EXAMINATION OF AN UNUSUAL STRAIN 0F SI'ANIIYLUCUUCUS AUREUS, 342- A Thesis for the Degree of M, S. NIICNIUAN STATE UNIVERSITY WILLIAM L. MUTH 1970 LIBRARY 6] Michigan State: University ABSTRACT EXAMINATION OF AN UNUSUAL STRAIN 0F STAPHYLOCOCCUS AUREUS, 342-A by William L. Muth The purpose of this study was; (i) to examine some of the“ characteristics of the cells exhibiting the high coagulase production, unique colony formation, and pigment alteration; and (ii) to provide evidence using established techniques for the extrachromosomal location of the genes responsible for 'these traits. Staphylococcal cells found to produce large quantities of free coagulase were also noted to produce a white pigmented colony of unique morphology which included reduced size, rough character, and tenaciousness when teased with a needle. All three traits were simultaneously genetically linked and un- stable. Reversion of these traits was not evident. The cells producing these altered characteristics displayed an intolerance for atmospheric oxygen in broth culture which precluded duplication of cells. This intolerance was relieved by lowering the oxygen tension artificially. Q The instability of these traits in actively growing broth Kahlil -7. William L. Muth cultures appeared to be relieved to some degree by incubating these cells in media of higher pH (7.5 to 8.0). The three markers considered here did not segregate with any of the known staphylococcal plasmid markers, but appeared to be cured from the cells by either incubation at elevated temperature (42 to 43 C) or incubation with acridine orange (20 ug/ml). Selective pressure could not be ruled out because of the natural gross instability of the markers and the inability of these cells to grow under aerated shake flask conditions. Nevertheless, these factors taken together support the hypothesis that.the genetic markers responsible for the high coagulase production, pigment alteration, and unique colonial morphology are linked on an extrachromosomal element. EXAMINATION OF AN UNUSUAL STRAIN OF STAPHYLOCOCCUS AURBUS, 342—A by f‘\ William L? Muth A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF.SCIENCE Department of Microbiology and Public Health 1970 ACKNOWLEDGEMENTS The author wishes to thank his major advisor, Dr. Charles L. San Clemente, for the support, enthusiasm, and advice given during the course of this investigation. Thanks are also extended to Dr. D.E. Schoenhard, Dr. R.R. Brubaker, and the Staphylococcus group for suggestions and encouragement which assisted in.the completionrof this thesis. ii .li“! ll ll.‘l' II‘ l 1' III I. TABLE OF CONTENTS Page ACKNOWLEDnglENTS O O O O O O O O O O O O O O O O O 0 ii LIST OF TABLES O O O O O O O O O O O O O O O O O O C v LIST OF FIGURES C O O O O O O O O O O O O O O O O 0 Vi INTRODUCTION 0 O O C O O O O _. O O O O O O O O O O O 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3 Isolation and Cultivation of Organism . . . . . 3 Colonial Morphology . . . . . . . . . . . . . . 3 Coagulase Production and Assay . . . . . . . . 4 Extrachromosomal Elements in S. aureus . . . . 5 _Stability of Extrachromosomal Elements to oxygen 0 O O O O I C O O O O O O O O O O O 7 Stability of Plasmids . . . . . . . . . . . . . 7 Segregation with Known Markers . . . . . . . . 8 Agents for the Selective Elimination of Extrachromosomal Elements . . . . . . . . 9 MATERIALS AND METHODS O O O O O O O O O O O O O O O 12 Isolation and Cultivation of Organism . . . . . 12 Colonial Morphology . . . . . . . . . . .,. . . 12 coagulase Assay O O O O O O O O O O O O O O O O 13 Spontaneous Reversion . . . . . . . . . . . . . 13 Media 0 o o o I o o o o o o o o o o o o o o o o 14 Growth in a Liquid Medium under Various Gas Phases . . . . . . . . . . . . . . . . 14 Effect of pH on the Stability of the Selected Markers . . . . . . . . . . . . . . . . . 16 Segregation of Coagulase-Pigment—Colonial Morphology with Known Staphylococcal Plasmid Markers . . . . . . . . . . . . . l7 Curing with Temperature . . . . . . . . . . . . 18 Curing with Acridine Orange . . . . . ... . . . 18 RESULTS . . . . . . . . . . . . . . . . . . . . . . 20 iii Page Cultivation of Organism . . . . . . . . . . . . 20 Colonial Morphology . . . . . . . . . . . . . . 20 Coagulase Assay . . . . . . . . . . . . . . . . 22 Correlation of Morphology, Pigmentation, and Coagulase Production . . . . . . . . . . . 22 Spontaneous Reversion . . . . . . . . . . . . . 22 Media . . . . . . . . . . . . . . . . . . . . . 22 Growth in a Liquid Medium under Various Gas Phases . . . . . . . . . . . . . . . . 23 'Effect of pH on the Stability of th Selected Markers . . . .-. . . . . . . . . 36 Segregation of Coagulase-Pigment-Colonial Morphology with Known Staphylococcal Plasmid Markers . . . . . . . . . . . . . 39 Curing with Temperature . . . . . . . . . . . . 39 Curing with Acridine Orange . . . . . . . . . . 41 DISCUSSION 0 O O O 0 O O O O O O O O O O O O O O O O 43 BIBLIOGRAPHY O O O O O O O O O O O C ' O O O O O O O O 4 8 iv Table LIST OF TABLES Effect of blood on the final number of colony forming units of each colony type . . . . . . Effect of a mixed gas on the final number of colony forming units of each colony type . . ' Effect of air or a gas mixture on the final number of colony forming units of each colony type . . . . . . . . . . . . . . . . . Effect of pH on the final number of colony forming units of each colony type . . . . . . Effect of pH on the final number of colony forming units of each colony type . . . . . . Correlation of markers under study with markers known to be extrachromosomally located in Staphylococcus aureus . . . . . . . . . . . . Effect of elevated temperature or acridine orange on the final number of colony forming units of each colony type . . . . . . . . . . Page 25 32 33 37 38 4O 42 I‘ll! lull III III 'I'I A III LIST OF FIGURES Figure Page l.- Diagram of flasks used in gas phase experiments 15 2. Photograph of high and low coagulase producing cell types 0 O 0 O O O O I O O O I O I O O O 21 3. 'Growth in shake flask culture of large and small colony producing cell types with air as the gas phase 0’ o o o o o o o o o o o o o 27 4. Theoretical growth plot of large colony producing cell type . . . . . . . . . . . . . 29 5. Growth in shake flask culture of large and small colony types in the presence of natural gas . 31 6. Growth in shake flask of large colony producing cell type with a mixture of 88 per cent N , 5 2 per cent C02, and 7 per cent 02 as the gaseous phase . . . . . . . . . . . . . . . . 