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Bacillus Proteus: Clinical Laboratory Classification
THE JOURNAL OF UROLOGY
Vol. 89, No. 1 January 1963 Copyright © 1963 by The Williams & Wilkins Co. Printed in U.S.A.
BACILLUS PROTEUS: CLINICAL LABORATORY CLASSIFICATION HARRY SENECA, P. PEER
AND
R. NALLY
From the Department of Urology, College of Physicians and Surgeons, Columbia University and the Presbyterian Hospital, New York, N. Y.
In Greek mythology Proteus was a sea god who had the power of assuming different forms or shapes. In English the word means a person or thing capable of taking on various aspects or characters. The protean nature of the genus Proteus justifies the name it was given by Hauser in 1885. In most clinical bacteriology laboratories and hospitals, the clinician and bacteriologist are not yet conditioned or indoctrinated into attempting to type or classify B. proteus in much detail. Both are justified in knowing or identifying it as such. The clinicians are not aware of the importance of knowing the various types, while the bacteriologists have lost their interest in this genus since it is no longer needed as a typing tool for Rickettsia. The importance of accurately classifying this bacterium is only now becoming apparent. Further investigations of the genus Proteus have been in progress at the Bacteriological Research Laboratories of the Department of Urology, Columbia University College of Physicians and Surgeons for the past 10 years.
be utilized as the sole source of carbon. Putrefactive odor is the characteristic of Proteus cultures. The antigenic composition of Proteus is of vital importance in rickettsial infections. The flagellar H antigen is inherent in the flagella while the somatic O antigen is located in the body (soma) of the organism. In the classical Weil Felix agglutination reaction Proteus OX 19, OX 2 and OX K are the non-motile variants. Agglutination with the H antigens or motile variants is specific for infection with Proteus but has no relationship to rickettsial infections. Bergey1 recognizes 5 types of Proteus. Those which hydrolyze urea are Proteus vulgaris, P. mirabilis, P. morganii and P. rettgeri. Proteus vulgaris is mannitol negative but produces acid and gas in dextrose, maltose and saccharose. Proteus mirabilis differs in failing to ferment maltose and is indole negative. In case acid or acid and gas are produced in mannitol and dextrose, it is P. rettgeri. Proteus inconstans does not hydrolyze urea. Proteus morganii is the only member which does not utilize carbon in citrate. P. OX 2 and OX 19, being maltose fermenters, are classified under the P. vulgaris species, while Proteus OX K, being a non-maltose fermenter, is classified as P. mirabilis.
CLASSICAL CHARACTERISTICS
Proteus is a gram-negative straight rod which is motile by means of peritrichous flagella. Proteus vulgaris and P. mirabilis produce ameboid colonies which manifest swarming on moist agar and gelatin or solid media devoid of bile salts. Pleomorphism is prominent in young, actively swarming cultures. Acid and gas are produced in glucose, fructose, galactose, maltose and sucrose, but there is no fermentation of lactose, mannitol or dextrin. Litmus milk is peptonized, indole is positive, acetylmethylcarbinol negative, hydrogen sulfide positive, nitrites are produced from nitrates, phenylpyruvic acid is produced from phenylalanine, leucine is rendered alkaline, urea is hydrolyzed (except by P. inconstans) and sodium citrate can
SUGGESTED NEW CLASSIFICATION
The present studies indicate that there is a simple, practical and quick procedure in typing Proteus. It is also of help to the clinician in planning chemotherapy. This study or investigation was evolved during the course of an investigation of the nature of resistance to chemotherapy and a study of mutagenicity among pathogenic bacteria which give rise to urinary tract infections. 1 Breed, R. S., Murray, E. G. D. and Smith, N. R.: Bergey's Manual of Determinative Bacteriology, 7th ed. Baltimore: The Williams and Wilkins Co., 1957.
Accepted for publication May 1, 1962. 89
90
H. SENECA, P. PEER AND R. NALLY MATERIAL AND METHODS
Preparation of media. 1. Bacto urea agar2 2. Bacto Simmon's citrate agar dehydrated (Difco) 2 3. Columbia Presbyterian urea citrate synthetic medium: Solution A Magnesium sulfate ............. Disodium phosphate. . . . . . . . . . . Monopotassium phosphate ...... Sodium chloride ................ Sodium citrate ................. Agar Noble ....................
0.2 gm. 1. 0 2.0 5.0 2.0 15 to 20
Boil in 960 ml. distilled water to dissolve, and sterilize in the autoclave at 15 lbs. for 15 minutes at 121C. Solution B Urea C.P ..................... 20.00 gm. Phenol red................ . . . 0.012 Distilled water. . . . . . . . . . . . . . 40. 0 ml. Dissolve and sterilize by filtering through Seitz. Cool the solution A to 45 degrees C. and add to it solution B under sterile conditions. Distribute in slants, 1 inch butt and 1.5 inches slant. Urea should be of pure quality and should not be autoclaved or overheated, otherwise it will be broken down. The final pH should be 6.8 and the color of the slants is no different from that of plain agar slants. In case the pH is not correct, or urea is split the color is pink, in which case the medium cannot be used. The butt should not be stabbed; the slant is streaked. The changes to be observed are growth, no growth and change of color of slant, or slant and butt to red. The readings are taken after 24, 48, and 72 hours and 1 week after incubation at 37C. 4. Synthetic citrate medium: Magnesium sulfate ............... Disodium phosphate . . . . . . . . . . . . . Monopotassium phosphate ....... Sodium chloride ................. Sodium citrate ................... Agar Noble ...................... Distilled water..... . . . . . . . . . . . . . .
0.2 gm. 1. 0 2.0 5.0 2.0 15 to 20 1000 ml.
2 Difeo Manual, Microbiological and Clinical Laboratory Procedures, Difeo Laboratories, Inc., Detroit, Mich., 1953.
Boil to dissolve, tube and autoclave, cool into slants. The final pH. is 6.8. Do not stab the butt; simply streak the slant. Incubate and observe growth. 5. Synthetic urea medium: Solution A has the same ingredients as solution A of the urea citrate medium minus sodium citrate. Solution B is the same as solution B of urea citrate medium. The remaining procedure is the same as for urea citrate. 6. The following substrates were also used as the sole source of carbon in the synthetic citrate medium, in place of sodium citrate: Sodium lactate D. L malic acid Ketoglutaric acid Isocitric acid Succinic acid Fumaric acid Sodium acetate Aconitic acid Pyruvic acid Oxalosuccinic acid Oxaloacetic acid Similarly these ingredients were used in the synthetic urea citrate medium in place of sodium citrate. This combination did not show any additional advantage over the plain carbon substrate and therefore it was not routinely used. Aconitic acid in synthetic urea medium failed to solidify and became black when the pH was adjusted. All the aforementioned media can also be prepared in liquid form by omitting agar. Since growth was difficult to determine in liquid medium, the liquid media were not routinely used. SOURCE OF PROTEUS CULTURES
Columbia-Presbyterian Medical Center, U. S. Army, U. S. Navy, Food and Drug Administration, Mount Sinai Hospital, in New York City; include both stock cultures and recent isolations from hospital patients. RESULTS
Table 1 shows the clinical classification of Proteus into 5 groups according to the enzyme patterns (profiles) in relation to the growth of the bacilli on synthetic media. Profile 1 includes P. vulgaris, P. mirabilis, P. rettgeri and P. OX 2. Proteus columbiensis
TABLE
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u
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0
l:J
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2
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5~
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-
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Profile 1
Proteus colurnbiensis a) P. vulgaris b) P. mirabilis c) P. rettgeri d) P. OX 2
"°,..._
+ SB + SB + SB + SB
+
s
+ SB + SB + SB + SB
+ SB
-
+
s
+
s
+
s
+
+ + +
+
s
+
s
+
s
-
-
+
-
+
+
+
+
+
-
-
AG
-
AG
+
+
AG
-
+
-
+
-
AG AG
+
+
+ +
-
+
+
-
+
-
+
+
+
+
+
-
-
AG
-
-
-
+ +
+
+
+
+
-
+
+
+
+
+
-
-
-
A AG
AG
-
+
+
+
+
-
+
-
s
+
-
+
-
+
+
-
+
+
-
+
+
+
+
+
-
-
A AG AG
+
-
+
+
+
+
+
-
-
AG
-
-
-
+
-
-
Profile 2
Proteus morganii
s
s
+
-
-
-
+
+
+
-
+
-
-
+
+
+
+
-
-
AG
-
-
-
-
+
-
-
+
-
+
+
+
+
+
-
-
AG
AG
-
AG
+
+
+
-
-
A AG
-
-
-
+
+
-
-
Profile 3
Proteus OXK
Profile
+ to SB
-
-
to
to
s
-
-
+
-
to
s
s
4
Proteus OX 19
+ to SB
-
-
+
-
-
to SB
-
+
-
to
-
-
+
+
s
-
-
Profile 5
Proteus inconstans
s
+
s
-
-
+
+
+
+
-
+
-
-1+
- means no growth;+ means growth; S means color change in slant and Bin slant and Butt A means acid production and AG acid and gas.
