Single-strain Regression Analysis Research Articles

Single-strain regression analysis evaluating disc potencies of flumequine and enrofloxacin for testing Aeromonas sobria and Vibrio anguillarum.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum. There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.

Setting interpretive breakpoints for antimicrobial susceptibility testing using disk diffusion

Antimicrobial susceptibility testing plays a key role in clinical microbiology. The disk diffusion test dates back to the 1940s and became standardised from the 1950s, with the International Collaborative Study (ICS) and National Committee for Clinical Laboratory Standards (NCCLS) as the two major standards. Interlaboratory variation of disk test results was recognised early but has never been dealt with in a satisfactory manner. The error-rate bounded method was described in 1974 and its role is discussed. Species-specific susceptibility interpretation was coined in 1980 for Proteus mirabilis and chloramphenicol. In the late 1970s, more extensive use of species-specific breakpoints was introduced in Lund (Sweden). At the same time, P. Mouton constructed species-specific regression lines and pointed out the difficulties with narrow ranges of minimal inhibitory concentration (MIC) values. A more general use of species-specific regression lines was made possible with single-strain regression analysis, using one well-defined strain tested in disk diffusion with a range of disk contents. This method made it possible to calibrate the disk test in an individual laboratory. Other methods to achieve such calibration are also described. A recent method, ‘MIC-coloured zone diameter histogram-technique’, has proven useful for the validation of species-specific interpretive breakpoints. The microbiological breakpoint proposed by Williams in 1990 has experienced a renaissance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value (ECOFF). MIC and zone diameter distributions with accompanying ECOFFs for species–antimicrobial combinations are published on the EUCAST website. A method for the reconstruction of wild-type zone diameter populations, namely normalised resistance interpretation, is described. This method can produce resistance figures that are truly comparable between laboratories.

Meropenem susceptibility breakpoint for Pseudomonas aeruginosa strains hyperproducing mexB mRNA

Twenty-five isolates of Pseudomonas aeruginosa with different meropenem susceptibilities were subjected to quantitative RT-PCR for analysis of transcription levels of oprD, mexB and mexD, and, in selected isolates, PA3720, which is hyper-expressed in nalC efflux mutants. Regulator genes of efflux pump MexAB-OprM, mexR and PA3721 (putative) were sequenced in selected isolates. The potential for mathematical reconstruction of the ideal susceptible population using normalised resistance interpretation (NRI) was also studied. In three isolates with intermediate susceptibility to meropenem (according to Swedish breakpoints), a reduction in MIC from 4 to 2 mg/L was observed with efflux inhibitor MC-207,110. These isolates would be considered susceptible according to British Society for Antimicrobial Chemotherapy and NCCLS breakpoints. These three isolates had between 4.6- and 5.0-fold increases in mexB transcription. None of these isolates had significant nalB mutations, but an Ala145-->Val mutation was observed in PA3721 in two of the isolates. However, these isolates had moderately increased production of PA3720 only. Single-strain regression analysis did not detect any major biological differences between the different groups. Using NRI, a disk-diffusion susceptibility breakpoint of >/= 28 mm was generated. Isolates with intermediate susceptibility to meropenem, which are considered fully susceptible in many countries, displayed possible low-grade meropenem resistance mechanisms, implying that the susceptibility breakpoint should be reconsidered. The increased transcription of mexB mRNA in such isolates seems unrelated to nalB or nalC mutations.

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.

Fluconazole and voriconazole multidisk testing of Candida species for disk test calibration and MIC estimation.

Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of susceptibility to fluconazole of < or =8 microg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-microg fluconazole disk was calculated to be > or =18 mm for C. albicans and > or =22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-microg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 microg/ml, whereas a 2.5-microg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.

MIC determination of fusidic acid and of ciprofloxacin using multidisk diffusion tests

To investigate the possibility of estimating the MICs of fusidic acid and ciprofloxacin for bacterial isolates using series of antibiotic disk concentrations in diffusion tests, so-called M-tests. Thirty Staphylococcus aureus and S. epidermidis strains were tested for fusidic acid susceptibility. Sixty-one clinical isolates of eight bacterial species were tested for ciprofloxacin susceptibility. Disk diffusion was standardized according to the Swedish reference group for antibiotics (SRGA). For fusidic acid, a series of disks (1.5, 5.0, 15, 50 and 150 microg) was used. Ciprofloxacin was applied in four different diffusion sources (1, 3, 10 and 30 microg) on a single strip, the M-strip, and used. True MIC values were determined using the standardized agar dilution method according to the SRGA. Single-strain regression analysis (SRA) was employed to calculate critical concentration equivalents (Qzero). Fusidic acid and ciprofloxacin critical concentrations were determined for the bacterial isolates. The mean conversion factors for Qzero to yield the true MIC were 2.06 (range 0.34-8.9) for fusidic acid and 2.05 (range 0.37-8.1) for ciprofloxacin. There was a correlation between true MIC values (all MICs expressed as 2 log + 9) and the calculated MIC values (Qzero x conversion factor) for both fusidic acid (R = 0.9822) and ciprofloxacin (R = 0.9696). MIC values of clinical isolates can be estimated using SRA calculations on zone measurements in disk tests with several concentrations of the antibiotic in diffusion sources.

Calibration of the disk diffusion test for trovafloxacin susceptibility testing of four anaerobic species

To study trovafloxacin susceptibility among clinical isolates of four anaerobic bacterial species using minimum inhibitory concentrations (MIC) determinations, E test assays and disk diffusion test results and to calibrate the disk diffusion method for these species using single strain regression analysis (SRA). One-hundred and eighty-seven clinical isolates of four anaerobic bacterial species were included. Trovafloxacin MIC determinations were performed using the agar dilution technique and MIC estimations using the E test. The disk diffusion test was performed according to Swedish Reference Group for Antibiotics standardization. NCCLS limits for susceptibility categories were applied. SRA was performed using 1, 3, 10, 30, and 100 microg trovafloxacin disk contents and ATCC control strains. The regression lines obtained permitted the calculation of zone equivalents to MIC limits as well as an evaluation of various disk potencies. Trovafloxacin susceptibility (S + I) was noted in 98.9, 100, 100, and 97% of Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium perfringens, and Peptostreptococcus magnus strains, respectively, as judged by MIC determinations. Agar dilution and E test estimations gave the same results, but E test values were consistently lower than MIC values by the reference method. Regression lines calculated for the four species using SRA showed different equation constants indicating species-related differences. Interpretive zone diameter breakpoints were calculated for the four species and used for the interpretation of susceptibility. The disk diffusion test was successfully calibrated for trovafloxacin susceptibility testing of four anaerobic species using single strain regression analysis, SRA. There was a good agreement between the results of MIC-tests and disk testing. Interpretive errors of type I are prone to occur among Bacteroides isolates and might require species-related MIC limits. SRA calculations permitted the testing of the effect of different disk potencies on inhibition zones produced at the interpretive MIC limits. Criteria for the selection of a minimal disk content showed that 5 microg trovafloxacin is sufficient, but a 10 microg disk will safeguard against residual laboratory variation without producing too large inhibition zones for very susceptible strains.

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.

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.

Comparison of 30 μg and 120 μg gentamicin disks for the prediction of gentamicin resistance in Enterococcus faecalis

