Effects of Kanamycin Administration to Poultry on the Proliferation of Drug-Resistant Salmonella1

ENVIRONMENT AND HEALTH Effects of Kanamycin Administration to Poultry on the Proliferation of Drug-Resistant Salmonella1 RICHARD K. GAST2 and J. F. STEPHENS Department of Poultry Science, The Ohio State University, Columbus, Ohio 43210 (Received for publication June 30, 1987)

1988 Poultry Science 67:689-698 INTRODUCTION

The effectiveness of antibiotics as growthpromoting feed additives for domestic animals was first reported nearly four decades ago (Jukes et al., 1950). This information led to the routine incorporation of subtherapeutic levels of various antibiotics into the diets of many food-producing animals. Extensive antibiotic use has been implicated as the source of selective pressure favoring drug-resistant bacterial strains (Stabler et al., 1982; Scioli et al, 1983; Dawson et al., 1983; Langloisefa/., 1983). Gastand Stephens (1986) provided evidence of a direct relationship between experimental antibiotic administration to turkeys and increased frequency of transfer of drug resistance plasmids to drug-sensitive Salmonella. Extensive use of antibiotics as feed additives in poultry production has been accompanied by the emergence of widespread resistance to these drugs in both the normal flora (Kanai et al., 1983) and in many of the characteristic pathogens of commercially raised birds (Blackburn

'Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center. The Ohio State University; Journal Article Number 129-87. "Present address: US Department of Agriculture, Agricultural Research Service. Southeast Poultry Research Laboratory. 934College Station Road. Athens. GA 30605.

689

et al., 1984; Gast and Stephens, 1985). As a consequence of the high frequency of isolation of Salmonella from poultry and poultry products (Cox et al., 1978), the acquisition of multiple drug resistance by these organisms is of particular concern. Various Salmonella species are also among the pathogens most often communicated from domestic animals to humans via the food chain (Holmberg et al., 1984a,b; Spika et al., 1987). Any relationship between the feeding of antibiotics to domestic animals and the frequency of antibiotic resistance in Salmonella cells carried by these animals would thus have particularly consequential implications from both agricultural and medical perspectives (Cohen and Tauxe, 1986). The objectives of the present study were to evaluate the effects of antibiotic administration to poultry on the in vivo transfer of antibiotic resistance from members of the normal intestinal microflora (Escherichia coli) to pathogenic species (Salmonella arizonae and 5. typhimurium) and on the frequency of isolation of Salmonella transconjugants from edible viscera (liver). MATERIALS AND METHODS

Experimental Animals. Four in vivo experiments were conducted, using newly hatched chickens or turkeys. Single Comb White Leghorn (DeKalb-XL) chicks were used in Experiment 1. Turkey poults for Experiments 2,

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ABSTRACT Four experiments were conducted to examine the relationship between antibiotic administration to poultry and the in vivo proliferation of Salmonellae. The frequency of isolation of drug-resistant transconjugant S. arizonae from the livers of chicks inoculated per os with multiply drug-resistant Escherichia coli and drug-sensitive S. arizonae was directly related to the concentration of kanamycin administered to the chicks in their drinking water. Kanamycin administration was also associated with a significant (P<.05) increase in the frequency of isolation of drug-resistant transconjugant S. typhimurium from the intestines and livers of poults inoculated with drug-sensitive S. typhimurium and multiply drug-resistant E. coli. Kanamycin administration significantly reduced the spread of drug-sensitive 5. typhimurium to the livers of poults inoculated only with that strain. These experiments demonstrate that antibiotic administration to poultry can enhance the proliferation of drug-resistant Salmonella. (Key words: Salmonella, antibiotic resistance, kanamycin, transconjugant, Escherichia coli)

690

GAST AND STEPHENS

and three liver samples were removed. One sample of each type of tissue was examined for the presence of EC, one for ST (or SA in Experiment 1), and one for STT or SAr. After removal, samples were transferred directly to tubes of broth media. The EC broth plus kan was used to test for EC, Selenite Cystine broth plus nal was used to select for ST or SA, and Selenite Cystine broth plus nal and kan was used to identify STT or SAr. Antibiotics were obtained from Sigma Chemical Company (St. Louis, MO) and incorporated into media at 50 (Jig/mL. After incubation of the broth tubes for 18 to 24 h at 41 C, a loopful of each broth culture was streaked onto an appropriate agar medium. The EC broth culture was transferred to MacConkey agar and each Selenite Cystine broth culture was transferred to SS agar. Each agar plate contained the same combination of antibiotics (at the same concentrations) as were in the corresponding broth medium. Agar plates were incubated for 18 to 24 h at 41 C. Biochemical and serological confirmation of the identity of colonies from these plates was then performed as described by Williams et al. (1981). Antibiotic administration involved the addition of kan to the drinking water of the birds. Although kan is not approved for use as a feed additive in poultry, it was most readily dissolved in the various bacteriological media. Bacterial cultures that grew on kan-containing media in preliminary studies were invariable identified as resistant to kan, tet, and amp in disc susceptibility testing, whereas similar correlations for media containing tet or amp were not as high. Experiment 1. The relationship between the level of antibiotic administered and the frequency of in vivo transfer of antibiotic resistance was investigated. One hundred and twenty newly hatched chicks were randomly allocated to six treatment groups of 20 chicks each on the day of hatching (Day 0). Each group in this and subsequent experiments was housed in a separate isolator unit. On Days 1 and 2, four groups (designated C, D, E, and F) were inoculated twice with EC (6 to 8 h apart). On Day 3, all six groups were inoculated twice with SA (6 to 8 h apart). One h after the second SA inoculation, kan was added to the drinking water of Groups B (80 mg/L), C (20 mg/L), D (40 mg/L), E (80 mg/L), and F (120 mg/L). These antibiotic levels were maintained in the water until the end of the experiment. On Day 5, half of the chicks in each group were killed for bacteriological examination as described above. The remaining

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3, and 4 were from an egg line (Nestor, 1980) maintained at the Ohio Agricultural Research and Development Center in Wooster, OH. Each group of newly hatched chicks or poults was housed in a modified Horsfall-Bauer isolation unit and provided water and antibiotic-free starter feed ad libitum. Bacterial Cultures. In all experiments, an E. coli culture (EC) was used as a donor of drug resistance determinants. This culture, obtained from Colorado Animal Research Enterprises (Fort Collins, CO) was resistant to kanamycin (kan), tetracycline (tet), and ampicillin (amp). In Experiment 1, a strain of S. arizonae (SA) obtained from the National Veterinary Services Laboratories (Ames, I A) was used as a plasmid recipient. This culture demonstrated resistance to nalidixic acid (nal) and streptomycin (str). In Experiments 2, 3, and 4, a strain of S. typhimurium (ST) obtained from Colorado Animal Research Enterprises was used as the plasmid recipient. This culture was resistant only to nal. Preliminary in vitro testing, by the method of Gast and Stephens (1985), established that all three resistant determinants of EC were conjugatively transferable to both SA and ST (yielding multiply resistant transconjugant cultures designated SAr and STT). The three determinants were always transferred together, indicating a close degree of genetic linkage. All testing for antibiotic susceptibility was by the standardized single disc method of Bauer et al. (1966). All media were obtained from BBL (Cockeysville, MD). Bacteriological Methods. Inoculations of birds with bacterial strains were performed by dispensing .5 mL of the appropriate overnight culture, in Antibiotic Assay broth, directly into the crop of each chick or poult with a sterile 1-mL pipet. The EC inocula contained approximately 1.22 x 109 colony forming units (cfu)/ mL; the ST inocula contained approximately 7.3 x 108 cfu/mL. After chicks or poults were killed by cervical dislocation, tissues were removed using aseptic procedures and tested for the presence of EC and Salmonella. Intestinal samples consisted of approximately 2.5-cm segments removed from the proximal ends of the ceca and adjacent regions of the large and small intestines. Liver samples of approximately 2 g were also removed. The bacteriological examination of tissues was performed according to a standardized protocol described by Gast and Stephens (1986). From each bird killed, three intestinal samples

