World Journal of Microbiology & Biotechnology 10, 230-231
Short Communication
Antimicrobial susceptibility of non-O1 Vibrio cholerae isolated from wastewater stabilization ponds in Marrakesh, Morocco B. Imziln* and L. Hassani The 36$ strains of Vibrio cholerae, isolated in Marrakesh from raw sewage and stabilization pond effluent, were all identified as non-O1 Vibrlo cholerae. When tested for their susceptibilities to ampicillin, amoxicillin, cephalothin, streptomycin, novobiocin, chloramphenicol, nalidixic acid and trimethoprim-sulphamethoxazole, 13% of the strains from raw sewage and 20% of those from stabilization pond effluent were found to be resistant to one or more of the antibiotics. There were no significant differences, in terms of drug resistance, between isolates from the new sewage and those from the ponds' effluent. Key words: Antimicrobial susceptibility, stabilization ponds, Vibrio cholerae, wastewater.
Non-O1 Vibrio cholerae has recently been recognized as an important human pathogen (Morris & Black 1985). It can cause sporadic human diseases, including gastroenteritis associated with diarrhoea (Shehabi et al. 1986). It is ubiquitous in water, both marine and freshwater and, of course, sewage (Lesne et al. 1991). When sewage effluents are used, without health protection measures, to irrigate vegetables, as they are in Marrakesh, people consuming the vegetables are exposed to pathogenic bacteria. This is particularly a problem when antibiotic-resistant bacteria are present (Morozzi et al. 1988; Hassani et al. 1992a). In Mexico, large water-bome outbreaks involving antibioticresistant Salmonella typhi (Baine et al. 1977) led to a large number of deaths, with patients failing to respond to selected antibiotics. In a previous study (Hassani et al. 1992b), we reported that antibiotic-resistant Escherichia coli were more frequent in treated sewage than in raw sewage. The present study was to elucidate how the sewage stabilization pond system that is used in the arid region of Marrakesh affects the frequency of antibiotic-resistant non-O1 Vibrio cholerae.
The authors are with the Universite Cadi Ayyad, Faculte des Sciences Semlalia, Departetnent de Biologie, Laboratoire de Microbiologie, BP S/15, Marrakesh, Morocco; fax: (212) 443 6769. *Corresponding author.
© 1994 Rapid Communications of Oxford Ltd
9.30
World Journalof Microbiology& Biotechnology,Vol I0, I994
Materials and Methods Study Site and Sampling Programme The experimental sewage treatment pond system is located near Marrakesh, Morocco. It comprises two successive ponds, each of 2500 m 2. The average depth of the first pond is 2.3 m and that of the second is 1.6 m. The flow of incoming waste is maintained at 380 m3/day. The effluent of the plant is used for irrigation. Water samples were taken, both from the raw sewage and from the pond effluent, in sterilized glass bottles and analysed within 2 h. The samples were kept on ice until analysed. Isolation and Identification of Vibrio cholerae strains Strains of Vibrio cholerae were isolated and identified according to the technique described by Lesne et al. (199I). Antibiotic Susceptibility Testing Antibiotic susceptibility of the non-OI Vibrio cholerae strains was determined using the procedure of Bauer et al. (1966). Antibiotic discs (Bio-M6rieux), which were placed on overlayed MuellerHinton agar (Bio-M6rieux), contained (~g/disc): ampicillin, 10; amoxicillin, 25; cephalothin, 30; streptomycin, 10; novobiocin, 30; chloramphenicol, 30; nalidixic acid, 30; or trimethoprimsulphamethoxazole (1.25 + 23.75). The antibiotics were chosen as those more widely used in the treatment of Gram-negative bacterial infections. Statistical Analysis The significance of the differences in antibiotic-resistance of the strains isolated from raw sewage and of those from the stabilization pond effluent were evaluated with the non-parametric Wilcoxon signed rank test.
Antimicrobial susceptibility of non-O1 Vibrio cholerae Table 1. Incidence of antibiotic resistance in non-01 Vibrio cholerae strains isolated from raw sewage and stabilization pond effluent,* Source
No. of isolates
Drug resistance (%)1"
Resistance:[:
(%)
examined
AM
AMX
S
SXT
Sewage Effluent
179 186
6.1 9.1
6.1 9.1
7.8 11.8
0 0.5
13 20
Totals
365
7.7
7.7
9.9
0.3
17
* No resistance to chloramphenicol, novobiocin, nalidixic acid or cephalothin was observed. l A M - - A m p i c i l l i n ; AMX--amoxicillin; S--streptomycin; SXT-trimethoprim-sulphamethoxazole. :I: Resistance to at least one antibiotic.