35 vi INTRODUCTION During a search for a strain of Staphylococcus aureus which would produce large amounts of extracellular free coagulase, a strain (342-A) was isolated that produced six to twelve times the usual level of free coagulase. The strain was unstable insofar as it could not maintain this high production level in broth to broth passage. Also there were noted along with the very high coagulase levels distinct morphological traits and pigment characteristics which were as unstable as the high coagulase trait. Unstable traits of another sort in S; aureus such as resis- tance to certain antibiotics and salts of heavy metals have been unequivocally shown to be coded for by genes located on an extrachromosomal element (Novick, 1963; Richmond and John, 1964; Hashimoto, Kono, and Mitsuhashi, 1964). Possibly coagu- lase, fibrinolysin, murine toxin, and pesticin are coded for by genes all located on an episome (Brubaker, Surgalla, and Beesley, 1965; Beesley, Brubaker, Janssen, and Surgalla, 1967). Several criteria are currently used to validate the plasmid nature of a genetic element. In organisms for which a well— mapped chromosome exists one can show the lack of linkage with the chromosome. If several genetic elements have no homology with the chromosome, these markers often show close linkage to one another during transduction (Novick and Roth, 1968). Also these closely linked markers occasionally show high rates of coelimination (Novick, 1966). Isolation of the extra chro- mosomal element by buoyant density centrifugation and/or sub- ' sequent electron microscopy on such isolates provide good evidence for a non-chromosomal genetic element (Rush, Gordon, NovickznuiWarner, 1969a and 1969b). Good evidence for an extrachromosomal element is also obtained if one can show that the genetic element of interest replicates separately from the chromosome. This replication can be shown by UV inactivation of transducing phages (Asheshov, 1966; Novick, 1967) or plasmid curing (Bouanchaud, Scavizzi, and Chabbert, 1969). The purpose of this study was: (i) to examine some of the characteristics of the cells exhibiting the high coagulase production, unique colony formation, and pigment alteration; and (ii) to provide evidence using some of the aforementioned techniques for the extrachromosomal location of the genes resPonsible for these traits. LITERATURE REVIEW Isolation and Cultivation of Ogganism In the past, the identification of isolates of S; aureus has depended in large measure on growth of the purified culture on mannitol and the production of coagulase. How- ever, the isolation of strains exhibiting very high capaci- ties for producing coagulase has been largely overlooked. Except for an earlier description of the nontypical colonies that are associated with high coaguiase production, Smith, Morrison and Lominski (1952) are the only authors to have des- cribed an isolation procedure for the selection of high coagu- lase producers. Their technique depended on cycling a culture of S; aureus first through a small tube of broth, then onto solid media, preferably blood agar. By inoculating the result- ing small rough colonies back into broth and repeating the cycle they isolated several strains having very distinct colonial morphology and producing high levels of coagulase. In these developed strains these two traits were inseparable. Colonial Morphology Bigger and Boland (1927) first described the colonial mor- phology of strains which were high coagulase producers. They 3 were concerned only with the colonial morphology and found that upon repeated subculture of fresh isolates the rough viscid nature of the plated colonies eventually completely disappeared. Some subcultures took longer than others to become typical staphylococcal colonies of the 'aureus, smooth, non-viscid type.’ Pigmentation was noted by Bigger and Boland (1927) to be typically 'aureus' or yellow and serial subculti- vation eventually produced 'albus' or white colonies when the' cultures were plated. Barber (1955) also noted this pheno- menon and added that the white to yellow alteration never occurred. Smith, Morrison and Lominski (1952) made no comments on the pigment at all. That these characteristics could pos- sibly be linked with extrachromosomal elements was shown by Richmond (1968) when he demonstrated that certain pemicillinase plasmid-containing cells develOped a unique colonial morphology. Coagulase Production and Assay The production of coagulase by microorganisms other than ‘S. aureus is limited to two genera, certain strains of Haemo- philus pertussis and Pasteurella pestis (Eisler, 1961). Other gram-negative bacilli were shown by Bayliss and Hall (1965) to utilize citrate, thus freeing calcium ions which were then available to the normal clotting process. A quantitative assay of true coagulase was initially devised by Tager and Hales (1948) and has been revised several 5 times. Another and a more recent quantitative assay devised by Muth and San Clemente (1969) based on‘a method reported by Stutzenberger and San Clemente (1966) was useful for purified preparations-of coagulase but has found little use in measuring coagulase levels in broth supernatant fluids due to the high salt content usually present. However, the source of coagulase reacting factor (CRF), which in the assay of Tager and Hales was rabbit or human plasma, has been efficiently supplanted with a commercially available mixture (KonyneTM, Cutter Labs) suggested by Soulier and Prou-Wartelle (1967) who favorably compared prothrombin-coagulase complexes with CRF-coagulase complexes.l W.H. Seegers (personal communication) actually~ made us aware of the availability of the product. Extrachromosomal Elements in S. aureus Barber (1949) observed that certain Strains of S. aureus which were designated as penicillinase-positive harbored a small proportion of cells which were penicillin sensitive (penicillinase negative) and that these newly recognized sensi— tive cells could not revert to the positive trait. Using this information Novick (1963) examined by transductional analysis a number of strains 0f.§- aureus with mutations in some aspect of penicillinase production. He found that while the frequency of mutations in the control region area caused by ethylmethane sull’onnte hum iIH‘I-I-nm-(i ova‘ thx- r- gum. {HM MAI 1. I‘I-I‘IIH’IH ."-'.