Effects of Tylosin Administration Routes on the Prevalence of Antimicrobial Resistance Among Fecal Enterococci of Finishing Swine

Abstract

Antibiotics can be administered orally or parenterally in swine production, which may influence antimicrobial resistance (AMR) development in gut bacteria. A total of 40 barrows and 40 gilts were used to determine the effects of tylosin administration route on growth performance and fecal enterococcal AMR. The antibiotic treatments followed Food and Drug Administration label directions and were as follows: (1) no antibiotic (CON), (2) 110 mg tylosin per kg feed for 21 d (IN-FEED), (3) 8.82 mg tylosin per kg body weight through intramuscular injection twice daily for the first 3 d of each week for 3 weeks (IM), and (4) 66 mg tylosin per liter of drinking water (IN-WATER). Antibiotics were administered during d 0 to 21 and all pigs were then fed the CON diet from d 21 to 35. Fecal samples were collected on d 0, 21, and 35. Antimicrobial susceptibility was determined by microbroth dilution method. No evidence of route × sex interaction (p > 0.55) was observed for growth performance. From d 0 to 21, pigs receiving CON and IN-FEED had greater (p < 0.05) average daily gain (ADG) than those receiving IM, with the IN-WATER group showing intermediate ADG. Pigs receiving CON had greater (p < 0.05) gain-to-feed ratio (G:F) than IM and IN-WATER, but were not different from pigs receiving IN-FEED. Overall, enterococcal isolates collected from pigs receiving IN-FEED or IM were more resistant (p < 0.05) to erythromycin and tylosin than CON and IN-WATER groups. Regardless of administration route, the estimated probability of AMR to these two antibiotics was greater on d 21 and 35 than on d 0. In summary, IM tylosin decreased ADG and G:F in finishing pigs, which may be because of a response to the handling during injection administration. Tylosin administration through injection and feed resulted in greater probability of enterococcal AMR to erythromycin and tylosin compared with in-water treatment.

Introduction

In the swine industry, antimicrobial feed additives have traditionally been used to prevent enteric infections, promote growth, and improve production efficiency (Muhl and Liebert, 2007).

However, the continued expansion of antimicrobial resistance (AMR) among commensal and pathogenic bacteria constitutes a major public health concern. Therefore, in swine production systems, there is considerable interest and effort in identifying feeding and management practices that maintain and improve production efficiency without promoting AMR in bacteria.

Antibiotics are administered either in-feed, in-water, or parenterally. The oral route, through either feed or water, is by far the most common route of administration of antibiotics in pigs (Callens et al., 2012; Merle et al., 2012). Oral administration is more convenient when treating a large number of pigs compared with individual treatment through the injectable route. Nevertheless, oral administration exposes gut bacteria directly to high concentrations of antibiotics and thus has been hypothesized to have a greater potential in promoting the emergence and amplification of AMR in the gut. A study using a mouse model suggests that oral administration of antibiotics has a greater impact on promoting and amplifying AMR in gut microbiota compared with intravenous injection (Zhang et al., 2013). However, to our knowledge, no study has been conducted to compare the impacts of oral administration of antibiotics through feed or water versus injectable administration on the development of AMR among gut bacteria in pigs.

Tylosin is used to treat or prevent swine dysentery, and other bacterial infections, including arthritis, ileitis, and erysipelas in swine (Dritz et al., 2002). Tylosin was selected as the antibiotic treatment because of its widespread use in the U.S. swine industry and its varying formulations that can be administered through different routes. The use of tylosin in swine production is ubiquitous. The understanding of how the oral route of administration affects resistance selection in the gut is fundamental to our use of this drug in swine production, and the way to evaluate the effect is to compare it with other routes. Therefore, the objective of this study was to determine the effects of tylosin administration route on the growth performance and the development of AMR in fecal enterococci of finishing pigs.

Materials and Methods

All experimental procedures in this study were approved by the Kansas State University Institutional Animal Care and Use Committee (IACUC #3529.10; Manhattan, KS).

