J. Anim Sci. 2008. 86:2385-2391. doi:10.2527/jas.2007-0705
© 2008 American Society of Animal Science
Technical note: Occurrence in fecal microbiota of genes conferring resistance to both macrolide-lincosamide-streptogramin B and tetracyclines concomitant with feeding of beef cattle with tylosin1
J. Chen*,
F. L. Fluharty*,
N. St-Pierre*,
M. Morrison*,
and
Z. Yu*,2
* Department of Animal Sciences, The Ohio State University, Columbus 43210;and
CSIRO Livestock Industries, St. Lucia, Australia
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Abstract
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Development of antimicrobial resistance in food animals receiving antimicrobials has been well documented among bacterial isolates, especially pathogens, but information on development of antimicrobial resistance at the microbial community level during long-term feeding of antimicrobials is lacking. The objective of this study was to examine the association between inclusion of tylosin in feed and occurrence of resistance to macrolide-lincosamide-streptogramin B (MLSB) in the entire fecal microbial communities of beef cattle over a feeding study of 168 d. A completely randomized design included 6 pens housed together in 1 barn, with each pen housing 10 to 11 steers. The control and tylosin groups each had 3 pens, with the former receiving no antimicrobial whereas the latter received both tylosin and monensin (11 and 29.9 mg/ kg of feed, respectively, DM) in feed. The abundance of genes conferring resistance to MLSB (erm genes) and tetracyclines (tet genes) were quantified using class-specific, real-time PCR assays. The abundances of erm and tet genes were analyzed with pens as experimental units using the MIXED procedure of SAS. Correlations between abundance of different resistance genes were calculated using the CORR procedure of SAS. We identified 4 classes (B, F, T, and X) of erm genes in fresh fecal samples collected at wk 2, 17, and 21 of feeding. From wk 2 to 17, the abundance of erm(T) and erm(X) increased (P < 0.05), whereas that of erm(B) and erm(F) did not. The abundance of the erm genes did not further change from wk 17 to 21. The tet(A/C), tet(G), and tet gene variants encoding ribosomal protection proteins (including classes M, O, P, Q, S, T, and W) appeared to be co-selected by tylosin feeding. Such co-selection of multiresistance at community level by one antimicrobial drug used in animals has the important implication that future studies should examine resistance to not only the antimicrobials used in animals, but also other antimicrobials, especially those used in human medicine, to fully assess the potential risk associated with antimicrobial use in animals. Both the erm and tet genes appeared to be disseminated among the microbial populations in all steers housed together.
Key Words: antimicrobial resistance • erm • real-time polymerase chain reaction • tet • tylosin
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INTRODUCTION
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Use of antimicrobials in food animals is under increasing scrutiny due to concerns over potential risk to human health from increasingly widespread antimicrobial resistance (AR; Smith et al., 2002
; Anderson et al., 2003). Tylosin is among the most commonly used antimicrobials in food animals, including beef cattle (US Department of Agriculture, 2000). It is typically administered via feed to reduce liver abscesses caused by bacteria such as Fusobacterium necrophorum and Arcanobacterium pyrogenes. Although tylosin is exclusively used on food animals, erythromycin is another drug of the same macrolide-lincosamide-streptogramin B (MLSB) superfamily used in both humans and food animals (Mellon et al., 2001). Being members of the same MLSB superfamily, tylosin and erythromycin cross-select for resistance to all drugs of this superfamily, including several drugs (clarithromycin, clindamycin, azithromycin) used in the treatment of human infections. Use of tylosin in animals increased resistance to MLSB antibiotics among intestinal bacteria (Christie et al., 1983; Jackson et al., 2004), and this resistance is encoded by erm genes (Jost et al., 2004). However, such a positive correlation between use of antimicrobials and AR development was mainly demonstrated in bacterial isolates, especially pathogens (Agustin et al., 2005; Berge et al., 2006; Inglis et al., 2006). This is one limitation in public risk assessment of antimicrobial use in farm settings (Bailar and Travers, 2002), because commensal bacteria, most of which are nonculturable, constitute a much larger AR reservoir than pathogens and may play a role in spreading AR (Andremont, 2003; Luo et al., 2005), not only among commensal bacteria, but also to pathogens (Witte, 2000). Increased horizontal AR gene transfer can also facilitate development of multidrug resistance (Courvalin, 1994; Dzidic and Bedekovic, 2003). Here we examined the occurrence of resistance to MLSB in the entire fecal microbial communities in steers receiving a mixture of tylosin and monensin in feed. Possible co-selection for resistance to tetracyclines was also assessed by analyzing tet genes.
