Microb Ecol (1995) 29:259-267

MICROBIAL ECOLOGY © 1995Springer-VerlagNewYorkInc.

M a x i m u m G r o w t h Temperature Ranges of Aeromonas Spp. Isolated F r o m Clinical or Environmental Sources M.-L. H~inninen,1 S. Salmi, 1 A. Siitonen2 ~College of Veterinary Medicine, Department of Food and Environmental Hygiene, H~rneent.57, 00580 Helsinki, Finland 2National Public Health Institute, Mannerheimint.166 Helsinki, Finland Received: 10 June 1994; Revised." 15 August 1994

Abstract.

Only a limited number of phenotypic tests are available for the differentiation of all 13 known hybridization groups (HG) of Aeromonas spp. These organisms have a wide spectrum of warm-blooded and cold-blooded hosts. In the present study, the maximum growth temperatures (tmax) of the most common HGs of Aeromonas spp. originating from human fecal samples, food, water, and healthy and diseased fish were determined with a plate-type continuous temperature-gradient incubator. We observed that determination of the tmaxcan be applied for differentiation of HG 1 from HG 2 and 3 (phenospecies A. hydrophila); HG 6 from HG 4, 5A, and 5B (phenospecies A. caviae); HG 7 from HG 8/10 (phenospecies A. sobria); and HG 11 from HG 8/10 (phenospecies A. veronii). HG 1, 4, 8/10, and 13 strains occurring also in human clinical samples had a high tmax,about 40°C or higher. Hybridization group 2, 3, 5A, and 5B strains, which in most cases originated from water or food, had tmaxvalues in the range of about 36-39°C, while HG 6, 7, and 11 had tmaxvalues in the range of about 33-37°C. Fish pathogenic strains of A. salmonicida subsp, salmonicida and subsp, achromogenes had the lowest tm,x values from about 30 to 35°C.

Introduction Aeromonas spp. have a wide environmental distribution. The organisms have been isolated from freshwater as well as from drinking water and foods [21, 22]. Aeromonas spp. are also pathogenic, causing infections in cold-blooded animals, such as frogs, snakes, or fish, and in warm-blood animals and humans [2, 9, 19]. There is increasing evidence that Aeromonas spp. are also human enteropathogens, causing sporadic infections in all age groups [2, 9, 12, 16]. Mesophilic Aeromonas spp. consist of at least nine phenotypically characterized

Correspondence to: M.-L. H~inninen

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species: A. hydrophila, A. caviae A. media, A. eucrenophila, A. veronii biotype sobria, A. veronii biotype veronii, A. jandaei, A. schubertii, A. trota, and A. allosaccharophila [9, 14]. DNA-DNA hybridization, however, differentiates 13 genetic species (genomospecies, hybridization groups, [HGs]) [9]. Psychrothrophic A. salmonicida group have typical phenotypic characteristics, but genetically they are members of HG 3 [19]. The complex taxonomy and the need for genetic methods in the identification of Aeromonas spp. to the species level are factors hampering the understanding of the pathogenicity and epidemiology of the organisms. Studies conducted on a limited number of Aeromonas strains indicate that the optimum growth temperature ofA. salmonicida and A. hydrophila is 22-25°C and 28°C, respectively. The maximum growth temperature (tmax) of A. salmonicida is reported to be approximately 35°C, and that of mesophilic Aeromonas spp. is 38--41°C [19]. The t~ax is an important factor determining the possibility for an organism to colonize, multiply, and cause infections either in cold-blooded or warm-blooded animals. It also may be of value in the taxonomy of a species [18]. In the present study, the tm,x of Aeromonas spp. that were identified to the genospecies level by phenotypic and genotypic methods were determined. The organisms were isolated from human fecal samples, foods, freshwater, drinking water, and healthy or diseased fish. Methods

