Increase in the prevalence of oxolinic acid resistant Acinetobacter spp. observed in a stream receiving the effluent from a freshwater trout farm following the treatment with oxolinic acid-medicated feed

Abstract

Acinetobacter spp. were used as bacterial indicators to monitor antimicrobial resistance in a freshwater trout farm before and after treatment with oxolinic acid (OA)-medicated feed. The level of susceptibility to four antimicrobial agents was determined in 331 Acinetobacter isolates obtained by a selective procedure from a pond (n=100), the inlet channel (n=105), and the stream receiving the farm effluent (n=126). Before treatment, OA resistance was detected only among isolates from the pond (40%), in which the last medication with OA was dated back to 6 months before. Following treatment, a high prevalence of OA resistance was observed among isolates from both the pond (33–53%) and the effluent recipient (21–55%). In contrast, no OA resistant strains were isolated from the inlet channel. OA resistant strains were significantly more resistant to oxytetracycline (OT) compared with OA sensitive strains (P<0.0001, odds ratio=43.6). Furthermore, OA resistant isolates showed an increase of at least 10-fold times in the MIC values for ciprofloxacin (CIP) resistance compared with sensitive isolates. Phenotypic characterisation and PCR-based fingerprinting analysis showed lower diversity among Acinetobacter isolates from the pond and the stream receiving the farm effluent in comparison with isolates from the inlet channel. Since the two biotypes predominant in the pond and the effluent recipient included the vast majority of OA resistant isolates, the low diversity of Acinetobacter spp. observed at these sites could have been enhanced by exposure to the drug. The results of this study indicate that the use of OA in inland farms can affect the levels of antimicrobial resistance and diversity in the microflora of natural aquatic habitats situated downstream. Since the ecological consequences deriving from changes of the indigenous aquatic microflora are unknown, the careful consideration of the use of OA in aquaculture was suggested.

Introduction

Oxolinic acid (OA) is an older member of the quinolones, a class of synthetic antimicrobial agents which include important agents for control of bacterial disease in human medicine. Although new generations of quinolones out-perform OA with respect to both bactericidal activity and bioavailability, its relatively modest cost, low fish toxicity and satisfactory performance render it a widely used drug in aquaculture (Wells, 1995).

Both laboratory and field experiments have shown that the concentration of OA in marine sediment is not significantly reduced 180 days after medication Samuelsen et al., 1994, Hektoen et al., 1995. The exposure to OA has been shown to affect the bacterial community of marine sediment with respect to bacterial numbers, degradation of organic material and development of resistance Nygaard et al., 1992, Hansen et al., 1993.

Previous studies have shown that higher numbers of antibiotic resistant bacteria are likely to be present in aquaculture habitats treated with antimicrobial agents compared with the same habitats prior to treatment (DePaola et al., 1995), similar aquaculture habitats treated with a smaller amount of antibacterials (Hervig et al., 1997), or aquaculture habitats with no recent history of antimicrobial use (McPhearson et al., 1991). The method traditionally used for the measurement of antimicrobial resistance at the population level consists in bacteriological counts on agar plates containing a particular concentration of the antimicrobial agent. The main drawback of this method is the use of a single arbitrary breakpoint for the determination of antimicrobial resistance in different bacterial taxa. In fact, the use of a single breakpoint, corresponding to the amount of antimicrobial agent added to the medium, does not take into account the variability in the levels of antimicrobial susceptibility existing among different bacterial taxa. Furthermore, the selective count of resistant bacteria on agar plates containing antimicrobial agents is affected by the bacterial composition of the sample and seems to overestimate the number resistant bacteria (Jones et al., 1986).

Bacteria belonging to the genus Acinetobacter are non-motile, non-fermentative, Gram-negative coccobacilli that can be easily isolated from soil, water, sewage, human skin and a large variety of foodstuffs (Towner, 1996). Acinetobacter spp. have been found at densities of 10⁴ organisms per 100 ml in freshwater ecosystems (LaCroix and Cabelli, 1982) and described as one of the major taxa among aerobic heterotrophic bacteria isolated from freshwater fish farms (Allen et al., 1983). In the present study, Acinetobacter spp. were used as bacterial indicators for monitoring antimicrobial resistance in a freshwater trout farm undergoing treatment with OA. Spatial and temporal differences in the levels of antimicrobial resistance of aquatic Acinetobacter spp. were considered indicative of the selective pressure exerted on the entire aquatic microflora . The choice of Acinetobacter spp. as bacterial indicators was prompted by their ubiquitous distribution and particular ability to develop antibiotic resistance in environments subjected to antibiotic selective pressure Towner, 1997, Guardabassi et al., 1998. Acinetobacter spp. were isolated using a genus-specific selective procedure and the levels of susceptibility to OA and three other antimicrobial agents were compared between different sampling sites and times. In addition, phenotypic characterisation and PCR fingerprinting were used to detect temporal and spatial variations in the distribution of Acinetobacter spp.