ANTIMICROBIAL AGENTs AND CHEMOrHERAPY, May 1978, p. 710-715 0066-4804/78/0013-0710$02.00/0 Copyright X) 1978 American Society for Microbiology
Vol. 13, No. 5
Printed in U.S.A.
Chloramphenicol Resistance Plasmids in Escherichia coli Isolated from Diseased Piglets SIGRID TUE J0RGENSENt Department of Animal Genetics, Royal Veterinary and Agricultural University, Copenhagen V, Denmark Received for publication 7 December 1977
The plasmids in 19 chloramphenicol-resistant Escherichia coli strains of three pig pathogenic antigen types were studied in conjugation and transduction experiments. The plasmids had identical resistance patterns: streptomycin, spectinomycin, sulfonamides, and chloramphenicol (Sm, Sp, Su, Cm) and belonged to IncFII. One plasmid carried ampicillin resistance in addition. Restriction enzyme analysis of the deoxyribonucleic acid from five of the plasmids originating from the same herd showed that their digestion patterns with EcoRI were indistinguishable. EcoRI cleaved the deoxyribonucleic acid of a sixth plasmid from the same herd and displayed nine of the ten bands of the other five plasmids plus an additional six. It appears that the five plasmids with identical restriction patterns have a common origin and may be copies of the same plasmid from which the sixth may have developed. Four strains carried two plasmids each. In two of these strains, a plasmid with a tetracycline marker (Tc), or possibly the tetracycline marker alone, recombined frequently with the Sm Sp Su Cm plasmid without destroying any known function of the latter. The possibility that Tc is carried on a translocation sequence is discussed.
In a study in 1976 (9) it was found that chloramphenicol (Cm) resistance plasmids occur with low, but noticeable frequency in Escherichia coli isolated from cases of diarrhea in Danish piglets. This situation has developed within the past few years. Except for some closed herds where antibiotics were used routinely (11), Cm resistance plasmids in E. coli from Danish piglets-whether healthy or diseased-were scarce until a few years ago (S. T. J0rgensen, Ph.D. thesis, Royal Vetr. & Agric. University, Copenhagen, 1974). The importance of Cm in the treatment of systemic salmonellosis in humans and animals and typhoid fever in humans, and its characteristic of being a "last resort drug" against cholera, render it necessary to watch, in animals, for the development of plasmid-borne resistance against this drug.
MATERIALS AND METHODS
Strains, plasmids, and bacteriophages. The organisms consisted of 19 naturally occurring Cm-resistant strains, 15 of which have been mentioned in a previous publication (9). The source as well as details of the isolation procedure was reported in the same paper. That description also applies to the four new strains. Three antigenic types were represented among the strains: 08:K87, 0141:K85ac, and 0149:K91. The reference strains were those already mentioned (9) plus a streptomycin (Sm)-resistant recA derivative of E. coli K-12. This strain and E. coli C600, as well as the reference plasmids listed in Table 1, and phage Plkc were obtained from Y. Chabbert, Institute Pasteur, Paris. Strain UB281 nalr pro met X lys recA was a gift from P. M. Bennet, University of Bristol, Bristol, England. Phage MS2 was from laboratory stock. Media In addition to those earlier reported (9), Miiller-Hinton agar (Difco) containing the appropriate antibiotics was used for selection of transconjugants, The aim of the present work was to begin a and L broth (12), phage buffer, and soft agar (5) were characterization of plasmids from 19 Cm-resist- applied in the preparation of lysates of phage M82 Plkc. ant E. coli strains of three different antigen andAntibiotics. The concentrations of the antibiotics types. Such a characterization might indicate in the selection plates were as follows: Ampicillin (Ap), whether the emergence of plasmids mediating Cm, dihydrostreptomycin sulfate (SM), kanamycin Cm resistance in pigs can be ascribed to the (Ka), and tetracycline (Tc), 12.5 ,g/ml; sodium nalispread of a few plasmid types, or even one in dixate, 20 ,ug/ml; and SM 1,000 ,ug/ml. The latter SM particular, analogous with the dissemination of concentration refers to selection for chromosomal Sm trimethoprim resistance among humans de- resisitance. All amounts mentioned above were pure substances. Cm, Sm, and Tc were commercially availscribed by Datta and Hedges (7). able compounds. Ka and Ap were obtained from H. t Present address: Department of Bacteriology, The Med- Lundbeck & Co. A/S, sodium nalidixate was obtained ical School, University Walk, Bristol BS8 1TD, England. from Winthrop Medicinalkompagni A/S, and rifamy710
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
VOL. 13, 1978
TABLE 1. Reference plasmids for incompatibility studies Plasmid
Ripl62/2 Rldrd-16 Rip1l2 Ripll3 RP4 Ripl35/1 R391 Rtsl Rip72
Phenotypic anti biotic resistance
markers
Ap Tc Ka Ka Tc Ap Ka Tc Tc Ka Ka Ka
Incompatibil- Referity group
IncFI IncFII IncIa IncN IncP IncM IncJ IncT InclO
ence