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MATERIALS AND METHODS
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The Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1988) were followed during the feeding study, which was approved by the OSU Institutional Animal Care and Use Committee.
Feeding Study
A completely randomized design experiment was conducted using 64 crossbred steers of primarily Angus and Charolais parentage housed in 6 experimental pens. Steers were 12 to 15 mo old and had received no antibiotics in their feed before the feeding study. Steers were allotted, by BW, to 6 pens (6.1 x 24 m) in a manner that allowed for a similar initial average pen BW, and pens served as the experimental units. The 6 pens were allotted randomly to either control or tylosin groups. The control group received no antimicrobial, whereas the tylosin group received tylosin and monensin at a concentration of 11 and 29.9 mg/kg of feed DM, respectively. Two pens of each feeding group had 11 steers/ pen and the third pen had 10 steers. Pens were adjacent to each other in an open-sided barn where feed bunks were under a roofed area. Two of the 3 pens in each feeding group shared a common source of water from an automatic waterer during the entire feeding study. All steers were fed the same base diet consisting of whole-shelled corn (70.0%), timothy-orchardgrass mixed hay (10.0%), ground corn (4.1%), soybean meal (7.8%), limestone (1.3%) on a DM basis. The diet was supplemented with 0.5% (DM basis) trace mineral salts of NaCl (>93% of total salts), Zn (0.35%), Mn (0.28%), Fe (0.175%), Cu (0.035%), and Co (0.007%). Additionally, vitamins A (0.01% on DM basis), vitamin D (0.01%), vitamin E (0.03%), selenium (0.05%), dynamate (0.4%), and animal-vegetable blend fat (0.3%) were provided. On a DM basis, the diet was calculated to contain 12.5% CP, 0.59% Ca, 0.36% P, 0.77% K, and to have NEm of 2.0 Mcal/kg and NEg of 1.4 Mcal/kg. Steers in each pen had access to their own concrete feed bunk, and water was available ad libitum. This study was a part of a large study and some pens were excluded from the AR analysis. The feeding trial lasted for 168 d of the finishing phase. One fresh fecal grab sample was collected from the bowel of each steer after 2, 17, and 21 wk of feeding. Approximately 50 g of each sample was immediately stored at –80°C until DNA extraction.
DNA Extraction, PCR, and Real-Time PCR
Individual fecal samples were thawed on ice and thoroughly mixed with a sterilized spatula. One composite fecal sample was prepared for each pen by pooling an equal amount (1 g, based on wet weight) of individual fecal samples of each pen. Each of the pen-based composite fecal samples was thoroughly mixed using a sterile spatula. Community DNA was extracted from each of these pen-based composite fecal samples using the method described previously (Yu and Morrison, 2004). The abundance of erm(A), erm(B), erm(C), erm(F), erm(T), erm(X), tet(A/C), tet(G), the tet genes encoding ribosomal protection proteins (RPP), as well as total bacteria was quantified for each of the pen-based composite fecal samples using the class- or groups-specific real-time PCR assays as described previously (Yu et al., 2005; Chen et al., 2007). Briefly, each class- or group-specific PCR primer set was used to prepare a sample-derived standard for that class or group from a pooled community DNA sample that contains an equal amount of genomic DNA from each of the DNA samples to be quantified. This standard was purified, quantified fluorometically, diluted serially, and used in real-time PCR to quantify the abundance of respective gene class or group. The abundance of total bacteria was quantified against a sample-derived standard prepared using the universal primers 27f and 1525r as described previously (Yu et al., 2005; Chen et al., 2007). All real-time PCR assays were performed in triplicate. The absolute abundance (gene copies/gram of fecal samples) and the relative abundance (gene copies per million copies of bacterial rrs genes) of erm and tet genes were calculated for each pen from the gene copy number per reaction quantified by each real-time PCR (Yu et al., 2005; Chen et al., 2007).
