VALIDITY OF THE GENUS ALCALIGENESH. J. CONN'

New York Agricultural Experimen Station, Geneva, New York2

Received for publication December 20, 1941

A recent paper by Conn, Wolfe and Ford (1939) discussed the question as towhether certain soil bacteria are related to the genus Alcaligenes as recognized inrecent editions of Bergey's Manual of Determinative Bacteriology. So far asconcerns the relationships between the soil bacteria and plant parasites discussedin that paper, the reasoning still seems sound. In one or two particulars, how-ever, modification of the conclusions seems necessary in light of further evidencethat has been obtained. This evidence relates to the identity of the type species,Alcaligene,s faecali. The statement was made in that paper: "Apparently,therefore, there is a commonly recognized organism to which the name Alcali-genes faecalis can be assigned." Evidence is now at hand which makes thatstatement doubtful. Before presenting this evidence, however, a few wordsare necessary as to the history of this name, and of the specific designation.

HISTORICAL

Petruschky (1896) applied the name Bacillus faecalis alcaligenes to a peri-trichous, non-spore-forming rod of intestinal origin, producing no acid in sugarbroths and causing alkalinity in milk; no detailed description was given. It wasrenamed BaciUlls alcaligenes by Migula (1900) and Alcaligenes faecalis by Cas-tellanni and Chalmers (1919). This generic name has been criticized as beingan adjective instead of a noun, thus having been formed contrary to a recom-mendation of authorities on nomenclature. It can, however, hardly be calledinvalid on this account and was accepted in the first edition of Bergey's Manual,with the following definition:

"Motile or non-motile rods, generally occurring in the intestinal canal of nor-mal animals. Do not form acetyl-methyl-carbinol. Do not ferment any of thecarbohydrates."In this edition of Bergey's Manual eight other species were included with it,

only two of which were stated to occur in the intestinal canal; three were de-scribed as milk or udder bacteria; three (A. melitensi, A. abortus, and A. bronchi-septicus) have since beel\ transferred to the genus Brucella, one becoming thetype species of the latter.The genus Brucella was first recognized in the fourth edition of the Manual,

and the first two of the three above mentioned species were then transferred tothis other genus. The number of Alcaligenes species recognized, however, was

1 Acknowledgement is made to Jean E. Conn and to Mark Ford for much of the data uponwhich this paper is based.

2 Approved by the Director of the New York State Agricultural Experiment Station forpublication as Journal Paper No. 481, December 17, 1941.

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now ten, since additional species had been placed in it; just half of these tenspecies are intestinal forms.

In the fifth edition of the Manual (1939) A. bronchisepticus was transferred tothe genus Brucella because of protests of students of the latter group, and onesoil form (A. radiobacter) was added; the latter had not been included in anygenus in earlier editions of the Manual and it was now placed in Atcaligenesbecause this was the only genus whose description it would fit. In this lastedition of the Manual, the writer undertook to revise the definition of the genusand the statement concerning habitat was made to read "which generally occurin the intestinal canal, in decaying materials, dairy products and soil." Justhalf of the species were still described as intestinal forms.The writer first became interested in this genus when it proved that it was the

only place in the Bergey system (until, in the 5th edition, Bacterium was againrecognized) where the numerous soil bacteria which do not ferment sugars andtend to produce alkalinity in milk can be placed. Recognition of this fact raisedtwo questions which have not proved easy to answer: (1) Are the soil forms andintestinal forms, which do not produce acid or gas from sugars, closely related toeach other? (2) Can the type species Alcaligenes faecalis be recognized?

THEORETICAL

There is one theoretical point which must be first discussed, because failure torecognize it can cause considerable confusion in the understanding of organismssimilar to these under discussion.When an organism produces acid and gas from a sugar, or even when it pro-

duces no gas but sufficient acid to be detectable in the presence of peptone, thereis no question as to its fermentative ability. When, however, neither acid norgas is evident on a peptone-sugar medium, there is danger of confusion. Oftenall such organisms are grouped together as "inert" or "non-fermenting" bacteria.Actually, however, there may be two distinctly different classes included in thiscategory; (1) organisms which are unable to use sugar; (2) organisms whichutilize sugar (especially glucose) so completely that there are no by-products,and the only end-product capable of giving an acid reaction to the medium iscarbon dioxide-a change in reaction which can be detected only on media of lowbuffer content. It will be seen that theoretically these two classes of bacteriastand far apart in physiology and yet the generally recognized tests for fermenta-tion would group them together.