Animals and housing

The study was conducted at the Kansas State University Swine Teaching and Research Center in Manhattan, KS. Pigs were housed in an environmentally controlled barn with completely slatted concrete floor. Each pen (1.52 m × 1.52 m) was equipped with a single-hole stainless steel feeder and a cup waterer for ad libitum access to feed and water. Each drinker was equipped with an individual water reservoir allowing for independent water treatment. Each two pens (one barrow pen and one gilt pen sharing the same treatment) were segregated by solid pen dividers to minimize nasal contact and manure cross contamination among pigs from different treatment groups; the combination of these two pens served as the experimental unit. A total of 40 barrows and 40 gilts (Line 600 × 241; DNA, Columbus, NE) were individually housed and used in a 35-d trial. Pigs were individually weighed, blocked by initial body weight (93.9 ± 3.57 kg), sex, and barn location, and assigned to pens 17 d before the start of the experiment. Early allotment was carried out to avoid pig movement across pens on d 0 and minimize cross contamination for fecal sample collection. Pigs were weighed and feed disappearance was recorded on d 0, 21, and 35 to determine average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F). The water reservoir was weighed and refilled twice daily to determine daily water consumption for each pig.

Diets and experimental design

On d 0, immediately after fecal collection, experimental treatments were assigned to the animals. The antibiotic treatments followed Food and Drug Administration (FDA) label directions for swine dysentery control and were as follows: (1) a corn-soybean meal-based diet with no antibiotic (CON), (2) a basal diet with 110 mg tylosin (Tylan^®100; Elanco Animal Health, Indianapolis, IN) per kg feed for 21 d (IN-FEED), (3) an average target dose of 8.82 mg tylosin (Tylan^®200; Elanco Animal Health, Indianapolis, IN) per kg body weight through intramuscular injection twice daily for the first 3 d of each week during the 3-week treatment period (IM), and (4) 66 mg tylosin (Tylan^®Soluble; Elanco Animal Health, Indianapolis, IN) per liter of drinking water for the first 3 d of each week during the 3-week treatment period (IN-WATER). Antibiotic treatments were terminated on d 21 and all pigs were fed the CON diet from d 21 to 35.

Complete diet samples were obtained at manufacture and delivered to the Kansas State University Swine Laboratory, Manhattan, KS, and stored at −20°C until analysis (NRC, 2012). Feed samples were analyzed for dry matter, crude protein, ether extract, calcium, and phosphorous at Ward Laboratories, Inc. (Kearney, NE). Standard procedures from AOAC International (2006) were followed for analysis of moisture (Method 934.01), crude protein (Method 990.03), ether extract (Method 920.39), calcium and phosphorous (Method 985.01).

Fecal sample collection

Fecal samples from each pig were collected into individual Whirl-Pak^® bags (Nasco, Ft. Atkinson, WI) on d 0 (baseline), 21 (end of treatment period), and 35 (end of post-treatment period). Samples were transported on ice to the laboratory at Kansas State University (Manhattan, KS) and stored at 4°C before processing within 24 h.

Bacterial isolation, identification, and polymerase chain reaction detection of erm(B) gene

For bacterial isolation, ∼1 g of feces from each sample was suspended in 9 mL of phosphate-buffered saline. Fifty microliters of the fecal suspension was then spread-plated onto M-Enterococcus agar plates for the selective isolation of Enterococcus spp. from each fecal sample. Unless otherwise specified, all the culture media were obtained from Difco (Becton-Dickinson and Company, Sparks, MD). M-Enterococcus plates were incubated at 42°C for 24–36 h. Two putative colonies (pin-point red, pink, or metallic red) were selected from each M-Enterococcus agar; next, each was individually streaked onto a blood agar plate (Remel, Lenexa, KS) and incubated at 37°C for 24 h. Preliminary genus confirmation of each of the enterococcal isolates was performed by esculin hydrolysis. Two confirmed Enterococcus isolates per original fecal sample were preserved using cryoprotect beads (Cryocare; Key Scientific Products, Round Rock, TX) and stored at −80°C for future use.

