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The Bacterial Flora of Urine Specimens from Normal Adults
THE JOURNAL OF UROLOGY
Vol. 75, No. 3, March 1956 Printed in U.S.A.
THE BACTERIAL FLORA OF URINE SPECIMENS FROM NORMAL ADULTS V. B. PHILPOT, JR.
From the Department of Pathology, University Hospitals, Madison, Wis.
There are inconsistencies in the literature regarding the diagnostic significance of bacteria found in urine properly collected for bacteriological examination. Gould states that "Organisms isolated on bacteriological examination are significant when the methods of collecting the urine are· satisfactory and the technique of the laboratory is of high standard. Under these conditions, no organisms are isolated from the urine of healthy persons and patients who have no disease of the urinary tract." Kleinschmidt, on the other hand, reported that thirty-five (17 per cent) of 200 females studied were found to have bacteriuria not associated with urinary tract disease. Approximately one third of this group of thirty-five were completely healthy and asymptomatic in every respect. Lazarus stresses the importance of detailed bacteriological examination of urine requiring culture methods for species identification. Cook states, "The general practitioner as well as the specialist can satisfactorily examine, diagnose, and treat the great majority of the patients he sees by using Gram's stain of the urinary sediment to ascertain the general type of infection present." All of the aforementioned authorities agree that urine from females should be obtained by the use of a sterile catheter and the three (Cook, Lazarus, Gould) who dealt with males agree that a midstream voided specimen from males after proper cleansing of the external genitalia is satisfactory. The differences in opinion, therefore, reflect variations in bacteriological technique and interpretation. In order to establish a base line of normals that will aid in determining the diagnostic significance of bacteria in the urine and clarifying the above mentioned disagreements, a study has been made of the bacterial flora of urine from 100 normal adults, 50 of which were male and 50 female. PROCEDURES
The volunteers whose urine was studied varied in age from 18 to 67 years, but the majority were between 20 and 30 years. Fourteen of the total number had mild symptoms of urinary tract disorder such as occasional burning on urination or nocturia, but none of them were sufficiently annoyed to seek medical attention or to alter their daily routine of activities. The remainder (86) were found to be completely free of symptoms of urinary tract disease after careful questioning and were actively engaged in employment. Procedure for collecting urine samples from males was as follows: 1) The foreskin was reflected backwards and the glans penis rinsed in tap water. Accepted for publication July 26, 1955. This work was done with the help and advice of W. D. Stovall, M.D., Dorothy Colien, Ph.D., and Miss Ann Knell, and other members of the staff of the University Hospitals. 562
BACTli;lUAL FLOHA OF URINE SPECIMENS
:!) The glans ,Yas washed carefully with tincture of green soap mid a sterile sponge, and rinsed 1vith a 1- .5000 aqueous solution of potnssium mercuric: 10dich A.t this point the penile meatus of 21 volunteers v,as swabbed with n, ::;terile cotton applicator which was then immersed in J per cent lactose broth and incubated at 87°C. The eultures were examined at n,.,,,,,""' of 24 and 48 hour,;; after inoculation. :3) From :30 to lOO cc of urine was voided and discarded. Approximalely :30 ec of bladder urine vvas then colleeted in a sterile contamer and cultures ~taned within two hours. All specimens from males were collected the author t,rJ ascertain that standard methods were observed in each case. The procedure of collecting samples from females was a8 follmrn: 1) Using titerile rubber the labia ·were spread apart exposing llw external urethral orifice. 2) The exposed area was washed with a sterile sponge and tincture of green soap (diluted 1 :4 in sterile water to avoid irritation) and ri11sed 1Yith a I 5000 aqueous solution of potassium mercuric iodide. 3) A sterile -···~·"'"" rnbber catheter was inserted mto tbe bladder and approximately 30 cc of urine collected in a sterile container. Cult.urP;-; \YPre started within 2 hours. All specimens from females were collected one well quaJi[ird uurse who used the same procedure on all volunteers. One ml. of each urine sample was placed in a pour plate with Bacto plate ,,ount agar containing yeast extract, glucose, and tryptose, and buffered at pH 7.2. Ten ml. of each urine sample were centrifuged at :-s,000 .p.m. for 10 mi11· utes and the residue examined for cellular content and stained ({ram's method for bacteria. fo addition, a loopful of the sediment of centrifuged urine specimens from males was placed in 1 per cent lactose broth with brom blue indicator and another loopful streaked out on a blood agar Initial agar plate cultures were incubated at ::l7°C for 2-t to 28 hours. Lactose broth ,ms held for a total of 48 hours before it was considered negative. The term, lVIicrococcaceae refers to the family of bacteria that i11elmle:,; th0 genera Micrococcus (Staphylococcus), Gaffkya, and Sanana. These s,vere identified on the basis of colonial morphology on a blood agar plate and <'ellnlar morphology after Gram staining. All streptococci recovered were placed in 1 per cent glucose broth ovt:rnight and transferred to ti.ti per cent sodium chloride broth at pH 7.2. Organisirn, grmving 011 the latter medimn 1rnre classified as enterococci. Streptococci I.hat failed to grow in (\.5 per cm1t sodium chloride broth were classified on the ha,~is of h<-m10lysis on a blood agar plate. Gram-negative rods that ferment 1 per cent lactose broth ,rnre clac1silied as members of the genus Escherichia or Aerobacter. The two strains of gramnegative Bacterium anitratum were identified on tho ba::;is of the charnc:tcristic fermentation reaction,, particularly their ability to ferment JO per erni" !mt not 1 per cent lactrn,e.
