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Published online 12 October 2005
Published in J Environ Qual 34:1952-1957 (2005)
DOI: 10.2134/jeq2005.0017
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Ecological Risk Assessment

Antibacterial Activity of Soil-Bound Antibiotics

Yogesh Chandera, Kuldip Kumara, Sagar M. Goyalb and Satish C. Guptaa,*

a Department of Soil, Water and Climate, 1991 Upper Buford Circle, University of Minnesota, Saint Paul, MN 55108
b Department of Veterinary Population Medicine, 1333 Gortner Avenue, University of Minnesota, Saint Paul, MN 55108

* Corresponding author (sgupta{at}umn.edu)

Received for publication January 18, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
There is some concern that antibiotic residues in land-applied manure may promote the emergence of antibiotic resistant bacteria in the environment. The goal of this study was to determine whether or not soil bound antibiotics are still active against bacteria. The procedure involved sorbing various amounts of tetracycline or tylosin on two different textured soils (Webster clay loam [fine-loamy, mixed, superactive, mesic Typic Endoaquolls] and Hubbard loamy sand [sandy, mixed, frigid Entic Hapludolls]), incubating these soils with three different bacterial cultures (an antibiotic resistant strain of Salmonella sp. [SalmonellaR], an antibiotic sensitive strain of Salmonella sp. [SalmonellaS], and Escherichia coli ATCC 25922), and then enumerating the number of colony forming units relative to the control. Incubation was done under both static and dynamic conditions. Soil-adsorbed antibiotics were found to retain their antimicrobial properties since both antibiotics inhibited the growth of all three bacterial species. Averaged over all other factors, soil adsorbed antimicrobial activity was higher for Hubbard loamy sand than Webster clay loam, most likely due to higher affinity (higher clay content) of the Webster soil for antibiotics. Similarly, there was a greater decline in bacterial growth with tetracycline than tylsoin, likely due to greater amounts of soil-adsorbed tetracycline and also due to lower minimum inhibitory concentration of most bacteria for tetracycline than tylosin. The antimicrobial effect of tetracycline was also greater under dynamic than static growth conditions, possibly because agitation under dynamic growth conditions helped increase tetracycline desorption and/or increase contact between soil adsorbed tetracycline and bacteria. We conclude that even though antibiotics are tightly adsorbed by clay particles, they are still biologically active and may influence the selection of antibiotic resistant bacteria in the terrestrial environment.

Abbreviations: CFU, colony forming units • MIC, minimum inhibitory concentration • SalmonellaR, antibiotic resistant strain of Salmonella sp. • SalmonellaS, antibiotic sensitive strain of Salmonella sp


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
WIDESPREAD USE of antibiotics in food animal production is viewed as one of the major reasons for the emergence of antibiotic resistant bacteria (ARB) in the environment (Jørgensen and Halling-Sørensen, 2000; Rooklidge, 2004). According to some estimates, as much as 80% of the orally administered antibiotics may pass through the animals unchanged (Levy, 1992; Thiele-Bruhn, 2003) and end up in manure. The concentrations of antibiotics in manure have been reported from trace levels to be as high as 200 mg L–1 (Kumar et al., 2004, 2005). These antibiotics find their way into the terrestrial environment when manure from antibiotic-fed animals is land applied as a source of crop nutrients.

Persistence of antibiotics in soil depends on many factors including soil type, climate, and class of antibiotics (Boxall et al., 2004). Most antibiotics are biodegradable in soils but some of them have a long half-life. Weerasinghe and Towner (1997) reported a half-life of 87 to 173 d for virginiamycin in sandy silt and silty sand soils under laboratory conditions. Kümmerer (2003) found that cyclosporin-A persisted in a garden soil for many months in spite of the presence of several degrading bacteria. Marengo et al. (1997) determined that less than 1% of sarafloxacin, an antibiotic used widely in poultry production, degraded in the soil after 80 d of incubation.

Antibiotics in manure-applied land may leach to ground water or move to surface waters via surface runoff. De Liguoro et al. (2003) detected oxytetracycline in soil (<5 µg kg–1 of soil) at a 60-cm depth after treatment with cattle manure while Hamscher et al. (2002) reported the presence of tetracycline (170 µg kg–1) at a 30-cm soil depth, 6 mo after liquid swine manure was applied. Antibiotics have also been detected in rivers (Kolpin et al., 2002, 2004) and marine sediments (Nygaard et al., 1992; Hektoen et al., 1995; Lai et al., 1995).