~ "1"" 6 occurrence of penicillinase negative cells under similar treat- ment remained unchanged. Also, while the control region mutants behaved as point mutants reverting occasionally or yielding wild-type reCombinants in transductional crosses, the penicil- linase negative cells did not revert and when crossed trans- ductionally with the same type or control region mutants yielded- no recombinants. Novick further showed that after UV irradia- tion of transducing phage lysate carrying the penicillinase 3 marker, the loss of that marker mimicked the exponential de- crease in phage titer rather than the increased transduction of chromosomal markers in E, 931;. This experiment provided streng evidence that the penicillinase marker was located on an autonomously replicating genetic element. Later, it was shown that the penicillinase marker was closely linked to resistance to certain inorganic salts such as cadmium nitrate, mercury nitrate and sodium silicate (Richmond and John, 1964; Novick, 1967; Novick and Roth, 1968; Peyru, Wexler and Novick, 1969). Resistance to several other antibiotics (Barber, 1960) are also thought to be coded for by extrachromosomally located genes. In some strains, however, occasionally the genetic determinants (penicillinase,streptomycin, tetracycline) have been shown to be chromosomally located (Asheshov, 1966b; Poston, 1966). Though Lampen (1965) postulated the cell membrane as the functioning organelle for the liberation of extracellular penicillinase, the genetic determinant for this function has been recently shown by Peyru, Wexler, and Novick (1969) to reside on the typical penicillinase plasmid. While these plasmids are quite similar to the resistance factors described by Watanabe (1963) for the enteric microorganisms, Novick (1969) points out that staphylococcal plasmids (i) are not known to integrate with the host chromosome, and (ii) are transferred from cell to cell not by conjugation, but by certain transducing phages. . \ Stability of Extrachromosomal Elements to Oxygen In the genus Pasteurella the virulence of these organisms and their virulence antigens are coded for by an extrachromoso- mal element (Higuchi and Smith, 1961). Fukui, Ogg, Wessman, and Surgalla (1957) and Ogg, Friedman, Andrews and Surgalla (1958) showed that virulent cells, when inoculated into rich broth and aerated while incubating at 37 C, rapidly became avirulent. They attributed this phenomenon to a selective pro- cess where the avirulent cells could grow faster than the viru- lent cells in the high oxygen tension of the aerated medium. The exact extent of this phenomenon was not determined. The selective pressure was reversed if spent medium was added or the pH was adjusted to 7.8 prior to incubation. Similar cases have not been reported for the genus Staphylococcus. Stability of Plasmids .The stability of various extrachromosomal elements is a trait apparently coded for either by the plasmid itself, as is the case with certain of the staphylococcal penicillinase plas- mids (Richmond, 1966), or perhaps by the cell chromosome itself (Hatanabe and Ogata, 1970). The penicillinase plasmid in‘g. aureus may be stable; that is, lost at'a frequency less than 1/25000 colonies observed (Richmond, 1966) or more commonly at 3 a frequency of more like 10- per cell division (Novick and Richmond, 1965). Uncommonly, a strain may harbor a plasmid so unstable it is most difficult to maintain. The frequency of \ ,4 loss may be as high as 10-1 per cell division (Novick, 1966). Seggegation with Known Markers One way to establish the suspected gene locus as being extrachromosomal is to show that in nearly every case it segre- gates with a gene already known to reside on such an element. Novick (1963) showed that resistance to,mercuric ion-was extra- chromosomally located since the marker segregated with the penicillinase marker and was spontaneously lost at exactly the same frequency as was the penicillinase marker. While there has been close correlation between presence or absence of coa- gulase and extracellular nuclease in the same strain of g. aureus, Omenn and Friedman (1970) believe that the genes for these molecules reside on the chromosome and the concomitant loss is due to a coordinated control mechanism of some sort. On the other hand in Pasteurella pgstis_coagulase production is closely linked with fibrinolysin and pesticin I production. That one of these three substances might be an activator for the other two, or that some other molecule might activate all three, was eliminated by Brubaker, Surgalla, and Beesley (1965). But the remaining possibility that the three molecules are coded for by a genetic element distinct from the cell chromo— some is consistent with the evidence described by Beesley, Brubaker, Janssen and Surgalla (1967) insofar as they were able to show that the three factors were closely linked and had no relationship with virulence or any other property uniquely asso- ciated with E. pestis. Spontaneous or induced supressor muta- tions causing reversion to pesticin formation were not found. By analogy it would be of interest to consider the possi- bility of a close link between staphylocoagulase and a peculiar staphylococcal toxin, staphylococcin, described by Lachowicz (1965). Agents for the Selective Elimination of Extrachromosomal Elements A variety of agents, both selective and non-selective, have been used for the elimination of a plasmid or episome. Hirota (1960) described the use of proflavin and acridine for curing of the F+ state in several strains of E, coil. Using acriflavin or acridine orange Watanabe and Fukasawa (1961) cured antibiotic resistance in several strains of E3.£21£: Salmonella typhimurium, and Sl‘liqella flexneri. Willetts (1967) used acridine orange, UV, nitrosoguanidine, or ethylmethane 10 sulfonate to remove the Flggf episome from a strain of g, £213. 