APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1990, p. 1999-2006
0099-2240/90/071999-08$02.P0/0 Copyright 0 1990, American Society for Microbiology
Vol. 56, No. 7
Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a Sewage Treatment Pond P. MONFORT* AND B. BALEUX Laboratoire d'Hydrobiologie Marine et Continentale, Unite de Recherche Associee, Centre National de la Recherche Scientifique 1355, Universite des Sciences et Techniques du Languedoc, F-34060 Montpellier Cedex, France Received 22 November 1989/Accepted 5 March 1990
The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased'at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at al stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic. Motile Aeromonas species (A. hydrophila, A. caviae, and A. sobria) (32) are ubiquitous bacteria in continental aquatic environments (17). These bacteria'can be found in both polluted and unpolluted fresh water, in sewage, and in drinking water (8, 33, 36). The abundance of Aeromonas species in fresh water has been found to be seasonally distributed, with a maximum occurring during summer through to early fall (8, 16, 33). Aeromonas spp. can cause infections and epizootics in a variety of animals. It is the etiological agent for red-sore disease in fish and for red-leg disease in amphibians (18, 19). At one time, Aeromonas spp. were thought to cause disease only in immunocompromised humans. Thus, they are usually considered to be secondary or opportunistic pathogens (30, 41). In recent years, a number of studies have demonstrated that Aeromonas spp. can infect wounds exposed during aquatic activity (4, 12, 22, 38). Aeromonas spp. have also been found to be important enteropathogens in-Australia (8), Thailand (31), and Europe (13, 27). A number of potential virulence factors related to the pathogenicity of Aeromonas spp. infections have been described. Aeromonas spp. have been found to produce a variety of extracellular products, including enterotoxins, hemolysins, and cytotoxic proteins (42). The enteropathogenic potential of Aeromonas spp. is mediated by the cytotoxic enterotoxin (10), and enterotoxin production is significantly correlated with hemolysin production (7, 10). The works of Asao et al. (1) support the notion that Aeromonas hemolysin is a cytotoxic enterotoxin. The production of cytotoxin and hemolysin is significantly related to A. hydrophila and A. sobria (3, 21, 40). Thus, both A. hydrophila and A. sobria are major aeromonad enteric pathogens (20, 27). The results of different studies have revealed a significant correlation between hemolysin production, enterotoxin production, and diarrheal disease (6, 7, 10). Hemolysin *
production is associated with enterotoxigenicity (1, 20). Daily et al. (11) suggest that A. sobria is the predominant human pathogen of the genus. In contrast, the decreasing frequency of reports of A. caviae-associated infections supports the idea of a less pathogenic role for this species (7, 21). Also, it is generally agreed that A. caviae is not an enteric pathogen and has a less pathogenic role than the other two species (21, 24, 27). Because Aeromonas spp. are increasing in importance as opportunistic pathogens, indeed as primary pathogens, and because their presence in aquatic environments could pose a problem for public health (37), the aim of the present investigation is to report on the spatiotemporal dynamics of A. hydrophila, A. caviae, and A. sobria in a sewage treatment pond. Isolation and identification of Aeromonas species was undertaken at the inflow and the outflow of this sewage treatment pond to know about the behavior of these species through this treatment sy$tem. These strains were tested for hemolytic activity (potential virulence factor), because this test is suitable for use without facilities for enterotoxin testing such as suckling mouse or ileal loop assays. MATERIALS AND METHODS Study site. The sewage treatment pond of the city of Meze (7,500 inhabitants) is located on the Northern shore of the Thau brackish water lagoon, which is open to the Mediterranean sea in the Languedoc-Roussillon area of southern France. The overflowing pond system comprises three successive ponds with a total surface area of 8 ha (first pond 4 ha and second and third ponds 2 ha each). These waste ponds are algal-bacterial systems in which algal photosynthesis and bacterial oxidation actions combine to treat the wastewaters (29). The average depth varies from 1.40 m (first pond) to 1.10 m (third pond). The flow of incoming waste varies from 1,200 m3 per day in winter to 2,000 m3 per day in summer, and the total detention time is about 70 days in winter and 40
Corresponding author. 1999
MONFORT AND BALEUX
2000
APPL. ENVIRON. MICROBIOL.
