Lactams and Florfenicol Antibiotics Remain Bioactive in Soils While Ciprofloxacin, Neomycin, and Tetracycline Are Neutralizedᰔ Murugan Subbiah,1 Shannon M
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2011, p. 7255–7260 Vol. 77, No. 20 0099-2240/11/$12.00 doi:10.1128/AEM.05352-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved. -Lactams and Florfenicol Antibiotics Remain Bioactive in Soils while Ciprofloxacin, Neomycin, and Tetracycline Are Neutralizedᰔ Murugan Subbiah,1 Shannon M. Mitchell,2 Jeffrey L. Ullman,4 and Douglas R. Call1,3* Departments of Veterinary Microbiology and Pathology1 and Biological Systems Engineering2 and Paul G. Allen School for Global Animal Health,3 Washington State University, Pullman, Washington 99164, and Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida 326114 Received 3 May 2011/Accepted 13 August 2011 It is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assump- tion was tested by separately adding 10 different antibiotics (>200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitive Escherichia coli strain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicator E. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dom- inated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, Thus, residual antibiotics in soil do not .(0.6 ؍ but sensitive bacteria were still unaffected by tetracycline (P necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment. Antibiotic resistance in bacterial populations is an inevitable Importantly, antibiotic residues are distributed heteroge- outcome of using antibiotics, and consequently prudent-use neously both at a microscale (animal pen) and at a regional practices are strongly advocated in both human and veterinary scale (e.g., feedlot versus pasture). Thiele-Bruhn (34) summa- medicine. Antibiotics have been used for therapeutic, prophy- rized a range of reported antibiotic residue concentrations in lactic, and growth promotion purposes in livestock production soils that varied for macrolides (0.0085 to 0.067 ppm), sulfon- (1). It is difficult to determine how much of the world’s anti- amides (0.001 to 0.011 ppm), trimethoprim (0.0005 ppm), fluo- biotic production is used in agriculture, but the World Health roquinolones (0.006 to 0.052 ppm), and tetracyclines (0.039 to Organization estimates that at least half of all antibiotics are 0.9 ppm). With the exception of those for tetracyclines, these used in food animals (36). In 2009, the U.S. Food and Drug values are below what has been proposed as a biologically Administration estimated that 13,067 metric tons of antimicro- effective concentration (0.1 ppm or 100 g/kg) for feces and bials were sold or distributed in the United States for use in soil but well above the 0.0001 ppm considered the biological food-producing animals; over 60% was represented by tetra- threshold for groundwater (32, 34). Hospital effluents may cyclines (Tets) and ionophores (8). contain higher concentrations of antibiotics (e.g., 0.02 to 0.08 Many of the antibiotics used in food animals are at least ppm ampicillin [Amp], 0.0007 to 0.125 ppm ciprofloxacin partially excreted as biologically active compounds in urine and [Cip]) than soil (11, 19). Using an in vitro model, Chander et al. feces, where they presumably can continue to exert a selective (4) found that high concentrations (500 to 2,500 ppm) of tet- effect on soil- and waterborne microflora. For example, Kumar racyclines in soil can select for antibiotic-resistant strains of et al. (18) reported that 10 to 90% of the antibiotics adminis- Salmonella sp. and Escherichia coli, but it is unclear if concen- tered to feedlot animals are excreted unaltered through feces trations this high are found in the environment because most and urine (manure) and thus potentially reach soil and water. studies report lower concentrations (12). Kemper et al. (13) reviewed several studies that reported vet- It remains difficult to ascertain the true biological impact of erinary antibiotic residues in the aquatic and terrestrial envi- antibiotics in the environment when residual concentrations ronments associated with agricultural lands. are the primary metric of concern. Furthermore, several stud- ies have shown that the proximal-spatial distribution of resis- tance genes is consistent with animal production facilities be- * Corresponding author. Mailing address: Department