'Other intercalcating dyes such as ethidium bromide (Waring, 1965) have been shown to eliminate resistance to various antibiotics in both the enterobacteria and staphylo- cocci. Bouanchaud, Scavizzi, and Chabbert (1969) were able to cure three strains of Salmonella, two strains of Escherichia, and one strain of Shigella of streptomycin, ampicillin, kana- mycin, chloramphenicol, tetracycline, and sulfonamide resis- ~ tances at frequencies far greater than the spontaneous frequen- cy and in one case actually obtained a 100 per cent cure. Furthermore, in nine strains of Staphylococcus they found that if mercury resistance was carried by the strain it could be cured of both mercury and penicillin resistance with ethidium bromide. On the other hand, strains of Staphylococcus sensitive to mercury could not be cured of penicillin resistance using the same level of ethidium bromide. Hashimoto, Keno, and Mitsuhashi (1964) effectively cured several strains of S. aureus of a penicillinase plasmid using acridine orange, although Novick (1963) was not successful. A discussant of the paper given by Novick (1966), J. Borowski, pointed out that he could cure penicillin resistance with several non-selective agents such as quinine, thymol, or tween. Tomoeda, Inuzuke, Kubo and Nakamura (1968) removed both sex factors and resistance factors from E. ggli'using sodium dodcéyl sulfate. These findings are interesting in 11 terms of the possible role of attachment of either plasmids or episomes to membrane surfaces in either replication or distribution of progeny elements. A novel curing agent, rifampicin, has been shown by Johnston and Richmond (1970) to remove the penicillinase plas- mid from §. aureus. It is interesting insofar as rifamycin, the parent compoundhas been shown to directly inhibit RNA polymerase in g, 221;. .s Both May, Houghton and Perret (1964) and Asheshov (1966a) showed that plasmids containing the penicillinase and tetra- 'b ucycline resistance genes could be cured by extended growth of §, aureus at 43 to 44 C. The extent of curing depended on the strain and the rate of cure for the two markers was different. MATERIALS AND METHODS Isolation and Cultivation of Organism A slight modification of the technique of Smith, Morri— son, and Lominski (1952) was used in which an'§. aureus cul- ture was first inoculated in 4 ml brain heart infusion (BHI) in 13 x 100 mm styrofoam—stoppered culture tubes, incubated 2 to 4 days at 37 C until differentiable colonies appeared (16 to 20 hours). We picked and examined 37 strains of S. aureus quantitatively for coagulase production. One strain, obtained as a fresh clinical isolate from a local hospital, yielded a subculture with unique morphology and a very high coagulase titer. Colonial Morphology The colonial morphology of both the high and low coagu- lase—producing colonies was examined on a variety of media including BHI agar, nutrient agar, tryptic soy agar, and tryptose blood agar base either with or without blood. Several batches of each agar were used to insure the relia- bility of the observations. ‘ 13 Coagulase Assay A modification of the original quantitative assay for coagulase by Tager and Hales (1948) was used. The spent medium was initially diluted 1:100 in sterile 0.15 M NaCl (saline). From this working stock dilution serial two-fold dilutions were made in saline containing 2 per cent Bacto-peptone (Difco) with Merthiolate 1:1000 (Lilly). The final volume of each of these dilutions was 1.0 ml. To each dilution there was added 1.0 ml ‘ of a solution containing 0.3 per cent bovine fibrinogen (ci- trated fraction I; Sigma) and 0.002 per cent Human Factor IX Complex (KonyneTM; Cutter), an excellent, constant reproducible source of coagulase reacting factor (CRF). 'The mixture was. agitated slightly and incubated at 37 C for 24 hours. The re- ciprocal of the highest dilution containing any detectable clot was considered the titer for that sample. Spontaneous Reversion Tubes of BHI were inoculated with low coagulase, large creamy colony producing cells and incubated at 37 C. A sample of this culture was inoculated into fresh broth and again incu- bated. This culture in turn was diluted in saline and 0.1 m1 aliquots were spread on blood agar plates so as to give about 1000 colonies. After ineubation'of the plates at 37 C for 24 hours, each was examined under a dissecting micrOSCOpe for the presence of high coagulase producing small rough,whitc colonies. 14 Media Brain heart infusion (BHI) broth or agar, nutrient agar (RA), tryptic soy agar (TSA), heart infusion broth (HIB), tryp- tose blood agar base (TBAB) and DNase test agar were all purchas- ed from Difco Labs and were generally used as the directions specified. The tryptose blood agar base was usually supplem- ented with 5 per cent outdated, sterile, defibrinated, whole human blood. In one study 16 mm tubes containing 10 m1 heart infusion broth were supplemented with 5 per cent blood of the same quality as before. ISome of the blood-broth tubes were heated to 80 C for 5 minutes, then cooled to room.temperature. After inoculation with 0.1 ml of a 48 hour culture of S. aureus, 342-A, the cultures were incubated at 37 C for 48 hours, then diluted in saline. A 0.1 ml sample of this final dilution was spread on blood agar. After incubation'at 37 C for 24 hours the two colony types were counted and compared. Growth in a Liquid Medium under Various Gas Phases In these experiments a 125 m1 Erlenmeyer flask containing 20 ml sterile BHI, pH 7.2, was fitted with a two-hole rubber stopper through which a short and a longer piece of glass tubing was inserted. The longer tube was placed in the flask so that the lower tip was just short of the gas-liquid inter— face.-(F:ig. l). The gas in use was pumped through this tube and exhausted through the shorter tube. Natural gas was .15 .munesauemxe emmnm mam smwm+xmm¢m¥m$w as news mxmmam mo amummfia 3.” 5..th 16 burned; other gases were vented to the atmosPhere. The gas and medium equilibrated through agitation of the flask in a gyratory water bath shaker at 37 C (New Brunswick). The gases used included air, natural gas, N contaminated with approxi- 2 mately 3 per cent oxygen, and a certified gas mixture contain- ing 88 per cent N 5 per cent C0 and 7 per cent 0 One 2’ 2’ 2' per cent inoculum of S, aureus 342—A for the flasks was ob- tained from 1 to 2 day static tube cultures. Hourly samples were taken, diluted in saline, and plated on blood agar plates. After 24-hour incubation at 37 C large colonies were counted on a standard colony counter, and the Small colonies were enumerated with the aid of a dissecting microscope. Effect ofng on the Stability of the Selected Markers In the first study, 50 ml BHI in 25 x 200 mm screwcapped tubes at five pH values ranging from pH 6.0 in half pH unit increments to pH 8.0 were tested in duplicate. After 1 per cent inoculum from an overnight culture was added, the tubes were incubated at 37 C for 2 days. Each tube was then agitated, and the contents were diluted in saline and plated in duplicate on blood agar. The second study included only duplicate tubes ' of BHI broth at pH 7.2 and 7.9, but the general procedure was the same. Again, samples were diluted and plated in duplicate on blood agar. l7 Seggegation of Coagulase-Pigment-Colonia1 Morphology with Known