CfiROMOSOMAL LOCATION FOR A GENETIC DETERMINANT 0F CHLORTETRACYCUNE RESTSTANCE IN A STRAIN OF STAPHYLOCOCCUS AUREUS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY DORIS J; BECK 1970 1... ‘L—r ‘ '-_-., *r—‘r—E -mn . '—'_‘i-:¢_;— —- .O.-.'.“‘M -W ' "w: I LIBRARY Michigan 515356 University TV W ABSTRACT Chromosomal Location For A Genetic Determinant Of Chlortetracycline Resistance In A Strain 0f Staphylococcus Aureus by Doris Beck The genetic determinant for chlortetracycline resis- tance in strain U9 of Stanhylococcus aureus was found to have both chromosomal and ex rachronosomal nature. Strain U9 was also resistant to both erythromycin and streptomycin which were studied for comparison to the tetracycline resistance. Chlortetracycline resistance was apparently stable in strain U9 because no sensitive colonies were detected during a series of curing experiments which utilized growth in solutions of acriflavine, ethidium bromide, and also growth at elevated temperatures. Low doses of ultraviolet radiation on transducing phage lysate propagated on strain U9 caused an increase in transduction frequency for all three antibio- tic resistances. The transduction frequency for chlortetracycline resis- tance with phage lysate propagated on U9 was more than a log higher than for the other chromosomal resistances. This transduction frequency was higher than that for chlortetra- cycline resistance obtained when phage 80 was prepagated on strain 2 (pen, tet), an example in which the antibiotic resistances are known to he plasmid linked. Transduction frequencies for extrachromosomal markers are generally Ihigher than for those of chromosomal markers because extra- chromosomal material does not depend on recombination within the host for gene expression. Transductants obtained with nonirradiated phage prepagated on U9 were not as stable as those obtained with irradiated phage because sensitive colonies were found in curing eXperiments. Three sensitive colonies were isolated with a concommitant loss of penicillinase production. Three additional colonies of strain 2 (pen, tet) also showed this coelimination. Chlortetracycline resistance may be due to a hetero- diploid state in U9 as both chromosomal and extrachromosomal prOperties were observed. An alternative explanation could be that a resident plasmid within the transductants interacted with the chromosome extracting the tetracycline resistance which yielded variants with plasmid linked resistance. CHROMOSOMAL LOCATION FOR A GEFETIC DETETNIKAUT OF CHLORTETRACYCLINE RESISTANCE IN A STRAIN OF STAPHYLOCOCCUS AUREUS By I' 4 Doris J. Beck A TIES IS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health I970 ,,/ 0 77A; \ AC KI‘IO’. IL LED G14 EI‘TT S The author wishes to express gratitude to Dr. C. L. San Clemente for his guidance during the time of this inves- tigation. ‘ A special note of thanks is ext-nded to Dr. R. R. Brubaker for his assistance and to my friends, Donna Muirhead and Aline Garrctson for their encouragement and advice. This thesis is dedicated to my family for their understanding. ii TABLE OF 23""TS AC IG‘IOL'LEGI: EIITS o o o o o o o o o o 0 LIST OF TILL! ‘38 o o o o o o o o o o o ' LIST OF FIG? :8 o o o o o o o o o o o IIITRODLTCTIOM . - o o o o o o o o o o o HIST OP ICAL REVIEW . . . . . . . . . . Chromosome mapping in Staphylococcus aureus . . . . . . . Plasmids in seer1"lococci . . . . . Evidence for plasmid linkage of somatic traits , o o o o o o o o o 0 Current genetic aspects of antibiotic reSiS 13311095 9 o o o o Mechanism of tetracycline activity . . LJYDERIALS AND M3T ZODS . . . . . . . '. . I'iedia o o o o o o o . o o Cultivation of organisms . . Selective media. . . . . . Curing agents . . . . . Strains of Starllzlococcus aureus and pelageS do 0 o o o o o o o o 0 Identification and sources . . homenclature. . . . . . . Phage typing. . . . . . . Determination of drug resistance. . . Disc assay . . . . . . . Graded plate assay. . . Selection of drug rcsi tent variants by gradient plates . . . Curing strains of anti biotic resistance Growth at elevated temperatures Growth in acriflavine. . . . Growth in ethidium bromide . . Transduction of chlortetracycline r9315 St’u’lce o o o o o o Iropagation of phage . . . . Transduction procedure . . . Time required for phenotypic expression . . . . . . 111 U) 0000 \0 “VI -F" WU) Ultraviolet irradiation of phage Transduction frequency . . . RESTJLTS O O O O O O O O O O O O 0 Phage trpe and drug resistance . . . Selection for drug resistant varic nts . Curing 9:5“31‘11’3 53an o o o o 0 Kinetics of ultraviolet irradiation of phage lysates . . . . . . . . PhenOtyI‘iC 13g 0 o o o o o o o o DISCT:SS:OII O O O O O 0 O O O O O 0 LITERATURE chED . . . . . . . . . . . iv Page Table l. 2. LIST OF TABLES Preparation of assay discs for drug resistance. ‘Phage type and chemical resistance of strains of Stanhylococcus aureus . . . . . . . . Level of kanamycin resistance after passages through a series of gradient plates with diff- erent concentrations of the antibiotic . . . Level of chlortctracycline resistance after passages serially through two gradient plates containing chlortetracycline (3ug/ml). . . . Stability of chlortetracycline resistance in strain U9 of Stanhylococcus aureus. . . . . Stability of chlortetracycline resistance in strain 2(pen, tet) of Staphylococcus aureus. . Stability of chlortetracycline resistance in transductants produced with irradiated rhage 80 propagated on Starfiylococcus aureus strain 0 O O O O O O C O O O O O O 0 O Stability of chlortetra rclino resistance in transductants produced with nonirradiated phage 80 propagated on Stapiylococcus aureus strain T79 \' o o o o o o o o o o o o o o o Transduction freauency for antibiotic resis- tances by phage 30 propagated on Staphylococcus aureus strain U9 and 2(pen, tet) in which Staphylococcus aureus strain 80 was the recip- Te n1 0 O O O C O O O O O O O O O Page 13 23 26 3O 31 32 j4 Ml Figure 1. 