Connecticut State Department of Health, Hartford, Conn. mannitol-negative strains were obtained.

Transcription

1 BIOCHEMICAL AND ANTIGENIC RELATIONSHIPS OF THE PARACOLON BACTERIA C. A. STUART, K. M. WHEELER, ROBERT RUSTIGIAN sid ALICE ZIMMERMAN Biological Laboratory, Brown University, Providence, Rhode Island, and the Bureau of Laboratories, Connecticut State Department of Health, Hartford, Conn. Received for publication June 6, 942 In the bacteriological laboratories of Brown University and the Connecticut State Department of Health, Bureau of Laboratories, we have been engaged in a systematic study of the biochemical and serological relationships of the coliform and related bacteria since April 937. During this time over 2,000 normal and aberrant coliform cultures have been isolated. The IMViC reactions and in addition cellobiose fermentation were determined for all these cultures. A wide variety of biochemical types were encountered ranging from those positive in all five tests to a few weakly positive in only one of the IMViC tests. The ability of more than 3,500 of these cultures to ferment glucose, lactose, sucrose, salicin, maltose and mannitol was determined. Normal coliform cultures negative in sucrose or salicin or both were commonly found. Strains failing to ferment maltose were not anticipated but several were isolated from widely different sources. Mannitol fermentation appeared to be a constant characteristic of normal coliforms but among the last 500 isolations two, otherwise normal, mannitol-negative strains were obtained. Coliform cultures fermenting lactose slowly or not at all, but normal in other respects, were frequently encountered. We have obtained from both rapid and slow lactose-fermenting coliforms variants which were anaerogenic in lactose only or in all carbohydrates. Some of these variants have remained constant for 2 years, one for more than 3 years. In addition motile and nonmotile, indolepositive cultures were found which produced no gas from carbohydrates when originally isolated. Many such cultures would be classed as Eberthella or Shigella, particularly when the fermentation of lactose, sucrose or salicin was slow or absent. After varying periods of laboratory cultivation many of these strains produced gas. Cultures that appeared to be non-gas-forming but which could not be identified as known Eberthella or Shigella have been included in the paracolon group since they could be anaerogenic variants of coliforms. These exceptions and others are a serious handicap to the practical bacteriologist because strains not infrequently defy classification in any of the recognized sections of the coliform group. The tendency to disregard such exceptions for the most part is justifiable but occasionally this might be a mistake. Stuart, Mickle and Borman (940) proposed the term "aberrant" coliforms for cultures not fermenting lactose, producing acid only, or requiring more than 48 hours for the production of 20 per cent gas when the unusual fermenting characteristics of such strains are relatively stable. Aberrant coliforms are frequently isolated from such sources as water with an excellent sanitary survey, soils from rigidly 0

2 02 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN posted areas and from cereals. Many aberrant strains from these sources appear to be more primitive than normal strains si sce they grow abundantly at room temperature but sparsely or not at all when inoculated into media preheated to 37 C. and incubated at 37 C. On thae other hand aberrant coliforms are often isolated from fecal specimens esphcially during gastroenteritis epidemics. Some distinction should be made between aberrant coliforms from nature and those from the intestinal tract of ma. In 897 Widal and Nobecourt described a slow lactose-fermenting strain isofated from a gastroenteritis patient as a "paracolon" organism and this term as applied to aberrant coliforms from man has been firmly established in British literature for many years. It has been used seldom in this country but will doubtless become more common since recent editions of bacteriological text books employ the term (e.g., Jordan and Burrows 94). In the present work the biochemical and serological relationships of the paracolon organisms among themselves, and to the normal coliforms and to Salmonella have been studied. A total of 465 (including 75 used by Stuart et al., 940) paracolon cultures isolated from 438 individuals were studied. The cultures were isolated from fecal, urine or blood specimens of gastroenteritis patients (77.4 per cent), from the fecal specimens of food handlers (8. per cent) and others (4.5 per cent). Of the 465 cultures 22 were obtained from one large institution and 22 from several smaller institutions all in the city of Providence or immediate vicinity. The remaining 3 cultures came from various laboratories as far west as Illinois and as far south as Tennessee. The cultures will be discussed under the headings Paracolon Aerobacter, Paracolon Intermediates, Paracolon Escherichia and Anaerogenic Paracolon. Observations on pathogenicity and the presence of a Vi like antigen in paracolon will be discussed under separate headings since the presence of Vi antigen and pathogenicity in bacteria do not appear to be correlated. PARACOLON AEROBACTER The biochemical and serological relationships of 40 cultures were studied. The frequency with which such cultures were isolated is noteworthy. Of 363 paracolon Aerobacter and paracolon Escherichia isolated 38.5 per cent were Aerobacter. A search of the literature reveals that only 4.6 per cent Aerobacter were obtained from a total of 6,973 normal Aerobacter and Escherichia isolated from man in twenty different investigations. Normal and paracolon Aerobacter show a marked difference in motility. Of 2,765 normal cultures isolated from milk, grain, water, dust, soil and feces in our work on the coliforms 36.6 per cent were motile. Variation in the per cent of motility from the various sources was only 48. Of 200 fecal strains 30.5 per cent were motile (unpublished data). In the present work 97.8 per cent of the 40 paracolon Aerobacter cultures were actively motile. Paracolon Aerobacter can be separated into 2 divisions on the basis of their IMViC reactions. Division consists of 48 strains which gave a strong Voges- Proskauer reaction, grew on citrate agar and fermented cellobiose in 24 hours

3 Totals RELATIONSHIPS OF PARACOLON BACTERIA 03 TABLE Biochemical and serological relationships of 40 paracolon Aerobacter strains hi U) NO.0OF NO. OFCUL- TYPE DASO LUOE h hi hi CUL- TURES CULTURE INCUBA- GAS (PER 0 00 O P TURES CLOSELY TION CENT) 0 INTHERELATED OR : X; g j Q Q o8 GROUP EDENTICAL First division Second division I B A Totals * * Exclusive of cultures homologous to antiserums. = Positive reaction, acid and gas in carbohydrate broths. - = no reaction. A = acid. B = bubble of gas. i = some strains negative; some strains positive.

4 04 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN (table ). Such cultures usually failed to grow on S S agar (Difco Salmonella and ShigeUa agar). On eosin methylene-blue agar some cultures in 24 hours produced large, raised, mucoid and confluent colonies. The colonies from other strains could not be distinguished with any certainty from paracolon Escherichia or Salmonella but after an additional 24 to 48 hours of incubation colonies from all except 3 of the 48 cultures could be easily differentiated. All cultures produced approximately 30 per cent gas from glucose in 24 hours and about 00 per cent in 48 hours. All required from 3 to 9 days to ferment lactose. Some were slow in lactose and salicin and a few in lactose and sucrose. The carbohydrate media remained very turbid and the alkaline color of the indicator (brom-cresol-purple) was frequently completely restored after several days incubation. Most cultures grew abundantly on agar and many were encapsulated. All 48 strains liquefied gelatin slowly but never completely. Hydrogen sulphide was not produced in lead acetate medium. (In the early part of this work lead acetate tubes were read daily for 5 days. So many intergrading degrees of positive reactions were recorded that finally tests were read as follows; if at the end of 24 hours at 37 C. there was marked blackening along the line of inoculation with some diffusion into the medium the reaction was recorded as positive and all others as negative.) Paracolon organisms of thil division despite their slow fermentation of lactose are easily placed in the Aerobacter section. Division of the paracolon Aerobacter is made up of 3 biochemical groups. One culture of each of the first two biochemical groups, 46 and 72 respectively (table ), was used to prepare antiserums in rabbits. All immunizations were done with living cultures; The 48 cultures were tested in the two antiserums in dilutions of 50, 250, 250 and Cultures agglutinating to high titers were used to adsorb the antiserums in which they agglutinated. Table shows that 3 strains in the first biochemical group, including the homologous strain, were antigenically identical or closely related to culture 46. (Cultures agglutinating to titer and removing all agglutinins upon adsorption were recorded as identical while cultures significantly reducing the homologous titer of an antiserum were recorded as closely related to the culture used to produce the antiserum.) Two cultures in the second biochemical group, including the homologous, reacted similarly in antiserum 72. Paracolon Aerobacter strains of the second division were readily distinguished from those of the first division. Gas volumes were usually lower and quite comparable to Escherichia or Salmonella, the alkaline color of the indicator in carbohydrate broths was never strongly restored and after 48 hours in glucose broth the organisms were almost always completely sedimented. With freshly isolated cultures the V. P. reaction was always weak and occasionally negative with Levine's reagent (98). Two type 375 cultures (table ) not fermenting lactose, sucrose, salicin or cellobiose were carried for nearly a year as unidentified Salmonella. After several IMViC tests their ability to produce acetyl-methylcarbinol was restored and they were immediately typed as 375. None of the cultures in this division fermented cellobiose or grew on citrate agar in 5 days,

5 RELATIONSHIPS OF PARACOLON BACTERIA some showed only a few colonies on citrate after 6 weeks. Growth on agar was seldom abundant and capsules were not formed. No culture liquefied gelatin. Occasionally strains were weakly motile or nonmotile when first isolated but after a few transplants were vigorously motile. Three strains appear to be genuinely nonmotile. One has been plated repeatedly for 2 years in semisolid agar where thousands of colonies have been observed without any trace of motility being developed. For the most part these cultures grew readily on S S agar and the colonies of a great majority could not be distinguished from Escherichia or Salmonella. In this division the two most important types on the basis of frequency of isolation from patients, 320 and 375, are differentiated easily from all others. Type 320 in 24 hours produces a weak acid reaction in maltose and a bubble of gas in mannitol. Of 35 strains of this type 32 were antigenically identical or closely related. Type 375 when first isolated may be easily mistaken for Salmonella. All 4 strains of this type were antigenically identical or closely related. Antiserums were prepared against all biochemical types of division 2 except 72 (table ). Only cultures with biochemical reactions similar to the culture used for immunization were tested in the antiserums. Consequently the incidence of agglutination and average titers for all cultures in all antiserums cannot be given. Table 2 shows that 47.8 per cent of all paracolon Aerobacter cultures were antigenically identical or closely related to one or another of the 8 cultures used to produce the antiserums. Hereinafter unless stated to the contrary reference to the paracolon Aerobacter will include both divisions. To study further the antigenic relationships of paracolon Aerobacter the 8 cultures used to prepare antiserums and 49 cultures not closely related were tested in 4 antiserums produced from normal Aerobacter. Table 2 shows that the incidence of agglutination was 7.0 per cent (57 cultures times 4 antiserums = 798 opportunities for cross reactions of which 7.0 per cent were realized) with an average titer of 34. No culture agglutinated to sufficiently high titer in any antiserum to warrant adsorption. The same paracolon Aerobacter cultures were tested in 25 Salmonella antiserums so selected that all somatic antigens from I to XXX were represented. Since antiserums were prepared from formalized antigens flagellar antibodies were present. The incidence of agglutination was 7.6 per cent with an average titer of 396 (table 2). Adsorptions of Salmonella antiserums were made with paracolon cultures when significant agglutinations occurred. None of the paracolon Aerobacter were identical with any of the Salmonella since adsorption failed to remove all the homologous agglutinins. When tested with single factor antiserums, however, 2 cultures agglutinated to titer in somatic XXV antiserum and reduced the titer for the homologous S. onderstepoort. Both cultures completely ad sorbed the I and VI fractions fromthis antiserum. One of the 2 cultures reacted strongly with S. tel-aviv antiserum but failed to reduce the titer and a third cultue reactred similarly in S. hvittingfoss antiserum. Titers for H agglutinins were not altered by adsorptions. The results obtained from 05