Single strain regression analysis was performed on PDM II medium for E. faecalis with 10, 30 and 120 micrograms gentamicin disks using E. faecalis, strain ATCC 29212 as the reference. This method permits the calculation of zone diameters corresponding to different MIC values for different disk contents. The lack of discrimination between normal low-level resistant strains and high-level resistance using the 10 micrograms disk was confirmed. However, both the 30 micrograms and 120 micrograms disks seemed to provide a separation of the normal low-level gentamicin-resistant population from strains with increased resistance. Since the 30 micrograms disk is used routinely in some countries, there should be no need for an extra high content disk in these laboratories. This was confirmed when 96 clinical isolates of E. faecalis were analysed and the results of routine disk diffusion tests were compared with the MIC values. Two of the strains showed high-level gentamicin resistance (greater than 2000 mg/l) and produced no zone of inhibition. The other 94 isolates showed gentamicin MIC values between 4-16 mg/l, and 72 of the MIC results were 8 mg/l. The zone diameters for these strains ranged between 15 and 25 mm with a mean of 18.2 and a median value of 18 mm. In order to include statistical considerations of the zone size populations for setting of breakpoints, a study of gentamicin zone size distributions was performed for several bacterial species. Inhibition zone diameter values around the 30 micrograms gentamicin disk for 2079 clinical isolates of E. faecalis, 2268 S. aureus, 3201 E. coli and 547 strains of P. mirabilis from different years were plotted as histograms. Tests for agreement with a Gaussian distribution showed that the histograms were slightly peaked and skewed towards higher zone values. Parametric and non-parametric statistical tests were compared and the results showed that means and medians were very similar and that parametric fractile estimations at the lower end of the histogram populations were conservative and could be used in view of a slightly lower rate of false resistance. The 1% parametric fractile of 12 mm was selected as a suitable breakpoint for the identification of normal, low-level resistant isolates of E. faecalis using the standardized disk test of the Swedish Reference Group for Antibiotics.

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.

Antimicrobial susceptibility testing of Haemophilus influenzae Improvement of accuracy of the disc diffusion test

A national quality control study was performed in 1986 to investigate the standard of performance of the disc diffusion antimicrobial susceptibility testing of Haemophilus influenzae in Sweden. The accuracy of susceptibility interpretations was unacceptably low. A new standardized method for susceptibility testing of H. influenzae was then worked out, and a new method of setting interpretive zone breakpoints was introduced. The susceptibility category of the main population of clinical isolates was determined according to the MIC50 of the strains. The zone histograms of clinical isolates from five reference laboratories were used for the calculation of interpretive breakpoints. For instance, for susceptible strains the mean of the combined zone values from these laboratories +/- 2 S.D. covered the zone range of the susceptible group, and one more S.D. below covered the intermediate/indeterminate group. The new zone breakpoints would place the main population of clinical isolates in the correct susceptibility group, and even make it possible to detect strains with different degrees of reduced susceptibility in the routine test. In a follow-up quality control study in 1988 the interpretive errors for clinical isolates were eliminated for all antibiotics except doxycycline in some laboratories. Laboratory-related zone breakpoints for doxycycline calculated by the single strain regression analysis method led to correct susceptibility interpretations also in these cases.

Types of interpretive errors in susceptibility testing. Zone breakpoints for norfloxacin disk diffusion testing.

A total of 548 strains of the eleven most common urinary tract pathogens were investigated for possible errors in norfloxacin susceptibility tests comparing MIC determinations with disk diffusion assays. Most strains were found to be sensitive with MIC-90 values below 1.0 for the Enterobacteriaceae while the classical nalidixic acid resistant species, the gram-positive bacteria and Pseudomonas aeruginosa, were less susceptible to norfloxacin with MIC-90 above 1.0 mg/l. MIC-values close to interpretive MIC-limits were recorded for S. faecalis and S. agalactiae using the recommendations of the national Committee for Clinical Laboratory Standards (NCCLS) (susceptible, S less than or equal to 4.0) and for P. aeruginosa and S. aureus using the Swedish Reference Group for Antibiotics (SRGA) standards (S less than or equal to 1.0). Susceptibility interpretations for these species showed a lack of accuracy consistent with methodological problems of reproducibility, an error called type I. The changes in the MIC-limits required for these strains to correct the error would be S less than or equal to 4 for P. aeruginosa and S. aureus, S less than or equal to 8 for S. agalactiae and S less than or equal to 0.5 for S. faecalis. A type II error, occurring when a bacterial species shows a regression line different from the regular line, was also identified for S. saprophyticus. The use of breakpoints derived from single strains regression analysis corrected this error and also reduced the frequency of similar misinterpretations in other species. The term "species-specific MIC-limits" should be introduced along with the established concept of "species-specific interpretive zone breakpoints" to allow for the correction of type I interpretive errors. Type II errors can be corrected by using species-specific interpretive breakpoints, either issued by reference laboratories or derived by calculations from single-strain regression analysis in the individual laboratory.