FIG. 1. Aerial photograph of the MWze sewage treatment ponds showing sampling stations 1 (raw sewage) to 5 (final effluent). summer (2, 25). The treated effluent flows into a channel 360 m long that runs into a brackish creek in the Thau Lagoon. Sampling strategy. To determine the distributions of Aeromonas spp. and fecal coliforms (indicators of fecal contamination) in the waste ponds between the initial raw sewage and the final effluent, we used the sampling program of Legendre et al. (25). The sampling stations, numbered 1 to 5, are shown in Fig. 1. These stations were placed according to the direction of the flow between the inflow and the outflow. The samples were taken at intervals of approximately 28 days between November 1984 and June 1986. This was done to determine whether the seasonal distributions of the bacterial concentrations observed previously in the water of the ponds persist (2, 25). To know whether seasonal distributions also exist among the species, isolation and identification of Aeromonas strains were carried out over four seasons (July 1985, October 1985, January 1986, and April 1986) at station 2 (inflow of the sewage treatment pond) and station 5 (outflow of the effluent). Water samples were collected by using sterile bottles (500 ml), preserved at 4°C, and analyzed within 4 h of collection. Media and culture procedure. Bacteria were isolated and counted either by the spread-plate procedure after dilution in sterile water with 9%00 NaCl or by the membrane (HAWG 047, 0.45-,um pore size; Millipore Corp.) filtration procedure.
days in narrow
A. hydrophila, A. caviae, and A. sobria were counted after incubation for 48 h at 37°C (28) in Pril ampicillin xylose agar (PXA agar) (34). Counts for fecal coliforms were achieved by using tergitol and T.T.C. agar (Institut Pasteur Production) after incubation for 24 h at 44.5°C. To estimate the proportion of the three Aeromonas spp. in a water sample, about 35 characteristic colonies were picked from the PXA medium. This colony sample was taken from around 50 colonies in a single petri dish. Each colony was subcultured onto nutrient agar (bioMdrieux) for 24 h at 37°C before the identification. A total of 247 isolates with the following reactions were confirmed to be in the genus Aeromonas: motility (+), Gram stain (-), cytochrome oxidase (+), D-glucose fermentation (+) (37°C, 24 h), arginine dihydrolase (+) (30°C, 24 to 48 h), ornithine decarboxylase (-) (30°C, 24 to 48 h), o-nitrophenyl-,-D-galactopyranoside test (+) (37°C, 2 h), and sensitivity to 0/129 (-) (30°C, 24 h). The species was determined by using the following screening tests (30°C, 24 to 48 h) (32): esculin hydrolysis, L-arabinose utilization, fermentation of salicin, acetoin from glucose (Voges-Proskauer), gas from glucose, and H2S from cysteine (Table 1). Hemolysin assays. Hemolysin activity was determined by the demonstration of zones of ,B-hemolysis around colonies on blood agar plates (bioMdrieux) containing 5% (vol/vol) horse blood after 18 to 24 h of incubation at 37°C (21).
VOL.
AEROMONAS SPP. IN A SEWAGE TREATMENT POND
56, 1990 TABLE 1. Differential characteristics of the motile Aeromonas speciesa Characteristic
A. hydrophila
A. caviae
A. sobria
Esculin hydrolysis L-Arabinose utilization Fermentation of salicin Acetoin from glucose (Voges-Proskauer) Gas from glucose H2S from cysteine
+ + + +
+ + + -
D
+ +
-
+ +
a The system of Popoff (32) was used: +, typically positive; negative; D, differs among strains.
