Antimicrobial susceptibility testing of Arcobacter butzleri: development and application of a new protocol for broth microdilution.

Objectives: To develop a standard reference broth microdilution method for antimicrobial susceptibility testing (AST) of Arcobacter butzleri. The protocol was subsequently applied to a collection of A. butzleri isolates from different sources. Methods: Broth microdilution susceptibility testing was performed on eight A. butzleri isolates in three media: non-supplemented CAMHB, CAMHB + 2% FBS and CAMHB + 5% FBS. The MIC values were read after 24 and 48 h of incubation at 35 ±âŸ2 °C in ambient air. A logistic regression model was used to determine the combination of medium and incubation time yielding the most homogeneous results. Subsequently, the protocol was applied to 65 A. butzleri isolates to determine their MICs of 31 antimicrobial agents. Results: The statistical analysis revealed that the most homogeneous MIC values were obtained with CAMHB + 5% FBS and reading of MIC values after 24 h of incubation. The standardized method was successful for AST of all 65 A. butzleri isolates. MIC values were distributed unimodally for most antimicrobial agents. However, one field isolate showed elevated MIC values of gentamicin, streptomycin, tetracycline and trimethoprim/sulfamethoxazole. Conclusions: This study presents a new protocol for AST of A. butzleri by broth microdilution and shows the distribution of MIC values of 31 antimicrobial agents for a collection of A. butzleri isolates from different origins.

MICs of 32 antimicrobial agents for Rhodococcus equi isolates of animal origin.

OBJECTIVES: The aim of this study was to determine the MICs of 32 antimicrobial agents for 200 isolates of Rhodococcus equi of animal origin by applying a recently described broth microdilution protocol, and to investigate isolates with distinctly elevated rifampicin MICs for the genetic basis of rifampicin resistance. METHODS: The study included 200 R. equi isolates, including 160 isolates from horses and 40 isolates from other animal sources, from the USA and Europe. MIC testing of 32 antimicrobial agents or combinations thereof followed a recently published protocol. A novel PCR protocol for the joint amplification of the three rpoB regions in which rifampicin resistance-mediating mutations have been reported was applied to isolates with elevated rifampicin MICs. The amplicons were sequenced and screened for mutations. RESULTS: Susceptibility testing revealed a rather uniform distribution of MICs for most of the antimicrobial agents tested. The lowest MICs were seen for clarithromycin, rifampicin and imipenem. Six isolates (3%) exhibited distinctly higher MICs of rifampicin than the remaining 194 isolates. In five of these six isolates, single bp exchanges, which resulted in the amino acid exchanges Gln513Leu, Asp516Val, His526Asp or Ser531Leu, were detected in the rifampicin resistance-determining region 1 of the rpoB gene, with Gln513Leu representing a novel substitution for R. equi. CONCLUSIONS: This study shows the MIC distribution of 32 antimicrobial agents for a large collection of R. equi isolates of animal origin from two continents. Isolates that exhibited distinctly elevated MICs of rifampicin were only rarely detected.

Susceptibility testing of Rhodococcus equi: An interlaboratory test.

Due to the importance of antimicrobial susceptibility testing (AST) for veterinary diagnostics, a standardised protocol for AST of Rhodococcus equi by broth microdilution has recently been developed and approved by the Clinical and Laboratory Standards Institute (CLSI). The aim of the present study was to test this protocol in an interlaboratory comparative study for its fitness for use in routine laboratory diagnostics. All of the 18 participating laboratories determined the minimum inhibitory concentrations (MIC) of two R. equi strains against 24 antimicrobial agents. The modal MIC values were determined and the acceptable ranges were set as the modal MIC ±1 dilution step. The R. equi field strain Rh110 showed a slightly better performance than the type strain R. equi ATCC® 25729. For the different antimicrobial agents tested, the percentage of MIC values within the acceptable ranges varied from 75.9 to 100% for R. equi ATCC® 25729, and from 85.2 to 100% for R. equi Rh110. The most homogeneous MIC results (i.e. modal MIC ±1 dilution step) were obtained for oxacillin and vancomycin, while the most divergent results were seen with cefotaxime and ceftiofur. Using a success rate of at least 80% of the strain-specific MICs being within the acceptable ranges as an arbitrary cut-off, only one of the participating laboratories failed to reach this cut-off value for one of the two R. equi strains. Thus, we consider the new protocol fit for use in routine AST of R. equi.

[Antimicrobial resistance, ESBL and MRSA--definitions and laboratory diagnostics]. / Antimikrobielle Resistenz, ESBL und MRSA--Definitionen und Labordiagnose.

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms "antimicrobial resistance", "ESBL", and "MRSA" are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria--clinical breakpoints and epidemiological cut-off values--including their applications are explained in detail. Furthermore, currently valid diagnostic procedures--as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI)--for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and--as a consequence--for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions.