Zone Breakpoints Research Articles

Comparative invitro activity of carbapenems against clinical isolates of Acinetobacter baumannii.

The aim of this multi-hospital study was to assess the invitro activity of doripenem and its comparators, imipenem and meropenem, using the new CLSI breakpoints against a large population of a frequently isolated nosocomial pathogen, Acinetobacter baumannii. During a 2-year period, four referral or tertiary hospitals submitted 400 isolates of Ac.baumannii for susceptibility testing using imipenem, meropenem and doripenem via disc diffusion and E-test methods. A subset of 390 isolates was resistant to all three tested carbapenems. Doripenem and meropenem (MIC50 , 32μgml(-1) ) had comparable activity, albeit doripenem's activity was greater than imipenem (MIC50 , >32μgml(-1) ). A significantly higher proportion of the isolates were inhibited by doripenem than by imipenem at MIC values of 12, 16, 24 and 32μgml(-1) (P<0·05). The cumulative percentage of imipenem MICs was lower compared to its comparators. The comparison of resistance rate to imipenem and meropenem based on old and new breakpoints showed <1% difference. The overall agreement between the two susceptibility testing methods was ≥95%. Doripenem has a slightly greater invitro activity than imipenem in terms of zone breakpoints and MIC values, but its activity is comparable to meropenem. Doripenem should be considered as a therapeutic option for monotherapy or combination therapy, particularly when the therapeutic options are limited.

Gentamicin in vitro activity and tentative gentamicin interpretation criteria for the CLSI and calibrated dichotomous sensitivity disc diffusion methods for Neisseria gonorrhoeae.

XDR Neisseria gonorrhoeae imposes the threat of untreatable gonorrhoea. Gentamicin is considered for future treatment; however, no interpretation criteria for the CLSI and calibrated dichotomous sensitivity (CDS) disc diffusion (DD) techniques are available for N. gonorrhoeae. We investigated the in vitro gentamicin activity by MIC and DD methods, proposed DD breakpoints and determined DD ranges for 10 international quality control (QC) strains. Gentamicin susceptibility of 333 N. gonorrhoeae isolates, including 323 clinical isolates and 10 QC strains, was determined. MIC determination (Etest) and DD methods (CLSI and CDS) were performed. The relationship between MIC, inhibition zone diameter and annular radius was determined by linear regression analysis and the correlation coefficient (r) was calculated. Gentamicin MICs for the QC strains were within published ranges. Of the 323 clinical isolates, according to published breakpoints 75.9%, 23.5% and 0.6% were susceptible, intermediately susceptible and resistant, respectively. Based on error minimization with MICs of ≤4, 8-16 and ≥32 mg/L, breakpoints proposed are susceptible ≥16 mm, intermediately susceptible 13-15 mm and resistant ≤12 mm for the CLSI method and susceptible ≥6 mm, less susceptible 3-5 mm and resistant ≤2 mm for the CDS technique. Low resistance to gentamicin was identified and gentamicin might be a future treatment option for gonorrhoea. Tentative gentamicin zone breakpoints were defined for two DD methods and QC ranges for 10 international reference strains were established. Our findings suggest that in resource-poor settings where MIC testing is not a feasible option, the DD methods can be used to indicate gentamicin resistance.

Tigecycline and intravenous fosfomycin zone breakpoints equivalent to the EUCAST MIC criteria for Enterobacteriaceae

Tigecycline and intravenous (i.v.) fosfomycin could be alternative therapeutic options for the treatment of carbapenemase-possessing Enterobacteriaceae bacterial infections. However, routine laboratories are forced to test these drugs using minimum inhibitory concentration (MIC) methods as zone breakpoints are not available for the disc diffusion technique. Clinical and Laboratory Standards Institute methods for agar dilution and disc diffusion were compared to determine tentative zone breakpoints that best correlate to tigecycline and i.v. fosfomycin MIC breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing. A total of 195 Enterobacteriaceae with defined mechanisms of resistance were tested in duplicate assays. Half of the strains were characterized as carbapenemase producers (KPC-2, OXA-48, OXA-163, VIM-1, VIM-2, IMP-8, NDM-1). Corresponding zone diameters of susceptible ≥ 15 mm, resistant ≤ 12mm and susceptible ≥ 17 mm, resistant ≤ 15 mm for the 50 µg fosfomycin plus 50 µg glucose-6-phosphate and 200 µg fosfomycin plus 50 µg glucose-6-phosphate discs, respectively, allowed categorization of the strains with an acceptable level of error (< 10% minor errors, < 1.5 % major errors, < 1% very major errors and categorical agreement > 90%). For the 15 µg tigecycline disc, the best performance was achieved with the corresponding zone diameters of susceptible ≥ 21 mm and resistant ≤ 16 mm, which eliminated the very major and major errors but not the minor errors (34.4%). Based on these results, tigecycline and fosfomycin can be included in the routine panel of antibiotics for susceptibility testing by disc diffusion to provide fast and reliable information for the selection of treatment alternatives, especially for strains with extreme resistance, as carbapenemase producers.