6 06 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN testing the paracolon cultures in normal Aerobacter and in Salmonella antiserums clearly indicate that the paracolon type of this section are no more closely related to normal Aerobacter than to Salmonella. In their own antiserums on the other hand paracolon Aerobacter showed a marked continuity of antigens since 47.8 per cent were identical or closely related. To complete the antigenic picture in the entire Aerobacter section 5 nonencapsulated, motile and nonmotile normal Aerobacter were tested in the 25 Sal- TABLE 2 Serological relationships of normal coliform, paracolon and Salmonella organisms ANTIGENS EMPLOYED Para. Aero. Para. Aero. Para. Aero. Norm. Aero. Norm. Aero. Para. inte. Para. inte. Norm. inte. Para. Esch. Para. Esch. Para. Esch. Norm. Esch. Norm. Esch. ANTISERUMS PRE- PARED FROM Para. Aero. Norm. Aero. Salmonella Salmonella Norm. Aero. Para. inte. Salmonella Norm. inte. Para. Esch. Norm. Esch. Salmonella Salmonella Norm. Esch. NUMBER OF CULTURES TESTED NUM- BER OF ANTI- SERUMS PER CENT INCIDENCE OF AGGLU- TINATION AVER- AGE TITER PER CENT OF CUILT. CLOSELY RELATED PER CENT OF CULT. IDENTICAL PER CENT OF CULT. CLOSELY RELATED OR IDENTI- CAL * * * 22.Ot 3.0 Totals and averages Para. = paracolon, Aero. - aerobacter, Norm. = normal, inte. = intermediate, Esch. escherichia. * Stuart et al., 940. t Brown, 94. monella antiserums. In these tests a marked difference between normal and paracolon Aerobacter was noted. After 2 to 5 hours at the normal Aerobacter in many tests and controls were strongly lysed. Some cultures which showed the usual turbidity at were only slightly opalescent at 550C. Several cultures which agglutinated in the 250 dilution and 3 in the 6250 dilution at were negative in the 50 dilution after 2 hours at With Aerobacter, intermediate and Escherichia paracolon cultures a few positive in the 50 or 250 dilutions at were negative at 55 C. but without any apparent lysis. In previous work with normal coliforms (Stuart et al., 940) tests were

7 RELATIONSHIPS OF PARACOLON BACTERIA incubated at 37 C. for 2 hours and then at 2 C. over night. The same procedure was followed in this work with normal Aerobacter in Salmonella antiserums. The incidence of agglutination with these cultures was 6.2 per cent with an average titer of 923. The higher average titer of normal Aerobacter than paracolon Aerobacter in Salmonella antiserums was due primarily to one culture which agglutinated strongly in several antiserums. This culture adsorbed all somatic agglutinins from S. paratyphi and the somatic I from S. senftenberg and S. onderstepoort antiserums. Other partial antigens were present since the culture was strongly agglutinated by but did not completely adsorb agglutinins from single factor antiserums for antigens XIX, XXIII, XXV, XXVII and XXVIII. Several relatively strong reactions occurred with other strains when tested in single factor or with unadsorbed Salmonella antiserums but adsorptions of the single factor antiserums failed to identify these normal Aerobacter antigens with the known Salmonella components. Four cultures agglutinated in S. hvittingfoss, 3 in S. tel-aviv and one in S. worthington but the common antigens are apparently "minor" or additional and unlabeled Salmonella somatic antigens. Six of the normal Aerobacter strains were isolated from cereals. Of the 694 cultures used in this work these 6 were the only cultures not isolated from human beings and it is noteworthy that 3 agglutinated in the 250 dilution of S. hvittingfoss or S. tel-aviv antiserums. PARACOLON INTERMEDIATES The biochemical and serological relationships of 40 such cultures were studied. These organisms frequently failed to produce gas in lactose. Some formed strong acid in 24 hours but failed to produce any gas in 6 weeks. Of 75 normal intermediates isolated from various sources 83.0 per cent, and of 63 cultures from feces 88.3 per cent were motile. In the present work 00 per cent of the paracolon intermediates were strongly motile. Many of these cultures reduced the brom-cresol-purple indicator in lactose, maltose and occasionally salicin but not in glucose, sucrose or mannitol. Paracolon intermediates grew as well on S S agar as on E. M. B. agar and could not be distinguished from normal Escherichia or Salmonella. Table 3 shows the biochemical reactions of the paracolon intermediates. Nearly half the cultures liquefied gelatin slowly and sparsely, never forming more than a drop or two of liquid. All cultures acting on gelatin failed to produce hydrogen sulphide. A few cultures were negative in both tests. All strains were positive on citrate agar and in cellobiose after 6 weeks incubation. Antiserums were prepared against cultures from 5 of the biochemical groups (table 3) and of the 40 cultures isolated 52.5 per cent were identical as shown by adsorptions (table 2). This is in marked contrast to the results obtained by Stuart et al. (940) who found that of 08 normal intermediates tested in 9 normal intermediate antiserums only 8.2 per cent were identical or closely related. The relationships of paracolon intermediates to normal and of normal to paracolon 07

