# Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains

Lucie Bardet, Jean-Marc Rolain

► To cite this version:

Lucie Bardet, Jean-Marc Rolain. Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains. Canadian Journal of Infectious Diseases and Medical Microbiology, 2018, 2018, pp.1 - 25. 10.1155/2018/3095249. hal-04811087

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## Review Article

# Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains

Lucie Bardet and Jean-Marc Rolain

Aix-Marseille Université, IRD, AP-HM, MEPHI, IHU-Méditerranée Infection, Marseille, France

Correspondence should be addressed to Jean-Marc Rolain; jean-marc.rolain@univ-amu.fr

Received 31 October 2017; Accepted 11 January 2018; Published 5 December 2018

Academic Editor: Elisabetta Caselli

Copyright © 2018 Lucie Bardet and Jean-Marc Rolain. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The recent discovery of the plasmid-mediated *mcr-1* gene conferring resistance to colistin is of clinical concern. The worldwide screening of this resistance mechanism among samples of different origins has highlighted the urgent need to improve the detection of colistin-resistant isolates in clinical microbiology laboratories. Currently, phenotypic methods used to detect colistin resistance are not necessarily suitable as the main characteristic of the *mcr* genes is the low level of resistance that they confer, close to the clinical breakpoint recommended jointly by the CLSI and EUCAST expert systems (S ≤ 2 mg/L and R > 2 mg/L). In this context, susceptibility testing recommendations for polymyxins have evolved and are becoming difficult to implement in routine laboratory work. The large number of mechanisms and genes involved in colistin resistance limits the access to rapid detection by molecular biology. It is therefore necessary to implement well-defined protocols using specific tools to detect all colistin-resistant bacteria. This review aims to summarize the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms and describe new tools specifically developed to assess plasmid-mediated colistin resistance. Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

## 1. Introduction

Multidrug-resistant (MDR) bacteria are of a global concern, notably with the description of carbapenemase-producing Enterobacteriaceae [1]. Colistin is an old antibiotic that regained popularity as a last resort treatment to face the worldwide emergence of these pathogens [2]. Colistin is a polycationic and bactericidal drug that targets the lipid A moiety of lipopolysaccharide (LPS), moving its cationic charges, leading to cell wall lysis and bacterial death [3]. The increasing use of colistin has led to emerging resistance, a phenomenon that represents a clinical source of worry [4]. Enterobacteriaceae are Gram-negative bacteria that are often described as the pathogens responsible for human infectious diseases, particularly the *Escherichia coli* and *Klebsiella pneumoniae* species. Until recently, all mechanisms described were of chromosomal origin, mostly mediated by the two-component systems PmrAB and PhoPQ, leading to the addition of positively charged carbohydrates

on the negatively charged lipid A (Figure 1), a phosphoethanolamine by a phosphoethanolamine transferase or a 4-amino-4-arabinose by surexpression of *arnBCADTEF* operon, leading to the loss of polymyxin affinity for the LPS [5]. In November 2015, Liu et al. reported the first plasmid-mediated gene which they named *mcr-1* [6], which encodes for a phosphoethanolamine transferase, and this was followed by the description of variants (*mcr-1.2*, *mcr-1.3*,...) and the genes *mcr-2*, *mcr-3*, *mcr-4*, *mcr-5*, *mcr-6*, *mcr-7*, and *mcr-8* [7–15]. This recent discovery raised concern about the increase and spread of resistance among the Enterobacteriaceae [16] and led to new recommendations for laboratory diagnosis and clinicians [17]. Specifically, the majority of these *mcr-1* strains exhibited a low minimal inhibitory concentration (MIC) of colistin, around 4 µg/ml [6], which is close to the MIC breakpoint according to the EUCAST guidelines (susceptibility ≤ 2 µg/ml and resistance > 2 µg/ml) (http://www.eucast.org). Moreover, several studies have reported the detection of the *mcr-1* gene in carbapenemase-producing
<figure><img src="image_3.png" /><figcaption>FIGURE 1: Molecular mechanisms of acquired resistance to polymyxins. L-Ara4N: 4-amino-4-arabinose; pEtN: phosphoethanolamine.</figcaption></figure>

Enterobacteriaceae strains, describing coproduction with other plasmid-mediated genes (*bla*<sub>NDM-1</sub>, *bla*<sub>NDM-5</sub>, *bla*<sub>NDM-9</sub>, *bla*<sub>KPC-2</sub>, *bla*<sub>KPC-3</sub>, *bla*<sub>OXA-48</sub>, and *bla*<sub>OXA-181</sub>) [18–24].

The emergence of antibiotic resistance of clinical interest usually conduces to the development of new tools in clinical microbiology laboratories [25]. Currently, the detection of carbapenemase-producing bacteria is well determined, combining specific culture media, phenotyping testing, antibiotic susceptibility testing, and molecular biology [26–28]. As colistin resistance is a recent global phenomenon, the implementation of rapid and reliable screening tools to detect and analyze colistin-resistant pathogens in such a way as to isolate the patient and adapt the treatment is a necessary approach [29]. Moreover, heteroresistance to colistin is a common phenomenon that is widely underestimated, requiring specific methods [30–32]. Here, we propose an overview of all the screening and analysis methods developed to assess colistin resistance among bacterial pathogens causing infectious diseases in hospitalized patients. This review summarizes the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms, and describes new tools specifically developed to assess plasmid-mediated colistin resistance [33].

Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

## 2. Phenotypic Detection Methods

2.1. *Selective Culture Media.* Culture remains the benchmark method for isolating pathogens within clinical samples, and

selective media are continuously developed to isolate specific bacteria [25]. Until recently, there was no specific culture medium for the detection of colistin-resistant strains, and current polymyxin-containing culture media were not able to detect low-level resistant strains because the concentrations of polymyxin in their composition are too high or because they contain other antimicrobial drugs [34–61] (Table 1). Therefore, some in-house media have been developed for colistin-resistant strain screening studies, including strains carrying the *mcr* genes (Supplementary Table S1). These selective culture media were developed by adding low concentrations of colistin (2 or 4 mg/L) to LB nonselective agar or a MacConkey medium, which is selective of Gram-positive contaminants [62, 63]. The chromogenic and nonselective CHROMagar Orientation medium (Biomérieux, Marcy l'Étoile, France) was also used with 4 mg/ml of colistin [64]. They were used in studies to detect the growth of colistin-resistant isolates by directly culturing samples [65–67] or following an enrichment step [68] which could also be selective with the addition of 2 mg/L of colistin to the broth medium [62, 64, 69]. Other anti-infective drugs could be added to avoid contaminants: vancomycin for Gram-positive contaminants [64, 66, 68] and/or amphotericin B for fungal pathogens [67, 68]. For some other studies, such media were developed to screen colistin resistance in bacterial isolates by subculturing them on agar with 2 mg/L of colistin: MH agar [9], COS medium [70], or MacConkey medium [65]. Wong et al. named their medium MHC1 for Mueller–Hinton colistin 1 [71]. Lastly, the selective CNA medium (colistin and nalidixic acid-containing agar), containing 10 mg/L of colistin, could
<table><caption>TABLE 1: Composition of polymyxin-containing agar.</caption><thead><tr><th rowspan="2">Targeted bacteria</th><th rowspan="2">Culture medium</th><th colspan="2">Antibiotics (µg/mL) targeting</th><th rowspan="2">Yeast</th><th rowspan="2">References</th></tr><tr><th>Gram-negative strains<br>Polymyxins</th><th>Gram-positive strains<br>Others</th></tr></thead><tbody><tr><td>Colistin-resistant<br>Gram-negative strains<br>VancoR Gram-positive strains<br>Gram-negative strains</td><td>LBJMR<sup>a</sup></td><td>4 (C)</td><td>Vancomycin 50</td><td></td><td></td></tr><tr><td>Colistin-resistant</td><td>SuperPolymyxin</td><td>3.5 (C)</td><td>Daptomycin 10<br>BM 65<br>Éosine 400</td><td>5 (AB)</td><td>[75]</td></tr><tr><td rowspan="4">Neisseria sp.</td><td>Martin-Lewis agar</td><td>7.5 (C)</td><td></td><td>Vancomycin 4</td><td>20 (A)</td><td>[34]</td></tr><tr><td>Thayer-Martin agar</td><td>7.5 (C)</td><td>5 (T)</td><td>Vancomycin 3</td><td>2.57 (N)</td><td>[35]</td></tr><tr><td>MTM<sup>b</sup> agar</td><td>7.5 (C)</td><td>5 (T)</td><td>Vancomycin 3</td><td>2.57 (N)</td><td>[36]</td></tr><tr><td>NYC<sup>c</sup> agar</td><td>7.5 (C)</td><td>3 (T)</td><td>Vancomycin 2</td><td>20 (A)</td><td>[37]</td></tr><tr><td rowspan="4">Burkholderia cepacia</td><td>Cepacia medium</td><td>30 (B)</td><td></td><td>Ticarcillin 100</td><td></td><td>[38]</td></tr><tr><td>OFPB<sup>d</sup> agar</td><td>30 (B)</td><td>Bacitracin 3</td><td></td><td></td><td>[39]</td></tr><tr><td>Burkholderia cepacia agar</td><td>17.8 (B)</td><td>5 (GEN)</td><td>Ticarcillin 100</td><td></td><td>[38]</td></tr><tr><td>Burkholderia cepacia<br>selective agar</td><td>71.4 (B)</td><td>10 (GEN)</td><td>Vancomycin 2.5</td><td></td><td>[40]</td></tr><tr><td rowspan="6">Legionella sp.</td><td>BCYE<sup>e</sup> selective agar with<br>GVPC<sup>f</sup></td><td>9.4 (B)</td><td>Glycine 3000</td><td>Vancomycin 1</td><td>80 (CH)</td><td>[41]</td></tr><tr><td>CCVC<sup>g</sup></td><td>16 (C)</td><td></td><td>Vancomycin 0.5<br>Cefalotin 4</td><td>80 (CH)</td><td>[42]</td></tr><tr><td>GPVA<sup>h</sup></td><td>11.9 (B)</td><td>Glycine 3000</td><td>Vancomycin 1</td><td>80 (A)</td><td>[43]</td></tr><tr><td>PAV<sup>i</sup></td><td>4.76 (B)</td><td></td><td>Vancomycin 0.5</td><td>80 (A)</td><td>[44]</td></tr><tr><td>PAC<sup>j</sup></td><td>9.52 (B)</td><td></td><td>Cefamandole 2</td><td>80 (A)</td><td>[45]</td></tr><tr><td>DGVP<sup>k</sup></td><td>8 (B)</td><td>Glycine 3000</td><td>Vancomycin 1</td><td></td><td>[46]</td></tr><tr><td rowspan="4">Campylobacter sp.</td><td>Campylobacter agar</td><td></td><td></td><td>Novobiocin 5<br>Cefazolin 15</td><td>50 (CH)</td><td>[47]</td></tr><tr><td>Butzler</td><td>0.33 (C)</td><td>Bacitracin 338</td><td></td><td></td><td>[48]</td></tr><tr><td>Skirrow</td><td>0.25 (B)</td><td>2.5 (T)</td><td>Vancomycin 5</td><td></td><td>[48]</td></tr><tr><td>Blaser-Wang</td><td>0.125 (B)</td><td>2.5 (T)</td><td>Vancomycin 5<br>Cefalotin 15</td><td>2 (AB)</td><td>[49]</td></tr><tr><td>Brucella spp.</td><td>Brucella selective medium</td><td>0.125 (B)</td><td>5 (T)</td><td>Rifampicin 5</td><td>50 (CH)</td><td>[50]</td></tr><tr><td rowspan="2">Vibrio sp.</td><td></td><td>1 (B)</td><td>Bacitracin 500</td><td></td><td>100 (CH)</td><td>[51]</td></tr><tr><td>CPC<sup>l</sup></td><td>66.34 (C)<br>11.9 (B)</td><td></td><td></td><td></td><td>[52]</td></tr><tr><td>Gram-positive strains<br>Streptococcus sp. and<br>Gram-positive strains</td><td>ANC<sup>m</sup></td><td>10 (C)</td><td>Nalidixic acid 10</td><td></td><td></td><td>[53]</td></tr><tr><td rowspan="2">Listeria monocytogenes</td><td>Oxford medium</td><td>20 (C)</td><td>Fosfomycin 10</td><td>Cefotetan 2<br>Acriflavine 5</td><td>400 (CH)</td><td>[54]</td></tr><tr><td>Modified Oxford</td><td>10 (C)</td><td></td><td>Moxalactam 15<br>Ceftazidime 8<br>Acriflavine 5</td><td></td><td>[55]</td></tr><tr><td>Listeria spp.</td><td>PALCAM<sup>n</sup></td><td>10 (B)</td><td></td><td></td><td></td><td>[56]</td></tr><tr><td>Bacillus cereus</td><td>MYP<sup>o</sup></td><td>10 (B)</td><td></td><td></td><td></td><td>[57]</td></tr><tr><td>Mycobacteriaceae</td><td>Middlebrook 7H11</td><td>25 (B)</td><td>20 (T)</td><td>Carbenicillin 50</td><td>10 (AB)</td><td>[58]</td></tr><tr><td rowspan="3">Clostridium perfringens</td><td>SPSP<sup>p</sup> agar</td><td>10 (B)</td><td>Sulfadiazine 120</td><td></td><td></td><td>[59]</td></tr><tr><td>TSN<sup>q</sup> agar</td><td>20 (B)</td><td>Neomycin 50</td><td></td><td></td><td>[60]</td></tr><tr><td>SFP<sup>r</sup> agar</td><td>3.57 (B)</td><td>Kanamycin 12</td><td></td><td></td><td>[61]</td></tr></tbody></table>