2. LIST OF FIGURES Selected example illustrative of colony distribution on gradient plates . . . . . Effect of number of gradient plate colony sel- ections on levels of resistance to two antibio- tics. The highest concentration of antibiotic uninhibitive to crowth was determined for col- onies selected from gradient rlntes by cultiv- ation on different graded plates of the antibiOtiC o o o o o o o o o o o 0 Effect of dose of ultraviolet light on the transduction frequency of chlortetracycline resistance by phage JO prepagated on Stan v- lococcus aureus strain U9 in which gtarnvlo- coccus aureus strain 30 was the recipient . . Effect of dose of ultraviolet light on the transduction frequency of chlortetracycline resistance by phase 80 pronagwted on Starhvlo- coccus aureus strain U9 in which Stnrhyloceccus aureus strain 30 was the recipient. . . . . Effect of dose of ultraviolet light on the transduction frequency of chlortetracyclise resistance by phase 30 propagated on Startho- coccus aureus strain 2(pen, tet) in which Starhylococcus nireus strain p0 was the recipient. . . . . . . . . . . . . Phenotypic lag of transduced resistance to three antibiotics as sdoun by the tine needed for gene expression after transduction . . . vi Page 27 28 37 39 H0 42 INTRODUCTION Since the advent of antibiotic therapy many of the enterococci and staphylococci have developed resistance to various antibiotics (Barber, 1966). The rapidity in which high levels of resistance are attained and the wideSpread occurrence of such resistances invoke more than merely the operation of evolutionary processes. Mutations occur- ring i vitro are observed to consist generally of sligat inoreases in resistance requiring a long time ‘to achieve any high level of drug resistance (Florey, l9h9). Uatanabe (1961) reports that high level multiple drug resistance in a strain of Shigella flexneri can be transferred via episomes to strains of Escherichia coli, Salmonella tyrhimurium, and Salmonellaaenteritidis. A conjugal relationship is not observed in staphylococci and thus the only means for transnitting resistances between strains appears to be by bacteriophage. Jarolnen et a1. (1965) succeeded in transducing tetracycline resis- tance into sensitive strains of Staphylococcus aureus in vivo by injecting infected mice with phage prOpagated on a tetracycline resistant strain of Staphylococcus aureus. The mechanism for tetracycline resistance varies with the particular resistance transfer factor within strains of Escherichia coli (Unowsky and Rachmeler, 1966) or with the nature of the resistance determinants, ie., whether they are of chromosomal or extrachronosonal origin (Weisblum and Davies, 1968). Asheshov (1966) and Poston (1966) have demonstrated that tetracycline resistance is extrachromosomal in certain curable strains of Staphylo- coccus aureus. The purpose of this study is to determine whether the genetic locus for chlortetracycline resistance in strain U9 of Staphylococcus aureus is located on chromo- somal or extrachromosomal DfiA and to observe any relation- ships or interactions of chromosomal and extrachronosomal markers. . HIST OR IC AL R EV I31! Chromosome mapping in Staphylococcus aureus Chromosome mapping of Staphylococcus aureus has been limited due to the absence of sexuality in this organism. Altenbern (1969) has mapped some markers on the chromosome of Staphylococcus aureus by using phenethyl alcohol to synchronize chromosomal replication and then subjecting the bacteria to treatment with N~methyl-H'~nitro-N-nitrosoguan- idine at various times after resumption of DNA synthesis. His results indicate that there is a definite locus on the chromosome for the initiation of chromosomal replication and that the gene order is essentially the same in randomly selected strains of Staphylococcus aureus: Altenbern reports that strains.containing multiple- resistance factors (MRF) show a delay in the gene replication time possibly because the KRF duplicates first after which chromosomal replication is permitted. Plasmids in staphylococci The type of plasmid found in the staphylococci are nontransmissible plasmids which are manifest by the estab- lishment of drug resistance in the host. It appears that plasmids are dispensable for the maintenance of cell viability but are genetically stable with some possible exceptions. Novick (1963) has found the spontaneous rate of loss for the penicillinase plasmid to be about 10-3 per cell generation. Evidence for plasmid linkage of somatic traits Novick (1969) gives a list of criteria for determin- ing plasmid linkage. Coelimination studies such as curing by growth in solutions containing acriflavine (Mitsuhashi et al., 1963), ethidium bromide (Bouanchaud et al., 1969), or rifampicin (Johnston and Richmond, 1970), and growth at elevated temperatures provide evidence for the autonomous nature of plasmids and those somatic traits carried on them. Irradiation of phage lysate utilized in transduction reveals the extent of genetic homology‘of markers to the host chromosome by the ability of the chromosome to rescue these markers (Garen and Zinder, 1955 and Arbor, 1960). Low doses of ultraviolet irradiation produce an increase in transduction frequency for markers carried by DFA homologous to the host chromosome and thus provide a means of differentiating between episomal and chromosomal locations -of markers. Plasmid DNA is not homologous to the chromosome and as increasing dosages of irradiation damage the plasmid carried by the transducing phage the less likely the survival of the plasmid and its somatic characteristics. Replicative autonomy of somatic traits may be demon- strated by radiation target size as the plasmid is much larger than a chromosomal gene. Incompatibility with a known plasmid constitutes evidence of plasmid linkage (Uovick and Richmond, 1965). Plasmids belonging to the same compatibility group can not exist as stable diploids within a cell so that the establishment of the incoming plasmid requires the loss of the resident plasmid. Rush et al. (1969) have demonstrated the autonomy of staphylo- coccal plasmids by isolating the plasmid DNA and preparing electron micrographs of the DhA by autoradiography. Their isolating of plasmid DNA was based on the resistance to alkali denaturation of the covalently closed circular duplex DNA. Current gpnetic aspects 9: antibiotic resistances O The incidence of drug resistant staphylococci has been extensively investigated by Barber (1966) who revealed the increasing numbers of resistant strains in clinical infections since the beginning of antibiotic therapy. She asserts that staphylococci develop resistance to erythromycin and streptomycin more readily than to most antibiotics. This ability would appear to reflect the chromosomal locus for these antibiotic resistances as shown for streptomycin by Korman and Berman (1962) and Poston (1966) and for erythromycin by Richmond (1969). Mutations occurring in bacteria are most likely changes of the chromosomal DNA as the chromosomal DNA is