Antibiotics have both quantitative and qualitative effects on the native microbial communities in the terrestrial environment (Nygaard et al., 1992). Although antibiotic concentrations in most soils are not at therapeutic levels to cause inhibitory effects on bacterial population, they may still influence the selection of antibiotic resistant bacteria in the environment (USEPA, 2002; Nygaard et al., 1992; Gavalchin and Katz, 1994; Kümmerer, 2003). In a Danish study, Sengeløv et al. (2003) observed a temporary increase (after 3–5 d) in tetracycline resistant bacteria in soil following the addition of pig manure. Jensen et al. (2001) reported increased antibiotic resistance among Pseudomonas sp. and Bacillus cereus isolates from fields after pig manure application. Onan and LaPara (2003) isolated a higher proportion of tylosin resistant bacteria from fields that had been treated with cattle and swine manure, as compared to fields where no manure was applied. An increase in antibiotic resistance in soil bacteria after manure application may be due to (i) exchange of genetic elements between soil bacteria and antibiotic resistant manure bacteria, (ii) exchange of genetic elements among antibiotic producing soil microorganisms, or (iii) in situ selection pressure from a low level of antibiotics in manure.

Many antibiotics have a strong tendency to bind with soil particles (Tolls, 2001; Kumar et al., 2005). Distribution coefficients (Kd,solid) as high as 2300, 6310, and 128 L kg–1 have been reported for tetracycline, enrofloxacin, and tylosin, respectively (Kumar et al., 2005). Although studies have shown the persistence of antibiotics in soil after manure application, not much is known about the antimicrobial properties of soil-bound antibiotics. This study was designed to determine whether soil-bound antibiotics retain their antibacterial activity or are inactivated on binding with soil. This is one of the first questions that needs to be addressed before studying the role of antibiotic laden manure application on emergence of antibiotic resistant bacteria in soil.

The antibiotics studied were tetracycline and tylosin, two widely used growth promoters in food animal production, especially swine and turkeys. Both these antibiotics also bind strongly to many different soils (Kumar et al., 2005).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soils and Antibiotics
The soils used in the study were: a Hubbard loamy sand (78% sand and 10% clay; 2.2% organic matter content) from Becker, MN, and a Webster clay loam (30% sand and 34% clay; 4.4% organic matter content) from Lamberton, MN. These soils represent two different extremes of clay and organic matter contents. Hubbard loamy sand is a typical soil of the Central Sands of Minnesota where turkey production and dairy industry dominate. Water table in this area is relatively shallow (<5 m) and as a result recreational lakes abound. There is strong evidence of contamination of shallow ground water from nonpoint sources (Myette, 1984). Webster clay loam is a typical soil of the west central and southern Minnesota, an area that drains to the Minnesota River, considered as one of the 20 most polluted rivers in the United States (Payne, 1994). Beef and swine production dominate this region.

Both soils were sterilized by autoclaving three times to ensure sterility. Both tetracycline and tylosin were of chemical grade (Sigma Chemicals, St. Louis, MO) and their stock solutions (1 mg mL–1) were made in deionized water.

Antibiotic Adsorption
Various amounts of antibiotic were adsorbed on a given soil by mixing 10 g of sterilized soil with 50 mL of antibiotic solution of different concentrations (0, 50, 100, 200, and 500 µg mL–1). These concentrations were selected to cover the range of antibiotic concentrations in manure as reported in the literature. Soil–antibiotic suspensions were equilibrated in the dark at room temperature on an orbital shaker (250 rpm) for 24 h. After equilibration, soil particles were separated from the suspension by centrifugation at 3000 x g for 15 min and the supernatant was collected for antibiotic analysis. The centrifuged soil was then washed twice with deionized water to remove any loosely bound antibiotic as well as the original antibiotic solution in soil pores. Each washing was followed by centrifugation to separate the soil from the suspension. After washings, the soil was air dried in a Petri dish for overnight at room temperature.

