CHARACTERIZATION OF A. FAECALIS ISOLATED FROM AN IODINE-DISINFECTED

John D. Marshall, M.S. Claire B. Wolford, M.S. John E. Faber, Ph.D. SOME 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 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 (1). mately 99 percent of the 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 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 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 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. 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 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 (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. In addition, 1 and 0.1 ml. of large inoculums. In addition, in a previous these treated suspensions were plated in milk study (1) no adverse effects were observed on protein hydrolysate agar (Baltimore Biological swimmers when the pool water contained as Laboratory) and incubated 48 hours at 35° C. many as 6 X IO3 organisms per milliliter. with counts being made after both 24 and 48 One of the most important results of this hours. Results of these experiments are pre¬ study was the discovery that the organism sented in tables 1 and 2. possesses an extremely high resistance to halo¬ Table 1 shows that at least 2.54 ppm of iodine gen . The tolerance to iodine and was required to kill 100 percent of the organ¬ chlorine was not only of a greater magnitude isms within 10 minutes as determined by failure than has been hitherto reported in the literature to grow in glucose-phenol red broth. Iodine in for bacteria, but also was approximately six the form of hypoiodous acid gave similar re¬ times greater than that required to kill the "in¬ sults. A residual-free chlorine concentration of dicator bacteria," namely, Escherichza coli at least 0.75 ppm was required to kill 100 per¬ and Streptococcus faecalis. Additionally, the cent of the organisms within 10 minutes as resistance of our isolant to halogen disinfect¬ determined in the same manner. Combined ants was approximately four times greater than available chlorine was completely ineffective that of various species of the Salmonella- unless the concentration was at least 19.59 ppm. Shigella group. Results of the plating experiments are shown It has been stated above that at no time in in table 2. In order to kill 99.99 percent of the this study were fecal streptococci or coliform test organism in 10 minutes, the following ini¬ bacteria detected. About 1 percent of the col¬ tial concentrations in ppm were required: onies appearing on standard plates were not iodine, 1.8; hypoiodous acid, 1.04; free chlorine, A. faecalis. This group, mainly pseudo- 0.88; and combined available chlorine, 10.37. monads, and chromogenic gram-negative rods, was found to be killed by the normal operating Discussion of Results concentrations of 0.2 to 0.6 ppm of iodine and The characteristics of the organism described chlorine. above most closely approximate those detailed The source of A. faecalis in swimming pools for Alcaligenes faecalis in Bergey's Manual of is obscure, although Bergey's reports that it has Determinative Bacteriology. The only excep¬ occurred in decomposing organic matter.

Table 2. Initial concentration of halogen required to kill at least 99.99 percent of Alcaligenes faecalis

Time and concentration (ppm) to kill

Halogen 1 minute 5 minutes 10 minutes

Concen¬ Percent Concen¬ Percent Concen¬ Percent tration kill tration kill tration kill

Iodine as I21_ 1.8 99. 991 1.8 99. 999 1.8 99. 9999 Iodine as hypoiodous acid_ 2.08 99. 990 2.08 99. 998 1.04 99. 992 Chlorine (free)_ 1.60 99. 996 .88 99. 992 .88 99. 9999 Chlorine (combined available) 49.99 99. 991 19.86 99. 993 10.37 99. 992

1 The next lower dilution, 1.4 ppm, killed 98.25 percent of the organisms in 10 minutes. Note: Temperature, 28° C; pH, 7.85. Initial concentration, 5X106/ml. 532 Public Health Reports Studies are in progress in our laboratory to Summary determine possible modes of entry of this or- ganism in such waters. Stevenson and Wetzler An organism comprising approximately 99 investigated the environmental contamination percent of the total bacteria count of swimming of pool water by total plate counts (personal pool water has been identified as Alcaligene8 communication). Pool decks, overshoes, duck- faecali.. This organism was found highly re- boards, stair wells, shower stalls, and locker sistant to halogen disinfectants. Apparently rooms were found to be reservoirs of A. faecalis. it has no great significance in the sanitary Such investigation pointed up the absolute analysis of swimming pool water. need for total environmental sanitation of all pool premises, often neglected in pool man- REFERENCES agement. (1) Marshall, J. D., Faber, J. E., and Campbell, W. R.: The significance of A. faecacis in total counts Advantages and limitations of iodine disinfec- of swimming pool water is difficult to assess. tion of a large controlled indoor swimming pool. If, as in our experience, 99 percent of the col- Am. J. Pub. Health. In press. onies appearing on standard plates are of this (2) Chambers, C. W., Kabler, P. W., Malaney, G., and type, it would be presumptuous to condemn Bryant, A.: Iodine as a bactericide. Soap and such water for sanitary reasons, even if the San. Chem. 28: 149-151, 153, 163, 165 (1952). numbers of colonies exceeded generally accepted (3) Chang, S. L., and Morris, J. K.: Elemental iodine as a for drinking water. Indust. values for total bacterial counts. We would & Eng. Chem. 45: 1009-1012 (1953). stress the qualitative rather than the quantita- (4) Chang, S. L.: The use of active iodine as a water tive aspects of such water, particularly in the disinfectant. J. Am. Pharm. A. 47: 417-423 light of the results presented here. (1958).

Uniform Personal Health Record for Migrant Workers A uniform personal health record form for for records of immunizations, laboratory migrant agricultural workers has been pro- tests and results, obstetrical history, and duced by the Public Health Service. The form clinical conditions, which are important in was recommended by the Association of State future care of the worker. In the last two and Territorial Health Officers sections, the name and address at their 1960 conference in San Gof the person making the entry Francisco. are requested. On the cover, space is pro- The forms are 101/2 by 4 vided for the worker's name, \ inches and have space on both home address, sex, and birth | sides for information pertinent date or age. A note in English to the worker's health. They and in Spanish advises him to fold, as illustrated, to 25/8 x 4 show the record whenever he inches, a size which the worker goes to a doctor or a nurse. Physicians and can carry easily in his billfold or in his pocket. nurses are requested to enter pertinent infor- Local health departments and volun- mation and return the record to the bearer. tary agencies may obtain these forms from The entry sections of the form provide space their State health departments.

Vol. 76, No. 6, June 1961 533 It is contrary to the custom of this journal to announce awards. An exception is made for Pupone, 2- year-old pointer, awarded a medal by the National Society for The Connecticut State Depart¬ Mothers who smoked a pack or Medical Research for contributions ment of Health is providing new more of cigarettes a day had a rate to the study of leptospirosis at the educational and consultant services of premature births more than double PHS Communicable Disease Center. to assist administrators of nursing that among nonsmoking mothers, it A carrier of leptospires, Pupone is homes and other long-term care in¬ was observed in a 2^-year study of personally in excellent health. As stitutions in improving restorative 2,736 Negro women by the Balti¬ for the disease, he says, "It shouldn't and dietary services to their pa¬ more City Health Department aided happen to a man." tients. by a PHS grant. a » The restorative services program Three possible reasons were cited: is recreational curb the and Following the pattern set on the surveying existing Smoking may appetite State New York and occupational activities and reduce weight of mother and level, City's execu¬ baby; tive director of medical care services needs in licensed nursing homes and it may constrict blood vessels which in the health will serve chronic disease facilities as a first nourish the a decrease department baby, causing also as medical in blood supply to the and welfare administra¬ step. placenta; tor in the welfare department. «c » smoking itself may be linked to other characteristics which contribute to « » A leaflet issued in Spanish, by the premature births. The New Jersey State Public Minnesota of to Department Health, « » Health Council has set forth what advise mothers on preparing their are to be considered the A report on the Conference on recognized children for school, has been repub- public health activities of local lished in revised form by the Mich¬ Leprosy, at Carville, La., November 7-10, 1960, is being printed by the health departments in New Jersey igan Health Department for the and the minimum standards of per¬ of that Star, published by patients in the Spanish-speaking population Public Health Service Hospital. formance they must follow. This migrant action was in State, including agricul¬ There is no accordance wTith 1947 tural workers and industrial work¬ charge for reprints re¬ quested from the Star, Carville, La. legislation which also provides for ers of Mexican or Puerto Rican the enforcement of the requirements origin. « » by the State commissioner of health. Minnesota also has published a The Minnesota Health Depart¬ The detailed requirements were ini¬ book of recipes in English and Span¬ ment's last budget statement used tially prepared by a committee of ish to help Spanish-speaking fam¬ charts to demonstrate the investment local health officers. order for potential outlays for gen¬ ilies ingredients Spanish alcholism, « » dishes in American markets. etics, and fluoridation. The first care The alcoholism chart contrasted a progressive patient to be the Hill- request for $35,000 for a preventive hospital sponsored by Burton is now in The National Science Foundation program with a disbursement of program operation on March 20, 1961, announced the $435,000 for institutional treatment in Grand Junction, Colo., with 15 beds for intensive 57 beds for award of 1,537 graduate fellowships of alcoholics, $600,000 spent on alco¬ care, in the sciences, mathematics, and holics in two city workhouses, and intermediate care, and 12 beds for engineering for the academic year the uncalculated number of accidents self-care. 1961-62. Of the awards, 347 were associated with drunken driving. « » in the made the life sciences; 1,156 in Taxes levied on alcohol within the In 1955, when the LeRoy, N.Y., physical sciences, including a num¬ State total $45 million a year. sewage program was defeated at the ber in interdisciplinary fields; and The chart for the genetics budget polls, the project would have cost 34 in certain areas of the social presents one example of an inherited $1,380,000. Today, the cost is sciences. disease, phenylketonuria. Faribault $2,150,000. State has 46 with Hospital patients « » The Doctor's Association of the this disease, representing a lifetime loss of Ohio's Health, published by the Department of Health has been earning approximately $6,- State of health devotes 900,000 and cost to the State for care department formed in New York City. Bargain¬ an of about $3,450,000. entire issue to dental health. ing collectively, the association will Articles on dental care for needy work for salary increases, pension « » children, emphasis on dental health rights, relief of dentists and physi¬ A census of public health nurses, in 4-H clubs, mouth protectors in cians from clerical work, more nurse compiled for the first time by the athletics, and school programs and assistance, and reestablishment of Public Health Service, appears in one titled "Fluoridation.the 65 Per¬ reorientation courses for public The American Journal of Nursing, cent Answer to Tooth Decay" are health doctors. February 1961. included.

534 Public Health Reports