4 13 3 3 7 18 6 6 17
was obtained from A/S Dumex. Antibiotic disks were purchased from AB Biodisk, Sweden, and con-
cin
tained 30 ug of each of the above-mentioned antibiotics except Ap, the concentration of which was 10 lAg/disk. Sulfdimidine (Su) and spectinomycin (Sp) were used only in disks. The concentrations were 250 and 30,ug/disk, respectively. Sp-containing disks were used to distinguish between the two main types of Sminactivating enzymes-the adenylyltransferase, which also inactivates Sp, and the phosphotransferase, which operates on Sm only (2). Conjugation and incompatibility experiments. Exponentially growing cultures of recipient and donor strains were mixed in a ratio of 5:1 in 10 ml of nutrient broth and incubated either for 2 h or overnight at 37°C. Samples of 0.1 ml of the mating mixtures and suitable dilutions were plated on appropriate antibiotic agar, selecting for the chromosomal resistance of the recipient and each of the plasmid resistance markers. A total of 50 transconjugants from each selection plate were patched on nutrient agar after purification and then replicated on antibiotic agar in search of unselected markers. Incompatibility between a reference and a test plasmid was studied by introducing either one into E. coli recA carrying the other. Three single colonies were tested for the resistance markers of both plasmids and, if both plasmids were present, each clone was subcultured up to four times in antibiotic-free broth with dilution when the cell number reached 107. The subcultures were kept overnight on ice, and when all reached 107 cells per ml, they were diluted and plated on antibiotic-free nutrient agar. Colonies obtained this way were replicated onto plates, each containing one antibiotic according to the determinants being tested for. Between 200 and 300 colonies from each original clone were thus examined for coexistence of the test and reference plasmids. Test for Fi character. The plasmid under study was introduced into E. coli HfrH, and its ability to repress production of F pili was studied by spot tests with phage MS2. Loss of sensitivity to MS2 was taken as evidence of the unknown plasmid's Fi+ character. Transduction experiments. Transduction experiments were carried out by means of phage Plkc propagated by the method of Adams (1) on E. coli nair harboring the plasmid under study. Transduction to E. coli C600 was as described by Lennox (12). per disk,
711
Isolation of plasmid deoxyribonucleic acid (DNA), digestion with restriction endonuclease EcoRI, and separation of the fiagments in 0.7% agarose gels was done essentially as described by Petrocheilou et al. (14). Phage A DNA, which was used as an internal marker in all gels, was a generous gift from J. Grinsted, University of Bristol, who also kindly donated the restriction enzymes. Agarose type HSA was purchased by Litex, Tastrup, Denmark.
RESULTS Demonstration of separate linkage groups. Table 2 shows that all 19 strains carried resistance to Su, Sm, and Sp in addition to Cm. Moreover, two strains were resistant to Tc and one was resistant to Ap. In 17 strains the resistance markers Sm Sp Su Cm were not observed to separate during either conjugal transfer or transduction and, therefore, are assumed to be on the same replicon. Strains 3734 and 2742 behaved differently. Strain 3734 transferred Sm Sp Su Cm Ap when selection was made for AP or Cm, but Ap Cm Sp were rarely seen as unselected markers with Sm selection. In this case the great majority of transconjugants had the phenotype Sm Su. Transduction experiments were then carried out with phage Plkc grown on cells with either phenotype. Only the Sm Sp Su Cm Ap pattern could be transduced. In spite of several attempts, transduction of the Sm Su markers was not accomplished. These results are compatible with the inactivation of Sm in strain 3734 by two different enzymes (2), as shown by the two different resistance patterns transferred, and make it highly probable that strain 3734 carries two plasmids, one of them, pHG17 (Sm Su), presumably too large to be packed in phage Plkc. In strain 2742 the resistance markers behaved in much the same way. Cm selection gave transconjugants carrying all markers (Sm Sp Su Cm), whereas only half of the transconjugants selected on Sm coinherited Sp Cm with Sm Su. All transductants exmined carried the fourmarker pattern and had retained transferability of the markers. Since the results from strain 2742 were analogous with those from strain 3734, it is assumed that strain 2742 harbors two plasmids, pHG18 (Sm Sp Su Cm) and pHG20 (Sm Cu). This assumption was later confirmed by electrophoresis of plasmid DNA in agarose gels. The resistance markers in strains 1324 and 1325 originally constituted two separate linkage groups, one mediating resistance to Sm, Sp, Su, and Cm, the other mediating resistance to Tc. The first group of markers was located on a
derepressed plasmid, which upon direct selection transferred Tc marker at a very low fre-
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VOL. 13, 1978 quency.
