IIHIIIIIHIIIIIIII I 115 964 __THS THE ACTION OF ANTIMICROBIAL SUBSTANCES ON SOME ENTERIC AND RELATED ORGANISMS 'I‘heais for flu: Dagroe cf M. S. MICHIGAN STATE COLLEGE Muriel Yin Fung Ling 1947 ‘ II -.N.O.A“..u.:l,.4vwl "1.. "Hump N H.EL*M“ E « V,...3.&.u .1. .. V. . . I ,. .. I .. .nv. {,I . . It. ,u.....u. «A. your record. move this chockout 1mm MSU In An Namath. ActionlEqual Opportunity Institution E U D m D E U D E m E U D m PLACE II RETURN BOX to m m m m m —I 9‘ _ I This is to certifg that the thesis entitled The Action of Antimicrobial Agents on Some Enteric and Related Organisms presented by Lhriel Ling has been accepted towards fulfillment of the requirements for “If... 51 ”degree in--- Bacteriology Dates J:@j_2_h4_l_9_lg_- - __ 56-795 THE5‘5 I—I II III IV VI * 3 s U.) I.“ D.- 0 bid Q :1 Introduction The Salmonella A Cultural and biochemica cha acteristics B The serOIOQy of the grouo The antimicrobial agents Experimental data A Organisms used B Antimicrobial agents used C Procedure D Results Summary Bibliography Acknowledgment 183102 Introduction Antimicrobial agents are of chemical or biologic ori- gin. The latter includes the products of a variety of higher plants, higher animals and microorganisms; to the substances obtained from the microorganisms is specifically ascribed the term "antibiotic". From the plants are obtained quinine, eme- tine, and chaulmoogra oil; from the animals, lactenin, lyso- zyme, histones and protamines. The study of antibiotic substances is a comparatively new one, the initial impetus having been given by Fleming (14) in 1929 with his discovery of penicillin. The phenomenon of antagonisms exhibited among microorganisms has led to the dis- covery of these antibiotic substances, for it is known that certain organisms cannot grow in the presence of another spe- cies of organism. Such substances have been obtained from both spore-forming and non-snore-forming bacteria, actino- mycetes and fungi. The antibiotic substances differ from the ordinary bactericidal agents because of their selective action upon different bacteria. Some bacteria may be affected by a very low concentration of a given substance whereas others are not acted upon at all or only by very high concentration. Each substance therefore is characterized by a specific bac- teriostatic or antibiotic spectrum. The other antimicrobial agents also seem to show definite spectra of action. Knowing that the antimicrobial agents, whether chemical l or biologic in origin, do show selectivity in their action, it was thought that a study of the action of several of these agents on some enteric organisms might lead to a method of early differentiation of the various organisms. Special interest was laid on the salmonellae because that group is exceedingly complex. The Salmonella Cultural and Biochemical Characteristics Since the isolation of Salmonella choleraesuis by Salmon and Smith in 1885, paratyphoid bacilli have been found by many workers in a variety of diseases of animals and in enteric fever and gastroenteritis of man. The organisms have been re- covered from many sources; isolations from such diverse sources have led inevitably to confusion. The International Associa- tion of Microbiologists in 1940 (50) defined the genus thusly: "A large genus of serologically related, gram-negative and non-eporing bacilli; 0.4-0.6 microns by 1-5 microns in usual dimensions but occasionally forming short filaments; showing, with certain exceptions, a motile peritrichous phase in which they normally occur; in fact, adhering to the pattern of ‘E. typhosus in staining properties and morphology. Failing to ferment sucrose or to clot milk and rarely fermenting lactose, liquefying gelatin or producing indol, they regularly attack glucose with, but occasionally without, gas production. All the known species are pathogenic for man, animals, or both." The bacteriologist normally finds little difficulty in identifying an organism as belonging to the genus Salmonella, for he can utilize various cultural, biochemical and agglu- tination reactions. Final species or type identification of the culture involves the use of known specific sera and what agglutinin absorbed sera necessary to distinguish one Species 5 from another. In an average laboratory the preliminary iden- tification of Salmonella-like colonies is based on the reac- tion of the organism on Kligler's iron agar with 0.1% sucrose, a gram stain, tests for motility, Voges-Proskauer reaction, indol and hydrogen sulfide production, fermentation of glucose, lactose, sucrose, salicin and other carbohydrates. A positive indol, Vegas-Proskauer test or fermentation of lactose or suc- rose would exclude the organism from the Salmonella group. Mere identification of the organism as belonging to the genus Salmonella may be sufficient information but in the larger laboratories, such as the laboratories of the state health de- partments, it is of especial epidemiological value to know of what species the organism is. The following is a review of the methods described by various authors for the differentiation of the Salmonella organisms. In 1924 Brown, Duncan and Henry (4) advocated the substitution of organic salts for "sugars" in cases where sugar reactions are untrustworthy or fail altogether to dif- ferentiate certain serologically well defined types of bac- teria. Salts of a few organic acids are utilized as food by some members of the paratyphoid group. Utilization of the salts was measured in a liquid medium by the precipitation of the residual acid with lead acetate; thus, observing the re- actions on tartaric, fumaric, citric and mucic acids, they were able to find seven different groupings of Salmonella types as compared to the four types differentiated by the sugar re- 4 actions. In 1926 Simmons (56) put forth a medium; his was a citrate agar medium with brom-thymol blue added as an indicator, re- sulting in an olive-green medium. He found that Sal. paratyphi and Sal. pgllorum failed to grow in the medium and did not change the color of the medium, whereas Sal. schottmuelleri, Sal. enteritidis and Sal. typhimurium grew luxuriantly and formed translucent greenish blue-green colonies on the citrate agar and produced sufficient alkali to color the medium a deep Prussian blue. Jordan and Harmon (21) in 1928 described another differ- ential medium for the paratyphoid group. Sodium.potassium tartrate was the salt incorporated into the medium which con- tained phenol red as an indicator. An acid reaction was given by 87 strains of Sal. typhimurium and an alkaline reaction was given by 45 strains of Sal. schottmuelleri. Following the idea set forth by Brown, Duncan and Henry, Mallman (26) in 1951 reported that he could differentiate Sal. pullorum from Sal. gallinarum with the addition of d-tartaric and mucic acids in the culture medium. Sal. pullorum constant- ly gave an alkaline reaction on sodium d-tartarate and sodium mucate whereas Sal. gallinarum yielded acid reactions on both. In 1954 Vogelsang (45) experimented with Sal. schottmuel- leri in western Norway and described its definite cultural and biological characteristics, such as "slime wall" formation at 5 room temperature, formation of papillae on raffinose agar, formation of hydrogen sulfide in lead acetate agar, negative Bitter reaction, negative Pesch-Maschke reaction, and the de- colorization of neutral red agar. More recently in 1941 Bornstein and Strauss (2), while utilizing the agar strip method in their in gitgg experiments, found that sulfanilyl-guanidine inhibited Sal. choleraesuis quite markedly and affected Sal. paratyphi to some slight de- gree but was ineffective against the other organisms of the group. Hinshaw (18) then reported that 89 out of 91 strains of Sal. ggllinarum produced a characteristic yellowish-white or grayish turbidity when incubated at 370 C for 72 hours in a gelatin medium containing 0.15% cysteine hydrochloride. None of 19 other types of Salmonella studied gave this typical reaction. The sequence of phenomena in litmus milk (47) is also a valuable guide in the differentiation of the Salmonella or- ganisms. No member of the group sets up sufficient acidity to produce clotting of the milk although all of the members cause the indicator to change within 24 hours at 57° C from the violet of neutrality to a more or less definite pink, re- gistering the formation of acid in small amounts due to the presence of traces of readily fermentable and glucose-like sugar. Further events then divide the Salmonella into two groups. One group is composed of Sal. schottmuelleri, Sal. typhimurium, Sal. enteritidis, Sal. abortivoeguina and Sal. 6 choleraesuis. In the case of these organisms, the acid indi- cator undergoes the so-called "chameleon" change; in the course of two to five days it assumes the blue color of alkalinity. Associated with the definite alkalinity there also occurs a clearing of the hitherto turbid medium with the formation of a deposit. The second group of strains is composed of §El° paratyphi and Sal. abortusovis. In the case of these organisms the acid reaction developed during the first 24 hours is main- tained. Neutral red agar (Rothberger) has already been mentioned for its purpose in Salmonella differentiation. The reactions seemingly run parallel with the aerogenic or anaerogenic be- havior of the organism. The gas-forming species such as Sal. schottmuelleri and Sal. typhimurium cause first fluorescence and then decolorization of the medium. Anaerogenic forms such as Eberthella typhosa, Sal. gallinarum and anaerogenic strains of types which normally form gas cause no indicator change. Differentiation of the Salmonella is also possible by their production of or lack of hydrogen sulfide as detected in media using soluble salts of lead or iron as indicators. Sal. schottmuelleri, Sal. enteritidis and Sal. typhimurium are hydrogen sulfide positive while Sal. paratyphi and Sal. abor- tivoequina are hydrogen sulfide negative. The carbohydrates for which differential value has been claimed are the pentoses -- arabinose, xylose, and rhamnose; the disaccharides -- maltose and trehalose; the trisaccharide 7 raffinose; the polysaccharide dextrin, and the alcohols -- mannitol, dulcitol, inositol, and glycerol. The outstanding reactions of differential value may be noted. Sal. paratyphi does not ferment xylose while most of the other members do. Sal. schottmuelleri does not ferment d-tartrate while Sal. typhimurium and most of the food-poisoning bacilli do. Dulcitol is almost always readily attacked by members other than Sal. pullorum and Sal. choleraesuis. Maltose is apparently attacked by all types save occasionally Sal. pullorum. Sal. choleraesuis