8 08 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN intermediates were not studied because of the small number of normal intermediate cultures and antiserums available. The 5 paracolon intermediates used to produce antiserums and 4 not closely related to any of the 5 were tested in the 25 Salmonella antiserums. The incidence of agglutination was 7.5 per cent with an average titer of 467 (table 2). Three cultures agglutinated in S. cerro, two in S. urbana and one in S. hvittingfoss antiserums to high titers but adsorptions failed to identify these paracolon antigens with any of the known Salmonella antigens. hi 0A TABLE 3 Biochemical and serological relationships of 40 paracolon intermediates 4~~~~~~~~~~~~~~~~~~~~~O F0 R i j4. - OtNO. hi~~ CUL. ~CLOSELY hi hi hi0 o >4 TURES RELATED Z 42 n hinthe o < MENTIPWCAL 33 2 A A A - A A - A 80 2 A A A A A A A- - - A A - A - Totals * * Exclusive of cultures homologous to antiserums. = positive reaction, acid and gas in carbohydrate broths; - = no reaction; -A = some strains negative; others producing acid only; others acid and gas; d = some strains negative; some strains positive. PARACOLON ESCHERICHIA The reactions of 223 cultures were studied. Whereas 97.8 and 00 per cent of the Aerobacter and intermediate paracolon cultures respectively were motile only 7.6 per cent of the paracolon Escherichia strains were motile. Of 3295 normal Escherichia from various sources 8. per cent and of 338 from feces 79. per cent were motile. The lower incidence of motility in the paracolon Escherichia is not surprising since the nonmotile type 3 (table 4) of which there

9 RELATIONSHIPS OF PARACOLON BACTERIA 09 TYPE CUL- TURE * TABLE 4 Biochemical and serological relationships of 22S paracolon Escherichia DAYS OF INCUBA- TION A A A 0 A A - A A _ - hi Pi co 0 0 A MOTILITY NO. OF CUL- TURES IN THE GROUP NO. Or CULTURES CLOSELY RELATED OR IDENTICAL Totals * * Exclusive of cultures homologous to antiserums. - positive reaction, acid and gas in carbohydrate broths; - no reaction; A = some strains producing acid only; others, acid and gas; A = some strains producing acid; others, negative.,,, = degrees of motility. Pe Ij _ l

10 0 STUART, WHEELER, RUSTIGIAN AND ZTIMERMAN are 5 antigenically identical strains possesses all the antigens of ShigeUa alkalescens. The nonmotile types 69 and 76 which are biochemically the same but antigenically different, include 2 cultures which, being positive on citrate agar, could be classed as intermediates and are recorded as Escherichia because they have antigens in common with type 3 and Shigella alkalescens. All but 3 of the paracolon Escherichia produced indole. All grew readily on S S agar and their colonies were indistinguishable from those of Salmonella. Table 4 shows the biochemical reactions of the paracolon Escherichia. Antiserums were prepared against all of these types. Of 223 cultures tested (table 2) 67.2 per cent were identical or closely related. Of 3 normal Escherichia tested in 0 normal Escherichia antiserums 39.8 per cent were identical or closely related. On the assumption that normal Escherichia which occasionally grow on S S agar might be more closely interrelated than inhibited strains, Brown (94) isolated 50 strains from fecal specimens of 200 individuals from S S agar. A few strains grew as readily on S S agar as on E. M. B. agar but with most strains only a few colonies developed on the S S agar plates. Antiserums were prepared from 6 of the 50 strains. The results of the serological tests are shown in table 2. In the present work an additional 00 normal Escherichia isolated from different mediums and individuals were tested in 5 normal Escherichia antiserums. In all, 263 normal Escherichia strains have been tested in an average of 0 antiserums: 23. per cent were closely related and 9.8 per cent were identical whereas of 223 paracolon Escherichia cultures tested in 3 such antiserums 9.4 per cent were closely related and 58.2 per cent were identical. The 3 cultures used to prepare paracolon Escherichia antiserums and 5 cultures neither identical nor closely related were tested in 25 normal Escherichia antiserums. The incidence of agglutination was 6.6 per cent with an average titer of 770 (table 2). Five cultures agglutinated to high titer and were used for adsorptions. One culture completely removed both H and 0 agglutinins from one antiserum; two others reduced the H and 0 titer of another antiserum; a fourth culture reduced the 0 but not the H titer in a third antiserum while the fifth culture, 267, completely removed the 0 without reducing the H titer of a fourth normal Escherichia antiserum. The 64 paracolon Escherichia strains were tested also in the 25 Salmonella antiserums. The incidence of agglutination was 2.4 per cent with an average titer of 635 (table 2). Adsorptions with paracolon Escherichia and tests in single factor antiserums were done. Culture 267 which had the complete somatic structure of a normal Escherichia removed all but the VI somatic agglutinins from S. onderstepoort antiserum. Another culture completely adsorbed the XI agglutinins from S. rubislaw antiserum. Many other reactions could not be attributed to any of the known antigenic components of Salmonella. Seventy-eight normal Escherichia strains were tested in the Salmonella antiserums. The incidence of agglutination was 9.6 per cent with an average titer of 780. Adsorptions and tests in single factor antiserums were done. Two cultures completely removed the somatic XI agglutinins from S. rubislaw antiserum; 3 cultures had a component associated but not identical with somatic I

