentified Aeromonas strains produced haemolysin. Therefore, our study shows that, as among the clinical strains, haemolysin production is related only to A. hydrophila and A. sobria species among the environmental strains. Haemolysin production clearly separates A. curiae from both A. hydrophila and A. sobria (Janda et al., 1984 ; Janda, 1985 ; Brenden and Janda, 1987). Among the 42 unidentified Aeromonas strains, about 30070 are haemolytic. These strains may belong to A. veronii or A. schubertii which are also haemolytic species (Hickman-Brenner et al., 1987 and 1988). In mouse lethality assays, a study of the virulence of 32 A e r o m o n a s spp. isolates mainly from clinical sources showed that, of the A. sobria isolates tested, 82070 fell into the h~ghly virulent category (p 2 is considered. These authors suggest that haemolysin assay is the most reliable method of discriminating enterotoxigenic A e r o m o n a s spp. and is applicable to both fecal and nonfecal isolates. According to Burke et al. (1983), about 50070 of both A. hydrophila and A. sobria strains (HT > 2) of the present study may be enterotoxigenic strains.

As Aeromonas spp. have been linked with wound infections and have been identified as important water-borne human enteric pathogens (see works cited in Introduction), these bacteria may be considered to pose a problem for public health (Seidler et aL, 1980 ; Kirov et al., 1986). Also, it may be necessary to enumerate these bacteria in water, since the faecal contamination indicators cannot be used to estimate the presence in water of Aeromonas spp. which are not faecal bacteria (Van Graevenitz and Mensch, 1968 ; Figura et al., 1986) and which belong to the bacterial flora of freshwater (Hazen et aL, 1978). Ecological studies, like water quality controls, require the analysis of numerous samples. For routine use, it is necessary to possess simple techniques easily suitable for diagnostic laboratories. Therefore, we suggest the following screening tests to enumerate both A. hydrophila/A, sobria and A . caviae groups in environmental water. In a previous paper, we recommended the use of P X A agar (Rogol et aL, 1979) to isolate and count motile Aeromonas in sewage water and in polluted and non-polluted fresh and marine water (Manfort and Baleux, 1988). With this medium, >_.80°-/0 of presumptive colonies are Aeromonas spp. The use of cytochrome oxidase and glucose fermentation tests is adequate for confirming Aeromonas spp. Also, spreading the presumptive colonies of Aeromonas spp. from P X A agar onto HB agar and using cytochrome oxidase and glucose fermentation tests enable o b t a i n i n g h a e m o I y s i n and presumptive species responses : cytochrome oxidase +, glucose fermentation +, haemolysin ÷ = A . h y d r o p h i l a / A . sobria group that may produce an enterotoxin and may be a waterborne pathogen ; cytochrome oxidase ÷, glucose fermentation +, h a e m o l y s i n - = A , caviae, rarely pathogen. With this schedule, it is possible to undertake studies of A. hydrophila/A, sobria group and A. caviae distribution in water and to clarify the role from the point of epidemiological view of these potential enteric pathogens.

HAEMOLYSIN OCCURRENCE AMONG AQUATIC AEROMONAS

Presence d'hdmolysine chez des souches de A c r o m o n a s h y d r o p h i l a , A e r o m o n a s caviae et A e r o m o n a s sobria isol~es de diff~rents ~cosyst~mes aquatiques La production de l'h~molysine a ~t~ recherch~e parmi 909 souches de Aeromonas spp. provenant de diff6rents ~cosyst6mes aquatiques. La mise en 6vidence de l'activit6 h6molytique a 6t6 faite par la technique sur g61ose au sang de mouton ou de cheval et par le microtitrage en plaque avec des &ythroeytes de lapin. Une comparaison est faite entre ces diff&entes m&hodes pour appr~cier leur capacit6 /~ mettre en 6vidence l'aetivit6 h6molytique de Aeromonas spp. dans des souches isol~es des milieux aquatiques. II est montr6 qu'environ 95% des souehes de A . hydrophila et de A . sobria isol6es d ' u n environnement aquatique produisent une h6molys!ne alors qu'environ 95% des souches de A . caviae isol6es du m~me environnement ne sont pas h6molytiques. II est propos6 une m6thode microbiologique de routine pour quantifier le groupe A . hydrophila/A, sobria et A . caviae dans les eaux. Mots-clds : H~molysine, Aeromonas hydrophila, Aeroraonas caviae, A e r o m o n a s sobria, Eau ; Eeosyst6mes, Num&ation.