B: polymyxin B; C: colistin; AB: amphotericin B; A: anisomycin; CH: cycloheximide; MB: methylene blue; N: nystatin; GEN: gentamicin; T: trimethoprim.
ªLBJMR: Lucie Bardet-Jean-Marc Rolain; ᵇMTM: modified Thayer-Martin; ᶜNYC: New York City; ᵈOFPB: oxidation/fermentation, polymyxin B, bacitracin, and lactose; ᶜBCYE: buffered charcoal and yeast extract; ᶜGPVC: glycine, polymyxin B, vancomycin, and cycloheximide; ᵈCCVC: cefalotin, colistin, vancomycin, and cycloheximide; ᵈGPVA: glycine, polymyxin B, vancomycin, and anisomycin; ᵈPAV: polymyxin B, anisomycin, and vancomycin; ᵈPAC: polymyxin B, anisomycin, and cefamandole; ᵈDGVP: dyes, glycine, vancomycin, and polymyxin B; ᵈCPC: cellobiose, polymyxin B, and colistin; ᵈCNA: colistin and nalidixic acid; ᵈPALCAM: polymyxin B, acriflavine, lithium, ceftazidime, esculin, and mannitol; ᵈMYP: mannitol, egg yolk, and polymyxin B; ᵈSPS: sulfite, polymyxin B, and sulfadiazine, ᵈTSN: trypticase, sulfite, and neomycin; ᵈSFP: Shahidi-Ferguson perfringens.

detect *mcr-1*-positive isolates, one *E. coli* [72] and one *K. pneumoniae* [73], and was also used with CLED (cysteine lactose electrolyte deficient) medium (BioMérieux, Marcy

l'Étoile, France) for screening samples that had or had not been precultured on Trypticase Soy Broth ±2 mg/L of colistin [74].
More specifically, the SuperPolymyxin medium (Elitech Microbio, Signes, France) was developed and intended to specifically detect colistin-resistant strains, including those with a low MIC of colistin and harboring the *mcr-1* gene [75]. The SuperPolymyxin medium has the advantage of facilitating the visualization of *E. coli* strains because it is composed of EMB agar, meaning that they exhibit a metallic green reflect. Its specificity is enabled by 3.5 µg/ml of colistin, 10 µg/ml of daptomycin, and 5 µg/ml of amphotericin B in its composition.

