This dissertation has been Mic 61-2840 microfilmed exactly as received

PATTEE, Peter Arthur. TRANSDUCTION OF ANTIBIOTIC RESISTANCE IN STAPHYLO­ COCCUSAUREUS. The Ohio State U niversity, Ph.D., 1961 Bacteriology

University Microfilms, Inc., Ann Arbor, Michigan TRANSDUCTION OF ANTIBIOTIC RESISTANCE

IN STAPHYLOCOCCUS AUREUS

DISSERTATION

Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

PETER ARTHUR PATTEE, B.S., M.Sc.

The Ohio State University

1S61

Approved by

/ / Adviser Department of Bacteriology ACKNOWLEDGMENT

The author wishes to express his sincere appreci­ ation to Dr. Jack N. Baldwin for his Invaluable guidance and interest during the course of this investigation and in the preparation of the manuscript. A word of gratitude is also extended to the author's wife for her aid in the preparation of the manuscript.

ii TABLE OF CONTENTS

Page INTRODUCTION...... 1

HISTORICAL REVIEW...... 4

Genetic Exchange...... 4

Conjugation...... 5 Transformation...... 6 Transduction...... 7 Lysogenic conversion...... 15

Staphylococcal Bacteriophages and Antibiotic Resistance...... 17

Bacteriophages...... 17 Antibiotic resistance...... 24

MATERIALS AND METHODS...... 27

Media ...... 27 Antibiotics...... 28 Preparation of Media Containing the Antibiotics...... 29 Bacteriophages...... 30

Propagation of bacteriophages...... 31 Titration of bacteriophages...... 32

Source of Cultures...... 33 Characterization of Cultures...... 34

Determination of phage type...... 34 Determination of antibiotic resistance...... 35

Donor Strains...... 37

Incorporation of markers by transduction 37 Incorporation of markers by mutation...... 38 Principal donor strains...... 39

Recipient Strains ...... 43 Transduction Procedure ...... 44 Velveteen Replications...... 47

H i TABLE OF CONTENTS (contd.)

Page MATERIALS AND METHODS (contd.)

Viable Cell Counts...... 47 Detection of Lysogenic Cultures...... 48

RESULTS...... 50

Incidence of Recipient Strains...... 50 Transduction of Chlortetracycline Resistance.... 52 Transduction of Novobiocin Resistance...... 56 Transduction of Oleandomycin Resistance...... 58 Transduction of Erythromycin Resistance... 62 Effect of Donor Strains and Transducing Phages on the Transduction Frequencies of the 01 and Ery Markers...... 67 Transduction of Erythromycin Resistance from Donor Strain U9(Pase,Tet,Ery-X)...... 72 Transduction of Erythromycin Resistance from Mutants Isolated In Vitro...... 77 Comparative Viable Cell Counts of Erythromycin- Resistant Strains...... 78 Induction of Cross Resistance in Erythromycin- Resistant Strains by Growth in the Presence of Erythromycin...... 82 Effect of Chlortetracycline on the Frequency of Transduction of Penicillinase Production 86 Effect of the Medium Used for Selection on the Transduction Frequency...... 89 Effect of Multiplicity of Infection on the Transduction Frequency...... 91 Determination of the Lag Period After Plating Prior to Division of the Transduced Markers.. 95 Effect of Growth in Broth on the Multiplication and Survival of the Transductants...... 95 Transduction of Streptomycin Resistance...... 96 Characteristics of the Transductants...... 100

Stability of phage type ...... 100 Level of antibiotic resistance...... 100 Stability of transduced characteristics...... 101 Effect of transduction on lysogeniclty...... 102 Tests for linked transductions...... 106

Transduction Studies Using Different Donor Strains and Bacteriophages...... 108

iv TABLE OF CONTENTS (contd.)

Page DISCUSSION...... 119

SUMMARY...... 134

REFERENCES...... 137

AUTOBIOGRAPHY...... 144

v LIST OF TABLES

Table Page

1 Concentration of antibiotics used in the preparation of stock solutions 28

2 Phage types of recipient strains 44

3 Concentrations of antibiotics employed in the selective media 46

4 Phage type and frequency of transduction of penicillinase production of strains trans­ duced by phage 80/U9(PB.se,Tet,Ery) 51

5 Phage type of strains not transduced to penicillinase production by phage 80/U9(Pase,Tet,Ery) 53

6 Effect of concentration of chlortetracycline in the selective medium on the frequency of transduction using phage 80/U9(Pase,Tet,Ery) and recipient strain 152 54

7 Frequency of transduction of chlortetra­ cycline resistance by phage 80/U9(Pase,Tet, Ery) 55

8 Effect of concentration of novobiocin in the selective medium on the frequency of trans­ duction using phage 80/U9(Pase,Tet,01,Nov) and recipient strain Ch 50 57

9 Frequency of transduction of novobiocin resistance by phage P0/U9(Pase,Tet,01,Nov) 57

10 Effect of concentration of oleandomycin in the selective medium on the frequency of transduction using phage 80/U9(P&se,Tet,01, Nov) and recipient strain 112 59

11 Frequency of transduction of oleandomycin resistance by phage 80/U9(Pase,Tet,01,Nov) 60

vl LIST OF TABLES (contd.)

Table Page

12 Effect of concentration of oleandomycin employed In the selective medium on the frequency of transduction using recipient strain 1 and phage 80/U9(Pase,Tet,01,Nov) 61

13 Effect of macrolide antibiotics in the selective medium on the frequency of trans­ duction of oleandomycin resistance using recipient strain 152 transduced with phage 80/U9(Pase,Tet,01,Nov) 62

14 Effect of concentration of erythromycin in the selective medium on the frequency of transduction using phage 80/U9(Pase,Tet,Ery) and recipient strain 112 63

15 Frequency of transduction of erythromycin resistance by phage 80/U9(Pase,Tet,Ery) using the agar transfer technique 66

16 Phage type and frequency of transduction of erythromycin resistance of strains trans­ duced by phage 80/U9(Pase,Tet,Ery) 68

17 Frequency of transduction of the Ery and Tet markers to strains C 72 and 152 transduced by different phage preparations 69

18 Frequency of transduction of the Ery marker to strains 112 and 1 transduced by phages 80/1(Ery) and 8 0 / 1 1 2 (Ery) 71

19 Frequency of transduction of the 01 marker to strains 112 and 1 transduced by phages 80/1(01) and 80/112(01) 71

20 Frequency of transduction of erythromycin resistance by phage 80/U9(Pase,Tet,Ery-X) 73

21 Percentage of erythromycin-resistant trans­ ductants resistant to 100 ug per ml of oleandomycin, spiramycin, and carbomycin 75

22 Viable cell counts of strains U9(Pase,Tet, Ery), U9(Pase,Tet,Ery-X), U9(Pase,Tet,01,Nov) and U40(Pase,Tet) as determined on BHI agar with and without added oleandomycin, spiramycin, or erythromycin 79 vli LIST OF TABLES (contd.)

Table Page

23 Viable cell counts of transductant clones of U40(Pase,Tet,Ery') and U4o(Pase,Tet,Ery-X) obtained by transduction of strain U40(Pase, Tet) with phage 80/U9(Pase,Tet,Ery-X) 81

24 Frequency of transduction of erythromycin resistance to recipient strain 152 by phages 80/U9(Pase,Tet,Ery) and 80/US(Pase,Tet,Ery-X) 82

25 Phage type of strains showing the "dissoci­ ated" type of erythromycin resistance 85

26 Effect of erythromycin In the transduction mixture on the frequency of transduction of erythromycin resistance using recipient strain U40(Pase,Tet) transduced by phage 80/U9(Pase, Tet,Ery) 87

27 Effect of chlortetracycline in the selective medium on the transduction of the Pase marker to strains Ch 50 and Ch 50(Tet) by phage 80/U9(Pase,Tet,Ery) 89

28 Effect of the medium employed for selection on the frequency of transduction of Pase and Tet markers by phage 80/U9(Pase,Tet,Ery) 90

29 Effect of multiplicity of infection on the frequency of transduction of the Pase, Tet, 01, and Nov markers to strain 152 transduced by phage 86/U9(Pase,Tet,01,Nov) 93

30 Growth lag of various transduced populations as determined by re-spreading plates inocu­ lated with the transduced suspension 94

31 Multiplication of transductants and total viable cells in BHI broth Inoculated with a suspension of strain 152 transduced by phage 80/U9(Pase,Tet,Ery) 97

32 Phage type of 19 recipient strains and penicillinase-producing transductants of each strain transduced by phage 80/U9(Pase, Tet,Ery) 101

viii LIST OF TABLES (contd.)

Table Page

33 Phage type of Indicator strains used in the detection of lysogeny 103

34 Lytic reactions of the parent recipient strains and the Pase and Tet transductants obtained by transduction with phage 80/U9 (Pase,Tet,Ery) 104

35 Frequency of transduction of the Pase, Tet, and Ery markers by phage 79/655(Pase,Tet, Ery) 109

36 Frequency of transduction of the Pase, Tet, and Ery markers by phage 53/655(Pase,Tet, Ery) 110

37 Frequency of transduction of the Pase, Tet, and Er.y markers by phage 29/655(Pase,Tet, Ery) 111

38 Frequency of transduction of the Pase, Tet, 01, and Nov markers by phage 29/4?T6B (Pase, Tet,01,Nov) 1 12

39 Frequency of transduction of the Pase, Tet, and Ery markers by phage 52A/655(Pase,Tet, Ery) 114

40 Frequency of transduction of the Pase, Tet, 01, and Nov markers by phage 52A/4865(Pase, Tet,01,Nov) 115

41 Frequency of transduction of the Pase, Tet. 01, and Nov markers by phage 80/4865(Pase, Tet,01,Nov) 116

42 Frequency of transduction of the Pase, Tet, 01, and Nov markers by phage 80/U9(Pase,Tet, 01,Nov) 117

Ix INTRODUCTION

Studies concerned with bacterial genetics have been particularly rewarding, and this has been primarily due to the recent discovery of sexual recombination and mechanisms of genetic exchange which are apparently unique for bac­ teria,, Although sexual recombination has proved to be the most useful technique, studies employing transduction have also been of great significance. Not only have some fundamentals concerning the host-virus relationship been revealed, but transduction has also been Invaluable in the analysis of the fine structure of the bacterial chromosome.

The discovery of sexual recombination and trans­ duction, and the great majority of the studies employing these mechanisms, have been performed by using bacteria of the enteric group. The species of the enteric group lend themselves extremely well to laboratory manipulation, and their rather simple growth requirements have proved to be a distinct advantage. However, while a great deal of information has been obtained by studying these organisms, this Information is perhaps of limited significance because of the narrow taxonomic confines of the species involved.

When Ritz discovered the existence of a trans­ duction system in Staphylococcus aureus in 1957, the way

1 was open to analyze a species not only unrelated to the enteric organisms, but a species whose physiology and pathogenic potential are not well understood. The staphy­ lococci are nutritionally complex, and these requirements have not been determined completely. When a chemically defined medium for the growth of these bacteria is de­ veloped, then an application of transductional analysis to the nutritional aspects of the staphylococci will undoubtedly prove of value.

The occurrence of endemic and epidemic staphylo­ coccal infections has been widely reported. At present, there is no known cause for the apparent increased viru­ lence of certain strains of staphylococci, termed "epi­ demic" strains, which are often incriminated in these infections. Aside from their increased virulence, the

"epidemic" strains are further characterized by being resistant to the majority of the currently employed anti­ biotics.

The decision to study antibiotic resistance by transduction was made for two reasons. First, antibiotic resistance in the staphylococci is vitally important, and perhaps has some as yet unknown relationship to the viru­ lence of the organism. Secondly, the problem of selecting a few genetically altered cells from a large population of normal cells requires the use of highly selective techniques and antibiotic resistance is particularly well suited to this requirement. Neither nutritional require­ ments nor fermentative abilities can be successfully detected by selection in this manner, and a genetic analysis of these characteristics in the staphylococci must await the development of a synthetic minimal medium. HISTORICAL REVIEW

Genetic Exchange

In recent years, various mechanisms of bacterial genetic exchange have been reported and Investigated rather intensively. The three basic mechanisms whereby genetic material is transferred from one strain, designated a donor, to another, designated a recipient, are termed conjugation (sexual recombination), transformation, and transduction. As this study has been concerned with transduction, only brief mention of the other phenomena will be made.

The processes of genetic exchange all have certain common features. First, the exchange is always uni­ directional from donor to recipient bacterium. Second, only a portion of the genetic material of the donor cell is transferred during a single exchange. Third, in each

Instance there is either direct or indirect evidence that the material being exchanged is deoxyribonucleic acid

(DNA). Fourth, and finally, the DNA fragment of the donor strain transferred to the recipient strain is perpetuated or carried in one of several alternate ways, i.e., a functional, but non-replicating fragment; a functional, stable fragment which is not necessarily Incorporated directly into the chromosomal material of the recipient., bacterium; a functional, replicating, but unstable fragment; or a functional, replicating, stable fragment which is Incorporated into the recipient cell by replace­ ment of the homologous chromosome area of the recipient cell (Hartman and Goodgal, 1959). The mechanisms of genetic exchange are also separable on the basis of well- defined characteristics which are discussed in the following paragraphs.

Conjugation. In 194? Lederberg observed that when two strains of Escherichia coll which differed in a number of nutritional, antibiotic-resistant, and fer­ mentative characteristics were mixed, cells were isolated from the mixture of the two strains which possessed characteristics of both parent strains. It was found that several characteristics were transferred simul­ taneously, and that the exchange required intimate contact between the two parent strains. The exchange was always found to obey a polarity of transfer from the donor strain, designated F*-, to the recipient strain, designated

F~ (Jacob and Wollman, 1958). A large fragment of the donor cell chromosome was transferred during a single exchange, and the exchange always occurred in a pre­ dictable linear genetic sequence for a given donor strain.

The total size, or length, of the chromosomal fragment which was transferred was dependent upon the time of contact between the two cell types. The linear arrange­

ment of the genes on the chromosome could be determined by Interrupting the exchange at different time intervals

and analyzing the recombinants which were present. This

latter experiment, and many others, were possible only

after the discovery of Hfr (high frequency recombinant)

donor strains, which were isolated from the original re­

strains. It was with the Hfr strains that a cytologically demonstrable conjugation tube was discovered.

Transformation. Transformation, discovered by

Griffith in 1928, resembles transduction in that intimate

cell contact is not required between donor and recipient

cells (Avery et al., 1944). When DNA of the donor strains

was extracted and purified and recipient cells grown in

the presence of this DNA, a number of cells were isolated

which had acquired one of several characteristics of the

donor strain. The DNA preparations were found to be free

of cells and phage. The frequency of transformation was

found to be dependent upon the concentration of DNA and

upon the competence of the recipient cells. The identity

of the transforming agent with DNA was demonstrated by

the concurrent loss of transforming activity as the DNA

was depolymerized by the action of highly purified deoxy­

ribonuclease. Unlike conjugation, only single or very

closely linked genetic characters were transformed to a

cell during a given exchange. The maximum frequency of transformation was approximately six transformed cells per

100 cells treated with DNA, depending on a number of factors, many of which were very difficult to control

(Ravin, 1955).

Transduction. Zinder and Lederberg (1952), while attempting to demonstrate conjugation in Salmonella typhimurium, encountered a mechanism of genetic exchange which was resistant to deoxyribonuclease treatment and which did not require intimate cell contact. It was found that the exchange, termed transduction, was due to a temperate bacteriophage, designated PLT-22, which was produced by lysis of the donor strain and which subse­ quently infected recipient cells which possessed the XII somatic antigen. The frequency of transduction, which paralleled the titer of the phage, was approximately

2x10”^ per phage particle for any single characteristic.

Using phage PLT-22 and appropriate bacterial strains,

Zinder and Lederberg were able to transduce a variety of characteristics, including nutritional requirements, antibiotic resistance, fermentation capacity, and antigenic composition. Each characteristic was transduced inde­ pendently of the others, and the transduced cells, termed transductants, were stable after many generations of sub­ culture. Subsequent studies, reviewed by Zinder (1953), revealed that when phage PLT-22 was employed as the vector of transduction, nearly all of the transductants were lysogenized by the phage. However, using a virulent mutant of phage PLT-22 to transduce strains which had been previously lysogenized with temperate phage PLT-22, transductants were obtained which were still lysogenic for the original prophage. This was one of the first indi­ cations that the transduced fragment and the phage genome functioned independently after injection into the host cell. The transduced fragment was incorporated into the bacterial genome, while the phage genome was felt to undergo abortive infection.

Transduction was reported by Baron's group (1953) in Salmonella typhosa, using recipient strains which possessed the Vi antigen and were resistant to lysis by the Vi II transducing phage. Streptomycin resistance, xylose fermentation (Baron, et al., 1953), purine dependence (Baron, 1953), fermentative, nutritional, and flagellar antigen characteristics (Baron et al., 1955) were independently transduced by using a variety of Vi II typing phages propagated on the donor strains. Lysogen- izatlon was not observed among the transductants. (Baron,

1953). Approximately 90 per cent of the phage were adsorbed during transduction, and the frequency of trans­ duction was approximately 1x10”^ per phage particle.

The frequency of transduction was found to parallel the phage titer and was not influenced by treatment of the phage with deoxyribonuclease.

While evidence for the essential role of phage in transduction was available from the foregoing studies, it was not known by what mechanism the phage served to bring about transduction. Hershey and Chase (1952) presented evidence for the functional roles of phage protein and phage DNA during the Infective process. They demonstrated the passive role of the phage protein membrane in the

Intracellular replication process, during which the protein coat of the infecting particle remained on the surface of the cell and was dispensable. Garen and Zinder

(1955), using phage PLT-22 and virulent mutants of this phage, demonstrated that the lytic and lysogenic activities of both phages were equally radiosensitive to ultraviolet irradiation and P^ decay. In contrast, the transducing activities of the phages were found to be relatively resistant to these agents. They concluded that the phage genome comprised the major portion of the radiosensitive material (DNA) of the phage, while the radiosensitive phage contents responsible for transducing activity comprised a minor portion of the total DNA present.

Levine (1957) suggested that the absence of lyso­ genic cells among transductant populations was not signifi­ cant. Lysogenic cells which had been transduced continued to segregate out daughter cells which were not lysogenic. 10

Adams and Luria (1958) compared the frequencies of lyso- genlzation among Infected but not "selected" cells and among transduced cells, using phage P 1 and strains of

E. coll and Shigella dysenterlae, It was found that the infected but "unselected" cells which did not lyse were almost Invariably lysogenic, while the transduced cells were lysogenized at a much lower frequency. This was

Interpreted to mean that the transducing particles possessed an abnormal phage genome.

Using phage PLT-22 and a number of Independently isolated tryptophan-dependent mutants of S. typhlmurium,

Demerec and Hartman (1956) and Demerec and Demerec (1956) were able to determine the linear order of the mutated loci on the chromosome on the basis of reciprocal trans­ ductions. Furthermore, by biochemical analysis they demonstrated that the chromosomal sequence of the genes responsible for tryptophan synthesis corresponded precisely to the biochemical sequence of tryptophan synthesis.

Hartman (1956), using this same system and purine- dependent mutants, was able to place the sites of mutation in linear order on the chromosome and to demonstrate that the sequence of genes determining purine synthesis corre­ sponded to the biochemical sequence of synthesis of purines.

Stocker, Zinder, and Lederberg (1953) demonstrated the transduction of motility with phage PLT-22 and S. typhimurium. Using transduction, they found that there were at least six non-homologous genes, a mutation of any one of which resulted In the loss of motility, and conse­ quently of the H antigen. When a non-motile strain of known parentage with respect to the H antigen was trans­ duced with phage prepared on a motile donor strain pos­ sessing a different H antigen, the transductants which were isolated were motile and possessed the H antigen of the parent from which they had originated by mutation.

In rare instances, motility was transduced jointly with the "slow spreading character" or with the H antigen.