Laboratory- and species-specific interpretive breakpoints for disk diffusion tests of chloramphenicol susceptibility of Haemophilus influenzae.

A total of 601 clinical isolates of Haemophilus influenzae isolated in six different regions of Sweden were tested for chloramphenicol susceptibility by using agar dilution MIC determinations and disk diffusion tests. For seven strains MICs were 4 micrograms/ml or higher, and for one strain the MIC was 2 micrograms/ml. All eight strains produced chloramphenicol acetyltransferase. For the remaining 593 strains, MICs were less than or equal to 1 microgram/ml, and the MICs for 50% and 90% of the strains were both 0.5 microgram/ml. Disk diffusion tests carried out by using revised interpretive criteria introduced in 1984 by the Swedish Reference Group for Antibiotics correctly identified the 593 strains as susceptible and the 8 strains as resistant. Quality assessments were performed in 29 clinical microbiology laboratories. The revised criteria for chloramphenicol disk diffusion testing gave rise to false resistance results in some laboratories. The interpretive accuracy improved when the interlaboratory variation was compensated for by using adjusted breakpoints. Such revision was possible through peak correction, single-strain regression analysis, and standard curve regression analysis. Peak-corrected breakpoints improved the accuracy from an overall incidence of false-resistant isolates of 4.4% to 2.3%. Single-strain regression analysis and standard curve regression analysis provided laboratory- and species-specific breakpoints which reduced false resistance rates of 0.14% and 0%, respectively.

On the theory of the disk diffusion test

The procedure for disk diffusion susceptibility testing has been worked out for rapidly growing non-fastidious bacteria. Using the general zone diameter breakpoints for interpretation of susceptibility, it was found that clinical isolates of H. influenzae were assigned to the wrong SIR category in fifty per cent of the strains for erythromycin, and ten per cent for doxycycline. New species- and laboratory-specific interpretive zone diameter breakpoints corresponding to the recommended MIC limits were therefore worked out. The Standard Curve regression Analysis (SCA) method used for this purpose is based on the correlation between zone size and disk content, using two reference strains with different MICs. In its original version (the Single strain Regression Analysis, SRA) only one reference strain was used. This equation was found not to be generally valid since the relationship between MIC and the critical concentration is not constant, as was originally assumed. The slope and intercept of the regression line obtained by SCA is species related, while a general regression line based on results from many different species assumes that there is the same relation between the zone size and MIC for all species. Breakpoints for erythromycin and doxycycline calculated by the SCA equation gave more accurate results in routine susceptibility testing of H. influenzae and reduced the error rate from fifty three and ten per cent to four and three per cent for the two antibiotics.

Determination of species- and laboratory-related interpretive breakpoints for doxycycline susceptibility testing using single-strain regression analysis.

Single-strain Regression Analysis (SRA) was performed for doxycycline on a total of 68 bacterial strains representing 16 different species. Species- and laboratory-related zone diameter breakpoints were determined and compared with histograms of zone diameter values obtained from 942 routine susceptibility tests. Calculated breakpoints were similar within individual species. When considering the homogeneity of susceptibility groups within bacterial species, the calculated breakpoints gave rise to relatively few interpretive errors. In contrast, general breakpoints for doxycycline as recommended by the Swedish Reference Group (SRG) (R less than = 20 mm and S greater than = 26 mm) would give rise to a high proportion of false interpretations in the present laboratory. Fifty-three per cent of H.influenzae strains would have been assigned to the wrong susceptibility group. For E.coli and K.pneumoniae, 22 and 35 per cent, respectively, would have been erroneously categorized using SRG breakpoints. E.cloacae and E.aerogenes would have been assigned another category in 39 and 50 per cent, respectively. The procedure for setting species-specific and laboratory-related interpretive breakpoints is described. Determination of species- and laboratory-related interpretive breakpoints using SRA provides a new approach towards improved accuracy of disc-diffusion susceptibility testing.

