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Growth of Escherichia coli in Human Urine: Role of Salt Tolerance and Accumulation of Glycine Betaine Calvin M. Kunin, Tong Hua Hua, Laura Van Arsdale White, and Merna Villarejo
Department of Internal Medicine. Ohio State University, Columbus; Department ofBiochemistry and Biophysics. University ofCalifornia. Davis
Urine is a variable but generally good culture medium for bacteria. The rate and extent of microbial growth depend on the pH, tonicity, concentration of urea, and presence of dietary-derived organic acids [1-4], Urine also contains the osmoprotectant molecules glycine betaine and proline betaine [5, 6]. These substances accumulate within bacteria under conditions of osmotic stress and serve to counteract external osmotic forces [7, 8]. Glycine betaine is synthesized from choline by the renal inner medulla, and proline betaine is believed to be derived from the diet [5, 6, 9]. The presence of glycine betaine and proline betaine in urine may explain its osmoprotectant properties and the ability of Escherichia coli and other enteric bacteria to grow in urine under hypertonic conditions [10]. E. coli can also adapt to osmotic stress by accumulation ofpotassium [11, 12], synthesis of trehalose [13], and alteration of outer membrane protein composition [14-16]. In view of the multiple mechanisms by which bacteria are able to adapt to osmotic stress, it is difficult to determine which is most critical in permitting bacteria to grow in hypertonic urine. It is also possible that E. coli strains that cause urinary tract infections might be better adapted to resist osmotic stress than are other strains. The current study was designed to answer the following questions: Do clinical isolates of E. coli, obtained from patients with urinary tract infections, bacteremia, and gastroenteritis and from the stools of healthy subjects, differ in their ability to grow under conditions of osmotic stress? Do these
Received 23 June 1992; revised 28 July 1992. Financial support: Ohio State University. Reprints or correspondence: Dr. Calvin M. Kunin, Department of Medicine. Ohio State University. Starling Loving Hall Rm. Ml l O, 320 W. 10th Ave., Columbus. OH 43210-1240. The Journal of Infectious Diseases 1992;166:1311-5 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0015$01.00
isolates differ in their ability to respond to choline and glycine betaine? Are differences in osmotic tolerance and response to osmoprotectants among clinical isolates related to their ability to grow in hypertonic human urine? Can mutants of E. coli that are defective in the ability to accumulate glycine betaine grow in hypertonic urine? Two laboratory strains of E. coli were selected to help answer the fourth question. These mutants have specific genetic deletions that affect their ability to use choline, proline, and glycine betaine as exogenous osmoprotectants [17]. Strain MC41 00 can use glycine betaine and proline as osmoprotectants by uptake through the transport systems specified by proU and prol'. However, it cannot use choline because of a large deletion of the chromosome in the lac region that also deletes the bet genes responsible for the uptake of choline and its oxidation to glycine betaine. The mutant strain EF047, derived from MC41 00, carries additional mutations in the proU and proP loci that destroy the capacity to use glycine betaine or proline.
Materials and Methods E. coli strains. E. coli 31 (023:H IS) was isolated from a patient with a urinary tract infection. This strain was used in previous studies [5, 6, 10] as the standard to measure osmoprotectant activity, E. coli KID was provided by Linda Tombras Smith (University of California, Davis) [7]. E. coli ATCC 25922 was obtained from the American Type Culture Collection (Rockville, MD). E. coli MC4100 (bet) and mutant strain EF047 (bet putP proP proU) were obtained as described previously [17]. Urinary isolates were obtained from 10I young women with acute urinary tract infection, seen consecutively, at the Ohio State University Student Health Center. All urine specimens contained> 105 cfu of E. coli/mL. Blood culture isolates from 100 patients were provided by the clinical microbiology laboratories of the Ohio State University Hospitals and several
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on September 10, 2015
Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidneyand excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salttolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains wereexamined. Tolerance varied from 0.1 to 0.7 M NaCI (median, 0.5) in minimal medium. There wereno significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.
Table 1. Effect of 0.1 mM choline and glycine betaine on salt tolerance of control strains, urinary isolates, and mutant strains of Escherichia coli. Salt tolerance (M NaCl)* of strain grown in Strain
lID 1992; 166 (December)
Kunin et al.
1312
Minimal medium alone
Choline
Glycine betaine
0.4 0.4 0.6 0.1 0.6 0.2 0.5 0.6
0.7 0.8 0.8 0.8 0.9 0.4 0.5 0.6
0.9 0.8 0.9 0.9 1.0 0.4 0.9 0.6
fold dilutions were prepared in sterile distilled water. The specimens were inoculated with - 103 cfu/ml. of selected strains of E. coli that had been grown overnight in MM. The tubes were incubated at 37°C and were cultured quantitatively at intervals on trypticase soy agar plates.