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
The ratio of Sm Sp Su Cm transconju-
gants to Sm Sp Su Cm Tc transconjugants was 106:1. It was not possible to obtain Tc free of Sm
Sp Su Cm. When the rare transconjugants that carry the Tc marker in addition to Sm Sp Su Cm were crossed, a high proportion proved to transfer the Tc marker at the same elevated frequency as Sm Sp Su Cm. Transduction of such transconjugants gave only one type of transductants, namely transfer-proficient Sm Sp Su Cm Tc colonies. Transconjugants retaining the low transfer frequency for Tc when crossed restricted phage Plkc, so transduction information is unavailable. From these results it was concluded that each of the two wild strains harbors two plasmids. One carries the markers Tc Tra- and recombines frequently with the other, which carries Sm Sp Su Cm Tra+ drd, thereby losing its phage inhibition function. In transconjugants where the two plasmids proved to be independent, mobilization must be responsible for transfer of the Tc Tra- plasmid. After a couple of months the work with the plasmids in strain 1325 was resumed and the transfer frequency for pHG28 Tr Tra- Res (Plkc) (or rather the frequency with which it was mobilized by pHG25 Sm Sp Su Cm Tra+) was 1,000-fold higher than that previously found. This change in the transfer frequency of pHG28 proved to be stable, and no other character was altered. Presumably a mutation in some system regulating the mobilization process is responsible for the change in frequency. Incompatibility reactions. Separation of the plasmids originating from the same strain was followed by a determination of their incompatibility group. However, for the reasons mentioned above it was not possible to separate pHG27 and pHG28 from pHG24 and pHG25, respectively. Moreover, the two pairs often recombined, and further study of pHG27 and pHG28 was therefore postponed. pHG24 and pHG25, however, were both IncFII plasmids, and the same was true for their recombinants with pHG27 and 28. Like pHG24 and pHG25, the remaining 16 Sm Sp Su Cm plasmids and pHG17 were found to inhibit phage MS2-induced lysis of E. coli HfrH carrying the plasmid under study. This suggested that the plasmids belonged to the F incompatibility complex. Further studies proved that all were incompatible with Rldrd-16 and thus IncFII plasmids. Most of them separated extremely efficiently from the reference plasmid, i.e., 90-100% separation was obtained after one subculture in antibiotic-free broth. Only a few needed three or four successive subcultures to obtain the same results.
713
In spite of their lack of stability with Rldrd16 which clearly demonstrates their membership of IncFII, some of the plasmids also showed varying degrees of interaction with the IncFI reference plasmid. This interaction in some cases took the form of an early loss (dislodgment) of the nonselected plasmid so that on resistance determination of the transconjugants only the incoming plasmid was present. In other cases dislodgment appeared during subcultivation of cell lines, which initially harbored both plasmids so that 25 to 50% of the colonies had lost the resident plasmid. This happened most often when the reference plasmid was the incoming plasmid. No valid explanation can at present be offered for these irregularities. All Sm Sp Su Cm plasmids and PHG17 were tested for incompatibility with reference plasmids from IncIa, -N, -P, -M, -J, -T, and -10. No incompatibility or interaction was observed in any case. Restriction enzyme analysis. Because some of the plasmids showed interaction with the IncFI reference plasmid and others did not, it was decided to do restriction enzyme analyses on their DNA to obtain information on their
degree of similarity. Transduced colonies, which had been crossed to prove that their plasmids were still transfer proficient, were used for DNA isolation. The purified DNA was first run undigested in an agarose gel to determine that only one plasmid DNA was present. Digestion with EcoRI of DNA from plasmids pHG1, -4, -7, -9, and -10 generated 10 bands with each after electrophoresis in agarose gels. The migration patterns of the bands were identical (Fig. 1). Another plasmid from that series, pHG11, had nine of the bands of the five plasmids mentioned above plus six extra bands (Fig. 2).
DISCUSSION The fact that the nonconjugative plasmids pHG27 and pHG28 were mobilized at very low frequency by derepressed pHG24 and pHG25 leads to the assumption that this low frequency reflects the recombination frequency between each of the two plasmid sets. Examination of the total number of Tc transconjugants (four in all) from the conjugation strain 1324 with E. coli nalr revealed that three of them harbored recombinant plasmids. The fourth, however, carried pHG24 and pHG27, i.e., no recombinationtion had taken place. Accordingly, the occurrence of the fourth transconjugant argues against the above hypothesis. How much of pHG27 integrates into pHG24 remains obscure-perhaps only the Tc genes and cer-
714
JORGENSEN
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. EcoRI-digested DNA from pHG1, -4, -7, -9, and -10. Phage y DNA was cleaved with EcoRI and BamHI and used as an internal marker. Electrophoresis was carried out in 0. 7% (wt/vol) agarose gels for 2 h, 45 min. Q ploza-Idt bctvc.ts
x
a" as
FIG. 2. EcoRI-cleaved DNA from pHG4 and -11. Phage y DNA was digested as described in the legend of Fig. 1. - indicates bands in common; ---- indicates bands only in pHG11; .. indicates band only in pHG4.
tainly not the region coding for phage Plkc restriction. Since recombination occurred relatively often, the possibility that the Tc marker might be carried on a translocation sequence was considered. This is supported by the finding that pHG24 and pHG27 were able to form stable recombinants for the resistance markers in an recA background. This finding applies to two of nine colonies examined. There is therefore no doubt that tet integrates into pHG24 even in the absence of the recA function. Against the hypothesis is that, although TnT is strongly polar (10), pHG24 was not observed to lose any of its phenotypic markers on acquisition of Tc. This may, however, be interpreted as integration of tet at a place in the DNA that has no easily recognizable function. Molecular studies that are underway might give the final proof of Tc as carried on a translocation sequence. The genetic data presented in this paper show
that the Cm resistance plasmids found among Danish piglets are so similar that a common origin is very likely. Postulating a closer relationship than that would be audacious on the basis of the genetic knowledge, since these plasmids do not belong to any rare incompatibility group such as those described by Datta et al. (8), Roe and Lowbury (16), and Datta and Hedges (7). But restriction enzyme analysis of their DNA represents a means of obtaining further information of a putative close relationship. Preliminary data show that five plasmids in strains isolated from the same herd within a month generate identical restriction patterns with EcoRI. Another plasmid from that series, pHG11, shows 9 of the 10 bands of the five above mentioned plus another 6. It is therefore assumed that the five plasmids pHG1, -4, -7, -9, and -10 are in fact copies of the same plasmid from which pHG11 has developed. These results illustrate the dissemination of one plasmid, or
VOL. 13, 1978
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
rather of its host bacteria, within the small area constituted by one herd. What remains is an analysis of plasmid DNA from different antigenic types of E. coli because the finding of an identical restriction pattern in that case would present additional evidence (14, 15) for the spread of single plasmid clones among bacteria. ACKNOWLEDGMENTh This work was supported by grants 513-3691 and 513-5182 from the Danish Agricultural and Veterinary Research Council.
LITERATURE CITED 1. Adams, M. H. 1950. Methods of study of bacterial viruses.
Methods Med. Res. 2:1-73. 2. Beneveniste, R, and J. Davies. 1973. Mechanisms of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471506. 3. Bouanchaud, D. H., and Y. A. Chabbert. 1969. Stable coexistence of three resistance factors (fl) in Salmonellapanama and Escherichia coli K12. J. Gen. Microbiol. 58:107-113. 4. Chabbert, Y. A., and G. R Gerbaud. 1974. SurveiLance epidemiologique des plasmides responsables de la resistance au chloramphenicol de SalmoneUa typhi. Ann. Inst. Pasteur, Paris 125A:153-166. 5. Clowes, R. C., and W. Hayes. 1968. Experiments in microbial genetics, p. 188. Blackwell Scientific Publications, Oxford. 6. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. 7. Datta, N., and R. W. Hedges. 1972. Trimethoprim resistance conferrd by W plsamids in Enterobacteriacea. J. Gen. Microbiol. 72:349-355.
715
8. Datta, N., R. W. Hedges, E. J. Shaw, R. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. 9. Jorgensen, S. T., and A.-L. Poulsen. 1976. Antibiotic resistance and Mly plasmids in serotypes of Escherichia coli associated with porcine enteric disease. Antimicrob. Agents Chemother. 9:6-10. 10. Kleckner, N., R. K. Chan, B.-K. Tye, and D. Bot8tein. 1975. Mutagenesis by insertion of a drug resistance element carrying an inverted repetition. J. Mol. Biol. 97:561-575. 11. Lareen, J. L., and N. C. Nielsen. 1975. Influence of restrictive use of antibiotics on the development of drug resistance in intestinal Escherichia coli from pigs. Nord. Veternaermed. 27:353-364. 12. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 13. Meynell, E., and N. Datta. 1967. Mutant drug resistance factors of high transmissibility. Nature (London) 214:885-887. 14. Petrocheilou, V., J. Grinsted, and M. H. Richmond. 1976. R-plasmid transfer in vivo in the absence of antibiotic selection pressure. Antimicrob. Agents Chemother. 10:753-761. 15. Petrocheilou, V., M. H. Richmond, and P. M. Bennett. 1977. Spread of a single plasmid clone to an untreated individual from a person receiving prolonged tetracycline therapy. Antimicrob. Agents Chemother. 12:219-225. 16. Roe, E., and E. J. L. Lowbury. 1972. Changes in antibiotic sensitivity patterns of Gram-negative bacilli in burns. J. Clin. Pathol. 25:176-178. 17. Scavizzi, ML R. 1973. Nouveaux groups d'incompatibilite des plasmides Lnt6ret danm les epid6mies de creches a Eswherichia coli 0111:B4. Ann. Inst. Pasteur, Paris 124B:153-167. 18. Witchitz, J. L, and G. R Gerbaud. 1972. Classification de plasnides conferant la resistance a la gentamicine. Ann. Inst. Pasteur, Paris 123:333-339.