PROLIFERATION OF DRUG-RESISTANT SALMONELLA

teriological examination of liver and intestinal tissues. Statistical Analysis. Statistically significant differences (P< .05) between means of treatment groups were determined by a one-way analysis of variance for weight gain data. Application of Student's t distribution was used for analysis of bacterial recovery data (Snedecor and Cochran, iQsm RESULTS

Experiment 1. On Day 5, EC was recovered from the intestinal tracts of all chicks that were inoculated with that strain, and from livers of 40% of chicks in Group F (given 120 mg/L kan in their water), but from none of the livers of other groups (Table 1). On Day 10, EC was recovered from livers of 40%, 20%, 10%, and 0% of chicks in groups given 120, 80, 40, and 20 mg/L kan in their water, respectively. The difference between recovery rates in groups given 120 and 20'mg/L kan was significant. When recovery data from Days 5 and 10 were combined (data not shown), the frequency of isolation of EC from the livers of chicks in Group F (given the highest level of kan) was found to be significantly greater than that from any other group. The SA was isolated from all of the intestinal samples taken from chicks on both Days 5 and 10 (Table 1). This recovery rate represents the total S. arizonae population and thus may include cells carrying resistance determinants in addition to nal and str. The SA was also found in an average of 80% of the liver samples obtained on Day 5 and from 100% of the liver samples obtained on Day 10. The SAx was found in an average of 95% of the intestinal samples taken on Days 5 and 10 from chicks inoculated with both EC and SA (Table 1); significant differences between these groups were not observed. The SAx was isolated on Day 5 from 40% of the livers of chicks given 120 mg/L kan (Group F), from only 10% of those given 80 mg/L kan (Group E), and from none of the livers of chicks given 40 or 20 mg/L kan (Groups D and C, respectively). On Day 10, SAx was recovered from 60% of the livers from both Groups E and F. The recovery rates of SAx from livers of chicks in Group F on Day 5 and from both Groups E and F on Day 10 were significantly higher than those from any other group. When liver sample data from Days 5 and 10 were combined, the frequency of isolation of SAx from livers of chicks in Groups E

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chicks in each group were killed and examined on Day 10. Experiment 2. The suitability of an ST strain as a recipient culture for in vivo resistance transfer experiments was examined. Forty-eight newly hatched turkey poults were randomly allocated to two treatment groups (designated A and B) and housed in isolator units. Both groups were inoculated twice with EC on Day 1 (6 to 8 h apart). EC was also incorporated into the drinking water of both groups (at a level of 1.22 x 106 cfu/mL) from Day 1 until Day 4. Both groups were inoculated twice with ST on Day 5 (6 to 8 h apart). In addition, poults in Group B received 80 mg/L kan in their drinking water from Day 1 until the end of the experiment. One-half of the poults in each group were killed on Day 7 for bacteriological examination of liver and intestinal tissues. The remaining poults in each group were killed on Day 14. Experiment 3. The effects of inoculation with Salmonella, in the presence and absence of kan administration, on weight gain patterns of turkey poults was evaluated in this trial. Forty-eight poults were randomly allocated to four treatment groups of twelve. Groups designated B and D were given 80 mg of kan per L of drinking water beginning on Day 1. Poults in Groups C and D were inoculated twice with ST on Day 5 (6 to 8 h apart). One-half of the poults in each group were killed for bacteriological evaluation on Day 7; the remainder on Day 14. All surviving poults were weighed individually on Days 1,7, and 14. Experiment 4. The fourth experiment was designed to provide further information regarding the effects of antibiotic administration on the spread of drug-resistant Salmonella to liver tissue. Seventy poults were used for the first trial of this experiment; 65 for the second. The newly hatched poults were randomly allocated to six treatment groups and housed in isolator units. Groups designated E and F were inoculated twice with EC on Day 1 (6 to 8 h apart). Beginning on Day 1, and continuing until Day 4, EC was incorporated into the drinking water of Groups E and F (1 mL of overnight broth culture per liter of water). Groups A, B, E, and F were inoculated twice with ST on Day 5 (6 to 8 h apart). Groups C and D were inoculated twice on Day 5 with STr. Kan (80 mg/L) was incorporated into the drinking water of Groups A, B, and C from Day 1 until the end of the experiment. All poults were weighed on Days 1, 7, and 14, and were killed on Day 14 for bac-

