TRANSDUCTION OF RESISTANCE TO SOME IN STAPHYLOCOCCUS A UREUS1 P. A. PATTEE AND J. N. BALDWIN Department of Bacteriology, Iowa State University, Ames, Iowa, and Department of Microbiology, Ohio State University, Columbus, Ohio Received for publication June 21, 1962

ABSTRACT among members of this species. Morse (1959), PATTEE, P. A. (Iowa State University, Ames) Edgar and Stocker (1961), and Dowell and AND J. N. BALDWIN. Transduction of resistance Rosenblum (1962) also reported staphylococcal to some macrolide antibiotics in Staphylococcus transductions involving a variety of charac- aureus. J. Bacteriol. 84:1049-1055. 1962.-By use teristics. Pattee and Baldwin (1961) reported of phage 80 of the International Typing Series, that phages 29, 52A, 79, 80, and 53 of the Inter- propagated on appropriate strains of Staphy- national Typing Series were capable of trans- lococcus aureus, two related markers controlling ducing the capacity to produce penicillinase, as resistance to certain macrolide antibiotics well as resistance to and novo- (, , spiramycin, and biocin, among a variety of strains of S. aureus. carbomycin) were transduced among a variety The present report is concerned with the trans- of strains of S. aureus. Unlike the markers duction of resistance to several of the macrolide controlling penicillinase production and resistance antibiotics among strains of S. aureus. to chlortetracycline and novobiocin, the deter- Among the pathogenic staphylococci, resistance minants of resistance to the macrolide antibiotics to the macrolide antibiotics, which include were transduced at normal frequencies (at least erythromycin, oleandomycin, spiramycin, carbo- 300 transductants per 109 phage) only to certain mycin, and leucomycin (Murai et al., 1959), of the recipient strains. One of the markers appears to be controlled by two distinctly differ- studied appears to control an inducible enzyme ent general mechanisms. In vitro adaptation system which is specifically induced by sub- results in a step-wise development of resistance inhibitory concentrations of erythromycin and to the macrolide antibiotics as a group (Jones, which controls resistance to erythromycin, Nichols, and Finland, 1956). Resistance in this oleandomycin, spiramycin, and carbomycin. The instance probably is determined by a series of other marker examined confers resistance to independent mutations and may be similar to erythromycin, oleandomycin, spiramycin, and erythromycin resistance in the pneumococcus carbomycin, and shows no evidence of being (Ravin and Iyer, 1961). The other mechanism of dependent upon an inducible mechanism. resistance, which predominates among naturally resistant strains of S. aureus, was extensively studied by Garrod (1957). His results generally Transduction in Staphylococcus aureus was were in agreement with others (Jones et al., 1956; first reported in 1958 by Ritz and Baldwin Waterworth, 1960; Rantz et al., 1957); i.e., in (1961), who demonstrated that several phages of vitro adaptation to one of the macrolide anti- the International Typing Series were capable of biotics results in a considerable degree of cross transducing the capacity to produce penicillinase resistance to other antibiotics of the macrolide group. During these studies, Garrod (1957) 1 Portions of this study were presented at the encountered certain strains of S. aureus, called 60th Annual Meeting of the American Society dissociated which were resistant for Microbiology, Philadelphia, Pa., May, 1960. strains, naturally This paper is based on portions of a dissertation to erythromycin but apparently sensitive to the submitted by the senior author to the Graduate other macrolide antibiotics. Garrod (1957) was School of Ohio State University, in partial ful- able to show that, while populations of dissociated fillment of the requirements for the degree, Doctor strains consisted primarily of erythromycin- of Philosophy. sensitive cells, such populations did not readily 1049 1050 PATTEE AND BALDWIN J. BACTERIOL. yield erythromycin-sensitive mutants. Further TABLE 1. Designations and phage types examination of dissociated