ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1975, p. 606-607 Copyright C) 1975 American Society for Microbiology
Vol. 8, No. 5
Printed in U.SA.
NOTES Detection of Temperature-Sensitive Tetracycline Resistance Plasmids in Staphylococcus aureus J. W. MAY,'* R. H. HOUGHTON, AND C. J. PERRET Department of Microbiology, University of Western Australia, Nedlands, Western Australia, 6009 Received for publication 28 May 1975
A method is described for the detection of temperature-sensitive plasmids
determining tetracycline resistance in Staphylococcus aureus. Temperaturesensitive plasmids were detected in phage group I strains but not in phage group
III strains.
In 1964 we proposed that tetracycline resistin Staphylococcus aureus strain E169 was determined by a plasmid whose replication was inhibited during growth of the bacteria at 44 C (8). Subsequently, Novick and Bouanchaud (9) demonstrated that the tetracycline resistance determinant of S. aureus E169 was a circular duplex of extrachromosomal deoxyribonucleic acid with a contour length of 1.4 um. In studies of the tetracycline resistance plasmid of S. aureus E169 after transduction, temperature sensitivity of plasmid replication was ascertained by growing the bacteria in broth at the nonpermissive temperature, allowing the resulting cells to form colonies at 37 C, and then picking and testing these colonies for their tetracycline resistance; temperature-sensitive plasmids produced predominantly tetracyclinesensitive colonies (3, 4, 6). To reduce the time and labor involved in screening further isolates for the presence of temperature-sensitive tetracycline resistance plasmids, we devised the following procedure based upon the presumption that at the nonpermissive temperature, after dilution-out of the plasmids, an individual coccus carrying one such plasmid will be able to produce at each division on tetracycline agar only one effectively viable cell and hence be unable to form a visible colony under these conditions. S. aureus E169 was grown overnight in meat infusion broth (2) at 37 C, and then six serial 10fold dilutions of the culture were prepared in 0.1% peptone water. One drop of each dilution was placed separately on each of four meat infusion agar (MIA) plates, two of the plates ance
containing 10 ,ug of tetracycline per ml. One pair of plates (one MIA, the other MIA plus tetracycline) was incubated at 37 C, and the other pair at 44 C. (To reliably achieve suppression of plasmid replication but avoid inhibition of bacterial growth, the temperature should be controlled at 44.0 + 0.1 C: isolated cells of S. aureus E169 are able to form visible colonies on MIA plus tetracycline at 43.0 C but are unable to do so on MIA at 44.5 C.) Prior to incubation at 44 C, the plates were sealed with paraffin wax or with strips of Parafilm (Gallenkamp, London, England) to prevent evaporation and then placed in an aluminum tray (2.5 cm deep, 26 cm wide, and 38 cm long) which was covered with a close-fitting aluminum lid of the same gauge as the tray (at least 2 mm thick). The box containing the plates was placed in an anhydric incubator fitted with a fan to ensure rapid circulation of the air (type TV40L, W. Memmett, Schwabach, West Germany): this type of incubator has proved suitable provided the thermostat is replaced by a contact thermometer (type MS123, M. Juchheim, Fulda, West Germany) which operates the heating element of the incubator by means of a hot-wire vacuum switch relay (type HVR, Satchwell Sunvic Ltd., Motherwell, Scotland). Thermocouples (type F6, used with an Ellab universal precision thermometer, model TE3, Electrolaboratoreit, Copenhagen, Denmark) attached to plates showed that the temperature varied by less than 0.1 C during the on-off cycle of the heater or between different positions in the box. If care is taken with positioning within the incubator, two boxes can be incubated at the same time. Compared with MIA at 37 C, no significant difference could be detected in the ability of
I Present address: Department of Microbiology, Monash University Medical School, Prahran, Victoria, Australia, 3181. 606
VOL. 8, 1975
NOTES
TABLE 1. Temperature sensitivity of tetracycline resistance and phage group of 42 strains of Staphylococcus aureus No. of strains in phage group
Tetracycline resistance at 44 C
I
III
Suppressed Unaffected
5 5
0 18
Not typable 3 11
607
tor (7) can be used in either of two ways to make the procedure more economical. First, each cell suspension is serially diluted in a row of wells in a reservoir tray: in this way a complete set of dilutions for each of six separate suspensions can be applied to a single plate. Second, a series of dilutions of each of 52 separate cell suspensions is made by using a multipoint applicator to transfer a small volume of each suspension from one tray to another: in this way one of the dilutions of each of 52 separate cell suspensions can be applied to a single plate. The high degree of control and uniformity of temperature over as many as 24 plate cultures achieved in the foregoing procedure would seem to be of general value in the study of temperature-conditional mutants.