References Allen, D.A., Austin, B. & Colwell, R.R. (1983), Aeromonas media, a new species isolated from river water. Int. J. Syst. Bact., 33, 599-604. Asao, T., Kinoshita, Y., Kozaki, S., Uemura, T. & Sakaguehi, G. (1984), Purification and some properties of Aeromonas hydrophila hemolysin. Infect. Immun., 46, 122-127. Barer, M.R., Millership, S.E. & Tabaqchaii, S. (1986), Relationship of toxin production to species in the genus Aeromonas. J. med. Microbiol., 22, 303-309. Beaune, J., Llorca, G., Gonin, A., Bran, Y., Fleurette,. J. & Neidhart, J.P. (1978), Phlegmon n~crotique de la main droite, point de d~part d'une septic~mie due /t Aeromonas hydrophila. D~monstration de l'origine iehtyologique de l'infection. Nouv. Press. Mdd. (Paris), 7, 1206-1207. Brauer, C., Scheftel, J.M., Rihn, B. & Monteil, H. (1985), Isolement de Aeromonas hydrophila dans les diarrh~es. Caraet6risation des souches ent6ropathog6nes et relations cliniques. Ann. BioL Ciin., 43, 725-731. Brender, R. & Janda, J.M. (1987), Detection, quantification and stability of the fl-haemolysin of Aeromonas spp. J. reed. MicrobioL, 24, 247-251. Burke, V., Robinson, J., Berry, R.J. & Gracey, M. (1981), Detection of enterotoxins of Aeromonas

I01

hydrophila by a suckling-mouse test. J. reed. MicrobioL, 14, 401-408. Burke, V., Robinson, J., Beaman, J., Gracey, M., Lesmaria, M., Rockhill, R., Echeverria, P. & Janda, J.M. (1983), Correlation of enterotoxicity with biotype in Aeromonas spp. J. clin. microbiol., 18,

1196-1200. Burke, V., Robinson, J., Gracey, M., Peterson, D. & Partridge, K. (1984), Isolation of Aeromonas hydrophila from a metropolitan waier supply: seasonal correlation with clinical isolates. AppL Environ. MicrobioL, 48, 361-366. Cumberbatch, N., Gurwith, M.J., Langston, C., Sack, R.B. & Brunton, J.L. (1979), Cytotoxic enterotoxin produced by Aeromonas hydrophila : relationship of toxigenic isolates to diarrheal disease. Infect. lmmun., 23, 829-837. Delbeke, E., Demarcq, M.J., Roubin, C. & Baleux, B. (1985), Contamination aquatique de plaies par Aeromona.~ sobria apr~s bain de rivi&e. Presse Med. (Paris), 14, 1292. Figura, N., Marri, L., Verdiani, S., Ceccherini, C. & Barberi, A. (1986), Prevalence, species differenciation, and toxigenicity of Aeromonas strains in cases of childhood gastroenteritis and in controls. J. Clin. MicrobioL, 23, 595-599. Hazen, T.C., Fliermans, C.B., Hirsch, R.P.'& Esch, G.W. (1978), Prevalence and distribution of Aeromonas hydrophila in the United States. Appl. Environ. MicrobioL, 36, 731-738. Hazen, T.C., Raker, M.L., Esch, G.W. & Fliermans, C.B. (1978a), Ultrastructure of red-sore lesions on largemouth bass (Micropterus salmoides) : association of the ciliate Epistylis sp. and the bacterium Aeromonas hydrophila J. ProtozooL, 25, 351-355. Hickman-Brenner, F.W., MacDonald, K.L., Steigerwalt, A.G., Fanning, G.R., Brenner, D.J. & Farmer, J.J. III (1987), Aeromonas veronii, a new ornithine decarboxylase-positive species that may cause diarrhea. J. Clin. MicrobioL, 25, 900.906. Hickman-Brenner, F.W., Fanning, G.R., Arduino, M.J., Brenner, D.J. & Farmer, J.J. III (1988), Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens. J. Clin. MicrobioL, 26, 1561-1564. Hird, W., Diesch, S.L., McKinnel, R.G., Gorham, E., Martin, F.B., Kurtz, S.W. & Dubrovolny, C. (1981), Aeromonas hydrophila in wild-caught frogs and tadpoles (Rana pipiens) in Minnesota. Lab. Animal Sc., 31, 166-169. Janda, J.M., Reitano, M. & Bottone, E.J. (1984), Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum. J. clin. MicrobioL, 19, 44-47. Janda, J.M. (1985), Biochemical and exoenzymatic properties of Aeromonas species. Diagn. MicrobioL Infect. Dis., 3, 223-232. Janda, J.M., Clark, R.B. & Brenden, R. (1985), Virulence of Aeromonas species as assessed through mouse lethality studies. Curt. MicrobioL, 12, 163-168. Janda, J.M. & Duffey, P.S. (1988), Mesophilic aeromonads in human disease: current taxonomy, laboratory identification, and infectious disease spectrum. Rev. Infect. Dis., I0, 980-997.