Bacterial Strains Aeromonas strains used were isolated from foods and water at the College of Veterinary Medicine; the clinical strains were isolated from diarrheal or nondiarrheal human fecal samples at the National Public Health Institute, Helsinki. A. salmonicida were isolated at the National Veterinary Medical Institute from diseased and healthy salmonid fish. In the isolation of food isolates, ampicillin-dextrinagar (ADA) [7] was used. Clinical strains were isolated on Aeromonas Selective Medium (Difco Laboratories, Detroit, Mich.). The strains included were 109 A. hydrophila, 13 A. salmonicida, 87 A. caviae, 74 A. sobria, and 7 A. veronii biotype veronii. Reference strains of each hybridization group were included in the study, and they are shown in Table 1. They were obtained from The Centers of Disease Control and Prevention, Atlanta, USA, and from The University of Zurich, Department of Medical Microbiology, Zurich, Switzerland. After isolation, the strains were stored at -70°C.

Identification of Strains The mesophilic Aeromonas strains were identified to the species level by phenotypic methods as recommended [1, 3, 4, 5, 19]. Both conventional media and commercial tests API 20E, API NE and ID 32GN (bioMeriuex, SA Marcy l'Etoile, France) were used. The biochemical tests reported by Altwegg et al. [3] and Abbott et al. [1] in the identification of the three HGs ofA. hydrophila (HG 1, 2, 3) and four HGs of A. caviae (HG 4, 5A, 5B, and 6) were used. Similarly, A. salmonicida was identified as recommended [19]. All mesophilic species were incubated at 30°C, and A. salmonicida was incubated at 22°C. Ribotyping was applied for the identification of hybridization groups of phenotypically identified strains, as recommended by Martinetti Lucchini and Altwegg [13]. Chromosomal DNA was isolated by a miniprep method using phenol chloroform extraction and precipitated with ice-cold isopropanol [20]. DNA was digested with SmaI ribonuclease. Restriction fragments were electrophoresed in 1.0% agarose gels and transferred by Southern blotting to a nylon membrane (Boehringer Mannheim GmbH Mannheim, Germany). Then 16S and 23S rRNA (Boehringer Mannheim) was used to prepare the

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261

Table 1. Maximum growth temperatures of the reference strains of hybridization groups (HGs)1 through 12 and two strains of HG 13 ofAeromonas spp. isolated from human fecal samples Phenotypic name

ATCC* No.

CDC~ No.

Source of isolation

HG

A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. caviae A. caviae A. caviae A. eucrenophila A. sobria A. sobria A. sobria A. jandaei A. jandaei A. veronii A. veronii

7966

9079-79 1271-82 9533-76 9090-79 0434-84 0433-84 9083-79 0862-83 0435-84 0859-83 9538-76 0337-80 1364-80 0787-80 1530-81 35624 1067-84 1306-83 3136-78 2446-81 9180-81

Canned milk Human bile Fish Fish Freshwater Freshwater Guinea pig Fish Fish Fish ? Fecal specimen Wound isolate Fecal specimen Fecal specimen Human sputum Fecal isolate Ankle suture Surface water Abscess Human skin Fecal specimen Fecal specimen

1 1 2 2 3 3 4 5A 5B 6 7 8 8 9 9 10/8 10/8 11 11 12 12 13 13

14715

15468

Unnamed Unnamed

A. A. A. A.

schubertii schubertii trota trota

43700 43701

Maximum growth temperature (°C) 40.5 39.0 38.5 36.7 36.8 37.4 40.5 36.6 40.1 34.5 35.0 40.9

41.4 42.7 42.3 42.7 42.8 37.0 36.6 41.9 41.9 42.8 44.0

* American Type Culture Collection (Rockville, Md USA) ? Centers for Disease Control (Atlanta, Ga USA) digoxigenin-labeled cDNA by reverse transcriptase [20]. Smal fragments with a molecular weight less than 4 kb were used for the identification of a genetic species [13].