92
H. SENECA, P. PEER AND R. NALLY
includes those strains which produce urease under aerobic (slant) and anaeroic (butt) conditions and utilize carbon in the citrate molecule in Simmon's citrate where there is growth with change of color of the slant from green to blue (indicator bromthymol blue) and growth in plain citrate medium. In synthetic urea citrate medium and Bacto urea medium, there is no difference, thus indicating that urea is split both under optimum (bacto urea medium Difeo) and under starvation diet (synthetic urea medium). Urea is split under aerobic (red slant) and anaerobic conditions, manifested by red color (phenol red indicator) in slant and butt. In plain synthetic urea medium, urea is split only under aerobic conditions, manifested by red color in only the slant. P. columbiensis utilizes carbon in citric, isocitric, fumaric, acetic, oxalic, aconitic and oxalosuccinic, but fails to utilize pyruvic and succinic acids. Growth in media combining oxalacetic, lactic, DL and L malic and ketoglutaric acids is variable. P. vulgaris is the only member which cannot utilize oxaloacetic acid. Proteus OX 2 has the same fermentation reactions as P. vulgaris, namely, fermenting dextrose, maltose and saccharose with acid and gas production, but differs from the latter in utilizing oxaloacetic acid. Proteus OX 2, however, fails to utilize carbon in DL and L malic acids. The main feature of P. mirabilis is failure to utilize carbon in ketoglutaric acid, and producing acid and gas in dextrose and saccharose; it is indole negative. P. rettgeri has the characteristic of producing acid or acid and gas in dextrose and mannitol, and may produce acid and gas in saccharose. Its distinctive features are that it is H 2S negative and mannitol positive. Most of the clinical types of Proteus isolated from the patients at the Columbia-Presbyterian Medical Center are in this group and of the subtypes P. rettgeri and P. mirabilis. P. vulgaris is rarely isolated. Profile 2 is uniform and includes P. morganii. Under optimum conditions, it splits urea under aerobic and anaerobic conditions (in Bacto urea agar, the slant and butt are red). Under starvation conditions, in urea citrate medium and synthetic urea only the slant is red. In Simmon's citrate there is no growth. It utilizes carbon in lactic, isocitric, fumaric, acetic and aconitic
acids and fails to utilize it in citric, oxaloacetic, succinic and pyruvic acids. It may or may not utilize carbon in DL and L malic, ketoglutaric, oxalic and oxaloacetic acids. It produces acid and gas only in dextrose and is H 2S negative. It is fairly commonly isolated from clinical patients. Profile 4 includes Proteus OX K and Proteus OX 19. They may or may not split urea and fail to utilize carbon of the citrate molecule. On urea citrate medium Proteus OX K may change the color of the slant to red while Proteus OX 19 may change the color of slant and butt. Profile 4 may change the color of Simmon's citrate to blue. They have almost the same biological features except that profile 3 consistently utilizes carbon in L malic acid, while profile 4 fails to do so. Profile 3 (always) and profile 4 (may) fail to utilize carbon of ketoglutaric acid and they are the only groups where fumaric acid is not utilized. The fermentation reaction of profile 4 (OX 19) is identical with that of P. vulgaris; as a matter of fact, Bergey1 calls it P. vulgaris. On the other hand, the fermentation reaction of profile 3 (acid and gas in dextrose) is identical with l'. morganii of profile 2. Profile 3 is also closely related to profile 2, but differs by occasionally changing the slant of Simmon's citrate to blue and constantly fails to utilize carbon in fumaric acid. As of the writing of this manuscript, we have identified from patients three organisms which fall in profile 5, of Proteus inconstans. They are Micalosa, D-Elia and Photidis. In urea citrate medium the slant is reel, and in Simmon's citrate the slant is blue. In Bacto urea medium, simple citrate medium and simple urea medium it does not grow; otherwise it does not have any other specific characteristic except for the fact that it has very poor fermentative reactions, producing acid or acid and gas in dextrose only. DISCUSSION
The general features of the genus Proteus are: swarming on solid media containing moisture, failure to utilize the carbon from succinic and pyruvic acids and failure to ferment lactose. On the basis of the splitting of urea and the utilization of the carbon from the citrate structure, the suggested new classification includes 5 groups with different profiles. Group 1, Proteus columbiensis, includes most of the clinical cul-
93
BACILLUS PROTEUS
tures isolated from patients, namely P. mirabilis, P. rettgeri and P. vulgaris. These organisms produce potent urease under aerobic and anaerobic conditions, and actively utilize carbon in the citrate radical. Although Bergey classifies P. OX 2 and P. OX 19 as P. vulgaris, according to this scheme, P. OX 19 should be in group 4. Groups 2, 3, 4, and 5 fail to utilize the carbon in citrate medium. Group 5 fails to split urea in Bacto urea medium, yet it does so in synthetic urea medium where there is no source of nitrogen in the form of proteins. In Bacto urea medium there is protein. Groups 2, 3, and 4 could be grouped together because of common features in Bacto urea and urea citrate, citrate and urea media. However, there are other basic differences in that group 3 is the only one which fails to utilize carbon in fumaric acid, and it may change the slant of Simmon's citrate medium to blue. Group 4 (OX 19) fails to utilize carbon in DL malic acid consistently. Thus P. OX 2 of profile 1 and P. OX 19 are similar. The value of this scheme is to simplify the work for the clinician and tell him about the properties of urea splitters and utilization of the carbon of the citrate molecule. We have already observed that organisms which are potent urease and citrase producers are drug resistant or acquire resistance quickly when exposed to chemotherapy. 3-10 Profile 1 is made up of potent urease and citrase producers and since most clinical 3 Lattimer, J. K., Seneca, H., Zinsser, H. H. and Troe, 0. K.: Increasing seriousness of resistant urinary tract infections with Aerobacter aerogenes. J.A.M.A., 170: 938, 1959. 4 Seneca, H., Zinsser, H. H. and Lattimer, J. K.: Relation of drug resistance to enzyme activity among coliform bacteria. J.A.M.A., 172: 1015,
1960.