is unable to ferment trehalose whereas Sal. enteritidis, Sal. paratyphi and Sal. schottmuelleri do so readily. Inositol is fermented by the great majority of strains of Sal. schottmuel- leri and Sal. typhimurium but not by Sal. paratyphi, Sal. abor- tivoequina, Sal. choleraesuis or Sal. enteritidis. Sal. schott- muelleri and Sal. enteritidis have been termed "Stern-positive", for when they are cultured in fuchsin-sulphite-glycerol-meat- extract broth they cause the indicator to assume a deep lilac- red color while other forms such as Sal. paratyphi, Sal. chol— eraesuis and Sal. abortivoequina determine at most a pale- pink coloration. Much work has been done by Kauffmann and some German wor- kers on the Salmonella organisms. Their work is not readily translated by the writer of this paper and consequently no com- ments have been made about their findings. The SerolOgy of the Group Cultural and biochemical reactions give the worker pre- sumptive clues as to the species of the organism; final type identification of the suspected Salmonella must be based on a serological study which is by no means simple, for there are at least 155 types of Salmonella and also some 9 coliform or- ganisms which have been described as containing Salmonella antigens (35). The method of subjecting an organism to a serological study will not be discussed here but is presented in Edwards' paper (15). In 1926 White (46) recognized the importance of consider- ing the bacterial variations in relation to the antigenic ana- lysis of Salmonella. Later this pioneer work of White's was confirmed and extended by Kauffmann (22), efforts which re- sulted in the Kauffmann-White scheme of Salmonella classifi- c atione In the serological identification of the Salmonella group one must keep in mind the variations that the organisms can undergo, variations which do cause discrepancies in the serology. There is first the H-0 variation. In 1920 Weil and Felix (46) reported that the typical Salmonella presents two distinct classes of antigens, each stimulating in_zizg pro- duction of its own particular antibodies with which it exclu- sively reacts. The H antigen is associated with the flagella and is therefore not found in the non-motile organisms. It is heat labile, being progressively inactivated by tempera- 9 tures above 600 C; after exposure to 1000 C for 30 minutes or when treated with acids and alcohols it fails to function in serolOgical tests or as an antigen when injected into an animal. Its develOpment is inhibited by culture on certain media, notably the phenol-agar medium of Braun (5). When it reacts with its corresponding agglutinins it results in the agglutination of the bacilli in large, fluffy clumps which are readily dispersed. The 0 antigen is present in both motile and non-motile forms of the group. It resists prolonged heat— ing at 1000 C and treatment with alcohol and dilute acids. It agglutinates slower than the H antigen and it forms granular clumps which are dispersed with difficulty. In the reaction of an ordinary motile culture with its antiserum the aggluti- nation determined by the flagellar reagents exceeds in titre and occurs more rapidly than that due to the somatic reagents. Also the somatic (0) and flagellar (H) agglutinins seem to be entirely independent because in correctly performed absorption tests either type may be withdrawn from the serum, leaving the other intact. The S-R variation is not merely a morphological charac- teristic; the R colonies, besides possessing roughened sur- faces and irregular borders, producing granular growth in broth, giving a saline suspension of organisms which is less stable than those of the S races, also have a tendency to cross- agglutinate. The typical rough variant is characterized by failure to respond to the 0 agglutinins of the corresponding 10 smooth serum and by failure to produce agglutinins acting on the somatic antigens of the smooth organism; however, it is marked by a special somatic antigen which reacts with the homologous rough antiserum to cause clumping of the bacilli in fine granules. Consequently only smooth cultures should be used to prepare 0 antiserums. In the change from a smooth to a rough form, the flagellar elements of the organism evidently suffer little or no qualitative modifications. The change is gradual. There is also the V-W variation. Some cultures are in— agglutinable in pure 0 antiserum but agglutinate with a Vi (virulence) antiserum. The virulence antigen is so called be- cause the organisms possessing it are more virulent for mice; it is inactivated by heat and acids but is unaffected by abso- lute alcohol. The W colonies agglutinate with the 0 anti- serum and not with the pure Vi antiserum. So far as is known phase variation occurs only in motile Salmonella strains and not in other genera. Some organisms have specific and non-specific antigenic factors; the non- specific antigens cause different types to cross-agglutinate in high dilutions because these types are related; therefore a simple agglutination cannot be depended upon for differen- tiation. The following is an example of phase variation: Two ' Salmonella types with related H antigens are plated. Certain colonies will agglutinate only with their homologous serum whereas others will agglutinate with the serums of both. The 11 former is said to be in its specific phase and the latter in its non-specific phase or in phase 1 and phase 2 respectively. Not all Salmonella types are diphasic. NonOphasic colonies have identical H antigens; Sal. enteritidis has that charac- teristic. Certain minor 0 antigens of the genus overlap; that is, they occur in more than one of the major groups into which the salmonellae are divided on the basis of their 0 antigens. Antigens I and XII exhibit a phenomenon known as form varia— tion. When certain types containing either of those two an- tigens are plated, some colonies contain large amounts of the antigen and some contain slight amounts of the antigen. S-R variation, V—W variation and form variation affect only the 0 antigens of the bacilli and their reactions in 0 serums. They have no effect on the H antigens except as roughness af- fects the stability of the bacterial suspensions and consequent- ly their agglutination. A survey of the cultural and biochemical characteristics and the serolOgy of the Salmonella group has been briefly pre- sented. It is easy to see that difficulty early arises in any attempt to differentiate among the species; the serological identification is a task demanding the best technique. If some of the organisms are sensitive to an antimicrobial substance, then it would be simple to eliminate quite early in the iden- tification procedure the possibility of its being one which is resistant to the substance. 12 The Antimicrobial Agents The following discussion on the antimicrobial agents deals largely with the antibiotic substances, since such information can be readily applied to the other antimicrobial agents. The antibiotic substances may be classified according to their solubility, chemical nature, biological activity or tox- icity to animals (44). They are selective in their action, even as regards specific types or strains of the group of bac- teria being acted upon. The selectivity of the antimicrobial agent (11) may be due to the acidic and basic properties of the cell under consideration, the nature and property of its- membrane, its permeability, the relative importance for meta- bolism and viability of the specific biochemical systems af— fected by the agent, and to the activity of the autolytic en- zymes. Selectivity is exhibited not only with reference to the type of cell which is affected but also with reference to the metabolic systems or structural constituents with which the agents react. Many mechanisms of action have been suggested. Some claim the antibiotic substance interferes with the enzymatic systems, with the vitamin utilization of the organism, with bacterial cell division, and with the metabolic processes of the micro- bial cell by substituting for one of the essential nutrients. It may bring about the oxidation of a metabolic substance which must be reduced in the process of bacterial nutrition or it 13 otherwise modifies the intermediary metabolism of the bac- terial cell. The agent may combine With the substrate or with one of its constituents thereby rendering it inactive for bac- terial utilization. The antibiotic substance may compete for an enzyme which is needed by the bacteria to carry out an es- sential metabolic process; it may favor certain lytic mechan- isms in the bacterial cell or even affect the surface tension of the cell surface. It may also interfere with the sulfhydryl group in the makeup of the bacteria which is essential for cell multiplication. Fleming (14) made his great discovery of penicillin in 1929 while studying the growth and properties of Staphylococ- ggg, He grew the organism on a solid medium containing agar. In his examination of the culture plates he naturally exposed the surface to the air and the plates were thus contaminated with various microorganisms. Around a large colony of conta- minating mold he observed that the staphylococcus colonies became transparent and were obviously undergoing lysis. This observation was the key to the discovery of penicillin, an antibiotic substance produced by various.strains of Penicil- lium notatum and Penicillium chrys0genum and probably by a variety of other fungi. In 1938 Florey and his group (17) pursued the initial work done by Fleming and develoned a rapid assay method uti- lizing the glass cylinder cup. They uncovered properties of penicillin such as its exhibiting acidic properties, being 14 unstable in acid or alkaline media, being non-toxic for ani- mals and leukocytes and tissue cultures, and its activity not being affected by pus, blood or breakdown products of dead tissue. The salts of penicillin are inactivated by penicillinase; destroyed by acids and alkali, by oxidizing agents such as potassium permanganate, by some metals such as copper, lead, and mercury, and by methyl alcohol. Penicillin is soluble in ether, ethyl alcohol, acetone, esters and dioxane and in water at the rate of 5 mg. per ml. It forms water soluble salts with most of the heavy metals except iron; its barium salt is the most suitable form for use as it retains its antibacterial activity for an indefinite period. Penicillin's action on bacteria is chiefly bacteriostatic and not bacteri- cidal. It exhibits a high degree of specificity, acting largely on the pyogenic cocci, anaerobic clostridia, certain pathogenic gram-negative cocci -- the meningococcus and gono- coccus, and also the Salmonella; it has no effect on the or- ganisms causing tuberculosis, malaria, typhoid, measles or mumps. Hobby (19) from the results of her experiments on the antibacterial action of penicillin against gram-negative or- ganisms thinks that penicillin does exert that activity, it being more apparent in high potency penicillin preparations. Therefore possibly there may be a form of penicillin which shows greater activity against the gram-negative organisms than the present forms. 