JAC
Journal of Antimicrobial Chemotherapy (1999) 43, 313–316
Correspondence The influence of factors affecting the ‘critical population’ density of inocula on the determination of bacterial susceptibility to antibiotics by disc diffusion methods J Antimicrob Chemother 1999; 43: 313 Alan J. Hedges Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK Tel:
44-(0)117-928-7759; Fax:
44-(0)117-928-7896.
Sir, The recent article by Gosden et al.1 prompted me to reflect on the widespread use of disc diffusion methods for determining antibiotic susceptibility that ignore an important finding of the definitive studies of Cooper 2 and Linton 3 on the formation of inhibition zones. These investigators showed that, although the intrinsic susceptibility of a bacterial isolate, under the in-vitro conditions of the test, is one parameter that influences zone diameter, of even greater importance are factors that determine the time taken for the bacterial inoculum to reach a ‘critical population’ density. The disc diffusion techniques most commonly used (the Kirby–Bauer and Stokes’ tests) take account of important factors, including the inoculum density, the composition of the medium, the delay between application of the disc and incubation, the temperature of incubation, etc., but the resulting zone of inhibition is then compared with that produced by a standard strain and/or predetermined templates, thereby disregarding the growth rate of the isolate being tested. In other words, these methods assume that all clinical isolates exhibit the same rate of growth in vitro as that of the control or ‘average’ strain. Many years ago, I carried out studies relating to the determination of the susceptibilities of Staphylococcus aureus isolates to erythromycin by the Kirby–Bauer method. I observed that, if a fully susceptible strain with a lag phase and a generation time of 30 min each mutated to one with a lag phase of the same duration, but a generation time of 21 min, the latter would appear to be resistant to erythromycin, even though its MIC was the same as that of the parent strain; a change in the reverse direction would result in a resistant organism’s appearing susceptible. Methods which overcome this difficulty by using discs
containing different concentrations have been proposed,4,5 but have not become popular, despite requiring only slightly more effort than the techniques in common use. Although I would invert the calculations in the second method referred to above,5 in practice, calculations are seldom needed, a simple, hand-drawn graph yielding a sufficiently accurate result. Moreover, such methods make the need to control many of the other factors associated with susceptibility testing less critical. The preceding discussion poses an obvious question: how variable are the growth kinetics of strains isolated in clinical practice? The answer may be ‘not very variable’, but it is clearly not in a patient’s best interests to be infected with a bacterium that is amenable to eradication by the antibiotic of choice, yet to be denied treatment with that agent because the organism appears to be resistant when susceptibility is determined by the disc diffusion method, owing to its rapid growth rate; the reverse situation is equally undesirable. I am hopeful that my colleagues in Bristol might turn their attentions to this interesting and potentially important issue.
References 1. Gosden, P. E., Andrews, J. M., Bowker, K. E., Holt, H. A., MacGowan, A. P., Reeves, D. S. et al. (1998). Comparison of the modified Stokes’ method of susceptibility testing with results obtained using MIC methods and British Society of Antimicrobial Chemotherapy breakpoints. Journal of Antimicrobial Chemotherapy 42, 161–9. 2. Cooper, K. E. (1963). The theory of antibiotic inhibition zones. In Analytical Microbiology (Kavanagh, F., Ed.), pp. 1–86. Academic Press, New York. 3. Linton, A. H. (1961). Interpreting antibiotic sensitivity tests. Journal of Medical Laboratory Technology 18, 1–20. 4. Shannon, R., Hedges, A. J. & Edwards, R. J. (1975). Distribution of levels of penicillin resistance among freshly isolated strains of N. gonorrhoeae. Application of a novel sensitivity assay. British Journal of Venereal Diseases 51, 246–50. 5. Delignette-Muller, M. L. & Flandrois, J. P. (1994). An accurate diffusion method for determining bacterial sensitivity to antibiotics. Journal of Antimicrobial Chemotherapy 34, 73–81.