Staphylococcal Plasmid Markers On blood agar plates spread with a diluted overnight cul- ture sufficient to give approximately 1000 colonies, three sensitivity discs were placed in a triangular fashion, maxi- mizing the distance between'dises. Each plate contained only one type of disc. The discs were impregnated with 0.01 ml of one of the following: erythromycin base; 20 ug, penicillin G; 12.1 units, Na HAsO 1 u mole, Cd(N0 2 4; 0.01 u mole, Pb(N0 3),; 3)2; 1 u mole, Zn(NO3)2; 1 u mole, or Hg(N03)2; 0.01 u mole. .Examination of DNase production was made using Difco DNase test agar. Plates of the test agar were inoculated with heavy streaks of the large or small colony producing cell type. Plates were incubated for 24 hours at 37 C and then flooded with 1 N HCl to precipitate any unhydrolyzed DNA. Examination of the two cell types was made to determine whether staphylococcin was produced or not. On common tryptose blood agar base plates supplemented with 0.05 per cent glucose each cell type (large or small colony producer) was inoculated in a 1 cm circle. After 48 hours incubation at 37 C the plates were covered with 4 ml of the same agar containing 4 x 105 test strain organisms per m1. Thirty—nine test strains were used including the cell types being tested for staphylococcin. Lysis of the strain in the area of growth of the original strain was taken as evidence of staphylococcin production. 18 Curing with Temperature Because of the failure of the one cell type to replicate under highly aerobic conditions, it was impossible to use shake flasks in the curing experiments. This problem preclu- ded taking samples during the course of the experiment so samples were taken at the beginning and the end of the incu— bation. Four tubes of BHI, pH 7.2, were all inoculated with 0.01 ml of a 48-hour mixed culture of S. aureus, 342—A. Two tubes were incubated at 37 C under static conditions; while the other two were incubated at 42—43 C under the same condi- tions. After 17 hours growth, samples of each tube of broth were diluted out in saline and 0.1 m1 of the appropriate dilu- tion was spread on the surface of each of two blood agar plates. After incubation of the plates for 24 hours, the two colony types were scored and analyzed statistically with the Chi Square Test. Curing with Acridine Orange Two experiments attempting to cure these cells of a plas— mid were carried out. In the first, four shake flasks each containing 10 ml BHI, pH 7.2, were inoculated (10 per cent) with an overnight static tube culture (mixed) of S. aureus, 342-A. The flasks were incubated for 1 hour at 37 C on a gyratory water bath shaker prior to addition of 10, 20, or 100 ug acridine orange (A0) to each of three of the flasks, 19 and incubated another 24 hours prior to termination of the experiment. During this time hourly samples were taken and diluted in saline. Tenth ml portions of several of the final dilutions were then spread on duplicate blood agar plates. After incubation of the plates at 37 C for 24 hours, the two colony types were scored. In the second experiment, 6 tubes containing 4 ml BHI, pH'7.2, were all inoculated with 0.01 ml of a 48-hour mixed culture of §, aureus, 342-A. Two tubes served as controls; two received acridine to a final concentration of 10 ug per ml, and the last two tubes each received 20 ug acridine orange per ml final concentration. After 17 hours incubation samples of each tube of broth were diluted out in saline and 0.1 ml of the appropriate dilutions were spread on the surface of duplicate blood agar plates. After incubation of the plates for 24 hours the two colony types wereoscored and analyzed statistically with the Chi Square Test. RESULTS Cultivation of Organism The organism, S. aureus, 342-A, was routinely grown 2 to 4 days in BHI broth at 37 C, then diluted out with saline and spread on blood agar plates. The high coagulase-producing small white colony was picked and inoculated back into BHI in a continuous cycle. The traits just mentioned, that is, the colonial morphology, pigment, and coagulase production all were such unstable traits that this procedure was the only way to maintain the stock. Colonial Morphology On all solid media tested the low coagulase—producing cell type produced a colony that glistened, was smooth, was fairly large sized (2 to 4 mm in diameter) and was creamy (occasionally turning to yellow upon prolonged incubation) in color and consistency (Figure 2). on all the media tested except the tryptose blood agar base with 5 per cent blood (blood agar) the high coagulase-producing cell type produced a colony which was small (1mm in diameter), rough, white and which retained its shape when teased with a needle. 20 21 '¢—— FIGURE 2. Photograph of high coagulase producing small, white, rough colonies and low coagulase producing large, creamy, smooth colonies (the bright area at the upper left of each of .the large colonies is a reflectibn of the oblique lights used to obtain the photograph). 22 Coagulase Assay The low coagulase—producing cell type eventually produced a coagulase titer ranging from 200 to 800. The high coagulase- producing cell type produced a final titer ranging from 6400 to 12,800. The titer variability was probably due to differ- ing incubation periods. Correlation of Morphology,_Pigmentation, and Coagulase Production No specific study was undertaken to establish the link- age of these traits, but it can be stated that all three traits have always been found together and never separated in this strain. Spontaneous Reversion No evidence has been yet found that a low coagulase pro- ducing large, creamy, colony cell type has reverted to the high coagulase producing small, rough, white colony cell type. The numbers examined admittedly are small (several tens of thousands) but at this frequency at least no revertants have been found. Media Final growth of S. aureus, 342-A, was examined in heart infusion broth. Since blood agar allowed a unique colony 23 type to develop, it was thought that perhaps the blood would enhance the growth and stability of the high coagulase produ- cing small, rough, white colony cell type. The results in Table 1 indicate otherwise. While the tubes of broth that contained no blood supported equivalent numbers of small and large colonies, the tubes containing blood in addition to the broth supported 100 times fewer small colony-producing cells. In the latter case the final numbers of large colony-producing" cells increased threefold in the case of the blood addition with no heating and increased sixfold in the case of the blood addition followed by 80 C heat treatment. From the data reported it is impossible to ascertain whether the increase in large colony-producing cells in the tubes containing blood was due to the enriched growth medium or due to the possibility of the small colony-producing cells dividing with the uni- lateral passage of the extrachromosomal element, which would increase the number of large colony-producing cells by the number of small colony—producing cells at every cell division. It is certain that fewer small colony-producing cells event- ually grew indicating a more complicated cell physiology than the large colony-producing cell type. Growth in a Liquid Medium under Various Gas Phases Q In the first experiment under normal atmosphere the num— bers of the two cell types were followed by hourly sampling w 24 TABLE 1. Effect of blood on the final number of colony forming units of each colony type' Media Colony forming units per ml’ a la col , sm col HIBC 1.18 x 108 2.28 x 108 HIB with - 8 6 5% blood 3.07 x 10 ' 3.8 x 10 HIB with 5% blood heated 8 . 6 80 c, 5 min. 6.8 x 10 3.5 x 10 aLarge smooth creamy colony, low coagulase-producing cell type bSmall rough white colony, high coagulase-producing cell type cHeart infusion broth 25 with subsequent dilution and plating. As can be seen from Figure 3, the low coagulase, large creamy colony-producing cell type grew quite well reaching stationary phase within 6 hours. However, the'high coagulase producing small rough white colony cell type appeared to remain in a static condition. Whether these cells were dividing normally but passing the extrachromo- somal element unilaterally or were not dividing at all is theo- retically treated in Figure 4. The straight line represents the low coagulase-producing cells dividing alone at hourly in- tervals. The upper, curved line represents these same cells dividing but at each cell division, the high coagulase-producing. cells each provide one more low coagulase-producing cell to the former population. Since the relative ratio of old low coagu— lase-producing cell to new low coagulase-producing cells is lowest early in the growth cycle, the constant addition of a fixed quantity of "new" low coagulase—producing cells results in a smaller ratio of new:old as new generations occur (the third line shows the percentage change in the low coagulase producing population due to the addition of new, low-producing cells). Since the early slope of the large colony producing cell type in Figure 3 can only be interpreted as a straight line, it was concluded that the high coagulase—producing cells were in fact some sort of induced non—reproducing stage. ~In the next experiment the normal atmosphere was replaced with natural gas. The flask was not entirely equilibrated with 26 FIGURE 3.- Growth in shake flask culture of large colony—producing cell type (-()-C)-) and small colony- producing cell type (:£§1£S-) with air as gaseous phase. , - . : LOG CPU/ML . m . ml... ’ ram HOURS - f. 0“- 28 FIGURE 4. Theoretical growth plot of large colony- producing cell type (-C>-()-) and large colony-producing cell type with an equivalent number of small colony—pro- ducing cell type turned large colony type by the unilateral passage of a plasmid (5131£§-). Initial points are based on observed data from Figure 2. Theoretical percentage change (-[J—[]-) in the low coagulase producing cell popu- lation due to addition of new low coagulase producing cells. 29 M. 23.31.) .71....) ‘3. .J\. J 1.1.141..de unlit/Lyn“. ..0.. LP. .f Vmu...h....( CW. IL. 0 0 0 0 8 .e . E. . 2 If... . [-Jut. MW «mu... 1 n11 u m - u u - 30 the new gas when inoculation took place as can be seen from Figure 5. Growth of both cell types started immediately, rapidly entering the log phase of growth. But soon after three hours, apparently all traces of oxygen vanished and there was a rapid decline in the number of cells which stabilized by the sixth hour of incubation. In the third experiment the gas phase was nitrogen, pre— sumably contaminated with approximately 3 per cent oxygen. Two flasks were used in this case: one containing BHI at pH 7.0 and the other containing BHI at pH 7.9. Both flasks were gassed 2 hours some twelve hours prior to initiating the ex- periment and again for 1 hour just before inoculation. The results as seen in Table 2 indicate growth of both the low and the high coagulase-producing cells was inhibited over the 8-hour duration of this experiment: In the final experiment of this series, 2 gaseous phases were used. One flask was aerated while the other flask was and 7 per cent 0 gassed with 88 per cent N 5 per cent CO 2’ 2’ 2' Once again this flask was gassed sufficiently beforehand to exclude all atmospheric oxygen. Table 3 and Figure 6 indicate that 7 per cent 0 is quite adequate for the support of the 2 low coagulase—producing cell type, but that it inhibited the growth of the high coagulase—producing cell type as completely . as did the atmOSpheric level of oxygen. 31 LOG cry/ML l> 6 I I I I O 2 4 6 8 HOURS FIGURE 5. Growth in shake flask culture of large colony- producing cell type (-C)—C)-) and small colony-producing cell type (~13-Z§—) in the presence of natural gas. 32 TABLE 2. Effect of a mixed gasa on the final number of colony forming units of each colony type Hour ' Colony forming units (x 106) pH 7.0 pH 7.9 1a colb sm colc 1a col I sm col 0 1.6 2.2 1.8 ' 3.2 l 3.1. 3.4 2.4 0.5 2 2.6 1.6 . 2.6 0.6 3 1.8 0.9 2:; 0.6 4 2.1 0.3 2.5 0.5 5 2.1 0.4 2.9 0.9 6 1.4 0.2 3.9 0.8 7 1.9 0.3 3.9 0.8 8 2.7 0.5 4.3 2.5 aNitrogen, 97%, oxygen 3% Large smooth creamy colony, low—coagulase-producing cell type cSmall rough white colony, high coagulase-producing cell type coho Haoo mcflOSUOLnlomoHSMmoo amen .hcoaoo mafia: :MSOh Manama was» Haoo unflosnopaloemHSMmoo 30H .hsoaoo hsmoao spooEm owned 33 3 Rs «cemhxo .Rm otflXOflt Conamo .wa .cowOprzm .O.N .Ovofi O.N .owo h m.v .OONH o.m .0mw_ o 0.5 .005 m.~ .msm m 0.2 .ove 0.“ .men e m.~ o.mo m.~ m.om m m.H o.ofi e.H c.e~ N mm.v ~.v O.N ©.m a s.m , mN.H m.m v~.