Journal of Antimicrobial Chemotherapy Advance Access published April 19, 2013
J Antimicrob Chemother doi:10.1093/jac/dkt134
Harmonization of antimicrobial susceptibility testing by broth microdilution for Rhodococcus equi of animal origin
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Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany; 2Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Food and Veterinary Institute Oldenburg, Oldenburg, Germany; 3Department of Biometry, Epidemiology and Information Processing, WHO Collaborating Centre for Research and Training in Veterinary Public Health, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany *Corresponding author. Tel: +49–5034-871-241; Fax: +49-5034-871143; E-mail:
[email protected]
Keywords: test medium, incubation time, lysed horse blood
supplement Sir, Rhodococcus equi is an important veterinary pathogen. It mainly affects foals, where it often causes fatal pneumonia.1 R. equi can also cause a wide range of diseases in other animals, such as pigs, ruminants, cats and dogs, as well as in immunocompromised humans.1 Antimicrobial agents are commonly used to treat R. equi infections in both humans and animals.1 Prudent use guidelines in veterinary medicine require that the antimicrobial susceptibility of a causative bacterial pathogen shall be determined by adequate diagnostic procedures prior to starting antimicrobial therapy. The only information on how to perform antimicrobial susceptibility testing of R. equi by broth microdilution is available in document M24-A2 (2011) from the CLSI.2 Although this document is not veterinary specific, it provides information on the inoculum density (1 –5×105 cfu/mL), the incubation temperature (35+28C) and the medium that shall be used [cation-adjusted Mueller – Hinton broth (CAMHB)]. The usual incubation time is indicated as 24 –48 h, which, however, can be extended to up to 5 days if necessary.2 Analysis of published studies and doctoral theses on susceptibility testing of mostly equine R. equi showed that the test conditions differed in some of the CLSI-approved test parameters (Table S1, available as Supplementary data at JAC Online).3 – 6 The aim of the present study was to compare the most frequently used test conditions in order to provide a recommendation for a harmonized protocol for susceptibility testing of R. equi of animal origin.
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Anne Riesenberg1,2, Andrea T. Feßler1, Cornelia Fro¨mke3, Kristina Kadlec1, Dieter Klarmann2, Lothar Kreienbrock3, Christiane Werckenthin2 and Stefan Schwarz1*
For this comparison, the R. equi reference strain ATCC 25729 and six epidemiologically unrelated R. equi field isolates of equine (n¼ 4), feline (n¼ 1) and bovine (n¼1) origin were used. These isolates were confirmed as R. equi by the R. equi-specific choE-PCR.7 Two different media, CAMHB (Oxoid, Wesel, Germany) and CAMHB supplemented with 2% (v/v) lysed horse blood (LHB) (Oxoid), were used and the results were read visually after 24 and 48 h. The inoculum was prepared by the direct colony suspension method according to CLSI document M07-A9 (2012),8 resulting in the recommended inoculum of 1– 5×105 cfu/mL as confirmed by plate counts of diluted aliquots. In addition, an aliquot of the final inoculum was streaked on a blood agar plate for purity confirmation. The susceptibility testing was performed by broth microdilution using custom-made microtitre plates (MCS Diagnostics, Swalmen, The Netherlands), which were inoculated according to the manufacturer’s instructions with 50 mL per well. Twenty-nine antimicrobial agents were tested (Table S2, available as Supplementary data at JAC Online), including members of all antimicrobial classes recommended in document M24-A2 for primary and secondary susceptibility testing of R. equi—except linezolid and rifampicin, both of which are currently not included in test panels for veterinary pathogens.2 Incubation of the microtitre plates was performed at 35+28C in ambient air. The susceptibility testing was conducted six times on independent occasions for each isolate. To prove which medium –incubation time combination yielded the most stable susceptibility testing results, the MIC data consisting of the six measurements per isolate were dichotomized to 0 (MIC values were heterogeneous) or 1 (MIC values were homogeneous). The odds of homogeneous measurements were modelled with a multiple logistic regression analysis,9 including the event of equal MIC measurements depending on the medium–incubation time combination, the antibiotic agent and the bacterial isolate. Interactions (e.g. differences between medium– incubation time depending on the antibiotic agent) were omitted to generalize the usability. In addition to the global test, pairwise comparisons of the medium– incubation time combination with CAMHB after 24 h as the reference were computed. The type I error was set to 5% (two-sided). SAS software version 9.3 (SAS Institute, Cary, NC, USA) was used for all analyses.10 For each medium– incubation time combination, dichotomized information of varying MIC data was collected for 29 antimicrobial agents and 7 R. equi isolates resulting in 203 measurements per medium –incubation time combination. For CAMHB– 24 h, 68 of the 203 MIC measurements (33.50%) yielded the same MIC for a specific antimicrobial agent and R. equi isolate in all six tests. For CAMHB–48 h, CAMHB+ LHB–24 h and CAMHB+ LHB– 48 h, 71 (34.98%), 84 (41.38%) and 54 (26.60%) measurements, respectively, resulted in the same MICs. Multiple logistic regression revealed differences in the medium– incubation time combinations (P ¼0.0074) and the antimicrobial agents tested (P,0.0001), whereas differences among the R. equi isolates could not be shown (P¼ 0.1392), although they were epidemiologically unrelated and, in part, of
Research letter
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Funding
Odds (homogeneous MIC)
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This study was financially supported by the BMELV through the DART project (grant number FKZ 2811HS020).
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None to declare.
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Supplementary data
0.1 CAMHB 24 h
CAMHB 48 h
CAMHB + 2% LHB 24 h
CAMHB + 2% LHB 48 h
Figure 1. Odds and corresponding 95% CIs to achieve homogeneous MIC measurements depending on medium– incubation time. The odds and the intervals were computed with multiple logistic regression and are stratified for antibiotic agents and bacterial isolates.
different animal origin. The odds of achieving homogeneous MIC measurements depending on medium–incubation time were the highest for CAMHB+LHB –24 h (0.648) followed by CAMHB –48 h (0.463), CAMHB–24 h (0.427) and CAMHB+ LHB–48 h (0.286) (Figure 1). Pairwise comparisons of odds with CAMHB–24 h as the reference method showed higher homogeneity of the results when using CAMHB+ LHB–24 h (OR¼1.517, 95% CI¼0.968–2.377, P¼0.0689), higher heterogeneity when using CAMHB+LHB–48 h (OR¼0.671, 95% CI¼0.419–1.073, P¼0.0958) and no difference when using CAMHB–48 h (OR¼1.084, 95% CI¼0.688–1.707, P¼0.7282). Based on these results, we suggest testing R. equi of animal origin using the standard inoculum size and incubation temperature, but adding 2% (v/v) LHB to the CAMHB test medium and reading the results after 24 h. The addition of LHB also facilitated the reading (e.g. the decision for growth versus no growth). Compared with a 48 h incubation period, reading the tests after 24 h also saves time and space in the incubators, which are important aspects in routine veterinary diagnostics.
Acknowledgements We wish to thank Vivian Hensel and Regina Ronge for excellent technical assistance.
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Tables S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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