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Growth and survival of clinical vs. environmental species of Aeromonas in tap water
International Journal of Food Microbiology 69 Ž2001. 191–198 www.elsevier.comrlocaterijfoodmicro
Growth and survival of clinical vs. environmental species of Aeromonas in tap water Patrice Mary a,b,) , Guillaume Buchet b, Claude Defives a , Jean-Pierre Hornez a a
Laboratoire de Microbiologie, UniÕersite´ des Sciences et Technologies de Lille. Batiment SN2. F-59655 VilleneuÕe d’Ascq cedex, France ˆ b Institut UniÕersitaire de Technologie A AB . Departement Genie ´ ´ Biologique. F-59653 VilleneuÕe d’Ascq cedex, France Received 14 July 2000; received in revised form 28 December 2000; accepted 9 February 2001
Abstract The ability of four species of Aeromonas Žtwo of clinical and two of environmental origin. to survive andror grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 = 10 5 cfu mly1 after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, AultramicrocellsB were detected and final populations of ca. 10 6 cfu mly1 after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 10 4 cfu mly1. All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10 5 –10 6 cfu mly1 was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and AultramicrocellsB bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit Ži.e. 1 log cfu mly1 . in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios Ž100r1 in unfiltered-non-autoclaved and 1000r1 in filtered microcosms. were favourable for aeromonads, at least for 1 to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of Atail phage-like particlesB bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Tap water; Autochthonous bacteria; Aeromonas spp.; Clinical species; Environmental species; Growth; Survival
) Corresponding author. Institut Universitaire de Technologie AAB, Departement Genie ´ ´ Biologique, Bd Paul Langevin, Cite´ Scientifique BP 179, F-59653 Villeneuve d’Ascq CEDEX, France. Fax: q33-3-20-43-44-72. E-mail address: [email protected] ŽP. Mary..
0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 4 9 1 - 3
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1. Introduction The genus Aeromonas is comprised of Gramnegative, glucose-fermenting, oxidase-positive, O129-resistant and DNase-positive rod-shaped bacteria which are generally motile by a single polar flagellum. The genus Aeromonas is quite complex with 14 DNA hybridization groups ŽHG. and three additional suspected ones. These HG are further separated in several genotypes and phenotypes ŽJoseph and Carnahan, 2000.. Only five genotypes are currently recognized as human pathogens associated with gastrointestinal ŽKirov, 1993. and numerous extra intestinal infections ŽJanda and Abbott, 1998.. Members of the Aeromonas genus are found primarily in aquatic environments. They have been observed frequently in surface water and groundwater ŽKersters et al., 1995., estuarine and sea water ŽKaper et al., 1981., chlorinated and non-chlorinated drinking water ŽGavriel et al., 1998. and, in some countries, in bottled mineral water ŽGonzalez et al., 1987; Tsai and Yu, 1997.. All phenospecies are found in sewage-contaminated water ŽBoussaid et al., 1991. and activated sludge ŽKampfer et al., ¨ 1996.. They are also common contaminants of a wide range of foods ŽKirov, 1993.. Because Aeromonas spp. are distributed widely in aquatic environments, there is considerable speculation about their role as contaminants. However, evidence for waterborne human Aeromonas infections and contamination of foods is still controversial ŽJoseph and Carnahan, 2000.. Indeed, it has been reported that pathogenic Aeromonas Žmainly HG1, HG4 and HG8Y. are rarely isolated in drinking and fresh water ŽHavelaar et al., 1992; Hanninen and Siitonen, 1995; Hanninen et al., 1997. and on the contrary that potentially pathogenic Aeromonas are frequently recovered from aquatic environments ŽKuhn ¨ et al., 1997; Imziln et al., 1998; Borell et al., 1998; Goni-Urriza et al., 1999.. Survival of ˜ Aeromonas in water microcosms is also the subject of much debate since high ŽWarburton et al., 1994; Tsai and Yu, 1997; Brandi et al., 1999. and low survival rates ŽLowcock and Edwards, 1994; Kersters et al., 1996; Janakiraman and Leff, 1999. have been reported.
Thus, in this study, the survival and ror growth capacities of two clinical and two environmental well characterized strains were investigated in tap water microcosms.