Determination of interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing using single-strain regression analysis.

Interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing were determined using single-strain regression analysis (SRA). Regression lines were determined for a total of 58 strains representing 15 species, from inhibition zone diameters obtained for discs containing six different ceftazidime concentrations. Statistical analysis for excluding non-linearity of test-results was performed. A minimum of five tests on consecutive days was required for maximal precision of regression analysis according to the SRA-method. Calculated regression lines showed similarities within individual and groups of bacterial species. A minimum of five strains could be used to represent these groups. Interpretive breakpoints according to recommended MIC-limits were determined for each species taking into consideration confidence limits for zone correlates of MIC-values. Single-strain regression analysis for the determination of interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing in individual laboratories.

Single-strain regression analysis for quality control of cephalothin-susceptibility testing and determination of interpretive breakpoints.

Histogram analysis of inhibition zone diameters around the 30 micrograms cephalothin disk for E. coli, P. mirabilis, and K. pneumoniae in samples from 1975 to 1982 showed a marked reproducibility of the disk-diffusion antibiotic-susceptibility test in the routine laboratory. A comparison of interpretive breakpoints with histograms for E. coli, P. mirabilis, K. pneumoniae, S. aureus, coagulase-negative staphylococci, and S. faecalis showed a higher proportion of possible misinterpretations using the breakpoints of the Swedish Reference Group, SRG, as compared to international (NCCLS) breakpoints. Further analysis using single-strain regression analysis revealed two major causes of interpretive errors. Firstly, the laboratory-related regression line for a bacterial species can be different from the general regression line of the reference laboratory. This difference has to be corrected by using species-related breakpoints. For E. coli, a species-specific breakpoint was determined to R = greater than 13 mm. Secondly, MIC limits recommended for the susceptibility categories of cephalothin by SRG are lower than the international limits and close to the true MIC values of many bacterial isolates, leading to misinterpretations due to the methodological variation. These studies suggest an adoption of international MIC limits for the susceptibility categories of cephalothin in Scandinavia. The "I" category should denote an indeterminate zone. A multi-laboratory quality control assessment using histogram analysis is recommended with optional single-strain regression analysis to determine breakpoints for problem combinations of bacterial species and antibiotics.

Single-strain regression analysis for determination of interpretive breakpoints for cefoperazone disk diffusion susceptibility testing.

A novel approach for setting interpretive breakpoints in disk diffusion antibiotic susceptibility testing according to determined minimum inhibitory concentration (MIC) limits is described, using the method of single-strain regression analysis. The procedure was tested on reference strains Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), using published results from cefoperazone disk diffusion experiments. The correlation between logarithm of the disk content and inhibition zone diameter squared was linear, excluding three endpoint values. When constants A and B in the new regression line equation were calculated for the four strains, all four showed different regression lines. Zone diameters corresponding to various MICs were calculated for a disk content of 75 micrograms. The values obtained for the four strains were 20.1, 20.9, 24.9, and 25.8 mm, respectively, for an MIC of 16 micrograms/ml, and 15.7, 15.7, 22.3, and 17.9 mm, respectively, for an MIC of 64 micrograms/ml. The following zone diameter breakpoints were determined for the "I" (intermediate) category, using a 75-micrograms disk: S. aureus, 18 to 15 mm; S. faecalis, 23 to 13 mm; E. coli, 20 to 17 mm; and P. aeruginosa, 20 to 17 mm.