313 © 1999 The British Society for Antimicrobial Chemotherapy
Correspondence reported as sensitive if the zone radius was equal to, wider than, or not 7 mm smaller than the control; intermediate if the zone radius was 2 mm but smaller than the control by 7 mm; and resistant if the zone radius was 2 mm.4 The Etest (AB Biodisk, Solna, Sweden) was performed on Iso-Sensitest agar (Unipath Ltd, Basingstoke, UK) using J Antimicrob Chemother 1999; 43: 314–315 the manufacturer’s instructions. A comparison was made A. Gallowaya*, J. Wrightb, O. Murphya and between the MIC result obtained by the Etest and the G. Dickinsonb sensitivity result obtained from the Stoke’s method. The results are given in the Table. No major errors were identia Public Health Laboratory, Newcastle General fied, i.e. no resistant strains were reported as sensitive. Hospital, Newcastle upon Tyne NE4 6BE; However, one minor error (i.e. sensitive strain reported as b Department of Microbiology, Hexham General resistant) was noted using the higher BSAC set of breakHospital, Hexham, Northumberland NE46 1QJ, UK points ( 1, 1–4 and 4 mg/L) and no errors using the lower BSAC set of breakpoints ( 0.5, 1–2 and 2 mg/L). Corresponding author. *Tel: 44-191-273-8811; A total of 17 isolates had an intermediate MIC, i.e. 1 and Fax: 44-191-226-0365; 4 mg/L (low-level resistance). In this group the greatest E-mail:
[email protected] number of misclassifications occurred. This is perhaps to be expected, as P. aeruginosa shows a continuous distribution Sir, of MICs and does not divide into two distinct (resistant and Gosden et al. reported on the comparison of the modified sensitive) populations; a disc test would therefore not Stokes’ method with the results of MIC testing by an agar clearly classify isolates in the same way as an MIC test, and incorporation method.1 We would like to report our exper- categorization errors do occur.5 Overreporting of ciproience of a similar comparison with Pseudomonas aeru - floxacin resistance for P. aeruginosa by the disc method has ginosa isolates, comparing the modified Stokes’ method been previously highlighted as a particular problem by with the MIC result obtained by the Etest. Evaluation of other workers.6 the Etest for antimicrobial sensitivity testing of P. aerugi Our laboratory serves a district general hospital but also nosa isolates to ciprofloxacin has been shown to be reliable houses the Regional Spinal Injuries Unit. P. aeruginosa is a compared with the agar dilution method.2,3 One hundred frequent cause of colonization and infection in patients and four isolates of P. aeruginosa (47 sensitive, four with spinal cord injury and we are therefore particularly intermediate, and 53 strains resistant to ciprofloxacin as interested in ensuring that ciprofloxacin sensitivity to P. defined by the modified Stokes’ method) from hospital aeruginosa isolates is reported accurately. The quinolones in-patients and out-patients and from general practitioners are the only effective oral anti-pseudomonal agents availwere tested. The disc diffusion test with a ciprofloxacin 1 g able and their use may allow patients who require treatdisc was carried out using Diagnostic Sensitivity Test ment to return home rather than stay in hospital for (DST) agar (Mast Diagnostics, Merseyside, UK) and the intravenous therapy. Routine determination of MICs for rotary modified Stokes’ method with the control organism all P. aeruginosa isolates in our laboratory would not be (P. aeruginosa NCTC 10662) on the outside and the test feasible. As a result of our study and because of the diffiorganism in the centre. culty in reading small zone sizes, we developed a local proInterpretation of the results of sensitivity testing for the tocol in which P. aeruginosa isolates that show a zone of Stokes’ method used the British Society for Antimicrobial inhibition to ciprofloxacin, but this radius is 3 mm, have Chemotherapy (BSAC) criteria, i.e. an isolate was an MIC determined using the Etest strip. This has reduced
Sensitivity testing of Pseudomonas aeruginosa to ciprofloxacin: comparison of the modified Stokes’ method with MIC results obtained by the Etest
Table. Performance of susceptibility testing to ciprofloxacin of 104 isolates of P. aeruginosa established by determination of MIC (Etest) and use of modified Stokes’ test results Susceptibility determined by MIC (Etest) BSAC breakpoint
Ciprofloxacin 1 g
S
I
0.5 1
1–2 1–4
R 2 4
sensitive (S) Stokes’ test results
intermediate (I) Stokes’ test results
resistant (R) Stokes’ test results
S
I
R
S
I
R
S
I
R
43 46
0 1
0 1
4 1
4 3
11 13
0 0
0 0
42 39
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Correspondence the number of isolates misclassified. We would agree with Gosden et al.1 that the modified Stokes’ method for sensitivity testing may show an unacceptable number of major errors for some organisms; however, for P. aeruginosa susceptibility to ciprofloxacin we have found the modified Stokes’ method to be acceptable provided its limitations are appreciated. We will be interested to compare our previous results with those obtained using the standardized disc method of sensitivity testing recommended in the BSAC Summer Newsletter, 1998.
References 1. Gosden, P. E., Andrews, J. M., Bowker, K. E., Holt, H. A., MacGowan, A. P., Reeves, D. S. et al. (1998). Comparison of the modified Stokes’ method of susceptibility testing with results obtained using MIC methods and British Society for Antimicrobial Chemotherapy breakpoints. Journal of Antimicrobial Chemotherapy 42, 161–9. 2. Jones, R. N., Erwin, M. E. & Croco, J. L. (1996). Critical appraisal of Etest for the detection of fluoroquinolone resistance. Journal of Antimicrobial Chemotherapy 38, 21–5. 3. Di Bonaventura, G., Ricci, E., Loggia, N. D., Catamo, G. & Piccolomini, R. (1998). Evaluation of the Etest for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from patients with long-term bladder catheterization. Journal of Clinical Microbiology 36, 824–6.
