Urinary Tract Infections

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1 JOURNAL OF CLINICAL MICROBIOLOGY, June 98, p Vol., No /8/6-654/6$./ Aerobic and Anaerobic Urethral Flora of Healthy Females in Various Physiological Age Groups and of Females with Urinary Tract Infections THOMAS J. MARRIE,'* CAROL A. SWANTEE, AND M. HARTLEN Departments of Medicine' and Microbiology, Dalhousie University, Halifax, Nova Scotia B3H V8, Canada We characterized the aerobic and anaerobic urethral flora of healthy females in three physiological age groups (premenarcheal, reproductive, and postmenopausal) and of females with urinary tract infections. The mean number of species per sample was 6.5, 7.7, and.3 for each of the physiological age groups, respectively, and 6.5 for the urinary tract infection group. Marked quantitative changes were seen with age and disease. Aerobes accounted for 65.6 and 73.8% of the flora of the premenarcheal and reproductive age groups, respectively, whereas anaerobes were dominant in the postmenopausal age group, accounting for 65.5% of the flora. Aerobic gram-negative rods were not isolated from any of the premenarcheal or reproductive-age subjects. Of the postmenopausal subjects, 5 carried aerobic gram-negative rods, but these organisms accounted for only % of the flora. In contrast, they constituted 95% of the urethral flora of the urinary tract infection group. Bacteroides melaninogenicus was commonly isolated and was the dominant isolate in the postmenopausal age group. Bacteroides fragilis was recovered from 7 of the postmenopausal subjects, but these organisms constituted only 3% of the total flora. The factors responsible for the changes in urethral flora with age remain to be determined. In a previous study (5), we showed that the anaerobic urethral flora of healthy females in aerobic and anaerobic urethral flora could be three physiological age groups (premenarcheal, delineated by culturing the first ml of voided reproductive, and postmenopausal) and compared it qualitatively and quantitatively with urine (urethral urine). In that study, results obtained with urethral urine cultures correlated the flora of six reproductive-age females with well with those obtained with urethral swabs. urinary tract infections. We found that the urethral flora in reproductiveage females consisted of both aerobes and an- Subjects and patients. Urethral urine samples MATERIALS AND METHODS aerobes. Such a rich endogenous urethral flora (the first to 5 ml of voided urethral urine) were raises the question of its role as a host defense obtained from nine healthy premenarcheal subjects mechanism. Various pieces of evidence suggest ranging in age from 3 to 5 years, with a mean age of that normal flora is important in this regard. 9 years. An additional reproductive-age females and Previous investigators (7) have shown that viridans group streptococci will inhibit the growth tured once. Only one of the healthy reproductive-age postmenopausal females had urethral urine cul- of group A beta-hemolytic streptococci. Murray females was on oral contraceptives. The postmenopausal females were all living in apartments in a and Rosenblatt have shown that Bacteroides home for senior citizens. They were self-sufficient, melaninogenicus in vitro will inhibit the growth were on no medications, and had no history of a of several strains of aerobic gram-negative rods urinary tract infection within the past 5 years. Twelve (6). The incidence of asymptomatic bacteriuria reproductive-age females with symptoms of frequency increases with age (), as does the incidence of of urination, urgency, and dysuria had urethral and colonization of the oropharynx by aerobic gramnegative rods (), suggesting that changes in pathogen in their midstream urine (five had Esche- midstream urines cultured. The six with a single uro- normal flora are dynamic and influenced by richia coli, and one had Staphylococcus saprophyticus) were included in this study. The remaining six several variables. had negative midstream urine cultures and were presumed to have the urethral syndrome. Their urethral If we are to elucidate the role of the urethral flora as a host defense mechanism, it is important to know how the normal aerobic and anaer- tract infections had pyuria (more than one leukocyte flora was not delineated. All six patients with urinary obic flora change with physiological age. In this per high-power field in an uncentrifuged urine specimen), and three were on oral contraceptives. study, we have characterized the aerobic and 654 Downloaded from on July, 8 by guest