In agreement with earlier transduction studies with phage

PLT-22, the transductants were found to be commonly, but not invariably, lysogenic for phage PLT-22. In subsequent studies, Stocker (1956) demonstrated abortive transduction of motility with this same system. Abortive transduction occurred when a gene allowing the synthesis of flagella was transduced, although the gene was not replicated in the transduced cell. Consequently, the gene was passed on to one daughter cell at each division, although the flagella which were synthesized were passed on to both daughter cells. Those daughter cells which did not receive the gene responsible for flagella synthesis eventually gave rise to non-motile progeny. Ozeki (1956) reported abortive transduction of adenine synthesis in

S. typhlmurium. His findings were in agreement with the 12 observations of Stocker. Moreover, Ozeki demonstrated that if the transduced fragment was not incorporated and replicated shortly after transduction, such an incorporation did not occur during subsequent cell divisions. Abortive transduction was not observed between non-identicar alleles.

Cavallo and Terranova (1955) isolated a phage from a strain of Staphylococcus aureus which was lysogenic, penicillin-resi stant, and streptomycin-resistant. When the phage from this strain was used to lysogenize a peni­ cillin and streptomycin-sensitive strain, an increase in the incidence of streptomycin-resistant cells resulted.

The number of resistant cells was felt to be in excess of those occurring through mutation, and the observations were ascribed to transduction, In 1957, Rltz presented the first well-documented evidence for transduction in

S. aureus. He propagated a virulent phage on a strain of S. aureus which produced penicillinase. Subsequent adsorption of the phage by a penicillin-sensitive strain which was susceptible to lysis by the phage resulted in approximately one penicillinase-producing cell per 10? cells treated. The transductants were found to be identical with the original recipient strain in every respect except for penicillinase production. The use of a medium Inhibiting lysis prevented the lysis of the transductants in the presence of virulent phage. The frequency of transduction was found to parallel the titer of the transducing phage, and was not affected by adding deoxyribonuclease to the phage lysate. Linked or Joint transduction was not observed. In 1959, Morse reported the transduction of streptomycin and novobiocin resistance in S. aureus. He employed a lysogenic donor strain which, after exposure to ultraviolet Irradiation, released a phage capable of transduction of the characteristics. The phage were adsorbed with phage-sensitive recipient strains, and all but one of the transductants examined were found to be lysogenic for the transducing phage. Linked trans­ duction and the formation of heterogenotes were not observed. The frequency of transductions was found to be one transductant per 10*^ to 10® phage particles employed.

In 1955 Lennox, using phage P 1 and E. coll K 12, confirmed the linkages previously determined by conjugation studies in this strain. Lennox (1955) and Jacob (1955) transduced prophage lambda with phage P 1, and by selecting the galactose-fermenting transductants were able to detect

Joint transduction of lambda prophage and the galactose characteristic. In 1960, Arber reported the transduction of prophage lambda and the fertility factor, F*, using

E. coll K 12. Both of these factors are considered to be episomes in view of their transient nature In the cell and their doubtful structural attachment to the chromosome of

the host cell. Specialized transduction was first observed by-

Morse (195^) in E. coli K 12. This strain occasionally

carried an ultraviolet-inducible prophage designated lambda. Irradiation of such a strain with ultraviolet light Induced lysis with the liberation of mature lambda.

Titers usually were found to be approximately 10^® particles per ml. Rare particles present in induced lysates of lambda transduced only a cluster of genes

controlling galactose fermentation (designated the Gal

region). When lambda was produced by external infection

and subsequent lysis of sensitive strains, this transducing

activity was absent. Prophage lambda had previously been

observed to -be closely linked to the Gal region during

conjugation studies (Lederberg and Lederberg, 1953). Many

of the galactose-fermenting transductants obtained by

transduction with induced lambda were unstable because of

the diploid nature of the chromosome at the Gal region.

When such a heterogenetlc transductant carried a second

"active" lambda prophage, or was superinfected with such

a phage, the transducing lambda genome reproduced, matured,

and .lysed the cell with the release of mature lambda

particles, the majority of which were transducing phage

for the Gal character (Morse, Lederberg, and Lederberg,

1956; Weigle, 1957). These HPT (high frequency trans­

ducing) phage lysates were found to be composed of two

different types of particles. Some were non-transducing, 15

"active" particles possessed of a complete phage genome, while others were transducing, defective particles (Weigle,

1957). Weigle pointed out that the total number of trans­ ducing particles was far in excess of the number of cells from which they were released. This was especially important, and Indicated that the Gal and lambda regions were not only closely associated on the chromosome, but that after induction the Gal fragment replicated either with, or as a part of, the phage genome.

Specialized transduction was also observed by

Lurie's group (1958) using phage P 1 and strains of E. coll and Sh. dysenterlae. Phage P 1 was propagated on a lactose fermenting strain of E. coli, and the resulting phage lysate used to transduce a lactose-deficient strain of Sh. dysenterlae. Among the lactose-fermenting trans­ ductants there were rare, unstable, lactose-fermenting clones which were immune to lysis by phage P 1. The loss of immunity to phage P 1 and the loss of lactose-fermenting ability always occurred simultaneously. If such an unstable clone was superinfected with normal phage P 1, a lysate was obtained which was capable of transducing lactose-fermenting ability only, and at a frequency approximately 10,000 times as high as that observed with normally prepared P 1 from E. coll.

Lysogenic conversion. A phenomenon associated with bacteriophage, but distinctly different from transduction, 16 has been termed lysogenic conversion. Freeman (1951,

1952) observed that when a non-toxigenic strain of Coryne- bacterium dlphtherlae was lysogenlzed with a particular bacteriophage, the strain became toxigenic. Conversely, when lysogeny was lost, the ability to produce toxin was also lost. The presence of the bacteriophage in the prophage state in the genome of the host cell rendered the cell toxigenic. Toxigenicity was not due to a fragment of a genome which was carried from another strain by the phage. Baron, Formal, and Washington (1957) observed a similar phenomenon in Salmonella. When a phage iso­ lated from a strain of S. kentucky lysogenlzed other strains, it conferred the ability on that strain to produce somatic antigen XX. Phage prepared from strains which lacked the XX somatic antigen also induced the formation of antigen XX in the lysogenlzed recipient cells. The ability of a phage to lysogenlze and change a characteristic of the strain, therefore, was independent of the last host which that phage occupied. The authors concluded that this was a situation analogous to the lysogenic conversion observed in C. dlphtherlae. Uetake et al. (1958) observed lysogenic conversion in Salmonella anatum involving a change in the somatic antigen pattern.

Using both virulent and temperate phages, they were able to demonstrate that the new antigen was formed by the cells within a few minutes following infection. The cells which were lysogenlzed segregated into lysogenic and non- lysogenic types. The stable lysogenic cells retained the new serotype, while those strains which were non-lysogenic retained the antigen for only a few generations, after which they reverted to the original serotype. This was interpreted to mean that both prophage and vegetative phage were able to convert the cell, but that the conversion was stable only when the phage was reduced to the stable prophage state.

Staphylococcal Bacteriophages and Antibiotic Resistance

Bacteriophages. The use of bacteriophage typing for the identification and differentiation of strains of

Staphylococcus aureus began with the studies of Fisk

(1942a), who Isolated 24 phages from lysogenic, coagulase positive strains. It was found that the non-pathogenic

(coagulase negative) strains were resistant to lysis by these phages and were not lysogenic. The phage reactions were not found to be influenced by the environment, and

Fisk was able to divide a number of different strains of

S. aureus into groups on the basis of their response to

the phages employed (Fisk, 1942b). The phage types of

these strains were found to be stable for some strains and variable for others over a period of approximately three years after their initial isolation (Fisk, 1944). Fisk

maintained his phages in suspension with the sensitive strains on which he propagated them, but Wilson and

Atkinson (194-5) were able to obtain stable phage prepa­ rations by filtering the mixture after propagation. These

Investigators isolated 18 phages, 11 by the cross-culture technique used by Fisk (1942a), and seven by adaptation to different propagating strains. They designated 21 different phage types based on confluent lysis by their phages. Subsequently, Wilson and Atkinson added three additional phages to the group, which were designated

42D, 42E, and 29A. Allison added phages 31A, 53, and 54.

Using these phages and 56? independently isolated strains of S. aureus, Williams and Rlppon (1952) made a compre­ hensive study of phage typing techniques and the interpre­ tations of results. They found that only 60 per cent of the 56? strains were lysed with the test dilution of typing phages. The use of concentrated phages allowed 50 per cent of the remaining strains to be typed. They also observed that certain phages tended to lyse the same strains, and on this basis the authors were able to distinguish three main groups. Group "3A" included phages 3A, 3B, 30, and

51; group 116/47” Included phages 47, 53, 54, 7, 6, 42B,

47B, 470, 42E, and 47A; while group "52" consisted of phages 44, 44A, 52, 52A, 31, 29, 31A, and 29A. Typable staphylococci were also classified into these same groups.

Rountree (1949a), using phage-neutralizing antibodies prepared in rabbits, was able to divide 39 phages into 6 serologic groups. Group A Included phages 3A, 3B, 3C,

51, 6, 7, 42B, and 470, while group B was comprised of phages 29, 29A, 31, 31A, 44, 44A, 52, 52A, 42C, and 42D.

The latter two phages were used by Williams and Rippon, but were not placed in a group by them. Groups C through F

consisted of only a few phages, none of which were used

routinely for typing purposes. Levy, Rippon, and Williams

(1953) could not correlate the phage types of a number of

strains with any other known characteristic. DiSalvo

(1960) was unable to correlate the phage type and anti­ biotic resistance of 102 strains susceptible to lysis by phage 52, 52A, 80, and 81. The staphylococcal bacterio­

phages are now placed in groups on the basis of the lysis

of related strains of S. aureus (Shaffer et al., 1958),

and these groups are now accepted terminology in the

classification of typing phages. Group I consisted of phages 29, 52, 52A, 29A, and 79 (group "52" of Williams

and Rippon); Group II consisted of phages 3A, 3B, 3C, 51,

55, and 523 (group "3A" of Williams and Rippon); and

Group III consisted of phages 6, 7, 42E, 47, 53, 54, 70,

73, 75, 77, 42B, 47B-, 47C, 76, and Va4 (group "6/47" of

Williams and Rippon). Phage Va4 is now designated as

phage 83. Group IV consisted of phages 42D, and the

Miscellaneous group consisted of phages 31, 420, 44, 44A,

47A, 57, 80, 81, and 142.

In 1955, Rountree and Freeman reported the occurrence of a new phage type which was encountered in increasing numbers among hospital patients in Australia.

The strain was reported to cause an epidemic of furuncu­ losis among hospital patients. The same strain caused

19 out of 24 outbreaks of neonatal staphylococcal in­ fections in hospitals throughout Australia, and was iso­ lated primarily from lesions of the newborn. Ninety-four per cent of these strains were resistant to penicillin, and the authors stated at the time that the strain was of increased virulence and was lysed only by phage 80. Phage

80 was obtained by the adaptation of phage 52A to one of the newly encountered strains, and was found to be sero­ logic group B (Rountree, 1959). Bynoe et al. (1956) encountered a large number of untypable strains which were resistant to penicillin. By adapting phage 42B to one of these strains, they obtained phage 81 (serologic group A).

Phage 81 was found to be lytic for 16 per cent of the previously untypable strains. The authors felt that the

strains encountered by them and lysed by phage 81 were similar but not identical with the strains encountered by

Rountree which were typed with phage 80. Eynoe observed that the strains were prone to produce boils and abcesses, and felt that strains typed by phages 80 and 81 were

classifiable into Group I. In 1957, Shaffer's group reported a study of 19 epidemics caused by an Identical

strain of staphylococci. This strain was characterized by being susceptible to lysis by phages 42B, 47C, 44A, 52,

80, and 81; produced coagulase and alpha toxin; was sensi­ tive to erythromycin, bacitracin, neomycin, chloramphenicol, novobiocin, and oleandomycin; was resistant to penicillin, the tetracyclines, and streptomycin; and was referred to as the "80/81" or ''epidemic" strain. Caswell et^ al.

(1958) reported the occurrence of an "epidemic" strain in the Temple University Hospital, although this strain was found to be resistant to erythromycin. During one year,

Caswell's group observed 164 antibiotic-resistant staphylo­ coccal infections in surgical wounds, 50 per cent of which were due to a staphylococci of the phage type 42B/80/81.

In 1958, Shaffer et al. reported the results of a study of "epidemic" strains which had been received from Rountree in Australia as phage type 80 strains, from Bynoe in

England as phage type 81 strains, and from several other sources, both domestic and foreign. A comparative study of these strains revealed that they were all identical with respect to their phage pattern, which was reported as being 42B/47C/44A/52/80/81. After the completion of this study, it was realized that phages 42B, 44A, and 52 used in the United States, as well as phage 47C used by

Schaffer's group, had undergone mutations to broader lytic specificity. They concluded that cultures previously reported in the United States as being of phage types

42B/47C/44A/52/80/81, 42B/44A/52, 42B/52/81, or 22

42B/52/80/81 were all Identical and should be designated as phage type 80/81. Phage 80 and 81 were both classified in the Miscellaneous group by these authors. By 1959,

Koch's group spoke of the widespread occurrence of

M80/81" strains, and of their severity in causing epidemic staphylococcal sepsis in nurseries and maternity wards.

Rountree (1949b) reported on a study of 30 coagulase positive staphylococci, of which 27 were found to be lysogenic. A number of these strains were found to carry as many as five different temperate bacteriophages. Smith

(1948a) isolated a number of phages from coagulase positive staphylococci. He then employed these phages to type several strains, and encountered a number of strains which were non-typable. All of the resistant strains were found to be lysogenic. Smith (1948b) felt that the staphylococci could be classified on the basis of their phage type, but that changes in the phage type could result from lyso- genlzation. Rountree (1956) studied the effect of lyso- genization on the susceptibility of a number of strains to the typing phages. She concluded that lysogenizatlon does alter the phage type, and that the change in type brought about by lysogenizatlon with a given phage was unpredictable, and resulted in either immunity to the lysogenizlng phage or immunity to a number of unrelated phages. Rountree (1959) artificially lysogenlzed epidemic strains whose phage type was 47C/42B/52/52A/80/81 with phages of different serological groups. She found that

Immunity to serologic B phages 52, 52A, and 80 was obtained after lysogenizatlon with a serological B phage. Immunity to phage 81, a serologically group A phage, was brought about by lysogenizatlon with a serologically unrelated F phage. Immunity to phage 47C and susceptibility to phages

52 and 52A resulted from lysogenizatlon with a number of

serologic A phages. Rountree concluded that the gene loci

controlling susceptibility to phages 52, 52A, and 80 were

closely linked, but not identical.

Lowbury and Hood (1953) studied the secondary growth

observed on plates of propagating strains infected with their homologous phages. This secondary growth was found to be either resistant or susceptible to the lytic activity

of the homologous phage, or to be a mixture of both

resistant and susceptible cells. Secondary growth was

readily obtained from the propagating strains for phages

of Groups I and III, but was not obtained from propagating

strains for phages of Group II. While changes in the phage

types of the propagating strains were observed, only rarely

was the group classification changed. For Instance, Ps

31/44 was Initially susceptible to lysis by phage of

Groups I and III. The resistant growth obtained after

incubation with phage 31 was found to be resistant to all

of the phages in Group I which were previously active,

and resistant to some of the Group III phages which were 24 previously active. Ps 29, Initially susceptible to lysis by phages 29 and 31, was resistant to both phages after incubation with phage 29. Gorrill (1957) observed the host modification of typing phages, and was able to replace one prophage with another. However, doubly lysogenic strains were unstable, and always segregated in subsequent gener­ ations into strains which were singly lysogenic for either phage.

Antibiotic resistance. As early as 1949, Barber et al. reported a hospital nursery epidemic caused by a penicillin-resistant staphylococci, and since that time the reports of antibiotic resistance and epidemics caused by antibiotic-resistant staphylococci have been plentiful in the literature. It also has been observed that following the introduction of new antibiotics, a short lag in time

ensues before the incidence of resistant strains begins to

increase (Lepper et al., 1954). The epidemic strains which have been described previously were resistant to penicillin,

the tetracyclines, and streptomycin, and some were resistant

also to erythromycin. The data of Spink (1956) serves to

illustrate the clinical significance of antibiotic-resistant

strains. His data was based on the minimum inhibitory

concentration of various antibiotics for coagulase positive

staphylococci isolated at the Minneapolis General Hospital

during 1955. Among 209 strains, approximately 70 per cent

were resistant to 50 units or more of penicillin and 25

50 ug of streptomycin. Approximately 50 per cent of the strains were resistant to 50 ug or more of the tetra­ cyclines. Only about two per cent of 200 strains were resistant to chloramphenicol, while 22 per cent and 14 per cent, respectively, were resistant to erythromycin and spiramycin.

Cross-resistance between penicillin, the tetra­ cyclines, streptomycin, chloramphenicol, novobiocin, and the macrolide antibiotics is not normally observed. However, cross-resistance within two of these groups is encountered in the majority of instances. Fusillo et al. (1951) reported reciprocal cross-resistance between chlortetra- cycline and terramycln, and it is generally accepted that resistance to all of the tetracycline antibiotics occurs simultaneously. Jones et al. (1956) reported that in vitro mutations to erythromycin, carbomycin, spiramycin, or oleandomycin resistance resulted in complete cross-resist­ ance to all four of these antibiotics. These antibiotics are classified as macrolide antibiotics due to their

common structure and characteristics (Mural et al., 1959).

Rantz et al. (1957) found that cross-resistance between

erythromycin and oleandomycin was observed among naturally

occurring erythromycin-resistant strains of S. aureus.

but that this was not invariably the ease. Resistance to

either antibiotic produced in vitro yielded complete

cross-resistance, however. Ross (1956) found 22 strains 26 resistant to erythromycin among 140 strains of S. aureus, but all were sensitive to oleandomycin.

Garrod (1957) extensively studied the resistance of S. aureus to the macrolide antibiotics, and found that naturally occurring erythromycin-resistant strains were only rarely resistant to oleandomycin and spiramycin.

However, selection in vitro of strains resistant to erythromycin, carbomycln, oleandomycin, or spiramycin resulted in complete cross-resistance to all four anti­ biotics. He termed a naturally occurring strain which was resistant to erythromycin and oleandomycin as a "double resistant" strain, while a strain which was naturally resistant to erythromycin but which was sensitive to oleandomycin was termed a "dissociated" strain. When a culture of a "dissociated" strain was examined, it was found that the culture was made up of cells with varying degrees of resistance to erythromycin. Maximum erythromycin resistance was exhibited by less than one per cent of the total population. Growth of a "dissociated" strain in broth containing as little as 0.0015 ug of erythromycin per ml bred a population uniformly resistant to 64 ug of erythromycin per ml or more. The high resistance of such a culture was unstable, but a mutant which was sensitive to erythromycin was never isolated from a "dissociated" strain. MATERIALS AND METHODS

Media

Brainheart infusion (BHI, Difco) agar slants con­ taining 1.5 per cent agar were employed for the growth of cultures for phage propagation, and also for the growth of recipient strains to be used in transduction experi­ ments. Brainheart infusion agar slants containing 7.5 per cent sodium chloride (BHS slants) were employed for the maintenance of stock cultures. Trypticase soy agar

(TSA, Baltimore Biological Co.) was used routinely for the titration of phages and for the detection of lysogenic cultures. The other medium employed routinely was phosphate and dextrose broth (P and D broth) which consisted of nutrient broth (Difco) to which 0.2 per cent glucose and

0.25 per cent dipotassium phosphate were added (Rltz,

1957). Phosphate and dextrose broth was used for the suspension of cells from slant cultures, for the washing of the transduction mixtures, and for the dilution of phage suspensions. Agar was added to P and D broth to prepare both the base and soft agar layers employed for the propagation of phages.

27 28

Antibiotics

The antibiotics employed were obtained from several

manufacturers. The manufacturer, concentration of the

antibiotics, and the diluent used in the preparation of

the stock solutions of the antibiotics are indicated in

" table 1. Aqueous solutions of the antibiotics were pre-

Table 1

Concentration of antibiotics used in the preparation of stock solutions

Stock solution Antibiotic Manufacturer Diluent (concentration per m l )

Penicillin G E. R. Squibb distilled 20,000 units Potassium and Sons water

Chlortetra- American water for 5,000 ug cycline Cyanamld Co. injection, U.S.P.

Novobiocin Upjohn Co. P and D 10,000 ug Sodium broth

Erythromycin Abbott distilled 10,000 ug lactoblonate Laboratories water

Oleandomycin Charles Pfizer distilled 10,000 ug phosphate and Co. water

Carbomycin Charles Pfizer 95^ ethanol 10,000 ug and Co.

Spiramycin Ciba Pharma- 95% ethanol 10,000 ug ceutical Products pared in sterile diluent or were dissolved and sterilized by passage through an 02 Selas filter if the antibiotic was not obtained as a sterile preparation. The aqueous solutions of the antibiotics were dispensed in one ml quantities in sterile plugged tubes which were then frozen and stored at -40 C. The antibiotics which were dissolved in 95 per cent ethanol were stored at 4 C in screw-capped tubes. With the exception of carbomycin, all of the antibiotics were obtained as crystalline products which were completely soluble in either ethanol or in an aqueous diluent. Carbomycin was available only in gelatin-coated tablets. These tablets were crushed in a mortar with a pestle, after which the powdered tablet was dissolved in ethanol. A fine insoluble precipitate remained after dissolution of the antibiotic which, however, did not affect the activity of the preparation. As a precaution, solutions of the antibiotics which were prepared in ethanol were replaced at monthly intervals. The antibiotics which were frozen as aqueous solutions were found to retain their

full activity after storage at -40 C for a year or more.

Preparation of Media Containing the Antibiotics

Media containing the various antibiotics were

prepared by the addition of appropriate amounts of the

antibiotic to sterile BHI agar which had been previously

melted and cooled to 45 C. After dispensing the media into petri dishes, it was allowed to solidify and then stored at 4 C until needed. As a precaution against contamination and the loss of antibiotic activity, media containing the antibiotics were not stored for more than one week after preparation. Although the majority of the antibiotics were stable under these conditions for several weeks or more, spiramycin and chlortetracycline were found to lose their inhibitory activity after storage for one week. When agar plates containing the antibiotics were required with a dry surface, they were dried either in an upright position at 37 C with porcelain lids for four to eight hours or overnight at 37 C.

Bacteriophages

The bacteriophages employed in this study were all phages of the International Typing Series. Phages 29,

52A, 79, 80, and 53 were employed as transducing phages after propagation on appropriate donor strains. Bacterio­ phages 29, 52, 52A, 79, 80, 81, 3A, 3B, 30, Ms 39, 51, 55,

70, 73, 75, 77, 6, 7, 83(Va4), 53, 54, 42B, 42E, 47, 47B, and 47C were routinely employed for the characterization of staphylococci by their patterns of lysis. These phages originally had been obtained from Dr. John E. Blair, and were available in the Department of Bacteriology.

The designation of the transducing phage Included both the

International number designation of the phage and the 31 designation of the donor strain. Therefore, phage 80 propagated on strain U9(P&se,Tet,Ery) was designated as phage 80/U9(Pase,Tet,Ery).

Propagation of bacteriophages. The propagation of all phages was performed by the agar layer method of

Swanstrom and Adams (1951). The base layer consisted of

P and D agar (1.5 per cent agar), and the soft agar overlay consisted of P and D broth containing 0.3 per cent agar. The P and D pour plates were prepared in lots of

20 or more and stored in the cold until needed. The soft agar was prepared and dispensed in two ml amounts in

Wassermann tubes which were also stored in the cold until needed. For propagation, a tube of soft agar which had been melted and cooled to 45 C was inoculated with 0.1 ml of a phage lysate containing at least 1x1010 plaque forming units (pfu) per ml. This tube was also inoculated with

0.2 ml of a cell suspension of the propagating strain obtained by suspending the growth from a BHI slant culture grown at 37 C for 18 to 24 hours in one ml of P and D broth. This mixture was then gently mixed and poured over the surface of a chilled P and D pour plate. After solidi­ fication of the soft agar layer, the plate was incubated at 37 C. After Incubation for six hours, the soft agar layer was harvested into five ml of P and D broth with the aid of a ten ml pipette. This mixture was then homogenized with the pipette and placed in a centrifuge tube. The surface of the P and D plate was then rinsed with an additional five ml of P and D broth, which was added to the homogenate in the tube. The harvested mixture was centrifuged at 5000 rpm for approximately ten minutes to sediment the cells and agar. The supernatant fluid was removed and passed through an 02 Selas filter. The resulting filtrate of concentrated sterile phage was then stored in sterile screw-capped tubes at 4 C until needed.

The sterility of the phage lysate was verified by spreading a few drops over the surface of a BHI agar plate, which was then incubated overnight at 37 C. Phage filtrates were not used for typing or transduction when stored for more than one month, but were retained as inocula for propagation for much longer periods.

Titration of bacteriophages. The titer of a phage filtrate (pfu per ml) was determined by preparing ten-fold dilutions of the filtrate in 1.8 ml of P and D broth from

1 510 through 1s100,000,000. The entire surface of a dry

TSA plate was inoculated with a cotton swab moistened in a heavy suspension of cells of the propagating Btrain. When the surface of the agar was dry, 0.01 ml of each phage dilution was placed on an appropriate area of the inocu­

lated agar surface. When these drops were dry, the plate was inverted and incubated at 37 C for 12 to 18 hours.

The titer of the original lysate wa 3 calculated from the

number of plaques obtained from the higher phage dilutions. While the aforementioned method for the titration of phages was suitable for routine use, a more accurate method was required when studies were made to determine the effect of multiplicity of infection on the frequency of transduction. The dilutions of phage were prepared as before. To each of the higher three or four dilutions several billion cells of the propagating strain suspended in 0.1 ml were added and the tube shaken. One-tenth ml of each dilution was then removed and spread over the entire surface of a TSA plate with the aid of a sterile bent glass rod. The plates were then Incubated at 37 C for 12 to 18 hours, porcelain lids being used during the initial

four to six hours Of incubation. After incubation, the number of plaques developing on each plate was determined and the titer of the concentrated phage filtrate calculated.

Invariably, the titer obtained by the agar layer method was in excess of 101® pfu per ml, with a maximum titer of

approximately 6x10 1® pfu per ml.

Source of Cultures

The strains of Staphylococcus aureus employed in

this study were obtained from several different sources

and were of human origin. However, with the exception of

the propagating strains for the phages of the International

Typing Series (designated by the prefix Ps), no attempt was

made to characterize the strains with respect to origin. 34

Many of these strains were obtained from a culture collec­ tion acquired during earlier epidemiological studies

(Baldwin et a l ., 1957; Shaffer et a l ., 1957, 1958) while other strains were obtained as fresh isolates from carriers or infected individuals. All cultures were maintained by growing them exclusively on BHS slants (Chapman, 1945) and storing them at 4 C. All stock cultures were transferred to fresh media at intervals of three months.

Characterization of Cultures

The phage types and patterns of antibiotic re­ sistance of the strains were among the characteristics which were determined as an integral part of this study.

Determination of phage type. The phage types of the strains were determined by a modification of the method of Wilson and Atkinson (1945). Cultures to be typed were grown overnight at 37 C on BHI agar. The following morning, each culture was used to prepare a dense cell suspension in P and D broth. The entire surface of a TSA plate previously dried at 37 C for 24 hours was then inoculated with a cotton swab moistened in the cell

suspension, and the surface allowed to dry again. Using tuberculin syringes fitted with 26 or 28 gauge needles, a drop of each phage suspension was added to an appropriate

area on the surface of the plate. The suspensions of

phage used routinely for typing were prepared by diluting the concentrated stock suspensions of phage 1?200 In P and

D broth. The drops of phage inocula were allowed to dry, and the plate Incubated at 37 C. After Incubation for six to eight hours, the plate was examined for evidence of lysis by each phage and the degree of lysis was recorded.

The plates were then incubated for an additional 10 to 18 hours and a final determination of lytic activity was made. Confluent lysis was recorded as 4+, semiconfluent lysis as 3+, many Individually discernable plaques as 2+, approximately ten or less plaques as 1+, and the lack of lytic activity as negative (-). The phage type of a culture was determined by the 3+ and 4+ reactions only, and those phages which were less active were not Included in the phage type.

Determination of antibiotic resistance. Routinely, the determination of the resistance or sensitivity of strains to the various antibiotics was made using filter paper discs and the agar diffusion technique. The strains to be tested were grown overnight on BHI agar and resus­ pended in the manner employed for the determination of phage types. The entire surface of a dried BHI agar plate was Inoculated with a cotton swab moistened in a dense cell suspension of the strain to be tested. When the surface of the agar was dry again, antibiotic discs were placed on the surface of the Inoculated agar with the aid of sterile forceps. The plates were then Inverted and Incubated at 37 C. After incubation for 24 hours, the plates were examined for zones of inhibited growth at the periphery of each antibiotic disc. Only discs containing the higher concentration of each antibiotic were used.

Therefore, sensitive strains yielded broad zones of in­ hibition while resistant strains failed to show any evidence of inhibition at the periphery of the discs. The strains were designated as resistant or sensitive with no attempt to record differences in the degree of reaction.

In view of the fact that the selection of antibiotic- resistant transductants was performed with an agar medium containing antibiotics, an agar medium was also employed to determine the level of resistance or sensitivity of the strains to the antibiotics. Brainheart infusion agar plates were prepared containing appropriate graded concen­ trations of the antibiotic to be employed. Each strain to be tested was grown for 24 hours on a BHI agar slant. The slant culture was then suspended in one ml of P and D broth, and a 0.05 ml quantity pipetted onto the surface of each of the agar plates containing the various concen­ trations of the antibiotic. Each inoculum was spread over the entire agar surface with the aid of a sterile bent glass rod. The plates were then Incubated at 37 C for 48 hours, porcelain lids being used during the initial four to six hours of incubation to facilitate drying of the agar surface. After incubation for 24 and 48 hours, 37 the plates were examined and the amount of growth at each concentration of antibiotic recorded. The level of resistance of strains resistant to the antibiotic was recorded as the maximum concentration of antibiotic allowing uninhibited growth to occur. The level of sensi­ tivity of sensitive strains was the minimum concentration of antibiotic necessary to inhibit the growth of spon­ taneous mutants present in the population under test.

Donor Strains

A number of donor strains were employed during the course of this investigation. Some strains with desirable, stable characteristics were employed without altering their genotype. Antibiotic resistance markers were added to the genotype of certain strains by transduction of the desired marker from another strain. Also, antibiotic- resistant mutants were isolated from some strains, and these mutant strains employed as donors.

Incorporation of markers by transduction. To incorporate an antibiotic resistance marker into the genome of a strain, that strain was transduced with an

appropriate phage preparation. The desired transductant was selected and subsequently purified by streaking onto

a medium containing the appropriate antibiotic. A single

colony was then picked and inoculated onto a BHS slant, 38 which was stored at 4 C as a stock culture after growth had occurred.

Incorporation of markers by mutation. To select mutants which were resistant to the desired antibiotics, the gradient plate technique of Szybalskl and Bryson

(1952) was employed. A gradient plate was prepared by pouring 25 ml of melted and cooled BHI agar into a pressed- bottom petri dish with a diameter of 100 mm. The agar was allowed to solidify while the dish was supported at the maximum angle just allowing the agar to cover the entire bottom of the dish. The plate was then placed in a horizontal position and an additional 25 ml of melted and cooled BHI agar containing the desired concentration of antibiotic was poured over the initial agar layer and allowed to harden. The strain from which the mutant was to be isolated was grown for 24 hours at 37 C on a BHI agar slant. The growth from the slant was suspended in one ml of P and D broth, and 0.05 ml of the cell sus­ pension spread over the entire surface of the gradient plate with the aid of a sterile bent glass rod. The

Inoculated plate was then Incubated at 37 C for 48 hours, a porcelain lid being used during the initial four to six hours of incubation. The plate was examined for the presence of colonies growing in the presence of concen­ trations of the antibiotic inhibitory to the parent strain. One of these colonies was inoculated onto a BHI 39 agar slant, and the resulting growth on the slant used to assay the level of resistance of the mutant. When the level of resistance of the mutant was sufficiently high, a single colony of the mutant was obtained by streaking the culture for Isolation on a BHI agar plate containing the antibiotic. The culture was stored at 4 C on a BHS agar slant.

Principal donor strains. A strain which was initially Isolated at Temple University, Philadelphia,

Pa., and which was incriminated in an outbreak of staphylo­ coccal Infections In a hospital, was employed as a donor and as the parent strain for two other donor strains. The parent strain was pigmented, produced alpha toxin, fermented glucose, glycerol, fructose, maltose, trehalose, sucrose, lactose, mannltoi, and galactose, was susceptible to lysis by phages 80 and 81, and was not detectably lyso- genie. This strain produced sufficient penicillinase to grow on BHI agar containing 100 units of penicillin per ml of BHI agar, was resistant to 75 ug of chlortetracycllne,

800 ug of erythromycin, and 2000 ug of streptomycin per ml BHI agar. This strain was designated U9(Pase,Tet,Ery), the abbreviations in parenthesis referring to penicillinase production (Pase marker), and resistance to chlortetra­ cycllne (Tet marker) and erythromycin (Ery marker) respectlvely. These and other abbreviations have been used 40 throughout the text to refer to the antibiotic resistance markers.

Through the use of gradient plates, a mutant of parent strain U9(P&se,Tet,Ery) was selected which was resistant to 20 ug of novobiocin per ml of BHI agar.

This mutant was obtained from the parent strain by inocu­ lating, in consecutive order, gradient plates containing

3.0, 8.0, and 18.0 ug of novobiocin per ml of BHI agar in the upper layer. At each concentration a mutant was isolated which was Inoculated in the prescribed manner onto the plate containing the next higher concentration of novobiocin. The final mutant was found to be resistant to 20 ug of novobiocin per ml of BHI agar (Nov marker) and was designated U9(Pase,Tet,Ery,Nov).

From mutant donor strain U9(P&se,Tet,Ery,Nov), a mutant was then selected by the gradient plate technique which was resistant to oleandomycin. This mutant was obtained directly from a gradient plate containing 10 ug of oleandomycin per ml of BHI agar in the upper layer, and was resistant to 800 ug of oleandomycin, 400 ug of spiramycin, and 400 ug of carbomycin per ml of BHI agar

(01 marker). The designation of this mutant was

U9(Pase,Tet,01,Nov). The Ery marker was masked by the 01 marker and was consequently deleted from the strain designation.

A mutant which arose on a BHI agar plate containing 41

100 ug of spiramycin per ml of media was also isolated from parent strain U9(Pase,Tet,Ery). This mutant, desig­ nated U9(Pase,Tet,Ery-X), was found to be resistant to

800 ug of oleandomycin per ml of BHI agar and to 400 ug of spiramycin and carbomycln per ml of BHI agar (Ery-X marker). The mutant strains derived from parent strain

U9(Pase,Tet,Ery) resembled the parent strain in every respect except for the newly acquired resistance to the antibiotics described.

Another donor strain which was used rather ex­ tensively was designated U40(Pase,Tet). This strain had been incriminated in an epidemic of staphylococcal infections in a hospital in Frederick, Maryland. Strain

U40(Pase,Tet) was pigmented, produced alpha toxin, fer­ mented glucose, glycerol, fructose, maltose, trehalose, sucrose, lactose, mannitol, and galactose, was susceptible to lysis by phageB 80 and 81, and was not detectably lysogenic. This strain produced sufficient penicillinase to grow on media containing 100 units of penicillin per ml of BHI agar, and was resistant to 75 ug of chlortetra- cycline and 2000 ug of streptomycin per ml of BHI agar.

The other strain which was employed as a donor and as the parent for two other donor strains was designated

4865(Pase). This strain was capable of growing on media containing 100 units of penicillin per ml of BHI agar, fermented glucose, glycerol, fructose, maltose, trehalose, 42 sucrose, lactose, mannltol, and galactose, and was sus­ ceptible to lysis by phages 2S, 52A, and 80.

A mutant of strain 4865(Pase) was Isolated by a series of gradient plates containing novobiocin similar to those used to obtain strain U9(Pase,Tet,Ery,Nov).

This strain was designated 4865(Pase,Nov), and was re­ sistant to 20 ug of novobiocin per ml of BHI agar.

Strain 4865(Pase,Nov) was transduced with phage

80/U9(Pase,Tet,01,Nov) and an oleandomycin-resistant transductant selected. This transductant was designated

4865(Pase,Nov,01).

Strain 4865(Pase,Nov,01) was transduced with phage 80/U9(Pase,Tet,01,Nov) and a chlortetracycllne- resistant transductant was selected. This transductant was designated 4865(Pase,Tet,01,Nov). All of the afore­ mentioned strains which were derived from strain 4865(Pase) were identical with the parent in every respect except for the altered characteristics controlling antibiotic resis­ tance. The Nov marker acquired by this strain was identical in all respects with the Nov marker of U9(Pase,Tet,01,Nov).

The remaining strain which was employed extensively as a donor was designated 655(Pase,Tet,Ery). This strain was capable of growing in the presence of 100 units of penicillin per ml of BHI agar, was resistant to 800 ug of erythromycin, 75 ug of chlortetracycline and 2000 ug of streptomycin per ml of BHI agar. This strain fermented 43 glucose, glycerol, fructose, maltose, trehalose, sucrose, lactose, mannltol, and galactose, and was susceptible to lysis by phages 29, 52A, 79, 7, 83, 47, 53, 54, 73, and

77.

Recipient Strains

The strains which were used routinely as recipient strains were selected for their sensitivity to the commonly employed antibiotics and for their suscepti­ bility to lysis by phages of Group I. The principal recipient strains and their phage types have been listed in table 2. These strains were all sensitive to 0.12 units or less of penicillin, 0.5 ug or less of chlor- tetracycline, 3.0 ug or less of novobiocin, 7.0 ug or less of oleandomycin, 6.0 ug or less of erythromycin,

100 ug or less of streptomycin, and approximately 10 ug of spiramycin and 10 ug of carbomycln per ml of BHI agar. Moreover, as determined by the agar diffusion technique, all strains were sensitive to 10 ug of baci­ tracin, 30 ug of chloramphenicol, and 30 ug of neomycin.

With the exception of strain C 72, which failed to ferment mannltol, and strain Ch 50, which failed to ferment trehalose, all of the recipient strains fermented glucose, glycerol, trehalose, fructose, maltose, sucrose, lactose,

mannltol, and galactose. With the exception of strains 44

Table 2

Phage types of recipient strains

Strain designation Phage type

Ps 42B 42B Ps 29 29 Ps 52 52 248 52A/80 608 52A/80/81 C 72 29/52A/79/80 W 26 29/80 1 52A/80/81 152 52A/79/80 720 52A/80 M-1 79/55 N 135 29 N 203 29 112 29/52A/79/83/42E/80/81 456 52A/80/81 569 52A/80 616 52A/80/81 745 81 769 81 Ch 50 80 D-1 52A/80

608, 152, 720, M-1, N 203, 112, Ps 29, Ps 52, and Ps 42B, all strains produced alpha toxin.

Transduction Procedure

Cells of the recipient strains were prepared by growth on a BHI agar slant incubated at 37 C for 18 to

24 hours. The resulting cells were suspended uniformly

in 1.0 ml of P and D broth in a centrifuge tube. The

concentration of cells varied from 2x1010 to 6x1010 cells

per ml. To the centrifuge tube was then added 1.0 ml of 45 sterile phage filtrate with a minimum titer of 1x1010 pfu per ml. When a control was desired, 1.0 ml of sterile

P and D broth was substituted for the phage. The centri­ fuge tube was then placed on a Burrell Wrist Action

Shaker with the arm mounted over a 37 C water bath in such a manner that the lower one-third of the tube was sub­ merged. The mixture was then shaken for one hour with the shaker adjusted to a maximum setting of ten. After shaking, the contents of the tube were centrifuged at

5000 rpm for approximately ten minutes to sediment the cells, after which the supernatant fluid was aseptically removed. The cells were then resuspended in one ml of

P and D broth and again centrifuged to sediment the cells.

