Folia Microbiol. 40 (5), 547-550 (1995)

Constituents of Human Urine Alter Siderophore Production of Escherichia coli S. SHARMA a*, N. PURl a and R. GUPTA b

aDepartment of Microbiology, Panjab University, Chandigarh - 160 014, India bDepartment of Microbiology, Government Medical College, Chandigarh - 160014, India

~,caved Yuly 27, 1~4 Revised version November 28, 1994

ABSTRACT. Four uroisolates and four faecal isolates of Escher/ch/a coli were randomly selected for studying the siderophore production in an iron-deficient, chemically defined, medium. G I ~ , lactose, urea and creatinine were added individually, as well as in combination, to estimate their influence on siderophore production. No difference in siderophore production was observed between stool and urinary isolates of E. co//. Alterations in phenolate production were observed to be constituent- dependent while a uniformly significant increase in hydmxamate production (p < 0.05) was recorded after addition of the con- stituents, either each individually or in combination.

To procure the iron required by microorganisms for their metabolic activities, they are now known to produce siderophores (phenolates and hydroxamates), which help them to sequester iron from the environment and the host (Hartman and Braun 1981). In the case of E. coli, phenolates are synthesized by most strains (de Lorenzo and Martinez 1988) while only a few uropathogenic strains of E. coli elaborate aerobactin, this being now considered an important virulence property. Aerobactin was found to be superior to enterochelin in stimulating growth because, in contrast to enterochelin, aerobactin seemed not to be hydrolyzed or modified during delivery of iron to the cell (Carbonetti and Williams 1984). The role of iron in regulating siderophore production is well known (Williams and Carbonetti 1986). However, little is known about the precise effect of constituents of human urine on virulence properties of urinary tract infection (UTI) pathogens. In the present investigation we report on the influence of glucose, lactose, urea and creatinine on siderophore production.

MATERIALS A N D METHODS

Bacterial strains. Four uroisolates of 17,. coli from patients with urinary tract infections and four faecal isolates of E. coli from healthy individuals were used in the study. All these organisms were identified as E. coli according to Bergey's Manual of Determinative Bacteriology and serotyped at National Salmonella and E. coli Typing Centre, Kasauli (India). These strains were maintained on nutrient agar (pH 7.4, 4 ~ and subjected to minimum number of passages before carrying out the experiments. The organisms were grown overnight in nutrient broth before inoculating into the test medium. Three passages were given to each strain in Fe-CDM (with or without additional constituents) before estimating the siderophores.

Growth medium. All strains were grown in an iron-deficient, chemically defined (Fe-CDM), medium (Geoffrey et al. 1985). Fe-CDM consists of a basal medium which is made up of (mmol/L): NH4CI 25, glucose 35, KCI 1.5, MgSO4 0.4, NaCI 0.045, NaeHPO4 66, NaH2PO4 86. To this basal medium (1 L) 100 ~tL of microdiluent solution (in nmol/L: CaCI2 500, HBO3 500, COC12 50, CaSO4 10, ZnSO4 10, MnSO4 100) were added. Glucose, lactose, urea and creatinine were added individually in three different concentrations. The concentration of each constituent which showed maximum hydrox- amate production was chosen for studying the combined effect of the constituents. Glucose and lactose in one set of experiments and urea and creatinine in another were added together.

Estimation of siderophores. Cell-free supernatants of the third-passage cultures from the medium (with and without additional substances) were used for siderophore estimation (Sharma et al. 1991). For hydroxamate estimation, the method of Gibson and Magrath (1959) and for phenolates, the method of Arnow (1937) was followed as employed by Harjal et al. (1990).

Statistical analysis. The F-test was applied to establish whether the difference in the means of samples in more than two groups was statistically significant or not.

*Corresponding author.

