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Effect of Combined Therapy on Emergence of Drug Resistant Bacteria in Urinary Tract Infections: Observations on Origin of Resistant Strains
THE JOURNAL OF UROLOGY
Vol. 65, No. 4, April 1951
Printed in U.S .A.
EFFECT OF COMBINED THERAPY ON EMERGENCE OF DRUG RESISTANT BACTERIA IN URINARY TRACT INFECTIONS: OBSERVATIONS ON ORIGIN OF RESISTANT STRAINS LEONARD J. KIMMELMAN, HANS H. ZINSSER
AND
MORTON KLEIN
From the Department of Bacteriology, University of Pennsylvania School of Medicine, the Department of Urology (Surgery) of the Hospital of the University of Pennsylvania, and the Genito-Urinary Service of the Philadelphia General Hospital, Philadelphia
The development of resistance of bacteria to chemotherapeutic and antibiotic agents has been a limiting factor in therapy. 1 The problem of the development of resistance to streptomycin had been a particularly serious one. It was found in vitro that such resistance could be inhibited by the combined use of streptomycin, penicillin and sulfadiazine. 2 Therapeutically, the overall effect of two or more antibacterial agents has frequently been found superior to that obtained with one drug3 and some clinical studies have been carried out indicating that this therapeutic effect was correlated with the inhibition of drug resistance by combined therapy. 4 Development of resistance has been a common finding in the treatment of urinary tract infections with streptomycin. 6 In the present study we were concerned with 1) the effectiveness of combined streptomycin, penicillin and sulThis investigation was supported (in part) by a research grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service, and also from the Theresa F. Felsen Memorial Fund. The streptomycin for the patients was supplied by E. R. Squibb and Sons, Inc., and by Merck and Co., Inc. · The authors wish to thank Dr. Stuart Mudd for his continued interest and helpful criticism during the course of this study and preparation of the manuscript. 1 Buggs, C. W., Bronstein, B., Hershfeld, J. W. and Pilling, M . A.: J. A. M.A., 130: 64, 1946. Finland, M., Murray, R., Hartis, H. W., Kilham, L ., and Meads, M.: J . A. M.A., 132: 16, 1946. Youmans, G. P., Williston, E. H., Feldman, W. H. and Hinshaw, H. C.: Proc. Staff Meet. Mayo Clin., 21: 126, 1946. Alexander, H. E., Leidy, G., Rake, G. and Donovick, R.: J. A. M.A., 132: 434, 1946. Hall, W. H. and Spink, W.W.: Proc . Soc. Exper. Biol. & Med., 64: 403, 1947. 2Klein, M. and Kimmelman, L. J.: J. Bact., 54: 363, 1947. Pulaski, E. J. and Baker, H.J.: J. Lab. & Clin . Med., 34: 186, 1949. 8 Dowling, H . F., Hussey, H. H ., Hirsh, H. L. and Wilhelm, F.: Ann . Int. Med., 25: 950, 1946. Eisele, C. W. and MacCullough, N. B . : J. A. M . A., 135: 1053, 1947. MacLean, J. T. and Smith, F.: Canad. M.A. J., 57: 131, 1947. Lincoln, E . M., Kirmse, T. W. and DeVito, E.: J. A. M.A., 136: 593, 1948. Kolmer, J. A.: Am. J.M. Sc., 215: 136, 1948. Thatcher, F. S. and MacLean, J. T.: J. Urol., 57: 902, 1947 . .. Cocchi, E. and Pasquinucci, G.: Quoted in Pfuetze, K. and Myle, M.: J . A. M.A., 139: 634, 1949. b Spink, W.W., Hall, W. H., Shaffer, J. and Braude, A. I.: J. A. M.A., 139: 352, 1949. • Delaude, A., Karlson, A.G., Carr, D. T., Feldman, W. H. and Pfeutze, K. H.: Proc. Staff Meet. Mayo Clin ., 24: 342, 1949. d Karlson, A.G., Pfeutze, K. H., Carr, D. T., Feldman, W. H. and Hinshaw, H. C .: Proc. Staff Meet. Mayo Clin., 24: 85, 1949. 6 Bondi, A., Ottenberg, D., Dietz, C. C. and Brown, C . L. : J . A. M.A., 132: 634, 1946. Adcock, J. D . and Plumb, R. T. : J. A. M.A., 133: 579, 1947. Harrell, G. T., Herndon, E.G., Gillikin, C. M. and Aikawa, J. K .: J . Clin. Investigation, 26: 577, 1947. 668
DRUG RESISTAN'l' BACTERIA IN URINARY TRACT
669
fonamide in inhibiting the development of resistant strains during the treatment of urinary infections, and 2) the origin of the resistant strains in the urinary tract. MATERIALS AND METHODS
Cases and drug dosages.-A total of 37 cases from the urological services of the Hospital of the University of Pennsylvania and the Philadelphia General Hospital were studied. These included both uncomplicated urinary tract infections (cystitis, pyelonephritis, urethritis, etc.) and those complicated by additional pathological changes, such as calculi, strictures and obstructions. All cases, unless otherwise indicated, had not received previous therapy with streptomycin, penicillin or sulfonamides as far as could be determined. AH patients (unless otherwise indicated) received the following daily dosage for a total of 7 days: streptomycin, 1.0 gm., intramuscularly; penicillin, 200,000 units, intramuscularly; sulfadiazine, 2.0 gm., orally. Alkali was not routinely given (except for patients numbers 11, 25, 27, 29 (tables ]. and 2) and the pH of the urines varied between 5.5 and 6.0. As will be indicated later, therapeutic response to combined therapy was excellent in all the simple urinary tract infections, in the presence or absence of alkalL When strains of Proteus were present in the urine, the pH varied between 8.0 and 8.2. Leukocyte counts, hemoglobin determinations, and microscopic urine analyses were followed during therapy and for 3 days to several :months after therapy. Urine specimens were obtained using aseptic precautions. In the male, preliminary sponging of the meatus or catheter tip with 70 per cent alcohol was carried out and samples were collected after urine had first rinsed the alcohol away. Samples were collected from the female by catheterization after perineal preparation. Urines were obtained before therapy and every 48 hours during the 7 days of therapy. Laboratory procedures. Isolations: All specimens were centrifuged at high speed in an angle centrifuge for 20 minutes, and the supernate was discarded. If the specimen ·was taken during therapy, the sediment was washed in ssJine to remove any residual drugs. The washing procedure was found to eliminate the necessity of using drug-antagonists in the medium, since the addition of penicillinase, P ABA and cysteine to the medium did not increase the amount of above that obtained from washed residues. Highly susceptible organisms were v"'"·""-"'-' isolated from washed specimens obtained from urines containing streptomycin, penicillin and sulfadiazine. All specimens were 1) gram stained, 2) inoculated into beef extract broth, 3) streaked on brom thymol blue lactose agar or MacConkey's agar, and blood extract agar ·when gram positive cocci were observed in smear. All cultures were incubated aerobically at 37 C. Since mixed coliform, infections regularly occur in urinary tract infections, 4a, 6 at least 5 colonies of each coliform colony type were picked and identified. When the organisms were identified, they were placed on extract agar under sterile, heavy mineral oil for subsequent study of drug resistance. Susceptibility tests: After completion of therapy, tests on all the organisms
670
L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
from each case were done simultaneously to determine the degree of susceptibility to each of the drugs. Dilutions of streptomycin (10 µ gm./ml. to 500 µ TABLE
1. Bacteriological summary of urinary tract infections. Cases in which negative cultures were obtained after therapy a IN VITRO SUS-
CASE
NO.
DIAGNOSIS
ORGANISMS ISOLATED BEFORE THERAPY
CEPTIBILITY TO
SM.
--
P.
--
SD.
1
Cystitis; pyelonephritish
E. coli Staphylococcus aureus
s R s s s R
2 3
Cystitis Cystitis Pyelonephritis (right) Pyelonephri tis Bilateral renal calculi Renal (staghorn) calculi Cystitis; pyuria Acute right pyelonephritis Cystitis Acute cystitis (post-cystoscopy) Epididymitis Cystitis and prostatitis
E.coli E.coli Paracol. coliforme A. aerogenes E.coli Kl. pneumoniae E. coli E.coli Staphylococcus albus E . coli
s s s s s s s s s
4
5 6 7 9 10 11* 12
13
14 15 16 17
Acute pyelonephritis Acute cystitish Chronic pyelonephritis and glomerular nephritis
Paracol. aerogenoides E.coli Paracol. coliforme E . coli E. coli Alpha hemolytic streptococcus
BACTERIOWGY
AFTER THERAPY
R
R R
R R R R R R R R
s R s R s R s R s R s s
s s s s s 0
s s s R
s s s s s 0
negative (Staphylococcus-resist. to SM. and SD .)• negative negative negatived negative negative negative negative negative negative negative negative negative negative negative• negative 1
* Received sodium bicarbonate 5.0 gm. every six hours in addition to the three agents . • Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P., penicillin; and SD ., sulfadiazine. 0 = no growth in medium (casein hydrolysate); S = susceptible; and R = resistant. b Previous therapy given: case 16 received penicillin and case 1, penicillin and sulfadiazine. • Probable contaminant; only few scattered colonies. d Paracolobactrum coliforme persisted for more than four days, still susceptible to streptomycin and sulfadiazine, and eventually disappeared. • E . coli recovered one month later, still suscept ible, and retreated wit h permanent success. 1 Streptomycin-susceptible alpha hemolytic streptococcus isolated two weeks later. Note: Case 8 was omitted because no organisms were isolated before or after therapy.
gm./ ml.) and penicillin (0.1 unit/ml. to 5.0 units/ml.) were made in beef extract broth pH 7.2 and seeded with 0.1 ml. of 10-2 dilution of a 20-24 hour brot h culture of the organism. A casein hydrolysate medium was used for determining
671
DRUG RESISTANT BACTERIA IN URINARY TRACT TABLE
2. Bacteriological summary of urinary tract infections. Cases in which resistant organisms were found after therapy a IN VITRO SUS-
CASE
NO.
DIAGNOSIS
ORGANISMS ISOLATED BEFORE THERAPY
CEPTIBILITY TO
P.
SD.
s s
R
s
R
R
R
R
R
SM.
18 19
20 21 22 23
24
25*
Pyelonephritis Pyelitis; prosta ti tis
E. coli Proteus vulgaris Pseud. aeruginosa
Chronic prostatitis and cystitis Benign prostatic hypertrophy; cystitits Benign prostatic hypertrophy Chronic pyelonephritis Calculus at ureteropelvic junction Bilateral renal calculi
Non-hemolytic Streptococcus A. fecalis Staph. aureus
s
R
Ob
R
R
R
E. coli
s
R
A. aerogenes
s
R
E. coli Paracol. coliforme Proteus mirabilis Pseud. aeruginosa Proteus rettgeri A. fecalis
s s
R R
R
R
R
R
R
R
R R
R R
R R
s s s
s s s
s
Hydronephrosis (congenital band)
27*
Calculus
Proteus morgani
s
R
R
28
Renal carbuncle; pyelonephritis Renal and ureteral calculi Bilateral calculi
Anaerogenic Paracol. species A. aerogenes A. fecalis Proteus mirabilis E. coli A. aerogenes
s
s
O•
s
R
30 31
32
33
Neurogenic bladder; carcinoma; cystitis; calculus U reteral stricture; pyelonephritis Hemorrhagic cystitis; diverticulum
R
s s s
s s s s
s
R R
s
s
E. coli
s
R
s
Staph. aureus
s
R
R
m IN VITRO SUSCEPTIBILITY TO
P.
SD.
R R
R R
R
R
R R R
R R
R R
R R
R
R
R
R
A. aerogenes
R
R
R
A. aerogenes
R
R
R
A. aerogenes Proteus mirabilis Proteus mirabilis
R
R R
R
R R
R
R
Proteus rettgeri Paracol. coliforme A. aerogenes Proteus mirnbilis Anaerogenic Paracol. species A. aerogenes Proteus rettgeri Proteus morganid
R R
R R
R R
SM.
-- --
26
29*
ORGANISMS ISOLATED AFTER THERAPY
A. aerogenes Proteus rettgeri Pseud. aeruginosa A. aerogenes Non-hemolytic Streptococcus Staph. aureus Staph. aureus
-- --
R R
R
R
R
R
R
R R
R
R
O•
R R
R R R
R
R
R R R
R R R
R
R
R
Pseud. aeruginosa R A. aerogenes R
R
R R
A. aerogenes Proteus rettgeri Pseud. aeruginosa Staph. albus
s R R
R
R
s
672
L . J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
TABLE 2- Cvntinued a IN VITRO SUS-
a IN VITRO SUSCASE NO.
DIAGNOSIS
Benign prostatic hypertorphy; multiple myeloma Benign prostatic hypertrophy Post-herniorrhaphy cysti tis and/or prostatitis Acute pyelonephritis; urethral stricture
35 36
37
Benign prostatic hypertrophy; surgery
38
CEPTIBILITY TO
SM.
-34
ORGANISMS ISOLATED BEFORE THERAPY
P.
ORGANISMS ISOLATED AFTER THERAPY
SD.
SM.
P.
--
A. aerogenes
- R R R
SD.
-- - A. aerogenes R R R P aracol. aeroge- R R R
CEPTIBILITY TO
noides Staph. aureus
s
R
s
A. aerogenes
R
R
R
A. aerogenes
R
R
R
A. aerogenes
R
R
R
Proteus mirabi]is
R
R
R
Proteus mirabilis A. aerogenes Coliform-intermediate Proteus rettgeri A. aerogenes
R
R
R
R R
R R
R R
R
R
R
R
E.coli
s
R
s
O• R
* Received sodium bicarbonate in addition to the other drugs. Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P. , penicillin; and SD., sulfadiazine. S = susceptible and R = resistant. b Previous therapy given: case '2:7 received penicillin; case 36, penicillin and sulfadiazine; and case 34, a full course of streptomycin, penicillin and sulfadiazine, 3 weeks before. °ી Could not be grown in casein hydrolysate medium with special vitamin additions. d One colony only on isolation plate. Note: Cases 19, 31, 33, and 37 were from the Philadelphia General Hospital through the courtesy of Dr. Earl W. Clawater. These were given a triple sulfonamide preparation (Lilly) consisting of sulfapyrazine, sulfamerazine and sulfadiazine (0.167 gm. of each per tablet). a
sulfonamide susceptibility of the organisms. 6 All tubes were seeded with 0.1 ml. of a 10-4 dilution of a 20- 24 hour broth culture. The lowest concentration of drugs showing complete inhibition of growth after 24 hours at 37 C was taken as the inhibitory concentration. Resistant strains are arbitrarily defined in the present study as those growing in at least 25.0 µ/ml. streptomycin, 1.0 unit/ml. penicillin and 1 :25,000 sulfadiazine. RESULTS
Bacteriological findings. Comparative success in simple and complicated infections:- The results obtained in the 37 cases are shown in tables 1 and 2. In table 1 are listed the cases in which no organisms were isolated after 7 days of combined therapy. It may be noted that of 16 cases, 14 were uncomplicated urinary tract infections. The organisms generally found before therapy were 6
Strauss, E., Dingle, J. H. and Finland, M.: J. Immunol., 42: 331, 1941.
673
DRUG RESISTANT BACTERIA IN UPJNARY TRACT
gram-negative bacilli, usually susceptible to streptomycin and sulfadiazine and resistant to penicillin. Gram-positive cocci (staphylococci and enterococci) were found only occasionally in the urines. Table 2 lists the cases of bacteriological failure. Nineteen of the 21 cases were complicated by the presence of calculi, hypertrophy, strictures, etc. With one exception (case 30) in all of the 21 cases, bacteria present after 7 days' therapy were found resistant to all three drugs. It is quite evident, therefore, that in the presence of factors complicating the infectious process, combined therapy did not permanently eliminate bacteria from the urinary tract. Although success was obtained. in eliminating bacteria in 15 of the 16 cases of simple urinary tract infections (table 1), the series is too TABLE
3. Incidence of urinary strains and susceptibility to streptomycin. Isolations before and after combined therapy
ORGANISM:
BEFORE THERAPY
AFTER THRRAPV
Number of Strains
Number of strains
Susceptible
Escherichia coli .. , .......... .. .. Aerobacter aerogenes ...... . ' ..... Proteus species ..... .. , .. . . . . . . Pseudomonas aeruginosa .... ... Paracolobactrum species ... ... Staphylococcus species ... .... Alkaligenes fecalis ... ' .... . . . . Streptococcus species .. Klebsiella pneumoniae .. . . .. ... . . Coliform-intermediate. ...... . . .. .
Resistant
4
l 2•
3
3
0 5
2
15
I Total II i
Susceptible
Resistanta
16
l
0
6 6
0
14 9
2
0 0 0
6
I
3
2
Total
-lb 14 10 3, 2 3 0
5
l 0
0
3
3
X
X
2 1
0
2
0
1
1
l 0
X
X
0
0
1
1
0
0 0
,5
I
I
3d
x = No strains encountered at all after therapy. • All resistant to penicillin, streptomycin, and sulfadiazine excepting one strain of Proteus susceptible to sulfadiazine and one strain of staphylococcus susceptible to penicillin. 0 One susceptible and apparently identical strain isolated 2 weeks later, also. " One case found subsequently to have had one course of combined therapy before th.is course. d Status of one or more strains as contaminants very likely.
small to determine whether or not combined therapy is superior to the results obtained by the use of one drug. In approximately 55 per cent of all these cases the init~al flora was eliminated by the third day. This is an indication of the effectiveness of combined therapy in eliminating the initial flora, although not a measure of the eventual. bacteriological outcome. Resistant urinary strains.-The incidence of the development of resistant strains is shown in tables 1 and 2 and summarized in table 3. It is quite clear that the use of combined therapy did not consistently prevent the ultimate ape· pearance of resistant bacteria. It should be noted, however, that the originally susceptible strains were usually eliminated and the resistant strains emerging represented new species, resistant to all three drugs. In 14 of 21 cases in table 2, the resistant strains isofated after therapy were
TABLE
BEFORE THERAPY
CASE NO.
DIAGNOSIS
Urine
24
25
36
33
34
•susceptibility To SM.
-23
-Chronic pyelone- A. aerogenes' phritis; previous nephrectomy Calculus and in- E. coli fection at ure- Paracol. colitero-renal juncforme tion Bilateral renal calculi; pre vious surgery; surgery now also before therapy Post-herniorrhaphy; cystitis and prostatitis Hemorrhagic cystitis, diverticu!um; surgery after therapy Benign prostatic hypertrophy, surgery, mu!ti pie myelomah
~
4. Comparison of urinary and fecal flora before and after combined therapy
s s s
Proteus mirab- R ilis Pseud. aeru- R ginosa
A. aerogenes
Staph. aureus
R
s
P.
-
SD .
s
R R
s s
R
R
R
R
R
R R
A. aerogenes
R
R
R
Paracol. aerogenoides
R
R
R
•susceptibil:ty To SM. P.
--
R
R
Feces•
AYTEJt THERAPY
-A. aerogenes s E . coli s Coliform-ins termediate A. aerogenes s Paracol. inter- s medium Coliform-ins termediate Proteusmirab- R ilis E . coli s
A. aerogenes Coliform-intermediate E . coli A. aerogenes Coliform-intermediate Proteus vu!garis Paracol. aerogenoides
-
SD .
--
R R R
s s s
R R
s s
R
s
Urine
•susceptibility To SM.
SD.
•susceptibility To SM.
P.
SD.
---- - -A. aerogenes• R R R A. aerogenes• R R R
Proteus mirab- R ilis A. aerogenes R
Proteus mirab- R ilis
R
R
R
s
R R
R R
s
s s s
R R R
s s s
A. aerogenes Pseud . aeruginosad
R R
s
R
s
A. aerogenes
R
s
R
s
R
P.
-
Feces•
A. aerogenes
R
R
R
R
R
R
R
R
R
Proteusmirab- R ilis A. aerogenes R
R
R
R
R
E . coli s Proteus mirab- R ilia E.coli R
R R
R R
R
s
A. aerogenes
R
R
R
~
:"
i
~I Fl ~ N
I ~ ~
R R R
R R R
A. aerogenes E. coli Paracol. intermedium A. aerogenes Proteus rettgeri
R R
R R R
R R R
R R
R R
R
s
0
ez
37
Urethral stric- Proteusmirab- R ture, febrile pyilis• elonephritis
R
R
Proteusmirab- R ilis
R
R
Proteusmirabilis A. aerogenes Coliform-intermediate Proteus rettgeri
R R
R
R R R R .R. R
R
R
Proteusmirab- R ilis Paracol. coli- s forme
R
R
R
s
0
* Since the great majority of strains isolated from the urines were gram-negative bacilli, the study of the feces was limited to the isolation of gram-negative bacilli. • Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P., penicillin; and SD., sulfadiazine. S = susceptible; R = resistant; 0 = no growth. b Previous therapy given: case 34 received a full course of streptomycin, penicillin and sulfadiazine, 3 weeks before. • The initial A. aerogenes differs biochemically and serologically from the final A. aerogenes; the final urine and fecal strains of A. aerogenes are apparently identical, however. d Isolated after 2 days of therapy but not present in final culture. • Isolated from the blood; no urine received.
t::l
g ~
I
I z
I I
~
Ot
676
L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
not recovered prior to therapy. This pattern of change in flora has previously been observed by others during treatment of urinary tract infections. 7 E. coli, which was present initially in 16 of the cases (tables 1 and 2), was eventually eliminated in all cases and never emerged as a resistant strain (table 3). A. aerogenes on the other hand, though initially present in only 6 cases, was found in 14 cases to be resistant to all three drugs after therapy. This striking difference in the development of resistance of E. coli and A. aerogenes will be considered further in the relationship of the urinary to the fecal flora. Proteus species and Pseudomonas aeruginosa were associated with the complicated cases. The species of Proteus often changed during the course of therapy, but when a strain was initially resistant, it remained as a final resistant strain. Resistance was observed in virtually all these strains after therapy. A Pseudomonas strain (case 19), although initially resistant, was not present at the end of therapy. The alkali apparently was of no added help in the 3 complicated cases (table 2). The simple infections did well without alkali. Correlation between fecal and urinary flora:-The presence and origin of resistant bacteria in the urine were of particular interest. It has been felt by many that the bacteria in the urine have their origins in the fecal flora .8 Bacteriological study of the fecal flora is obviously a complex problem. It was believed, however, that study of the correlation between the fecal and urinary flora would be simpler in patients receiving combined therapy, since there would be 1) a marked reduction of the total number of organisms in the feces, and 2) the development of specific resistance to three drugs in the strains, would offer a method for correlating the flora of the urine and feces. A total of 7 cases were studied in which an attempt was made to correlate the fecal and urinary flora. The experimental procedure was as follows : a peasized portion of feces was emulsified in 0:5 ml. of sterile water, and approximately 0.05 ml. of the suspension spread over media for isolation; the procedures for identification and susceptibility tests were as carried out in the study of the flora of the urinary tract. Since the smears and the isolations from the urines after therapy rarely revealed gram-positive cocci, no attempt was made to recover them from the feces. The criteria for establishing a correlation between the flora of the colon and the urine are considered to be the following: 1) an organism found in the feces resistant to all three drugs should be present in the urine; 2) an organism found in the feces susceptible to any of the three drugs should not be present in the urine; 3) an organism found resistant in the urine should be present in the feces. In cases 33, 34,. 37 (table 4) the correlation between the fecal and urinary flora is at best suggestive ; a resistant strain in the feces was not found in the 7
Harris,H. W., Murray, R. , Paine, T . F ., Kilham, L . and Finland, M. : Am. J . Med., 2:
229, 1947.
Bandier, C. G., Roen, P.R. and Mulaire, V. J .: J . Urol., 69 : 96, 1948. Redewill , F . H. , Potter, J.E. and Garrison, H. A.: References in the J. A. M . A., 94: 688, 1930. 8
DRUG RESISTANT BACTERIA IN URINARY TRACT
677
urine, e.g., the Paracolobactrum strain in case 33. Also, resistant strains in the urine, e.g., case 37, A. aerogenes and Coliform-intermediate were not found in the feces. The results in these three cases may well indicate a lack of correlation between the fecal and urinary flora. It should be pointed out, however, that the failure to isolate bacteria, particularly from the feces, may not be due to their absence but to the technical difficulties inherent in fecal isolations. Further, in those cases in which bacteria were found in the urine and not in the feces, the results may also be interpreted as indicating sources of gram-negative bacilli other than directly from the feces (catheterization, cystoscopies, as well as open procedures). In cases 23, 24, 25, 36, the correlation between the flora of the urine and feces is complete; strains of resistant bacteria found in the urine were found in the feces. E. coli isolated in the feces of 24, 25, 33, and the Paracolobactrum organism in 37, were still susceptible to one of the chemotherapeutic agents and did not establish themselves in the urinary tract. It is believed that these results do permit the conclusion that there is a definite correlation between the urine and fecal flora and that the feces probably represent a major source of resistant strains isolated from the urine after therapy. Clinical findings.-N otice11ble clinical improvement was found in almost every case within a day or two after therapy was instituted. Even in the cases complicated by calculi, intubations, etc., dramatic symptomatic improvement was often observed. Fever, symptoms, leukocyte counts, and urine white cells were usually reduced before 48 hours. These improvements were obtained in the face of very obvious resistant bacillurias. Except for one patient (case 27), who received several courses, no toxicity ascribable to the use of this combined therapy was noted. This patient showed a transitory deafness and tinnitus, which may have been due to the streptomycin alone, DISCUSSION
If the colon is the source of infection, as indicated in our study, then complete therapeutic success theoretically requires the permanent elimination of the fecal bacteria involved in complicated urinary tract infections, Since there is little reason to believe that any therapeutic agent or combination of agents can permanently sterilize the intestinal tract, one should not expect that chemotherapy alone would effect lasting cure in obstrnctive cases. This interpretation is quite consistent with the usual failure of any chemotherapeutic agents t,o eliminate bacteria permanently from complicated urinary tract infections. It should follmv, therefore, that in the presence of urinary tract infections the temporary elimination of bacteria in the colon should be reflected in their temporary disappea,rance from the urinary tract. This has been observed with sulfasuxidine 9• and sulfathalidine 9b where the action of the compounds is essentially limited to the flora of the colon. Experimental evidence that bacteria 9•
Everett, H. S., Scott, R. B. and Steptoe, P. P.: Am. J. Obst. & Gynec., 49: 114, 1945. Everett, H. 8., Vosberg, G. A. and Davis, .J.M.: J. Urol., 59: 83, 1948. • Bandler, C. G. a.nd Roen, P.R.: Ann. Surg., 128: 80, 1948.
b
678
L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
can pass the intact colon is found in a study of nephrectomized dogs, injected intraperitoneally with a dialyzing solution, in which transmural escape of coliform organisms occurred, unless sulfathalidine and oral streptomycin were given. 10 A striking difference was observed in our study between E. coli and A. aerogenes strains in their susceptibilities and development of resistance. In the 16 strains of E.coli isolated from the urine, none became resistant during therapy and all were successfully eliminated from the urine. In those cases in which the fecal flora was studied no strain of E. coli isolated during therapy was found to have become resistant to all three drugs in the feces. On the other hand, A. aerogenes frequently emerged in the urine and feces resistant to all three drugs. In another recent study, 11 when streptomycin alone was given orally or intramuscularly, the resistant fecal organisms recovered were most commonly E. coli. It is interesting that virtually all of these cases responded well clinically, at least temporarily. Although we have no data concerning this phenomenon, it is possible that the original flora was more toxic than the immediately suceeding flora. It has been shown12 that certain E. coli strains from peritoneal and urinary infections were more apt to cause toxic manifestations (mouse pathogenicity, hemolysis, skin necrosis) than E. coli strains from normal feces. This, of course, does not explain cases of clinical response where no change in flora took place. As long as the obstruction remained the possibility of reinfection with a toxic strain remained. Resistant strains generally appeared 2-3 days after the beginning of therapy, and the resistance was usually very great; at least 50-fold increase in streptomycin resistance (growth in at least 500 µ/ml.); at least 100-fold increase in sulfadiazine resistance (growth in 1: 1000 dilution). This high streptomycin resistance is very interesting in view of the low level of streptomycin in the feces expected after intramuscular administration. 13 Almost all of the gram-negative bacilli were initially susceptible to streptomycin and sulfadiazine and resistant to penicillin. It has been observed in urinary tract infections that changes in flora occur during therapy. 7 We believe that the obvious interpretation is that susceptible bacteria are inhibited or destroyed and the resistant strains survive and multiply. We considered as an alternate possible interpretation of this phenomenon that an organism such as A. aerogenes which was often found following therapy might actually play a role in inhibiting the multiplication of those bacteria which had disappeared during therapy. However, in vitro tests showed no evidence of inhibition of these organisms by A. aerogenes. In addition to changes in flora we have noted that even in the few cases in which ostensibly there is no change in species, e.g., A. aerogenes constantly present in the urine (case 23), 10 Schweinburg, H.B., Frank, F. A., Frank, E. D., Heimberg, F. and Fine, J.: Proc. Soc. Exper. Biol. & Med., 71: 150, 1949. 11 Hamburger, M. and Berman, J. : J. Clin. Investigation, 28: 1042, 1949. 12 Sjostedt, S., quoted by Kaufmann, F.: J . Immunol., 57: 71, 1947. 13 Zintel, H. A., Flippin, H . F., Nichols, A. C., Wiley, M. M. and Rhoads, J.: Am. J. M . Sc., 210: 421, 1945.
DRUG RESISTANT BACTERIA IN URINARY TRACT
679
biochemical and serological studies revealed that a different strain of A. aerogenes had appeared. Success in combined therapy has been reported in a number of infections, but there have been only a limited number of studies concerned with the inhibition of the development of drug resistance. In brucellosis, for example, a combination of streptomycin and sulfadiazine has led to an inhibition of drug resistance. 4b Combined streptomycin and para-aminosalicylic acid have been effective against M. tuberculosis in vitro4d and in vivo, 15 and have prevented development of streptomycin resistance. The inhibition of the development of drug resistance has also been observed in tuberculosis with streptomycin and promin. 4a ' d One may ask why resistant strains arise, in view of in vitro and in vivo evidence that combined therapy can inhibit the development of resistance. The colon is unique in that there are a far greater number of bacterial individuals than are ever present in any infection. It may therefore be possible that, following the elimination of the majority of susceptible strains from this enormous population, there may be present or there may emerge a few bacteria simultaneously resistant to two or more drugs, 14 thus providing a potential source of resistant urinary organisms. It is felt, then, that in view of the effectiveness of combined therapy in inhibiting the development of resistance in vitro,-and in vivo as referred to above-that the failure to inhibit the development of resistance in the present work should not be considered representative for the reasons that have been discussed. In the present study it was found that bacteria initially present as drugsensitive strains in the urinary tract were generally eliminated during therapy and did not develop into resistant strains. These results may be interpreted as indicating that combined therapy inhibited development of drug resistance. The finding of new strains of resistant bacteria during or after treatment appears to represent, as we have indicated, a problem peculiar to urinary tract infections. It is felt, therefore, that although resistant strains do emerge during combined therapy, they do not originate as susceptible organisms in the urinary tract, but that they can be traced to the immense intestinal flora, in which similar resistant strains have been found. Studies are now being carried out on fecal flora changes under streptomycin (and combined drugs) in vitro. SUMMARY AND CONCLUSIONS
The therapeutic effects of combined streptomycin, penicillin and sulfadiazine were studied in 37 cases of urinary tract infection. Following therapy, sterile urine cultures were obtained in 16 cases, 14 of which were uncomplicated urinary tract infections. Dalton, H.: Nature, Ul2: 227, 1948. Spaulding, E. H., Madajewski, D.S., Rowe, R. J. and Bacon, H. E.: J. Bact., 58: 279, 1949. . 16 Vennesland, K., Ebert, R.H. and Bloch, R. G.: Proc. Soc. Exper. Biol. & Med., 68: 250, 1948. Graessle, 0. E., and Pietrowski, J. J.: J. Bact., 57: 459, 1949. 14
680
L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
Therapy was bacteriologically unsuccessful in 21 cases, 19 of which were characterized by some complication. In these 19 cases, bacteria resistant to all three drugs regularly emerged. A study of resistant strains in the urine and feces of 7 patients strongly indicated that many of the resistant strains in the urine had their origins in the fecal flora of the patient. Good clinical response was obtained in virtually all of the cases in spite of persistent bacillurias. Patients under therapy showed no evidence of toxicity due to the combined use of the three drugs. Efforts to remove the source of obstruction or complication must be made in order for combined therapy to be of any permanent value. Department of Bacteriology and Immunology, School of Medicine, University of Buffalo, 24 High St., Buffalo, N. Y. (L. J. K.) Department of Bacteriology, School of Medicine, Temple University, Broad and and Ontario, Philadelphia, Penna. (M. K.) 851 Adelaide Drive, Pasadena, Calif. (H. H. Z.)
Vol. 65, No. 4, April 1951
Printed in U.S .A.
EFFECT OF COMBINED THERAPY ON EMERGENCE OF DRUG RESISTANT BACTERIA IN URINARY TRACT INFECTIONS: OBSERVATIONS ON ORIGIN OF RESISTANT STRAINS LEONARD J. KIMMELMAN, HANS H. ZINSSER
AND
MORTON KLEIN
From the Department of Bacteriology, University of Pennsylvania School of Medicine, the Department of Urology (Surgery) of the Hospital of the University of Pennsylvania, and the Genito-Urinary Service of the Philadelphia General Hospital, Philadelphia
The development of resistance of bacteria to chemotherapeutic and antibiotic agents has been a limiting factor in therapy. 1 The problem of the development of resistance to streptomycin had been a particularly serious one. It was found in vitro that such resistance could be inhibited by the combined use of streptomycin, penicillin and sulfadiazine. 2 Therapeutically, the overall effect of two or more antibacterial agents has frequently been found superior to that obtained with one drug3 and some clinical studies have been carried out indicating that this therapeutic effect was correlated with the inhibition of drug resistance by combined therapy. 4 Development of resistance has been a common finding in the treatment of urinary tract infections with streptomycin. 6 In the present study we were concerned with 1) the effectiveness of combined streptomycin, penicillin and sulThis investigation was supported (in part) by a research grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service, and also from the Theresa F. Felsen Memorial Fund. The streptomycin for the patients was supplied by E. R. Squibb and Sons, Inc., and by Merck and Co., Inc. · The authors wish to thank Dr. Stuart Mudd for his continued interest and helpful criticism during the course of this study and preparation of the manuscript. 1 Buggs, C. W., Bronstein, B., Hershfeld, J. W. and Pilling, M . A.: J. A. M.A., 130: 64, 1946. Finland, M., Murray, R., Hartis, H. W., Kilham, L ., and Meads, M.: J . A. M.A., 132: 16, 1946. Youmans, G. P., Williston, E. H., Feldman, W. H. and Hinshaw, H. C.: Proc. Staff Meet. Mayo Clin., 21: 126, 1946. Alexander, H. E., Leidy, G., Rake, G. and Donovick, R.: J. A. M.A., 132: 434, 1946. Hall, W. H. and Spink, W.W.: Proc . Soc. Exper. Biol. & Med., 64: 403, 1947. 2Klein, M. and Kimmelman, L. J.: J. Bact., 54: 363, 1947. Pulaski, E. J. and Baker, H.J.: J. Lab. & Clin . Med., 34: 186, 1949. 8 Dowling, H . F., Hussey, H. H ., Hirsh, H. L. and Wilhelm, F.: Ann . Int. Med., 25: 950, 1946. Eisele, C. W. and MacCullough, N. B . : J. A. M . A., 135: 1053, 1947. MacLean, J. T. and Smith, F.: Canad. M.A. J., 57: 131, 1947. Lincoln, E . M., Kirmse, T. W. and DeVito, E.: J. A. M.A., 136: 593, 1948. Kolmer, J. A.: Am. J.M. Sc., 215: 136, 1948. Thatcher, F. S. and MacLean, J. T.: J. Urol., 57: 902, 1947 . .. Cocchi, E. and Pasquinucci, G.: Quoted in Pfuetze, K. and Myle, M.: J . A. M.A., 139: 634, 1949. b Spink, W.W., Hall, W. H., Shaffer, J. and Braude, A. I.: J. A. M.A., 139: 352, 1949. • Delaude, A., Karlson, A.G., Carr, D. T., Feldman, W. H. and Pfeutze, K. H.: Proc. Staff Meet. Mayo Clin ., 24: 342, 1949. d Karlson, A.G., Pfeutze, K. H., Carr, D. T., Feldman, W. H. and Hinshaw, H. C .: Proc. Staff Meet. Mayo Clin., 24: 85, 1949. 6 Bondi, A., Ottenberg, D., Dietz, C. C. and Brown, C . L. : J . A. M.A., 132: 634, 1946. Adcock, J. D . and Plumb, R. T. : J. A. M.A., 133: 579, 1947. Harrell, G. T., Herndon, E.G., Gillikin, C. M. and Aikawa, J. K .: J . Clin. Investigation, 26: 577, 1947. 668
DRUG RESISTAN'l' BACTERIA IN URINARY TRACT
669
fonamide in inhibiting the development of resistant strains during the treatment of urinary infections, and 2) the origin of the resistant strains in the urinary tract. MATERIALS AND METHODS
Cases and drug dosages.-A total of 37 cases from the urological services of the Hospital of the University of Pennsylvania and the Philadelphia General Hospital were studied. These included both uncomplicated urinary tract infections (cystitis, pyelonephritis, urethritis, etc.) and those complicated by additional pathological changes, such as calculi, strictures and obstructions. All cases, unless otherwise indicated, had not received previous therapy with streptomycin, penicillin or sulfonamides as far as could be determined. AH patients (unless otherwise indicated) received the following daily dosage for a total of 7 days: streptomycin, 1.0 gm., intramuscularly; penicillin, 200,000 units, intramuscularly; sulfadiazine, 2.0 gm., orally. Alkali was not routinely given (except for patients numbers 11, 25, 27, 29 (tables ]. and 2) and the pH of the urines varied between 5.5 and 6.0. As will be indicated later, therapeutic response to combined therapy was excellent in all the simple urinary tract infections, in the presence or absence of alkalL When strains of Proteus were present in the urine, the pH varied between 8.0 and 8.2. Leukocyte counts, hemoglobin determinations, and microscopic urine analyses were followed during therapy and for 3 days to several :months after therapy. Urine specimens were obtained using aseptic precautions. In the male, preliminary sponging of the meatus or catheter tip with 70 per cent alcohol was carried out and samples were collected after urine had first rinsed the alcohol away. Samples were collected from the female by catheterization after perineal preparation. Urines were obtained before therapy and every 48 hours during the 7 days of therapy. Laboratory procedures. Isolations: All specimens were centrifuged at high speed in an angle centrifuge for 20 minutes, and the supernate was discarded. If the specimen ·was taken during therapy, the sediment was washed in ssJine to remove any residual drugs. The washing procedure was found to eliminate the necessity of using drug-antagonists in the medium, since the addition of penicillinase, P ABA and cysteine to the medium did not increase the amount of above that obtained from washed residues. Highly susceptible organisms were v"'"·""-"'-' isolated from washed specimens obtained from urines containing streptomycin, penicillin and sulfadiazine. All specimens were 1) gram stained, 2) inoculated into beef extract broth, 3) streaked on brom thymol blue lactose agar or MacConkey's agar, and blood extract agar ·when gram positive cocci were observed in smear. All cultures were incubated aerobically at 37 C. Since mixed coliform, infections regularly occur in urinary tract infections, 4a, 6 at least 5 colonies of each coliform colony type were picked and identified. When the organisms were identified, they were placed on extract agar under sterile, heavy mineral oil for subsequent study of drug resistance. Susceptibility tests: After completion of therapy, tests on all the organisms
670
L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
from each case were done simultaneously to determine the degree of susceptibility to each of the drugs. Dilutions of streptomycin (10 µ gm./ml. to 500 µ TABLE
1. Bacteriological summary of urinary tract infections. Cases in which negative cultures were obtained after therapy a IN VITRO SUS-
CASE
NO.
DIAGNOSIS
ORGANISMS ISOLATED BEFORE THERAPY
CEPTIBILITY TO
SM.
--
P.
--
SD.
1
Cystitis; pyelonephritish
E. coli Staphylococcus aureus
s R s s s R
2 3
Cystitis Cystitis Pyelonephritis (right) Pyelonephri tis Bilateral renal calculi Renal (staghorn) calculi Cystitis; pyuria Acute right pyelonephritis Cystitis Acute cystitis (post-cystoscopy) Epididymitis Cystitis and prostatitis
E.coli E.coli Paracol. coliforme A. aerogenes E.coli Kl. pneumoniae E. coli E.coli Staphylococcus albus E . coli
s s s s s s s s s
4
5 6 7 9 10 11* 12
13
14 15 16 17
Acute pyelonephritis Acute cystitish Chronic pyelonephritis and glomerular nephritis
Paracol. aerogenoides E.coli Paracol. coliforme E . coli E. coli Alpha hemolytic streptococcus
BACTERIOWGY
AFTER THERAPY
R
R R
R R R R R R R R
s R s R s R s R s R s s
s s s s s 0
s s s R
s s s s s 0
negative (Staphylococcus-resist. to SM. and SD .)• negative negative negatived negative negative negative negative negative negative negative negative negative negative negative• negative 1
* Received sodium bicarbonate 5.0 gm. every six hours in addition to the three agents . • Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P., penicillin; and SD ., sulfadiazine. 0 = no growth in medium (casein hydrolysate); S = susceptible; and R = resistant. b Previous therapy given: case 16 received penicillin and case 1, penicillin and sulfadiazine. • Probable contaminant; only few scattered colonies. d Paracolobactrum coliforme persisted for more than four days, still susceptible to streptomycin and sulfadiazine, and eventually disappeared. • E . coli recovered one month later, still suscept ible, and retreated wit h permanent success. 1 Streptomycin-susceptible alpha hemolytic streptococcus isolated two weeks later. Note: Case 8 was omitted because no organisms were isolated before or after therapy.
gm./ ml.) and penicillin (0.1 unit/ml. to 5.0 units/ml.) were made in beef extract broth pH 7.2 and seeded with 0.1 ml. of 10-2 dilution of a 20-24 hour brot h culture of the organism. A casein hydrolysate medium was used for determining
671
DRUG RESISTANT BACTERIA IN URINARY TRACT TABLE
2. Bacteriological summary of urinary tract infections. Cases in which resistant organisms were found after therapy a IN VITRO SUS-
CASE
NO.
DIAGNOSIS
ORGANISMS ISOLATED BEFORE THERAPY
CEPTIBILITY TO
P.
SD.
s s
R
s
R
R
R
R
R
SM.
18 19
20 21 22 23
24
25*
Pyelonephritis Pyelitis; prosta ti tis
E. coli Proteus vulgaris Pseud. aeruginosa
Chronic prostatitis and cystitis Benign prostatic hypertrophy; cystitits Benign prostatic hypertrophy Chronic pyelonephritis Calculus at ureteropelvic junction Bilateral renal calculi
Non-hemolytic Streptococcus A. fecalis Staph. aureus
s
R
Ob
R
R
R
E. coli
s
R
A. aerogenes
s
R
E. coli Paracol. coliforme Proteus mirabilis Pseud. aeruginosa Proteus rettgeri A. fecalis
s s
R R
R
R
R
R
R
R
R R
R R
R R
s s s
s s s
s
Hydronephrosis (congenital band)
27*
Calculus
Proteus morgani
s
R
R
28
Renal carbuncle; pyelonephritis Renal and ureteral calculi Bilateral calculi
Anaerogenic Paracol. species A. aerogenes A. fecalis Proteus mirabilis E. coli A. aerogenes
s
s
O•
s
R
30 31
32
33
Neurogenic bladder; carcinoma; cystitis; calculus U reteral stricture; pyelonephritis Hemorrhagic cystitis; diverticulum
R
s s s
s s s s
s
R R
s
s
E. coli
s
R
s
Staph. aureus
s
R
R
m IN VITRO SUSCEPTIBILITY TO
P.
SD.
R R
R R
R
R
R R R
R R
R R
R R
R
R
R
R
A. aerogenes
R
R
R
A. aerogenes
R
R
R
A. aerogenes Proteus mirabilis Proteus mirabilis
R
R R
R
R R
R
R
Proteus rettgeri Paracol. coliforme A. aerogenes Proteus mirnbilis Anaerogenic Paracol. species A. aerogenes Proteus rettgeri Proteus morganid
R R
R R
R R
SM.
-- --
26
29*
ORGANISMS ISOLATED AFTER THERAPY
A. aerogenes Proteus rettgeri Pseud. aeruginosa A. aerogenes Non-hemolytic Streptococcus Staph. aureus Staph. aureus
-- --
R R
R
R
R
R
R
R R
R
R
O•
R R
R R R
R
R
R R R
R R R
R
R
R
Pseud. aeruginosa R A. aerogenes R
R
R R
A. aerogenes Proteus rettgeri Pseud. aeruginosa Staph. albus
s R R
R
R
s
672
L . J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
TABLE 2- Cvntinued a IN VITRO SUS-
a IN VITRO SUSCASE NO.
DIAGNOSIS
Benign prostatic hypertorphy; multiple myeloma Benign prostatic hypertrophy Post-herniorrhaphy cysti tis and/or prostatitis Acute pyelonephritis; urethral stricture
35 36
37
Benign prostatic hypertrophy; surgery
38
CEPTIBILITY TO
SM.
-34
ORGANISMS ISOLATED BEFORE THERAPY
P.
ORGANISMS ISOLATED AFTER THERAPY
SD.
SM.
P.
--
A. aerogenes
- R R R
SD.
-- - A. aerogenes R R R P aracol. aeroge- R R R
CEPTIBILITY TO
noides Staph. aureus
s
R
s
A. aerogenes
R
R
R
A. aerogenes
R
R
R
A. aerogenes
R
R
R
Proteus mirabi]is
R
R
R
Proteus mirabilis A. aerogenes Coliform-intermediate Proteus rettgeri A. aerogenes
R
R
R
R R
R R
R R
R
R
R
R
E.coli
s
R
s
O• R
* Received sodium bicarbonate in addition to the other drugs. Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P. , penicillin; and SD., sulfadiazine. S = susceptible and R = resistant. b Previous therapy given: case '2:7 received penicillin; case 36, penicillin and sulfadiazine; and case 34, a full course of streptomycin, penicillin and sulfadiazine, 3 weeks before. °ી Could not be grown in casein hydrolysate medium with special vitamin additions. d One colony only on isolation plate. Note: Cases 19, 31, 33, and 37 were from the Philadelphia General Hospital through the courtesy of Dr. Earl W. Clawater. These were given a triple sulfonamide preparation (Lilly) consisting of sulfapyrazine, sulfamerazine and sulfadiazine (0.167 gm. of each per tablet). a
sulfonamide susceptibility of the organisms. 6 All tubes were seeded with 0.1 ml. of a 10-4 dilution of a 20- 24 hour broth culture. The lowest concentration of drugs showing complete inhibition of growth after 24 hours at 37 C was taken as the inhibitory concentration. Resistant strains are arbitrarily defined in the present study as those growing in at least 25.0 µ/ml. streptomycin, 1.0 unit/ml. penicillin and 1 :25,000 sulfadiazine. RESULTS
Bacteriological findings. Comparative success in simple and complicated infections:- The results obtained in the 37 cases are shown in tables 1 and 2. In table 1 are listed the cases in which no organisms were isolated after 7 days of combined therapy. It may be noted that of 16 cases, 14 were uncomplicated urinary tract infections. The organisms generally found before therapy were 6
Strauss, E., Dingle, J. H. and Finland, M.: J. Immunol., 42: 331, 1941.
673
DRUG RESISTANT BACTERIA IN UPJNARY TRACT
gram-negative bacilli, usually susceptible to streptomycin and sulfadiazine and resistant to penicillin. Gram-positive cocci (staphylococci and enterococci) were found only occasionally in the urines. Table 2 lists the cases of bacteriological failure. Nineteen of the 21 cases were complicated by the presence of calculi, hypertrophy, strictures, etc. With one exception (case 30) in all of the 21 cases, bacteria present after 7 days' therapy were found resistant to all three drugs. It is quite evident, therefore, that in the presence of factors complicating the infectious process, combined therapy did not permanently eliminate bacteria from the urinary tract. Although success was obtained. in eliminating bacteria in 15 of the 16 cases of simple urinary tract infections (table 1), the series is too TABLE
3. Incidence of urinary strains and susceptibility to streptomycin. Isolations before and after combined therapy
ORGANISM:
BEFORE THERAPY
AFTER THRRAPV
Number of Strains
Number of strains
Susceptible
Escherichia coli .. , .......... .. .. Aerobacter aerogenes ...... . ' ..... Proteus species ..... .. , .. . . . . . . Pseudomonas aeruginosa .... ... Paracolobactrum species ... ... Staphylococcus species ... .... Alkaligenes fecalis ... ' .... . . . . Streptococcus species .. Klebsiella pneumoniae .. . . .. ... . . Coliform-intermediate. ...... . . .. .
Resistant
4
l 2•
3
3
0 5
2
15
I Total II i
Susceptible
Resistanta
16
l
0
6 6
0
14 9
2
0 0 0
6
I
3
2
Total
-lb 14 10 3, 2 3 0
5
l 0
0
3
3
X
X
2 1
0
2
0
1
1
l 0
X
X
0
0
1
1
0
0 0
,5
I
I
3d
x = No strains encountered at all after therapy. • All resistant to penicillin, streptomycin, and sulfadiazine excepting one strain of Proteus susceptible to sulfadiazine and one strain of staphylococcus susceptible to penicillin. 0 One susceptible and apparently identical strain isolated 2 weeks later, also. " One case found subsequently to have had one course of combined therapy before th.is course. d Status of one or more strains as contaminants very likely.
small to determine whether or not combined therapy is superior to the results obtained by the use of one drug. In approximately 55 per cent of all these cases the init~al flora was eliminated by the third day. This is an indication of the effectiveness of combined therapy in eliminating the initial flora, although not a measure of the eventual. bacteriological outcome. Resistant urinary strains.-The incidence of the development of resistant strains is shown in tables 1 and 2 and summarized in table 3. It is quite clear that the use of combined therapy did not consistently prevent the ultimate ape· pearance of resistant bacteria. It should be noted, however, that the originally susceptible strains were usually eliminated and the resistant strains emerging represented new species, resistant to all three drugs. In 14 of 21 cases in table 2, the resistant strains isofated after therapy were
TABLE
BEFORE THERAPY
CASE NO.
DIAGNOSIS
Urine
24
25
36
33
34
•susceptibility To SM.
-23
-Chronic pyelone- A. aerogenes' phritis; previous nephrectomy Calculus and in- E. coli fection at ure- Paracol. colitero-renal juncforme tion Bilateral renal calculi; pre vious surgery; surgery now also before therapy Post-herniorrhaphy; cystitis and prostatitis Hemorrhagic cystitis, diverticu!um; surgery after therapy Benign prostatic hypertrophy, surgery, mu!ti pie myelomah
~
4. Comparison of urinary and fecal flora before and after combined therapy
s s s
Proteus mirab- R ilis Pseud. aeru- R ginosa
A. aerogenes
Staph. aureus
R
s
P.
-
SD .
s
R R
s s
R
R
R
R
R
R R
A. aerogenes
R
R
R
Paracol. aerogenoides
R
R
R
•susceptibil:ty To SM. P.
--
R
R
Feces•
AYTEJt THERAPY
-A. aerogenes s E . coli s Coliform-ins termediate A. aerogenes s Paracol. inter- s medium Coliform-ins termediate Proteusmirab- R ilis E . coli s
A. aerogenes Coliform-intermediate E . coli A. aerogenes Coliform-intermediate Proteus vu!garis Paracol. aerogenoides
-
SD .
--
R R R
s s s
R R
s s
R
s
Urine
•susceptibility To SM.
SD.
•susceptibility To SM.
P.
SD.
---- - -A. aerogenes• R R R A. aerogenes• R R R
Proteus mirab- R ilis A. aerogenes R
Proteus mirab- R ilis
R
R
R
s
R R
R R
s
s s s
R R R
s s s
A. aerogenes Pseud . aeruginosad
R R
s
R
s
A. aerogenes
R
s
R
s
R
P.
-
Feces•
A. aerogenes
R
R
R
R
R
R
R
R
R
Proteusmirab- R ilis A. aerogenes R
R
R
R
R
E . coli s Proteus mirab- R ilia E.coli R
R R
R R
R
s
A. aerogenes
R
R
R
~
:"
i
~I Fl ~ N
I ~ ~
R R R
R R R
A. aerogenes E. coli Paracol. intermedium A. aerogenes Proteus rettgeri
R R
R R R
R R R
R R
R R
R
s
0
ez
37
Urethral stric- Proteusmirab- R ture, febrile pyilis• elonephritis
R
R
Proteusmirab- R ilis
R
R
Proteusmirabilis A. aerogenes Coliform-intermediate Proteus rettgeri
R R
R
R R R R .R. R
R
R
Proteusmirab- R ilis Paracol. coli- s forme
R
R
R
s
0
* Since the great majority of strains isolated from the urines were gram-negative bacilli, the study of the feces was limited to the isolation of gram-negative bacilli. • Susceptibility defined in METHODS AND MATERIALS: SM. denotes streptomycin; P., penicillin; and SD., sulfadiazine. S = susceptible; R = resistant; 0 = no growth. b Previous therapy given: case 34 received a full course of streptomycin, penicillin and sulfadiazine, 3 weeks before. • The initial A. aerogenes differs biochemically and serologically from the final A. aerogenes; the final urine and fecal strains of A. aerogenes are apparently identical, however. d Isolated after 2 days of therapy but not present in final culture. • Isolated from the blood; no urine received.
t::l
g ~
I
I z
I I
~
Ot
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L. J. KIMMELMAN, H. H. ZINSSER AND M. KLEIN
not recovered prior to therapy. This pattern of change in flora has previously been observed by others during treatment of urinary tract infections. 7 E. coli, which was present initially in 16 of the cases (tables 1 and 2), was eventually eliminated in all cases and never emerged as a resistant strain (table 3). A. aerogenes on the other hand, though initially present in only 6 cases, was found in 14 cases to be resistant to all three drugs after therapy. This striking difference in the development of resistance of E. coli and A. aerogenes will be considered further in the relationship of the urinary to the fecal flora. Proteus species and Pseudomonas aeruginosa were associated with the complicated cases. The species of Proteus often changed during the course of therapy, but when a strain was initially resistant, it remained as a final resistant strain. Resistance was observed in virtually all these strains after therapy. A Pseudomonas strain (case 19), although initially resistant, was not present at the end of therapy. The alkali apparently was of no added help in the 3 complicated cases (table 2). The simple infections did well without alkali. Correlation between fecal and urinary flora:-The presence and origin of resistant bacteria in the urine were of particular interest. It has been felt by many that the bacteria in the urine have their origins in the fecal flora .8 Bacteriological study of the fecal flora is obviously a complex problem. It was believed, however, that study of the correlation between the fecal and urinary flora would be simpler in patients receiving combined therapy, since there would be 1) a marked reduction of the total number of organisms in the feces, and 2) the development of specific resistance to three drugs in the strains, would offer a method for correlating the flora of the urine and feces. A total of 7 cases were studied in which an attempt was made to correlate the fecal and urinary flora. The experimental procedure was as follows : a peasized portion of feces was emulsified in 0:5 ml. of sterile water, and approximately 0.05 ml. of the suspension spread over media for isolation; the procedures for identification and susceptibility tests were as carried out in the study of the flora of the urinary tract. Since the smears and the isolations from the urines after therapy rarely revealed gram-positive cocci, no attempt was made to recover them from the feces. The criteria for establishing a correlation between the flora of the colon and the urine are considered to be the following: 1) an organism found in the feces resistant to all three drugs should be present in the urine; 2) an organism found in the feces susceptible to any of the three drugs should not be present in the urine; 3) an organism found resistant in the urine should be present in the feces. In cases 33, 34,. 37 (table 4) the correlation between the fecal and urinary flora is at best suggestive ; a resistant strain in the feces was not found in the 7
Harris,H. W., Murray, R. , Paine, T . F ., Kilham, L . and Finland, M. : Am. J . Med., 2:
229, 1947.
Bandier, C. G., Roen, P.R. and Mulaire, V. J .: J . Urol., 69 : 96, 1948. Redewill , F . H. , Potter, J.E. and Garrison, H. A.: References in the J. A. M . A., 94: 688, 1930. 8
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urine, e.g., the Paracolobactrum strain in case 33. Also, resistant strains in the urine, e.g., case 37, A. aerogenes and Coliform-intermediate were not found in the feces. The results in these three cases may well indicate a lack of correlation between the fecal and urinary flora. It should be pointed out, however, that the failure to isolate bacteria, particularly from the feces, may not be due to their absence but to the technical difficulties inherent in fecal isolations. Further, in those cases in which bacteria were found in the urine and not in the feces, the results may also be interpreted as indicating sources of gram-negative bacilli other than directly from the feces (catheterization, cystoscopies, as well as open procedures). In cases 23, 24, 25, 36, the correlation between the flora of the urine and feces is complete; strains of resistant bacteria found in the urine were found in the feces. E. coli isolated in the feces of 24, 25, 33, and the Paracolobactrum organism in 37, were still susceptible to one of the chemotherapeutic agents and did not establish themselves in the urinary tract. It is believed that these results do permit the conclusion that there is a definite correlation between the urine and fecal flora and that the feces probably represent a major source of resistant strains isolated from the urine after therapy. Clinical findings.-N otice11ble clinical improvement was found in almost every case within a day or two after therapy was instituted. Even in the cases complicated by calculi, intubations, etc., dramatic symptomatic improvement was often observed. Fever, symptoms, leukocyte counts, and urine white cells were usually reduced before 48 hours. These improvements were obtained in the face of very obvious resistant bacillurias. Except for one patient (case 27), who received several courses, no toxicity ascribable to the use of this combined therapy was noted. This patient showed a transitory deafness and tinnitus, which may have been due to the streptomycin alone, DISCUSSION
If the colon is the source of infection, as indicated in our study, then complete therapeutic success theoretically requires the permanent elimination of the fecal bacteria involved in complicated urinary tract infections, Since there is little reason to believe that any therapeutic agent or combination of agents can permanently sterilize the intestinal tract, one should not expect that chemotherapy alone would effect lasting cure in obstrnctive cases. This interpretation is quite consistent with the usual failure of any chemotherapeutic agents t,o eliminate bacteria permanently from complicated urinary tract infections. It should follmv, therefore, that in the presence of urinary tract infections the temporary elimination of bacteria in the colon should be reflected in their temporary disappea,rance from the urinary tract. This has been observed with sulfasuxidine 9• and sulfathalidine 9b where the action of the compounds is essentially limited to the flora of the colon. Experimental evidence that bacteria 9•
Everett, H. S., Scott, R. B. and Steptoe, P. P.: Am. J. Obst. & Gynec., 49: 114, 1945. Everett, H. 8., Vosberg, G. A. and Davis, .J.M.: J. Urol., 59: 83, 1948. • Bandler, C. G. a.nd Roen, P.R.: Ann. Surg., 128: 80, 1948.
b
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can pass the intact colon is found in a study of nephrectomized dogs, injected intraperitoneally with a dialyzing solution, in which transmural escape of coliform organisms occurred, unless sulfathalidine and oral streptomycin were given. 10 A striking difference was observed in our study between E. coli and A. aerogenes strains in their susceptibilities and development of resistance. In the 16 strains of E.coli isolated from the urine, none became resistant during therapy and all were successfully eliminated from the urine. In those cases in which the fecal flora was studied no strain of E. coli isolated during therapy was found to have become resistant to all three drugs in the feces. On the other hand, A. aerogenes frequently emerged in the urine and feces resistant to all three drugs. In another recent study, 11 when streptomycin alone was given orally or intramuscularly, the resistant fecal organisms recovered were most commonly E. coli. It is interesting that virtually all of these cases responded well clinically, at least temporarily. Although we have no data concerning this phenomenon, it is possible that the original flora was more toxic than the immediately suceeding flora. It has been shown12 that certain E. coli strains from peritoneal and urinary infections were more apt to cause toxic manifestations (mouse pathogenicity, hemolysis, skin necrosis) than E. coli strains from normal feces. This, of course, does not explain cases of clinical response where no change in flora took place. As long as the obstruction remained the possibility of reinfection with a toxic strain remained. Resistant strains generally appeared 2-3 days after the beginning of therapy, and the resistance was usually very great; at least 50-fold increase in streptomycin resistance (growth in at least 500 µ/ml.); at least 100-fold increase in sulfadiazine resistance (growth in 1: 1000 dilution). This high streptomycin resistance is very interesting in view of the low level of streptomycin in the feces expected after intramuscular administration. 13 Almost all of the gram-negative bacilli were initially susceptible to streptomycin and sulfadiazine and resistant to penicillin. It has been observed in urinary tract infections that changes in flora occur during therapy. 7 We believe that the obvious interpretation is that susceptible bacteria are inhibited or destroyed and the resistant strains survive and multiply. We considered as an alternate possible interpretation of this phenomenon that an organism such as A. aerogenes which was often found following therapy might actually play a role in inhibiting the multiplication of those bacteria which had disappeared during therapy. However, in vitro tests showed no evidence of inhibition of these organisms by A. aerogenes. In addition to changes in flora we have noted that even in the few cases in which ostensibly there is no change in species, e.g., A. aerogenes constantly present in the urine (case 23), 10 Schweinburg, H.B., Frank, F. A., Frank, E. D., Heimberg, F. and Fine, J.: Proc. Soc. Exper. Biol. & Med., 71: 150, 1949. 11 Hamburger, M. and Berman, J. : J. Clin. Investigation, 28: 1042, 1949. 12 Sjostedt, S., quoted by Kaufmann, F.: J . Immunol., 57: 71, 1947. 13 Zintel, H. A., Flippin, H . F., Nichols, A. C., Wiley, M. M. and Rhoads, J.: Am. J. M . Sc., 210: 421, 1945.
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biochemical and serological studies revealed that a different strain of A. aerogenes had appeared. Success in combined therapy has been reported in a number of infections, but there have been only a limited number of studies concerned with the inhibition of the development of drug resistance. In brucellosis, for example, a combination of streptomycin and sulfadiazine has led to an inhibition of drug resistance. 4b Combined streptomycin and para-aminosalicylic acid have been effective against M. tuberculosis in vitro4d and in vivo, 15 and have prevented development of streptomycin resistance. The inhibition of the development of drug resistance has also been observed in tuberculosis with streptomycin and promin. 4a ' d One may ask why resistant strains arise, in view of in vitro and in vivo evidence that combined therapy can inhibit the development of resistance. The colon is unique in that there are a far greater number of bacterial individuals than are ever present in any infection. It may therefore be possible that, following the elimination of the majority of susceptible strains from this enormous population, there may be present or there may emerge a few bacteria simultaneously resistant to two or more drugs, 14 thus providing a potential source of resistant urinary organisms. It is felt, then, that in view of the effectiveness of combined therapy in inhibiting the development of resistance in vitro,-and in vivo as referred to above-that the failure to inhibit the development of resistance in the present work should not be considered representative for the reasons that have been discussed. In the present study it was found that bacteria initially present as drugsensitive strains in the urinary tract were generally eliminated during therapy and did not develop into resistant strains. These results may be interpreted as indicating that combined therapy inhibited development of drug resistance. The finding of new strains of resistant bacteria during or after treatment appears to represent, as we have indicated, a problem peculiar to urinary tract infections. It is felt, therefore, that although resistant strains do emerge during combined therapy, they do not originate as susceptible organisms in the urinary tract, but that they can be traced to the immense intestinal flora, in which similar resistant strains have been found. Studies are now being carried out on fecal flora changes under streptomycin (and combined drugs) in vitro. SUMMARY AND CONCLUSIONS
The therapeutic effects of combined streptomycin, penicillin and sulfadiazine were studied in 37 cases of urinary tract infection. Following therapy, sterile urine cultures were obtained in 16 cases, 14 of which were uncomplicated urinary tract infections. Dalton, H.: Nature, Ul2: 227, 1948. Spaulding, E. H., Madajewski, D.S., Rowe, R. J. and Bacon, H. E.: J. Bact., 58: 279, 1949. . 16 Vennesland, K., Ebert, R.H. and Bloch, R. G.: Proc. Soc. Exper. Biol. & Med., 68: 250, 1948. Graessle, 0. E., and Pietrowski, J. J.: J. Bact., 57: 459, 1949. 14
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Therapy was bacteriologically unsuccessful in 21 cases, 19 of which were characterized by some complication. In these 19 cases, bacteria resistant to all three drugs regularly emerged. A study of resistant strains in the urine and feces of 7 patients strongly indicated that many of the resistant strains in the urine had their origins in the fecal flora of the patient. Good clinical response was obtained in virtually all of the cases in spite of persistent bacillurias. Patients under therapy showed no evidence of toxicity due to the combined use of the three drugs. Efforts to remove the source of obstruction or complication must be made in order for combined therapy to be of any permanent value. Department of Bacteriology and Immunology, School of Medicine, University of Buffalo, 24 High St., Buffalo, N. Y. (L. J. K.) Department of Bacteriology, School of Medicine, Temple University, Broad and and Ontario, Philadelphia, Penna. (M. K.) 851 Adelaide Drive, Pasadena, Calif. (H. H. Z.)