Microb Ecol (1992) 23:227-237
MICROBIAL ECOLOGY © Springer-VerlagNew York Inc. 1992
Seasonal Incidence of and Antibiotic Resistance Among Aeromonas Species Isolated from Domestic Wastewater Before and After Treatment in Stabilization Ponds L. Hassani,1 B. Imziln,~ A. Boussaid,~ and M. J. G a u t h i e r 2 ~Universit6 Cadi Ayyad, Facult6 des Sciences, D6partement de Biologie, Laboratoire de Microbiologie, BP S/15 Marrakech, Morocco; and qNSERM, Unit6 303 "Mer et Sant6," F-06300 Nice, France Received." July 8, 1991; Revised."January 29, 1992
Abstract. The efficiency o f stabilization pond treatment o f domestic wastewater in removing culturable cells o f motile A e r o m o n a s and its influence on the incidence o f resistance to seven antibiotics were investigated in this study. R e m o v a l efficiency was higher (P < 0.001) in the warm m o n t h s (98.8%) than in the cold m o n t h s (97%). A m o n g the 264 isolates, 163 were A e r o m o n a s caviae, 24 were A. hydrophila, and 54 were A. sobria. Twenty-three isolates could not be identified to the species level. In the influent, A. caviae d o m i n a t e d in both cold and warm months. In the water samples originating from the influent, A. sobria was present at higher percentages in the warm period. All the isolates were resistant to amoxicillin and most o f them (73%) exhibited resistance to cephalothin. O f the three species tested, A. sobria was m o r e susceptible to antibiotics than either A. caviae or A. hydrophila. The most striking difference a m o n g the species was seen in resistance to cephalothin. There were 91% ofA. caviae strains and 96% ofA. hydrophila isolates that were resistant to cephalothin. However, only 9% o f A. sobria strains exhibited resistance to this drug. The high incidence o f resistance in raw sewage was connected with a high p r o p o r t i o n o f A. caviae, whereas in the water samples collected from the effluent during the warm months, a high p r o p o r t i o n ofA. sobria decreased the total a m o u n t o f multiple-resistant bacteria. Results demonstrated the need for identification to the species level.
Introduction Since 1984, the Faculty o f Science o f Marrakech, in collaboration with the University o f Science o f Languedoc (Montpellier, France) and Municipal and Regional offices, has tested the efficiency o f stabilization ponds in removing pathogenic bacteria from local wastewaters [8, 32]. Offprint requests to: L. Hassani.
228
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Local sewage effluents are used without health protection measures to irrigate vegetables. So people consuming these crops can be exposed to surviving pathogenic bacteria. This problem could further be complicated by the presence of antibiotic-resistant bacteria in the effluents. Indeed, large waterborne outbreaks involving antibiotic-resistant bacteria have led to a large number of deaths, due partly to the failure of the patients to respond to antibiotics of choice [3, 18]. It is then important to evaluate the influence of water treatment on antibiotic resistance among surviving bacteria in the effluents. There are a few reports on the influence of sewage treatment on antibiotic resistance in fecal coliforms [5, 6, 19, 29, 36, 46], but no information has been reported concerning drug resistance among Aeromonas isolates from stabilization ponds. The present study was undertaken to examine the distribution of Aeromonas species in the wastewater treatment ponds and to determine the effect of this treatment on drug resistance among Aeromonas species.
Materials and Methods
Study Site and Sampling Program The wastewater treatment system is situated at the sewage spreading field of the city of Marrakech, Morocco. It receives only part of the city sewage. It is composed of two successive experimental stabilization ponds with an area of 2,500 m 2 each and an average depth of 2.3 m in the first basin and 1.6 m in the second basin. The retention times in each pond were thus 13 days in the first pond and 9 days in the second pond. The raw sewage flow to the system is maintained at 4.4 liter/ sec. The sites used in this study were the entry canal in the first basin (Station E) and the exit of the system (Station 6). Surface water samples were collected twice monthly over a 17-month period, from February 1988 to June 1989. Water temperature during the investigation period varied between 9.9°C (January 1989) and 34.8°C (July 1988) with an average of 21°C. Following Demillac et al. [12], the period when the water temperature was below 21°C was defined as the cold period, whereas the warm period was defined for the months when water temperature was equal to or above 2 I°C. Water samples were obtained aseptically in presterilized bottles, placed in ice, and processed for enumeration and isolation of bacteria within 2 hours after sampling.