D e t e r m i n a t i o n o f M a x i m u m G r o w t h T e m p e r a t u r e (tmax) The inoculum was a bacterial suspension grown either at 30°C (mesophilic Aeromonas spp.) or at 22°C (A. salmonicida) for 24 h in BHI (Brain Heart Infusion Broth) broth and diluted to 10 5 to 10-6 colony-forming units (CFU) per milliliter. Then 20 Ixl of the suspension were run as a droplet in a straight line across BHI agar, which was in a specific cuvette. Extra fluid was collected with a pipette [17, 18]. Purity of the culture was verified by subculture on BHI blood agar. The cuvettes were incubated in a plate-type temperature-gradient incubator (Gradiplate W 10, Biodata, Helsinki, Finland) for 24 h (mesophilic Aeromonas spp.) or 48 h (A. salmonicida) in the continuous temperature gradient of 5-6°C. The temperature gradient area was chosen to be close to the expected tm~x.The strains were pretested on BHI agar plates at 35, 37, 39, 41, and 44°C to find the preliminary maximum growth temperature ranges. For mesophilic Aeromonas spp., the temperature range was 33-46°C, and for A. salmonicida it was 25-36°C. Readings were recorded to an accuracy of 0.1°C. The length of the growth was measured. The limit at which no colonies were visible under a dissecting microscope was called the tmax.The distance readings were converted to temperatures with the aid of the following empirical calibration equation [17]: tn -- tL tm,x=tL+~X(y--24)

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in which, tL = temperature at lower reference point; tH = temperature at higher reference point; 50 = distance of two temperature sensors (mm); y = distance of the end of the growth from the edge of the lowest temperature (mm). Each strain was tested at least twice.

Statistical Analysis Student's t-test was used for the comparison of the means of tmaxS.

Results

Species Identification More than 20 tests were used for the identification of the strains to the phenospecies level (A. hydrophila, A. caviae, A. sobria, A. veronii, A. jandaei, A. trota). Usually, the three generally accepted Aeromonas species, A. hydrophila, A. caviae, and A. sobria were confirmed by analysis of ribopatterns to be members of H G 1, 2, or 3 (A. hydrophila), H G 4, 5A, 5B, or 6 (A. caviae), and H G 7 or 8/10 (A. sobria), as recommended [13]. Ribotyping differentiated A. sobria group as A. sobria (HG 7) and as A. veronii biotype sobria (HG 8/10). The ribopatterns of each H G matched with the ribopatterns of the respective reference strain in the molecular area < 4 kb [13].

Maximum Growth Temperatures of the Reference Strains The maximum growth temperatures of the reference strains were determined, and they are presented in Table 1. Because the reference strains of A. trota (HG 13) were not available, the results of H G 13 represent tmaxvalues of tWO human fecal strains. All results represent the average tmax values of two determinations. The values varied between two determinations with 0.3 to 1.0°C. The tmaxvalue of H G 1 was higher than that of H G 2 or 3. In the A. caviae phenospecies, the tmaxSof HG 4 and 5B reference strains were higher than the tmaxSof H G 5A or 6. A. sobria HG 7 reference strain had a lower tmaxthan did the reference strains of A. sobria H G 8/10. The reference strains of H G 9, 10, 12, H G 13 had all a high tmax of 41-44°C. The tmaxof H G 11 strains, which phenotypically cannot be differentiated from A. veronii biotype veronii, was lower than that of H G 10 strains.

Maximum Growth Temperatures of Aeromonas Spp. Isolated From Different Sources The maximum growth temperature ranges ofAeromonas spp., representing different HGs and various isolation sources, are given in Fig. 1 (phenospecies A. hydrophila and A. salmonicida), Fig. 2 (phenospecies A. caviae), and Fig. 3 (phenospecies A. sobria). The distribution of tmaxis illustrated as multiple box-and-whisker plots, where a box describes the middle 50% of the temperature values, and the vertical lines describe the temperature range in which 25% of the values are distributed.

Growth Temperature Ranges of Aeromonas Spp.

263

T (°(2)

39

"

37

Fig. 1. Maximum growth temperature distribution of mesophilic Aeromonas hydrophila group A (17 HG 1 strains isolated from ground beef, chicken, or fish); group B (38 HG 2 strains from ground beef, chicken, freshwater or well water); group C (36 HG 3 strains from ground beef, chicken, freshwater or well water); group E (18 HG 1 strains from human fecal samples); and 6 strains of psychrophilic A. salmonicida subsp, salmonicida (group D) and 7 strains of subsp, achromogenes (group F). The ranges are described as multiple box-and-whisker plots.

33

31

B

A

(~ D GROUP

E

F

T(°C)

43--

I

A

B

C

GROUP

D

B

Fig. 2. Maximum growth temperature distribution of phenospecies A. caviae representing HG 4 isolated from ground beef or chicken (group A; No. of strains, 7); HG 5A and 5B isolated from ground beef and chicken (group B; No. of strains, 20), well water (group d; No. of strains, 21) or freshwater (group E; No. of strains, 7); and HG 4 isolated from human fecal samples (group C; No. of strains, 32).

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T (°C) m

.i[ -

--

33 A

B

C

D

E

GROUP

Fig. 3. Maximum growth temperature distribution of A. veronii biotype sobria HG 8/10 (groups A, C, D, and E) and A. sobria HG 7 isolated from water (group B). Group A: from ground beef or chicken (No. of strains, 26). Group C: from freshwater (No. of strains, 3). Group D: from fecal samples of Finnish tourists in Morocco (No. of strains, 21). Group E: from human fecal samples in Finland (No. of strains, 10).

Table 2. Maximum growth temperatures (t~ax)of certain hybridization groups (HGs) of mesophilic Aeromonas spp. isolated from different sources Phenospecies (No. of strains) A. A. A. A. A. A. A.

hydrophila (35) hydrophila (38) hydrophila (36) caviae (39) caviae (48) sobria (14) sobria (60)

Hybridization group 1 2 3 4 5A and 5B 7 8/10

tmax;°C (mean; range of values) 41.0 (39.0-42.3) 38.0 (36.5-39.8) 38.5 (36.2-39.8) 40.8 (38.0-42.5) 38.3 (36.5-39.5) 35.0 (33.2-36.0) 41.2 (39.1-43.6)

The mean t~ax o f A . hydrophila H G 1 strains was significantly higher (P < .001) than those of HG 2 or 3 or A. salmonicida (Fig. 1, Table 2). The ranges o f tm,xS o f HG 2 and 3 were similar (Table 2). Aeromonas caviae H G 4 strains displayed a wide range o f temperature distribution from 38 to 42.5°C. The means of tmaxS o f H G 4 groups were significantly higher (P < .001) than those of H G 5 A and 5B, which originated either from food, well water, or freshwater. The strains o f H G 5A and 5B were included in the same group because their tmaxvalues distributed similarly. Overlapping was seen between the tmaxS o f H G 4, 5A, and 5B in the temperature range o f 38-39°C. However, 80% of the strains o f H G 4 had a tm~xhigher than 40°C (Fig. 2, Table 2). Clinical and food strains o f A. veronii biotype sobria strains (HG 8/10) had a high tmax of 4 2 4 4 ° C . Aeromonas sobria H G 7 isolated f r o m freshwater had a low tm~x in the range o f 3 3 - 3 6 ° C (Fig. 3, Table 2).