2. Materials and methods 2.1. Bacteria Aeromonas hydrophila ATCC 7966 ŽHG1. and A. caÕiaercaÕiae LMG 13455 ŽHG4. were selected as representative of clinical isolates. A. mediarmedia LMG 13464 ŽHG5B. formerly known as A. caÕiaermedia and A. popoffii LMG 17541 ŽHG 17?. ŽJoseph and Carnahan, 2000. were considered to be representative of environmental strains. 2.2. Water microcosms Samples of tap water were collected in 500-ml acid-washed sterile screw-capped glass bottles. In order to inactivate chlorine ŽAmerican Public Health Association, 1992. sterile sodium thiosulphate ŽS1830, Fisher Scientific 78996 Elancourt Cedex, France. solution was added Žfinal concentration: 13.2 mg ly1 .. The water samples were used without further treatment Žunfiltered–non-autoclaved., were filtered through a 0.2-mm cellulose nitrate filter ŽSartoriusr A70.800.684, Fisher Scientific. to remove autochthonous microflora Žfiltered. or were filtered and autoclaved at 1218C for 15 min Žfiltered–autoclaved. to destroy biological and heat sensitive antimicrobial agents that might pass through the 0.2-mm filters. 2.3. Inoculation of microcosms Early stationary phase cultures of Aeromonas in Tryptic Soy Broth ŽDifcorA50.370.179, Fisher Scientific. were harvested and washed twice in 0.85% Žwrv. NaCl ŽA48.909.518, Fisher Scientific. solution by centrifugation Ž14,000 = g, 5 min, 48C.. The different water microcosms Ž250 ml. were inoculated with 1 ml of washed Aeromonas suspension to give initial cell concentrations of ca. 5 =
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10 3 –10 4 cfu mly1 . The flasks were incubated at ambient temperature Žabout 208C., in the dark and without shaking. 2.4. Growth and surÕiÕal in inoculation studies Viable counts were determined immediately after inoculation, at regular intervals Ž1 day. over a 10-day period and after 14 days by the spread plate procedure. Samples were serially diluted in a 0.85% Žwrv. NaCl solution. Undiluted sample and y1 to y4 dilutions were plated on Tryptic Soy Agar ŽDifcor A50.369.171, Fisher Scientific. and R2A Agar ŽDifcorA51.826.179, Fisher Scientific.. Aeromonas populations were determined on TSA after 2 and 7 days incubation at 308C. The autochthonous microflora of tap water was enumerated on R2A Agar after 3 and 7 days incubation at 258C. All experiments were independently repeated three times and mean log cfu mly1 " SD were calculated. Bacteriological counts were equated with the detection limit Ži.e. 1 log cfu ml y1 . when no colony appeared on media. 2.5. Transmission electron microscopy
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toclaved microcosms ŽFig. 1.. After a short lag phase Ž1 day., this microflora grew actively to reach 6 = 10 5 cfu mly1 . Five to seven bacterial phenotypes were observed. The filtration through the 0.2-mm filter retained the great majority of autochthonous bacteria Žinitial cell concentration - 1 log cfu mly1 ., however, after a 3 days lag phase, the growth of AultramicrocellsB was observed in filtered microcosms and this population reached 10 6 cfu mly1 after 7 days ŽFig. 1.. Only two phenotypes were detected. Indigenous Aeromonas species were not detected in this tap water. The further evolution of these two types of microflora were not significantly affected by the presence of the inoculated Aeromonas Ždata not shown.. 3.2. Growth of the Aeromonas strains in filtered and autoclaÕed tap water All four strains of Aeromonas grew in filtered-autoclaved tap water ŽFig. 2.. However, growth varied. A ranking in growth rates was observed in the following order: A. caÕiaer caÕiae HG4 ) A. media r media HG5B) A. popoffii HG17?) A. hydrophila HG1. No clear relationship between ability to grow in nutrient poor water and the origin of the
Filtered microcosms were centrifuged at 150 000 = g for 3 h at 48C. Pellets were resuspended in 1 ml of a 0.85% Žwrv. NaCl solution. Samples of 50 ml were placed on collodion-coated copper grids which were then negatively stained with 1% sodium phosphotungstate ŽpH 7.4.. Preparations were examined with a JEM 100CX transmission electron microscope ŽJapan Electronic Optical Laboratory, Parc Technologique Claude Monet 78290 Croissy sur Seine, France. operating under standard conditions at 80 kV.
3. Results 3.1. Growth of autochthonous microflora in unfiltered–non-autoclaÕed and filtered tap water Just after bottling and inactivation of chlorine, the initial plate counts of the autochthonous bacteria were 2.39 " 0.19 log cfu mly1 in unfiltered–non-au-
Fig. 1. Growth of autochthonous bacteria Žv . in unfiltered–nonautoclaved tap water and indigenous AultramicrocellsB ŽB. in filtered tap water. Vertical bars represent SEM Ž P s 0.05..
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Fig. 2. Growth curves of Aeromonas spp. in filtered–autoclaved tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv .. Vertical bars represent SD Ž ns 3..