The CHROMagar COL-APSE medium was also developed to detect colistin-resistant strains and was compared to the SuperPolymyxin [76]. Its composition is not precisely described, based on commercial CHROMagar compounds containing colistin sulfate and oxazolidonone antibiotics. The CHROMagar COL-APSE medium presents the advantage to be chromogenic, with the capacity to differentiate colistin-resistant nonfermentative Gram-negative strains as well as Enterobacteriaceae.

The LBJMR medium was also developed to detect all the colistin-resistant bacteria, including those harboring *mcr-1* genes [77]. The LBJMR medium presents the advantage of being versatile, combining colistin-resistant and vancomycin-resistant bacteria screening tools, conferred by 4 µg/ml of colistin sulfate and 50 µg/ml of vancomycin. In particular, the LBJMR medium can be used to detect vancomycin-resistant enterococci (VRE), which represents another emerging field of clinical concern. Both colistin-resistant Enterobacteriaceae and VRE strains are easy to detect on the LBJMR medium with the presence of bro-mocresol purple and glucose: fermentative strains exhibit yellow colonies on a purple agar. Lastly, it can be used to specifically detect pathogens that are often diagnosed in cystic fibrosis patient samples.

The sensitivities of these three media were excellent to detect colistin-resistant strains.

## 2.2. Qualitative Detection of Colistin Resistance with Phenotypic Tests

### 2.2.1. Rapid NP Polymyxin Test for Enterobacteriaceae.
The rapid polymyxin NP test (Elitech, Signes, France) is based on a simple pH test, and detection of colistin resistance is obtained by a color change within 2 hours [78, 79]. The test was evaluated on 200 isolates of Enterobacteriaceae and can be used directly on blood samples [80]. A recent review proposed a diagnosis plan integrating this phenotypic test to confirm colistin resistance of Enterobacteriaceae strains after their growth on a selective medium [29], and its reliability is discussed in several studies [81, 82]. Compared to the broth microdilution (BMD) susceptibility testing method, agreements were excellent to detect *mcr-1* and *mcr-2* strains [83, 84]. The rapid polymyxin test has a good sensitivity to detect *Hafnia* sp. colistin-resistant isolates [79] but failed to detect *Enterobacter* sp. isolates, surely due to their heteroresistance to colistin [85]. This test has to be evaluated with nonfermentative colistin-resistant strains, such as *Acinetobacter baumannii* and *Pseudomonas aeruginosa*.

### 2.2.2. Micromax Assay for A. baumannii.
The Micromax assay (Halotech DNA SL, Madrid, Spain) is based on the detection of DNA fragmentation and cell wall damage in the presence of colistin [86]. Bacteria are incubated for 60 min with 0.5 µg/ml of colistin, trapped in a microgel, and then incubated with a lysis solution to remove weakened cell walls. The presence of DNA fragments is detected after staining by SYBR Gold fluorochrome and observed by fluorescence microscopy. Resistance corresponds to ≤11% of bacteria with cell wall damage. This method is rapid (3 h 30 min) and showed an excellent sensitivity for the detection of colistin resistance on the 70 A. baumannii tested isolates (50 susceptible and 20 resistant), but it is not specific for determining the resistance type.

## 2.3. Specific Phenotypic Screening Methods for the Detection of MCR-1

### 2.3.1. Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).
The detection of polymyxin-resistant bacteria by MALDI-TOF is a promising and costless approach, as the majority of clinical microbiology laboratories own the required equipment to routinely identify clinical isolates [87]. Currently, the use of MALDI-TOF for detecting the carbapenemase-producing bacteria is described, with the detection of carbapenem hydrolysis [88–90]. As a specific peak was described for lipid A at 1796.2 m/z [91], the MALDI-TOF could be used for the detection of lipid A modifications [92]. Very recently, the MALDIxin test was developed for *E. coli* strains, based on the detection of phosphoethanolamine addition on lipid A, and could specifically detect the *mcr*-positive isolates [93]. Indeed, an additional peak at 1919.2 m/z was observed for all polymyxin-resistant strains, and a second additional peak at 1821.2 m/z was observed for all the *mcr*-positives. The MALDIxin test could detect polymyxin-resistant *E. coli* and also differentiate the chromosome- and plasmid-encoded resistance in 15 minutes, and should be evaluated on other species for which phosphoethanolamine addition is involved in polymyxin resistance.

