Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria

Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated... Abstract Objectives Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. Methods Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time–kill study was performed on five strains carrying different colistin resistance mechanisms. Results Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time–kill study showed the combination was bacteriostatic on strains tested. Conclusions These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans. Introduction Efflux in bacteria is a ubiquitous mechanism to combat antibiotic therapy.1 Multidrug pumps are often non-specific, leading to cross-resistance with several antimicrobial compounds, and can interact synergistically with other resistance mechanisms in order to increase the resistance level. Colistin resistance mechanisms are complex and involve many genes that have not yet been fully identified.2 A well-known, identified mechanism involves reducing the negative charge of the outer membrane by adding positive charges such as sugars (phosphoethanolamine, aminoarabinose) to lipid A of the lipopolysaccharide, resulting in the decreased electrostatic attraction of polymyxins.3 Several mutations in genes such as the two-component systems pmrA/pmrB and phoP/phoQ, the negative regulator of PhoP/PhoQ, mgrB and the aminoarabinose biosynthesis operon arnBCADTEF have been involved in colistin resistance.3 Recently, a transferable colistin-resistance gene mcr-1, which codes for a phosphoethanolamine transferase, has been discovered and appears to have already spread worldwide.2 Colistin resistance in Gram-negative bacteria has emerged over the last few years with its increasing use in human medicine, but also its wide use in agriculture, especially in animal breeding.4,5 In some bacteria, efflux pump mechanisms have been reported to play a role in colistin resistance. Many efflux pumps have been identified as reducing colistin susceptibility, such as KpnEF and the AcrAB–TolC complex.2,3,6 These mechanisms often lead to resistance to other antimicrobial compounds such as aminoglycosides and fluoroquinolones. Various efflux pump inhibitors (EPIs) have been tested on Gram-negative bacteria such as CCCP, 2,4-dinitrophenol (DNP), PABN, reserpine, omeprazole and verapamil.1 Only CCCP and DNP, which inhibit the energy of efflux pumps, showed a significant decrease in colistin resistance.7 The effect of EPIs such as CCCP in the colistin resistance reversion of Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia7 supported the hypothesis that this mechanism plays a role in colistin resistance. More recently, colistin resistance reversion by CCCP has also been demonstrated in the naturally colistin-resistant species Serratia marcescens.8 In our laboratory, we isolated a collection of well-defined colistin-resistant strains including naturally resistant Gram-negative bacteria and acquired-resistance bacteria (mcr-1, mgrB, pmrAB, etc.). The mechanisms of resistance for some of these strains are, however, still unknown. The objective of our study was to evaluate the effect of CCCP on this group in order to evaluate whether the mechanism of resistance can influence the response to efflux pump inhibition and to evaluate its effect on mcr-1 plasmid-mediated colistin-resistant strains. Materials and methods Bacterial strains and colistin resistance mechanisms Strains used in this study are listed in Table 1. Ninety-two strains isolated from various human and pig samples were included in this study. Samples came from France, Laos, Thailand, Nigeria, Algeria and Saudi Arabia (Table 1). Some of these strains have already been published (Table 1). Mechanisms of resistance to colistin have been studied previously with the analysis of phoP, phoQ, pmrA, pmrB, mgrB and mcr-1 performed by real-time PCR, standard PCR and/or sequencing.9–11 Two reference strains of Escherichia coli, one susceptible to colistin (ATCC 25922) and one mcr-1-positive strain (NCTC 13846), were used as controls. The mcr-1-positive strain has been included in the statistical analysis. Table 1. Summary of strains used in this study. Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 ND, not detected; NG, no growth; del, deletion. a A. O. Olaitan and J. M. Rolain, unpublished data. b S. A. Baron, N. Cassir and J. M. Rolain, unpublished data. c S. A. Baron and J. M. Rolain, unpublished data. d Performed using the Etest method (mg/L). Table 1. Summary of strains used in this study. Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 ND, not detected; NG, no growth; del, deletion. a A. O. Olaitan and J. M. Rolain, unpublished data. b S. A. Baron, N. Cassir and J. M. Rolain, unpublished data. c S. A. Baron and J. M. Rolain, unpublished data. d Performed using the Etest method (mg/L). Effect of CCCP on colistin MIC using the broth microdilution method Exploration of the effect of the oxidative phosphorylation uncoupler CCCP on colistin MIC was performed as follows: MIC determination of colistin was performed according to EUCAST recommendations using the broth microdilution method. Colistin sulphate salt (MP-Biomedicals, LLC, Illkirch Graffenstaden, France) concentrations ranged from 0.25 to 256 mg/L. Two tests were performed in parallel: one without adding CCCP and one adding CCCP to each CAMHB well. A stock solution of CCCP was prepared at 5 mg/mL in DMSO. The final concentration of CCCP in the CAMHB was 10 mg/L with a DMSO concentration of 0.2%.7 A growth control well containing 10 mg/L CCCP in CAMHB was also added for each strain, in order to check the absence of effect of CCCP alone on these strains. Visualization of bacterial growth was done by adding iodonitrotetrazolium to wells. The resulting MIC fold changes after the addition of CCCP was calculated as the ratio of the CCCP-free antibiotic’s MIC level to that of the CCCP-added antibiotic. As previously described,8 the positive criterion for the presence of efflux pumps in isolates was an ≥8-fold decrease in colistin MIC after adding CCCP. The mean fold change was calculated by species and by colistin resistance mechanism identified as previously described:8 [1/total sample size(n)] × Σ(MIC fold change × frequency of fold change) where the ‘frequency of fold change’ is the number of times a particular MIC fold change was recorded for that species. The symbol of superiority for MIC fold changes was considered as an equal for the mean fold change analysis. Statistical analyses were performed using non-parametric one-way analysis of variance (ANOVA) (GraphPad Software Inc., La Jolla, CA, USA). A P value <0.05 was taken into account for the MIC fold change. The resulting MIC after adding CCCP was compared to the EUCAST cut-off (established at 2 mg/L) and the effect was considered reversed if the strain became susceptible to colistin again. Effect of CCCP on colistin MIC on Mueller–Hinton (MH) agar plates Colistin susceptibility testing was performed according to EUCAST recommendations using the Etest (bioMérieux, Marcy-l’Étoile, France) or the disc diffusion method on MH agar with commercial antibiotic discs (bioMérieux). Initially, Etests were performed on S. marcescens in order to better visualize the ‘cocarde’ effect,12 which corresponds to an absence of inhibition next to the disc surrounded by a ring of inhibition close to the edge of the bacterial growth unaffected by the antibiotic. The withdrawal from the market of Etests forced us to use the disc method for the other species (http://ansm.sante.fr/var/ansm_site/storage/original/application/7e5c29808ae0bc4d37761e98cc8eeb06.pdf). Colistin MIC was determined on two types of media: MH agar with DMSO, and MH agar with 10 mg/L CCCP (Sigma–Aldrich, Saint-Quentin-Fallavier, France) dissolved in DMSO. When a strain was totally inhibited by colistin + CCCP, a plate containing 10 mg/L CCCP only was used to confirm the absence of effect of the EPI alone. The