15 Fleming (14) found that penicillin was bacteriostatic with regard to streptococci, pneumococci, and diphtheroids and other organisms found in the respiratory tract while no inhibitory action was exhibited on the influenza bacillus. Thus he was able to isolate the hemOphilic organisms, Hemophi- lus influenzae and gemOphilus pertussis, from specimens which also contained the aforementioned contaminating organisms. Potassium tellurite (16) has an extraordinarily selective bac- teriostatic action on such gram-negative bacteria as the coli- typhoid group, Pasteurella, Brucella and the hemonhilic bac- teria whereas it exerts little action on the streptococci, staphylococci and diphtheroids. one can immediately note that the penicillin sensitive microbes are potassium tellurite insensitive and vice-versa. Fleming (15) also used gentian violet as a spread over a blood agar plate and was able to iso- late a few streptococci from a multitude of staphylococci. Following Fleming's statement that penicillin had a more pronounced action on the staphylococcus than on the diph- theroids and since the acne bacillus belongs to the diphtheroid group, Craddock (8) used glucose broth with penicillin double the concentration which inhibits the staphylococcus. In that way he was able to obtain pure cultures of acne bacilli from 47 specimens. Osborn (28) in 1945 reported on the investigation for an- tibacterial substances of approximately 2500 species of plants belonging to 166 families. The test organisms were §tgp§. l6 aureus and g. 321}, Of these plants 65 genera were found to contain substances which inhibited the growth of one or both of the test organisms. The inhibitory substances were some- times distributed throughout the different parts of the plant and at other times, restricted to a single part. Tsuchiya, et a1 (41), discussed an antibacterial substance isolated from a plant; it is called nordihydroguiaretic acid and Was found to be active 32 vitro against Stagh. aureus, Sal. paratyphi, Sal. schottmuelleri and Sal. enteritidis. Both in legend and in the scientific literature Allium sativum, the common garlic, has been endowed with therapeutic virtues. The claims of its remarkable characteristic have been attributed to a number of substances in its makeup -- diallyl sulfide, the unstable sulfur in the alkyl polysulfides, a bacteriophage, acrolein or some other unstable aldehyde, and to some undefined group of substances designated as the phy- toncides. Cavallito (5) has isolated a substance which he calls allicin and he reports that to be the garlic antibacter- ial substance. It is a colorless oil, approximately 2.5% soluble in water and relatively unstable. It is equally effec- tive against gram-positive and gram-negative organisms. Gar- lic oil can achieve some interesting results, such as preven- ting meat decay 12 23339, killing some types of worms, stimu- lating the intestinal mucosa and peristalsis, inhibiting pep- tic digestion, and oxidizing hemoglobin to methemoglobin i2 vitro. ———-——_—_— 17 Pederson and Fisher (29) discovered that certain undes- irable gram-negative bacteria present on the surface of the cabbage leaves ordinarily disappear shortly after the cabbage is cut. The presence of a bactericidal substance in the cab- bage tissues causes a marked reduction in the number of gram- negative bacteria within 6 to 24 hours. This substance is less active against gram-positive bacteria and is inactivated by heating. Osborn (27) stated in his article that the in- hibitory substance of Brassica oleracea, the cabbage, was of greater concentration in the seed than other parts of the plant. Thalhimer (58) in 1911 recognized the bactericidal action of quinones and thought they compared with other disinfectants. In the chemical analysis of the antibiotic substances some were found to have the quinone structure. Investigators therefore tested compounds with a known quinone structure to see what their effect on bacteria would be. Colwell and McCall (6) reported that synthetic 2-methyl-1-4-naphthoquinone was bacteriostatic and bactericidal for both the gram-positive and gram-negative organisms. The introduction of the sulfonamide grbup of drugs as antibacterial chemotherapeutic agents and its clinical appli- cation dates back to the time of Domagk (10), when he repor- ted on the curative effects of "prontosil" in streptococcal infections in mice in 1955; in the same year the Trefouels, Nitti and Bovet (40) announced the antibacterial activity 18 of the simple organic compound, sulfanilamide, which is related to prontosil. Dubos (12) reports that the bacteriostatic action of the sulfonamides is due to the competition of the drug with p- aminobenzoic acid at some vital stage of metabolism in which the latter substance is concerned. P-aminobenzoic acid is an essential growth factor or metabolite for