The cells were again resuspended in 1.0 ml of fresh P and

D broth, and 0.05 ml quantities of the suspension pipetted onto the surface of the desired agar media. Each inoculum was spread over the surface of the agar with the aid of a sterile bent glass rod, and the plates Incubated at 37 C

for approximately 18 to 24 hours. Porcelain lids were employed during the first four to six hours of incubation

to facilitate the drying of the agar surface. At the

conclusion of the incubation period the colonies were

scored and further studies made when desired.

To select transductants carrying the Pase, Nov, 01,

or Tet marker, the transduced cell suspension was inocu­

lated directly onto an agar medium containing the selective 46 concentration of the appropriate antibiotic. To select the Ery and Ery-X transductants, it was necessary to inoculate the transduced cell suspension onto the surface of an agar plate containing 25 ml of BHI agar (two per cent agar) in a pressed-bottom petri dish with a diameter of 100 mm. After incubation at 37 C for one hour, the agar was removed with a sterile spatula and placed, inoculum side up, onto the surface of the selective medium, care being taken to avoid breakage of the agar.

This procedure has been termed the agar transfer technique.

Table 3 indicates the level of each antibiotic employed in the selective media.

Table 3

Concentration of antibiotics employed in the selective media

Antibiotic Concentration per ml of BHI agar

Penicillin 0.12 units

Chlortetracycllne 3.0 ug

Novobiocin 10.0 ug

Erythromycin 25.0 ug*

Oleandomycin 25.0 ug

^Concentration of erythromycin in base layer for use with agar transfer technique. Velveteen Replications

The replica plating technique of Lederberg and

Lederberg (1952) was employed to test the transductant populations for linkages. The templates were prepared by tying swatches of velveteen over the top of metal cans with flat tops. The templates were then autoclaved for approximately one-half hour and then dried in a hot air oven at 170 C for two or three hours. All media used in conjunction with the replica platings were incubated at

37 C for 24 hours to insure a dry surface. Plates to be used to inoculate the templates were sufficiently dry when the colonies were large enough for replication. A template was inoculated with an agar plate containing the inoculum by gently placing the agar surface in' contact with the velveteen surface in such a manner that a uniform and complete contact was obtained between the template and agar. The plate was then removed, care being taken to maintain a constant orientation between the agar and template surfaces during the entire process. Agar plates were then inoculated in turn from the template in a similar manner.

Viable Cell Counts

To determine the number of viable cells in a cell suspension, ten-fold dilutions of the suspension were prepared in 4.5 ml quantities of saline. Each of the appropriate dilutions was assayed for viable cells in

duplicate. One-tenth ml of each dilution was spread over

the entire surface of a dried BHI agar plate with the aid

of a sterile bent glass rod. The plates were then incu­ bated at 37 C for 24 hours and the number of colonies on

each plate determined. The number of viable cells in the

original suspension was then calculated in the routine

manner.

Detection of Lysogenic Cultures

The detection of lysogenic cultures was performed by a modification of the cross-culture technique of Fisk

(1942a). Cultures to be examined for lysogeny were grown

overnight at 37 C in P and D broth. The indicator strains

were grown overnight at 37 C on BHI agar. The P and D

broth cultures were then centrifuged to sediment the cells

and the supernatant fluid removed from each culture. A

dense cell suspension of each indicator strain was prepared

in P and D broth and used to inoculate the entire surface of

a dried TSA plate with a cotton swab. When the agar surface

was dry again, a drop of the supernatant fluid of each

culture to be tested for lysogeny was placed onto a

predetermined area of the surface of each plate of TSA,

using a tuberculin syringe fitted with a 26 or 28 gauge

needle. When these drops were dry, the plates were Incubated at 37 C. After incubation for 8 and 24 hours, each plate was examined for the presence of lytic reactions.

The presence of lysis of the indicator strain was in­ terpreted as evidence of lysogeny of the strain being examined. RESULTS

Incidence of Recipient Strains

Ritz (1957), using donor strain U4o (Pase, Tet), phage 52, and recipient strains W 26, M-1, C 72, and

Ch 50, was able to transduce the ability to produce peni­

cillinase by inoculating the transduction mixture directly

onto BHI agar containing 0.12 units of penicillin per ml.

The method of transduction used by Ritz was applied directly in this study to determine the competence of a

larger number of strains of Staphylococcus aureus to

serve as recipient strains of the Pase marker. A total

of 48 strains which were sensitive to penicillin were

tested for their competence as recipient strains by

transduction with phage 80/U9(Pase,Tet,Ery). A control

suspension of each of the 48 strains was also Inoculated

onto the selective medium. In each instance, a concen­

tration of 0.12 units of penicillin per ml of BHI agar

was capable of inhibiting the growth of these control

suspensions. Twenty-six of these strains were found to be

competent recipients of the Pase marker. The phage type

and frequency of transduction of the Pase marker of each

strain is given in table 4. it was observed that ten of

the 26 competent recipient strains were not susceptible

50 52 ducing phage, only strains Ps 44 and Ps 31 were trans­ duced at relatively low frequencies.

Strains M-1, Ps 31, and Ps 44 were transduced with phage 80/U9(Pase,Tet,Ery), and the penicillinase-producing transductants assayed on BHI agar containing 0.06, 0.08,

0.10, and 0.12 units of penicillin per ml. The resulting transduction frequencies at each concentration of peni­ cillin were nearly identical. This indicated that the frequency of transduction observed with these strains was not limited by the concentration of penicillin routinely employed in the selective medium.

The phage types of the remaining 22 strains which were found to be incompetent recipients or were trans­ duced at frequencies below those detectable by the methods used are presented in table 5.

Among the strains which were not transducable, only strains 771 and 1 C were susceptible to lysis by phages in Group I.

Transduction of Chlortetracycllne Resistance

The choice of a strain for use as a donor of resistance to chlortetracycllne was variable as several of the principal donor strains were resistant to 75 ug or more of this drug per ml of BHI agar. However, as with all markers except the Pase marker, it was necessary to determine a suitable concentration of chlortetracycllne 51

Table 4

Phage type and frequency of transduction of penicillinase production of strains transduced by phage 80/US(Pase,Tet,Ery)

Strain designation Frequency of Phage type transduction*

Ps 42B 100 42B Ps 29 300 29 621 400 29 N 135 500 29 N 203 600 29 Ps 52 600 52 Ch 50 500 80 745 600 81 769 300 81 8 S 400 29/80 W 26 1000 29/80 C 72 100 29/52A/79/80 Ps 31 40 29/52A/79/53/80 Ps 44 4 29/52A/79/53/80 112 100 29/52A/79/83/42E/80/8 ‘ Ps 47 8 29/47/75/77 D-1 800 52A/80 569 600 52A/80 248 600 52A/80 720 200 52A/80 456 100 52A/80/81 608 700 52A/80/81 616 700 52A/80/81 1 600 52A/80/81 152 300 52A/79/80 M-1 4 79/55

*Transductants recovered per 10^ phage particles employed.

to lysis by phage 80/U9(Pase,Tet,Ery). However, among

these ten strains, only Ps 47 and M-1 were transduced at

relatively low frequencies. Among the remaining 16

strains which were susceptible to lysis by the trans- 53

Table 5

Phage type of strains not transduced to penicillinase pro­ duction by phage 80/U9(Pase,Tet,Ery)

Strain designation Phage type

601 80 771 52A 484 (44a )* 1 C 79 Ps 55 55 Ps 47B 47B 1 L 79/3B/55/71 Ps 79 79/3B/3C/55/53 Ps 3A 3A/3B/3C/51 Ps 3B 3B/3C/Ms39/51 Ps 3C 3A/3B/3C/Ms39/51 Ps 83 3A/3B/3C/6/83/47/53/77 Ps Ms 39 3A/3B/3C/MS39/51/55/53 Ps 51 3B/3C/MS39/51 Ps 523 Ms39/51 Ps 42C 3B/55 Ps 29A 3B/3C/55 Ps 6 6/7/83/47/53/54/75/77 Ps 7 7/42B/42E/47 4S 7/83/47/53/54/75/77 Ps 70 6/7/42B/42E/47/47B/47C/53/54/70/77 Ps 42E 42B/42E

*Phage 44A not used in routine determinations of phage patterns. for use in the selective medium. A concentration which inhibited the growth of spontaneous mutants present in cell populations of sensitive strains, and yet which did not significantly depress the frequency of transduction, was considered as optimum.

A series of plates was prepared in duplicate con­ taining chlortetracycllne at concentrations varying from 0.5 to 15.0 ug per ml of BHI agar. One series of plates was then inoculated with a control suspension of recipient strain 152. The remaining series of plates was inoculated with a suspension of recipient strain 152 which had been transduced with phage 80/U9(Pase,Tet,Ery). The plates were then examined after incubation at 37 C for 24 hours for the numbers of colonies occurring at each antibiotic concentration. The results of this experiment are given in table 6.

Table 6

Effect of concentration of chlortetracycllne in the selec­ tive medium on the frequency of transduction using phage 80/U9(Pase,Tet,Ery) and recipient strain 152 ug chlortetra- Numbers of colonies recovered per 10$ cells cycline per ml BHI agar Control suspension Transduced suspension

0.5 TMTC* TMTC 1.0 0 3800 3.0 0 4000 5.0 0 2800 10.0 0 640 15.0 0 150

#Too many colonies to count.

After evaluating the results of this experiment,

a concentration of 3.0 ug of chlortetracycllne per ml of

BHI agar was adopted for the detection of transductants

resistant to chlortetracycllne. A series of 14 recipient

strains was then transduced with phage 80/U9(Pase,Tet,Ery), 55 and the chlortetracyeline-resistant transductants selected.

Control suspensions of each recipient strain were also inoculated onto plates of the selective medium. The results of these transductions are given in table 7. An

Table 7

Frequency of transduction of chlortetracycllne resistance by phage 80/U9(Pase,Tet,Ery)

Recipient strain Frequency of transduction of chlortetracycllne resistance

Ps 42B 1000 112 9000 248 1000 N 135 8500 1 3000 152 8000 Ch 50 6000 W 26 8200 C 72 7800 M-1 34 769 8000 608 8000 Ps 52 2000 Ps 29 8000

inspection of the results of this experiment indicated that the frequency of transduction of chlortetracycllne re­ sistance was approximately ten-fold higher than the trans­ duction frequencies previously observed for the Pase marker. The results also indicated-that the concentration of chlortetracycllne in the selective medium was suitable for use with a large number of recipient strains, as the growth of all control suspensions was inhibited. 56

Transduction of Novobiocin Resistance

As all of the available strains were sensitive to novobiocin, it was necessary to select novobiocin-resistant mutants from sensitive strains for use as donors of novo­ biocin resistance. The procedure used to obtain these strains has been described in a previous section.

A series of plates was prepared in duplicate containing from 0.4 to 10.0 ug of novobiocin per ml of

BHI agar. One series of plates was then inoculated with a control suspension of recipient strain Ch 50. The other series of plates was Inoculated with a suspension of recipient strain Ch 50 which had been transduced with phage 80/U9(Pase,Tet,01,Nov). The plates were examined after Incubation at 37 C for 24 hours, and the number of

colonies on each plate determined. The results of this experiment, designed to determine the optimal concentration of novobiocin for use in the selective medium, are given in table 8. From the results of this experiment, a

concentration of 10.0 ug of novobiocin per ml of BHI agar was adopted for use in the selective medium for the Nov marker. A series of 14 recipient strains was then trans­ duced with phage 80/U9(Pase,Tet,01,Nov), and the novobiocin-

resistant transductants selected. Control suspensions of

each recipient strain were also Inoculated onto plates

of the selective medium. The results of this experiment

are given in table 9. An examination of the results of 57

Table 8

Effect of concentration of novobiocin in the selective medi­ um on the frequency of transduction using phage 80/U9(Pase, Tet,01,Nov) and recipient strain Ch 50 ug novobiocin Number of colonies recovered per 10-" cells per ml of BHI ------agar Control suspension Transduced suspension

0.4 160 620 3.0 26 550 5.0 10 300 7.0 0 280 10.0 0 230

Table 9

Frequency of transduction of novobiocin resistance by phage 80/U9(Pase,Tet,01,Nov)

Recipient strain Frequency of transduction of novobiocin resistance

Ps 42B 10 112 2000 248 600 N 135 350 1 400 152 1000 Ch 50 400 W 26 1300 C 72 340 M-1 8 769 900 608 770 Ps 52 360 Ps 29 400 Table 10

Effect of concentration of oleandomycin in the selective medium on the frequency of transduction using phage 80/U9(Pase,Tet,01,Nov) and recipient strain 112

o ug oleandomycin Number of colonies recovered per 10'' cells per ml of BHI ------agar Control suspension Transduced suspension

3.0 22 1200 5.0 20 1000 7.0 0 1000 9.0 0 1000 12.0 0 1500 25.0 0 1000

selective medium. A concentration of 25 ug of oleandomycin

per ml of BHI agar was adopted for routine use. Phage

80/U9(Pase,Tet,01,Nov) was then employed to transduce a

series of 14 recipient strains and the transductants

resistant to oleandomycin were selected. Control sus­

pensions of each recipient strain were also inoculated

onto plates of the selective medium. The results of

these transductions are given in table 11. It was found

that all control suspensions were inhibited by the con-,

centratlon of oleandomycin employed in the selective

medium. An examination of the results of this experiment

Indicated that only recipient strains 112, 152, 248, and

D-1 were transduced at relatively high frequencies with

the 01 marker. An experiment was performed to determine

whether the low rates of transduction were due to the this experiment revealed that novobiocin resistance was successfully transduced to the 14 recipient strains using phage 80/U9(Pase,Tet,01, Nov). All but strains M-1 and

Ps 42B were transduced at relatively high frequencies.

In each experiment, the concentration of novobiocin used for the selection of the transductants was capable of inhibiting the growth of the control suspensions of the recipient strains.

Transduction of Oleandomycin Resistance

The strains employed as donors of oleandomycin

resistance have been previously described in detail. In

order to determine the optimum concentration of oleando­

mycin to be used in the selective medium, a series of plates was prepared in duplicate containing from 3.0 to

25.0 ug of oleandomycin per ml of BHI agar. One series

of plates was inoculated with a control suspension of

recipient strain 112. The remaining set of plates was

inoculated with a suspension of recipient strain 112

which had been transduced with phage 80/U9(Pase,Tet,01,

Nov). All plates then were incubated at 37 C for 24 hours,

after which the number of colonies on each plate was

determined. The results of this experiment are given In

table 10. An examination of the results of this experi­

ment Indicated that any concentration of oleandomycin

from 7.0 to 25.0 ug per ml was suitable for use in the 60

Table 11

Frequency of transduction of oleandomycin resistance by phage 80/U9(Pase,Tet,01,Nov)

Recipient strain Frequency of transduction of oleandomycin resistance

Ps 42B 0 Ps 29 0 Ps 52 32 C 72 12 Ch 50 0 W 26 6 N 135 4 608 6 1 2 769 14 D-1 3000 152 3000 112 2000 248 800

concentration of oleandomycin employed in the selective medium. The results of this experiment, which are shown in table 12, Indicated that the frequency of transduction was not appreciably Influenced by the concentration of oleandomycin employed in the selective medium. In experi­ ments to be described in subsequent sections, the frequency of transduction of erythromycin resistance was found to vary considerably, and was found to be relatively high only with strains 112, 152, 248, and D-1.

In view of the resistance of strain U9(Pase,Tet,01,

Nov) to oleandomycin, erythromycin, spiramycin, and

carbomycin, phage 80/U9(Pase,Tet,01,Nov) was used to 61

Table 12

Effect of concentration of oleandomycin employed in the se­ lective medium on the frequency of transduction using recipient strain 1 and phage 80/U9(Pase,Tet,01,Nov) ug oleandomycin Number of colonies recovered per 10^ cells per ml BHI agar Control suspension Transduced suspension

3.0 6 19 5.0 4 19 7.0 4 18 9.0 0 12 12.0 0 14 15.0 0 8 25.0 0 5

transduce recipient strain 152. The transduced suspension was then inoculated onto plates of selective media, each plate containing 25 ug of oleandomycin, erythromycin,

spiramycin, or carbomycin per ml of BHI agar. The results of this experiment are given in table 13. It was found that the frequency of transduction varied only about three-fold by substituting another macrolide antibiotic

for oleandomycin in the selective medium. 'When the trans­ ductants were examined, they were found to be equally

resistant to 100 ug or more of each macrolide antibiotic per ml of BHI agar regardless of which macrolide anti­ biotic had been employed in the selective medium.

Phage 80/U9(Pase,Tet,01,Nov) was employed to trans­

duce recipient strain 152. Transductants resistant to

oleandomycin were selected by inoculating the transduction 62

Table 13

Effect of macrolide antibiotics in the selective medium on the frequency of transduction of oleandomycin resistance using recipient strain 152 transduced with phage 80/U9(Pase,Tet,01,Nov)

Antibiotic employed in selective Frequency of transduction medium (25 ug per ml BHI agar) of oleandomycin resistance

Oleandomycin 6200

Erythromycin 3800

Spiramycin 1900

Carbomycin 2500

suspension onto BHI agar containing from 25 to 100 ug of oleandomycin per ml and onto BHI agar containing from 100 to 800 ug of both oleandomycin and erythromycin per ml.

The transduction frequency of the 01 marker was found to be the same when the transduced clones were isolated on selective media containing these concentrations and combinations of antibiotics.

Transduction of Erythromycin Resistance

Strain U9(Pase,Tet,Ery), which is naturally re­ sistant to 800 ug of erythromycin per ml of BHI agar, was used as the donor of the Ery marker in these experiments.

A series of plates was prepared in duplicate containing from 0.5 to 10.0 ug of erythromycin per ml of BHI agar.

A control suspension of strain 112 was inoculated onto 63 one series of these plates. The remaining series was inoculated with a suspension of recipient strain 112 which had been transduced with phage 80/U9(Pase,Tet,Ery). All plates were then incubated at 37 C for 24 hours, after which the number of colonies on each plate was determined.

The results of this experiment are presented in table 14.

In the selection of an appropriate concentration of

Table 14

Effect of concentration of erythromycin in the selective medium on the frequency of transduction using phage 80/U9(Pase,Tet,Ery) and recipient strain 112 ug erythromycin Number of colonies recovered per 10^ cells per ml of BHI agar Control suspension Transduced suspension

0.5 200 1000 1.0 40 6 0 0 2.0 20 5 0 4.0 10 20 6.0 4 4 8.0 0 0 10.0 0 0

erythromycin for use in the selective medium, an attempt was made to use a concentration which inhibited the growth of spontaneous mutants present in control suspensions and still allowed the fullest possible expression of the transductants present in the transduced suspensions. The results of the foregoing experiment indicated clearly that this objective was not obtainable by inoculating the transduced suspensions directly onto a selective medium.

Two observations, however, indicated that erythromycin- resistant transductants were present in the transduced suspensions. First, at 0.5 and 1.0 ug of erythromycin per ml of BHI agar, the transductant populations yielded approximately ten-fold more colonies than did the control suspensions. Second, from plates inoculated with the transduced suspension colonies were obtained which grew well on BHI containing 50 ug of erythromycin per ml, while all colonies derived from the control suspension were sensitive to this concentration of erythromycin.

The agar transfer technique was developed for use in the selection of erythromycin-resistant transductants, and has been described in detail in an earlier section.

Six plates were prepared which contained 25 ug of erythro­ mycin per ml of BHI agar. Six plates of BHI agar (two per cent agar) were also prepared which did not contain erythromycin. A control suspension of recipient strain

112 was inoculated onto three of the BHI agar plates.