-,
typically
Statistical analyses. All bacterial counts were first log transformed (base 10). This transformation eliminates a good deal of the asymmetry in the frequency distributions of the variables (25). The time series of bacterial data for each sampling station were studied by chronological clustering (26). This method has been described and used to analyze aerobic heterotrophic bacteria and pollution-indicator bacteria in the waste ponds (25). The chronological clustering method allows to search for homogeneous steps along a succession of events for a considered significance level. This statistical analysis was performed by using the programs of the R package (P. Legendre, Departement de Sciences Biologiques, Universite de Montrdal, Canada) on an IBM 3081 computer (Centre National Universitaire Sud de Calcul, Montpellier, France). RESULTS Temporal distribution of Aeromonas spp. and fecal coliforms in the waste pond water. Figure 2 shows the abundance distributions of Aeromonas spp. and fecal coliforms at each sampling station in the waste ponds. Chronological clustering was run for several significance levels. A significance level of 10% was used for chronological clustering of the Aeromonas spp. time series, and a significance level of 5% was used for the fecal coliform time series, because these levels delineated as many groups as were clearly discernible along the data series themselves. In Fig. 2, each step computed by the chronological clustering method is demonstrated by the logarithm of the mean value of the points contained in it (height of each horizontal line segment). Table 2 shows the logarithm of the mean values of Aeromonas spp. and the fecal coliform concentrations at each sampling station, for every time period illustrated by the chronological clustering method, as well as the mean temperatures of pond water. The distributions of the Aeromonas spp. time series showed the same behavior at every station from sewage water to treated effluent; that is, higher abundances in summer periods (mean temperatures of pond water, 20.5 and 21°C) than in winter periods (mean temperatures of pond water, 9.5 and 9°C). The significant clusters (significance level of 10%) provided by the chronological clustering method illustrated this phenomenon and also showed greater regular clustering in the final effluent than in the raw sewage. However, the amplitude between summer and winter concentration curves increased from the sewage water (station 1) (6.7 and 5.78 log1o Aeromonas spp. cells 100 ml-' in winter, 7.69 and 7.45 log1o cells 100 ml-1 in summer) through to the final effluent (station 5) (3.85 and 3.44 log1o Aeromonas spp. cells 100 ml-' in winter, 6.07 and 5.29 log1o cells ml-' in summer).
2001
The distributions of the fecal coliform time series in the sewage water and in the first pond were fairly stable throughout all seasons (6.76 to 7.31 log1o fecal coliforms 100 ml-' at station 1; 6.27 to 6.74 log1o fecal coliforms 100 ml-' at station 2; 5.77 to 6.46 log1o fecal coliforms 100 ml-' at station 3). This was shown by three significant clusters (significance level, 5%) whose differences were slight (
0099-2240/90/071999-08$02.P0/0 Copyright 0 1990, American Society for Microbiology
Vol. 56, No. 7
Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a Sewage Treatment Pond P. MONFORT* AND B. BALEUX Laboratoire d'Hydrobiologie Marine et Continentale, Unite de Recherche Associee, Centre National de la Recherche Scientifique 1355, Universite des Sciences et Techniques du Languedoc, F-34060 Montpellier Cedex, France Received 22 November 1989/Accepted 5 March 1990
The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased'at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at al stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic. Motile Aeromonas species (A. hydrophila, A. caviae, and A. sobria) (32) are ubiquitous bacteria in continental aquatic environments (17). These bacteria'can be found in both polluted and unpolluted fresh water, in sewage, and in drinking water (8, 33, 36). The abundance of Aeromonas species in fresh water has been found to be seasonally distributed, with a maximum occurring during summer through to early fall (8, 16, 33). Aeromonas spp. can cause infections and epizootics in a variety of animals. It is the etiological agent for red-sore disease in fish and for red-leg disease in amphibians (18, 19). At one time, Aeromonas spp. were thought to cause disease only in immunocompromised humans. Thus, they are usually considered to be secondary or opportunistic pathogens (30, 41). In recent years, a number of studies have demonstrated that Aeromonas spp. can infect wounds exposed during aquatic activity (4, 12, 22, 38). Aeromonas spp. have also been found to be important enteropathogens in-Australia (8), Thailand (31), and Europe (13, 27). A number of potential virulence factors related to the pathogenicity of Aeromonas spp. infections have been described. Aeromonas spp. have been found to produce a variety of extracellular products, including enterotoxins, hemolysins, and cytotoxic proteins (42). The enteropathogenic potential of Aeromonas spp. is mediated by the cytotoxic enterotoxin (10), and enterotoxin production is significantly correlated with hemolysin production (7, 10). The works of Asao et al. (1) support the notion that Aeromonas hemolysin is a cytotoxic enterotoxin. The production of cytotoxin and hemolysin is significantly related to A. hydrophila and A. sobria (3, 21, 40). Thus, both A. hydrophila and A. sobria are major aeromonad enteric pathogens (20, 27). The results of different studies have revealed a significant correlation between hemolysin production, enterotoxin production, and diarrheal disease (6, 7, 10). Hemolysin *
production is associated with enterotoxigenicity (1, 20). Daily et al. (11) suggest that A. sobria is the predominant human pathogen of the genus. In contrast, the decreasing frequency of reports of A. caviae-associated infections supports the idea of a less pathogenic role for this species (7, 21). Also, it is generally agreed that A. caviae is not an enteric pathogen and has a less pathogenic role than the other two species (21, 24, 27). Because Aeromonas spp. are increasing in importance as opportunistic pathogens, indeed as primary pathogens, and because their presence in aquatic environments could pose a problem for public health (37), the aim of the present investigation is to report on the spatiotemporal dynamics of A. hydrophila, A. caviae, and A. sobria in a sewage treatment pond. Isolation and identification of Aeromonas species was undertaken at the inflow and the outflow of this sewage treatment pond to know about the behavior of these species through this treatment sy$tem. These strains were tested for hemolytic activity (potential virulence factor), because this test is suitable for use without facilities for enterotoxin testing such as suckling mouse or ileal loop assays. MATERIALS AND METHODS Study site. The sewage treatment pond of the city of Meze (7,500 inhabitants) is located on the Northern shore of the Thau brackish water lagoon, which is open to the Mediterranean sea in the Languedoc-Roussillon area of southern France. The overflowing pond system comprises three successive ponds with a total surface area of 8 ha (first pond 4 ha and second and third ponds 2 ha each). These waste ponds are algal-bacterial systems in which algal photosynthesis and bacterial oxidation actions combine to treat the wastewaters (29). The average depth varies from 1.40 m (first pond) to 1.10 m (third pond). The flow of incoming waste varies from 1,200 m3 per day in winter to 2,000 m3 per day in summer, and the total detention time is about 70 days in winter and 40
Corresponding author. 1999
MONFORT AND BALEUX
2000
APPL. ENVIRON. MICROBIOL.
FIG. 1. Aerial photograph of the MWze sewage treatment ponds showing sampling stations 1 (raw sewage) to 5 (final effluent). summer (2, 25). The treated effluent flows into a channel 360 m long that runs into a brackish creek in the Thau Lagoon. Sampling strategy. To determine the distributions of Aeromonas spp. and fecal coliforms (indicators of fecal contamination) in the waste ponds between the initial raw sewage and the final effluent, we used the sampling program of Legendre et al. (25). The sampling stations, numbered 1 to 5, are shown in Fig. 1. These stations were placed according to the direction of the flow between the inflow and the outflow. The samples were taken at intervals of approximately 28 days between November 1984 and June 1986. This was done to determine whether the seasonal distributions of the bacterial concentrations observed previously in the water of the ponds persist (2, 25). To know whether seasonal distributions also exist among the species, isolation and identification of Aeromonas strains were carried out over four seasons (July 1985, October 1985, January 1986, and April 1986) at station 2 (inflow of the sewage treatment pond) and station 5 (outflow of the effluent). Water samples were collected by using sterile bottles (500 ml), preserved at 4°C, and analyzed within 4 h of collection. Media and culture procedure. Bacteria were isolated and counted either by the spread-plate procedure after dilution in sterile water with 9%00 NaCl or by the membrane (HAWG 047, 0.45-,um pore size; Millipore Corp.) filtration procedure.
