Urea Utilization by Leptospira

Transcription

1 INFECTION AND IMMUNITY, OCt. 1974, p Vol. 10, No. 4 Copyright American Society for Microbiology Printed in U.S.A. Urea Utilization by Leptospira SOLOMON KADIS AND WILLIAM L. PUGH' Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia Received for publication 11 April 1974 One representative of each of five different pathogenic serotypes of Leptospira as well as one saprophytic strain were capable of growing on medium containing urea in place of an ammonium salt as a nitrogen source. Growth of all of the organisms tested on 1% urea was substantial, but only those that exhibited strong urease activity could grow to any appreciable extent on urea at a concentration as high as 2%. Intact urea-grown cells of the pathogenic serotypes tested (grippotyphosa and icterohaemorrhagiae) exhibited urease activity, with the level of activity of the former being considerably greater. No urease could be detected in cells of the saprophytic strain. When the pathogenic leptospires were sonicated or treated with toluene, the urease activity was greatly enhanced. When cultivated on NH4Cl, neither intact nor disrupted cells of any of the strains tested exhibited any urease activity. Cells of the grippotyphosa and icterohaemorrhagiae strains exhibited diauxic growth when cultivated in the presence of both NH4Cl and urea, whereas only monophasic growth could be detected for the saprophytic test strain. The experimental data on urea utilization and urease activity, when considered in the light of previously reported findings on leptospiral pathology, renal physiology, and the role of urease in other bacterial infections, suggests a significant role for leptospiral urease (in addition to other factors) in determining localization of the organism in the kidney and contributing to the resultant kidney pathology. There is ample evidence for the involvement of the kidneys during the various stages of leptospirosis in both man and animals. The acute infection is accompanied by renal malfunction, which is believed to be responsible for many of the clinical manifestations and possibly death of the host (1). During chronic leptospirosis, the spirochetes often localize in the lumen of the convoluted tubules, thereby establishing either a temporary or permanent carrier state (2). Nonspecific factors such as inhibition of phagocytic response by high salt and urea concentrations (9, 27, 29) and probably inactivation of complement by ammonia (3) contribute to the general susceptibility of kidney tissue to infection. However, studies with Corynebacterium renale (22, 23), Proteus mirabilis (4, 6, 24), and various other bacteria (5, 8, 15) have suggested that the possession of a highly active urease, which is associated with the rapid hydrolysis of urea and ability to utilize high concentrations of urea, appears to influence the persistence of each of these organisms in the kidney medulla of the respective hosts and the severity of the kidney damage inflicted. Previ- I Present address: Laboratory Unit, Georgia Department of Human Resources, Atlanta, Ga ous interest in urea utilization by Leptospira, on the other hand, has been restricted to two cursory references (17, 30). The former suggests that a good source of nitrogen for growth of Leptospira interrogans serotype pomona in serum may be urea that is present in adequate concentrations. The latter merely states that urea replaced (NH,2SO, as the nitrogen requirement for growth of serotype canicola in a synthetic medium. There is no indication in the literature that any attempt has been made to determine whether leptospires are capable of hydrolyzing urea enzymatically. The present study was undertaken to determine whether representatives of five different pathogenic leptospiral serotypes, as well as one saprophytic serotype, were capable of growing on urea as a nitrogen source and exhibiting urease activity, with a view towards eliciting some relationship between leptospiral utilization of urea in vitro and the pathogenesis of leptospirosis. It was envisaged that indications could be obtained as to whether growth on urea is directly related to urease activity and whether distinct differences in urea utilization could be detected between pathogenic and saprophytic organisms. (This paper is based on a thesis submitted by 79:3

