APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2005, p. 5865–5872 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.10.5865–5872.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 10
Effects of Orally Administered Tetracycline on the Intestinal Community Structure of Chickens and on tet Determinant Carriage by Commensal Bacteria and Campylobacter jejuni A. S. Fairchild,1 J. L. Smith,1 U. Idris,1 J. Lu,1 S. Sanchez,2 L. B. Purvis,1 C. Hofacre,1 and M. D. Lee1* Department of Population Health1 and Athens Diagnostic Laboratory,2 College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 Received 2 September 2004/Accepted 28 April 2005
There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 g/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at 16 g/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment. avoparcin, but VRE remains persistent in Danish broiler houses despite the withdrawal (19). The urgent need to understand the processes underlying the spread of antibiotic resistance among major food-borne pathogens is illustrated by the emergence of resistant strains of Campylobacter. The fluoroquinolone resistance seen in Campylobacter spp. may have increased over time by the management practice of using a poultry fluoroquinolone to treat bacterial respiratory infections in chickens and turkeys (12). Fluoroquinolone resistance in Campylobacter spp. occurs by way of a chromosomal mutation of the DNA gyrase (18) and resistance to macrolides through a mutation of the 23S rRNA at the erythromycin binding site (14). However, food-borne pathogens can acquire a variety of resistance genes from the reservoir of commensal bacteria in the animals’ intestines (35). Campylobacter spp. exhibit resistance to aminoglycosides through the acquisition of resistance genes, such as aacA4, aphA-3, and aphA-7, that are commonly carried by intestinal genera of bacteria (20, 41). Similarly, resistance to trimethoprim has been acquired through mobile dfr genes encoding dihydrofolate reductases (16) and tetracycline resistance by obtaining a mobile tetO gene also found in gram-positive organisms such as enterococci (26, 34, 44). Tetracycline-resistant bacteria have been isolated from humans, animals, and the environment, with the majority of tet genes associated with mobile plasmids or transposons (10). Since tetracycline is the most common resistance reported in the human and veterinary Campylobacter databases of the National Antimicrobial Resis-
Over the last 50 years, broiler chicken production has increased from about 5 billion to over 40 billion pounds annually (42). This increase in production has been achieved through integration of the industry, with fewer companies controlling the majority of facilities. After integration, poultry companies implemented standardized management practices to decrease the morbidity and mortality associated with high-density growout operations. However, with the incorporation of antimicrobials into standard management practices, there has also been an increase in the observance of antibiotic-resistant bacteria, some of which are pathogenic to humans (1, 9, 12, 35). Researchers armed with resistance data and consumers exerting the power of purchasing choice are strongly encouraging changes in how the United States currently grows poultry for food consumption. Many European countries have taken the stance of reduced antibiotic usage for growth promotion purposes in an effort to reduce the prevalence of resistant bacteria (3, 9). The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) reported a decline in resistance in E. faecium cultured from broiler farms after halting the use of virginiamycin (1). The prevalence of vancomycin-resistant enterococci (VRE) has declined in some areas of Europe after withdrawal of the related veterinary drug
* Corresponding author. Mailing address: Department of Population Health, The University of Georgia, 953 College Station Rd., Athens, GA 30602-4875. Phone: (706) 583-0797. Fax: (706) 542-5630. E-mail:
[email protected]. 5865
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tance Monitoring System (NARMS) and because tetracyclines (oxytetracycline and chlortetracycline) are arguably the most commonly used therapeutic antibiotic used in food animal production, the aim of the present study was to determine whether Campylobacter sp. subsequently acquired tetracycline resistance genes from the intestinal commensal community of broiler chickens that had been given oxytetracycline.
MATERIALS AND METHODS Sample collection. Freshly voided cecal droppings were collected from a flock house (⬃22,000 chickens per house) on each of three commercial broiler farms (CF-1, CF-2, and CF-3) when the birds (Ross/Cobb hybrids) were 3 and 6 weeks of age. Two flocks were sampled from each farm. Oxytetracycline was administered on farm CF-2 in the water supply of flock 1 at 6.5 weeks of age to treat an outbreak of airsacculitis therefore these birds were also sampled after the treatment. Farms CF-2 and CF-3 had an antibiotic usage history of sarafloxacin and oxytetracycline during the year before sampling; farm CF-1 had not used therapeutic antibiotics for over a year. At each sampling, approximately 120 cecal droppings were collected from the top of the chicken litter by using sterile cotton-tipped swabs. The 120 swabs were pooled into 30 tubes, each of which contained 1 ml of brain heart infusion broth, and placed on ice for transport back to the laboratory. The 30 tubes were then pooled into 10 tubes, the contents were diluted with saline, and the following dilutions were plated and incubated: 10⫺3 and 10⫺5 on MacConkey agar for 24 h at 38°C for the selection of gram-negative enterics and on m-Enterococcus agar for 48 h at 38°C for the selection of Enterococcus species. Campylobacter spp. were cultured from 10⫺1 and 10⫺2 dilutions by membrane filtration as described by Engberg et al. (13). Membrane filtration was done by placing sterile cellulose acetate membrane filters (0.45-mm pore size; diameter of 25 mm) on the surface of blood agar plates. Then, 100 l of diluted sample was carefully placed on top of a filter, avoiding spillage around the edges of the filter, and the plates were incubated at room temperature for 30 min until the liquid was absorbed by the agar. The filters were removed by using sterile tweezers, and the plates were placed in ZipLoc bags that were flushed with a gas mixture consisting of 10% CO2, 5% O2, and 85% N2. The plates were incubated at 37C and observed over 72 h for growth. Thirty isolated Campylobacter-like colonies (gray, watery) were selected for further study. In order to detect phenotypes and genes at a flock prevalence rate of 5% or greater, 30 isolated colonies were selected across the 10 plates for each type of bacteria and streaked to isolation, stocked in freezer medium (15% glycerol, 1% peptone), and stored at ⫺80°C. Fresh cecal droppings were also collected from a university research broiler flock (Ross/Cobb hybrids) at 3, 5, and 7 weeks of age. At placement (1 day of age), the chicks were inoculated with Campylobacter jejuni by adding bacteria at a concentration of 62.5 CFU/ml of deionized drinking water. Chicks were allowed full access for 1 day. Fresh water was supplied to the birds for the remainder of the study. An oxytetracycline treatment was administered at 4 weeks of age via the birds’ water supply at a dose of 25 mg/lb for 5 days. Ten chickens were euthanatized with CO2 gas at 3, 5, and 7 weeks of age, and the ceca were removed. The contents of the ceca were expressed into tubes containing 2 ml of brian heart infusion broth and vortexed. Culturing of the various types of bacteria was done using the methods described above for culture of bacteria from the commercial flocks. To improve the detection of resistant Campylobacter strains of low prevalence, samples were also plated on blood agar containing 1 g of tetracycline/ml. Antimicrobial susceptibility testing. The MICs of tetracycline and oxytetracycline (range, 0 to 8 g/ml) were determined for gram-negative enterics and Enterococcus spp. by using the avian plate (CVM1BAVF) of the Sensititre automated antimicrobial susceptibility system (Trek Diagnostics Systems, Westlake, OH). The appropriate quality control organisms were used for every trial, and the results were interpreted according to the criteria established by the National Committee for Clinical Laboratory Standards (27) for E. coli and Enterococcus spp. and by using the NARMS tentative breakpoints for Campylobacter spp. (29). The tetracycline MIC (range, 0 to 256 g/ml) for C. jejuni was determined by the agar dilution method as described by the National Committee for Clinical Laboratory Standards (28) with C. jejuni ATCC 33560 as the quality control organism. Trials were accepted if this isolate’s tetracycline MIC was 1 to 4 g/ml. Characterization of bacterial isolates. Campylobacter isolates were identified by species-specific PCR (22) and identified as C. jejuni or C. coli. C. jejuni isolates obtained from the research flock and from a commercial farm that used oxytet-
APPL. ENVIRON. MICROBIOL. TABLE 1. DNA templates used in PCR to produce probes for the detection of tetracycline resistance determinants in C. jejuni, E. coli, and Enterococcus sp. isolated from broiler chickens tet determinant
Source of gene
Source or reference
tetA tetB tetC tetD tetK tetL tetM tetO
pSL18 Tn10 (3419) pBR322 pSL106 pAT102 pVBA15 pJ13 C. jejuni
30 24 8, 39 30 30 30 30 This studya
a The tetO probe identity was confirmed by DNA sequencing of the PCR amplicon.
racycline were strain typed by using pulsed-field gel electrophoresis (PFGE) after digestion of genomic DNA with SmaI or KpnI. Isolates were grown on a blood agar plate for 48 h and harvested by using an inoculating loop. DNA was prepared as described by Barrett et al. (5) and digested for 6 h with KpnI (Roche Applied Science, Indianapolis, IN) or overnight with SmaI (Roche). DNA was also digested in order to evaluate restriction enzyme activity and determine minimum time needed for complete digestion. Fragments were separated in a 1.2% agarose gel in 0.5⫻ TBE buffer (45 mmol of Tris, 45 mmol of boric acid, 1 mmol of EDTA) using a CHEF PFGE unit (Bio-Rad, Hercules, CA). SmaI fragments were separated with 5- to 30-s pulses (6V/cm) at 14°C for 25 h; KpnI fragments were separated by using 0.5- to 25-s pulses for 19 h. E. coli were identified by using a panel of biochemical tests, which included triple sugar iron agar, gas production, indole production, citrate fermentation, ornithine decarboxylase, and oxidase reaction (4). Enterococcus isolates were identified by using a panel of biochemical tests, which included growth in the presence of bile and acid production from carbohydrate fermentation (mannitol, sorbose, arabinose, sorbitol, and raffinose) and enzymatic hydrolysis of esculin, L-pyrrolidonyl-b-naphthylamide, and arginine (11). Isolates producing ambiguous results using classical methods were identified by using the BBL Crystal gram-positive ID system (Sparks, MD). Detection of tetracycline resistance determinants. Isolates used as templates for generating digoxigenin-labeled DNA probes for DNA-DNA hybridizations are listed in Table 1. Probes were generated by PCR with primers specific for tetA, -B, -C, -D, -K, -L, and -M as described by Ng et al. (30). A 505-bp fragment of tetO (26) was amplified by using the primers tetOF (5⬘-ATA ATT AAC TTA GGC ATT CTG GCT C-3⬘) and tetOR (5⬘-CGT CAT TGT CCG TTA CAT TTA TAT G-3⬘). Campylobacter and E. coli DNA template for PCR was prepared as described by Woods et al. (43). Enterococcus DNA was extracted as described by Pitcher et al. (32). The DNA template was stored at ⫺20°C. Then, 1 l of working template (in deionized distilled H2O) was used in a 10 ml of PCR mixture that consisted of 0.2 mM deoxynucleoside triphosphates, 2.0 mM MgCl2, PCR buffer (50 mM Tris [pH 7.4]), 50 pmol (each) of forward and reverse PCR primers, and 0.5 U of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN) and deionized distilled H2O. The program parameters for the Idaho Technology RapidCycler (Idaho Falls, ID) for tetO were 94°C for 15 s, 94°C for 1 s, 54°C for 1 s, and 72°C for 15 s for 30 cycles, after which the reactions were incubated at 72°C for 4 min. The program parameters for the other tet determinants were 94°C for 5 min, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s for 35 cycles. DNA amplicons were analyzed by gel electrophoresis with a 1.5% agarose gel. In order to screen the isolates for carriage of tet genes, Campylobacter and E. coli cells were transferred onto a nylon membrane by using toothpicks, and the DNA was liberated by alkaline lysis of the bacterial cells (36); 1 g of Enterococcus genomic DNA was placed on a nylon membrane using a vacuum manifold. The DNA/DNA hybridizations were performed as described in the protocol for the DIG DNA labeling and detection kit (Roche Applied Science); the annealing temperature for hybridizations and washes was 68°C. Characterization of the effect of oxytetracycline on the cecal bacterial community. The bacterial community structure was determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA gene amplicons after digestion with BsuRI and CfoI (23). The bacterial fraction was recovered from the cecal contents of pooled samples from research birds as described previously (25). Genomic DNA was extracted as follows. Bacterial cells were lysed by using the beads, Solution 1, and IRS of Mo Bio soil DNA isolation
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TABLE 2. Tetracycline susceptibility of C. jejuni cultured from commercial and research broiler chicken flocks at various ages Tetracycline MIC90 (g/ml)a Farm or group
Farm CF-1 CF-2 CF-3
Flock 1
Flock 2
Research flock
3–5 wk
6 wk
7 wk
3 wk
6 wk
NC 1 (30; 0) NC
64 (24; 100) 1 (29; 0) 1 (7; 0)
ND 2* (29; 0) ND
128 (30; 100) 64 (21; 100) 64 (22; 100)
128 (25; 100) 64 (30; 100) 64 (26; 100)
Group Control Oxytetracycline
3 wk
5 wk
7 wk
1 (30; 0)
1* (30; 0) 1 (30; 0)
ND 2 (4; 0)
a A total of 273 C. jejuni isolates were cultured from the cecal droppings of consecutive flocks of commercial broiler chickens located on three farms. The birds in the research flock were raised in one pen until they were separated at 4 weeks of age. Ninety-four C. jejuni isolates were cultured from the research flock. Tetracycline susceptibility was determined by agar dilution. All isolates from the same sampling expressed the same MIC. NC, no C. jejuni was detected by culture of the cecal droppings; ND, no samples were collected at this time. Text in parentheses indicates “number of isolates; % tetO-positive isolates.” ⴱ, the birds in flock 1 on CF-2 were given oxytetracycline in their drinking water after the 6-week sampling. The research flock control group received no antibiotics; the treatment group was administered oxytetracycline at 4 weeks of age.
kit (Mo Bio Laboratories, Inc., Solana Beach, CA). The solution was treated with sodium dodecyl sulfate (final concentration of 0.5%) and proteinase K (final concentration 0.1 mg/ml) and then incubated at 37°C for 30 min. The sample was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1) then the DNA was harvested by using alcohol precipitation. After resuspension in water, the DNA was quantitated using a Beckman DU640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). The 16S rRNA ribotypes present among cecal community DNA were amplified by PCR with the primers 8F and 926R as previously described (25) except that the 8F primer was labeled with 5⬘FAM (carboxyfluorescein-N-hydroxysuccinimide ester–dimethyl sulfoxide). PCR conditions included template DNA at 2.5 ng/l, PCR buffer, 2.0 mM MgCl2, 0.20 mM deoxynucleoside triphosphate, 1 M concentrations of each primer, and 0.05 U of Taq DNA polymerase (Roche Diagnostics Corp., Indianapolis, IN)/l in a final reaction volume of 18 l. Initial DNA denaturation and enzyme activation steps were performed at 94°C for 2 min in a RapidCycler (Idaho Technology), followed by 18 cycles of denaturation at 94°C for 15 s, annealing at 54°C, for 30 s, and elongation at 72°C for 45 s, which was followed by a final incubation at 72°C for 7 min. Three replicate PCRs were performed for each sample to increase the DNA yield. The PCR products were loaded onto a gel from which bands were cut and eluted in 35 l of sterile filtered distilled water by using a QIAquick gel extraction kit (QIAGEN, Chatsworth, CA). The concentrations of the fluorescently labeled PCR products were measured on a spectrophotometer (DU Series 500; Beckman, Fullerton, CA). A total of 100 ng of purified PCR products was digested in a 10-l volume for 4 h at 37°C with 10 U of BsuRI (HaeIII isoschizomer; Fermentas/MBI) or with CfoI, and then the digests were desalted with the QIAquick nucleotide removal kit (QIAGEN). The DNA fragments were separated by electrophoresis on an automated sequence analyzer (ABI Prism 310 DNA Sequencer; PE Biosystems, Foster City, CA) in GeneScan mode. Aliquots (2 l) of DNA fragments were mixed with 2 l of deionized formamide and 0.5 l of GS-500 size standard (PE Biosystems). The DNA fragments were denatured by incubation at 94°C for 5 min and immediately chilled on ice prior to electrophoresis. After electrophoresis, the lengths of the fluorescently labeled fragments were determined by comparison with internal standards by using GeneScan software (ABI). For each sample, peaks over a threshold of 50 U above background fluorescence were analyzed by manually aligning fragments to the size standard. To avoid detection of primers and uncertainties of size determination, terminal fragments smaller than 50 bp and larger than 350 bp were excluded from the analysis. The identity of T-RF peaks was determined by comparison to HaeIII/CfoI T-RFLP databases produced from previous studies (25). The changes in phylotype contribution to the community structures was evaluated by using the Shannon and Weaver index (38) calculated by considering each peak as an individual phylotype (species) and the area of the peaks as indicators of abundance. Similarly, differences in the composition of communities were evaluated by using Morishita’s index of community similarity and Simpson’s (37).
RESULTS Did oxytetracycline usage in broiler chickens select for tetracycline-resistant C. jejuni? Table 2 shows the tetracycline susceptibility profiles and tetO gene carriage for 273 C. jejuni isolates cultured from the commercial chicken flocks and 94 isolates from a research flock. We found that 65% of the C. jejuni isolates from commercial chickens expressed tetracycline MICs in excess of 16 g/ml, and all of these resistant isolates harbored tetO. Tetracycline resistance breakpoints have not yet been established for C. jejuni (28, 29). In the present study, isolates that express MICs of ⱖ16 g/ml are considered resistant. No isolates with an MIC of ⬎2 g/ml were cultured from the commercial chickens treated with oxytetracycline to resolve an outbreak of airsacculitis (farm CF-2). On the other hand, tetracycline-resistant (MIC ⫽ 64 g/ml), tetO-harboring isolates were cultured from the subsequent flock on this farm. We sought to determine whether these flocks contained the same strain(s) of C. jejuni because the persistence of a strain would suggest that oxytetracycline usage selected for resistance but the resistant isolates were present at a prevalence or density below the detection level of our sampling protocol. Strain typing by PFGE indicated that the two flocks were colonized with different C. jejuni strains (Fig. 1). Using a research flock of broiler chickens, we sought to experimentally determine the effect of oxytetracycline administration on the prevalence of resistant Campylobacter. Day-ofhatch broiler chicks, housed in research floor pens, were orally administered a tetracycline-susceptible C. jejuni isolate, and oxytetracycline was administered when the birds were 4 weeks old. In the present study, we removed a cecum from each bird and our isolate screening strategy was designed to detect resistant isolates if they represented at least 5% of the total Campylobacter population of each cecum. However, to facilitate the likelihood of detecting resistant campylobacters present at a low density, we also plated diluted and undiluted samples on media containing tetracycline. No growth occurred on plates containing 1 g of tetracycline/ml; however, 94 C. jejuni isolates were stocked from plates that did not contain
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APPL. ENVIRON. MICROBIOL. TABLE 3. Bacterial community structure of the cecal contents of research chickens before and after oxytetracycline treatmenta Control animals at:
Oxytetracycline-treated animals at:
Abundant species
Bacteroides spp. Fusobacterium spp. Clostridium orbiscindens Clostridium sp. strain 1 Clostridium sp. strain 2 Ruminococcus spp. Oscillospira spp. Alcaligenes spp. Lactobacillus crispatus Lactobacillus reuteri Lactobacillus aviarius Eubacterium spp.
5 wk
7 wk
5 wk (2 days posttreatment)
7 wk (2 wk posttreatment)
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹
a Antibiotic was administered to the oxytetracycline group for 5 days beginning when the birds were 4 weeks of age. The bacterial community structure of the cecum was determined by T-RFLP analysis of the 16S rRNA gene amplicons digested with BsuRI and CfoI (23). Bacterial genera were identified by using a T-RFLP database composed of 16S rRNA sequences retrieved from a cecal community DNA clone library (25). ⫹, presence of organism; ⫺, absence of organism.
FIG. 1. PFGE strain typing of tetracycline-susceptible (MIC ⫽ 1 g/ml) C. jejuni isolates cultured from oxytetracycline-treated commercial broiler chickens in flock 1 and their relationship to the tetracycline-resistant (MIC ⫽ 64 g/ml) isolates from an untreated sequential flock raised in the same house on farm CF-2. Lane 1 contains the S. cerevisiae molecular weight marker; lane 2 shows the SmaI pattern common to isolates cultured from flock 1; lane 3 represents the SmaI pattern detected in isolates cultured from flock 2; lane 4 contains the molecular weight marker, lane 5 shows the KpnI pattern for flock 1; lane 6 shows the KpnI pattern for flock 2.
tetracycline. All of these isolates expressed tetracycline MICs of 1 to 2 g/ml, indicating that no resistant isolates were detected. All of the chickens were colonized with the same C. jejuni strain that was administered (PFGE strain-typing data is not shown). The C. jejuni isolates from the research flocks were also screened by DNA hybridization in order to determine whether there was silent carriage of tetA, -B, -C, -D, -K, -L, -M, and -O determinants. No tet determinants were detected by DNA hybridization, indicating that no transfer of tetracycline resistance genes had occurred at a rate detectable by our sampling protocol. These data suggest that oral administration of oxytetracycline did not amplify resistant strains of C. jejuni within the chicken intestine or appreciably increase gene transfer to C. jejuni. Effect of oxytetracycline administration on intestinal commensal bacteria. In order to detect the effects of oral administration of oxytetracycline on the bacterial community structure of the chicken cecum, we characterized bacterial pellets from the cecum by using 16S rRNA community analysis. Table 3 shows the comparison of the abundant genera of the cecal bacterial community, of control and treated groups, 2 days and 2 weeks after oxytetracycline was administered. There were no differences in the distribution of the abundant genera among
the treated and untreated birds. The community structure of the flocks was further characterized and compared by using the Shannon-Weaver diversity index (38) and a modification of Simpson’s (37). Diversity indices combine species (phylotype) richness and the abundance of each species (evenness) as a way to assess and compare the composition of a community; the parameters are weighed differently by different indices. In addition, we compared the communities’ composition by using the Morishita’s index of similarity to quantify the degree of similarity between the treated and untreated communities (37). Thirty-five different terminal restriction fragments (phylotypes) were detected among the samples. If a sample contained all of these fragments, it would have a Shannon index of 5.13. Both treated and untreated cecal bacterial communities demonstrated high diversity and displayed similar Shannon indices (control ⫽ 4.38, treated ⫽ 4.6) and similar values (0.04 to 0.05), indicating that the treatment did not reduce community diversity. In addition, the communities possessed a high Morishita’s index (0.72 of a possible 1.0), indicating significant similarity between them. These data suggest that oral oxytetracycline administration exhibited little effect on the cecal bacterial community structure of our research chickens. Effect of oxytetracycline administration on prevalence of tetracycline resistance of E. coli. Ninety-nine percent of the colonies isolated on MacConkey agar, cultured from cecal droppings of both the commercial and research flocks, were identified as E. coli. This finding correlates with their abundance as detected by molecular methods (25). Isolates collected from both commercial and research flocks displayed resistance to tetracycline and oxytetracycline, with MIC values at which 90% of the isolates tested are inhibited (MIC90) at ⬎8 g/ml (Table 4). In addition, several isolates were evaluated for resistance to tetracycline at higher concentrations and many were cultivable in tetracycline concentrations of at least 30 g/ml. In order to investigate the genetic basis of the resistance, the carriage of tet determinants A, B, C, and D was
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TABLE 4. Tetracycline and oxytetracycline MICs of Enterococcus sp. and E. coli isolates cultured from cecal droppings of broiler chickens before and after oxytetracycline treatmenta MIC (g/ml) Bacteria
Flock
Enterococcus sp.
Commercial Research
E. coli
Commercial Research
Treatment
b
Tetracycline
Oxytetracycline
MIC90
Range
MIC90
Range
Pre Post Pre Post
⬎8 ⬎8 ⬎8 ⬎8
⬎8 ⬎8 ⬎8 8–⬎8
⬎8 ⬎8 ⬎8 ⬎8
8–⬎8 8–⬎8 4–⬎8 4–⬎8
Pre Post Pre Post
⬎8 ⬎8 ⬎8 ⬎8
0.5–⬎8 0.5–⬎8 ⬎8 0.5–⬎8
⬎8 ⬎8 ⬎8 ⬎8
0.5–⬎8 0.5–⬎8 ⬎8 0.5–⬎8
a MICs were determined by microbroth dilution using the Sensititre avian plate (Trek Diagnostic Systems, Westlake, OH); the range of concentrations was 0.25 to 8 g of tetracycline and oxytetracycline/ml. b Pre, before administration of oxytetracycline; Post, after oxytetracycline administration.
determined (Table 5). Over 53% of the research flock E. coli isolates possessed the tetB determinant, followed by a lesser prevalence (25%) of tetA. None of the E. coli isolates from the research flock harbored either tetC or tetD. Many E. coli isolated from commercial chickens possessed tetA (32%) or tetB (30%) although two isolates cultured from 3-week-old birds harbored tetC or tetD. Each E. coli isolate possessed only one tetracycline resistance determinant. Effect of oxytetracycline administration on the prevalence of antibiotic resistance of Enterococcus spp. Among the Enterococcus isolates detected, E. faecium, E. faecalis, and E. gallinarum were the most prevalent cultivable species; however, E. hirae, E. durans, E. casseliflavus, and E. raffinosus were also
TABLE 5. Tetracycline resistance determinant distribution among Enterococcus spp. and E. coli isolates cultured from the cecal droppings of commercial and research broiler chickens before and after oxytetracycline administrationa Farm Source
Tet determinant
% Positive (% pretreatment, % posttreatment) Enterococcus sp.
b
E. coli
Commercial
tetA tetB tetC tetD tetK tetL tetM tetO
ND ND ND ND 1.3 (0, 1.3) 25.4 (0, 50) 61.0 (44.8, 76.7) 52.5 (58.6, 46.7)
32.2 (3.6, 58.1) 30.5 (53.6, 9.7) 1 1 ND ND ND ND
Researchc
tetA tetB tetC tetD tetK tetL tetM tetO
ND ND ND ND 0 70.0 (50, 90) 96.7 (93.3, 100) 2.3 (6.7, 0)
25.6 (33.3, 21.7) 53.3 (50.0, 55.0) 0 0 ND ND ND ND
a The presence of determinant was detected by DNA hybridization; ambiguous results were confirmed by PCR. tetO and tetL were only detected among E. faecium and E. faecalis isolates; tetK was detected in E. durans, whereas tetM was found among all Enterococcus species. ND, not determined. b n ⫽ 59 (Enterococcus sp.) and n ⫽ 59 (E. coli). c n ⫽ 86 (Enterococcus sp.) and n ⫽ 90 (E. coli).
detected. Aerococcus viridans, Streptococcus vestibularis, Staphylococcus spp., and Lactococcus spp. were detected by culture in some samples from young birds. E. faecium (65.5%) and E. gallinarum (4.5%) were the most common species isolated from the commercial chickens immediately before the oxytetracycline treatment, but E. faecalis (86.7%) was predominant posttreatment. Table 6 shows the susceptibility of the Enterococcus isolates to 14 antimicrobial agents. Resistance was commonly detected to streptomycin and neomycin among Enterococcus isolates from both the commercial and the research flocks prior to antibiotic administration. Enterococcus isolates cultured from the research broiler chickens at 5 weeks of age were less susceptible to erythromycin and novobiocin than were the isolates collected from the commercial flock at 6 weeks of age. However, differences in species prevalence were also found between the two flocks; E. faecium (65.5%; n ⫽ 29) was primarily isolated from the commercial flock at 6 weeks of age, but E. faecalis (86.7%; n ⫽ 30) was the most cultivable species from the research flock at 5 weeks of age. Enterococcus isolates collected from both commercial and research flocks displayed resistance to tetracycline and oxytetracycline, with MIC90 values at ⬎8 g/ml (Table 4), the highest concentration present in the avian Sensititre plate. In addition, several isolates were evaluated for resistance to tetracycline at higher concentrations, and many were cultivable in tetracycline concentrations of at least 30 g/ml. In order to investigate the genetic basis of the resistance, the carriage of tet determinants K, L, M, and O was determined (Table 5). The majority (66.7%) of commercial Enterococcus isolates possessed the tetM determinant, but tetO (41.3%), tetL (21.3%) and tetK (1.3%) were also detected. tetL and tetK carriage were always associated with tetM carriage, although isolates harboring tetO tended not to possess the other tet genes. tetL and tetO were only detected among the E. faecium and E. faecalis isolates; tetM was detected among all of the Enterococcus species, as well as A. viridans (three of four isolates). At least one tet determinant was detected among 130 of the 134 Enterococcus isolates that were screened. However, no tet genes were detected among the Streptococcus vestibularis isolates (n ⫽ 7). Enterococci cultured from the research flock were also fre-
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APPL. ENVIRON. MICROBIOL.
TABLE 6. MICs of for 14 antimicrobial agents for Enterococcus isolates cultured from the cecal droppings of broiler chickens that were given oxytetracyclinea MIC (g/ml) for birds at age: Flock
Antibiotic
5 or 6 wkb
3 wk MIC90
Range
MIC90
7 wk Range
MIC90
Range
Commercial
Enrofloxacin Gentamicin Ceftiofur Neomycin Erythromycin Oxytetracycline Tetracycline Amoxicillin Sarafloxacin Streptomycin Novobiocin Clindamycin Penicillin Spectinomycin
2 ⬎8 ⬎4 32 ⬎4 ⬎8 ⬎8 0.5 ⬎0.25 1,024 4 ⬎4 8 64
0.25–⬎2 0.5–⬎8 0.25–⬎4 2–32 0.12–⬎4 4–⬎8 ⬎8 0.25–1 0.25–⬎0.25 8–⬎1,024 0.5–4 0.25–⬎4 0.06–⬎8 8–⬎64
1 ⬎8 ⬎4 32 0.5 ⬎8 ⬎8 4 0.25 1,024 0.5 ⬎4 ⬎8 64
0.25–⬎2 ⬎8 0.5–⬎4 4–32 0.12–⬎4 8–⬎8 ⬎8 0.25–4 ⬎0.25 32–⬎1,024 0.5 0.25–⬎4 0.12–⬎8 8–64
1 ⬎8 ⬎4 ⬎32 ⬎4 ⬎8 ⬎8 0.5 ⬎0.25 1,024 4 ⬎4 4 64
0.25–1 4–⬎8 0.25–⬎4 8–⬎32 0.12–⬎4 8–⬎8 ⬎8 0.25–1 ⬎0.25 32–⬎1,024 0.5–⬎4 4–⬎4 0.06–4 64–⬎64
Research
Enrofloxacin Gentamicin Ceftiofur Neomycin Erythromycin Oxytetracycline Tetracycline Amoxicillin Sarafloxacin Streptomycin Novobiocin Clindamycin Penicillin Spectinomycin
1 8 ⬎4 32 2 ⬎8 ⬎8 0.5 ⬎0.25 ⬎1,024 ⬎4 ⬎4 4 64
0.5–2 0.5–⬎8 1–⬎4 2–32 0.25–4 0.25–⬎8 0.25–⬎8 0.25–1 ⬎0.25 8–⬎1,024 0.5–⬎4 0.25–⬎4 0.5–⬎8 16–⬎64
1 8 ⬎4 ⬎32 ⬎4 ⬎8 ⬎8 0.5 ⬎0.25 ⬎1,024 ⬎4 ⬎4 ⬎8 64
0.5–⬎2 2–⬎8 2–⬎4 2–32 0.12–⬎4 4–⬎8 ⬎8 0.25–1 ⬎0.25 32–⬎1,024 0.5–⬎4 0.25–⬎4 2–⬎8 32–⬎64
2 ⬎8 ⬎4 16 ⬎4 ⬎8 ⬎8 1 ⬎0.25 ⬎1,024 ⬎4 ⬎4 ⬎8 64
0.5–⬎2 0.5–⬎8 1–⬎4 2–32 0.12–⬎4 4–⬎8 8–⬎8 0.25–1 ⬎0.25 8–⬎1,024 0.25–⬎4 0.25–⬎4 0.5–⬎8 16–64
a The antibiotics used and the ranges of concentrations tested were as follows (g/ml): enrofloxacin, 0.12 to 2; gentamicin, 0.5 to 8; ceftiofur, 0.25 to 4; neomycin, 2 to 32; erythromycin, 0.12 to 4; oxytetracycline, 0.25 to 8; tetracycline, 0.25 to 8; amoxicillin, 0.25 to 16; sarafloxacin, 0.03 to 0.25; streptomycin, 8 to 1,024; novobiocin, 0.5 to 4; clindamycin, 0.25 to 4; penicillin, 0.06 to 8; and spectinomycin, 8 to 64. Oxytetracycline was administered to birds in the commercial flock after we collected samples from 6-week-old birds. The samples at 7 weeks were collected after oxytetracycline administration. The research birds were given oxytetracycline when they were 4 weeks of age. b That is, 5 weeks for the research flock and 6 weeks for the commercial flock.
