Correspondence
521
Downloaded from http://jac.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 8, 2015
Bacteroides fragilis infections of the urinary tract Sir, The susceptibility or resistance to antimicrobial agents of aerobic organisms that produce urinary tract infections is well known, but the situation is less clear in infections caused by anaerobic bacteria. Although anaerobic urinary tract infections are uncommon, anaerobes are occasionally implicated as urinary pathogens (Finegold, Miller, Merril & Posnick, 1965; Headington & Beyerkein, 1966; Segura, Kelalis, Martin & Smith, 1972; Garcia, Prieto, Garcia & Gomez, 1975). With the notable exception of Bacteroides fragilis most anaerobes are susceptible to P-lactam agents; all anaerobes, however, are resistant to the aminoglycosides. Since antibiotics are frequently used impirically in the treatment of urinary tract infections, we undertook an in vitro study of the activity against B. fragilis of four urinary chemotherapeutic agents. The susceptibilities of 124 human isolates of B. fragilis to nitrofurantoin, nitro-5-hydroxy8-quinoline (Nibiol), nalidixic acid and oxolinic acid were examined by agar disc diffusion and by minimum inhibitory concentration determinations. For disc diffusion studies the inoculum was prepared as a standard dilution from a 24-h culture in cysteine brain heart infusion broth, and applied to laked sheep blood agar plates D. I. ANNEAR with a sterile cotton swab. After application of Department of Microbiology, sensitivity discs the cultures were incubated at Royal Perth Hospital, Perth, 37°C anaerobically for 48 h. Western Australia, Minimum inhibitory concentrations were Australia determined using twofold serial dilutions of the antimicrobial agent incorporated in laked sheep blood agar plates. The inoculum was a 24-h culture in cysteine brain heart infusion References broth adjusted to the No. I McFarland Annear, D. I. & Grubb, W. B. Linked and un- nephelometer standard; it was applied to the stable resistance to kanamycin and penicillin, agar surface with a Steer's replicator. Control and diffusible pigment production, in an isolate of Staphylococcus aureus. Journal of Medical cultures without antimicrobial agents were incubated under aerobic and anaerobic conMicrobiology 5: 109-11 (1971). ditions, and in an atmosphere of air plus 10% Evans, R. J. & Waterworth, P. M. Naturallyoccurring fusidic acid resistance in staphylo- COS. After anaerobic incubation for 48 h at cocci and its linkage to other resistances. 37°C the MIC was recorded as the minimum Journal of Clinical Pathology 19: 555-60 amount of antimicrobial agent that showed visible growth. (1966). Grubb, W. B. & Annear, D. I. Linked extraAs is clear from Table I nitrofurantoin and chromosomal resistance to streptomycin and 'Nibiol' were active against all of the strains kanamycin in Staphylococcus aureus. Journal of of B. fragilis at concentrations easily achieved Medical Microbiology 5: 529-32 (1972). in urine; at concentrations 3s 12-5 ug/ml there Soussy, C. J., Bouanchaud, D. H., Fouace, J., was no growth of any of the strains tested. On Dublanchet, A. & Duval, J. A gentamicin plates containing naladixic acid and oxolinic resistance plasmid in Staphylococcus aureus. Annals of Microbiology {Paris) 126B: 91-4 acid all of the anaerobes showed uninhibited growth at the highest concentrations used (1975).
25°C). At various intervals fresh broth subcultures were made and spread for single colonies. These were screened by methods previously described (Annear & Grubb, 1972) on Oxoid Diagnostic Sensitivity Testing Agar (DST) containing tetracycline (5 ug/ml) and gentamicin (5 ug/ml). A gradually increasing proportion of sensitive variants was detected in all cultures. After about 20 days of ageing these levels were usually more than 95 % of the total population. Several hundred colonies of each category were screened and in all of them linkage between the two drugs was demonstrated. Minimum inhibitory concentration values for the resistant and sensitive organisms were determined in DST agar using one drop inocula containing approximately 10* cells. For tetracycline, the values for the resistant and sensitive organisms were 25 and 0-3 ng/ml respectively. The corresponding values for gentamicin were 12-5 and 0-2 ug/ml. The results thus reveal another clearly defined example of linked and unstable drug resistance to add to those already demonstrated for S. aureus. It should be of interest to investigate the transducibility of the determinant concerned in this dual resistance and to characterize the resistance mechanisms involved.
