3% WWW \HHI, A STUDY OF THE CULTURAL AND SEROLOGICAL REACTIONS OF SOME TYPICAL AND ATYPICAL COLIFORM ORGANISMB Thesis forfhc chrce of M. S. MiCHlGAN STATE COLLEGE | ' Mary Jane Washburn . 1943 I _ THFSIS u'lil‘ll ll A STUDY OF THE CULTURAL AND SEROLOGICAL REACTIONS OF SOME TYPICAL AND ATYPICAL COLIFORM ORCANISMS by Mary Jane Washburn / A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology 1943 THESIS ACKNOWLEDGEJENT The writer w'shes to thank Professor W. L. Mallmann for his advice and guidance, and Mr. R. J. Patrick for his able technical assistance. ,J‘ 4 " "”5, "" - L' I u 'r' ; l . a J! i!”- "';J a K INTRODUCTION Some years ago Bergey and his colleagues (5) described organisms of the genera Escherichia and Aerobacter as aerobic, Gram negative, non-spore forming short rods which ferment lactose and dextrose with the production of acid and gas. These organisms, now collectively designated as coliform, (7) have . risen in importance. For a number of years the coliform group has been used as the criterion of polluted water in routine laboratory analysis all over the country and, indeed, all over the world. For this reason the coliform organisms have been and are being studied more extensively than probably any other one group of organisms. As a result of these wide detailed studies, observers are continually finding organisms apparently coliform but which vary in some characteristic which hag been acdepted as standard for the colon—aerogenes group. Various authors have designated those organisms as "irregulars" which ferment lactose but do not fall into either the Escherichia or Aerobacter group according to the citrate, methyl red and Voges Proskauer tests for the differentiation of Escherichia coli from Aerobacter aeroggngg. Those organisms which are seemingly coliform but do not ferment lactose with the production of acid or acid and gas in 48 hours will be called atypical. In addition to a discussion of "irregular" and atypical organisms, a consideration of The Standard Methods for the Examinatign 9: Water and Sewggg (2) from the point of view of the efficiency of eosin.methylene blue agar as a means of differentiating E. coli from A, aerogenes. will be presented. HISTORICAL "Irregular" Coliform Organisms Coliform organisms which could not be classified as Escherichia or Aerobacter according to the methyl red, Voges Proskauer, and citrate tests were mentioned by MacConkey (24) as early as 1905. France (10) isolated $3.221; from both feces and polluted water. He found that 28.7 per cent of the organisms from water gave irregular results as compared with 2.2 per cent from feces. Others have found also a large percentage of "irregular" coliform organisms outside of the animal body. Bahlman (3) states that in water he found 15.3 per cent "irregular" out of 1223 strains tested. From a total of 4297 strains Bradsley (6) found that 581 or 12 per cent were "irregular." Koser (18) stated that "irregular" forms were found mostly among soil strains but were absent in feces. Again in 1926 he (19) found 31.9 per cent of the organisms in non-polluted pasture soil to be "irregular" and a very small number in polluted pasture soil. Yale (39) isolated 204 colon-aerogenes cultures from milk. He reported that 33 per cent fell into the "irregular" group. Hicks (13) found 10.6 per cent "irregular" from human feces, Car- penter and Fulton (8) 13.3 per cent, and Parr (28) found 7.7 per cent from fresh human fecal samples. These researches indicate that large numbers of "irregular" organisms are commonly present in soil and water samples. The Accuracy of Eosin Methylene Blue Agar The accuracy with which eosin methylene blue agar can be used to differ- entiate g. 5511; and A. aerogenes has been contested. Levine (22) believed it to be highly satisfactory. He stated that in 96.8 per cent of the cases eosin methylene blue agar gave typical colonies from E. 991;, 82.4 per cent were typical from A, aerogenes and that "irregup lar" colonies showed characteristics similar to A, aerogenes. Georgia and Morales (ll) concluded that in general the coliform organisms could be differentiated on E. M. B. agar. They reported, however, that in a number of instances colonies which were picked for one type proved to be another. Poe (31) testing the dependability of the E. M. B. agar in 195 cultures belonging to the colon group found that 88.2 per cent gave typical Escheri- chia colonies. 23 of these cultures were "irregular" and 13 had sufficient characteristics to put them in the colon group. He stated that there was a 94.9 per cent correlation for Escherichia. 4.3 per cent of the 164 aero- genes cultures were not characteristic, giving an accuracy of 95.7 per cent. Ruchhoft (33) in a study of both pure and mixed cultures stated that macroscopic interpretation of first streaked isolation plates is unscientific and in many cases conjecture. In this paper an attempt has been made to determine the accuracy of this widely used method for differentiating g, coli and A. aerogene . 