Tigecycline activity: low resistance rates but problematic disc breakpoints revealed by a multicentre sentinel survey in the UK

A multicentre surveillance of tigecycline activity against relevant pathogens in the UK. Forty-three representative UK hospitals were each asked to test 150 consecutive, clinically significant isolates from hospitalized patients, excluding those from urine or faeces. The sentinel laboratories tested all these isolates against a panel of antibiotics by the BSAC disc method and a selection of isolates were retested centrally by the BSAC agar dilution method. The isolates collected comprised Staphylococcus aureus (39.9%), Escherichia coli (13.3%), streptococci (11.9%), enterococci (6.3%), Klebsiella (6.1%), coagulase-negative staphylococci (6.1%), Enterobacter spp. (3.9%), Proteus spp. (2.2%) and other Gram-negative species (10.3%). The laboratories' disc susceptibility testing found that 4% of isolates were resistant to tigecycline, using the zone breakpoints in place at that time. However, centralized retesting by agar dilution showed that many of these 'resistant' isolates were susceptible, with resistance only confirmed in a range of Gram-negative isolates and one S. aureus. These findings reduced the estimated resistance rate to 2.4%, or to 0.8% if Proteus spp. were ignored as intrinsically resistant to tigecycline. Sixty-two percent of the isolates found resistant by the disc method but susceptible by agar dilution had zones of inhibition within experimental error (4 mm) of the published breakpoints. Tigecycline resistance was rare in isolates causing clinically significant infections in the UK and was overestimated ∼2-fold by the BSAC disc method. Adjustment of the breakpoints might overcome this problem but at the risk of increasing the false susceptibility rate. The best advice is to perform dilution tests, e.g. by gradient strip test on isolates with borderline results, especially in severe infection and when tigecycline use is intended.

Zone breakpoints, by the CLSI disc method, for 15 g tigecycline discs corresponding to EUCAST MIC breakpoints

Zone breakpoints, by the CLSI disc method, for 15 g tigecycline discs corresponding to EUCAST MIC breakpoints

Proposed disc zone breakpoints for doripenem for use with the BSAC disc susceptibility testing method

Sir, Doripenem is a new carbapenem, recently licensed in the European Union for treatment of nosocomial pneumonia (including ventilator-associated pneumonia), complicated intraabdominal infections and complicated urinary tract infections. A 10 mg doripenem disc has been proposed for routine susceptibility testing. We determined provisional zone breakpoints corresponding to European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints for these 10 mg doripenem discs in the BSAC disc method. Isolates (n1⁄4810) were selected from the BSAC Bacteraemia Resistance Surveillance Project (2005–06), supplemented with additional, more-resistant, isolates from other survey collections and reference submissions held by the UK HPA’s Antibiotic Resistance Monitoring and Reference Laboratory. Care was taken, whenever possible, to select isolates for which the MICs corresponded with or were within one or two dilutions of the breakpoints. Zones and agar dilution MICs were determined by the BSAC methods. Results were reviewed against EUCAST/ European Medicines Agency (EMEA) MIC breakpoints, as also adopted by the BSAC, of: susceptible, ≤1 mg/L and resistant, .1 mg/L for streptococci; and susceptible, ≤1 mg/L and resistant, .4 mg/L for staphylococci, enterococci, Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. Error minimization was used to optimize the values. Zone breakpoints corresponding to these MIC breakpoints were as follows:

Evaluation of the susceptibility of Aeromonas salmonicida to oxytetracycline and tetracycline using antimicrobial disk diffusion and dilution susceptibility tests

