Comparison of cation-adjusted Mueller–Hinton broth with Iso-Sensitest broth for the NCCLS broth microdilution method

L. M. Koetha,*, A. Kingb, H. Knighta, J. Mayb, L. A. Millerc, I. Phillipsb and J. A. Poupardc

a Laboratory Specialists, Inc., 1651A Crossings Parkway, Westlake, OH 44145, USA; b St Thomas' Hospital, London SE1 7EH, UK; c SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparison of MIC results obtained in different parts of the world is currently difficult because of variations in methods. In this study, cation-adjusted Mueller–Hinton broth, the NCCLS-recommended medium, was compared with Iso-Sensitest broth, which is widely used in Europe. Microbroth dilution testing, using the NCCLS procedure, was performed on 124 Gram-positive (staphylococci and enterococci) and Gram-negative (Enterobacteriaceae and Pseudomonas aeruginosa) isolates from the CDC reference set, with the only variable being the medium used. Twelve antimicrobial agents were tested: amoxycillin–clavulanic acid, ampicillin, ciprofloxacin, erythromycin, gentamicin, imipenem, levofloxacin, oxacillin, gemifloxacin, trimethoprim– sulphamethoxazole, tetracycline and vancomycin. Vancomycin, erythromycin and oxacillin were only evaluated for the Gram-positive organisms. Trimethoprim–sulphamethoxazole was only evaluated for a subset of Gram-negative organisms because of off-scale results. The 124 isolates were tested in one American and one UK laboratory with two batches of cation-adjusted Mueller–Hinton broth and two of Iso-Sensitest broth. A statistical evaluation of the data used a 24 fully specified factorial analysis to determine if there were significant differences in results owing to Gram reaction, site of testing and type and/or batch of broth. In addition, the cumulative results for each antimicrobial agent in each broth were plotted against the range of MIC dilutions tested. MICs of ciprofloxacin, levofloxacin, gemifloxacin, gentamicin and tetracycline were slightly higher (half a doubling dilution) with Iso-Sensitest broth than with Mueller–Hinton broth. MIC results for the other antimicrobial agents were equivalent. Essential and category agreement rates were comparable for all agents (88.4–100% and 88.2–99.0%, respectively).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the earliest comprehensive surveillance studies of antibiotic resistance, an American study using standardized disc testing, was published in 1964.1 Since then, local, national and worldwide studies have proliferated. A recent compilation of surveillance studies identified 48 major studies conducted on both national and worldwide levels, not including the many smaller local and national studies that have not been published or widely promoted.2 In addition, pharmaceutical companies developing new antimicrobial agents perform worldwide studies for regulatory submissions, which include susceptibility testing of the new and existing agents against a wide range of pathogens.

The optimal way of monitoring resistance trends would be to combine data from as many studies as possible. However, it is difficult to compare susceptibility results because of variation in testing methods and antimicrobial concentrations tested. In addition, some studies lack proper quality control and patient demographic information. Finally, standardized reporting is required for the successful combination and analysis of data.

If standardized testing methods were used for surveillance studies, one of the major variables would be controlled. The combination of European data alone presents its own challenges, as multiple methods are used throughout Europe. For example, the British Society for Antimicrobial Chemotherapy (BSAC) method is used in the UK,3 the Deutsches Institut für Normung (DIN) method in Germany,4,5 the Société Française de Microbiologie (SFM) method in France6 and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden (http:/www.ltkronoberg.se/ext/raf).

One primary objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) is to formulate guidelines for a standardized reference susceptibility method among the European Union participants. Most studies in the USA use the standardized methods of the NCCLS. As a first step in harmonizing efforts, a EUCAST representative is an adviser to the NCCLS Subcommittee on Antimicrobial Susceptibility Testing and has requested NCCLS representation at EUCAST meetings.