4 Atypical 90.1.1393!!! Organisms. Atypical coliform organisms have been referred to as non-lactose fermenters, slow lactose fermenters or late lactose fermenters. Some workers believe that atypical coliform organisms are not derived from g. £21; and therefore are of no sanitary significance. Klein and Houston (14) found atypical organisms on grain.' Savage (35) stated that atypical E, 291; were of little sanitary significance. He showed that hold— ing typical g. 29;; in mud did not change its characteristics. MacConkey (24) showed that E, ggli_retained all of its characteristics unchanged after an unfavorable environment of 258 days. Later some evidence was brought forth to show the importance of atypi- cal coli. Karstrom (17) demonstrated that the enzyme lactase in coliform organisms is adaptive. It therefore may vary in amount depending upon the conditions for growth or synthesis of protOplasm. Stokes, Weaver and Scherago (36)reported the conversion of late lactose fermenters to rapid lactose fermenters and again to late lactose fermenters. They concluded that the strains studied were variants of different members of the colon-aerogenes group. Kriebel (20) agreed that such organisms as these are as definitely fecal contamination as E, 321;. That same year Lewis (23) studied the phenomenon of dissociation in mutable strains of Escherichia and Aerobacter by the use of synthetic media containing lactose. He stated that non-lactose fermenting variants gave rise to lactose fermenting strains. Ziegler (40) in 1939 suggested that since lactase is adaptive, a reversal may have taken place in late lactose fermenters. In a polluted water the concentration of nutrient material is usually too low to permit active multiplication of cells. Conditions may exist which would cause a decrease in lactase activity even in the absence of cell multiplication. In this paper an attempt has been made to identify these atypical organisms by the use of cultural reactions such as the fermentation of various carbohydrates. Serological Relationship Among Coliform Organisms Many investigators have tried using serological methods to determine the relationship between atypical coliform organisms and Escherichia.ggli. A rather startling fact was mentioned by Pfaundler- (30) in 1898 when he stated that there was no serological homogeneity among typical coliform cultures. He also noted that sera of the immunizing animals did not always agglutinate their homologous strains. In other words, he found it diffi- cult to produce antibodies for these organisms. One year later Radzrevsky (32) concluded that the colon bacteria can be divided into a large number of serological groups. Jatha (15), Mackie (25), Van Loghem (38), Herrold (l2), and Magheru (26) confirmed the work of Bad- Vzievsky. Strunz (37) immunized rabbits with 14 out of 23 strains of fecal coli; form organisms and by cross agglutination classified these 23 strains into 3 major groups independent of their origin. Meyer (27) obtained similar results. He dividedlg.‘ggli into 3 types on the basis of type—specific and species-specific'antigens. ven thvujh the various strains 9€.§-.£2li were shown to be serologi— ca3_3y heteiogenous many tried to show Serological homogeneity between the non—la tos- fe WI enterL or atypical coli. Fothergill (9) shoaed that non— lactoee fermenters were serologiC'lly heterogeneous and Abdoosh (1) con- firrrxec’. this . Jones and Little (16) found that the slow lactose fermenters shoved no im unolorice l specizic; t} for paratynhoids. Kriebel (20) and Sandiford (34) confirme3 this and Sandiford also stLtec that the at. niczl coli are heocro— geneoue tith a small degree of common antigen bet as n indivi uzl st aims. Kriebel (20) attempted to classify the e.ty -Mi a1 coli from feces. Antisera were used from 2 Escherichia typees, 4 Salmone33 a, 3 Shi e3la and 0‘.) l Eaerthe la t3pe. Ove r 65 per c nt of +he ctrzinsz were nega tive nd the nositive ones agglutinated non-Specifically. Parr (29) using slow lactose, ran-lactose and non—dextroL e fez mentars testiu teem sarolori c 33_y \ith an 1 ans 0; fiffi’ F‘V‘l‘ 1" = ‘ 1‘ '5. g, .aranL entciiae, g. naict phi, S. scnottmallcii, S. .. x - . .g. aertrycke, Protc s 2, ana Alcaligenes feca H P U) C :11 (D A cross reactions occurred slirhuly for only E. typhosa a It i these researches Les succeeded in linkin" ((3 easily seen that none 0; the a typical coli with any other one group or organisms serolosicelly. All workers do agree upon one fa t, i.e., that various typical strains of E. coli are not serological3y homoteneous. ntly, very few workers, if a1,, have determined the serological d Apnar (U I ~- . h': D h~~n ~- -~ 1‘ r- e v , relrtionfi in between various L*r. n: o! A. hero_anes nor hale they attempted to def ermine the relationship between A. ~ero~cncs and the aty5ical oliform W org??? nal S O 7 EXPERIMENTAL The cultures with which this work was done were obtained from various bacteriological laboratories over the country. They were isolated from water supplies and were identified as coliform organisms according to The Standard Methods for the Examination of Water and Sewage (2). The eXperimantal work which follows is divided into two parts. First, an analysis was made of the typical cultures (those coliform organisms which ferment lactose and dextrose with the production of acid and gas in 24 or 48 hours). These organisms were classified as E, coli, A, aero enes, or an "irregular" type according to the citrate, methyl red and Voges—Proskauer tests. A correlation was made between the original classification of these cultures on eosin methylene blue agar by workers in various laboratories according to results obtained with the citrate, M. R., and V. P. tests. Secondly, the atypical coliform organisms (slow lactose fermenters or non lactose fermenters) were considered. They were tested for acid and gas production on the following media: inositol, inulin, dextrin, xylose, mannose, sorbitol, raffinose, mannitol, dulcite, trehalose, galactose, levu- lose, and salicin broths. Gelatin liquefaction and the ability to produce hydrogen sulfide were noted. A correlation was also made between the nature of the organism isolated and the source of the water sampled. In addition an attempted classification was made by serological methods. Antiserum for typical E. coli and g. aerogenes cultures was obtained from rabbits. Agglutination and absorption tests were run with typical antigens of E, coli, A. aerogenes, and atypical coliform organisms. TECHWIQUE The coliTorn cultures received vere checked for purity and were tested on the Koscr's citrate medium, and for methyl red and Vogcs Proshuuer reac- tions on dextrose phosnhete medium. Loser's citrate test and the methyl red tests tare carried out in the usual manner. A nexcr modi- LCCtion of the Vores Fresnnuer test we {0 used. To 1 ml. of the culture, 0.6 ml. of 5 per cent aloha nupthol in abs olute eth"l alcohol and .2 ml. of 40 per cent KOH were added. The tubes more “ncrh‘ ed at 3700 end the results mere reed from 3 to 4 hours later. A positive reaction was czsi n tx by the formation of. a red ring (A). The atyoicel cultures V re tested on verious cerbohydr .t.es for tee production of acid and gas, on gelatin for liquefaction, and on iron citrate gear For the production of hydrogen sulfide. The following carbohydrates were us:ed: inositol, inulin, dextrin, mannose, sorbitol, raftinose, munnitol, du-01te, trehnlose, selectose, levulose, and selicin. Five tenths per cent 0 each of these UL: added to nutrient broth and eutecleved at 10 pounds pressure for 10 minutes. One-tenth per cent Andrade's indicator tee used to designate a . all in pH. Durham tubes were used to check gas production. Observations were made after 245 M( 48 hours incubation. Gel: tin liquefaction t8 5 te Hte by incubzfi in.: the cultures for 3 days at 370C and chillin: to determine whether the gelatin would solidiTy. Hydro: n sulfide production “as deter- mined by using stew cultures 0 iron citrate agar and obs ervinq a blackening along the line of inocu_ation. The medium contained 2 pe‘ cent Difco p“oteose peptone, 0.1 per cent KZEWOA and 0.05 per cent iron citre te in egrr. ‘ 0 -\ The seroloficul tort ta carried on with antisera from rabtits. The U; ' Y antigens used for the production of the antisera mere onenolized (.5 per cent) usnel.1ons in phvsi olefical saline but were not heat killed. The antigens mere 0? a density conoarhble to a No. IcTar‘ rcl Nepholometer. Intravenous injections were made on three successive days of euch Leek until the maximum titre was obtained. The cuantity of antQ 1n v-5 increased from .5 ml. to 3.0 ml. over a period of three weeks. The ent1seruum x.as preserved by repriserstion. The antigens used in the agelutination rezc ions MC re made in the same manner as those For inj ction s. The agglutination reactions were made in the follomizg anner. The antigen in saline was added to 8 tfil -es, 2 ml. in the First and 1 ml. in the rest. One tenth of the anti— serum was added to the First tube containine 2 ml. 0 antifien. Serial dilutions xere made by t king 1 ml. from the irst tube and adding it to the sszcond. In this may, di]-u+ions o; 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1230 were obtained. T.e eighth tube served as a control. Tests for soecies—soecific and erouo-soecidic anticens were run. The L .& ~— - ‘ _.. q H or species—snecific antigen was prepared as .ollows: the wenty-Four hour 1’ 1 growth :rom bee_—in? usion agar in pint B13? bottles was tusp oed in 20 to (D :5 30 ml. of .85 per cent salt solution containing 0.2 p r cent formalin. After the micro-organisms had been killed the turbidity was adjusted to corr;snond to 1351804 standard No. 3 by the addition of salt solution containing 2.0 per cent ferns .lin. The 0 or group— —specif ic antigen v.25 prepared by washing the grouth in 10 ml. of 0.85 per cent salt solution containing 0.5 per cent phenol. The growth from seve‘al bottles was combined anc one-half+ he volume of absolute 10 alcohol or a proportional amount of 95 per cent alcohol was added slowly, while the suSpension was constantly stirred. It was then allowed to remain at a temperature of 37°C for about 18 hours, after which the supernatant fluid was decanted and tested for bacterial growth and agglutinability. After determining the dilution necessary to secure a density equal to that of BaSOA standard No. 3, sufficient alcohol was added to the concentrated suspension to give 2.5 per cent in the diluted antigen, which should contain not more than 0.04 per cent phenol. The agglutination reactions with these antigens were made in the same manner as those previously described. ll .ynical Coliform Organisms "Irregular" Colif orm Orgm n1s ms A total of 254 coliform