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. 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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.
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0305-7453
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1460-2091
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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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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)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 26, 2018

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