### 2.3.2. Inhibition of MCR-1 Activity.
Several studies on the structure of the catalytic domain of the *MCR-1* protein have demonstrated that the phosphoethanolamine transferase is a zinc metalloprotein [94–96], and that zinc deprivation could reduce the colistin MIC in *E. coli* isolates [97]. Screening tests were developed to specifically detect *MCR-1*, based on the difference of colistin susceptibility obtained in the presence or absence of chelators of zinc ion.

The colistin-MAC test consists of the addition of dipicolinic acid (DPA) in the BMD method, leading to a colistin MIC reduction of ≥ 8-fold in case of *MCR-1*-positive strain [98]. 74 colistin-resistant Enterobacteriaceae strains were tested, and 59 of the 61 strains carrying *mcr-1*-like genes were detected by the colistin-MAC test, while the 13 *mcr*-negative strains exhibited discrepancy in results (increase, maintain, or slow decrease) giving a sensitivity of 96.7% and specificity of 100%. Interestingly, the two *mcr-1* strains that
were negative with the colistin-MAC test were *K. pneumoniae* strains.

More recently, four assays were tested, based on in-
hibition by EDTA [99]. The specific detection of *MCR-1* was
assessed with the following tests: combined-disk method
with diameter differences ≥3 mm, BMD with a reduction of
colistin MIC of ≥4-fold, modified rapid polymyxin NP test
with the absence of color change, and the alteration of zeta
potential *R<sub>ZP</sub>* ≥ 2.5. These assays were performed on 109
Enterobacteriaceae including 59 *mcr-1*-positive *E. coli* and
one *mcr-1*-positive *K. pneumoniae*. The modified rapid NP
test and zeta potential methods showed excellent sensitivity
and specificity and could be inexpensive and simple methods
to detect the presence of the *mcr-1* gene.

These tests should be performed on other species har-
boring the *mcr-1* gene, in particular *K. pneumoniae*, and also
on strains harboring other *mcr* genes, to validate their ability.

**3. New Recommendations on Polymyxins Susceptibility Testing**

Polymyxin susceptibility testing is challenging, as these large
and cationic molecules poorly diffuse into the reference
cation-enriched Mueller-Hinton (MH2) agar, giving dis-
crepant results, and much more since the description of the
*mcr* genes that confer low MICs. Moreover, even in MH2
broth medium, the concentration of cation could largely
influence the polymyxin MIC results [64], notably by
interacting with the acquired resistance mechanisms of the
tested isolates. Defining a reference method for colistin
susceptibility testing is a priority, along with the increasing
use of polymyxin as last-line therapies.

3.1. *Reference Method.* Broth microdilution (BMD) is the only approved method for colistin MIC determination by both the European Committee on Antibiotic Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) [100, 101]. BMD has to be performed with colistin sulfate in untreated polystyrene plates without addition of any surfactant (polysorbate 80) (Table 2). The Mueller-Hinton broth medium has to be cation-adjusted, with a final composition of 20–25 mg/L of calcium and 10–12.5 mg/L of magnesium [102]. EUCAST and CLSI joined their recommendations on the polymyxin breakpoint for MIC of Enterobacteriaceae, *P. aeruginosa* and *Acinetobacter* spp. isolates: susceptible (S) if ≤2 µg/ml and resistant (R) if >2 µg/ml [100, 103]. In 2017, EUCAST added a new quality control (QC) strain that has to be used to control the performances of a colistin susceptibility method: *E. coli* NCTC 18853 that harbors the *mcr-1* gene, in addition to *E. coli* ATCC 25922 and *P. aeruginosa* ATCC 27853 [104] (Table 2).

Dilution methods consist of adding colistin to the
culture medium in such a way as to obtain twofold dilutions
and are prepared according to the CLSI guide M07-A10
[101] and ISO 20776-1 standard (International Standard
Organization). Broth macrodilution is performed in tubes
when reference broth microdilution (BMD) is performed

in 96-well trays. Only colistin sulfate can be used and
particular care is required, as the powder is expressed in
IU/mg, meaning that the concentrations need to be ad-
justed according to the CLSI M100 and the manufacturer's
instructions [103]. The antibiotic is suspended in sterile water
and then diluted in MH2 broth medium before its distri-
bution into 96-well trays. The final bacterial inoculum is
5 × 10⁵ CFU/ml (colony-forming unit) or 5 × 10⁴ CFU/well for
the BMD method, prepared using the 0.5 McFarland standard
(corresponding to approximately 1 to 2 × 10⁸ CFU/ml) [101].
Trays are then incubated at 35 ± 1°C for 18 ± 2 hours
[100, 102]. Results are read visually or with
a spectrophotometer.

Broth microdilution is a time-consuming and fastidious
way to assess MIC in clinical routines [105, 106]. Many
errors can occur, such as an incorrect colistin concentration
or dilution. This technique is not well suited to clinical
microbiology routines and needs to be automated. More-
over, this method exhibits limitations for assessing heter-
oresistance. Indeed, the presence of resistant subpopulations
can give uninterpretable results due to the presence of
skipped wells and has been described for the *Enterobacter*
species, as presented in a study of 114 *Enterobacter cloacae*
[107]. Population analysis profiling is recommended to
confirm heteroresistance [108]. For now, heteroresistance to
polymyxins is not correlated with the presence of *mcr* genes.