many times the plasmid DNA and because the chromosome is an essential feature of all cells. In contrast the extrachromosomal nature of erythromycin resistance has been demonstrated by Mitsuhashi et a1. (1965) by joint elimination of penicillinase production and erythromycin.resistance utilizing ultraviolet irradiation and growth in acridine. May et al. (196%) and Asheshov (1966) have shown that loss of tetracycline resistance can be accelerated by growth at elevated temperatures illustrating an extrachromosomal inheritance in some strains. Asheshov (1966) and Foston (1966) utilized irradiation inactivation kinetics of transducing phage lysates to demonstrate the plasmid linkage for tetracycline resistance and the chromosomal location for penicillinase production in some strains of Staphylococcus aureus. The strains used by these investigators had unstahle resistance to tetracyclines but stable determinants for producing penicillinase. Asheshov (1969) in reference to penicillinase production and Richmond (1969) for erythromycin resistance refer to the transition of these genes between integrated chromosomal loci and the extrachromosomal state. These investigators are finding that resistance to some drugs may show both chromosomal and extrachromosomal inheritance within a strain of staphylococci at different periods of time. Richmond (1969) demonstrates that there is a reversible transition between the extrachromosomal and the integrated state for ervthromycin resistance in . some strains of Staphylococcus aureus. Mechanism of tetracvcline activity The tetracyclines are a group of antibiotics composed of four fused rings including the following: aureomycin-(7-chlortetracycline), terramycin (5-hydroxy- tetracycline), and declomycin (demethylchlortetracycline). The microbial spectra and biochemical activities for these antibiotics are essentially the same so that effects using one antibiotic usually are paralleled by the others. Chlortetracycline is somewhat unstable in liquid media so that determinations of resistance levels for strains of bacteria are overestimated. The tetracyclines are inhibitors of protein synthesis that effect the ribosome (Ueisblum and Davis, 1968). Tetracycline has been shown by Day (1966) to bind toboth the 50 S and 30 S subunits of the ribosome. Suarez (1965) has reported that tetracycline inhibits the binding of aminoacyl-tRNA to 30 8 subunits in_vit£9 and by this mechanism perhaps inhibits polypeptide synthesis. Day (1966) has found that if either the 50 S or 30 8 subunits are treated with tetracycline, washed by dialysis and centrifugations, and then studied in cell free extracts of Escherichia coli that protein synthesis is inhibited and that the tetracycline- molecules are distributed afterwards between both subunits. Gurgo et a1. (1969) have studied the synthesis and location of mRNA in cells treated with tetracyclines and other inhibitors of protein synthesis. They report that peptide bond formation and polyribosome formation are "uncoupled" by the antibiotics. Their observation that newly formed mRNA continually appeared in pelyribosomes suggests that the ribosomes must add to and move along the mRNA without the occurrence of protein synthesis. In any event the site of action for tetracycline has not been localized and the reaction of the tetracyclines may be more extensive than binding to the ribosomes. Resistance to tetracyclines may be due to a permea- bility factor or other protective barriers such as drug inactivation depending upon the organism ahd the genetic determinant for resistance as reported by Unowsky and Rachmeler (1966). They were able to show reduced uptake of tetracycline by Escherichia gel; containing the negative fertility inhibition episome but not for those cells containing the positive fertility inhibition episome. Unowsky and Rachmeler were unable to isolate an enzyme active on tetracycline for Escherichia coli containing the positive fertility inhibition resistance-transfer (R) factor. MATERIALS AND {ETTODS sage Cultivation 9: organisms. Dacto Brain Heart Infus- . ion (BHI), BHI agar, nutrient broth, and nutrient agar (Difco Laboratories, Detroit, fiich.) were used in routine cultivation of bacteria. For phage propagation nutrient agar and nutrient broth were enriched with dextrose and phosphate (K2HFOu), described and named P and D media by Pattee and Baldwin, 1961 (cf. pp 17). Selective media. Chlortetracycliue hydrochloride and erythromycin (Sigma Chemical Co., St. Louis, he.) were sterilized with a membrane filter (Hillipore Filter Corp- oration,1hdford, Kass.) having a pore diameter of O.h5 u. The solutions of antibiotics were frozen and stored at -20 C until needed and then added to sterile tempered (molten agar between HS-SC C) media before pouring plates. Dihydrostreptomycin sulfate (3. R. Squibb & Sons, Div. of Olin Fatnieson Chemical Corp., New York, N. Y.) and Kantrex (Bristol Laboratories, Div. of Bristol-Myers Co., Syracuse, N. Y.) were purchased sterilized so that sterile solutions of these antibiotics were simply added to hot sterile BHI agar. Curing agents. The curing agents used were Ethidium bromide, 2,7,-diamino-D-ethyl-9-phenylpenantridium bromide 10 (Calbiochem in Los Angeles, Calif.) and Acriflavine HCl, a mixture of the hydrochlorides of 2,8(3,6) Diamino-D-meth acridinium chloride and 2,8(3,6) Diaminoacridine (Sigma Chemical Co. in St. Louis, Ho.). Stock solutions containing 5000 ug/ml were sterilized by membrane filtration as described previously and stored at h C. The curing agents were added to BHI broth to achieve desired concentrations. Strains of Staphylococcus aureus and phages Identification and sources. Strains U9(ary, pen, str,tet) and 8325 (str, tet) were supplied by Dr. Richard P. Novick of the Public Health Research Institute of the City of New '-’or3-:, Inc., New "Tork, ‘r’. Dr. I-f. 11'. Richmond of Bristol University hedical School in Bristol sent strain 147(str). Dr. 3. H. Asheshov of the Captral Public Health Laboratory in London kindly submitted a culture of strain 2(pen, tet). Strain P855 and A3, prepagating strains for phages of the International Typing Series came from our stock. Phage prepagating strains 53 and 80 along with phages 53 and so of the International Typing Series were donated by the hichigan State Public Health Department. Strains h(1-5) are clinical isolates that were received from Edward T. Sparrow Hospital in Lansing, Mich. Strains A-G are tetracrcline resistant transductants of strain 