SIMILARITY TO SOIL FORMS

As explained above, one of the questions to be answered is: Are the soil formsand the intestinal forms which produce no acid from sugars closely related? Toanswer this question, a series of soil and intestinal forms were collected.For the soil forms, Bacterium radiobacter L6hnis was selected as typical. In-

cluded with it,were miscellaneous short rod forms isolated from soil in the writer'slaboratory.To secure intestinal forirF known to be Alcaligenes faecalis proved difficult.

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VALIDITY OF GENUS ALCALIGENES

Four cultures under this name were secured from the American Type CultureCollection; one of them was quickly discarded, as it proved to have been over-grown by one of the fluorescent pseudomonads. Four were obtained from theNational Collection of Type Cultures at the Lister Institute. A collection of14 cultures was obtained from the laboratory of Dr. Carl Nyberg in Helsinki,5 of which were labelled "A. faecalis" the others "similar to A. faecatis." Twostrains were obtained from Dr. Pribram at Chicago. A series of 11 cultures wassecured from Dr. E. G. D. Murray of Montreal; these were freshly isolated non-acid-formers, but were not all regarded as typical representatives of this species.A strain was obtained from the University of Rochester Medical School, whichwas a stock culture that had been used for illustrative purposes in class work forsome time, but whose origin was not known. Of these various cultures, a fewproved to be lophotrichous; they obviously must belong elsewhere, as this specieswas originally described as peritrichous and has generally been accepted as such.The difficulty in securing authentic cultures of this species seems to arise from

the fact that bacteriologists are not specially interested in it. Any non-fer-menting culture of fecal origin is regarded as Alcaligenes faecalis and of no sig-nificance; hence the culture is discarded without thought of preserving it as astock culture for comparison with authentic strains.

In comparing the soil forms with the intestinal forms only a few characteristicsproved available for making distinctions. The morphology did not vary; acidand gas tests on ordinary media are always negative; production of nitrite fromnitrate proved a variable character. Two features, however, did prove useful:(1) ability to grow on synthetic media; (2) production of CO2 from glucose. Thelatter character seemed particularly worth investigating, as it has been shownby Conn and Darrow (1935) and by Hofer (1941) that such soil forms as Bac-terium globiforme and Bacterium radiobacter, although producing no organic acidfrom glucose, do give rise to very appreciable,quantities of carbon dioxide.

Ability to grow on synthetic media. To test this point two synthetic mediawere prepared, of the following composition:

Medium I

(NH4)2HP04....................................................... 1.0 g.KCl....................................................... 0.2MgS04.7H20....................................................... 0.2Yeast extract....................................................... 0.2Agar. 15H20....................................................... 1000 ml.

Medium IISame but with the addition of glucose.............................. 1.5 g.

The soil forms showed scanty growth on Medium I, but very vigorous growth onMedium II. Only five of the cultures received as examples of Alcaligenesfaecaliswere the same in this respect. Two of these five were from the collection re-ceived from Montreal, but showed evidence of contamination. The other threewere the strains obtained from the American Type Culture Collection and on

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the basis of which the earlier conclusions were drawn; two of them were reportedto have come originally from Dr. E. 0. Jordan, the other (isolated from blood)from Dr. Alice C. Evans.Three of the strains obtained from Dr. Nyberg showed fair growth in both

media, no better with glucose than without; but these three strains were amongthose labelled by him as merely "similar to A. faecalis."

TABLE 1CO2- Production in 20 ml. of ammonium phosphate media

C02 FROm 30 mGM. GLUCOSE

ORGANISM SOURCEMedium III, Medium IV, Medium V,

complete no yeast extract no glucose

mgm. mgm. mgm.

B. radiobacter Hofer 27.5 23.2? Soil 14.0? Soil 12.5

A. faecalis 1 ATCC 17.38 15.84 0.88A. faecalis 2 ATCC 16.72 15.73A. faecalis 3 ATCC 4.62 2.75 0.88A. faecalis 4 U. of Rochester 1.43 0.11 0.22A. faecalis 5 Murray 1.76 0.55A. faecalis 6 Murray 4.62 1.21A. faecalis 7* Murray 20.24 22.84A. faecalis 8 Murray 5.39 ? tA. faecalis 9 Murray 0.66 1.65A. faecalis 10 Murray 0.66 1.43A. faecalis 11 Murray 5.06 2.42 0.44A. faecalis 12 Murray 0.55 0.33A. faecalis 13* Murray 19.69 4.62A. faecalis 14 Murray 4.62 0.44 0.22A. faecalis 16 Lister Inst. 2.86 2.42A. faecalis 17 Lister Inst. 1.54 1.98A. faecalis 18 Lister Inst. 2.75 0.22A. faecalis 19 Lister Inst. 2.20 1.54A. faecalis 20 Pribram 2.75 0.99 0.44A. faecalis 22 Nybergt 2.42 0.44A. faecalis 23 Nyberg 21.45 9.46 0.88

* Culture apparently overgrown by contaminant before this test was made.t Disagreement of duplicates.I See Nyberg (1935).

All the rest of the strains showed poor or no growth in either medium. Amongthem were included: Nine of the cultures from Dr. Murray; 4 from the ListerInstitute; the University of Rochester culture, and the 2 from Dr. Pribram; all4 of the cultures from Dr. Nyberg claimed to be authentic, and 6 of those sub-mitted by him as being similar organisms.The weight of evidence from this comparison of cultures is that A. faecalis, as

generally recognized, does not grow on the particular synthetic medium selected,

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and unlike the soil forms, cannot make use of glucose on such a medium. Thereis no evidence at hand as yet to indicate that A. faecalis, as represented by thebulk of cultures received, can grow without organic nitrogen. Apparentlystrains from the American Type Culture Collection are a different organism.

Production of CO2 from glucose. This problem was investigated by the El-dredge tube technic. The media employed were as follows:Medium III: Like medium I above but without agar.Medium IV: Like Medium III but without yeast extract.Medium V: Like III but without glucose (used merely as a check on 7 of the

cultures). The results, with 21 strains of the so-called "A. faecalis", in com-parison with similar determinations for certain soil forms are given in table 1.It will be seen that abundant CO2 is produced by the soil forms. The only"A. faecalis" cultures, however, which showed appreciable C02 in the absenceof yeast-extract were two of the three from the American Type Culture Collec-tion and one or two others of doubtful purity.3

CONCLUSIONS

It is evident that the similarity of A. faecalis to certain soil bacteria whichwas previously pointed out by Conn, Wolfe, and Ford, may not actually exist;in pointing out this similarity, too much significance was attached to the culturesreceived from the American Type Culture Collection. If instead of regardingthese cultures as representative of the species, one accepts as typical thoseobtained from other collections, very different conclusions must be drawn. Thecultures from the former source are very much like the soil forms; but a majorityof those from other sources require organic nitrogen and show no evidence ofutilizing glucose-while the soil forms in question utilize inorganic nitrogen andglucose, using the latter so economically and completely that they do not produceappreciable acid in ordinary media. Following the scheme of classification ofthe Eubacteriales given on pp. 20-21 of the 5th edition of Bergey's Manual, thiswould put the soil forms in a widely different section of this Order from thatwhere one would have to place most of the A. faecalis cultures studied (exceptthose from the American Type Culture Collection).

These findings also raise serious question as to whether anyone knows just whatAlcaligenes faecalis is. The original description by Petruschky is vague; andeven the recent emendations are incomplete, as evidenced by the fact that thepublished descriptions would fit either the type growing on synthetic media orthat which requires organic nitrogen. Both of these types are being distributedunder the same name; and it is difficult, if not impossible, to decide which isauthentic. The writer of this paper, as a soil bacteriologist, hardly feels com-petent to venture an opinion.

' Abundant CO2 production from all of these cultures has been observed in a veal-infusionmedium containing glucose. This, however, is of no significance, as the break-down ofveal infusion has been shown to yield C02, and those of the organisms included in this workwhich were tested on this same medium without glucose were found to give off almost, ifnot quite, as much of this gas as in its presence.

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Still further evidence that bacteriologists are in disagreement as to the char-acteristics of this species is furnished by the fact that at least one of the culturesfurnished under this name proves to have polar flagella. This organism canhardly be the true A. faecalis, as this species has always been described (even byPetruschky) as being peritrichous; but the fact that it was obtained under thatname is further indication of the confusion that exists.