691

A

EC

A B C D E F

SArb

SA SA EC, EC, EC, EC,

EC, EC, EC, EC,

SA SA

SA SA EC, EC, EC, EC,

SA SA SA SA

SA SA SA SA

SA SA SA SA

Bacterial inoculum

0 80 20 40 80 120

0 80 20 40 80 120

0 80 20 40 80 120

(mg/L)

treatment2

Kan

SA resistant to nalidixic acid (nal) and streptomycin (str).

EC resistant to kan, tetracycline (tet), and ampicillin (amp).

Kan added to drinking water.

Twenty chicks per group.

SAr resistant to nal, str, kan, tet, and amp.

s

4

3

2

1

a ' b Values (within columns) with no common superscripts differ significantly (P<.05).

A B C D E F

SA"

B C D E F

Group 1

Strain recovered

100 a 100 a

80a

100 a

Ob Ob

100 a 100 a 100 a 100 a 100 a 100 a

100 a 100 a 100 a 100 a

Ob Ob

Intestine

Day 5

TABLE 1. Effect of level of kanamycin (kan) administration on the recovery rate of Es Salmonella arizonae (SA), and transconjugant SA (SAr) from chick tissue samples i

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A B

A B

ST"

STr 5

0 80 0 80

:, ST :, ST :, :, ST ST

0 80

(mg/L)

Kan treatment2

ST ST

:, :,

Bacterial inoculation

EC resistant to kan, tetracycline (tet), and ampicillin ( a m p ) .

Kan added t o drinking water.

T w e n t y chicks per g r o u p .

STr resistant to nal, str, kan, tet, and a m p .

s

*ST resistant t o nalidixic acid (nal).

3

2

1

a b ' Values (within c o l u m n s ) with no c o m m o n superscripts differ significantly ( P < . 0 5 ) .

A B

Group1

EC3

Strain recovered

Ob 42a

100a 100a

100a 100a

Intestine

Day 7

T A B L E 2. Effect of level of kanamycin (kan) administration on the recovery rate of Salmonella tymphimurium (ST), and transconjugant ST (STr) from poult tissue samp

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694

GAST AND STEPHENS

7 to 14, however, poults treated with kan but not inoculated with ST (Group B) gained significantly more weight than did those inoculated with ST but given no kan (Group C). Weight gains of uninoculated birds from Days 7 to 14 (Groups A and B combined), moreover, were significantly greater than those of poults inoculated with ST (Groups C and D combined, data not shown). There were no significant differences in weight gains for Days 1 to 14. Administration of kan in the drinking water of ST-inoculated poults reduced the frequency of recovery of ST from intestinal samples on both sampling dates in Experiment 3 (Group D vs. Group C in Table 4). When data for Days 7 and 14 were combined, the ST recovery rate from the livers of poults inoculated with ST and not given kan (58%) was found to be significantly greater than that from ST-inoculated poults given kan (17%). Experiment 4. Poults inoculated with ST and treated with kan (Group B) gained significantly more weight than poults inoculated with ST but given no kan (Group A) during each interval measured (Table 5). Kan administration had no apparent effect, however, on the weight gains of poults inoculated with S7r (Group C vs. Group D). Moreover, the provision of kan had no significant effect on the weight gains of poults inoculated with both EC and ST (Group E vs. Group F). When data were pooled, weight gains of poults in the three groups given kan were found to be significantly greater between Days

TABLE 3. Effect of Salmonella typbimurium1 (ST) inoculation and kanamycin (kan) administration on weight gains of poults in Experiment 1 Weight gain, X Group 2

Treatments

Weight (Day 1)

Days 1 to 7

A B C D

Control Kan3 ST 4 Kan, ST

45.08 a 45.64 a 45.50 a 45.67 a

39.34 a 38.09 a 39.50 a 39.31 a

Days 7 to 14

Days 1 to 14

95.83 a b 104.17 a 86.00 b 91.33 a b

137.00 a 144.67 a 130.50 a 134.00 a

(g)

ab ' Values (within columns) with no common superscripts differ significantly (P<.05). Resistant to nalidixic acid (nal).

1 2

Twelve poults per group.

3

Kanamycin added to drinking water at 80 mg/L.

"Inoculated with nal-resistant ST.

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and F was found to be significantly greater (42.5%) than from those in the groups given lower levels of kan or not inoculated with EC (0%). Experiment 2. The EC was isolated from all intestinal samples and from 8% of liver samples taken on Days 7 and 14 from poults inoculated with EC and ST and given kan (Group B in Table 2). The ST (resistant to at least nal) was recovered from all the intestinal samples taken on Days 7 and 14 (Table 2). The ST was also detected on Day 7 in all the liver samples taken from poults not given kan (Group A), but from none of the liver samples taken from kan-treated poults (Group B) on that day. On Day 14, however, ST isolates were obtained from 75% of liver samples from poults in Group A and from 92% of those from poults in Group B. The 5Tr was isolated on Day 7 (Table 2) from a significantly higher percentage of intestinal samples taken from kan-treated poults (42%) than from those of poults given no kan (0%). On Day 14, S7r was isolated from the intestines of all kan-treated poults, but from significantly fewer (17%) of those from poults given no kan. No STr was found in liver samples taken on Day 7. On Day 14, STr was recovered from livers of 83% of the kan-treated poults, but from livers of none of the poults given no kan. Experiment 3. No significant differences in weight gains between groups of poults were observed during Days 1 to 7 (Table 3). From Days

PROLIFERATION OF DRUG-RESISTANT SALMONELLA

695

TABLE 4. Effect of kanamycin (kan) administration on recovery rate of Salmonella typhimurium from poult tissue samples in Experiment 3 Day 7 Group 2

Treatments

A B C D

Control Kan3

Intestine

1

(ST)

Day 14 I ntestine

Liver

Liver

(%) Ob Ob

100 a

ST4

Kan, ST

Ob Ob

0" 0" 50" 0"

83"

100" 83"

Ob Ob 66"

3 3"b

a ' b Values (within columns) with no common superscripts differ significantly (P<.05). 1 2 3

Resistant to nalidixic acid (nal). Twelve poults per group. Kanamycin added to drinking water at 80 mg/L.

1 and 14 than were those of poults in the remaining groups. The EC was recovered from the intestines of all £C-inoculated poults (Groups E and F), but from none of the livers. The ST (resistant to at least nal) was recovered from an average of 98% of intestinal samples, although no significant differences between recovery rates of groups were observed (Table 6). Among poults inoculated only with ST (Groups A and B), administration of kan was associated with a significantly

reduced rate of recovery of ST from livers (18% in Group B vs. 71% in Group A). The frequency of recovery of ST from livers of poults given ST but no kan (Group A) did not differ significantly from the 50% recovery rate from those given STr and no kan (Group C), but the 76% recovery rate from poults inoculated with STr and given kan (Group D) was significantly higher than that from poults given ST and kan (Group B). The recovery rates of ST from livers of poults given no kan and inoculated with either

TABLE 5. Effect of bacterial inoculations and kanamycin (kan) administration on weight gains of poults in Experiment 4 Weight gain, X Group 1

Treatments

Weight (Day 1)

Days 1 to 7

A B C D E F

ST2 ST, kan 3 STr STr, kan ECS, ST EC, ST, kan

49.38" 48.91" 50.14" 50.14" 50.54" 49.84"

47.29 b 58.14" 54.00 a b 53.76" b 49.25" b 53.40" b

g)

Days 7 to 14

Days 1 to 14

81.19 b 96.68" 86.91ab 86.62" b 75.08 b 83.76" b

128.48b 154.82" 140.91" b 140.38"b 124.33b 137.16"b

ab ' Values (within columns) with no common superscripts differ significantly (P<,05). 'Twenty-one poults in Group A; 22 in B; 22 in C; 21 in D; 24 in E; and 25 in F. 2

ST = Salmonella typhimurium

3

resistant to nalidixic acid (nal).

Kan added to drinking water at 80 mg/L.

4

STr resistant to nal, kan, tetracycline (tet), and ampicillin (amp).

5

EC = Escherichia coli resistant to kan, tet, and amp.

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"inoculated with nal-resistant ST.

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GAST AND STEPHENS

TABLE 6. Effect of kanamycin (kan) administration on the recovery rate of Salmonella typhimurium strains resistant to nalidixic acid (nal), kan, tetracycline (tet), and ampicillin (amp) from poult tissue samples in Experiment 4

(ST)

Nal, kan, tet, amp

Nal Group 1

Treatments

Intestine

Liver

A B C D E F

ST2 ST, kan3 STr STr, kan EC, ST EC, ST, kan

100a 86a 100a 100a 96a 100a

71 a 18 b 5 0 ab 76a 54 ab 64 a

Intestine

Liver

(%) 0a 0a 100b 100b 17a 100b

oaa ob

50 76 b 4a 48 b

ab ' Values (within columns) with no common superscripts differ significantly (P<.05). ST resistant to nalidixic acid (nal).

3

Kan added to drinking water at 80 mg/L.

4

STr resistant to nal, kan, tet, and amp.

5

EC = Escherichia coli resistant to kan, tet, and amp.

ST (Group A) or both EC and ST (Group E) did not differ significantly (71% and 54%, respectively). However, the 64% recovery rate from poults inoculated with both EC and ST and also given kan (Group F) significantly exceeded the 18% recovery rate from poults inoculated with ST and given kan (Group B). The 5Tr was recovered from the intestines of all poults given EC, ST, and kan (Group F in Table 6) or inoculated with STr and given kan (Group C), but from significantly fewer (17%) of those from poults inoculated with EC and ST and not given kan (Group E). A similar treatment effect upon recovery rates of STr from liver samples was observed. The 5Tr was isolated from livers of a significantly higher percentage of poults in groups either inoculated with STr (Groups C and D) or inoculated with both EC and ST and given kan (Group F) than from livers of poults inoculated with EC and ST but not given kan (Group E). DISCUSSION

Observations made in this study can be summarized in two general statements. First, kan administration significantly reduced the occurrence of drug-sensitive Salmonella in livers of inoculated birds, but had no effect on the occurrence of such organisms in the birds' intestines. Second, antibiotic administration significantly

increased the frequency of recovery of drug-resistant Salmonella from livers of chicks and poults inoculated with a drug-sensitive strain and drug-resistant E. coli. In the absence of kan administration, SA and ST persisted in the intestinal tract and invaded the liver at a high frequency. The diminished frequency of recovery of ST from livers of kantreated poults in Experiments 3 and 4 may have resulted from interference by kan with colonization or penetration of the intestinal epithelium by ST cells. Alternatively, a reduction in the number of animals carrying Salmonella following antibiotic administration (as reported by Evangelisti etal., 1975) may have led to a reduction in Salmonella levels in individual animals. As the probability of colonization and penetration of the intestinal epithelium is to some extent a function of the number of bacterial cells present in the gut, reducing the number of ST cells in the intestinal tract should reduce the number of cells that reach the liver. In Experiment 4, the kan-treated poults inoculated with ST gained significantly more weight than did those not given kan. Poults in the group given no kan probably harbored a larger intestinal Salmonella population, and thus suffered a higher degree of morbidity than did kan-treated poults. The effectiveness of kan in restricting the spread of ST to the poults' livers prior to Day 7 of Experiment 2 was probably due to the fact

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'Twenty-one poults in Group A; 22 in B; 22 in C; 21 in D; 24 in E; and 25 in F. 2

PROLIFERATION OF DRUG-RESISTANT SALMONELLA

by multiply resistant STx was particularly responsive to antibiotic administration, which enabled the transconjugants to colonize and invade the intestinal epithelium and thereby spread to the liver in greater numbers than in birds given no kan. In the absence of antibiotic pressure, drug-sensitive and drug-resistant Salmonella strains of the same species have been found to demonstrate virtually identical degrees of invasiveness (Lakhotia and Stephens, 1974). Gast and Stephens (1986) similarly reported that antibiotic treatment was associated with an increased frequency of recovery of antibiotic-resistant transconjugant Salmonella. The rate of recovery of SAr from poults given no antibiotic in that study was much higher than that observed for the S7r recipient culture in the present study, suggesting that ST is a less effective plasmid recipient than SA. Variations in recipient competence between Salmonella strains have been previously reported (Lakhotia et al., 1972; Gast and Stephens, 1985). Differences in the abilities of various bacterial strains to act as recipients for the transfer of resistance genes can be due to a number of factors, including the presence of restriction systems that encode enzymes to degrade unmodified incoming DNA molecules (Lyon and Skurray, 1987). Growth promotion by antibiotics has been hypothesized to result from inhibition of deleterious intestinal microorganisms (Jukes, 1975), although feed additive levels of antibiotics have been found by some researchers to only minimally affect the shedding rate of Salmonella (Abou-Youssef et al., 1982). The growth-promoting effectiveness of kan, which perhaps was due to inhibition of drug-sensitive Salmonella, was thus lost when the EC resistance plasmid was introduced into the ST population. Two observations based on these experiments have particularly important implications in regard to the possible transmission of drug-resistant Salmonella through the food chain. First, kan-resistant Salmonella was recovered from edible viscera (livers) of chicks and poults orally inoculated with kan-resistant Salmonella or both EC and kan-sensitive Salmonella. Second, the frequency of such recovery was increased by kan administration to the birds at therapeutic levels. Data obtained from the present experiments support the concept that administration of antibiotics to animals, particularly at therapeutic levels, exerts a selective pressure that can alter

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that few ST cells had acquired the resistance plasmid by that time. By Day 14 a larger proportion of the total intestinal ST population had apparently acquired the resistance plasmid, as the provision of kan in the water seems to have actually promoted the invasion of the liver by ST. The increase over time in the rate of recovery of ST from livers of poults given kan may thus have been largely a function of the relative abundance of drug-resistant cells in the intestinal ST population. Subtherapeutic administration of antibiotics to poultry has been found, in several instances, to reduce the shedding rate of drugsensitive Salmonella (Jarolmen et al., 1976; Williams, 1985). Persistent shedding, however, may depend on the colonization and invasion capabilities of particular Salmonella strains. The recovery rate of SAr from the intestinal tracts of chicks inoculated with both EC and SA in Experiment 1 was consistently high, suggesting that conjugative transfer of the resistance plasmid proceeded at a fairly high frequency. Although kan administration had no significant effect on the recovery rate of SAr from the intestines, the influence of kan was evident in the rate of recovery of SAr from livers. As with the EC strain, the recovery rate of SAr from livers was proportional to the level of kan provided to chicks, suggesting that the antibiotic conferred a selective advantage on resistant cells in colonizing or invading the intestinal epithelium. In chicks given lower levels of kan, other drug-sensitive microflora may have more successfully competed with the drug-resistant Salmonella. Such competition has been noted by Weinack etal. (1982). Evangelisti et al. (1975) theorized that, although subtherapeutic levels of antibiotics might increase the portion of the bacterial flora that is drug-resistant, such antibiotic pressure is insufficient to give resistant strains any meaningful selective advantages. In an experiment conducted with the same bacterial strains used in the present study, Gast and Stephens (1986) isolated transconjugant 5. arizonae from 40% of the intestines, but from none of the livers, of poults inoculated with EC and SA and given no antibiotic. The recovery of STx from intestinal and liver samples in Experiments 2 and 4 was also dramatically affected by provision of kan to poults. The presence of the antibiotic may act to shift the equilibrium between costs and benefits of plasmid maintenance to favor plasmid retention over spontaneous plasmid loss by bacterial cells (Sansonetti et al., 1980). Invasion of the liver

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GAST AND STEPHENS

the composition of the intestinal bacterial flora in favor of drug-resistant strains. Furthermore, linkage of resistance genes on particular plasmids makes it possible to indirectly select for several resistance determinants via the administration of a single antibiotic. In the present study, administration of kan was correlated with increased rates of recovery of Salmonella resistant to tet and amp as well as kan, as all three of the resistance determinants were carried on the same plasmid. REFERENCES

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