The antibiotic concentrations in the supernatants were determined by high performance liquid chromatography (HPLC) (Beckman Coulter, Fullerton, CA) with diode array detector. Isocratic separation was achieved using a 150- x 4.6-mm C18 Adsorbosphere OPA HR 5-µm column (Alltech, Deerfield, IL). The chromatographic conditions were as follows: flow rate 1 mL min–1; mobile phase consisting of 40% acetonitrile (mobile phase A) and 60% water/methanol containing 10 mM oxalic acid (mobile phase B); injection volume 50 µL; and detection at wavelength 280 nm. All solvents used were of HPLC grade. Tetracycline and tylosin concentrations in the supernatants were determined from a linear standard curve. Antibiotic concentration in the supernatant was used to calculate the amount of soil adsorbed antibiotic:

[1]
where Csoil = antibiotic adsorbed per g of soil; Csolution = antibiotic concentration in solution, mg L–1; Vsolution = volume of the solution added, L; Csupernatant = antibiotic concentration in the supernatant after equilibration, mg L–1; Vsupernatant = volume of the supernatant, L; and Wsoil = weight of the soil, g.

Antimicrobial Activity of Adsorbed Antibiotics
Antimicrobial activity of soil-bound antibiotics was tested against three bacterial strains: an antibiotic resistant strain of Salmonella sp. (SalmonellaR), an antibiotic sensitive strain of Salmonella sp. (SalmonellaS), and Escherichia coli ATCC 25922. Salmonella sp. was selected because it is an important food borne pathogen and is often present in the animal manure. E. coli ATCC 25922, a quality control strain, was included in the study because it has a standardized antimicrobial susceptibility pattern.

Minimum inhibitory concentrations (MIC) of tetracycline and tylosin for the above three microorganisms was determined with sensititer plates (TREK Diagnostic Systems, Cleveland, OH) using the procedure described by Chander et al. (2005). For determining the antimicrobial activity of soil bound antibiotics, 0.5 g of antibiotic equilibrated soil was inoculated with a given bacterial culture. The soil–bacterium mixture was then incubated at 37°C under static or dynamic conditions. For static conditions, the soil was inoculated with 0.1 mL bacterial inoculum adjusted to 0.5 McFarland turbidity (1 x 108 colony forming units) and incubated in a stationary condition. For dynamic conditions, the soil was inoculated with 0.5 mL of tryptic soy broth (TSB) (Becton, Dickinson and Company, Sparks, MD) containing 0.1 mL of 0.5 McFarland adjusted bacterial inoculum and incubated on a shaker (200 rpm) at 37°C. Each experiment was repeated three times.

Static and dynamic conditions were included in the experiment to simulate field soil conditions. Static condition represents the field situation when there is no mixing of manure with the soil and there is no additional input of nutrients. Dynamic condition represents circumstance when there is mixing of soil and manure or when there is additional inputs of nutrients such as during rainfall after manure application.

Enumeration of Bacterial Colonies
After 24 h of incubation, soil samples were plated on solid media for enumeration of bacterial colonies. For this, 10-fold serial dilutions of soil–bacterium mix were made in buffered peptone water (BPW, pH 7.0) and mixed by vortexing. Appropriate dilutions were plated on solidified agar media (without antibiotics). For enumeration of Salmonella sp. and E. coli, brilliant green agar and MacConkey agar (Becton, Dickinson and Company) were used, respectively. Inoculated plates were incubated at 37°C and examined for number of colony forming units (CFU) after 24 h of incubation.

Data Analysis
Antimicrobial activity of soil-bound antibiotics was calculated as percent decline in CFU on mixing of a bacterial culture with antibiotic-equilibrated soil relative to the control (without antibiotic):

[2]
Differences in antibiotic adsorption by soil was tested at p = 0.05 using the paired t test (SYSTAT Version 6.0; SPSS, 1996). Differences in percent decline in CFU of three bacterial species with soil adsorbed antibiotics were analyzed using the analysis of variance (ANOVA) with Statistical Analysis System (SAS Institute, 1996) software. Coefficients of the power function describing percent decline in CFU vs. soil adsorbed antibiotic were obtained by fitting a linear regression to the log transformed data on both axes.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Sorption of Antibiotics
Amount of tetracycline and tylosin adsorbed on two soils is given in Table 1. Each data point represents one observation. Paired t test analysis of the data showed that irrespective of antibiotic type, antibiotic sorption was significantly (p ≤ 0.05) higher on the Webster than the Hubbard soil. Results on higher sorption of antibiotics on clay rich soil are similar to those reported earlier (Rabølle and Spliid, 2000; Loke et al., 2002; Kumar et al., 2005). Higher antibiotic adsorption on Webster soil is mainly due to its greater exchange capacity because of higher clay (34% vs. 10%) and organic matter (4.4% vs. 2.2%) contents. Loke et al. (2002) also reported that binding of oxytetracycline (a tetracycline class antibiotic) was influenced by ionic binding to divalent metal ions such as Mg2+ and Ca2+ and because of this metal ion–soil–oxytetracycline complex antibiotic binding to soil was stronger than manure.