522
Correspondence Table I. Susceptibility of B. fragilis (124 strains) to antimicrobial agents Sub-species of B. fragilis
distasonis (12 strains) fragilis (61 strains) ovatus (4 strains) thetaiotaomicron (24 strains) vulgatus (23 strains)
Cumulative per cent susceptible to indicated concent (ug/ml) 0-8
1-6
31
6-2
12-5 0-8i
9
22 50
58 86 75
100 100 100
25 4
50 30
83 86
100 100
9
4
(100 ug/ml). F o r the two active compounds there was poor correlation between the M I C and the inhibition zone diameter in disc difiusion tests. Although the frequency of urinary infections involving anaerobes is much lower than with aerobes, their role should not be wholly disregarded. Finegold et al. (1965) referred to 56 urinary tract infections due to anaerobes, of which 10 were produced by B. fragilis, and Segura et al. (1972) attributed an anaerobic bacterial aetiology t o 1 to 2 % of all urinary infections. Of 100 clean-voided mid-stream specimens of urine examined by Garcia et al. (1975) 38 yielded anaerobes; B. fragilis was isolated on 13 occasions, once in pure culture. Although we recognize that urinary infections due t o anaerobes represent only a small fraction of the total, it is clearly important that these uncommon cases should not be overlooked, and that an adequate antimicrobial therapeutic regime for them is defined. F o r this purpose both nitrofurantoin and 'NibioF seem t o be well suited. J. A. GARCIA J. PRIETO J. E. GARCIA M. C. SAENZ Department of Microbiology and Hygiene, Faculty of Medicine, University of Salamanca, Spain References Finegold, S. M., Miller, L. G., Merril, S. L. & Posnick, D. Significance of anaerobic and capnophilic bacteria isolated from the urinary tract. Progress in Pyelonephritis Kass, J. (ed.) F. A. Davis Co., Philadelphia (1965). Finegold, S. M., Harada, N. E. & Miller, L. G. Antibiotic susceptibility patterns as aids in classification and characterization of Gram negative anaerobic bacilli. Journal of Bacteriology 94: 1443-50 (1972).
4 13
31
6-2
12-5 100 or more
50 9
91 68 50
100 100 100
100 100 100
79 73
91 95
100 100
100 100 Nalidixic and oxolinic acids
Nitro-5 hidroxy-l3 quinoline
Garcia, J. A., Prieto, J., Garcia, J. E. & Gomez, A. C. Germenes anaerobios en infecciones urinarias. Ada Medica iv: 39-44 (1975). Headington, J. T. & Beyerlein B. Anaerobic bacteria in routine urine culture. Journal of Clinical Pathology 19: 573-6 (1966). Segura, J. W., Kelalis, P. P., Martin, W. J. & Smith, L. M. Anaerobic bacteria in the urinary tract. Mayo Clinical Proceedings 47: 30-3 (1972). Susceptibility of Bacteroides fragilis to fusidic acid Sir, The choice of antibacterial agents available for the treatment of systemic infection with Bacteroides fragilis is limited. In this hospital, of 122 isolates of Bacteroides spp. only 41 % were sensitive to co-trimoxazole and 47 % to tetracycline but 89 % were sensitive to fusidic acid by disc diffusion (Goodwin, 1975). The percentage of strains sensitive to fusidic acid has been reported by other workers as 42% of 19 strains (Mackenzie & Litton, 1974) 70% of 200 strains (Leigh, 1974) and 100% of 794 strains (Mitchell, 1973). Metronidazole is effective (Report, 1975) but must usually be given by mouth and its mutagenic properties may give some cause for concern. Phillips & Warren (personal communication) found the MIC of fusidic acid for B. fragilis to be 0-25 to 4 ug/ml. We determined the MIC of fusidic acid in solid medium with and without blood for 14 clinical isolates of B. fragilis (Table I). B. fragilis multiplies more rapidly at pH 6-0 than at pH 7-4 but the results for each strain were the same at both pH values. In liquid medium the results without serum were comparable but the addition of serum raised the MIC considerably although in all but one of the strains the MIC was still
521
Downloaded from http://jac.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 8, 2015
