Scandinavian Journal of Infectious Diseases
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
Urinary Tract Pathogens-Sensitivity to Trimethoprim/Sulphamethoxazole Helga Laursen & Mogens Lykkegaard Nielsen To cite this article: Helga Laursen & Mogens Lykkegaard Nielsen (1970) Urinary Tract Pathogens-Sensitivity to Trimethoprim/Sulphamethoxazole, Scandinavian Journal of Infectious Diseases, 2:3, 205-209 To link to this article: http://dx.doi.org/10.3109/inf.1970.2.issue-3.09
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Date: 13 November 2015, At: 00:15
Scand J Infect Dis 2: 205-209, 1970
URINARY TRACT PATHOGENS-SENSITIVITY TO TRIMETHOPRIMjSULPHAMETHOXAZOLE Helga Laursen and Mogens Lykkegaard Nielsen
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From Statens Seruminstitut, Laboratory of Clinical Microbiology at Fredriksberg Hospital, and the Department K of Surgery, Fredriksberg Hospital, Copenhagen, Denmark
ABSTRACT. The sensitivity to sulphamethizole, sulphamethoxazole (SM), trimethoprim (TMP) and the combination SM/TMP of freshly isolated urinary tract pathogens from hospital patients was studied. Out of 798 strains tested primarily, 47% were found to be fully sensitive to sulphamethizole (tablet method), and 81 % fully sensitive to the SM/TMP combination (disk method), Out of 423 strains with reduced sensitivity to sulphamethizole, 65% were fully sensitive to SM/TMP. Out of 391 strains tested secondarily (disk method), 39% were fully sensitive to SM, 68% fully sensitive to TMP, and 91 % to SM/ TMP. Of 283 strains presenting a reduced sensitivity to SM, 85% were fully sensitive to SM/TMP. In both series the isolated Streptococcus faecalis strains were highly resistant to sulphamethizole and SM, but presented a very high degree of sensitivity to TMP and SM/TMP. In contrast, nearly all isolated Pseudomonas aeruginosa strains were found to be resistant both to suiphonamides, TMP and SM/TMP. Of the 18 Staphylococcus aureus strains tested secondarily, 10 out of 12 multiresistant strains were fully sensitive to TMP. Synergy between SM and TMP could be demonstrated in 61 % of the 391 strains tested secondarily. The synergic effect depended on the degree of sensitivity to SM.
The description of trimethoprim (fMP) (2,4diamino-5-(3 1 , 41,51-trimethoxybenzyl)-pyrimidine) as being an antibacterial agent alone and in combination with sulphonamides, is due to Hitchings and others (for survey, see 4). TMP is an inhibitor of dihydrofolate reductase, the enzyme being responsible for the conversion of dihydrofolic acid to tetrahydrofolic acid. Differential studies indicate that bacterial dihydrofolate reductase is 10 000 times as sensitive to the inhibitory action of TMP as is the corresponding mammalian enzyme (3, 5). Several investigations in vitro have shown that, when acting alone, TMP inhibits the growth of a wide range of bacteria generally in much lower
concentrations than the sulphonamides do (1, 2, 6, 8). The combined action of the bacteriostatic TMP and a sulphonamide produces a bactericidal effect, and the action of TMP with a sulphonamide is often synergic (2, 6). Studies in vitro with TMP and sulphonamides have often been performed using sulphamethoxazole (SM) because rates of absorption and elimination are almost identical with those of TMP (1, 2, 8). Sulphafurazole has also been used (6). Several clinical trials in various types of infection with TMP and SM in varying combinations have been performed and the pharmacodynamics of TMP are well known (for survey, see 7). The compound available for clinical use consists of a combination of TMP and SM at a ratio of 1 : 5, on a weight basis. The purpose of the present study was to determine the in vitro effect of trimethoprim alone and in combination with sulphamethoxazole against freshly isolated bacterial strains from the urinary tract in hospital patients. Furthermore, the aim was to determine the synergic effect of TMP and SM and to determine the possible value of the combination of TMP and SM in cases with sulphonamide-resistant strains. MATERIAL AND METHODS The study comprised 798 bacterial strains, tested prim-. arily, collected over a period of 2 months. All urine samples received in the laboratory with;;, 100000 colonies. per ml were included in the study. The final series consisted of 684 samples of urine, 97 samples presenting mixed flora with up to 3 different species. All strains were tested primarily for sensitivity to sulphamethizole using the tablet method (Neo-Sensitabsta). Furthermore, the strains were tested by the disk method for sensitivity to Scand I Infect Dis 2·
206
H. Laursen and M. Lykkegaard Nielsen
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Table I. Sensitivity to sulphamethizole (tablet method) and Bactrim (sulphamethoxazole + trimethoprim) (disk method) of 798 bacterial strains tested primarily Sulphamethizole (zone of inhibition in mm)"