days in narrow
A. hydrophila, A. caviae, and A. sobria were counted after incubation for 48 h at 37°C (28) in Pril ampicillin xylose agar (PXA agar) (34). Counts for fecal coliforms were achieved by using tergitol and T.T.C. agar (Institut Pasteur Production) after incubation for 24 h at 44.5°C. To estimate the proportion of the three Aeromonas spp. in a water sample, about 35 characteristic colonies were picked from the PXA medium. This colony sample was taken from around 50 colonies in a single petri dish. Each colony was subcultured onto nutrient agar (bioMdrieux) for 24 h at 37°C before the identification. A total of 247 isolates with the following reactions were confirmed to be in the genus Aeromonas: motility (+), Gram stain (-), cytochrome oxidase (+), D-glucose fermentation (+) (37°C, 24 h), arginine dihydrolase (+) (30°C, 24 to 48 h), ornithine decarboxylase (-) (30°C, 24 to 48 h), o-nitrophenyl-,-D-galactopyranoside test (+) (37°C, 2 h), and sensitivity to 0/129 (-) (30°C, 24 h). The species was determined by using the following screening tests (30°C, 24 to 48 h) (32): esculin hydrolysis, L-arabinose utilization, fermentation of salicin, acetoin from glucose (Voges-Proskauer), gas from glucose, and H2S from cysteine (Table 1). Hemolysin assays. Hemolysin activity was determined by the demonstration of zones of ,B-hemolysis around colonies on blood agar plates (bioMdrieux) containing 5% (vol/vol) horse blood after 18 to 24 h of incubation at 37°C (21).
VOL.
AEROMONAS SPP. IN A SEWAGE TREATMENT POND
56, 1990 TABLE 1. Differential characteristics of the motile Aeromonas speciesa Characteristic
A. hydrophila
A. caviae
A. sobria
Esculin hydrolysis L-Arabinose utilization Fermentation of salicin Acetoin from glucose (Voges-Proskauer) Gas from glucose H2S from cysteine
+ + + +
+ + + -
D
+ +
-
+ +
a The system of Popoff (32) was used: +, typically positive; negative; D, differs among strains.
-,
typically
Statistical analyses. All bacterial counts were first log transformed (base 10). This transformation eliminates a good deal of the asymmetry in the frequency distributions of the variables (25). The time series of bacterial data for each sampling station were studied by chronological clustering (26). This method has been described and used to analyze aerobic heterotrophic bacteria and pollution-indicator bacteria in the waste ponds (25). The chronological clustering method allows to search for homogeneous steps along a succession of events for a considered significance level. This statistical analysis was performed by using the programs of the R package (P. Legendre, Departement de Sciences Biologiques, Universite de Montrdal, Canada) on an IBM 3081 computer (Centre National Universitaire Sud de Calcul, Montpellier, France). RESULTS Temporal distribution of Aeromonas spp. and fecal coliforms in the waste pond water. Figure 2 shows the abundance distributions of Aeromonas spp. and fecal coliforms at each sampling station in the waste ponds. Chronological clustering was run for several significance levels. A significance level of 10% was used for chronological clustering of the Aeromonas spp. time series, and a significance level of 5% was used for the fecal coliform time series, because these levels delineated as many groups as were clearly discernible along the data series themselves. In Fig. 2, each step computed by the chronological clustering method is demonstrated by the logarithm of the mean value of the points contained in it (height of each horizontal line segment). Table 2 shows the logarithm of the mean values of Aeromonas spp. and the fecal coliform concentrations at each sampling station, for every time period illustrated by the chronological clustering method, as well as the mean temperatures of pond water. The distributions of the Aeromonas spp. time series showed the same behavior at every station from sewage water to treated effluent; that is, higher abundances in summer periods (mean temperatures of pond water, 20.5 and 21°C) than in winter periods (mean temperatures of pond water, 9.5 and 9°C). The significant clusters (significance level of 10%) provided by the chronological clustering method illustrated this phenomenon and also showed greater regular clustering in the final effluent than in the raw sewage. However, the amplitude between summer and winter concentration curves increased from the sewage water (station 1) (6.7 and 5.78 log1o Aeromonas spp. cells 100 ml-' in winter, 7.69 and 7.45 log1o cells 100 ml-1 in summer) through to the final effluent (station 5) (3.85 and 3.44 log1o Aeromonas spp. cells 100 ml-' in winter, 6.07 and 5.29 log1o cells ml-' in summer).
2001
The distributions of the fecal coliform time series in the sewage water and in the first pond were fairly stable throughout all seasons (6.76 to 7.31 log1o fecal coliforms 100 ml-' at station 1; 6.27 to 6.74 log1o fecal coliforms 100 ml-' at station 2; 5.77 to 6.46 log1o fecal coliforms 100 ml-' at station 3). This was shown by three significant clusters (significance level, 5%) whose differences were slight (