cul ures which fe1ment lac ose and dextrose with the procuction of acid and gas in 24 to 48 hours was classified accor— o. H- s on to the methyl red, Voges Proskauer and Koser' s citrate test. As shown in tables 1 and 2, a total of 89 (34.6 per c>nt) of these orsa nisms was found to be g, 2211: 94 (36.5 per cent).g. aerog s, and 71 (28.8 per cent) were "irregular" to these tests. This rela ively his :h percentag of "irregular" organisms confirms the orh of France (10) who found 28.7 per cent of the "irregular" coliform organisms in water. Previously, Bahlman (3) had found only 15.3 per cent to be "irregu er" of the cultures isolated from vx ter. The variation in the results found by France (10) and the author as compared with that of Bahl— man (3) 1s probably caused by a basic difference in the source of the tater s believed that there Ho from which the samples ts re taken. For e sample, it is a smaller percentage of "irregular" organisms in fresh feces than in cultures isola stew fr om water suiplies. Ko oser (19) found a smaller number of "irregular" organisms in polluted pasture soil than in virgin soil, and Parr(28) in 1936 stated that there were less "irreg1lar" organisms in fresh “3 eces the an in.water. Table 3 shows that the majority (54.9 per cent) of the 71 "irregular" organisms found in this group isolated from various water supplies were citrate positive, methyl red positive and Vegas Prosl:auer negative. \s high as 35- 3 per cent were positive to all three tests. Seven per cent were 12 Table 1 Classification of coliform organisms which ferment lactose and dextrose * Culture Kosar's citrate Methyl red Vegas-Proskauer l - + - 3 + + - 5 - + — 7 + - + 8 + - + 9 + - + 10 - + .. 11 — + _ 12 - + _ 13 - + _ 14 + - + 15 - + .. 16 _ .. + 17. - + — 19 - + . _ 20 - - + — 21 + - + 23 + + - 25 + + .. 26 + — + 33 - + - 35 + + + 13 __Culture Koser's citrate Methyl red Vgggs-Proskauer 40 + + .- 41 + + .. 42 + + .- 43 + - + U. - + - 46 + - + 55 - + - 56 - + — 61 + + - 62 - + .. '70 - + - '75 - + - 90 + - - 91 + .. + 92 + — + 102 .. + _ 104 — + _ 107 + + + 108 + + - 109 - + - 115 - + + 132 + - - 142 + - + 143 + - + Culture Koser's citrate Methyl red ches—Proskauer 11.4 + - + 145 + .. + 146 + _ + 149 + _ + 160 + .. + 164 + + + 165 + - + 167 + .. + 171 + - + 172 + — + 176 + + + 178 + - + 179 + .. + 182 + - + 185 + - + 189 + — + 203 + - + 205 — + - 208 + - + 215 + - + 229 .. .. 1 221 + - + 223 + - + 239 + + + 15 Culture Koser's citrate Methyl red Voges-Proskauer 226 + + — 245 + - + 278 + + - 291 + - + 241 + - + 292 + + + 299 + + - 231 + - + 298 — + - 295 + + — 258 + + + 296 + + - 291. + + + 261 + - + 26 7 - + '- 260 + + - 281 - + - 283 — + - 297 + - + 2'79 + - + 280 + - + 285 + + " 287 + + + 286 + + " 16 Culture Kgser's citrate Methyl red Voges—Proskauer 288 + + + 289 + + + 290 - + — 277 - + - 2'75 - + _. 242 + — + 275 - + - 268 + + - 269 - + - 274 - + - 238 — - + 192 + - + 193 + _ + 342 - + ‘ 343 - + ‘ 31.4 - + " 345 - + - 346 - + - 348 + - - 349 r + ' 350 - + ‘ 351 - + ’ 352 - + ’ - + - 353 17 " Voges-Proekauer flture Koser's citrate Methyl red 354 - + - 355 + - + 356 .. + _ 357 + + _ 358 + _. + 359 + - + 360 + + _ 361 - + .. 362 + .. 4. 363 + - + 36!. + - + 365 - + .. 368 .. + __ 367 + + + 369 + .. .. 370 + + .— 371 .. + + 372 + + + 374 - + _ 375 + + .. 376 + + + 3’77 - + " 278 - + - 279 + — + .. .nl'. .nll Hiya, 18 Culture Koser's_gitrate Methyl red. Voges—Proskauer 380 + + + 381 .. + _ 382 + - + 384 + + _ 385 + - + 386 + + .- 387 + '+ - 3 88 + + _ 389 + + - 390 + + __ 391 + + _ 392 + + __ 393 + + _ 399 + - + 400 + + - 301 .. _ + 302 + .. + 304 - + - 306 - + — 307 + + - 308 - + - 310 .. + _ 311 + - + 300 .. + _ 19 " Cglture Koser's citrate Methyl red_> Voges-Proskauer 312 + .. .. 313 + + — 314 - + .. 315 - + - 316 + + - 318 _ + _ 319 - + - 320 - + - 317 + + - 322 - + - 323 - + - 324 - + - 325 - + - 326 - + - 328 + + + 327 - + - 329 + + + 330 - + r 331 + + - 321 + + r 340 ' + ‘ 339 - + ' 338 - + ' 337 - + ' 2O Culture Koser ' s citrate Methyl red Voges—Proskauer 336 - + - 335 - + - 334. - + - 333 - + -_ 332 - + - 513 — + _ 512 .. + __ 510 _ + _ 509 - + _ 507 - + - 501+ - + - 523 + .. + 518 - +' _ 517 - + .. 516 .. + _ 522 .. + _ 532 + + + 531 - + - 530 + - - 529 — -- + 528 + — + 527 - + - 526 .. + _ 542 + + _ ‘ {naho w. 4‘ 21 __¥Cu1ture Koser's citrate Methyl red Voges-Proskauer 544 + — + 545 + + _ 303 + — + 597 + - + 585 + + + 593 + - + 588 + + + 594 + - + 583 + — + 595 + + + 596 + - - 607 + - + 612 + + + 625 + - + 629 + + + 601 + .. + 600 + .. + 620 + _ + 592 + .. + 590 + _ + 589 + _ + 615 + .. + 631 + - + 609 + - + 22 Culture Koser's citrate Methyl red Voges-Proskauer 622 + + + 602 + .. + 614 + + + 599 + - + 628 + - + 623 + + + 618 + + + * Source of cultures used: Connecticut State Department of Health, Hartford, Connecticut. Detroit Board of Water, Detroit, Michigan. The Flint Water Filtration Plant, Flint, Michigan. Highland Park Filtration Plant, Hirhland Park, Michigan. Indianapolis Water Board, Indianapolis, Indiana. Maryland State Department of Health, Baltimore, Maryland. Minnesota State Department of Health, Minneapolis, Minnesota. Quebec Province of Health, Montreal, Quebec. Pennsylvania State College, State College, Pennsylvania. Saginaw Water Filtration Plant, Saginaw, Michigan. Department