Bacteroides fragilis infections of the urinary tract Sir, The susceptibility or resistance to antimicrobial agents of aerobic organisms that produce urinary tract infections is well known, but the situation is less clear in infections caused by anaerobic bacteria. Although anaerobic urinary tract infections are uncommon, anaerobes are occasionally implicated as urinary pathogens (Finegold, Miller, Merril & Posnick, 1965; Headington & Beyerkein, 1966; Segura, Kelalis, Martin & Smith, 1972; Garcia, Prieto, Garcia & Gomez, 1975). With the notable exception of Bacteroides fragilis most anaerobes are susceptible to P-lactam agents; all anaerobes, however, are resistant to the aminoglycosides. Since antibiotics are frequently used impirically in the treatment of urinary tract infections, we undertook an in vitro study of the activity against B. fragilis of four urinary chemotherapeutic agents. The susceptibilities of 124 human isolates of B. fragilis to nitrofurantoin, nitro-5-hydroxy8-quinoline (Nibiol), nalidixic acid and oxolinic acid were examined by agar disc diffusion and by minimum inhibitory concentration determinations. For disc diffusion studies the inoculum was prepared as a standard dilution from a 24-h culture in cysteine brain heart infusion broth, and applied to laked sheep blood agar plates D. I. ANNEAR with a sterile cotton swab. After application of Department of Microbiology, sensitivity discs the cultures were incubated at Royal Perth Hospital, Perth, 37°C anaerobically for 48 h. Western Australia, Minimum inhibitory concentrations were Australia determined using twofold serial dilutions of the antimicrobial agent incorporated in laked sheep blood agar plates. The inoculum was a 24-h culture in cysteine brain heart infusion References broth adjusted to the No. I McFarland Annear, D. I. & Grubb, W. B. Linked and un- nephelometer standard; it was applied to the stable resistance to kanamycin and penicillin, agar surface with a Steer's replicator. Control and diffusible pigment production, in an isolate of Staphylococcus aureus. Journal of Medical cultures without antimicrobial agents were incubated under aerobic and anaerobic conMicrobiology 5: 109-11 (1971). ditions, and in an atmosphere of air plus 10% Evans, R. J. & Waterworth, P. M. Naturallyoccurring fusidic acid resistance in staphylo- COS. After anaerobic incubation for 48 h at cocci and its linkage to other resistances. 37°C the MIC was recorded as the minimum Journal of Clinical Pathology 19: 555-60 amount of antimicrobial agent that showed visible growth. (1966). Grubb, W. B. & Annear, D. I. Linked extraAs is clear from Table I nitrofurantoin and chromosomal resistance to streptomycin and 'Nibiol' were active against all of the strains kanamycin in Staphylococcus aureus. Journal of of B. fragilis at concentrations easily achieved Medical Microbiology 5: 529-32 (1972). in urine; at concentrations 3s 12-5 ug/ml there Soussy, C. J., Bouanchaud, D. H., Fouace, J., was no growth of any of the strains tested. On Dublanchet, A. & Duval, J. A gentamicin plates containing naladixic acid and oxolinic resistance plasmid in Staphylococcus aureus. Annals of Microbiology {Paris) 126B: 91-4 acid all of the anaerobes showed uninhibited growth at the highest concentrations used (1975).
25°C). At various intervals fresh broth subcultures were made and spread for single colonies. These were screened by methods previously described (Annear & Grubb, 1972) on Oxoid Diagnostic Sensitivity Testing Agar (DST) containing tetracycline (5 ug/ml) and gentamicin (5 ug/ml). A gradually increasing proportion of sensitive variants was detected in all cultures. After about 20 days of ageing these levels were usually more than 95 % of the total population. Several hundred colonies of each category were screened and in all of them linkage between the two drugs was demonstrated. Minimum inhibitory concentration values for the resistant and sensitive organisms were determined in DST agar using one drop inocula containing approximately 10* cells. For tetracycline, the values for the resistant and sensitive organisms were 25 and 0-3 ng/ml respectively. The corresponding values for gentamicin were 12-5 and 0-2 ug/ml. The results thus reveal another clearly defined example of linked and unstable drug resistance to add to those already demonstrated for S. aureus. It should be of interest to investigate the transducibility of the determinant concerned in this dual resistance and to characterize the resistance mechanisms involved.