many species of or- ganisms. Numerous i3 2:333 studies (27) have been made of the effect of various sulfonamides against bacteria. It was found that the concentration of the drug necessary to produce bacteriostatic or bactericidal action i§_yitgg_may have abso- lutely no apparent relation to that concentration necessary for its $2 vivo activity. Sulfapyridine is about two to three times as active as sulfanilamide in_zit£g but the two drugs show an equal activity in 3332 in the mouse against some strains of streptococci. Sulfanilamide (25) is active for hemolytic streptococcal infections produced by Lancefield's Group A organisms, also for chancroid, Welch bacillus infections, meningococcal infec- tions and for infections (other than subacute bacterial endo- carditis) produced by the viridans streptococci. SulfapYri— dine and sulfathiazole are superior to sulfanilamide in the treatment of gonococcal infections. Sulfathiazole is the drug of choice in most staphylococcal infections. It is the best of the group for the treatment of colon bacillary infection of the tissues, such as peritonitis and pyelonephritis. 19 Cooper and Keller (7) obtained results from an 1g vitro test which indicate that sodium sulfathiazole is more bac- teriostatic and bactericidal for the Flexner strains of Shig- ella paradysenteriae than for the Sonne strains and still less bactericidal for E. typhosa and Sal. paratyphi. Streptomycin is a recent discovery by Schatz (51) in 1944. It is isolated from two strains of an actinomyces re- lated to an organism described as Actinomyces griseus. Strep- tomycin resembles streptothricin, which is a substance produced by Streptomyces lavendulae, in its selective action against gram-negative bacteria; streptomycin differs in its greater activity against various gram-negative bacteria. Schatz (52) showed that streptomycin inhibits the growth of Mycobacterium Jr tuberculosis and Mycobacterium phlei. Jones', et al (20), eXperiments on chick embryos indicate that streptomycin, if used in sufficient concentration, offers full protection to the chick against fowl typhoid caused by Shig. gallinarum; even the dead embryos showed complete freedom from the disease. Schoenbach (54) was able to isolate gram—negative patho— genic organisms from the nasopharynx with the incorporation of tyrothricin in the medium, which selectively killed the staphylococcus. Stokinger (57) also utilized tyrothricin in his work. He found that chocolate agar containing tyrothricin in a con- centration of 1:15,000 aided in the primary isolation of EEEET seria gonorrhoeae, since the tyrothricin acted against the 20 gram-positive organisms which are present in the genito-uri- nary tract. There have been reports on the synergic action obtained when sulfonamides are used with certain other substances. Bigger (1) reported that the presence of sulfathiazole in the broth greatly increased the dilution at which the inhibitory action of penicillin on staphylococci could be demonstrated. Thatcher (59) noted a pronounced synergistic effect when sul- fonamides which normally have little if any effect on g mn- negative bacteria would in the presence of a l:28,000 dilu— tion of methylene blue or brilliant cresyl blue completely inactivate 10 million cells of a 24 hour culture of §°.22l£’ the final concentration of sulfapyridine, sulfathiazole or sodium sulfathiazole being l:l4,000. Schwartzman (55) says that the addition of methionine in combination with threonine and methionine sulfoxide enhanced the activity of penicillin against gram—negative organisms in the presence of antipeni- cillin factors. He believes the enhancement is a result of synergistic rather than additive action of the amino acids. Thus far investigators have utilized the antimicrobial agents for means of differentiating between groups of bacteria as an aid in the isolation of organisms. Little has been done to attempt to differentiate among species with the use of these agents. EXperimental Data Organisms Used The following salmonellae were used in the experiment: Sal. choleraesuis, var. Kunzendorf, Sal. paratyphi, Sal. schottmuelleri, Sal. enteritidis, Sal. abortivoequina, Sal. typhimurium and four strains of Sal. pullorum. Also tested were two strains of E. coli, three strains of Proteus and one strain of g. aerogenes. Sal. pullorum 5, Sal. pullorum 4, E. coli, Proteus and g. aerogenes were tested with three anti- microbial agents only, penicillin, streptomycin hydrochloride and 2—methyl—l-4-naphthoquinone. with the exception of three strains of Sal. pullorum, all the Salmonella cultures were ob- tained from the Michigan Department of Health; the other or- ganisms were obtained from the Michigan State College bacterio- lOgy department. Antimicrobial Agents Used The antimicrobial agents tested were garlic oil, a cab- bage seed extract, cabbage oil, hydroquinone, Hykinone —- a commercial product which contains a vitamin K derivative; 2- methyl-l-4-naphthoquinone, which is vitamin K; penicillin, sulfanilamide, sodium sulfapyridine, sodium sulfathiazole and streptomycin hydrochloride. Procedure A number of methods have been developed for determining 22 the activity of antibiotic substances. They vary greatly, each having its limitations and advantages. Results obtained with one method cannot be accurately compared with those ob- tained by