11 RELATIONSHIPS OF PARACOLON >BACTERIA antigen; another had part of the VI complex; 4 cultures agglutinating to titer failed to reduce the titer of S. tel-aviv; 2 cultures reacted similarly in S. hvittingfoss antiserum. One strain, 485, agglutinated to titer but failed to reduce the titer of S. urbana antiserum upon adsorption. Reciprocal tests with an antiserum against culture 485 showed that S. urbana agglutinated to titer and reduced the homologous 0 titer from 0,240 to 320 upon adsorption. The H titers of the Salmonella antiserums were not altered by any adsorptions. ill ANAEROGENIC PARACOLON Many anaerogenic Aerobacter and a few anaerogenic intermediates were isolated. All were motile, some produced acid in lactose rapidly, others slowly, and all fermented cellobiose and grew on citrate agar in 48 hours. Biochemical TABLE 5 Biochemical and serological relationships of 62 anaerogenic paracolon 04 ~~~NO.0OF NO.0OF TURE TION 8IN THE OREAD IDOPENTICAL 0~~~~~. 98 A A - - A A A A - - A A 33 AB - - -A - A - - S AB - A -A - A 299 AB W t AB - A A - -A -A A A -A A -A AA _ Totals.62 26* * Exclusive of cultures homologous to antiserrms. = positive reaction; A = acid; -A = some strains negative, others produce acid; B - bubble of gas; AB = some strains produce acid only, others, a bubble of gas; S = strong reaction on urea; -W = some strains do not attack urea; others attack it weakly.,, = degrees of motility. and serological studies were limited, however, to 62 strains which were indolepositive and motile. Some of the 62 strains grew on citrate agar in 24 hours but none fermented cellobiose. One type attacked urea strongly in from 8 to 2 hours. One type fermented lactose rapidly and other types slowly or not at all. (Rapid lactose-fermenting variants are obtained easily from a great majority of slow lactose-fermenting cultures and to determine whether parent and variant coexisted in feces both red and white colonies from S S agar and black and white from E.M.B. were fished during a part of this work. If the slow fermenter was identified by any antiserum the rapid fermenter was tested in the same antiserum.

12 2 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN No antigenically related slow and rapid fermenting strains were isolated from any specimen.) From 8 gastroenteritis patients the white colonies proved to be extraneous bacteria or yeasts while the colonies fished as normal coliforms had the reactions of type 98 (table 5). One culture failed to ferment lactose but all were antigenically identical. Previous to the first isolation such strains may have been missed but for nearly 2 years no other culture of this type has been isolated. All strains were encountered in one institution in a period of 5 weeks. Type 33 appears to be more closely related to Proteus than to paracolon since it attacks urea readily (Rustigian and Stuart 94). The serological reactions of these strains are peculiar in that some agglutinated to titer, others to partial titer and two not at all; yet upon adsorption all strains reduced the homologous titer of 33 antiserum. Moreover some strains, after being carried for from one to three years on standard agar, appear to have lost a part of their original antigenic structure. Strains with the reaction of type 299 are serologically heterogeneous since only the homologous cultures agglutinated in antiserums prepared from 2 cultures in this biochemical group. PARACOLON CULTURES AND THE VI ANTIGEN Among the Salmonella antiserums used in this work was one prepared from S. ballerup and one from Eberthella typhosa S 07 both of which contain VI antigen. An antiserum derived from E. typhosa H 90 lacking Vi antigen was also used. All tests of normal or paracolon strains in E. typhosa S 07 antiserum were incubated 4 hours at and then placed at 2 0. over night. All other tests after 2 hours at 37 C. were incubated over night at Twenty-three strains which agglutinated in the 50 or 250 dilutions of S. ballerup antiserum after 2 hours at showed complete reversal of agglutination at 550. Thirty-seven strains, only 2 of which agglutinated in E. typhosa H 90 antiserum, agglutinated in E. typhosa S 07 antiserum in dilutions of from 50 to 250. The 37 strains were tested in serial dilutions of from 50 to 800 in pure VI antiserum prepared by adsorbing S 07 antiserum with H 90 until no agglutination occurred with H 90, heated S 07 or a rough strain of Salmonella. Of the 37 cultures 5 failed to agglutinate in the 50 dilution at either 37 or 2 C., 7 agglutinated in dilutions of 50 to 200 and 5 to titers of 400 to 800. The control titer of S 07 in the pure Vi antiserum was 800 and of S. ballerup 400. The agglutinability of all cultures agglutinating in the pure VI antiserum was destroyed by heating to for one hour. The 5 cultures, two paracolon Escherichia, one paracolon Aerobacter and two normal Escherichia agglutinating to titers of 400 to 800 were used to adsorb the pure Vi antiserum. Two adsorptions were made using the growth from 2 plates for each adsorption. The antiserum was tested after each adsorption with E. typhosa S 07 and S. ballerup. Two adsorptions with each of the 5 strains failed to reduce the titer for S 07 or S. ballerup. The same results were obtained when the adsorptions were repeated. The pure Vi antiserum was then adsorbed twice with 2 and 36, two of the 5 strains agglutinating to titer, with S 07 and with S. ballerup. Tests were made with all four cultures after each adsorption. One adsorption with either the Eberthella

13 RELATIONSHIPS OF PARACOLON BACTERIA or Salmonella culture removed all agglutinins for all four cultures. One adsorption with 2. or 36 removed agglutinins for both cultures but 2 adsorptions again failed to reduce the titer for S 07 or S. ballerup. All cultures reacting in the pure Vi antiserum showed the same fine granular agglutination as the Eberthella and Salmonella cultures with Vi antigen. The pure Vi antiserum apparently contained agglutinins for two thermolabile antigenic components, S 07 and S. ballerup contained both while 2 and 36 had only one. E. typho8a S 07, S. ballerup and 2 gave the same fine granular agglutination to a titer of 800 to,600 in an antiserum against 36. Heating the three antigens to 70 C. for one hour destroyed their agglutinability in 36 antiserum. One adsorption of this antiserum with any of the three cultures removed the agglutinins for all three cultures. It should-be remembered that the unusual reactions obtained in the pure Vi antiserum resulted from testing 269 strains which would not ordinarily be tested in such an antiserum. PATHOGENICITY OF THE PARACOLON TYPE The question of the pathogenicity of coliform organisms has received considerable comment in recent literature and several outbreaks of gastroenteritis have been attributed to normal or aberrant types (Parr 939). Proof of pathogenicity is difficult to obtain because of the lack of susceptible experimental animals. Our opinion after nearly four years work in this field is that paracolon organisms are often associated with, and that under certain conditions some types probably can cause, a mild or acute gastroenteritis of short duration. In the present work, evidence indicating the pathogenicity of paracolon is mostly circumstantial. When a particular type was present in the stools from a large number of patients in an outbreak in an institution the same type nearly always could be isolated from one or more food handlers. From small outbreaks, usually one type of paracolon was found but in large outbreaks often several types were isolated. Paracolon forms from patients frequently were in almost pure culture whereas from food handlers, with one exception, the organisms never comprised more than a very small percentage of the coliform flora. One food handler who yielded a nearly pure culture of type 8 appeared to have been involved in 3 outbreaks in one institution and carried large numbers of the organisms for several months after the last outbreak. Clinical symptoms usually correlated with the presence of almost pure cultures in the stools and case histories of patients from which paracolon were isolated for the most part were similar. One piece of direct evidence implicates the paracolon organisms as a cause of gastroenteritis. Shortly after the start of this investigation all serological work was confined to one room and aseptic technic was not used in agglutination and adsorption tests. Among 6 persons working for from one to nearly 4 years with paracolon strains four infections resulted, two with 36 and one each with 8 and 320. Four persons working with Shigella sonnei under the same conditions were infected within a relatively short time. Infections with S. sonnei were, however, no more severe than with paracolon.. This might indicate that 3

14 4 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN paracolon bacteria lack invasiveness and that some predisposing factor for the host or massive infection is necessary before the invader can become sufficiently established to produce symptoms. Case histories tend to show that colds, improper or inadequate diets or excessive fatigue may predispose an individual to attack by paracolon bacteria. The question of the pathogenic nature of the paracolon group must remain open for the present. In any event the paracolons are not a serious problem except possibly to industry in the number of man hours lost during a national emergency. Hay (939) reports that among the workers in a large steel plant more man hours were lost through gastroenteritis than any other cause. DISCUSSION The limitations of morphology as a criterion on which to build a system of bacterial classification need no discussion. In the 5th edition of Bergey's Manual of Determinative Bacteriology greater emphasis has been placed upon physiological characteristics. This gives much greater flexibility to classification but increases the weight of physiological reactions, a burden which the bacterial physiologist may assume with some trepidation. In genera and species especially, as well as in larger divisions of the system, divergence of opinion and some confusion exists as to the most suitable criteria for taxonomic purposes. For example Shigella gallinarum is nonmotile, produces no gas and ferments maltose. Salmonella pullorum is nonmotile, gas production varies from normal to a bubble and maltose is not fermented. Because pullorum has somatic antigens in common with some Salmonella types Kauffmann considers this organism a Salmonella despite its nonmotile characteristic. Rather than disregard a cardinal characteristic, failure to produce gas, the American Committee considers gallinarum a Shigella even though it is antigenicallyidentical with pullorum. From several coliform cultures Stuart et al. (940) isolated variants differing from the parents in one or two physiological reactions and with one exception parent and variant were antigenically identical. Subsequently from one paracolon culture 5 variants were isolated differing from the parent in motility, indole production, growth on citrate, and fermentation of lactose and cellobiose. Except for the lack of H antigens in the nonmotile variant and one somatic antigen in the indole negative variant parent and variants were identical. An antigenic analysis of any cultures under question may well be the final "court of appeal" but unfortunately such analysis, particularly of the Salmonella group, reveals increasing complexity of structure. The "major and minor" agglutinins of yesterday have become; () the somatic antigens of which a species may possess several, some which may be present in one strain and not another, (2) the alpha and beta phases of the specific flagellar antigens and finally (3) the several flagellar components of the nonspecific phase. Craigie and Yen (938) found that the Vi antigen of E. typhosa could be separated into some 7 components on the basis of bacteriophage susceptibility. The value to the epidemiologist of Vi and other antigens for strain typing of E. typhosa cannot be questioned (Lazarus 94). Kauffmann's discovery of Vi in a normal

15 RELATIONSHIPS OF PARACOLON BACTERIA coliform and our finding another thermolabile antigen in normal coliform and paracolon in common with E. typhosa S 07 indicates that emphasis should not be placed on Vi and Vi-like antigens as indicators of virulence. Typing the paracolon bacteria according to their flagellar and somatic antigens seems impracticable. Of 355 paracolon (exclusive of the 48 division I Aerobacter and all anaerogenic cultures) 72.9 per cent were antigenically identical or closely related. Unfortunately this fairly close relationship may be more apparent than real since there is some evidence of geographical segregation with paracolon strains. Of the 50 strains of 36 every one was isolated in Providence or the immediate vicinity. All 9 strains of type isolated during two years came from a single institution. The only notable exceptions were types 375 and 320 (paracolon Aerobacter) and particularly 33 (anaerogenic paracolon) which were isolated in the laboratories of several states. Cope and Kilander (942) described an organism producing acid in glucose and mannitol but not in lactose, sucrose, salicin or maltose and usually nonmotile which was isolated from gastroenteritis patients over a period of years. Many of our 33 strains had the same biochemical reactions when first isolated but after varying periods of time all fermented sucrose slowly (2 to 3 weeks) and became motile, while some fermented salicin rapidly. Five strains received from Cope were identical or closely related to 33. This organsm can be identified easily by its rapid utilization of urea and merits the attention of diagnostic bacteriologists because of its widespread distribution and the frequency of isolation from gastroenteritis patients. The finding of antigens common to Salmonella and other enteric groups of bacteria is not new. Nonspecific H and the specific e, h flagellar antigens were found in 4 normal Escherichia and one paracolon strains (Gard 937 and Gard and Ericson 939). Schiff et al. (94) found one indole-negative paracolon with the complete somatic antigens of S. onderstepoort and 4 others with all or a part of the somatic antigens of other Salmonella. Bornstein et al. (94) have demonstrated somatic antigens VI and XIII in a Shigella paradysenteriae strain. In the present work 29 normal coliform cultures were tested in Salmonella antiserums. Two cultures had the complete somatic antigens of S. rubislaw and one of S. paratyphi. Many others gave strong reactions but could not be identified with any known SalmoneUa somatic antigens. Of 40 paracolon cultures the somatic antigens of one was identical with S. rubislaw and 3 were closely related to S. onderstepoort. Other important antigens are common to normal coliform, paracolon and Salmonella cultures. Antiserums prepared from S. hvittingfoss, S. tel-aviv, S. urbana and S. cerro al of which have only one labeled somatic antigen, agglutinated coliform cultures in 30 instances to high titers. In no case did adsorption with the coliforms significantly affect the homologous titer. On the other hand the homologous titer of an antiserum prepared from a normal coliform culture which reacted in S. urbana antiserum was markedly reduced upon adsorption with S. urbana. It is, therefore, evident that these Salmonella cultures have unlabeled somatic components. As more information is collected it seems 5

16 6 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN likely that a continuous series of antigenic relationships can be shown for the bacteria included in the coliform, paracolon, Salmonella and Shigella groups. While a given set of antigens would be most commonly found in organisms possessing the same physiological characteristics these antigens will also occur in cultures with different physiological reactions. From this it seems probable that serologically there is no sharp line of division between Salmonella and coliforms of all kinds and that many intermediate types exist just as intermediate physiological types are found. This assumption is confirmed by the fact that 4 cultures, originally from the Jordan collection, received by one of us as Proteus morgani failed to attack urea. Further biochemical tests revealed that three of the cultures consistently gave a weakly positive V. P. reaction while the fourth was weakly positive on some tests and negative on others. The culture could not be typed in the paracolon Aerobacter antiserums. In single factor Salmonella antiserums they were found to possess the VI and VII somatic and the nonspecific flagellar, 5 antigens of group C. These cultures definitely seem to be acetylmethyl-carbinol-producing Salmonella organisms in nonspecific phases. TAXONOMY IN THE FAMILY ENTEROBACTERIACEAE When the American Committee on Classification grouped numerous species into the single species, Escherichia coli, they must have recognized the futility of speciation in the coliform bacteria. Designating a group of bacteria as a species seems awkward at first but we venture to predict that it will not be uncommon in the future. In fact one may well wonder why this procedure was limited to the coliform bacteria. Speciation on the basis of physiological reactions seems quite impossible with the Enterobacteriaceae. Speciation on the basis of antigenic analyses can be done but it is wholly impracticable except for such genera as Salmonella and Shigella and even here practicability may be lost. The discontinuity between groups upon which taxonomy depends is lacking for physiological characteristics and to a lesser extent at the present time for antigenic properties. However, further antigenic analysis of Salmonella and an extension of the antigenic types of normal coliforms and paracolon would probably show as continuous a series of intergrading antigenic types as has already been shown for physiological properties. Practical and theoretical considerations necessitate the use of physiological characteristics, antigenic relationships and habitat in forming any classification of the organisms in the Enterobacteriaceae. One recent American text book has retumed to "Bacterium" as a generic name for many groups of enteric bacteria. In a certain sense this is unfortunate but we feel that the use of Bacterium in this way may increase unless further simplification is made. Although the present work and other investigations cited show antigenic fractions common to Aerobacter, intermediates, Escherichia, paracolon, Salmonella and Shigella we feel that one genus for all these groups is too restiicted. We have not found adequate biochemical or serological evidence for retaining cloacae in the genus Aerobacter. Cloacae has been suggested as a possible pro-