Results and Discussion There have been few studies on the occurrence and dynamics of non-O1 V. cholerae in sewage stabilization ponds. In a previous study on the same site at Marrakesh, Lesne eta]. (1991) reported a strong annual periodicity in the abundance of non-O1 V. cholerae in the effluent from the ponds, with low levels (40 MPN/100 ml) during the cold season and high levels (2 x 104 MPN/ml) during the hot season. During the course of the present investigation, 179 strains were isolated from raw sewage and 186 from the pond effluent. All isolates were identified as Vibrio cholerae strains. None showed agglutination with polyvalent V. cholerae O1 antiserum (Diagnostics Pasteur) and so the O i serovar of V, cholerae was undetectable in our analysed wastewater samples. This result is in agreement with that of Lesne et al. (1991). All 365 non-O1 V. cholerae strains were tested for their resistance to each of the eight antibiotics used. Most were sensitive to all eight but 62 (17%) isolates were found to be resistant to at least one antibiotic (Table 1). Of these, 28 (7.7%) were resistant to ampicillin and amoxicillin, 36 (9.9%) to streptomycin and one isolate was found to be resistant to trimethoprim-sulphamethoxazole. There was no apparent significant difference in the drug resistance of the raw sewage isolates and that of the effluent isolates although a larger proportion of the isolates from the effluent were resistant. It can be concluded that treatment of sewage using stabilization ponds does not affect the anfibiotic resistance of the Vibrio cholerae population.
Comparison of these results with those of earlier studies is difficult as so little relevant research has been done. Several studies on the evolution of antibiotic-resistant bacteria in sewage treatment plants were limited to coliform bacteria. Although treatment of sewage in stabilization ponds may directly affect the selection of antibiotic-resistant bacteria (Hassani et aI. 1992b), this remains in doubt. Morozzi et al. (1988) found, as in the present study, no significant difference between the proportion of antibioticresistant bacteria in stabilization ponds effluent and that in r a w sewage.
Acknowledgements This study was financially supported by the FrenchMoroccan Cooperation Program (Action Int6gr6e No. 75). We thank A. Bakri for his help in the correction of the text.
References Baine, W.B., Farmer, J.J., Gangerosa, E.J., Hermann, G.T., Thomsberry C. & Rice, P.A. 1977 Typhoid fever in the United States associated with the 1972-73 epidemic in Mexico. ]ournal of Infectious Diseases 135, 649--653. Bauer, A.W., Kirby, W.M.M., Sherris, J.C. & Turck, M. 1966 Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology 45, 493-496. Hassani, L., Imziln, B., Boussaid, A. & Gauthier, J.M. 1992a Seasonal incidence of and antibiotic resistance among Aeromonas species isolated from domestic wastewater before and after treatment in stabilization ponds. Microbial Ecology 23, 227-237. Hassani, L., Imziln, B. & Gauthier, J.M. 1992b Antibiotic-resistant Escherichia coli from wastewater before and after treatment in stabilization ponds in the arid region of Marrakesh, Morocco. Letters in Applied Microbiology 15, 228-231. Lesne, J., Baleux, B., Boussaid, A. & Hassani, L. 199i Dynamics of non-O1 Vibrio cholerae in experimental sewage stabilization ponds under arid Mediterranean climate. Water Science and Technology 24, 387-390. Morozzi, G., Sportolari, R., Caldini, G., Cenci, G. & Morosi, A. I988 The effect of anaerobic and aerobic wastewater treatment on faecal coliforms and antibiotic-resistant faecal coliforms. Zentralblatt ff~r Bakteriologie, Mikrobiologie und Hygiene B185, 340-349. Morris, J.G. & Black, R.E. 1985 Cholera and other vibrioses in the United States. New England Journal of Medicine 312, 343-350. Shehabi, A.A., Drexler, H. & Richardson, S.H. 1986 Virulence mechanisms associated with clinical isolates of non-O1 Vibrio cholerae. Zentralblatt f~r Bakteriologie, Mikrobiologie und Hygiene A261, 232-239.