2 794 KADIS AND PUGH W. L. Pugh to the graduate faculty of the University of Georgia in partial fullfillment of the requirements for the degree of Master of Science. Portions of the work were presented at the 73rd Annual Meeting of the American Society for Microbiology, 6-11 May 1973, Miami Beach, Fla.) MATERIALS AND METHODS Test organisms. The following organisms obtained from the Center for Disease Control, Atlanta, Ga., and received as cultures in Ellinghausen and McCullough liquid medium (14), were used in this study: L. interrogans serotype icterohaemorrhagiae strain co- *penhageni M20, serotype grippotyphosa strain Andaman, serotype pomona strain Pomona, serotype canicola strain Hond Utrecht IV, serotype hardjo strain hardjoprajitno, and L. biflexa strain LT430. All strains represent pathogenic serotypes, capable of initiating infection, except for L. biflexa LT430. This water isolate has never been shown to produce disease and thus was considered to be a saprophytic organism, and was used for comparative purposes. Measurement of growth. Growth studies were conducted in 10-ml volumes contained in optically matched glass tubes (19 by 150 mm) capped with stainless-steel closures. Cell turbidity was measured daily as change in percent light transmittance relative to uninoculated control tubes set at 100% with a Coleman model 14 spectrophotometer at a wave length of 630 nm. Cell counts were performed using a Petroff-Hauser counting chamber under dark-field illumination at a magnification of x970 by the procedure of VanEseltine and Staples (32). A standard curve was constructed relating cell number to percent light transmittance, which was found to be linear over the range used and valid for all strains used in this study. A reading of 85% light transmittance corresponded to 2.6 x 108 cells/ml. Cultivation conditions. All experiments were conducted with leptospiral cells cultivated in the bovine serum albumin-tween 80 medium of Ellinghausen and McCullough (14) with the addition of trace elements as described by Johnson and Harris (18). When urea was substituted for or included together with NH4Cl in the complete medium, solutions of ultra-pure urea (Schwarz-Mann) were filter-sterilized and added aseptically. For the initial experiments involving growth of leptospires on various concentrations of urea, unwashed inocula consisting of cells cultivated for 8 days in complete Ellinghausen and McCullough medium were used. Each inoculum contained approximately 5 x 101 to 1 x 106 cells/ml. These investigations were performed by serial transfers of a 10% inoculum weekly for a total of 15 transfers. The bacteria were incubated in stationary cultures under normal atmospheric conditions at 30 C. Studies concerning determination of urease activity requiring the use of relatively large numbers of cells were conducted by dispensing 225 ml of medium into 1-liter Erlenmeyer flasks. Ten percent inocula were used. Each inoculum was washed three times with INFECT. IMMUNITY growth medium devoid of a nitrogen source by centrifugation at 18,000 x g for 15 min. Washed inocula were used for all experiments subsequent to the determination of the ability of each leptospiral strain tested to grow on urea as a nitrogen source. Measurement of urease activity. The urease activity of leptospiral cells was measured by two different methods. The first of these entailed a modification of the procedure of Magana-Plaza and Ruiz-Herrera (25). A Coming model 12 ph meter with an expanded scale coupled to a Sargent model SRL recorder was used to give a system capable of detecting accurately changes in ph as small as 0.01 ph unit. The enzymatic activity was determined by following the increase of ph in a nonbuffered cell suspension or cell-free enzyme preparation resulting from the liberation of ammonia accompanying the hydrolysis of urea. Leptospiral cells were cultivated for 5 days in 1-liter flasks in media containing either 1% urea or 0.025% NH4Cl, harvested, and washed three times in deionized water by centrifugation. The net weight of the cells was determined and the pellet obtained after centrifugation was resuspended in sufficient water to give a cell concentration of 1 mg/ml. Dry weight determinations were also made on these cells after they were dried at 80 C for 24 h. Measurements of ph were made on 1 ml of a cell suspension contained in a test tube (8 by 50 mm). To this suspension, the ph of which ranged initially from 6.7 to 7.0, was added 1 ml of an aqueous solution of urea (24 umol). The change in ph was recorded continuously until a final ph had been established. The effect of disruption of the cell membranes on the expression of urease activity was determined in a similar manner. Cells were toluene-treated by adding 1 ml of toluene to 10 ml of cell suspension and shaking for 20 min at 37 C. Also, 10 ml of the cell suspension was subjected to sonication for 1 min at a power of 40 W in a Branson model W140D sonifier equipped with a microtip. This was done while maintaining the sonically treated material in a small beaker in an ice bath. Urease activity was measured with 1 ml of each of the treated suspensions and compared to that of jack bean urease (Sigma Chemical Co., type III) containing 1.6 U/mg. A unit of urease activity was defined as the Amoles of ammonia liberated per minute at 27 C. Measurement of urease activity was also performed spectrophotometrically by using a modification of the reduced nicotinamide adenine dinucleotide (NADH)- dependent coupled enzyme assay described previously (19, 20). This method is based on the ability to measure the decrease in the absorption of NADH due to its oxidation in the reaction by which glutamate is synthesized from a-ketoglutarate in the presence of NADH-.dependent glutamic acid dehydrogenase. Urease activity can be measured by this procedure because the ammonia liberated by the enzymatic hydrolysis of urea is necessary for this reaction to take place. A cell suspension containing approximately 100 mg (wet weight)/ml was sonicated as described previously and centrifuged at 4 C at 105,000 x g for 1 h in a

