Toxigenic studies with the antibiotic pigments from Serratiu marcescens 3 2
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G. S. KALESPERIS, K. V. PRAHLAD, AND D. L. LYNCH Department ofBiologica1 Sciences, Northern Illinois University, DeKalb, Illinois 60115 Accepted September 13, 1974 KALESPERIS, G . S., K. V . F'RAHLAD, and D. L. LYNCH.1975. Toxigenic studies with the antibiotic pigments from Serratia rnarcescens. Can. J . Microbiol. 21: 213-220. Prodigiosin, obtained from the bacterium, Serratia marcescens, was extracted in five organic solvents, petroleum ether, chloroform, acetone, ethanol, and methanol, and the fractions were labeled PE-I, C-2, A-3, E-4, and M-5 respectively. The effects of prodigiosin and its fractions on embryogenesis showed the whole pigment and C-2 fraction to be highly toxigenic while other fractions demonstrated toxicities approaching LDso values of 26-30 pglegg when dissolved in 100% dimethyl sulfoxide. The E-4 fraction in DMSO was least toxic. Ninety-five percent ethanol proved to be highly toxic at a dose level of 0.1 ml/egg indicating that it was an unsuitable solvent for studies of this nature. Disc-agar diffusion sensitivity studies were performed against E. coli, E. aerogenes, S . aureus,B. s~rbtilis,and P. aeruginosa with prodigiosinand fractions dissolved in 100% DMSO. The solvent was found to have no diffusible bacteriostatic activity in vitro. However, prodigiosin and the ethanol (E-4) and methanol (M-5) fractions produced inhibition zones with every organism tested. Data presented below indicate that prodigiosin extracts have toxigenic effects on chick embryos and inhibit the growth of several species of bacteria. KALESPERIS, G . S., K. V. F'RAHLAD et D. L. LYNCH.1975. Toxigenic studies with the antibiotic pigments from Serratia tnarcescens. Can. J . Microbiol. 21: 213-220. Une prodigiosine, produite par Serratia rnarcescens, a ete extraite dans cinq solvants organiques soit I'tther de g t r o l e , le chlorofome, I'acetone, I'ethanol et le methanol et les fractions ont ete Ctiquetees dans l'qrdre PE-I, C-2, A;3, E-4 et M-5. L'effet de la prodigiosine et de ses fractions montre que le pigment intact et la fraction C-2 sont hautement toxiques et que les autres fractions ont des toxicites voisines des valeurs de la LDso soit 26-30 pgloeuf lorsque dissoutes dans du dimethyl sulfoxyde B 100%. Dans ce DMS0,c'est la fraction E-4qui est lamoins toxique. L'ethanol a 95% est hautement toxique a la dose de 0.1 mllceuf, ce qui indique que ce solvant ne convient pas B des etudes de ce genre. Par la mkthode des disques sur gelose, on a v6rifi6 la sensibilite d'E. coli, d'E. aerogenes, deS. aureus, deB. subtilis et de P. aeruginosa vis-a-vis de la prodigiosine et des fractions dissoutes dans du DMSO B 100%. In vitro ce solvant n'a aucune activite bacteriostatique diffusable. Par contre la prodigiosine et les fractions de I'ethanol (E-4) et du methanol (M-5) produisent des zones d'inhibition avec chacun des microorganismes 6tudiCs. Les resultats present& ici indiquent que les extraits de prodigiosine ont des effets toxiques sur les embryons de poulet et inhibent la croissance d e quelques esptces bactiriennes. [Traduit par le journal]
Introduction Prodigiosin is the characteristic red pigment commonly produced by Serratia marcescens. The nomenclature of this water-insoluble, 20-carbon tripyrrole was ultimately derived by Kraft (13), who extracted the red "prodigiosine" from Bacillus prodigiosus. Before the actual isolation and identification of prodigiosin, observations of antibiotic properties of pigmented cultures of S. marcescens were first reported in 1885. These early investigations (1) described antagonistic effects both in vitro and in vivo of pigmented cultures against many bacterial and fungal 'Received March 22, 1974. ZPublicationno. 518, Department ofBiological Sciences, Northern Illinois University, DeKalb, Ill. 601 15.
species. Since that time, the pigment has been evaluated for its antibiotic, antigenic, and antitumor effects. Another characteristic first observed (3) and later refined by a number of workers indicated that prodigiosin was probably a complex of closely related components which could be separated into several different fractions with antibiotic properties (4, 7). Considering the antibiotic nature of prodigiosin and in spite of the importance many other antibiotics have served in current chemotherapy, it is interesting to note that only a select few are recommendedto be used durini pregnancy. This is due the fact that many antibiotics as as other drugs, which are comparatively well
214
CAN. J. MICROBIOL. VOL. 21, 1975
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tolerated by the maternal organism, may have adverse effects on the fetus at certain stages of embryogenesis. The most feared outcome deterring the usage of these drugs is the range of adverse and toxigenic effects which might result in fetal death or malformations (18, 19).
Materials and Methods The pigment extracts were obtained from cultures of S . niarcescens wild-type strain 75. The cells were grown and harvested according to techniques previously described (14). The prodigiosin pigment was extracted in glacial acetic acid, taken to dryness, and the fractions were obtained with the following successive extracting solvents: 100% petroleum ether, 100% chloroform, 100% acetone, 95% ethanol, and 100% methanol, respectively. The fractions from the various solvents were designated as PE-1, C-2, A-3, E-4, and M-5. Enlbryotoxic Studies Freshly laid fertilized eggs from White Leghorn breed were obtained from a commercial hatchery and were separated into seven groups according to substance injected. All eggs were incubated in a Jamesway incubator (model No. 252 B) at 38.3"C at 60% humidity (16). After 27-28 h of incubation (embryonic development about 24 h) all eggs were candled and control and experimental media were injected into the yolk sac if fertility was confirmed by the presence of a blastodisc. All eggs were candled daily for the first 5 days and then once every 5 days until hatched. Throughout the course of incubation. eggs with defective or stunted embryos were isolated from their respective groups and allowed to incubate an additional 24 h before they were opened for examination. This was to ensure that no error in candling had occurred thus killing embryos that may have been retarded to some degree in growth. Estimated time of embryonic death was determined using Hamburger and Hamilton stages (10) and all abnormalities were similarly recorded, labeled, and fixed in Bouin's or 10% formalin. After the chicks had hatched, they were kept for 72 h in a heated box with water and food to determine posthatching mortality. Antibiotic Sensitivity The antimicrobial activity of prodigiosin and its fractions were determined by the disc-agar diffusion method (2). Organisms used in this study were Escherichia coli, Enterobacter aerogenes, Staphylococcus arrreus, Bacill~s subtilis, and Pse~rdomorras aerrrginosa. Cultures were first grown in nutrient broth with 0.1% dextrose for 24 h on a rotary shaker a t 50 rpm and incubated at 37°C. An inoculum size of 0.1 ml from each stock culture was aseptically pipetted into 15 ml of sterile, liquid TGEA3 held at 45°C in a water bath, lightly vortexed, and plated. After the medium solidified, all plates were held at 5"C until use. Five sets of plates were incubated for 18-20 h at 37°C. The presence of zones of growth inhibition around these discs was used as the parameter to assess the antimicrobial activity of prodigiosin and its fractions.