,564
V. B. PHILPOT, JR.
TAB LE
1. N wnber of urine specimens from 100 normal volunteers showinu ·.bacteria
by various techniques ·-
Specimens from 50
Method
l. Plate count agar 1-30 colonies/cc .. 30-400 colonies/cc Over 400 colonies/cc
Total.
Males Showing Bacteria
Specimens from 50 Females Showing Bacteria
Number
% of total
Number
% of total
27 10 4
54 20 8
14 3 0
28 6 0
41
82
17
34
26
52
not done
-
2. 1% lactose broth.
not done
--
3. Blood agar streak plate .. Less than 16 colonies per plate .. Over 16 colonies per plate .. Total.
11 1 12
22 2 24
1
2
not done
-----·--·-
·---
4. Gram stain.
2
4
RESULTS
As shown in table 1, 82 per cent of midstream urine specimens from 50 apparently normal males showed bacteria, when 1.0 cc of undiluted urine was cultured in a pour plate with plate count agar. Only 28 per cent showed more than 30 organisms per cubic centimeter and the highest number found in a single specimen was 5,700. This figure can be regarded as only an approximation since accurate plate counts of bacteria can be obtained only when the number of bacteria is within the range of 30-400 per plate. Using a loopful of centrifuged urine as an inoculum, bacteria were recovered from 52 per cent of urine specimens from males cultured in 1 per cent lactose broth, and from only 24 per cent of specimens streaked on a blood agar plate. The single specimen containing bacteria revealed by Gram's stain of centrifuged urine was the one showing approximately 5,700 organisms per cubic centimeter. Organisms seen by direct examination of uncentrifuged urine are probably present in numbers of 100,000 per cubic centimeter or greater. Table 1 also shows that a catheterized urine specimen from a normal female is much more likely to be sterile than is a midstream specimen from a normal male. By the most sensitive method of detecting bacteria (plate count) only 34 per cent of urine specimens from females contained bacteria in contrast to 82 per cent from males. The average number of bacteria per cubic centimeter from females is also significantly less than that from males. On direct microscopic exarnination of centrifuged urine from females, bacteria were seen in only 2 of the 50: gram-positive cocci in one and gram-positive bacilli in the other. Neither of these organisms grew on plate count agar indicating that although it ·was the most sensitive of the media used in these experiments, plate count agar is by no means a perfect medium for the growth of all bacteria. The organisms recovered from "normal" urine listed in table 2 include many
TABLE
2. Surnl>er of -unne samples frmn 100 normal iJolunt,,ers .,howinu uan,,,,.., types of bacteria !vJalcs r,Total 50) Type of Bacteria
i
No. ol _____ ] specimens
Betlt hemolytic strnptococci Alpha, hemolytic ~I n•ptococci ,Hierococcaceae Enterococci .. Escherichia--Aerobac tcr _ ;wicrococcace8,e -+ Eschcrichia-Aerobac1.er. .iVIicrococcaceae + panwolon J\ficrococcaceae + ;'1.lph,1 hemolyiic streptococci l\Iicrococca.ceae Bei.n. hemolytic streptococci. '\i[icrococca.ceae diphtheroids l\Iierococcn.ceae Grnrn positive bacilli Micrococcaceae Bacterium anitratnm Micrococca.c()ae enteror;occi. Total
+ + + + +
of toiaf
0 0
()
0
2
22
4~
()
2
2 --i
18
2 2
-±
2 2
2
2 2
2 5 11
(i
cl
10 82
\7
of those capable of cansing disease of the genitourinary tract. Bacieria rnosL frequently found were :vricrococcaceae, a family that includes staphylococci ,i.nd gram-positive cocci occurring in packetR or tetrads frequently rnfern:d to as "rnicrococei ." Although Goldstein reported that micrococci were uot, capahle of producing Pxperimeutal renal lesions in rabhit.s, lVInnger states, "The micro-cocci may in themselv(,s produce a pyuria which picture, both objectively and suhjcctively, will rnpidly be relieved under a few injections of neoarsphenarnine." Detailed cla&sification of staphylococci has not been done since the p,11.hogenicity of these organisms is not necPssarily related to pigrnent production, hcrnolysi.s, or results of the coagulase test. Levine considern the eoagula;-;e te,t Io be thP most, significant of the three methods. Other t.ypes of orgamsms isolated i11 this series: Enterococci, alpha and beta hemolytic streptoc:occi, Bacterium auitraturn, and members of the genus Escherichia or Aerobact.er (ofteu referred to as coliform organisms m· the "coliaerogenes group"), have all been rc:ported as the causr of genitourinary tract infect.ion. Table 3 shmvs that cotton swabs of 19 of a total of 21 penile meatm-:ei3 taken immediately after thorough cleansing and rinsing 11·ith disinfectant ,;]w,n~cl bacteria. Many of these organisms are potential pathogens of the ttrirnuy tract, alld in 9 of the 21 voiunt.eerP the same organisms were isolated from urmc t,hat were r;ultivated from meatal swabs. Only 2 of the JOO urine specimens examined showed abnormal cellular coutent on direct mieroscopie examination aft.er cent.rifngaljon. Both wen: from males; one showed 15-20 white blood cells per high powered field; and the other l 5 20 red blood cells per high powered field. There was no correlation bet.ween thec;e findings and bact.eriuria or 1;he slight syrnpLomatology rnenLioued under Procedure. The man whose urine contained 5,700 organisms per cubic centinwter ,vas complPt.ely asymptornatic and the cellular content of his nrine was 11nnrml.
566 TABLE
V. B. PHILPOT, JR.
3. Correlation of bacteria found in urine and meatal swabs from 21 normal males Bacteria Found in Urine
Micrococcaceae No growth Micrococcaceae Bact. anitratum. No growth. Micrococcaceae A. hem. Strep .. Micrococcaceae. Micrococcaceae A. hem. strep .. Micrococcaceae. Enterococci. Escherichia-Aerobacter .. Micrococcaceae B. hem. strep. No growth ..
+ + +
+
Bacteria Found on J\lleatal Swabs
Micrococcaceaf' JVIicrococcaceae Micrococcaceae No growth B. hemolytic streptococci Gram-positive bacilli Micrococcaceae No growth Enterococci Escherichia-Aerobacter Micrococcaceae B. hem. strep. Gram-positive bacilli
+
No. of Volunteers
6 3
2
1 2
1 1 1 1 1 l
DISCUSSION
The variations in sensitivity of the four methods listed in table 1 for detecting the presence or absence of bacteria in urine can be explained at least partially on the mechanics of handling the urine. Ordinary glass centrifuge tubes have been broken in this laboratory when spun at speeds greater than 3,000 r.p.m. It is doubtful that at this speed for a period of 10 minutes all the bacteria present in 10.0 cc of urine will be completely sedimented. Even if one assumes, however, that all the bacteria present in the original specimen are concentrated in the sediment, other factors still must be taken into consideration. The usual procedure in most laboratories is to decant the supernatant fluid leaving approximately 0.2--0.5 cc of sediment. A platinum loop measuring 5 111111. in diameter will transfer approximately 0.003 cc of water. Depending upon such variable factors as viscosity of urinary sediment and the angle at which the loop is held, an ordinary platinum loop may not transfer more than .003/.2 or 1.5 per cent of the bacteria contained in the original urine sample. One cubic centimeter of uncentrifuged urine will, on the other hand, contain 10 per cent of a 10.0 cc sample. Bacteria adhering to the platinum loop are much more likely to be removed when immersed into a liquid broth than when one side of the loop is streaked on a solid agar plate. Therefore, even if the nutritional factors of all media are the same, methods employing 1.0 cc of uncentrifuged urine should be more likely to reveal bacteria, than methods employing a loopfu] of centrifuged urine and when the latter method is employed liquid media should reveal a higher percentage of positives than solid. Variations in methodology probably account for some of the discrepancies in the literature. The overwhelming majority of specimens reported herewith would undoubtedly have been "sterile" if only a loopful of uncentrifuged urine had been streaked on a blood agar plate. The present work is consistent with that of Levine who reported, "it is evident that the presence of staphylococci both coagulase positive and coagulase negative, on all media is associated i.n most cases with leukocytes and erythro-
BACTJ,;HIAL FLORA OF URINE SPE:CDIIDNS
5ti7
cytes in the urine .... vVe see that both the coagulase positive and tlegati.ve 8trains when on liquid media alone. are associated with fe,v or 110 leukocytes or erythrocytes in the urine as well as with few cases of albuminuria.'' LevinE inoculated a loopful of urine on 5 per cent human blood agar and eosin mothykne blue agar while he placed 0,5--1.0 cc in liver peptone broth. The latter met.hod is much 1nore likely to reveal presence of bacteria in small nurnber than is the method for inoculating the two solid media. McGlane and Trout 3 studied the urine of 30 obstetrical patients duriug various stage8 of their antenatal and puerperal periods. Of 115 caLheterized urine specirnens obtained from the right ureter of these patients, 15 per cent showed bacteria and of 58 specimens of bladder urine, 55 per cent showed bacteria. Since the ureteral urine was plated directly on an ascitic agar slant and the bladder urine (the amount ,vas not sta.ted) was cultured in beef infusion broth, the variations in the percentage of specimens in the two groups showing bacteria, may be due to variation in technique rather than source from which nrines were derived, 'J'he species Bacterium auitra.tum was described in 1H48 by Schaub and Haubi11ger as a gram-negative, nonmotile rod capable of fermenting 10 per cent lactose, glucose, and galactose. It does not ferment l per cent lactose or other carbohydrates used in the identification of gram-negative bacilli. Schaub and I-Iaubinger isolated this organism from nine urine specimens, five of which were from individuals known to have genitourinary tract infection. Flicitories were not available on the other four. Bacterium anitratum was also found in pleural fiuid, pericardial fluid, heart blood, lungs, and cervix. The dernons1ration of ihis organism in urine from two nonnal individuals emphasizes that its isolat.ion from urine is not per 8C diagnostic of infection. It is not known whether the bacteria isolated from the urine of normal individuals comes from the urinary tract or represents contaminant,, frorn the external urethral orifice. Table B shmvs that the meatus of the male rwuis has noL been sterilized by the cleansing operation employed prior to thP voiding of urme. Seastone reporiPd that saline washings of the hands clea11sed with the best available snrgical tec:hniqucs contain bacteria. It is not likely that the g1an8 peuis can he morn easily 8tcrilized than human hands and urine passing through a contaminated meatus is just as likely to contain bacteria as is saline passing over contaminated hands. The correlation of bacteria found on the meatus with that found in the urine further suggests contamination. ::VIorse reports that Corynebacterium renale, a causative agent of infectious cyc1titis and pyelonephri-tis of cattle, was isolated from the urine of l7J\ per cent of fi23 appare11tly normal dairy cattle. Vaginal or penile swabs of 108 normal cattle rnade imnwdiateJy after slaughter proved positive for C. renale in 4 instances, but no diphi.heroid bacilli were isolatPd from the bladder urine. Regardless of y,:hether the bacteria reported herewith. represent contamina11ts or inhabitants of the genitourinary tract, the fact that they were present in a certain prr cent of urme samples collected from apparently healthy indi,iduals
568
V. B. PHILPOT, JR.
by methods widely accepted as satisfactory, must be recognized in the clinical interpretation of urine cultures. The length of time and temperature of the storage of these specimens must also be considered. SUMMARY AND CONCLUSIONS
Bacteria were found in 82 per cent of 50 midstream voided specimens from normal human males and 34 per cent of 50 catheterized specimens from normal human females when 1 cc of urine was placed in a plate count agar pour plate. ·when a loopful of centrifuged urine was placed in 1 per cent lactose broth, 52 per cent of the specimens from males showed bacterial growth and only 22 per cent were positive when a loopful of centrifuged urine was streaked on a blood agar plate. Bacteria were seen on Gram's stain of centrifuged urine from one of the 50 specimens from males and two from_ females. Many of the types of bacteria isolated have been reported as pathogenic for the genitourinary tract. It is concluded that the demonstration of potentially pathogenic bacteria in a properly collected urine sample is not in itself diagnostic of infection. Since bacteria in most of the urine samples studied in this series were present in small numbers, the bacteriologist may be of additional aid to the clinician by giving him some idea of the number of bacteria present in a given sample of urine within a specified time limit as well as identifying species and determining sensitivities. Direct microscopic examination of urine stained by Gram's method is of considerable value in this regard since bacteria seen in uncentrifuged urine by this procedure are probably present in numbers of 100,000 per cubic centimeter or greater. REFERENCES BREED, R. S. AND DOTTERER, W. D.: The number of colonies allowable on satisfactory agar plates. J. Bact., 1: 321-331, 1916. CooK, E. N.: Symptoms, diagnosis and treatment of infections of the urinary tract. Med. Clin. N. Am., 33: 1071-1078, 1949. GOLDSTEIN, M.: Pathogenicity of cocci (Micrococcus, Staphylococcus aureus, and Streptococcus faecalis) isolated from urine. J. Urol., 41: 237-250, 1939. GouLD, J. C.: The bacteriological approach to the treatment of nontuberculous urinary infection. Edinburgh Med. J., 58: 62-71, 1951. KLEINSCHMIDT, A.: Die bedeutungslosigkeit der reinan bakteriurie ohne pyurie bei weiblicken krankan. Deutsch. Med. Wochenschr., 65: 11-13, 1939. LAZARUS, J. A. AND SCHWARZ, L. H.: Importance of precise bacteriologic data in treatment of infections of the urogenital tract. Surgery, 21: 713-719, 1947. LEVINE, M. AND CREEVY, C. D.: Pathogenicity of staphylococci isolated from urine. J. Urol., 47: 515-521, 1942. McCLANE, C. M. AND TROUT, H. F.: Relationship between infected urine and etiology of pyelitis in pregnancy. Am. J. Ob. and Gyn., 33: 828-834, 1937. MORSE, E. V.: An ecological study of Corynebacterium renale. Cornell Vet., 40: 178-187, 1950. MUNGER, A. D.: Newer concepts in the diagnosis and treatment of urinary tract infections. J. Iowa ::\Ied. Soc., 32: 112-115, 1942. SCHAUB, I. G. AND HA UBER, F. D.: A biochemical and serologic study of a group of identical unidentifiable gram-negative bacilli from human sources. J. Bact., 56: 379-385, 1948. SEASTONE, C. V.: Observations on the use of G-11 in the surgical scrub. Surg. Gynec. & Obst., 84: 355-360, 1947.
Vol. 75, No. 3, March 1956 Printed in U.S.A.
THE BACTERIAL FLORA OF URINE SPECIMENS FROM NORMAL ADULTS V. B. PHILPOT, JR.
From the Department of Pathology, University Hospitals, Madison, Wis.
There are inconsistencies in the literature regarding the diagnostic significance of bacteria found in urine properly collected for bacteriological examination. Gould states that "Organisms isolated on bacteriological examination are significant when the methods of collecting the urine are· satisfactory and the technique of the laboratory is of high standard. Under these conditions, no organisms are isolated from the urine of healthy persons and patients who have no disease of the urinary tract." Kleinschmidt, on the other hand, reported that thirty-five (17 per cent) of 200 females studied were found to have bacteriuria not associated with urinary tract disease. Approximately one third of this group of thirty-five were completely healthy and asymptomatic in every respect. Lazarus stresses the importance of detailed bacteriological examination of urine requiring culture methods for species identification. Cook states, "The general practitioner as well as the specialist can satisfactorily examine, diagnose, and treat the great majority of the patients he sees by using Gram's stain of the urinary sediment to ascertain the general type of infection present." All of the aforementioned authorities agree that urine from females should be obtained by the use of a sterile catheter and the three (Cook, Lazarus, Gould) who dealt with males agree that a midstream voided specimen from males after proper cleansing of the external genitalia is satisfactory. The differences in opinion, therefore, reflect variations in bacteriological technique and interpretation. In order to establish a base line of normals that will aid in determining the diagnostic significance of bacteria in the urine and clarifying the above mentioned disagreements, a study has been made of the bacterial flora of urine from 100 normal adults, 50 of which were male and 50 female. PROCEDURES
The volunteers whose urine was studied varied in age from 18 to 67 years, but the majority were between 20 and 30 years. Fourteen of the total number had mild symptoms of urinary tract disorder such as occasional burning on urination or nocturia, but none of them were sufficiently annoyed to seek medical attention or to alter their daily routine of activities. The remainder (86) were found to be completely free of symptoms of urinary tract disease after careful questioning and were actively engaged in employment. Procedure for collecting urine samples from males was as follows: 1) The foreskin was reflected backwards and the glans penis rinsed in tap water. Accepted for publication July 26, 1955. This work was done with the help and advice of W. D. Stovall, M.D., Dorothy Colien, Ph.D., and Miss Ann Knell, and other members of the staff of the University Hospitals. 562
BACTli;lUAL FLOHA OF URINE SPECIMENS
:!) The glans ,Yas washed carefully with tincture of green soap mid a sterile sponge, and rinsed 1vith a 1- .5000 aqueous solution of potnssium mercuric: 10dich A.t this point the penile meatus of 21 volunteers v,as swabbed with n, ::;terile cotton applicator which was then immersed in J per cent lactose broth and incubated at 87°C. The eultures were examined at n,.,,,,,""' of 24 and 48 hour,;; after inoculation. :3) From :30 to lOO cc of urine was voided and discarded. Approximalely :30 ec of bladder urine vvas then colleeted in a sterile contamer and cultures ~taned within two hours. All specimens from males were collected the author t,rJ ascertain that standard methods were observed in each case. The procedure of collecting samples from females was a8 follmrn: 1) Using titerile rubber the labia ·were spread apart exposing llw external urethral orifice. 2) The exposed area was washed with a sterile sponge and tincture of green soap (diluted 1 :4 in sterile water to avoid irritation) and ri11sed 1Yith a I 5000 aqueous solution of potassium mercuric iodide. 3) A sterile -···~·"'"" rnbber catheter was inserted mto tbe bladder and approximately 30 cc of urine collected in a sterile container. Cult.urP;-; \YPre started within 2 hours. All specimens from females were collected one well quaJi[ird uurse who used the same procedure on all volunteers. One ml. of each urine sample was placed in a pour plate with Bacto plate ,,ount agar containing yeast extract, glucose, and tryptose, and buffered at pH 7.2. Ten ml. of each urine sample were centrifuged at :-s,000 .p.m. for 10 mi11· utes and the residue examined for cellular content and stained ({ram's method for bacteria. fo addition, a loopful of the sediment of centrifuged urine specimens from males was placed in 1 per cent lactose broth with brom blue indicator and another loopful streaked out on a blood agar Initial agar plate cultures were incubated at ::l7°C for 2-t to 28 hours. Lactose broth ,ms held for a total of 48 hours before it was considered negative. The term, lVIicrococcaceae refers to the family of bacteria that i11elmle:,; th0 genera Micrococcus (Staphylococcus), Gaffkya, and Sanana. These s,vere identified on the basis of colonial morphology on a blood agar plate and <'ellnlar morphology after Gram staining. All streptococci recovered were placed in 1 per cent glucose broth ovt:rnight and transferred to ti.ti per cent sodium chloride broth at pH 7.2. Organisirn, grmving 011 the latter medimn 1rnre classified as enterococci. Streptococci I.hat failed to grow in (\.5 per cm1t sodium chloride broth were classified on the ha,~is of h<-m10lysis on a blood agar plate. Gram-negative rods that ferment 1 per cent lactose broth ,rnre clac1silied as members of the genus Escherichia or Aerobacter. The two strains of gramnegative Bacterium anitratum were identified on tho ba::;is of the charnc:tcristic fermentation reaction,, particularly their ability to ferment JO per erni" !mt not 1 per cent lactrn,e.
,564
V. B. PHILPOT, JR.
TAB LE
1. N wnber of urine specimens from 100 normal volunteers showinu ·.bacteria
by various techniques ·-
Specimens from 50
Method
l. Plate count agar 1-30 colonies/cc .. 30-400 colonies/cc Over 400 colonies/cc
Total.
Males Showing Bacteria
Specimens from 50 Females Showing Bacteria
Number
% of total
Number
% of total
27 10 4
54 20 8
14 3 0
28 6 0
41
82
17
34
26
52
not done
-
2. 1% lactose broth.
not done
--
3. Blood agar streak plate .. Less than 16 colonies per plate .. Over 16 colonies per plate .. Total.
11 1 12
22 2 24
1
2
not done
-----·--·-
·---
4. Gram stain.
2
4
RESULTS
As shown in table 1, 82 per cent of midstream urine specimens from 50 apparently normal males showed bacteria, when 1.0 cc of undiluted urine was cultured in a pour plate with plate count agar. Only 28 per cent showed more than 30 organisms per cubic centimeter and the highest number found in a single specimen was 5,700. This figure can be regarded as only an approximation since accurate plate counts of bacteria can be obtained only when the number of bacteria is within the range of 30-400 per plate. Using a loopful of centrifuged urine as an inoculum, bacteria were recovered from 52 per cent of urine specimens from males cultured in 1 per cent lactose broth, and from only 24 per cent of specimens streaked on a blood agar plate. The single specimen containing bacteria revealed by Gram's stain of centrifuged urine was the one showing approximately 5,700 organisms per cubic centimeter. Organisms seen by direct examination of uncentrifuged urine are probably present in numbers of 100,000 per cubic centimeter or greater. Table 1 also shows that a catheterized urine specimen from a normal female is much more likely to be sterile than is a midstream specimen from a normal male. By the most sensitive method of detecting bacteria (plate count) only 34 per cent of urine specimens from females contained bacteria in contrast to 82 per cent from males. The average number of bacteria per cubic centimeter from females is also significantly less than that from males. On direct microscopic exarnination of centrifuged urine from females, bacteria were seen in only 2 of the 50: gram-positive cocci in one and gram-positive bacilli in the other. Neither of these organisms grew on plate count agar indicating that although it ·was the most sensitive of the media used in these experiments, plate count agar is by no means a perfect medium for the growth of all bacteria. The organisms recovered from "normal" urine listed in table 2 include many
TABLE
2. Surnl>er of -unne samples frmn 100 normal iJolunt,,ers .,howinu uan,,,,.., types of bacteria !vJalcs r,Total 50) Type of Bacteria
i
No. ol _____ ] specimens
Betlt hemolytic strnptococci Alpha, hemolytic ~I n•ptococci ,Hierococcaceae Enterococci .. Escherichia--Aerobac tcr _ ;wicrococcace8,e -+ Eschcrichia-Aerobac1.er. .iVIicrococcaceae + panwolon J\ficrococcaceae + ;'1.lph,1 hemolyiic streptococci l\Iicrococca.ceae Bei.n. hemolytic streptococci. '\i[icrococca.ceae diphtheroids l\Iierococcn.ceae Grnrn positive bacilli Micrococcaceae Bacterium anitratnm Micrococca.c()ae enteror;occi. Total
+ + + + +
of toiaf
0 0
()
0
2
22
4~
()
2
2 --i
18
2 2
-±
2 2
2
2 2
2 5 11
(i
cl
10 82
\7
of those capable of cansing disease of the genitourinary tract. Bacieria rnosL frequently found were :vricrococcaceae, a family that includes staphylococci ,i.nd gram-positive cocci occurring in packetR or tetrads frequently rnfern:d to as "rnicrococei ." Although Goldstein reported that micrococci were uot, capahle of producing Pxperimeutal renal lesions in rabhit.s, lVInnger states, "The micro-cocci may in themselv(,s produce a pyuria which picture, both objectively and suhjcctively, will rnpidly be relieved under a few injections of neoarsphenarnine." Detailed cla&sification of staphylococci has not been done since the p,11.hogenicity of these organisms is not necPssarily related to pigrnent production, hcrnolysi.s, or results of the coagulase test. Levine considern the eoagula;-;e te,t Io be thP most, significant of the three methods. Other t.ypes of orgamsms isolated i11 this series: Enterococci, alpha and beta hemolytic streptoc:occi, Bacterium auitraturn, and members of the genus Escherichia or Aerobact.er (ofteu referred to as coliform organisms m· the "coliaerogenes group"), have all been rc:ported as the causr of genitourinary tract infect.ion. Table 3 shmvs that cotton swabs of 19 of a total of 21 penile meatm-:ei3 taken immediately after thorough cleansing and rinsing 11·ith disinfectant ,;]w,n~cl bacteria. Many of these organisms are potential pathogens of the ttrirnuy tract, alld in 9 of the 21 voiunt.eerP the same organisms were isolated from urmc t,hat were r;ultivated from meatal swabs. Only 2 of the JOO urine specimens examined showed abnormal cellular coutent on direct mieroscopie examination aft.er cent.rifngaljon. Both wen: from males; one showed 15-20 white blood cells per high powered field; and the other l 5 20 red blood cells per high powered field. There was no correlation bet.ween thec;e findings and bact.eriuria or 1;he slight syrnpLomatology rnenLioued under Procedure. The man whose urine contained 5,700 organisms per cubic centinwter ,vas complPt.ely asymptornatic and the cellular content of his nrine was 11nnrml.
566 TABLE
V. B. PHILPOT, JR.
3. Correlation of bacteria found in urine and meatal swabs from 21 normal males Bacteria Found in Urine
Micrococcaceae No growth Micrococcaceae Bact. anitratum. No growth. Micrococcaceae A. hem. Strep .. Micrococcaceae. Micrococcaceae A. hem. strep .. Micrococcaceae. Enterococci. Escherichia-Aerobacter .. Micrococcaceae B. hem. strep. No growth ..
+ + +
+
Bacteria Found on J\lleatal Swabs
Micrococcaceaf' JVIicrococcaceae Micrococcaceae No growth B. hemolytic streptococci Gram-positive bacilli Micrococcaceae No growth Enterococci Escherichia-Aerobacter Micrococcaceae B. hem. strep. Gram-positive bacilli
+
No. of Volunteers
6 3
2
1 2
1 1 1 1 1 l
DISCUSSION
The variations in sensitivity of the four methods listed in table 1 for detecting the presence or absence of bacteria in urine can be explained at least partially on the mechanics of handling the urine. Ordinary glass centrifuge tubes have been broken in this laboratory when spun at speeds greater than 3,000 r.p.m. It is doubtful that at this speed for a period of 10 minutes all the bacteria present in 10.0 cc of urine will be completely sedimented. Even if one assumes, however, that all the bacteria present in the original specimen are concentrated in the sediment, other factors still must be taken into consideration. The usual procedure in most laboratories is to decant the supernatant fluid leaving approximately 0.2--0.5 cc of sediment. A platinum loop measuring 5 111111. in diameter will transfer approximately 0.003 cc of water. Depending upon such variable factors as viscosity of urinary sediment and the angle at which the loop is held, an ordinary platinum loop may not transfer more than .003/.2 or 1.5 per cent of the bacteria contained in the original urine sample. One cubic centimeter of uncentrifuged urine will, on the other hand, contain 10 per cent of a 10.0 cc sample. Bacteria adhering to the platinum loop are much more likely to be removed when immersed into a liquid broth than when one side of the loop is streaked on a solid agar plate. Therefore, even if the nutritional factors of all media are the same, methods employing 1.0 cc of uncentrifuged urine should be more likely to reveal bacteria, than methods employing a loopfu] of centrifuged urine and when the latter method is employed liquid media should reveal a higher percentage of positives than solid. Variations in methodology probably account for some of the discrepancies in the literature. The overwhelming majority of specimens reported herewith would undoubtedly have been "sterile" if only a loopful of uncentrifuged urine had been streaked on a blood agar plate. The present work is consistent with that of Levine who reported, "it is evident that the presence of staphylococci both coagulase positive and coagulase negative, on all media is associated i.n most cases with leukocytes and erythro-
BACTJ,;HIAL FLORA OF URINE SPE:CDIIDNS
5ti7
cytes in the urine .... vVe see that both the coagulase positive and tlegati.ve 8trains when on liquid media alone. are associated with fe,v or 110 leukocytes or erythrocytes in the urine as well as with few cases of albuminuria.'' LevinE inoculated a loopful of urine on 5 per cent human blood agar and eosin mothykne blue agar while he placed 0,5--1.0 cc in liver peptone broth. The latter met.hod is much 1nore likely to reveal presence of bacteria in small nurnber than is the method for inoculating the two solid media. McGlane and Trout 3 studied the urine of 30 obstetrical patients duriug various stage8 of their antenatal and puerperal periods. Of 115 caLheterized urine specirnens obtained from the right ureter of these patients, 15 per cent showed bacteria and of 58 specimens of bladder urine, 55 per cent showed bacteria. Since the ureteral urine was plated directly on an ascitic agar slant and the bladder urine (the amount ,vas not sta.ted) was cultured in beef infusion broth, the variations in the percentage of specimens in the two groups showing bacteria, may be due to variation in technique rather than source from which nrines were derived, 'J'he species Bacterium auitra.tum was described in 1H48 by Schaub and Haubi11ger as a gram-negative, nonmotile rod capable of fermenting 10 per cent lactose, glucose, and galactose. It does not ferment l per cent lactose or other carbohydrates used in the identification of gram-negative bacilli. Schaub and I-Iaubinger isolated this organism from nine urine specimens, five of which were from individuals known to have genitourinary tract infection. Flicitories were not available on the other four. Bacterium anitratum was also found in pleural fiuid, pericardial fluid, heart blood, lungs, and cervix. The dernons1ration of ihis organism in urine from two nonnal individuals emphasizes that its isolat.ion from urine is not per 8C diagnostic of infection. It is not known whether the bacteria isolated from the urine of normal individuals comes from the urinary tract or represents contaminant,, frorn the external urethral orifice. Table B shmvs that the meatus of the male rwuis has noL been sterilized by the cleansing operation employed prior to thP voiding of urme. Seastone reporiPd that saline washings of the hands clea11sed with the best available snrgical tec:hniqucs contain bacteria. It is not likely that the g1an8 peuis can he morn easily 8tcrilized than human hands and urine passing through a contaminated meatus is just as likely to contain bacteria as is saline passing over contaminated hands. The correlation of bacteria found on the meatus with that found in the urine further suggests contamination. ::VIorse reports that Corynebacterium renale, a causative agent of infectious cyc1titis and pyelonephri-tis of cattle, was isolated from the urine of l7J\ per cent of fi23 appare11tly normal dairy cattle. Vaginal or penile swabs of 108 normal cattle rnade imnwdiateJy after slaughter proved positive for C. renale in 4 instances, but no diphi.heroid bacilli were isolatPd from the bladder urine. Regardless of y,:hether the bacteria reported herewith. represent contamina11ts or inhabitants of the genitourinary tract, the fact that they were present in a certain prr cent of urme samples collected from apparently healthy indi,iduals
568
V. B. PHILPOT, JR.
by methods widely accepted as satisfactory, must be recognized in the clinical interpretation of urine cultures. The length of time and temperature of the storage of these specimens must also be considered. SUMMARY AND CONCLUSIONS
Bacteria were found in 82 per cent of 50 midstream voided specimens from normal human males and 34 per cent of 50 catheterized specimens from normal human females when 1 cc of urine was placed in a plate count agar pour plate. ·when a loopful of centrifuged urine was placed in 1 per cent lactose broth, 52 per cent of the specimens from males showed bacterial growth and only 22 per cent were positive when a loopful of centrifuged urine was streaked on a blood agar plate. Bacteria were seen on Gram's stain of centrifuged urine from one of the 50 specimens from males and two from_ females. Many of the types of bacteria isolated have been reported as pathogenic for the genitourinary tract. It is concluded that the demonstration of potentially pathogenic bacteria in a properly collected urine sample is not in itself diagnostic of infection. Since bacteria in most of the urine samples studied in this series were present in small numbers, the bacteriologist may be of additional aid to the clinician by giving him some idea of the number of bacteria present in a given sample of urine within a specified time limit as well as identifying species and determining sensitivities. Direct microscopic examination of urine stained by Gram's method is of considerable value in this regard since bacteria seen in uncentrifuged urine by this procedure are probably present in numbers of 100,000 per cubic centimeter or greater. REFERENCES BREED, R. S. AND DOTTERER, W. D.: The number of colonies allowable on satisfactory agar plates. J. Bact., 1: 321-331, 1916. CooK, E. N.: Symptoms, diagnosis and treatment of infections of the urinary tract. Med. Clin. N. Am., 33: 1071-1078, 1949. GOLDSTEIN, M.: Pathogenicity of cocci (Micrococcus, Staphylococcus aureus, and Streptococcus faecalis) isolated from urine. J. Urol., 41: 237-250, 1939. GouLD, J. C.: The bacteriological approach to the treatment of nontuberculous urinary infection. Edinburgh Med. J., 58: 62-71, 1951. KLEINSCHMIDT, A.: Die bedeutungslosigkeit der reinan bakteriurie ohne pyurie bei weiblicken krankan. Deutsch. Med. Wochenschr., 65: 11-13, 1939. LAZARUS, J. A. AND SCHWARZ, L. H.: Importance of precise bacteriologic data in treatment of infections of the urogenital tract. Surgery, 21: 713-719, 1947. LEVINE, M. AND CREEVY, C. D.: Pathogenicity of staphylococci isolated from urine. J. Urol., 47: 515-521, 1942. McCLANE, C. M. AND TROUT, H. F.: Relationship between infected urine and etiology of pyelitis in pregnancy. Am. J. Ob. and Gyn., 33: 828-834, 1937. MORSE, E. V.: An ecological study of Corynebacterium renale. Cornell Vet., 40: 178-187, 1950. MUNGER, A. D.: Newer concepts in the diagnosis and treatment of urinary tract infections. J. Iowa ::\Ied. Soc., 32: 112-115, 1942. SCHAUB, I. G. AND HA UBER, F. D.: A biochemical and serologic study of a group of identical unidentifiable gram-negative bacilli from human sources. J. Bact., 56: 379-385, 1948. SEASTONE, C. V.: Observations on the use of G-11 in the surgical scrub. Surg. Gynec. & Obst., 84: 355-360, 1947.