The development of resistance to antibiotics commonly used in animal production is an increasing concern for agriculture industries and public health. This study examined the utilisation of proposed standard methods for antimicrobial susceptibility testing as described by Alderman and Smith [Alderman, D.J., Smith, P. 2001. Development of draft protocols of standard reference methods for antimicrobial agent susceptibility testing of bacteria associated with fish diseases. Aquaculture. 196, 211–243.] for oxytetracycline (OTC) and tetracycline (TET). Fifty strains of Aeromonas salmonicida subsp. salmonicida were tested using disk diffusion and MIC techniques, with comparison of incubation periods of 24 and 48 h. The relationship between the MIC and the disk diffusion data was also used to evaluate possible breakpoints for the isolates utilised in this experiment. Susceptible and non-susceptible MIC breakpoints of ≤1 μg/ml and >16 μg/ml, and ≤1 μg/ml and >4 μg/ml fit the datasets for OTC and TET respectively. The calculation of the inhibition zone breakpoints using best-fit line equations yielded values of 15 and 26 mm for OTC and 21 and 28 mm for TET, for the non-susceptible and susceptible breakpoints respectively. The apparent differences in the inhibition zone distributions of the two tetracycline derivatives presented in this study argue for further investigation of breakpoints, with the inclusion of more bacterial strains of intermediate resistance as well as methodology development of susceptibility testing for both oxytetracycline and tetracycline.

Extended antimicrobial resistance screening of the dominant faecal Escherichia coli and of rare resistant clones

Fifty faecal samples from healthy adults were grown on MacConkey agar and three pink colonies were subcultured, identified to species level and their antimicrobial susceptibility determined. Forty-seven samples yielded 141 isolates of Escherichia coli that were susceptible to most antimicrobials. Resistance was noted for ampicillin (30.5%), chloramphenicol (12.1%), tetracycline (23.4%), trimethoprim (24.8%) and co-trimoxazole (22.7%). A direct faecal plating method was used for extended resistance screening with E. coli as the indicator organism. Zone breakpoints were determined using normalised resistance interpretation and gave similar susceptibility results. Eighty-eight isolates of E. coli from within the zones of inhibition revealed four times more antimicrobial resistance. Extended antimicrobial resistance screening both provides the susceptibility profile of the dominant E. coli isolate and detects greater resistance in rare isolates.

Patterns of mutations in target genes in septicemia isolates of Escherichia coli and Klebsiella pneumoniae with resistance or reduced susceptibility to ciprofloxacin.

Thirty-two Escherichia coli and 21 Klebsiella pneumoniae septicemia isolates with varying degrees of resistance to ciprofloxacin were analyzed for the presence of point mutations within the quinolone-resistance target genes. The number of mutations observed in the resistant isolates agreed with the level of ciprofloxacin resistance in both species. Such isolates were also resistant to nalidixic acid. Isolates with borderline susceptibility to ciprofloxacin, on the other hand, behaved differently in the two species. In E. coli all the isolates harbored at least one mutation and these isolates were also resistant to nalidixic acid, while no mutations were detected in the K. pneumoniae isolates, and susceptibility to nalidixic acid was unpredictable. Therefore, nalidixic acid cannot be used as a class representative. Time-kill curve studies on an isolate with borderline susceptibility from each species showed higher degrees of resistance to ciprofloxacin in comparison to that of the wild-type E. coli. A previously unreported parC mutation, S57-->T, was detected in a resistant E. coli isolate and might expand the QRDR of this gene. Normalized resistance interpretations of histograms confirmed the setting of microbiological zone breakpoints for ciprofloxacin testing.

Determination of the real standard distribution of susceptible strains in zone histograms

The procedure for NRI, a method for normalised resistance interpretation, is presented. By means of its promising autocalibration system the method generates zone breakpoints for resistance in zone diameter histograms for bacterial species–antibiotic combinations. These breakpoints give a normalised interpretation of resistance which is independent of disc test standard chosen, disc potency, medium used, inoculum size, etc. of the individual laboratory as long as the performance shows good reproducibility and precision. The method should be further tested as a tool for setting ‘bacteriological breakpoints’ or ‘epidemiological breakpoints’, for calculating resistance rates for comparative purposes, e.g. in antibiotic resistance surveillance, and for quality control of the disc diffusion test.