80 obtained using five minute ultraviolet irradiated phage 80 ll propagated on Strain U9. Strains 1-8 are tetracycline resistant transduntants of strain 835 obtained with nonirrad- iated phage 3O propagated on strain U9. Nomenclature. Strains of Staphylococcus aureus have been identified by using the first three letters of the antibiotics to *1ich the strain is resistant following the strain number. Phage typing. Sensitivities and/or resistances t0' phage 30 and 53 were determined for most strains utilized. When needed, relationship of strains was ascertained by a complete typing carried out by the hichinan State Public Health Department in Laising, Kich. Cultures to be phag typed were cultivated on BHI agar slants containing 7.5% haCl for 16 to 18 hours at 37 C. The bacteria were suspended in 5 ml of P and D broth and diluted so that a 0.1 m1 sample contai cd apprw xi mately 5 x 108 cells. Cell concentrations were determined by measuring absorbence with a Bausch & Lomb Spectronic 20. Tempered soft P and D agar (2.5nfl) was inoculated with 5 x 108 bacteria and poured over a P and D base agar. The plates were allowed to dry and then spotted with 0.1 ml of typing phage. The plates were incuo ted at 37 C and checked for lysis after 3, 16, and 2% hour periods. This procedure is a modification of the method used by Pattee and Baldwin (1961). 12 Determination gf drug resistance Disc assay. Bact sensitivity discs for antibiotics (Difco) were used to assay for resis ane e to antibiotics. Inorganic assay discs were made using discs (6.35 mm in diameter manlfactured by Carl Schleic1cr & Schuell Co., Keene, Y. H.) which were sterilized by autoclaving for 25 minutes at 20 pounds pressure, dried, and impregnated with 0.01 ml of inorganic ion solutions steri ized previously by autoclaving. The amounts of inhibitors impregnate on discs are listed in Table 1. The amounts of inorganic ions were the concentrations found to be most suitable in demonstrating variability of staphylococci by Iovick and Roth (1968). Four inhibitor impregnated-discs'were placed on BTI agar plates seed ed with 108 cells of the strain to be tested. The plates were subsequently incu ated at 37 C for 16 to 2% hours. Resistances and/or sensitivities were determined by measuring the radius of the inhib iti ion zone around the disc. Graded plate assay.- A series of graded plates was made up by dding suitable dilutions of the antibiotic to BHI agar. An overnight BHI culture of the organism to be tested was spotted with an inoculating loop on the prepared set of graded plates. Overnight broth cultures were also suitably diluted so that about 200 cfu (colony forming units) would be obtained when 0.1 ml was spread over the surface of each plate. The level of res istance was recorded as the highest concentration of antibiotic 13 Table 1. Preparation of assay discs for drug resistance. Inhibitor . Amount of inhibitor impregnated per disc U9 Kanamycin sulfate ' 30.00 Streptomycin 10.00 Penicillin , 10.00 Erythromycin . 15.00 ‘Novobiocin 30.00 Neomycin 30L00 Tetracycline 30.00 Ehloromycetin - 30.00 Sodium arsenite l3.00 Sodium orthoarsenate 3.!0 Lead nitrate 3.30 Sodium metasilicate - 12.20 Zincznitrate 0.45 fiadmium nitrate 0.03 lMercuric nitrate 0.05 1% in which isolated colonies were able to grow or that highest concentration of antibiotic in which growth was not imraired for spotted cultures. Any effect of inoculum size was observed and noted. Selection of drug resistant variants b, cud. : The method of Szylbalski and Bryan (1952) for gradient plate construction was utilized to create a gradient of antibiotic concentration across a petri dish. BHI agar was 4.11 used as t1e slanted base aaar and a top ar;a.r ms de up of B1 II agar contai ining antibiotic ULiCl was added after solidi- fication of the slanted base layer. For selection of calor- tetracycline resistant colonies the top agar contained 3 ug/ml chlortetracycline. Selection for kana ycin resis ant organ- isns utilized kanamvcin gr radie nts of increasine concentra- d U - tion' employing concentrations of 3, 10, 20, 30, and 40 ug/ml kanamycin sulfate in the top agar. By meca ns of a sterile glue ss rod durlicate gradient plates '.1. re spr read with 107 sensitive organisms of an over- night ELI broth culture and then incubated at 37 C for 16 to 2% hours. Colonies growing in the highest concentration of anti- biotic were selected, inoculated into BTI broth, and incubated at 37 C for 16 to 2% hours. The overnight broth cultures were assayed for level of drug resist.nce by culturing on a series of graded plates and also inoculated onto another 15 set of gradient plates of the same or aigher concentration of antibiotic as in the previous gradient. Selection and cultivation of most resistant colonies was repeated with other passages through gradient plates to obtain. drug resistant strains. Levels of resistance were measured and noted after passage through each gradient. Curins strains 2; antibiotic resistance Growth at elevated tenperatures. This curing procé edure was slightly modified from the method used by Ashes- hov, 1966. Resistant colonies were selected from BET agar plates containing 15 ug/ml calortetracycline which had been incubated at room temperature for three days previous to use, inoculated into SKI broth, and incubated at 37 C for 16 to 2% hours. The overnight cu tures were diluted lzh in fresh EII broth and incubated 1% hours at 37 C in a gyrotory water bath shaking 123 cycles/min. Samples (0.15 ml) of this log phase culture were inoculated into 20 ml of B21 broth prewarned to 37 and #3 C and incubated 5% hours in water baths of 37 and 42.5 to #3.5 C reSpectively without shaking. After the incubation 5 ml samples of the cultures were shaken vigorously with 3 ml of ballotini in a vortex blender to separate clunps of cells into single cell units. The cultures were then diluted so that 50 to 150 cfu in 0.1 ml samples were spread onto nutrient agar plates (dried 16 previously for three days at room temperature. The plates were incubated overnight at 37 C and replicated onto nutrient agar containing 15 ug/ml chlortetracycline. To diminish spreading of colonies all plates to be used in the replicating ' proceSs were routinely dried by incubating at room temper- ature three days and additionally at 37 C overnight rrior to use. Due to this drying period selective media is not as concentrated as indicated for chlortetracycline. Sensi- tive colonies which grow only on media uitlout antibiotic were 0 isolated and restreaked on ~elective tetracycline medium to [ reconfirm tetracycline sensitivity. Cured colonies were comrared with parent strains by disc assays and phage tvring so that relationship was ascertained and linkage groups of tetracycline and other markers could be disclosed. Growth in acriflavine. Solutions of acriflavine TN-T ‘ were made up in :s~.I in concentration of 25.00, 12.50, 6.25, and 3.12 ug/ml according to the method of Hashi- moto et al. (196%). A colony of the resistant strain was selected from an antibiotic containing 331 agar plate, inocul- ated into 331 broth, and incubated at 37 C for a period of 16 to 2% hours. Samples of 0.1 ml of the overnight broth culture were then inoculated into the series of acriflavine dilutions and incubated 2% to 3C hours at 37 C. The culture of highes acriflavine concentration yielding uninhibited growth was ‘17 selected and a sample of 5 ml blended with 3 m1 ballotini as previously described. Suitable dilutions were made so that 50 to 150 cfu were spread onto nutrient agar plates and incubated overnight at 37 C. Suitable plates containing isolated colonies were then replicated on nutrient agar containing 15 ug/ml chlortetracyeline. Cured colonies were tested as before to confirm drug sensitivity, to ascertain relationship with parent strain, and to detect link- age with other markers. Growth in ethidium bromide. Ethidium bromide was added to 831 broth to give final concentrations of 6, h, 2, and l ug/ml (Pouanchaud et al., 1969). The experiment was performed as described for acriflavine in the preceding section. . Transduction of chlortetracvcline resistance Propagation of phage. P and D agar and P and D broth were used in phage propagation and transduction procedures following the methods of Pattee and Baldwin (1961). P and D broth is composed of nutrient broth enriched with 0.2% dextrose and 0.25% phosphate (K2HP04). P and D base agar and soft agar contain 1.5% and 0.3 % agar respect- ively. Typing phage 80 was propagated on strain U9 (pen, str, tet) using the agar-layer technique of Swanstrom and Adams (1951). 18 The prepagating strain was grown overnight on BYI agar slants containing 7.5% NaCl and the growth from this slant was suspended in 5 ml of F and D broth giving a suspension of approximately 2 to 6 x 109 cells/ml. Soft P and 8 cells of the propagating ' D agar was inoculated with 5 x 10 strain and 105 to 106 phage. The soft agar mixture was poured over a P and D agar base, allowed to solidify, and incubated overnight at 37 C. Two ml of P and D broth were then added to each plate and the soft agar layers scraped into a sterile centrifuge tube with a sterile glass rod. The mixture was shaken vigorously to extract the phage from the soft agar and allowed to incubate 30 minutes at room temperature. The soft agar was removed from the phage lysate by centrifuging 10 minutes at 1100 rpm and collecting the 'supernatant lysate. To sterilize the lysate 0.5 ml of chloroform was added for each 10 ml of so”ution, shaken 'vigorously, and incubated 30 minutes at room temperature. Before use,the lysatexem decanted from the chloroform layer and any remaining chloroform blown off. Titers of about 109 pfu were achieved by this method. For assay, S("-1111‘1‘33 of 0.1 m1 of suitably diluted ,phage were added to 2.5 ml of warm tempered soft P and D agar containing 5 x 108 cells of strain 30. The soft agar was poured over a P and D base agar, allowed to solidify, and incubated overnight at 37 C prior to counting plaque forming units (pfu). l9 Transduction procedure. Recipient strains, 30 and 8H5 were grown overnight on BHI agar slants containing 7.5% NaCl and the growth from these slants was suspended in 10 ml of P and D broth giving a suspension of approximately 1 to 3 x 109 cells/ml. One ml of the recipient bacteria suspension was mixed with 1 ml of the phage lysate yielding a multiplicity or infection (moi) 0f 0.5 to 1.0. The cells were incubated in a gyrotory water bath shaker at 37 C shaking 123 cycles/min. After the incubation period the cells were diluted 1:10 to retard phage adsorption and centrifuged 10 minutes at 1100 rpm to remove unadsorbed phage. The cells were resuSpended in P and D broth and assayed for antibiotic resistrnce by plat— ing 0.1 to 0.2 ml samples on selective media. The three selective media consisted of B31 agar containing 1 ug/ml erythromycin, 15 ug/ml chlortetracycline, or 25 ug/ml streptomycin sulfate. Chlortetracycline plates were used three to ten days after preparation and were kept in refrig- eration at h C until needed. 331 agar was used as it inhibits lysis of transductants by virulent phage. Controls consis- ting of recipient bacteria plated on selective media and sterility tests for ph:ge plated on BHI agar were run with each transduction period. Suitable dilution and plating of recipient cells on B3: agar at time of assay was done to determine the fraction of recipient cells. 20 Time reguired £2£_phenotypic expression. Transduced cells were resuspended in 10 ml of P and D broth after the transduction period and incubated at 37 C in the gyrotory water bath shaking 123 cycles/min. Samples of 0.1 ml were plated on chlortetracycline selective agar and 0.2 ml were plated on erythromycin and streptomycin selective agar each hour for a total assay period of 5 hours. Ultraviolet irradiation 2: phage. Four ml of phage lysate suspended in P and D broth rere gently swirled under an ultraviolet lamp at a distance of #3 cm at which the light intensity was 13 ergs/mmZ/sec as determined by the killing effect on g. £91; K12. One ml of the radiated phage suspension was used in transduction and another ml in titer- Q ing the phage. The remaining 2 ml of lysate were refriger- - ated at h C for repetition of experiment if needed. All procedures were carried out in yellow light to diminish possibilities of photoreactivation. Tetracycline transduct- ants were assayed after resuspension of centrifuged transduced bacteria but erythromycin and streptomycin transductants were assayed after a h hour incubation in the gyrotory water bath since there was a rhenotypic expression lag “ith these resistances. Transduction frequen z. Transduction frequencies were calculated as the number of transductants/input rhige. The total number of transductants was calculated by a 21 multiplying the number of resistant colonies growing on antibiotic selective media by the ratio of input recipient bacteria to the number of bacteria assayed. This method