17 RELATIONSHIPS OF PARACOLON BACTERIA genitor of Proteus but work in this laboratory shows, in regard to the cardinal characteristic of Proteus-ability to attack urea-that Aerobacter aerogenes rather than cloacae could be the progenitor of Proteus. Our previous suggestion that all coliform bacteria be placed in a single genus "presumably Escherichia" was wrong, as kindly pointed out to us by Dr. Breed in a personal communication, since Aerobacter has a priority. We, therefore, recommend that three species aerogenes, freundii and coli be included in one properly named genus and that cloacae be dropped. In the 5th edition of Bergey 2 species are recognized in the genus Eberthella besides typhosa and sendai. The biochemical description of some of these is so limited as to make their identification impossible. Fermentation of lactose is not given for 5 species but considering litmus milk it would seem that all but 3 of the 2 species might have some action on lactose. All but 3 species produce indole and 5 liquefy gelatin, 2 of these rapidly. Stuart, Griffin and Baker (938) isolated a number of anaerogenic coliform cultures from various sources. Some appeared to be identical with one or another of the 2 Eberthella species while others differed by one or two reactions. One type of culture with the principal characteristic of Eberthella dubia, abundant yellowish growth on agar, was occasionally isolated from decayed vegetables. Attempts to secure authentic cultures of the various Eberthella species for biochemical and serological comparisons failed. It is recommended that E. typhosa and E. sendai, the latter a gas-former, be classified in Salmonella and that the remaining species of the genus be considered collectively as anaerogenic coliforms or paracolon. The careful work of Osterman and Rettger (94) on the morphology, biochemical and serological reactions and pathogenicity of "Friedlainder" and coli-aerogenes groups resulted in the conclusion, "That valid criteria have not yet been established for the differentiation of organisms of the "Friedlander" and coli-aerogenes groups". All the "Friedlander" cultures used by these investigators could be placed in the normal coliform group or in the paracolon or anaerogenic paracolon groups herein suggested. Possibly the time has come to question the validity of the genus Klebsiella. It is recommended that the term "aberrant coliform" be used to include all slow or non-lactose-fermenting cultures but that the term "paracolon" be applied to such cultures isolated from feces, also that the paracolons be adequately described as para-aerogenes, para-freundii and para-coli under their respective normal species. Non-gas-producing cultures fermenting lactose rapidly, slowly or not at all can be grouped as anaerogenic aerogenes, freundii or coli or collectively as anaerogenic paracolon. They have not been studied sufficiently, however, to warrant more than brief mention under a general description of the coliform group or under their respective normal species. SUMMARY Paracolon organisms which are defined as a group of aberrant coliform organisms isolated from man, especially in gastroenteritis outbreaks, comprise a distinct biological group of bacteria. Specialization in the paracolon group 7

18 8 STUART, WHEELER, RUSTIGIAN AND ZIMMERMAN proceeds independently in the Aerobacter, intermediate and Escherichia sections and a high degree of physiological specialization can be reached even by Aerobacter types. For example type 375 although resembling Aerobacter fails to ferment lactose, sucrose, salicin or cellobiose, gives a weak or negative V. P. reaction upon isolation, occasionally fails to grow on citrate agar and does not liquefy gelatin. Other members of the three sections differ to a greater or lesser extent from normal coliforms and on the basis of physiological properties the paracolon forms are intermediate between normal coliforms and Salmonella. A marked difference was found in the antigenic continuity of the normal and paracolon groups. Normal Aerobacter tested in normal Aerobacter antiserums, normal intermediates tested in normal intermediate antiserums and normal Escherichia in their own antiserums showed that, 8.2 and 33.0 per cent respectively were identical or closely related. Similar tests with Aerobacter, intermediate and Escherichia paracolon cultures in their respective paracolon antiserums showed 47.8, 52.5 and 67.2 per cent respectively to be identical or closely related. Known somatic Salmonella antigens were occasionally found in normal coliforms and in paracolon. Unidentified somatic antigens common to normal coliforms (all sections), to paracolon (all sections) and Salmonella were frequently encountered. A Vi like somatic antigen common to Eberthella typhosa S 07, Salmonella ballerup and a number of normal coliform and paracolon types was also found. There is some evidence of geographical segregation in the paracolon group since with few exceptions strains from one locality were frequently identical with other cultures from the same, but seldom with strains from different localities. Although 72.9 per cent of the more specialized paracolon cultures were identical with or closely related to one or another of the cultures used to produce the paracolon antiserums there remained 96 cultures which could not be identified by the 26 antiserums. Because of their complex serological structure classification of paracolon bacteria on the basis of H and 0 antigens is impracticable. From serological and physiological properties the paracolon group is intermediate between normal coliforms and Salmonella. With both properties there appears to be no sharp distinction between the groups and a more or less continuous series of types exist. The pathogenicity of the paracolon bacteria has not been proven. They have been found very often associated with mild or acute gastroenteritis of short duration. Some evidence was found indicating an etiological role for types 8, 36, 320, and especially the proteus-like 33 which appear to possess some pathogenic properties. ACKNOWLEDGMENTS We are indebted to Dr. F. L. Mickle for revising the manuscript, to Miss Betty Brown and particularly to M. A. Gallagher for their assistance in this work.

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