Vol. 29, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1991, p. 1078-1080
0095-1137/91/051078-03$02.00/0 Copyright © 1991, American Society for Microbiology
Survival of Feline Mycoplasmas in Urinet MARY B. BROWN,'* MARGARET STOLL,' JEFF MAXWELL,' AND DAVID F. SENIOR2 Department of Infectious Diseases' and Department of Small Animal Clinical Sciences,2 College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611-0633 Received 5 December 1990/Accepted 15 February 1991
The effects of length of incubation and urine osmolality on the survival of feline mycoplasmas and ureaplasmas and representative gram-positive and gram-negative bacteria in synthetic urine which approximated the osmolality of normal cat urine were investigated. Both Escherichia coli and Staphylococcus aureus withstood the effects of increasing osmotic pressure. In the most concentrated urine, significant decreases (P < 0.001) in CFU were observed for E. coli at exposure times of 30 min and longer. S. aureus was not affected by longer exposure or increased osmotic strength. Both Mycoplasma felis and Mycoplasma gateae were affected adversely by longer exposure times and high osmotic strength (P < 0.001). A Ureaplasma sp. was not adversely affected except at very high (.2,980 mosM) osmotic strengths or after prolonged incubation (120 min) at relatively high (1,976 mosM) osmotic strengths (P < 0.001). The failure of both M. felis and M. gateae to survive under osmotic conditions present in normal feline urine suggests that it is unlikely that these mycoplasmas are involved in urinary disorders in cats.
Mycoplasmas and ureaplasmas have been implicated in urinary tract disorders in either natural or experimental infections in a number of hosts, including humans, rodents, sheep, and dogs (3, 12, 13, 18). Because of the common occurrence of lower urinary tract infection in cats (1, 21) and frequent colonization of cats by mycoplasmas (2, 7, 14, 19), it was of interest to determine the relationship between lower urinary tract infection and mycoplasmas in the urine. However, cats generally produce very concentrated urine, with mean normal values of 2,270 + 407 mosM (11, 15). In cats with urinary tract disease, even greater concentrations might occur. Mycoplasmas lack a cell wall, thus rendering the cells at risk for osmotic damage. The ability of the mycoplasmas to survive under osmotic conditions encountered in feline urine would be relevant to any studies which attempt their isolation from the urine. The goal of the present study was to compare the survival of feline mycoplasmas and ureaplasmas with that of representative gram-positive and gram-negative bacteria in synthetic urine which approximated the physiological osmolality of mycoplasmas as well as the osmotic range observed in normal cat urine. Microorganisms. Clinical isolates of Mycoplasma felis UF26, Mycoplasma gateae UF-JF1, and Ureaplasma sp. strain UF-LR2 were obtained from urogenital swabs of cats presented to the University of Florida Veterinary Medical Teaching Hospital. Informed consent was obtained from all pet owners. M. felis and M. gateae were confirmed by an immunobinding assay as previously described (3). The Ureaplasma sp. was identified by urease reaction and colony morphology on A8 differential agar (16). Clinical feline isolates of Escherichia coli UF-GG1 and Staphylococcus aureus UF-F7 were obtained from the clinical microbiology laboratory of the University of Florida Veterinary Medical Teaching Hospital. Synthetic urine. Synthetic urine (lx) contained 0.065 g of CaCl2 2H20, 0.065 g of MgCl2 6H20, 0.46 g of NaCl, 0.23
g of Na2SO4, 0.065 g of sodium citrate, 0.002 g of sodium oxalate, 0.28 g of KH2PO4, 0.16 g of KCl, 0.1 g of NH4Cl, 2.5 g of urea, and 0.11 g of creatine in a final volume of 100 ml, pH 5.7. Preparations of 2x, 3 x, 5 x, and 10x synthetic urine were done by appropriate adjustment of the salts with one exception. For 5 x and 10x urine, the CaCI2 was kept at 0.26 g because of solubility problems. All urine preparations were sterilized by passage through a 0.2-p.m filter. The milliosmolalities of the synthetic urine solutions were determined by freezing point depression by using an Osmette A automatic osmometer (Precision Systems, Sudbury, Mass.). The milliosmolalities of the urine preparations were as follows: 1 x, 671 mosM; 2x, 1,340 mosM; 3x, 1,976 mosM; 5x, 2,980 mosM; and 1Ox, 5,976 mosM. Survival assay. All samples were tested in duplicate, and the experiment was repeated on three separate occasions. M. felis and M. gateae were grown overnight in SP4 broth (20), the Ureaplasma sp. was grown overnight in 10B broth (17), and E. coli and S. aureus were grown overnight in brain heart infusion broth. A CFU determination was performed on each overnight culture, and 1-ml aliquots were transferred to sterile Eppendorf tubes. The microorganisms were centrifuged at maximum speed for 10 min in an Eppendorf centrifuge. The supernatant was removed, and the cell pellet was resuspended in 1 ml of synthetic urine or phosphatebuffered saline (PBS). Tubes were placed in a 37°C water bath. Immediately, 0.12 ml was withdrawn, serially diluted, and plated in duplicate on SP4 (M. felis and M. gateae), A8 (the Ureaplasma sp.), or brain heart infusion (E. coli and S. aureus) agar for CFU determination. The elapsed time between the time of resuspension, dilution, and plating was 3 min. Therefore, the initial CFU prior to centrifugation was used as the zero time point. Additional CFU determinations were made after 30, 60, and 120 min of incubation. It was felt that these time points would reflect the potential intervals between sample collection and culture of clinical urine
samples. Statistical analysis. The effects of time of incubation and urine osmolality on the survival of the microorganisms were analyzed by analysis of variance by using the Statview computer program for the MacIntosh computer. A P value of c0.05 was accepted as significant.
Corresponding author. t Journal series article no. R-01372 of the Florida Agricultural Experiment Station. *
0.' E _ _
0 o LAI
* O min
3 min 30 min o 60 min 0] 120 min
* Omin Ea 3 min M 30 min Eo 60 min El 120 min
4 E1) _ b
FIG. 1. Survival of E. coli (top) and S. aureus (bottom) in synthetic urine. Microorganisms were incubated in PBS (300 mosM) or in 671 mosM (lx), 1,340 mosM (2x), 1,976 mosM (3x), 2,980 mosM (5x), or 5,976 mosM (10x) synthetic urine for 0, 3, 30, 60, and 120 min.
Both E. coli and S. aureus withstood the effects of increasing osmotic pressure (Fig. 1). In the most concentrated urine (lOx), significant decreases (P < 0.001) in CFU were observed for E. coli at exposure times of 30 min and longer. Similar decreases were not observed for S. aureus. Results obtained with M. felis, M. gateae, and the Ureaplasma sp. (Fig. 2) were markedly different from results obtained for E. coli and S. aureus (Fig. 1). Both M. felis and M. gateae were affected adversely by both increased time of exposure and increased osmotic strength (P < 0.001). M. gateae maintained viability in PBS for the duration of the experiment; however, after 30 min of exposure to 1 x urine, the viability began to decline and dropped precipitously with additional exposure. Increased osmotic strength resulted in decreased survival, and effectively no survival was observed after 30 min at osmotic strengths greater than 671 mosM. Although similar trends were observed for M. felis, the
FIG. 2. Survival of M. felis (top), M. gateae (center), and the Ureaplasma sp. (bottom) in synthetic urine. Microorganisms were incubated in PBS (300 mosM) or in 671 mosM (lx), 1,340 mosM (2x), 1,976 mosM (3x), 2,980 mosM (5x), or 5,976 mosM (10x) synthetic urine for 0, 3, 30, 60, and 120 min.
J. CLIN. MICROBIOL.
effects were less severe than those for M. gateae. Although marked decreases in viability occurred with increased exposure time and increased osmotic strength, M. felis was able to tolerate slightly greater osmotic strengths. Effectively, no survival was observed after 30 min at 1,976 mosM. Surprisingly, the Ureaplasma sp. was not adversely affected except at very high (.2,980 mosM) osmotic strengths or after prolonged incubation (120 min) at relatively high (1,976 mosM) osmotic strengths (P < 0.001). In fact, the viability patterns for the Ureaplasma sp. more closely resembled those of the gram-negative E. coli than those of M. felis and M. gateae. The rigid bacterial cell wall can withstand pressures, which renders it relatively resistant to hypoosmotic shock, but it is still affected by hyperosmotic conditions (8-10). Mycoplasmas, which lack a cell wall, do not have the added protection afforded to gram-positive and gram-negative bacteria. Therefore, it was not surprising that M. felis and M. gateae were deleteriously affected by exposure to hyperosmotic conditions. The relative resistance of the Ureaplasma sp. to the same conditions was an unexpected finding. Ureaplasmas use urea as a carbon source and may be isolated from the urinary tracts of a variety of hosts (5, 12, 13, 18). It is possible that specific transport mechanisms are present which allow ureaplasmas to adjust more readily to alterations in osmotic pressure. In many bacteria, the mechanism for resistance to osmotic damage is based on selective accumulation of compatible solutes, which counteracts the spontaneous loss of water (6, 8). Although it has yet to be determined, it is possible that ureaplasmas have improved mechanisms for concentration or transport of these osmoprotective compounds. It is possible that growth of M. felis and M. gateae in vitro resulted in the failure of the microorganisms to produce proteins which might be osmoprotective under in vivo conditions. However, we do not consider that this is a likely explanation because of the failure to isolate these microorganisms from feline urine, even when the urogenital tract is colonized (4). We have been able to isolate both E. coli and Ureaplasma spp. from urine samples from a limited number of cats (4). The failure of both M. felis and M. gateae to survive under osmotic conditions present in normal feline urine suggests that it is unlikely that these mycoplasmas are involved in urinary disorders in cats. Ureaplasmas, on the other hand, were capable of surviving under conditions similar to those encountered in feline urine. In view of the association of ureaplasmas with urinary tract disorders in other hosts and their ability to survive under osmotic conditions comparable to those in normal feline urine, these microorganisms should be considered as potential pathogens in feline urinary disorders. We thank Debbie Sundstrom for technical assistance. Support for this study was provided by a grant from the Morris Animal Foundation to M.B.B. and D.F.S.