of Bacteriology, Stan”ord University, California. Department of Bacteriology, University of Pennsylvania, Philadelphia, Pennsylvania. West Virginia State Department of Health, Charleston, West Virginia. Table 2 Classification by percentages of which ferment lactose and dextrose the coli?orm organisms (summarized from table 1) Organism Total number Per cent Escherichia coli 89 34.6 Aerobacter aerogenes 94 36.5 "Irregulars" 71 28.8 Table 3 Relative percentages for the different types of "Irregular" organisms (summarized from Table l) Tests used Number of Citrate Methyl red Voses-Proskauer cultures Per cent + + — 39 54.9 + + + 25 35.3 - + + 2 2.8 - _ + 5 7.0 24 citrate negative, methyl red negative and Voges Proskauer positive. Very few (2.8 per cent) were citrate negative, methyl red positive and Vegas Proskauer positive. This confirms the work of Parr (29) who in 1938 also found the citrate positive, methyl red positive and Voges Proskauer negative group to be the one in which most "irregular" organisms are classified. The Accuracy of Eosin Methylene Blue Agar A correlation was made between the original classification of the typical coliform organisms by the eosin methylene blue agar plate method and the results obtained by the citrate, methyl red and Voges Proskauer tests. According to tables 4 and 5, out of a total of 89 typical g. ggli cultures only 60 (67k5 per cent) formed characteristic colonies as observed by various technicians in laboratories over the country. Out of a total of 75 g, aerogenes cultures only 34 (45.3 per cent) had produced characteristic colony formations on E. M. B. agar. These results are much lower than those found by Levine (22) in 1921. He stated that g. 99;; could be identified in 96.9 per cent of the times in contrast to 67k5 per cent as found by the author. Levine (22) also found an accuracy of 82.4 per cent for A, aerogenes in contrast to the 45.3 per cent reported here. Levine (22) reported that eosin methylene blue agar could be used to identify colonies of E, 29;; and‘g. aerogenes with almost 100 per cent accuracy. This may easily be the case with the experienced worker. However, this differential medium is being used by laboratory technicians all over 25 the country and, as the data of these workers compiled here show, they are not getting the accurate results that Levine (22) obtained. These tech— nicians were 67.5 per cent accurate in the identification of E. ggli, which is only 17.5 per cent better than pure Speculation. In the identification of A. aerogenes colonies they were accurate in 45.3 per cent of the cases, which is not as good as guessing. Since the data are compiled from the work of many technicians, it clearly shows that eosin.methylene blue agar should not be used routinely in the laboratory as a means of differentiating‘fi. coli from A. aerogenes. 26 Table 4 Comparison of the designation of coliform organisms on eosin methylene blue agar and their complete identification Organisms Original identification Final identification 1 E. coli E. coli 5 E. coli E. coli 7 E. coli A. aerogenes 8 A. aerogenes A. aerogenes 9 E. coli A. aerogenes 10 E. coli E. coli 11 E. coli E. coli 12 E. coli E. coli 13 E. coli E. coli 14 ? A. aerogenes l5 ? E. coli 17 A. aerogenes E. coli 19 E. coli E. coli 20 E. coli E. coli 21 Confluent A. aerogenes 26 E. coli A. aerogenes 33 Atypical E. coli 43 Confluent A. aerogenes 44 E. coli E. coli 46 Atypical A. aerogenes 55 E. coli E. coli 27 Organisms Original identification Final identification 56 E. coli E. coli 62 E. coli E. coli 70 E. coli E. coli 75 A. aerogenes E. coli 91 A. aerogenes A. aerogenes 92 Atypical A. aerogenes 102 E. coli E. coli 104 E. coli E. coli 109 Atypical E. coli 142 E. coli A. aerogenes 143 Atypical A. aerogenes 144 Atypical A. aerogenes 145 Atypical A. aerogenes 146 Atypical A. aerogenes 149 E. coli A. aerogenes 160 E. coli A. aerogenes 165 E. coli A. aerogenes 167 E. coli A. aerogenes 171 A. aerogenes A. aerogenes 172 Atypical A. aerogenes 178 Atypical A. aerogenes 179 A. aerogenes A. aerogenes 182 E. coli A. aerogenes 205 A. aerogenes E. coli 28 Organisms Original identification Final identification 267 278 279 281 298 300 302 303 304 306 308 310 315 318 319 320 322 323 325 326 327 A. aerogenes Confluent Confluent A. aerogenes E. coli E. coli A. aerogenes E. coli E. coli E. coli E. coli A. aerogenes A. aerogenes A. aerogenes A. aerogenes E. coli E. coli A. aerogenes E. coli E. coli E. coli Confluent E. coli E. coli coli coli aerogenes coli coli coli aerogenes aerogenes coli coli coli coli . aerogenes coli coli coli coli coli coli coli coli coli coli coli 29 Organisms Original identification Fipal identification 330 E. coli E. coli 332 E. coli E. coli 333 E. coli E. coli 334 E. coli E. coli 335 Atypical E. coli 336 A. aerogenes E. coli 337 E. coli E. coli 338 Atypical E. coli 339 E. coli E. coli 340 E. coli E. coli 382 Atypical A. aerogenes 399 E. coli A. aerogenes 504 E. coli E. coli 507 E. coli E. coli 509 E. coli E. coli 510 E. coli E. coli 512 E. coli E. coli 513 E. coli E. coli 516 Atypical E. coli 517 A. aerogenes E. coli 518 E. coli E. coli 522 E. coli E. coli 523 E. coli A. aerogenes 526 E. coli A. aerogenes 30 Organisms Original identification Final identification 527 E. coli A. aerogenes 528 Atypical E. coli 531 E. coli E. coli 544. A. aerogenes A. aerogenes 583 A. aerogenes A. aerogenes 592 A. aerogenes A. aerogenes 594 A. aerogenes A. aerogenes 597 A. aerogenes A. aerogenes 599 Dew drop colonies A. aerogenes 600 A. aerogenes A. aerogenes 601 A. aerogenes A. aerogenes 602 A. aerogenes A. aerogenes 605 A. aerogenes A. aerogenes 607 A. aerogenes A. aerogenes 615 Confluent A. aerogenes 620 A. aerogenes A. aerogenes 628 A. aerogenes A. aerogenes 31 Table 5 Comparison of the designation of coliform organisms on eosin methylene blue agar and complete cultural identification (Summarized from table 4) Cultures Per cent Number of correctly correctly Organisms cultures identified identified _E_o c011 89 60 6705 'A. aerogenes 75 34 45.3 I'Er rd... . . II . 