S. S H A R M A et al. Vol. 40

R E S U L T S

Table I. shows the phenolate and hydroxamate production in a chemically defined iron- deficient medium (Fe-CDM) and Fe-CDM supplemented with glucose and lactose. Glucose (0.5 %) siL, nificantly increased (d7 < 0.05) the production of phenolates while hydroxamate production was reduced significantly (p < 0.05). Further increase in the concentration of glucose did not alter the pro- duction of phenolates but hydroxamates increased in a dose-related manner. However, lactose (0.5 %) significantly enhanced the production of both phenolates and hydroxamates. This increase was inde- pendent of the concentration of lactose for phenolates but in the case of hydroxamates again a concentration-dependent increase was observable.

Table L Effect of glucoc~ and lactose on siderophore production by urinary and faecal strains of E. colt s,b

Medium supplemented with

Without

Strains supplement Glucoc, e (%) Lactose (%)

03 1 2 03 1 2

Phenolates, A515 x 103

Urinary 47 • 5 78 • 7* 71 -+ 8* 65 • 8* 98 _+ 6* 83 _ 3* 114 _* 16"

Faecal 46 -+ 8 72 _+ 9* 65 • 5* 56 • 1" 88 • 9* 80 • 7* 101 • 8*

Hydroxamate, A526 x 103

Urinary 367 • 70 212 • 75* 535 • 1780 + 81" 1190 • 133" 1350 • 92* 1510 • 28*

Faecal 381 _+ 91 203 • 26* 402 • 15"** 1790 • 82* 1060 • 373* 1400 • 50* 1620 • 158

aMean -+ SD of the values obtained for four urinary or four faecal strains. bp-values fort-test: *p < 0.005; "*p < 0.05; ***p Z 0.05.

Table 11. Effect of urea and creatinine on siderophorr production by urinary and faecal strains of E. coil a'b

Medium supplemented with

Without

Strains supplement Urea (%) Creatinine (%)

1.25 5 10 0.075 0.6 1.2

Phenolates, As15 x 103

Urinary 47 • 5 83 • 9* 80 • 11" 85 • 12" 37 _+ 7** 61 • 6*** 14 + 3* Faecal 46 • 8 86 __. 6* 68 • 9* 71 • 4* 35 • 6** 52 • 13"** 10 • 4*

Hydroxamate, A526 x 103

Urinary 367 • 70 1050 _+ 226* 1850 _+ 94" 1970 • 41" 455 • 81"* 214 • 71" 811 • 77*

Faecal 381 _+ 91 976 _+ 156" 1810 • 201" 1950 • 33* 424 • 95** 290 • 70* 664 _+ 41"

aMean -+ SD of the values obtained for four urinary or four faecal strains. bp-values for t-test: *p < 0.005; **p < 0.01; ***p < 0.025.

Table II shows the effect of different concentrations of urea and creatinine on phenolate and hydroxamate production. Both too low (0.075 %) and too high (1.2 %) concentrations of creatinine were found to reduce significantly (p < 0.05 and p < 0.005, respectively) the amount of phenolates and hydroxamates. The optimum concentration (0.6 %) of creatinine significantly enhanced the production of both siderophores. Increase in production of phenolates with increasing concentration of urea was found to be random but was significant (p < 0.005) at all the concentrations examined. In contrast, the

1995 SIDEROPIIORE PRODUCTION OF E. coli 549

increase in hydroxamate production was concentration-dependent. Urea, however, enhanced the pro- duction of both phenolates and hydroxamates to a greater extent than did creatinine.