Fig. 3. Survival curves of Aeromonas spp. in unfiltered–non autoclaved tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv ..Vertical bars represent SD Ž ns 3..
strains i.e. environmental or clinical was thus observed. It was noteworthy that the lowest rate of growth was observed for A. hydrophila.
caÕiaer caÕiae remained at a constant level over a 9-day period and thereafter declined slowly. No clear relationship was observed between the origin of the bacteria and their ability to survive in the presence of autochthonous microflora.
3.3. SurÕiÕal of the Aeromonas strains in unfiltered– non-autoclaÕed tap water Since the colonial morphology of the Aeromonas spp and those of the autochthonous bacteria were quite distinct, it was possible to distinguish easily the two populations in inoculated unfiltered–non-autoclaved microcosms. On TSA plates, the Aeromonas grew more rapidly and the colonies appeared as Chinese hat and orange-coloured. Furthermore, the different autochthonous bacteria did not exhibit a DNase activity in contrast to the Aeromonas strains. Although the inoculation ratio Ž100 Aeromonasr1 autochtonous bacteria. was favourable, at least for day 1 the Aeromonas strains did not grow and a decrease in viable cell counts of the organism were observed in this microcosm ŽFig. 3.. Levels of A. popoffii fell below the detection limit Ž1 log cfu mly1 . after only 40 h incubation. Numbers of A. mediar media and A. hydrophila declined linearly and approached the detection limit in 14 days. A.
Fig. 4. Survival curves of Aeromonas spp. in filtered tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv .. Vertical bars represent SD Ž ns 3..
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3.4. SurÕiÕal of the Aeromonas strains in filtered tap water Despite being introduced at a very favourable ratio Ž10,000 Aeromonasr-10 autochthonous AultramicrocellsB ., which was maintained at least for 3 days, all Aeromonas strains disappeared rapidly in filtered microcosms ŽFig. 4.. The declines in viable cell counts were even more pronounced in this microcosm than in unfiltered–non-autoclaved microcosms. Thus, the filtration step seemed to have purified or concentrated a biological or an antimicrobial substance highly active against Aeromonas. On other hand, the removal of filter-retained autochthonous bacteria led to a reduction in the number of potential targets for these agents. After the disappearance of the Aeromonas in filtered microcosms, bacteriophages were not detected in culture filtrates by the
Fig. 5. Electron micrographs of membrane vesicle ŽA. and Atail phage-like particlesB bacteriocins ŽB. negatively stained with 1% sodium phosphotungstate ŽpH 7.4.. Bars represent 100 nm.
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spot test or by enrichment in liquid medium with the corresponding Aeromonas strain. However, structures evocative of membrane vesicles and Atail phage-like particlesB bacteriocins ŽFig. 5A and B. have been observed by electron microscopy of negatively stained samples.
4. Discussion The level of autochthonous bacteria is low in tap water Žca. 250 cfu mly1 . according to Brandi et al. Ž1999. and van der Kooij Ž1992.. However, regrowth of bacteria just after bottling was observed. A final concentration of 6 = 10 5 cfu mly1 was obtained and this value seems to be the upper limit obtainable in different nutrient-poor waters for the indigenous microflora ŽAmy and Hiatt, 1989; Deere et al., 1996; Miettinen et al., 1997; Tsai and Yu, 1997; Leclerc and Da Costa, 1998.. The availability of assimilable organic carbon Žvan der Kooij, 1992. and also phosphorus ŽMiettinen et al., 1997. is considered the key factor in the regulation of microbial regrowth in drinking water. The presence of AultramicrocellsB in tap water, capable of passing through a 0.2 mm cellulose nitrate filter, has been demonstrated. AUltramicrocellsB are frequently observed in different types of water ŽRoszak and Colwell, 1987; Morita, 1990; Schut et al., 1997; Jones et al., 1999.. The cell numbers of AultramicrocellsB increased to ca. 10 6 cfu mly1 after a 7-day incubation period according to Jones et al. Ž1999.. In these two types of microcosm, inoculated Aeromonas were thus in the presence of actively growing indigenous bacteria. All the Aeromonas strains tested were able to grow in filtered and autoclaved tap water and reached a maximum of 10 5 –10 6 cfu mly1 within a 1.5–15day incubation period according to the species tested. Maximal cell densities observed were of the same order of magnitude as those previously reported for A. hydrophila HG3 in nutrient poor waters ŽRippey et al., 1994; Kersters et al., 1996; Tsai and Yu, 1997.. Among the four species tested, A. hydrophila HG1 was the less able to grow in this microcosm. Delayed growth of A. hydrophila in tap water has been already described Žvan der Kooij and Hijnen,