3.2. *Comparative Evaluations of Polymyxin Susceptibility Testing Methods.* Evaluating antimicrobial susceptibility testing (AST) methods is performed using a comparison with the reference method, as per the ISO 20776-2 standard [109]: a categorical agreement (CA) is obtained when the strain is in the same clinical category (R, I, S), and an essential agreement (EA) is obtained when the MIC is within plus or minus one doubling dilution from the reference MIC. A very major error (VME) corresponds to a false susceptibility result and is calculated using the resistant strains tested, and a major error (ME), in the case of false resistance, is calculated on the number of susceptible strains. Finally, a minor error (MiE) occurs when a strain is classified as Intermediate (I) instead of S or R, or S or R instead of I. A reliable method will obtain the following scores: CA ≥ 90%, EA ≥ 90%, VME ≤ 3%, and ME ≤ 3% [109]. The results of all the comparative studies performed on colistin susceptibility testing are summarized in Table 3 (in Table S2 for polymyxin B). MIC50 and MIC90 correspond to the MIC that inhibits 50 or 90%, respectively, of the tested strains of the same species.

The surfactant polysorbate 80 was previously added to
trays to limit polymyxin adherence to polystyrene and is not
yet recommended; however, it could induce VME and *mcr*
strains might not be detected [31, 110–112]. Albur et al.
demonstrated that the polystyrene trays used also have an
influence: using tissue-culture-coated round-bottom trays
gave a 5.3-fold increase in MIC values compared to non-
coated V-bottom trays [113], for the material used [106]
(Table S3). A very recent study compared polystyrene coated
trays to glass coated trays and also showed very few dif-
ferences (Table 3) [114].
<table><caption>TABLE 2: Joint EUCAST-CLSI recommendations on colistin susceptibility testing.</caption><thead><tr><th>Reference method</th><th colspan="3">Broth microdilution</th></tr></thead><tbody><tr><td>Preparation according to ISO 20776-1 standard [102]</td><td colspan="3">(i) Cation-adjusted Mueller-Hinton medium (MH2)<br>(ii) Colistin sulfate<br>(iii) Polystyrene trays without pretreatment<br>(iv) Absence of polysorbate 80 or any surfactant</td></tr><tr><td>MIC breakpoint (µg/ml)</td><td>Enterobacteriaceae</td><td>P. aeruginosa</td><td>Acinetobacter spp.</td></tr><tr><td>EUCAST [100]</td><td>S≤2 and R>2</td><td>S≤2 and R>2</td><td>S≤2 and R>2</td></tr><tr><td>CLSI [103]</td><td>ECV*: WT≤2 and NWT≥4</td><td>S≤2 and R≥4</td><td>S≤2 and R≥4</td></tr><tr><td>Quality control [104] (µg/ml)</td><td>E. coli ATCC 25922</td><td>P. aeruginosa ATCC 27853</td><td>E. coli (mcr-1) NCTC 18853#</td></tr><tr><td>Target</td><td>0.5–1</td><td>1–2</td><td>4</td></tr><tr><td>Range</td><td>0.25–2</td><td>0.5–4</td><td>2–8</td></tr></tbody></table>

Until 2013, many comparative studies used agar dilution (AD) as the reference method for polymyxins susceptibility testing, a method that differs from the BMD only because the polymyxins are added to a solid MH2 medium [31, 32, 115-126]. Compared to BMD, agar dilution generally gave concordant results for colistin and polymyxin B [31, 110, 127, 128]. VMEs were very uncommon with AD, and this pointed to the AD's potential role in screening, as it presents the advantage to test several strains on the same plates [117, 129]. A recent study compared agar dilution to broth macro- and microdilution on 8 strains and concluded that agar dilution was the most reproducible method, with an excellent distribution of colistin in agar, but that colistin-containing agar plates could be only stored for 7 days [130].

Diffusion methods based on the antibiotic diffusion in agar, whether with the Kirby-Bauer disk diffusion [131] or with the gradient strips, are not reliable for polymyxin testing and should not be used as a large number of studies have obtained high rates of VMEs or poor EA [32, 120, 125, 128, 132-135]. Some studies showed good results but contained only susceptible strains [136-138]. The influence of MH2 agar composition was assessed: agreement was not affected with agar dilution, but important differences were highlighted with Etest (BioMérieux, Marcy l'Étoile, France) [31, 139]. The advantage of the agar diffusion method is the detection of heteroresistance: colonies present within the inhibition zone correspond to resistant subpopulations [140]. One study compared disc diffusion to Etest method, and a large rate of minor errors occurred [141]. The ColiSpot test consists of replacing the disk of colistin by a drop of a calibrated colistin solution (8 µg/ml). Colistin resistance is revealed in the absence of the inhibition zone. This technique was evaluated with 89 colistin-resistant and 52 colistin-susceptible strains and was developed for veterinarian laboratories [142].

**3.3. Commercial Devices Based on Broth Microdilution Reference Method.** Several commercial devices based on BMD reference methods were developed to easily assess the reference method by offering ready-to-use systems. Their advantage is the elimination of critical preparation steps of MH2 medium and antibiotic dilutions. These systems were used to detect *mcr-1* strains and were evaluated by EUCAST,

giving correct results, with essential agreement ranging from 82% to 96%, and few MEs or VMEs (http://www.eucast.org/ast_of_bacteria/warnings/) [143].