Onto the remaining three plates of BHI agar, a suspension of strain 112 which had been transduced with phage 80/U9

(Pase,Tet,Ery) was inoculated. All of the plates were

then Incubated at 37 C. At hourly Intervals for three hours after inoculation, the agar from one plate inocu­

lated with each cell suspension was removed and placed,

inoculum side up, onto the surface of a plate containing 25 ug of erythromycin per ml of BHI agar. Incubation of

the plates was then continued for a total of approximately

24 hours, at which time the total number of colonies on

each plate was determined. The results of this experi­

ment Indicated that a relatively high transduction

frequency was obtained by the use of this technique, while

the growth of control suspensions was completely inhibited.

It was also observed that a small increase In the frequency

of transduction of the Ery marker resulted when the

antibiotic-free agar layer was incubated for one hour before transferring it onto the medium containing erythro­

mycin. The incubation of the antibiotic-free layer could

not be continued beyond two hours as the growth of the

Inoculum became excessive. When the agar transfer tech­

nique was employed using lower concentrations of erythro­

mycin in the base layer, no significant increase in the

transduction frequency was observed. When the concen­

tration of erythromycin in the base layer was increased

to 50 or 100 ug per ml of BHI agar, the frequency of

transduction of the Ery marker was reduced drastically.

Consequently, a concentration of 25 ug of erythromycin

per ml of BHI agar was adopted for routine use In the

base layer.

Phage 80/U9(Pase,Tet,Ery) was then employed to

transduce a series of 14 recipient strains, and the Ery

transductants were selected by the agar transfer tech- 66 nique. The results of this experiment are given in table

15. An examination of the results of this experiment

Table 15

Frequency of transduction of erythromycin resistance by phage 80/U9(Pase,Tet,Ery) using the agar transfer technique

Recipient strain Frequency of transduction of erythromycin resistance

Ps 42B 0 Ps 29 0 Ps 52 50 c 72 4 Ch 50 6 W 26 10 N 135 6 608 6 1 6 769 20 D-1 800 152 1000 112 1200 248 1000

revealed that a relatively high frequency of transduction

was obtained only with recipient strains D-1, 248, 112,

and 152. It was pointed out in an earlier section that

these four strains appear to have been unique among the

14 strains tested in being transduced at relatively high

frequencies with the 01 and Ery markers. It was also

noted during this series of transductions that the control

suspensions were completely inhibited by using 25 ug of

erythromycin per ml of BHI agar in the base layer. The 6? agar transfer technique was employed routinely for the isolation of transductants resistant to erythromycin in all of the following studies unless otherwise indicated.

Effect of Donor Strains and Transducing Phages on the Transduction Frequencies of the 01 and Ery markers

In view of the marked difference in the frequencies of transduction of the 01 and Ery markers to the recipient

strains, further studies were performed using a variety of donor and recipient strains. Phage 80/U9(Pase,Tet,Ery) was used to transduce five recipient strains which previ­

ously had not been examined for their ability to be

transduced by the Ery marker. Seven "epidemic" strains

which were sensitive to erythromycin were also transduced,

as was strain 4865(Pase) from which one of the principal

donor strains was derived. The results of these trans­

ductions, and the phage types of the recipient strains,

are Indicated In table 16. Among the strains which were

tested, only "epidemic" strains were capable of relatively

high frequencies of transduction of the Ery marker using

phage 80/U9(Pase,Tet,Ery). The remaining strains tested,

including strain 4865(Pase), were only capable of a

relatively low frequency of transduction with this marker

when transduced with phage 80/U9(Pase,Tet,Ery). Strain

569 was not transduced, at least not at a frequency

detectable by the methods employed. 68

Table 16

Phage type and frequency of transduction of erythromycin resistance of strains transduced by phage 80/U9(Pase,Tet,Ery)

Strain Phage type Frequency of transduction of designation erythromycin resistance

N 203 29 52 569 52A/80 0 720 52A/80 28 616 52A/80/81 4 456 52A/80/81 12 4 8 6 5 (Pase) 29/52A/80 72 U35(Pase,Tet) 80/81 700 U40(Pase,Tet) 80/81 520 U61(Pase,Tet) 80/81 620 588(Pase,Tet) 80/81 360 4l6(Pase,Tet) 80/81 720 U1l4(Pase,Tet) 80/81 320 U91(Pase,Tet) 80/81 820

Three strains which were naturally erythromycin-

resistant were then employed as donor strains, using

a variety of transducing phages. These strains, and their phage types, are as follows;

Designation Phage type

6 5 5 (Pase, T e t ,Er y ) 29/52A/79/7/83/47/53/54/73/77 U66(Pase,Tet,Ery) 80/81 4A(Pase,Tet,Ery) 79/83/53/54/77

Two recipient strains were transduced with several phages

propagated on these strains, and erythromycin-resistant

and chlortetracycline-resistant transductants were

selected In each instance. One recipient strain, C 72,

had been found to be a relatively low frequency recipient 69 of oleandomycin resistance when transduced with phage

80/U9(Pase,Tet,01,Nov), and of erythromycin resistance when transduced with phage 80/U9(Pase,Tet,Ery). The other recipient strain, 152, was a relatively high fre­ quency recipient of the Ery and 01 markers from these two phages. The frequency of transduction of the Ery and Tet markers, as well as the transducing phages employed, are indicated in table 17. The results of this experiment indicated that the transduction frequencies of erythromycin

Table 17

Frequency of transduction of the Ery and Tet markers to strains C 72 and 152 transduced by different phage preparations

Frequency of transduction of the Tet and Ery markers using recipi- ent strains;______Transducing phage C 72 152 Tet Ery Tet Ery

53/655(Pase,Tet,Ery) 560 0 190 500 52A/655(Pase,Tet,Ery) 4000 0 4000 600 79/655(Pase,Tet.Ery) 40 0 60 400 29/655(Pase,Tet,Ery) 6000 0 8000 1000 80/U66(Pase,Tet,Ery) 5000 2 1000 600 53/4A( Pase, Tet, Ery) 500 28 330 600 79/4A( Pase, Tet, Ery) 400 0 140 200

resistance to the^e two recipient strains were not altered by the donor strain employed. The relative competence of the transducing phage is indicated by the transduction frequencies of the Tet marker. All of the donor strains 70 which were used In this experiment were Isolated from natural sources as erythromycin-resistant strains.

Recipient strains 1 and 112 were then Individually transduced with phages 80/U9(Pase,Tet,Ery) and 80/U9(Pase,

Tet,01,Nov). Oleandomycin-resistant and erythromycin- resistant transductants were selected from the appropriate transduced suspensions. One erythromycin-resistant trans- duct ant derived from each recipient strain transduced with phage 80/U9(Pase,Tet,Ery) was then purified by streaking for isolation onto a BHI agar plate containing 100 ug of erythromycin per ml. Similarly, an oleandomycin-resistant transductant from each strain transduced with phage

80/U9(Pase,Tet,01,Nov) was purified by streaking onto a

BHI agar plate containing 100 ug of oleandomycin per ml.

Phage 80 was then propagated on each of the four trans­ ductants, and the resulting phage preparations employed to transduce recipient strains 112 and 1. From the trans­ duced suspensions obtained by transducing strains 112 and

1 with phages 80/l(Ery) and 80/l12(Ery), the erythromycin- resistant transductants were assayed. The results of the transductions of the Ery marker are indicated in table 18.

The results of transducing strains 1 and 112 with phages

80/1(01) and 80/112(01) are indicated in table 19. The results of these transductions revealed that the 01 and

Ery markers behaved In a similar manner. An oleandomycin or erythromycin-resistant transductant derived from strain 71

Table 18

Frequency of transduction of the Ery marker to strains 112 and 1 transduced by phages 80/"f'(Ery) and 80/112(Ery)

Frequency of transduction of the Ery marker toj Transducing phage ----■------*---- Strain 112 Strain 1

8 0 / 1 (Ery) 800 950

80/112(Ery) 700 22

112, a relatively high frequency recipient, served as a donor of the marker to the recipient strains at frequencies similar to those observed with phages 80/U9(Pase,Tet,Ery) and 80/U9(Pase,Tet,01,Nov). However, an oleandomycin or erythromycin-resistant transductant derived from strain 1, a relatively low frequency recipient, served as a donor of

Table 19

Frequency of transduction of the 01 marker to strains 112 and 1 transduced by phages 807T(01) and 80/112(01)

Frequency of transduction of the 01 marker tos Transducing phage Strain 112 Strain 1

80/1(01) 1100 1700

80/112(01) 1000 70 72

the marker to the recipient strains at relatively high

frequencies.

Donor strain 4865(Pase,Tet,01,Nov) was derived from

strain 4865(Pase). The latter strain was found to be a relatively low frequency recipient of the Ery marker

(table 16). However, as table 41 Indicates, the recipient

strains were all transduced at relatively high frequencies

to oleandomycin resistance by phage 80/4865(Pase,Tet,01,

Nov).

Transduction of Erythromycin Resistance from Donor Strain U9(Pase,Tet,Ery-X)

The agar transfer technique, developed for use in

the transduction of erythromycin resistance from donor

strain U9(Pase,Tet,Ery), was also employed for the trans­ duction of erythromycin resistance from donor strain

U9(Pase,Tet,Ery-X). Phage 80/U9(Pase,Tet,Ery-X) was

employed to transduce 14 recipient strains, and the

erythromycin-resistant transductants were isolated from

each transduced suspension by the agar transfer technique.

Control suspensions of each strain were also employed in

this experiment. Table 20 Indicates the frequencies of

transduction of erythromycin resistance observed in this

experiment. The results of this experiment indicated that

the frequencies of transduction of erythromycin resistance

to the recipient strains were relatively high only with

recipient strains D-1, 112, 152, and 248. These were the 73

Table 20

Frequency of transduction of erythromycin resistance by phage 80/U9(Pase, Tet,Ery-X)

Recipient strain Frequency of transduction of erythromycin resistance

Ps 42B 0 Ps 29 0 Ps 52 14 C 72 2 Ch 50 2 W 26 8 N 135 6 608 4 1 12 769 38 D-1 270 152 760 112 430 248 560

only strains among those tested which were relatively high frequency recipients of the 01 and Ery markers.

Donor strain U9(Pase,Tet,Ery-X) was resistant not only to erythromycin, but also to oleandomycin, spiramycin, and carbomycin. Therefore, transductants derived from this donor strain were examined for resistance to the related macrollde antibiotics. Strains 112, 152, D-1,

248, W 26, and 1 were transduced with phage 80/U9(Pase,

Tet,Ery-X) and the transductants resistant to erythromycin selected. Quadruplicate platings were made from the trans­ duced suspensions of strains W 26 and 1 in order to obtain a sufficient number of transductants. A number of trans- duetants were picked from each transductant population and

inoculated at spaced intervals on the surface of BHI agar plates (master plates) free of erythromycin. The master plates were used in order to obtain clearly separated transductant clones for replication, and also to avoid induction of resistance to the other macrolide antibiotics by growing the strains on a medium containing erythro­ mycin. After incubation of the master plates for 24 hours at 37 C, each population was replicated onto plates of BHI agar containing 100 ug of erythromycin, oleando­ mycin, spiramycin, or carbomycin per ml. The plates

containing erythromycin were Inoculated last in order to

avoid possible Induction of cross resistance by trace

amounts of erythromycin on the template. After incubation

of the replica plates for 24 hours at 37 C, it was found

that approximately 85 per cent of the erythromycin-

resistant transductants were sensitive to oleandomycin,

spiramycin, and carbomycin, while approximately 15 per

cent of the transduced clones were resistant to all four

antibiotics. Table 21 indicates the number of erythromycin-

resistant clones examined from each recipient strain, and

the percentage of the erythromycin-resistant transductants which were also resistant to oleandomycin, spiramycin, and

carbomycin. The results of this experiment indicated that

the per cent of erythromycin-resistant transductants

which were resistant to the other macrolide drugs did not Table 21

Percentage of erythromycin-resistant transductants resistant to 100 ug per ml of oleandomycin, spiramycin, and carbomycin

Percentage of trans­ Recipient Erythro mycln-resistant ductants resistant to strain transductants tested oleandomycin, spiramycin and carbomycin

1 36 17

W 26 30 13

D-1 100 15

152 100 13

112 100 13

248 100 17

vary significantly, regardless of recipient strain from which they were derived.

The observations of cross resistance among the transductants were suggestive of a chromosomal linkage between a factor controlling resistance to erythromycin

(Ery*) and a factor controlling resistance to spiramycin, oleandomycin, and carbomycin (X). To test this hypothesis, an erythromycin-resistant transductant (Ery1) and an erythromycin, oleandomycin, spiramycin, carbomycin- reslstant transductant (Ery-X) were obtained by trans­ ducing strains 1 and 152 with phage 80/U9(Pase,Tet,Ery-X).

After purification by streaking onto BHI agar containing 100 ug erythromycin per ml, these transductants were used as donors of erythromycin resistance to recipient strains

1 and 152. Phages 80/1(Ery'), 80/1(Ery-X), 80/l52(Ery•), and 80/152(Ery-X) were used to transduce recipient strains

1 and 152. From the transduced suspensions the erythro­ mycin-resistant clones were isolated by the agar transfer technique. Master plates were prepared from the transduced suspensions of each recipient, which after incubation were replicated onto BHI agar plates containing 100 ug per ml of oleandomycin, spiramycin, carbomycin, or erythromycin.

After Incubation of the replica plates, it was observed that the transductant populations obtained by transduction with phages 80/1(Ery-X) and 80/l52(Ery-X) were uniformly resistant to all four antibiotics. The erythromycin- resistant populations obtained by transduction with phages

80/1(Ery') and 80/l52(Ery’) were resistant only to erythro­ mycin.

The experiment described above was then repeated with erythromycin-resistant transductants obtained by transducing strain U40(Pase,Tet) with phage 80/U9(Pase,

Tet,Ery-X). An Ery1 and an Ery-X transductant were puri­ fied and used as donors to transduce strain U40(Pase,Tet).

When the erythromycin-resistant clones were examined, those obtained by transduction with phage 80/U40(Pase,

Tet,Ery’) were uniformly sensitive to oleandomycin, spiramycin, and carbomycin, while the erythromycin- 77 resistant clones obtained by transduction with phage

8 0 /u 4 0Pase,Tet,Ery-X) ( were completely resistant to all four macrolide antibiotics.

Finally, a variety of techniques were used in an attempt to isolate spiramycin, carbomycin, or oleandomycin- resistant transductants directly from the transduced suspension by using phage 80/U9(Pase,Tet,Ery-X) as the transducing phage. However, In all Instances it was possible to isolate only the erythromycin-resistant transductants from these suspensions.

Transduction of Erythromycin Resistance from Mutants Isolated In Vitro

In an attempt to extend further the studies con­

cerned with erythromycin resistance, the gradient plate technique was employed to Independently isolate erythromycin and oleandomycin-resistant mutants from strains 1 and U40

(Pase,Tet). In each Instance, mutants were obtained

through a series of three to five distinct steps which were

resistant to 400 ug per ml of erythromycin, oleandomycin,

spiramycin, and carbomycin.

Phage 80 was propagated on each mutant, and the

resulting lysates employed to transduce strains 112, 152,

1, and U40(Pase,Tet). Transduced and control suspensions

of each strain were plated by the agar transfer technique

and by direct plating onto BHI agar containing 25 ug of

erythromycin or oleandomycin per ml. In no instance 78 was there an indication that these donor strains were able to transduce resistance to oleandomycin or erythromycin.

These erythromycin and oleandomycin-resistant mutants of strain U40(Pase,Tet) were capable of serving as donors of the Pase and Tet markers, however, indicating that donor competence per se was not lost during the mutations to antibiotic resistance.

Comparative Viable Cell Counts of Erythromycin-Resistant Strains

In view of the unexpected nature of the erythro­ mycin-resistant transductants derived from donor strain

U9(Pase,Tet,Ery-X), viable cell counts were performed using strains U9(Pase,Tet,Ery), U9(Pase,Tet,Ery-X),

U9(Pase,Tet,01,Nov) and U40(Pase,Tet). The viable cell counts were made by inoculating dilutions of the cultures onto BHI agar with and without added erythromycin, oleandomycin, or spiramycin at a concentration of 25 ug per ml of medium. The results of these counts are given in table 22.

Strain U9(Pase,Tet,Ery) was found to yield approximately 0.3 per cent of the total viable cells on

BHI agar containing erythromycin. This is in agreement with similar findings reported by Garrod (1957). The percentage of total viable cells of strain U9(Pase,Tet,

Ery) capable of growth on BHI agar containing oleando­ mycin or spiramycin was extremely low, suggesting that Table 22

Viable cell counts of strains U9(Pase,Tet,Ery), U9(Pase,Tet, Ery-X), U9(Pase,Tet,01,Nov) and U40(Pase,Tet), as determined on BHI agar with and without added oleandomycin, spiramycin, or erythromycin

Viable cells per ml determined on BHI agar containing 25 ug per ml ofs Donor strain - Erythro­ Oleando­ Spiramycin mycin mycin

U 9 (Pa s e ,Te t ,E r y ) 1.7x1010 6.0x10? 1.OxIO2 2.0x102

U9(Pase.Tet, Ery-X) 2.0x1010 1.8x1010 1.0x105 2.0x102

U9(Pase,Tet, 01,Nov) 2 .3 x 1 0 10 1.8x10*° 2.0x1010 1.9x10*°

U40(Pase,Tet) 2.2x10*° 0 0 0

the colonies observed on these media represented mutant cells In the population.

Strain U9(Pase,Tet,Ery-X) was found to yield similar counts on BHI agar and on BHI agar which contained erythromycin. The cell counts obtained on media containing oleandomycin were found to be a small per cent of the total count, suggesting that In strain U9(Pase,Tet,Ery-X) oleandomycin resistance was similar to the erythromycin resistance of strain U9(Pase,Tet,Ery). Strain U9(Pase,

Tet,Ery-X) yielded extremely low counts on media con­ taining spiramycin.

Strain U9(Pase,Tet,01,Nov) was found to consist only of resistant cells, all of which grew readily in 80 the presence of each antibiotic employed. Strain U40(Pase,

Tet), which resembled strain U9(Pase,Tet,Ery) except for the erythromycin resistance of the latter strain, failed to yield colonies when inoculated onto BHI agar containing any of the three antibiotics.

To determine whether the observations concerning viable cell counts were due to the Ery-X marker itself, phage 80/U9(Pase,Tet,Ery-X) was employed to transduce

strain U4o(Pase,Tet). The erythromycin-resistant clones were isolated by the agar transfer technique, and after

subculturing the clones on a master plate they were replicated onto BHI agar plates containing 25 ug of erythro­ mycin, oleandomycin, spiramycin, or carbomycin. When it was determined which clones on the master plate were

resistant only to erythromycin (Ery *) and which were

resistant to all four antibiotics (Ery-X), two colonies

of each type from the master plate were suspended in

saline, and viable cell counts made as before. The results

of this experiment are given in table 23. An examination

of the cell counts indicated that the two Ery * trans­

ductants yielded only about one per cent of the total

viable cells when plated on media containing erythromycin.

The two Ery1 transductants were also essentially sensitive

to oleandomycin and spiramycin. In comparison, the Ery-X

transductants yielded similar cell counts on BHI agar and

BHI agar containing erythromycin or oleandomycin. The 81

Table 23

Viable cell counts of transductant clones of U40(Pase,Tet, Ery') and U40(Pase,Tet,Ery-X) obtained by transduction of strain U40(Pase,Tet) with phage 80/U9(Pase,Tet,Ery-X)

Viable cells determined on BHI Transductant agar containing 25 ug per ml of: designation - Erythro­• Oleando­ Spira­ mycin mycin mycin

U40(Pase,Tet,Ery1) 1.4x109 6.4x106 0 0

U40(Pase,Tet,Ery*) 2.4x109 3.7x107 4.0x101 0

U40(Pase,Tet,Ery-X) 1,2x109 8.0x108 6.0x10S 3.0x102

U40 (Pas e, Te t, Ery-X) 1,7x109 1.2x109 7.1x10S 4.0x102

counts of the Ery-X transductants on media containing spiramycin were much lower than the total cell counts determined on BHI agar.

