World Journal of Microbiology & Biotechnology 12, 385-390
Effect of wastewater stabilization ponds on antimicrobial susceptibility and haemolysin occurrence among motile Aeromonas strains B. Imziln,* Y.M.O. Lafdal and M. Jana Aeromonas strains (total = 953) isolated from raw wastewater, stabilization pond effluent and sediments were evaluated for their susceptibilities to 17 antibiotics and for their ability to produce haemolysins. Stabilization ponds did not seem to select highly resistant strains of aeromonads. There were no differences in the resistance patterns of isolates from raw sewage, stabilization pond effluent and sediments. All strains were found to possess multiple resistance, most commonly to ampicillin, amoxicillin and novobiocin. Almost 90% of the strains of A. hydrophila and A. caviae were resistant to cephalothin, whereas more than 80% of A. sobria isolates were found to be susceptible to this antibiotic. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed 10%. Moreover, no strain was found to be resistant to gentamycin and only 9 of the 953 isolates exhibited resistance to cefotaxim. The percentage of haemolytic strains was significantly higher in the stabilization pond effluent than in raw sewage. This high incidence of haemolytic activity was connected with a high proportion of A. sobria whereas, in samples from the raw sewage or stabilization pond sediments a high proportion of A. caviae decreased the total amount of haemolytic aeromonads. The high incidence of haemolytic activity (a + d) was associated particularly with A. sobria (93.3%) and A. hydrophila (88.7%) whereas A. caoiae was found to be the lowest haemolytic species (16.3%). Key words: Aeromotzas, antibiotic resistance, haemolytic activity, stabilization ponds, wastewater.
Several wastewater treatment systems have been tested in many regions of the world and under various climates. The experience of Marrakech in this field has pointed out the suitability of the stabilization pond system for purifying wastewater under arid climates (Mandi et al., I993). Among their advantages, stabilization ponds eliminated 100% of helminth eggs (Schwartzbrod et al. 1989). Moreover, satisfactory results were obtained by this system in removing pathogenic bacteria such as Salmonella, Vibrio cholerae, Pseudomonas aeruginosa and Aeromonas (Boussaid et al. 1991; Lesne et al. 1991; Hassani et al. 1992). However, the B. Imziln and Y.M.O. Lafdal are with Cadi Ayyad University, Faculty of Sciences Semlalia, Department of Biology, Laboratory of Microbiology, BP S/15 Marrakech, Morocco; fax: (212) 443 6769/(212) 443 7412. M. Jana is with the H6pital Millitaire Avicenne Marrakech, Morocco. *Corresponding author.
removal ratio of these bacteria did not reach 100% and non-negligible quantities of them were still present in the treated effluent. Among these microorganisms, motile Aeromonas spp. are more often recovered from these kinds of waters. Furthermore, in many cases their concentrations exceeded 103 c.f.u./m] (Boussaid et al. 1991; Hassani et al. 1992; Stecchini & Domenis 1994). These species have long been known to cause different infections in poikilothermic animals (Shotts et al. 1972). In recent years the clinical importance of motile Aeromonas isolates has been recognized. They are associated with several categories of infections of humans (Burke & Gracey 1986; Janda et al. 1988; Altwegg et al. 1991; Ebrad & Gerry 1992; Ghanem et al. 1993) and of warm-blooded animals (Andr6-Fontaine et al. 1995). Numerous putative virulence factors have been ascribed to Aeromonas spp. to explain the process of patho-
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B. Irnziln, Y.M.O. Lafdal and M. ]ana genicity of these bacteria, including the production of endotoxins, extracellular enterotoxins, haemolysins, cytotoxins and proteases, the ability to adhere to cells and the possession of certain surface proteins (Cahill 1990; Janda 1991). Recent attention has been focused on the haemolysin of motile Aeromonas spp. because of its potential diagnostic, epiderniologic and pathogenic significance (Aoki & Hirono I991). Several investigators have demonstrated significant association between haemolysin production and elaboration of the enterotoxigenicity in Aeromonas spp. (Burke et al. 1984; Gray et aL 1990; Mateos et al. 1992). Many investigators have described multiple uses of antibiotics in agriculture as therapeutic or prophylactic agents to treat and prevent diseases, or as growth promoters (DePaola el al. 1988; Frost 1990). This widespread use of antibiotics has resulted in the evolution of resistant strains of bacteria. Moreover, there remains the possibility that resistance may be transmitted from antibiotic-resistant bacteria to the susceptible ones (Linton 1984). In the current study we investigated the effect of the sewage stabilization pond treatment on antibiotic resistance and haemolytic activity among Aeromonas species, in order to confirm the quality of treated effluent which is used directly for irrigation of various crops in Marrakech.
