Manure-Based Amendments Influence Surface-Associated Bacteria and Markers of Antibiotic Resistance on Radishes Grown in Soils with Different Textures

Abstract

A controlled greenhouse study was performed to determine the effect of manure or compost amendments, derived during or in the absence of antibiotic treatment of beef and dairy cattle, on radish taproot-associated microbiota and indicators of antibiotic resistance when grown in different soil textures. Bacterial beta diversity, determined by 16S rRNA gene amplicon sequencing, bifurcated according to soil texture (P < 0.001, R = 0.501). There was a striking cross-effect in which raw manure from antibiotic-treated and antibiotic-free beef and dairy cattle added to loamy sand (LS) elevated relative (16S rRNA gene-normalized) (by 0.9 to 1.9 log₁₀) and absolute (per-radish) (by 1.1 to 3.0 log₁₀) abundances of intI1 (an integrase gene and indicator of mobile multiantibiotic resistance) on radishes at harvest compared to chemical fertilizer-only control conditions (P < 0.001). Radishes tended to carry fewer copies of intI1 and sul1 when grown in silty clay loam than LS. Composting reduced relative abundance of intI1 on LS-grown radishes (0.6 to 2.4 log₁₀ decrease versus corresponding raw manure; P < 0.001). Effects of antibiotic use were rarely discernible. Heterotrophic plate count bacteria capable of growth on media containing tetracycline, vancomycin, sulfamethazine, or erythromycin tended to increase on radishes grown in turned composted antibiotic-treated dairy or beef control (no antibiotics) manures relative to the corresponding raw manure in LS (0.8- to 2.3-log₁₀ increase; P < 0.001), suggesting that composting sometimes enriches cultivable bacteria with phenotypic resistance. This study demonstrates that combined effects of soil texture and manure-based amendments influence the microbiota of radish surfaces and markers of antibiotic resistance, illuminating future research directions for reducing agricultural sources of antibiotic resistance.IMPORTANCE In working toward a comprehensive strategy to combat the spread of antibiotic resistance, potential farm-to-fork routes of dissemination are gaining attention. The effects of preharvest factors on the microbiota and corresponding antibiotic resistance indicators on the surfaces of produce commonly eaten raw is of special interest. Here, we conducted a controlled greenhouse study, using radishes as a root vegetable grown in direct contact with soil, and compared the effects of manure-based soil amendments, antibiotic use in the cattle from which the manure was sourced, composting of the manure, and soil texture, with chemical fertilizer only as a control. We noted significant effects of amendment type and soil texture on the composition of the microbiota and genes used as indicators of antibiotic resistance on radish surfaces. The findings take a step toward identifying agricultural practices that aid in reducing carriage of antibiotic resistance and corresponding risks to consumers.

Keywords: antibiotic resistance; antibiotics; beef; compost; dairy; greenhouse; manure; soil microbiology; vegetables.

FIG 1

Effects of soil amendments and soil texture on the average relative abundances of the 20 most abundant families of bacteria represented by OTUs recovered from the surfaces of radish taproots (n = 3, except DC turned compost in loamy sand, where n = 2) grown in either loamy sand (LS) or silty clay loam (SCL) mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with chemical fertilizer only as a control. There were no statistically significant differences in relative abundance of taxonomic groups for radishes grown among all treatments (Kruskal-Wallis, P > 0.05).

FIG 2

Nonmetric multidimensional scaling plot of unweighted UniFrac distance matrices comparing the effect of soil amendment type, i.e., manure (triangles), static compost (squares), turned compost (circles), or fertilizer control (diamonds), on the microbiota associated with radish taproot surfaces grown in either loamy sand (open symbols) or silty clay loam (closed symbols). Each data point represents an individual radish taproot grown in the corresponding conditions and are labeled as follows: F, chemical fertilizer; BC, BA, DC, and DA, manure-derived amendments originating from control beef cattle, antibiotic-treated beef cattle, control dairy cattle, or antibiotic-treated dairy cattle, respectively. Soil texture, crossed with amendment types, resulted in significant differences in bacterial beta diversity (P < 0.001, R = 0.501; ANOSIM).