(Received in revised form 6 ]uly 1993; accepted 7 August 1993)
World ]ournal of Microbiology & Biotechnology, Vol I O, 1994
231
Short Communication
Antimicrobial susceptibility of non-O1 Vibrio cholerae isolated from wastewater stabilization ponds in Marrakesh, Morocco B. Imziln* and L. Hassani The 36$ strains of Vibrio cholerae, isolated in Marrakesh from raw sewage and stabilization pond effluent, were all identified as non-O1 Vibrlo cholerae. When tested for their susceptibilities to ampicillin, amoxicillin, cephalothin, streptomycin, novobiocin, chloramphenicol, nalidixic acid and trimethoprim-sulphamethoxazole, 13% of the strains from raw sewage and 20% of those from stabilization pond effluent were found to be resistant to one or more of the antibiotics. There were no significant differences, in terms of drug resistance, between isolates from the new sewage and those from the ponds' effluent. Key words: Antimicrobial susceptibility, stabilization ponds, Vibrio cholerae, wastewater.
Non-O1 Vibrio cholerae has recently been recognized as an important human pathogen (Morris & Black 1985). It can cause sporadic human diseases, including gastroenteritis associated with diarrhoea (Shehabi et al. 1986). It is ubiquitous in water, both marine and freshwater and, of course, sewage (Lesne et al. 1991). When sewage effluents are used, without health protection measures, to irrigate vegetables, as they are in Marrakesh, people consuming the vegetables are exposed to pathogenic bacteria. This is particularly a problem when antibiotic-resistant bacteria are present (Morozzi et al. 1988; Hassani et al. 1992a). In Mexico, large water-bome outbreaks involving antibioticresistant Salmonella typhi (Baine et al. 1977) led to a large number of deaths, with patients failing to respond to selected antibiotics. In a previous study (Hassani et al. 1992b), we reported that antibiotic-resistant Escherichia coli were more frequent in treated sewage than in raw sewage. The present study was to elucidate how the sewage stabilization pond system that is used in the arid region of Marrakesh affects the frequency of antibiotic-resistant non-O1 Vibrio cholerae.
The authors are with the Universite Cadi Ayyad, Faculte des Sciences Semlalia, Departetnent de Biologie, Laboratoire de Microbiologie, BP S/15, Marrakesh, Morocco; fax: (212) 443 6769. *Corresponding author.
© 1994 Rapid Communications of Oxford Ltd
9.30
World Journalof Microbiology& Biotechnology,Vol I0, I994
Materials and Methods Study Site and Sampling Programme The experimental sewage treatment pond system is located near Marrakesh, Morocco. It comprises two successive ponds, each of 2500 m 2. The average depth of the first pond is 2.3 m and that of the second is 1.6 m. The flow of incoming waste is maintained at 380 m3/day. The effluent of the plant is used for irrigation. Water samples were taken, both from the raw sewage and from the pond effluent, in sterilized glass bottles and analysed within 2 h. The samples were kept on ice until analysed. Isolation and Identification of Vibrio cholerae strains Strains of Vibrio cholerae were isolated and identified according to the technique described by Lesne et al. (199I). Antibiotic Susceptibility Testing Antibiotic susceptibility of the non-OI Vibrio cholerae strains was determined using the procedure of Bauer et al. (1966). Antibiotic discs (Bio-M6rieux), which were placed on overlayed MuellerHinton agar (Bio-M6rieux), contained (~g/disc): ampicillin, 10; amoxicillin, 25; cephalothin, 30; streptomycin, 10; novobiocin, 30; chloramphenicol, 30; nalidixic acid, 30; or trimethoprimsulphamethoxazole (1.25 + 23.75). The antibiotics were chosen as those more widely used in the treatment of Gram-negative bacterial infections. Statistical Analysis The significance of the differences in antibiotic-resistance of the strains isolated from raw sewage and of those from the stabilization pond effluent were evaluated with the non-parametric Wilcoxon signed rank test.
Antimicrobial susceptibility of non-O1 Vibrio cholerae Table 1. Incidence of antibiotic resistance in non-01 Vibrio cholerae strains isolated from raw sewage and stabilization pond effluent,* Source
No. of isolates
Drug resistance (%)1"
Resistance:[:
(%)
examined
AM
AMX
S
SXT
Sewage Effluent
179 186
6.1 9.1
6.1 9.1
7.8 11.8
0 0.5
13 20
Totals
365
7.7
7.7
9.9
0.3
17
* No resistance to chloramphenicol, novobiocin, nalidixic acid or cephalothin was observed. l A M - - A m p i c i l l i n ; AMX--amoxicillin; S--streptomycin; SXT-trimethoprim-sulphamethoxazole. :I: Resistance to at least one antibiotic.