Bactrim (zone of inhibition in mrn)"
;;>28
15-27
.-;;14
;;>18
13-18
173 10 43 10 86 2 0 5 4
22 2 14 2 10 4 3 1 0
96 9 48 5 63 15 1 3 1
268 19 77 14 122 1 2 5 3
0 4 2 0 15 15
1 0 0 0 8 1
95 0 0 1 8 6
Aerobic corynebacterium Anaerobic corynebacterium
3 1
1 0
Bact. fragilis CI. Welchii
1 1 375
Escherichia Enterobacter Klebsiella Citrobacter Proteus + Providencia Ps. aeruginosa Alcaligenes faecalis Moraxella + Anitratum Other aerobic gram-negative rods Str, faecalis fJ-haem. streptococci Q(- haem. streptococci Anaerobic streptococci Staph. aureus Staph. albus
Total
.-;; 12
Total
19 2 24 3 30 3 2 3 0
4 0 4 0 7 17 0 1 2
291 21 105 17 159 21 4 9 5
79 3 2 0 30 20
14 1 0 0 1 1
3 0 0 1 0 1
96 4 2 1 31 22
3 0
4 1
0 0
3 0
7 1
0 0
0 0
0 0
1 1
0 0
1 1
69
354
650
105
43
798
a Tablet method: ;;> 28 mm= sensitive; 15-27 mm= slightly sensitive; .-;; 14 mm e- resistant. b
Disk method (1,2): > 18 mm-e sensitive; 13-18 mm e-slightly sensitive; .-;; 12 mm-s resistant.
the TMP ISM combination (Bactrim®). Peptone-free 10% horse blood agar plates were used in primary sensitivity tests. Quantitative urine cultivation was carried out according to the principles laid down by Vejlsgaard (9). Qualitative cultivation with determination of species was made applying routine bacteriological methods. Out of the 798 bacterial strains, 391 strains from a medical and a surgical ward were furthermore tested secondarily for sensitivity to TMP, SM and Bactrim, using disks containing 1.25 ftg TMP, 23.75 ftg SM, and disks containing the above-mentioned amounts of the two agents per disk. Sensitivity tests were made on freshly prepared suspensions of bacteria, using the technique described by Bohni (I, 2). According to Bohni, the use of the two agents in combination in one disk is justified by the existence of an almost identical diffusion time in the agar of the agents. The ratio 1 : 19 (1.25 flg TMP and 23.75 flg SM) in the disks is based on the fact that TMP often has a 20-fold higher activity in vitro and that the agents show a relationship of 1 : 20 in human plasma during treatment with the combination Bactrim (1, 2). The plates were left for pre-diffusion for 1 hour and then incubated at 35°C for 18-20 hours.
RESULTS The samples tested primarily are shown in Table I and those tested secondarily in Table II. To Scand J Infect Dis 2
simplify a review, the samples shown in the tables have generaIly been classified into genera, except cases of particular diagnostic interest. Alcaligenes and others have also been listed under species, since only one species was found in these groups. It appears from Table I that 44 % of the strains were resistant to sulphamethizole and 5 % to Bactrim. It should be noted specifically that only 3 of 96 Streptococcus faecalis strains were resistant to Bactrim. Out of 423 strains presenting reduced sensitivity to sulphamethizole, 275 (65 %) were fully sensitive to Bactrim. Table II shows that 54 % and 13 % of all strains were resistant to SM and TMP, respectively. The 51 Str. faecalis strains were resistant to SM, whereas 7 strains only were not fully sensitive to TMP. Out of 238 strains presenting reduced sensitivity to SM, 36 had a reduced sensitivity to the SM jTMP combination, i.e. 85 % of the sulphonamide-resistant strains were fully sensitive to the combination. The synergy appears from Tables II and III and Fig. 1. Table II shows that definite synergy could be demonstrated in 61 % of all strains. The
Urinary tract pathogens-sensitivity to trimethoprim / sulphamethoxazole
207
Table II. Sensitivity to sulphamethoxazole (SM) and trimethoprim (TMP) (disk method) of 391 bacterial strains tested secondarily SM (zone of inhibition in mm)
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> 18
13-18
TMP (zone of inhibition in mm)
';;;12
> 18
SM+TMP (zone of inhibition inmm)
13-18
';;;\2
>18
13-18
,;;; 12
Total
Synergy"
Escherichia Enterobacter Klebsiella Citrobacter Proteus + Providencia Ps. aeruginosa Alcaligenes faecalis Moraxella
66 4 13 10 40 0 1 1
13 1 6 0 5 0 0 0
47 9 35 3 28 12 2 0
102 8 24 11 50 0 0 0
16 6 17 1 20 0 1 1
8 0 13 1 3 12 2 0
122 14 44 13 71 0 2 1
0 0 5 0 2 0 1 0
4 0 5 0 0 12 0 0
126 14 54 13 73 12 3 1
90 12 33 8 36 2 2 1
Str. faecalis p-haem. streptococci a-haem. streptococci Anaerobic streptococci Staph. aureus Staph. a1bus
0 3 1 0 4 7
0 0 0 0 1 2
51 0 0 1 13 6
44 0 1 0 14 10
3 2 0 0 4 3
4 1 0 1 0 2
48 3 1 0 18 14
0 0 0 0 0 0
3 0 0 1 0 1
51 3 1 1 18 15
26 0 1 0 13 11
Corynebacterium Total
3
0
3
2
0
4
4
0
2
6
2
153
28
210
266
74
51
355
8
28
391
237
a Zone of inhibition for the agents tested in combination at least 5 mm larger than the largest zone of inhibition for the agents tested separately (I, 2).
Table III. Synergic effect of sulphamethoxazole (SM) and trimethoprim (TMP) assessed on the basis of the sensitivity of the bacterial strains to the agents separately (disk method) z~zone
of inhibition in mm; Bactv--Bactrim (SM+TMP) SM z> 18 SyTMP z> 18 nergy
Escherichia 56 Enterobacter 2 Klebsiella 7 Citrobacter 9 Proteus + Providencia 26 Ps, aeruginosa 0 Alcaligenes faecalis 0 Moraxella 0 Str. faecalis p-haem. streptococci a-haem. streptococci Anaerobic streptococci Staph. aureus Staph. a1bus Corynebacterium Total
SM z';;; 18 SyTMP z> 18 nergy
SM z> 18 SyTMP z';;; 18 nergy
SM z «: 18 SyBact. TMP z';;; 18 nergy z> 18
SM z';;; 12 SyBact. TMP z';;; 12 nergy z> 18
50 2 5 6
46 6 17 2
22 4 8 1
10 2 6 1
9 2 6 0
14 4 24 1
9 4 14 1
10 4 11 1
6 0 12 1
2 0 7 1
2 0 2 1
14 0
24 0
9 0
14 0
9 0
9 12
4 2
7 0
1 12
1 2
1 0
0 0
0 0
0 0
1 1
0 1
2 0
2 0
1 0
1 0
1 0
1 0
0
0
44
22
0
0
7
4
4
4
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 12 5
0 8 4
0 2 2
0 2 1
1 2 3
0 2 2
0 2 2
1 0 1
0 0 0
0 0 0
0 2 5 2 110
0 1 4 1 84 (76%)
0 156
0 78 (50%)
0 40
30 (70%)
3 82
1 45 43 (55 %) (52 %)
3 42
1 17 9 (40%)(21%)
Scand J Infect Dis 2
208 40
H. Laursen and M. Lykkegaard Nielsen ,
mm
38
/
/
36
I gram negative rods
34
• gram positive cocci
32 30 28 26 24 22
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20
from the table that the synergic effect seems to be less dependent on the TMP-sensitivity. There is a significant difference in the incidence of synergy in strains with complete sensitivity to SM and in strains with reduced sensitivity to this agent. Table IV shows the correlation between the sensitivity of Staphylococcus aureus strains tested secondarily to SM /TMP and other chemotherapeutics. It should be observed that 10 out of 12 multiresistant strains were fully sensitive to TMP.
18
16 14 12 12 14 16 18 20 22 24 26 28 30 32 34 36 38 /.0 42 44 mm
1. The degree of synergy between trimethoprim (TMP) and sulphamethoxazole (8M) in 391 bacterial strains. Ordinate: the largest zone of inhibition for either 8M or TMP. Abscissa: the zone of inhibition for Bactrim (8M + TMP). The full line represents the same zone of inhibition for one of the agents (the largest zone for 8M or TMP) and for Bactrim. The dotted line indicates the transition to "definite" synergy, i.e. a difference in zone of inhibition of more than 4 mm. Fig.
degree of synergy appears from Fig. 1, in which all strains tested secondarily are shown graphically. It appears from the figure that there is a very marked deviation and that often pronounced synergy is found. Table III shows the occurrence of synergy as compared with the sensitivity of the bacteria to the individual agents SM and TMP. It appears Table IV. Sensitivity to sulphamethoxazole (SM) and trimethoprim (TMP) of 18 Staph. aureus strains tested secondarily compared with sensitivity to other chemotherapeutics P~ resistant to penicillin; S ~ resistant to streptomycin; T~ resistant to tetracycline; M ~ resistant to methicillin; Ce ~ resistant to cephalosporins
SM TMP (zone of inhibition (zone of inhibition in mm) in mm) > 18
13-18
Sensitive 1 P 3 PST PSTM PSTMCe Total
4
Scand J Infect Dis 2
~
12 > 18
13-18
12 Synergy
8
7
1 1
2 4 1 1 5
13
14
4
13
1 3
1 3
~
3
I
DISCUSSION From a bacteriological point of view the SM / TMP combination should be a promising antibacterial agent, firstly because, during studies in vitro on a weight basis, TMP presents a considerably higher activity than sulphonamides, secondly because the two agents act synergically, and thirdly because the combination is bactericidal (1, 2, 6). If it is taken into consideration that the bac-
teria were isolated from the urinary tract in hospital patients, a fairly low number of resistant Staph. aureus and Pseudomonas aeruginosa strains was seen. In 4 cases anaerobic bacteria were isolated. One of these was Clostridium Welchii which was isolated in pure culture and in significant number from one patient. This patient presented pronounced symptoms of cystitis which disappeared after 4 days of Bactrim therapy, during which the urine became sterile. The results obtained in the series tested primarily and secondarily showed that there was a high frequency of sulphonamide-resistant strains, but that a relatively low number of strains were resistant to TMP. Bohni (2) examined 354 isolates, mainly cultures from the urinary tract. This material compares well with ours as far as the distribution into species is concerned, but Bohni's material comprised a much lower number of sulphonamideresistant strains (31 %). The proportion of TMPresistant strains (15 %) in Bohni's series was of the same order of magnitude as that found in our study. From a clinical point of view, the interest is centred on the sulphonamide-resistant pathogens and on the synergic effect of the two agents in combination.
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Urinary tract pathogens-sensitivity to trimethoprimjsulphamethoxazole If the family Enterobacteriaceae is viewed collectively, it appears from Table II that 46 %, 67 % and 90 % of the strains, respectively, were fully sensitive to SM, TMP and Bactrim. On the basis of Table II, it seems justifiable to conclude that Bactrim therapy, from this "in vitro" point of view, might be indicated in cases with sulphonamide-resistant pathogens within the genera Escherichia, Enterobacter, Klebsiella and Proteus-Providencia. Relatively higher numbers of sulphonamide-resistant strains appear within the genera Enterobacter and Klebsiella, only 29 and 24 %, respectively, being sensitive to SM. Similarly, 100 and 81 %, respectively, were found to be sensitive to the agents in combination. In contrast, infections produced by Ps. aeruginosa do not seem to be well suited for Bactrim treatment. As will be seen from Table II, all strains were found to be resistant to either agent and, in these cases, only moderate synergy was seen. The series comprise 51 Str. faecalis strains. 44 and 48 strains were found to be fully sensitive to TMP and Bactrim, respectively. Hence, Bactrim might be valuable in these urinary tract infections. The same considerations can be applied in cases of infections with Staph. aureus (fable IV), in which 10 out of 12 multi-resistant strains were found to be fully sensitive to TMP. The synergic effect (Fig. 1) will cause the greater majority of strains to be sensitive to the SM jTMP combination, with zones of inhibition within therapeutic range. This agrees well with the findings of Darrell et al. (6) who, by means of activity determinations (by the plate-dilution method), were able to demonstrate that inefficient concentrations of one agent reduced the MIC of the other, often by 8 times and frequently more. The synergy (fable III) seems mainly to depend on the sensitivity to sulphonamides. Bohni (1, 2) found similar conditions. However, synergy was demonstrated in fewer cases in Bohni's material than in our series.
4.
5. 6.
7. 8.
9.
209
analysis of dihydrofolate-reductase from various species. Molec Pharmacol 1: 126, 1965. Bushby, S. R. M. & Hitchings, G. H.: Trimethoprim, a sulphonamide potentiator. Brit J Pharmacol 33: 72, 1968. Clinical data sheet-trimethoprim. La Roche & Co. AG, Basel 1969. Darrell, J. H., Garrod, L. P. & Waterworth, P.: Trimethoprim: laboratory and clinical studies. J Clin Path 21: 202, 1968. Lykkegaard Nielsen, M. & Laursen, H.: Trimethoprim. Nord Med 84: 1070, 1970. Reeves, D. S., Faiers, M. C., Pursell, R E. & Brumfitt, W.: Trimethoprim-sulphamethoxazole: comparative study in urinary infections in hospital. Brit Med J 1: 541, 1969. Vejlsgaard, R: Urinundersegelse. Ugeskr Laeg 131: 545, 1969.
H. Laursen, M.D •• Iunggreensvej 9, 2000 Kebenhavn F. Denmark
REFERENCES 1. Bohni, E.: Sensitivity testing and outcome of therapy with the combination trimethoprim/sulphamethoxazole in patients with urinary tract infection. 6th International Congress of Chemotherapy, Tokyo 1969. 2. - Personal communication. 3. Burchall, J. J. & Hitchings, G. H.: Inhibitor binding 14-701954
Scand J Infect Dis 2
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
Urinary Tract Pathogens-Sensitivity to Trimethoprim/Sulphamethoxazole Helga Laursen & Mogens Lykkegaard Nielsen To cite this article: Helga Laursen & Mogens Lykkegaard Nielsen (1970) Urinary Tract Pathogens-Sensitivity to Trimethoprim/Sulphamethoxazole, Scandinavian Journal of Infectious Diseases, 2:3, 205-209 To link to this article: http://dx.doi.org/10.3109/inf.1970.2.issue-3.09
Published online: 02 Jan 2015.
Submit your article to this journal
View related articles
Citing articles: 6 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=infd19 Download by: [University of California, San Diego]
Date: 13 November 2015, At: 00:15
Scand J Infect Dis 2: 205-209, 1970
URINARY TRACT PATHOGENS-SENSITIVITY TO TRIMETHOPRIMjSULPHAMETHOXAZOLE Helga Laursen and Mogens Lykkegaard Nielsen
Downloaded by [University of California, San Diego] at 00:15 13 November 2015
From Statens Seruminstitut, Laboratory of Clinical Microbiology at Fredriksberg Hospital, and the Department K of Surgery, Fredriksberg Hospital, Copenhagen, Denmark
ABSTRACT. The sensitivity to sulphamethizole, sulphamethoxazole (SM), trimethoprim (TMP) and the combination SM/TMP of freshly isolated urinary tract pathogens from hospital patients was studied. Out of 798 strains tested primarily, 47% were found to be fully sensitive to sulphamethizole (tablet method), and 81 % fully sensitive to the SM/TMP combination (disk method), Out of 423 strains with reduced sensitivity to sulphamethizole, 65% were fully sensitive to SM/TMP. Out of 391 strains tested secondarily (disk method), 39% were fully sensitive to SM, 68% fully sensitive to TMP, and 91 % to SM/ TMP. Of 283 strains presenting a reduced sensitivity to SM, 85% were fully sensitive to SM/TMP. In both series the isolated Streptococcus faecalis strains were highly resistant to sulphamethizole and SM, but presented a very high degree of sensitivity to TMP and SM/TMP. In contrast, nearly all isolated Pseudomonas aeruginosa strains were found to be resistant both to suiphonamides, TMP and SM/TMP. Of the 18 Staphylococcus aureus strains tested secondarily, 10 out of 12 multiresistant strains were fully sensitive to TMP. Synergy between SM and TMP could be demonstrated in 61 % of the 391 strains tested secondarily. The synergic effect depended on the degree of sensitivity to SM.
The description of trimethoprim (fMP) (2,4diamino-5-(3 1 , 41,51-trimethoxybenzyl)-pyrimidine) as being an antibacterial agent alone and in combination with sulphonamides, is due to Hitchings and others (for survey, see 4). TMP is an inhibitor of dihydrofolate reductase, the enzyme being responsible for the conversion of dihydrofolic acid to tetrahydrofolic acid. Differential studies indicate that bacterial dihydrofolate reductase is 10 000 times as sensitive to the inhibitory action of TMP as is the corresponding mammalian enzyme (3, 5). Several investigations in vitro have shown that, when acting alone, TMP inhibits the growth of a wide range of bacteria generally in much lower
concentrations than the sulphonamides do (1, 2, 6, 8). The combined action of the bacteriostatic TMP and a sulphonamide produces a bactericidal effect, and the action of TMP with a sulphonamide is often synergic (2, 6). Studies in vitro with TMP and sulphonamides have often been performed using sulphamethoxazole (SM) because rates of absorption and elimination are almost identical with those of TMP (1, 2, 8). Sulphafurazole has also been used (6). Several clinical trials in various types of infection with TMP and SM in varying combinations have been performed and the pharmacodynamics of TMP are well known (for survey, see 7). The compound available for clinical use consists of a combination of TMP and SM at a ratio of 1 : 5, on a weight basis. The purpose of the present study was to determine the in vitro effect of trimethoprim alone and in combination with sulphamethoxazole against freshly isolated bacterial strains from the urinary tract in hospital patients. Furthermore, the aim was to determine the synergic effect of TMP and SM and to determine the possible value of the combination of TMP and SM in cases with sulphonamide-resistant strains. MATERIAL AND METHODS The study comprised 798 bacterial strains, tested prim-. arily, collected over a period of 2 months. All urine samples received in the laboratory with;;, 100000 colonies. per ml were included in the study. The final series consisted of 684 samples of urine, 97 samples presenting mixed flora with up to 3 different species. All strains were tested primarily for sensitivity to sulphamethizole using the tablet method (Neo-Sensitabsta). Furthermore, the strains were tested by the disk method for sensitivity to Scand I Infect Dis 2·
206
H. Laursen and M. Lykkegaard Nielsen
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Table I. Sensitivity to sulphamethizole (tablet method) and Bactrim (sulphamethoxazole + trimethoprim) (disk method) of 798 bacterial strains tested primarily Sulphamethizole (zone of inhibition in mm)"
Bactrim (zone of inhibition in mrn)"
;;>28
15-27
.-;;14
;;>18
13-18
173 10 43 10 86 2 0 5 4
22 2 14 2 10 4 3 1 0
96 9 48 5 63 15 1 3 1
268 19 77 14 122 1 2 5 3
0 4 2 0 15 15
1 0 0 0 8 1
95 0 0 1 8 6
Aerobic corynebacterium Anaerobic corynebacterium
3 1
1 0
Bact. fragilis CI. Welchii
1 1 375
Escherichia Enterobacter Klebsiella Citrobacter Proteus + Providencia Ps. aeruginosa Alcaligenes faecalis Moraxella + Anitratum Other aerobic gram-negative rods Str, faecalis fJ-haem. streptococci Q(- haem. streptococci Anaerobic streptococci Staph. aureus Staph. albus
Total
.-;; 12
Total
19 2 24 3 30 3 2 3 0
4 0 4 0 7 17 0 1 2
291 21 105 17 159 21 4 9 5
79 3 2 0 30 20
14 1 0 0 1 1
3 0 0 1 0 1
96 4 2 1 31 22
3 0
4 1
0 0
3 0
7 1
0 0
0 0
0 0
1 1
0 0
1 1
69
354
650
105
43
798
a Tablet method: ;;> 28 mm= sensitive; 15-27 mm= slightly sensitive; .-;; 14 mm e- resistant. b
Disk method (1,2): > 18 mm-e sensitive; 13-18 mm e-slightly sensitive; .-;; 12 mm-s resistant.
the TMP ISM combination (Bactrim®). Peptone-free 10% horse blood agar plates were used in primary sensitivity tests. Quantitative urine cultivation was carried out according to the principles laid down by Vejlsgaard (9). Qualitative cultivation with determination of species was made applying routine bacteriological methods. Out of the 798 bacterial strains, 391 strains from a medical and a surgical ward were furthermore tested secondarily for sensitivity to TMP, SM and Bactrim, using disks containing 1.25 ftg TMP, 23.75 ftg SM, and disks containing the above-mentioned amounts of the two agents per disk. Sensitivity tests were made on freshly prepared suspensions of bacteria, using the technique described by Bohni (I, 2). According to Bohni, the use of the two agents in combination in one disk is justified by the existence of an almost identical diffusion time in the agar of the agents. The ratio 1 : 19 (1.25 flg TMP and 23.75 flg SM) in the disks is based on the fact that TMP often has a 20-fold higher activity in vitro and that the agents show a relationship of 1 : 20 in human plasma during treatment with the combination Bactrim (1, 2). The plates were left for pre-diffusion for 1 hour and then incubated at 35°C for 18-20 hours.
RESULTS The samples tested primarily are shown in Table I and those tested secondarily in Table II. To Scand J Infect Dis 2
simplify a review, the samples shown in the tables have generaIly been classified into genera, except cases of particular diagnostic interest. Alcaligenes and others have also been listed under species, since only one species was found in these groups. It appears from Table I that 44 % of the strains were resistant to sulphamethizole and 5 % to Bactrim. It should be noted specifically that only 3 of 96 Streptococcus faecalis strains were resistant to Bactrim. Out of 423 strains presenting reduced sensitivity to sulphamethizole, 275 (65 %) were fully sensitive to Bactrim. Table II shows that 54 % and 13 % of all strains were resistant to SM and TMP, respectively. The 51 Str. faecalis strains were resistant to SM, whereas 7 strains only were not fully sensitive to TMP. Out of 238 strains presenting reduced sensitivity to SM, 36 had a reduced sensitivity to the SM jTMP combination, i.e. 85 % of the sulphonamide-resistant strains were fully sensitive to the combination. The synergy appears from Tables II and III and Fig. 1. Table II shows that definite synergy could be demonstrated in 61 % of all strains. The
Urinary tract pathogens-sensitivity to trimethoprim / sulphamethoxazole
207
Table II. Sensitivity to sulphamethoxazole (SM) and trimethoprim (TMP) (disk method) of 391 bacterial strains tested secondarily SM (zone of inhibition in mm)
Downloaded by [University of California, San Diego] at 00:15 13 November 2015
> 18
13-18
TMP (zone of inhibition in mm)
';;;12
> 18
SM+TMP (zone of inhibition inmm)
13-18
';;;\2
>18
13-18
,;;; 12
Total
Synergy"
Escherichia Enterobacter Klebsiella Citrobacter Proteus + Providencia Ps. aeruginosa Alcaligenes faecalis Moraxella
66 4 13 10 40 0 1 1
13 1 6 0 5 0 0 0
47 9 35 3 28 12 2 0
102 8 24 11 50 0 0 0
16 6 17 1 20 0 1 1
8 0 13 1 3 12 2 0
122 14 44 13 71 0 2 1
0 0 5 0 2 0 1 0
4 0 5 0 0 12 0 0
126 14 54 13 73 12 3 1
90 12 33 8 36 2 2 1
Str. faecalis p-haem. streptococci a-haem. streptococci Anaerobic streptococci Staph. aureus Staph. a1bus
0 3 1 0 4 7
0 0 0 0 1 2
51 0 0 1 13 6
44 0 1 0 14 10
3 2 0 0 4 3
4 1 0 1 0 2
48 3 1 0 18 14
0 0 0 0 0 0
3 0 0 1 0 1
51 3 1 1 18 15
26 0 1 0 13 11
Corynebacterium Total
3
0
3
2
0
4
4
0
2
6
2
153
28
210
266
74
51
355
8
28
391
237
a Zone of inhibition for the agents tested in combination at least 5 mm larger than the largest zone of inhibition for the agents tested separately (I, 2).
Table III. Synergic effect of sulphamethoxazole (SM) and trimethoprim (TMP) assessed on the basis of the sensitivity of the bacterial strains to the agents separately (disk method) z~zone
of inhibition in mm; Bactv--Bactrim (SM+TMP) SM z> 18 SyTMP z> 18 nergy
Escherichia 56 Enterobacter 2 Klebsiella 7 Citrobacter 9 Proteus + Providencia 26 Ps, aeruginosa 0 Alcaligenes faecalis 0 Moraxella 0 Str. faecalis p-haem. streptococci a-haem. streptococci Anaerobic streptococci Staph. aureus Staph. a1bus Corynebacterium Total
SM z';;; 18 SyTMP z> 18 nergy
SM z> 18 SyTMP z';;; 18 nergy
SM z «: 18 SyBact. TMP z';;; 18 nergy z> 18
SM z';;; 12 SyBact. TMP z';;; 12 nergy z> 18
50 2 5 6
46 6 17 2
22 4 8 1
10 2 6 1
9 2 6 0
14 4 24 1
9 4 14 1
10 4 11 1
6 0 12 1
2 0 7 1
2 0 2 1
14 0
24 0
9 0
14 0
9 0
9 12
4 2
7 0
1 12
1 2
1 0
0 0
0 0
0 0
1 1
0 1
2 0
2 0
1 0
1 0
1 0
1 0
0
0
44
22
0
0
7
4
4
4
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 12 5
0 8 4
0 2 2
0 2 1
1 2 3
0 2 2
0 2 2
1 0 1
0 0 0
0 0 0
0 2 5 2 110
0 1 4 1 84 (76%)
0 156
0 78 (50%)
0 40
30 (70%)
3 82
1 45 43 (55 %) (52 %)
3 42
1 17 9 (40%)(21%)
Scand J Infect Dis 2
208 40
H. Laursen and M. Lykkegaard Nielsen ,
mm
38
/
/
36
I gram negative rods
34
• gram positive cocci
32 30 28 26 24 22
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20
from the table that the synergic effect seems to be less dependent on the TMP-sensitivity. There is a significant difference in the incidence of synergy in strains with complete sensitivity to SM and in strains with reduced sensitivity to this agent. Table IV shows the correlation between the sensitivity of Staphylococcus aureus strains tested secondarily to SM /TMP and other chemotherapeutics. It should be observed that 10 out of 12 multiresistant strains were fully sensitive to TMP.
18
16 14 12 12 14 16 18 20 22 24 26 28 30 32 34 36 38 /.0 42 44 mm
1. The degree of synergy between trimethoprim (TMP) and sulphamethoxazole (8M) in 391 bacterial strains. Ordinate: the largest zone of inhibition for either 8M or TMP. Abscissa: the zone of inhibition for Bactrim (8M + TMP). The full line represents the same zone of inhibition for one of the agents (the largest zone for 8M or TMP) and for Bactrim. The dotted line indicates the transition to "definite" synergy, i.e. a difference in zone of inhibition of more than 4 mm. Fig.
degree of synergy appears from Fig. 1, in which all strains tested secondarily are shown graphically. It appears from the figure that there is a very marked deviation and that often pronounced synergy is found. Table III shows the occurrence of synergy as compared with the sensitivity of the bacteria to the individual agents SM and TMP. It appears Table IV. Sensitivity to sulphamethoxazole (SM) and trimethoprim (TMP) of 18 Staph. aureus strains tested secondarily compared with sensitivity to other chemotherapeutics P~ resistant to penicillin; S ~ resistant to streptomycin; T~ resistant to tetracycline; M ~ resistant to methicillin; Ce ~ resistant to cephalosporins
SM TMP (zone of inhibition (zone of inhibition in mm) in mm) > 18
13-18
Sensitive 1 P 3 PST PSTM PSTMCe Total
4
Scand J Infect Dis 2
~
12 > 18
13-18
12 Synergy
8
7
1 1
2 4 1 1 5
13
14
4
13
1 3
1 3
~
3
I
DISCUSSION From a bacteriological point of view the SM / TMP combination should be a promising antibacterial agent, firstly because, during studies in vitro on a weight basis, TMP presents a considerably higher activity than sulphonamides, secondly because the two agents act synergically, and thirdly because the combination is bactericidal (1, 2, 6). If it is taken into consideration that the bac-
teria were isolated from the urinary tract in hospital patients, a fairly low number of resistant Staph. aureus and Pseudomonas aeruginosa strains was seen. In 4 cases anaerobic bacteria were isolated. One of these was Clostridium Welchii which was isolated in pure culture and in significant number from one patient. This patient presented pronounced symptoms of cystitis which disappeared after 4 days of Bactrim therapy, during which the urine became sterile. The results obtained in the series tested primarily and secondarily showed that there was a high frequency of sulphonamide-resistant strains, but that a relatively low number of strains were resistant to TMP. Bohni (2) examined 354 isolates, mainly cultures from the urinary tract. This material compares well with ours as far as the distribution into species is concerned, but Bohni's material comprised a much lower number of sulphonamideresistant strains (31 %). The proportion of TMPresistant strains (15 %) in Bohni's series was of the same order of magnitude as that found in our study. From a clinical point of view, the interest is centred on the sulphonamide-resistant pathogens and on the synergic effect of the two agents in combination.
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Urinary tract pathogens-sensitivity to trimethoprimjsulphamethoxazole If the family Enterobacteriaceae is viewed collectively, it appears from Table II that 46 %, 67 % and 90 % of the strains, respectively, were fully sensitive to SM, TMP and Bactrim. On the basis of Table II, it seems justifiable to conclude that Bactrim therapy, from this "in vitro" point of view, might be indicated in cases with sulphonamide-resistant pathogens within the genera Escherichia, Enterobacter, Klebsiella and Proteus-Providencia. Relatively higher numbers of sulphonamide-resistant strains appear within the genera Enterobacter and Klebsiella, only 29 and 24 %, respectively, being sensitive to SM. Similarly, 100 and 81 %, respectively, were found to be sensitive to the agents in combination. In contrast, infections produced by Ps. aeruginosa do not seem to be well suited for Bactrim treatment. As will be seen from Table II, all strains were found to be resistant to either agent and, in these cases, only moderate synergy was seen. The series comprise 51 Str. faecalis strains. 44 and 48 strains were found to be fully sensitive to TMP and Bactrim, respectively. Hence, Bactrim might be valuable in these urinary tract infections. The same considerations can be applied in cases of infections with Staph. aureus (fable IV), in which 10 out of 12 multi-resistant strains were found to be fully sensitive to TMP. The synergic effect (Fig. 1) will cause the greater majority of strains to be sensitive to the SM jTMP combination, with zones of inhibition within therapeutic range. This agrees well with the findings of Darrell et al. (6) who, by means of activity determinations (by the plate-dilution method), were able to demonstrate that inefficient concentrations of one agent reduced the MIC of the other, often by 8 times and frequently more. The synergy (fable III) seems mainly to depend on the sensitivity to sulphonamides. Bohni (1, 2) found similar conditions. However, synergy was demonstrated in fewer cases in Bohni's material than in our series.
4.
5. 6.
7. 8.
9.
209
analysis of dihydrofolate-reductase from various species. Molec Pharmacol 1: 126, 1965. Bushby, S. R. M. & Hitchings, G. H.: Trimethoprim, a sulphonamide potentiator. Brit J Pharmacol 33: 72, 1968. Clinical data sheet-trimethoprim. La Roche & Co. AG, Basel 1969. Darrell, J. H., Garrod, L. P. & Waterworth, P.: Trimethoprim: laboratory and clinical studies. J Clin Path 21: 202, 1968. Lykkegaard Nielsen, M. & Laursen, H.: Trimethoprim. Nord Med 84: 1070, 1970. Reeves, D. S., Faiers, M. C., Pursell, R E. & Brumfitt, W.: Trimethoprim-sulphamethoxazole: comparative study in urinary infections in hospital. Brit Med J 1: 541, 1969. Vejlsgaard, R: Urinundersegelse. Ugeskr Laeg 131: 545, 1969.
H. Laursen, M.D •• Iunggreensvej 9, 2000 Kebenhavn F. Denmark
REFERENCES 1. Bohni, E.: Sensitivity testing and outcome of therapy with the combination trimethoprim/sulphamethoxazole in patients with urinary tract infection. 6th International Congress of Chemotherapy, Tokyo 1969. 2. - Personal communication. 3. Burchall, J. J. & Hitchings, G. H.: Inhibitor binding 14-701954
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