Rapid Detection of Polymyxin Resistance in Enterobacteriaceae

Patrice Nordmann; Aurélie Jayol; Laurent Poirel

Disclosures

Emerging Infectious Diseases. 2016;22(6):1038-1043. 

In This Article

Results

Of the 200 enterobacterial isolates tested to evaluate the performance of the rapid polymyxin NP test (online Technical Appendix), 5 isolates belonged to bacterial species that are intrinsically resistant to colistin (M. morganii, P. mirabilis, P. vulgaris, P. stuartii, and S. marcescens) and 130 isolates of various species displayed an acquired mechanism of resistance to colistin. For 87 Klebsiella spp. isolates, resistance to colistin was associated with various chromosomal gene changes responsible for lipopolysaccharide modifications (online Technical Appendix): 10 isolates had mutations in the PmrAB two-component system (n = 3 in pmrA gene, n = 7 in pmrB gene); 2 isolates had structural modifications in the PhoPQ two-component system (n = 1 in phoP gene, n = 1 in phoQ gene); and 75 isolates had various alterations in mgrB gene, the negative regulator of the PhoPQ system (online Technical Appendix). Seven nonduplicate E. coli isolates harbored a plasmid-mediated mcr-1 gene. Pulsed-field gel electrophoresis revealed that isolates with identical mechanisms of colistin resistance (chromosomal or plasmid-encoded) were not clonally related (data not shown). The mechanism(s) of colistin resistance remained unknown for the 43 remaining enterobacterial isolates (online Technical Appendix). With regard to performance of the rapid polymyxin NP test with colistin-susceptible strains, the 65 colistin-susceptible isolates tested (MICs of colistin 0.12–2 μg/mL) gave negative results, except for 3 isolates (isolates FR-180, 181, and 182) for which colistin MICs were 1–2 μg/mL (just below the EUCAST breakpoint) and which gave a positive (false-positive) result (online Technical Appendix).

As expected, isolates that were intrinsically resistant to colistin (n = 5), such as Proteus spp., P. stuartii and S. marcescens, gave a positive test result (online Technical Appendix). Colistin-resistant enterobacterial isolates (n = 130, MICs of colistin ranging from 4 to >128 μg/mL) also gave positive results, except for 1 colistin-resistant E. coli isolate (isolate FR-119) for which colistin MIC was 8 μg/mL and which gave a negative (false-negative) result (online Technical Appendix).

Correlation was high between colistin resistance and positive rapid polymyxin NP test results and, conversely, colistin susceptibility and negative test results (online Technical Appendix). Sensitivity (99.3%) and specificity (95.4%) of the test were also high, compared with the standard broth microdilution method.

By reading the color change of the wells every hour, we determined that final results were obtained 2 h after incubation when the tray was incubated at 35 ± 2°C under an ambient atmosphere. However, positive results (frank color change) were obtained as early as 1 h after incubation for Klebsiella spp. and E. coli isolates. Half of the Enterobacter spp. isolates gave positive results within 1 h of incubation and the other half within 2 h. In addition, all resistant isolates gave positive results after 1 h of incubation when trays were incubated at 35 ± 2°C under 5% CO2. Agitating the tray did not improve the speed with which results were obtained.

The rapid polymyxin NP test results were the same whether performed with polymyxin B or with colistin (data not shown). Testing of several agar media revealed that 30% of the colistin-susceptible tested isolates gave false-positive results when bacterial colonies were recovered from acidifying media such as Drigalski, MacConkey, or bromocresol purple agar. Media that were adequate for culturing bacteria before performing the rapid polymyxin NP test were Luria Bertani agar, Mueller-Hinton agar, Columbia agar + 5% sheep blood, chocolate agar, UriSelect 4 agar, and eosin methylene blue agar.

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