Similarities and differences between the methods must first be established to determine a baseline for standardization. One of the major variables between methods is the medium used. This study was undertaken as an initial evaluation to determine how Iso-Sensitest broth (ISB), which is recommended by several European National Committees, compares with cation-adjusted Mueller– Hinton broth (CAMHB) defined in the NCCLS method. To control all other variables, testing was performed by the NCCLS method with CAMHB and by the NCCLS method with ISB instead of CAMHB.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates

One hundred and twenty-four CDC reference strains with established MICs, comprising 20 Pseudomonas aeruginosa, 20 Enterococcus spp., 19 Staphyloccus aureus, 15 coagulase-negative Staphyloccus spp. and 50 Enterobacteriaceae (20 Escherichia coli, eight Klebsiella spp., eight Enterobacter spp., seven Serratia spp. and seven Proteus spp.) were tested. Ten strains from the CDC challenge set were chosen for reproducibility testing.

Testing sites

The two testing laboratories were St Thomas' Hospital in London, UK (laboratory 1) and Laboratory Specialists, Inc., Westlake, OH, USA (laboratory 2).

Susceptibility test methods and materials

The organisms and antimicrobial agents used in this study have been previously reported as having been most affected by variations in the medium.7–19 MIC custom dehydrated microtitre panels (Dade, Sacramento, CA, USA) containing the antimicrobial agents (amoxycillin– clavulanic acid, ampicillin, ciprofloxacin, erythromycin, gentamicin, imipenem, levofloxacin, oxacillin, gemifloxacin, trimethoprim–sulphamethoxazole, tetracycline and vancomycin) were used. Microdilution MICs were tested according to current NCCLS guidelines,20 with the only variation being the prepared media that were used for plate rehydration and inoculation: in each tube, 25 mL of CAMHB (PML, Tualatin, OR, USA) or ISB (PML) was used. Each strain was tested with two batches of CAMHB and two batches of ISB by both laboratories. All panels were rehydrated and inoculated with the MicroScan Renok (Dade) using disposable inoculators and incubated under ambient conditions at 35°C for 16–24 h. Enterococcus spp. and Staphylococcus spp. were incubated for 24 h. The lowest concentration of antimicrobial agent at which organisms showed no growth was read as the MIC. The 10 reproducibility strains were tested in triplicate on three occasions at laboratory 2 and in triplicate on one occasion at laboratory 1 using the same batch of CAMHB and ISB at both laboratories. Quality control organisms were tested for each microdilution run using both CAMHB and ISB for five quality control organisms: S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922 and ATCC 35218 and P. aeruginosa ATCC 27853.

Data collection and analysis

The data for the 124 CDC reference strains were analysed by four different methods. The cumulative MICs for each broth for each antimicrobial agent were plotted against the range of MIC dilutions tested. For statistical evaluation of any differences between broths, a 24 fully specified factorial analysis was performed for each antimicrobial agent.21 For those antimicrobial agents that differed, the MICs from laboratory 1 and batch 1 were further analysed according to organism by determining the log2 difference (doubling dilution difference) between the ISB MIC and the CAMHB MIC. Essential and category agreement rates and number of errors were calculated for each antimicrobial agent.

Because of off-scale trimethoprim–sulphamethoxazole results (<=;0.5 mg/L) with the Gram-positive organisms and the majority of the Gram-negative organisms, only 26 isolates (six Serratia marcescens and 20 P. aeruginosa) were included in the data analysis. Only Gram-positive isolates (a total of 54) were included in the analysis of erythromycin, oxacillin and vancomycin. For all other drugs, all isolates were analysed.

The independent factors included in the statistical analysis were Gram's stain (positive or negative), laboratory (1 or 2), broth (CAMHB or ISB) and batch of medium (1 or 2). The dependent variable was MIC, transformed to a log2 scale.22

The percentage essential agreement is the percentage of results that were within one doubling dilution of each other. As there were no significant differences between laboratories and batches of media, essential agreement rates, category agreement rates and error analyses were performed on CAMHB batch 1 and ISB batch 1 from laboratory 1. Essential agreement rates were determined from on-scale MICs only. Category agreement rates were based on those results that could be interpreted according to current NCCLS breakpoints.23 An error was considered minor when the results obtained with CAMHB and ISB differed by one category (e.g. susceptible versus intermediate). An error was considered major when the result obtained with CAMHB was susceptible and that with ISB was resistant. An error was considered very major when the result with CAMHB was resistant and that with ISB was susceptible. Agreement and error calculations were based on Staphylococcus spp. only for oxacillin and erythromycin, and Gram-positive organisms only for vancomycin. For all other antimicrobial agents, both Gram-negative and Gram-positive results were included.