was used to normalize for multiplication of bacteria during assays or for loss of bacteria in centrifugation. RESULTS Phage type and drug resistance Strains of Staphylococcus aureus were typed for susceptibilities to lytic action of phage 80 and 53. Drug resistance was determined using inhibitor disc assays and series of graded plates. Table 2 lists the results of disc U assays and phage typing. Selection for drug resistant variants To select for kanamycin resistant mutants, cultures were passed through a series of five gradient plates of increasing kanamycin concentration and the level of resistance determined by testing the most resistant colonies isolated from each gradient on a series of graded plates containing various amounts of kanamycin. Two subcultures of strain 8325, A3, and lh7 and one subculture of U9 and P855 were each observed in duplicate. The initial level of resistance for all strains was equal to or less than 5 ug/ml kanamycin sulfate. The levels of resis- tance after each passage through gradient plate selections are shown in Table 3. There was much fluctuation and varia- tion occurring within strains so that subcultures showed a large standard deviation when results were compared. Due to this large variation in kanamycin resistance within strains it 22 23 Table 2. Phage type and chemical resistance of strains of Staphylococcus aureus. Strain Inorganic ion . Antibiotic Susceptibility resistance resistance to phagea 80 53 09 Cd, Hg, Zn. Ery, Pen, Str, Tet S R lh7 Cd, Hg, Zn. Str. S S 8325 Pb. Str. P555 A3 80 Hg. Pen. S R 53 . S SHl Cd, Zn. Ery, Str, Tetu. S R 5H2 Cd, Hg, Zn. Ery, Pen, Str R R 5H3 R R SHh Cd, Zn. Chl. R R SHS Cd, Hg, Zn. Pen. S R 5H6 R R aSymbols R and 5 denote cell resistance and sensitivity respectively to lytic action by phage. Blanks for susceptibility to phage indicate that the strain was not phage typed. 2h o + o. a. « ca __ e m. o « om : H mm __ a m. o « om o a o. o: w o a o. o a on __ a m. o a on a. « oN . _. H m. o n on o a o. On a . o h o. . o H cm 0 e o. o « om o a o_ o a o. o H ow o h 0. cm 9 o e o. o « om o a o. o a mm o h o. __ M w. o H o_ o n o. o. _ m o a m on « mm _ a a o « m o a u o a m o a on o a o. m < _E\m: _E\m: _E\m: _E\m: _E\m= _E\m: N _ _ N _ _ N _ co_umcucoucou mNMm mmma .u:_ m: m< c_u>smcmx mo_tom A_E\mnv c_u>Emcmx cu w>_u_mcom mc_mcum mo AN to _V mmc:u_:un:m noun—a ucu_vmgo . .u_uo_n_ucm egg mo «co—uncucoucou ucocomu_v zu_3 noun—a ace_pocm mo momcum smsoccu mommmmma Loumm oocmum_muc c_u>Emcex mo _o>oq .m o_nmh 25 was relatively easy to obtain a more resistant strain by gradient plate selection and an isolate of 1H7 was able to survive a concentration of 100 ug/ml kanamycin when a broth culture was spotted on a graded plate. This isolate was phage typed by the Michigan State Public Health Department ascertaining its relationship to strain 147. Strain A3, 1%7, F855, and 8325 were similarly observed for their mutation to chlortetracycline resistance. The results are tabulated in Table h and show that there is little variation of resistance in these sensitive strains. Antibiotic agar used in making gradient plates contained 3 ug/ml chlortetracycline for all experiments as growth was continuously inhibited at this concentration and colony forming units were uniformly restricted to the least concen- trated region of the plate. Lack of growth on gradient plates past regions of a particular antibiotic concentration or lack of variability in resistances prevented isolation of any resist nt variants for chlortetracycline resistance. Two to six samples of each gradient were observed. There was a contrast of growth on the kanamycin and chlortetracycline gradient plates (Fig. 1). Fig. 2 is a graph of the average level of resistance for all strains tested versus the numb- er of gradient plates in which the strains had been passaged. 26 N e m N a m _ a J _ a J m uce_pmco N a m N a m N a m N a m . < aco_emcu Na. Na. Nam in: 2o: _s\ma _E\m: _E\ma _E\m: 33 39. Ni Q 32.. 2285 A_E\m:v oc__u>umcueueo_zu ou o>_u_mcem mc_meum _ A_E\m:mV ecm_u>umcuouco_;u mc_c_mucou moue_a uco_vmcm ozu canoes“ >__m_com mommmmma coumo oucmum_moc ec__u>umcueuao_cu mo _e>oq .: o.nmh 27 .n—. Q's“ a u o R. .mOlea acohvmcm co comuanhcum_p >c0_ou mo u>.umnum:___ o_ameo veuue_em ._ ocam.u O x 5.2: .fEEEeE: . .. 2:2: 3:335 .l. ' Cab ‘ . l 0 ' Ilmflmmn ‘mm!Jm-.M' "I m. “M" ii 1-2 '3 fit: Cu iii}. 6‘? Still. PE 4—: Z: 3 21".: C23 Figure 2. Effect of number of gradient plate colony selections on levels of resistance to two antibiotics. The highest concentration of antibiotic uninhibitive to growth was determined for colonies selected from gradient p iates by cultivation on different graded plates of the antibiotic. Symbols: ([3) ievel of resistance to chlortetra- cyCIine and ((3) leveh of resistance to kanamycin. ,a- 29 Curinc experiments Procedures in which growth in solutions of acriflavine or ethidium bronide as well as in normal 311 broth at elevated temperatures were utilized to possibly eliminate resistance to chlortetracycline in strain U9(ery, pen, tet). The success- ful curing of strain 2(pen, tet) with etdidiun bromide pre- _cluded using growth under the other conditions. The results of these experiments are tabulated in Tables 5 and 6. Curing experiments were carried out also on chlor- tetracycline resistant transductants of strain 80 and SH Phage 80 was propagated on U9 and H ml of the transducing .hage lysate radiated with ultraviolet radiation having an intensity of 13 ergs/mn2/sec at a distance of 48 cm. The set of transcuctants A to G were transduced with the irradiated' phage and the set of transductants l to 8 obtained with non- irradiated phage. These results are tabulated in Table 7 and 8. Curing of U9 and those transduetants produced with irrad- iated phage was not achieved, but chlortetracycline resistance was lost by strain 2(pen, tet) and by transductants 3 and 6 which were a product of nonirradiated phage. Kinetics g§_ultraviolet irradiation 9g pha:e lysates Phage suspension was irradiated for O, l, 2, 3, 5 and 7 minutes and then the bacteriophage was used to trans- duce chlortetracycline, erythromycin, and streptomycin resis- tance into strain 835 and 80. The frequency of transduction is 30 o o S: o o mom 0 0 mm? o . . o mmm_ Axv oocmum_mon mm_co~0u no:_mem mc_mo_ o>_u_mcmm mo_co~oo mo_co_0u mo LonEJZ Loganz mu_eoen e:_n_£sm c_ Lazaro oc_>m_m_uom c_ Luzouw u m.m:-m.