Materials and Methods Study Area and Sample Collection The experimental sewage treatment pond system is located in Marrakech, (Morocco, 31°36 north, 08°02 west, altitude 471m). It consists of two successive ponds each of 2500 m 2 area. The average depth of the first pond is 2.3 m and that of thesecond is 1.6 m. The flow of incoming wastewater is maintained at 500 m ~ per day which corresponds to about 12 days of hydraulic retention time. The treated effluent is used for irrigation. Water samples were taken from plant inlet and outlet. Sediment samples were taken from the first pond. The frequency of sampling was twice a month from January 1992 to June 1993. Samples were collected in sterilized glass bottles and were kept on ice until analyzed. Time between collection and analysis never exceeded 3 hours. Isolation and Identification of Aeromonas Strains Samples were shaken for a period of 30 min. and then diluted in sterile saline solution (0.85% (w/v) NaC1 in demineralized water). Aliquots of 0.1 ml from suitable dilutions were spread onto ADA agar (Havelaar et al. 1987). Inoculated plates were then incubated at 37°C for 24h. Typical Aeromonas colonies were purified on trypto-casein soy agar (TSA, Diagnostic Pasteur 64554) plates and confirmed as belonging to the motile aeromonad group by standard biochemical reactions on the basis of the following characteristics: Gram-negative rods; motile organisms; oxidase and catalase positive; fermentative glucose metabolism (O/F test with Hugh and Leifson medium, Merck 10282); arginine dihydrolase positive (ADH, M611er); omithine decarboxylase negative (ODC, M611er) [ODC positive strains were also accepted as Aeromonas on the basis of the recommendation of Hickman-Brenner et aL (1987)]; resistance to the vibriostatic agent, 2,4-diamino-6,7-di-
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isopropylpteridine phosphate (O/129, 150 /2g disc, Diagnostic Pasteur 53872); no growth on TSA with 6.5% NaCI. Isolates which did not satisfy these criteria were considered not to be aeromonas. Seven additional tests that have been suggested to be useful in the identification of the confirmed motile aeromonads isolates to species level (Popoff I984) were added to this range. These additional tests included fermentation of salicin; esculin hydrolysis; L-arabinose utilization; gas from glucose; production of acetoin from glucose (Voges-Proskauer); lysine decarboxylase (LDC, M611er) and production of H2S from L-cycteine. Strains which did not satisfy the biochemical profiles of the motile Aeromonas spp. described by Popoff (I984) were considered as other unknown Aeromonas species.
Antibiotic Susceptibility Testing Antibiotic susceptibility of Aeromonas isolates was determined by the methods of Calomiris et al. (1984). Purified isolates were inoculated by multipoint inoculation on Mueller-Hinton agar (Mueller-Hinton 2, Bio-M6rieux 51861, France) plates supplemented with the following antibiotics (,ug/mt): Ampicillin, 10; Amoxicillin, 25; Cephalothin, 30; Streptomycin, 10; Novobiocin, 30; Chloramphenicol, 30; Nalidixic acid, 30; Kanamycin, 30; Gentamycin, 10; Erythromycin, 15; Polymyxin B; 37.5 (300 units); Tetracycline, 30; Oxytetracycline, 30; Cefotaxim, 30; Trimethoprim, 5; Sulphamethoxazol, I00; Trirnethoprim-sulphamethoxazol, 1.25 + 23.75. These antibiotics were chosen because they are widely used for the treatment of Gram-negative bacterial infections. Inoculated plates were incubated overnight at 35°C. Haemolysin Assays Haemolytic activity was determined by a zone of haemolysis around colonies on Columbia agar plates (Columbia Blood agar, Difco 0792-01-1) containing human blood, O-group (6% v/v). Plates were inoculated by spotting up to eight strains per plate, and incubated for 24 h at 37°C. Beta-haemolysis was indicated by total lysis of the erythrocytes surrounding the colonies, alphahaemolysis was indicated by a partial [ysis of the erythrocytes and gamma-haemolysis means no haemolysis of erythrocytes around colonies. Data Analysis In order to compare the level of antibiotic resistance of Aeromonas species isolated from different samples, an antibiotic resistance index (ARI) was calculated according to Hinton & Linton (1983) using the following formula: ARI = x/ny, where x is the number of resistance determinants in a population y, and n is the number of antibiotics tested. The statistical significance of differences in antibiotic resistance and haemolytic activity of the strains isolated from different locations (raw sewage, stabilization pond sediments and stabilization pond effluent) or between Aeromonas species were evaluated with the non parametric Wilcoxon-signed rank test (Schwartz 1963), with a Star View TM SE + Graphics from Abacus Concepts, Inc. (VI.03, 9/15/88) program.
Results Of the 953 Aeromonas strains isolated from the experimental wastewater stabilization ponds (Table 1), 393 (41.2%) strains were identified as A. caviae, 53 (5.6%) as A. hydrophila and 223 (23.4%) as A. sobria. On the basis of criteria defined by Popoff (1984), biochemical profiles of the 284 (29.8%)
Wastewater aeromonads Table 1. Origin and distribution of the 953 tested Aeromonas isolates Sample source
Raw sewage Stabilization pond effluent Stabilization pond sediments Total
No. of examined isolates 360 397 196 953
remaining isolates did not agree with any of the three above-mentioned species. Domestic raw sewage of Marrakech city harboured the three Aeromonas species: A. caviae, A. hydrophila and A. sobria, with a clear domination of A. caviae. The same results were found in the sediments from the ponds, whereas at the treatment plant outlet water samples showed high frequencies of A. sobria in comparison with the concentrations of the two other species. Antibiotic Resistance All isolates were tested for their resistance to each of the seventeen antibiotics used. Frequencies of antibiotic-resistance among these isolates are shown in Table 2. All 953 isolates were resistant to ampicillin and amoxicillin. More than 98% of them were found to be resistant to novobiocin. Resistance to cephalothin occured in more than 70% of all Aeromonas isolates. Percentages of sulphametoxazol and streptomycin-resistant isolates were 41 and 23 respectively. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed I0%. None of the isolates was resistant to gentamycin and only 9 of 953 tested strains exhibited resistance to cefotaxim. Results of antibiotic resistance among motile aeromonads in relation to source of isolation of strains are shown in Table 3. According to the calculated antibiotic resistance index, levels of global resistance among aeromonads isolated from influent, effluent and sediments were similar and did not show any significant difference (P > 0.05). Levels of antibiotic resistance among the three motile Aeromonas species (Table 2) are compared and according to the antibiotic resistance index (ARI) calculated for each species, A. sobria (ARI = 0.22) seemed to be the lowest antibiotic resistant species. This is related to its higher susceptibility to cephalothin, streptomycin, sulphamethoxazol and trimethoprim than the two other species (A. caviae and A. hydrophila). Haemolytic Activity Of the 393 A. caviae isolates tested, 35 (8.9%), 29 (7.4%) and 329 (83.7%) were observed to be alpha, beta and gamma haemolytic, respectively. Among 53 isolates of A.
No. of Aeromonas species A. caviae
A. hydrophila
A. sobria
A. sp. (Others)
177 92 124 393
12 20 21 53
50 159 14 223
121 126 37 284
hydrophila 13 (24.5%) were alpha, 34 (04.2%) were beta and 6 (11.3%) were found to be gamma haemolytic. For 223 strains belonging to A. sobria species, I9 (8.5%) were alpha, 189 (84.8%) were beta and only 15 (6.7%) were found to be gamma haemolytic (Table 4). The production of haemolysin by Aeromonas isolates in relation to source of isolation is shown in Table 5. Among 360 isolates sampled from raw sewage I9 (5.3%) and 63 (17.5%) were found to be alpha and beta haemolytic respectively. Among 196 strains isolated from sediment samples I7 (8.7%) were alpha and 3I (15.8%) were beta haemolytic. In contrast of 397 isolates obtained from stabilization pond effluent 51 (12.8%) and 222 (55.9%) were found to be alpha and beta haemolytic respectively. When data relative to these results were analyzed using the Wilcoxon signedrank test, frequencies of haemolytic strains among isolates originating from raw sewage did not differ significantly from those obtained for isolates from sediment samples. However, levels of haemolytic activity (particularely flhaemolysis) among bacteria isolated from stabilization ponds outlet were significantly higher (P< 0.05) than those observed for raw sewage and stabilization pond sediment.
Discussion Our study has shown that A. caviae dominated in both raw sewage and stabilization pond sediments. At the exit of the treatment plant, the motile aeromonad group was dominated by A. sobria. This is in agreement with other workers (Monfort et Baleux 1990; Boussaid et al. 1991; Stecchini et al. 1994), and suggests that the three Aeromonas species were affected differently during the sewage treatment. A. sobria seemed to be relatively less effectively removed by this kind of wastewater treatment in comparison with the other two species (Boussaid et al. 1991; Hassani et aL 1992). In order to investigate the fate and the behaviour of different Aeromonas species in wastewater treatment plant further, studies of survival of these species in wastewater are underway in our laboratory. All Aeromonas isolates were resistant to amoxicillin, as expected since resistance to ampicillin is highly correlated with amoxicillin resistance (Hassani eta]. 1992) and since the isolation medium relies on ampicillin as a selective
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B, lmziln, Y,M,O. Lafdal and M. ]ana Table 2. Resistance to different antibiotics among Aeromonas species Isolated from wastewater stabilization ponds plant. Aeromonas species A. caviae A. hydrophila A. sobria A. sp. (others) Mean
No. of isolates examined
Percentage of isolates resistant to:* AM
AMX
Nv
CF
Sx
Sr
Tc
393 53 223 284 953
100 100 100 100 100
100 100 100 100 100
99 100 98 99 99
96 93 20 81 73
46 34 31 42 41
28 21 11 27 23
3 8 5 3 4
Cm OxT NA Trm 9 8 6 10 9
6 17 4 12 8
9 8 4 15 9
ARI**
Ery
7 8 2 7 6
3 6 1 1 2
PB SXT Km CFX Gm 0 8 3 2 2
4 2 1 7 4
1 8 1 0 1
0 2 1 2 1
0 0 0 0 0
0.3004 0.3052 0.2276 0.2988 0.2832
* AM, ampicillin; AMX, amoxicillin; CF, cefalothin; CFX, cefotaxim; Cm, chloramphenicol; Ery, erythromycin; Gm, gentamycin; Km, kanamycin; NA, nalidixic acid; Nv, novobiocin; Oxt, oxytetracycline; PB, polymyxin B; Sr, streptomycin; Sx, sulphamethoxazol; SXT, trimethoprimsulphamethoxazol; Tc, tetracycline; Trm, trimethoprim. ** ARI, antibiotic resistance index.
Table 3. Antibiotic resistance among Aeromonas Isolates in relation to source of isolation of strains. Source
R.S. S.P.E. S.P.S. Total
No. of Isolates examined
Percentage of isolates resistant to:* AM
AMX
Nv
CF
Sx
Sr
Tc
Cm
OxT
360 397 196 953
100 100 100 100
100 100 100 100
98 100 98 99
87 60 91 77
41 43 25 39
27 23 28 25
8 4 7 3
4 8 10 7
3 9 9 6
ARI**
NA Trm Ery 5 9 7 7
6 7 4 6
3 1 2 2
PB SXT Km 1 2 4 2
1 5 3 3
1 1 2 1
CFX Gm 0 1 1 1
0 0 0 0
0.2809 0.2794 0.2890 0.2819
For abbreviations, see Table 2. ** ARI, antibiotic resistance index. R.S., Raw sewage; S.P.E., Stabilization pond effluent; S.P.S., Stabilization pond sediment.
Table 4, Haemolytlc activity among motile Aeromonas species Isolated from wastewaler stabilization. Aeromcnas species
No. of isolates examined
A. caviae A. hydrophila A. sobria A. sp. (others) Mean
Percentage of: ahaemolysis
phaemolysis
yhaemolysis
(a + ~ haemolysis
9 25 8 11 10
7 64 85 34 37
84 11 7 55 53
16 89 93 45 47
393 53 223 284 953
Table 5. Types of haemolysis among Aeromonas strains in relation to source of isolation. Source
R.S. S.P.E. S.P.S. Total
No. of Isolates examined
360 397 196 953
Percentage of: a haemolysis
phaemolysis
yhaemolysis
5 13 9 9
18 56 16 33
77 31 75 58
(a +,8) haemolysis 23 69 25 42
R.S., Raw sewage; S.P.E., Stabilization pond effluent; S.P.S., Stabilization pond sediment.
agent. The high rate of ampicillin resistance among Aeromonas isolates is confirmed by other reports (Rippey & Cabelli 1979; Ansary et al. 1992). More than 98% of Aeromonas isolates exhibited resistance to novobiocin,
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which confirmed the finding of Rahim & Sulnanul Aziz (1994), who have reported that all Aeromonas strains tested were resistant to novobiocin. Aeromonas sobria seemed to be the species most suscepti-
Wastewater aeromonads
ble to antibiotics. The most striking difference among the three species was seen in resistance to cephalothin followed by streptomycin and sulphamethoxazol. This difference in the distribution of susceptibility to cephalothin among the Aeromonas species has been described previously (Hassani et al. 1992; Urbaskova et al. 1993). Moreover, Janda & Motyl (1985) suggesi:ed cephalothin susceptibility as an additional potential marker useful in the identification of the A. sobria group. More than 90% of tested strains were found to be sensitive to 11 antibiotics used in this study (cefotaxim, chloramphenicol, erythromycin, gentamycin, kanamycin, nalidixic acid, oxytetracycline, tetracycline, poiymyxin B, trimethoprim or trimethoprim-sulphamethoxazol), which agrees with many recent reports (Hassani et al. 1992; Urbaskova et al. 1993). However, Ansary et al. (1992) have reported that more than 23, 38, 40 and 58% of their Aeromonas strains isolated from fish were resistant respectively to gentamycin, kanamycin, tetracycline and erythromycin. Rahim & Sultanul-Aziz (1994) analyzed 11 enterotoxigenic Aeromonas strains isolated from flesh water prawn and noted that all isolates were resistant to erythromycin, neomycin, novobiocin, streptomycin and vancomycin. More than 90% were found to be resistant to ampicillin and trimethoprim-sulfamethoxazol, more than 70% were resistant to polymyxin B and more than 25% were resistant to tetracycline. These differences may be related to the technique of isolation, the sources of isolation, number of isolates examined in each report, the proportion of different species composing the Aeromonas group, the frequency of use of certain drugs in a specific geographic area or to other unknown factors. The impact of stabilization pond treatment of raw wastewater on the distribution of antibiotic-resistant Aeromonas was investigated and no significant difference (P > 0.05) was recorded between the three locations (Table 3). However, when comparing each individual drug resistance, a difference was observed with cephalothin (resistance expressed in percentage : raw sewage = 86.6%; sediments = 92.8% and effluent = 60.4%). This difference may be related to the ratio of A. sobria strains in samples from different sources. This species was the most susceptible to cephalothin among the motile aeromonads group. Most isolates of A. sobria (93.3%) and A. hydrophila (88.7%) produced haemolysin. In contrast, fewer isolates of A. caviae (16.3%) showed haemolytic activity. This is in agreement with the results of many workers from different parts of the world (Janda et al. 1984; Pin et al. 1994). However, Parveen et al. (1992) have found low frequencies of isolates belonging to the three motile Aeromonas species with haemolytic activity (25 of 155 A. hydrophila strains, 56 of 244 A. veronii bv. sobria strains and 5 of 133 A. caviae strains showed haemolytic activity). Sing & Sanyal (1992) have reported opposite results with Aeromonas caviae
strains, with more than 65% of their isolates showing haemolytic activity. Differences in occurrence of haemolysin-producing Aeromonas species reported by different workers may be due to several factors including differences in methodology, the source of isolates and other unknown factors. But the most likely explanation is the physiological state of strains. According to Husslein et al. (1988) who described the evidence of homologous sequences of the haemolysin gene in all motile species of Aeromonas including A. caviae, the expression of the haemolysin gene may be controlled by a repression-derepression phenomenon. Furthermore, Sing & Sanyal (1992) have succeded in converting 0¢ and non-haemolytic strains of Aeromonas to fl-haemolytic strains after one to three consecutive passages through rabbit ileal loops. The frequencies of haemolytic strains of Aeromonas isolated from both raw sewage (22.8%) and stabilization pond sediments (24.5%) were similar (P > 0.05) but they are significantly lower (P < 0.05) than those obtained with isolates from the plant effluent (68.8%). This may be due to high dominance of A. caviae (o~ + fl haemolysis = 16.3%) in the raw sewage and sediments. In contrast, in stabilization pond effluent a high proportion of A. sobria (or + fl haemolysis = 93.3%) were responsible for the increase in the frequencies of haemolysis among isolates of motile aeromonads taken from the effluent. We conclude that the stabilization pond plant did select A. sobria from the Aeromonas population. However, it did not favour either antibiotic-resistant or haemolytic strains within each species of Aeromonas. Our results also show that substitution of Aeromonas species in the stabilization pond plant (from the inlet to the outlet of the plant) may be the most likely explanation for the variation in the incidence of some factors among the Aeromonas population in this ecosystem. Despite its higher susceptibility to antibiotics, A. sobria is the dominant species recovered from the treated wastewater, and exhibited the highest haemolytic activity. Although Aeromonas species are not considered to be among the very dangerous pathogens, the presence of A. sobria in high numbers in the treated effluent is certainly a potential health concern.
Acknowledgements The authors wish to thank Dr Abdellatif Boussaid for the revision of this manuscript.
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603. Sing, D.V. & Sanyal, S.C. 1992 Haemolysin and enterotoxin production by Aeromonas caviae isolated from diarrhoeal patients, fish and environment. Journal of diarrhoeal disease and research 10, 16-20. Stecchini, M.L. & Domenis, C. 1994 Incidence of Aeromonas species in influent and effluent of urban wastewater purification plants. Letters in Applied Microbiology 19, 237-239. Urbaskova, P., Schindler, J., Aldova, E. & Vemec, A. 1993 Antibiotic susceptibility of mesophilic aeromonads isolated in Czechoslovakia. Medical Microbiology Letters 2, 152-158.
(Received as revised form 5 February 1996; accepted 5 February t996)
Effect of wastewater stabilization ponds on antimicrobial susceptibility and haemolysin occurrence among motile Aeromonas strains B. Imziln,* Y.M.O. Lafdal and M. Jana Aeromonas strains (total = 953) isolated from raw wastewater, stabilization pond effluent and sediments were evaluated for their susceptibilities to 17 antibiotics and for their ability to produce haemolysins. Stabilization ponds did not seem to select highly resistant strains of aeromonads. There were no differences in the resistance patterns of isolates from raw sewage, stabilization pond effluent and sediments. All strains were found to possess multiple resistance, most commonly to ampicillin, amoxicillin and novobiocin. Almost 90% of the strains of A. hydrophila and A. caviae were resistant to cephalothin, whereas more than 80% of A. sobria isolates were found to be susceptible to this antibiotic. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed 10%. Moreover, no strain was found to be resistant to gentamycin and only 9 of the 953 isolates exhibited resistance to cefotaxim. The percentage of haemolytic strains was significantly higher in the stabilization pond effluent than in raw sewage. This high incidence of haemolytic activity was connected with a high proportion of A. sobria whereas, in samples from the raw sewage or stabilization pond sediments a high proportion of A. caviae decreased the total amount of haemolytic aeromonads. The high incidence of haemolytic activity (a + d) was associated particularly with A. sobria (93.3%) and A. hydrophila (88.7%) whereas A. caoiae was found to be the lowest haemolytic species (16.3%). Key words: Aeromotzas, antibiotic resistance, haemolytic activity, stabilization ponds, wastewater.
Several wastewater treatment systems have been tested in many regions of the world and under various climates. The experience of Marrakech in this field has pointed out the suitability of the stabilization pond system for purifying wastewater under arid climates (Mandi et al., I993). Among their advantages, stabilization ponds eliminated 100% of helminth eggs (Schwartzbrod et al. 1989). Moreover, satisfactory results were obtained by this system in removing pathogenic bacteria such as Salmonella, Vibrio cholerae, Pseudomonas aeruginosa and Aeromonas (Boussaid et al. 1991; Lesne et al. 1991; Hassani et al. 1992). However, the B. Imziln and Y.M.O. Lafdal are with Cadi Ayyad University, Faculty of Sciences Semlalia, Department of Biology, Laboratory of Microbiology, BP S/15 Marrakech, Morocco; fax: (212) 443 6769/(212) 443 7412. M. Jana is with the H6pital Millitaire Avicenne Marrakech, Morocco. *Corresponding author.
removal ratio of these bacteria did not reach 100% and non-negligible quantities of them were still present in the treated effluent. Among these microorganisms, motile Aeromonas spp. are more often recovered from these kinds of waters. Furthermore, in many cases their concentrations exceeded 103 c.f.u./m] (Boussaid et al. 1991; Hassani et al. 1992; Stecchini & Domenis 1994). These species have long been known to cause different infections in poikilothermic animals (Shotts et al. 1972). In recent years the clinical importance of motile Aeromonas isolates has been recognized. They are associated with several categories of infections of humans (Burke & Gracey 1986; Janda et al. 1988; Altwegg et al. 1991; Ebrad & Gerry 1992; Ghanem et al. 1993) and of warm-blooded animals (Andr6-Fontaine et al. 1995). Numerous putative virulence factors have been ascribed to Aeromonas spp. to explain the process of patho-
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B. Irnziln, Y.M.O. Lafdal and M. ]ana genicity of these bacteria, including the production of endotoxins, extracellular enterotoxins, haemolysins, cytotoxins and proteases, the ability to adhere to cells and the possession of certain surface proteins (Cahill 1990; Janda 1991). Recent attention has been focused on the haemolysin of motile Aeromonas spp. because of its potential diagnostic, epiderniologic and pathogenic significance (Aoki & Hirono I991). Several investigators have demonstrated significant association between haemolysin production and elaboration of the enterotoxigenicity in Aeromonas spp. (Burke et al. 1984; Gray et aL 1990; Mateos et al. 1992). Many investigators have described multiple uses of antibiotics in agriculture as therapeutic or prophylactic agents to treat and prevent diseases, or as growth promoters (DePaola el al. 1988; Frost 1990). This widespread use of antibiotics has resulted in the evolution of resistant strains of bacteria. Moreover, there remains the possibility that resistance may be transmitted from antibiotic-resistant bacteria to the susceptible ones (Linton 1984). In the current study we investigated the effect of the sewage stabilization pond treatment on antibiotic resistance and haemolytic activity among Aeromonas species, in order to confirm the quality of treated effluent which is used directly for irrigation of various crops in Marrakech.
Materials and Methods Study Area and Sample Collection The experimental sewage treatment pond system is located in Marrakech, (Morocco, 31°36 north, 08°02 west, altitude 471m). It consists of two successive ponds each of 2500 m 2 area. The average depth of the first pond is 2.3 m and that of thesecond is 1.6 m. The flow of incoming wastewater is maintained at 500 m ~ per day which corresponds to about 12 days of hydraulic retention time. The treated effluent is used for irrigation. Water samples were taken from plant inlet and outlet. Sediment samples were taken from the first pond. The frequency of sampling was twice a month from January 1992 to June 1993. Samples were collected in sterilized glass bottles and were kept on ice until analyzed. Time between collection and analysis never exceeded 3 hours. Isolation and Identification of Aeromonas Strains Samples were shaken for a period of 30 min. and then diluted in sterile saline solution (0.85% (w/v) NaC1 in demineralized water). Aliquots of 0.1 ml from suitable dilutions were spread onto ADA agar (Havelaar et al. 1987). Inoculated plates were then incubated at 37°C for 24h. Typical Aeromonas colonies were purified on trypto-casein soy agar (TSA, Diagnostic Pasteur 64554) plates and confirmed as belonging to the motile aeromonad group by standard biochemical reactions on the basis of the following characteristics: Gram-negative rods; motile organisms; oxidase and catalase positive; fermentative glucose metabolism (O/F test with Hugh and Leifson medium, Merck 10282); arginine dihydrolase positive (ADH, M611er); omithine decarboxylase negative (ODC, M611er) [ODC positive strains were also accepted as Aeromonas on the basis of the recommendation of Hickman-Brenner et aL (1987)]; resistance to the vibriostatic agent, 2,4-diamino-6,7-di-
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isopropylpteridine phosphate (O/129, 150 /2g disc, Diagnostic Pasteur 53872); no growth on TSA with 6.5% NaCI. Isolates which did not satisfy these criteria were considered not to be aeromonas. Seven additional tests that have been suggested to be useful in the identification of the confirmed motile aeromonads isolates to species level (Popoff I984) were added to this range. These additional tests included fermentation of salicin; esculin hydrolysis; L-arabinose utilization; gas from glucose; production of acetoin from glucose (Voges-Proskauer); lysine decarboxylase (LDC, M611er) and production of H2S from L-cycteine. Strains which did not satisfy the biochemical profiles of the motile Aeromonas spp. described by Popoff (I984) were considered as other unknown Aeromonas species.
Antibiotic Susceptibility Testing Antibiotic susceptibility of Aeromonas isolates was determined by the methods of Calomiris et al. (1984). Purified isolates were inoculated by multipoint inoculation on Mueller-Hinton agar (Mueller-Hinton 2, Bio-M6rieux 51861, France) plates supplemented with the following antibiotics (,ug/mt): Ampicillin, 10; Amoxicillin, 25; Cephalothin, 30; Streptomycin, 10; Novobiocin, 30; Chloramphenicol, 30; Nalidixic acid, 30; Kanamycin, 30; Gentamycin, 10; Erythromycin, 15; Polymyxin B; 37.5 (300 units); Tetracycline, 30; Oxytetracycline, 30; Cefotaxim, 30; Trimethoprim, 5; Sulphamethoxazol, I00; Trirnethoprim-sulphamethoxazol, 1.25 + 23.75. These antibiotics were chosen because they are widely used for the treatment of Gram-negative bacterial infections. Inoculated plates were incubated overnight at 35°C. Haemolysin Assays Haemolytic activity was determined by a zone of haemolysis around colonies on Columbia agar plates (Columbia Blood agar, Difco 0792-01-1) containing human blood, O-group (6% v/v). Plates were inoculated by spotting up to eight strains per plate, and incubated for 24 h at 37°C. Beta-haemolysis was indicated by total lysis of the erythrocytes surrounding the colonies, alphahaemolysis was indicated by a partial [ysis of the erythrocytes and gamma-haemolysis means no haemolysis of erythrocytes around colonies. Data Analysis In order to compare the level of antibiotic resistance of Aeromonas species isolated from different samples, an antibiotic resistance index (ARI) was calculated according to Hinton & Linton (1983) using the following formula: ARI = x/ny, where x is the number of resistance determinants in a population y, and n is the number of antibiotics tested. The statistical significance of differences in antibiotic resistance and haemolytic activity of the strains isolated from different locations (raw sewage, stabilization pond sediments and stabilization pond effluent) or between Aeromonas species were evaluated with the non parametric Wilcoxon-signed rank test (Schwartz 1963), with a Star View TM SE + Graphics from Abacus Concepts, Inc. (VI.03, 9/15/88) program.
Results Of the 953 Aeromonas strains isolated from the experimental wastewater stabilization ponds (Table 1), 393 (41.2%) strains were identified as A. caviae, 53 (5.6%) as A. hydrophila and 223 (23.4%) as A. sobria. On the basis of criteria defined by Popoff (1984), biochemical profiles of the 284 (29.8%)
Wastewater aeromonads Table 1. Origin and distribution of the 953 tested Aeromonas isolates Sample source
Raw sewage Stabilization pond effluent Stabilization pond sediments Total
No. of examined isolates 360 397 196 953
remaining isolates did not agree with any of the three above-mentioned species. Domestic raw sewage of Marrakech city harboured the three Aeromonas species: A. caviae, A. hydrophila and A. sobria, with a clear domination of A. caviae. The same results were found in the sediments from the ponds, whereas at the treatment plant outlet water samples showed high frequencies of A. sobria in comparison with the concentrations of the two other species. Antibiotic Resistance All isolates were tested for their resistance to each of the seventeen antibiotics used. Frequencies of antibiotic-resistance among these isolates are shown in Table 2. All 953 isolates were resistant to ampicillin and amoxicillin. More than 98% of them were found to be resistant to novobiocin. Resistance to cephalothin occured in more than 70% of all Aeromonas isolates. Percentages of sulphametoxazol and streptomycin-resistant isolates were 41 and 23 respectively. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed I0%. None of the isolates was resistant to gentamycin and only 9 of 953 tested strains exhibited resistance to cefotaxim. Results of antibiotic resistance among motile aeromonads in relation to source of isolation of strains are shown in Table 3. According to the calculated antibiotic resistance index, levels of global resistance among aeromonads isolated from influent, effluent and sediments were similar and did not show any significant difference (P > 0.05). Levels of antibiotic resistance among the three motile Aeromonas species (Table 2) are compared and according to the antibiotic resistance index (ARI) calculated for each species, A. sobria (ARI = 0.22) seemed to be the lowest antibiotic resistant species. This is related to its higher susceptibility to cephalothin, streptomycin, sulphamethoxazol and trimethoprim than the two other species (A. caviae and A. hydrophila). Haemolytic Activity Of the 393 A. caviae isolates tested, 35 (8.9%), 29 (7.4%) and 329 (83.7%) were observed to be alpha, beta and gamma haemolytic, respectively. Among 53 isolates of A.
No. of Aeromonas species A. caviae
A. hydrophila
A. sobria
A. sp. (Others)
177 92 124 393
12 20 21 53
50 159 14 223
121 126 37 284
hydrophila 13 (24.5%) were alpha, 34 (04.2%) were beta and 6 (11.3%) were found to be gamma haemolytic. For 223 strains belonging to A. sobria species, I9 (8.5%) were alpha, 189 (84.8%) were beta and only 15 (6.7%) were found to be gamma haemolytic (Table 4). The production of haemolysin by Aeromonas isolates in relation to source of isolation is shown in Table 5. Among 360 isolates sampled from raw sewage I9 (5.3%) and 63 (17.5%) were found to be alpha and beta haemolytic respectively. Among 196 strains isolated from sediment samples I7 (8.7%) were alpha and 3I (15.8%) were beta haemolytic. In contrast of 397 isolates obtained from stabilization pond effluent 51 (12.8%) and 222 (55.9%) were found to be alpha and beta haemolytic respectively. When data relative to these results were analyzed using the Wilcoxon signedrank test, frequencies of haemolytic strains among isolates originating from raw sewage did not differ significantly from those obtained for isolates from sediment samples. However, levels of haemolytic activity (particularely flhaemolysis) among bacteria isolated from stabilization ponds outlet were significantly higher (P< 0.05) than those observed for raw sewage and stabilization pond sediment.
Discussion Our study has shown that A. caviae dominated in both raw sewage and stabilization pond sediments. At the exit of the treatment plant, the motile aeromonad group was dominated by A. sobria. This is in agreement with other workers (Monfort et Baleux 1990; Boussaid et al. 1991; Stecchini et al. 1994), and suggests that the three Aeromonas species were affected differently during the sewage treatment. A. sobria seemed to be relatively less effectively removed by this kind of wastewater treatment in comparison with the other two species (Boussaid et al. 1991; Hassani et aL 1992). In order to investigate the fate and the behaviour of different Aeromonas species in wastewater treatment plant further, studies of survival of these species in wastewater are underway in our laboratory. All Aeromonas isolates were resistant to amoxicillin, as expected since resistance to ampicillin is highly correlated with amoxicillin resistance (Hassani eta]. 1992) and since the isolation medium relies on ampicillin as a selective
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B, lmziln, Y,M,O. Lafdal and M. ]ana Table 2. Resistance to different antibiotics among Aeromonas species Isolated from wastewater stabilization ponds plant. Aeromonas species A. caviae A. hydrophila A. sobria A. sp. (others) Mean
No. of isolates examined
Percentage of isolates resistant to:* AM
AMX
Nv
CF
Sx
Sr
Tc
393 53 223 284 953
100 100 100 100 100
100 100 100 100 100
99 100 98 99 99
96 93 20 81 73
46 34 31 42 41
28 21 11 27 23
3 8 5 3 4
Cm OxT NA Trm 9 8 6 10 9
6 17 4 12 8
9 8 4 15 9
ARI**
Ery
7 8 2 7 6
3 6 1 1 2
PB SXT Km CFX Gm 0 8 3 2 2
4 2 1 7 4
1 8 1 0 1
0 2 1 2 1
0 0 0 0 0
0.3004 0.3052 0.2276 0.2988 0.2832
* AM, ampicillin; AMX, amoxicillin; CF, cefalothin; CFX, cefotaxim; Cm, chloramphenicol; Ery, erythromycin; Gm, gentamycin; Km, kanamycin; NA, nalidixic acid; Nv, novobiocin; Oxt, oxytetracycline; PB, polymyxin B; Sr, streptomycin; Sx, sulphamethoxazol; SXT, trimethoprimsulphamethoxazol; Tc, tetracycline; Trm, trimethoprim. ** ARI, antibiotic resistance index.
Table 3. Antibiotic resistance among Aeromonas Isolates in relation to source of isolation of strains. Source
R.S. S.P.E. S.P.S. Total
No. of Isolates examined
Percentage of isolates resistant to:* AM
AMX
Nv
CF
Sx
Sr
Tc
Cm
OxT
360 397 196 953
100 100 100 100
100 100 100 100
98 100 98 99
87 60 91 77
41 43 25 39
27 23 28 25
8 4 7 3
4 8 10 7
3 9 9 6
ARI**
NA Trm Ery 5 9 7 7
6 7 4 6
3 1 2 2
PB SXT Km 1 2 4 2
1 5 3 3
1 1 2 1
CFX Gm 0 1 1 1
0 0 0 0
0.2809 0.2794 0.2890 0.2819
For abbreviations, see Table 2. ** ARI, antibiotic resistance index. R.S., Raw sewage; S.P.E., Stabilization pond effluent; S.P.S., Stabilization pond sediment.
Table 4, Haemolytlc activity among motile Aeromonas species Isolated from wastewaler stabilization. Aeromcnas species
No. of isolates examined
A. caviae A. hydrophila A. sobria A. sp. (others) Mean
Percentage of: ahaemolysis
phaemolysis
yhaemolysis
(a + ~ haemolysis
9 25 8 11 10
7 64 85 34 37
84 11 7 55 53
16 89 93 45 47
393 53 223 284 953
Table 5. Types of haemolysis among Aeromonas strains in relation to source of isolation. Source
R.S. S.P.E. S.P.S. Total
No. of Isolates examined
360 397 196 953
Percentage of: a haemolysis
phaemolysis
yhaemolysis
5 13 9 9
18 56 16 33
77 31 75 58
(a +,8) haemolysis 23 69 25 42
R.S., Raw sewage; S.P.E., Stabilization pond effluent; S.P.S., Stabilization pond sediment.
agent. The high rate of ampicillin resistance among Aeromonas isolates is confirmed by other reports (Rippey & Cabelli 1979; Ansary et al. 1992). More than 98% of Aeromonas isolates exhibited resistance to novobiocin,
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which confirmed the finding of Rahim & Sulnanul Aziz (1994), who have reported that all Aeromonas strains tested were resistant to novobiocin. Aeromonas sobria seemed to be the species most suscepti-
Wastewater aeromonads
ble to antibiotics. The most striking difference among the three species was seen in resistance to cephalothin followed by streptomycin and sulphamethoxazol. This difference in the distribution of susceptibility to cephalothin among the Aeromonas species has been described previously (Hassani et al. 1992; Urbaskova et al. 1993). Moreover, Janda & Motyl (1985) suggesi:ed cephalothin susceptibility as an additional potential marker useful in the identification of the A. sobria group. More than 90% of tested strains were found to be sensitive to 11 antibiotics used in this study (cefotaxim, chloramphenicol, erythromycin, gentamycin, kanamycin, nalidixic acid, oxytetracycline, tetracycline, poiymyxin B, trimethoprim or trimethoprim-sulphamethoxazol), which agrees with many recent reports (Hassani et al. 1992; Urbaskova et al. 1993). However, Ansary et al. (1992) have reported that more than 23, 38, 40 and 58% of their Aeromonas strains isolated from fish were resistant respectively to gentamycin, kanamycin, tetracycline and erythromycin. Rahim & Sultanul-Aziz (1994) analyzed 11 enterotoxigenic Aeromonas strains isolated from flesh water prawn and noted that all isolates were resistant to erythromycin, neomycin, novobiocin, streptomycin and vancomycin. More than 90% were found to be resistant to ampicillin and trimethoprim-sulfamethoxazol, more than 70% were resistant to polymyxin B and more than 25% were resistant to tetracycline. These differences may be related to the technique of isolation, the sources of isolation, number of isolates examined in each report, the proportion of different species composing the Aeromonas group, the frequency of use of certain drugs in a specific geographic area or to other unknown factors. The impact of stabilization pond treatment of raw wastewater on the distribution of antibiotic-resistant Aeromonas was investigated and no significant difference (P > 0.05) was recorded between the three locations (Table 3). However, when comparing each individual drug resistance, a difference was observed with cephalothin (resistance expressed in percentage : raw sewage = 86.6%; sediments = 92.8% and effluent = 60.4%). This difference may be related to the ratio of A. sobria strains in samples from different sources. This species was the most susceptible to cephalothin among the motile aeromonads group. Most isolates of A. sobria (93.3%) and A. hydrophila (88.7%) produced haemolysin. In contrast, fewer isolates of A. caviae (16.3%) showed haemolytic activity. This is in agreement with the results of many workers from different parts of the world (Janda et al. 1984; Pin et al. 1994). However, Parveen et al. (1992) have found low frequencies of isolates belonging to the three motile Aeromonas species with haemolytic activity (25 of 155 A. hydrophila strains, 56 of 244 A. veronii bv. sobria strains and 5 of 133 A. caviae strains showed haemolytic activity). Sing & Sanyal (1992) have reported opposite results with Aeromonas caviae
strains, with more than 65% of their isolates showing haemolytic activity. Differences in occurrence of haemolysin-producing Aeromonas species reported by different workers may be due to several factors including differences in methodology, the source of isolates and other unknown factors. But the most likely explanation is the physiological state of strains. According to Husslein et al. (1988) who described the evidence of homologous sequences of the haemolysin gene in all motile species of Aeromonas including A. caviae, the expression of the haemolysin gene may be controlled by a repression-derepression phenomenon. Furthermore, Sing & Sanyal (1992) have succeded in converting 0¢ and non-haemolytic strains of Aeromonas to fl-haemolytic strains after one to three consecutive passages through rabbit ileal loops. The frequencies of haemolytic strains of Aeromonas isolated from both raw sewage (22.8%) and stabilization pond sediments (24.5%) were similar (P > 0.05) but they are significantly lower (P < 0.05) than those obtained with isolates from the plant effluent (68.8%). This may be due to high dominance of A. caviae (o~ + fl haemolysis = 16.3%) in the raw sewage and sediments. In contrast, in stabilization pond effluent a high proportion of A. sobria (or + fl haemolysis = 93.3%) were responsible for the increase in the frequencies of haemolysis among isolates of motile aeromonads taken from the effluent. We conclude that the stabilization pond plant did select A. sobria from the Aeromonas population. However, it did not favour either antibiotic-resistant or haemolytic strains within each species of Aeromonas. Our results also show that substitution of Aeromonas species in the stabilization pond plant (from the inlet to the outlet of the plant) may be the most likely explanation for the variation in the incidence of some factors among the Aeromonas population in this ecosystem. Despite its higher susceptibility to antibiotics, A. sobria is the dominant species recovered from the treated wastewater, and exhibited the highest haemolytic activity. Although Aeromonas species are not considered to be among the very dangerous pathogens, the presence of A. sobria in high numbers in the treated effluent is certainly a potential health concern.
Acknowledgements The authors wish to thank Dr Abdellatif Boussaid for the revision of this manuscript.
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603. Sing, D.V. & Sanyal, S.C. 1992 Haemolysin and enterotoxin production by Aeromonas caviae isolated from diarrhoeal patients, fish and environment. Journal of diarrhoeal disease and research 10, 16-20. Stecchini, M.L. & Domenis, C. 1994 Incidence of Aeromonas species in influent and effluent of urban wastewater purification plants. Letters in Applied Microbiology 19, 237-239. Urbaskova, P., Schindler, J., Aldova, E. & Vemec, A. 1993 Antibiotic susceptibility of mesophilic aeromonads isolated in Czechoslovakia. Medical Microbiology Letters 2, 152-158.
(Received as revised form 5 February 1996; accepted 5 February t996)
