a Department of Bacteriology, St Mary's Hospital, London W2 1NY b PHLS Statistics Unit, CDSC, 61 Colindale Avenue, London NW9 5EQ c Clinical Research and Development, SmithKline Beecham Pharmaceuticals UK Ltd, Welwyn Garden City, AL7 1EY, UK
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Abstract |
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Introduction |
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Materials and methods |
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Hospital microbiology laboratories in the following towns in Britain were asked to supply 80 consecutive bacterial isolates from urine samples submitted from community (general practice) patients: Aberdeen, Belfast, Chester, Leicester, Oldham, Plymouth, Reading, Southend, Sunderland, Swansea, West Bromwich and Worthing. The laboratories were asked to provide only isolates considered significant by their usual criteria. At the same time, the laboratories were asked to provide results of the bacterial identification and antimicrobial sensitivity tests they had performed on the isolates. Details of test methods used and number of urine samples received per head of community population were also requested.
Isolates were subcultured on to nutrient agar slopes and sent to the microbiology department of St Mary's Hospital (London, UK) and stored for batched testing. When all isolates had been received, the cultures were checked for purity by subculture on to cysteine lactose electrolyte deficient (CLED) agar (Gram-negative isolates) or blood agar (Gram-positive isolates). Sending laboratories' provisional identifications were confirmed as follows. Staphylococci were checked for clumping factor production (Staphylase, Oxoid, Basingstoke, UK), and enterococci were subcultured onto bileaesculin agar. The sending laboratories' identification was accepted for other streptococci. Proteus spp. were checked by API 20E (API-bioMérieux, Basingstoke, UK), coliforms were grown in peptone water at 37°C for 3 h and then multipoint inoculated on to xylose and ß-glucuronidase agars; the indole test was performed by adding Kovac's reagent to the peptone water after overnight incubation. Organisms which gave positive results in all three tests were designated Escherichia coli; otherwise they were classed as `coliforms'.
MICs were determined using a microdilution method. The following control strains were examined in parallel with the test organisms: E. coli NCTC 10418 and ß-lactamase-producing E. coli 11560, Pseudomonas aeruginosa NCTC 10662 and Staphylococcus aureus NCTC 657. Bacteria were grown on either CLED or blood agar incubated aerobically overnight at 37°C and then inoculated into IsoSensitest broth (Oxoid) and incubated for 4 h to provide sensitivity test inocula. Sensitivities to antimicrobials were investigated in accordance with the recommendations of the British Society for Antimicrobial Chemotherapy (BSAC) Working Party5 using a microdilution method. Stock solutions of the following antimicrobials were freshly prepared in IsoSensitest broth using Adatabs (Mast Diagnostics, Bootle, Merseyside, UK): amoxycillin, cephalexin, cephradine and nitrofurantoin, 128 mg/L; norfloxacin and trimethoprim, 32 mg/L; ciprofloxacin, 8 mg/L; co-amoxiclav, 128 mg/L for Gram-negative organisms, 16 mg/L for Gram-positive organisms. One-hundred microlitres of antimicrobial solution were placed in the first two wells of each row and doubling dilutions made along the 12 wells of the row. Five microlitres of inoculum were added to each well, and the trays were sealed and incubated at 37°C overnight. Growth was assessed as turbidity visible on transillumination. The minimum inhibitory concentration (MIC) of a particular antimicrobial was recorded as the lowest concentration to inhibit growth. To interpret MICs as sensitive or resistant, the following breakpoints, suggested by the British Society for Microbial Technology,6 were taken: 32 mg/L of amoxycillin, cephalexin, cephradine, co-amoxiclav and nitrofurantoin; 8 mg/L of ciprofloxacin and trimethoprim; and 16 mg/L of norfloxacin. These were used rather than the values given by the BSAC,5 which are listed as commonly used breakpoints rather than formal recommendations for urinary isolates. The only difference is the citing of 4 mg/L for ciprofloxacin in the BSAC document.
Methicillin resistance of staphylococci was assessed by streaking organisms on a nutrient agar plate to which a methicillin strip was applied. S. aureus NCTC 657 was used as a control organism. Plates were incubated at 30°C aerobically overnight.
