Abstract
Objectives:
Owing to the emergence of colistin resistance in nonfermenting Gram negative bacteria, reliable and rapid techniques for testing colistin susceptibility are needed. We evaluated the performances of the Rapid Polymyxin Acinetobacter and Pseudomonas tests using a collection of Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates.
Methods:
Colistin susceptibility of A. baumannii and P. aeruginosa isolates (colistin susceptible and colistin resistant) was tested with the Rapid Polymyxin Acinetobacter and Pseudomonas tests and compared with the broth microdilution method.
Results:
The Rapid Polymyxin Acinetobacter and Pseudomonas tests were able to detect all colistin-resistant and all colistin-susceptible A. baumannii and P. aeruginosa isolates within 4 hours.
Conclusion:
The Rapid Polymyxin Acinetobacter and Pseudomonas tests are reliable techniques for detecting colistin resistance. Overall, both techniques allow an accurate and a rapid screening (<4 hours) of colistin resistance in A. baumannii and P. aeruginosa.
Introduction
A
The current standard method of detection for colistin susceptibility in Gram negatives is the manual determination of minimum inhibitory concentration (MIC) by the broth microdilution (BMD) method. 8 However, this procedure is technician dependent (partly due to the fact that colistin must be weighted for each experiment), is time consuming, and results are obtained in 24 hours.
Recently, Nordmann et al. developed the Rapid Polymyxin Nordmann-Poirel (NP) test that categorizes colistin-susceptible from colistin-resistant enterobacterial isolates in <2 hours. 9 However, this test based on visualization of glucose metabolization cannot be applied to nonfermenting Gram negative bacteria, such as A. baumannii and P. aeruginosa.
Using the same principle as the Rapid Polymyxin NP test, the Rapid Polymyxin Acinetobacter and Pseudomonas tests have recently been developed by Elitech Microbiology and are liquid-based techniques that rely on the colorimetric detection of a rapid metabolism related to bacterial growth, in the presence of a defined concentration of colistin. The acidification of the medium related to bacterial growth is visualized by the color shift of the pH indicator (red to yellow or orange with the red phenol in the Rapid Polymyxin Acinetobacter test and green to violet with bromocresol purple in the Rapid Polymyxin Pseudomonas test).
The objective of this study was to evaluate the performance of those novel tests by comparison with the BMD method using a collection of colistin-susceptible and colistin-resistant A. baumannii and P. aeruginosa clinical isolates.
Materials and Methods
Bacterial strains
This study was carried out using 38 clinical isolates of A. baumannii (n = 21) and P. aeruginosa (n = 17) identified at the species level using the Microflex benchtop matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (Brücker, Champs-sur-Marne, France). Nine out of the 21 A. baumannii isolates and 10 out of the 17 P. aeruginosa isolates were susceptible to colistin. Twelve out of the 21 A. baumannii and 7 out of the 17 P. aeruginosa isolates were colistin resistant according to BMD testing. Isolates were grown on Luria Bertani (LB; Sigma, Saint Louis, MO) agar plates at 35°C ± 2°C for 18 hours. The colistin-susceptible strain P. aeruginosa ATCC 27853 and the colistin-resistant Escherichia coli R2739 were used as negative and positive controls, respectively, for the determination of MIC of colistin by the BMD method. None of the colistin-resistant isolates carried a plasmid-encoded MCR-like (MCR-1 to -4) colistin resistance determinant, as assessed by the negative polymerase chain reaction results (data not shown).
Susceptibility testing
The BMD method was performed in triplicate and interpreted according to the EUCAST/CLSI joined guidelines 8 as described. 10
Rapid Polymyxin Acinetobacter and Pseudomonas tests
The Rapid Polymyxin Acinetobacter and Pseudomonas tests from Elitech Microbiology were performed according the manufacturer instructions. In brief, a standardized suspension of each isolate is prepared using a medium specific to each species. A specific volume of this suspension is then placed in the different wells of the kit containing defined amounts of colistin to obtain final concentrations of 0 (positive control), 2, and 4 mg/L for both species, and in addition of 8 mg/L for P. aeruginosa. A supplementary well is used as a negative control in which a suspension with only NaCl is prepared. After 3–4 hours of incubation at 37°C, a valid result is obtained when a color shift is observed in the positive control well, and no color shift in the negative control well. The result is then read for each colistin containing well as an MIC reading. For this evaluation, the results of susceptibility/resistance to polymyxins for each isolate were observed after 2 hours every 15 minutes until 4 hours.
Result analysis
The results obtained with the Rapid Polymyxin tests were compared with those obtained with the reference BMD method. In brief, discrepancies were determined to assess their performance to detect colistin susceptibility. Very major errors (VME) and major errors (ME) corresponding to false-susceptible and false-resistant results, respectively, were calculated as described elsewhere.11,12
Results
A total of 21 A. baumannii and 17 P. aeruginosa isolates were included to evaluate the performances of the Rapid Polymyxin Acinetobacter and Pseudomonas tests (Table 1). All of the nine A. baumannii isolates defined as colistin susceptible according to the result of the BMD method (MICs of colistin ranging from <0.125 to 0.25 mg/L) were identified as susceptible by the Rapid Polymyxin Acinetobacter test (Table 1). All of the 12 colistin-resistant A. baumannii isolates (MICs of colistin ranging from 4 to >128 mg/L) were detected as colistin resistant by the Rapid Polymyxin Acinetobacter test (Table 1). Out of the 10 colistin-susceptible P. aeruginosa isolates (MICs of colistin ranging from <0.125 to 0.25 mg/L), all were found susceptible using the Rapid Polymyxin Pseudomonas test. All of the seven colistin-resistant P. aeruginosa isolates (MICs of colistin ranging from 4 to 128 mg/L) were identified as colistin resistant by the Rapid Polymyxin Pseudomonas test (Table 1). Interpretation of the results for all isolates was obtained between 3 and 4 hours; no positive result was observed before 3 hours.
Minimum Inhibitory Concentrations of Colistin (mg/L) Using the Broth Microdilution Method and Results of the Rapid Polymyxin Acinetobacter and Pseudomonas Tests
The colistin-resistant isolates are shaded in gray.
BMD, broth microdilution; MIC, minimum inhibitory concentration; R, resistant; S, susceptible.
Discussion
Out of the 19 colistin-susceptible A. baumannii and P. aeruginosa isolates, the Rapid Polymyxin Acinetobacter test identified correctly all susceptible isolates, hence no ME (i.e., false resistance) was detected for both tests (specificities of 100%). Out of the 19 colistin-resistant A. baumannii and P. aeruginosa isolates, the Rapid Polymyxin Acinetobacter and Pseudomonas tests were excellent with no VME (i.e., false susceptibility; sensitivities of 100%).
This study showed that the Rapid Polymyxin Acinetobacter and Pseudomonas tests are reliable tools for detecting resistance to colistin in A. baumannii and P. aeruginosa. In comparison, BMD systems (Sensititre [ThermoFischer Diagnostics] and UMIC [Biocentric and MicroScan]) have been evaluated and showed VME for the three systems and ME for the MicroScan system. 13 Moreover, these are the first tests that are available for determination of colistin susceptibility in those species in <4 hours. However, other evaluations with a higher number of isolates should be performed to confirm our results as well as at least a multicenter study and its possible interest directly from clinical samples. Although MICs of colistin are only determined in ranges (≤ 2, comprised between 2 and 4 mg/L, and >4 mg/L for A. baumannii and ≤ 2, comprised between 4 and 8 mg/L, and >8 mg/L for P. aeruginosa) using those tests, they give results of susceptibility/resistance categorization very rapidly, which is the most important feature with respect to the treatment strategy and may contribute to optimize antibiotic stewardship. Colistin is indeed often used to treat infections caused by MDR A. baumannii and P. aeruginosa isolates, mostly remaining susceptible to colistin. 14 However, the increasing trend of acquired colistin resistance observed in those species, and particularly in A. baumannii, 15 highlights the interest to use such rapid tests able to efficiently detect that resistance trait.
Footnotes
Acknowledgments
We thank the Elitech Microbiology company for providing us kits of Rapid Polymyxin Acinetobacter and Pseudomonas tests. We thank Javier Fernandez, Véronique Dubois, and Deniz Güneşer for providing several resistant strains. This work has been funded by the University of Fribourg and by the Swiss National Reference Center for Emerging Antibiotic Resistance, Fribourg, Switzerland. It has also been funded by the Swiss National Science Foundation (projects FNS-407240_177381 and FNS-407240_177382).
Disclosure Statement
No competing financial interests exist.