3TGEA-tryptone glucose extract agar.
Results Embryo Toxicity Tables 1 and 2 illustrate the embryotoxic effects induced by various fractions, whole prodigiosin, and the solvents used. As illustrated in Table 1, dimethyl sulfoxide (DMSO) alone surpasses ethanol as a suitable injection solvent at both concentrations of 0.05 and 0.1 ml with hatchabilities of 100% and 75% respectively for DMSO and 83.3% and 25% for ethanol. Prodigiosin in DMSO (65 pgl0.05 ml and 130 pg/ 0.1 ml) gave hatching percentages of 94.4 and 72.2, whereas these values were lower with prodigiosin in ethanol (58.8 and 22.2). Because of these results, DMSO was used as the solvent for all trials thereafter. Data from all trials (Table 2) illustrate the high percentage of survival in the E-4 treated group and in comparison a high mortality in animals injected with all other fractions and whole pigment. Also listed in this table are hatchability results from a total of 42 eggs that were not injected (89.0%) to compare with those results of our controls. Typical toxigenic effects of solvent, prodigiosin, and its fractions on chick embryo are depicted in Fig. 1, A through L. As described in the figure legends it is seen that both DMSO and E-4 fraction in DMSO are least embryotoxic. However, solvent ethanol itself and other fractions of the pigment prodigiosin proved to be more detrimental with respect to the survival of a greater number of chick embryos as indicated by both pre- and post-natal deaths (Table 2). Antibiosis Data from the observations of antimicrobial activity of prodigiosin and its fractions in all solvent systems used are listed in Table 3. From these results, it was observed that all solvents, with the exception of glacial acetic acid, demonstrated no microbial inhibition even at the dose level of 0.05 ml. Although glacial acetic acid would obviously never be used as an injection media, it was interesting to note that prodigiosin in this particular solvent as well as at this extreme in \pH still possessed greater inhibitor) effects than the acid alone. This is especiall) outstanding with E. coli at the dose level 01 0.03 ml with the acid alone giving a zone diameter of 21.3 & 1.4 mm and 5 1 pg prodigiosin in 0.03 ml acid showing a zone of growth inhibitior
215
KALESPERIS E T AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
TABLE 1 Comparative analysis of abnormalities and toxicity of prodigiosin in ethanol vs. prodigiosin in DMSO Injected substance, pg injected/ml solvent
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95% ethanol
Total eggs injected A. Prenatal deaths (abnormal) B. Hatched (no abnormalities) % Percentage of abnormal occurrence
.
Pr;digiosinl 95 ethano
DMSO
12 2
12 9
11 0
12 3
10 83.3
(2) 3 25.0
11 100.0
0.0
16.7
0.0
18 14
18 1
18 5
9 75.0
17 7 (4) 10 58.8
22.2
17 94.4
13 72.2
0.0
23.5
16.7
0.0
0.0
6)
TABLE 2 Composite of treatments and solvents used in the antibiotic studies Substance injected, pg1O.l ml DMSO
Total eggs injected Percentage hatched Survival percentage of those hatched Percentage - mortality*
Prod
PE- I
C-2
A-3
E-4
M-5
None
DMSO
44
28
26
26
48
30
42 89.0
46 71.7
34 58.8
36 58.3
36 61.1
36 55.6
35 82.9
36 66.7
85.7 14.3
71.7 28.3
50.0 50.0
52.8 47.2
55.6 44.4
41.7 58.3
77.1 22.9
50.0 50.0
*Includes all embryos which did not hatch and those which died within 48 h after they had hatched.
+
amounting to 41.7 8.9 mm, a difference of 20.4 f 7.5 mm. None of the discs charged with pigment fractions in petroleum ether, chloroform, acetone, ethanol, Bnd methanol exhibited any growth inhibition for all organisms tested. It should be noted at this point that Pseudomonas was used only in the sensitivity studies incorporating the solvent DMSO, thus accounting for the blanks in Table 3. From these results, it can be seen that PE-I, C-2, and A-3 in DMSO also failed to exhibit any in vitro antimicrobial activity, thus eliminating these fractions as possible bacteriocidal substances. However, prodigiosin, E-4, and M-5 in DMSO showed definite growth inhibition for all organisms tested including Pseudomonas with comparable antibiotic properties between the whole pigment and E-4 fraction. Least zonal inhibition occurred with M-5. The above observations were compared with commercially prepared discs from Difco laboratories using the antibiotics chloromycetin (chloramphenicol), penicillin, tetracyclin, aureomycin,
and ampicillin. It was apparent that the most effective of these was aureomycin with respect to overall inhibitory action against E. coli, E. aerogenes, S. aureus, and B. subtilis; however, in spite of its efficiency against these organisms, it failed to exhibit any zonal occurrence against Pseudomonas (Table 4). Both cultures of P. aeruginosa with the five commercial discs showed no zonal inhibition in comparison to the inhibition demonstrated by the E-4 and M-5 fraction and prodigiosin against this microorganism.
Discussion Although several investigators have observed morphological alterations with DMSO in fowl and mammalian embryos (5, 6 , 8, and 15), the occurrence of abnormal embryos induced in the controls was limited to 1 out of 79 eggs injected with 100% DMSO. Furthermore, the one abnormal embryo had been injected with 0.1 ml DMSO which is equivalent to 100 mg compared with 10-12 mg reported by Caujolle et al. (5). This deviation is not due to a difference in breed,
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216
CAN. J. MICROBIOL. VOL. 21, 1975
ABBREV!ATIONS USED:
(0)anterior,
(be) beak, (by) brain, ( e ) eye, (11) heart, (Ib) limb bud, ( n ) nasal aperture,
( p ) poster~or.
F I G . IA-F. Embryotoxicity of pigments in 95% ethanol. A. Abnormality induced by the injection of 0.1 nil ar was dead after 70 h of incubation. Of p a r t i c ~ ~ l interest ar is the enlarged 95% EtOH. This p a r t i c ~ ~ l embryo al Two other embryos developed similar features. B. (a) This embryo heart and stunted n e ~ ~ r development. n. injected with 27 pg C-2 in 0.05 nil EtOH, it developed twinned died after about 15 days of i n c ~ ~ b a t i oInitially tr~lnkswith a common cephalic region having a n inverted beak and exposed brain. (b) Injected with 41 pg C-2 in 0.05 ml EtOH, the animal had bilateral anophthalmia with inverted superior beak. This embryo was full-term and had pecked at shell, yet died without completing hatch. C. Embryo injected with 207 pg E-4 in 0.05 nil EtOH was alive when opened after 96 h incubation. Aside from lack of posterior differentiation and abnormal optic development, this embryo appeared to be retarded in morphological development by about 36 h. D. Embryo injected with 361 pg M-5in 0.05 ml EtOH died between 72 and 96 h of incubation. The only morphological development that was recognizable was the eye pigment. E. Embryo injected with 120 pg prodigiosin in 0.1 ml EtOH. Egg was opened at 6 days, heart was still beating yet enibryonic developnlent appeared to have only reached 55 h. Note again the enlarged heart and stunted posterior development similar to Fig. IA. F. This embryo exemplified three such abnormal trunk flexures induced by 60 and 120 pg prodigiosin in ethanol. Limb bud and optic development also appeared to be inhibited in these embryos with all dying between 64 and 76 h.
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KALESPERIS ET AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
FIG.IG-L. Embryotoxicity of pigments in 100x DMSO. G. Embryo injected with 0.1 ml DMSO dead at about 3-5 days. This was the only abnormality induced by the solvent and was characterized by abnormal optic development and lack of abdominal wall. H. Dead at about 3 days, this embryo was injected with 13 pg C-2 in 0.05 ml DMSO. Note the similarity of posterior twinning with Fig. 1, (a),enlarged heart, and stunted cranial development. I. Four-day embryo with bilateral anophthalmia, no limb buds, and retarded heart development was injected with 26 pg C-2 0.1 ml DMSO. J. Only abnormality induced by E-4 in DMSO was injected with 48 pg. Age of death was estimated between 48 and 72 h. K. Chick exemplifies condition induced by both 30 pg M-5 in 0.1 ml DMSO and 44 pg prodigiosin in 0.1 ml DMSO characterized by extreme cervical flexure. Both animals died within 48 h after they were hatched. L. Abnormality induced by 44 pg prodigiosin in 0.1 ml DMSO. Cranial and cardiac development of this 43-day embryo appeared normal; however, no budding or posterior trunk development was noted.
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CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 3 In vitro bacterial inhibition by prodigiosin and its fractions in several solvents with zone diameters expressed in milliliters
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Disc Nos.
5-23
24
un
ml solvent
Listed below
0.01 0.02 0.03 0.05 0.01 0.02 0.03
4.9 9.8 14.7
E. coli 1 . 9 x lo7
E. aerogenes 1 . 8 lo9 ~
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.7k0.4 9.8k0.7
S. aureus
2.8 x lo8
B. subtilis 2 . 8 lo6 ~
-
8.4k0.5
8.6k0.3
P . aeruginosa 1.3 x lo7*
-
10.8k0.6
TABLE 4 Comparative zone diameters of prepared Bacto sensitivity discs Chloromycetin (chloramphenicol) 30 pg Eschericllia coli Etiterobacter aerogenes Stapliylococcus aweus Bacill~ssubfilis Psertdot?iot?asaerl~ginosa
32.0k2.0 20.0k0.5 19.0+ 1 .O 30.0k0.5 -
Penicillin 10units=16pg 12.0k2.0 -
7.5k0.5 20.0k0.5 -
for Caujolle compared the results of Rhode Island chickens with those of Leghorns and found a similar sensitivity. Moreover, these concentrations used by Caujolle represent LD,, values, values which we never observed since survival at 0.05 ml(55 mg DMSO/embryo) for 33 control embryos approached a mean of 90.0 5.4% and survival at 0.1 ml (1 10 mg DMSO/embryo) for 46 control embryos approached 71.7 +_ 1.7%.
+
Aureomycin Tetracycline (chlortetracycline) 30pg 30
Ampicillin 10 ~g
31.0k1.0 18.0k0.5 20.0k0.5 38.0k2.0
38.0k0.5 12.0k0.5 20.0k0.5
30.0k2.0 20.0k0.5 27.0k 1 .O 40.0k0.5 -
-
The observations by Caujolle et al. (5) of malformations induced by DMSO were similar in a few cases to those induced by the whole pigment and two of the pigment fractions, malformation of beak (parrot beak in prodigiosin, C-2 and A-3) and anophthalmia of the left eye (C-2 and A-3). However, if one takes into consideration the differences in DMSO concentrations, the higher percentage of abnormal embryos seen in the
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KALESPERIS ET AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
219
prodigiosin, C-2, and A-3 groups (Table I), and observation eliminated the possibility of the the incidence of posterior twinning induced by solvent being the diffusible static agent, it was C-2 in 0.05 ml ethanol as well as in 0.05 ml also noted that no growth had occurred beneath DMSO indicate that the pigment and some of the discs charged with DMSO, i.e., inhibition the fractions and not the solvent are indeed was limited to the diameter of the disc. Previous responsible for the induction of malformations investigators have reported a bacteriostatic or bacteriocidal effect of DMSO at concentrations and toxicity in the chick embryo. The most outstanding feature of our investiga- from 1 to 50% for a variety of bacteria (1 1, 12, tion was the consistency of greatest survival of and 17). However, standard dilution assays were the E-4 chicks over all other experimental used in their studies. The difference in results groups. Not only did this fraction demonstrate when this technique is compared with our a greater survival than the whole pigment and method is evident since with standard dilution other fractions with both 0.05 and 0.1 ml of there is a large margin of error in attributing solvent, but hatching as well as survival percent- static effects to a certain antibiotic when the ages exceeded the control group injected with solvent may be equally capable of inhibiting bacterial growth. 0.1 ml DMSO. Burgova et al. (4) and Efimenko et al. (7) have A number of embryonic deaths also occurred during the last several days of incubation and reported that they found a fraction that posseswithin 48 h after hatching; this may have been sed antibiotic activity, which appears to be differdue to the absorbance of the remaining yolk ent in many physical properties from our fracand in turn to the toxicity of the residual pig- tions. Both of them stated that their antibiotic ment. DMSO was ruled out as a causative agent fraction showed a red coloration; however, in since there were no posthatch deaths in our the present study the only fraction that came close to fitting this description was the C-2 fraccontrols. From the results of our sensitivity studies, it tion which proved to be totally worthless as an is apparent that the E-4 fraction in 100x DMSO antimicrobial agent in vitro and was highly was more bacteriostatic than any of the other toxigenic during embryogenesis. In conclusion the E-4 fraction from the pigfractions except for B. subtilis which showed the greatest inhibition of growth with the crude ment prodigiosin has been shown to be least prodigiosin in DMSO. In spite of the fact that embryotoxic and to inhibit the growth of several concentrations greater than 14.7 pg were not species of bacteria. tested, it is interesting to note from Table 3 that at a similar concentration the whole pigment was 1. ABRAHAM, E . P., and H. W . FLOREY. 1949. Antibioless effective than the E-4 fraction except for tics fromchromogenic bacteria.In Antibiotics. Vol. 1. Edited by H . W. Florey, E . Chain, N . G . Heatley, M. B. subtilis. Special consideration should be A. Jennings, A . G. Snaders, E . P. Abraham, and M. E . directed toward the inability of prodigiosin to Florey. Oxford University Press, London. pp. 558-562. inhibit Staphylococcus aureus at a concentration 2. BARRY,A . L. 1964. The routine antibiotic disc-plate of 12.9 pg in 0.03 ml DMSO, whereas E-4 demonsensitivity test. 11. Technological variations. Am. J. strated a positive zonal inhibition. Furthermore, Med. Technol. 30(5): 333-342. 3. BUNTING,M. 1. 1940. A description of some color the definite inhibition of P. aeruginosa by provariants produced by Serratia marcescens strain 274. digiosin as well as E-4 places these antibiotics in J. Bacterial. 40: 57-68. a position of distinction since relatively few M. P., E. V . LOVYAGINA, N. N. FAL~NA, 4. BURGOVA, and A. L. GOL'DENBERG. 1957. A study of theabsorpantibiotics are capable of suppressing this organtion suectra of the antibiotic from Bacteri~rm oroism. Current drugs of choice against Pseudodigios;m. Primenenie Metodov Spektroskipii v 6 o m . Prodovol'stven. 1955: 173-179. monas include colistin, polymyxin, carbenicillin, 5. CAUJOLLE, F . M. E . , D. H. CAUJOLLE, S . B. CROSS, and gentamicin (9); however, since none of these and M. M. Y . CALVET.1967. Limits of toxic and substances has yet been extensively studied in teratogenic tolerance of dimethyl sulfoxide. Ann. N . Y . Sci. 141(1): 100-126. regard to their effects on embryogenesis, it is 6. CROS.S . . M. C. V O I S I N . TOLLON. ~. and J. OREGLIA. necessary to view them as a possible risk if taken 1 9 7 0 . ' ~ a m a e eof limb'buds of chicken embrvo bv dimethylsu16xide. (In French, English sumr;laryj. during pregnancy. Ann. Pharm. Fr. 28(4): 263-270. Prior determination of the effects of 100% 7 . EFIMENKO, 0. M., G. A . KUZNETSOVA, and P. A. DMSO in our studies showed no zonal inhibition YAKIMOV.1956. Prodigiosin - an antibiotic from for any of the organisms tested. Although this Bacterium prodigiosum. Biokhimiya, 21(3): 4 1 9 4 2 3 .
220
8. FERM,V. H. 1966. Teratogenic effect of DMSO. Lancet, 7430: 208. 9. GOODMAN. L . S.. and A. GILMAN.1970. The ~ h a r macologicil basis bf therapeutics. 4th ed. The ~ ; ~ m i l Ian Co., London. 10. HAMBURGER, V., and H. L. HAMILTON.1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92. 11. JACOB.S. W.. R. J. HERSCHLER. and M. D. BISCHEL. 1964. ~ i m e t h )sulfoxide ~ (DMSO): a new concept in parmoco-therapy. Curr. Ther. Res. Clin. Exp. 6: 134-135. 12. KLIG;;~, A. M. 1965. Dimethyl sulfoxide. Part 2. J. Am. Med. Assoc. 193: 923-928. 13. KRAFT,E . 1902. Beitrage zur biologie des B. prodigios~tsund zum chemischen verhalten seines pigments. Thesis, Wurzburg, Germany. (Cited by Williams and Hearn 1967). and G. C. KRESHECK. 14. LYNCH,D. L., T. E. WORTHY, 1%8. Chromatographic separation of the pigment ~
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CAN. J. MICROBIOL. VOL. 21. 1975
-
-
-
-
~
-
-
15. 16. 17.
18. 19.
fractions from a Serratia marcescens strain. Appl. Microbiol. l6(1): 13-20. MARIN-PADILLA. M. 1966. Mesodermal alterations induced by dimethyl sulfoxide. R o c . Soc. Exp. Biol. Med. 122: 717-720. NEW, D. A. T. 1966. The culture of vertebrate embryos. Academic Press, New York. POTTZ,G. E., J. H. RAMPEY, and F. BENJAMIN. 1967. The effect of dimethyl sulfoxide (DMSO) on antibiotic sensitivity of a group of medically important microorganisms: preliminary report. Ann. N.Y. Acad. Sci. 141(1): 261-272. ROBSON,J. M. 1963. The action of drugs on the embryos. Pharmacological considerations. Proc. R. Soc. Med. 56: 600605. WOLLAM,D. H. M., and J. W. MILLEN.1963. The action of drugs on the embryo. Experimental mammalian teratology and the effects of drugs on the embryo. Proc. R. Soc. Med. 56: 597601.
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G. S. KALESPERIS, K. V. PRAHLAD, AND D. L. LYNCH Department ofBiologica1 Sciences, Northern Illinois University, DeKalb, Illinois 60115 Accepted September 13, 1974 KALESPERIS, G . S., K. V . F'RAHLAD, and D. L. LYNCH.1975. Toxigenic studies with the antibiotic pigments from Serratia rnarcescens. Can. J . Microbiol. 21: 213-220. Prodigiosin, obtained from the bacterium, Serratia marcescens, was extracted in five organic solvents, petroleum ether, chloroform, acetone, ethanol, and methanol, and the fractions were labeled PE-I, C-2, A-3, E-4, and M-5 respectively. The effects of prodigiosin and its fractions on embryogenesis showed the whole pigment and C-2 fraction to be highly toxigenic while other fractions demonstrated toxicities approaching LDso values of 26-30 pglegg when dissolved in 100% dimethyl sulfoxide. The E-4 fraction in DMSO was least toxic. Ninety-five percent ethanol proved to be highly toxic at a dose level of 0.1 ml/egg indicating that it was an unsuitable solvent for studies of this nature. Disc-agar diffusion sensitivity studies were performed against E. coli, E. aerogenes, S . aureus,B. s~rbtilis,and P. aeruginosa with prodigiosinand fractions dissolved in 100% DMSO. The solvent was found to have no diffusible bacteriostatic activity in vitro. However, prodigiosin and the ethanol (E-4) and methanol (M-5) fractions produced inhibition zones with every organism tested. Data presented below indicate that prodigiosin extracts have toxigenic effects on chick embryos and inhibit the growth of several species of bacteria. KALESPERIS, G . S., K. V. F'RAHLAD et D. L. LYNCH.1975. Toxigenic studies with the antibiotic pigments from Serratia tnarcescens. Can. J . Microbiol. 21: 213-220. Une prodigiosine, produite par Serratia rnarcescens, a ete extraite dans cinq solvants organiques soit I'tther de g t r o l e , le chlorofome, I'acetone, I'ethanol et le methanol et les fractions ont ete Ctiquetees dans l'qrdre PE-I, C-2, A;3, E-4 et M-5. L'effet de la prodigiosine et de ses fractions montre que le pigment intact et la fraction C-2 sont hautement toxiques et que les autres fractions ont des toxicites voisines des valeurs de la LDso soit 26-30 pgloeuf lorsque dissoutes dans du dimethyl sulfoxyde B 100%. Dans ce DMS0,c'est la fraction E-4qui est lamoins toxique. L'ethanol a 95% est hautement toxique a la dose de 0.1 mllceuf, ce qui indique que ce solvant ne convient pas B des etudes de ce genre. Par la mkthode des disques sur gelose, on a v6rifi6 la sensibilite d'E. coli, d'E. aerogenes, deS. aureus, deB. subtilis et de P. aeruginosa vis-a-vis de la prodigiosine et des fractions dissoutes dans du DMSO B 100%. In vitro ce solvant n'a aucune activite bacteriostatique diffusable. Par contre la prodigiosine et les fractions de I'ethanol (E-4) et du methanol (M-5) produisent des zones d'inhibition avec chacun des microorganismes 6tudiCs. Les resultats present& ici indiquent que les extraits de prodigiosine ont des effets toxiques sur les embryons de poulet et inhibent la croissance d e quelques esptces bactiriennes. [Traduit par le journal]
Introduction Prodigiosin is the characteristic red pigment commonly produced by Serratia marcescens. The nomenclature of this water-insoluble, 20-carbon tripyrrole was ultimately derived by Kraft (13), who extracted the red "prodigiosine" from Bacillus prodigiosus. Before the actual isolation and identification of prodigiosin, observations of antibiotic properties of pigmented cultures of S. marcescens were first reported in 1885. These early investigations (1) described antagonistic effects both in vitro and in vivo of pigmented cultures against many bacterial and fungal 'Received March 22, 1974. ZPublicationno. 518, Department ofBiological Sciences, Northern Illinois University, DeKalb, Ill. 601 15.
species. Since that time, the pigment has been evaluated for its antibiotic, antigenic, and antitumor effects. Another characteristic first observed (3) and later refined by a number of workers indicated that prodigiosin was probably a complex of closely related components which could be separated into several different fractions with antibiotic properties (4, 7). Considering the antibiotic nature of prodigiosin and in spite of the importance many other antibiotics have served in current chemotherapy, it is interesting to note that only a select few are recommendedto be used durini pregnancy. This is due the fact that many antibiotics as as other drugs, which are comparatively well
214
CAN. J. MICROBIOL. VOL. 21, 1975
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tolerated by the maternal organism, may have adverse effects on the fetus at certain stages of embryogenesis. The most feared outcome deterring the usage of these drugs is the range of adverse and toxigenic effects which might result in fetal death or malformations (18, 19).
Materials and Methods The pigment extracts were obtained from cultures of S . niarcescens wild-type strain 75. The cells were grown and harvested according to techniques previously described (14). The prodigiosin pigment was extracted in glacial acetic acid, taken to dryness, and the fractions were obtained with the following successive extracting solvents: 100% petroleum ether, 100% chloroform, 100% acetone, 95% ethanol, and 100% methanol, respectively. The fractions from the various solvents were designated as PE-1, C-2, A-3, E-4, and M-5. Enlbryotoxic Studies Freshly laid fertilized eggs from White Leghorn breed were obtained from a commercial hatchery and were separated into seven groups according to substance injected. All eggs were incubated in a Jamesway incubator (model No. 252 B) at 38.3"C at 60% humidity (16). After 27-28 h of incubation (embryonic development about 24 h) all eggs were candled and control and experimental media were injected into the yolk sac if fertility was confirmed by the presence of a blastodisc. All eggs were candled daily for the first 5 days and then once every 5 days until hatched. Throughout the course of incubation. eggs with defective or stunted embryos were isolated from their respective groups and allowed to incubate an additional 24 h before they were opened for examination. This was to ensure that no error in candling had occurred thus killing embryos that may have been retarded to some degree in growth. Estimated time of embryonic death was determined using Hamburger and Hamilton stages (10) and all abnormalities were similarly recorded, labeled, and fixed in Bouin's or 10% formalin. After the chicks had hatched, they were kept for 72 h in a heated box with water and food to determine posthatching mortality. Antibiotic Sensitivity The antimicrobial activity of prodigiosin and its fractions were determined by the disc-agar diffusion method (2). Organisms used in this study were Escherichia coli, Enterobacter aerogenes, Staphylococcus arrreus, Bacill~s subtilis, and Pse~rdomorras aerrrginosa. Cultures were first grown in nutrient broth with 0.1% dextrose for 24 h on a rotary shaker a t 50 rpm and incubated at 37°C. An inoculum size of 0.1 ml from each stock culture was aseptically pipetted into 15 ml of sterile, liquid TGEA3 held at 45°C in a water bath, lightly vortexed, and plated. After the medium solidified, all plates were held at 5"C until use. Five sets of plates were incubated for 18-20 h at 37°C. The presence of zones of growth inhibition around these discs was used as the parameter to assess the antimicrobial activity of prodigiosin and its fractions.
3TGEA-tryptone glucose extract agar.
Results Embryo Toxicity Tables 1 and 2 illustrate the embryotoxic effects induced by various fractions, whole prodigiosin, and the solvents used. As illustrated in Table 1, dimethyl sulfoxide (DMSO) alone surpasses ethanol as a suitable injection solvent at both concentrations of 0.05 and 0.1 ml with hatchabilities of 100% and 75% respectively for DMSO and 83.3% and 25% for ethanol. Prodigiosin in DMSO (65 pgl0.05 ml and 130 pg/ 0.1 ml) gave hatching percentages of 94.4 and 72.2, whereas these values were lower with prodigiosin in ethanol (58.8 and 22.2). Because of these results, DMSO was used as the solvent for all trials thereafter. Data from all trials (Table 2) illustrate the high percentage of survival in the E-4 treated group and in comparison a high mortality in animals injected with all other fractions and whole pigment. Also listed in this table are hatchability results from a total of 42 eggs that were not injected (89.0%) to compare with those results of our controls. Typical toxigenic effects of solvent, prodigiosin, and its fractions on chick embryo are depicted in Fig. 1, A through L. As described in the figure legends it is seen that both DMSO and E-4 fraction in DMSO are least embryotoxic. However, solvent ethanol itself and other fractions of the pigment prodigiosin proved to be more detrimental with respect to the survival of a greater number of chick embryos as indicated by both pre- and post-natal deaths (Table 2). Antibiosis Data from the observations of antimicrobial activity of prodigiosin and its fractions in all solvent systems used are listed in Table 3. From these results, it was observed that all solvents, with the exception of glacial acetic acid, demonstrated no microbial inhibition even at the dose level of 0.05 ml. Although glacial acetic acid would obviously never be used as an injection media, it was interesting to note that prodigiosin in this particular solvent as well as at this extreme in \pH still possessed greater inhibitor) effects than the acid alone. This is especiall) outstanding with E. coli at the dose level 01 0.03 ml with the acid alone giving a zone diameter of 21.3 & 1.4 mm and 5 1 pg prodigiosin in 0.03 ml acid showing a zone of growth inhibitior
215
KALESPERIS E T AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
TABLE 1 Comparative analysis of abnormalities and toxicity of prodigiosin in ethanol vs. prodigiosin in DMSO Injected substance, pg injected/ml solvent
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95% ethanol
Total eggs injected A. Prenatal deaths (abnormal) B. Hatched (no abnormalities) % Percentage of abnormal occurrence
.
Pr;digiosinl 95 ethano
DMSO
12 2
12 9
11 0
12 3
10 83.3
(2) 3 25.0
11 100.0
0.0
16.7
0.0
18 14
18 1
18 5
9 75.0
17 7 (4) 10 58.8
22.2
17 94.4
13 72.2
0.0
23.5
16.7
0.0
0.0
6)
TABLE 2 Composite of treatments and solvents used in the antibiotic studies Substance injected, pg1O.l ml DMSO
Total eggs injected Percentage hatched Survival percentage of those hatched Percentage - mortality*
Prod
PE- I
C-2
A-3
E-4
M-5
None
DMSO
44
28
26
26
48
30
42 89.0
46 71.7
34 58.8
36 58.3
36 61.1
36 55.6
35 82.9
36 66.7
85.7 14.3
71.7 28.3
50.0 50.0
52.8 47.2
55.6 44.4
41.7 58.3
77.1 22.9
50.0 50.0
*Includes all embryos which did not hatch and those which died within 48 h after they had hatched.
+
amounting to 41.7 8.9 mm, a difference of 20.4 f 7.5 mm. None of the discs charged with pigment fractions in petroleum ether, chloroform, acetone, ethanol, Bnd methanol exhibited any growth inhibition for all organisms tested. It should be noted at this point that Pseudomonas was used only in the sensitivity studies incorporating the solvent DMSO, thus accounting for the blanks in Table 3. From these results, it can be seen that PE-I, C-2, and A-3 in DMSO also failed to exhibit any in vitro antimicrobial activity, thus eliminating these fractions as possible bacteriocidal substances. However, prodigiosin, E-4, and M-5 in DMSO showed definite growth inhibition for all organisms tested including Pseudomonas with comparable antibiotic properties between the whole pigment and E-4 fraction. Least zonal inhibition occurred with M-5. The above observations were compared with commercially prepared discs from Difco laboratories using the antibiotics chloromycetin (chloramphenicol), penicillin, tetracyclin, aureomycin,
and ampicillin. It was apparent that the most effective of these was aureomycin with respect to overall inhibitory action against E. coli, E. aerogenes, S. aureus, and B. subtilis; however, in spite of its efficiency against these organisms, it failed to exhibit any zonal occurrence against Pseudomonas (Table 4). Both cultures of P. aeruginosa with the five commercial discs showed no zonal inhibition in comparison to the inhibition demonstrated by the E-4 and M-5 fraction and prodigiosin against this microorganism.
Discussion Although several investigators have observed morphological alterations with DMSO in fowl and mammalian embryos (5, 6 , 8, and 15), the occurrence of abnormal embryos induced in the controls was limited to 1 out of 79 eggs injected with 100% DMSO. Furthermore, the one abnormal embryo had been injected with 0.1 ml DMSO which is equivalent to 100 mg compared with 10-12 mg reported by Caujolle et al. (5). This deviation is not due to a difference in breed,
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216
CAN. J. MICROBIOL. VOL. 21, 1975
ABBREV!ATIONS USED:
(0)anterior,
(be) beak, (by) brain, ( e ) eye, (11) heart, (Ib) limb bud, ( n ) nasal aperture,
( p ) poster~or.
F I G . IA-F. Embryotoxicity of pigments in 95% ethanol. A. Abnormality induced by the injection of 0.1 nil ar was dead after 70 h of incubation. Of p a r t i c ~ ~ l interest ar is the enlarged 95% EtOH. This p a r t i c ~ ~ l embryo al Two other embryos developed similar features. B. (a) This embryo heart and stunted n e ~ ~ r development. n. injected with 27 pg C-2 in 0.05 nil EtOH, it developed twinned died after about 15 days of i n c ~ ~ b a t i oInitially tr~lnkswith a common cephalic region having a n inverted beak and exposed brain. (b) Injected with 41 pg C-2 in 0.05 ml EtOH, the animal had bilateral anophthalmia with inverted superior beak. This embryo was full-term and had pecked at shell, yet died without completing hatch. C. Embryo injected with 207 pg E-4 in 0.05 nil EtOH was alive when opened after 96 h incubation. Aside from lack of posterior differentiation and abnormal optic development, this embryo appeared to be retarded in morphological development by about 36 h. D. Embryo injected with 361 pg M-5in 0.05 ml EtOH died between 72 and 96 h of incubation. The only morphological development that was recognizable was the eye pigment. E. Embryo injected with 120 pg prodigiosin in 0.1 ml EtOH. Egg was opened at 6 days, heart was still beating yet enibryonic developnlent appeared to have only reached 55 h. Note again the enlarged heart and stunted posterior development similar to Fig. IA. F. This embryo exemplified three such abnormal trunk flexures induced by 60 and 120 pg prodigiosin in ethanol. Limb bud and optic development also appeared to be inhibited in these embryos with all dying between 64 and 76 h.
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KALESPERIS ET AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
FIG.IG-L. Embryotoxicity of pigments in 100x DMSO. G. Embryo injected with 0.1 ml DMSO dead at about 3-5 days. This was the only abnormality induced by the solvent and was characterized by abnormal optic development and lack of abdominal wall. H. Dead at about 3 days, this embryo was injected with 13 pg C-2 in 0.05 ml DMSO. Note the similarity of posterior twinning with Fig. 1, (a),enlarged heart, and stunted cranial development. I. Four-day embryo with bilateral anophthalmia, no limb buds, and retarded heart development was injected with 26 pg C-2 0.1 ml DMSO. J. Only abnormality induced by E-4 in DMSO was injected with 48 pg. Age of death was estimated between 48 and 72 h. K. Chick exemplifies condition induced by both 30 pg M-5 in 0.1 ml DMSO and 44 pg prodigiosin in 0.1 ml DMSO characterized by extreme cervical flexure. Both animals died within 48 h after they were hatched. L. Abnormality induced by 44 pg prodigiosin in 0.1 ml DMSO. Cranial and cardiac development of this 43-day embryo appeared normal; however, no budding or posterior trunk development was noted.
217
CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 3 In vitro bacterial inhibition by prodigiosin and its fractions in several solvents with zone diameters expressed in milliliters
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Disc Nos.
5-23
24
un
ml solvent
Listed below
0.01 0.02 0.03 0.05 0.01 0.02 0.03
4.9 9.8 14.7
E. coli 1 . 9 x lo7
E. aerogenes 1 . 8 lo9 ~
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.7k0.4 9.8k0.7
S. aureus
2.8 x lo8
B. subtilis 2 . 8 lo6 ~
-
8.4k0.5
8.6k0.3
P . aeruginosa 1.3 x lo7*
-
10.8k0.6
TABLE 4 Comparative zone diameters of prepared Bacto sensitivity discs Chloromycetin (chloramphenicol) 30 pg Eschericllia coli Etiterobacter aerogenes Stapliylococcus aweus Bacill~ssubfilis Psertdot?iot?asaerl~ginosa
32.0k2.0 20.0k0.5 19.0+ 1 .O 30.0k0.5 -
Penicillin 10units=16pg 12.0k2.0 -
7.5k0.5 20.0k0.5 -
for Caujolle compared the results of Rhode Island chickens with those of Leghorns and found a similar sensitivity. Moreover, these concentrations used by Caujolle represent LD,, values, values which we never observed since survival at 0.05 ml(55 mg DMSO/embryo) for 33 control embryos approached a mean of 90.0 5.4% and survival at 0.1 ml (1 10 mg DMSO/embryo) for 46 control embryos approached 71.7 +_ 1.7%.