quently tetracycline resistant and displayed a similar distribution of tet determinants as isolates from the commercial flocks (Tables 4 to 6). The research flock Enterococcus isolates harbored the tetM determinant with very high prevalence (96.7%), although tetL (70%) was also commonly detected; all tetLpositive isolates also possessed tetM. Only one Enterococcus isolate from the research flock possessed tetO and tetK was not detected. Although tetL and tetM carriage appeared to increase after treatment, the control flock (which received no antibiotic treatment) also demonstrated increased tetL gene carriage with increasing age of the birds. DISCUSSION The NARMS reported that for the past 6 years, 13 to 30% of C. jejuni isolates obtained from chicken carcasses after processing expressed an MIC to tetracycline of ⬎16 g/ml (29). A U.S. Food and Drug Administration study detected a high prevalence of tetracycline-resistant isolates in retail meat (15). Oxytetracycline is used in broiler chicken production to treat E. coli airsacculitis that occurs secondary to viral respiratory infections. Outbreaks of viral infections are most likely to occur in areas of high bird density and these areas are most likely
to use therapeutic antibiotic treatment. The high prevalence of tetracycline-resistant C. jejuni from poultry suggested that tetracycline usage may have selected for resistant strains. Most tetracycline resistance determinants confer decreased susceptibility to the typical tetracycline compounds such as tetracycline, oxytetracycline, and chlortetracycline (34); administration of oxytetracycline should select for determinants conferring resistance to both oxytetracycline and tetracycline. Therefore, in the present study, we investigated whether C. jejuni acquired tetracycline resistance subsequent to oxytetracycline administration to broiler chickens. We did not detect emergence of resistant C. jejuni in treated commercial or research flocks, although we did culture many resistant isolates from untreated commercial flocks. In order to determine the prevalence of tetracycline-resistant commensals and to investigate potential reservoirs of tetracycline determinants, we cultured Enterococcus spp. and E. coli from the cecum as well. Our previous studies had shown that these bacteria were present among the abundant genera in the cecum of young broiler chickens (25) and would potentially occupy adjacent intestinal microhabitats with the C. jejuni. However, we were unable to experimentally detect in vivo transfer
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ORAL TETRACYCLINE ADMINISTRATION IN CHICKENS
of resistance genes to C. jejuni even after finding commensal bacteria harboring tetracycline resistance determinants and after the selective pressure of oral oxytetracycline administration. Information regarding the transmissibility of tet determinants to Campylobacter is fairly limited. Some plasmid-borne tetO alleles have been transferred by conjugation among C. jejuni isolates in vivo and in vitro (2, 40). However, some tetO-harboring plasmids are not mobilizable, and some C. jejuni isolates contain chromosomally encoded alleles that are not transferable (17, 33). Chromosomal and plasmid-borne tetO genes have been detected among multiple species of streptococci and enterococci and tetO-harboring plasmids have varied in their ability to be transferred by conjugation (44). It is unknown whether tet determinants from commensal enterococci or coliforms are transmissible to C. jejuni or whether they would be expressed by Campylobacter if acquired. Some tet determinants, such as tetM, are encoded on conjugative elements and many are encoded on transposons, but the vast majority are present on transferable plasmids that may have a broad host range (34). Gene transfer to Campylobacter has been shown to occur in the lumen of the intestine of chickens, and C. jejuni has been shown to undergo natural transformation in the intestine (2, 7). In vivo transfer of antibiotic resistance has also been demonstrated in the intestine among some bacteria colonizing adjacent microhabitats (21, 35), suggesting that conjugation events are not infrequent in this environment. However, in the present study we also did not detect silent carriage of tet determinants by Campylobacter, indicating that these isolates of C. jejuni did not readily acquire determinants from the broiler intestinal bacterial community. The pharmacokinetics and instability of oxytetracycline may be limiting factors in its ability to produce a selective pressure on bacteria in the intestine of chickens. Oxytetracycline administered to poultry by the oral route is fairly well absorbed into the circulation and is excreted primarily by the kidneys (6). There is also some biliary excretion of serum oxytetracycline into the small intestine of chickens, but