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1988.. The maximum cell densities attained by A. hydrophila in oligotrophic freshwaters can be increased by as much as 100-fold with a 70% increase in growth rate by the addition of both carbon and phosphorus ŽRippey et al., 1994. or by the addition of mixtures of carboxylic acids, carbohydrates or amino acids each at concentration of 1 mg of Crl Žvan der Kooij and Hijnen, 1988.. Thus, the maximum cell densities observed in our study might be a consequence of carbon and phosphorus limitations and the A. hydrophila was more susceptible to these limitations than the three other species tested. The Aeromonas species tested were unable to initiate growth in the presence of autochthonous microflora. This finding is in agreement with the results of Kersters et al. Ž1996. for tap water microcosms. The minor contribution of the aeromonads to the bacterial populations of tap water, in which low concentrations Ž- 10 mg of Crl. of substrates is available, may be explained by the presence of many autochthonous bacteria having transport systems with higher affinity than the aeromonads Žvan der Kooij et al., 1980; van der Kooij, 1991.. Drinking water contains heterotrophic bacteria that have the ability to utilize compounds such as carbohydrates, carboxylic acids, peptides and amino acids at concentrations of a few micrograms per litre Žvan der Kooij and Hijnen, 1981, 1984; van der Kooij et al., 1982.. The strain of A. popoffii tested disappeared rapidly in unfiltered–non-autoclaved microcosm although this species has been isolated from drinking water production plants and reservoirs ŽHuys et al., 1997.. In contrast, the A. caÕiaer caÕiae Žof clinical origin. maintained a constant level for at least 9 days. The A. hydrophila Žclinical strain. and the A. mediar media Ženvironmental strain. underwent a similar reduction in viable counts. Thus, no correlation was found between strain origin and the survival capacity. In our study, the declines in cell counts for A. hydrophila HG1 and A. media r media HG5B Žformerly A. caÕiaer media. were similar or slightly lower than that previously reported for Aeromonas HG1, HG3 and HG5B ŽLowcock and Edwards, 1994; Kersters et al., 1996; Brandi et al., 1999; Janakiraman and Leff, 1999.. The higher survival rate of A. caÕiaer caÕiae ŽHG4. compared with the survival of A. hydrophila ŽHG1. reported here might explain the relatively higher isolation rate of this species in
drinking water ŽHanninen and Siitonen, 1995. and its high survival in treated sewage samples ŽImziln et al., 1998.. Surprisingly, when autochthonous bacteria were removed from the tap water by filtration, the declines in cell numbers were more rapid than those observed in unfiltered–non-autoclaved microcosms. Although, AultramicrocellsB were detected in the microcosm, they grew actively only after 3 days and thus competition for nutrients was an unlikely cause for the limitation of aeromonads. Kersters et al. Ž1996. have also reported a rapid decrease in cultivability when A. hydrophila HG3 is introduced into filtered tap water. It seems that a biological andror a thermolabile antimicrobial substance synthesised by AultramicrocellsB or by the autochthonous bacteria retained by the filter might affect the survival of aeromonads. The autochthonous microflora Ž Pseudomonas and related genera. of mineral water produce siderophores and bacteriocins showing activity against A. hydrophila and other bacteria of significance to health ŽVachee ´ et al., 1997.. Bacteriophages were not detected in the present study, however Atail phage-like particlesB bacteriocins and membrane vesicles were observed by electron microscopy. Phage-like particles, described in numerous host bacteria, are able to kill sensitive cells without multiplying in them ŽReanney and Ackermann, 1982.. Many Gram-negative bacteria that produce membrane vesicles ŽMVs. during normal growth are capable of killing other bacteria in the surrounding medium ŽKadurugamuwa and Beveridge, 1996; Li et al., 1998..These MVs can be isolated by filtration to separate them from the producing cells and are more potent on host bacteria that are not actively growing ŽLi et al., 1998.. Thus, MVs could be a good candidate to explain the rapid disappearance of non-growing aeromonads in the filtered microcosms. In unfiltered–non-autoclaved microcosms, MVs could be naturally released at a much lower rate while the potential targets were multiple Žautochthonous non-producer and introduced Aeromonas. and thus the MVs were less active in this microcosm than in filtered microcosm. The present study showed that environmental non-pathogenic Aeromonas species did not present higher specific growth rates, stationary phase densities and survival abilities than clinical strains. Thus,
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waterborne infections and contaminations of foods by pathogenic Aeromonas could not be discounted.
Acknowledgements The authors acknowledge the helpful comments of the reviewers and wish to thank Loıc ¨ Brunet for technical assistance with the TEM.