5 Seneca, H., Lattimer, J. K. and Zinsser, H. H.: Chemotherapy of urease and citrase producing bacteria of the urinary tract. Ann. Intern. Med.,
63: 468, 1960. 6 Seneca, H., Lattimer, J. K. and Zinsser, H. H.: Chemotherapy of urinary tract infections, past and present. New York J. Med., 60: 3630, 1960. 7 Seneca, H., Lattimer, J. K., Peer, P. and Stuart, P.: Urease activity of sonic lysates of pathogenic bacteria Lancet, 2: 1166, 1961. 8 Seneca, H., Peer, P., Nally, R. and Stuart, P. F.: Relationship between drug susceptibility patterns among pathogenic Gram-positive cocci. Am. J. Med., 32: 56, 1962. 9 Lattimer, J. K., Seneca, H., Zinsser, H. H. and Donovan, J. T.: Drug resistant bacteria made drug susceptible by enzyme inhibitors. J.A.M.A.
178: 764, 1961. 10 Seneca, H., Peer, P. and Nally, R.: Microbial urease. Nature, 193: 1106, 1962.
cultures of Proteus fall in this group, the clinician has a tremendous problem in the management of P. columbiensis. In case the organism splits only urea, as is the case with profile 2 (P. morganii), the drug resistance pattern of this organism is definitely less serious than that of profile 1. In case the organism fails to split urea, as is the case with profile 5 (P. inconstans) the management should not be difficult. Additional data are necessary in this case. It is interesting to note that Proteus OX 2 and OX 19 were isolated in 1916 by Weil and Feli'C in Rumania. Since then they have been kept in cultures. Proteus OX K is a mutant of P. OX 19. This mutation took place in 1934 when Kingsbury was carrying the original strain of P. OX 19 to Malaya. They definitely differ from one another on the basis of antigenic structure, effect of Simmon's citrate, growth on L malic acid and fermentation reactions as well as indole production. P. OX 2 is a potent urease and citrase producer and is in profile 1. It is definitely more primitive than P. OX 19 and P. OX K. As previously reported, 4 these test tube Proteus strains are drug sensitive. It would be interesting if these organisms were exposed to increasing increments of antibiotics in vitro. Probably OX 2 will mutate and become very drug resistant, while the other two may be less mutagenic because they are less active in the production of citrase and urease. In this study we obtained Proteus X strains from the U.S. Navy, U.S. Army, U.S. Public Health Service, Food and Drug Administration and Mt. Sinai Hospital in New York. We have studied 5 strains of OX 19, five of OX 2 and four of OX K. The enzyme profile of OX 2 is uniform, but the other two are very variable concerning urease and citrase. The utilization of carbon in the other substrates is also very variable. Apparently, this is a very mutagenic group and that is the reason why Weil-Felix antigens should be prepared from a standard and constant strain of Proteus X strains otherwise the diagnosis of typhus group of fevers should be based on a complement fixation test, using rickettsial antigens. SUMMARY
On the basis of the urease and citrase production by Proteus, five different subgroups or "profiles" were observed: Profile 1, urease and citrase actively pro-
94
H. SENECA, P. PEER AND R. NALLY
duced by the general group "Proteus columbiensis", which includes P. vulgaris, P. mirabilis, P. rettgeri and P. OX 2. Profile 2, urease actively produced but no citrase activity, includes P. morganii. Profile 3, or Proteus OX K, urease produced best under optimum conditions, and citrase activity may be present in Simmon's citrate but not on synthetic citrate medium, and constantly fails to utilize carbon or fumaric acid. Profile 4, or Proteus OX 19, is similar to profile 3 except that citrase is never produced and fumaric carbon may or may not be utilized. Profile 5, includes P. inconstans, which fails to grow in Bacto urea medium, synthetic urea,
and synthetic citrate but grows in Simmon's citrate and urea citrate media. Most of the clinical strains are profile 1 or 2 or P. rettgeri, P. mirabilis, P. vulgaris or P. morganii. All Proteus strains fail to grow on succinic and pyruvic synthetic media. The carbon in ketoglutaric is very poorly utilized. They all utilize carbon of isocitric acid, despite the fact that only profile 1 utilizes citric acid. In the management of Proteus infections, the clinician should have at his disposal the urease and citrase profiles of this genus so that he can scientifically manage the control of this refractory organism.
Vol. 89, No. 1 January 1963 Copyright © 1963 by The Williams & Wilkins Co. Printed in U.S.A.
BACILLUS PROTEUS: CLINICAL LABORATORY CLASSIFICATION HARRY SENECA, P. PEER
AND
R. NALLY
From the Department of Urology, College of Physicians and Surgeons, Columbia University and the Presbyterian Hospital, New York, N. Y.
In Greek mythology Proteus was a sea god who had the power of assuming different forms or shapes. In English the word means a person or thing capable of taking on various aspects or characters. The protean nature of the genus Proteus justifies the name it was given by Hauser in 1885. In most clinical bacteriology laboratories and hospitals, the clinician and bacteriologist are not yet conditioned or indoctrinated into attempting to type or classify B. proteus in much detail. Both are justified in knowing or identifying it as such. The clinicians are not aware of the importance of knowing the various types, while the bacteriologists have lost their interest in this genus since it is no longer needed as a typing tool for Rickettsia. The importance of accurately classifying this bacterium is only now becoming apparent. Further investigations of the genus Proteus have been in progress at the Bacteriological Research Laboratories of the Department of Urology, Columbia University College of Physicians and Surgeons for the past 10 years.
be utilized as the sole source of carbon. Putrefactive odor is the characteristic of Proteus cultures. The antigenic composition of Proteus is of vital importance in rickettsial infections. The flagellar H antigen is inherent in the flagella while the somatic O antigen is located in the body (soma) of the organism. In the classical Weil Felix agglutination reaction Proteus OX 19, OX 2 and OX K are the non-motile variants. Agglutination with the H antigens or motile variants is specific for infection with Proteus but has no relationship to rickettsial infections. Bergey1 recognizes 5 types of Proteus. Those which hydrolyze urea are Proteus vulgaris, P. mirabilis, P. morganii and P. rettgeri. Proteus vulgaris is mannitol negative but produces acid and gas in dextrose, maltose and saccharose. Proteus mirabilis differs in failing to ferment maltose and is indole negative. In case acid or acid and gas are produced in mannitol and dextrose, it is P. rettgeri. Proteus inconstans does not hydrolyze urea. Proteus morganii is the only member which does not utilize carbon in citrate. P. OX 2 and OX 19, being maltose fermenters, are classified under the P. vulgaris species, while Proteus OX K, being a non-maltose fermenter, is classified as P. mirabilis.
CLASSICAL CHARACTERISTICS
Proteus is a gram-negative straight rod which is motile by means of peritrichous flagella. Proteus vulgaris and P. mirabilis produce ameboid colonies which manifest swarming on moist agar and gelatin or solid media devoid of bile salts. Pleomorphism is prominent in young, actively swarming cultures. Acid and gas are produced in glucose, fructose, galactose, maltose and sucrose, but there is no fermentation of lactose, mannitol or dextrin. Litmus milk is peptonized, indole is positive, acetylmethylcarbinol negative, hydrogen sulfide positive, nitrites are produced from nitrates, phenylpyruvic acid is produced from phenylalanine, leucine is rendered alkaline, urea is hydrolyzed (except by P. inconstans) and sodium citrate can
SUGGESTED NEW CLASSIFICATION
The present studies indicate that there is a simple, practical and quick procedure in typing Proteus. It is also of help to the clinician in planning chemotherapy. This study or investigation was evolved during the course of an investigation of the nature of resistance to chemotherapy and a study of mutagenicity among pathogenic bacteria which give rise to urinary tract infections. 1 Breed, R. S., Murray, E. G. D. and Smith, N. R.: Bergey's Manual of Determinative Bacteriology, 7th ed. Baltimore: The Williams and Wilkins Co., 1957.
Accepted for publication May 1, 1962. 89
90
H. SENECA, P. PEER AND R. NALLY MATERIAL AND METHODS
Preparation of media. 1. Bacto urea agar2 2. Bacto Simmon's citrate agar dehydrated (Difco) 2 3. Columbia Presbyterian urea citrate synthetic medium: Solution A Magnesium sulfate ............. Disodium phosphate. . . . . . . . . . . Monopotassium phosphate ...... Sodium chloride ................ Sodium citrate ................. Agar Noble ....................
0.2 gm. 1. 0 2.0 5.0 2.0 15 to 20
Boil in 960 ml. distilled water to dissolve, and sterilize in the autoclave at 15 lbs. for 15 minutes at 121C. Solution B Urea C.P ..................... 20.00 gm. Phenol red................ . . . 0.012 Distilled water. . . . . . . . . . . . . . 40. 0 ml. Dissolve and sterilize by filtering through Seitz. Cool the solution A to 45 degrees C. and add to it solution B under sterile conditions. Distribute in slants, 1 inch butt and 1.5 inches slant. Urea should be of pure quality and should not be autoclaved or overheated, otherwise it will be broken down. The final pH should be 6.8 and the color of the slants is no different from that of plain agar slants. In case the pH is not correct, or urea is split the color is pink, in which case the medium cannot be used. The butt should not be stabbed; the slant is streaked. The changes to be observed are growth, no growth and change of color of slant, or slant and butt to red. The readings are taken after 24, 48, and 72 hours and 1 week after incubation at 37C. 4. Synthetic citrate medium: Magnesium sulfate ............... Disodium phosphate . . . . . . . . . . . . . Monopotassium phosphate ....... Sodium chloride ................. Sodium citrate ................... Agar Noble ...................... Distilled water..... . . . . . . . . . . . . . .
