1. Colistin MIC Testing: The Effect of Different Types of Microtitre Trays and Surfactant Tween 80
in Broth Microdilution Method. M. Albur, A. Noel, K. Bowker, A. MacGowan BCARE, Department of Microbiology, North Bristol NHS Trust, Bristol, UK
D-763
Introduction:
Table 1. MIC distribution of the isolates
Fig. 3. Effect of MITT & Tw80 on MIC
Colistin has re-emerged in recent times due to lack of alternative therapeutic options against carbapenem-resistant Gram-negative bacteria. Despite >50years of clinical use, there are uncertainties regarding optimal dosage of colistin particularly in critically ill patients. Also, the degree of colistin binding to organic (e.g. protein, tissues) and inorganic materials, both in vivo as well as in vitro remains an unresolved issue. Colistin exhibits a concentration-dependent bactericidal activity, and hence achieving a higher Cmax/MIC or AUC/MIC ratio improves therapeutic efficacy. Therefore an accurate estimation of the “key” component of the PK/PD index i.e. MIC is important. The objective of this study was to evaluate the influence of surfactant Tween80 (Tw80), and different types of the micro-titre trays (MITT) on the MIC result in broth-dilution method (BMD).
MIC distribution
Type of Isolate
Micro-titre Tray
<0.015
0.015
0.03
0.06
0.12
0.25
0.5
1.0 2 4 8 16
<16
PSAE
N=56
NMTT 31 14 3
NMTT+T80 9 32 1
TCMTT 44 6
TCMTT+T80 7 38
Acinetobacter
N=29 13 5 10
Enterobacteriaceae
N=61 20 34 11 28 17
Methodology:
Fig. 4. Effect of MTT & Tw80 on MIC of
different groups of isolates
Results:
Bacterial Isolates: A total of 146 clinical isolates collected from a variety of sources and stored at
-70 0C were evaluated in this study. The isolates included 56 Pseudomonas spp., 29 Acinetobacter spp., and 61 Enterobacteriaceae.
Microtitre plates: The MIC testing was carried out on two different types of micro titre trays, namely non-coated V-bottom microtitre tray NMTT (costar 3896, Corning, NY) and Tissue-culture-coated round bottom microtitre tray TCMTT (costar 3799, Corning, NY).
Tween 80: Tween 80 is a surfactant widely deployed as a dispersing agent in the preparation of broth micro-dilution panels used in the susceptibility testing.
MIC determination: The colistin MIC of the isolates was determined by CLSI broth dilution method using colistin sulphate, because colistin sulphate is the most stable (in vitro) & active component amongst the polymyxin group. The MIC determination was carried out using an initial inoculum of 0.5 McFarland’s in Muller-Hinton broth with or without Tw80 (final Tw80 conc. of 0.002%) on both types of microtitre trays (NMTT & TCMTT). The experiments were done in triplicates, and the quality control was assured by concurrent testing of P.aeruginosa ATCC as control with all results within the range published by the CLSI (M100-S10).
Statistics: The statistical analysis including the boxplot and scatterplot graphics were carried out by using SPSS (version 18, Chicago, Illinois).
Fig. 1 Scatterplot: Effect of MITT
MIC distribution of the isolates in both types of microtitre trays with or without Tween 80 is shown in Table 1.
Effect of microtitre tray on MIC result:
The NMTT MICs were significantly lower than TCMTT MICs (p< % CI –2.6 to –2.0).
Similar findings were noted between NMTT and TCMTT containing Tween80 (p<0.0001, 95% CI –2.9 to –2.3) [Fig. 1 & Fig. 3].
Effect of Tween80 on MIC result:
Addition of Tween80 significantly decreased the MIC by 3 to 4 dilutions in NMTT plates (p< , 95% CI to 0.54) [Fig. 2].
Although there was slight decrease in the mean MIC of TCMTT by the addition of Tween80, but this was not statistically significant (p= % CI to 0.38) [Fig. 3].
Effect on different types isolates:
The effect of microtitre tray & the presence of Tween80 on MIC results were similar amongst all types/groups of isolates i.e. PSAE, Acinetobacter or Enterobacteriaceae [Fig. 4].
Fig. 2 Scatterplot: Effect of Tween 80
Discussion:
This study shows the significant variations in colistin binding to various surfaces and the effect of surfactant Tween80 on the MIC result. Both of these features could have major clinical implications in terms of accurate estimation of MIC – the “key” component of PK/PD index, and also in assessing serum drug concentration of colistin.