Bacterial strains and samples cultured on LBJMR
A total of 143 bacterial strains were used in this study, of which 101 were Enterobacteriaceae: 75 with an acquired mechanism of resistance to colistin (including 32 strains harboring the mcr-1 gene [6, 19, 29,30,31,32,33,34,35,36,37] and 43 to another resistance mechanism), six with an intrinsic colistin resistance mechanism , and 20 colistin-susceptible strains (Additional file 1: Table S1). This set also comprised 17 non-fermentative Gram-negative bacteria: ten were colistin-resistant strains  including one Pseudomonas aeruginosa, one Acinetobacter baumannii  and other pathogens that are frequently diagnosed in samples from patients with cystic fibrosis, and seven colistin-susceptible strains [40, 41]. Finally, 25 Gram-positive strains, including eight vancomycin-resistant enterococci , three intrinsically vancomycin-resistant genera and 14 vancomycin-susceptible strains  were also included in this study (Additional file 1: Table S1).
The colistin-resistance genes which were potentially involved were previously screened by RT-PCR for Enterobacteriaceae [6, 44], as were the vancomycin resistance genes vanA and vanB for the VRE strains . MICs of colistin and vancomycin were determined using E-test® strips (BioMérieux, Marcy l’Étoile, France) for Gram-negative and Gram-positive strains, respectively, and MICs of daptomycin were determined for the four reference strains of Enterococcus faecium (DSM 17050, 25,698, 25,697 and 13,590), and were evaluated according to the EUCAST guidelines .
In addition, 68 samples were cultivated on LBJMR medium, including 66 stool samples (56 from humans and ten from chickens) that were previously screened for mcr-1 by RT-PCR (56 positive and ten negative) and two clinical rectal swabs obtained from patients in the La Timone hospital, from which a vancomycin-resistant E. faecium VRE strain was isolated. Colonies with different morphologies isolated by culture of samples on LBJMR were isolated by subculture on Trypticase Soy Agar (TSA, Becton-Dickinson, Heidelberg, Germany), and the species were identified using Matrix-Assisted Laser Desorption-Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS), as described previously .
In order to choose the most suitable medium for selecting colistin-resistant Enterobacteriaceae, including strains harboring the mcr-1 gene, five commercial differential media usually used for the detection of Enterobacteriaceae were supplemented with a range of concentration of colistin sulfate salt (MP Biomedicals, Illkirch, France) from 0 to 32 μg/mL: Drigalski Lactose Agar (Biokar diagnostics, France), BBL™ Eosin Methylene Blue Agar (EMB; Becton-Dickinson, Heidelberg, Germany), Difco™ Violet Red Bile Agar (Becton-Dickinson, Heidelberg, Germany), BBL™ MacConkey Agar II (Becton-Dickinson, Heidelberg, Germany), and Difco™ Purple Agar Base (Becton-Dickinson, Heidelberg, Germany) supplemented with 7.5 g /L of glucose (MP Biomedicals, Illkirch, France). Four Klebsiella pneumoniae, including two colistin-resistant, and four Escherichia coli, including two which were colistin-resistant with the mcr-1 gene, were inoculated at 106 CFU on each prepared agar .
The Purple Agar Base supplemented with glucose (7.5 g/L), colistin sulfate (0, 4 and 8 μg/mL) and vancomycin (0 and 50 μg/mL) (Sandoz, Levallois-Perret, France) was evaluated with 24 representative strains, including 20 Enterobacteriaceae (five intrinsic genera, eight with acquired resistance and seven colistin-susceptible strains as control) and four Gram-positive strains susceptible to vancomycin, which were inoculated at 106 CFU. This experiment was also conducted by replacing glucose with lactose (Laboratoires Humeau, La Chapelle-sur-Erdre, France). The optimum concentration of vancomycin for the medium composition was then determined using 20 Gram-positive strains representative of vancomycin resistance (three intrinsically-resistant genera and 17 enterococci, including seven VRE) and ten chicken stool samples from Algeria, five of which were positive for mcr-1 by RT-PCR, and five of which were negative [19, 44].
The sensitivity and specificity of the LBJMR medium to detect all colistin-resistant Gram-negative bacteria and all vancomycin-resistant Gram-positive bacteria were evaluated with all the strains listed in Additional file 1: Table S1 and samples listed in Additional file 1: Table S2, which were incubated on LBJMR in an aerobic atmosphere at 37 °C for between 24 and 48 h.