1.1 F.’ .I it... . finish .1. .c. . 32 Atypical Coliform Organisms Cultural Characteristics A number of so—called atypical coliform organisms were studied both culturally and serologically in an attempt to discover whether these organisms should be considered important from a public health standpoint. Atypical coliform organisms are unlike the true coliform organisms in that they either do not ferment lactose with the production of acid and gas or they ferment it slowly (after 48 hours). The 38 atypical organisms studied showed in general the same cul- tural reactions on the carbohydrates as that for the typical organisms. Gelatin was liquified by only two of the cultures and none produced H S. 2 1 Table 6 shows that some of the cultures react the same as E, col or A. agrgggggg to the carbohydrates tested. However, some of the strains in addition to lactose do not attack one or more of the carbohydrates which are usually fermented by the coliform organisms. The results were more irregular on sorbitol and xylose than on any of the other media tested. fter studying the reactions of these organisms on the carbohydrates it is conceivable that all of these organisms are typical coliform organ— isms in different stages of losing their fermentative abilities. They IhaVe not only lost their ability to ferment lactose but many of them have lost the ability to ferment also such things as sorbitol and xylose. The reason for this gradual loss in fermenting power is evident when the source of these atypical cultures is noted. 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QOH \TCOW Mb- \0 meOr-‘l O'HNm mmmmmmxmmmmmmm 35 cowpospoam new new wave monocmp e soapospoam pace moaocop + COHPOde OS mwubGOmu .I .x. I e a l a a I a I a e e a e + e I PS I e e a a a a e o e o a e I I I a. I + + I I I I I I a I + + I I I am I + + I I + I a I e + I + + I I I an I a o e o o e e e a I e a I + I mom. I a e e e o e e e e e e e I + + me. new nee omoH i omop omoa opao Iwop moo: Hopan omos moa can» QHH onwm omoap mop mama ImHmo Iwaem Isbmq omawo recess IHSQ Tandem Iammmm Inom Inez Ihx Ixom FsmH Ion Imehomq rswmno 36 the samples from which these atypical organisms were obtained were taken from deep wells or chlorinated water supplies. Raw polluted water supplies seldom showed atypical organisms. These atypical organ— isms are probably forms which have degraded after a long absence from their natural habitat. The idea of a degraded form of‘§.'ggli and ‘5. aerogenes is not new for it was mentioned by Parr (29) in 1936. He suggested that A. aerogenes changes more than E, 291;. This was con— firmed by the author and is shown in tables 7 and 8. According to the citrate, methyl red and Vegas Proskauer tests 57.1 per cent of the atypical cultures were found to be A. aerogenes, 2.9 per cent Q..ggli and 40.0 per cent "irregular". Serological Relationship Among Coliform Organisms In order to show more conclusively that the non—lactose fermenters are truly coliform, serological methods were used. First of all, the serological relationShip between various strains of typical‘E..ggli and A, aerogenes was determined in order to know what could be eXpected from the atypical cultures. It was discovered that various strains of .§-.£Qll are serologically heterogenous with a high degree of common antigen shown only in 35 per cent of the cultures. The same was found true among typical A. aerogenes cultures. I Next, agglutination reactions were run with atypical antigens and antiserum from various strains of typical E. coli and A. aerogenes. 'Using E. coli culture No. l and 42 atypical antigens, agglutination was <5btained in a dilution of 1:80 or higher in only 1726 per cent of the 37 Table 7 Cultural identification of atypical coliform organisms Organisms Koser's citrate Methyl red Voges-Proskauer 514 + - + 80 + - — 250 + - + 201 + - + 515 + _ + 183 + - + 556 - .. + 521. - + _ 540 + + - 541 -:- - + 6 + _ + 555 + - - 4 - - + 573 + - - 366 + .. - 251 . + . - + 536 — - + 568 + .. ' + 577 + - + 563 + - + 567 + .. + 578 + - + 38 Organisms Koser's citrate Methyl red Voges-Proskauer 566 + _ + 572 + - + 570 + — + 564 + - - 565 + - + 562 + - + 5'76 + _ + 561 + - + 273 — + + 265 + .. + 37 _ _ + 50 - — _ 72 - — - Taole 8 Cultural identification by percentages of organisms (summarized from Table 7) atypical coliform Organisms Number of cultures Per cent A. aerogenes 20 57.1 E. coli 1 2.9 Irregular 14 40.0 39 cultures. Using other typical E. gal; antisera the percentage was even