522
Correspondence Table I. Susceptibility of B. fragilis (124 strains) to antimicrobial agents Sub-species of B. fragilis
distasonis (12 strains) fragilis (61 strains) ovatus (4 strains) thetaiotaomicron (24 strains) vulgatus (23 strains)
Cumulative per cent susceptible to indicated concent (ug/ml) 0-8
1-6
31
6-2
12-5 0-8i
9
22 50
58 86 75
100 100 100
25 4
50 30
83 86
100 100
9
4
(100 ug/ml). F o r the two active compounds there was poor correlation between the M I C and the inhibition zone diameter in disc difiusion tests. Although the frequency of urinary infections involving anaerobes is much lower than with aerobes, their role should not be wholly disregarded. Finegold et al. (1965) referred to 56 urinary tract infections due to anaerobes, of which 10 were produced by B. fragilis, and Segura et al. (1972) attributed an anaerobic bacterial aetiology t o 1 to 2 % of all urinary infections. Of 100 clean-voided mid-stream specimens of urine examined by Garcia et al. (1975) 38 yielded anaerobes; B. fragilis was isolated on 13 occasions, once in pure culture. Although we recognize that urinary infections due t o anaerobes represent only a small fraction of the total, it is clearly important that these uncommon cases should not be overlooked, and that an adequate antimicrobial therapeutic regime for them is defined. F o r this purpose both nitrofurantoin and 'NibioF seem t o be well suited. J. A. GARCIA J. PRIETO J. E. GARCIA M. C. SAENZ Department of Microbiology and Hygiene, Faculty of Medicine, University of Salamanca, Spain References Finegold, S. M., Miller, L. G., Merril, S. L. & Posnick, D. Significance of anaerobic and capnophilic bacteria isolated from the urinary tract. Progress in Pyelonephritis Kass, J. (ed.) F. A. Davis Co., Philadelphia (1965). Finegold, S. M., Harada, N. E. & Miller, L. G. Antibiotic susceptibility patterns as aids in classification and characterization of Gram negative anaerobic bacilli. Journal of Bacteriology 94: 1443-50 (1972).
4 13
31
6-2
12-5 100 or more
50 9
91 68 50
100 100 100
100 100 100
79 73
91 95
100 100
100 100 Nalidixic and oxolinic acids
Nitro-5 hidroxy-l3 quinoline
Garcia, J. A., Prieto, J., Garcia, J. E. & Gomez, A. C. Germenes anaerobios en infecciones urinarias. Ada Medica iv: 39-44 (1975). Headington, J. T. & Beyerlein B. Anaerobic bacteria in routine urine culture. Journal of Clinical Pathology 19: 573-6 (1966). Segura, J. W., Kelalis, P. P., Martin, W. J. & Smith, L. M. Anaerobic bacteria in the urinary tract. Mayo Clinical Proceedings 47: 30-3 (1972). Susceptibility of Bacteroides fragilis to fusidic acid Sir, The choice of antibacterial agents available for the treatment of systemic infection with Bacteroides fragilis is limited. In this hospital, of 122 isolates of Bacteroides spp. only 41 % were sensitive to co-trimoxazole and 47 % to tetracycline but 89 % were sensitive to fusidic acid by disc diffusion (Goodwin, 1975). The percentage of strains sensitive to fusidic acid has been reported by other workers as 42% of 19 strains (Mackenzie & Litton, 1974) 70% of 200 strains (Leigh, 1974) and 100% of 794 strains (Mitchell, 1973). Metronidazole is effective (Report, 1975) but must usually be given by mouth and its mutagenic properties may give some cause for concern. Phillips & Warren (personal communication) found the MIC of fusidic acid for B. fragilis to be 0-25 to 4 ug/ml. We determined the MIC of fusidic acid in solid medium with and without blood for 14 clinical isolates of B. fragilis (Table I). B. fragilis multiplies more rapidly at pH 6-0 than at pH 7-4 but the results for each strain were the same at both pH values. In liquid medium the results without serum were comparable but the addition of serum raised the MIC considerably although in all but one of the strains the MIC was still