strains revealed that of strains of Staphylococcus aureus growth in broth containing subinhibitory con- Strain designation Phage type centrations of erythromycin resulted in a popula- tion which consisted entirely of cells resistant not U9 (Pase, Tet, Ery) 80/81 only to erythromycin, but also to oleandomycin, U9 (Pase, Tet, Ery, spiramycin, and carbomycin. Reversion of Nov) 80/81 dissociated strains to the original phenotype U9 (Pase, Tet, 01, occurred after several transfers in the absence of Nov) 80/81 erythromycin. Waterworth (1960) reported that U35 (Pase, Tet) 80/81 dissociated strains acquired temporary resistance U40 (Pase, Tet) 80/81 to leucomycin after growth in the presence of U61 (Pase, Tet) 80/81 U66 (Pase, Tet, Ery) 80/81 erythromycin. U91 (Pase, Tet) 80/81 MATERIALS AND METHODS U114 (Pase, Tet) 80/81 461 (Pase, Tet) 80/81 The methods employed for determining the 588 (Pase, Tet) 80/81 characteristics possessed by strains of S. aureus, 655 (Pase, Tet, Ery) 29/52A/79/7/83/47/ the methods used for the maintenance of bacteria 53/54/73/77 and bacteriophages, and the transduction 4A (Pase, Tet, Ery) 79/83/53/54/77 techniques have been described in detail (Pattee Ps 42B 42B and Baldwin, 1961). The strains of S. aureus Ps 29 29 employed were originally isolated from several Ps 52 52 C-72 29/52A/79/80 different sources, and all were of human origin. Ch-50 80 The genetic markers possessed by these strains W-26 29/80 are included in the strain designations, and N-135 29 include the capacity to produce sufficient peni- 608 52A/80/81 cillinase to grow on Brain Heart Infusion (BHI; 1 52A/80/81 Difco) agar containing 100 units of per 769 81 ml (Pase marker), and the ability to grow on BHI D-1 52A/80 agar containing either 75 ,ug of chlortetracycline 152 52A/79/80 per ml (Tet marker) or 20 jig of novobiocin per 112 29/52A/79/83/42E/ ml (Nov marker). Strains exhibiting erythromycin 80/81 resistance of the dissociated type possess the 248 52A/80 Ery marker. From strain U9(Pase, Tet, Ery, Nov), a mutant was isolated by the gradient- phage 80 propagated on donor strain U9(Pase, plate technique (Szybalski, 1952), which was Tet, Ery) was designated 80/U9(Pase, Tet, Ery). resistant to oleandomycin. This mutant was Briefly, the transduction procedure involved resistant to 800 MAg of oleandomycin per ml of combining 1.0 ml of a cell-free phage lysate BHI agar (01 marker) and was designated (1010 plaque-forming units per ml) with between U9(Pase, Tet, 01, Nov). The designations and 2 and 6 X 1010 cells of the recipient strain sus- phage types of the strains employed are given pended in 1.0 ml of P and D broth (nutrient in Table 1. broth containing 0.25% K2HPO4 and 0.2 % The bacteriophages of the International dextrose). The mixture was then shaken for 1 Typing Series used in this study were originally hr at 37 C, after which the cells were washed once obtained from John E. Blair and were main- 1 in 1.0 tained in the Department of Microbiology, Ohio with ml of P and D broth, resuspended State University. Phages 29, 52A, 79, 80, and 53 ml of P and D broth, and 0.05-ml quantities were employed for transduction after propagation spread over the surface of each plate of the on appropriate donor strains. The designation of selective medium with the aid of a sterile bent the transducing phages includes both the inter- glass rod. Control suspensions consisted of an national number designation of the phage and the identical suspension of recipient cells shaken with designation of the donor strain. Accordingly, 1.0 ml of sterile P and D broth. VOL. 84, 1962 TRANSDUCTION OF RESISTANCE IN S. AUREUS 1051

