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Waters Containing Between 0.2 and 0.8 Ppm Iodine Ranged from 8 To

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Waters Containing Between 0.2 and 0.8 Ppm Iodine Ranged from 8 To CHARACTERIZATION OF A. FAECALIS ISOLATED FROM AN IODINE-DISINFECTED SWIMMING POOL John D. Marshall, M.S. Claire B. Wolford, M.S. John E. Faber, Ph.D. SOME bacteria whose sanitary significance in diameter occurred after 48 hours. Standard is obscure have been found to resist concen¬ plate counts made on samples of swimming pool trations of halogens bactericidal to fecal strep¬ waters containing between 0.2 and 0.8 ppm tococci and coliform bacteria, the accepted in¬ iodine ranged from 8 to 17X102. Approxi¬ dicators of swimming pool sanitation (1). mately 99 percent of the organisms encountered Many instances of high plate counts in the ab¬ were of the colony type. During the entire 8 sence of coliforms are known. The identifica¬ months of this study, not a single positive test tion of species within these populations is for fecal streptococci or coliform bacteria was lacking in the literature, although there are obtained. In view of the fact that the flora several reports concerning the bactericidal recovered on TGE agar appeared to be almost properties of iodine disinfection on known in¬ homogeneous, our interest was directed toward testinal pathogens in water (0-4) . We have further characterizing these organisms. It been unable to discover any information con¬ should be pointed out that similar observations cerning the occurrence in water of a micro¬ have been made on waters from pool samples organism highly resistant to halogens. taken at Baltimore, Md., Buffalo, N.Y., Denver, This paper supplies data on the basic physi¬ Colo., and elsewhere. ological characteristics of an organism identi¬ fied as Alcaligenes faecalis, together with infor¬ Macroscopic Morphology mation on its resistance to various levels of As stated above, after 48 hours at 35° C. the iodine and chlorine disinfection. The opera¬ colonies were about 1.0 mm. in diameter. They tion of a large swimming pool and the rates were glistening, convex, mucilaginous, and with and methods of feeding are discussed in an¬ no apparent pigmentation. Plates containing other paper (1). pure cultures of these organisms possessed an unclean, characteristic odor upon removing the Occurrence and Characterization covers initially. After 10 days at 20° C. the colonies increased in size to 2 to routine of approximately During analysis (1) swimming 3 mm. in diameter. The centers were dome water disinfected with pool iodine, pinpoint and a faint buff coloration was appar¬ those less than 0.25 mm. in shaped, colonies, diameter, ent. In liquid media, the organism grew with were observed after 24 hours of in¬ consistently the formation of a slight pellicle and a heavy cubation at 35° C. on trypticase glucose extract sediment. The sediment formed An increase to 1.0 mm. mucilaginous (TGE) agar plates. a characteristic strand when the tube was swirled. The authors are with the department of microbiol¬ ogy, University of Maryland, College Park, Md. Microscopic Morphology This investigation was supported by a grant from Twenty-four cultures, grown on either TGE the Chilean Iodine Educational Bureau, Inc. agar or in phenol red broth (Baltimore Biolog- Vol. 76, No. 6, June 1961 529 Alcaligenes faecalis (X35,600) ical Laboratory), were found to be gram-nega¬ Citrate was used as the sole carbon source, and tive, bipolarly staining rods, occurring singly or the organism grew on desoxycholate agar but in pairs. The organisms were approximately 2/* not on tellurite agar. Litmus milk was made in length and 0.9/x in width, as determined by alkaline without digestion. Gelatin was not electron microscopy. They possessed a distinct liquefied after 21 days' incubation. Tests for capsule when stained by Maneval's technique. indol, nitrate reduction, and hydrogen sulfide The organism was actively motile when ob¬ were negative. Urea was hydrolyzed, and the served in wet preparations and on hydrogen methyl red and Voges-Proskauer tests were sulfide indole motility medium (Baltimore Bio¬ negative. The organism was not hemolytic on logical Laboratory). Spores were never ob¬ horse blood agar, and it failed to grow in the served. An electron micrograph showing the presence of 3 percent sodium chloride. Colori- morphology and flagellar arrangement of these metric tests for cytochrome oxidase were posi¬ organisms is presented in the photograph. tive after 30 seconds. Growth was optimum between 25° and 35° C, and slight at 20° C. Physiological Reactions There was no growth at 10° or 45 ° C. Routine Physiological reactions were obtained by sensitivity tests against various antibiotics and tests described in the Manual of Microbiologi¬ sulfa compounds gave the following results: cal Methods. The organism may be character¬ not inhibited by bacitracin, oleandomycin, peni¬ ized as follows: There was no acid production cillin, erythromycin, altafur, or furadantin; from xylose, arabinose, rhamnose, glucose, inhibited by dihydrostreptomycin, triple sulfa, mannose, galactose, fructose, sorbose, glycerol, sulfathiazole, albamycin, neomycin, polymixin mannitol, sorbitol, inositol, sucrose, maltose, B, terramycin, mandelamine, dimethylchlortet- lactose, raffinose, salicin, inulin, or dextrin. racycline, and novobiocin. 530 Public Health Reports Pathogenicity Tests was diluted to give 0.3, 0.5, 0.8, and 1.6 ppm Five 30-gm. white mice were inoculated intra¬ solutions of free chlorine. Combined available chlorine solutions were from a stock venously with 0.2 ml. of an 18-hour trypticase prepared soy broth culture of the All of the solution of (NH4)C1 and NaOCl to provide organism. and mice survived, and none showed symptoms of final concentrations of 1.0, 5.0, 10.0, 20.0, any kind. The test dose was then raised to 0.5 50.0 ppm. were to ml., and a second set of five mice was inoculated Halogen levels determined prior inoc¬ intraperitoneally. Again all mice survived and ulation of the test suspension of bacteria and were asymptomatic. One-half milliliter of again at the end of the experiments by amperi- broth culture was placed under the eyelids and metric and iodometric titrations. in the ears of each of three white rabbits weigh¬ To 400 ml. of test solution, 0.25 ml. of a ing 6 to 7 pounds each, and no irritation was suspension of the test organism was added so observed in any of the sites. Intradermal in¬ that the final count was approximately 4 to oculation of. rabbits with 0.1 ml. of broth 6 X106 organisms per milliliter. One-ml. sam¬ culture resulted in a slight reddening of the ples were withdrawn at intervals of 1, 5, and skin at the inoculation site, but this disappeared 10 minutes and placed into 2 ml. of N/4 sodium in 5 days without ulceration. thiosulfate solution. At the conclusion of the The from a 24-hour culture on a growth Table 1. Bactericidal of TGE agar slant was suspended in 0.85 percent efficiency halogens saline, and standardized to the No. 3 tube of against Alcaligenes faecalis the McFarland Two-tenths nephelometer. Growth after milliliter of this solution was injected into the Halogen concentration (ppm) exposure yolk sacs of embryonated eggs, as follows: (minutes) six eggs with 5-day embryos, seven eggs with 9-day embryos, and four eggs with 12-day em¬ Start 30 minutes 10 bryos. Among the 5-day embryos, one died within 24 two died within 48 and Iodine: hours, hours, 0.32 0.02 + + + the remaining three survived. Among the 9- .69 .35 + + two died within 48 one died 1.39 .67 + + + day embryos, hours, 1.86 1.20 + + after 72 hours, and the remaining 4 survived. 3.42 2.54 + 0 0 the one died within 24 4.52 3.49 0 0 0 Among 12-day embryos, Hypoiodous acid: hours, and the remaining three survived. 1. 04/0. 77 i 1. 04/0. 48 + + + 2. 08/0. 89 2. 08/0. 74 + + 0 4. 16/1. 72 4. 16/1. 47 0 0 0 Tolerance Chlorine (free): Halogen 0.36 0. 18 + + + .49 .27 + + + Stock chlorine demand-free water was pre¬ .88 .45 + + + in accordance with Standard Methods for 1.60 .75 + + 0 pared Chlorine (combined the Examination of Water and Waste Water available): Scientific Co. standard iodine solution 1.22 1.24 + + (Fisher 5. 14 4.96 + I2/I"). Stock solutions of iodine and chlorine 10.37 10. 10 + + were as follows: iodine solution 19.86 19.59 + 0 prepared N*/50 49.99 48.04 0 0 was diluted with chlorine demand-free water to a concentration of give approximately 0.3, 0.6, 1 Numerator refers to actual iodine concentration; 1.2, 1.8, 3.6, and 4.8 ppm iodine. Iodine solu¬ denominator, to residual available chlorine from di- chlorodimethylhydantoin. tions in the form of hypoiodous acid were pre¬ Note: Temperature, 28° C; pH, 7.85; initial cell 4 ml. of a iodide concentration, 5X10 6/ml. pared by adding potassium Plus indicates in red solution to an solution of sign growth glucose-phenol (0.416 mg./ml.) aged broth after 72 hours at 35° C; 0, no growth. l,3,dichloro-5,5-dimethylhydantoin (8.54 mg./ The large decrease in halogen concentration during the 30-minute period was due to the addition of washed 1.) and diluting to provide 1.0, 2.0, and 4.0 ppm bacteria to the flasks (1.6-2.4X109 organisms). Dupli¬ iodine. cate flasks treated in like manner but without addition of bacteria showed no drop in halogen concentration Five percent sodium hypochlorite (NaOCl) during the 30-minute period. Vol. 76, No. 6, June 1961 531 experiment, 0.1 ml. of these treated suspensions tion noted was the ability of the isolated organ¬ was inoculated into glucose-phenol red broth ism to hydrolyse urea. This isolant apparently base medium, incubated for 72 hours at 35° C, possesses no pathogenic properties, as shown and observed for visible growth after 24, 48, by the tolerance of rabbits and mice to relatively and 72 hours.
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