Analysis of parameters and validation of method for normalized interpretation of antimicrobial resistance

The method of normalized resistance interpretation (NRI), uses the high-zone side of the susceptible peak in a zone diameter histogram as an internal calibrator to construct the real standard distribution of susceptible isolates even in the presence of resistant isolates. NRI parameters were optimized using control strain histograms from microbiology laboratories in Stockholm, Argentina, and the Philippines. A moving average based on four-zone values was slightly better than based on two-zone average values. The optimal peak adjustment from the switch position of the moving average was 1.0 for two-zone averages and 2.5 for four-zone averages. A comparison between true means and NRI-calculated means showed a highly significant correlation ( R 2=0.963). Coefficients of variation (CV), comparing the CV of the true distribution of control strain test results with the NRI calculated distribution, identified two types of aberrant histograms. NRI calculations on clinical isolates of Escherichia coli and Staphylococcus aureus from selected laboratories showed a good agreement between the local resistance interpretations with the NRI calculated levels. One type of deviation was most marked with cephalothin histograms for E. coli isolates where the regular zone breakpoints used cut through the population of susceptible strains. With proper markers for required quality of disc test results, the NRI method might be a valuable tool for both resistance surveillance and for quality control of the disc diffusion method.

A new method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes

To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test.

Calibration of fusidic acid disk diffusion susceptibility testing of Staphylococcus aureus

Single strain regression analysis, SRA, was used to calibrate disk diffusion fusidic acid susceptibility testing of Staphylococcus aureus in two laboratories using different standard methods but the same interpretative MIC limits. SRA equation constants were calculated using five different fusidic acid disk contents (1.5, 5, 15, 50, 150 microg). These disks were tested on five separate occasions against quality control strain S. aureus ATCC 29213. The National Committee for Clinical Laboratory Standards (NCCLS) method was employed in Tartu, Estonia (TE) and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden at the Karolinska Hospital (KS). SRA constants obtained were used for calculating zone breakpoints corresponding to MIC breakpoints recommended by the SRGA (S < or = 0.5 mg/L, R > or = 1 mg/L). Zone diameter histograms from KS, performed with a 50 microg disk, and from TE, using a 10 microg disk, showed a clustering of wild type strains around 41 mm and 30 mm, respectively, reflecting differences in methodology. Zone breakpoints calculated from the equations were validated by comparison with the histograms. Breakpoints were also calculated for a suggested lower disk content in Sweden, 10 microg, and validated in tests of clinical isolates and by histogram analysis.

Fusidic acid disk diffusion testing of clostridium difficile can be calibrated using single-strain regression analysis.

Single-strain regression analysis (SRA) was employed to calibrate the disk diffusion antibiotic susceptibility test for fusidic acid and Clostridium difficile. MIC determinations of 40 clinical isolates of C. difficile were performed with the E-test. The disk diffusion test was standardized according to the Swedish Reference Group for Antibiotics (SRGA). Disks used for SRA contained 1.5, 5, 15, 50 and 150 microg fusidic acid and the routine disk contained 50 microg fusidic acid. A control strain, ATCC 9689, was also tested. SRA constants A and B of the regression lines were calculated. This permitted the determination of zone breakpoints for C. difficile. When applying the pharmacological MIC S and R limits set by SRGA to the E-test results I strain of C. difficile was interpreted as resistant. Zone breakpoints corresponding to the pharmacological MIC limits and calculated using the mean SRA constants for the 40 clinical isolates lead to all strains being interpreted as susceptible. SRA calculations enable laboratories to set up calibrated disk tests with species-related and laboratory-specific interpretations.

Quality of antimicrobial susceptibility testing in the UK: a Pseudomonas aeruginosa survey revisited.