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Discussion The t~ax is one of the factors determining the host spectrum of a potentially pathogenic organism. Usually, environmental organisms that have a lower tm,xthan the body temperature of warm-blooded animals cannot colonize or cause infections in those animals. Aeromonas spp. are interesting because they have a wide spectrum of both warm-blooded and cold-blooded hosts. The tmax spectrum of Aeromonas spp., from approximately 27 to 44°C, reflects the wide distribution of the organisms in different environmental niches and human clinical specimens. The lowest tmax values were found in the A. salmonicida group, indicating the host specificity of the group as a fish-adapted species in arctic and subarctic regions. Also, a few strains ofA. eucrenophila (HG 6) and A. sobria (HG 7) had a low tmaxof 33-36°C, and they were isolated from a water environment (results not shown). Four strains ofA. hydrophila (HG 1), however, which had tmaxhigher than 40°C, were isolated from the intestines or kidneys of fish originating from an arctic area. All strains of HG 1, 4, 8/10, 9, 12, or 13, which have been shown to be the predominating Aeromonas spp. isolated from human clinical specimens [9], had a high tm,x of approximately 38 44°C. Two strains of A. trota isolated from tourists who had visited Morocco had a high tm,xof 43-44°C. Aeromonas trota is probably associated with subtropical or tropical regions, because all the strains described in the literature have originated from subtropical regions [5]. The t~,x is a stable characteristic and is of value in the taxonomy of microbes. For example, Niemi et al. [18] used tm,x in the studies on the taxonomy of fecal streptococci. Recently, Havelaar et al. [8] found indirect evidence on the taxonomic role of tmaxbecause their Aeromonas strains isolated from human fecal samples grew more often at 41°C than the strains isolated from drinking water. In the A. hydrophila group, the strains of HG 1 had a significantly higher tmax than the strains of HG 2 or 3. This suggests that a growth temperature of 41°C in a controlled temperature incubator may be used as a test in the separation of HG 1 strains from those of HG 2 or 3. However, the range of tmaxSof HG 1, 2, and 3 indicated that about 10-15% of the strains had overlapping growth temperatures of 39-40°C. In the A. caviae group, HG 4 was represented by the strains isolated from fecal samples of humans or from food sources. HG 4 organisms had tmax values similar to those of the reference strains of this group. The strains isolated from foods had a higher tm,x than did the clinical strains. This can be explained by the isolation source, because most of the food strains were from chicken, which has a high body temperature of 43°C. The organisms of HG 5A and 5B are known to be phenotypically similar and genetically closely related [3, 11]. The distribution of their tm,xvalues was similar, suggesting a close relatedness. The distribution of the t~x of 5A and 5B was wide, 36-40°C. Because some clinical strains of HG 4 may have a rather low tmaxof 38-39°C, a growth temperature may not be used in the separation of HG 4 from 5. The organisms of HG 5A and 5B are known in most cases to be of environmental origin, although HG 5B has also been associated with human diarrhea [9]. Only a few strains of HG 6 were available, and they had a low tm0xsimilar to that of the reference strains of this group (results not shown). The highest t ~ of Aeromonas spp. was found among the strains of A. veronii biotype veronii (results now shown) and A. veronii biotype sobria (HG 8/10).