Despite the differences in the percentage of total viable cells which were obtained on BHI agar containing erythromycin, strains U9(Pase,Tet,Ery) and U9(Pase,Tet,

Ery-X) were found to transduce erythromycin resistance at nearly identical frequencies. The transduction frequencies observed with phages propagated on both of these strains are given in table 24. Table 24

Frequency of transduction of erythromycin resistance to recipient strain 152 by phages 80/U9(Pase,Tet,Ery) and 80/U9(Pase,Tet,Ery-X)

Transducing phage Frequency of transduction of erythromycin resistance

80/U9(Pase,Tet,Ery) 450

80/U9(Pase,Tet,Ery-X) 500

Induction of Cross Resistance in Erythromycin-Resistant Strains by Growth in the Presence of Erythromycin

The transduction of erythromycin resistance was done primarily with strains U9(Pase,Tet,Ery) and U9(Pase,

Tet,Ery-X). As previously described, the Ery marker conferred resistance to erythromycin but not to oleando­ mycin, spiramycin, or carbomycin, while the Er.y-X marker conferred resistance to erythromycin, oleandomycin, spiramycin, and carbomycin. In view of Garrod’s obser­ vations (1957) concerning the "dissociated" type of erythromycin resistance, both of the aforementioned strains were examined to determine the effect of growth In the presence of erythromycin on the pattern of drug resistance.

Strains U9(Pase,Tet,Ery) and U9(Pase,Tet,Ery-X) were inoculated from BHI agar onto the surface of BHI agar plates containing 100 ug of erythromycin per ml. The plates were then Incubated at 37 C and observed at Inter­

vals. Both strains Initiated growth in the presence of

erythromycin only after an initial lag period of several hours duration when compared with the same strains inocu­ lated onto BHI agar plates containing no antibiotic.

After incubation for 24 hours, however, both strains had grown well. Strain U9(Pase,Tet,Ery-X) was observed to undergo this same lag period preceeding complete growth when inoculated from BHI agar onto BHI agar plates con­

taining 100 ug per ml of oleandomycin, spiramycin, or

carbomycin. Strain U9(Pase,Tet,Ery) was incapable of

growing on BHI agar containing 100 ug per ml of oleando­

mycin, spiramycin, or carbomycin. When the above pro­

cedure was repeated with the plates containing the anti­

biotics inoculated from cultures prepared on BHI agar

containing 10 ug of erythromycin per ml, the results were

strikingly different. Both strains initiated growth on

BHI agar plates containing 100 ug per ml of erythromycin,

oleandomycin, spiramycin, or carbomycin without any

detectable lag period. Strain U9(Pase,Tet,Ery), moreover,

grew equally well on media containing any of the four

antibiotics. Therefore, when an erythromycin-resistant

strain was cultivated on a medium containing erythromycin,

it was Induced and was capable of growth on a medium con­

taining normally inhibitory concentrations of the related

macrolide antibiotics. In addition, growth on a medium 84 containing erythromycin induced the strain and no lag period in growth occurred on media containing any of the four macrolide antibiotics. When an induced strain was cultured on a medium containing no antibiotic and then examined, it was found that the effects of induction were no longer detectable, the process being a temporary modification.

Strains U9(Pase,Tet,01,Nov), U9(Pase,Tet,Ery),

U9(Pase,Tet,Ery-X), and eight other naturally erythro­ mycin-resistant strains were Inoculated from BHI agar ont.o BHI agar plates containing 100 ug of oleandomycin, spiramycin, or carbomycin per ml. A filter paper disc containing two ug of erythromycin was then placed on the surface of each plate and the plates incubated at 37 C.

After incubation for eight hours, all strains except

U9(Pase,Tet,01,Nov) were found to have initiated growth only at the periphery of the disc. Strain U9(Pase,Tet,01,

Nov) initiated growth over the entire agar surface and appeared to have grown more rapidly than the other strains.

When the plates were examined after Incubation for 24 hours, strains U9(Pase,Tet,01,Nov) and U9(Pase,Tet,Ery-X) were found to have grown equally well over the entire surface of each plate. The remaining strains grew only in an area within ten mm of the periphery of the filter paper disc on each medium. It appeared that all strains except U9(Pase,Tet,01,Nov) and U9(Pase,Tet,Ery-X) were 85 of the ’'dissociated" type of erythromycin resistance.

Moreover, strains U9(Pase,Tet,01,Nov) and U9(Pase,Tet,

Ery-X) differed in that the latter was still induced by erythromycin, although on extended incubation the stimu­ latory effect of erythromycin was not in evidence. The phage types of the strains tested in the foregoing experi­ ment are given in table 25. Strains related to U9(Pase,

Tet,Ery) have been omitted from the table as their phage types have been reported elsewhere.

Table 25

Phage type of strains showing the "dissociated" type of erythromycin resistance

Strain designation Phage type

655(Pase,Tet,Ery) 29/52A/79/7/83/47/53/54/73/77 11l8(Pase,Tet,Ery) n.t.# 359(Pase,Tet,Ery) 83/53/54/75/77 U7t(Pase,Tet,Ery) n.t. 1031(Pase,Tet,Ery) n.t. U66(Pase,Tet,Ery) 80/81 4A(Pase,Tet,Ery) 79/83/53/54/77 4S(Tet,Ery) 7/83/42E/47/53/5V73/75/77

♦Strain not typable with phages employed.

As all of the foregoing experiments to determine the induction of resistance were performed with strains which were naturally resistant to erythromycin, tests were performed on a variety of erythromycin-resistant trans­ ductants. The erythromycin resistance of the trans- 86 ductants examined were all found to be inducible, thus resembling the donor strain from which they were derived.

When the agar transfer technique was first developed, it was assumed that this method functioned by allowing time for the expression of the newly acquired Ery characteristic before selective concentrations of the antibiotic were applied to the environment. During the later phases of this study, and with a fuller understanding of the induction phenomenon, some doubt arose as to the true mode of action of the agar transfer technique. Therefore, the following experiment was performed. Strain u40(Pase,

Tet) was employed as the recipient strain, and phage

80/U9(Pase,Tet,Ery) was employed as the transducing phage.

Eight separate transduction suspensions were prepared, and immediately after the phage and cells were combined, graded concentrations of erythromycin were added to the suspensions. The preparations were then shaken and washed in the routine manner. From each transduced sus­ pension, the Ery transductants were assayed by the agar transfer technique and by direct plating onto BHI agar containing 25 ug of erythromycin per ml. The results of the transductions are indicated in table 26. The results of this study indicated that by adding as little as

0.025 ug of erythromycin per ml to the transduction suspensions, the Ery transductants were recovered by 87

Table 26

Effect of erythromycin in the transduction mixture on the frequency of transduction of erythromycin resistance using recipient strain U4o(Pase,Tet) transduced by phage 80/U9(Pase,Tet,Ery)

Method of Frequency of transduction when erythromycin selection of was added at concentrations of! (ug per ml Ery trans­ suspension) ductants 0 .025 .05 .1 .5 .75 1.0 2.0

Agar transfer 350 300 400 400 1000 800 1000 1000

Direct plating* 0 600 800 800 600 400 230 86

*BHI agar containing 25 ug of erythromycin per ml. direct plating. It appears that the agar transfer tech­ nique did not operate primarily as a technique to obtain delayed selection, but served as a means for allowing the transductants to be exposed to gradually increasing

concentrations of erythromycin as the antibiotic diffused upward through the upper layer of agar.

Spiramycin, oleandomycin, and carbomycin were unable to bring about any of the changes previously

described as being due to erythromycin.

Effect of Chlortetracycline on the Frequency of Transduction of Penicillinase Production

A comparison of the transduction frequencies of the

various markers revealed that the frequencies observed

for the Tet marker were the highest, and were on the average ten-fold higher than the transduction frequencies for the

Pase marker. Chlortetracycline has been reported to be a chelating agent (Saz et al., 1956), and chelating agents have been shown to increase the frequency of genetic transfer in bacterial systems (Kirchner et al., 1957).

Therefore, the effect of this antibiotic on the trans­ duction frequency of the Pase marker was determined.

Strain Ch 50 was transduced with phage 80/U9(Pase,Tet,

Ery), and a Tet transductant selected and purified by streaking onto a BHI agar plate containing 50 ug of chlortetracycline per ml. Phage 80/U9(Pase,Tet,Ery) was then employed to transduce strains Ch 50 and Ch 50(Tet).

The penicillinase-producing transductants were selected from each transduced suspension by plating onto BHI agar containing 0.12 units of penicillin per ml, and onto BHI agar containing 0.12 units of penicillin and 3.0 ug of chlortetracycline per ml. The results of this experiment are indicated in table 27. The results of this experi­ ment revealed that the addition of chlortetracycline to the selective medium did not increase the frequency of transduction of the Pase marker. This indicated that neither chelation nor Inhibition of lysis by the trans­ ducing phage was a probable cause of the high frequencies observed for the transductions of the Tet marker. 89

Table 27

Effect of chlortetracycline in the selective medium on the transduction of the Pase marker to strains Ch 50 and Ch 50(Tet) by phage 80/U9(Pase,Tet,Ery)

Frequency of transduction of the Pase marker when the selective medium contained: Recipient strain 0.12 units 0.12 units penicillin and 3.0 penicillin ug chlortetracycline per ml per ml

Ch 50 400 0

Ch 5 0 (Tet) 510 530

Effect of the Medium Used for Selection on the Transduction Frequency

As BHI agar was inhibitory to the lytic activity

of all of the transducing phages, this medium was employed

routinely as the base for the preparation of the selective media. This inhibition of the lytic activity of the

transducing phage was essential in order to recover the

Pase, 01, Nov, Ery and Ery-X transductants when they were"

derived from recipient strains sensitive to the lytic

activity of the transducing phage. Transductants re­

sistant to chlortetracycline were recoverable from phage-

sensitive recipient strains when plated on selective media

prepared with TSA as this antibiotic inhibited the lytic

activity of the phage.

The results of transducing seven recipient strains

with phage 80/U9(Pase,Tet,Ery) and selecting Pase and Tet 90 transductants on BHI agar and on TSA containing the selective concentration of antibiotics are shown in table

28. The phage type of each recipient strain has been

Table 28

Effect of the medium employed for selection on the fre­ quency of transduction of Pase and Tet markers by phage 80/U9(Pase,Tet,Ery)

Frequency of transduction ofs Recipient Phage strain type Pase Tet BHI agar TSA BHI agar TSA

C 72 29/52A/79/80 100 0 4200 6500 W 26 29/80 1100 0 4400 5800 152 52A/80/81 340 0 2200 2200 769 81 270 300 2600 4300 Ps 42B 42B 150 82 1000 1900 Ps 29 29 300 360 2300 2800 N 135 29 . 500 720 5400 3900

included in the table to indicate those strains which were sensitive to lysis by the transducing phage. This experi­ ment clearly demonstrated that those strains which were resistant to lysis by phage 80 were capable of yielding

Pase transductants on TSA or BHI agar, while strains sensitive to lysis by the transducing phage were capable of yielding Pase transductants only on BHI agar. The recovery of Tet transductants from all recipient strains when plated on selective media prepared from BHI agar and

TSA indicated that the chlortetracycline in the selective medium was active in inhibiting the lysis of the trans- ductant clones. This was verified when mixtures of phage

80/U9(Pase,Tet,Ery) and several recipient strains which were sensitive to lysis by the phage, but which were resistant to chlortetracycline, were spread over the surface of TSA plates. A filter paper disc containing

30 ug of chlortetracycline was then placed on the surface of each plate. After Incubation for 24 hours, growth occurring at the periphery of the disc was free of plaques, while the growth occurring at a distance from the disc was liberally contaminated with phage, as indicated by the presence of many plaques.

When the Pase, Ery. Ery-X, and Nov transductants of recipient strains sensitive to lysis by the transducing phage were selected on a medium prepared from TSA, some clones of the transductants were recovered although all were heavily contaminated with the transducing phage as evidenced by their motheaten appearance. The frequency of transduction in each instance was also reduced con­ siderably below that observed on selective media prepared with BHI agar.

Effect of Multiplicity of Infection on the Transduction Frequency

Phage 80/U9(Pase,Tet,01,Nov) was employed to determine the effect of the multiplicity of infection on the frequency of transduction of the Pase. Tet, 01, and

Nov markers. The multiplicity of infection has been defined as the average number of phage particles adsorbed per bacterium. Strain 152, ivhich was sensitive to lysis by the transducing phage, and strain 769, which was re­ sistant to lysis by the transducing phage, were employed in this study. The transduction suspensions were pre­ pared from a phage lysate with a titer of 2.7x10 1® pfu per ml. The recipient cell suspensions were prepared in the routine manner, and diluted in P and D broth to obtain the desired numbers of cells per ml for the preparation of the transduction suspensions. The transduction suspensions were then shaken and washed in the routine manner, and the frequency of transduction for the Pase, Tet, 01, and Nov markers determined. The results of these experiments are represented by those in table 29, which were obtained with recipient strain 152. Similar results were obtained by using recipient strain 769, which was resistant to lysis by the transducing phage. An examination of the results revealed that a multiplicity of infection of approximately 1.0 resulted in the maximum frequency of transduction of each marker. When the multiplicity of

infection exceeded a value of 3.6, the transduction fre­ quency for each marker was markedly reduced. There also

appeared to be a slight decrease in the frequency of

transduction when the multiplicity of infection was lower

than 1.0. Within experimental limitations, the trans­

duction frequencies with both recipient strains were found 93

Table 29

Effect of multiplicity of Infection on the frequency of transduction of the Pase, Tet, 01, and Nov markers to strain 152 transduced by phage 80/U9(Pase,Tet,01,Nov)

Transduction suspension Frequency of transduction ofs

- m o i w w pfu per Cells per Pase Tet 01 Nov ml* ml

2.7x1010 6.3x1°]° 0.4 1040 3000 1500 960 it 2.1x10].° 1.3 2000 5000 2000 1500 it 7.0x10^ 3.6 410 1500 1000 250 it 2.1x10-; 12.8 70 300 160 40 it 1.0x10* 25.7 16 200 50 4 ii 7.0x10° 38.5 9 48 14 5 •

#Plaque forming units. ^Multiplicity of infection. to respond similarly to changes in the multiplicity of

Infection used in the transduction suspensions.

Determination of the Lag Period After Plating Prior to Division of the Transduced Markers

The spreading technique of Newcombe (194-9) was employed in order to determine the approximate length of time which elapsed after plating the transduced suspension until the division of the transduced characteristic. Using a multiplicity of Infection of 0.9, recipient strain 152 was transduced with phage 80/U9(P&se,Tet,01,Nov), and

0.05 ml quantities of a 1 110,000,000 dilution of the transduced suspension spread over the surfaces of eight

BHI agar plates. Five-hundredth ml quantities of a 1?20 94 dilution of the transduced suspension were spread over the surfaces of eight plates of each medium selective for the

Pase, Tet. 01, and Nov markers. All plates were incubated at 37 C for 24 hours. At hourly intervals from one through seven hours, one drop of P and D broth was added to a plate of each selective medium and to one plate of BHI agar, and the surface vigorously rubbed with a sterile bent glass rod. After incubation, the plates were ex­ amined and the numbers of colonies on each plate determined as indicated in table 30. The results of this experiment

Table 30

Growth lag of various transduced populations as determined . by re-spreading plates Inoculated with the transduced suspension

Colonies per plate of BHI agar Time after inoculation selective for: before spreading - Pase Tet 01 Nov

0 hr 21 19 68 42 18

100 1 hr 19 15 46 7 2 hr 70 18 134 55 5

3 hr 183 22 560 42 15

4 hr 560 141 2000 54 21

5 hr 1280 560 6000 55 71

6 hr TMTC TMTC TMTC 53 196

7 hr TMTC TMTC TMTC 400 800 95 indicated that the cells inoculated onto BHI agar and which were assumed to he infected with the transducing phage initiated growth soon after the first hour of incubation.

The transductants selected for chlortetracycline resistance were also observed to initiate growth rapidly after a lag period of approximately two hours. The penicillinase- producing transductants were found to undergo division after a lag of approximately three hours. The Nov marker was not replicated until a lag period of four hours had elapsed, while the 01 marker was not replicated until incubation had continued for six hours.

Effect of Growth in Broth on the Multiplication and Survival of the Transductants

Two observations led to a study of whether the transductants were recoverable from the transduced sus­ pension after dilution and subsequent multiplication of the population in BHI broth. These observations were; the ability to recover erythromycin-resistant transductants only by the agar transfer technique, and the inability to detect streptomycin-resistant transductants even when the lag period prior to expression was compensated for by dilution and incubation of the transduced suspension in

BHI broth.

Phage 80/U9(Pase,Tet,Ery) was employed to transduce strain 152. The transduced suspension was assayed for the Pase. Tet. and Ery markers and for total viable cells.

The transduced suspension was then diluted 1:1000 by inoculating 0.05 ml of the suspension into 50 ml of BHI broth contained in a 100 ml volumetric flask. The contents of the flask were mixed and assayed for total viable cells and for each type of transduetant. The flask was then shaken at 37 C for five hours and samples assayed at intervals for viable cells and transductants. After shaking for five hours, 10 ml of the broth culture was centrifuged to sediment the cells, which were then resus­ pended in 1.0 ml of P and D broth and assayed for total viable cells and transductants. The Pase. Tet. and Ery transductants were assayed routinely. The Ery trans­ ductants were also assayed on BHI agar containing 25 ug of erythromycin per ml. The results of this experiment, shown in table 31, indicated that the transductants were quantitatively recoverable, and also indicated that they actively multiplied. The Ery transductants were not recoverable by plating directly on BHI agar containing erythromycin.

Transduction of Streptomycin Resistance

Donor strains U9(Pase,Tet,Ery), U40(Pase,Tet), and

655(Pase,Tet,Ery) were resistant to 2000 ug of strepto­ mycin per ml of BHI agar. All of the recipient strains were inhibited by approximately 10 ug of streptomycin per 97

Table 31

Multiplication of transductants and total viable cells in BHI broth inoculated with a suspension of strain 152 transduced by phage 80/U9(Pase,Tet,Ery)

Frequency of transduction of: Source and time Viable of incubation cells Pase Tet Erx of sample per ml Agar Direct transfer plating** transduction suspension 4.2x1010 1764 3880 2260 0

0 hr BHI broth* 4.2x107 0 0 2 0

1 hr BHI broth - 0 4 0 0

2 hr BHI broth - 4 6 6 0

3 hr.BHI broth 4.8x10® 8 20 6 0

4 hr BHI broth - 44 220 28 0

5 hr BHI broth 1.7x 109 48 348 46 0

5 hr BHI broth conc. 10x 9.2x 109 642 3800 438 0

♦Represents a 1:1000 dilution of the transduced suspension into 50 ml of BHI broth.

♦♦BHI agar containing 25 ug of erythromycin per ml. ml of BHI agar, although as many as 30 spontaneous streptomycin-resistant mutants per 109 cells were obtained on plates containing either 100 or 1000 ug of strepto­ mycin per ml.

Phage 80 was propagated on donor strains U9(Pase,

Tet,Ery) and U40(Pase,Tet), and phage 53 propagated on 9 8 strain 655(Pase,Tet,Ery). A one-step mutant which was resistant to 2000 ug of streptomycin per ml was isolated from recipient strain 112, and phage 80 also propagated on this mutant strain. The four phage preparations were employed in attempts to transduce streptomycin resistance to recipient strains 112, 152, 29, and 769. In all instances, a control suspension of each recipient strain was also employed. All media employed was incubated for five days at 37 C after inoculation.

When the transduced suspensions and control suspen­ sions were inoculated directly onto BHI agar plates contain­ ing either 100 or 1000 ug of streptomycin per ml, only equal numbers of streptomycin-resistant colonies were 'obtained from the control and transduced cell suspensions. Morse

(1959) and Wanatabe and Wanatabe (1959) have reported a phenomlc lag associated with the expression of strepto­ mycin resistance by the transductants. In view of these reports, a variety of techniques were employed to allow for the phenomic lag preceding expression of resistance.