As part of a programme to assess the usefulness of routine antimicrobial susceptibility data as a surveillance tool, we reviewed the results of a national survey of resistance in Pseudomonas aeruginosa, undertaken in 1993. Twenty-four UK laboratories contributed isolates for centralized MIC testing, indicating also their own susceptibility test data. As reported previously (Chen et al. (1995) Journal of Antimicrobial Chemotherapy 35, 521-34), the rate of false resistance (isolates reported susceptible, but found resistant on MIC testing/all isolates reported susceptible) was 0.6-8%, according to the antimicrobial and breakpoint. Review showed that this favourable position reflected the fact that >88% of isolates were susceptible to any given antimicrobial and--in most cases--were correctly reported as such. Reporting was more erratic for resistant isolates: for beta-lactams and amikacin, isolates resistant at the highest MIC breakpoints were equally likely to be reported as 'susceptible' or 'resistant'; such misreporting was less common with ciprofloxacin and gentamicin but still occurred in 9-20% of cases. Conversely, up to 73% of the isolates reported as resistant proved to be susceptible at high breakpoints, and up to 44% were susceptible at low breakpoints. Miscategorizations did not reflect failure to detect particular mechanisms but, rather, the fact that MIC and zone breakpoints for P. aeruginosa serve to cut 'tails' of resistant organisms from continuous distributions, not to distinguish discrete populations. In this situation, some disagreement between routine tests and MICs is inevitable, but the frequency at which highly resistant isolates were reported as sensitive is disturbing. For surveillance, we conclude that resistance rates based on routine tests are unreliable for P. aeruginosa. This situation may improve with greater standardization of routine testing, but the continuous susceptibility distributions without discrete resistant and susceptible populations militate against perfect agreement. Despite these deficiencies, routine data should allow trend analysis.

Calibration of disk diffusion antibiotic susceptibility testing: species-related trovafloxacin interpretive zone breakpoints and selection of disk potency.

International comparisons of antibiotic susceptibility require the use of common minimum inhibitory concentration (MIC) limits. Disk diffusion test results are not directly suitable for such comparisons, since different standards are often used and zone breakpoints issued might reflect different MIC limits. We have used single strain regression analysis (SRA) for the calibration of the disk test, both according to species and individual laboratory, and for quality control of trovafloxacin disk diffusion tests in 5 laboratories in Sweden. Preliminary controls using histogram analysis including subtraction histograms of reference strains revealed marked differences between different laboratories. SRA was performed on 4 reference strains, S. aureus, E. faecalis, E. coli and P. aeruginosa, using disks containing 1, 3, 10, 30 and 100 microg trovafloxacin. The results using SRA showed a difference between laboratories using Biodisk PDM medium, which produced smaller zones, and those using Oxoid IsoSensitest. Species-related regression lines for laboratories using either medium were calculated and corresponding interpretive zone breakpoints determined for MIC limits. Rational criteria for the selection of a suitable disk content of an antibiotic were also defined and applied to trovafloxacin. The 10 microg disk selected by NCCLS (National Committee for Clinical Laboratory Standards) proved optimal.

Evaluation of different methods for the detection of methicillin resistance in coagulase-negative staphylococci.

The efficacy of 19 agar diffusion methods for the detection of methicillin resistance among coagulase-negative staphylococci (CoNS) within 24 h was evaluated. A total of 359 CoNS isolates were tested, of which 204 were Staphylococcus epidermidis. In 164 isolates, the presence of mecA was investigated; 61 strains were mecA-positive and 103 were mecA-negative by Southern blot analysis. Based on the best agreement shown with the mecA determination (94%) among four agar dilution assays for determining methicillin MIC, an assay with Columbia agar supplemented with NaCl and incubation with a heavy bacterial inoculum of 10(5)-10(6) cfu/spot was used as the reference MIC method. The best agar diffusion results were obtained with a 1 microg oxacillin disc on Columbia agar with 4.5% NaCl supplement. With this method, 99% of S. epidermidis and 94% of non-S. epidermidis were in agreement with the MIC determination. However, Columbia (without NaCl), Mueller-Hinton and Isosensitest agars were almost as useful when a 1 microg oxacillin disc was used. The zone breakpoints for S. epidermidis were, in general, considerably larger than those for other CoNS species and, consequently, differentiation according to species is recommended. Furthermore, resistance to other antibiotics, such as gentamicin and erythromycin, makes methicillin resistance highly likely.

Sensitivity testing of ciprofloxacin for Pseudomonas aeruginosa.