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Aeromonas jandaei (HG 9) was represented only by the reference strains, which also had rather high t~,xs. The tmaxvalues of the reference strains of HG 11 were much lower than in the other HGs ofA. veronii/A, sobria group. Aeromonas veronii biotype sobria strains either isolated from foods or from clinical samples had similar t~,x values. Kuijper et al. [10] also found that Aeromonas strains of HG 8 have a high growth temperature, because 64% of their strains grew at 42°C. Aeromonas sobria HG 7 isolated from freshwater or well water had a low tmaxof approximately 35°C. The tm,xvalues can be used in the differentiation ofA. sobria HG 7 from biochemically similar A. veronii biotype sobria HG 8. The tm~ is also reflected in the results of some biochemical tests. Clinical laboratories usually isolate and identify Aeromonas spp. at 35-37°C. The strains that have a rather low tmax of 35-39°C may produce false-negative results in biochemical tests [3, 6, 15]. In most cases, the clinical strains have a rather high tm,~ of more than 40°C, and they may produce typical reactions also at 37°C. In conclusion, the tm,xSof different Aeromonas spp. differed, and this characteristic may be used as an additional test in the differentiation of certain HGs within a phenospecies. This study suggests that Aeromonas spp. that occur in the environment, such as HG 7, 6, and 11, have a low tm,x of about 33-37°C and that HGs that may be of human clinical significance have a rather high tm,~of close to 40°C. Although HG 5A and 5B are also common in the water environment and in foods, their wide tma~distribution indicates that they may have potential to colonize in human intestine. References 1. Abbott SL, Cheung WKW, Kroske-bystr6m S, Malekzadeh T, Janda MJ (1992) Identification of Aeromonas strains to the genospecies level in the clinical laboratory. J Clin Microbio130:1262-1266 2. Altwegg M, Geiss HK (1989) Aeromonas as human pathogen. Crit Rev Microbiol 16:253-286 3. Altwegg M, Steigerwalt AG, Altwegg-Bissig R, Ltuhy-Hottenstein J, Brenner DJ (1990) Biochemical identification of Aerornonas genospecies isolated from humans. J Clin Microbiol 28:258-264 4. Carnahan AM, Behram S, Joseph SW (1991) Aerokey II: a flexible key for identifying clinical Aeromonas species. J Clin Microbiol 29:2843-2849 5. Carnahan AM, Chakrabotry T, Fanning GR, Verma D, Ali A, Janda JM, Joseph SW (1991) Aeromonas trota sp. nov., an ampicillin-suscepfible species isolated from clinical specimens. J Clin Microbiol 29:1206-1210 6. George WL, Jones MJ, Nakata MM (1986) Phenotypic characteristics of Aeromonas species isolated from adult humans. J Clin Microbiol 23:1026-1029 7. Havelaar AH, During M, Versteegh JFM (1987) Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration. J Appl Bactefiol 62:279-287 8. Havelaar AH, Schets FM, van Silhout A, Jansen WH, Wieten G, van der Kooij D (1992) Typing of Aeromonas strains from patients with diarrhoea and from drinking water. J Appl Bactefiol 72:435-444 9. Janda JM (1991) Recent advances in the study of the taxonomy, pathogenicity and infections syndromes associated with the genus Aeromonas. Clin Microbiol Rev 4:397-410 10. Kuijper EJ, Steigerwalt AG, Schoenmakers BSIM, Peeters MF, Zanen HC, Brenner DJ (1989) Phenotypic characterization and DNA relatedness in human isolates of Aeromonas spp. J Clin Microbiol 27:132-138 11. K~mpfer P, Altwegg M (1992) Numerical classification and identification ofAeromonas genospecies. J Appl Baeteriol 72:341-351 12. Liesenfeld O, Weinke T, Hahn H (1993) Three-year prevalence of enteropathogenic bacteria in an urban patient population in Germany. Infection 21:101-105

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13. Martinetti Lucchini G, Altwegg M (1992) rRNA restriction patterns as taxonomic tools for the genus Aeromonas. Int J Syst Bacteriol 42:384-389 14. Martinez-Murcia AJ, Esteve C, Garay E, Collins MD (1992) Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. FEMS Microbiol Lett 91:199-206 15. Mateos D, Anquita J, Naharro G, Paniaqua C (1993) Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas. Appl Bacteriol 74:111-118 16. Moyer M (1987) Clinical significance of Aeromonas species isolated from patients with diarrhea. J Clin Microbiol 27:2044-2048 17. Niemelfi SI, Tubb R, Suominen H (1990) Temperature-gradient incubator, Gradiplate ® Wl0 user's guide. Biodata Oy (Ltd) Helsinki, Finland 18. Niemi RM, Niemelfi SI, Bamford DM, Hantula J, Hyvarinen T, Forsten T, Raateland A (1993) Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Appl Environ Microbiol 59:2190-2196 19. Popoff M (1984) Genus III. Aeromonas Kluyver and Van Niel 1936, 398 AL. In: Krieg NR, Holt JG (eds) Bergey's manual of systematic bacteriology, vol 1. Williams & Wilkins, Baltimore, pp 545-548 20. Popovic T, Bobb CA, Olsvik O, Kielbauch JA (1993) Ribotyping in molecular biology. In: Peshing DH, Smith TF, Tenover FC (eds) Molecular microbiology. Principles and application. American Society of Microbiology, Washington, D.C., pp 573-583 21. Schubert RHW (1991) Aeromonads and their significance as potential pathogens in water. J Appl Bacteriol Symp Suppl 70:131S-135S 22. Wadstr0m T, Ljungh A (1991) Aeromonas and Plesiomonas as food- and waterborne pathogens. Int J Food Microbiol 12:303-312