Results and Discussion There have been few studies on the occurrence and dynamics of non-O1 V. cholerae in sewage stabilization ponds. In a previous study on the same site at Marrakesh, Lesne eta]. (1991) reported a strong annual periodicity in the abundance of non-O1 V. cholerae in the effluent from the ponds, with low levels (40 MPN/100 ml) during the cold season and high levels (2 x 104 MPN/ml) during the hot season. During the course of the present investigation, 179 strains were isolated from raw sewage and 186 from the pond effluent. All isolates were identified as Vibrio cholerae strains. None showed agglutination with polyvalent V. cholerae O1 antiserum (Diagnostics Pasteur) and so the O i serovar of V, cholerae was undetectable in our analysed wastewater samples. This result is in agreement with that of Lesne et al. (1991). All 365 non-O1 V. cholerae strains were tested for their resistance to each of the eight antibiotics used. Most were sensitive to all eight but 62 (17%) isolates were found to be resistant to at least one antibiotic (Table 1). Of these, 28 (7.7%) were resistant to ampicillin and amoxicillin, 36 (9.9%) to streptomycin and one isolate was found to be resistant to trimethoprim-sulphamethoxazole. There was no apparent significant difference in the drug resistance of the raw sewage isolates and that of the effluent isolates although a larger proportion of the isolates from the effluent were resistant. It can be concluded that treatment of sewage using stabilization ponds does not affect the anfibiotic resistance of the Vibrio cholerae population.
Comparison of these results with those of earlier studies is difficult as so little relevant research has been done. Several studies on the evolution of antibiotic-resistant bacteria in sewage treatment plants were limited to coliform bacteria. Although treatment of sewage in stabilization ponds may directly affect the selection of antibiotic-resistant bacteria (Hassani et aI. 1992b), this remains in doubt. Morozzi et al. (1988) found, as in the present study, no significant difference between the proportion of antibioticresistant bacteria in stabilization ponds effluent and that in r a w sewage.
Acknowledgements This study was financially supported by the FrenchMoroccan Cooperation Program (Action Int6gr6e No. 75). We thank A. Bakri for his help in the correction of the text.
References Baine, W.B., Farmer, J.J., Gangerosa, E.J., Hermann, G.T., Thomsberry C. & Rice, P.A. 1977 Typhoid fever in the United States associated with the 1972-73 epidemic in Mexico. ]ournal of Infectious Diseases 135, 649--653. Bauer, A.W., Kirby, W.M.M., Sherris, J.C. & Turck, M. 1966 Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology 45, 493-496. Hassani, L., Imziln, B., Boussaid, A. & Gauthier, J.M. 1992a Seasonal incidence of and antibiotic resistance among Aeromonas species isolated from domestic wastewater before and after treatment in stabilization ponds. Microbial Ecology 23, 227-237. Hassani, L., Imziln, B. & Gauthier, J.M. 1992b Antibiotic-resistant Escherichia coli from wastewater before and after treatment in stabilization ponds in the arid region of Marrakesh, Morocco. Letters in Applied Microbiology 15, 228-231. Lesne, J., Baleux, B., Boussaid, A. & Hassani, L. 199i Dynamics of non-O1 Vibrio cholerae in experimental sewage stabilization ponds under arid Mediterranean climate. Water Science and Technology 24, 387-390. Morozzi, G., Sportolari, R., Caldini, G., Cenci, G. & Morosi, A. I988 The effect of anaerobic and aerobic wastewater treatment on faecal coliforms and antibiotic-resistant faecal coliforms. Zentralblatt ff~r Bakteriologie, Mikrobiologie und Hygiene B185, 340-349. Morris, J.G. & Black, R.E. 1985 Cholera and other vibrioses in the United States. New England Journal of Medicine 312, 343-350. Shehabi, A.A., Drexler, H. & Richardson, S.H. 1986 Virulence mechanisms associated with clinical isolates of non-O1 Vibrio cholerae. Zentralblatt f~r Bakteriologie, Mikrobiologie und Hygiene A261, 232-239.
(Received in revised form 6 ]uly 1993; accepted 7 August 1993)
World ]ournal of Microbiology & Biotechnology, Vol I O, 1994
231