another. Some of the methods are the agar plate- dilution method, serial dilution method, the classic agar diffusion or agar cup method, turbidimetric method and the filter paper disc method. For its simplicity and practicability in the performance of the test in this paper, the filter paper disc method des- cribed by Vincent and Vincent (42) and with the modifications by Lucas, et al (24), was used. DeBeer and Sherwood (9) listed some worthwhile advantages of the filter paper disc method. Those applicable to this experiment are the following: Measurement of the volume of antimicrobial substance is accom- plished simply by touching the edge of the paper disc to the material to be assayed because discs of the same size absorb surprisingly uniform amounts of solution; only small amounts of solution are necessary; and the labor of washing and caring for the small cylinders and pipettes is eliminated. The technique for the preparation of plates is as follows: Ten ml. of tryptose agar was pipetted into each specially selected flat-bottomed Petri dish. The plates were allowed to stand at room temperature for two hours so that some of the surface moisture may be removed. They were stored in the re- frigerator at 40 C for at least 1 hour and for not more than three days before seeding. One ml. of an 18-24 hour tryptose 25 broth culture of the test organism was pipetted onto each plate. The excess of inoculum was drained off by placing the inverted dish on a metal rack at an angle of 45-500 for twen- ty minutes; the little drOps of inoculum that collected on the lower rim of the inverted dish were wiped off with sterile gauze. Thus the plate was ready for the test. Filter paper discs about 15.0 mm. in diameter were the conveyors of the antimicrobial substance. They were first sterilized in the autoclave and dried overnight in a 570 C incubator. With the use of sterile forceps each disc was placed on a seeded agar plate after having been immersed in the test fluid. The plates were incubated for 18-24 hours in a 570 C incubator and the zones of inhibition were then recorded. Results Tables 1 and 2 show the results of the action of various antimicrobial agents on all the Salmonella organisms, with the exception of Sal. pullorum §_and Sal. pullorum 4, which were tested with only penicillin, streptomycin hydrochloride, and 2—methyl-l-4-naphthoquinone. The results of the organ- isms tested with only three substances appear on Table 5. Each result is an average of the zones of inhibition produced by eight separate filter paper discs. The results are the diameters of the inhibition zones recorded in millimeters; the results include the diameter of the filter paper disc 24 qoauapanqa on conceded“ . .. EH .. an .. mam ‘ 3d 3m 5233.5 .3 o. N n “we . o.om. m.m: m asked .Ham , , HOH ,nhuJHWJ . m . om o o. mun m.mw, a auuoaaan .aum 39:36 Man ~.ma o.m~ - H.wn . n.mm m.~n1, m.ma «uoaaogaaaono. .aam «.ma H.om a m.nn . n.on o.~n, n.o~ «nmmeaaca .Hum . . ~.ma . n.nm, . m.mm w.om o.ma cacauauquo .aam , n.aw m.m~ u m.o: n H.om a.mn m.ma .as-ouaoaoau .Hau m.ma m.am u H.mn - m.an m.mm m.~m A anaueoopauuogu .Hum «ea» .mn .n» Mg .m» nu mu mu m m m a.» a m a. m m m a a." W m. .m m. o .m .m mm m m u . n u n 0 a... O O O O 1. O 0 m w n m m w T. .9 T. I m . . H Bugs 8.3.255: on 0098.35 I goém .. - .. .. .99 mém .. Saganfia 48 ._ aim .. .. .. .. fidm don .. m 5.3% .1» a 0.9.... u a .. .. mdm 9mm .. a 5.3.39 43 _ «and .. u .. .. 93 :.mm w‘ .. 2318.332 Jun 13 .. .. .. .. ”.8 1mm .. 395:3. 0.3 .. a .. .. 0.8 w.n~ .. .332an .1» 1......“ .. t u ... «.mm 95 .. 338.835 .1» 98 .. n .. .. Him mfim .. 3390522... .16 I O T. T. a .d 0 O a. m. a, .. .. a. w... a, a \1 m. I I W m M... m. m. m I r m m r n m w m n m .. m m m m 1. T. o u aw .. . m... mm. mm W l.\ m. m W W W Wm Wm... B .m P To T. m m . m m u m m p o m. m m u 0 O O n. N Handy 83335 on .3885. u 3.3 was 3: m 3...... 3.5 n 0.: m 338m T9 0.3 0.: a 338m .. .. T3 m :8 .n .. .93 .19 a :8 .n I him." mid 0030.36 .4 .18 :.~m Tam : .5333 .10 0.9 0.3m Him n .5533.“ .16 J T. 0 a... a a... m w MW. .3 m m m m m .... _. u. .m a. .c u m m m 0 m m w m an: which is approximately 15.0 mm. The cabbage extract was an aqueous emulsion of the oils obtained from the cabbage seeds. There was a white zone of precipitate surrounding each filter paper disc; the zones of inhibition usually extended beyond that Opaque zone. The sal- monellae were not markedly differentiated since the largest zone of inhibition Was no more than 5 mm. greater than the smallest zone. The 1% cabbage oil solution used in this experiment showed negative results. That is contrary to what was anti- cipated, since the cabbage extract produced inhibition and the cabbage oil solution contains the essential oil from that extract. Dr. E. H. Lucas (25) explains the lack of action to an interruption during the extraction procedure, an inter- ruption which may have cost the loss or destruction of the antimicrobial agent. Different lots of 0.1% of garlic oil solutions -- A and B, were tested. B is the same as that used with the 175 hydro- quinone solution in an attempt to illustrate synergistic ac- tion. A 0.5% solution of an oxalic acid preparation of garlic oil was also tested. The results for each organism, when tested with the separate solutions, show about the same degree of sensitivity. Sal. pullorum seems to be the most