RESULTS expression of resistance to erythromycin in a Characteristics of strains possessing the Ery and strain possessing the Ery marker. When the 01 markers. When a heavy (109 cells) inoculum of colonies obtained on BHI agar containing strain U9(Pase, Tet, Ery), prepared from an erythromycin were replicated with velveteen to overnight BHI agar slant culture, was inoculated BHI agar containing 100 ,ug of oleandomycin, onto BHI agar containing 800 Mg of erythromycin spiramycin, or carbomycin per ml, they all were per ml, apparent uninhibited growth was ob- found to be resistant to these antibiotics. When tained. Oleandomycin, spiramycin, and carbo- similar colonies were replicated serially three mycin (10 ,g per ml of BHI agar) were inhibitory times on BHI agar at 24-hr intervals, and then to identical inocula, except for a few resistant replicated to BHI agar containing 100 Mg of mutants present in the cell population. In con- oleandomycin, spiramycin, or carbomycin per trast, strain U9(Pase, Tet, 01, Nov) was capable ml, the majority of the colonies failed to develop. of uninhibited growth on BHI agar containing Those few colonies which retained resistance to either 800 ,g per ml of oleandomycin or erythro- these antibiotics were resistant mutants, a result mycin, or 400 Mg per ml of spiramycin or carbo- which was expected in view of the high mutation mycin. Strains U9(Pase, Tet, Ery) and U9(Pase, frequency previously observed with strain Tet, 01, Nov) were then examined for resistance, U9(Pase, Tet, Ery). by performing plate counts on BHI agar and Transduction of oleandomycin resistance. Trans- BHI agar containing 25 Mug per ml of erythro- duction of resistance to oleandomycin was mycin, oleandomycin, or spiramycin. The results performed by using as a selective medium BHI of this experiment (Table 2) indicate that strain agar containing 25 Mug of oleandomycin per ml. U9(Pase, Tet, Ery) consisted primarily of Control suspensions of erythromycin-sensitive erythromycin-sensitive cells. In contrast, strain recipient strains yielded approximately 20 U9(Pase, Tet, 01, Nov) consisted entirely of cells resistant mutants per 109 cells when BHI agar resistant to all of the macrolide antibiotics containing either 3 or 5 Mig of oleandomycin per examined. It was also observed that strain ml was used. When from 10 to 800 ,g of oleando- U9(Pase, Tet, Ery) possessed a significant muta- mycin per ml were employed, the transduction tion frequency, conferring resistance to oleando- frequencies for the 01 marker were not changed mycin and spiramycin. When such mutants significantly, and control suspensions were were further examined, approximately 30% were completely inhibited. The frequencies of trans- found to possess the 01 marker. duction of the 01 marker obtained by use of 15 To verify the fact that erythromycin resistance recipient strains transduced with phage 80/ possessed by strain U9(Pase, Tet, Ery) was of the U9(Pase, Tet, 01, Nov) are shown in Table 4. dissociated type, an overnight BHI agar slant All control suspensions were completely in- culture of this strain was suspended uniformly in hibited, with the exception of strain U9(Pase, 10 ml of P and D broth. The cell suspension was Tet, Ery) which yielded 10 mutants per 109 cells. then shaken at 37 C, and 1 Mg of erythromycin When erythromycin, spiramycin, or carbomycin added per ml. Immediately before and for several (25 ,g per ml) was employed in place of oleando- hours after the addition of erythromycin, samples mycin in the selective medium, no significant were removed and plate counts made by using changes in the transduction frequencies were BHI agar and BHI agar containing 50 Mg of observed, although overgrowth of strain U9(Pase, erythromycin per ml. The results (Table 3) Tet, Ery) was obtained with both control and clearly indicate that a brief exposure to low transduced suspensions on BHI agar containing concentrations of erythromycin caused a rapid erythromycin. Replication experiments revealed

TABLE 2. Plate counts of strains U9(Pase, Tet, Ery) and U.O(Pase, Tet, 01, Nov) as determined on BHI agar and BHI agar containing macrolide antibiotics Viable cells per ml on BHI agar containing (25 lAg/ml): Strain - Erythromycin Oleandomycin Spiramycin

