composition: glycerol, 1.00 g; glycine, 0.60 g; Irleucine, 0.60 g; K2HPO4,

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1 A STUDY OF THE INCIDENCE OF PSEUDOMONAS AERUGINOSA FROM VARIOUS NATURAL SOURCES1 LEIF M. RINGEN' AND CHARLES H. DRAKE Department of Bacteriology and Public Health, Washington State College, Pulman, Washington Received for publication May 1, 1952 The seeming ubiquity of Pseudomonas aerumtnosa ramses the question as to its normal habitat. This organism has been isolated from soil, animal feces, water, and many other sources. The aquatic and soil habitat of the pseudomonads as a group suggests water or soil as a possible habitat, and many species of Pseudomonas can be isolated from these sources. Some species are plant pathogens, and a few may be animal pathogens. Many standard texts list various sources for this organism, but there seems to be very little or no information about sources from which it can be isolated with sufficient consistency so that they can be considered, in part at least, the natural habitat of the organism. This raises the question as to whether the animal infections represent invasion of the body by organisms that naturally occur elsewhere with a nonparasitic mode of existence, or whether these infections result from invasion of the tissues by organisms that are usually parasitic in the intestinal tract or elsewhere in the body. The following investigation was made in order to determine those sources from which P. aeruginosa could be isolated with the greatest regularity and coincidentally to study some of the identifying characteristics of this microorganism. MATERIALS AND METHODS The medium used in the following experiments is a modification of the synthetic medium developed by Burton et al. (1948). The basal medium has the following composition: glycerol, 1.00 g; glycine, 0.60 g; Irleucine, 0.60 g; K2HPO4, 0.04 g; MgSO-7H20, 2.00 g; FeSO4, g; tap water, 80 ml. To the basal medium was added acid pyocyanin solution, 20 ml. Ten ml of this medium were placed in a 50 ml Erlenmeyer flask and adjusted to ph 7.4 to 7.6. The basal medium favors the production of pyocyanin while the complete medium is highly selective, the pyocyanin almost completely inhibiting the competitive growth of all other microorganisms. The use of flasks has the advantage over the use of culture tubes in that they provide an increased surface area, thus increasing the aerobic conditions of the culture medium. The pyocyanin solution was prepared by inoculating 250 ml of the sterilized 1 This investigation was supported in part by a grant from the Upjohn Company, Kalamazoo, Michigan. 2 A part of this investigation was used in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Department of Pathology and Hygiene, School of Veterinary Medicine, Pullman, Washington. 841

2 842 LEIF M. RINGEN AND CHARLES H. DRAKE [VOL. 64 basal medium, contained in penicillin bottles, with 2 ml of a 24 hr broth culture of P. aeruginosa. After 1 to 2 weeks' incubation at 37 C, the pyocyanin was extracted from the broth with chloroform and then separated from the chloroform by dilute HCl. This acid form of the pyocyanin was standardized to give a light transmission of 5.0 per cent or less on a Coleman Junior electrophotometer; light wavelength-625 A. This corresponds to a minimum of 0.45 mg of pyocyanin per ml of solution. The sample to be examined was placed in the flask and incubated at 37 C for a total of 96 hr or until growth occurred. Transfers were made every 24 hr from this culture onto agar slants made from the basal medium, and the identification of P. aeruginosa was based on the production of the chloroform soluble pigment, pyocyanin, and growth at 42 C (Seleen and Stark, 1943). The quantity of the sample varied from 0.5 to 1.0 g if a solid and 1 to 2 ml if a liquid. TABLE 1 Incidence of Pseudomona8 aeruginosa from various natural sources SOURtcE NAYI]:S NUICXI OF PER CENT Soil Barnyards* Human feces Sewage Natural waters Miscellaneoust Total * Includes both fresh and dried sheep, cow, and horse feces. t Includes swabs taken from table tops, sinks, floors, etc. The two isolations were made from fermenting fruit. EXPERIMENTAL RESULTS The results of the previously described experiments indicate that although P. aeruginosa can be isolated from several different sources its normal habitat, in part at least, is human feces and sewage (table 1). Cultures made from soils from which P. aeruginosa had been isolated were negative upon subsequent culturing. That they will survive in soil, however, was shown by their isolation from soils which had been aliowed to dry for several months at room temperature. The isolations made from the barnyard samples were made from a sheep dropping and one from horse feces. The two isolations made from the miscellaneous group were from a fermenting apple and a fermenting banana. Their presence here seems to be one of chance as no reports of isolations from this type of environment were found in the literature, and this type of laboratory experiment, often run in a general bacteriology class, has never before given cultures of this organlsm. Isolations of P. aeruginosa were made from raw and clarified sewage, including both the primary and final clarifiers, but not from sludge. Thirteen additional

