Many investigators have recognized that wastewater treatment plants are the principal recipients of enteric bacteria with multiple antibiotic resistance (Iversen et al. 2002; Selvaratnam and Kunberger, 2004) and an important site for horizontal gene transfer, by containing nutrients and high concentrations of microorganisms (Barberio et al. 2001; Vilanova et al. 2004). Biological treatment processes at sewage treatment plants could produce selective elimination, and / or changes in the proportions of phenotypes within effluent bacterial populations (Andersen,1993; Mezrioui and Baleux, 1994). Furthermore the disposal of treated sewage into rivers, lakes, or elsewhere  may or may not influence environmental bacterial populations (Vilanova et al. 2002). Some studies have found that wastewater treatment can raise or lower the proportions of antibiotic resistant bacteria which carry antibiotic resistance plasmids (Andersen, 1993; Ohlsen et al. 2003). Antibiotic resistant coliforms obtained from inflow and effluent waters were studied to determine influences of treatment on the species composition, resistance patterns and transferable resistance in these bacteria.

Samples of wastewater were collected from an activated sludge system at an urban sewage treatment plant, located in Antofagasta, Chile. This plant has a biological carrying capacity of 100,000-120,000 person-equivalents. The sewage spends a total retention time in the system of about 3-4 hours prior to the (final) chlorination step, with a discharge rate of 50 L/seg of treated effluent. Samples were collected on four occasions, including September and December 2001, and March and July 2002. Antibiotic-resistant coliform bacteria from raw and treated water from activated sludge of a sewage treatment plant were isolated on eosin methylene blue agar (EMB) plates in the separate presence of ampicillin, tetracycline, chloramphenicol and streptomycin. The isolates were identified by biochemical tests(Kelly et al. 1995). A total of 248 coliform isolates from raw and treated sewage were tested for antibiotic resistance by a standard disc diffusion technique (NCCLS, 2000). Patterns of antibiotic resistance were determined. Transference of bacterial resistance by conjugation experiments were carry out in broth (Silva et al. 1987). Some multi-resistant coliform strains NA^S isolated from treated sewage were used as donor organisms, and Escherichia coli K-12 Lac^- NA^R, as a recipient strain. Then transconjugants were subcultured on non-selectives medium and the resistance pattern was determined by the disc dilution technique.

The results of this study suggested that wastewater treatment could reduce the total number of enteric bacteria in sewage, but may increase the proportion of antibiotic resistant coliforms in effluent water at the Antofagasta plant. The high values of resistance found to ampicillin was in general agreement with that reported by others (Andersen and Sandaa, 1994; Boon and Cattanach, 1999), and is very common in coliforms isolated from the human and animal intestines. Climatic conditions in summer months probably influenced increases the proportion of resistant coliforms as demonstrated in our study, as shown by the higher percentages of resistant isolates observed in the December samples. 

Eight different species were identified by the biochemical tests, with E. coli the most frequent species isolated from both raw and treated sewage. The resistant coliform species were eliminated at different rates by the treatment Escherichia coli decreased, whereas the proportion of Klebsiella sp increased. A high resistance was found to ampicillin, followed by tetracycline and sulphamethoxazole-trimethoprim. Moderate resistance was observed to streptomycin, chloramphenicol and cephradine. The lowest resistance was observed with kanamycin and nalidixic acid. The percentage of multiple-resistant isolates increased at the end of the treatment process, exhibiting resistance to as many as 8 antibiotics. Similar results have been reported in coliform isolates from sewage treatment plant (Andersen, 1993). The bacterial conjugation experiments showed that a high percentage of multi-resistant coliforms tested (89%) were able to partially or completely transfer their resistance patterns to E. coli K-12. A high proportion of the multi-resistance was mediated by conjugative plasmids, which were easily transferred to E. coli K-12. These findings agree with other studies which established that  sewage treatment plants  are potential sites for the genetic transfer of antibiotic resistance in some species of bacteria (Andersen, 1993; Goñi-Urriza et al. 2000; Schluter et al. 2003). The increase of multi-resistance in coliforms isolated from treated wastewater is explainable by bacterial conjugation or other genetic mechanisms of change from antibiotic resistant to antibiotic sensitive bacteria by horizontal gene transfer within the treatment plant.

The Antofagasta treatment plant is apparently working well, reducing total coliform and resistant coliforms to acceptable levels from a sanitary standpoint, but the release of treated water containing bacteria having increased antibiotic resistance levels is an undesirable outcome, which should be remediated (Andersen, 1993).

The wastewater treatment plant constitutes an important reservoir of enteric bacteria which carry potentially transferable resistance genes. This study has demonstrated that sewage treatment may result in the increase the proportion of antibiotic resistant bacteria in effluent water. The use of the treated sewage may contribute to spread antibiotic resistance in the environment, specially when regions poor in water resources re-use it in watering agricultural crops, such as in northern part of Chile. This represents a dangerous public health risk, which needs future evaluation and control.

ANDERSEN, Sigrid Rita. Effects of waste water treatment on the species composition and antibiotic resistance of coliform bacteria. Current Microbiology, February 1993, vol. 26, no. 2, p. 97-103. [CrossRef]

ANDERSEN, Sigrid Rita and SANDAA, R.A. Distribution of tetracycline resistance among gram-negative bacteria isolated from polluted and unpolluted marine sediments. Applied and Environmental Microbiology, March 1994, vol. 60, no. 3, p. 908-912.

BARBERIO, Claudia; PAGLIAI, Letizia; CAVALIERI, Duccio and FANI, Renato. Biodiversity and horizontal gene transfer in culturable bacteria isolated from activated sludge enriched in nonylphenol ethoxylates. Research in Microbiology, January-February 2001, vol. 152, no. 1, p. 105-112. [CrossRef]

BOON, P.I. and CATTANACH, M. Antibiotic resistance of native and faecal bacteria isolated from rivers, reservoirs and sewage treatment facilities in Victoria, south-eastern Australia. Letters in Applied Microbiology, March 1999, vol. 28, no. 3, p. 164-168. [CrossRef]

GOÑI-URRIZA, Marisol; CAPDEPUY, Michèle; ARPIN, Corinne; RAYMOND, Nathalie; CAUMETTE, Pierre and QUENTIN, Claudine. Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp. Applied and Environmental Microbiology, January 2000, vol. 66, no. 1, p. 125-132.

IVERSEN, Aina; KÜHN, Inger; FRANKLIN, Anders and MÖLLBY, Roland. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Applied and Environmental Microbiology, June 2002, vol. 68, no. 6, p. 2838-2842. [CrossRef]

KELLY, M.T.; BRENNER, J. and FARMER III, J.J. Enterobacteriaceae. In: HAUSLER, William J.; HERRMANN, Kenneth L.; ISENBERG, Henry D. and SHADOMY, H. eds. Manual of Clinical Microbiology. 4^th ed. American Society for Microbiology, Washington DC, 1995, p. 347-352.

MEZRIOUI, N. and BALEUX, B. Resistance patterns of E. coli strains isolated from domestic sewages before treatment in both aerobic lagoon and activated sludge. Water Research, November 1994, vol. 28, no. 11, p. 2399-2406. [CrossRef]

NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS (NCCLS). Performance standards for antimicrobial disk susceptibility test-approved standard (document M2-A7). 7^th ed. NCCLS, Pennsylvania, USA, 2000. ISBN 1-56-238208-X.

OHLSEN, Knut; TERNES, Thomas; WERNER, Guido; WALLNER, Ursulla; LÖFFLER, Dirk; ZIEBUHR, Wilma; WITTE, Wolfgang and HACKER, Jörg. Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage. Environmental Microbiology, August 2003, vol. 5, no. 8, p. 711-716. [CrossRef]

SCHLÜTER, A.; HEUER, H.; SZCZEPANOWSKI, R.; FORNEY, L.J.; THOMAS, C.M.; PÜHLER, A. and TOP, E.M. The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group. Microbiology, November 2003, vol. 149, no. 11, p. 3139-3153. [CrossRef]

SELVARATNAM, Shivi and KUNBERGER, J.D. Increased frequency of drug-resistant bacteria andfecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge. Canadian Journal of Microbiology, August 2004, vol. 50, no. 8, p. 653-656.

SILVA, J.; ZEMELMAN, R.; MANDOCA, M.A.; HENRÍQUEZ, M.; MERINO, C. and GONZÁLEZ, C. Antibiotic- resistant gram negative bacilli isolated from sea water and shellfish. Possible epidemiological implications. Revista Latinoamericana de Microbiología, April-June 1987, vol. 29, no. 2, p.165-169.

VILANOVA, X.; MANERO, A.; CERDÀ-CUÉLLAR, M. and BLANCH, A.R. The effect of a sewage treatment plant effluent on the faecal coliforms and enterococci populations of the reception river waters. Journal of Applied Microbiology, February 2002, vol. 92, no. 2, p. 210-214. [CrossRef]

VILANOVA, X.; MANERO, A.; CERDÀ-CUÉLLAR, M. and BLANCH, A.R. The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants. Journal of Applied Microbiology, February 2004, vol. 96, no. 2, p. 279-288. [CrossRef]

Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.