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Growth and survival of clinical vs. environmental species of Aeromonas in tap water Patrice Mary a,b,) , Guillaume Buchet b, Claude Defives a , Jean-Pierre Hornez a a
Laboratoire de Microbiologie, UniÕersite´ des Sciences et Technologies de Lille. Batiment SN2. F-59655 VilleneuÕe d’Ascq cedex, France ˆ b Institut UniÕersitaire de Technologie A AB . Departement Genie ´ ´ Biologique. F-59653 VilleneuÕe d’Ascq cedex, France Received 14 July 2000; received in revised form 28 December 2000; accepted 9 February 2001
Abstract The ability of four species of Aeromonas Žtwo of clinical and two of environmental origin. to survive andror grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 = 10 5 cfu mly1 after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, AultramicrocellsB were detected and final populations of ca. 10 6 cfu mly1 after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 10 4 cfu mly1. All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10 5 –10 6 cfu mly1 was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and AultramicrocellsB bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit Ži.e. 1 log cfu mly1 . in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios Ž100r1 in unfiltered-non-autoclaved and 1000r1 in filtered microcosms. were favourable for aeromonads, at least for 1 to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of Atail phage-like particlesB bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Tap water; Autochthonous bacteria; Aeromonas spp.; Clinical species; Environmental species; Growth; Survival
) Corresponding author. Institut Universitaire de Technologie AAB, Departement Genie ´ ´ Biologique, Bd Paul Langevin, Cite´ Scientifique BP 179, F-59653 Villeneuve d’Ascq CEDEX, France. Fax: q33-3-20-43-44-72. E-mail address: [email protected] ŽP. Mary..
0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 4 9 1 - 3
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1. Introduction The genus Aeromonas is comprised of Gramnegative, glucose-fermenting, oxidase-positive, O129-resistant and DNase-positive rod-shaped bacteria which are generally motile by a single polar flagellum. The genus Aeromonas is quite complex with 14 DNA hybridization groups ŽHG. and three additional suspected ones. These HG are further separated in several genotypes and phenotypes ŽJoseph and Carnahan, 2000.. Only five genotypes are currently recognized as human pathogens associated with gastrointestinal ŽKirov, 1993. and numerous extra intestinal infections ŽJanda and Abbott, 1998.. Members of the Aeromonas genus are found primarily in aquatic environments. They have been observed frequently in surface water and groundwater ŽKersters et al., 1995., estuarine and sea water ŽKaper et al., 1981., chlorinated and non-chlorinated drinking water ŽGavriel et al., 1998. and, in some countries, in bottled mineral water ŽGonzalez et al., 1987; Tsai and Yu, 1997.. All phenospecies are found in sewage-contaminated water ŽBoussaid et al., 1991. and activated sludge ŽKampfer et al., ¨ 1996.. They are also common contaminants of a wide range of foods ŽKirov, 1993.. Because Aeromonas spp. are distributed widely in aquatic environments, there is considerable speculation about their role as contaminants. However, evidence for waterborne human Aeromonas infections and contamination of foods is still controversial ŽJoseph and Carnahan, 2000.. Indeed, it has been reported that pathogenic Aeromonas Žmainly HG1, HG4 and HG8Y. are rarely isolated in drinking and fresh water ŽHavelaar et al., 1992; Hanninen and Siitonen, 1995; Hanninen et al., 1997. and on the contrary that potentially pathogenic Aeromonas are frequently recovered from aquatic environments ŽKuhn ¨ et al., 1997; Imziln et al., 1998; Borell et al., 1998; Goni-Urriza et al., 1999.. Survival of ˜ Aeromonas in water microcosms is also the subject of much debate since high ŽWarburton et al., 1994; Tsai and Yu, 1997; Brandi et al., 1999. and low survival rates ŽLowcock and Edwards, 1994; Kersters et al., 1996; Janakiraman and Leff, 1999. have been reported.
Thus, in this study, the survival and ror growth capacities of two clinical and two environmental well characterized strains were investigated in tap water microcosms.