~: um Luzoeu noummeuca .mco_p_ucou mc_L:u .mamL3m mzuoooo_>cawum mo m: c_muum c_ oucmum_mon oc__o>omeuoueo_£o mo >u___nmum .m o_nmh .._ N mm_ _ ovmeoLn E:_n_£uo 2. LuzoLc 0.0 o moo umumoeucn Axv mocmum_mou mm_co_ou voc_mem mc_mo_ o>mummcmm mo_co_oo mo_co~0u wo LmnEJZ mo gossaz mco_u_ncou mc_c:u .msmLJm msououo_xnamum mo Auou .coav N c_mcum c_ oucmum_mou oc__o>umLumuLo_cu mo >u___nmum .m e_nmh 32 0.0 0 3m: 0cm)m—$—LUM C— SHSLU J 0.0 0 new vaumouuca : 0.0 o 00: ocp>mztum c. 9:395 M m _ N mm_ o m.m:-m.~: um zygote m o.o o mom voumucuc: m 0.0 0 mm ocw>mTZLum c. LuzoLo N o.o o m:. . u m.m:nm.~¢ um LuzoLu N o.o o m_~ noumoeuca N 0.0 o ___ oc_>m_m_gum c. Luzono l o o o me. u m.mq-m.~: um zuzoto _ o.o o . mm voumoguc: _ 68v oucmum_moL mo_co_ou voc_meo mc_mo_ o>_u_mcom mm_co_ou mo_co_ou mo gossaz mo Lucasz mcomu_vc0u mc_csu c_mcum .m: c_meum macezm mauuouo_>LQMum co voummmaoLa om omega wean—nuce_ ;u_3 vuuavoea mucmuoavmcmgu c. museum—mug oc__u>umeumuco_su mo >um__amum .m o_nmh 33 0.0 0.0 0.0 0.0 0.0 .2 0.0 o.o. 0.0 0.0 0.0 A5 oucmum _ m9. mc_mo_ mo_co_ou 0 mu_co_ou o>_u_mcom uo consaz mm mm 0N. mwu Sm oqm mm o_m com :4 ~04 vocmsmxo mo_co_ou mo Lucas: 0:30:23 5 539.0 0 m.m::m.N4 um gazebo uuumucuca oc_>o_m_cuu c_ Luzogu noumoLuc: oc_>m_m_eum c_ Luzoco u m.m:-m.~: um guzotu woumonuca oc.>m_m_num c_ :uZOgo u m.m: m.~: um zygote noumouuca mco_u_ucou mc_ezu .voac_ucou :_meum .m o_nmh 3h- 0 GS «ocean—mm... mc_mo_ mo_co_ou 0 mo_co_ou o>_u_mcom mo Lonsaz .m: c_mLum msocam m _. mN mow 0_N om mu. N¢_ m 0N :0. mo. vmc_meo mo_co_ou $0 Lonszz on_sots e=_u_;ua mc_>m_mwgum u m.m:-m.~: oc_>m_m_gum u m.m:um.~: ov_soen E:_u_cuo oc_>m_m_eum u m.m¢um.~¢ 5 5395 E 5.55 an zuzoco voumoeuc: c_ gazebo um zuzoeo vaumucuc: c_ guzoto a. cuzotu um guzoeu woumoeuc: mco_u_vcou mc_L:u nmwuouo_xzmmum co voummmaoLn om omega vuuupvmcc.c01 :u_3 vouavoLa mucmuuavmcmeu c. vacuum_mon oc__u>umcuouco_su mo >u___nmum c_mcum .m o_nmh 35 0 0 ARV oucmum _ m9. mc_mo_ mo_co_ou 0 0 mo_co_ou o>_u_mcom mo consaz on— :m. we. 00 mm. 0NN 8. 5 mo. mm. voc_meo mo_co_ou mo gossaz on_eots e=_n_;sm c_ guzotu u m.ma-m.~: um Lazaro voumeeuc: uu_eota s=_n_;no c. zygote u m.m:-m.~: um cuzota noumocuca 0 m.m:um.~: um zuzoco voumoeuc: u m.m:nm.N: um Luzouo woumoLuc: mco_u_vcoo mc_Lau .uo:c_ucou c_mgum .w o.g~» 36 plotted against dose of ultraviolet light (Fig. 3 and h) for strain 80 as recipient strain. When strain SHS was utilized as the recipient strain similar results were obtained. Small doses of ultraviolet radiation increased the frequency of transduction for resistances to_chlortetracycline, ery- thronycin, and streptomycin with phage 30 when propagated on strain U9. A similar experiment was done utilizing a phage lysate prepagated on strain 2(pen, tet) in which the tet- racycline marker had been shown by Asheshov (l966-b) to be extrachronosomal. Fig. 5 is the graph of transduction frequency for chlortetracycline resistance versus dosage of ultraviolet radiation of phage lysate propagated on strain 2(pen, tet). Table 9 is a list of strains utilized in transduc- tion experiments and the transduction frequencies for the different antibiotic resistances which were assayed. henotypic lag Time required for expression of transduced genes was determined by assaying for erythronycin, streptomycin, and chlortetracyclinc resistances at hourly intervals after transduction. The number of transductants for each antibiotic resistance is plotted versus time after trans- duction (Fig. 6). There is a two hour lag before genes 37 Figure 3. Effect of dose of ultraviolet light on the transduction frequency of chlortetracycline resistance by phage SO prepacated on Staphylococcus aureus strain U9 (0 in which Staphylococcus aureus strain 80 was the recipient. ymbols: (E1) transduction frequency of erythromycin resistance, (CD) transduction frequency of streptomycin resistance, and (A) phage survival. 38 _¢,i.==a iug=i.a=. I 1 9 a , 7 fl — — .8 . ) .OA inm . .m ( l 5" . . H _V _. an. s l I . ..l a... E . 0 l A O "I. l. A . , n r i I O '21 ‘A O E s 1 n . u 4 . .3, . . . _ . . . luai==izi ==__u==a=.=_ a=_, ‘ . on :3- can :3 :sa (:3 .;4 EL! Ii n3: ‘33 II . " :2-' ts. ;;: m - 3:; can can ‘ h“- I.” as d C) [J “ 7E; :33 Q; 33 O (3.. p.2- . ‘2: c2} .12: . . '-' D . Si: in! .g :23 ~ ._5 H , II *5 w. ‘—- " "- 4 .' M394; .5 ._ n, 2. '4 a . 8 ' mm unmwm um (min) Figure #. Effect of dose of ultravioiet iight on the transduction frequency of chsortetracyciine resistance by phage 80 propagated on Staphyiococcus aureus strain U9 in which §£§phylococcus aureus strain 80 was the recipient. Symbois: -(())-transduction frequency of chzortetracyciine, ([1) 'phage survival. ' iasriim raratrnsv l 1 ; tat talc H5512. iii 1D 88 RAGE. El- (.353 Hi 0" 2 4‘ B I O ' _ m ‘ _! q I ., 8 ‘ '18 ‘ DBSE llll'élillllfllfi llfilli (min) : Figure 5; Effect of dose of ultraviolet light on the transduction frequency of chlortetracycline resistance by phage 80 propagated on Staphylococcus aureus strain 2(pen, tet) in which "Staphylococcus aureus strain 80 was the recipient. Symbols:A (C)). transduction frequency of chlortetracycline, (£3) phage survival 1+1 Table 9. Transduction frequency for antibiotic resistances by phage 80 propagated on Staphylococcus aureus strain U9 and 2( pen, tet) in which Staphylococcus aureus strain 80 was the recipient. Propagating Transduction of resistance strain Frequency Antibiotic ,U9 l.0 x l0"6 Erythromycin U9 . 5.8 x 10‘9 Streptomycin U9 7.9 x l0"6 Chlortetracycline U9 9.9 x 10"6 Chlortetracycline 2 5.6 x l0"6 Chlortetracycline Figure 6. Fhenotvpic lag of transduced resistance to three antibiotics as shown by the time needed for gene expression after transduction. Graphs indicate the number of transductants resistant to ohlortetracycline ((3), erythromvcin (D), and. streptonycin (I) determined at hourly intervals after transduction and the number of recipient bacteria per ml (O) as determined by viable cell count at hourly intervals after transduction. a...” a: mafia... swag: -19 Li3 9 00 ’9 . .U ”a” I/ I2 . .11 I p .. . . .. - s .E 23:22:: 3.: a: . Hill? Mitt? ifliiilSEiiiSiliiil (Elli) 1+1. determining erythromvcin and streptomycin resistances were expressed. Genes for chlortetracycline resistance in contrast were expressed immediately so that the number of transductants paralleled the viable cell count Which was determined hourly. D ISO 73 SIGN By the use of gradient plate tech ique bacteria resistant to kanarycin can be selected and isolated but the same method is ineffective for chlortetracycline. On kanamycin gradient plates the number of colonies inversely reflects the gradient of antibiotic