it is believed that little of the drug is biologically active in the lumen of the intestine because of pH and chelation by divalent cations (6). We evaluated the effect of oxytetracycline on the cecal bacterial community by evaluating community structure changes posttreatment. Comparative analysis suggested that the cecal bacterial community of treated and untreated research flocks had very similar compositions. This finding indicates that the distribution of the most abundant species of bacteria was not significantly affected, but it is also possible that the most abundant members of the cecal community were already resistant to tetracycline. The majority of the E. coli and Enterococcus isolates cultured in our study were resistant prior to oxytetracycline administration, suggesting that tetracycline resistance occurs at a high prevalence among poultry commensal bacteria. Nevertheless, whereas we failed to detect selection of resistant Campylobacter in vivo, we did detect resistant C. jejuni isolated from the next flock raised in the house. Strain typing showed that the flocks were colonized with different strains. Although oxytetracycline administration may not have been adequate for selection of resistance within the chicken intestine, the chickens may have acquired the tetracycline-resistant Campylobacter strain from an environmental source in the flock house such as contaminated nipple drinkers or litter. The
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oxytetracycline was orally administered to the birds through a housewide waterline; a situation that could potentially select for resistant biofilm strains that were disseminated to the subsequent flocks. C. jejuni isolates harboring a tobramycin-resistance gene have been reported among the biofilm microflora of nipple drinkers in a poultry house (20), and water has been shown to be a source of campylobacters for both humans and chickens (31, 45). Oxytetracycline selection may be more effective in the poultry house environment resulting in an environmental reservoir of resistant strains. Because the microbial ecology of gene transfer and antibiotic resistance is multifactorial, animal production management practices that increase the frequency of transfer events or the stability of elements encoding resistance determinants could increase the prevalence of resistance in bacteria associated with meat and poultry. Risk assessment of production practices, including antibiotic administration and litter/manure management, may be very useful in identifying those practices that can reduce the overall prevalence of resistant strains present in the animal’s intestine or environment. ACKNOWLEDGMENTS Financial support for this study was provided by a grant from the U.S. Food and Drug Administration. We thank the members of the Athens Veterinary Diagnostic Laboratory who assisted in the identification of the Enterococcus isolates. We especially thank Tongrui Liu for assistance in evaluating and confirming the PFGE results. REFERENCES 1. Aarestrup, F. M., A. M. Seyfarth, H. Emborg, K. Pedersen, R. S. Hendriksen, and F. Bager. 2001. Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark. Antimicrob. Agents Chem. 45:2054–2059. 2. Avrain, L., C. Vernozy-Rozand, and I. Kempf. 2004. Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens. J. Appl. Microbiol. 97:134–140. 3. Bager, F., M. Madsen, and J. Christensen. 1997. Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms. Prev. Vet. Med. 31:95–112. 4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Enterobacteriaceae, p. 370. In Bailey and Scott’s diagnostic microbiology, 9th ed. Mosby, St. Louis, Mo. 5. Barrett, T. J., H. Lior, J. H. Green, R. Khakhria, J. G. Wells, B. P. Bell, K. D. Greene, J. Lewis, and P. M. Griffin. 1994. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsedfield gel electrophoresis and phage typing. J. Clin. Microbiol. 32:3013–3017. 6. Black, W. D. 1977. A study of the pharmacodynamics of oxytetracycline in the chicken. Poult. Sci. 56:1430–1434. 7. Boer, P., J. A. Wagenaar, R. P. Achterberg, J. P. Putten, L. M. Schouls, and B. Duim. 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44:351–359. 8. Bolivar, F. 1978. Construction and characterization of new cloning vehicles. 3. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA-molecules. Gene 4:121–136. 9. Casewell, M., C. Friis, E. Marco, P. McMullin, and I. Phillips. 2003. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J. Antimicrob. Chem. 52:159–161. 10. Chopra, I., and M. Roberts. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 65:232–260. 11. Devriese, L. A., B. Pot, and M. D. Collins. 1993. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75:399–408. 12. Endtz, H. P., G. J. Ruijs, B. van Klingeren, W. H. Jansen, T. van der Reyden, and R. P. Mouton. 1991. Quinolone resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. J. Antimicrob. Chemother. 27:199–208. 13. Engberg, J., L. W. Stephen, C. Harrington, and P. Gerner-Smidt. 2000. Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for campylobacters. J. Clin. Microbiol. 38:286–291.
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