+
Aureomycin Tetracycline (chlortetracycline) 30pg 30
Ampicillin 10 ~g
31.0k1.0 18.0k0.5 20.0k0.5 38.0k2.0
38.0k0.5 12.0k0.5 20.0k0.5
30.0k2.0 20.0k0.5 27.0k 1 .O 40.0k0.5 -
-
The observations by Caujolle et al. (5) of malformations induced by DMSO were similar in a few cases to those induced by the whole pigment and two of the pigment fractions, malformation of beak (parrot beak in prodigiosin, C-2 and A-3) and anophthalmia of the left eye (C-2 and A-3). However, if one takes into consideration the differences in DMSO concentrations, the higher percentage of abnormal embryos seen in the
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KALESPERIS ET AL.: ANTIBIOTIC PIGMENTS FROM SERRATIA
219
prodigiosin, C-2, and A-3 groups (Table I), and observation eliminated the possibility of the the incidence of posterior twinning induced by solvent being the diffusible static agent, it was C-2 in 0.05 ml ethanol as well as in 0.05 ml also noted that no growth had occurred beneath DMSO indicate that the pigment and some of the discs charged with DMSO, i.e., inhibition the fractions and not the solvent are indeed was limited to the diameter of the disc. Previous responsible for the induction of malformations investigators have reported a bacteriostatic or bacteriocidal effect of DMSO at concentrations and toxicity in the chick embryo. The most outstanding feature of our investiga- from 1 to 50% for a variety of bacteria (1 1, 12, tion was the consistency of greatest survival of and 17). However, standard dilution assays were the E-4 chicks over all other experimental used in their studies. The difference in results groups. Not only did this fraction demonstrate when this technique is compared with our a greater survival than the whole pigment and method is evident since with standard dilution other fractions with both 0.05 and 0.1 ml of there is a large margin of error in attributing solvent, but hatching as well as survival percent- static effects to a certain antibiotic when the ages exceeded the control group injected with solvent may be equally capable of inhibiting bacterial growth. 0.1 ml DMSO. Burgova et al. (4) and Efimenko et al. (7) have A number of embryonic deaths also occurred during the last several days of incubation and reported that they found a fraction that posseswithin 48 h after hatching; this may have been sed antibiotic activity, which appears to be differdue to the absorbance of the remaining yolk ent in many physical properties from our fracand in turn to the toxicity of the residual pig- tions. Both of them stated that their antibiotic ment. DMSO was ruled out as a causative agent fraction showed a red coloration; however, in since there were no posthatch deaths in our the present study the only fraction that came close to fitting this description was the C-2 fraccontrols. From the results of our sensitivity studies, it tion which proved to be totally worthless as an is apparent that the E-4 fraction in 100x DMSO antimicrobial agent in vitro and was highly was more bacteriostatic than any of the other toxigenic during embryogenesis. In conclusion the E-4 fraction from the pigfractions except for B. subtilis which showed the greatest inhibition of growth with the crude ment prodigiosin has been shown to be least prodigiosin in DMSO. In spite of the fact that embryotoxic and to inhibit the growth of several concentrations greater than 14.7 pg were not species of bacteria. tested, it is interesting to note from Table 3 that at a similar concentration the whole pigment was 1. ABRAHAM, E . P., and H. W . FLOREY. 1949. Antibioless effective than the E-4 fraction except for tics fromchromogenic bacteria.In Antibiotics. Vol. 1. Edited by H . W. Florey, E . Chain, N . G . Heatley, M. B. subtilis. Special consideration should be A. Jennings, A . G. Snaders, E . P. Abraham, and M. E . directed toward the inability of prodigiosin to Florey. Oxford University Press, London. pp. 558-562. inhibit Staphylococcus aureus at a concentration 2. BARRY,A . L. 1964. The routine antibiotic disc-plate of 12.9 pg in 0.03 ml DMSO, whereas E-4 demonsensitivity test. 11. Technological variations. Am. J. strated a positive zonal inhibition. Furthermore, Med. Technol. 30(5): 333-342. 3. BUNTING,M. 1. 1940. A description of some color the definite inhibition of P. aeruginosa by provariants produced by Serratia marcescens strain 274. digiosin as well as E-4 places these antibiotics in J. Bacterial. 40: 57-68. a position of distinction since relatively few M. P., E. V . LOVYAGINA, N. N. FAL~NA, 4. BURGOVA, and A. L. GOL'DENBERG. 1957. A study of theabsorpantibiotics are capable of suppressing this organtion suectra of the antibiotic from Bacteri~rm oroism. Current drugs of choice against Pseudodigios;m. Primenenie Metodov Spektroskipii v 6 o m . Prodovol'stven. 1955: 173-179. monas include colistin, polymyxin, carbenicillin, 5. CAUJOLLE, F . M. E . , D. H. CAUJOLLE, S . B. CROSS, and gentamicin (9); however, since none of these and M. M. Y . CALVET.1967. Limits of toxic and substances has yet been extensively studied in teratogenic tolerance of dimethyl sulfoxide. Ann. N . Y . Sci. 141(1): 100-126. regard to their effects on embryogenesis, it is 6. CROS.S . . M. C. V O I S I N . TOLLON. ~. and J. OREGLIA. necessary to view them as a possible risk if taken 1 9 7 0 . ' ~ a m a e eof limb'buds of chicken embrvo bv dimethylsu16xide. (In French, English sumr;laryj. during pregnancy. Ann. Pharm. Fr. 28(4): 263-270. Prior determination of the effects of 100% 7 . EFIMENKO, 0. M., G. A . KUZNETSOVA, and P. A. DMSO in our studies showed no zonal inhibition YAKIMOV.1956. Prodigiosin - an antibiotic from for any of the organisms tested. Although this Bacterium prodigiosum. Biokhimiya, 21(3): 4 1 9 4 2 3 .
220
8. FERM,V. H. 1966. Teratogenic effect of DMSO. Lancet, 7430: 208. 9. GOODMAN. L . S.. and A. GILMAN.1970. The ~ h a r macologicil basis bf therapeutics. 4th ed. The ~ ; ~ m i l Ian Co., London. 10. HAMBURGER, V., and H. L. HAMILTON.1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92. 11. JACOB.S. W.. R. J. HERSCHLER. and M. D. BISCHEL. 1964. ~ i m e t h )sulfoxide ~ (DMSO): a new concept in parmoco-therapy. Curr. Ther. Res. Clin. Exp. 6: 134-135. 12. KLIG;;~, A. M. 1965. Dimethyl sulfoxide. Part 2. J. Am. Med. Assoc. 193: 923-928. 13. KRAFT,E . 1902. Beitrage zur biologie des B. prodigios~tsund zum chemischen verhalten seines pigments. Thesis, Wurzburg, Germany. (Cited by Williams and Hearn 1967). and G. C. KRESHECK. 14. LYNCH,D. L., T. E. WORTHY, 1%8. Chromatographic separation of the pigment ~
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CAN. J. MICROBIOL. VOL. 21. 1975
-
-
-
-
~
-
-
15. 16. 17.
18. 19.
fractions from a Serratia marcescens strain. Appl. Microbiol. l6(1): 13-20. MARIN-PADILLA. M. 1966. Mesodermal alterations induced by dimethyl sulfoxide. R o c . Soc. Exp. Biol. Med. 122: 717-720. NEW, D. A. T. 1966. The culture of vertebrate embryos. Academic Press, New York. POTTZ,G. E., J. H. RAMPEY, and F. BENJAMIN. 1967. The effect of dimethyl sulfoxide (DMSO) on antibiotic sensitivity of a group of medically important microorganisms: preliminary report. Ann. N.Y. Acad. Sci. 141(1): 261-272. ROBSON,J. M. 1963. The action of drugs on the embryos. Pharmacological considerations. Proc. R. Soc. Med. 56: 600605. WOLLAM,D. H. M., and J. W. MILLEN.1963. The action of drugs on the embryo. Experimental mammalian teratology and the effects of drugs on the embryo. Proc. R. Soc. Med. 56: 597601.