lower. On the other hand, A. aerogenes was found to have a much closer relationship. In table 9 it is shown that A. aerogenes culture No. l with as high as 19 cultures out of 42 tested (45.2 per cent) showed agglutination in 1:80 or higher. Table 10 shows that A; aerogenes culture No. 43 showed 10 out of 42 or 23.8 per cent of the cultures with close antigenic relationship. This shows that in general the atypical coliform organisms are much more closely related to A. aerogenes than Q. ggli. On an average, atypical coliform organisms are agglutinated in 34.5 per cent of the time by typical g. aerogenes antiserum. This is the same relationship which was found to exist between A. aerogenes serum and typical aerogenes antigens. A.macroscopic agglutinin absorption test was run with the atypical antigens which agglutinate the A. aerogenes No. l antiserum. Species Specific or formalin treated antigens and group specific or alcohol treated antigens were used. According to table 11 both species specific and group specific antigens were found to be present in 10 out of the 16 cultures tested. 4O .11. +++ .TTI. +1... ++.I. +I+ i++ .TTI. .TTI. +I+ .TTI. ++++ + ++.I.. 1.1. +1... ++.I. +I+ + + ++++ ... ++.I. a. a. .H 3m mam mom mom Rm 9% rpm hm com Ohm «om 0m mum Honpcoo Houpnoo omeNH oqw\H 8% omwNH ow\H 0M\H 8K». adhomflpnm H .02 monowonmm .< mo oppap soapsHHn mOHn—VHHHO HwOfiahpm mo mammfipn< * msmflcemno snowflaoo Hmoflgmpw was H .02 mmmmwonoe hopomn onm¢ Hwofimhp Goosvmn mazmnoflpmamh edcmmwpnm as» wnasonm mpmop soapmmapdawm¢ a canoe +++ +++ +++ 'E1HI'HHI' 'iii Hi '1$"$""$$' «hm 0mm mam 0mm com «mm ovm mg g mm om 8m 3m as an as... we... mam Hospcoo omNH\H ommh oqwa wcmuflpcd HF 42 +++... mowpmcflpdammd on I soapmaflpsammw mo momnmmw hmmmma + .++ .+++ +++ i+ ++++ ++ + +++ doapoCMpSHmmw mpmamsoo ++++ * me Honvcoo cams} o¢w\a 8me SHE ow} 0st ON H mnemwwcd 43 _ .1. .TI. .TI. .TT+ ++.I.. .TTI. mom Hon mam new mom bum mom rem pm On mum Ohm «on Hohpaoo Honpcoo 8mm}! o ~¢ C) (V asymmecw H .02 mmcmmonmw .d mo mthp mOHpSHHQ mmHSpHSo onhpw mo mammapna * ashomHenw monomonod .¢.Hm0Hmhp can mnowfinnm HHOMHHoo HmOHmhpd mo m:0Hpowon nOHpmhomnm HOHpmaHpsHmw< HH oHndH Ii Ix‘L [llll PIIIDILI! .llnlll-IIIIYIIBEVIE lg illii .u ..Eiiaga‘tc .24 ‘r 55...!va .lpdnls .4. . u I. .v .P.. 47 soapwcavzammw on I moauwcflpsamww mo mmmhmoc HmmmmH + .++ .+++ noapmdapsamwd mpmamsoo ++++ * I I I I I I + I + I ++ I +1 i... .1 +++ own I I I I I I I I I I I I I I + mum I A I I I I I ++ I I I + I + + + 3m m o m o m o m o m o m o m m o 3.338 H228 8~H\H 0% H Jfiomn H 8H H SNH omeH A339?” .8 annomwpaw H .02 manomonow .4 mo ougap sea»: a maomdpn¢ 48 CONCLUSION A total of 254 typical coliform cultures were classified according to the citrate, methyl red and Voges Proskauer tests. As high as 28.8 per cent were found to be irregular. This almost exactly confirms the work of Parr (29). The "irregular" organisms most commonly found are of the citrate positive, methyl red positive, and Voges Proskauer negative group. The accuracy of eosin methylene blue agar for the differentiation of E. coli from A. aerogenes by laboratory technicians was determined. It was found that E. M. B. agar is not satisfactory for the differentiation of these organisms in routine laboratory analyses. Most of the atypical coliform fultures were found to be identical culturally with the typical organisms except for their fermentation of lactose. Some of the strains have also lost the ability to ferment one or more of the other carbohydrates. It was concluded that these so—called atypical coliform organisms are merely a degraded form of the typical organ- isms and, therefore, have just as much sanitary significance. According to the citrate, methyl red and Voges Proskauer tests, it was found that these atypical organisms largely react as‘g. aerogenes. That atypical coliform organisms are mainly‘g. aerogenes was also shown by using serological methods. In addition, agglutinin absorption tests showed both group specific and Species Specific antigens present for A. aerogenes. I~ —'.T" 4. 5. 49 SUI-m-IARY 29 per cent of the coliform organisms in water supplies are "irregular" types. The citrate positive, methyl red positive and Voges Proskauer negative organisms are the most common group found. Eosin methylene blue agar cannot be accurately used by laboratory tech- nicians as a means of differentiating g. 93;; from A. aerogenes. Atypical coliform cultures are probably degraded coliform organisms. Serologically, atypical cultures are frequently shown to be A, aerogenes and seldom E. coli. ‘ fTJ‘flfl'" I ' . TC. Ada {mmwifis -' '5? 2. 7. 8. 10. 5O LITERATURE CITED Abdoosh, Y. B., Observations on certain atypical coliform bacilli. J. Egyptian M. A., 17:700-728, 1934. American Public Health Association, Standard methods for the examina- tion of water and sewage. 