2. Materials and methods 2.1. Bacteria Aeromonas hydrophila ATCC 7966 ŽHG1. and A. caÕiaercaÕiae LMG 13455 ŽHG4. were selected as representative of clinical isolates. A. mediarmedia LMG 13464 ŽHG5B. formerly known as A. caÕiaermedia and A. popoffii LMG 17541 ŽHG 17?. ŽJoseph and Carnahan, 2000. were considered to be representative of environmental strains. 2.2. Water microcosms Samples of tap water were collected in 500-ml acid-washed sterile screw-capped glass bottles. In order to inactivate chlorine ŽAmerican Public Health Association, 1992. sterile sodium thiosulphate ŽS1830, Fisher Scientific 78996 Elancourt Cedex, France. solution was added Žfinal concentration: 13.2 mg ly1 .. The water samples were used without further treatment Žunfiltered–non-autoclaved., were filtered through a 0.2-mm cellulose nitrate filter ŽSartoriusr A70.800.684, Fisher Scientific. to remove autochthonous microflora Žfiltered. or were filtered and autoclaved at 1218C for 15 min Žfiltered–autoclaved. to destroy biological and heat sensitive antimicrobial agents that might pass through the 0.2-mm filters. 2.3. Inoculation of microcosms Early stationary phase cultures of Aeromonas in Tryptic Soy Broth ŽDifcorA50.370.179, Fisher Scientific. were harvested and washed twice in 0.85% Žwrv. NaCl ŽA48.909.518, Fisher Scientific. solution by centrifugation Ž14,000 = g, 5 min, 48C.. The different water microcosms Ž250 ml. were inoculated with 1 ml of washed Aeromonas suspension to give initial cell concentrations of ca. 5 =
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10 3 –10 4 cfu mly1 . The flasks were incubated at ambient temperature Žabout 208C., in the dark and without shaking. 2.4. Growth and surÕiÕal in inoculation studies Viable counts were determined immediately after inoculation, at regular intervals Ž1 day. over a 10-day period and after 14 days by the spread plate procedure. Samples were serially diluted in a 0.85% Žwrv. NaCl solution. Undiluted sample and y1 to y4 dilutions were plated on Tryptic Soy Agar ŽDifcor A50.369.171, Fisher Scientific. and R2A Agar ŽDifcorA51.826.179, Fisher Scientific.. Aeromonas populations were determined on TSA after 2 and 7 days incubation at 308C. The autochthonous microflora of tap water was enumerated on R2A Agar after 3 and 7 days incubation at 258C. All experiments were independently repeated three times and mean log cfu mly1 " SD were calculated. Bacteriological counts were equated with the detection limit Ži.e. 1 log cfu ml y1 . when no colony appeared on media. 2.5. Transmission electron microscopy
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toclaved microcosms ŽFig. 1.. After a short lag phase Ž1 day., this microflora grew actively to reach 6 = 10 5 cfu mly1 . Five to seven bacterial phenotypes were observed. The filtration through the 0.2-mm filter retained the great majority of autochthonous bacteria Žinitial cell concentration - 1 log cfu mly1 ., however, after a 3 days lag phase, the growth of AultramicrocellsB was observed in filtered microcosms and this population reached 10 6 cfu mly1 after 7 days ŽFig. 1.. Only two phenotypes were detected. Indigenous Aeromonas species were not detected in this tap water. The further evolution of these two types of microflora were not significantly affected by the presence of the inoculated Aeromonas Ždata not shown.. 3.2. Growth of the Aeromonas strains in filtered and autoclaÕed tap water All four strains of Aeromonas grew in filtered-autoclaved tap water ŽFig. 2.. However, growth varied. A ranking in growth rates was observed in the following order: A. caÕiaer caÕiae HG4 ) A. media r media HG5B) A. popoffii HG17?) A. hydrophila HG1. No clear relationship between ability to grow in nutrient poor water and the origin of the
Filtered microcosms were centrifuged at 150 000 = g for 3 h at 48C. Pellets were resuspended in 1 ml of a 0.85% Žwrv. NaCl solution. Samples of 50 ml were placed on collodion-coated copper grids which were then negatively stained with 1% sodium phosphotungstate ŽpH 7.4.. Preparations were examined with a JEM 100CX transmission electron microscope ŽJapan Electronic Optical Laboratory, Parc Technologique Claude Monet 78290 Croissy sur Seine, France. operating under standard conditions at 80 kV.
3. Results 3.1. Growth of autochthonous microflora in unfiltered–non-autoclaÕed and filtered tap water Just after bottling and inactivation of chlorine, the initial plate counts of the autochthonous bacteria were 2.39 " 0.19 log cfu mly1 in unfiltered–non-au-
Fig. 1. Growth of autochthonous bacteria Žv . in unfiltered–nonautoclaved tap water and indigenous AultramicrocellsB ŽB. in filtered tap water. Vertical bars represent SEM Ž P s 0.05..
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Fig. 2. Growth curves of Aeromonas spp. in filtered–autoclaved tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv .. Vertical bars represent SD Ž ns 3..