TAXONOMIC RECOMMENDATIONS

The above data seem to invalidate the recent recommendations, made in partby the author and associates, that some of these soil bacteria belong in the genusAlcaligenes. The same objection can be raised to including the other non-para-sitic forms which have been put in the genus-notably the species Alcaligenesviscosus, which proves to be closely related to the soil types under consideration.If the three species occurring in milk and soil, listed in this genus in the lastedition of Bergey's Manual, be removed from it, there remain six parasiticspecies, including the type species A. faecalis. Three pertinent questions arise:(1) Should the Genus Alcaligenes be retained for these six species? (2) If not,in what genus should they be placed? (3) In any event, what should be donewith the soil and milk forms now bearing this generic name?The writer wishes to venture the following opinions:1. The genus Alcaligenes can be retained only if its type species can be recog-

nized. As explained above, at least two different organisms are being distributedby culture collections under the name A. faecalis. Although it seems entirelyprobable that the true intestinal type is the one which requires organic nitrogen,the matter certainly needs clarification. It is apparently impossible to deter-mine the exact nature of the original organism studied by Petruschky; and gen-eral agreement as to the nature of the species would be very difficult to secureunder present world conditions. Hence, the final decision may be to drop thename entirely. Further justification for doing this could be found in the factthat the generic name is an adjective, not a noun as recommended in codes ofnomenclature.

2. There are at present five intestinal inhabitants listed under this genus inBergey's Manual: A. faecalis, A. metalcaligenes, A. ammoniagenes, A. bookeri andA. recti. These might well make a genus by themselves, especially if it provesthat A. faecatis is the organism which requires organic nitrogen, and that theother species are like it in this respect.

Undoubtedly, the most convenient arrangement, in the next revision of Ber-gey's Manual, will be to leave them in this genus, with the thought of its being atemporary grouping until further study of species included can be made. It isclear that all five species, even the type species, require such study before theiridentity can be definitely learned. Meanwhile those who are unwilling to putthem in a genus whose type species is not definitely recognized can call themBacterium alcaligenes, B. metalcaligenes, B. ammoniagenes, B. bookeri, and B. recti,respectively. None of these names are new combinations, and the definition ofBacterium in the 5th edition of the Manual is sufficiently broad to cover theseorganisms.

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3. At present only one soil organism has been placed in Alcaligenes, namelyA. radiobacter. This organism is plainly out of place in the genus if the typespecies is the intestinal organism which requires organic nitrogen. It is, on theother hand, very similar to the legume nodule organisms, Rhizobium spp. andto the bacteria causing crown gall and hairy root (now in Phytomonas). Stu-dents of these groups are coming to think that all these species should be closetogether in the classification. The legume nodule organisms undoubtedlyconstitute a definite and easily recognized genus; but after consultation withbacteriologists who have studied both the genus Rhizobium, and the closelyrelated plant pathogens, it seems that a new genus is called for. This genusshould contain the plant pathogens closely related to the crown gall organism,the soil species now in Alcaligenes, and other saprophytic organisms that may befound to be similar in morphology and physiology. For this new genus the nameAgrobacterium is now proposed, with A. tumefaciens as its type species4. Forthis genus the following description is hereby given:

Agrobacterium. Small, short, non-spore-forming rods, which are typically motile with1-4 peritrichous flagella; (if only one flagellum, lateral attachment is as common as polar).Occur primarily in the soil or as pathogens attacking roots or producing hypertrophies onthe stems of plants. Are ordinarily gram-negative. Do not produce acid or gas6 in glucose-peptone media, although a certain amount of acid is often evident in synthetic media;this latter observation is ordinarily due merely to presence of C00 which may be producedin considerable abundance. Liquefy gelatin slowly or not at all.

The type species Agrobacterium tumefaciens is the cause of crown-gall of plants,and may be described as in the 5th edition of Bergey's Manual. Placed withthis should be Agrobacterium radiobacter (syn. Bacterium radiobacter, Achromo-bacter radiobacter, Alcaligenes radiobacter)5 and Agrobacterium rhizogenes (syn.Phytomonas rhizogenes), the cause of hairy root, both described as in the 5thedition of Bergey.