The reproducibility results, from 10 of the CDC reference isolates, were analysed by combining both CAMHB and ISB results and determining the percentage of results within three doubling dilutions. Quality control results were analysed according to NCCLS expected ranges.23

A qualitative determination of growth was also evaluated by both laboratories. The growth on each batch was compared with that in CAMHB batch 1 and recorded as similar, better or less.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reproducibility studies

For all on-scale results, all of the results for all antimicrobial agents were within a three-dilution range.

Media comparison studies

For all three quinolones (ciprofloxacin, levofloxacin and gemifloxacin), gentamicin and tetracycline, slightly higher results were obtained with ISB than with CAMHB, according to both statistical and cumulative MIC methods of analysis (Table IGo). There were no significant differences with amoxycillin–clavulanic acid, ampicillin, imipenem, oxacillin, trimethoprim–sulphamethoxazole and vancomycin. There were no significant differences between the laboratories and batches of broth.


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Table I. Correlation of MICs determined with CAMHB and ISB for 12 antimicrobial agents: statistical and cumulative MIC analyses
 
For those antimicrobial agents that differed, the cumulative MIC analysis allowed determination of the differences at specific MICs (Table IGo). Representative graphs for gentamicin and gemifloxacin are shown in Figures 1 and 2GoGo, respectively. For those antimicrobial agents that were similar, the cumulative MIC analysis showed comparable results throughout the dilution range. A representative graph for one of these antimicrobial agents (amoxycillin– clavulanic acid) is shown in Figure 3Go.



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Figure 1. Cumulative MICs of gentamicin for (a) 54 Gram-positive isolates and (b) 70 Gram-negative isolates using Mueller– Hinton batches 1 ({blacksquare}) and 2 ({blacktriangleup}) and Iso-Sensitest batches 1 ({circ}) and 2 ({square}).

 


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Figure 2. Cumulative MICs of gemifloxacin for (a) 54 Gram-positive isolates and (b) 70 Gram-negative isolates. Symbols as in Figure 1Go.

 


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Figure 3. Cumulative MICs of amoxycillin–clavulanic acid for 124 Gram-positive and Gram-negative isolates. Symbols as in Figure 1Go.

 
An analysis of the dilution difference by organism for the five antimicrobial agents that differed demonstrated an upward shift in the ISB MICs by approximately half a dilution overall (Table IIGo). This was more pronounced for Enterobacteriaceae tested with quinolones and tetracycline and for Staphlococcus spp. tested with gentamicin. The shift was less pronounced for P. aeruginosa with all antimicrobial agents tested. Although the shift was similar for all five antimicrobial agents, ciprofloxacin was slightly more affected overall.


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Table II. Comparison of MIC results obtained in ISB with those in CAMHB taken as standard for five antimicrobial agents that differed
 
With the exception of P. aeruginosa, many of the trimethoprim–sulphamethoxazole results were off-scale (less than the lowest concentration tested or greater than the highest concentration tested) and therefore could not be evaluated. However, an analysis of the results obtained with P. aeruginosa indicated that MICs were approximately one dilution lower with ISB than with CAMHB. Of the 17 on-scale results, 53% of the ISB results were one to two dilutions lower than those obtained with CAMHB.

Essential agreement for all antimicrobial agents was >90%, with the exception of ampicillin (88.4%). Category agreement for all antimicrobial agents was >93%, with the exception of erythromycin (88.2%). There were very few errors, the majority of which were within one dilution (Table IIIGo).