U9(Pase, Tet, Ery) ...... 1.7 X 1010 6.0 X 107 1.0 X 102 2.0 X 102 U9(Pase, Tet, 01, Nov) ...... 2.3 X 1010 1.8 X 1010 2.0 X 1010 1.9 X 1010 1052 PATTEE AND BALDWIN J. BACTERIOL. that all of the transductants were uniformly TABLE 5. Effect of erythromycin concentration in resistant to at least 100 ,ug of any of the four the selective medium on the frequency of transduc- macrolide antibiotics per ml of BHI agar. The tion of erythromycin resistance using phage transductants were stable after many subcul- 80/U9 (Pase, Tet, Ery) and recipient strain 112 tures, and no evidence of linkage between the 01 No. of colonies recovered marker and the Pase, Tet, and Nov markers was Erythromycin per 109 cells obtained. (ug per ml of BHI agar) Control Transduced Transduction of erythromycin resistance. The suspension suspension concentration of erythromycin required to 0.5 200 1,000 inhibit the growth of spontaneous mutants 1.0 40 600 present in control suspensions of erythromycin- 2.0 20 50 sensitive recipient strains was determined, after 4.0 10 20 which the isolation of Ery transductants was 6.0 4 4 attempted by using an appropriate concentration 8.0 0 0 10.0 0 0 TABLE 3. Effect of erythromycin on the incidence of erythromycin-resistant cells in a population of strain U9 (Pase, Tet, Ery) of erythromycin. Typical results of such experi- ments (Table 5) indicated that there existed a Viable cells per ml as determined on Time of exposure to BHI agar containing: higher incidence of erythromycin-resistant cells 1.0 jg of erythro- mycin per ml _ Erythromycin in the transduced population than in the control. (50 Ag/ml) However, those concentrations of erythromycin allowing the direct isolation of erythromycin- hr resistant transductants also permitted the growth 7.9 0 X 109 8.6 X 107 mutants in 2 8.1 X 109 8.8 X 109 of numerous the control suspensions. 4 1.2 X 1010 1.1 X 1010 A modified technique for the selection of Ery 5½fi 1.0 X 1015 1.2 X 10lo transductants, termed the agar-transfer tech- nique, was therefore devised. After the cell sus- pensions were washed with P and D broth, 0.05- TABLE 4. Frequency of transduction of oleandomycin ml quantities of the transduced and control resistance by phage 80/U9 (Pase, Tet, suspensions were inoculated onto BHI agar 01, Nov) (2% agar) plates and incubated at 37 C for 1 hr. The agar was then removed intact from the dish Recipient strain transduction9Frequency of with a sterile 6-in. spatula and placed, inoculated surface uppermost, onto the surface of a BHI U9 (Pase, Tet, Ery) 4,100 agar plate containing 25 ,ug of erythromycin per 112 2,000 ml. Incubation was continued for 24 hr at 37 C. 152 3,000 Transductants were readily 248 800 obtained by this D-1 3,000 technique, while spontaneous mutants present Ps 42B 0 in control suspensions were inhibited. The initial Ps 29 0 1-hr incubation period prior to transfer and the Ps 52 32 time required after transfer for the erythromycin C-72 12 to diffuse into the upper agar layer resulted in a Ch-S0 0 light film of background growth which, however, W-26 6 did not interfere with the scoring and isolation N-135 4 of Ery transductants. When the concentration of 608 6 erythromycin in the base layer was reduced to 1 2 12 a 769 14 ,ug per ml, numerous mutants and heavy background growth were obtained. Increasing * Transductants recovered per 109 phage the concentration of erythromycin in the base particles employed in the transduction sus- layer to 50 or 100 jAg per ml caused a significant pension. depression in the observed transduction fre- VOL,. 84, 1962 TRANSDUCTION OF RESISTANCE IN S. AUREUS 1053

TABLE 6. Frequency of transduction of erythromycin TABLE 7. Frequency of transduction of the Ery and resistance using phage 80/U9 (Pase, Tet, Tet markers by use of recipient strains C-72 Ery) and the agar-transfer technique and 152 and various transducing phages