4In Bergey's Manual Polymonas tumefaciens (Lieske, 1928) is given among the synonymsof this species, and would seem to be called for by rules of priority. This is not the case,however, because a consultation of Lieske's paper shows that it is not a true synonym.Lieske was of the school who believed crown-gall to be identical with human and animalcancer, and to be caused by a microorganism. The above-cited name he assigns to a poly-morphic organism which he claims to be the cause of cancer, and he adds that Bacteriumtumefaciens is one phase of this organism. His theory is not accepted today, and no organ-ism is known which possesses the pleomorphic characteristics he assigns to Polymonastumefaciens.

6 In this definition the expression "acid or gas" is used in the rather indefinite sense inwhich it is ordinarily employed in manuals of determinative bacteriology; thus "acid"essentially means a sufficient change in reaction to be detected by litmus, and "gas" meanssufficient gas to be visible as a bubble in the liquid. Hence the statement "do not produceacid or gas" does not exclude the organisms which produce C02 without saturating themedium with the gas or lowering its pH beneath approximately 6.4, nor those which produceorganic acids without greater change in pH than this.

6 Hofer (1941) mentions as a possible synonym of this organism Pseudohizobium ramosumHartleb, 1900. This name would have priority if it could stand; but Hartleb's descriptionis too vague for recognition, and it seems very unwise to emend the description so that itcould be recognized-for this would mean abandoning the specific as well as the genericname under which this organism has long been known.

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Of the milk forms in the genus Alcaligenes the only one which seems definitelyrecognizable is Alcaligenes viscosum. This seems to lie closer to those speciesdiscussed in the preceding paragraph than it does to the intestinal forms whichrequire organic nitrogen; until its relationships are more definitely establishedit can be called Bacterium vigcosum after Weldin and Levine (1923). The othermilk species are very poorly known and have never been put in the genus Bac-terium except under the trinomials Bacterium lactis album and Bacterium lactismarshalli. It is recommended that these be transferred together with the equallypoorly known species Alcaligenes dernieri (obtained from rubber latex) to anappendix of indefinitely described species.For the present the writer prefers to make no further new combinations by

putting other species in this new genus. There are several other plant pathogens,listed in Appendix II of Phytomonas in the 5th edition of Bergey's Manual,which probably belong in the same genus with the crown-gall organism; but itseems well to leave it for phytopathologists to rename them if they are to betransferred to Agrobacterium. There are, moreover, various soil species, someof which have been named and others still unnamed, which seem equally closelyrelated to the species which it is now proposed to call Agrobacterium radiobacter.It is hoped that the new genus will prove a convenient place for some formswhich do not fit well into any of the genera recognized in past editions of Bergey'sManual; it is also hoped that those adding species to this genus will include onlythose types covered by the above definition.

REFERENCES

BERGEY, D. H. ET AL. 1939 Manual of Determinative Bacteriology. 5th ed. Baltimore,Williams & Wilkins Co.

CASTELLANI, A., AND CHALMERS, A. 1919 Manual of Tropical Medicine. 3rd ed. Lon-don, Balliere, Tyndall and Cox.

CONN, H. J., AND DARROW, MARY A. 1935. Characteristics of certain bacteria belongingto the autochthonous microflora of soil. Soil Sci., 39, 95-110.

CONN, H. J., WOLFE, GLADYS E., AND FORD, M. 1940 Taxonomic relationships of Alcali-genes 8pp. to certain soil saprophytes and plant parasites. J. Bact., 39, 207-226.

HARTLEB, R. 1900 Die Morphologie und systematische Stellung der sogenannten Kn6ll-chenbakterien. Chem. Ztg., 24, 2d. sem. 887-888.

HOFER, A. W. 1941 A characterization of Bacterium radiobacter (Beijerinck and VaDDelden) L6hnis. J. Bact., 41, 193-224.

LIESKE, R. 1928 Untersuchungen uiber die Krebskrankheit bei Pflanzen, Tieren, undMenschen. Zentr. Bakt. Parasitenk., I, Orig., 19, 118-146.

MIGULA, W. 1909 System der Bakterien. Fischer, Jena., 2, 737.NYBERG, C. 1935 Bacillus faecalis alcaligenes Petruschky. Zentr. Bakt. Parasitenk.,

I, Orig., 133, 443-449.PETRUSCEKY, J. 1896 Bacilul faecalis atcaligenes (n.sp.). Zentr. Bakt. Parasitenk.,

I, 19, 187-91.

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