Results KIO ATCC 25922 3]t
* Maximum concentration that permitted growth in minimal medium at 48 h. t Isolated from patients with urinary tract infections: 31 is standard strain used in previous studies [5, 6, 10): SSU and SRU are salt-sensitive and -resistant urinary isolates. t Enteropathogenic strain (salt-tolerant, heat stabile). 'Mutant strains that lack ability to synthesize or transport glycine betaine.
local community hospitals. Stool isolates were obtained from 100 healthy volunteers. They were collected by rectal swab, diluted in I mL of PBS, pH 7.2, and inoculated onto MacConkey agar plates. Isolates of enteropathic E. coli were provided by Richard Guerrant (University of Virginia, Charlottesville). Stock cultures, obtained from a single colony of each isolate, were stored at -40°C, after overnight growth, in Schaedler's broth containing 15% glycerin (Difco, Detroit). Media. Minimal medium (MM) [18] was composed of glucose, 2.0 g, dipotassium phosphate, 10.5 g. monopotassium phosphate, 4.5 g, sodium citrate, 0.5 g, magnesium sulfate, 0.264 g, and ammonium sulfate, 1.0 g, per liter. The pH was adjusted to 7.2 before use. The osmolarity was 231 rrrOsm/kg. The medium was supplemented with NaCI, in increments of 0.1 M. to assess salt tolerance. Growth conditions. A single colony was isolated on MacConkey agar plates and grown overnight at 37°C in MM. The inoculum consisted of 0.1 mL of a I:200 dilution of a 0.5 McFarland standard (final concentration, -5 X 105 cfu/rnl.). This was added to tubes containing 0.9 mL ofMM or to tubes of MM containing O.I-M stepped concentrations of NaCI (range, 0.1-1.0 M). Growth at 24 h was assessed visually for turbidity and at 48 h by measurement of optical density at 600 nm with a spectrophotometer (Spectronic 60 I; Milton Roy, Rochester, NY). The end point was the maximal concentration of NaCI at which there was 50% growth (absorbance of -0.5) compared with the density in tubes with MM alone. E. coli 31, KID, and ATCC 25922 were included as controls in each experiment. Collection of human urine. Maximally concentrated firstmorning-voided urine samples were obtained from healthy adult volunteers. The subjects had not taken fluids for the preceding 16 h. The osmolality was determined by the freezing point depression method at the clinical laboratories of the Ohio State University Hospitals. The urine was filter sterilized. Serial two-
50 o BLOOD (99) • URINE (85) • STOOL(96)
en 40 z
Growth of Escherichia coli in Human Urine: Role of Salt Tolerance and Accumulation of Glycine Betaine Calvin M. Kunin, Tong Hua Hua, Laura Van Arsdale White, and Merna Villarejo
Department of Internal Medicine. Ohio State University, Columbus; Department ofBiochemistry and Biophysics. University ofCalifornia. Davis
Urine is a variable but generally good culture medium for bacteria. The rate and extent of microbial growth depend on the pH, tonicity, concentration of urea, and presence of dietary-derived organic acids [1-4], Urine also contains the osmoprotectant molecules glycine betaine and proline betaine [5, 6]. These substances accumulate within bacteria under conditions of osmotic stress and serve to counteract external osmotic forces [7, 8]. Glycine betaine is synthesized from choline by the renal inner medulla, and proline betaine is believed to be derived from the diet [5, 6, 9]. The presence of glycine betaine and proline betaine in urine may explain its osmoprotectant properties and the ability of Escherichia coli and other enteric bacteria to grow in urine under hypertonic conditions [10]. E. coli can also adapt to osmotic stress by accumulation ofpotassium [11, 12], synthesis of trehalose [13], and alteration of outer membrane protein composition [14-16]. In view of the multiple mechanisms by which bacteria are able to adapt to osmotic stress, it is difficult to determine which is most critical in permitting bacteria to grow in hypertonic urine. It is also possible that E. coli strains that cause urinary tract infections might be better adapted to resist osmotic stress than are other strains. The current study was designed to answer the following questions: Do clinical isolates of E. coli, obtained from patients with urinary tract infections, bacteremia, and gastroenteritis and from the stools of healthy subjects, differ in their ability to grow under conditions of osmotic stress? Do these
Received 23 June 1992; revised 28 July 1992. Financial support: Ohio State University. Reprints or correspondence: Dr. Calvin M. Kunin, Department of Medicine. Ohio State University. Starling Loving Hall Rm. Ml l O, 320 W. 10th Ave., Columbus. OH 43210-1240. The Journal of Infectious Diseases 1992;166:1311-5 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6606-0015$01.00
isolates differ in their ability to respond to choline and glycine betaine? Are differences in osmotic tolerance and response to osmoprotectants among clinical isolates related to their ability to grow in hypertonic human urine? Can mutants of E. coli that are defective in the ability to accumulate glycine betaine grow in hypertonic urine? Two laboratory strains of E. coli were selected to help answer the fourth question. These mutants have specific genetic deletions that affect their ability to use choline, proline, and glycine betaine as exogenous osmoprotectants [17]. Strain MC41 00 can use glycine betaine and proline as osmoprotectants by uptake through the transport systems specified by proU and prol'. However, it cannot use choline because of a large deletion of the chromosome in the lac region that also deletes the bet genes responsible for the uptake of choline and its oxidation to glycine betaine. The mutant strain EF047, derived from MC41 00, carries additional mutations in the proU and proP loci that destroy the capacity to use glycine betaine or proline.