3 VOL. 10, 1974 Spinco model L preparative ultracentrifuge. The supernatant fluid was used for the enzyme assay. Jack bean urease served as the standard. The reaction was initiated by the addition of 0.2 ml of a urea solution containing 30,mol of this substrate in a total volume of 3 ml. Decrease in optical density at 340 nm was measured by using a Bausch and Lomb Spectronic 505 recording double-beam spectrophotometer. The reference cuvette contained the complete reaction mixture except for NADH. Continuous measurements were made until no further decrease in absorbancy occurred. RESULTS Growth on urea. All strains tested exhibited a similar response to growth on modified Ellinghausen and McCullough medium with urea as the nitrogen source. The initial studies were UREA UTILIZATION BY LEPTOSPIRA conducted with L. interrogans serotypes grippotyphosa and icterohaemorrhagiae and L. biflexa strain LT430. Transfers were made into media containing one of three different comparatively low concentrations of urea. After indications were obtained that the NH4Cl carried over from the inoculum was depleted and the cells could grow on these concentrations of urea, a substantially higher concentration was used. Cells of serotype grippotyphosa as well as those of serotype icterohemorrhagiae were transferred in media with 0.007, 0.014, and 0.028% urea for 11 serial transfers. Although limited growth was obtained in all three of these concentrations, it was observed that growth increment was a function of urea concentration. A typical adaptation to growth on urea is depicted for serotype grippotyphosa (Fig. 1). Growth after the flrst transfer was due primarily to the residual NH4Cl carried over from the stock cultures and not to a significant response to the urea. The amount of growth diminished over the next several serial transfers to the minimal level seen in transfer 5. Thereafter, a slight increase was noted, and the maximal level for growth on 0.028% urea was observed in transfer 10. A dramatic increment was noted in transfer 12 due to the substitution of a higher concentration of urea (1%). The growth continued to increase over the next two transfers and achieved the maximal level on 1% urea shown in transfer 15. Results similar to those just described were obtained with the strain of serotype icterohaemorrhagiae used in this study. Whereas appreciable growth was obtained with 1% urea for both of these strains, it should be noted that for these as well as other strains tested, the growth was always less when the organism was utilizing urea than when NH4Cl was supplied as the nitrogen source. 'x' 9r,iUIXO X 10p -J U 5x O8k 5X107 II TIME (DAYS) FIG. 1. Growth of the grippotyphosa strain on urea. Transfers 1 (0), 5 (V), and 10 (U) were performed on modified Ellinghausen and McCullough medium with 0.028% urea, whereas 12 (0) and 15 (A) were conducted in medium containing 1% urea. The control curve (0) refers to growth on medium with 0.025% NH4CI as the nitrogen source and each point represents the average value from 15 consecutive serial passages. It should be noted that a control consisting of leptospiral cells grown on the designated culture medium in the absence of either an ammonium salt or urea was included for each strain examined. The number of cells in these control cultures did not increase above that in the inoculum for any stage of any of the 15 serial transfers. The saprophytic test organism, L. biflexa strain LT430, was carried for six transfers on 0.007, 0.014, and 0.028% urea before 1% urea was substituted. 795 The growth response of this strain to 0.028% as well as 1% urea was somewhat similar to that of serotypes grippotyphosa and icterohaemorrhagiae. Upon depletion of NH4Cl from the medium, increases in cell numbers attributable to the utilization of urea supplied at a concentration of 0.028% were noted through transfer 6. Increasing the urea concentration to 1% resulted in a corresponding elevation of the growth response, which by transfer 11 had reached the maximal level obtained in transfer 15. The difference between growth on urea and that on NH4C1 was considerably greater for the strain of L. biflexa used than for any other strain tested.