The agar transfer technique was adapted for use by employing base layers of BHI agar containing either 100 or 1000 ug of streptomycin per ml. When the pre-incubation period prior to agar transfer was extended beyond three hours, the amount of unrestricted growth of the Inoculum was sufficiently heavy to prevent the detection of strepto­ mycin-resistant transductant clones. When the time of 99 transfer was sufficiently early to prevent overgrowth, equal numbers of streptomycin-resistant colonies were obtained from the control and from the transduced cell suspensions.

A variation of the agar transfer technique was also employed. The transduced and control suspensions were spread over the surface of Millipore Filter membranes previously placed on the surface of BHI agar plates.

At intervals from one through five hours, a membrane

Inoculated with each suspension was removed to the surface of a BHI agar plate containing 100 ug of streptomycin per ml. Although control experiments indicated that the

Pase. Tet, 01, and Nov markers were quantitatively re­ covered by this technique, streptomycin-resistant trans­ ductants were not detected.

Finally, the transduced and control suspensions were diluted 1:1000 in 50 ml quantities of BHI broth, which were then shaken at 37 C for 14 hours. Strepto­ mycin-resistant cells were assayed at intervals on BHI agar plateB containing 1000 ug of streptomycin per ml.

When assayed at intervals for 14 hours, Pase, Tet, and

Ery transductants were recovered quantitatively by this method, and were found to Initiate growth shortly after inoculation Into broth (table 31). The streptomycin- resistant colonies were recovered In only equal numbers from both the control and transduced suspensions, however. 100

Characteristics of the Transductants

Stability of phage type. Ritz (1957) studied the effect of transduction on the phage types of the recipient strains. While some changes were observed, in general, transduction did not significantly alter the phage type.

Phage 80/U9(Pase,Tet,Ery) was employed to transduce 19 recipient strains, and the penicillinase-producing trans­ ductants were selected. A single Pase transductant from each recipient strain, after purification by streaking onto a BHI agar plate containing 50 units of penicillin per ml, was tested for the phage type, as was each parent recipient strain. The results of this experiment are shown in table 32. The results indicated that with few exceptions, penicillinase-producing transductants possessed the same phage type as the recipient strains from which they originated. Detailed phage .type analyses were not performed with other antibiotic-resistant transductants.

Level of antibiotic resistance. An examination of the level of resistance conferred on a strain by the transduction of the Pase, Ery, 01, Nov, and Tet markers was made. It was found that the markers conferring anti­ biotic resistance, when present in donor strain or trans­ ductant strain genome, enable that strain to grow on

BHI agar containing the same maximal level of the ap­ propriate antibiotic. 101

Table 32

Phage type of 19 recipient strains and penicillinase-pro­ ducing transductants of each strain transduced by phage 80/U9(Pase,Tet,Ery)

Phage type of: Strain designation Parent strain Penicillinase- producing transductant

Ps 29 29 29 Pa 52 52 52/52A/80 Ps 42B 42B 42B C 72 29/52A/79/80 29/52A/79/80 W 26 29/80 29/80 Ch 50 80 80 M-1 79/55 53/77/29/52A/79/ 80/55 1 52A/80/81 52A/80/81 248 52A/80 52/80 608 52A/80/81 52A/80/81 769 81 n.t. N 135 29 29 N 203 29 29 745 81 n.t. 1 12 29/52A/79/83/ 29/52A/79/83/ 42E/80/81 42E/80/81 569 52A/80 52A/80 152 52A/79/80 52A/79/80 616 52A/80/81 52A/80/81 720 52A/80 52A/80

Stability of transduced characteristics. The stability of the transductants was determined by several observations. When colonies of the transductants were

streaked for isolation on BHI agar containing high con­

centrations of the appropriate antibiotics, the resulting growth was always heavy and complete. Also, during the

course of this study transductants were employed in turn as donor strains of the transduced marker. The trans­ duction frequencies of these markers were found to equal the frequencies observed when using donor strains of known genetic homogeneity. Donor strain 4865(Pase,Tet,

01 ,Nov), which has been described previously, acquired the Tet and 01 markers from strain U9(Pase,Tet,01,Nov) by transduction. When these two donor strains were compared with respect to their competence as donors of these markers,

they were found to transduce the 01 and Tet markers at

very similar frequencies. ThiB finding is illustrated in

tables 38, 40,.41, and 42, using phages 29, 52A, and 80.

Effect of transduction on l.ysogenlcit.y. To

determine the effect of transduction on the lysogeniclty

of the recipient strains, 12 strains were transduced with

phage 80/U9(Pase,Tet,Ery), and the Pase and Tet trans­

ductants selected from each transduced suspension. Two

Tet and two Pase transductants from each population were

purified on BHI agar plates containing either 50 ug of

chlortetracycline or 50 units of penicillin per ml. The

purified transductants and the parent recipient strains

were then tested for lysogeny. Seven strains which were

not detectably lysogenlc and which possessed a variety

of phage types were employed as Indicator strains. Table

33 indicates Jthe phage types of the Indicator strains. 103

Table 33

Phage type of indicator strains used in the detection of lysogeny

Strain designation Phage type

1 52A/80/81 U9(Pase,Tet,Ery) 80/81 112 29/52A/79/83/42E/80/81 2S(Pase) 29/6/7/83/42E/47/54/75/77/81 535(Pase) 80/81 Ps 31 29/52A/79/53/80 Ps 73 79/3A/3B/3C/Ms39/51/55/6/7/83/42E/ 42B/47/47B/47C/53/54/70/73/75/ 77/31/44/80/81

Table 34 indicates the lytic reactions of the transductants

and recipient strains determined by using the seven indi­

cator strains shown in table 33. An examination of the

lytic reactions indicated that many of the transductants

were lysogenic, while several of the recipient strains

from which they were derived were not lysogenic. However,

the lack of detectable lysogenlcity among many of the

transductants was also commonly observed.

The phages from transductants 1(Pase), 152(Pase),

and 608(Pase) were Isolated and propagated on strain

U9(Pase,Tet,Ery). When the resulting lysates, all of

which yielded titers of 1x1010 pfu per ml or higher, were

used in an attempt to transduce the Pase marker from

strain U9(Pase,Tet,Ery) to recipient strains Ch 50, 1, 608,

and 152, no evidence of transducing activity was detected. 104

Table 34

Lytic reactions of the parent recipient strains and the Pase and Tet transductants obtained by transduction with phage 80/U9(Pase,Tet,Ery)

Lytic reactions* observed on Strain tested for indicator strains .ysogeny 1 U9 112 2S 535 Ps31 Ps73

608 6 0 8 (Tet) — — - — — -- 6 0 8 (Tet) 2+ 2+ 2+ 2+ — - 2+ 6 0 8 (Pase) 4+ 4+ 4+ — 2+ 2+ 4+ 6 0 8 (Pase) — 4+ 4+ 2+ 2+ - 4+ 769 2+ — 2+ 2+ _ 1+ — 7 6 9 (Tet) 2+ — 2+ 1+ - 1+ 2+ 7 6 9 (Tet) 4+ 4+ 4+ 4+ 3+ 3+ 4+ 769(Pase) 4+ 3+ 4+ 2+ 2+ 2+ 3+ 769(Pase) 4+ 4+ 4+ 3+ 3+ 2+ 3+

1 (Tet) —_ — 1 (Tet) 2+ 2+ 2+ — —- 2+ 1(Pase) 4+ 4+ 4+ 2+ 2+ - 3+ 1(Pase) 3+ 4+ 4+ 2+ 1+ - 2+ 248 — 2+ — —— - 2 4 8 (Tet) — _ 2+ —- -— 2 4 8 (Tet) — 2+ - —-— 2 4 8 (Pase) — — 2+ — — - - 2 4 8 (Pase) -- 2+ “- - - ivl“*M — 1 1 M-1(Tet) — _ _ M-1(Tet) — _— „ ~ - — M-1(Pase) — — - - --- M-1(Pase) ------152 —_ -— --- 152(Tet) — — 2+ - - - 152(Tet) — - 2+ - -- 1+ 152(Pase) — 3+ 2+ 1 + --- _ 152(Pase) 3+ 2+ 1 + 2+ - 3+ Ps 42B 2+ 3+ 2+ 1 + ~ 1 + 3+ Ps 42B(Tet) 2+ 2+ 3+ 1 + - 1+ 3+ Ps 42B(Tet) - 2+ 2+ 3+ 1 + - 1+ 3+

*Lytic reactions recorded as 4+, 3+, 2+ , 1+, and -, indicating confluent through the absence of lysis, respectively. 105

Table 34 (contd.)

Lytic reactions* observed on Strain tested for Indicator strain: lysogeny 1 U9 112 2S 535 Ps31 Ps73

Ps 42B(Pase) 3+ 3+ 3+ 2+ 3+ Ps 42B(Pase) 3+ 3+ 3+ 2+ — - 3+ C 72 — — 2+ —— - C 7 2 (Tet) 2+ 2+ — — — - C 7 2 (Tet) - 2+ 2+ -- - - C 7 2 (Pase) 2+ 2+ —-- C 7 2 (Pase) - 2+ 2+ - --- Ch 50 — — -- - -- Ch 5 0 (Tet) —- —-—-- Ch 5 0 (Tet) — ------Ch 50(Pase) — — - - - -- Ch 50(Pase) ------N 135 — 2+ * —-- N 135(Tet) — - 2+ - -- - N 135(Tet) - - 2+ - - -- N 135(Pase) — - 2+ • -- N 135(Pase) — - 2+ - - -- Ps 29 — — 2+ — -- Ps 2 9 (Tet) — — 2+ — -- Ps 2 9 (Tet) _ - 2+ -- Ps 29(Pase) — — 2+ - — -- Ps 29(Pase) —— 2+ * - - W 26 — 2+ _— -- W 2 6 (Tet) — — 2+ - -- - W 2 6 (Tet) — — 2*4* — — —— — W 26(Pase) — 2+ - - - - W 2 6 (Pase) — 2+ — —— —

#Lytic reactions recorded as 4+, > , 2+ , 1+ , and ~ 9 Indicating confluent through the absence of lysis, respectively.

In conjunction with these studies, strains U9(Pase,

Tet,Ery) and U40(Pase,Tet) were tested for lysogeny on 160

randomly selected indicator strains. No evidence of

lysogeny was detected in these tests. 106

Tests for linked transductions. Strains 152 and 112 were transduced with phage 80/U9(Pase,Tet,01,Nov), and the

Pase. Tet, 01, and Nov transductants selected. Each popu­ lation, consisting of at least 500 transductants, was repli­ cated onto BHI agar containing 50 units of penicillin, 50 ug of chlortetracycline, 100 ug of oleandomycin, 15 ug of novobiocin, or 100 ug of streptomycin per ml. After incu­ bation of the replica plates, all transductants were found to grow only on media containing the antibiotic employed in their initial selection. This procedure was repeated several times using a variety of donor and recipient strains, and Included tests to detect linkages with the Ery marker. However, in no instance were the markers determin­ ing resistance to two antibiotics transduced together.

Donor strain U9(Pase,Tet,01,Nov) was found to be hemolytic on BHI agar containing whole rabbit blood

(alpha hemolysin), cleared BHI agar containing milk

(proteolytic enzymes), cleared TSA containing heat-

coagulated rabbit plasma (flbrinolysin), was pigmented,

and caused an increase in opacity when grown on BHI agar

containing egg yolk (lipase). Recipient strains Ch 50

and Ps 52 were unable to bring about the above changes,

although they grew normally on all test media. Strains

Ch 50 and Ps 52 were transduced with phage 80/U9(Pase,Tet,

01,Nov), and the Pase, Tet, 01, and Nov transductants

selected. At least 500 transductants possessing each marker except 01 were replicated onto each of the afore­ mentioned test media, as were 15 01 transductants from strain Ps 52. An examination of all transductants failed to reveal any deviation from the characteristics of the parent recipient strains. Also, 0.05 ml quantities of ten-fold dilutions of the transduced suspensions from 1s10 through 1510,000 were spread on BHI agar plates containing whole rabbit blood. At dilutions of 1s10 and 1s100 the blood was altered, making the detection of individual hemolytic clones impossible. At the higher dilutions, the medium appeared normal and no hemolytic centers were evident.

Strain Ch 50, which did not ferment trehalose, and strain C 72, which failed to ferment mannitol, were trans­ duced with phage 80/U9(Pase,Tet,01,Nov), and the Pase,

Tet, 01, and Nov transductants selected. Concentrated and diluted samples of the transduced suspensions were also plated onto an appropriate medium containing trehalose or mannitol. The antibiotic-resistant transductants were replicated onto the same medium. The antibiotic-resistant transductants did not differ in fermentation capacity from the parent recipient strain. Also, attempts to detect mannitol or trehalose-fermenting transductants directly in the transduced suspension were unsuccessful. 108

Transduction Studies Using Different Donor Strains and Bacteriophages

Tables 35 through 42 indicate the transduction

frequencies observed by using 14 recipient strains trans­ duced with phages 29, 79, and 52A propagated on strain

655(Pase,Tet,Ery); phages 52A, 29, and 80 propagated on

strain 4865(Pase,Tet,01,Nov); and phage 80/U9(Pase,Tet,

01,Nov). The results given in these tables indicate the

relative competence of the transducing phages, using a

series of 14 recipient strains which were originally

selected for their competence to be transduced by phage

80/U9(Pase,Tet,Ery). Table 35 indicates the transduction

frequencies with phage 79/655(Pase,Tet,Ery). Strains 152,

C 72 and 112 were the only strains sensitive to lysis by

phage 79, but were found to be transduced at frequencies

no higher than the other strains which were transduced.

In general, phage 79/655(Pase,Tet,Ery) was not a highly

competent transducing phage..

Table 36 indicates the transduction frequencies

with phage 53/655(Pase,Tet,Ery). While none of the

recipient strains were susceptible to lysis by phage 53,

the majority of them were transduced with this phage. The

transduction frequencies which were observed with phage

53/655(Pase,Tet,Ery) were in some cases very similar to

those observed with several of the phage 80 preparations.

Tables 37 and 38 indicate the transduction frequencies with 109

Table 35

Frequency of transduction of the Pase. Tet. and Ery markers by phage 79/655(Pase,Tet,Ery)

Frequency of transduction ofs Recipient strain ------Pase Tet Ery

42 B 0 0 0

112 0 30 4

248 0 0 0

N 135 0 40 6

1 0 0 0

152 50 60 400

Ch 50 0 50 0

W 26 0 30 0

C 72 0 40 0

7 69 0 0 0

608 0 0 0

52 0 0 0

29 0 2 0

D-1 0 60 8 110

Table 36

Frequency of transduction of the Pase, Tet, and Ery markers by phage 53/655(Pase,Tet,Ery)

Frequency of transduction ofi Recipient strain ------Pase Tet Ery

42B 0 10 0

112 2 600 10

248 0 0 0

N 135 10 200 20

1 0 0 0

152 300 200 2000

Ch 50 8 600 20

W 26 2 200 10

C 72 4 200 10

769 0 0 0

608 0 0 0

52 0 0 2

29 0 200 0

D-1 80 200 300 Table 37

Frequency of transduction of the Pase. Tet. and Ery markers by phage 29/655(Pase,Tet,Ery)

Frequency of transduction of i Recipient strain Pase Tet Ery:

42B 0 0 0

112 10 6000 6

248 0 2 0

N 135 4 6000 20

1 0 0 0

152 700 8000 1000

Ch 50 4 4000 6

W 26 8 4000 0

C 72 0 6000 0

7 69 0 2 0

608 0 0 0

52 0 4 0

29 4 2000 0

D-.1 60 6000 200 112

Table 38

Frequency of transduction of the Pase. Tet. 01. and Nov markers by phage 29/4865(Pase,Tet,01,Nov)

Frequency of transduction of: Recipient strain ------Pase Tet 01 Nov

42B 0 . 0 0 2

112 100 6000 600 400

248 0 8 10 0

N 135 10 3000 600 300

1 0 20 50 6

152 30 6000 800 300

Ch 50 10 6000 600 300

W 26 30 8000 600 300

C 72 40 6000 400 300

769 0 4 10 2

608 0 10 20 4

52 0 0 10 6

29 2 2000 400 80

D-1 20 6000 800 300 113 phages 29/655(Pase,Tet,Ery) and 29/4865(Pase,Tet,01,Nov), respectively. In general, a recipient strain which was transduced with phage 29/655(Pase,Tet,Ery) was also trans­ duced with phage 29/4865(Pase,Tet,01,Nov). The frequencies observed with these phages were extremely low or inde- tectable only among those recipient strains which were resistant to lysis by phage 29, but high frequency trans­ ductions were observed with strains which were either resistant or sensitive to lysis by this phage.

Tables 39 and 40 indicate the transduction fre­ quencies of phages 52A/655(Pase,Tet,Ery) and 52A/4865

(Pase,Tet,01,Nov), respectively. A comparison of the frequencies of transduction of the 01 and Ery markers by these strains revealed the effect of the donor strain on the transduction frequency with these markers. Strain

655(Pase,Tet,Ery) was naturally resistant to erythromycin.

Strain 4865(Pase,Tet,01,Nov) was derived from an erythro­ mycin-sensitive strain which was transduced with the Ery and 01 markers at a relatively low frequency, and when used as a donor strain transduced the 01 marker at

relatively high frequency to all recipient strains. Tables

41 and 42 represent the transduction frequencies observed

with phages 80/4865(Pase,Tet,01,Nov) and 80/U9(Pase,Tet,

01,Nov), respectively. The same phenomenon of recipient

strain specificity with the 01 marker was observed with

these transducing phages as was observed with phage 52A Table 39

Frequency of transduction of the Pase. Tet. and Ery markers by phage 52A/655(Pase,Tet,Ery)

Frequency of transduction of: Recipient strain Pase Tet Ery

42B 0 400 0

112 V- 0 2000 0

248 300 2000 500

N 135 20 2000 8

1 8 2000 8

152 400 4000 600

Ch 50 2 4000 6

W 26 10 6000 4

C 72 2 4000 0

' 7 69 4 2000 6

608 0 2000 2

52 50 1000 10

29 2 1000 0

D-1 10 4000 20 115

Table 40

Frequency of transduction of the Pase, Tet, 01, and Nov markers by phage 52A/4865(Fase,Tet,01,Nov)

Frequency of transduction ofs Recipient strain Pase Tet 01 Nov

42B 2 700 200 50

112 80 2000 600 500

248 10 2000 700 400

N 135 20 3000 700 300

1 2 4000 1000 200

152 10 4000 700 400

Ch 50 20 4000 800 200

W 26 20 4000 800 400

C 72 40 6000 500 600

7 69 30 6000 700 800

608 8 2000 600 300

52 10 2000 400 500

29 0 2000 300 200

D-1 10 2000 800 500 116

Table 41

Frequency of transduction of the Pase, Tet. 01, and Nov markers by phage 80/4865(Pase,Tet,01,Nov)

Frequency of transduction of: Recipient strain Pase Tet 01 Nov

42B 0 600 300 90

112 300 6000 800 600

248 70 3000 900 600

N 135 30 2000 700 500

1 30 3000 900 500

152 40 3000 900 500

Ch 50 50 4000 800 500

W 26 60 2000 800 500

C 72 80 2000 700 600

769 100 2000 800 700

608 20 2000 800 600

52 60 2000 600 700

29 10 2000 600 400

D-1 40 2000 800 500 Table 42

Frequency of transduction of the Pase, Tet, 01, and Nov markers by phage 80/U9(Pase,Tet,01,Nov)

Frequency of transduction of: Recipient strain Pase Tet 01 Nov

42B 600 1000 0 10

112 40 9000 2000 2000

248 600 1000 800 600

N 135 600 9000 4 400

1 800 3000 2 400

152 600 8000 3000 1000

Ch 50 “700 6000 0 400

W 26 1000 8000 6 1000

C 72 90 8000 10 300

769 800 8000 10 900

608 3000 8000 6 800

52 300 2000 30 400

29 1000 8000 0 400

D - 1 800 6000 3000 700 In tables 39 and 40. Also, in the transductions employing phage 80, there was a distinct difference between the two donor strains in the frequency of transduction of the

Pase marker, phage 80/4865(Pase,Tet,01,Nov) transducing this marker at a ten-fold lower frequency than phage

80/U9(Pase,Tet,01,Nov).