UK clinical laboratories overestimate ciprofloxacin resistance amongst Pseudomonas aeruginosa isolates, relative to the MIC breakpoint of 1 mg/L. Most tests leading to this overestimation use 1 microg discs and are by Stokes' method with the breakpoint taken as the zone radius for P. aeruginosa NCTC 10662 minus 3 mm. Aiming to reduce this error rate, we examined alternative disc breakpoints. Tests were performed for 100 P. aeruginosa isolates on three media, with breakpoints selected (i) as the zone for P. aeruginosa NCTC 10662 minus 7 mm, as recommended for ciprofloxacin by the BSAC; (ii) with reference to MIC/zone correlation lines; (iii) from natural divisions in zone distribution histograms; and (iv) so as to minimize categorization errors. Breakpoints from regression lines, and those optimized to the susceptibility distribution, reduced the proportion of susceptible organisms misreported as resistant, but the improvement was not significant (P > 0.05, chi2 test). The breakpoint of the zone radius for P. aeruginosa NCTC 10662 minus 7 mm significantly reduced (P < 0.05) the number of susceptible organisms reported as resistant, but led to 50-75% of those with low level resistance (MIC 2-4 mg/L) and 4-10% of those with high-level resistance (MIC > 4 mg/L) being classed as susceptible. Irrespective of the medium and the basis of choosing breakpoints, 5 microg ciprofloxacin discs gave a lower rate of susceptible organisms being reported as resistant than did 1 microg discs; however, the improvement was not significant (P > 0.05, chi2 test) and the 5 microg discs had the disadvantages of forming very large zones for susceptible isolates and giving some--albeit small--zones for highly resistant organisms. In conclusion, the over-reporting of resistance could be reduced by use of zone breakpoints optimized to the MIC distribution and by the use of 5 microg discs, but the case for these changes is not overwhelming; taking the breakpoint as the zone for NCTC 10662 minus 7 mm led to unacceptable numbers of resistant organisms being reported as susceptible. More fundamentally, ciprofloxacin zones and MICs are continuously distributed for P. aeruginosa isolates, so susceptibility tests cannot divide the species into discrete populations. In these circumstances, it is optimistic to expect disc and MIC categorizations to agree perfectly.

Susceptibility testing of anaerobic bacteria

The relationship between susceptibility testing by an agar dilution test and a tablet diffusion test was studied for 60 anaerobic bacteria (20 B. fragilis, 20 anaerobic cocci, 20 Clostridium species). For cefoxitin, no prediffusion and prediffusion times of one h, three h, 12 h, 24 h and 48 h were examined. For metronidazole, erythromycin, clindamycin, penicillin and imipenem, only 24 h prediffusion and no prediffusion were studied. Measurements were made after incubation for 24 h and 48 h. Prediffusion improved the correlation for all antibiotics tested, and 24 h prediffusion gave the best results. The slope of the regression line increased and the influence of the individual growth parameters on zone size was reduced. Prediction of susceptibility based on three zone breakpoints to estimate MIC was also better with 24 h prediffusion. However, the variation about the regression line for many of the antibiotics was still extremely high. Measurements after 24 h and 48 h incubation times showed almost identical regression equations, except for erythromycin, where the regression lines differed.

Antibiotic disk diffusion testing revisited. Single strain regression analysis

The standardized (NCCLS, ICS, DIN etc.) disk diffusion method is the most widespread technique for antibiotic susceptibility testing. Interpretive zone breakpoints are calculated from the regular regression line between minimum inhibitory concentrations (MIC) of bacterial isolates and the corresponding inhibition zone diameters around the disk containing the antibiotic. Studies of the regression line has revealed marked differences between different bacterial species. A newly described equation, the single strain regression analysis (SRA) equation, can be used to determine the regression line constants for individual strains. This method was applied to ciprofloxacin and S. aureus, E. faecalis, E. coli, P. mirabilis, P. aeruginosa, and P. maltophilia. The slope and intercept constants were determined for all 40 strains and showed a strong similarity within each species. A close similarity was also observed between the two Pseudomonas species and between S. aureus and E. faecalis. When the regression lines calculated by SRA for individual strains were extrapolated towards higher MIC values, the lines obtained for the more susceptible strains predicted the zones of more resistant strains within the species. The applications of SRA to several other antibiotics and bacterial species in earlier studies were reviewed. One exception to the predictive power of SRA has been detected earlier, H. influenzae and erythromycin. This led to the formulation of the standard curve regression analysis (SCA) equation which requires the use of two or more strains. Methodological aspects of SRA/SCA applications were presented. Three areas are particularly well suited for the use of SRA/SCA: 1. Calculation of interpretive zone breakpoints corresponding to recommended MIC limits in the individual laboratory. 2. Analysis of the effects of various disk contents of antibiotic on the resulting inhibition zones for various bacteria when new antibiotics are introduced. 3. Analytical tool as part of external quality control programmes.