Vol. 13, No. 5
Printed in U.S.A.
Chloramphenicol Resistance Plasmids in Escherichia coli Isolated from Diseased Piglets SIGRID TUE J0RGENSENt Department of Animal Genetics, Royal Veterinary and Agricultural University, Copenhagen V, Denmark Received for publication 7 December 1977
The plasmids in 19 chloramphenicol-resistant Escherichia coli strains of three pig pathogenic antigen types were studied in conjugation and transduction experiments. The plasmids had identical resistance patterns: streptomycin, spectinomycin, sulfonamides, and chloramphenicol (Sm, Sp, Su, Cm) and belonged to IncFII. One plasmid carried ampicillin resistance in addition. Restriction enzyme analysis of the deoxyribonucleic acid from five of the plasmids originating from the same herd showed that their digestion patterns with EcoRI were indistinguishable. EcoRI cleaved the deoxyribonucleic acid of a sixth plasmid from the same herd and displayed nine of the ten bands of the other five plasmids plus an additional six. It appears that the five plasmids with identical restriction patterns have a common origin and may be copies of the same plasmid from which the sixth may have developed. Four strains carried two plasmids each. In two of these strains, a plasmid with a tetracycline marker (Tc), or possibly the tetracycline marker alone, recombined frequently with the Sm Sp Su Cm plasmid without destroying any known function of the latter. The possibility that Tc is carried on a translocation sequence is discussed.
In a study in 1976 (9) it was found that chloramphenicol (Cm) resistance plasmids occur with low, but noticeable frequency in Escherichia coli isolated from cases of diarrhea in Danish piglets. This situation has developed within the past few years. Except for some closed herds where antibiotics were used routinely (11), Cm resistance plasmids in E. coli from Danish piglets-whether healthy or diseased-were scarce until a few years ago (S. T. J0rgensen, Ph.D. thesis, Royal Vetr. & Agric. University, Copenhagen, 1974). The importance of Cm in the treatment of systemic salmonellosis in humans and animals and typhoid fever in humans, and its characteristic of being a "last resort drug" against cholera, render it necessary to watch, in animals, for the development of plasmid-borne resistance against this drug.
MATERIALS AND METHODS
Strains, plasmids, and bacteriophages. The organisms consisted of 19 naturally occurring Cm-resistant strains, 15 of which have been mentioned in a previous publication (9). The source as well as details of the isolation procedure was reported in the same paper. That description also applies to the four new strains. Three antigenic types were represented among the strains: 08:K87, 0141:K85ac, and 0149:K91. The reference strains were those already mentioned (9) plus a streptomycin (Sm)-resistant recA derivative of E. coli K-12. This strain and E. coli C600, as well as the reference plasmids listed in Table 1, and phage Plkc were obtained from Y. Chabbert, Institute Pasteur, Paris. Strain UB281 nalr pro met X lys recA was a gift from P. M. Bennet, University of Bristol, Bristol, England. Phage MS2 was from laboratory stock. Media In addition to those earlier reported (9), Miiller-Hinton agar (Difco) containing the appropriate antibiotics was used for selection of transconjugants, The aim of the present work was to begin a and L broth (12), phage buffer, and soft agar (5) were characterization of plasmids from 19 Cm-resist- applied in the preparation of lysates of phage M82 Plkc. ant E. coli strains of three different antigen andAntibiotics. The concentrations of the antibiotics types. Such a characterization might indicate in the selection plates were as follows: Ampicillin (Ap), whether the emergence of plasmids mediating Cm, dihydrostreptomycin sulfate (SM), kanamycin Cm resistance in pigs can be ascribed to the (Ka), and tetracycline (Tc), 12.5 ,g/ml; sodium nalispread of a few plasmid types, or even one in dixate, 20 ,ug/ml; and SM 1,000 ,ug/ml. The latter SM particular, analogous with the dissemination of concentration refers to selection for chromosomal Sm trimethoprim resistance among humans de- resisitance. All amounts mentioned above were pure substances. Cm, Sm, and Tc were commercially availscribed by Datta and Hedges (7). able compounds. Ka and Ap were obtained from H. t Present address: Department of Bacteriology, The Med- Lundbeck & Co. A/S, sodium nalidixate was obtained ical School, University Walk, Bristol BS8 1TD, England. from Winthrop Medicinalkompagni A/S, and rifamy710
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
VOL. 13, 1978
TABLE 1. Reference plasmids for incompatibility studies Plasmid
Ripl62/2 Rldrd-16 Rip1l2 Ripll3 RP4 Ripl35/1 R391 Rtsl Rip72
Phenotypic anti biotic resistance
markers
Ap Tc Ka Ka Tc Ap Ka Tc Tc Ka Ka Ka
Incompatibil- Referity group
IncFI IncFII IncIa IncN IncP IncM IncJ IncT InclO
ence
4 13 3 3 7 18 6 6 17
was obtained from A/S Dumex. Antibiotic disks were purchased from AB Biodisk, Sweden, and con-