DNA was extracted from enterococcal isolates by suspending a single colony from the blood agar plate in nuclease-free water with Chelex^® 100 Resin (Bio-Rad Laboratories, Hercules, CA) and boiling for 10 min. Species identification was carried out to identify Enterococcus faecium and Enterococcus faecalis using multiplex polymerase chain reaction (PCR) (Jackson et al., 2004). E. faecium ATCC19434 and E. faecalis ATCC29212 (American Type Culture Collection, Manassas, VA) isolates served as reference strains for speciation. The primer and PCR condition for detection of erm(B) gene was as per Amachawadi et al. (2010). E. faecium BAA-2127 strain served as positive control for detection of erm(B) gene. The primers were supplied by Integrated DNA Technologies (IDT, Coralville, IA).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing, as outlined by the Clinical and Laboratory Standards Institute (CLSI, 2018), was performed on one of the two stored isolates per fecal sample to determine the minimal inhibitory concentrations to each of 16 antimicrobials using the Sensititre^® (TREK Diagnostic Systems, Oakwood Village, OH) microbroth dilution procedure. The enterococcal isolate preserved in cryoprotect beads was streaked onto a blood agar plate and incubated at 37°C for 24 h. Individual colonies were selected and suspended in demineralized water (TREK Diagnostic Systems) and turbidity was adjusted to 0.5 McFarland turbidity standards. Then, 10 μL of the bacterial inoculum was added to cation-adjusted Mueller–Hinton broth and vortexed. The Sensititre automated inoculation delivery system (TREK Diagnostic Systems) was used to dispense 100 μL of the broth into National Antimicrobial Resistance Monitoring System panel plates (CMV3AGPF; TREK Diagnostic Systems) designed for Gram-positive bacteria. A table of resistance breakpoints and evaluated concentrations for antimicrobials of National Antimicrobial Resistance Monitoring System panel were presented in a previous study (Feldpausch et al., 2016). E. faecalis ATCC 29212 (American Type Culture Collection) strain was included as the quality control for the susceptibility testing. Plates were incubated at 37°C for 18 h and then bacterial growth was assessed using Sensititre ARIS and Vizion^® systems (TREK Diagnostic Systems). CLSI (2018) guidelines were used to classify each bacterial isolate as resistant or nonresistant (intermediate and susceptible) according to the breakpoints established for each antimicrobial.

Statistical analysis

Responses on growth performance, water intake, and tylosin intake were measured at the pen (pig) level and were analyzed using general linear mixed models. The linear predictors included the fixed effects of tylosin administration route (CON, IN-FEED, IM, and IN-WATER), sex (gilt and barrow), and their interaction. The model also included the random effects of block and block × route cross product. The latter random effect specified the pair of pens with one barrow pen and one gilt pen sharing the same treatment as the level of replication for tylosin administration route. Residual assumptions were checked using studentized residuals.

For AMR data, frequency tables of resistant and nonresistant isolates for each antibiotic were initially evaluated. For gentamicin, kanamycin, streptomycin, and vancomycin, none of the fecal isolates were categorized as resistant and thus no further statistical analyses were performed for these antibiotics. For each remaining antibiotic, frequency tables on resistant and nonresistant isolates were further evaluated by tylosin administration route, sampling day, and their combination. These tables were used to identify potential extreme category problems during model fitting. Subcategories with all resistant or nonresistant isolates or frequencies close to these extremes can lead to model fitting problems because of quasicomplete separation of data points, also known as extreme category problem.

For each antibiotic, the probability of AMR was estimated using a generalized linear mixed model with a Bernoulli distribution on the AMR responses and a logit link function. The linear predictor included the fixed effects of tylosin administration route, sex, sampling day, enterococcal species, and their interactions, as well as the random blocking effect and its cross products with tylosin administration route and with gender to identify the proper level of replication for each fixed-effect factor. Because of the presence of extreme category problems, it was not possible to fit the three-way interaction for chloramphenicol, linezolid, nitrofurantoin, penicillin, quinupristin/dalfopristin, tigecycline, ciprofloxacin, daptomycin, erythromycin, lincomycin, tetracycline, and tylosin. For similar reasons, it was also not possible to fit two-way interactions between administration route and sampling day for linezolid, nitrofurantoin, penicillin, quinupristin/dalfopristin, and tigecycline, as well as any interaction involving sex for ciprofloxacin, daptomycin, erythromycin, lincomycin, tetracycline, and tylosin. Overdispersion was assessed using the maximum-likelihood-based fit statistic Pearson chi-square over degree of freedom. In all cases, final models used for inference showed no evidence for overdispersion.

Pairwise comparisons were conducted using a Tukey–Kramer or Bonferroni adjustment, as appropriate in each case. Statistical models were fit using the GLIMMIX procedure of SAS (version 9.4; SAS Institute, Inc., Cary, NC). In all cases, the final model used for inference was fit using residual (pseudo-) likelihood implemented with a Newton–Raphson optimization with ridging. Least-square mean estimates of growth responses and of probability of AMR are presented, along with corresponding standard error of mean or 95% confidence intervals. Results were considered significant at p ≤ 0.05, and marginally significant at 0.05 < p ≤ 0.10.

Results

Growth performance

No evidence of route × sex interaction (p > 0.55) was observed for any of the growth responses during treatment, post-treatment, or overall periods (Table 1). During the treatment period (d 0–21), the main effect of administration route marginally contributed to ADG response (p = 0.098). Pigs that received CON and IN-FEED had greater (p < 0.05) ADG than those receiving IM tylosin, with IN-WATER pigs showing intermediate ADG. For the main effect of sex, barrows grew marginally faster (p = 0.094) than gilts during the treatment period regardless of tylosin administration route. ADFI was greater (p = 0.031) in barrows than in gilts, but there was no evidence for any effect of tylosin administration route on ADFI (p = 0.219). Overall, there was no evidence of any effect of IN-FEED tylosin on G:F relative to CON pigs. In contrast, administration of tylosin through IM or IN-WATER decreased G:F (p < 0.05) compared with pigs from CON. No evidence of sex effect was observed for G:F during the treatment period. During the post-treatment period (d 21–35), no evidence for any effects of administration route or sex was observed for any growth responses (p > 0.26). Overall (d 0–35), there was no evidence that growth performance was influenced by the tylosin administration route; barrows had marginally greater (p = 0.068) ADFI than gilts but no evidence of differences in ADG or G:F were observed.

Table 1.

Effects of Tylosin Administration Route and Sex on Growth Performance of Finisher Pigs

	Tylosin administration route^*					Sex			p<
	CON	IN-FEED	IM	IN-WATER	SEM	Barrow	Gilt	SEM	Route	Sex	Route × sex
Treatment (d 0–21)
ADG, kg	1.26^a	1.26^a	1.15^b	1.22^ab	0.034	1.25	1.20	0.023	0.098	0.094	0.554
ADFI, kg	3.64	3.72	3.55	3.82	0.099	3.78	3.59	0.071	0.219	0.031	0.822
G:F	0.347^a	0.339^ab	0.324^b	0.322^b	0.0067	0.331	0.335	0.0046	0.041	0.606	0.652
Post-treatment (d 21–35)
ADG, kg	1.20	1.21	1.16	1.17	0.033	1.19	1.18	0.024	0.601	0.844	0.987
ADFI, kg	3.74	3.69	3.53	3.67	0.087	3.70	3.61	0.067	0.292	0.269	0.879
G:F	0.322	0.330	0.327	0.322	0.0079	0.322	0.329	0.0060	0.844	0.381	0.750
Overall (d 0–35)
ADG, kg	1.23	1.24	1.15	1.20	0.027	1.23	1.19	0.018	0.117	0.155	0.756
ADFI, kg	3.68	3.71	3.54	3.76	0.086	3.75	3.60	0.066	0.262	0.068	0.837
G:F	0.337	0.335	0.326	0.322	0.0057	0.328	0.332	0.0035	0.195	0.257	0.472
Water intake, L/d^†	6.14	6.45	6.87	6.06	0.287	6.56	6.20	0.241	0.179	0.310	0.566
Tylosin intake, g	—	8.61^b	18.00^a	3.69^c	0.148	10.20	10.01	0.123	0.001	0.262	0.425

There were 40 barrows and 40 gilts (Line 600 Duroc × Line 241; DNA, Columbus, NE; initially 94 ± 3.6 kg) housed with 1 pig per pen and 10 replicate pens per treatment per sex.

CON = pigs received no antibiotic; IN-FEED = pigs received 110 mg tylosin per kg feed for 21 d; IM = pigs received 8.82 mg tylosin per kg body weight through intramuscular injection twice daily for the first 3 d of each week during the 3-week treatment period; IN-WATER = 66 mg tylosin per liter of drinking water for the first 3 d of each week during treatment period.

†

Measured during treatment period only.

abcd

Mean values with different superscripts within a row differ (p < 0.05).

ADFI, average daily feed intake; ADG, average daily gain; G:F, gain-to-feed ratio; SEM, standard error of mean.

Concerning average daily water intake, there was no evidence (p > 0.10) for any effects of tylosin administration route or sex (Table 1). Among the medicated pigs, total tylosin dose administrated per pig was the greatest through IM, second highest through IN-FEED, with the IN-WATER route being the lowest (p < 0.01).