4 796 KADIS AND PUGH Studies were conducted on the previously designated strains of serotypes pomona, canicola, and hardjo by transferring from the stock cultures maintained on medium containing NH,Cl directly into medium with 1% urea and thereafter subculturing on this same urea concentration. The response for all three strains was similar. Since growth after depletion of NH4Cl was much better for the pomona strain as well as the canicola and hardjo strains than for those initially cultured on 0.028% urea, it is likely that urea played a substantial role in the initial growth response. It should also be noted that the maximal growth levels for these strains were substantially higher than those observed for the grippotyphosa and icterohaemorrhagiae strains. Although differences in growth response to urea or NH,Cl as the nitrogen source depended on the leptospiral strain used, it was consistently observed that urea supported less growth than NH,Cl, even if the cells were initially subcultured into the optimal concentration of urea. The best growth on the modified Ellinghausen and McCullough medium containing NH4Cl was obtained with the strains of the pomona, hardjo, and icterohaemorrhagiae serotypes as well as with L. biflexa strain LT430. Although the canicola and grippotyphosa strains grew well in this medium, the cell yield was appreciably less than that obtained with the other four strains. When 1% urea was substituted for NH4Cl, the hardjo, pomona, and grippotyphosa strains exhibited the best response, whereas the maximal growth level for the icterohaemorrhagiae and canicola strains was slightly lower. L. biflexa displayed the lowest growth level on urea of any strain tested. Optimal concentration of the nitrogen source. Determinations of the optimal concentrations of NH,Cl or urea required for growth on modified Ellinghausen and McCullough medium were performed only with the designated strains of serotypes icterohaemorrhagiae and grippotyphosa as well as that of L. biflexa, and these experiments were initiated with washed inocula. As shown in Fig. 2, a broad range of NH4Cl concentrations (0.004 to 0.125%) would support optimal growth, and differences between the strains are depicted. Studies with urea-containing medium showed that the range of concentrations required to support growth was restricted as compared with NH4Cl (Fig. 3). These cells are apparently utilizing only a small fraction of the urea added for growth. It is interesting to note Q002 CONCENTRATION OF NH4CI (%) FIG. 2. Optimal concentration of NH4Cl for growth of the grippotyphosa and icterohaemorrhagiae strains and L. biflexa. All cells were cultivated on modified Ellinghausen and McCullough medium containing different concentrations of NH4CI. Turbidity was measured after day 5 of growth. Each point represents the average value of three determinations. -i w u ixio9r 5XIo8l- 5X lo7. /11 * I, / Q5 - BIFLEXA - ICTERO. GRIPPO. INFECT. IMMUNITY... \ 0.. ***0 *- -~. \ CONCENTRATION OF UREA (%) FIG. 3. Optimal concentrations of urea for growth of the grippotyphosa and icterohaemorrhagiae strains and L. biflexa. Experimental conditions were the same as those indicated in the legend for Fig. 2 except that varying concentrations of urea were substituted in the culture medium for NH4CI.

5 VOL. 10, 1974 UREA UTILIZATION BY LEPTOSPIRA 797 ixi 9r A. - 5XI08 I.A/..- _ A A. that of the three strains tested, the one belonging to serotype grippotyphosa demonstrated the least amount of growth at optimal NH4Cl concentrations, but the most favorable response to optimal urea concentrations. The situation for the saprophytic organism, L. biflexa, was completely reversed. The strain of grippotyphosa used, unlike those of icterohaemorrhagiae and L. biflexa, could grow on concentrations of urea as high as 4%. Growth of grippotyphosa on urea concentrations ranging from 0.5 to 2.0% was superior to that of the other two strains. The icterohaemorrhagiae strain grew reasonably well at the lower concentrations (0.032 to 0.25%), whereas the response of the grippotyphosa strain was by comparison rather poor. The range of urea concentrations that would support optimal growth was broader for L. biflexa than for the other two organisms tested, but it should be noted that the highest urea concentration at which these saprophytic cells would grow effectively was approximately 1%. Effect of ammonium salt on urea utilization. These