~ .o Amoaxv H00 in Aoofixv Hoe mH Amofixv oaoo an Aoofixv nHoo mg otspxwe mow aw<. muss: mafiELom hcoaoo haom mo tones: Hmcwm esp no N vamp hcoaoo some mo mews: mcfishom heedoo canvass new a no use mo uoomum .m mqm§0 $0.. . House 36 Effect of pH on the Stability of the Selected Markers In these studies the pH of the culture medium was examined as a possible means of stabilizing these very unstable markers. The results of the first experiment are in Table 4. The large colony-producing cell type grew well over the whole range of pH values tested as evidenced by the final cell counts, and appeared to grow to highest yield at pH 7.0 or greater. The small colony—producing cell type yielded final cell counts ‘ similar to the other cell type though lower, with an indica- tion that pH 8.0 gave the highest yield of cells. Whether these data indicate that low pH inhibits growth or that high pH stabilizes this cell type, or perhaps both, cannot be ascertained. A In the second experiment only two pH values were examined. The results are depicted in Table 5, Large colony-producing cells appeared uneffected by either pH 7.2 or 7.9 as evidenced by the final growth yield. But by this same criterion, the small colony—producing cells appeared to be more stable at pH 7.9 as can be seen from the significant difference in the final cell yield. There are other possibilities for this dif— ference, but they cannot be separated out by this experiment. Actually separating the possibilities can only be done when the small colony—producing cell type can be routinely grown in shake culture and periodically sampled. TABLE 4. 37 Effect of pH on the final number of colony forming units of each colony type Average no. Average no. pH large colonies per ml small colonies per ml 7 7 6.0 5.25 x 10 less than 10 6.5 9.5 x 107 less than 107 . 8 7 7.0 6.17 x 10 6.25 x 10 7.5 5.1 Ax 108 1.5 x 107 8.0 4.9 x 108 8.0 x 107 38 TABLE 5. Effect of pH on the final number of . . a colony forming units of each colony type Tube Average no. Average no. number pH large colonies per ml small colonies per ml 1 7;2 6.5 x 108 3.0 x 108 8 8 2 7.2 8.3 x 10 2.0 x 10 q ..'\Vro, 2.5 X I()~ 3 7.9 7.0 x 108 . 4.1 x 108 4 7.9 8.8 x 108 6.3 x 108 8 Av., 5.2 x 10 aCalculated X2 value = 14.1; Table value (p 0.01) = 6.63 39 Segregation of Coagulase-Pigment-Colonial Morphology with Known Staphylococcal Plasmid Markers This experiment was conducted as a preliminary to a more fully developed design. The results in Table 6 indicate that further experimentation is not warranted. Both the high coa- gulase-producing cell type and the low coagulase-producing cell type exhibited exactly the same pattern to the sensitivity discs. Both cell types grew well up to the periphery of the' discs impregnated with the As, Cd,le, and Hg ions, while both cell types were inhibited to some extent by Zn ions, erythro- mycin, and penicillin. Thus, there is no segregation of the markers being studied here and certain known staphylococcal plasmid markers. Extracellular nuclease appeared to be produced equally well by both strains insofar as the cleared area in the test agar was the same for both strains. lStaphylococcin was produced by neither cell type. Thus, neither marker resided exclusively with one cell type or the other. Curing with Temperature Increasing the rate at which a specified culture yields a negative variant is usually called "curing" and can be done a number of ways in the genus Staphylococcus. In this experi- ment, a temperature increase (42 to 43 C) was used. While samples could not be taken during the course of the experiment, 40 TABLE 6. Correlation of markers under study with markers known to be extrachromosomally located in ' Staphylococcus aureus Antibacterial as Inhibitiongpattern‘ known marker Amount/disc 1a cola sm colD Erythromycin 20 ug 4+c ’ 4+. Penicillin G 12.1 units 4+' 4+ N82HA804 I u mole o o Cd(N03)2 0.01 u mole o o Pb(NO3)2 1 u mole o o Zn(N03)2 I u mole 2+ 2+ Hg(NO3)2 0.01 u mole o o aLarge smooth creamy colony, low coagulase-producing cell type type bSmall rough white colony, high coagulase-producing cell cRelative degree of inhibition varied from zero to 4+ 41 the number of small colony forming units in the final samples were significantly (p< 0.01) smaller that the number in the control samples (Table 7). This amounts to 51.61 per cent fewer small colony-producing cells in the tubes grown at 42 to 43 C than the control tubes. Curing with Acridine Orange In the last experiment, also depicted in Table 7, two con— centrations of acridine orange (A0) were used. The lower (10 ug/ml) had no curing effect whatsoever. The X2 value was 0.259 compared with a table value of 6.635. However, the higher level of A0 (20 ug/m.) significantly (p< 0.01) reduced the number of small colony-producing cells. The actual per— centage reduction of small colony—producing cells in 20 ug/ml A0 was 54.84. 42 TABLE 7. Effect of elevated temperature or acridine orange on the final number of colony forming units of each colony type \ Set Temp (c) Additions 1:02:22 foggigglgnitiémi 1 _ 37 -- 1.90 x 109 6.2 x 107 -- 2 43 -- 1.24 x 109 3.0 x 107 17.2 3 37 10 ug AOd/ml 1.56 x 109' 5.8 x 107 0.259 . 4 37 20 ug AO/ml 1.95 x 109 2.8 x 107 19.4 aLarge smooth creamy colony, low coagulase-producing cell type ' bSmall rough white colony, high coagulase-producing cell type \- cThe table value forix2 with one degree of freedom calcu- lated from the means is 6.635 dAcridine orange DISCUSSION The assortment of traits found to segregate together in this=strain of §p aureus appears to be unusual. There seems to be no functional connection between coagulase production, pigment formation, and a unique colonial merphology; yet these three markers segregate together during growth of this Strain. The high coagulase production can be accounted for in two alternative ways. The first and moSt reasonable explanation is that a second structural gene for coagulase is present in the high producing cells. That the second.type of coagulase might be substantially different from the chromosomally—coded molecule is evident since plasmids presumably do not show suffi- cient homology with the chromosome to integrate it. Miale, Winningham, and Kent (1964) showed the existence of multiple electrophoretic forms of coagulase from a single strain of S. aureus which supports the contention that there may be more than one molecular form of coagulase in a strain of S. aureus. The second eXplanation is that the gene in high coagulase producing cells codes for an effector molecule which in turn greatly potentiatcs the action of the existing staphylocoagu].use. The multifunctioning of this enzyme (Drummond and Tager, 1962) 43 44 strengthens this possibility. The possibility that the extreme potentiation is due to a multiple component enzyme is dismissed since the entire molecule has a molecular weight of about 44,000. Pigment formation reported in the literature (Barber, 1955; Bigger and Boland, 1927) has