Statistical Analysis
The pen-based real-time PCR data of erm and tet gene abundances were log10 transformed to normalize errors and then analyzed as a completely randomized design with repeated measurements using the MIXED procedure (SAS Inst. Inc., Cary, NC), with pens serving as experimental units. The mixed model included the fixed effects of treatments, weeks, their interaction effects, and the random effect of pens within treatments. Error correlations due to the repeated measures on pens were modeled using an unstructured covariance structure. Normality of residuals was tested using the Shapiro-Wilk test of the UNIVARIATE procedure of SAS (Shapiro and Wilk, 1965). Means separation was conducted using the Fisher’s protected least significant difference test of SAS, with significance declared at P 
0.05. The association between the abundance of 2 gene classes was expressed as a linear relationship measured by Pearson product-moment correlations, which were calculated using the CORR procedure of SAS. The absolute abundance (gene copies/gram of fecal sample) and relative abundance (gene copies per million copies of bacterial rrs gene) of erm genes and tet genes were graphed using the GraphPad Prism 4 (GraphPad Software, San Diego, CA).
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RESULTS AND DISCUSSION
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Using class-specific real-time PCR assays, we detected erm(B), erm(F), erm(T) and erm(X) in all samples analyzed, whereas erm(A) and erm(C) were either detected at very low levels or not detected (data not shown). Overall, erm(B) had greater (P < 0.02) abundance, in both absolute and relative terms, than the other classes of erm gene (Figure 1). Among the tet genes, RPP tet genes exhibited greater (P < 0.05) abundances (on both absolute and relative bases) than the other 2 tet gene groups (Figure 2). These results were in accordance with the previous finding in both beef cattle manure and swine manure samples (Yu et al., 2005; Chen et al., 2007). These results also corroborate the widespread presence of these genes in beef cattle feces.
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Figure 1. Abundance of the erm gene in feces of beef cattle: I) Absolute abundance (least squares means of log-transformed gene copies/gram of wet feces) and II) relative abundance (least squares means of log-transformed gene copies per million copies of bacterial rrs genes, cpmc) at 2, 17, and 21 wk from the start of the feeding study. Panels A and a = erm(B); panels B and b = erm(F), panels C and c = erm(T); panels D and d = erm(X). a–cDifferent letters denote differences (P 0.05) among all samples within a panel.
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Figure 2. Abundance of the tet gene in feces of beef cattle: I) Absolute abundance (least squares means of log-transformed gene copies/gram of wet feces) and II) relative abundance (least squares means of log-transformed gene copies per million copies of bacterial rrs genes, cpmc) at 2, 17, and 21 wk from the start of the feeding study. Panels A and a = tet(A/C); panels B and b = tet(G); panels C and c = RPP tet. a,bDifferent letters denote differences (P 0.05) among all samples within a panel.
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Occurrence of erm Genes
Because this study was part of a larger study, no fecal sample was, unfortunately, collected at the beginning of the feeding study. The absolute abundance of erm(B) and erm(F) did not increase over time within either feeding group (P > 0.11 and P > 0.10, respectively). Except the tylosin group having more erm(B) than the control group at wk 2 (P = 0.03), the 2 feeding groups had similar abundance of erm(B) and erm(F) (P > 0.11) at wk 2, 17, and 21 (Figure 1
, panels A and B). From wk 2 to 17, erm(T) increased in absolute abundance in both the control (P = 0.04) and the tylosin (P = 0.01) groups (Figure 1, panel C). For erm(X), only the absolute abundance increased (P = 0.05) in the tylosin group over the same period (Figure 1, panel D). From wk 17 to 21, no increase (P > 0.13) in abundance was observed in either feeding group in any of the 4 classes of erm genes except erm(X). The total bacterial abundance, as determined as rrs copies/gram of fecal sample, changed over time in both the feeding groups (ranging from 2.1 x 109 to 4.0 x 1010 rrs copies/g), but no clear trend was observed among them. Nonetheless, the relative abundance of these erm genes showed similar patterns of increase as their absolute abundance (Figure 1, panels a to d). It is interesting to note that erm(B) and erm(F) had greater (P < 0.05) abundance than erm(T) and erm(X) at wk 2, and the former 2 erm classes did not increase (P > 0.10) over the course of the study. Although this observation cannot be fully explained by the data of this study, it may reflect the possibility that bacterial populations carrying erm(B), erm(F), or both, had been rapidly selected for by the feeding of tylosin during the first 2 wk so that they might have reached the maximal population sizes sustainable in the fecal community. Collectively, these results suggested that the feeding of beef cattle with tylosin at a concentration (11 mg/kg of feed) below the minimum inhibitory concentration of most isolated resistant bacteria had selected for erm genes in the entire fecal microbial communities in steers. This observation corroborates the positive association of antimicrobial use and development of AR, which was primarily observed among bacterial isolates, in entire microbial communities of beef cattle feces. Additionally, future studies should sample more frequently during the first few weeks of feeding to fully determine the dynamics of AR development.