Prevalence of fecal enterococci and erm(B) gene

A total of 480 enterococcal isolates consisting of 120 isolates per treatment group (control, feed, water, and injectable) and sampling day (d 0, 7, 14, 21, 28, and 35) were obtained. Of these, a total of 292 (292/480; 60.8%) and 188 (188/480; 39.2%) isolates were E. faecium and E. faecalis. Both, treatment and sampling days did not affect the prevalence of either species significantly (p > 0.05). No evidence of route × day interaction or the main effect of administration route was observed for the prevalence of erm(B) gene among treatments (p > 0.54). The prevalence of erm(B) gene increased (p < 0.001) during the treatment period (22.7% and 59.6% on d 0 and 21, respectively) but then decreased (p < 0.001) to baseline level on d 35 (13.8%; Table 2).

Table 2.

Effects of Tylosin Administration Route and Sampling Day on the Prevalence of erm(B) Gene

Antibiotics and treatment period	Tylosin administration route ^a				Probability, p<
Antibiotics and treatment period	CON	IN-FEED	IM	IN-WATER	Route	Day	Route × day
erm(B)					0.661	0.001	0.545
Baseline (d 0)	24 [10–49]^b	24 [10–49]	15 [5–38]	30 [13–54]
Treatment (d 21)	45 [24–68]	66 [41–84]	75 [51–90]	50 [28–72]
Post-treatment (d 35)	9 [2–32]	19 [7–43]	20 [7–44]	10 [2–33]

Values represent the estimated prevalence of erm(B) gene among 20 enterococcal isolates per sampling day (d 0, 21, or 35); susceptibility was determined according to National Antimicrobial Resistance Monitoring System (CLSI, 2018; https://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM581395.pdf) established breakpoints. One fecal sample was collected per pen per day and 1 enterococcal isolate per fecal sample was assessed. There was a total of 80 pigs (Line 600 × 241; DNA, Columbus, NE; initially 94 ± 3.6 kg) housed with 1 pig per pen and 10 replicates per treatment route.

Values in parenthesis indicate 95% confidence intervals.

Antimicrobial resistance

There was no evidence for any effects of either E. faecium and/or E. faecalis on the antimicrobial susceptibilities of all antibiotics tested. Table 3 illustrates the estimated probability of AMR—among enterococcal isolates in response to tylosin administration route and sampling day—to antibiotics critically important to human medicine (WHO, 2012), namely, ciprofloxacin, daptomycin, erythromycin, gentamicin, kanamycin, linezolid, penicillin, streptomycin, tigecycline, tylosin, and vancomycin. No enterococcal isolates showed resistance to gentamicin, kanamycin, streptomycin, or vancomycin for the duration of the study. For ciprofloxacin, there was no evidence of interaction or main effects involving tylosin administration route, sex, or sampling day on AMR in the study period. For daptomycin, only the main effect of sampling day was evident on AMR (p < 0.001), whereby the probability of AMR decreased during the treatment period and increased thereafter regardless of administration route or sex. For erythromycin, no evidence of route × sampling day interaction was apparent; however, both main effects significantly (p < 0.05) contributed to explain AMR. Overall, the probability of AMR to erythromycin was marginally greater (p < 0.10) when pigs received tylosin through either IN-FEED or IM relative to IN-WATER, with that of CON pigs being intermediate. Moreover, the probability of AMR to erythromycin increased from d 0 to 21 and d 35 regardless of tylosin administration route. For linezolid, penicillin, and tigecycline, there was no evidence for any effects of tylosin administration route, sex, or sampling day on AMR. For tylosin, the main effect of administration route marginally contributed to explain AMR (p = 0.068), whereby the probability of AMR to tylosin was greater (p < 0.05) in enterococcal isolates collected from pigs receiving tylosin through IN-FEED and IM (69% and 70% of isolates, respectively) compared with CON pigs and those receiving tylosin through IN-WATER (50% and 50%, respectively). The probability of AMR to tylosin increased (p < 0.01) from d 0 to 21 and d 35.

Table 3.