3 1952] INCIDENCE OF PSEUDOMONAS AERUGINOSA 843 samples were taken from the final effluent as it left the chlorinator. Of these, one sample taken immediately after the effluent passed through the chlorinator was positive; the remaining samples were negative. Ten of these samples were taken after the chlorine had acted upon the effluent for a short period of time, the chlorine content of the effluent ranging from 0.2 to 0.25 ppm. The sewage samples were taken on various days and at different times of the day. Because of the large number of isolations, counts were made in order to determine the most probable number of organisms (P. aeruginosa) per ml of sewage. These counts were made by inoculating the various dilutions of sewage into each of 10 flasks containing the complete medium. The cultures were incubated at 37 C for 48 hr, then transferred onto agar slants made from the basal medium. The most probable number of organisms per ml was determined by use of the Halverson-Ziegler (1933) tables. The results of these experiments indicate that the most probable number of organisms per ml of raw sewage is 760 while in the final clarifier this number is reduced to 17. The identification of P. aeruginosa is based primarily upon the production of water-soluble pigments. This organism can produce at least three different types: pyocyanin, pyorubin, and fluorescin. Pyocyanin is considered as specific for P. aeruginosa; however, the greenish fluorescent pigment, fluorescin, is produced by other species in this genus, particularly P. fluorescens. The type of pigment produced varies according to the composition of the medium and cultural conditions. A carbon source which is suitable for growth is not necessarily suitable for maximum pigment production. Various carbon sources were tested in the basal medium, and it was found that mannitol, glycerol, and fructose were the only carbon sources which favored maximum pigment production. Other carbon sources tested included glucose, lactose, sucrose, galactose, dulcitol, arabinose, maltose, xylose, inulin, sorbitol, and mannose. All of these showed a trace of pigment after 72 hr incubation at 37 C. The use of gelatin liquefaction and biochemical reactions has not proven satisfactory for the identification of P. aeruginosa. Glucose is the only carbohydrate that can be attacked by this organism with the production of enough acid to give a color change in the usual media employed, that is, a liquid medium. Furthermore, this reaction is a variable one. However, if an agar slant is used in place of the broth, acid can be produced from various carbon sources. Various carbon sources were added to phenol red broth base (Difco) and to this basal medium plus 1.5 per cent agar. These tubes were inoculated and incubated for 48 hr at 24 C, and another set of tubes incubated at 37 C; the broth tubes were placed on a continuous shaker in an attempt to increase aeration. It was found that both P. aeruginosa and P. fluorescens can produce acid from glucose, galactose, mannose, arabinose, and xylose. It is of interest to note that acid will be produced from galactose agar slants when incubated at 37 C, but not at 24 C. Only one strain each of P. aeruginosa and P. fluorescens was used in these experiments. Salvin and Lewis (1946) found these same carbon sources, plus glycerol, could give acid production in a liquid medium if the mediuim was low in organic nitrogen.

4 844 LEIPF M. RINGEN AND CHARLES H. DRAKE [vol. 64 DISCUSSION The results of the experiments described in this paper indicate that although P. aeruginosa can be isolated from various sources its normal habitat, in part at least, is the human intestinal tract and sewage. Human feces probably serves as the inoculuim for the sewage and, therefore, could be considered as a normal habitat. However, the most probable number of organisms (P. aeruginosa) per ml of sewage represents less than one per cent of the total numbers of microorganisms present in sewage. The percentage of human carriers of P. aeruginosa as found by the authors is in agreement with that of Hunter and Ensign (1947). These workers found 15.5 per cent of the individuals examined to be carriers and suggested that healthy individuals may become temporary carriers. It is not known if this organism is a permanent inhabitant of the human intestinal tract or if it occurs in large or small numbers. The isolations of P. aeruginosa from barnyards and soil seem to be one of chance. It is of interest to note that the percentage of isolations from soil is approximately the same as that obtained from the barnyard samples. Rochaiz et al. (1933) isolated it only four times in their bacteriological examination of 207 eggs. The pathogenicity of this organism for domestic animals suggests that it would not be found normally in these animals. The natural water supplies examined included mountain springs and deep wells. Presumably these supplies were not fecally contaminated which may explain the negative results obtained. The reports found in the literature concerning the isolation of P. aeruginosa from private water supplies always indicated that these supplies were subject to contamination from waste materials of human origin. One of the difficulties involved in a study of P. aeruginosa is its identification, especially when comparing this organism to P. fluorescens. The authors found that these two species could give the same biochemical reactions and could not be distinguished on this basis. Haynes (1951) reported that P. aeruginosa can be identified on the basis of growth at 42 C, oxidation of potassium gluconate, and the production of slime in a medium containing potassium gluconate as the principal carbon source. Apyocyaninigenic strains also can be identified on this basis. In addition to pyocyanin and fluorescin, P. aeruginosa can produce at least one other pigment. This is the red, water soluble, chloroform insoluble pigment, pyorubin, first described by Leonard (1924). The authors isolated several strains from sewage which produced pyorubin upon subculturing. Unlike pyocyanin, it is not an indicator, remaining red in both alkaline and acid conditions. It should be noted that the color change of pyocyanin occurs at ph 5.1 to 5.2 rather than at neutrality. SUMMARY AN CONCLUSIONS A selective medim for the isolation of Pseudomonas aerugino8a has been described. This medium was used in a study of the incidence of this organism from various natural sources. It was found that this organism can be isolated con-

5 1952] INCIDENCE OF PSEUDOMONAS AERUGINOSA 845 sistently from sewage and from all parts of the sewage plant except the sludge digestors. Although only 11 per cent of the individuals examined proved to be carriers, human feces probably serves as the inoculum for sewage and should be considered as a normal habitat. Relatively few isolations were made from soil or samples taken from around barnyards. Subsequent cultures made from soils from which the organisms had been previously isolated were negative. It was shown that P. aeruginosa can produce acid from glucose, galactose, mannose, arabinose, and xylose if an agar slant is used instead of the usual liquid medium. REFERENCES BURTON, M. O., CAMPBELL, J., AND EAGLES, B. A The mineral requirements for pyocyanin production. Can. J. Research, C, 26, HALVERSON, H. O., AND ZIEGLER, N. R Application of statistics to problems in bacteriology. I. A means of determining bacterial populations by the dilution method. J. Bact., 26, HAYNES, W. C Pseudomonas aeruginosa-its characterization and identification. J. Gen. Microbiol., 5, HUNTER, C. A., AND ENSIGN, P. R An epidemic of diarrhea in a new-borne nursery caused by Pseudomonas aeruginosa. Am. J. Pub. Health, 37, LEONARD, V Pyorubin, a new pigment produced by Bacillus pyocyaneus. Am. J. Hyg., 4, RocHIMz, A., TOPERNOux, A., AND COUTuRE, E Oeufs et bacille pyocyanique. Bull Soc. Vet. Lyon, 36(4), In Biol. Abst., 9, 147. SALVIN, S. B., AND LEwIs, M. L External otitis, with additional studies on the genus Psudomonas. J. Bact., 51, SELEEN, W., AND STARK, C Some characteristics of green-fluorescent pigment producing bacteria. J. Bact., 46,