8th Ed. Am. Pub. Health Ass., 50 West 50th St. New York, 1936. I Bahlman, C., and Henry Sohn, Colon-aerogenes differentiation. Jour. A. W. W. A., 11:416—433, 1924. Barritt, N. W., The intensification of the Voges-Proskauer reaction by the addition of alpha—naphthol. J. Path. and Bach/+2, 1.41, 1936. Bergey, David, Robert Breed, E. G. D. Murray, and A. Parker Hitchens, Manual of determinative bacteriology. Williams and Wilkins Co. New York, 5th Ed., 1939. Bradsley, Doris, The destruction and sanitary significance of‘B..ggli, .B. lactis aerogenes and intermediate types of coliform bacilli in water, feces and ice cream. J. Hyg., 34:38-68, 1934. Breed, R. S. and John Norton, Nomenclature for the colon group. Am. J. Pub. Health, 27:560-563, 1937. Carpenter, P. L. and M. Fulton, Escherichia-aerobacter intermediates from human feces. Am. J. Pub. Health, 27:822—827, 1937. Fothergill, L. D., Unusual types of non-lactose fermenting, gram negative bacilli from acute diarrhea in infants. J. Infect. Dis., 45:393— 403, 1929. France, R. L., Studies of Bacterium coli in privately owned rural water supplies. J. Bact. 25:623-635, 1933. "» if”: 51 11. Georgia, H. and L. Morales, The diagnostic value of neutral red lactose peptone media for the coli-aerogenes group. Jour. A. W. T. A., 16:6314641, 1926. 12. Herrold, a. D. and H. Culver, A study f the gram negative bacilli of renal infections. J. Inf. Dis., 24:114-119, 1919. 13. Hicks., E. P., The value of methods for the difierentiation of bacilli of he coli-aerogenes group when applied in shanghai. J. Hyg., 26:357-361, 1927. 14. Houston, A. 0., Chemical and bacteriological examination of soils with reference to the amount and nature of organic matter and number and character of bacteria contained in them. Suppl. 27th Ann. Rep. of Lee. Gov. Bd., 251, 1897. 15. Jatta, M., Experimentelle Untersuchung uber die Agglutination des Typhus— bacillus und der Mikroorganism der Coligrupye. Z. Hyg. Infektions~ krankh., 33:185—934, 1900. .4 16. Jones, E. W. an (i L R. B. Little, Etiology of infectious diarrhea in caotle. J. Exner. Ned., 53:835—843, 1931. 17. Korstiom, H., Uber die enzymbelding in bakterien. Dissertation, Hel— sinfors, 1930. 18. Koser, S. A., Citrate utilization by coli-aerogenes group. J. Bact., 9:59—77, 1924. 19. Koser, S. A., Ccli aerogenes group in soil. Jour. A. W. H. A., 15:641—646, 20. Kriebel, R. M., Incidence and behavior of non—lactose fermenting bacteria from normal stools. A.. J. Pub. Health, 26:793—798, 1936. ___—_ 'us‘ mun-1 arm ‘ i \ 52 f‘ 21. Levine, Max, A ('1 tatistical classification of the colon—aerogenes group. J. Bact., 3:253-276, 1918. 22. Levine, Max, Bacteria fermenting lactose and their significance in water analysis. Bull. Iowa State College, 62:72, 1921. 23. Lewis, I. M., Bacterial variation with special reference to behavior of some mutable strains of colon bacteria on synthetic media. J3 Bact., 28:619-638, 1934. 24. MacConkey, A., Lactose-fermenting bacteria in faeces. J. of H"g., 5:333—379, 1905- 3 if b. 25. Beckie, T. J., The inmunity reactions of the coli group. J. Path. and Bact., 18:137Ll44, 1913. 26. Hagheru, G. et a1, Recherches sur 1'antirene somatique complet (antigene O) des coli bacilles. Arch. roumaines de path. exper. et de micro- biol., 10:29-65, 1937. 27. Meyer, K., Zur Theorie dur coliagclutination. Z. Immunitats., 61:232- g) 239, 1929. lo 8. Parr, L. W., Cultural characters, relationships, significance and oc- currence of the coli—aerogenes intermediates, tith particular refer- ence to feces, fresh and stored at various temperatures. J. Bact., 31:23, 1936. 29. Parr, L. W., Coliporm intermediates in human feces. Ibid, 36:1—15, 1938. 30. Pfaundler, M., Eine neue Form de Serumreaktion auf Coli— und Proteus- bacillosen. Zentr. Bakt. Parasitenk., 23:9—15, 71-79, 131-138, 1898. 31. Poe, C. P., Differential tests for the colon—aerogenes groups. Jour. A. v. I. A., 23:1:1ee1226, 1931. 34. 35. 360 37. 40. 53 Radzieviky, M., Bertrag zur Kenntnis des Bakterium coli. Zentr. Baht. arasitenk., 26:753-757, 1899. Ruchhoft, C. C. et a1, Coli—aerogenes differentiation in water analysis. J. Bact.,621:407L440, 1o31. Sandiford, B. B., The paracolon group of bacteria. J. Path. and Bact., 41:77-88, 1935. E Savage, V. G., Bacteriological examination of tidal mud as an index of E pollution of the river. J. Hyg., 5:146-174, 1905. E .‘ Stokes, J. H., R. H. Weaver and M. Scherago, A study of the paracolon % ,1 group. J. Bact., 35:20, 1938. Strunz, P., Coli—Agglutinationem mit tierschem Immunseren. Zentr. Parasitenk., 99:223-234, 1926. Van Loghem, J. J., Variabilitat und parasitismus. Zentr. Bakt. Para— sitenL., 83:401—409, 1919. Yale, M. W., The Escherichia-aerobacter group. J. Dairy Science, 16:481-494, 1933. Ziegler, N. B., Late lactose fermenting organisms of the coli-aerogenes group. Amer. J. Pub. Health, 29:257L26O, 1939. . .3. ......1. . .. .-....fl?&_ ... . h...»l1..t..&1a....‘. . §Yv ”.51.. \ , I. W..- . "1111‘ 01.4‘ 1735’.” . o ., . . . #75.....1M..\1.€1%I". .f... - 0-1 _ C8,. ..1...v.....1.“‘\.dfld.;nfi\ ”...-u . \. ...V‘. ..1L.§.1....I.J1... l . tla . A. .-.... . .. .. . .. .. .....1... .. .32... t1.. ..- ..‘VL. .‘ .01 v r .’ 17.73.! . ...I'flu ..v.1.. v. . -.\. s ' 1'. n . 1 fi .114 .7\.§l"1rg§ Iyx. . .5.‘.l. . «5 .. .13. O..1Hwk1gr..r11n M. 1.... .C out, VP. .. ..‘A . ..g. EQNVS. . .i‘xf ._....\ .P- 1 . .. ...;f..... "n 3....117 ... Ji}ofi}«ltlla .uI-Mlh.l...la‘... I ....n17... .., in? $8.3. W . new. é’lg .n’ “Vt/7 .. ... .1. ”$51.. «376.... . .IV. _ . ...“. . .. .. _ we»... :31 . ...l.’ ... ....F...1\r amuV1... fif’rwaS _ . . . .... . .01 x J. ...! .v .... _ . .. r M3 . .... .... u, .. 3 . .. 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