isolated cocci ofS. aureus E169 to form colonies within 18 h on MIA plus tetracycline at 37 C and MIA at 44 C. However, on MIA plus tetracycline at 44 C, as many as 1,000 cocci failed to produce any visible colonies in the same period. Preliminary experiments with liquid batch cultures had revealed several strains of S. aureus We wish to thank D. Birch, Royal Perth Hospital, for whose resistance to tetracycline was not elimi- performing the phage typing. nated by growth at 44 C; the colony-forming ability of these strains on MIA plus tetracycline LITERATURE CITED was unaffected by incubation at 44 C. 1. Asheshov, E. H. 1966. Loss of antibiotic resistance in The foregoing procedure and criteria were Staphylococcus aureus resulting from growth at high temperature. J. Gen. Microbiol. 42:403-410. applied to 42 tetracycline-resistant strains of S. 2. Cruickshank, R. (ed.). 1965. Medical microbiology, 11th aureus isolated from clinical sources. Table 1 ed. E. and S. Livingstone, Edinburgh. shows the distribution of the two types of tetra- 3. Grubb, W. B., and R. J. O'Reilly. 1971. Joint transduccycline resistance (suppressed and unaffected tion of separate extrachromosomal drug resistance determinants in Staphylococcus aureus E169. Bioat 44 C) within phage groups of S. aureus; alchem. Biophys. Res. Commun. 42:377-383. though none of the 18 phage group III strains 4. Grubb, W. B., R. J. O'Reilly, and J. W. May. 1972. carried temperature-sensitive tetracycline reSegregation of co-transduced streptomycin and tetrasistance plasmids, 5 of the 10 phage group I cycline resistance in Staphylococcus aureus. Genet. Res. 20:43-50. strains appeared to do so. The failure to induce loss of tetracycline resistance in strains of 5. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring phage group III by growth at 44 C was also Harbor Symp. Quant. Biol. 28:329-343. found by Asheshov (1) and indicates either that 6. Kasuga, T., and S. Mitsuhashi. 1968. Drug resistance of Staphylococci. vyI. Genetic properties of resistance temperature-sensitive tetracycline resistance to tetracycline in Staphylococcus aureus E169. Jpn. plasmids are relatively rare in strains of phage J. Microbiol. 12:269-273. group III or that the temperature sensitivity of 7. May, J. W., and R. H. Houghton. 1965. A simple multithe plasmids is suppressed when present in point inoculator. Lab. Pract. 14:168. strains of this phage group. One mechanism 8. May, J. W., R. H. Houghton, and C. J. Perret. 1964. The effect of growth at elevated temperatures on permitting such a suppression would be the some heritable properties of Staphylococcus aureus. integration of the plasmid into the bacterial J. Gen. Microbiol. 37:157-169. chromosome (see 5). 9. Novick, R. P., and D. Bouanchaud. 1971. Extrachromosomal nature of drug resistance in Staphylococcus When a large number of cultures or of coloaureus. Ann. N. Y. Acad. Sci. 182:279-294. nies have to be screened, a multipoint inocula-
Vol. 8, No. 5
Printed in U.SA.
NOTES Detection of Temperature-Sensitive Tetracycline Resistance Plasmids in Staphylococcus aureus J. W. MAY,'* R. H. HOUGHTON, AND C. J. PERRET Department of Microbiology, University of Western Australia, Nedlands, Western Australia, 6009 Received for publication 28 May 1975
A method is described for the detection of temperature-sensitive plasmids
determining tetracycline resistance in Staphylococcus aureus. Temperaturesensitive plasmids were detected in phage group I strains but not in phage group
III strains.
In 1964 we proposed that tetracycline resistin Staphylococcus aureus strain E169 was determined by a plasmid whose replication was inhibited during growth of the bacteria at 44 C (8). Subsequently, Novick and Bouanchaud (9) demonstrated that the tetracycline resistance determinant of S. aureus E169 was a circular duplex of extrachromosomal deoxyribonucleic acid with a contour length of 1.4 um. In studies of the tetracycline resistance plasmid of S. aureus E169 after transduction, temperature sensitivity of plasmid replication was ascertained by growing the bacteria in broth at the nonpermissive temperature, allowing the resulting cells to form colonies at 37 C, and then picking and testing these colonies for their tetracycline resistance; temperature-sensitive plasmids produced predominantly tetracyclinesensitive colonies (3, 4, 6). To reduce the time and labor involved in screening further isolates for the presence of temperature-sensitive tetracycline resistance plasmids, we devised the following procedure based upon the presumption that at the nonpermissive temperature, after dilution-out of the plasmids, an individual coccus carrying one such plasmid will be able to produce at each division on tetracycline agar only one effectively viable cell and hence be unable to form a visible colony under these conditions. S. aureus E169 was grown overnight in meat infusion broth (2) at 37 C, and then six serial 10fold dilutions of the culture were prepared in 0.1% peptone water. One drop of each dilution was placed separately on each of four meat infusion agar (MIA) plates, two of the plates ance