REFERENCES 1. Barsanti, J. A., D. R. Finco, E. B. Shotts, J. Blue, and L. Ross. 1982. Feline urologic syndrome: further investigation into etiology. J. Am. Anim. Hosp. Assoc. 18:391-395. 2. Blackmore, D. K., A. Hill, and 0. F. Jackson. 1971. The
3. 4. 5. 6.
9. 10. 11.
14. 15. 16.
18. 19. 20. 21.
incidence of mycoplasmas in pet and colony maintained cats. J. Small Anim. Pract. 12:207-216. Brown, M. B., P. Gionet, and D. F. Senior. 1990. Identification of Mycoplasma felis and Mycoplasma gateae by an immunobinding assay. J. Clin. Microbiol. 28:1870-1873. Brown, M. B., and D. F. Senior. Unpublished data. Cassell, G. H., and B. C. Cole. 1981. Mycoplasmas as agents of human disease. N. Engl. J. Med. 304:80-89. Chambers, S., and C. M. Kunin. 1985. The osmoprotective properties of urine for bacteria: the protective effect of betaine and human urine against low pH and high concentrations of electrolytes, sugars, and urea. J. Infect. Dis. 152:1308-1316. Cole, B. C., L. Golightly, and J. R. Ward. 1967. Characterization of mycoplasma strains from cats. J. Bacteriol. 94:14511458. Csonka, L. N. 1989. Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53:121-147. Koch, A. L. 1984. Shrinkage of Escherichia coli cells by osmotic stress. J. Bacteriol. 159:919-924. Koch, A. L., and M. F. S. Pinette. 1987. Nephelometric determination of turgor pressure in growing gram-negative bacteria. J. Bacteriol. 169:3654-3668. Lees, G. E., C. A. Osborne, and J. B. Stevens. 1979. Antibacterial properties of urine: studies of feline urine specific gravity, osmolality, and pH. J. Am. Anim. Hosp. Assoc. 15:135-141. Livingston, C. W., M. C. Calhoun, B. B. Gauer, and B. C. Baldwin, Jr. 1984. Effect of experimental infection with ovine ureaplasma upon the development of uroliths in feedlot lambs. Isr. J. Med. Sci. 20:958-961. McDonald, M. I., M. H. Lam, D. F. Birch, A. F. D'Arcy, K. F. Fairley, and E. R. Pavillard. 1982. Ureaplasma urealyticum in patients with acute symptoms of urinary tract infections. J. Urol. 128:517-519. Rosendal, S. 1979. Canine and feline mycoplasmas, p. 217-234. In J. G. Tully and R. F. Whitcomb (ed.), The mycoplasmas, vol. II. Academic Press, New York. Ross, L. A., and D. R. Finco. 1981. Relationship of selected clinical renal function tests to glomerular filtration rate and renal blood flow in cats. Am. J. Vet. Res. 4:1704-1710. Shepard, M. C., and C. D. Lunceford. 1976. Differential agar medium (A7) for identification of Ureaplasma urealyticum in primary cultures of clinical material. J. Clin. Microbiol. 3:613625. Shepard, M. C., and C. D. Lunceford. 1978. Serological typing of Ureaplasma urealyticum isolates from urethritis patients by an agar growth inhibition method. J. Clin. Microbiol. 8:566-574. Takebe, S., A. Numata, and K. Kobashi. 1984. Stone formation by Ureaplasma urealyticum in human urine and its prevention by urease inhibitors. J. Clin. Microbiol. 20:869-873. Tan, R. J. S., and J. A. R. Miles. 1974. Incidence and significance of mycoplasmas in sick cats. Res. Vet. Sci. 16:27-34. Tully, J. G., R. F. Whitcomb, H. F. Clark, and D. L. Williamson. 1975. Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195:829-894. Wiileberg, P. 1981. Epidemiology of the feline urologic syndrome. Adv. Vet. Sci. Comp. Med. 25:311-344.