sensitive of the group. A 1% Hykinone solution did not show any effect on any of the organisms, so a 5% solution was then prepared. There was 25 no marked sensitivity on the part of any one of the organisms to the substance although each one was inhibited. The 1% acetone solution of 2-methyl-l-4-naphthoquinone (vitamin K) gave interesting results with several of the Sal- monellg organisms. Sal. enteritidis and Egl. typhimurigm were not inhibited at all or had zones of inhibition which were too small to read. Sal. abortivoeqpina, Sal. paratyphi and Sal. schottmuelleri all had zones which were less than 20 mm. Sal. choleraesuig and the four strains of Sal. ppllorum exhi- bited zones of inhibition of 20.0 mm. or more. The other or- ganisms tested showed either a slight or no sensitivity to the antimicrobial agent. The sensitivity Varied among strains of the same organism; thus one g. 331; strain was inhibited slight- ly and the other was not. One strain of Proteus was also in- sensitive. When 2% Hykinone in 0.2% garlic oil solution was tested with the organisms, there were no zones of inhibition. Normally a 0.2% garlic oil solution would give a noticeable zone of inhibition. It is surmised that the Hykinone behaves as a depressant rather than as a synergist. The 13 hydroquinone in 0.1% garlic oil solution (B) in- hibited the organisms but again, the quinone substance acted as a depressant, for the zones of inhibition obtained from the 0.1% garlic oil solution alone were approximately 10 mm. lar- ger. Another phenomenon which may be of differential value was noticed. On all the plates, save those which were seeded 26 with Sal. pullorum, the pads and zones of diffusion of the sub- stance into the agar were a definite brown color. Hydroquinone is a reducing agent and may have reacted with the products of those organisms, and Sal. pullorum may have been able to pre- vent the hydroquinone from reducing any such substance. The penicillin results offer another worthwhile differen— tiation of the organisms. Sal. choleraesuis and one strain of Sal. pullorum are the most sensitive organisms of the group tested. The difference between the two strains of Sal. pullo- rum is quite remarkable and must be attributed to the marked selectivity of these antibiotic substances. é. aerogenes and g. ggli_were not inhibited by this concentration of penicil- lin. All the sulfonamides gave no zones of inhibition al- though there were a few instances where an initial inhibition could be detected. If the plates had been read after fewer hours of incubation -- six hours, for instance, the zones of inhibition could possibly have been distinguished. Reports from investigators indicate that streptomycin is strongly active against the gram-negative organisms. It is not known how much streptomycin there is in the streptomycin hydrochloride tested. Two lots of the streptomycin hydro- chloride were obtained from Merck & Co. The 1:500 (0.2% solution of streptomycin hydrochloride was arbitrarily used and gave no spectacular results. The two lots of streptomy- cin hydrochloride gave comparable results. 27 Summary The sulfonamides and the cabbage oil solution were not active against the Salmonella. The solutions may have been too weak and the method of testing may not have been sensitive enough to detect any slight action of the sub- stances. The cabbage extract, Hykinone solution and streptomycin hydrochloride acted on the organisms but did not re- veal any differential characteristics. Sal. pullorum was the most sensitive to the garlic oil solution. The 1% acetone solution of 2-methyl-l-4-naphthoquinone produced a variety of results. Sal. enteritidis, Sal. typhimurium, one strain of E. coli and one strain of Proteus were resistant to the substance. The other or— ganisms were slightly, moderately or markedly inhibited. Penicillin was most active against Sal. choleraesuis and Sal. pullorum. There was also a marked difference in the degree of sensitivity of the four strains of Sal. pullorum. g. aerogenes and E. coli were not inhibited by this con- centration of penicillin. Two per cent Hykinone and 1% hydroquinone in garlic oil solutions both acted as depressants to the activity of the garlic oil solutions. With the exception of the two strains of Sal. pullorum, 28 the 1% hydroquinone in 0.1% garlic oil solution produced a brown color on the filter paper discs and the zones of diffusion on all the plates. 29 10. ll. 12. 15. Bibliography Bigger, J. W. 1944. Synergic action of penicillin and sulphonamides. Lancet, 2:142. Bornstein, S. and Strauss, L. 1941. Selective action of sulfanilyl—guanidine on different Salmonella types and its practical importance. Proc. Soc. Exper. Biol. Med., 31:112. Braun, H. and Cahn—Bronner, C. E. 1921. Zbl. Bakt., Abt. I, 0rig., 22:1, 196 and 580. Brown, H. C., Duncan, J. T., and Henry, T. A. 1924. 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Klin. Wchnschr., 51:896. White, P. B. 1929. The Salmonella group. Gt. Brit. Med. Res. Coun., A System of Bacteriology in Relation to Medi- cine, Vol. IV, page 86. 55 Acknowledgment The author wishes to eXpress her sincere appre- ciation to Dr. H. J. Stafseth for his generous assistance and guidance, to Dr. B. H. Lucas for supplying the antimicrobial substances, and to Betty Vincent for her valuable aid in the labo- ratory. "lillllllfillll Mill