0.2 gm. 1. 0 2.0 5.0 2.0 15 to 20 1000 ml.
2 Difeo Manual, Microbiological and Clinical Laboratory Procedures, Difeo Laboratories, Inc., Detroit, Mich., 1953.
Boil to dissolve, tube and autoclave, cool into slants. The final pH. is 6.8. Do not stab the butt; simply streak the slant. Incubate and observe growth. 5. Synthetic urea medium: Solution A has the same ingredients as solution A of the urea citrate medium minus sodium citrate. Solution B is the same as solution B of urea citrate medium. The remaining procedure is the same as for urea citrate. 6. The following substrates were also used as the sole source of carbon in the synthetic citrate medium, in place of sodium citrate: Sodium lactate D. L malic acid Ketoglutaric acid Isocitric acid Succinic acid Fumaric acid Sodium acetate Aconitic acid Pyruvic acid Oxalosuccinic acid Oxaloacetic acid Similarly these ingredients were used in the synthetic urea citrate medium in place of sodium citrate. This combination did not show any additional advantage over the plain carbon substrate and therefore it was not routinely used. Aconitic acid in synthetic urea medium failed to solidify and became black when the pH was adjusted. All the aforementioned media can also be prepared in liquid form by omitting agar. Since growth was difficult to determine in liquid medium, the liquid media were not routinely used. SOURCE OF PROTEUS CULTURES
Columbia-Presbyterian Medical Center, U. S. Army, U. S. Navy, Food and Drug Administration, Mount Sinai Hospital, in New York City; include both stock cultures and recent isolations from hospital patients. RESULTS
Table 1 shows the clinical classification of Proteus into 5 groups according to the enzyme patterns (profiles) in relation to the growth of the bacilli on synthetic media. Profile 1 includes P. vulgaris, P. mirabilis, P. rettgeri and P. OX 2. Proteus columbiensis
TABLE
_oo"' ,,~
.8 a'1 ~~
>'I
.I~ U)
"'
1. Growth of Bacillus proteus on various synthetic media and sugars u
u
~
E:
ed."'.::::
~u
-- -- -
"E:
6
"'
'·~
~
-
"' ~e .su ~ -0. "' .-'I"' ~
-
"'::-1"'
u
A
"
+
+
.-'I
::-1
.-'I
~ ~u
,,,,~ ·5u
0
>-<
~"' "'~
14
-- -- -
·o0
u
-~
::l
u
-~
~
.s" ~
~ fa< <" -- -- -- -- U)
u
"go·~ ·,: 1. ·s ea.s .,..[ 0 0 !:I
u 0
u
u
u
.. u
< -- -- -
1;i
t
1;i
"
."
ol
AG
0
l:J
00
2
0
·i::-1
"' ~ 00
5~
"Z"O
..g u
"' U) A .-'I ::-1 "' -- - - - -·-- - -
-
..:1
~<,::j
tl>,UJ::i ~
--
Profile 1
Proteus colurnbiensis a) P. vulgaris b) P. mirabilis c) P. rettgeri d) P. OX 2
"°,..._
+ SB + SB + SB + SB
+
s
+ SB + SB + SB + SB
+ SB
-
+
s
+
s
+
s
+
+ + +
+
s
+
s
+
s
-
-
+
-
+
+
+
+
+
-
-
AG
-
AG
+
+
AG
-
+
-
+
-
AG AG
+
+
+ +
-
+
+
-
+
-
+
+
+
+
+
-
-
AG
-
-
-
+ +
+
+
+
+
-
+
+
+
+
+
-
-
-
A AG
AG
-
+
+
+
+
-
+
-
s
+
-
+
-
+
+
-
+
+
-
+
+
+
+
+
-
-
A AG AG
+
-
+
+
+
+
+
-
-
AG
-
-
-
+
-
-
Profile 2
Proteus morganii
s
s
+
-
-
-
+
+
+
-
+
-
-
+
+
+
+
-
-
AG
-
-
-
-
+
-
-
+
-
+
+
+
+
+
-
-
AG
AG
-
AG
+
+
+
-
-
A AG
-
-
-
+
+
-
-
Profile 3
Proteus OXK
Profile
+ to SB
-
-
to
to
s
-
-
+
-
to
s
s
4
Proteus OX 19
+ to SB
-
-
+
-
-
to SB
-
+
-
to
-
-
+
+
s
-
-
Profile 5
Proteus inconstans
s
+
s
-
-
+
+
+
+
-
+
-
-1+
- means no growth;+ means growth; S means color change in slant and Bin slant and Butt A means acid production and AG acid and gas.
