Staphylococcus saprophyticus as a Cause of Urinary Tract

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1982, p /82/ $02.00/0 Copyright 1982, American Society for Microbiology Vol. 16, No. 3 Staphylococcus saprophyticus as a Cause of Urinary Tract Infections THOMAS J. MARRIE,l.2* CAROL KWAN,1 MICHAEL A. NOBLE,'2 ANN WEST,2 AND LENORA DUFFIELD3 Department of Medicine1 and Department of Microbiology,2 Dalhousie University and Victoria General Hospital, and Dalhousie Student Health Service,3 Dalhousie University, Halifax B3H I V8, Nova Scotia Received 4 March 1982/Accepted 1 June 1982 Our objectives in this study were to elucidate various aspects of the epidemiology of Staphylococcus saprophyticus. This organism was isolated from the midstream urine specimens of 7.5% of 145 college women with frequency and urgency of urination and dysuria, but from only 0.07% of 14,835 urine specimens from adult inpatients at the Victoria General Hospital. It was found to be part of the urethral flora of only 2% of healthy women. Other staphylococci which formed part of the urethral flora of 100 healthy women included S. epidermidis (59 women), S. hominis (15 women), S. haemolyticus (13 women), S. warneri (9 women), and S. aureus (6 women). Finally, we determined that resistance to the 5-,ug novobiocin disk has a 93% positive predictive accuracy as a presumptive test for S. saprophyticus. Coagulase-negative staphylococci have long been considered of little significance as a cause of urinary tract infections. However, during the 1970s, starting in Europe and later in the United States and Canada, a particular subgroup of coagulase-negative staphylococci, Staphylococcus saprophyticus (formerly Micrococcus subgroup 3), was shown to be an important cause of urinary tract infections in young females (1, 2, 7, 9, 11, 12, 16, 17, 19, 22, 26). However, the reservoir and pathogenesis of infection of the urinary tract with S. saprophyticus are poorly understood in contrast to our understanding of the pathogenesis of urinary tract infections with Enterobacteriaceae, which is as follows: endogenous fecal organisms colonize the introitus and then the urethra and finally ascend to the bladder (8, 23). Our objectives in this study were: (i) to determine the incidence of S. saprophyticus as a cause of urinary tract infections in two patient populations, young women with symptoms of urinary infection and inpatients whose urine specimens were submitted for culture; (ii) to determine the reservoir of S. saprophyticus by identifying coagulase-negative Staphylococcus species from the urethras of 100 healthy college women and by obtaining rectal swabs from women with S. saprophyticus urinary infection; and (iii) to determine the positive predictive accuracy of resistance to 5,ug of novobiocin as a presumptive identification of S. saprophyticus. MATERIALS AND METHODS Subjects and patients. (i) Outpatients. We studied 145 women with symptoms of frequency and urgency of urination and dysuria who presented to the Dalhousie Student Health Service between September 1980 and February (ii) Inpatients. The results of all urine specimens submitted to the Victoria General Hospital Microbiology Laboratory during 1981 were recorded. The charts of 11 patients from whom S. saprophyticus organisms were isolated were reviewed, and the following data were abstracted: age, sex, underlying disease, symptoms related to the urinary tract, and treatment, if any. (iii) Subjects. A total of 100 healthy college women volunteers (informed consent was obtained) completed a questionnaire and supplied a specimen of urethral urine (we have previously shown that urethral urine specimens correlate with urethral swab specimens). The questionnaire inquired about previous urinary tract infections and present medications, including oral contraceptives. Organisms. Isolates of coagulase-negative staphylococci, presumptively identified as members of the S. saprophyticus group by resistance to the 5-p.g novobiocin disk, were obtained and identified as described later. 0. C. McIntosh, St. Martha's Hospital, Antigonish, Nova Scotia, provided 66 isolates, and M. A. Noble, Victoria General Hospital, Halifax, Nova Scotia, provided 41 isolates. Specimen collection and transport. Midstream urine specimens were obtained from both in- and outpatients, and samples were refrigerated until transferred to the laboratory. In addition, for the outpatient group, a Dipslide (Nocu Dip Pharma-Medica of Scandinavia, Canada, Ltd., Toronto, Ontario, Canada) was inocu- 427

2 428 MARRIE ET AL. TABLE 1. Results of culture of midstream urine specimens from 145 college women with frequency and urgency of urination and dysuria Organism isolated Concn No. (%) (CFU/ml) of women E. coli (24.8) Gram-negative organisms (40) S. saprophyticus (7.5) Staphylococcus sp (14.5) No growth 19 (13.2) lated immediately and held at room temperature until transferred to the laboratory. Urethral urines were obtained by having the subject collect the first 15 to 20 ml of voided urine into a sterile container. No cleansing was carried out beforehand. Of the 11 college women with S. saprophyticus urinary infection, 9 provided a sample of their anal canal flora by means of a rectal swab inserted until it was stained with feces. Inoculation. When urine specimens arrived at the clinical laboratory, they were inoculated onto blood agar (Trypticase soy agar [BBL Microbiology Systems, Cockeysville, Md.] containing 5% sheep blood) and MacConkey agar plates with quantitative loops (0.001 ml for midstream urine specimens; 0.1 ml for urine specimens obtained through the cystoscope). After overnight incubation, organisms growing in significant numbers as defined by the following criteria were identified: (i) any number of organisms isolated from midstream urine specimens from renal transplant patients and from catheter and cystoscopy urine specimens; and (ii) organisms growing in pure culture with colony counts of 5 x 104 colony-forming units (CFU) per ml. Urine specimens exhibiting mixed growth were considered individually, and a decision to identify the organisms was based on the patient's history, the total colony count, and the numbers of different morphological types of organisms present and their colony counts. Urethral urine samples were inoculated onto blood and MacConkey agar with and ml quantitative loops. Only colonies morphologically suggestive of staphylococci were enumerated and picked for identification (3, 15). The rectal swabs were blended in a Vortex mixer for 15 s in 2 ml of Trypticase soy broth (BBL Microbiology Systems), and then 0.5 ml of this material was plated onto Trypticase soy agar containing 1.5 mg of novobiocin (Upjohn Company of Canada, Don Mills, Ontario) per liter and 25 mg of nalidixic acid (Winthrop Laboratories, Aurora, Ontario, Canada) per liter. Colonies morphologically resembling staphylococci (3, 15) were picked and identified as described below. Identification. Gram-negative bacteria were identified as previously described (15). Coagulase-negative staphylococci from midstream urine specimens were presumptively identified as members of the S. saprophyticus group if the diameter of the zone of inhibition of the 5-,ug novobiocin disk was less than 14 mm (5). These isolates were later identified as outlined below. Five colonies of coagulase-negative staphylococci from each urethral urine sample were identified as follows. The modified oxidase test of Faller and J. CLIN. MICROBIOL. Schleifer (6) was used to separate staphylococci from micrococci. The coagulase-negative staphylococci were then identified by using our modification of the scheme of Kloos and Schleifer (13), which consists of performing the carbohydrate fermentation reactions in glass tubes (13 by 100 mm) rather than incorporating sugars into agar. Organisms were inoculated into 2.5 ml of 0.5% of each of the following carbohydrates in Purple Broth Base (Difco Laboratories, Detroit, Mich.): D-(+)-mannose, xylitol, L-(+)-galactose, D- (-)-ribose, D-(+)-turanose (Sigma Chemical Co., St. Louis, Mo.), maltose, saccharose (sucrose), trehalose, D-fructose, melezitose, D-mannitol, D-xylose, and lactose (Difco). The phosphatase, nitrate reduction, and hemolysis tests were performed as described by Kloos and Schleifer (13). S. saprophyticus ATCC 15305, S. xylosus ATCC 29971, and S. cohnii ATCC were included with each run. Tube coagulase tests were performed as outlined by Paik (18). RESULTS Outpatients. The mean age of the 145 college women who presented to the Student Health Service with frequency and urgency of urination and dysuria was 23 years (range, 17 to 39 years). Of these, 79 (53%) had a history of previous urinary tract infection, and 69 (48%) were taking oral contraceptives. Table 1 shows that Escherichia coli in counts of s105 CFU/ml was the most frequent cause of urinary tract infection in this population. S. saprophyticus caused 11 (7.5%) of the urinary tract infections; in 10 of these, the organism was present in counts of a105 CFU/ml. Low counts of Gram-negative bacteria were isolated from 40% of these patients. Only 19 (13.2%) had no organisms isolated from their midstream urine specimens. Of the 11 women with S. saprophyticus urinary tract infections, 4 were taking oral contraceptives, compared with 16 of the 36 women with an E. coli urinary tract infection (X2 = 0.015, P = not significant). TABLE 2. Results of culture of 14,835 urine specimens from inpatients at the Victoria General Hospital during 1981 No. (%) Organism isolated of urine specimens Gram-negative organisms... 2,340 (15.8) Yeasts.388 (2.6) Enterococci.346 (2.3) S. epidermidis.312 (2.1) Streptococci other than enterococci (0.52) S. aureus.70 (0.47) S. saprophyticus.11 (0.07) No growth.7,535 (50.8) Various organisms (<104 CFU/ml). 3,743 (25.2)

3 VOL. 16, 1982 Of the 11 patients with S. saprophyticus urinary infections, 9 provided rectal swabs. S. saprophyticus was part of the rectal flora of two patients, and S. cohnii was isolated from one other. Inpatients. Table 2 shows the results of the culture of 14,835 urine specimens from inpatients (all adults; no obstetrical patients) at the Victoria General Hospital during Note that 11 (0.07%) grew S. saprophyticus. The charts of the 11 patients from whom S. saprophyticus was isolated were reviewed. Four of these patients were male, and seven were female; their mean age was 57.1 years (range, 23 to 80 years). Only one patient was symptomatic. This was a 31-year-old female who developed frequency and urgency of urination and dysuria 2 days before admission to the Psychiatric Ward for treatment of various phobias. Six patients had abnormalities of the urinary tract: renal stones (two patients), and bladder carcinoma, renal cell carcinoma, Epstein's disease, and acute renal failure (one each). An additional two patients had indwelling urinary catheters. Two patients had low counts of S. saprophyticus (40 and 103 CFU/ml, respectively) in urine obtained at the time of cystoscopy and from an indwelling urinary catheter, respectively. The urine of the remaining nine patients showed counts of 1 CFU/ml. S. saprophyticus (106 CFU/ml) and Enterococcus (106 CFU/ml) were isolated from a midstream urine specimen of one patient. Three patients received antibiotics, including erythromycin, cephalexin, and trimethoprim-sulfamethoxazole, from their attending physicians for S. saprophyticus bacteriuria. One other patient received antibiotics for concomitant pneumonia and wound sepsis. In all instances, S. saprophyticus was cleared from the urine. Urethral staphylococci. The 100 healthy college women who provided urethral urine samples had a mean age of 25.2 years; 31 were taking oral contraceptives, and 11 had experienced a urinary tract infection in the past. Table 3 shows the frequency of the various types of staphylococci isolated from the urethras of these subjects. S. saprophyticus was the least TABLE 3. Staphylococci isolated from the urethral urine of 100 healthy college women Organism No. of women S. epidermidis S. hominis S. haemolyticus S. warneri... 9 S. simulans... 5 S. aureus... 5 S. saprophyticus... 2 S. SAPROPHYTICUS 429 common. It was isolated from only two subjects. Positive predictive accuracy of resistance to the 5-pg novobiocin disk as a presumptive identification of S. saprophyticus. The 66 novobiocinresistant, coagulase-negative staphylococcal isolates from St. Martha's Hospital were identified: 62 isolates were S. saprophyticus, 2 were S. warneri, and 2 were S. cohnii, for a positive predictive accuracy of 94%. Identification of the 41 novobiocin-resistant, coagulase-negative staphylococcal isolates from the Victoria General Hospital revealed that 38 were S. saprophyticus, 1 was S. cohnii, and 2 were S. epidermidis, for a positive predictive accuracy of 92%. Thus, the overall positive predictive accuracy of resistance to the 5-,ug novobiocin disk is 93%. DISCUSSION In this study, we have shown that S. saprophyticus is an important cause of urinary tract infections in young women. The 7.5% incidence of S. saprophyticus urinary tract infections among young college women in Nova Scotia is remarkably similar to the 6.6 and 6.9% incidences in York, England, and Vancouver, Canada, respectively (1). However, it is lower than the 20% incidence reported by Jordan et al. (11). Only two of the nine patients with S. saprophyticus urinary infection who provided rectal swabs carried this organism as part of their fecal flora. Other investigators have recovered S. saprophyticus from the mucosal surfaces of the urogenital and anal areas of 14% of patients with S. saprophyticus urinary tract infection (11). It is probably unlikely that the rectum is the reservoir of S. saprophyticus. Sellin et al. (21) recovered S. saprophyticus from the urethras of 4 (5.8%) of 68 healthy women. Walmark et al. (26) were unable to isolate S. saprophyticus from the urethras of 110 patients attending a venereal disease clinic and isolated it from the rectum of only 1 of 206 healthy females. Our recovery of S. saprophyticus from the urethras of 2% of healthy females, then, is further evidence suggesting that the female urethra is not the reservoir of this organism. In contrast, Hovelius et al. recovered S. saprophyticus from the urethras of 20.8% of 178 with symptoms of urethritis and from 14.9% of men without such symptoms who were attending a venereal disease clinic (10). Clearly, a case control study is the next step in elucidating the reservoir of S. saprophyticus. The urethras of contacts of patients and controls should be sampled for S. saprophyticus. In a previous study, we have shown that urethral flora varies with physiological age (15). Indeed, not only does the incidence of colonization of the urethra by aerobic Gram-negative rods increase in the elderly (15), but so does the inci-

4 430 MARRIE ET AL. dence of asymptomatic bacteriuria due to these organisms (25). The apparent limitation of S. saprophyticus urinary tract infections to young women (1, 11, 16, 17, 19, 21) suggests that hormonal factors may be important. Our data suggest that oral contraceptive use (exogenous hormones) does not predispose to S. saprophyticus urinary tract infections, since there was no difference in the number of women using oral contraception who had this infection as compared with those who had E. coli urinary tract infections. S. saprophyticus adheres to uroepithelial cells much better than to buccal or skin cells (4, 14). Whether there is differential adherence of this organism to uroepithelial cells from females of reproductive age remains to be determined. In view of the frequency of S. saprophyticus as a cause of urinary tract infections, it is important that clinical laboratories be able to identify this pathogen. Even the simplified schema of Kloos and Schleifer (13) is cumbersome for most diagnostic laboratories. The positive predictive accuracy of 93% for the 5-p.g novobiocin disks is sufficient as a means of presumptive identification of S. saprophyticus. S. cohnii and S. xylosus are also resistant to novobiocin (13, 20). We did not isolate either S. cohnii or S. xylosus from the urethras of healthy women; however, S. cohnii accounted for three of the seven novobiocin-resistant, non-s. saprophyticus isolates in our study of 107 novobiocinresistant, coagulase-negative staphylococci. Two isolates each of S. warneri and S. epidermidis were the other true false-positives. These isolates are usually sensitive to novobiocin and indeed were found to be sensitive by the agar dilution technique. Finally, it is worthwhile to examine the data on the remainder of the college women with frequency and urgency of urination and dysuria. The 40% of women with low counts (s104 CFU/ ml) of Gram-negative bacteria probably had urinary tract infections caused by these organisms, as recently shown by Stamm et al. (24; W. E. Stamm, G. W. Counts, K. Running, M. Turck, and K. K. Holmes, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 21st, Chicago, Ill. abstr. no. 145, 1981). The Staphylococcus species (S. epidermidis [16 women], S. hominis [4 women], and S. warneri [1 woman]) isolated from 14.5% of these women probably represent urethral contaminants, since the occurrence of these species parallels that of staphylococci isolated from urethral urine of healthy women (Table 3). ACKNOWLEDGMENTS This research was supported by a grant from National Health and Welfare Canada. J. CLIN. MICROBIOL. We thank the medical staff of the Dalhousie Student Health Service for their assistance. LITERATURE CITED 1. Anderson, J. D., A. M. Clarke, M. E. Anderson, J. L. Isaac-Renton, and M. G. 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