Frequency of transduction* Recipient strain transduction*Frequency of Transducing phage C-72 152 U35 (Pase, Tet) 700 U40 (Pase, Tet) 520 Tel Ery Tel Ery U61 (Pase, Tet) 620 U91 (Pase, Tet) 820 29/655 (Pase, Tet, Ery) 6,000 0 8,000 1,000 U114 (Pase, Tet) 320 52A/655 (Pase, Tet, 461 (Pase, Tet) 720 Ery) 4,000 0 4,000 600 588 (Pase, Tet) 360 79/655 (Pase, Tet, Ery) 40 0 60 400 112 1,200 53/655 (Pase, Tet, Ery) 560 0 190 500 152 1,000 80/U66 (Pase, Tet, Ery) 5,000 2 1,000 600 248 1,000 79/4A (Pase, Tet, Ery) 400 0 140 200 D-1 800 53/4A (Pase, Tet, Ery) 500 28 330 600 Ps 42B 0 Ps 29 0 * Transductants recovered per 109 phage Ps 52 50 particles employed in the transduction sus- C-72 4 pension. Ch-50 6 W-26 10 transducing phage or the donor strain, three N-135 6 additional chlortetracycline-resistant dissociated 608 6 strains obtained from natural sources were 1 6 20 examined by use of a variety of transducing 769 phages. The frequencies of transduction of the * Transductants recovered per 109 phage Tet and Ery markers were determined with particles employed in the transduction sus- recipient strains 152 and C-72 transduced with pension. each phage. The results (Table 7) indicated that neither the donor strain nor the transducing quency. The results of transducing 21 recipient phage significantly influenced the relative strains with phage 80/U9(Pase, Tet, Ery), by transduction frequencies obtained. In all cases, means of the agar-transfer technique, are shown the transduction frequencies were significantly in Table 6. All control suspensions were com- higher for the Ery marker when recipient strain pletely inhibited, the transductants were stable 152 was employed. after many subcultures, and no linkages were detected between the Ery marker and the Pase, DISCUSSION Tet, and Nov markers. The results of this study generally are in Frequencies of transduction. In contrast to the agreement with earlier studies concerning the transduction frequencies previously reported for transduction of the capacity to produce peni- the Pase, Tet, and Nov markers (Pattee and cillinase and resistance to novobiocin and Baldwin, 1961), the Ery and 01 markers were chlortetracycline in S. aureus (Pattee and transduced by phage 80 at normal frequencies Baldwin, 1961). None of the aforementioned (at least 300 transductants per 109 phage) only characteristics was detectably linked to either to recipient strains 112, 152, 248, and D-l, and the Ery or 01 markers, lysogenization of the seven 80/81 strains of independent origin. The transductants by the transducing phage was not remainder of the recipient strains, including observed, and the frequencies of transduction for strain C-72, were transduced at frequencies sig- all markers were similar in certain instances. nificantly lower than normal, but only with A logical interpretation of the characteristics respect to the Ery and 01 markers. To determine displayed by strains of the dissociated type is that whether the competence of a strain to serve as a resistance to erythromycin involves ani inducible recipient of the Ery marker was a function of the enzyme system. Garrod (1957) reported that as 1054 PATTEE AND BALDWIN J. BACTERIOL. little as 0.0015 ,ug of erythromycin per ml "bred" The Ery and 01 markers were transduced at a population which was uniformly resistant to at normal frequencies to strains 112, 152, 248, least 64 ,ig of erythromycin per ml. Although the and D-1, and to seven epidemic strains, whereas results of the present study indicate that low the majority of the recipient strains, including concentrations of erythromycin were capable of strain C-72, were transduced at low frequencies inducing resistance, no attempt was made to by both markers. When a variety of phages determine the minimal active concentration of were propagated on several naturally occurring this . Thus far, only erythromycin has chlortetracycline-resistant dissociated strains, proven to be capable of inducing resistance. In and employed to transduce strains 152 and C-72, agreement with Garrod (1957) is the finding that in every case the Ery marker was transduced at growth of an induced population in the absence normal frequencies only to strain 152. The Tet of erythromycin results in a population which no marker was transduced to both recipient strains longer is resistant to oleandomycin, spiramycin, at frequencies which were uniform within the and carbomycin, and which contains only a few capacities of the phages employed. The trans- cells capable of growth in the presence of inhibi- ducing phages used vary in their transductional tory concentrations of erythromycin. competence. Phage 79 was the least effective and When cells of a dissociated strain are induced, phage 80 the