concentration. This gradient of growth however does not occur on the chlor- tetracycline gradient plates and thus strains with increased resistance are not obtained. It appears that the chlor- tetracycline level of resistance is uniform within the strain and that mutations to tetracycline resistance are exceedingly rare whereas mutations to slight increases in kanamvcin resistance do occur and do yield a significant number of resistant variants. Such selected nutations have high rrob- ability of being chromosomal mutations as most of the DNA of the bacteria is chromosomal and the chromosome is an ess- ential part of all cells. Plasmids or erisomes are not essential for the maintenance of cell viability. Curing experiments are not successful in eliminating chlortetracycline resistance from strain U9 but do eliminate the resistance in strain 2(pen, tet) which has been shown by Asheshov (1966) to be extrachronosomal. It is concluded that tetracycline resistance is stable in U9 because #262 colonies were examined and no sensitive colonies were disclosed. 1+5 #6 Ultraviolet irradiation of transducing phage lysate increases the transduction frequency for resistance to chlortetracycline, erythromycin, and streptomycin when phage 80 has been prepagated on U9. If phage 80 has been propagated on strain 2(pen, tet), irradiation of phage suspension does not increase the transduction frequency of chlortetracycline resistance but instead there is a decrease of the transduct- ion frequency uhich parallels phag. inactivation. Arber (1960) has shown that ultraviolet irradiation of phage lysate pro- duces an increase in transduction frequency for chromosomal markers and a decrease for episone linked traits. It is apparent that the genetic determinant for chlortetracycline resistance in U9 is either linked to the chromosome or to a plasmid containing DNA homologous to the chromosome. The transduction frequency for chlortetracycline resistance with phage 90 propagated on U9 is approximately a log higher (Fig. 6) than the transduction frequency for resistances to erythromycin and streptomycin. The trans- duction frequency for chlortetracycline resistance is 1.5 times larger when Phage 80 is prepagated on U9 rather than on strain 9(pen, tet). Arber (1960) reports that the trans- duction frequency of extrachromosona mrrkers is much higher than that of chrom somal markers as the establishment of plasmid DNA within the recipient does not depend on recombin- ation with the host chromosome. I'll. 1'! ',I 1+7 Chlortetracycline resistant transductants produced with phage 80 propagated on U9 were tested by growing under the curing conditions described previously. A set of 8 transductants were a product of nonirradiated phage and a set of 7 of irradiated phase lysato. Sensitive colonies were isolated only from the group transduced with nonirrad- 5 iated ~hage. Since ultraviolet irradiation of transducing La- phage rroduces an increase in transduction frequency for chromosomal markers and a decrease in transduction frequency for plasmid linked markers, a heterodiploid having chlortet- racycline resistance determinants both chromosomally and extrachromosomally would yield nainly plasmid markers with nonirradiated transducing phage and chromosomal markers with irradiated transducing range. Such heterodirloidy in U9 would account for the nigh transduction frequency of chlortetracycline resistance, the homology of this genetic marker with the chromosome as noted by the increase in transduction frequency with low doses of ultraviolet irrad- iation of transducing _hage, and the curing of resistance in transductants produced with nonirradiated rhage lysate. A second alternative could be tlat chlortetracycline resistance is linked to the chromosome in U9 but that in the transductants there is an extraction of the DNA conferr- ing tetracycline resistance by an interaction of the trans- duetant chromosome with a resident_plasmid. The resulting transductants would then possess a plasmid linked resistance for chlortetracycline which mirht be curable. The coelimin- {1'1 #8 ation of resistances to tetracycline and penicillin su55est that the interacting plasmid could be a penicillinase plasmid. A third alternative is that U9 contains a stable plasmid which has extensive homology with the chromosome. This explanation avoids reasons for such homology such as a chromosomal origin of the plasmid or the incorporation of chromosomal material into the plasmid. The time required for gene expression of resistances to chlortetracvcline, erythronycin, and streptomycin were determined and compared with the results described by Poston (1966). The purpose here was to observe any deviation of chromosomal resistance for chlortetracvcline and erythro- mzfcin witii plasnid borne resistances. Dorr inance of chlor— tetracycline resistance linked to both chromosome and plasmid is shown by its immediate expression and inheritance in all da u5hter cells. Erythromycin and streptomycin require 2 hours after transduction for their expression and thus must be recessive traits. In summary strain U9 has an unusual genetic determin- ant for chlortetracycline resistance since it displays properties of both extrachromosomal and chromosomal linkage. Evidenoefor the extrac1roraosomal locus of tetracycline resis- tance in V9 is the high transduction frequency when phage is propagated on U9 and the instability of this tetracycline resistance in some transductants. The chromosomal linkage is evidenced by the stability of tetracycline resistance in #9 U9 and the rise in transduction frequency when transducing phage propagated on U9 is eXposed to low doses of ultraviolet light. U9 might therefore be a heterodiploid with respect to tetracycline resistance LITERATURE CITED Altenbern, R. A. 1969. A survey of genomic maps in strains of Staphylococcus aureus. Can. J. Microbiol. 12:959-962. Arber, U. 1960. Transduction of chromosomal genes and episomes in Escherichia coli. Virology 11:273-288. Asheshov, E. H. l966-a. Loss of antibiotic resistance in Staphylococcus aureus resulting from growth at high temperature. J. Gen. Microbiol. Egzho3-h10. Asheshov, E. H. 1966-b. Chromosomal location of the gene- tic elements controlling penicillinase production in a strain of Staphylococcus aureus. Nature 31938015806. ’ Asheshov, E. H. 1969. The genetics of penicillinase production in Staphylococcus aureus strain P880. J. Gen. Microbiol. 22:284-301. 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