92
H. SENECA, P. PEER AND R. NALLY
includes those strains which produce urease under aerobic (slant) and anaeroic (butt) conditions and utilize carbon in the citrate molecule in Simmon's citrate where there is growth with change of color of the slant from green to blue (indicator bromthymol blue) and growth in plain citrate medium. In synthetic urea citrate medium and Bacto urea medium, there is no difference, thus indicating that urea is split both under optimum (bacto urea medium Difeo) and under starvation diet (synthetic urea medium). Urea is split under aerobic (red slant) and anaerobic conditions, manifested by red color (phenol red indicator) in slant and butt. In plain synthetic urea medium, urea is split only under aerobic conditions, manifested by red color in only the slant. P. columbiensis utilizes carbon in citric, isocitric, fumaric, acetic, oxalic, aconitic and oxalosuccinic, but fails to utilize pyruvic and succinic acids. Growth in media combining oxalacetic, lactic, DL and L malic and ketoglutaric acids is variable. P. vulgaris is the only member which cannot utilize oxaloacetic acid. Proteus OX 2 has the same fermentation reactions as P. vulgaris, namely, fermenting dextrose, maltose and saccharose with acid and gas production, but differs from the latter in utilizing oxaloacetic acid. Proteus OX 2, however, fails to utilize carbon in DL and L malic acids. The main feature of P. mirabilis is failure to utilize carbon in ketoglutaric acid, and producing acid and gas in dextrose and saccharose; it is indole negative. P. rettgeri has the characteristic of producing acid or acid and gas in dextrose and mannitol, and may produce acid and gas in saccharose. Its distinctive features are that it is H 2S negative and mannitol positive. Most of the clinical types of Proteus isolated from the patients at the Columbia-Presbyterian Medical Center are in this group and of the subtypes P. rettgeri and P. mirabilis. P. vulgaris is rarely isolated. Profile 2 is uniform and includes P. morganii. Under optimum conditions, it splits urea under aerobic and anaerobic conditions (in Bacto urea agar, the slant and butt are red). Under starvation conditions, in urea citrate medium and synthetic urea only the slant is red. In Simmon's citrate there is no growth. It utilizes carbon in lactic, isocitric, fumaric, acetic and aconitic
acids and fails to utilize it in citric, oxaloacetic, succinic and pyruvic acids. It may or may not utilize carbon in DL and L malic, ketoglutaric, oxalic and oxaloacetic acids. It produces acid and gas only in dextrose and is H 2S negative. It is fairly commonly isolated from clinical patients. Profile 4 includes Proteus OX K and Proteus OX 19. They may or may not split urea and fail to utilize carbon of the citrate molecule. On urea citrate medium Proteus OX K may change the color of the slant to red while Proteus OX 19 may change the color of slant and butt. Profile 4 may change the color of Simmon's citrate to blue. They have almost the same biological features except that profile 3 consistently utilizes carbon in L malic acid, while profile 4 fails to do so. Profile 3 (always) and profile 4 (may) fail to utilize carbon of ketoglutaric acid and they are the only groups where fumaric acid is not utilized. The fermentation reaction of profile 4 (OX 19) is identical with that of P. vulgaris; as a matter of fact, Bergey1 calls it P. vulgaris. On the other hand, the fermentation reaction of profile 3 (acid and gas in dextrose) is identical with l'. morganii of profile 2. Profile 3 is also closely related to profile 2, but differs by occasionally changing the slant of Simmon's citrate to blue and constantly fails to utilize carbon in fumaric acid. As of the writing of this manuscript, we have identified from patients three organisms which fall in profile 5, of Proteus inconstans. They are Micalosa, D-Elia and Photidis. In urea citrate medium the slant is reel, and in Simmon's citrate the slant is blue. In Bacto urea medium, simple citrate medium and simple urea medium it does not grow; otherwise it does not have any other specific characteristic except for the fact that it has very poor fermentative reactions, producing acid or acid and gas in dextrose only. DISCUSSION
The general features of the genus Proteus are: swarming on solid media containing moisture, failure to utilize the carbon from succinic and pyruvic acids and failure to ferment lactose. On the basis of the splitting of urea and the utilization of the carbon from the citrate structure, the suggested new classification includes 5 groups with different profiles. Group 1, Proteus columbiensis, includes most of the clinical cul-
93
BACILLUS PROTEUS
tures isolated from patients, namely P. mirabilis, P. rettgeri and P. vulgaris. These organisms produce potent urease under aerobic and anaerobic conditions, and actively utilize carbon in the citrate radical. Although Bergey classifies P. OX 2 and P. OX 19 as P. vulgaris, according to this scheme, P. OX 19 should be in group 4. Groups 2, 3, 4, and 5 fail to utilize the carbon in citrate medium. Group 5 fails to split urea in Bacto urea medium, yet it does so in synthetic urea medium where there is no source of nitrogen in the form of proteins. In Bacto urea medium there is protein. Groups 2, 3, and 4 could be grouped together because of common features in Bacto urea and urea citrate, citrate and urea media. However, there are other basic differences in that group 3 is the only one which fails to utilize carbon in fumaric acid, and it may change the slant of Simmon's citrate medium to blue. Group 4 (OX 19) fails to utilize carbon in DL malic acid consistently. Thus P. OX 2 of profile 1 and P. OX 19 are similar. The value of this scheme is to simplify the work for the clinician and tell him about the properties of urea splitters and utilization of the carbon of the citrate molecule. We have already observed that organisms which are potent urease and citrase producers are drug resistant or acquire resistance quickly when exposed to chemotherapy. 3-10 Profile 1 is made up of potent urease and citrase producers and since most clinical 3 Lattimer, J. K., Seneca, H., Zinsser, H. H. and Troe, 0. K.: Increasing seriousness of resistant urinary tract infections with Aerobacter aerogenes. J.A.M.A., 170: 938, 1959. 4 Seneca, H., Zinsser, H. H. and Lattimer, J. K.: Relation of drug resistance to enzyme activity among coliform bacteria. J.A.M.A., 172: 1015,
1960.