Inocula of the strains were prepared at 0.5 McFarland (corresponding to 1.5 × 108 CFU/mL according to the EUCAST expert system) and serial 10-fold dilutions were then performed in Phosphate Buffer Saline. 10 μL of these suspensions were deposited on agars and simultaneously on TSA medium to determine the detection limits for each bacterium by assessing the viable concentration by colony count. Growth or inhibition of these microorganisms was observed after overnight incubation at 37 °C in an aerobic atmosphere. Those experiments were performed in duplicate.
Finally, as the LBJMR medium was developed so as to avoid Proteus swarming, Proteus mirabilis and Proteus vulgaris strains were inoculated alone or mixed with E. coli P10 (mcr-1) at the same concentration to evaluate the isolation ability of the LBJMR medium. Cultures were incubated at 37 °C for 72 h.
Mcr-1 screening on stool samples
A total of 1052 stool samples were used in this study, 899 from humans from different regions of the world: Marseille (n = 212) and Angers (n = 128) in France, Thailand (n = 212), Laos (n = 189), Nigeria (n = 143), and Senegal (n = 15). 153 samples were from animals, including chickens from Algeria (n = 10), rodents from Cambodia (n = 21), and rodents (n = 60), pigs (n = 16) and goats (n = 46) from Laos.
The presence of the mcr-1 gene in those stools was first screened using RT-PCR as previously described . Briefly, the DNA of the stools was extracted, after pre-treatment by proteinase K and incubation at 95 °C, using Biorobot EZ1 Advanced XL (Qiagen, Hilden, Germany). Positive DNA was confirmed using standard PCR. Samples with positive DNA were then cultured on LBJMR, after a prior enrichment step performed by inoculating approximately 1 g of the sample in tryptic soy broth (TSB) medium (BioMérieux, Marcy l’Étoile, France) and incubating for 24 h at 37 °C. 100 μL of these liquid media were then plated on agars that were also incubated at 37 °C for 24 h.
For each Gram-negative isolate identified by MALDI-TOF MS, colistin MIC was determined with a colistin E-test® strip. For each colistin-resistant isolate, the presence of mcr-1 was investigated by RT-PCR as previously described . Susceptibility testing to 26 antibiotics was performed on mcr-1-positive strains using the disk diffusion method, and interpreted according to the EUCAST guidelines .
Comparison of the LBJMR medium with other polymyxin-containing media
The sensitivity and specificity of the LBJMR medium was first compared to the BD™ Cepacia Medium (Cepacia, Becton-Dickinson, Heidelberg, Germany) medium with all the colistin-resistant Enterobacteriaceae strains listed in Additional file 1: Table S1. The LBJMR medium was then compared to the SuperPolymyxin medium with 103 bacterial strains, including 61 of the Enterobacteriaceae, 17 non-fermentative Gram-negative, and 25 Gram-positive strains (Additional file 1: Table S1). Other polymyxin-containing media were also compared concomitantly to the LBJMR medium, using EMB Agar (Sigma-Aldrich, Illkirch, Germany) as a control, with some of these strains as described below. Inocula were prepared following the same protocol as for the LBJMR evaluation.
Columbia Colistin Nalidixic Acid Agar +5% sheep blood (CNA) (BioMérieux, Marcy l’Étoile, France) and three mixed culture media: EMB with colistin sulfate (4 μg/mL) and vancomycin (50 μg/mL), LBJMR with Daptomycin (10 μg/ml, Novartis, Horsham, United Kingdom) in place of vancomycin and LBJMR with Amphotericin B (5 μg/ml, Bristol-Myers Squibb, Rueil-Malmaison, France) were concomitantly tested with 45 Enterobacteriaceae strains representative of colistin resistance (30 mcr-1, ten susceptible and five intrinsically resistant genera). LBJMR with Amphotericin B was also evaluated with the eight VRE strains. Finally, the LBJMR medium was compared to the SuperPolymyxin medium with 12 samples, including the two clinical rectal swabs from the La Timone hospital and ten human stools from Thailand, five of which had been identified as positive by culture for the detection of an isolate harboring the mcr-1 gene, and five controls that were negative for mcr-1 using RT-PCR.
The data were analyzed using a Chi-test to compare the Cepacia and LBJMR media and a Student-t test for a pairwise comparison of SuperPolymyxin and LBJMR selective media for the detection of non-fermentative Gram-negative colistin-resistant strains. Significance was assessed at p < 0.05.