Attempts to use phages 42B and 81 as transducing phage were unsuccessful even when a variety of donor and recipient strains of known competence were used. Both of these phages were capable of lysing sensitive strains growing on BHI agar, and this may have been the reason for their inability to participate in transduction. DISCUSSION

According to this investigation, the competence of the donor strains of Staphylococcus aureus was dependent upon only two major requirements. The donor strain had to possess the characteristic to be transduced. This is not only a very obvious requirement, but a very fundamental requirement to distinguish between transduction and lysogenic conversion. It was also necessary for the donor strain to support the propagation of the transducing phage and to yield titers of at least 1x101® phage parti­ cles per ml. When phage with titers lower than this were used, the resulting transduction frequencies were signifi­ cantly depressed. The titers of transducing phages em­ ployed by Morse (1959) in S. aureus and Baron et al.

(1953) in S. typhosa were between 1x10^ and 1x10^® parti­ cles per ml.

Among the phages of the International Typing Series which were examined for participation in transduction, phages 29, 52A, 79, 80, and 53 were found to be competent while phages 42B and 81 were Incompetent. The ability of phage 53 to serve as a transducing phage was of particular interest as this phage is classified into Group III, while phages 29, 52A, and 79 are classified into Group I. Phages

119 120

80 and 81, while classified into the Miscellaneous group by some authors,, are placed into Group I by others. The

finding that phages 42B and 81 were incompetent was also

of interest in view of the close relationship between

phages 80 and 81, and the fact that phage 81 was obtained

by adaptation of phage 42B. Phages 42B and 81 were capable

of lysing susceptible strains grown on BHI agar, which may be a partial explanation of the incompetence of these

phages.

It was not surprising that the degree of competence

of the transducing phages was found to vary considerably.

On the basis of the transduction frequencies and the

number of recipient strains successfully transduced, phage

79 was the least competent and phage 80 was the most

competent transducing phage. The competence of a phage

to participate in transduction was felt to be determined

as much by the phage genome as by the genetic makeup of

the donor and recipient strains.

Five phages of the International Typing Series were

employed to transduce the ability to produce penicillinase,

novobiocin resistance, chlortetracycline resistance,

erythromycin resistance, and oleandomycin resistance.

Some of the recipient strains were resistant while other

recipient strains were susceptible to lysis by the trans­

ducing phage. However, susceptibility to lysis by the

transducing phage did not appear to determine either the 121 competence of the recipient strain or the frequency of transduction. Although lower frequencies were occasionally encountered, the frequencies of transduction were found to vary between one and ten transductants per 10^ phage particles employed. Similar frequencies were reported by

Baron et al. (1953) in Salmonella and by Morse (1959) in

S. aureus. In view of the fact that the multiplicities of

Infection used in the transduction experiments were less than 1-.0, it is felt that the transduction frequencies were not limited by the number of recipient cells available per transducing particle.

Transduction did not significantly modify the phage types of the recipient strains, although occasional minor

changes in the phage type were observed. When the lyso- genlc states of recipient strains and of the transductants

obtained from them were examined, in most instances a

pre-existing lysogenlc state was not altered. Furthermore,

some non-lysogenlc recipients were not lysogenized follow­

ing transduction. However, when a change in the phage

type or lysogeny was observed following transduction, It

was not correlated with any other known characteristic of

S. aureus. As the phages which were Isolated from lyso-

genic transductants derived from non-lysogenlc recipient

strains were not capable of transduction, these lyso-

genizing phages were not considered to be the original

transducing phage. If the lysogenizing phages in these 122 instances were the transducing phages, they were markedly altered with respect to the spectrum of lytic activity and transduction competence. In conjunction with these studies, the principal donor strain, U9(Pase,Tet,Ery), was found to be non-lysogenic on the basis of tests performed with 160 indicator strains. Therefore, when phage 80/U9(Pase,Tet,Ery) was employed to transduce a non-lysogenic strain and the resulting transductants were lysogenlc, the lysogenlzing phage was of unknown origin.

The fate of the phage genome during transduction has not been determined. In almost every instance, it was apparent that the transducing phage did not become incorporated as prophage during transduction. When the recipient strain was resistant to lysis by the trans­ ducing phage, apparently the phage genome underwent a- bortive infection after injection into the host cell.

When the recipient strain was susceptible to lysis by the transducing phage, apparently growth of the Infected cells on BHI agar sufficiently altered the physiology of the

cells so that lysis was prevented and the phage genome

again underwent abortive Infection.

With few exceptions, all of the competent recipient

strains were susceptible to lysis by one or more of the

following phagess 29, 52, 52A, 79, 80, 81, and 42B. On

the other hand, nearly all of the incompetent recipient

strains were susceptible to lysis by phages of Groups II, Ill, or Miscellaneous. In general, the competent recipient strains encountered which were resistant to lysis by phages of Group I or phage 42B were transduced at a low frequency. This observation strengthens the contention that the staphylococci may be placed in groups on the basis of their phage types. It also Indicates that these groups of staphylococci are not mere artifacts of taxonomy, but are valid groups on the basis of genetic compatability.

The relationship of the transducing phage to the recipient strains was found to be Independent of the susceptibility of the strain to lysis by the specific transducing phage.

Phage 80 transduced 26 recipient strains, only 16 of which were susceptible to lysis by phage 80. Phage 29 trans­ duced the Tet marker at relatively high frequencies to eight recipient strains, three of which were resistant to lysis by phage 29. Similarly, phage 52A transduced the

Tet marker at relatively high frequencies to 14 recipient strains, seven of which were resistant to lysis by this phage. Finally, none of the recipient strains were susceptible to lysis by phage 53, but eight of these strains were transduced by phage 53.

The transduction frequencies of the Pase, Tet, and

Nov markers, as well as the Ery and 01 markers in specific instances, were all relatively uniform. However, the average frequency of transduction of chlortetracyclihe resistance was about ten-fold higher than the frequencies 124 observed with the Pase and Nov markers. Chlortetracycllne has been reported to be a chelating agent (Saz, 1956) and was found to inhibit the lytic activity of the transducing phages. Furthermore, Klrchner and Elsenstark (1957) observed that treatment of the recipient cells with chelating agents increased the frequency of transduction and sexual recombination. However, it was demonstrated in this study that the presence of chlortetracycllne in the selective medium did not increase the frequency of transduction of the Pase marker. Therefore, the high frequency of transduction of the Tet marker was not due to either the ability of chlortetracycllne to inhibit lysis or the chelating activity of this antibiotic.

Studies designed to determine the time lag prior to replication of the transduced marker revealed a distinct difference among the markers in this respect. The Tet marker was found to be expressed and replicated rapidly after transduction, while the Pase, Nov, and 01 markers all underwent varying lag periods prior to expression and replication. The reasons for this are not known, but several possibilities exist. The marker may have been

Incorporated rapidly by the cell, but for a period of time was not replicated because growth of the transduced cell was inhibited by the antibiotic. Alternatively, the marker may have been injected but not Incorporated immediately, and was passed on to one daughter cell at each cell division prior to final Incorporation and segre­ gation in a manner resembling abortive transduction.

Ozeki (1956) has presented evidence that a transduced fragment which is not incorporated during the first one or two cell divisions following injection'will not be incorporated during subsequent generations. These obser­ vations suggest that the transduced markers were incorpo­ rated immediately after injection, and that there was a lag period prior to phenotypic expression and segregation of some markers because the cell was inhibited by the antibiotic and did not divide. In the case of the Tet marker, expression appeared to occur Immediately after injection and cell growth did not appear to be interrupted.

This seems to indicate that the Tet marker is dominant and enabled the heterogenote to grow in the presence of the antibiotic before integration of the marker into the genome of the recipient and segregation in the daughter cells. As Geronimus and Cohen (1957) demonstrated that staphylococcal penicillinase is an inducible enzyme, it is felt that the expression of the Pase marker was followed by a time lag during which the cell was induced to form penicillinase in response to the penicillin in the medium. This lag not only accounts for the delayed replication of the new marker, but also explains the lower transduction frequencies observed with the Pase and

Nov markers, and the necessity of using small amounts of 126 penicillin in the selective medium. Some loss of the transductants may have resulted due to bactericidal action of the antibiotic before the transduced fragment was expressed sufficiently to protect the cell.

The failure to transduce streptomycin resistance may have been due to a number of factors. Morse (1959) and Wanatabe et al. (1959) have reported a distinct lag period associated with the expression of streptomycin resistance in a transduced population. Also, Lederberg

(1951) demonstrated that streptomycin resistance was recessive to streptomycin sensitivity in E. coll. If streptomycin resistance is recessive to sensitivity in

S. aureus. and if there is some delay in the development from the original transduced cell of a daughter cell which possesses only the recessive allele, then some delay would be expected to occur prior to phenotypic expression. On the other hand, if the transduction of streptomycin resistance resulted in the formation of a stable hetero- genote possessing both alleles, then expression would be dependent upon the degree of instability of the hetero- genote. Wanatabe et al. (1959) found that when a donor strain of intermediate streptomycin resistance was used to transduce a streptomycin-sensitive population, the streptomycin-resistant transductants grew much slower than the remainder of the cells in the transduced population.

Under these conditions, it would be extremely difficult 127 to isolate the streptomycin-resistant transductants. As a lag period of 14 hours or less was taken into consider­ ation by the methods used in the present study, it would seem that the inability to transduce streptomycin resistance must have been due to a factor such as the growth dis­ advantage of the transductants or the formation of a stable heterogendte. The detection of streptomycin- resistant transductants was further complicated by the high Incidence of streptomycin-resistant mutants which were encountered among, the recipient strains.

Using donor strains which were naturally resistant to erythromycin, only strains 112, 152, 248, and D-1 were transduced at relatively high frequencies with respect to the Ery marker. In addition, seven "epidemic" strains which were sensitive to erythromycin were found to serve as high frequency recipients of the Ery marker. All other

strains examined were competent recipients of the other

markers, but were low frequency recipients of the Ery

marker. However, when a low frequency recipient strain was transduced, arid one of the resulting Ery transductants

employed as a donor of the Ery marker, all of the recipient

strains were transduced at relatively high frequencies.

The specificity of the recipient strains with respect to

the Ery, Ery-X, and 01 markers was found to be independent

of the phage employed for transduction. The frequencies

of transduction of the Ery, Ery-X, and 01 markers were 128 also Independent of the donor strain employed, provided that the donor strain either was naturally resistant to erythromycin or was derived from a high frequency recipient of the Ery marker.

The most logical explanation for the specificity of the recipient strains is that the region of the chro­ mosome involved was similar in donor strains naturally resistant to erythromycin and in high frequency recipient strains of the Ery marker. On the other hand, the region of the chromosome involved in these transductions differed sufficiently between the donor strains and the low fre­ quency recipient strains so that incorporation of the transduced fragment occurred at a low efficiency in these recipient strains. When the Ery marker was incorporated into a low frequency recipient, however, some structural modification must have occurred so that when this strain was employed as a donor of the Ery marker, Incorporation occurred at a high frequency in all recipient strains.

The observation that all of the "epidemic" strains were high frequency recipients of the Ery marker may have some significance. Many authors have related genetic compatability with taxonomic relationships among species.

If the foregoing assumption concerning chromosomal compatability is correct, the "epidemic" strains and strains which were sensitive to the antibiotics but were high frequency recipients of the Ery marker may have 129 shared a common ancestor. Furthermore, the high fre­ quency recipients of the Ery marker may represent the descendants of strains from which the "epidemic" strains evolved during the early 1950's. All of the "epidemic" strains were shown to he nearly identical, differing mainly by the presence or absence of erythromycin re­ sistance (Caswell et al., 1958; Shaffer et al., 1957), and therefore probably were of common genetic origin.

The variety of phage types displayed by antibiotic- sensitive, high frequency recipient strains of the Ery marker is felt to be insignificant as among four recipient strains possessing the 52A/80 phage type, only two were found to be high frequency recipients of the Ery marker.

The results of this study indicated that the Ery,

Ery-X, and 01 markers all shared a nearly identical locus on the bacterial chromosome. This view is supported by their common behavior with respect to the specificity of the recipient strains and by their common mutational origin from strain U9(Pase,Tet,Ery). It was not possible, however, to study these relationships by transductlonal analysis. The 01 marker was transduced to strains possessing the Ery and Ery-X markers, but whether this occurred by replacement or by addition at another locus was unknown due to the phenotypic masking of the Ery and

Ery-X markers by the 01 marker. Attempts to transduce the 130

Ery-X marker into a strain possessing the Ery marker were unsuccessful.

The characteristics of erythromycin resistance displayed by strains possessing the Ery marker resemble an inducible enzyme system. Induction was brought about

specifically by erythromycin, low concentrations of

erythromycin were sufficient to cause induction, and the

effects of Induction were rapidly lost after growth of

the strain in the absence of erythromycin. After in­ duction, a strain possessing the Ery marker was resistant

to high concentrations of oleandomycin, spiramycin, and

carbomycin, and was capable of Initiating growth rapidly

in the presence of high concentrations of erythromycin.

These characteristics are considered to be due to an

inducible enzyme system responsible for resistance to the

macrolide antibiotics. The activity of the enzyme system

in uninduced cells was sufficient to allow the initiation

of growth in the presence of low concentrations of erythro­

mycin. As the concentration of erythromycin in the medium

Increased, however, the number of cells which possessed

a sufficiently active enzyme system to allow their

survival and induction decreased. The observation that

only a minority of the cells of an unlnduced strain

possessing the Ery marker were capable of initiating

growth in the presence of high concentrations of erythro­

mycin Is assumed to be due to a variation in the activity 131 of the enzyme system among the cells present In the population. As only the Induced cells were resistant to all of the macrolide antibiotics, it Is felt that the enzyme system responsible for resistance was less efficient

In conferring, resistance to oleandomycin, spiramycin, and carbomycin than in conferring resistance to erythromycin.

The mutation from Ery to Ery-X is felt to have resulted In an increase In the activity of the enzyme system responsible for resistance to the macrolide anti­ biotics in the unlnduced cells. Strains possessing the

Ery-X marker were inducible, but in the uninduced state each cell apparently possessed a sufficiently active enzyme system to Initiate growth in the presence of erythromycin at high concentrations. However, as only a minority of the cells possessing the Ery-X marker were capable of initiating growth in the presence of oleando­ mycin, spiramycin, and carbomycin, it is felt that a variation among the uninduced cells still existed in the degree of activity of the enzyme system responsible for resistance.

By transduction, it was shown that the inducible nature of the erythromycin resistance was due to either

the Ery marker or the Ery1 portion of the Ery-X marker.

The Ery and Ery1 markers are undoubtedly identical as both

markers conferred resistance only to erythromycin. The

identity of the Ery and Ery1 markers is also demonstrated 132 by the fact that while 100 per cent of the total viable cells of a strain possessing the Ery-X marker were re­ coverable on a medium containing high concentrations of erythromycin, only about one per cent of the total viable cells were recoverable from a strain possessing the Ery marker or from a transductant population possessing the

Ery' marker.

The mutation from Ery to 01 resulted in a pro­ nounced change from an inducible mechanism of erythro­ mycin resistance to an indifference to the presence or absence of any of the macrolide antibiotics. This in­ difference to all of the macrolide antibiotics was due specifically to the 01 marker as shown by the response of the transductants to a variety of selective media.

The importance of employing a naturally occurring erythromycin-resistant strain as the donor of the Ery marker was demonstrated by the failure of erythromycin- resistant mutants isolated in vitro to serve as donors.

The failure to serve as donors of resistance was in all probability due to their origin during selection which involved several distinct stepwise mutations.

The Ery-X marker was transduced from the original donor strain to the transductants in two forms, the Ery1 marker and the Ery-X marker. The division of the Ery-X marker observed when donor strain U9(Pase,Tet,Ery-X) was employed was not observed when the Ery-X marker was trans­ 133 duced from strains which acquired the marker from strain

U9(Pase,Tet,Ery-X) by transduction. The failure to observe this division when the Ery-X marker was trans­ duced from other donor strains indicated some unique characteristic of donor strain U9(Pase,Tet,Ery-X). If the Ery-X marker is assumed to represent two very closely spaced characteristics, designated Ery1 and X, which were found to dissociate only from the parent donor strain, then apparently the chromosome of strain U9(Pase,Tet,

Ery-X) had a tendency to fragment between Ery1 and X during phage replication. This would result in some phage particles which acquired the complete fragment, while others acquired only the Ery1 or the X factor.

Whether the fragmentation of the chromosome of strain

U9(Pase,Tet,Ery-X) was due to a salt bridge in the DNA, which would be a natural breakage point, or whether it was due to a dissociator gene present in the genome at a distance from the Ery-X locus is not known. SUMMARY

The ability to produce penicillinase, as well as chlortetracycllne resistance, novobiocin resistance, erythromycin resistance, and oleandomycin resistance were

Independently transduced by using a variety of donor and recipient strains of Staphylococcus aureus and five phages of the International Typing Series. Attempts to transduce streptomycin resistance were unsuccessful, although a variety of transducing phages, donor and recipient strains, and selective techniques were employed. The frequency of transduction was usually between one and ten transductants per 107 phage particles employed when the multiplicity of infection was 1.0 or less. Lower transduction fre­ quencies were occasionally encountered. Joint trans­ duction was not observed between two markers controlling resistance to the antibiotics, or between a marker con­ trolling antibiotic resistance and any of a variety of other characteristics. The transduced markers were found to be stable, and to be identical in both the donor and the recipient strain.

Phages 29, 52A, 79, 80, and 53 were capable of transduction while phages 42B and 81 were not. Lysogenl- zation of the transduced cells was observed, but this was

134 135 not a prerequisite for transduction. The recipient strains were either resistant or sensitive to lysis by the transducing phage, but this was not found to influence the transduction frequency. Lysis of susceptible strains by the transducing phage was prevented by preparing the selective media with brainheart infusion agar, a medium which was inhibitory to the lytic activities of the trans­ ducing phage.

Twenty-six of 48 strains examined were found to be transduced by phage 80. The majority of the competent strains were susceptible to lysis by one or more of the

following phagess 29, 52, 52A, 79, 80, 81, and 42B. The majority of the strains which were not transduced were

found to be susceptible to lysis by phages of Groups II,

III, or Miscellaneous.

With the exception of the erythromycin-resistant

transductants, all of the transductants were isolated by inoculating them directly onto media containing the

selective concentration of the antibiotic. The trans­ ductants resistant to erythromycin were isolated by a

technique which exposed them to gradually increasing con­

centrations of erythromycin. Evidence was presented that naturally occurring resistance to erythromycin was due to

an enzyme system specifically Induced by erythromycin.

Growth of an erythromycin-resistant, but oleandomycin,

spiramycin, carbomycin-sensitlve strain in the presence of 136 minute quantities of erythromycin induced a temporary resistance in the strain to oleandomycin, spiramycin, and carbomycin.

The transduction of resistance to the macrolide antibiotics occurred at a high frequency with only four recipient strains and seven "epidemic" strains. The possible significance of this observation has been discussed in terms of the relationship of the "epidemic" strains to other, antibiotic-sensitive, strains of S. aureus. The transduction of the other antibiotic resistance markers occurred at relatively uniform frequencies among the majority of the recipient strains. J

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I, Peter Arthur Pattee, was born In Garden City,

New York, November 15, 1932. I received my secondary school education In the public schools of Garden City, New

York, and my undergraduate training at the University of

Maine, which granted me the Bachelor of Science degree in

1955. From The Ohio State University, I received the

Master of Science degree In 1957. During my residence at

The Ohio State University I held a graduate assistantshlp in the Department of Bacteriology. I held this position for five years while completing the requirements for the

Master of Science and Doctor of Philosophy degrees.

144