To correlate resistance patterns with antimicrobial usage, regional prescribing data for the treatment of UTI was obtained from the Mediplus IMS patient database (1997). This database contains information on regional prescriptions from a representative panel of approximately 600 GP practices throughout the UK. The data produced is based on approximately one in 60 GP prescriptions.
Sensitivity protocols in submitting laboratories
Eight submitting laboratories measured antibiotic sensitivity by Stokes' disc diffusion method, with control organisms on individual plates. Only four of these used the recommended ß-lactamase-producing E. coli control for co-amoxiclav sensitivity, and there were also differences in disc antibiotic strengths employed. Three laboratories used breakpoint technology. Interpretation of sensitive breakpoints differed by up to two-fold between laboratories, and the two that tested for sensitivity to co-amoxiclav used a ß-lactamase-producing E. coli control. One laboratory used National Committee for Clinical Laboratory Standards (NCCLS) methodology with batch media control and zone size measurement, without organism controls. Four of the laboratories reported sensitivities that they judged as `intermediate', eight either did not recognize this category or reported such results as `resistant'.
Statistical methods
Overall sensitivities to the antimicrobials were calculated, with 95% confidence intervals obtained using an arc-sine transformation on each laboratory's isolates to allow normality assumptions. The effects of laboratory, bacterial species and prescribing patterns on sensitivities were examined together for each antimicrobial in multivariable logistic regression models. This enabled the independent effect of each factor to be assessed.
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Results |
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The 12 laboratories sent 972 viable isolates in which information for 962 (7983 per laboratory) could be analysed after discarding those with mismatching data. The identification of the organisms at St Mary's revealed that 65.1% were E. coli, 23.4% `coliforms', 4.6% Proteus and Morganella spp., 1.8% Pseudomonas spp., 2.4% enterococci, 0.7% group B streptococci, 1.5% coagulase-negative staphylococci and 0.5% S. aureus. This corresponded with the sending laboratories' identifications, with the exception of one specimen sent as Proteus spp., which was identified at St Mary's as E. coli. Many laboratories did not distinguish E. coli from other coliforms.
MICs and sensitivity results
Cumulative MIC results are shown graphically in Figures 1 and 2. In all cases, the MICs for control organisms were within one dilution of expected values. Taking the cut-off MICs given in Materials and methods, approximately 99% of all isolates were sensitive to norfloxacin and ciprofloxacin, 95% to co-amoxiclav, 87% to nitrofurantoin, 75% to trimethoprim and the cephalosporins and 50% to amoxycillin (Table I). Not all laboratories provided sensitivity reports for every antimicrobial for which MIC determinations were made. Where such information was provided, there were sometimes considerable discrepancies between the two sets of data(see Table I).
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Tables II and III show that there are significant regional differences in reported rates of resistance to the cephalosporins. This did not result from differences in the proportions of bacterial species found in the different centres and did not correlate with regional differences in prescription of cephalosporins. Adjustments were made for these factors in the multivariate logistic regression. Only in the case of amoxycillin was there a relationship between regional antibiotic prescribing and sensitivity (P= 0.03, coefficient of rank correlation = 0.64), with higher prescribing in regions with greater amoxycillin sensitivity. There was no apparent relationship between the rate at which urine samples were submitted from community patients and antibiotic prescription preferences or resistance rates (Table II).
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Discrepancies between sensitivity results from the submitting and central laboratories, particularly for cephalosporins, prompted examination of the methods used by the submitting laboratories. Cephalosporin results falsely reported as sensitive by central laboratory criteria came principally from four centres, where 3053% of such reports were judged false-positive. One of these laboratories used breakpoint technology, but with a cut off of 32 mg/L rather than the 16 mg/L used centrally. Two of the others used a 30 µg cephradine disc and one a 30 µg cephalexin disc. These laboratories reported a 02.5% false resistance rate for cephalosporins. These were the four laboratories judged to have the highest rates of genuine cephalosporin resistance in the central laboratory. The six other laboratories reporting cephalosporin results had false resistance rates of 014.5%, with false-sensitive rates of 012%. Of the 14 false resistances reported to ciprofloxacin, three occurred in two of the centres using breakpoint methodologies, one being due to a breakpoint of 4 mg/L, which would be correctly considered sensitive if the BSAC list of commonly used breakpoints was employed. Of the remainder, all used a 1 µg sensitivity disc and most discrepant results were from single laboratories. Another four were reported by the laboratory with the highest percentage of cephalosporin false-sensitive reports, and three from a second laboratory reporting a high degree of false positivity to cephalosporins. Four reports of false resistance to co-amoxiclav came from a laboratory using breakpoints, despite the sensitive breakpoint being set at 32 mg/L. The nine other laboratories reporting co-amoxiclav results used a 30 µg disc, and false-resistant reports were 015% of total co-amoxiclav reports. No major difference was attributable to the use of a ß-lactamase-producing control. An average of 5.3% of co-amoxiclav results were false-resistant if this control was used, and 7.8% if not.
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Discussion |
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The most relevant studies for comparison with the present work are the two multicentre reports,7,8 both of which relied upon submitting laboratories' own sensitivity determinations. These are likely to have been based mainly on Stokes' disc diffusion methods7 and were not confirmed by a single centre, or by breakpoint or MIC determination. These two reports gave mean sensitivity levels close to those of the present study for most antimicrobials but found higher sensitivity rates for cephalexin (87.7% versus 77.3% in this study) and lower ones for co-amoxiclav (78.8% versus 95.9%).
These differences may have arisen because the laboratories included in the other studies genuinely had different levels of sensitivity from those participating in the present study. It may also be that Stokes' results do not always correlate well with those determined by MIC or breakpoint. This was particularly evident in this study in the case of the cephalosporins, for which MICs suggested sensitivity levels 1520% lower than the sending laboratories' own reports. Apart from random erroneous reports, our results suggest that in a few laboratories there may be systematic underestimation of resistance to certain antibiotics, particularly if local resistance rates are elevated. There is no definitive guidance on sensitive/resistant designation for MICs of urinary antimicrobials. The BSAC guidelines5 make no recommendations for urine isolates, and different limits are suggested by various national schemes.6 This is of particular significance in the case of the oral cephalosporins, for which MICs of 832 mg/L have been suggested as resistance breakpoints. As shown in Figure 1, this is a range over which the proportion of strains inhibited by cephalosporins changes markedly, and where a change in breakpoint of one dilution will lead to a considerable change in the proportion reported as susceptible. Our findings suggest that many organisms with cephalosporin MICs > 32 mg/L are being reported as sensitive. This may explain the observation that failure rates with cephalosporins exceed those predicted by laboratory sensitivity reports.9,10 In contrast, Figure 1 does not appear to offer an explanation for the tendency to overestimate resistance to co-amoxiclav (Table I). This may be explained by the observation that many laboratories do not use the recommended ß-lactamase-producing E. coli control when evaluating co-amoxiclav sensitivity by Stokes' method.7
It has been suggested that misinterpretation of antimicrobial sensitivity may be unimportant, in-vitro sensitivity reports having no bearing on the response of patients with UTIs.11 If the given recommended breakpoints are assumed to be valid, then apart from interpretative differences, it appears that genuine differences in sensitivity levels to certain antimicrobials do exist between centres, notably for co-amoxiclav and the cephalosporins. Table II suggests that although different antimicrobials vary in their popularity as treatments for UTIs between regions, there is no obvious relationship with levels of sensitivity. This is confirmed as shown in Table III, except in the case of amoxycillin. It may be that the data concerning prescriptions is not sufficiently discriminatory to reveal differences over broad geographical regions.
This work supports the overall findings of other single-centre studies of the antimicrobial sensitivity of community UTI isolates in the British Isles,3,4 undertaken a few years ago using Stokes' methodology. These data indicate a further reduction in the value of ampicillin and also of the cephalosporins, although in the case of the latter this may result from interpretive differences between Stokes' and MIC methods.
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Acknowledgments |
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Notes |
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References |
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Received 25 August 1998; returned 7 December 1998; revised 16 March 1999; accepted 27 April 1999