significance of the change in diameter of colistin inhibition by the disc diffusion method with and without CCCP was evaluated by a Student’s t-test in a matched series. Results were considered as statistically significant when they had a P value <0.05. S. marcescens results were considered separately as the MIC was determined on an agar plate for this species and a fold change was calculated as described above. Time–kill study A time–kill study was performed on one strain of each colistin resistance mechanism group, namely, K. pneumoniae FHM169 (mgrB stop), E. coli 44A (mcr-1 positive), Proteus vulgaris P97 (intrinsically resistant to colistin), Enterobacter asburiae LH74 and K. pneumoniae KP4CR (unknown resistance). A fresh culture of bacteria was inoculated in the three following conditions: CAMHB + DMSO, MH broth + colistin (2 mg/L) and CAMHB + colistin (2 mg/L) + CCCP (10 mg/L) and incubated for 24 h at 37°C with shaking. At times 0 h, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h, several dilutions of each culture were spread on Trypticase soya agar and incubated for 24 h before colony counting. RNA expression In order to evaluate the impact of CCCP on mcr-1 gene transcription, we quantified mcr-1 RNA in two mcr-1-positive E. coli strains, 44A and P10. We inoculated fresh colonies into three different LB broth cultures, containing DMSO, 2 mg/L colistin + DMSO or 2 mg/L colistin + 10 mg/L CCCP, that were incubated at 37°C with shaking for 4 h. One millilitre of this culture, calibrated to an OD of 0.19 (corresponding to ∼1 × 108 cfu/mL), was extracted using the TRIzol® MaxTM Bacterial RNA isolation Kit (Thermo Fisher, Waltham, MA, USA). Briefly, after centrifugation, the pellet was resuspended with 700 μL of TRIzol and 50 μL of 4-bromoanisole (BAN). After 15 min of centrifugation at 4°C at 12 000 g, the aqueous phase containing the RNA was suspended in a new Eppendorf tube and precipitated using 500 μL of isopropanol. The RNA was then washed with 75% ethanol and suspended in 30 μL of water after centrifugation. This RNA was purified from the remaining DNA using the DNA-freeTM kit procedure (Thermo Fisher) following the recommendations of the manufacturer. Finally, 20 μg of the RNA was used to quantify gene expression using the SuperScriptTM III Platinum One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations. Each quantification was performed in duplicate. The ΔΔCt method was used to quantify gene expression. The dxS housekeeping gene was used as calibrator using the following primers: F-GCTTCACAATGCCTTTGCCA, R-TATAACGATGGCCCGTCAGC and the following probe: 6 FAM-CGTCAGTTCCACGCCGACCG whereas the mcr-1 primers and probe were those previously described.11 Results We tested the effect of CCCP on 91 Enterobacteriaceae including 32 K. pneumoniae, 26 E. coli, 6 Enterobacter spp., 2 Salmonella enterica, 1 Klebsiella oxytoca, 14 S. marcescens, 3 Morganella morganii, 3 Proteus spp., 4 Providencia spp. and 3 P. aeruginosa. All strains, except for the colistin-susceptible reference strain ATCC 25922, were found to be resistant to colistin, with an MIC between 4 and >256 mg/L (Table 1). Mechanisms of resistance to colistin had already been identified in some of the strains studied (Table 1). All isolates used in this study grew in the presence of CCCP at the concentration of 10 mg/L in DMSO without any colistin, in broth medium and on agar plates, confirming that this substance alone has no effect at this concentration on these Gram-negative bacteria (Figure 1).7 Figure 1. View largeDownload slide Agar method [left, colistin inhibition diameter on an MH + DMSO plate (photograph not to scale) and on an MH + 10 mg/L CCCP plate (photograph not to scale); right, colistin MIC determined using the Etest method with and without 10 mg/L CCCP]. Liquid method (colistin MIC determined using the microdilution method with and without 10 mg/L CCCP). NC, negative control. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1. View largeDownload slide Agar method [left, colistin inhibition diameter on an MH + DMSO plate (photograph not to scale) and on an MH + 10 mg/L CCCP plate (photograph not to scale); right, colistin MIC determined using the Etest method with and without 10 mg/L CCCP]. Liquid method (colistin MIC determined using the microdilution method with and without 10 mg/L CCCP). NC, negative control. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Of the 93 strains tested, 90 strains had an MIC fold change ≥8, confirming the presence of the efflux pump mechanism of resistance. Only two strains of K. pneumoniae showed an MIC fold change <8, but their colistin MICs were low (2 and 4 mg/L, respectively) and a reversal effect was still observed after adding colistin (0.5 and 1 mg/L, respectively). Likewise, the addition of CCCP led to reversed colistin resistance (MIC <2 mg/L) in all the strains studied. The mean fold change determined by species varied from 24 for S. enterica to 1024 for colistin-heteroresistant Enterobacter (Table 2). We noticed that the effect of CCCP on intrinsically resistant bacteria was much greater than on other Enterobacteriaceae (P < 0.0001). Table 2. Summary of mean fold change in colistin MIC after adding CCCP, by species and by colistin resistance mechanism Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Table 2. Summary of mean fold change in colistin MIC after adding CCCP, by species and by colistin resistance mechanism Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 We then decided to compare the impact of CCCP according to the mechanism of resistance identified. Bacteria were separated between ‘intrinsic’ or ‘natural’ resistance, ‘heteroresistance’, which describes a phenomenon where subpopulations of seemingly isogenic bacteria exhibit a range of susceptibilities to a particular antibiotic,13 ‘mcr-1 plasmid-mediated’ resistance, ‘other’ resistance when the resistance was caused by a known chromosomic mutation and ‘unknown’ when the resistance mechanism was still unknown. By comparing the resistance mechanism (Table 3), we observed that the efflux mechanism was significantly greater in bacteria naturally resistant to colistin and in bacteria with a heteroresistance mechanism than in bacteria with other resistance mechanisms (P < 0.0001) (Figure 2a). On the other hand, there were no significant differences between ‘intrinsic’ and ‘heteroresistance’ groups or between ‘mcr-1’, ‘other mutation’ or ‘unknown’ groups (Figure 2a). Table 3. Summary of mean fold change in colistin MIC after adding CCCP, by colistin resistance mechanism in Enterobacteriaceae Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 a Excluding S. marcescens. Table 3. Summary of mean fold change in colistin MIC after adding CCCP, by colistin resistance mechanism in Enterobacteriaceae Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 a Excluding S. marcescens. Figure 2. View largeDownload slide (a) Mean fold change of colistin MIC by species and mechanism of resistance. (b) Colistin inhibition diameter on MH agar plate without and with 10 mg/L CCCP. EIRC, Enterobacteriaceae intrinsically resistant to colistin. Figure 2. View largeDownload slide (a) Mean fold change of colistin MIC by species and mechanism of resistance. (b) Colistin inhibition diameter on MH agar plate without and with 10 mg/L CCCP. EIRC, Enterobacteriaceae intrinsically resistant to colistin. The effect of CCCP on agar plates showed significant CCCP activity on colistin resistance. The diameter of inhibition of colistin (mm) increased from 14.1 ± 4.4 mm without CCCP to 23.5 ± 5.3 mm with CCCP (P < 0.0001) (Figure 2b). For S. marcescens strains, the fold change on agar plates with Etest MIC determination varied from 1347-fold to 4000-fold (P < 0.0001). Strains were all resistant to colistin with MIC >256 mg/L before adding CCCP, whereas colistin MIC values after adding CCCP varied from 0.064 to 0.19 mg/L (mean 0.128 mg/L). However, no correlation between the disc diffusion assay and the microdilution method was observed. This can be explained by the fact that the disc diffusion method is not a relevant method to determine colistin susceptibility as the colistin does not diffuse correctly in an agar plate.14 Analyses of the time–kill study showed that the association of colistin + CCCP was bacteriostatic on the five strains tested (Figure 3). No difference was observed between the strains with different resistance mechanisms. This is concordant with a previous time–kill study done on K. pneumoniae7 that showed an inhibiting effect on bacterial growth, but no killing effect. Figure 3. View largeDownload slide Time–kill study of colistin (2 mg/L) + CCCP (10 mg/L) on five colistin-resistant strains with different mechanisms of resistance to colistin. Figure 3. View largeDownload slide Time–kill study of colistin (2 mg/L) + CCCP (10 mg/L) on five colistin-resistant strains with different mechanisms of resistance to colistin. Finally, the effect of the association of colistin + CCCP on mcr-1 gene expression was performed on two mcr-1-positive E. coli. The addition of 2 mg/L colistin led to a decrease of 1.65-fold for strain 44A and to an increase of 0.84-fold for strain P10 (Figure 4). However, the combination of 2 mg/L colistin + 10 mg/L CCCP decreased mcr-1 expression 40.5-fold and 26.9-fold, respectively, for strain 44A and strain P10. Figure 4. View largeDownload slide mcr-1 gene expression in E. coli 44A and P10 in the absence of antibiotic, after 2 mg/L colistin and after 2 mg/L colistin + 10 mg/L CCCP. Figure 4. View largeDownload slide mcr-1 gene expression in E. coli 44A and P10 in the absence of antibiotic, after 2 mg/L colistin and after 2 mg/L colistin + 10 mg/L CCCP. Discussion The role of efflux in colistin resistance is still unknown in enterobacteria. In our study, we showed a reversal effect on colistin resistance by CCCP on all the strains studied. This effect was more significant in ‘intrinsically resistant’ and ‘heteroresistant’ enterobacteria than in other Enterobacteriaceae, suggesting that the mechanism blocked by CCCP is critical for colistin resistance in these species. Although the main mechanism described for these species is the modification of lipid A by amino sugars, susceptibility to EPIs was significantly greater in this group than in the ‘other mutation’ and ‘mcr-1’ groups for which colistin resistance is also mediated by lipid A modification. Interestingly, the time–kill study showed no differences between the different strains carrying different colistin resistance mechanisms. The association of colistin + CCCP was bacteriostatic on all strains tested, supporting previous results observed on a colistin-resistant K. pneumoniae strain.7 Many mechanisms have been identified so far, with the most recent, mcr-1, carried by a transferable plasmid, mostly in E. coli and K. pneumoniae.2 The transcriptomic analysis of the mcr-1 gene in two mcr-1-positive E. coli isolates showed that the association of colistin and CCCP inhibits the transcription of the mcr-1 gene. This could likely be explained either by inhibition of efflux (CCCP increases the colistin concentration, which potentiates its effect and inhibits the transcription of the mcr-1 gene) or by an unknown action of CCCP. Efflux has been reported to play a role in colistin resistance, such as with AcrAB–TolC in E. coli15 and MexXY–OprM in P. aeruginosa.16 EPIs have been used to evaluate the effect of efflux pump up-regulation in resistance to colistin. Effects of EPIs on colistin resistance vary according to the type of EPI. For example, PABN and 1-(1-naphtylmethyl)-piperazine (NMP) are EPIs believed to act in competition with drugs in the pocket of the action site of AcrB17 and they failed to recover colistin susceptibility in colistin-resistant Gram-negative bacteria.7,18 This inefficiency has also been reported for other well-known EPIs such as verapamil, omeprazole and reserpine.7 On the other hand, CCCP and DNP, which are non-specific EPIs, showed good activity in restoring colistin susceptibility.7,8,18 Therefore, it is difficult to identify the efflux pump responsible for the resistance because they act on the energy source of efflux pumps, the proton motive force, and can modulate other protein activity in the membrane.1 Ni et al.7 have also suggested that the CCCP effect observed on colistin activity may be due to regeneration of negative charges on the cell membrane. In another study, Park and Ko18 hypothesized the decrease of ATP production caused by CCCP action could be responsible for increased colistin activity in these cells. Our study suggests that CCCP probably has an effect as an EPI. Moreover, the role of soxRS, a modulator of the efflux pump AcrAB-TolC, has recently been reported to be responsible for colistin resistance in Enterobacter spp.6 TolC is an outer membrane protein that can interact with many inner membrane proteins, especially membrane fusion proteins, in order to create a channel for various protein extrusions.19 Usually, bacteria use at least one efflux pump system in the inner membrane such as AcrAB in E. coli that, in association with TolC, enables antibiotic extrusion. Some studies showed that some drugs were dependent on TolC without being dependent on AcrAB, suggesting that TolC can be associated with other inner membrane proteins to evacuate drugs.19 But until now, none of the studied efflux pumps could prove this hypothesis. In our study, the action of CCCP was particularly important for the heteroresistant strains. In these strains, PABN, which is known to permeabilize the outer membrane of Gram-negative bacteria,20 showed an effect on colistin resistance in Enterobacter cloacae and in Enterobacter aerogenes, suggesting that AcrAB–TolC is up-regulated in these strains, but no effect was observed in S. marcescens strains (data not shown). Despite its resistance being similar to the heteroresistance of Enterobacter spp., some other genes seem to be involved in colistin resistance. In a study on S. marcescens, it has been shown that CCCP increases the accumulation of ciprofloxacin in a WT strain, but not in an sdeB-deficient mutant. The resistance–nodulation–division tripartite efflux pump SdeAB–HasF is homologous to AcrAB–TolC and seems to be the major efflux pump system in Serratia.21 This pump seems to extrude a wide variety of components, is dependent on proton motive force and may be a good candidate for a colistin-resistance target gene. Further studies are needed on efflux pump inhibition in colistin resistance to better understand the pathways of this complex mechanism, particularly by describing efflux pump genes and their expression in susceptible/resistant/heteroresistant mutant strains. Our study further suggests that several EPIs could be developed in the future to tackle the issue of emerging colistin resistance in Gram-negative bacteria as demonstrated in the past with β-lactamase inhibitors and resistance to β-lactams. Some studies are currently trying to develop associations of EPIs with other molecules22 to fight bacterial infections. Funding This work was supported by the French Government under the ‘Investissements d’avenir’ program managed by the Agence Nationale de la Recherche (reference: Méditerranée Infection 10-IAHU-03). Transparency declarations None to declare. References 1 Li X-Z , Plésiat P , Nikaido H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria . Clin Microbiol Rev 2015 ; 28 : 337 – 418 . Google Scholar CrossRef Search ADS PubMed 2 Baron S , Hadjadj L , Rolain J-M et al. Molecular mechanisms of polymyxin resistance: knowns and unknowns . Int J Antimicrob Agents 2016 ; 48 : 583 – 91 . Google Scholar CrossRef Search ADS PubMed 3 Olaitan AO , Morand S , Rolain J-M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria . Front Microbiol 2014 ; 5 : 643. Google Scholar CrossRef Search ADS PubMed 4 Kempf I , Jouy E , Chauvin C. Colistin use and colistin resistance in bacteria from animals . Int J Antimicrob Agents 2016 ; 48 : 598 – 606 . Google Scholar CrossRef Search ADS PubMed 5 Olaitan AO , Morand S , Rolain J-M. Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance . Int J Antimicrob Agents 2016 ; 47 : 1 – 3 . Google Scholar CrossRef Search ADS PubMed 6 Telke AA , Olaitan AO , Morand S et al. soxRS induces colistin hetero-resistance in Enterobacter asburiae and Enterobacter cloacae by regulating the acrAB-tolC efflux pump . J Antimicrob Chemother 2017 ; 72 : 2715 – 21 . Google Scholar CrossRef Search ADS PubMed 7 Ni W , Li Y , Guan J et al. Effects of efflux pump inhibitors on colistin resistance in multidrug resistant Gram-negative bacteria . Antimicrob Agents Chemother 2016 ; 60 : 3215 – 8 . Google Scholar CrossRef Search ADS PubMed 8 Osei SJ , Amoako DG. Carbonyl cyanide m-chlorophenylhydrazine (CCCP) reverses resistance to colistin, but not to carbapenems and tigecycline in multidrug-resistant Enterobacteriaceae . Front Microbiol 2017 ; 8 : 228 . Google Scholar PubMed 9 Olaitan AO , Diene SM , Kempf M et al. Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study . Int J Antimicrob Agents 2014 ; 44 : 500 – 7 . Google Scholar CrossRef Search ADS PubMed 10 Olaitan AO , Chabou S , Okdah L et al. Dissemination of the mcr-1 colistin resistance gene . Lancet Infect Dis 2016 ; 16 : 147 . Google Scholar CrossRef Search ADS PubMed 11 Chabou S , Leangapichart T , Okdah L et al. Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance . New Microbes New Infect 2016 ; 13 : 71 – 4 . Google Scholar CrossRef Search ADS PubMed 12 von Graevenitz A , Zollinger-Iten J. Reliability of the colistin disk test in identification of Serratia marcescens and Serratia liquefaciens . Eur J Clin Microbiol 1987 ; 6 : 70 – 1 . Google Scholar CrossRef Search ADS PubMed 13 El-Halfawy OM , Valvano MA. Antimicrobial heteroresistance: an emerging field in need of clarity . Clin Microbiol Rev 2015 ; 28 : 191 – 207 . Google Scholar CrossRef Search ADS PubMed 14 Maalej SM , Meziou MR , Rhimi FM et al. Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae . Lett Appl Microbiol 2011 ; 53 : 546 – 51 . Google Scholar CrossRef Search ADS PubMed 15 Warner DM , Levy SB. Different effects of transcriptional regulators MarA, SoxS and Rob on susceptibility of Escherichia coli to cationic antimicrobial peptides (CAMPs): Rob-dependent CAMP induction of the marRAB operon . Microbiology 2010 ; 156 : 570 – 8 . Google Scholar CrossRef Search ADS PubMed 16 Pérez A , Poza M , Fernández A et al. Involvement of the AcrAB-TolC efflux pump in the resistance, fitness, and virulence of Enterobacter cloacae . Antimicrob Agents Chemother 2012 ; 56 : 2084 – 90 . Google Scholar CrossRef Search ADS PubMed 17 Opperman TJ , Nguyen ST. Recent advances toward a molecular mechanism of efflux pump inhibition . Front Microbiol 2015 ; 6 : 421 . Google Scholar CrossRef Search ADS PubMed 18 Park YK , Ko KS. Effect of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on killing Acinetobacter baumannii by colistin . J Microbiol Seoul Korea 2015 ; 53 : 53 – 9 . 19 Zgurskaya HI , Krishnamoorthy G , Ntreh A et al. Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria . Front Microbiol 2011 ; 2 : 189 . Google Scholar CrossRef Search ADS PubMed 20 Lamers RP , Cavallari JF , Burrows LL. The efflux inhibitor phenylalanine-arginine β-naphthylamide (PAβN) permeabilizes the outer membrane of Gram-negative bacteria . PLoS One 2013 ; 8 : e60666. Google Scholar CrossRef Search ADS PubMed 21 Begic S , Worobec EA. The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis . Microbiology 2008 ; 154 : 454 – 61 . Google Scholar CrossRef Search ADS PubMed 22 Sinha D , Pandey S , Singh R et al. Synergistic efficacy of bisbenzimidazole and carbonyl cyanide 3-chlorophenylhydrazone combination against MDR bacterial strains . Sci Rep 2017 ; 7 : 44419. Google Scholar CrossRef Search ADS PubMed 23 Baron S , Bardet L , Dubourg G et al. mcr-1 plasmid-mediated colistin resistance gene detection in an Enterobacter cloacae clinical isolate in France . J Glob Antimicrob Resist 2017 ; 10 : 35 – 6 . Google Scholar CrossRef Search ADS PubMed 24 Olaitan AO , Thongmalayvong B , Akkhavong K et al. Clonal transmission of a colistin-resistant Escherichia coli from a domesticated pig to a human in Laos . J Antimicrob Chemother 2015 ; 70 : 3402 – 4 . Google Scholar PubMed 25 Leangapichart T , Gautret P , Brouqui P et al. Acquisition of mcr-1 plasmid-mediated colistin resistance in Escherichia coli and Klebsiella pneumoniae during Hajj 2013 and 2014 . Antimicrob Agents Chemother 2016 ; 60 : 6998 – 9 . Google Scholar CrossRef Search ADS PubMed 26 Berrazeg M , Hadjadj L , Ayad A et al. First detected human case in Algeria of mcr-1 plasmid-mediated colistin resistance in a 2011 Escherichia coli isolate . Antimicrob Agents Chemother 2016 ; 60 : 6996 – 7 . Google Scholar CrossRef Search ADS PubMed 27 Rolain J-M , Kempf M , Leangapichart T et al. Plasmid-mediated mcr-1 gene in colistin-resistant clinical isolates of Klebsiella pneumoniae in France and Laos . Antimicrob Agents Chemother 2016 ; 60 : 6994 – 5 . Google Scholar CrossRef Search ADS PubMed 28 Batah R , Loucif L , Olaitan AO et al. Outbreak of Serratia marcescens coproducing ArmA and CTX-M-15 mediated high levels of resistance to aminoglycoside and extended-spectrum β-lactamases, Algeria . Microb Drug Resist 2015 ; 21 : 470 – 6 . Google Scholar CrossRef Search ADS PubMed 29 Baron S , Leulmi Z , Villard C et al. Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of Proteus vulgaris . Int J Antimicrob Agents 2018 ; 51 : 450 – 7 . Google Scholar CrossRef Search ADS PubMed 30 Olaitan AO , Dia NM , Gautret P et al. Acquisition of extended-spectrum cephalosporin- and colistin-resistant Salmonella enterica subsp. enterica serotype Newport by pilgrims during Hajj . Int J Antimicrob Agents 2015 ; 45 : 600 – 4 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
ISSN
0305-7453
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1460-2091
D.O.I.
10.1093/jac/dky134
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Abstract

Abstract Objectives Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. Methods Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time–kill study was performed on five strains carrying different colistin resistance mechanisms. Results Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time–kill study showed the combination was bacteriostatic on strains tested. Conclusions These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans. Introduction Efflux in bacteria is a ubiquitous mechanism to combat antibiotic therapy.1 Multidrug pumps are often non-specific, leading to cross-resistance with several antimicrobial compounds, and can interact synergistically with other resistance mechanisms in order to increase the resistance level. Colistin resistance mechanisms are complex and involve many genes that have not yet been fully identified.2 A well-known, identified mechanism involves reducing the negative charge of the outer membrane by adding positive charges such as sugars (phosphoethanolamine, aminoarabinose) to lipid A of the lipopolysaccharide, resulting in the decreased electrostatic attraction of polymyxins.3 Several mutations in genes such as the two-component systems pmrA/pmrB and phoP/phoQ, the negative regulator of PhoP/PhoQ, mgrB and the aminoarabinose biosynthesis operon arnBCADTEF have been involved in colistin resistance.3 Recently, a transferable colistin-resistance gene mcr-1, which codes for a phosphoethanolamine transferase, has been discovered and appears to have already spread worldwide.2 Colistin resistance in Gram-negative bacteria has emerged over the last few years with its increasing use in human medicine, but also its wide use in agriculture, especially in animal breeding.4,5 In some bacteria, efflux pump mechanisms have been reported to play a role in colistin resistance. Many efflux pumps have been identified as reducing colistin susceptibility, such as KpnEF and the AcrAB–TolC complex.2,3,6 These mechanisms often lead to resistance to other antimicrobial compounds such as aminoglycosides and fluoroquinolones. Various efflux pump inhibitors (EPIs) have been tested on Gram-negative bacteria such as CCCP, 2,4-dinitrophenol (DNP), PABN, reserpine, omeprazole and verapamil.1 Only CCCP and DNP, which inhibit the energy of efflux pumps, showed a significant decrease in colistin resistance.7 The effect of EPIs such as CCCP in the colistin resistance reversion of Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia7 supported the hypothesis that this mechanism plays a role in colistin resistance. More recently, colistin resistance reversion by CCCP has also been demonstrated in the naturally colistin-resistant species Serratia marcescens.8 In our laboratory, we isolated a collection of well-defined colistin-resistant strains including naturally resistant Gram-negative bacteria and acquired-resistance bacteria (mcr-1, mgrB, pmrAB, etc.). The mechanisms of resistance for some of these strains are, however, still unknown. The objective of our study was to evaluate the effect of CCCP on this group in order to evaluate whether the mechanism of resistance can influence the response to efflux pump inhibition and to evaluate its effect on mcr-1 plasmid-mediated colistin-resistant strains. Materials and methods Bacterial strains and colistin resistance mechanisms Strains used in this study are listed in Table 1. Ninety-two strains isolated from various human and pig samples were included in this study. Samples came from France, Laos, Thailand, Nigeria, Algeria and Saudi Arabia (Table 1). Some of these strains have already been published (Table 1). Mechanisms of resistance to colistin have been studied previously with the analysis of phoP, phoQ, pmrA, pmrB, mgrB and mcr-1 performed by real-time PCR, standard PCR and/or sequencing.9–11 Two reference strains of Escherichia coli, one susceptible to colistin (ATCC 25922) and one mcr-1-positive strain (NCTC 13846), were used as controls. The mcr-1-positive strain has been included in the statistical analysis. Table 1. Summary of strains used in this study. Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 ND, not detected; NG, no growth; del, deletion. a A. O. Olaitan and J. M. Rolain, unpublished data. b S. A. Baron, N. Cassir and J. M. Rolain, unpublished data. c S. A. Baron and J. M. Rolain, unpublished data. d Performed using the Etest method (mg/L). Table 1. Summary of strains used in this study. Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 Strain (reference) Name Source/ country Mechanism of resistance to colistin Microdilution assay (mg/L) Conclusion effect Disc diffusion assay (mm) MIC of colistin MIC of colistin + CCCP MIC fold change MH agar MH agar + CCCP difference E. coli ATCC 25922 – colistin susceptible 2 0.5 4 – 19 27 8 E. coli NCTC 13846 human/England mcr-1 4 1 4 reverse 16 19 3 E. asburiaea TH66 human/Thailand unknown 32 0.5 64 reverse 6 22 16 E. asburiae6 LH74 human/Laos unknown >256 <0.25 >1024 reverse 6 27 21 E. cloacae6 NH52 human/Nigeria unknown >256 <0.25 >1024 reverse 18 22 4 E. cloacaea NH132 human/Nigeria unknown >256 0.25 >1024 reverse 17 22 5 E. cloacaea NH131 human/Nigeria unknown >256 0.25 >1024 reverse 17 25 8 E. cloacae23 SB1 human/France mcr-1 8 0.5 16 reverse 17 21 4 E. coli10,24 P17 pig/Laos mcr-1 4 0.25 16 reverse 12 21 9 E. coli10,24 LH30 human/Laos mcr-1 4 0.25 16 reverse 15 25 10 E. coli25 6R human/Saudi Arabia mcr-1 8 <0.25 >32 reverse 16 21 5 E. coli10,24 P6 pig/Laos mcr-1 8 0.5 16 reverse 15 22 7 E. coli25 44A human/Saudi Arabia mcr-1 8 0.5 >16 reverse 12 20 8 E. coli26 SE65 human/Algeria mcr-1 8 <0.25 >32 reverse 12 21 9 E. coli10,24 LH57 human/Laos mcr-1 8 0.5 16 reverse 15 20 5 E. coli10 TH176 human/Thailand unknown 8 0.5 16 reverse 17 26 9 E. coli10,24 LH257 human/Laos mcr-1 16 0.5 32 reverse 17 21 4 E. coli25 134R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 96R human/Saudi Arabia mcr-1 16 0.5 32 reverse 15 19 4 E. coli25 143R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 16 21 5 E. coli10 235 chicken/Algeria mcr-1 16 <0.25 >64 reverse 15 21 6 E. coli10,24 P10 pig/Laos mcr-1 8 0.5 16 reverse 15 21 6 E. coli25 117R human/Saudi Arabia mcr-1 16 <0.25 >64 reverse 12 19 7 E. coli10,24 LH1 human/Laos mcr-1 16 <0.25 >64 reverse 15 22 7 E. coli25 95R human/Saudi Arabia mcr-1 32 0.5 64 reverse 15 20 5 E. coli25 85R human/Saudi Arabia mcr-1 32 0.5 64 reverse 14 20 6 E. coli10 TH99 human/Thailand mcr-1 64 <0.25 >256 reverse 16 21 5 E. colib EC1CR human/France unknown 64 0.5 128 reverse 16 32 16 E. coli10,24 LH121 human/Laos mcr-1 16 0.5 32 reverse 17 23 6 E. coli25 1R 2014 human/Saudi Arabia mcr-1 4 <0.25 >16 reverse 15 23 8 E. colib EC2CR human/France unknown 4 0.5 8 reverse 17 22 5 E. colia TH66 human/Thailand unknown 4 <0.25 >16 reverse 16 25 9 K. oxytoca9 FHA124 human/France unknown 128 1 128 reverse 18 27 9 K.pneumoniae9 LH12 human/Laos mgrB stop 16 <0.25 >64 reverse 8 20 12 K. pneumoniae25 119R human/Saudi Arabia mcr-1 32 0.5 64 reverse 17 21 4 K. pneumoniae9 FHA60 human/France mcr-1 32 0.5 64 reverse 16 23 7 K. pneumoniae9 LH92 human/Laos mcr-1 32 0.5 64 reverse 11 18 7 K. pneumoniae9,27 LH17 human/Laos mcr-1 + PmrB T157P 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH34 human/Thailand PmrB S205P 32 0.5 64 reverse 10 20 10 K. pneumoniae9 LH375 human/Laos unknown 32 0.5 64 reverse 15 22 7 K. pneumoniae9 TH114 human/Thailand unknown 32 0.5 64 reverse 8 22 14 K. pneumoniae9,27 LH 131 human/Laos mcr-1 + mgrB stop 64 0.5 128 reverse 14 20 6 K. pneumoniae9 TH176 human/Thailand unknown 64 0.25 128 reverse 15 21 6 K. pneumoniae9 TH166 human/Thailand unknown 64 1 64 reverse 16 22 6 K. pneumoniae9 TH164 human/Thailand unknown 64 1 64 reverse 16 20 4 K. pneumoniae9 FHM77 human/France unknown 64 0.5 128 reverse 17 22 5 K. pneumoniae9 FHM169 human/France mgrB stop 64 1 64 reverse 16 23 7 K. pneumoniae9 LH102 human/Laos unknown 128 1 128 reverse 16 21 5 K. pneumoniae9,27 LH61 human/Laos mcr-1 + mgrB subs >256 <0.25 >1024 reverse 16 20 4 K. pneumoniaeb KP7CR human/France unknown 4 1 4 reverse 18 22 4 K. pneumoniaeb KP1CR human/France unknown 16 1 16 reverse 18 20 2 K. pneumoniaeb KP3CR human/France unknown 16 1 16 reverse 17 19 2 K. pneumoniae9 TH205 human/Thailand unknown 32 1 32 reverse 17 20 3 K. pneumoniaeb KP4CR human/France unknown 8 1 8 reverse 17 21 4 K. pneumoniaeb KP8CR human/France mgrB del 32 1 32 reverse 18 22 4 K. pneumoniaeb KP9CR human/France unknown >256 1 >256 reverse 16 20 4 K. pneumoniae9 TH68 human/Thailand unknown 16 1 16 reverse 17 22 5 K. pneumoniae9 TH21 human/Thailand unknown 64 1 64 reverse 17 23 6 K. pneumoniae9 LH94 human/Laos unknown 16 0.5 32 reverse 15 22 7 K. pneumoniaeb KP5CR human/France unknown 32 1 32 reverse 18 25 7 K. pneumoniae9 LH140 human/Laos unknown 4 0.5 8 reverse 12 22 10 K. pneumoniaeb KP2CR human/France unknown 4 0.5 8 reverse 14 25 11 K. pneumoniae9 FHA105 human/France mgrB ND 32 0.5 64 reverse 15 27 12 K. pneumoniae9 FHM120b human/France mgrB ND 64 1 64 reverse 16 24 8 K. pneumoniaeb KP6CR human/France unknown 8 1 8 reverse 12 31 19 M. morganiic KON human/France natural >256 0.5 >512 reverse 6 NG >30 M. morganiia FM102 human/France natural >256 1 >256 reverse 6 34 28 M. morganiia FHA60 human/France natural >256 1 >256 reverse 6 32 26 P. aeruginosaa FHM-PACOLR1 human/France unknown >256 0.5 >512 reverse 25 30 5 P. aeruginosac AMO human/France unknown >256 0.5 512 reverse 23 23 0 P. aeruginosac GON human/France unknown >256 <0.25 >1024 reverse 23 32 9 Providencia alcalifaciensa TH44 human/Thailand natural >256 1 >256 reverse 6 28 22 P. alcalifaciensa TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 28 22 Proteus mirabilisa FH112 human/France natural >256 0.5 >512 reverse 6 17 11 P. mirabilisa TH41 human/Thailand natural >256 <0.25 >1024 reverse 6 22 16 P.vulgaris29 P97 human/Algeria natural >256 <0.25 >1024 reverse 6 38 32 Providencia rettgeria HI734 human/France natural >256 1 >256 reverse 6 21 15 P. rettgeria TH66 human/Thailand natural >256 <0.25 >1024 reverse 6 30 24 Salmonella enterica30 100RC3 human/Saudi Arabia unknown 16 0.5 32 reverse 17 37 20 S. enterica30 65RC human/Saudi Arabia unknown 16 1 16 reverse 17 34 17 S. marcescens28 1237 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.064 4000 S. marcescens28 Oxa-48 human/France natural >256 <0.25 >1024 reverse 256d 0.094 2723 S. marcescens28 2186 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1332 human/Algeria natural >256 0.5 >512 reverse 256d 0.094 2723 S. marcescens28 1262 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 50 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 1036 human/Algeria natural >256 <0.25 >1024 reverse 256d 0.125 2048 S. marcescens28 122 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 200 human/Algeria natural >256 1 >256 reverse 256d 0.125 2048 S. marcescens28 1211 human/Algeria natural >256 0.5 >512 reverse 256d 0.125 2048 S. marcescens28 1072 human/Algeria natural >256 0.5 512 reverse 256d 0.125 2048 S. marcescens28 237 human/Algeria natural >256 0.5 >512 reverse 256d 0.19 1347 S. marcescens28 567 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 S. marcescens28 2701 human/Algeria natural >256 1 >256 reverse 256d 0.19 1347 ND, not detected; NG, no growth; del, deletion. a A. O. Olaitan and J. M. Rolain, unpublished data. b S. A. Baron, N. Cassir and J. M. Rolain, unpublished data. c S. A. Baron and J. M. Rolain, unpublished data. d Performed using the Etest method (mg/L). Effect of CCCP on colistin MIC using the broth microdilution method Exploration of the effect of the oxidative phosphorylation uncoupler CCCP on colistin MIC was performed as follows: MIC determination of colistin was performed according to EUCAST recommendations using the broth microdilution method. Colistin sulphate salt (MP-Biomedicals, LLC, Illkirch Graffenstaden, France) concentrations ranged from 0.25 to 256 mg/L. Two tests were performed in parallel: one without adding CCCP and one adding CCCP to each CAMHB well. A stock solution of CCCP was prepared at 5 mg/mL in DMSO. The final concentration of CCCP in the CAMHB was 10 mg/L with a DMSO concentration of 0.2%.7 A growth control well containing 10 mg/L CCCP in CAMHB was also added for each strain, in order to check the absence of effect of CCCP alone on these strains. Visualization of bacterial growth was done by adding iodonitrotetrazolium to wells. The resulting MIC fold changes after the addition of CCCP was calculated as the ratio of the CCCP-free antibiotic’s MIC level to that of the CCCP-added antibiotic. As previously described,8 the positive criterion for the presence of efflux pumps in isolates was an ≥8-fold decrease in colistin MIC after adding CCCP. The mean fold change was calculated by species and by colistin resistance mechanism identified as previously described:8 [1/total sample size(n)] × Σ(MIC fold change × frequency of fold change) where the ‘frequency of fold change’ is the number of times a particular MIC fold change was recorded for that species. The symbol of superiority for MIC fold changes was considered as an equal for the mean fold change analysis. Statistical analyses were performed using non-parametric one-way analysis of variance (ANOVA) (GraphPad Software Inc., La Jolla, CA, USA). A P value <0.05 was taken into account for the MIC fold change. The resulting MIC after adding CCCP was compared to the EUCAST cut-off (established at 2 mg/L) and the effect was considered reversed if the strain became susceptible to colistin again. Effect of CCCP on colistin MIC on Mueller–Hinton (MH) agar plates Colistin susceptibility testing was performed according to EUCAST recommendations using the Etest (bioMérieux, Marcy-l’Étoile, France) or the disc diffusion method on MH agar with commercial antibiotic discs (bioMérieux). Initially, Etests were performed on S. marcescens in order to better visualize the ‘cocarde’ effect,12 which corresponds to an absence of inhibition next to the disc surrounded by a ring of inhibition close to the edge of the bacterial growth unaffected by the antibiotic. The withdrawal from the market of Etests forced us to use the disc method for the other species (http://ansm.sante.fr/var/ansm_site/storage/original/application/7e5c29808ae0bc4d37761e98cc8eeb06.pdf). Colistin MIC was determined on two types of media: MH agar with DMSO, and MH agar with 10 mg/L CCCP (Sigma–Aldrich, Saint-Quentin-Fallavier, France) dissolved in DMSO. When a strain was totally inhibited by colistin + CCCP, a plate containing 10 mg/L CCCP only was used to confirm the absence of effect of the EPI alone. The significance of the change in diameter of colistin inhibition by the disc diffusion method with and without CCCP was evaluated by a Student’s t-test in a matched series. Results were considered as statistically significant when they had a P value <0.05. S. marcescens results were considered separately as the MIC was determined on an agar plate for this species and a fold change was calculated as described above. Time–kill study A time–kill study was performed on one strain of each colistin resistance mechanism group, namely, K. pneumoniae FHM169 (mgrB stop), E. coli 44A (mcr-1 positive), Proteus vulgaris P97 (intrinsically resistant to colistin), Enterobacter asburiae LH74 and K. pneumoniae KP4CR (unknown resistance). A fresh culture of bacteria was inoculated in the three following conditions: CAMHB + DMSO, MH broth + colistin (2 mg/L) and CAMHB + colistin (2 mg/L) + CCCP (10 mg/L) and incubated for 24 h at 37°C with shaking. At times 0 h, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h, several dilutions of each culture were spread on Trypticase soya agar and incubated for 24 h before colony counting. RNA expression In order to evaluate the impact of CCCP on mcr-1 gene transcription, we quantified mcr-1 RNA in two mcr-1-positive E. coli strains, 44A and P10. We inoculated fresh colonies into three different LB broth cultures, containing DMSO, 2 mg/L colistin + DMSO or 2 mg/L colistin + 10 mg/L CCCP, that were incubated at 37°C with shaking for 4 h. One millilitre of this culture, calibrated to an OD of 0.19 (corresponding to ∼1 × 108 cfu/mL), was extracted using the TRIzol® MaxTM Bacterial RNA isolation Kit (Thermo Fisher, Waltham, MA, USA). Briefly, after centrifugation, the pellet was resuspended with 700 μL of TRIzol and 50 μL of 4-bromoanisole (BAN). After 15 min of centrifugation at 4°C at 12 000 g, the aqueous phase containing the RNA was suspended in a new Eppendorf tube and precipitated using 500 μL of isopropanol. The RNA was then washed with 75% ethanol and suspended in 30 μL of water after centrifugation. This RNA was purified from the remaining DNA using the DNA-freeTM kit procedure (Thermo Fisher) following the recommendations of the manufacturer. Finally, 20 μg of the RNA was used to quantify gene expression using the SuperScriptTM III Platinum One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations. Each quantification was performed in duplicate. The ΔΔCt method was used to quantify gene expression. The dxS housekeeping gene was used as calibrator using the following primers: F-GCTTCACAATGCCTTTGCCA, R-TATAACGATGGCCCGTCAGC and the following probe: 6 FAM-CGTCAGTTCCACGCCGACCG whereas the mcr-1 primers and probe were those previously described.11 Results We tested the effect of CCCP on 91 Enterobacteriaceae including 32 K. pneumoniae, 26 E. coli, 6 Enterobacter spp., 2 Salmonella enterica, 1 Klebsiella oxytoca, 14 S. marcescens, 3 Morganella morganii, 3 Proteus spp., 4 Providencia spp. and 3 P. aeruginosa. All strains, except for the colistin-susceptible reference strain ATCC 25922, were found to be resistant to colistin, with an MIC between 4 and >256 mg/L (Table 1). Mechanisms of resistance to colistin had already been identified in some of the strains studied (Table 1). All isolates used in this study grew in the presence of CCCP at the concentration of 10 mg/L in DMSO without any colistin, in broth medium and on agar plates, confirming that this substance alone has no effect at this concentration on these Gram-negative bacteria (Figure 1).7 Figure 1. View largeDownload slide Agar method [left, colistin inhibition diameter on an MH + DMSO plate (photograph not to scale) and on an MH + 10 mg/L CCCP plate (photograph not to scale); right, colistin MIC determined using the Etest method with and without 10 mg/L CCCP]. Liquid method (colistin MIC determined using the microdilution method with and without 10 mg/L CCCP). NC, negative control. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1. View largeDownload slide Agar method [left, colistin inhibition diameter on an MH + DMSO plate (photograph not to scale) and on an MH + 10 mg/L CCCP plate (photograph not to scale); right, colistin MIC determined using the Etest method with and without 10 mg/L CCCP]. Liquid method (colistin MIC determined using the microdilution method with and without 10 mg/L CCCP). NC, negative control. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Of the 93 strains tested, 90 strains had an MIC fold change ≥8, confirming the presence of the efflux pump mechanism of resistance. Only two strains of K. pneumoniae showed an MIC fold change <8, but their colistin MICs were low (2 and 4 mg/L, respectively) and a reversal effect was still observed after adding colistin (0.5 and 1 mg/L, respectively). Likewise, the addition of CCCP led to reversed colistin resistance (MIC <2 mg/L) in all the strains studied. The mean fold change determined by species varied from 24 for S. enterica to 1024 for colistin-heteroresistant Enterobacter (Table 2). We noticed that the effect of CCCP on intrinsically resistant bacteria was much greater than on other Enterobacteriaceae (P < 0.0001). Table 2. Summary of mean fold change in colistin MIC after adding CCCP, by species and by colistin resistance mechanism Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Table 2. Summary of mean fold change in colistin MIC after adding CCCP, by species and by colistin resistance mechanism Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 Species Colistin resistance mechanism Mean fold change M. morganii natural 341.3 Proteus spp. natural 853.3 Providencia spp. natural 640.0 S. marcescens natural 621.7 Enterobacteriaceae intrinsically resistant to colistin natural 618.7 E. coli mcr-1 46.6 unknown 36.8 Enterobacter spp. unknown 64 heteroresistance 1024 mcr-1 16.0 Klebsiella spp. mcr-1 64.0 other mutation 181.3 unknown 60.4 S. enterica unknown 24.0 Other Enterobacteriaceae — 132.5 Total Enterobacteriaceae — 260.7 P. aeruginosa unknown 682.7 Total Gram-negative bacteria — 274.2 We then decided to compare the impact of CCCP according to the mechanism of resistance identified. Bacteria were separated between ‘intrinsic’ or ‘natural’ resistance, ‘heteroresistance’, which describes a phenomenon where subpopulations of seemingly isogenic bacteria exhibit a range of susceptibilities to a particular antibiotic,13 ‘mcr-1 plasmid-mediated’ resistance, ‘other’ resistance when the resistance was caused by a known chromosomic mutation and ‘unknown’ when the resistance mechanism was still unknown. By comparing the resistance mechanism (Table 3), we observed that the efflux mechanism was significantly greater in bacteria naturally resistant to colistin and in bacteria with a heteroresistance mechanism than in bacteria with other resistance mechanisms (P < 0.0001) (Figure 2a). On the other hand, there were no significant differences between ‘intrinsic’ and ‘heteroresistance’ groups or between ‘mcr-1’, ‘other mutation’ or ‘unknown’ groups (Figure 2a). Table 3. Summary of mean fold change in colistin MIC after adding CCCP, by colistin resistance mechanism in Enterobacteriaceae Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 a Excluding S. marcescens. Table 3. Summary of mean fold change in colistin MIC after adding CCCP, by colistin resistance mechanism in Enterobacteriaceae Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 Resistance mechanism Number of isolates Mean fold change mcr-1 24 51.8 Other mutation 9 181.3 Heteroresistance 18 711.1 Naturala 10 614.4 Unknown 32 112.9 a Excluding S. marcescens. Figure 2. View largeDownload slide (a) Mean fold change of colistin MIC by species and mechanism of resistance. (b) Colistin inhibition diameter on MH agar plate without and with 10 mg/L CCCP. EIRC, Enterobacteriaceae intrinsically resistant to colistin. Figure 2. View largeDownload slide (a) Mean fold change of colistin MIC by species and mechanism of resistance. (b) Colistin inhibition diameter on MH agar plate without and with 10 mg/L CCCP. EIRC, Enterobacteriaceae intrinsically resistant to colistin. The effect of CCCP on agar plates showed significant CCCP activity on colistin resistance. The diameter of inhibition of colistin (mm) increased from 14.1 ± 4.4 mm without CCCP to 23.5 ± 5.3 mm with CCCP (P < 0.0001) (Figure 2b). For S. marcescens strains, the fold change on agar plates with Etest MIC determination varied from 1347-fold to 4000-fold (P < 0.0001). Strains were all resistant to colistin with MIC >256 mg/L before adding CCCP, whereas colistin MIC values after adding CCCP varied from 0.064 to 0.19 mg/L (mean 0.128 mg/L). However, no correlation between the disc diffusion assay and the microdilution method was observed. This can be explained by the fact that the disc diffusion method is not a relevant method to determine colistin susceptibility as the colistin does not diffuse correctly in an agar plate.14 Analyses of the time–kill study showed that the association of colistin + CCCP was bacteriostatic on the five strains tested (Figure 3). No difference was observed between the strains with different resistance mechanisms. This is concordant with a previous time–kill study done on K. pneumoniae7 that showed an inhibiting effect on bacterial growth, but no killing effect. Figure 3. View largeDownload slide Time–kill study of colistin (2 mg/L) + CCCP (10 mg/L) on five colistin-resistant strains with different mechanisms of resistance to colistin. Figure 3. View largeDownload slide Time–kill study of colistin (2 mg/L) + CCCP (10 mg/L) on five colistin-resistant strains with different mechanisms of resistance to colistin. Finally, the effect of the association of colistin + CCCP on mcr-1 gene expression was performed on two mcr-1-positive E. coli. The addition of 2 mg/L colistin led to a decrease of 1.65-fold for strain 44A and to an increase of 0.84-fold for strain P10 (Figure 4). However, the combination of 2 mg/L colistin + 10 mg/L CCCP decreased mcr-1 expression 40.5-fold and 26.9-fold, respectively, for strain 44A and strain P10. Figure 4. View largeDownload slide mcr-1 gene expression in E. coli 44A and P10 in the absence of antibiotic, after 2 mg/L colistin and after 2 mg/L colistin + 10 mg/L CCCP. Figure 4. View largeDownload slide mcr-1 gene expression in E. coli 44A and P10 in the absence of antibiotic, after 2 mg/L colistin and after 2 mg/L colistin + 10 mg/L CCCP. Discussion The role of efflux in colistin resistance is still unknown in enterobacteria. In our study, we showed a reversal effect on colistin resistance by CCCP on all the strains studied. This effect was more significant in ‘intrinsically resistant’ and ‘heteroresistant’ enterobacteria than in other Enterobacteriaceae, suggesting that the mechanism blocked by CCCP is critical for colistin resistance in these species. Although the main mechanism described for these species is the modification of lipid A by amino sugars, susceptibility to EPIs was significantly greater in this group than in the ‘other mutation’ and ‘mcr-1’ groups for which colistin resistance is also mediated by lipid A modification. Interestingly, the time–kill study showed no differences between the different strains carrying different colistin resistance mechanisms. The association of colistin + CCCP was bacteriostatic on all strains tested, supporting previous results observed on a colistin-resistant K. pneumoniae strain.7 Many mechanisms have been identified so far, with the most recent, mcr-1, carried by a transferable plasmid, mostly in E. coli and K. pneumoniae.2 The transcriptomic analysis of the mcr-1 gene in two mcr-1-positive E. coli isolates showed that the association of colistin and CCCP inhibits the transcription of the mcr-1 gene. This could likely be explained either by inhibition of efflux (CCCP increases the colistin concentration, which potentiates its effect and inhibits the transcription of the mcr-1 gene) or by an unknown action of CCCP. Efflux has been reported to play a role in colistin resistance, such as with AcrAB–TolC in E. coli15 and MexXY–OprM in P. aeruginosa.16 EPIs have been used to evaluate the effect of efflux pump up-regulation in resistance to colistin. Effects of EPIs on colistin resistance vary according to the type of EPI. For example, PABN and 1-(1-naphtylmethyl)-piperazine (NMP) are EPIs believed to act in competition with drugs in the pocket of the action site of AcrB17 and they failed to recover colistin susceptibility in colistin-resistant Gram-negative bacteria.7,18 This inefficiency has also been reported for other well-known EPIs such as verapamil, omeprazole and reserpine.7 On the other hand, CCCP and DNP, which are non-specific EPIs, showed good activity in restoring colistin susceptibility.7,8,18 Therefore, it is difficult to identify the efflux pump responsible for the resistance because they act on the energy source of efflux pumps, the proton motive force, and can modulate other protein activity in the membrane.1 Ni et al.7 have also suggested that the CCCP effect observed on colistin activity may be due to regeneration of negative charges on the cell membrane. In another study, Park and Ko18 hypothesized the decrease of ATP production caused by CCCP action could be responsible for increased colistin activity in these cells. Our study suggests that CCCP probably has an effect as an EPI. Moreover, the role of soxRS, a modulator of the efflux pump AcrAB-TolC, has recently been reported to be responsible for colistin resistance in Enterobacter spp.6 TolC is an outer membrane protein that can interact with many inner membrane proteins, especially membrane fusion proteins, in order to create a channel for various protein extrusions.19 Usually, bacteria use at least one efflux pump system in the inner membrane such as AcrAB in E. coli that, in association with TolC, enables antibiotic extrusion. Some studies showed that some drugs were dependent on TolC without being dependent on AcrAB, suggesting that TolC can be associated with other inner membrane proteins to evacuate drugs.19 But until now, none of the studied efflux pumps could prove this hypothesis. In our study, the action of CCCP was particularly important for the heteroresistant strains. In these strains, PABN, which is known to permeabilize the outer membrane of Gram-negative bacteria,20 showed an effect on colistin resistance in Enterobacter cloacae and in Enterobacter aerogenes, suggesting that AcrAB–TolC is up-regulated in these strains, but no effect was observed in S. marcescens strains (data not shown). Despite its resistance being similar to the heteroresistance of Enterobacter spp., some other genes seem to be involved in colistin resistance. In a study on S. marcescens, it has been shown that CCCP increases the accumulation of ciprofloxacin in a WT strain, but not in an sdeB-deficient mutant. The resistance–nodulation–division tripartite efflux pump SdeAB–HasF is homologous to AcrAB–TolC and seems to be the major efflux pump system in Serratia.21 This pump seems to extrude a wide variety of components, is dependent on proton motive force and may be a good candidate for a colistin-resistance target gene. Further studies are needed on efflux pump inhibition in colistin resistance to better understand the pathways of this complex mechanism, particularly by describing efflux pump genes and their expression in susceptible/resistant/heteroresistant mutant strains. Our study further suggests that several EPIs could be developed in the future to tackle the issue of emerging colistin resistance in Gram-negative bacteria as demonstrated in the past with β-lactamase inhibitors and resistance to β-lactams. Some studies are currently trying to develop associations of EPIs with other molecules22 to fight bacterial infections. Funding This work was supported by the French Government under the ‘Investissements d’avenir’ program managed by the Agence Nationale de la Recherche (reference: Méditerranée Infection 10-IAHU-03). Transparency declarations None to declare. References 1 Li X-Z , Plésiat P , Nikaido H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria . Clin Microbiol Rev 2015 ; 28 : 337 – 418 . Google Scholar CrossRef Search ADS PubMed 2 Baron S , Hadjadj L , Rolain J-M et al. Molecular mechanisms of polymyxin resistance: knowns and unknowns . Int J Antimicrob Agents 2016 ; 48 : 583 – 91 . Google Scholar CrossRef Search ADS PubMed 3 Olaitan AO , Morand S , Rolain J-M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria . Front Microbiol 2014 ; 5 : 643. Google Scholar CrossRef Search ADS PubMed 4 Kempf I , Jouy E , Chauvin C. Colistin use and colistin resistance in bacteria from animals . Int J Antimicrob Agents 2016 ; 48 : 598 – 606 . 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Antimicrob Agents Chemother 2012 ; 56 : 2084 – 90 . Google Scholar CrossRef Search ADS PubMed 17 Opperman TJ , Nguyen ST. Recent advances toward a molecular mechanism of efflux pump inhibition . Front Microbiol 2015 ; 6 : 421 . Google Scholar CrossRef Search ADS PubMed 18 Park YK , Ko KS. Effect of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on killing Acinetobacter baumannii by colistin . J Microbiol Seoul Korea 2015 ; 53 : 53 – 9 . 19 Zgurskaya HI , Krishnamoorthy G , Ntreh A et al. Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria . Front Microbiol 2011 ; 2 : 189 . Google Scholar CrossRef Search ADS PubMed 20 Lamers RP , Cavallari JF , Burrows LL. The efflux inhibitor phenylalanine-arginine β-naphthylamide (PAβN) permeabilizes the outer membrane of Gram-negative bacteria . PLoS One 2013 ; 8 : e60666. Google Scholar CrossRef Search ADS PubMed 21 Begic S , Worobec EA. 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Acquisition of mcr-1 plasmid-mediated colistin resistance in Escherichia coli and Klebsiella pneumoniae during Hajj 2013 and 2014 . Antimicrob Agents Chemother 2016 ; 60 : 6998 – 9 . Google Scholar CrossRef Search ADS PubMed 26 Berrazeg M , Hadjadj L , Ayad A et al. First detected human case in Algeria of mcr-1 plasmid-mediated colistin resistance in a 2011 Escherichia coli isolate . Antimicrob Agents Chemother 2016 ; 60 : 6996 – 7 . Google Scholar CrossRef Search ADS PubMed 27 Rolain J-M , Kempf M , Leangapichart T et al. Plasmid-mediated mcr-1 gene in colistin-resistant clinical isolates of Klebsiella pneumoniae in France and Laos . Antimicrob Agents Chemother 2016 ; 60 : 6994 – 5 . Google Scholar CrossRef Search ADS PubMed 28 Batah R , Loucif L , Olaitan AO et al. Outbreak of Serratia marcescens coproducing ArmA and CTX-M-15 mediated high levels of resistance to aminoglycoside and extended-spectrum β-lactamases, Algeria . Microb Drug Resist 2015 ; 21 : 470 – 6 . Google Scholar CrossRef Search ADS PubMed 29 Baron S , Leulmi Z , Villard C et al. Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of Proteus vulgaris . Int J Antimicrob Agents 2018 ; 51 : 450 – 7 . Google Scholar CrossRef Search ADS PubMed 30 Olaitan AO , Dia NM , Gautret P et al. Acquisition of extended-spectrum cephalosporin- and colistin-resistant Salmonella enterica subsp. enterica serotype Newport by pilgrims during Hajj . Int J Antimicrob Agents 2015 ; 45 : 600 – 4 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 26, 2018

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