Occurrence of tet Genes and Co-Selection.
The absolute abundance of tet(A/C) tended to increase (P = 0.09) from wk 2 to 17, whereas its relative abundance increased (P = 0.05) over the same period (Figure 2, panels A and a). For tet(G), both its absolute and relative abundances increased (P 0.02) in both the control and the tylosin groups (Figure 2, panels B and b). The initially abundant RPP tet gene did not increase (P > 0.06) over the feeding period (Figure 2, panels C and c). Similarly to the erm genes, no increase (P > 0.26) was observed in either absolute or relative abundance of these tet genes from wk 17 to 21. Interestingly, the abundance of the 3 groups of tet genes exhibited similar dynamic patterns to that of the erm genes even though no tetracycline was fed to these cattle. Indeed, both the absolute and relative abundances of the 3 groups of tet genes were positively correlated to that of the erm genes, especially the erm(T) and erm(X) genes (Table 1). Positive correlation was also found for tet(A/C) and tet(G), as well as tet(G) and RPP tet genes. This observed positive correlation may be attributable to co-selection by the use of tylosin. Such co-selection for multiresistance by a single antimicrobial drug has been well demonstrated among resistant bacterial isolates (Guerra et al., 2003; Agustin et al., 2005; Burgos et al., 2005). A similar pattern of occurrence between erm genes and tet genes was also observed in beef cattle manures and swine manures (Yu et al., 2005; Chen et al., 2007). The co-residence of multiple resistance genes on the same mobile genetic elements such as plasmids and transposons was shown to be responsible for the development of most multiresistances in bacterial isolates (Walsh, 2006). The mechanism(s) for such co-selection for multiresistances at the microbial community level, however, remains to be elucidated. Nonetheless, the use of tylosin selected for not only erm genes, but also tet genes at a microbial community level. This finding implies that AR to other antimicrobials, especially those reserved for human medicine (e.g., expanded-spectrum cephalosporins, aminoglycosides, fluoroquinolones), also needs to be examined to better assess the potential risk imposed by the use of antimicrobials in food animals.
Dissemination of erm and tet Gene Among the Steers.
The control group surprisingly also showed increases, although in a delayed fashion [except for tet(A/C) and tet(G)], in the abundance of several erm genes and tet genes (Figures 1 and 2). The abundance of these genes in the control group reached a level comparable to that in the tylosin group by wk 17. Thus, no significant time by treatment interactions were observed. Because all pens were housed together randomly in 1 barn and shared a common manure scraper and drinking water (2 of the 3 pens within each group), it is hypothesized that the resistant bacteria initially selected for in the tylosin group probably had been disseminated to the control group by direct animal-to-animal contact and sharing of the same manure scraper and the same drinking water. To avoid such confounding factors in future studies, different feeding groups of animals have to be separated, spatially or temporally, to prevent or minimize possible dissemination of bacteria among different pens. The persistence of erm and tet genes in the antimicrobial-free groups also corroborates the previous finding that AR, once developed, can be persistent even without the selective pressure from the selecting antimicrobials (Gillespie, 2001; Andersson, 2003).
Results of this study documented the increased occurrence of both erm and tet genes in fecal microbial communities of beef cattle concomitant with feeding a low level of tylosin. Different treatment groups of animals need to be well separated spatially or temporally to avoid transmission of resistance between different treatment groups. To fully assess the potential risk imposed to humans by use of antimicrobials in animals, antimicrobial resistance to not only the antimicrobial drugs used in animals, but also other antimicrobials, especially those used in human medicine, needs to be examined closely.
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Footnotes
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1 This work was supported partially by a USDA-CSREES award (M.M., Z.Y., and F.F., 2003-45050-01616) as well as by an OARDC SEED award (Z.Y., 2005-OHOA1188). 
2 Corresponding author: yu.226{at}osu.edu
Received for publication November 2, 2007.
Accepted for publication April 28, 2008.
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Abstract
INTRODUCTION