most effective transducing phage resistance is acquired to erythromycin, oleando- when the Pase, Tet, and Nov markers were mycin, spiramycin, and carbomycin. Therefore, examined by using many of the same recipient in the induced state, dissociated strains are strains uised in the present study (Pattee and indistinguishable from strains possessing the 01 Baldwin, 1961). These results suggest that a marker. This observation, and the fact that strain degree of genetic incompatibility associated with U9(Pase, Tet, 01, Nov) was obtained as a mutant the region of the chromosome occupied by the of strain U9(Pase, Tet, Ery, Nov), make it Ery and 01 markers exists among certain strains reasonable to assume that the 01 marker repre- of S. aureus. The observation that the Ery sents a mutant of the Ery locus in which resis- marker was transduced to seven 80/81 strains tance no longer is due to an induced enzyme at normal frequencies simply adds to the avail- system but rather is due to the same enzyme able evidence that all 80/81 strains are closely system functioning constitutively. related (Shaffer, Baldwin, and Wheeler, 1958). In view of the nature of dissociated resistance to erythromycin, the necessity of employing the LITERATURE CITED agar-transfer technique to isolate the Ery DOWELL, C. E., AND E. D. ROSENBLUM. 1962. transductants is quite apparent. After incorpora- The nature of the staphylococcal transducing tion of the Ery marker by the recipient bacterium, particle. Bacteriol. Proc., p. 145. induction is necessary before complete phenotypic EDGAR, J. B., AND B. A. D. STOCKER. 1961. Meta- is An identical bolic and genetic investigations of nutri- expression of the marker obtained. tionally exacting strains of Staphylococcus situation concerning the phenotypic expression pyogenes. Nature 191:1121-1122. of resistance exists among naturally occurring GARROD, L. P. 1957. The erythromycin group of dissociated strains. The induction process is antibiotics. Brit. Med. J. 2:57-63. conveniently accomplished by means of the agar- JONES, W. F., JR., R. L. NICHOLS, AND M. FIN- transfer technique. When the appropriate LAND. 1956. Development of resistance and concentration of erythromycin is employed in the cross-resistance in vitro to erythromycin, base layer, the Ery transductants first are ex- carbomycin, spiramycin, oleandomycin, and posed to subinhibitory concentrations. During . Proc. Soc. Exptl. Biol. Med. subsequent incubation, the concentration of 93:388-393. gradually increases to a level which MORSE, M. L. 1959. Transduction by staphylococ- erythromycin cal bacteriophage. Proc. Natl. Acad. Sci. is inhibitory to the growth of all cells except the U.S. 45:722-727. induced transductants. When excessively high MURAI, K., B. A. SOBIN, W. D. CELMER, AND concentrations of erythromycin are used, then F. W. TANNER. 1959. PA-108, PA-133A, inhibition occurs prior to complete induction of PA-133B, and PA-148: new macrolide-type all cells genetically capable of dissociated resis- antibiotics. Antibiot. Chemotherapy 9:485-490. tance. PATTEE, P. A., AND J. N. BALDWIN. 1961. Trans- VOL. 84, 1962 TRANSDUCTION OF RESISTANCE IN S. A UREUS 1055

duction of resistance to chlortetracycline RITZ, H. L., AND J. N. BALDWIN. 1961. Transduc- and novobiocin in Staphylococcus aureus. J. tion of capacity to produce staphylococcal Bacteriol. 82:875-881. penicillinase. Proc. Soc. Exptl. Biol. Med. RANTZ, L. A., E. RANDALL, L. THUM, AND L. F. 107:678-680. BARKER. 1957. The effects of vancomycin, SHAFFER, T. E., J. N. BALDWIN, AND W. E. oleandomycin, and novobiocin on staphy- WHEELER. 1958. Staphylococcal infections lococci in vitro. Antibiot. Chemotherapy in nurseries. Advan. Pediat. 10:243-281. 7:399-409. SZYBALSKI, W. 1952. Gradient plates for the RAVIN, A. W., AND V. I. NYER. 1961. The genetic study of microbial resistance to antibiotics. relationship and phenotypic expression of Bacteriol. Proc., p. 36. mutations endowing pneumococcus with WATERWORTH, P. M. 1960. The antibacterial resistance to erythromycin. J. Gen. Microbiol. properties of leucomycin. Antibiot. Chemo- 26:277-301. therapy 10:101-108.