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  		Table 1. Adsorption of antibiotics on Hubbard sandy loam and Webster clay loam soils at various solution concentrations. Antibiotic adsorbed on the soil after equilibration was calculated using Eq. [1].

 
For a given soil type, differences between tetracycline and tylosin adsorption were not statistically significant (p ≤ 0.05). The lack of statistical significance between antibiotic adsorption for a given soil is mainly due to the small number of observations (n = 3). When the data were lumped together for both soils (n = 8), tetracycline sorption was significantly (p ≤ 0.05) higher than tylosin sorption. These results are similar to those obtained by Rabølle and Spliid (2000) in which they found stronger adsorption of oxytetracycline (Kd, slope of the linear adsorption isotherm = 417 for sand soil and 1027 for sandy loam soil) than tylosin (Kd = 8 for sand soil and 127 for sandy loam soil). Other research by the authors of this paper (Gupta et al., 2003) has also shown that larger tylosin molecules are adsorbed on the surface of the clay particles whereas the smaller tetracycline molecules tend to penetrate into the inter-clay spaces. Since a greater amount of tetracycline is retained in soil (Table 1) and MIC of tetracycline for most bacteria is lower than that of tylosin, it is possible that soil microorganisms may develop higher resistance to tetracycline than tylosin.

Antimicrobial Activity of Soil-Adsorbed Antibiotics
Figure 1 shows an example of percent decline in CFU of SalmonellaS as a function of soil-adsorbed tetracycline. The power function fitted to the data is described by Eq. [3]:

[3]
where a and b are empirical constants. The values of constants a and b for various combinations of three bacterial species, two antibiotics, and two soils are given in Table 2. The relationships and the coefficients show that soil adsorbed antibiotics are effective in inhibiting the growth of microorganisms but the rate of inhibition (slope of the line, b) depends on the soil type, bacterial species, and growth conditions.



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  		Fig. 1. Percent decline in colony forming units (CFU) of antibiotic sensitive strain of Salmonella sp. (SalmonellaS) at various concentrations of soil-adsorbed tetracycline. Soil adsorbed antibiotic concentration was calculated using Eq. [1]. Lines represent the best-fit values.

 

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  		Table 2. Parameters of the power function used to describe the percent decline in number of colony forming units (CFU) with increasing soil adsorbed antibiotic concentration.

 
In general, the rate of decline (b) with antibiotic concentration was generally higher with the Hubbard soil than the Webster soil (Table 2). This is likely due to stronger binding of antibiotics to high clay and high organic matter Webster clay loam soil and thus their slower release to the microorganisms. For cases where the slope (b) values are higher for the Webster soil than the Hubbard soil, there was greater variability (lower R2 values) in percent decline of CFU.

Table 3 shows the analysis of variance of various factors affecting the growth of three bacterial species when incubated with soil-adsorbed antibiotics. The differences between the two antibiotics, two soil types, three bacterial strains, and five antibiotic concentrations were found to be statistically significant at p = 0.05. Differences in percent decline of CFU between two growth conditions (static and dynamic) were also significant but at a lower probability level (p = 0.10). There were also two, two-way interactions, antibiotics x strains and antibiotics x growth conditions, that were significant but at different probability levels (p = 0.05 and p = 0.10, respectively). These interactions suggest that the soil-adsorbed antibiotic effects on percent decline in CFU are strain and growth conditions specific.


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  		Table 3. Analysis of variance of the data on percent decline in colony forming units (CFU) for three bacterial strains, two soils, two antibiotics, two growth conditions, and five antibiotic concentrations.

 
In Table 4 the mean percent decline for main effects is given. Averaged over all other factors, tetracycline was more active (46% decline) in inhibiting the growth of all three microorganisms as compared to tylosin (29% decline). This could be because there is more tetracycline retained in soil than tylosin (Table 1) and also because most bacteria have lower resistance (lower MIC) to tetracycline than tylosin. Apparently, both these factors eclipse the strong adsorption tendency of tetracycline (inter lattice adsorption) over tylosin (surface adsorption) in soil.


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  		Table 4. Mean percent decline in bacterial growth for main effects.