Fig. 3. Survival curves of Aeromonas spp. in unfiltered–non autoclaved tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv ..Vertical bars represent SD Ž ns 3..
strains i.e. environmental or clinical was thus observed. It was noteworthy that the lowest rate of growth was observed for A. hydrophila.
caÕiaer caÕiae remained at a constant level over a 9-day period and thereafter declined slowly. No clear relationship was observed between the origin of the bacteria and their ability to survive in the presence of autochthonous microflora.
3.3. SurÕiÕal of the Aeromonas strains in unfiltered– non-autoclaÕed tap water Since the colonial morphology of the Aeromonas spp and those of the autochthonous bacteria were quite distinct, it was possible to distinguish easily the two populations in inoculated unfiltered–non-autoclaved microcosms. On TSA plates, the Aeromonas grew more rapidly and the colonies appeared as Chinese hat and orange-coloured. Furthermore, the different autochthonous bacteria did not exhibit a DNase activity in contrast to the Aeromonas strains. Although the inoculation ratio Ž100 Aeromonasr1 autochtonous bacteria. was favourable, at least for day 1 the Aeromonas strains did not grow and a decrease in viable cell counts of the organism were observed in this microcosm ŽFig. 3.. Levels of A. popoffii fell below the detection limit Ž1 log cfu mly1 . after only 40 h incubation. Numbers of A. mediar media and A. hydrophila declined linearly and approached the detection limit in 14 days. A.
Fig. 4. Survival curves of Aeromonas spp. in filtered tap water: A. caÕiaer caÕiae Ž^., A. hydrophila Ž'., A. mediar media Ž`. and A. popoffii Žv .. Vertical bars represent SD Ž ns 3..
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3.4. SurÕiÕal of the Aeromonas strains in filtered tap water Despite being introduced at a very favourable ratio Ž10,000 Aeromonasr-10 autochthonous AultramicrocellsB ., which was maintained at least for 3 days, all Aeromonas strains disappeared rapidly in filtered microcosms ŽFig. 4.. The declines in viable cell counts were even more pronounced in this microcosm than in unfiltered–non-autoclaved microcosms. Thus, the filtration step seemed to have purified or concentrated a biological or an antimicrobial substance highly active against Aeromonas. On other hand, the removal of filter-retained autochthonous bacteria led to a reduction in the number of potential targets for these agents. After the disappearance of the Aeromonas in filtered microcosms, bacteriophages were not detected in culture filtrates by the
Fig. 5. Electron micrographs of membrane vesicle ŽA. and Atail phage-like particlesB bacteriocins ŽB. negatively stained with 1% sodium phosphotungstate ŽpH 7.4.. Bars represent 100 nm.
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spot test or by enrichment in liquid medium with the corresponding Aeromonas strain. However, structures evocative of membrane vesicles and Atail phage-like particlesB bacteriocins ŽFig. 5A and B. have been observed by electron microscopy of negatively stained samples.
4. Discussion The level of autochthonous bacteria is low in tap water Žca. 250 cfu mly1 . according to Brandi et al. Ž1999. and van der Kooij Ž1992.. However, regrowth of bacteria just after bottling was observed. A final concentration of 6 = 10 5 cfu mly1 was obtained and this value seems to be the upper limit obtainable in different nutrient-poor waters for the indigenous microflora ŽAmy and Hiatt, 1989; Deere et al., 1996; Miettinen et al., 1997; Tsai and Yu, 1997; Leclerc and Da Costa, 1998.. The availability of assimilable organic carbon Žvan der Kooij, 1992. and also phosphorus ŽMiettinen et al., 1997. is considered the key factor in the regulation of microbial regrowth in drinking water. The presence of AultramicrocellsB in tap water, capable of passing through a 0.2 mm cellulose nitrate filter, has been demonstrated. AUltramicrocellsB are frequently observed in different types of water ŽRoszak and Colwell, 1987; Morita, 1990; Schut et al., 1997; Jones et al., 1999.. The cell numbers of AultramicrocellsB increased to ca. 10 6 cfu mly1 after a 7-day incubation period according to Jones et al. Ž1999.. In these two types of microcosm, inoculated Aeromonas were thus in the presence of actively growing indigenous bacteria. All the Aeromonas strains tested were able to grow in filtered and autoclaved tap water and reached a maximum of 10 5 –10 6 cfu mly1 within a 1.5–15day incubation period according to the species tested. Maximal cell densities observed were of the same order of magnitude as those previously reported for A. hydrophila HG3 in nutrient poor waters ŽRippey et al., 1994; Kersters et al., 1996; Tsai and Yu, 1997.. Among the four species tested, A. hydrophila HG1 was the less able to grow in this microcosm. Delayed growth of A. hydrophila in tap water has been already described Žvan der Kooij and Hijnen,