experiments were performed to determine whether the presence of ammonium ion would alter the response of leptospires to growth on urea. It was observed that growth of cells of the icterohaemorrhagiae strain on medium containing 1% urea was stimulated slightly after the addition of or % NH,Cl (Fig. 4A). However, the growth levels did not reach that attained by the respective NH,Cl control cultures. All of these growth curves were monophasic. On the other hand, cells incubated in the presence of 0.1% urea plus 0.025% NH,Cl (Fig. 4B) exhibited a biphasic growth pattern. Growth on 2% urea was very low with or without the addition of any of the NH,Cl concentrations tested. The overall response of the grippotyphosa strain to growth on urea plus NH4Cl was similar to that of the icterohaemorrhagiae strain, but the concentrations of each nitrogen source required to elicit specific effects were quite different. Growth of grippotyphosa cells on medium containing 0.1% urea was enhanced by the addition of , 0.025, or 0.25% NH4Cl, but no biphasic pattern was elicited. Moreover, whereas growth in the presence of both 0.1% urea and NH,Cl followed the growth pattern of the respective NH4Cl controls, the cellular yields attained did not reach the levels observed for the respective NH4Cl control cultures. When incubated in medium containing both 1% urea and % NH4Cl, the grippotyphosa cells exhibited diauxic growth comparable to that observed for the icterohaemorrhagiae -i U, /~~~~~~ 5x o7 F 0.025% N %N... I% U 0.025%N %I#U, %N II !7 TIME (DAYS) FIG. 4. The effect of NH4CI on urea utilization by the icterohaemorrhagiae strain. All values are averages of duplicate cultures. N and U are the abbreviations for NH4CI and urea, respectively. strain. With either 0.25 or 0.025% NH4Cl in combination with 1% urea, no biphasic curve was noted. Growth on 2% urea with or without NH,Cl was appreciable but monophasic. The addition of NH4Cl did not alter the growth response on 2% urea. The failure to observe a diauxic effect in some of the experiments with each of the two pathogenic strains tested could have been due to the fact that the cells exhausted something else in the medium before exhausting the preferable nitrogen source, or the ratio of the two compounds investigated may be important; that is, in the case of 1% urea, % NH4Cl for the icterohaemorrhagiae strain, the urea concentration may be high enough to overcome any possible repression of urea catabolism by ammonia. For L. biflexa a quite different response was seen. Instead of a biphasic growth pattern, an additive effect was noted for some concentrations of urea and ammonium salt. The growth of cells cultured in medium containing 0.1% urea and 0.025% NH4CI exceeded that observed for either nitrogen source alone. The addition of 0.025% NH4Cl to 0.1% urea also resulted in a slight additive effect. This growth pattem was also observed when cells were cultured in the presence of 1% urea together with or 0.025% NH4Cl. In the presence of either 0.1 or 1% urea with 0.25% NH4C1, growth proceeded as if urea was not present. No appreciable increase in cell numbers was noted for cells cultured on 2% urea with or without the addition of NH4Cl.

6 798 KADIS AND PUGH Urease activity. When intact cells were used to measure the change in ph resulting from liberation of ammonia by the urease-catalyzed hydrolysis of urea, each strain tested exhibited a different degree of activity (Fig. 5). The increase in ph per milligram (dry weight) per unit of time exhibited by the grippotyphosa strain was approximately 10-fold greater than that elicited by the icterohaemorrhagiae strain. On the other hand, no increase in ph could be noted when intact L. biflexa cells were cultivated on 1% urea, washed free of culture medium, and incubated with urea for the purpose of the enzyme assay. Likewise, no significant urease activity could be detected for any of the three strains tested when these cells were cultured on medium with NH,Cl but no urea. When grippotyphosa cells were disrupted after treatment with toluene, the urease activity increased significantly during the first 3 min (Fig. 6). The activity of sonicated-cell suspensions was linear for approximately the same length of time but did not approach that observed with the toluene-treated preparations due perhaps to incomplete cell disruption. Neither intact nor disrupted NH,Cl-grown cells exhibited any urease activity at all. A comparison of urease activity for grippotyphosa, icterohaemorrhagiae, and L. biflexa whole and disrupted cells is depicted in Table 1. Whereas toluene treatment of grippotyphosa k 0.35 _ a 0.25 E I 0.20 k 0.15_ o01ok 0.05 /P GRIPPO0 */ 0/ / a-- V/ I/ I/ BIFLEXA.0**. 