Effects of Tylosin Administration Route and Sampling Day on the Probability of Antimicrobial Resistance of Fecal Enterococci Isolates to Critically Important Antimicrobials

Antibiotics and treatment period	Tylosin administration route ^a				Probability, p<
Antibiotics and treatment period	CON	IN-FEED	IM	IN-WATER	Route	Day	Route × day
Ciprofloxacin					0.318	0.904	0.986
Baseline (d 0)	10 [2–33]^b	20 [8–43]	20 [8–43]	0 [.]
Treatment (d 21)	10 [2–33]	25 [11–48]	20 [8–43]	15 [5–38]
Post-treatment (d 35)	10 [2–33]	25 [11–48]	10 [2–33]	15 [5–38]
Daptomycin					0.312	0.001	0.708
Baseline (d 0)	70 [47–86]	55 [33–75]	60 [38–79]	40 [21–62]
Treatment (d 21)	40 [21–62]	25 [11–48]	25 [11–48]	20 [8–43]
Post-treatment (d 35)	50 [29–71]	40 [21–62]	40 [21–62]	55 [33–75]
Erythromycin					0.025	0.004	0.258
Baseline (d 0)	55 [33–76]	65 [42–83]	45 [24–67]	35 [17–58]
Treatment (d 21)	50 [28–71]	80 [57–93]	95 [72–99]	50 [28–71]
Post-treatment (d 35)	65 [42–83]	80 [57–93]	75 [51–90]	70 [46–87]
Linezolid					0.688	0.942	—
Baseline (d 0)	0 [0]	20 [8–42]	10 [2–35]	0 [0]
Treatment (d 21)	20 [7–47]	10 [2–35]	0 [0]	0 [0]
Post-treatment (d 35)	15 [5–37]	10 [3–32]	10 [3–32]	0 [0]
Penicillin					0.697	0.187	—
Baseline (d 0)	5 [0.7–27]	10 [2–33]	0 [0]	0 [0]
Treatment (d 21)	0 [0]	5 [0.7–27]	0 [0]	0 [0]
Post-treatment (d 35)	10 [2–33]	10 [2–33]	10 [2–33]	0 [0]
Tigecycline					0.279	0.832	—
Baseline (d 0)	85 [63–95]	90 [68–98]	95 [71–99]	100
Treatment (d 21)	90 [68–98]	90 [68–98]	100	95 [74–99]
Post-treatment (d 35)	90 [68–98]	90 [68–98]	100	85 [62–95]
Tylosin					0.068	0.001	0.233
Baseline (d 0)	45 [24–68]	55 [32–76]	30 [13–54]	35 [17–58]
Treatment (d 21)	50 [28–72]	75 [51–90]	90 [67–98]	50 [28–72]
Post-treatment (d 35)	55 [32–76]	75 [51–89]	75 [51–89]	65 [41–83]

Values represent the estimated probability of resistance among 20 enterococcal isolates per sampling day (d 0, 21, or 35); susceptibility was determined according to National Antimicrobial Resistance Monitoring System (CLSI, 2018; https://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM581395.pdf) established breakpoints. One fecal sample was collected per pen per day and one enterococcal isolate per fecal sample was assessed. There was a total of 80 pigs (Line 600 × 241; DNA, Columbus, NE; initially 94 ± 3.6 kg) housed with 1 pig per pen and 10 replicates per treatment route; None of the enterococcal isolates were identified as resistant to gentamicin, kanamycin, streptomycin, and vancomycin.

Values in parenthesis indicate 95% confidence intervals.

Table 4 gives the estimated probability of AMR of enterococcal isolates to antibiotics considered highly important or important to human medicine, namely, chloramphenicol, quinupristin/dalfopristin, lincomycin, tetracycline, and nitrofurantoin (WHO, 2012). E. faecalis is intrinsically resistant to quinupristin/dalfopristin (synercid), so we removed these isolates from the final analyses. There was no evidence for any effects of tylosin administration route, sex, and sampling day on AMR to chloramphenicol, lincomycin, or tetracycline. For quinupristin/dalfopristin susceptibility data among E. faecium isolates, we did not find any evidence of tylosin administration route, sex, and sampling day (p > 0.05). For nitrofurantoin, only the main effect of sampling day significantly contributed to explain AMR (p = 0.002), whereby the probability of AMR to nitrofurantoin was not significantly modified during the treatment period but decreased (p < 0.01) thereafter (22, 27, and 2% on d 0, 21, and 35, respectively) regardless of sex or tylosin administration routes.

Table 4.