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1988.. The maximum cell densities attained by A. hydrophila in oligotrophic freshwaters can be increased by as much as 100-fold with a 70% increase in growth rate by the addition of both carbon and phosphorus ŽRippey et al., 1994. or by the addition of mixtures of carboxylic acids, carbohydrates or amino acids each at concentration of 1 mg of Crl Žvan der Kooij and Hijnen, 1988.. Thus, the maximum cell densities observed in our study might be a consequence of carbon and phosphorus limitations and the A. hydrophila was more susceptible to these limitations than the three other species tested. The Aeromonas species tested were unable to initiate growth in the presence of autochthonous microflora. This finding is in agreement with the results of Kersters et al. Ž1996. for tap water microcosms. The minor contribution of the aeromonads to the bacterial populations of tap water, in which low concentrations Ž- 10 mg of Crl. of substrates is available, may be explained by the presence of many autochthonous bacteria having transport systems with higher affinity than the aeromonads Žvan der Kooij et al., 1980; van der Kooij, 1991.. Drinking water contains heterotrophic bacteria that have the ability to utilize compounds such as carbohydrates, carboxylic acids, peptides and amino acids at concentrations of a few micrograms per litre Žvan der Kooij and Hijnen, 1981, 1984; van der Kooij et al., 1982.. The strain of A. popoffii tested disappeared rapidly in unfiltered–non-autoclaved microcosm although this species has been isolated from drinking water production plants and reservoirs ŽHuys et al., 1997.. In contrast, the A. caÕiaer caÕiae Žof clinical origin. maintained a constant level for at least 9 days. The A. hydrophila Žclinical strain. and the A. mediar media Ženvironmental strain. underwent a similar reduction in viable counts. Thus, no correlation was found between strain origin and the survival capacity. In our study, the declines in cell counts for A. hydrophila HG1 and A. media r media HG5B Žformerly A. caÕiaer media. were similar or slightly lower than that previously reported for Aeromonas HG1, HG3 and HG5B ŽLowcock and Edwards, 1994; Kersters et al., 1996; Brandi et al., 1999; Janakiraman and Leff, 1999.. The higher survival rate of A. caÕiaer caÕiae ŽHG4. compared with the survival of A. hydrophila ŽHG1. reported here might explain the relatively higher isolation rate of this species in
drinking water ŽHanninen and Siitonen, 1995. and its high survival in treated sewage samples ŽImziln et al., 1998.. Surprisingly, when autochthonous bacteria were removed from the tap water by filtration, the declines in cell numbers were more rapid than those observed in unfiltered–non-autoclaved microcosms. Although, AultramicrocellsB were detected in the microcosm, they grew actively only after 3 days and thus competition for nutrients was an unlikely cause for the limitation of aeromonads. Kersters et al. Ž1996. have also reported a rapid decrease in cultivability when A. hydrophila HG3 is introduced into filtered tap water. It seems that a biological andror a thermolabile antimicrobial substance synthesised by AultramicrocellsB or by the autochthonous bacteria retained by the filter might affect the survival of aeromonads. The autochthonous microflora Ž Pseudomonas and related genera. of mineral water produce siderophores and bacteriocins showing activity against A. hydrophila and other bacteria of significance to health ŽVachee ´ et al., 1997.. Bacteriophages were not detected in the present study, however Atail phage-like particlesB bacteriocins and membrane vesicles were observed by electron microscopy. Phage-like particles, described in numerous host bacteria, are able to kill sensitive cells without multiplying in them ŽReanney and Ackermann, 1982.. Many Gram-negative bacteria that produce membrane vesicles ŽMVs. during normal growth are capable of killing other bacteria in the surrounding medium ŽKadurugamuwa and Beveridge, 1996; Li et al., 1998..These MVs can be isolated by filtration to separate them from the producing cells and are more potent on host bacteria that are not actively growing ŽLi et al., 1998.. Thus, MVs could be a good candidate to explain the rapid disappearance of non-growing aeromonads in the filtered microcosms. In unfiltered–non-autoclaved microcosms, MVs could be naturally released at a much lower rate while the potential targets were multiple Žautochthonous non-producer and introduced Aeromonas. and thus the MVs were less active in this microcosm than in filtered microcosm. The present study showed that environmental non-pathogenic Aeromonas species did not present higher specific growth rates, stationary phase densities and survival abilities than clinical strains. Thus,
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waterborne infections and contaminations of foods by pathogenic Aeromonas could not be discounted.
Acknowledgements The authors acknowledge the helpful comments of the reviewers and wish to thank Loıc ¨ Brunet for technical assistance with the TEM.
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