't.t A'.. A TIME (MINUTES) FIG. 5. Urease activity of intact cells of the icterohaemorrhagiae and grippotyphosa strains and L. biflexa. Change in ph was measured continuously and recorded automatically. Cells of each strain were cultivated on modified Ellinghausen and McCullough medium with 1% urea as the nitrogen source. 1.9r Ii1 a a 0.90 E I rp _ - Z _ *-- -e- *~ *-*o- *t*o- TOLI(UI #...&,.A A...A....& SON.(U) a- ~~~~~~~~~~~TOL.(N) WHOLE (U) 1o@anr n a " XWHOLE (N) t TIME (MINUTES) FIG. 6. Effect of membrane disruption on the urease activity of the grippotyphosa strain. Cells were cultivated in modified Ellinghausen and McCullough medium with either 0.025% NH4CI (N) or 1% urea (U) as the nitrogen source. Membrane dissociation achieved by treatment with toluene (tol) or sonication (son.). TABLE 1. Comparison of urease activity of the icterohaemorrhagiae and grippotyphosa strains and L. biflexa Strain A- - INFECT. IMMUNITY ApH per mg (dry weight) Nitrogen per min source for growth Whole Toluene- cls treated cells Grippotyphosa Urea Grippotyphosa NH4C Icterohaemorrhagiae Urea Icterohaemorrhagiae NH4C L. biflexa Urea L. biflexa NH4C cells resulted in a 10-fold increase in activity during the linear portion of the experiment (3 min) as compared with whole cells, the magnitude of the increase as well as the total activity for the icterohaemorrhagiae strain was considerably less. Neither L. biflexa urea-grown intact cells nor those subjected to membrane dissociation exhibited any sign of urease activity. Lack of enzymatic activity was also observed when cells of each of the three strains tested were grown on NH4Cl instead of urea. Leptospiral urease activity was compared to that of jack bean urease used as a standard. Toluene-treated icterohaemorrhagiae cells ex-

7 VOL. 10, 1974 UREA UTILIZATION BY LEPTOSPIRA 799 hibited activity comparable to that of jack bean urease at a concentration of 10 ug/ml; that is, U/mg (dry weight). The activity of similar preparations of the grippotyphosa strain was equivalent to a urease concentration of 100,qg/ml or 0.36 U/mg (dry weight). Urease activity of the grippotyphosa and icterohaemorrhagiae strains as well as L. biflexa was also determined by a NADH-dependent coupled enzyme assay using cell-free preparations. The results obtained with this procedure were very similar to those attained using the ph method. DISCUSSION All six leptospiral strains studied, including the saprophyte, were capable of growth on urea, but the level attained was lower than that achieved on NH4Cl. Since all of these strains could be cultivated continuously on urea with a consistent level of growth shown, it appears that urea can serve as a utilizable nitrogen source for these leptospires. Furthermore, since no growth was observed in control cultures containing modified Ellinghausen and McCullough medium without urea or NH4Cl, it is apparent that urea rather than other potential sources of nitrogen present in the culture medium such as bovine serum albumin is required for growth. A pattern of adaptation to growth on urea was observed for strains initially cultivated on minimal urea concentrations. Also, the presence of ammonium ion in the first transfer could conceivably delay the synthesis of urease. Our studies in which the growth of icterohaemorrhagiae and grippotyphosa strains in medium containing both urea and NH4Cl exhibited a diauxic response, together with experiments showing that no urease activity could be detected in NH,Cl-cultivated cells, could indicate that urea utilization commences only after ammonium ion, which represses urease synthesis, has been consumed. It should be emphasized, however, that these data only suggest that ammonium ion represses urease synthesis. The fact that cells cultivated on NH.Cl alone contained no urease is the expected result if this enzyme is inducible and urea is the inducer, but no definite conclusions can be reached about possible ammonium repression. Induction by urea and repression by ammonium is one hypothesis which is consistent with the data. Other models such as the one involving exclusion of urea from the cells are also possible at this stage