Materials and Methods E. coli strains. E. coli 31 (023:H IS) was isolated from a patient with a urinary tract infection. This strain was used in previous studies [5, 6, 10] as the standard to measure osmoprotectant activity, E. coli KID was provided by Linda Tombras Smith (University of California, Davis) [7]. E. coli ATCC 25922 was obtained from the American Type Culture Collection (Rockville, MD). E. coli MC4100 (bet) and mutant strain EF047 (bet putP proP proU) were obtained as described previously [17]. Urinary isolates were obtained from 10I young women with acute urinary tract infection, seen consecutively, at the Ohio State University Student Health Center. All urine specimens contained> 105 cfu of E. coli/mL. Blood culture isolates from 100 patients were provided by the clinical microbiology laboratories of the Ohio State University Hospitals and several
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on September 10, 2015
Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidneyand excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salttolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains wereexamined. Tolerance varied from 0.1 to 0.7 M NaCI (median, 0.5) in minimal medium. There wereno significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.
Table 1. Effect of 0.1 mM choline and glycine betaine on salt tolerance of control strains, urinary isolates, and mutant strains of Escherichia coli. Salt tolerance (M NaCl)* of strain grown in Strain
lID 1992; 166 (December)
Kunin et al.
1312
Minimal medium alone
Choline
Glycine betaine
0.4 0.4 0.6 0.1 0.6 0.2 0.5 0.6
0.7 0.8 0.8 0.8 0.9 0.4 0.5 0.6
0.9 0.8 0.9 0.9 1.0 0.4 0.9 0.6
fold dilutions were prepared in sterile distilled water. The specimens were inoculated with - 103 cfu/ml. of selected strains of E. coli that had been grown overnight in MM. The tubes were incubated at 37°C and were cultured quantitatively at intervals on trypticase soy agar plates.
Results KIO ATCC 25922 3]t
* Maximum concentration that permitted growth in minimal medium at 48 h. t Isolated from patients with urinary tract infections: 31 is standard strain used in previous studies [5, 6, 10): SSU and SRU are salt-sensitive and -resistant urinary isolates. t Enteropathogenic strain (salt-tolerant, heat stabile). 'Mutant strains that lack ability to synthesize or transport glycine betaine.
local community hospitals. Stool isolates were obtained from 100 healthy volunteers. They were collected by rectal swab, diluted in I mL of PBS, pH 7.2, and inoculated onto MacConkey agar plates. Isolates of enteropathic E. coli were provided by Richard Guerrant (University of Virginia, Charlottesville). Stock cultures, obtained from a single colony of each isolate, were stored at -40°C, after overnight growth, in Schaedler's broth containing 15% glycerin (Difco, Detroit). Media. Minimal medium (MM) [18] was composed of glucose, 2.0 g, dipotassium phosphate, 10.5 g. monopotassium phosphate, 4.5 g, sodium citrate, 0.5 g, magnesium sulfate, 0.264 g, and ammonium sulfate, 1.0 g, per liter. The pH was adjusted to 7.2 before use. The osmolarity was 231 rrrOsm/kg. The medium was supplemented with NaCI, in increments of 0.1 M. to assess salt tolerance. Growth conditions. A single colony was isolated on MacConkey agar plates and grown overnight at 37°C in MM. The inoculum consisted of 0.1 mL of a I:200 dilution of a 0.5 McFarland standard (final concentration, -5 X 105 cfu/rnl.). This was added to tubes containing 0.9 mL ofMM or to tubes of MM containing O.I-M stepped concentrations of NaCI (range, 0.1-1.0 M). Growth at 24 h was assessed visually for turbidity and at 48 h by measurement of optical density at 600 nm with a spectrophotometer (Spectronic 60 I; Milton Roy, Rochester, NY). The end point was the maximal concentration of NaCI at which there was 50% growth (absorbance of -0.5) compared with the density in tubes with MM alone. E. coli 31, KID, and ATCC 25922 were included as controls in each experiment. Collection of human urine. Maximally concentrated firstmorning-voided urine samples were obtained from healthy adult volunteers. The subjects had not taken fluids for the preceding 16 h. The osmolality was determined by the freezing point depression method at the clinical laboratories of the Ohio State University Hospitals. The urine was filter sterilized. Serial two-
50 o BLOOD (99) • URINE (85) • STOOL(96)
en 40 z