of Veteri- ing a source of the resistance traits (15, 22, 23, 33). It is unclear, nary Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: however, if the higher prevalence of resistance genes proximal (509) 335-8529. E-mail: [email protected]. to production facilities is due to selection from environmental ᰔ Published ahead of print on 19 August 2011. exposure to residual antibiotics or simply due to the higher 7255 7256 SUBBIAH ET AL. APPL.ENVIRON.MICROBIOL. concentration of resistant organisms being shed from the ani- TABLE 1. Soil physicochemical propertiesa mals themselves. That is, the presence and abundance of re- Soil typec,d sistance genes themselves do not unequivocally demonstrate Parameterb Unit selection in the environment. Silt-loam Sand-loam Sandy The motivation for the current study was to further examine pH 4.71 (0.01) 5.54 (0.01) 7.75 (0.13) EC dS/g soil 5.78 (0.18) 6.22 (0.07) 1.53 (0.05) the biological impacts exerted by antibiotics in soils; i.e., to TOC %C 2.27 (0.09) 2.06 (0.03) 0.24 (0.007) move beyond the level of simply measuring antibiotic concen- Sulfur %S 0.03 (0.002) 0.05 (0.004) 0.0001 (0.0005) Nitrogen %N 0.17 (0.006) 0.17 (0.003) 0.0002 (0.0007) trations and assuming that the presence of a drug is equivalent Calcium cmol(ϩ)kgϪ1 5.70 5.20 0.02 to a selective pressure and therefore a public health concern. Magnesium cmol(ϩ)kgϪ1 1.20 1.00 0.02 Potassium cmol(ϩ)kgϪ1 1.70 0.68 0.08 This is a particularly important question for regulatory bodies Sodium cmol(ϩ)kgϪ1 0.09 0.17 0.05 that need to determine regulatory thresholds for antibiotic CEC cmol(ϩ)kgϪ1 22.00 15.00 7.30 residues or contamination in the environment. From public Sand % (wt) 4 50 98 Silt % (wt) 79 45 2 health and economic perspectives, there is no reason to incur Clay % (wt) 17 5 0 extra monitoring or mitigation costs for antibiotics if they do a Modified from Paternostre (21). not exhibit a significant biological impact. In contrast, antibi- b EC, electrical conductivity; TOC, total organic carbon (calculated from the otics that continue to exert a biological impact in the environ- total carbon and carbonate content); CEC, cation exchange capacity. c Soils were collected in the vicinities of Pullman, WA (silt-loam), Puyallup, ment may warrant considerably more attention. WA (sand-loam), and Paterson, WA (sandy). The model employed in this study was a simple one whereby d Values in parentheses are standard deviations. we added a relatively high concentration of antibiotics (Ն200 ppm) to soil slurries and measured the concentration and in- hibitory effect from the supernatant after a 24-h exposure pe- riod. We expected to find that most antibiotics would remain using the flow injection analysis described by Ruzicka and Hansen (27) while the biologically effective at these high concentrations, and our in- extractable cations were analyzed using the method described by Field et al. (9). tention was to characterize the decay of this activity over time Soil slurry experiments. Soil-water slurries (Ϸ20% soil [wt/vol]) were pre- and under different conditions. Contrary to our expectations, pared in glass tubes by mixing1gofsoil with 5 ml of 0.01 M calcium chloride- amended nanopure water (with and without 1 mM sodium azide). Antibiotic these experiments demonstrated that from a biological per- stocks were freshly prepared, separately, in dimethyl sulfoxide (DMSO) at a spective several of the antibiotics tested are effectively neutral- concentration of 10 mg/ml. Soil slurries were spiked with 200 ppm (equivalent to ized upon contact with soil in a process that appears to be 200 g/ml) of each antibiotic in separate tubes, but 1,000 ppm was used for dominated by adsorption. Importantly, however, representa- sulfadiazine and sulfadimethoxine to achieve growth inhibition (the MIC of sulfa tives from two classes of drugs (-lactams and florfenicol [Flo]) drugs is over 512 ppm for the E. coli K-12 strain). Then, the slurries were mixed well (vortexed for 30 s) and the tubes were wrapped in aluminum foil and retained biological activity after exposure to different soils and, incubated on a shaker (150 rpm) at room temperature (23 to 25°C) for 24 h. consequently, may represent an important and unappreciated After incubation the supernatants were collected by centrifuging the slurries at factor in the selection for antibiotic resistance in soils.