102

P. MONFORT A N D B. BALEUX

Joseph, S.W., Daily, O.P., Hunt, W.S., Seidler, R.J., Allen, D.A. & Colwell, R.R. (i979), Aeromonas primary wound infection of ~t diver in polluted waters. J. Clin. MicrobioL, 10~. 46-49. Kirov., S.M., Rees, B., Wellock, R.C., Goldsmid, J.M. & Van Galen, A.D. (1986), Virulence characteristics of Aeromonas spp. in relation to source and biotype. J. Clin. MicrobioL, 24, 827-834. Kuijper, E.J., Steigerwalt, A.G., Schoenmakers, B.S.C.I.M., Peeters, M.F., Za~ea, H.C. & Brenner, D.J. (1989), Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp. J. Clin. Microbiol., 27, 132-138. Megraud, F. (1986), Incidence and viru!ence of Aeromonas species in feces of children with diarrhea. Europ. J. Clin. MicrobioL, 5, 311-316. Monfort, P. & Baleux, B. (1988), Evaluation de deux milieux de culture pour l'isolement et le d~nombrement des Aeromonas mobiles dans diff~rents types d'eau. C.R. Acad. Sci. (Paris), 307, 523-527. Moyer, N.P. (1987), Clinical significance of Aeromonas species isolated from patients with diarrhea. J. Clin. MicrobioL, 25, 2044-2048. Picard, B., Arlet, G. & Goullet, Ph. (1984), Septic~mies Aeromonas hydrophila. Aspects ~pid~miologiques. Quinze observations. Presse Mdd. (Paris), 13, 1203-1205. Pitarangsi, C., Echeverria, P., Whitmire, R., Tirapat, C., Formal, S., Dammin, G.J. & Tingtalapong, M. (1982), Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides : prevalence among individuals with and without diarrhea in Thailand. lnfect, hnmun., 35, 666-673.

Popoff, M. (1984), Genus III, Aeromonas, in "Bergey's manual of systematic bacteriology, vol. 1" (N.R. Krieg & J.G. Holt) (pp. 545-548). Williams & Wilkins Co., Baltimore, London. Rogol, M., Sechter, I., Grinberg, L. & Gerichter, Ch.B. (1979), Pril xyIose Ampicillin agar, a new selective medium for the isolation of Aeromonas hydrophila. J. reed. iVIicrobiol., 12, 229-23I. Seidler, R.J., Allen, D.A., Lockman, H., Colwell, R.R., Joseph, S.W. & Daily, O.P. (1980), Isolation and characterisation of Aeromonas from polluted waters encountered in diving operations. AppL Environ. MicrobioL, 39, 1010-1018. Thonnon, J., Rives, J.M. & Laniel, D. (1987), Infection du cuir chevelu /l Aeromonas hydrophila, particularit6s. Presse Med. (Paris), 16, 1244. Turnbull, P.C.B., Lee, J.V., Miliotis, M.D., Van De WaUe, S., Koornhof, H.J., Jeffery, L. & Bryant, T.N. (1984), Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species. Z Clin. MicrobioL, 19, 175-180. Von Graevenitz, A. & Mensch, A.H. (1968), The genus Aeromonas in human bacteriology. New Engl. J. Med., 278, 245-249. Watson, I.M., Robinson, J.O., Burke, V. & Gracy, M. (1985), Invasiveness of ~eromonas spp. in relation to biotype, virulence factors, and clinical features. J. Clin. MicrobioL, 22, 48-51. Wretlind, B., Heden, L. & Wadstr6m, T. (1973), Formation of extracellular haemolysin by Aeromonas hydrophila in relation to protease and staphylolytic enzyme. J. Gen. MicrobioL, 78, 57-65.