5 Seneca, H., Lattimer, J. K. and Zinsser, H. H.: Chemotherapy of urease and citrase producing bacteria of the urinary tract. Ann. Intern. Med.,
63: 468, 1960. 6 Seneca, H., Lattimer, J. K. and Zinsser, H. H.: Chemotherapy of urinary tract infections, past and present. New York J. Med., 60: 3630, 1960. 7 Seneca, H., Lattimer, J. K., Peer, P. and Stuart, P.: Urease activity of sonic lysates of pathogenic bacteria Lancet, 2: 1166, 1961. 8 Seneca, H., Peer, P., Nally, R. and Stuart, P. F.: Relationship between drug susceptibility patterns among pathogenic Gram-positive cocci. Am. J. Med., 32: 56, 1962. 9 Lattimer, J. K., Seneca, H., Zinsser, H. H. and Donovan, J. T.: Drug resistant bacteria made drug susceptible by enzyme inhibitors. J.A.M.A.
178: 764, 1961. 10 Seneca, H., Peer, P. and Nally, R.: Microbial urease. Nature, 193: 1106, 1962.
cultures of Proteus fall in this group, the clinician has a tremendous problem in the management of P. columbiensis. In case the organism splits only urea, as is the case with profile 2 (P. morganii), the drug resistance pattern of this organism is definitely less serious than that of profile 1. In case the organism fails to split urea, as is the case with profile 5 (P. inconstans) the management should not be difficult. Additional data are necessary in this case. It is interesting to note that Proteus OX 2 and OX 19 were isolated in 1916 by Weil and Feli'C in Rumania. Since then they have been kept in cultures. Proteus OX K is a mutant of P. OX 19. This mutation took place in 1934 when Kingsbury was carrying the original strain of P. OX 19 to Malaya. They definitely differ from one another on the basis of antigenic structure, effect of Simmon's citrate, growth on L malic acid and fermentation reactions as well as indole production. P. OX 2 is a potent urease and citrase producer and is in profile 1. It is definitely more primitive than P. OX 19 and P. OX K. As previously reported, 4 these test tube Proteus strains are drug sensitive. It would be interesting if these organisms were exposed to increasing increments of antibiotics in vitro. Probably OX 2 will mutate and become very drug resistant, while the other two may be less mutagenic because they are less active in the production of citrase and urease. In this study we obtained Proteus X strains from the U.S. Navy, U.S. Army, U.S. Public Health Service, Food and Drug Administration and Mt. Sinai Hospital in New York. We have studied 5 strains of OX 19, five of OX 2 and four of OX K. The enzyme profile of OX 2 is uniform, but the other two are very variable concerning urease and citrase. The utilization of carbon in the other substrates is also very variable. Apparently, this is a very mutagenic group and that is the reason why Weil-Felix antigens should be prepared from a standard and constant strain of Proteus X strains otherwise the diagnosis of typhus group of fevers should be based on a complement fixation test, using rickettsial antigens. SUMMARY
On the basis of the urease and citrase production by Proteus, five different subgroups or "profiles" were observed: Profile 1, urease and citrase actively pro-
94
H. SENECA, P. PEER AND R. NALLY
duced by the general group "Proteus columbiensis", which includes P. vulgaris, P. mirabilis, P. rettgeri and P. OX 2. Profile 2, urease actively produced but no citrase activity, includes P. morganii. Profile 3, or Proteus OX K, urease produced best under optimum conditions, and citrase activity may be present in Simmon's citrate but not on synthetic citrate medium, and constantly fails to utilize carbon or fumaric acid. Profile 4, or Proteus OX 19, is similar to profile 3 except that citrase is never produced and fumaric carbon may or may not be utilized. Profile 5, includes P. inconstans, which fails to grow in Bacto urea medium, synthetic urea,
and synthetic citrate but grows in Simmon's citrate and urea citrate media. Most of the clinical strains are profile 1 or 2 or P. rettgeri, P. mirabilis, P. vulgaris or P. morganii. All Proteus strains fail to grow on succinic and pyruvic synthetic media. The carbon in ketoglutaric is very poorly utilized. They all utilize carbon of isocitric acid, despite the fact that only profile 1 utilizes citric acid. In the management of Proteus infections, the clinician should have at his disposal the urease and citrase profiles of this genus so that he can scientifically manage the control of this refractory organism.