consistently been shown to change from the gold/yellow/aureus color to white/albus spon- taneously. In our case the change was just the opposite; that is, from white to yellow. A possible explanation for this is that in the high producing state the cell routinely uses a chromogenic intermediate as a substrate for another cellular reaction. Upon loss of the ability to use this intermediate, i.e., loss of the plasmid, the chromogenic intermediate is con- centrated by the cells yielding a pigmented colony. That a suppressor gene is present on the suspected plasmid is unlikely, insofar as the former explanation is more probable. The unique colonial morphology appearing as a phenotypic expression of the suspected plasmid has been previously docu- mented in the literature. Richmond (1968) showed that some ‘S. aureus cultures harboring a penicillinase plasmid produce a colonial morphology distinct from the plasmid—negative cell type. Ionesco (1953) reported a strain of Bacillus megaterium, which when infected with a lysogenic phage developed a colonial morphology quite distinct from the uninfected type. The colo— nial morphology of S. aureus 342-A exhibiting high coagulase 45 production and white color is unique among staphylococci. Upon the elimination of this suspected extrachromosomal element the colony type becomes typical, neither reverting nor exhi- biting the unique morphology again. - The fact that the high coagulase producing, small white rough colonies do not grow in a well-aerated medium is surpris- ing but not unique. A similar phenomenon exists with the viru- lent form of‘g. pestis (Fukui, Ogg, Wessman, and Surgalla, 1957; Ogg, Friedman, Andrews, and Surgalla, 1958). However, these authors did not determine the extent of the selection of the avirulent cell type over the virulent type. By the use of gas mixtures containing various concentrations of oxygen, we were able to show the critical nature of oxygen tension on growth of the high coagulase-producing, small white rough colony-producing cell type. Below seven per cent and perhaps below three per cent oxygen, but above zero oxygen tension of the gas phase the high coagulase producing cell type was able to grow at a rate quite similar to the low coagulase producing cell type. At higher oxygen tension selection is obvious; the low coagulase producing cell type could grow while the other could not. This fact was shown to be the case via use of the theoretical growth curves in Figure 4. That this phenomenon is the cause for the altered colonial morphology is unlikely since there are no colonial differences observed between the virulent and avirulent E. pestis. Stability of the typically unstable small colony cell type was . J 46 enhanced at the high pH value in_a manner similar to that shown by Ogg, Friedman, Andrews, and Surgalla (1958) for the viru— lent form of g. pestis. The failure of the observed markers to segregate with known markers in the genus Staphylococcus was partially expec- ted, as these well investigated markers are currently under heavy investigation in many laboratories and the likelihood of another marker remaining obscure until now was remote. How—. ever, it was felt that the Segregation of these markers with extracellular nuclease or staphylococcin was a much better possibility. The close relationship that appears to exist between coagulase and extracellular nuclease appears to be a chromosomal linkage, since there was no correlation between the high coagulase production and extracellular nuclease pro- duction. The reason for examining this organism for a possible linkage of the high coagulase marker with staphylococcin pro- duction was the tantilizing similarity in 2.. estis, which has a close linkage group containing the code for coagulase, fibri— nolysin, and pesticin I production. Curing of staphylococcal plasmids with temperature or acridine orange has been adequately described in the literature (Asheshov, 1966; May, Houghton, and Perret, 1964; Hashimoto, Kono, and Mitsuhashi, 1964). But because of the lack of a fair- ly stable cell type (high preportion of large colony producing cells in every inoculum) and the sensitivity to oxygen, we could not take advantage of these techniques, which necessitated 47 the sampling-of shake flask cultures. Growth of the small oblony producing cell type would have been inhibited due to aeration of the medium. Instead, samples could be taken only at the termination of the experiment, which of course, did not rule out the selective processes. However, the actual percent reduction in the number of small colony pro— ducing cells, while larger than those percentages reported by Asheshov (1966) using temperature to cure—or Hashimoto, Kon0'and Mitsuhashi (1964) using acriflavine, are similar to those reported by May, Houghton and Perret (1964) who cured auK§§.aureus strain of resistance to tetracycline and peni- cillin by use of elevated temperature. BIBLIOGRAPHY Asheshov, E.H., 1966a. Loss of antibiotic resistance in Staphy— lococcus aureus from growth @ high temperature. J. Gen. Microbiol. 42:402-10. \ Asheshov, E.H., 1966b. Chromosomal locatidn of the genetic ele- ments controlling penicillinase production in a strain of Staphylococcus aureus. Nature 210:804-806. Barber, M., 1949. 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Winningham, and J.W. Kent, 1964. Staphylo- coccal isocoagulases. Nature 197:392I Muth, W.L., and C.L. San Clemente, 1969. Temperature studies on staphylococcal coagulase by the use of a turbidimetric assay. Bacteriol. Proc. 62;93. Novick, R.P., 1963. Analysis by transduction of mutations affecting penicillinase formation in Staphylococcus aureus. J. Gen. Microbiol. 33:121-136. Novick, R.P., and M.H. Richmond, 1965. Nature and interactions of the genetic elements governing penicillinase synthesis in Staphylococcus aureus. J. Bacteriol. 29:467—480. Novick, R.P., 1966. Extrachromosomal inheritance of antibiotic resistance in Staphylococcus aureus. Postepy Mikrobiologii to V, 20 2, 345-359. Novick, R.P., 1967. Penicillinase plasmids of Staphylococcus aureus. Federation Proc. 26:29-38. Novick, R.P., and C. Roth, 1968. Plasmid-linked resistance to inorganic salts is StaphyloCoccus aureus. J. Bacteriol. Q 951335-1342. 52 Novick, R.P., 1969. Extrachromosomal inheritance in bacteria. 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The elimination of Flac from Escherichia coli by mutagenic agents. Biochcm. BiOphys. Res. Comm. MICHIGAN STATE UNIVERSITY LIBRARIES II IIIIIII llllllli 3 1293 3196 6934 i