cin
tained 30 ug of each of the above-mentioned antibiotics except Ap, the concentration of which was 10 lAg/disk. Sulfdimidine (Su) and spectinomycin (Sp) were used only in disks. The concentrations were 250 and 30,ug/disk, respectively. Sp-containing disks were used to distinguish between the two main types of Sminactivating enzymes-the adenylyltransferase, which also inactivates Sp, and the phosphotransferase, which operates on Sm only (2). Conjugation and incompatibility experiments. Exponentially growing cultures of recipient and donor strains were mixed in a ratio of 5:1 in 10 ml of nutrient broth and incubated either for 2 h or overnight at 37°C. Samples of 0.1 ml of the mating mixtures and suitable dilutions were plated on appropriate antibiotic agar, selecting for the chromosomal resistance of the recipient and each of the plasmid resistance markers. A total of 50 transconjugants from each selection plate were patched on nutrient agar after purification and then replicated on antibiotic agar in search of unselected markers. Incompatibility between a reference and a test plasmid was studied by introducing either one into E. coli recA carrying the other. Three single colonies were tested for the resistance markers of both plasmids and, if both plasmids were present, each clone was subcultured up to four times in antibiotic-free broth with dilution when the cell number reached 107. The subcultures were kept overnight on ice, and when all reached 107 cells per ml, they were diluted and plated on antibiotic-free nutrient agar. Colonies obtained this way were replicated onto plates, each containing one antibiotic according to the determinants being tested for. Between 200 and 300 colonies from each original clone were thus examined for coexistence of the test and reference plasmids. Test for Fi character. The plasmid under study was introduced into E. coli HfrH, and its ability to repress production of F pili was studied by spot tests with phage MS2. Loss of sensitivity to MS2 was taken as evidence of the unknown plasmid's Fi+ character. Transduction experiments. Transduction experiments were carried out by means of phage Plkc propagated by the method of Adams (1) on E. coli nair harboring the plasmid under study. Transduction to E. coli C600 was as described by Lennox (12). per disk,
711
Isolation of plasmid deoxyribonucleic acid (DNA), digestion with restriction endonuclease EcoRI, and separation of the fiagments in 0.7% agarose gels was done essentially as described by Petrocheilou et al. (14). Phage A DNA, which was used as an internal marker in all gels, was a generous gift from J. Grinsted, University of Bristol, who also kindly donated the restriction enzymes. Agarose type HSA was purchased by Litex, Tastrup, Denmark.
RESULTS Demonstration of separate linkage groups. Table 2 shows that all 19 strains carried resistance to Su, Sm, and Sp in addition to Cm. Moreover, two strains were resistant to Tc and one was resistant to Ap. In 17 strains the resistance markers Sm Sp Su Cm were not observed to separate during either conjugal transfer or transduction and, therefore, are assumed to be on the same replicon. Strains 3734 and 2742 behaved differently. Strain 3734 transferred Sm Sp Su Cm Ap when selection was made for AP or Cm, but Ap Cm Sp were rarely seen as unselected markers with Sm selection. In this case the great majority of transconjugants had the phenotype Sm Su. Transduction experiments were then carried out with phage Plkc grown on cells with either phenotype. Only the Sm Sp Su Cm Ap pattern could be transduced. In spite of several attempts, transduction of the Sm Su markers was not accomplished. These results are compatible with the inactivation of Sm in strain 3734 by two different enzymes (2), as shown by the two different resistance patterns transferred, and make it highly probable that strain 3734 carries two plasmids, one of them, pHG17 (Sm Su), presumably too large to be packed in phage Plkc. In strain 2742 the resistance markers behaved in much the same way. Cm selection gave transconjugants carrying all markers (Sm Sp Su Cm), whereas only half of the transconjugants selected on Sm coinherited Sp Cm with Sm Su. All transductants exmined carried the fourmarker pattern and had retained transferability of the markers. Since the results from strain 2742 were analogous with those from strain 3734, it is assumed that strain 2742 harbors two plasmids, pHG18 (Sm Sp Su Cm) and pHG20 (Sm Cu). This assumption was later confirmed by electrophoresis of plasmid DNA in agarose gels. The resistance markers in strains 1324 and 1325 originally constituted two separate linkage groups, one mediating resistance to Sm, Sp, Su, and Cm, the other mediating resistance to Tc. The first group of markers was located on a
derepressed plasmid, which upon direct selection transferred Tc marker at a very low fre-
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VOL. 13, 1978 quency.
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
The ratio of Sm Sp Su Cm transconju-
gants to Sm Sp Su Cm Tc transconjugants was 106:1. It was not possible to obtain Tc free of Sm
Sp Su Cm. When the rare transconjugants that carry the Tc marker in addition to Sm Sp Su Cm were crossed, a high proportion proved to transfer the Tc marker at the same elevated frequency as Sm Sp Su Cm. Transduction of such transconjugants gave only one type of transductants, namely transfer-proficient Sm Sp Su Cm Tc colonies. Transconjugants retaining the low transfer frequency for Tc when crossed restricted phage Plkc, so transduction information is unavailable. From these results it was concluded that each of the two wild strains harbors two plasmids. One carries the markers Tc Tra- and recombines frequently with the other, which carries Sm Sp Su Cm Tra+ drd, thereby losing its phage inhibition function. In transconjugants where the two plasmids proved to be independent, mobilization must be responsible for transfer of the Tc Tra- plasmid. After a couple of months the work with the plasmids in strain 1325 was resumed and the transfer frequency for pHG28 Tr Tra- Res (Plkc) (or rather the frequency with which it was mobilized by pHG25 Sm Sp Su Cm Tra+) was 1,000-fold higher than that previously found. This change in the transfer frequency of pHG28 proved to be stable, and no other character was altered. Presumably a mutation in some system regulating the mobilization process is responsible for the change in frequency. Incompatibility reactions. Separation of the plasmids originating from the same strain was followed by a determination of their incompatibility group. However, for the reasons mentioned above it was not possible to separate pHG27 and pHG28 from pHG24 and pHG25, respectively. Moreover, the two pairs often recombined, and further study of pHG27 and pHG28 was therefore postponed. pHG24 and pHG25, however, were both IncFII plasmids, and the same was true for their recombinants with pHG27 and 28. Like pHG24 and pHG25, the remaining 16 Sm Sp Su Cm plasmids and pHG17 were found to inhibit phage MS2-induced lysis of E. coli HfrH carrying the plasmid under study. This suggested that the plasmids belonged to the F incompatibility complex. Further studies proved that all were incompatible with Rldrd-16 and thus IncFII plasmids. Most of them separated extremely efficiently from the reference plasmid, i.e., 90-100% separation was obtained after one subculture in antibiotic-free broth. Only a few needed three or four successive subcultures to obtain the same results.
713
In spite of their lack of stability with Rldrd16 which clearly demonstrates their membership of IncFII, some of the plasmids also showed varying degrees of interaction with the IncFI reference plasmid. This interaction in some cases took the form of an early loss (dislodgment) of the nonselected plasmid so that on resistance determination of the transconjugants only the incoming plasmid was present. In other cases dislodgment appeared during subcultivation of cell lines, which initially harbored both plasmids so that 25 to 50% of the colonies had lost the resident plasmid. This happened most often when the reference plasmid was the incoming plasmid. No valid explanation can at present be offered for these irregularities. All Sm Sp Su Cm plasmids and PHG17 were tested for incompatibility with reference plasmids from IncIa, -N, -P, -M, -J, -T, and -10. No incompatibility or interaction was observed in any case. Restriction enzyme analysis. Because some of the plasmids showed interaction with the IncFI reference plasmid and others did not, it was decided to do restriction enzyme analyses on their DNA to obtain information on their
degree of similarity. Transduced colonies, which had been crossed to prove that their plasmids were still transfer proficient, were used for DNA isolation. The purified DNA was first run undigested in an agarose gel to determine that only one plasmid DNA was present. Digestion with EcoRI of DNA from plasmids pHG1, -4, -7, -9, and -10 generated 10 bands with each after electrophoresis in agarose gels. The migration patterns of the bands were identical (Fig. 1). Another plasmid from that series, pHG11, had nine of the bands of the five plasmids mentioned above plus six extra bands (Fig. 2).
DISCUSSION The fact that the nonconjugative plasmids pHG27 and pHG28 were mobilized at very low frequency by derepressed pHG24 and pHG25 leads to the assumption that this low frequency reflects the recombination frequency between each of the two plasmid sets. Examination of the total number of Tc transconjugants (four in all) from the conjugation strain 1324 with E. coli nalr revealed that three of them harbored recombinant plasmids. The fourth, however, carried pHG24 and pHG27, i.e., no recombinationtion had taken place. Accordingly, the occurrence of the fourth transconjugant argues against the above hypothesis. How much of pHG27 integrates into pHG24 remains obscure-perhaps only the Tc genes and cer-
714
JORGENSEN
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. EcoRI-digested DNA from pHG1, -4, -7, -9, and -10. Phage y DNA was cleaved with EcoRI and BamHI and used as an internal marker. Electrophoresis was carried out in 0. 7% (wt/vol) agarose gels for 2 h, 45 min. Q ploza-Idt bctvc.ts
x
a" as
FIG. 2. EcoRI-cleaved DNA from pHG4 and -11. Phage y DNA was digested as described in the legend of Fig. 1. - indicates bands in common; ---- indicates bands only in pHG11; .. indicates band only in pHG4.
tainly not the region coding for phage Plkc restriction. Since recombination occurred relatively often, the possibility that the Tc marker might be carried on a translocation sequence was considered. This is supported by the finding that pHG24 and pHG27 were able to form stable recombinants for the resistance markers in an recA background. This finding applies to two of nine colonies examined. There is therefore no doubt that tet integrates into pHG24 even in the absence of the recA function. Against the hypothesis is that, although TnT is strongly polar (10), pHG24 was not observed to lose any of its phenotypic markers on acquisition of Tc. This may, however, be interpreted as integration of tet at a place in the DNA that has no easily recognizable function. Molecular studies that are underway might give the final proof of Tc as carried on a translocation sequence. The genetic data presented in this paper show
that the Cm resistance plasmids found among Danish piglets are so similar that a common origin is very likely. Postulating a closer relationship than that would be audacious on the basis of the genetic knowledge, since these plasmids do not belong to any rare incompatibility group such as those described by Datta et al. (8), Roe and Lowbury (16), and Datta and Hedges (7). But restriction enzyme analysis of their DNA represents a means of obtaining further information of a putative close relationship. Preliminary data show that five plasmids in strains isolated from the same herd within a month generate identical restriction patterns with EcoRI. Another plasmid from that series, pHG11, shows 9 of the 10 bands of the five above mentioned plus another 6. It is therefore assumed that the five plasmids pHG1, -4, -7, -9, and -10 are in fact copies of the same plasmid from which pHG11 has developed. These results illustrate the dissemination of one plasmid, or
VOL. 13, 1978
E. COLI: CHLORAMPHENICOL RESISTANCE PLASMIDS
rather of its host bacteria, within the small area constituted by one herd. What remains is an analysis of plasmid DNA from different antigenic types of E. coli because the finding of an identical restriction pattern in that case would present additional evidence (14, 15) for the spread of single plasmid clones among bacteria. ACKNOWLEDGMENTh This work was supported by grants 513-3691 and 513-5182 from the Danish Agricultural and Veterinary Research Council.
LITERATURE CITED 1. Adams, M. H. 1950. Methods of study of bacterial viruses.
Methods Med. Res. 2:1-73. 2. Beneveniste, R, and J. Davies. 1973. Mechanisms of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471506. 3. Bouanchaud, D. H., and Y. A. Chabbert. 1969. Stable coexistence of three resistance factors (fl) in Salmonellapanama and Escherichia coli K12. J. Gen. Microbiol. 58:107-113. 4. Chabbert, Y. A., and G. R Gerbaud. 1974. SurveiLance epidemiologique des plasmides responsables de la resistance au chloramphenicol de SalmoneUa typhi. Ann. Inst. Pasteur, Paris 125A:153-166. 5. Clowes, R. C., and W. Hayes. 1968. Experiments in microbial genetics, p. 188. Blackwell Scientific Publications, Oxford. 6. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. 7. Datta, N., and R. W. Hedges. 1972. Trimethoprim resistance conferrd by W plsamids in Enterobacteriacea. J. Gen. Microbiol. 72:349-355.
715
8. Datta, N., R. W. Hedges, E. J. Shaw, R. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. 9. Jorgensen, S. T., and A.-L. Poulsen. 1976. Antibiotic resistance and Mly plasmids in serotypes of Escherichia coli associated with porcine enteric disease. Antimicrob. Agents Chemother. 9:6-10. 10. Kleckner, N., R. K. Chan, B.-K. Tye, and D. Bot8tein. 1975. Mutagenesis by insertion of a drug resistance element carrying an inverted repetition. J. Mol. Biol. 97:561-575. 11. Lareen, J. L., and N. C. Nielsen. 1975. Influence of restrictive use of antibiotics on the development of drug resistance in intestinal Escherichia coli from pigs. Nord. Veternaermed. 27:353-364. 12. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 13. Meynell, E., and N. Datta. 1967. Mutant drug resistance factors of high transmissibility. Nature (London) 214:885-887. 14. Petrocheilou, V., J. Grinsted, and M. H. Richmond. 1976. R-plasmid transfer in vivo in the absence of antibiotic selection pressure. Antimicrob. Agents Chemother. 10:753-761. 15. Petrocheilou, V., M. H. Richmond, and P. M. Bennett. 1977. Spread of a single plasmid clone to an untreated individual from a person receiving prolonged tetracycline therapy. Antimicrob. Agents Chemother. 12:219-225. 16. Roe, E., and E. J. L. Lowbury. 1972. Changes in antibiotic sensitivity patterns of Gram-negative bacilli in burns. J. Clin. Pathol. 25:176-178. 17. Scavizzi, ML R. 1973. Nouveaux groups d'incompatibilite des plasmides Lnt6ret danm les epid6mies de creches a Eswherichia coli 0111:B4. Ann. Inst. Pasteur, Paris 124B:153-167. 18. Witchitz, J. L, and G. R Gerbaud. 1972. Classification de plasnides conferant la resistance a la gentamicine. Ann. Inst. Pasteur, Paris 123:333-339.