data provided by the Prescriptions Pricing Authority) are not amenable to interpretation in relation to clinical usage. None the less, there is widespread concern that the overall level of antibiotic prescribing is excessive. We recently analysed PACT data for antibiotic usage in the West Midlands and concluded that seasonal analysis might contribute to interpreting and setting targets for appropriate prescribing. Early fluoroquinolones (ciprofloxacin, ofloxacin and norfloxacin) have limited activities against pneumococci, streptococci, enterococci, anaerobes and methicillin-resistant Staphyloccocus aureus.1 For these reasons, it is widely regarded that these drugs should not be used routinely as treatment of patients with respiratory tract, soft tissue or bone infections without clear bacteriological indications; in contrast to ciprofloxacin and ofloxacin, norfloxacin is licensed solely for use as therapy of patients with urinary tract infections. The Figure shows the prescribing trends for fluoroquinolones in the West Midlands Health Authorities over a 3 year period between March 1995 and January 1998. As expected from the usage of norfloxacin being limited to the treatment of patients with urinary tract infections, there was no evidence of seasonal fluctuation in the prescribing of this agent, whereas fluctuations in prescriptions for ciprofloxacin and ofloxacin are consistent with them being
4. Working Party of the British Society for Antimicrobial Chemotherapy (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50. 5. Ibrahim-Elmagboul, I. B. & Livermore, D. M. (1997). Sensitivity testing of ciprofloxacin for Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 39, 309–17. 70,000
6. Chen, H. Y., Yuan, M., Ibrahim-Elmagboul, I. B. & Livermore, D. M. (1995). National survey of susceptibility to antimicrobials amongst clinical isolates of Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 35, 521–34.
60,000
Seasonal variation in fluoroquinolone prescribing J Antimicrob Chemother 1999; 43: 315–316 S. Abella, S. Chapmana*, L. Nadina and R. Warrenb a
Department of Medicines Management, Keele University, Keele, Staffordshire ST5 5BG; b Public Health Laboratory, Royal Shrewsbury Hospital, Shrewsbury, UK *Corresponding author. Tel: Fax: 44-(0)1782-713-586.
44-(0)1782-584-131;
Sir, National prescribing data for general practice in the UK (obtained from Prescribing Analysis and Cost (PACT)
Figure. Prescribing trends in the West Midlands (March 1995– January 1998) for ciprofloxacin ( ), norfloxacin ( ), ofloxacin ( ), and ciprofloxacin and ofloxacin combined ( ).
315
Correspondence administered as therapy of patients with seasonal infections, particularly those of the respiratory tract. While the quinolones have potent activities against Haemophilus influenzae, they exhibit only modest activities against pneumococci and there are several antibiotics that should be used in preference in patients with respiratory tract infections; these include the tetracyclines, co-amoxiclav, trimethroprim and second- and third-generation cephalosporins. The newer fluoroquinolones, grepafloxacin and levofloxacin, have superior activities against pneumococci, compared with earlier agents, but it should not be construed from this that either drug should be used routinely as first-line therapy. Fluoroquinolones are used widely in both human and veterinary medicine; indeed, ciprofloxacin is currently one of the world’s most frequently administered antibiotics.2 In order to minimize the emergence of resistant strains, crossresistance being a feature of these agents,3 they should be used rationally. Fluoroquinolones are described in the ninth World Health Organisation Model List of Essential Drugs as being among ‘the most important reserve agents’.4 Thus, the use of these antibiotics in patients with respiratory tract infections, as implied by the data reported here, and the rapid increase in ciprofloxacin prescribing in the West Midlands during 1997 are causes for concern. Ongoing education of both physicians and the public is essential, given that most episodes of upper and lower respiratory tract infections do not require antibiotic therapy. A public health policy on appropriate prescribing must be a priority and the ethics of promoting antibiotics in clinical settings where they are unnecessary should be given serious consideration. 4 Meanwhile, we suggest that a low seasonal fluctuation in fluoroquinolone prescribing is a good marker of restrained use.
References 1. Warren, R. (1995). Antibacterial agents: the fluoroquinolones. Prescribers’ Journal 35, 95–101.
2. Acar, J. F. & Goldstein, F. W. (1997). Trends in bacterial resistance to fluoroquinolones. Clinical Infectious Diseases 24, Suppl. 1, S67–73. 3. Eliopoulos, G. M. (1995). In-vitro activity of fluoroquinolones against Gram-positive bacteria. Drugs 49, Suppl. 2, 48–57. 4. Couper, M. R. (1997). Strategies for the rational use of antimicrobials. Clinical Infectious Diseases 24, Suppl. 1, S154–6.
Retraction: The failure of a once-daily vancomycin dosing regimen in patients with normal renal function J Antimicrob Chemother 1999; 43: 316 Paul E. Marik Department of Critical Care Medicine, St Vincent Hospital and the University of Massachusetts Medical School, 25 Winthrop Street, Worcester, MA 01604-4593, USA Tel:
1-508-798-6049; Fax:
1-508-798-1135.
Sir, In the Journal of Antimicrobial Chemotherapy 1997; 40 745–6, I published a letter to the editor describing two cases and suggesting that the two patients failed to respond to once-daily vancomycin dosing. In the interest of encapsulating two highly complex cases in a brief letter to the editor, certain liberties were taken with the representation of data, which may have served to mislead. Although this was not my intent, I nonetheless wish to retract this letter to the editor.
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