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Table III. Comparison of MICS determined with CAMHB and ISB for 12 antimicrobial agents: essential and category agreement rates and number of errors
 
Qualitative analysis of growth indicated better growth in ISB than in CAMHB, although the difference was considered more evident in laboratory 2. The growth for 19.8% of isolates tested by laboratory 1 was regarded as better in ISB than in CAMHB (batch 1) while nearly all of the isolates (96.5%) were considered to show better growth in ISB by laboratory 2. There were no differences in growth between CAMHB batches 1 and 2 at either laboratory.

Quality control

At least one out-of-range quality control result was obtained with all of the antimicrobial agents, except for ampicillin and oxacillin. With the exception of P. aeruginosa tested with tetracycline and S. aureus tested with erythromycin, >95% of CAMHB quality control results were within the expected ranges. The majority of the out-of-range results were obtained with ISB; <95% of ISB quality control results were within the expected ranges for at least one of the quality control organisms for amoxycillin–clavulanic acid, ciprofloxacin, gentamicin, imipenem, levofloxacin, gemifloxacin, trimethoprim–sulphamethoxazole and tetracycline. All outlying ISB results were higher than the expected range. Quality control results are summarized in Tables IV and VGoGo.


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Table IV. Percentage of results within expected quality control ranges (CAMHB/ISB)
 

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Table V. Summary of out-of-range quality control results below 95%
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although there were minimal differences with most of the drugs tested, the three quinolones tested (ciprofloxacin, levofloxacin and gemifloxacin), gentamicin and tetracycline showed slightly higher MICs in ISB. Although these differences were statistically significant, the majority of differences were not greater than ± one dilution. Therefore, essential and category agreement rates and numbers of errors were >90% for all agents with the exception of ampicillin and erythromycin. The lower essential agreement with ampicillin (88.4%) was because only 43 strains were on-scale and evaluable and a two-fold dilution difference was obtained with four of the S. aureus strains. The essential agreement calculation was based on data from laboratory 1. With the exception of one strain, similar discrepancies were not obtained at laboratory 2. The category agreement of erythromycin was lower (88.2%) because four of the 34 evaluable results differed by only one dilution, but differed by interpretive category. If ISB were to be used instead of CAMHB for a surveillance study and compared with results for other studies using NCCLS methods, the differences in MICs may affect MIC50 and MIC90 results by one dilution, but would not change susceptibility category rates.

The antimicrobial agents that differed were those most affected by varying concentrations of cations in the medium, namely quinolones, aminoglycosides and tetracycline.711 The calcium and magnesium ion levels in the CAMHB were equivalent to those recommended in NCCLS guidelines (20–25 and 10–12.5 mg/L, respectively). The concentrations of calcium and magnesium ions in the ISB were 3.3 and 53.0 mg/L, respectively. In a previous study, addition of 100 mg/L of magnesium and calcium ions to commercial media deficient in these cations increased ciprofloxacin MICs four-fold.24 The concentration of magnesium ions in this study was approximately half that used in the study by Blaser & Luthy,24 therefore the half dilution difference is consistent with earlier observations and is presumably a result of increased concentrations of magnesium ions.

This study indicates that ISB is an acceptable alternative to CAMHB when using the NCCLS MIC method. Although technologists at both laboratories noted better growth with ISB than with CAMHB, the technologists at laboratory 2 perceived ISB to be better more often. This is an important observation as laboratory 2 is more accustomed to using CAMHB than laboratory 1.

Modification of the Mg2+ concentration in the ISB to be consistent with the 10.0–12.5 mg/L in CAMHB seems appropriate. A further study would be needed to confirm that this cation adjustment would correct the slight shifts in MICs that were observed and bring quality control results within acceptable ranges. In addition, a greater number of on-scale results, together with the testing of additional antimicrobial agents, would be required for complete validation.


    Acknowledgments
 
We thank Dr Linda Quinn for her statistical analysis and Jeanna DiFranco for secretarial support. This work was supported by funds from SmithKline Beecham Pharmaceuticals, Inc., Philadelphia, PA, USA.


    Notes
 
* Corresponding author. Tel: +1-440-835-4458; Fax: +1-440-835-5786; E-mail: amdlmk{at}aol.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 17 November 1999; returned 25 January 2000; revised 9 March 2000; accepted 7 April 2000