in our studies. Investigations with a variety of bacterial organisms other than Leptospira have shown that the synthesis of urease in these cells is inducible and can be repressed by ammonium ion (11, 12, 25, 26; G. D. Vogels, Abstr. Meet. Neth. Soc. Microbiol. p. 225, 1966). Urease activity has been shown to be exhibited by strains of the pathogenic serotypes, icterohaemorrhagiae, and grippotyphosa, but not by the saprophyte, L. biflexa, even though the latter could utilize urea as a nitrogen source for growth. The activity of the grippotyphosa strain is much greater than that of the icterohaemorrhagiae strain. This is not surprising since the former can effectively utilize larger quantities of urea than the latter. Moreover, the portions of the renal tubule in which icterohaemorrhagiae is usually found (10) has a lower urea concentration (13) than the area in which grippotyphosa localizes predominately (16, 13). Thus, it may be advantageous for grippotyphosa to have the capability of hydrolyzing larger quantities of urea than icterohaemorrhagiae. Although it has been found that L. biflexa can utilize urea, no urease activity could be detected for this organism. No satisfactory explanation can be offered at this time for this apparent discrepancy. The role of the enzyme urease in bacteria other than Leptospira in the production of renal disease is well documented. Similarities can be noted between kidney infections initiated by Leptospira and those associated with other bacterial species. First of all, various pathogenic leptospiral serotypes localize predominately in the kidney during leptospirosis as do many other bacteria that are responsible for pyelonephritis. Secondly, there is some similarity between the overall renal pathology attributed to leptospiral infections and that associated with other types of bacterially induced renal disease. Since some of the bacteria capable of proliferation in the kidney medulla and causing renal damage (5, 6, 15, 22-24) possess urease, the demonstration of urease activity in representatives of at least two pathogenic leptospiral serotypes suggests a possible relationship between this enzymatic activity and the disease process itself. Studies on the role of urease in pyelonephritis caused by P. mirabilis have suggested that the alkalinity of the kidney tissues resulting from the presence of ammonia produced via the hydrolysis of urea may lead to local necrosis and the precipitation of salts in the renal tubule (6). The possession of an enzyme by leptospiral organisms allowing them to take advantage of in vivo growth conditions could also be a factor in producing a carrier state which, as a consequence of the shedding of leptospires in the urine, is of importance for the dissemination of the disease.

8 800 KADIS AND PUGH INFECT. IMMUNITY The results of experiments involving urea utilization and growth of leptospires can be correlated with previously reported findings from studies on renal physiology and leptospiral pathology. In the renal tubule the urea concentration is known to change due to the secretion of urea from the blood into the proximal convoluted tubule, addition and reabsorption of water, and finally loss of urea through the lower third of the collecting duct (13). The icterohaemorrhagiae and canicola serotypes have been reported to localize predominately in the proximal tubule and to a lesser extent in the distal tubule and collecting duct (10, 31). The concentration of urea that has been reported to be present in these areas (13) is well within the limits shown to promote good growth for selected strains of each of these two serotypes. The grippotyphosa and pomona serotypes are found more often in the interstitial spaces where the urea concentration is somewhat higher (7, 16, 21, 29). Again, this concentration is well within the range of urea concentrations found to support in vitro growth of these two organisms. In fact, as has been shown in this study, the grippotyphosa and pomona strains actuallv prefer a higher urea concentration than do those of icterohaemorrhagiae and canicola. ACKNOWLEDGMENTS We are grateful to Catherine B. Sulzer, Emmett B. Shotts, and James C. Feeley for their help in securing the cultures used in this study as well as to William P. VanEseltine and William L. Ragland for discussions of the manuscript. LITERATURE CITED 1. Alston, J. M., and J. C. Broom Leptospirosis in man and animals, p E. S. Livingstone, Ltd., Edinburgh and London. 