Effects of Tylosin Administration Route and Sampling Day on the Probability of Antimicrobial Resistance of Fecal Enterococci Isolates to Highly Important and Important Antimicrobials

	Tylosin administration route ^a				Probability, p<
	CON	IN-FEED	IM	IN-WATER	Route	Day	Route × day
Chloramphenicol					0.331	0.234	0.935
Baseline (d 0)	19 [7–44]^b	14 [4–38]	3 [0.3–26]	4 [0.4–28]
Treatment (d 21)	10 [2–33]	9 [2–32]	4 [0.4–28]	5 [0.4–28]
Post-treatment (d 35)	19 [7–44]	14 [4–38]	19 [7–44]	8 [2–32]
Lincomycin					0.996	0.555	0.340
Baseline (d 0)	95 [72–99]	86 [61–96]	76 [52–90]	91 [67–98]
Treatment (d 21)	100 [.]	91 [67–98]	95 [71–99]	81 [56–93]
Post-treatment (d 35)	86 [62–96]	95 [72–99]	95 [72–99]	95 [72–99]
Nitrofurantoin					0.331	0.002	—
Baseline (d 0)	20 [7–43]	10 [2–33]	35 [17–58]	25 [10–49]
Treatment (d 21)	25 [10–49]	30 [13–54]	15 [5–38]	40 [20–63]
Post-treatment (d 35)	0 [0]	0 [0]	0 [0]	10 [3–31]
Quinupristin/dalfopristin					0.688	0.942	—
Baseline (d 0)	0 [0]	20 [8–42]	10 [2–35]	0 [0]
Treatment (d 21)	20 [7–47]	10 [2–35]	0 [0]	0 [0]
Post-treatment (d 35)	15 [5–37]	10 [3–32]	10 [3–32]	0 [0]
Tetracycline					0.753	0.104	0.747
Baseline (d 0)	80 [55–93]	80 [55–93]	75 [50–90]	80 [55–93]
Treatment (d 21)	80 [55–93]	90 [65–98]	95 [70–99]	80 [55–93]
Post-treatment (d 35)	90 [65–98]	85 [60–96]	95 [70–99]	90 [65–98]

Values represent the estimated probability of resistance among 20 enterococcal isolates per sampling day (d 0, 21, or 35); susceptibility was determined according to National Antimicrobial Resistance Monitoring System (CLSI, 2018; https://www.fda.gov/downloads/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/UCM581395.pdf) established breakpoints for human medicine. Clindamycin breakpoints are used as an indicator for interpretation of lincomycin. One fecal sample was collected per pen per day and one enterococcal isolate per fecal sample was assessed. There was a total of 80 pigs (Line 600 × 241; DNA, Columbus, NE; initially 94 ± 3.6 kg) housed with 1 pig per pen and 10 replicates per treatment route.

Values in parenthesis indicate 95% confidence intervals.

Discussion

In this study, we evaluated the effects of tylosin administration route on the growth performance and the selection and expansion of AMR among fecal enterococci of finishing pigs. Tylosin was selected as the antibiotic treatment because of its widespread use in the U.S. swine industry and its varying formulations that can be administered through different routes. It has been reported in studies (NCR-89 Committee on Confinement Management of Swine, 1986; Pilcher et al., 2015) that feeding tylosin at a low dosage (44 or 22 ppm) promoted ADG and G:F of growing-finishing pigs. However, other studies (Lillie et al., 1997; Dritz et al., 2002; Van Lunen, 2003) have suggested a lack of growth-promoting response of tylosin when fed to finishing pigs according to these regimens. In this study, the tylosin in-feed regimen was approved for control of porcine proliferative enteropathies at 100 g/ton (110 mg/kg of feed). As of January 1, 2017, all indications for improved feed efficiency or rate of gain were removed from the labels of medically important antimicrobials used in food animals. At the label therapeutic dose used in this study, we did not observe any evidence for differences in growth performance among pigs fed tylosin-medicated feed and those with no antibiotic treatment. A potential reason for this observation is that pigs in this study were individually housed and had ∼15% greater ADFI and 20% greater ADG than the normally group-housed pigs of similar weight range and raised on the same research site. Moreover, the treatment period in the study was only 21 d, which is relatively a short duration to see differences in growth performance. In addition, the good hygienic condition of the university research environment may have also contributed to the lack of any observed growth response to this feed antibiotic because of lack of disease occurrence. Pigs from the IM group had decreased ADG and G:F than control pigs, which may be a result of pig reaction to the handling and injection procedure. However, it remains unclear why pigs offered medicated water were less feed efficient than control pigs.

