Receptiveness of Salmonella and Arizona Strains to R Factor from Escherichia Coli in Vitro1

Receptiveness of Salmonella and Arizona Strains to R Factor from Escherichia Coli in Vitro1


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RECEPTIVENESS OF SALMONELLA AND ARIZONA STRAINS TO R FACTOR FROM ESCHERICHIA COLI IN VITRO1 R. L. LAKHOTIA, J. F. STEPHENS AND SHARON HARPSTER Department 0/ Poultry Science, Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (Received for publication August 8, 1972) ABSTRACT Twelve antibiotic-sensitive strains of Arizona and ten of Salmonella were tested for ability to receive drug resistance in vitro from two multiple resistant, R factor-carrying strains of E. coli isolated from soiled eggs. Eleven of the 12 Arizona strains and 8 of the 10 Salmonella strains received the R factor from at least one of the donor E. coli strains. One of the donor strains transferred only part of its drug resistance pattern to some of the sensitive strains. POULTRY SCIENCE 51: 2106-2108,


A total of 56 Salmonella cultures from chickens (7), turkeys (29), feed or feed in1 Ohio Agricultural Research and Development Center Journal Article No. 71-72.

gredients (20) and 18 Arizona cultures from turkeys were obtained from several diagnostic and research laboratories. Each isolate was tested for sensitivity to nine antimicrobial drugs by a standardized singledisc method (Bauer et al., 1966). The ten Salmonella and 12 Arizona strains which were sensitive to the largest number of drugs were subsequently tested for ability to receive the R factor from donor strains of E. coli. The donor strains of E. coli were selected from several R factor-containing strains isolated from chicken and turkey eggs soiled with feces. The obvious original source of these E. coli cultures was the intestinal tracts of the birds. The two strains selected as "donors" were efficient in transferring their patterns of drug resistance to a drug-sensitive strain of E. coli (K-12NA) obtained from Dr. H. Jarolmen, American Cyanamid Co., Princeton, N.J. One of the donor strains, designated 1-19, is resistant to chlortetracycline (A), oxytetracycline (T), neomycin (N), and dihydrostreptomycin (Ds), and transfers resistance to all four antibiotics to E. coli K-12NA. The other strain (M-22) is resistant to A, T, Ds, colistin, and furazolidone, but only transfers resistance to A, T, and Ds to E. coliK-UNA (Table 1).

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Several workers have reported the occurrence of transferable drug resistance among Escherichia coli (Smith and Halls, 1966; Mitsuhashi et al., 1967; Kim and Stephens, 1972), and Salmonella spp. (Smith, 1970; Pocurull et al., 1971) isolated from poultry. It is possible that nonpathogenic E. coli in the alimentary tract can act as a reservoir of drug resistance transferable to human and animal pathogens. Special concern has been expressed concerning the possibility of transfer of antibiotic resistance from E. coli to Salmonella spp. because the latter are considered zoonotic pathogens. Little information on transferable drug resistance in cultures of Arizona is available, although this group of bacteria presents a considerable disease problem in turkey production. It was the purpose of this study to determine to what extent some enteric pathogens {Salmonella and Arizona) are capable of becoming drug-resistant through contact with drug-resistant members of the normal intestinal microflora (E. coli) having the resistance (R) factor.




TABLE 1.—In vitro transfer of drug resistance from Escherichia coli to Salmonella and Arizona strains Resistance Pattern Transferred

Donor strain of E. coli

To E. coli K-12NA

To Arizona

To Salmonella

1-19 M-22

A, T. Ds. N 1 A , T , Ds

A, T, Ds, N(10/12) 2 A, T(8/12)

A, T, Ds, N(6/10) A, T, Ds(2/10), Ds(4/10)


A=chlortetracycline, T = oxytetracycline, Ds=dihydrostreptomycin, N=neomycin. Denominator represents number of Arizona or Salmonella strains tested. Numerator represents number of strains that received resistance pattern indicated. 2

RESULTS AND DISCUSSION Results of this study indicate that a high percentage of drug-sensitive Salmonella and Arizona cultures of avian or feed origin can receive drug resistance by the mechanism of R factor transfer from E. coli (Table 1). Only one of the 12 Arizona cultures and two of the ten Salmonella cultures failed to receive R factor from either of the donor strains of E. coli. E. coli 1-19 transferred its entire resistance pattern to the recipient cultures of Arizona and Salmonella. Donor strain M22, which transferred only part of its resistance pattern to E. coli K-12NA, transferred only chlortetracycline-and oxytetracycline resistance to the eight recipient Arizona cultures. E. coli M-22 also transferred A, T, and Ds resistance to two of the six recipient Salmonella cultures, but trans-

ferred only Ds resistance to the other four cultures (Table 1). These results illustrate differences between strains of drug-resistant E. coli in efficiency of R factor transfer as well as differences between strains of Arizona and Salmonella as recipients. It was observed in the course of the experiment that after introduction of R factor from E. coli 1-19 into a strain of Salmonella injantis, the Salmonella developed characteristics of "rough" cultures. This strain formed a granular precipitate in tubes of liquid medium and no longer reacted with specific O group antiserum. It also clumped in the presence of neutral acriflavin-a characteristic of "rough" cultures described by Braun and Bonestell (1957). Similar observations have been reported by Jarolmen and Kemp (1968) with a strain of Salmonella cholera-suis var. kunzendorf. Such changes were not observed in any culture of Arizona used in this study. Further studies are needed to explain the cause of roughness in some strains after they receive the R factor, and to further evaluate the significance of R factor transfer between nonpathogenic and pathogenic bacteria associated with poultry. REFERENCES Bauer, A. W., W. M. M. Kirby, J. C. Sherris and M. Turck, 1966. Antibiotic susceptibility testing by a standardized single disk method. Amer. J. Clin. Pathol. 36: 493-496. Braun, W., and A. E. Bonestell, 1947. Independent variation of characteristics in Brucella abortus

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A 0.1 ml. quantity of 24 hr. broth culture of prospective recipient Arizona or Salmonella strain and 0.05 ml. of donor strain were added to 10 ml. of sterile antibiotic medium 3 (Difco); the mixed culture was incubated overnight at 37°C, centrifuged, and the precipitate streaked on one plate of Brilliant Green Agar containing A (30[Ag./ml.), one containing Ds (10 [j,g./ml.) and one plate containing no antibiotic. Salmonella or Arizona cultures growing on the agar containing either A or Ds were subsequently examined for resistance to the selected drugs by the singledisc procedure.



variants and their detection. Amer. J. Vet. Res. 8: 386-390. Jarolmen, H., and G. Kemp, 1969. Association of increased recipient ability for R factors and reduced virulence among variants of Salmonella cholera-sttis var. kunzendorf. J. Bacteriol. 97: 962-963.

Kim, T. K., and J. F. Stephens, 1972. Drug resistance and transferable drug resistance of Escherichia coli isolated from "ready-to-cook" broilers. Poultry Sci. S i : 1165-1170. Mitsuhashi, S., H. Hashimoto and K. Suzuki, 1967. Drug resistance of enteric bacteria. XIII. Distribution of R factors in Escherichia coli

strains isolated from livestock. J. Bacteriol. 94: 1166-1169. Pocurull, D. W., S. A. Gains and H. D. Mercer, 1971. Survey of infectious multiple drug resistance among Salmonella from animals in the United States. App. Microbiol. 2 1 : 3 58-362. Smith, H. W., 1970. The transfer of antibiotic resistance between strains of enterobacteria in chicken, calves and pigs. J. Med. Microbiol. 3 : 165-180. Smith, H. W., and S. Halls, 1966. Observations on infective drug resistance in Britain. Vet. Rec. 78: 415-420.


Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80521 (Received for publication August 8, 1972)

ABSTRACT Chicks were hatched from S.C.W.L. hens fed either a control ration or one supplemented with 1333 p.p.m. ascorbic acid. Chicks from each maternal ration were allotted to either a control ration or one supplemented with S00 p.p.m. ascorbic acid. Plasma total and free cholesterol were determined on blood obtained from 1, 7, 14 and 21 day-old chicks on each treatment. Day-old chicks from hens fed ascorbic acid had significantly higher (P < 0.01) plasma total and free cholesterol than did the controls. Plasma cholesterol levels decreased rapidly after hatching and plateaued at about 14 days of age in chicks from all treatments. Ascorbic acid supplementation did not significantly alter hatching or subsequent chick weight. POULTSY SCIENCE 51: 2108-2110, 1972



Feeding ascorbic acid to hypercholesteremic rabbits (Sokoloff et al., 1967), pigs (Hutagalung et al. 1969), and humans (Ginter et al., 1970) resulted in significantly reducing serum cholesterol levels. The purposes of this investigation were to determine if supplementing the hen's ration with ascorbic acid would alter chick plasma cholesterol level and if feeding ascorbic acid to the chick would change plasma cholesterol levels.

Single Comb White Leghorn pullets were fed the previously reported grower and layer diets (Dorr and Nockels, 1971). From 9 weeks of age treated hens received 1333 p.p.m. supplemental ascorbic acid in their rations. When the hens reached 35 weeks of age, eggs were incubated. The chicks produced were weighed prior to bleeding. At 1, 7, 14 and 21 days of age, blood samples were collected by heart puncture using a syringe rinsed with sodium heparin. Free and total plasma cholesterol were determined according to Zak (196S). Cholesterol ester was estimated by subtracting free from total cholesterol.

"Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series paper number 1726.

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