Bacteriological Analysis The samples were analyzed in triplicate for Aeromonas densities by spread plating serial dilutions on Pril-Xylose-Ampicillin (PXA) agar [43] at 37°C for 48 hours. Sampling of Aeromonas isolates for identification and antibiotic resistance studies was performed as recommended by Bianchi and Bianchi [7]. Each sample contained a single plate with at least 30 colonies. Four samplings were thus taken at stations E (influent) and 6 (effluent) as follows: June 1988, 33 isolates from E and 26 from 6; September 1988, 31 from E and 34 from 6; December 1988, 30 from E and 31 from 6; February 1989, 40 from E and 40 from 6. Isolates of aeromonads were first identified as presumptive motile Aeromonas species on the basis of the following characters: Gram-negative, fermentative, motile rods, oxidase positive, and resistant to the vibriostatic agent 0/129. Identification of Aeromonas species was performed according to Popoff and Veron [40]. Resistance to antibiotics was determined by using antibiotic disks (BioMrrieux, France) and Mueller-Hinton agar plates (Difco) according to the instructions of the manufacturer and standard procedures [4]. Resistance was tested to (concentrations in micrograms per disk, except where
Antibiotic Resistance of Aeromonas in Stabilization Ponds
229
indicated otherwise): amoxicillin (AMX 25), cephalothin (CF 30), streptomycin (S 10), chloramphenicol (C 30), nalidixic acid (NA 30), trimethoprim-sulfamethoxazole (SXT 1.25 + 23.75) and polymyxin B (PB 300 units).
Data Analysis Aeromonas species densities were log-transformed to avoid the problem posed by the asymmetry of density distribution. Comparisons were tested for statistical significance by using the nonparametric Wilcoxon's signed rank test on the Statview statistics program (Brain Power Inc., 1986) by using a Macintosh Plus computer (Apple Computer, France). Results of identification of Aeromonas isolates were analyzed using the weighted centroid cluster test (WPGMC) on the Progiciel R (P. Legendre and A. Vaudor, Universit6 de Montreal, Montreal, Quebec, Canada, 1987). Moving average and the eorrelogram methods [30] were used to analyze temporal changes in Aeromonas densities. In order to compare the level of antibiotic resistance of Aeromonas species isolated from the different water samples, an antibiotic resistance index (ARI) was calculated according to Hinton and Linton [24] using the formula ARI = x/ny, where x is the number of resistance determinants in a population, y, and n is the number of antibiotics tested.
Results S p a t i o - T e m p o r a l C h a n g e s o f A e r o m o n a s Densities T h e m e a n A e r o m o n a s p o p u l a t i o n o f the i n f l u e n t ( S t a t i o n E) a n d the effluent ( S t a t i o n 6) were 5.63 × 104 c o l o n y - f o r m i n g u n i t s ( c f u ) / m l a n d 1.04 x 103 cfu/ m l , r e s p e c t i v e l y , d u r i n g the e n t i r e p e r i o d o f i n v e s t i g a t i o n ( T a b l e 1). D e c r e a s e i n A e r o m o n a s p l a t a b i l i t y i n the s y s t e m was 5.53 x 104 c f u / m l , c o r r e s p o n d i n g to 9 8 . 2 % r e d u c t i o n i n A e r o m o n a s p o p u l a t i o n . T h e W i l c o x o n ' s s i g n e d r a n k test s h o w e d a s i g n i f i c a n t difference b e t w e e n A e r o m o n a s c o u n t s f r o m the i n f l u e n t a n d c o u n t s f r o m effluent (P < 0.001). A b u n d a n c e v a l u e s o f A e r o m o n a s species s h o w e d t e m p o r a l c h a n g e s i n all the sites (Fig. 1). T h e h i g h e s t d e n s i t i e s were o b s e r v e d d u r i n g the c o l d p e r i o d , w h e r e a s m i n i m u m c o u n t s were e n c o u n t e r e d at t h e w a r m p e r i o d ( T a b l e 1). M o v i n g a v e r a g e m e t h o d (Fig. 1) a n d the corr e l o g r a r n a n a l y s i s (Fig. 2) d e m o n s t r a t e d t h a t these c h a n g e s were cyclic w i t h a p e r i o d o f 28 t w o - w e e k i n t e r v a l s . I n d e e d , e x t r a c t i o n o f the s e a s o n a l c o m p o n e n t u s i n g a p e r i o d o f 28 i n t e r v a l s p e r m i t t e d t h e c a l c u l a t i o n o f the m o v i n g average,
Table 1. Arithmetic mean densities ofAeromonas (cfu/ml) and decrease in platability as cfu/ml in the treatment plant Warm period a Station E Station 6 Decrease in platability (% decrease)
Cold periodb
Entire periodc
2.6 × 104`/ 1.43 x 105 5.63 x 104 3.14 × 102 4.24 × 103 1.04 x 103 2.57 x 104 (98.8) 1.39 × 105 (97) 5.53 x 104 (98.2)
a Eight-month period from May 1988 to October 1988 and from May 1989 to June 1989 b Nine-month period from February 1988 to April 1988 and from November 1988 to April 1989 c 17-month period from February 1988 to June 1989 a Arithmetic mean determined from all the water samples processed during the period investigated
230
L, Hassani et al. 7 =
E It. ¢..) O~ 0
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35
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14 days intervals Fig. 1. Temporal evolution of the counts o f A e r o m o n a s in raw sewage ~1), and final effluent (A), and moving average calculated for the data from raw sewage ([]) and final effluent (A). Water samples were collected at 14 day intervals over a 17-month period (37 intervals, from February 1988 to June 1989) and analyzed in triplicate for A e r o m o n a s densities.