 
The percent decline in three strains followed the trend: E. coli (45%) > SalmonellaS (39%) > SalmonellaR (26%). Since SalmonellaR is inherently resistant, it showed lesser decline in percent CFU than SalmonellaS and E. coli. There was a significant interaction between antibiotics and strains (Fig. 2) . In the presence of tetracycline, percent decline in E. coli was much greater than that of either Salmonella sp. This may be because the MIC of tetracycline for E. coli is much lower (≤0.5 µg mL–1) than that for SalmonellaS (≤2 µg mL–1) and SalmonellaR (>16 µg mL–1). With tylosin, there was no statistical difference in percent decline in CFU between SalmonellaS (MIC < 32 µg mL–1) and E. coli (MIC > 32 µg mL–1) or between E. coli and SalmonellaR (MIC ≥ 20 µg mL–1). Also, percent decline in CFU of SalmonellaR and E. coli was significantly higher in presence of soil-adsorbed tetracycline than tylosin (Fig. 3) . This difference was not significant for SalmonellaS suggesting that this bacterial strain was equally sensitive to both the antibiotics in adsorbed phase.



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  		Fig. 2. Percent decline in colony forming units (CFU) of three microbial strains with two different antibiotics. Values are means over two soils, five different antibiotic concentrations, and two different growth conditions. Bars with same letter are not significantly different at p = 0.05. SalmonellaR, antibiotic resistant strain of Salmonella sp.; SalmonellaS, antibiotic sensitive strain of Salmonella sp.

 


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  		Fig. 3. Percent decline in colony forming units (CFU) for two growth conditions with two different antibiotics. Values are means over two soils, five different antibiotic concentrations, and three bacterial species. Bars with same letter are not significantly different at p = 0.10.

 
Percent decline in bacteria was higher for Hubbard sandy loam (43%) than Webster clay loam (28%) (Table 4), most likely due to stronger binding of antibiotic to Webster soil (Table 1). As expected, there was a greater decline (52%) of bacteria at higher antibiotic concentrations (solution concentration of 500 µg mL–1) as compared to the decline (26%) at lower antibiotic concentration (solution concentration of 50 µg mL–1) (Table 4).

Percent bacterial decline was also higher under dynamic (42%) than under static (33%) conditions (Table 4). However, this difference was mainly due to soil-adsorbed tetracycline and not due to tylosin (Fig. 3). Higher percent decline in CFU under dynamic condition with tetracycline is possibly because agitation helped desorb more tetracycline from inter-clay layers under dynamic than static conditions. Since tylosin is surface adsorbed, its desorption should be about same under both dynamic and static conditions. It is also likely that agitation (dynamic conditions) helped increase contact between the bacteria and the inter-lattice tetracycline.

For growth under dynamic conditions, bacterial culture was inoculated in a growth medium (3% tryptic soy broth), which allowed microbial culture to divide rapidly. Actively growing cultures have active protein synthesis machinery, and since both of these antibiotics inhibit protein synthesis (Fluit et al., 2001), the inhibitory effect of these antibiotics should be more pronounced under dynamic than static growth conditions. Tetracycline inhibits protein synthesis by binding to 30S subunit of the ribosome and blocking the binding of tRNA at the acceptor site, whereas tylosin binds to 50S subunit of the ribosome and inhibits the peptide bond formation (Chopra and Roberts, 2001; Fluit et al., 2001). It is thus possible that some of the differences in percent bacterial decline with soil-adsorbed tetracycline may be due to the presence of nutrients in growth medium. However, presence of nutrients in the growth medium (dynamic conditions) had little effect on percent decline in number of CFU with soil-adsorbed tylosin.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Results of this study showed that tetracycline and tylosin adsorbed to soils retained their antimicrobial activity suggesting that even if antibiotics are tightly sorbed to soil, they can still play a role in the emergence of antibiotic resistant bacteria in the environment. Since tetracycline has a longer half-life than tylosin (Kumar et al., 2005), soil microbes may be more induced to develop tetracycline resistance than tylosin resistance. The antibiotic solution concentrations used in this study are higher than those normally found in field soils after manure application, thus it remains to be seen what effect lower levels of antibiotic concentrations may have under field conditions.


   ACKNOWLEDGMENTS
 
This study was supported in part by a grant from the USDA-NC SARE program, Grant no. UNE/25 6205-0034-023/USDA SARE.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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