Table III shows the effect of Table IlL Siderophore production by urinary (U) and f~eal (F) isolated of E. coli in Fe-CDM medium without and with supplementation

Strain Without supplement

Supplemented with

lactose (2 %) urea (10 %) glucose (2 %) creatinine (1.2 %)

Phenolates, ASl 5 x 103

1U 57 60 61 2U 50 60 61 3U 34 43 61 4U 44 64 59 1F 51 57 60 2F 56 58 61 3F 55 52 63 4F 34 57 55

Hydroxamates, A526 x 103

1U 490 2000 680 2U 280 1800 610 3U 375 1890 630 4U 305 1800 630 1F 490 1450 540 2F 280 1800 580 3F 375 1800 610 4F 375 1820 610

these substances when added in combination, i.e. urea + creatinine and glucose + lactose. For the majority of strains, the phenolate production is lowest in the medium alone and highest in medium + 10 % urea + 1.2 % creatinine. On the other hand, for all the strains, the hydroxamate production is low- est in an iron-deficient medium (Fe-CDM), followed by Fe-CDM + 10% urea + 1.2 % creatinine and is highest in Fe-CDM con- taining 2 % lactose + 2 % glucose. The increase in hydroxamate pro- duction following combined addi- tion of glucose and lactose, as well as urea and creatinine, was significant (/9 < 0.05) in comparison to Fe-CDM alone.

DISCUSSION

Hydroxamate production is now recognized to be one of the virulence factors of uropathogenic E. coli (de Lorenzo and Martinez

1988). Neilands (1982) reported that pathogenic species when present in host tissues are derepressed with respect to iron- and high-affinity siderophore-mediated pathway which is functional. Urine is known to provide iron-limiting conditions and can, therefore, influence bacterial iron uptake systems. There are only few data showing the effect of growth media including urine on virulence properties of uropathogens. In an earlier study carried out in this laix)ratory we found that E. coli strains show enhanced production of siderophores when grown in normal human urine in comparison to trypticase soy broth (Sharma et al. 1991) and that urine-grown bacteria are more virulent in the mouse model of pyelonephritis. In the present investigation we used an iron-depleted medium to mimic the milieu of the iron-limiting environment which is provided by urine, in order to see the contribution of urinary constituents on siderophore production by E. co i l

Diabetic patients are more prone to UTI. The exact mechanism leading to enhanced multipli- cation of bacteria in these patients is not known. It has been reported that higher incidence and severity of UTI in diabetes may rather be due to the effect of metabolic status via other mechanisms and not due to altered adhesive ability (Raffel et al. 1981). However, we observed a greater enhancement in hydroxamate production by lactose than by glucose. Andriole (1987) reported that pregnancy is one of the major intrinsic risk factors leading to UTI, while Stamm et al. (1989) reported that both maternal and infant morbidity may occur in women who suffer from UTI during pregnancy. The role of physical factors as contributors to higher incidence of UTI in pregnant females is understandable. Pregnant females, excreting lactose in urine, could thus have enhanced siderophore production helping growth and establishment of uropathogens and this can contribute toward the process of infection.

Addition of urea enhanced siderophore production quite significantly but enhancement by urea plus creatinine was much less. In clinical situations, a rise in urea and creatinine in urine often go together, but in some situations a disproportionate rise in urea or creatinine can be observed. Urea and creatinine have been shown to increase the adhesion of E. coli to uroepithelial cells in vitro (Funfstuck et al. 1987) and a high concentration of urea inhibits phagocytosis of E. coli and staphylococci by leuko- cytes while this effect is less marked with creatinine (Chernew and Braude 1962).

5 5 0 S. S H A R M A et al. Vol. 40

While enterochelin is uniformly produced by almost all strains of E. coli, hydroxamate, recog- nized as a virulence factor in uropathogenic strains of E. coli, are produced by a few strains (deLorenzo and Martinez 1988). The enhancement observed in constituents individually as well as in combination enables the organism to sequester iron from the iron-limiting environment of the urinary tract. Though urinary isolates studied here do not belong to known uropathogenic serotypes, they show alterations in aerobactin production in the different experimental conditions studied. Therefore, it should be kept in mind that once the strain, endowed with a genetic make-up for aerobactin, gains entry into the urinary tract, will be influenced by environmental conditions prevailing there. It may then get derepressed for aerobactin production and contribute to the infective ability of an organism depending on the host's condition.

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