which became nearly invariate (Fig. 1). The mean difference in Aeromonas species abundance between sites was 2.57 x 104 cfu/ml (98.8%) at the warm period, whereas the average difference during the cold period was 1.39 x 105 cfu/ml (97%). These differences were statistically significant (P < 0.001).
Spatio-Temporal Changes in Proportion of Different Aeromonas Species O f the 264 isolates, 163 (62%) were identified as A. caviae, 24 (9%) as A. hydrophila, and 54 (20%) as A. sobria. Twenty-three isolates (9%) could not be identified to species level; they were considered to be atypical Aeromonas isolates. All the samples from the entry canal contained the three species, i.e., Aerornonas caviae, A. sobria, and A. hydrophila. At this station, A. caviae dominated in both cold and warm months (Table 2). In water samples originating from the effluent, A. caviae was present at higher percentages in the cold months: December 1988, 80.7% of the total isolates and February 1989, 77.5%. High frequencies ofA. sobria, however, were found in warm months, i.e., June 1988 (57.7%) and September 1988 (64.7%). A. hydrophila was present at lower levels (
MICROBIAL ECOLOGY © Springer-VerlagNew York Inc. 1992
Seasonal Incidence of and Antibiotic Resistance Among Aeromonas Species Isolated from Domestic Wastewater Before and After Treatment in Stabilization Ponds L. Hassani,1 B. Imziln,~ A. Boussaid,~ and M. J. G a u t h i e r 2 ~Universit6 Cadi Ayyad, Facult6 des Sciences, D6partement de Biologie, Laboratoire de Microbiologie, BP S/15 Marrakech, Morocco; and qNSERM, Unit6 303 "Mer et Sant6," F-06300 Nice, France Received." July 8, 1991; Revised."January 29, 1992
Abstract. The efficiency o f stabilization pond treatment o f domestic wastewater in removing culturable cells o f motile A e r o m o n a s and its influence on the incidence o f resistance to seven antibiotics were investigated in this study. R e m o v a l efficiency was higher (P < 0.001) in the warm m o n t h s (98.8%) than in the cold m o n t h s (97%). A m o n g the 264 isolates, 163 were A e r o m o n a s caviae, 24 were A. hydrophila, and 54 were A. sobria. Twenty-three isolates could not be identified to the species level. In the influent, A. caviae d o m i n a t e d in both cold and warm months. In the water samples originating from the influent, A. sobria was present at higher percentages in the warm period. All the isolates were resistant to amoxicillin and most o f them (73%) exhibited resistance to cephalothin. O f the three species tested, A. sobria was m o r e susceptible to antibiotics than either A. caviae or A. hydrophila. The most striking difference a m o n g the species was seen in resistance to cephalothin. There were 91% ofA. caviae strains and 96% ofA. hydrophila isolates that were resistant to cephalothin. However, only 9% o f A. sobria strains exhibited resistance to this drug. The high incidence o f resistance in raw sewage was connected with a high p r o p o r t i o n o f A. caviae, whereas in the water samples collected from the effluent during the warm months, a high p r o p o r t i o n ofA. sobria decreased the total a m o u n t o f multiple-resistant bacteria. Results demonstrated the need for identification to the species level.
Introduction Since 1984, the Faculty o f Science o f Marrakech, in collaboration with the University o f Science o f Languedoc (Montpellier, France) and Municipal and Regional offices, has tested the efficiency o f stabilization ponds in removing pathogenic bacteria from local wastewaters [8, 32]. Offprint requests to: L. Hassani.
228
L. Hassani et al.
Local sewage effluents are used without health protection measures to irrigate vegetables. So people consuming these crops can be exposed to surviving pathogenic bacteria. This problem could further be complicated by the presence of antibiotic-resistant bacteria in the effluents. Indeed, large waterborne outbreaks involving antibiotic-resistant bacteria have led to a large number of deaths, due partly to the failure of the patients to respond to antibiotics of choice [3, 18]. It is then important to evaluate the influence of water treatment on antibiotic resistance among surviving bacteria in the effluents. There are a few reports on the influence of sewage treatment on antibiotic resistance in fecal coliforms [5, 6, 19, 29, 36, 46], but no information has been reported concerning drug resistance among Aeromonas isolates from stabilization ponds. The present study was undertaken to examine the distribution of Aeromonas species in the wastewater treatment ponds and to determine the effect of this treatment on drug resistance among Aeromonas species.
Materials and Methods
Study Site and Sampling Program The wastewater treatment system is situated at the sewage spreading field of the city of Marrakech, Morocco. It receives only part of the city sewage. It is composed of two successive experimental stabilization ponds with an area of 2,500 m 2 each and an average depth of 2.3 m in the first basin and 1.6 m in the second basin. The retention times in each pond were thus 13 days in the first pond and 9 days in the second pond. The raw sewage flow to the system is maintained at 4.4 liter/ sec. The sites used in this study were the entry canal in the first basin (Station E) and the exit of the system (Station 6). Surface water samples were collected twice monthly over a 17-month period, from February 1988 to June 1989. Water temperature during the investigation period varied between 9.9°C (January 1989) and 34.8°C (July 1988) with an average of 21°C. Following Demillac et al. [12], the period when the water temperature was below 21°C was defined as the cold period, whereas the warm period was defined for the months when water temperature was equal to or above 2 I°C. Water samples were obtained aseptically in presterilized bottles, placed in ice, and processed for enumeration and isolation of bacteria within 2 hours after sampling.
Bacteriological Analysis The samples were analyzed in triplicate for Aeromonas densities by spread plating serial dilutions on Pril-Xylose-Ampicillin (PXA) agar [43] at 37°C for 48 hours. Sampling of Aeromonas isolates for identification and antibiotic resistance studies was performed as recommended by Bianchi and Bianchi [7]. Each sample contained a single plate with at least 30 colonies. Four samplings were thus taken at stations E (influent) and 6 (effluent) as follows: June 1988, 33 isolates from E and 26 from 6; September 1988, 31 from E and 34 from 6; December 1988, 30 from E and 31 from 6; February 1989, 40 from E and 40 from 6. Isolates of aeromonads were first identified as presumptive motile Aeromonas species on the basis of the following characters: Gram-negative, fermentative, motile rods, oxidase positive, and resistant to the vibriostatic agent 0/129. Identification of Aeromonas species was performed according to Popoff and Veron [40]. Resistance to antibiotics was determined by using antibiotic disks (BioMrrieux, France) and Mueller-Hinton agar plates (Difco) according to the instructions of the manufacturer and standard procedures [4]. Resistance was tested to (concentrations in micrograms per disk, except where
Antibiotic Resistance of Aeromonas in Stabilization Ponds
229
indicated otherwise): amoxicillin (AMX 25), cephalothin (CF 30), streptomycin (S 10), chloramphenicol (C 30), nalidixic acid (NA 30), trimethoprim-sulfamethoxazole (SXT 1.25 + 23.75) and polymyxin B (PB 300 units).
Data Analysis Aeromonas species densities were log-transformed to avoid the problem posed by the asymmetry of density distribution. Comparisons were tested for statistical significance by using the nonparametric Wilcoxon's signed rank test on the Statview statistics program (Brain Power Inc., 1986) by using a Macintosh Plus computer (Apple Computer, France). Results of identification of Aeromonas isolates were analyzed using the weighted centroid cluster test (WPGMC) on the Progiciel R (P. Legendre and A. Vaudor, Universit6 de Montreal, Montreal, Quebec, Canada, 1987). Moving average and the eorrelogram methods [30] were used to analyze temporal changes in Aeromonas densities. In order to compare the level of antibiotic resistance of Aeromonas species isolated from the different water samples, an antibiotic resistance index (ARI) was calculated according to Hinton and Linton [24] using the formula ARI = x/ny, where x is the number of resistance determinants in a population, y, and n is the number of antibiotics tested.
Results S p a t i o - T e m p o r a l C h a n g e s o f A e r o m o n a s Densities T h e m e a n A e r o m o n a s p o p u l a t i o n o f the i n f l u e n t ( S t a t i o n E) a n d the effluent ( S t a t i o n 6) were 5.63 × 104 c o l o n y - f o r m i n g u n i t s ( c f u ) / m l a n d 1.04 x 103 cfu/ m l , r e s p e c t i v e l y , d u r i n g the e n t i r e p e r i o d o f i n v e s t i g a t i o n ( T a b l e 1). D e c r e a s e i n A e r o m o n a s p l a t a b i l i t y i n the s y s t e m was 5.53 x 104 c f u / m l , c o r r e s p o n d i n g to 9 8 . 2 % r e d u c t i o n i n A e r o m o n a s p o p u l a t i o n . T h e W i l c o x o n ' s s i g n e d r a n k test s h o w e d a s i g n i f i c a n t difference b e t w e e n A e r o m o n a s c o u n t s f r o m the i n f l u e n t a n d c o u n t s f r o m effluent (P < 0.001). A b u n d a n c e v a l u e s o f A e r o m o n a s species s h o w e d t e m p o r a l c h a n g e s i n all the sites (Fig. 1). T h e h i g h e s t d e n s i t i e s were o b s e r v e d d u r i n g the c o l d p e r i o d , w h e r e a s m i n i m u m c o u n t s were e n c o u n t e r e d at t h e w a r m p e r i o d ( T a b l e 1). M o v i n g a v e r a g e m e t h o d (Fig. 1) a n d the corr e l o g r a r n a n a l y s i s (Fig. 2) d e m o n s t r a t e d t h a t these c h a n g e s were cyclic w i t h a p e r i o d o f 28 t w o - w e e k i n t e r v a l s . I n d e e d , e x t r a c t i o n o f the s e a s o n a l c o m p o n e n t u s i n g a p e r i o d o f 28 i n t e r v a l s p e r m i t t e d t h e c a l c u l a t i o n o f the m o v i n g average,
Table 1. Arithmetic mean densities ofAeromonas (cfu/ml) and decrease in platability as cfu/ml in the treatment plant Warm period a Station E Station 6 Decrease in platability (% decrease)
Cold periodb
Entire periodc
2.6 × 104`/ 1.43 x 105 5.63 x 104 3.14 × 102 4.24 × 103 1.04 x 103 2.57 x 104 (98.8) 1.39 × 105 (97) 5.53 x 104 (98.2)
a Eight-month period from May 1988 to October 1988 and from May 1989 to June 1989 b Nine-month period from February 1988 to April 1988 and from November 1988 to April 1989 c 17-month period from February 1988 to June 1989 a Arithmetic mean determined from all the water samples processed during the period investigated
230
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E It. ¢..) O~ 0
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14 days intervals Fig. 1. Temporal evolution of the counts o f A e r o m o n a s in raw sewage ~1), and final effluent (A), and moving average calculated for the data from raw sewage ([]) and final effluent (A). Water samples were collected at 14 day intervals over a 17-month period (37 intervals, from February 1988 to June 1989) and analyzed in triplicate for A e r o m o n a s densities.
which became nearly invariate (Fig. 1). The mean difference in Aeromonas species abundance between sites was 2.57 x 104 cfu/ml (98.8%) at the warm period, whereas the average difference during the cold period was 1.39 x 105 cfu/ml (97%). These differences were statistically significant (P < 0.001).
Spatio-Temporal Changes in Proportion of Different Aeromonas Species O f the 264 isolates, 163 (62%) were identified as A. caviae, 24 (9%) as A. hydrophila, and 54 (20%) as A. sobria. Twenty-three isolates (9%) could not be identified to species level; they were considered to be atypical Aeromonas isolates. All the samples from the entry canal contained the three species, i.e., Aerornonas caviae, A. sobria, and A. hydrophila. At this station, A. caviae dominated in both cold and warm months (Table 2). In water samples originating from the effluent, A. caviae was present at higher percentages in the cold months: December 1988, 80.7% of the total isolates and February 1989, 77.5%. High frequencies ofA. sobria, however, were found in warm months, i.e., June 1988 (57.7%) and September 1988 (64.7%). A. hydrophila was present at lower levels (