FIG 3

Relative abundances of (a) intI1, (c) sul1, and (e) tet(W) and absolute abundances of (b) intI1, (d) sul1, and (f) tet(W), measured by qPCR, on taproot surfaces of radishes (n = 3) grown in loamy sand (LS) and silty clay loam (SCL) mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with chemical fertilizer only as a control. Treatments for which the values for all 3 radishes were below the limit of quantification (asterisks) were excluded from analysis. Survival models were incorporated assuming a Weibull distribution to calculate the means and standard errors (indicated by the error bars). Significant differences (P < 0.050, survival analysis) are indicated as follows: #, for the same amendment, the soil texture that yielded significantly greater gene abundance; ^, for the same soil texture, amendments that yielded gene abundances significantly different from those of the fertilizer control; +, for the same cattle type (beef or dairy), same amendment type, and same soil texture, the antibiotic condition (antibiotic or control) that yielded significantly greater gene abundance. The letters A, B, and C show statistical groupings for the effect of amendment type (manure, static compost, and turned compost) for same cattle and same soil texture.

FIG 4

Abundances of 16S rRNA genes, enumerated by qPCR, on radish taproot surfaces (n = 3) grown in loamy sand (LS) and silty clay loam (SCL) mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with chemical fertilizer only as a control. Standard errors are indicated by the error bars. Significant differences (P < 0.050, survival analysis) are indicated as follows: #, for the same amendment, the soil texture that yielded significantly greater gene abundance; ^, for the same soil texture, amendments that yielded gene abundances significantly different from those of the fertilizer control; +, for the same cattle type (beef or dairy), same amendment type, and same soil texture, the antibiotic condition (antibiotic or control) that yielded significantly greater gene abundance. The letters A and B indicate statistical groupings for the effect of amendment type (manure, static compost, and turned compost) for the same cattle and same soil texture.

FIG 5

Aerobic heterotrophic bacterial counts (HPCs) from radish taproot surfaces (n = 3) grown in loamy sand (LS) or silty clay loam (SCL) mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with a chemical fertilizer only as a control on R2A control. Standard errors are indicated by the error bars.

FIG 6

Aerobic heterotrophic bacterial counts (HPCs) from radish taproot surfaces (n = 3) on R2A supplemented with (a) ceftazidime (25 μg/ml), (b) clindamycin (10 μg/ml), (c) erythromycin (25 μg/ml), (d) sulfamethoxazole (100 μg/ml), (e) tetracycline (3 μg/ml), and (f) vancomycin (11 μg/ml). Loamy sand (LS) and silty clay loam (SCL) were mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with chemical fertilizer only as a control. Survival models were incorporated assuming a Weibull distribution to calculate the means and standard errors for Em, Smz, Tc, and Vm (indicated by the error bars). Standard errors for Cm and Cz are indicated by the error bars. Significant differences (P < 0.050, survival analysis) are indicated as follows: #, for the same amendment, the soil texture that yielded significantly greater gene abundance; ^, for the same soil texture, amendments that yielded gene abundances significantly different from that of the fertilizer control; +, for the same cattle type (beef or dairy), same amendment type, and same soil texture, the antibiotic condition (antibiotic or control) that yielded significantly greater gene abundance. The letters A and B indicate statistical groupings for effect of amendment type (manure, static compost, and turned compost) for the same cattle and same soil texture.

FIG 7

Aerobic heterotrophic bacterial counts (HPCs) from radish taproot surfaces (n = 3) on R2A supplemented with (a) ceftazidime (25 μg/ml), (b) clindamycin (10 μg/ml), (c) erythromycin (25 μg/ml), (d) sulfamethoxazole (100 μg/ml), (e) tetracycline (3 μg/ml), and (f) vancomycin (11 μg/ml), relative to HPCs on R2A medium without antibiotics. Loamy sand (LS) and silty clay loam (SCL) were mixed with amendment from antibiotic-treated beef cattle (BA), antibiotic-free beef cattle (BC), antibiotic-treated dairy cattle (DA), or antibiotic-free dairy cattle (DC) or mixed with chemical fertilizer only as a control. Survival models were incorporated assuming a Weibull distribution to calculate the means and standard errors for Em, Smz, Tc, and Vm (indicated by the error bars). Standard errors for Cm and Cz are indicated by the error bars. Significant differences (P < 0.050, survival analysis) are indicated as follows: #, for the same amendment, the soil texture that yielded significantly greater gene abundance; ^, for the same soil texture, amendments that yielded gene abundances significantly different from that of the fertilizer control. The letters A and B indicate statistical groupings for effect of amendment type (manure, static compost, and turned compost) for the same cattle and same soil texture.