2 VOL., 98 None of the subjects or patients had douched in the week before collection of the urine cultures. All subjects and patients were sampled once only. Specimen collection. The subjects and patients were instructed to void the first to 5 ml of urine into a sterile container. No cleansing was carried out beforehand. In addition, the patients obtained midstream urine samples. Inoculation and incubation. The urine sample was immediately carried to the laboratory, where it was inoculated onto a prereduced Brucella blood agar plate containing,ug of vitamin K, per ml and 5% sheep blood and onto a prereduced kanamycin-vancomycin-laked-blood agar plate containing Brucella agar base, 5% laked sheep blood,,g of vitamin K per ml,,tg of kanamycin per ml, and 7.5 jg of vancomycin per ml. Quantitative loops delivering. and. ml were used to inoculate these plates. The plates were prepared in our laboratory and were held under anaerobic conditions before use. After inoculation, the plates were incubated anaerobically in GasPak jars (BBL Microbiology Systems, Cockeysville, Md.) for 48 h. The disposable GasPak hydrogencarbon dioxide generators were used in the jars to supply a gas mixture containing hydrogen and carbon dioxide. In addition, the sample was cultured aerobically onto blood agar (Trypticase soy agar [BBL Microbiology Systems] containing 5% sheep blood), MacConkey agar, and chocolate agar (GC agar base [BBL Microbiology systems] containing % hemoglobin [Difco Laboratories, Detroit, Mich.] and Iso- VitaleX [BBL Microbiology Systems] at ml/liter) with.- and.-ml quantitative loops. The blood and MacConkey agar plates were incubated aerobically for 8 h, and the chocolate agar plate was incubated in an atmosphere of 5 to % C for 36 h. Isolation and identification. (i) Aerobes. All aerobic isolates were identified by conventional techniques for gram-positive cocci and Enterobacteriaceae (, 7, 8, ). Except in the case of one patient with S. saprophyticus in her urethral and midstream urine cultures, no attempt was made to identify coagulasenegative staphylococci as to species. (ii) Anaerobes. All colonies growing on the Brucella blood agar and kanamycin-vancomycin-lakedblood agar plates were examined for fluorescence under ultraviolet light (365 nm). Plates that exhibited brick-red fluorescence were held for 3 weeks to detect pigment production. All colonial types were Gram TABLE. URETHRAL FLORA 655 stained and inoculated onto plates incubated anaerobically to obtain pure cultures and onto plates incubated aerobically in an atmosphere of 5 to % C to identify facultative and microaerophilic organisms. Organisms that grew only anaerobically were inoculated into 5 ml of chopped-meat carbohydrate medium (prereduced and anaerobically sterilized) containing 5 Lg of hemin per ml and. Lg of vitamin K per ml for subsequent inoculation of biochemicals and into peptone-yeast extract with % glucose for gas-liquid chromatography. All organisms were identified by the methods outlined previously (). The prereduced, anaerobically sterilized media were obtained from Carr-Scarborough Microbiologicals, Stone Mountain, Ga. RESULTS Table shows the number of subjects, age range, and mean age in each group and the mean, median, and range of species, aerobes, and anaerobes isolated. Great variation occurred from subject to subject. Aerobic microorganisms were recovered from all 35 subjects. Anaerobic microorganisms were recovered from 33 of the 35 subjects. One reproductive-age healthy female and one of the six females with urinary tract infections failed to grow anaerobes. There was an increase in the number of species per sample with an increase in physiological age from 6.5 in the premenarcheal age group to.3 in the postmenopausal age group. The number of aerobes per sample increased from the premenarcheal to the reproductive age group, but there was only a slight increase in the number of aerobes per sample from the reproductive to the postmenopausal age group. The number of species of anaerobes recovered remained the same, i.e., 3.4 per sample, for the premenarcheal and reproductive age groups but increased to 5.7 for the postmenopausal age group. The patients with urinary tract infections were all of reproductive age. The number of species per sample recovered from this group was slightly fewer than that from the healthy reproductive-age subjects. Aerobic and anaerobic urethral flora in conditions of health and disease: characteristics of the study population and an overview of the flora Species/sample Aerobes/sample Anaerobes/sample Group No. of subjects Age range (yr) Mean age (yr) Mean dian Me- Range Mean Me- dian Range Mean Me- dian Range Premenar cheal Reproductive Postmeno pausal Urinary tract infection Downloaded from on July, 8 by guest

3 656 MARRIE, SWANTEE, AND HARTLEN J. CLIN. MICROBIOL. Quantitatively (Table ), much the same relationship existed among the three physiological age groups. The mean numbers of colony-forming units per sample were 74,434, 98,493, and 8,65, respectively, for the three groups; however, there was a marked increase in the urinary tract infection group to 73,35 colony-forming units per sample. This was largely due to the number of aerobic gram-negative rods. In the postmenopausal age group, anaerobes were quantitatively more important, accounting for 65% of the total flora. In the premenarcheal and reproductive age groups, anaerobes accounted for 34 and 6% of the flora, respectively. Anaerobes accounted for only.97% of the flora in the infected group. The qualitative aspects of the aerobic urethral flora are shown in Table 3. The predominance of lactobacilli in the reproductive age group is evident. Two of the three premenarcheal subjects from whom lactobacilli were isolated were aged 4 and 5 years, respectively; both began to menstruate within to 3 months after determination of their urethral flora. The remaining premenarcheal subject from whom a Lactobacillus sp. was isolated was 6 years old. Quantitatively (Table ), lactobacilli were the dominant organisms isolated from the reproductive-age subjects, accounting for 8.3% of the total flora as compared with 4% in both of the other groups. Corynebacterium spp. and coagulasenegative staphylococci were isolated from nearly all healthy subjects in the three physiological age groups. However, quantitatively, there are important differences (Table ). Coagulase-negative staphylococci were recovered from 9 of the postmenopausal subjects but represented only.5% of the total flora, as compared with.6 and 7.8% of that in the premenarcheal and reproductive age groups, respectively. The quantitative changes in the Corynebacterium spp. were even more striking. In the premenarcheal age group, this species represented 33.4% of the total urethral flora but declined to 6.7 and 6.5% in the reproductive and postmenopausal age groups, respectively. Enterococci were infrequently isolated from the premenarcheal and reproductive age groups but were isolated from 5 of the postmenopausal subjects. Aerobic gram-negative rods were absent from the premenarcheal and reproductive age groups but were recovered from 5 of the postmenopausal subjects; however, quantitatively, these organisms represented only % of the total flora (Table ). Four of the five postmenopausal subjects with aerobic gram-negative rods carried E. coli only. The other subject had E. coli, Enter-. L. L. s - t Z ;. s U e_ O c -a C) rz. Ua. u co. O E X.~ m v. as* :*C (v b D r C4 o <. ; O V) 6 Z o.. 4. oku X as5 ~ CD C.-.. v! m N -4 (;- 6w CS cs-o o; -C's Uc m Les 3 4 M -Ct C's ~6 O Co CD- ) N o o e6 _; _- X I Co ) ulc li Oz - - I- Ci ll - C~ C6 l ) Co ;> C) 3 o..., C) o= E r -- 4)c ) rz = cd (L. E... ' N X X t. m. c X t Qom Downloaded from on July, 8 by guest

4 VOL., 98 TABLE 3. Aerobic microorganisms isolated from the urethras of healthy femakse^in three physiological age groups and offemales with a urinary tract infection No. of positive cultures Pre- Post- Urinary menar- dti meno- tract incheal ()a pausal fection (9)a ()a (6)a Lactobacillus spp. Corynebacterium spp. Coagulase-nega b tive staphylococci Streptococci Nonhemolytic 4 8 Alpha-hemo lytic GroupB Enterococci 3 5 Gram-negative 5c 5d bacilli anumber of subjects is given within parentheses. bone isolate was S. saprophyticus. e Four subjects carried E. coli, and one subject carited E. coli, Proteus spp., and Enterobacter spp. d All five isolates were E. coli. obacter sp., and Proteus sp. isolated. A marked difference was evident between the qualitative (Table 3) and quantitative (Table ) aspects of the aerobic urethral flora of the urinary tract infection group. Whereas a mean of 3.8 aerobes was isolated per sample, E. coli accounted for 95% of the total flora. In one of the six subjects in this group, the infection was due to S. saprophyticus. Anaerobic microflora. Table 4 shows that anaerobic gram-positive cocci were frequently recovered from subjects throughout the three physiological age groups. Anaerobic gram-negative cocci and anaerobic gram-positive bacilli were rarely recovered. Anaerobic gram-negative bacilli were frequently recovered, and B. melaninogenicus was the most prominent of these organisms. In the postmenopausal age group, B. melaninogenicus accounted for 36.4% of the total flora as compared with % of the total premenarcheal age group flora and.5% of the reproductive age group flora. The number of Bacteroides fragilis group isolates increased with physiological age (Table 4); however, quantitatively, these organisms accounted for only URETHRAL FLORA 657 TABLE 4. Anaerobic microorganisms isolated from the urethras of healthy females in three physiological age groups and offemales with a urinary tract infection No. of positive cultures Cocci Peptococcus prevotii Peptococcus magnus Peptococcus asaccharolyticus Peptostreptococcus productus Peptostreptococcus anaerobius Veillonella spp. Gaffkya anaerobia Gram-positive bacilli Eubacterium lentum Propionibacterium app- Clostridium spp. Gram-negative bacilli Bacteroides melaninogenicus Bacteroides fragilis group" Bacteroides capillosus Bacteroides amylophilus Bacteroides disiens Fusobacterium nucleatum Uri- Pre- Repro- Post- nary duc- meno- tract tive cheal ear pausal infec- ()" () tion (6)a "Number of subjects is given within parentheses. b Includes B. fragilis subsp. distasonis, B. fragilis subsp. vulgatus, and B. fragilis subsp. thetaiotaomicron. and 3% of the total flora in the reproductive and postmenopausal age groups, respectively. DISCUSSION In this study, we have shown striking qualitative and quantitative changes in urethral flora with physiological age. To obtain an accurate picture of the urethral flora, one must analyze it both qualitatively and quantitatively. This is illustrated best by considering the flora of the Downloaded from on July, 8 by guest

5 658 MARRIE, SWANTEE, AND HARTLEN urinary tract infection group. Whereas these subjects had a mean of 6.3 species isolated per sample, E. coli accounted for 95% of the total flora. Bollgren et al. (4) found that anaerobes accounted for 95% of the total periurethral flora of healthy girls and felt that the periurethral microenvironment is a distinctive ecological niche separate from the fecal and skin biotas. We found that aerobes, primarily Corynebacterium spp., dominated the urethral flora of our premenarcheal females. This would indicate that the urethral area is an ecological niche distinct from that of the periurethral areas. This is not surprising as other areas juxtaposed anatomically have distinctive bacterial populations (). Both healthy male and female infants are heavily colonized with E. coli and enterococci during the first few weeks of life, but such colonization is rare after 5 years of age (6). None of our premenarcheal subjects was colonized with aerobic gram-negative rods, and only two of nine carried enterococci as part of their urethral flora. The extremes of age seem to predispose even healthy individuals to colonization with gramnegative organisms both in the urethra, as seen in our postmenopausal females, and in the pharynx (). However, even though 5 of the postmenopausal subjects had aerobic gram-negative rods as part of their urethral flora, these organisms accounted for only % of the total flora. Hormonal factors also play a role in changes in the bacterial flora of the urogenital area. Group B streptococci and E. coli are more commonly isolated from the periurethral area during menstruation (5). The flora of the cervix changes during pregnancy, with a marked decrease in the number of anaerobes from the first to the third trimester and a rebound almost immediately postpartum (9). Variations in the vaginal flora of rats occur during the estrous cycle, with counts being, to, times greater during the estrus phase of the cycle than during J. CLIN. MICROBIOL. the metestrus or diestrus phase (3). The predominance of lactobacilli quantitatively and qualitatively during the reproductive years is probably also influenced by hormonal changes. Two of the three premenarcheal subjects who were colonized with lactobacilli began to menstruate 3 months after their urethral urine was cultured. The other subject was 6 years old. B. melaninogenicus was isolated from 66% of the premenarcheal age group, 7% of the reproductive age group, and % of the postmenopausal age group. Quantitatively, in the latter group, this species was the dominant organism, accounting for 36.4% of the total flora. Hammerschlag et al. () isolated B. melaninogenicus as part of the vaginal flora of 56% of girls aged months to 5 years. This organism has been isolated from the vagina in 36% of healthy reproductive-age females (3), and the isolation rate from the cervix has varied from 4% during the first trimester of pregnancy to 3.6% 3 days postpartum in one study (9). In a similar study, Thadepalli et al. found that B. melaninogenicus was the most frequent anaerobe isolated from the cervix. In their study, % of 5 women in labor carried this organism as part of their cervical flora, whereas 4% did so during the puerperium (9). The much higher isolation rate of B. melaninogenicus from the urethra would indicate that the urethra is an ecological niche for this organism. In contrast, only one of the six with urinary infections had B. melaninogenicus isolated from the urethra. Whether this is a true finding reflecting the role of this organism as part of the host defense against urinary tract infection deserves further study with a larger number of subjects. Adhesion of bacteria to uroepithelial cells has been studied extensively in patients with urinary tract infections (4, 8). In general, strains of bacteria causing acute pyelonephritis adhere better than those that cause asymptomatic bacteriuria. Whether blockage of adhesion of uropathogens to uroepithelial cells is one of the mechanisms whereby urethral flora acts as a host defense mechanism remains to be determined. Another unanswered question is whether differential adhesion with physiological age accounts for the changes that we have demonstrated in the urethral flora. ACKNOWLEDGMENT This study was supported by a grant from the Dalhousie University Internal Medicine Research Fund. LITERATURE CITED. Austrian, R Streptococcus pneumoniae (pneumococcus), p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, nd ed. American Society for Microbiology, Washington, D.C.. Bartlett, J. G., N. E. Moon, P. R. Goldstein, B. Goren, A. Onderdonk, and B. F. Polk Cervical and vaginal bacterial flora: ecological niches in the female lower genital tract. Am. J. Obstet. Gynecol. 3: Bartlett, J. G., A. B. Onderdonk, E. Drude, C. Goldstein, M. Anderka, S. Alpert, and W. M. Mc- Cormack Quantitative bacteriology of the vaginal flora. J. Infect. Dis. 36: Bollgren, I., G. Kallenius, C. E. Nord, and J. Winberg Periurethral anaerobic microflora of healthy girls. J. Clin. Microbiol. : Bollgren, I., V. Vaclavinkova, B. Hurvell, and G. Bergquivst Periurethral aerobic microflora of pregnant and non-pregnant women. Br. Med. J. : Downloaded from on July, 8 by guest

6 VOL., Bollgren, I., and J. Winberg The periurethral aerobic bacterial flora in healthy boys and girls. Acta Paediatr. Scand. 65: Ewing, W. H., and W. J. Martin Enterobacteriaceae, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, nd ed. American Society for Microbiology, Washington, D.C. 8. Facklam, R. R Streptococci, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, nd ed. American Society for Microbiology, Washington, D.C. 9. Goplerud, C. P., M. J. Ohm, and R. P. Galask Aerobic and anaerobic flora of the cervix during pregnancy and the puerperium. Am. J. Obstet. Gynecol. 6: Hammerschlag, M. R., S. Alpert, A. B. Onderdonk, P. Thurston, E. Drude, W. M. McCormack, and J. G. Bartlett Anaerobic microflora of the vagina in children. Am. J. Obstet. Gynecol. 3: Holdeman, L. V., and W. E. C. Moore (ed.) Anaerobe laboratory manual. Virginia Polytechnic Institute and State University, Blacksburg.. Ivler, D Staphylococcus, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, nd ed. American Society for Microbiology, Washington, D. C. 3. Larsen, B., A. J. Markovetz, and R. P. Galask Quantitative alterations in the genital microflora of female rats in relation to the Estrous cycle. J. Infect. URETHRAL FLORA 659 Dis. 34: Mardh, P. A., S. Colleen, and B. Hovelius Attachment of bacteria to exfoliated cells from the urogenital tract. Invest.Urol. 6: Marrie, T. J., G. K. M. Harding, and A. R. Ronald Anaerobic and aerobic urethral flora in healthy females. J. Clin. Microbiol. 8: Murray, P. R., and J. E. Rosenblatt Bacterial interference by oropharyngeal and clinical isolates of anaerobic bacteria. J. Infect. Dis. 34: Saunders, E Bacterial interference. I. Its occurrence among the respiratory tract flora and characterization of inhibition of group A streptococci by viridans streptococci. J. Infect. Dis. : Svanborg-Eden, C Attachment of Escherichia coli to urinary tract epithelial cells-an in vitro test system applied in the study of urinary tract infections. Scand. J. Infect. Dis. Suppl. 5: Thadepalli, H., W. H. Chan, J. E. Maidman, and E. C. Davidson, Jr Microflora of the cervix during normal labor and the puerperium. J. Infect. Dis. 37: Valenti, W. M., R. G. Trudell, and D. W. Bently Factors predisposing to oropharyngeal colonization with Gram negative bacilli in the aged. N. Engl. J. Med. 98:8-.. Walkey, F. A., T. G. Judge, J. Thompson, and N. B. S. Sarkari Incidence of urinary infection in the elderly. Scott. Med. J. :4-44. Downloaded from on July, 8 by guest