containing 10 ,ug of tetracycline per ml. One pair of plates (one MIA, the other MIA plus tetracycline) was incubated at 37 C, and the other pair at 44 C. (To reliably achieve suppression of plasmid replication but avoid inhibition of bacterial growth, the temperature should be controlled at 44.0 + 0.1 C: isolated cells of S. aureus E169 are able to form visible colonies on MIA plus tetracycline at 43.0 C but are unable to do so on MIA at 44.5 C.) Prior to incubation at 44 C, the plates were sealed with paraffin wax or with strips of Parafilm (Gallenkamp, London, England) to prevent evaporation and then placed in an aluminum tray (2.5 cm deep, 26 cm wide, and 38 cm long) which was covered with a close-fitting aluminum lid of the same gauge as the tray (at least 2 mm thick). The box containing the plates was placed in an anhydric incubator fitted with a fan to ensure rapid circulation of the air (type TV40L, W. Memmett, Schwabach, West Germany): this type of incubator has proved suitable provided the thermostat is replaced by a contact thermometer (type MS123, M. Juchheim, Fulda, West Germany) which operates the heating element of the incubator by means of a hot-wire vacuum switch relay (type HVR, Satchwell Sunvic Ltd., Motherwell, Scotland). Thermocouples (type F6, used with an Ellab universal precision thermometer, model TE3, Electrolaboratoreit, Copenhagen, Denmark) attached to plates showed that the temperature varied by less than 0.1 C during the on-off cycle of the heater or between different positions in the box. If care is taken with positioning within the incubator, two boxes can be incubated at the same time. Compared with MIA at 37 C, no significant difference could be detected in the ability of
I Present address: Department of Microbiology, Monash University Medical School, Prahran, Victoria, Australia, 3181. 606
VOL. 8, 1975
NOTES
TABLE 1. Temperature sensitivity of tetracycline resistance and phage group of 42 strains of Staphylococcus aureus No. of strains in phage group
Tetracycline resistance at 44 C
I
III
Suppressed Unaffected
5 5
0 18
Not typable 3 11
607
tor (7) can be used in either of two ways to make the procedure more economical. First, each cell suspension is serially diluted in a row of wells in a reservoir tray: in this way a complete set of dilutions for each of six separate suspensions can be applied to a single plate. Second, a series of dilutions of each of 52 separate cell suspensions is made by using a multipoint applicator to transfer a small volume of each suspension from one tray to another: in this way one of the dilutions of each of 52 separate cell suspensions can be applied to a single plate. The high degree of control and uniformity of temperature over as many as 24 plate cultures achieved in the foregoing procedure would seem to be of general value in the study of temperature-conditional mutants.
isolated cocci ofS. aureus E169 to form colonies within 18 h on MIA plus tetracycline at 37 C and MIA at 44 C. However, on MIA plus tetracycline at 44 C, as many as 1,000 cocci failed to produce any visible colonies in the same period. Preliminary experiments with liquid batch cultures had revealed several strains of S. aureus We wish to thank D. Birch, Royal Perth Hospital, for whose resistance to tetracycline was not elimi- performing the phage typing. nated by growth at 44 C; the colony-forming ability of these strains on MIA plus tetracycline LITERATURE CITED was unaffected by incubation at 44 C. 1. Asheshov, E. H. 1966. Loss of antibiotic resistance in The foregoing procedure and criteria were Staphylococcus aureus resulting from growth at high temperature. J. Gen. Microbiol. 42:403-410. applied to 42 tetracycline-resistant strains of S. 2. Cruickshank, R. (ed.). 1965. Medical microbiology, 11th aureus isolated from clinical sources. Table 1 ed. E. and S. Livingstone, Edinburgh. shows the distribution of the two types of tetra- 3. Grubb, W. B., and R. J. O'Reilly. 1971. Joint transduccycline resistance (suppressed and unaffected tion of separate extrachromosomal drug resistance determinants in Staphylococcus aureus E169. Bioat 44 C) within phage groups of S. aureus; alchem. Biophys. Res. Commun. 42:377-383. though none of the 18 phage group III strains 4. Grubb, W. B., R. J. O'Reilly, and J. W. May. 1972. carried temperature-sensitive tetracycline reSegregation of co-transduced streptomycin and tetrasistance plasmids, 5 of the 10 phage group I cycline resistance in Staphylococcus aureus. Genet. Res. 20:43-50. strains appeared to do so. The failure to induce loss of tetracycline resistance in strains of 5. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring phage group III by growth at 44 C was also Harbor Symp. Quant. Biol. 28:329-343. found by Asheshov (1) and indicates either that 6. Kasuga, T., and S. Mitsuhashi. 1968. Drug resistance of Staphylococci. vyI. Genetic properties of resistance temperature-sensitive tetracycline resistance to tetracycline in Staphylococcus aureus E169. Jpn. plasmids are relatively rare in strains of phage J. Microbiol. 12:269-273. group III or that the temperature sensitivity of 7. May, J. W., and R. H. Houghton. 1965. A simple multithe plasmids is suppressed when present in point inoculator. Lab. Pract. 14:168. strains of this phage group. One mechanism 8. May, J. W., R. H. Houghton, and C. J. Perret. 1964. The effect of growth at elevated temperatures on permitting such a suppression would be the some heritable properties of Staphylococcus aureus. integration of the plasmid into the bacterial J. Gen. Microbiol. 37:157-169. chromosome (see 5). 9. Novick, R. P., and D. Bouanchaud. 1971. Extrachromosomal nature of drug resistance in Staphylococcus When a large number of cultures or of coloaureus. Ann. N. Y. Acad. Sci. 182:279-294. nies have to be screened, a multipoint inocula-