Because tylosin has a significant Gram-positive antibacterial spectrum component, fecal enterococci were chosen to evaluate the impact of administration route on AMR development. Enterococci are considered as major nosocomial pathogens and also as a reservoir of AMR genes (Jackson et al., 2004). Macrolide resistance in swine enterococci and its cross resistance to erythromycin are thought to be the result of tylosin use (Jackson et al., 2004). In enterococci, resistance to macrolides has been very well documented (Aarestrup et al., 2000). Evidence from earlier studies suggests that erm(B) is the most widely distributed macrolide resistance gene in piglets (Jackson et al., 2004; Patterson et al., 2007). Consistent with this spectrum, in this study tylosin and erythromycin resistance were observed among enterococcal isolates and their prevalence was sensitive to tylosin administration route. Alteration in the efflux pumps that remove antibiotics from the cell or the modification of the bacterial target structure induces acquired resistance to macrolides, including tylosin and erythromycin (Roberts et al., 1999). Acquisition and expansion of macrolide resistance among enterococci as a result of tylosin use in swine production has been well documented (Aarestrup et al., 2000; Jackson et al., 2004).

With regard to administration route, we initially hypothesized that oral administration would expose gut bacteria to higher concentrations of antibiotics and thus would promote greater expansion of AMR. Indeed, using a mouse model, Zhang et al. (2013) reported that when the same doses of tetracycline or ampicillin were administered, enrichment of corresponding AMR gene pools in gut microbiota were greater and faster through oral administration compared with intravenous injection. However, results from this study suggest that IM or IN-FEED tylosin equally promote the development of enterococcal resistance to erythromycin and tylosin to a greater extent relative to oral water administration. Two readily identified reasons might explain this finding. The first is bile excretion of injected tylosin and its metabolites into the gastrointestinal tract of pigs that exerted selection pressure on gut bacteria. Both secretion from the liver into the gastrointestinal tract and urinary excretion of absorbed tylosin and the metobolite desmycosin have been reported (Worth, 1971; Wal and Bories, 1973). Second, the effects of administration route on the development of AMR in this study may be dose dependent (Zhang et al., 2013). The treatment dose and procedure administrated in each tested route followed the precise label regimen of the corresponding tylosin product formulation. Based on these dosages, pigs provided the WATER treatment received only 21% and 43% of the total tylosin doses administrated to those on the IM and FEED treatments, respectively (Table 1). However, label regimen for tylosin injection is not always followed in common practices, which results in a lower dose of tylosin intake. Future research is needed to verify the AMR response to lower dose of tylosin administration through IM. Moreover, a recent review by Pyörälä et al. (2014) suggested that applying macrolide antibiotics in feed or through injections creates long-acting concentrations of active substance in pigs that may contribute to the expansion of AMR. The slow absorption and release of tylosin in injected pigs and the uninterrupted tylosin administration through feed may have created a continuous selection pressure on resistant bacteria in contrast to the lower dosage and intermittently administered tylosin treatment effected through water.

In addition, it was unexpected that no evidence of a route × day interaction was apparent for the development of resistance to tylosin and erythromycin. Given the significant main effects of sampling day and route, this would suggest an increase in the resistance between sampling days among enterococcal isolates collected from pigs that received no tylosin treatment. It is possible that resistant bacteria could have been transmitted from the tylosin-treated pigs to control pigs through fecal contamination, although isolation measures were put in place between pens. Indirect physical contact of pigs through personnel movement across pens could also lead to cross contamination of resistant bacteria. Remaining unexplained is the reason why resistance of enterococcal isolates to daptomycin decreased from baseline (d 0) to the end of the treatment period (d 21) and then increased back to baseline levels after 2 weeks (d 35) of the wash-out period (Table 2).

Conclusions

In summary, we found no evidence that feeding tylosin promotes the growth performance of finishing pigs in the absence of the disease challenge for which it is labeled at the regimen administered in this study; in contrast, tylosin injection reduced ADG and G:F compared with untreated pigs. The probable reason for this is stress reaction to the injection and handling of pigs. Tylosin administration through injection and feed resulted in an increased probability of detecting resistance to erythromycin and tylosin among fecal enterococcal isolates compared with those collected from pigs that received either no or oral tylosin through water. However, no evidence of selection of resistance to other antimicrobial groups was apparent in the population of pigs and enteric bacteria in this study.

Footnotes

Acknowledgments

This study was supported in part by a grant from the National Pork Board (#16-053). Contribution no. 18-280-J Kansas Agric. Exp. Station, Manhattan.

Disclosure Statement

No competing financial interests exist.

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