2. Babudieri, B Animal reservoirs of leptospires. Ann. N.Y. Acad. Sci. 70: Beeson, P. B., and D. Rowley The anticomplementary effect of kidney tissue. Its association with ammonia production. J. Exp. Med. 110: Braude, A. I The role of bacterial urease in the pathogenesis of pyelonephritis, p In E. L. Quinn and E. H. Kass (ed.), Biology of pyelonephritis. Henry Ford Hospital, International Symposium, London. 5. Braude, A. I., A. P. Shapiro, and J. Siemienski Hematogenous pyelonephritis in rats. III. Relationship of bacterial species to the pathogenesis of acute pyelonephritis. J. Bacteriol. 77: Braude, A. I., and J. Siemienski Role of bacterial urease in experimental pyelonephritis. J. Bacteriol. 80: Burnstein, T., and J. A. Baker Leptospirosis in swine caused by Leptospira pomona. J. Infect. Dis. 84: Carroll, G., and R. V. Brennan The urea-splitting organisms in the formation of urinary calculi. J. Int. Coll. Surg. 17: Chernew, I., and A. I. Braude Depression of phagocytes by solutes in concentrations found in the kidney and urine. J. Clin. Invest. 41: DeBrito, T., E. Freymuller. D. 0. Penna, H. S. Santos, S. Soares De Almeida, P. A. Ayroza Galvao, and V. G. Pereira Electron microscopy of the biopsied kidney in human leptospirosis. Amer. J. Trop. Med. Hyg. 14: DeTurk, W. E The adaptive formation of urease by washed suspensions of Pseudomonas aeruginosa. J. Bacteriol. 70: DeTurk, W. E., and Bernheim, F The inhibition of enzy-ne induction and ammonia assimilation in Pseudomonas aeruginosa by sulfhydryl compounds and by cobalt, and its reversal by iron. Arch. Biochem. Biophys. 90: Dicker, S. E Mechanisms of urine concentration and dilution in mammals, p Williams & Wilkins Co., Baltimore, Md. 14. Ellinghausen, H. C., Jr., and W. G. McCullough Nutrition of Leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Amer. J. Vet. Res. 26: Gorrill, R. H., and S. J. DeNavasquez Experimental pyelonephritis in the mouse produced by Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. J. Pathol. Bacteriol. 87: Hanson, L. E., H. A. Reynolds, and L. B. Evans Leptospirosis in swine caused by serotype grippotyphosa. Amer. J. Vet. Res. 32: Johnson, R. C., and N. D. Gary Nutrition of Leptospira pomona. I. A chemically defined substitute for rabbit serum ultrafiltrate. J. Bacteriol. 83: Johnson, R. C., and V. G. Harris Differentiation of pathogenic and saprophytic leptospires. I. Growth at low temperatures. J. Bacteriol. 94: Kaltwasser, H., and H. G. Schlegel NADHdependent coupled enzyme assay for urease and other ammonia-producing systems. Anal. Biochem. 16: Konig, C., H. Kaltwasser, and H. G. Schlegel Measurement of urease activity. Arch. Mikrobiol. 53: Langham, R. F., E. V. Morse, and R. L. Morter Experimental leptospirosis. V. Pathology of Leptospira pomona infection in swine. Amer. J. Vet. Res. 19: Lister, A. J The kinetics of urease activity in Corynebacterium renale. J. Gen. Microbiol. 14: Lovell, R., and D. G. Harvey A preliminary study of ammonia production by Corynebacterium renale and some other pathogenic bacteria. J. Gen. Microbiol. 4: MacLaren, D. M The significance of urease in Proteus pyelonephritis: a bacteriological study. J. Pathol. Bacteriol. 96: Magana-Plaza, I., and J. Ruiz-Herrera Mechanisms of regulation of urease biosynthesis in Proteus rettgeri. J. Bacteriol. 93: Mehta, S. L., M. S. Naik, and N. B. Das Induced synthesis of urease in Azotobacter vinelandii. Ind. J. Biochem. 4: Miller, T. E., and J. D. K. North Effect of protein intake on bacterial growth in the kidney. Brit. J. Exp. Pathol. 47: Miller, N. G., and R. B. Wilson Electron microscopic study of the relationship of Leptospira pomona to the renal tubules of the hamster during acute and chronic leptospirosis. Amer. J. Vet. Res. 28: Rocha, N., and F. R. Fekety, Jr Acute inflammation in the renal cortex and medulla following thermal injury. J. Exp. Med. 119: Stalheim, 0. H. V., and J. B. Wilson Cultivation of

9 VOL. 10, 1974 UREA UTILIZATION BY LEPTOSPIRA 801 leptospirae. I. Nutrition of Leptospira canicola. J. Bacteriol. 88: Taylor, P. L., L. E. Hanson, and J. Simon Serologic, pathologic and immunologic features of experimentally induced leptospiral nephritis in dogs. Amer. J. Vet. Res. 31: VanEseltine, W. P., and S. A. Staples Nutritional requirements of leptospirae. I. Studies on oleic acid as a growth factor for a strain of Leptospira pomona. J. Infect. Dis. 108: