Leland W. Parr

Occurrence of Organisms Resembling Pathogenic Members of the Colon-Typhoid Group in Long Stored Feces

When saline suspensions of human feces, stored in the icebox for many months, are plated on Endos agar resultant colonial growth is different from that obtained with the same specimens when fresh. In most specimens, so stored and plated, there occurs an abundant growth of small, white colonies of Gram negative, non-sporing, generally motile, aerobic bacilli which exhibit a variety of biochemical activity, failing however to ferment lactose with acid and gas production, even after prolonged incubation. Some of these organisms culturally resemble paratyphoid, typhoid and dysentery bacilli and may be mistaken for them. Furthermore, certain of the strains give serological cross-reactions with these known pathogens. Possibly they may be involved in gastro-enteritis, apparently water borne, since organisms similar to those we have under study have been reported from such outbreaks. 1 Clemesha 2 was probably first to observe these organisms. For some reason he named them “Bacillus P”, although noting they were a group of organisms not a species. Clemesha found them in fecal suspensions exposed in shallow dishes to the action of sunlight and observed that they appeared when the conditions of the experiment had eliminated the typical coliform bacteria of feces. Parr and Caldwell 3 encountered similar organisms in great numbers in water deriving from wells evidencing old fecal pollution as shown by chemical tests. Parr 4 again mentions them in a study on the viability of coliform bacteria and elsewhere he 5 states that they may be found occasionally in small numbers in fresh feces. This report deals with 35 strains of the “Bacillus P” isolated from fecal suspensions stored in the icebox (4°C.) for a number of months and in a few instances isolated from fresh feces.

Action of Colicines on Shigella boydii and on their Rough and Mucoid Variants

Summary In 58 experiments involving comparisons of 6 smooth parent cultures of various Sh. boydii serotypes and their corresponding non-S variants, the variants were significantly more sensitive to colicine action than the smooth parent strains. Reversal of the culture from the non-S to the S form was accomplished by use of kinetin, and the “transformed” cultures reflected, in part, the colicine spectrum of the original S parent culture and in part the spectrum of the non-S variants from which the “transformed” S cultures were immediately derived.

Unrecorded Form of Bacterium aurescens, Sole Colon-Group Representative in a Fecal Specimen

Chromogenic members of the colon-group are not unknown. MacConkey 1 listed yellow colon-group liquefiers from horse feces, pond water, rain water, roof washings, oats, beans, malt and ears of corn, and he reported a yellow B. coli communis from rain water. Rogers, Clark, and Lubs 2 reported that but few grain cultures are without pigment, many being decidedly chromogenic. They stated that this property is correlated with other characters and consequently is of value in classification. In a collection of colon-bacteria from human feces they found chromogenesis almost entirely absent. All their fecal cultures gave a faint yellow color but this was so slight and showed so little variation that it was of no value in differentiation. They did state, however, that there were a few exceptions to this rule. Wood 3 reported that 7 of 20 colon-group strains isolated from grains, hay and dried eggs and milk produced yellow pigment. In his Pocomoke river series Perry 4 encountered 5 cultures of aërobic, non-sporulating bacteria producing gas from lactose which produced a distinct yellow pigment. He excluded these chromogenic strains from consideration as fecal coli. On January 14, 1936, a fecal specimen was received for study. Although the patient complained of certain general symptoms none referred to the gastro-intestinal tract and the analysis was under-taken as part of a routine rather than as an indicated procedure. A suitable saline suspension was at once prepared and adequately plated on citrated agar, blood agar, and Endos agar. A small bit of the stool was placed in a large tube containing 30 cc. of lactose-indicator-broth. In this enriched medium typical acid- and gas-formation was observed in 24 hours. No colonies appeared on the citrated agar although it was heavily inoculated.

Mucoid Bacterium Coli in Feces of Normal Subject

Theobald Smith 1 was first to call attention to colon bacilli so similar in colony form to B. aerogenes as to be indistinguishable morphologically. Smiths culture “29” derived from a sample of spring water. Mucoid forms of colon bacilli have since been reported under a variety of conditions. Kuwaraba 2 reported a cap-sulated organism from panophthalmitis at first identified as Fried-landers bacillus, but later as B. coli. Revis 3 contaminated soil with feces and after several months obtained mucoid colon bacilli which at 20°C. produced slimy colonies as much as half an inch in diameter. These forms reverted readily to typical colon bacilli. Bordet and Ciuca 4 obtained mucoid “modified B. coli” by the action on the colon bacillus of a guinea pig peritoneal exudate produced by injecting B. coli. Gratia 5 confirmed the wrork of Bordet and Ciuca. He also produced non-mucoid variants by aging a normal culture. Gory 6 obtained mucoid coli by the bacteriophagic action of filtered sewage. Theobald Smith and associates 7 in work on pathogenic B. coli from bovine sources (scours) dealt with mucoid forms and derivatives therefrom. Parr and Caldwell 8 recovered mucoid forms of various members of the colon-aerogenes group, including coli, from water samples from pumps near bored latrines. Lawson 9 reports the recovery of mucoid colon bacilli from diarrheal stools of infants. Dominick 10 encountered mucoid coli in well water regularly receiving chlorine. Furthermore the colon bacillus is ordinarily noncapsulated except by special technique, Churchman and Eme-lianoff. 11 We here report the occurrence of encapsulated B. coli, giving rise to large, moist mucoid colonies on plate culture, derived from the feces of a normal, adult male. During 1932-33, 156 strains were isolated on Endos agar from 16 fresh fecal suspensions in sterile saline in a preliminary attempt to determine the relative percentages of the different members of the colon-aerogenes group appearing on a medium on which all such members grow well. Unexpectedly it was found that on most platings all colonies were devoid of metallic sheen and were larger, more raised and more moist than the classical colon bacilli. A few times these mucoids were mixed on the plates with typical forms and once no mucoid types were encountered. Of the 156 isolations, 91% were B. coli. The mucoid colonies were made up of very short, encapsulated Gram negative bacilli which did not liquefy gelatin nor ferment saccharose, but which in all cases fermented dextrose and lactose with production of gas as well as acid were methyl-red positive, produced indol and failed to grow on citrate agar. No H2S was produced. Motility was negative in 24 hr. broth cultures. Flagellar stains have not yet been made. Milk was on first isolation only acidified but on sub-culture coagulated. Broth suspensions remained homogenous. Serological investigations have not yet been undertaken. These forms have thus far maintained their mucoid nature despite manipulation designed to revert them to a non-encapsulated state.

Sanitary Significance of the Succession of Colon-Aerogenes Organisms in Feces

A study has been made of 57 specimens of feces from normal humans, from which by direct plating, 1454 strains of bacteria have been isolated. Eight of the 57 were stored at 37°C. and plated 60 times during the period of their viability, 2 to 3 months, yielding 592 strains. Duplicates of these 8 and 4 others of the 57 were kept in the ice box and on, 162 platings have yielded 1086 strains. The period of viability for cold stored suspensions varies but may exceed 15 months. The feces were suspended in sterile saline to a heavy turbidity and platings have been made from these suspensions. The procedure diagrammed has been adopted. It provides for adequate purification of strains isolated, permits a comparison of the reactions of purified and original strains, and utilizes the tests now considered most significant for the group. Recognition of lactose-deficient organisms in normal feces 1 has led to the inclusion in the series of all organisms typical of the group which ferment dextrose. Strains failing to ultimately dissimulate lactose are dropped. Adequate sampling has been the rule, 11.2 organisms on average being picked from each of the 279 platings. The strains have been tested for their fermentation of dextrose, lactose, saccharose, dulcite, salicin, alpha-methyl-d-glucoside and cellose; for their methyl-red, Voges-Proskauer, citrate utilization, milk and gelatin reactions; and for the production of indol and hydrogen sulphide. Data pertinent to the topic of this paper are presented in Table I.∗ The only group member found in several fresh fecal specimens was some form of Coli (Escherichia). Of the 1262 strains in the corrected summary only 2 were Intermediate (Citrobacter). On storage the 2–3% of Aerogenes (Aerobacter) originally present in fresh feces greatly increase and the Coli disappear—a process more marked for specimens ice box stored and in such specimens complete in about 5 weeks. Almost coincident with the disappearance of typical coli intermediates types appear and they increase and persist with cold storage. They occur also but are not prominent at any