Evaluation of the susceptibility test of polymyxin B using the commercial test Policimbac®

Abstract

Broth microdilution (BMD), the reference method to determine bacterial susceptibility to polymyxins, is a laborious and time-consuming technique. Policimbac® is a commercial test panel which uses lyophilized polymyxin B to determine the minimum inhibitory concentration for Gram-negative isolates. This study evaluated the performance of Policimbac® in comparison with BMD for 110 isolates. Although the Policimbac® presented a very low essential agreement, the categorical agreement with BMD was optimal. Policimbac® is an alternative approach to BMD for evaluating the susceptibility to polymyxin B.

Carbapenems are considered the treatment of choice for serious infections caused by Gram-negative bacteria. However, the increasing use of this class of antibiotics worldwide has led to resistance to carbapenems. The polymyxins (polymyxin B and colistin) are considered last resorts for the treatment of infections caused by carbapenem-resistant bacteria [1–4].

Polymyxin resistance is an emerging problem, primarily due to chromosomal mutations that modify the lipopolysaccharide (LPS) molecules which form the external layer of the bacterial outer membrane [5]. Moreover, in November 2015, the first mobile polymyxin resistance mechanism was described and shown to be mediated by the mcr-1 (mobile colistin resistance) gene located in a plasmid [6, 7]. The mcr-1 gene has been described in several countries, including Brazil [8, 9].

The reference method commonly used to determine bacterial susceptibility to polymyxins is broth microdilution (BMD); BMD is a laborious and time-consuming procedure that requires material not often found in the routine microbiology laboratory. For this reason, commercial products have been developed to evaluate polymyxin susceptibility [10].

The Policimbac® (Probac do Brasil) is a commercial test developed to determine the minimum inhibitory concentration (MIC) of Gram-negative isolates in a panel with lyophilized polymyxin B antibiotic. This study was designed to evaluate the performance of Policimbac® in comparison with the reference method (BMD).

We tested a total of 110 Gram-negative isolates (87 Enterobacterales, 17 Acinetobacter spp., and 6 Pseudomonas aeruginosa) obtained from surveillance studies in southern Brazil between 2013 and 2016, including isolates from both animal and human origin [11, 12]. The susceptibility of the isolates to polymyxins was established by BMD reference method in a cation-adjusted Mueller-Hinton broth (MHB-CA-BD™) per the ISO 20776 at the time of the study.

The Policimbac® test consists of a plastic panel of twelve wells. Wells 1–10 contain dehydrated MHB-CA with decreasing concentrations of the lyophilized antibiotic polymyxin B (64 to 0.125 μg/mL). Well 11 was used as the positive control, containing only bacterial inoculum and dehydrated MHB-CA, and Well 12 served as the negative control containing only MHB-CA [13].

Three glass tubes were used to prepare each isolate for testing. Tube 1 contained a bacterial suspension with turbidity equivalent to 0.5 MacFarland (~ 10⁸ CFU/mL) obtained from growth on Mueller-Hinton agar plates after an overnight incubation. Tube 2 was prepared using a 100-fold dilution from tube 1 (~ 10⁶ CFU/mL). Tube 3 was prepared using a 10-fold dilution of tube 2, resulting in a final concentration of bacteria of 10⁵ CFU/mL [13].

A volume of 100 μL of the solution from tube 3 was added to wells 1–11 of the Policimbac® test panel, which was incubated at 35 ± 2 °C for 24 h in ambient air. After incubation, one drop of the revealing solution was added to each well. Bacterial growth was confirmed when the revealing solution became red after 20 min of incubation (1 h for Acinetobacter sp.) at 35 ± 2 °C. The MIC was defined as the lowest antibiotic concentration that inhibited visible growth of an isolate.

All tests were performed in duplicate and the results were interpreted according to the breakpoints of the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST), where isolates with polymyxin MICs ≤ 2 μg/mL were considered susceptible and those with MICs > 2 μg/mL were considered resistant [14].

A total of 110 isolates were evaluated. Fifty-one isolates were polymyxin-resistant (MIC by BMD and Policimbac from 4 to > 64 μg/mL) and 59 were polymyxin-susceptible (MICs by BMD and Policimbac ≤ 0.125 to 2 μg/mL). The resistant isolates included nine isolates with intrinsic resistance to polymyxins (6 S. marcescens, 2 M. morganii, and 1 P. rettgeri), 40 isolates belonging to Enterobacterales (32 Klebsiella sp., 7 Enterobacter sp., and 1 E. coli), and two non-fermenters (1 Acinetobacter sp. and 1 P. aeruginosa). Among the susceptible isolates, 38 belonged to Enterobacterales (17 Klebsiella sp., 10 Enterobacter sp., 8 E. coli, 3 C. freundii), 16 isolates were Acinetobacter sp., and five were P. aeruginosa.

There was poor essential agreement between the Policimbac® and BMD, as only 18 out of 110 isolates (16.4%) presented identical MIC with both methods (Table 1). In fact, most isolates with different MIC values between the tests presented higher MICs using the Policimbac® test compared with BMD. However, all isolates presented the same categorical classification with both Policimbac® and BMD (100% categorical agreement) (Table 1).

Table 1.

Correlation of Polymyxin B MICs between the BMD reference method and the Policimbac®

^aNumber of isolates with identical MIC values according to the Policimbac® and the BMD (highlighted in gray)

^bNumber of isolates with different MIC values according to the Policimbac® and the BMD (not highlighted)

The Policimbac® test is considered less laborious than BMD reference method, as there is no need to obtain the antibiotic salt used to prepare the solution for further dilution and distribution into a microtiter plate, the standard protocol for BMD. In addition, observation of the results is simplified by the use of a revealing solution, facilitating the visualization of bacterial growth with a prominent red color.

The Policimbac® presented a very low essential agreement (16.4%) in comparison with BMD. Even with a 1 log difference of MIC as an inherent error of the techniques, the essential agreement between Policimbac® and BMD occurred in only 50 isolates (45.5%). There was also a trend for the Policimbac® to present higher MIC values in comparison with BMD. The higher MIC of the Policimbac® might be due to the fact that the re-suspension of the lyophilized polymyxin B is not totally achieved with the addition of the solution with bacteria in the wells. This would lead to a well with less polymyxin B than expected and consequently to an increased MIC. It should be noted that this poor essential agreement did not affect the categorical agreement, as all 110 isolates presented the same categorical classification (susceptible or resistant) according to both Policimbac® and BMD.

Policimbac® proved to be a useful commercial test to classify Gram-negative isolates as susceptible or resistant to polymyxin B in comparison with BMD reference method. This commercial test is less laborious and does not require any special reagents, making it suitable for clinical microbiology laboratories with fewer resources. However, it is important to emphasize that the Policimbac® assay provides reliable results only for categorical classification of polymyxin resistance and the MICs obtained with this test were demonstrated to be higher than those obtained with BMD.

Funding information

This work was supported in part by Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) (project no. 16-0559).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.