**3.3.1. UMIC Colistine (Biocentric, Bandol, France).** UMIC colistine consists of unitary tests composed of 12-well polystyrene strips, one for growth control and 11 containing dehydrated colistin, with concentrations ranging from 0.06 to 64 µg/ml, provided with unitary MH2 tubes. Inoculation is performed simply, after diluting the 0.5 McFarland suspension by 200-fold into the MH2 tubes, by distributing 100 µL of this diluted suspension into the 12 wells of the strip, leading to the rehydration of the antibiotic. The strips are then incubated at 35 ± 1°C using the UMIC box to avoid any desiccation. One comparative evaluation on 71 *A. baumannii* isolates and one on 92 Gram-negative isolates including 76 Enterobacteriaceae highlighted the reliability of the UMIC colistine kit [144, 145].

**3.3.2. MIC Strip Colistin (MERLIN Diagnostika Bornheim-Hersel, Germany).** MIC Strip Colistin also consists in unitary 12-well strips with concentrations of dehydrated colistin ranging from 0.06 to 64 µg/mL, and Micronaut-S is a panel composed of different antibiotics on standard 96-well trays. Those systems can be automated with Micronaut ASTroID that concomitantly performs dilution for antimicrobial susceptibility testing (AST) and deposits on the MALDI-TOF target, simultaneously identifying the same colony being tested.

**3.3.3. Sentitest Colistin (Liofilchem, Roseto degli Abruzzi, Italy).** It consists of a compact panel of 4 tests containing 7 twofold dilutions of dehydrated colistin (0.25-16 µg/ml). It showed excellent correlation with BMD when tested on 353 isolates, including 259 Enterobacteriaceae, 83 harboring the *mcr-1* gene [146]. Recently, a combined Sentitest colistin/piperacillin-tazobactam was developed, with the same design, providing a unitary test for testing both antibiotics, with colistin concentrations ranging from 0.008 to 128 µg/ml.

<footnote>* Epidemiological cutoff values: clinical data and PK/PD are not sufficient to evaluate a clinical breakpoint for the following species: *E. aerogenes*, *E. cloacae*, *E. coli*, *K. pneumoniae*, and *R. ornithinolytica*. WT: wild type; NWT: non-wild type. #Recommended by EUCAST; MIC must be 4 µg/ml and only occasionally 2 or 8 µg/ml.</footnote>
<table><caption>TABLE 3: Comparison of different colistin susceptibility testing methods to detect colistin resistance in Gram-negative clinical isolates.</caption><thead><tr><th>Bacterial species</th><th>Reference method</th><th>MIC breakpoint</th><th>MIC range; % resistant</th><th>MIC50 (µg/ml)</th><th>MIC90 (µg/ml)</th><th>Methods</th><th>CA ≥ 90% EA ≥ 90% ME ≤ 3% VME ≤ 3% MIE</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">42 A. baumannii isolates</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>0.5–4 µg/ml</td><td rowspan="2">1 µg/ml</td><td rowspan="2">2 µg/ml</td><td>BMD in glass-coated plates</td><td>92.8</td><td>100</td><td>0</td><td>100</td></tr><tr><td>R > 2 µg/ml</td><td>0.07%</td><td>AD</td><td>78.5</td><td>92.8</td><td>15.4</td><td>100</td></tr><tr><td rowspan="2">353 isolates (83 mcr-1)</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>ND</td><td rowspan="2">ND</td><td rowspan="2">ND</td><td rowspan="2">Sensitest</td><td rowspan="2">98.9</td><td rowspan="2">96</td><td rowspan="2">1.46</td><td rowspan="2">0.93</td></tr><tr><td>R > 2 µg/ml</td><td>38.8%</td><td>E-test</td><td>92.8</td><td>16.6</td><td>0</td><td>100</td></tr><tr><td rowspan="2">219 isolates</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>ND</td><td rowspan="2">ND</td><td rowspan="2">ND</td><td rowspan="2">Phenix 100<sup>3</sup> NMIC-417</td><td rowspan="2">96.8</td><td rowspan="2">ND</td><td rowspan="2">0.6</td><td rowspan="2">10</td></tr><tr><td>R > 2 µg/ml</td><td>27.4%</td><td>E-test</td><td>92.8</td><td>61.9</td><td>0</td><td>100</td></tr><tr><td>14 E. coli isolates</td><td>BMD</td><td>S ≤ 2 µg/ml</td><td>0.25–128 µg/ml; 48%</td><td>2</td><td>16</td><td>Sensititre<sup>1</sup> SEMPA1</td><td>94.7</td><td>96</td><td>10.2</td><td>0</td></tr><tr><td>18 K. pneumoniae isolates</td><td></td><td></td><td></td><td></td><td></td><td>Micronaut-S</td><td>89.3</td><td>96</td><td>15.4</td><td>5.6</td></tr><tr><td>21 P. aeruginosa isolates</td><td></td><td></td><td></td><td></td><td></td><td>Micronaut-MIC</td><td>90.7</td><td>99</td><td>12.8</td><td>5.6</td></tr><tr><td rowspan="6">22 Acinetobacter spp. isolates</td><td></td><td></td><td></td><td></td><td></td><td>Etest, Oxoid MH</td><td>81.3</td><td>71</td><td>5.1</td><td>33.3</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Etest, BBL MH</td><td>78.7</td><td>43</td><td>2.6</td><td>41.7</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Etest, MHE</td><td>85.3</td><td>47</td><td>5.1</td><td>25</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>MTS, Oxoid MH</td><td>78.7</td><td>40</td><td>0</td><td>44.4</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>MTS, BBL MH</td><td>76</td><td>49</td><td>0</td><td>50</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Sensitest</td><td>89.3</td><td>88</td><td>17.9</td><td>2.8</td></tr><tr><td rowspan="2">117 A. baumannii isolates</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>≤0.5–16 µg/ml; 24.8%</td><td>≤0.5</td><td>8</td><td>Vitek 2 AST-XN05</td><td>89.7</td><td>88.9</td><td>1.1</td><td>37.9</td></tr><tr><td>R > 2 µg/ml</td><td></td><td></td><td></td><td></td><td>Phoenix 100 NMIC/ID-96 AD</td><td>88.9</td><td>91.5</td><td>1.1</td><td>41.4</td></tr><tr><td rowspan="2">123 Enterobacteriaceae isolates (14 mcr-1 and 1 mcr-2)</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>0.12–128 µg/ml; 67.5%</td><td></td><td></td><td>Phoenix 100 NMIC-93</td><td>91.8</td><td>ND</td><td>0</td><td>12.1</td></tr><tr><td>R > 2 µg/ml</td><td></td><td></td><td></td><td></td><td>Rapid NP</td><td>98.3</td><td>NA</td><td>2.5</td><td>1.2</td></tr><tr><td>15 Hafnia alvei isolates</td><td>BMD</td><td>S ≤ 2 µg/ml</td><td>0.125–32 µg/ml; 96%</td><td>8</td><td>16</td><td>DD</td><td>4</td><td>NA</td><td>0</td><td>100</td></tr><tr><td rowspan="2">10 Hafnia paralvei isolates</td><td></td><td></td><td></td><td>8</td><td>8</td><td>Etest</td><td>76</td><td>32</td><td>0</td><td>25</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Phoenix NMIC-93 Rapid NP</td><td>100</td><td>NA</td><td>0</td><td>0</td></tr><tr><td rowspan="2">76 Enterobacteriaceae isolates (21 mcr-1)</td><td rowspan="2">BMD</td><td>S ≤ 2 µg/ml</td><td>0.06–64 µg/ml; 32.9%</td><td>0.25</td><td>16</td><td>Vitek 2 AST N315</td><td>88.2</td><td>93.9</td><td>0</td><td>36</td></tr><tr><td>R > 2 µg/ml</td><td></td><td>2 (4)</td><td>8 (8)</td><td>Sensititre GNX3F</td><td>90.1</td><td>89.5</td><td>11.8</td><td>4</td></tr><tr><td></td><td></td><td></td><td>0.12</td><td>0.5</td><td>Etest</td><td>92.1</td><td>75</td><td>5.9</td><td>12</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>MicroScan<sup>4</sup> NM44</td><td>88.2</td><td>NA</td><td>15.8</td><td>4</td></tr></tbody></table>
<table><caption>TABLE 3: Continued.</caption><thead><tr><th>Bacterial species</th><th>Reference method</th><th>MIC breakpoint</th><th>MIC range; % resistant</th><th>MIC50 (µg/ml)</th><th>MIC90 (µg/ml)</th><th>Methods</th><th>CA ≥ 90% EA ≥ 90%</th><th>ME ≤ 3% VME ≤ 3%</th><th>MI&E</th><th>References</th></tr></thead><tbody><tr><td>246 isolates (absence of mcr genes)</td><td>Broth macrodilution</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>≤0.5-≥8 µg/ml; 12.6%</td><td>≤0.5</td><td>8</td><td>Etest</td><td>95.1</td><td>92.3</td><td>0.4</td><td>35.5</td><td>[160]</td></tr><tr><td>41 K. pneumoniae isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>2-≥128 µg/ml; 95.1%</td><td>8</td><td>32</td><td>BMD-P80</td><td>82</td><td>95.1</td><td>0</td><td>18.9</td><td>NA</td><td>[110]</td></tr><tr><td>20 A. baumannii isolates</td><td></td><td></td><td></td><td>8</td><td>32</td><td>AD<br>Etest<br>MTS</td><td>91.8<br>59<br>67.2</td><td>55.7<br>50.8<br>65.6</td><td>100<br>33.3<br>33.3</td><td>3.4<br>39.3<br>41.4</td><td></td><td></td></tr><tr><td>290 A. baumannii isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>1-128 µg/ml; 9.3%</td><td>2</td><td>2</td><td>Vitek 2 AST EXN8</td><td>96.7</td><td>70</td><td>66.6</td><td>0</td><td></td><td></td></tr><tr><td>213 Acinetobacter sp. isolates</td><td>AD</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>≤0.5-≥32 µg/ml; 6.1%</td><td>1</td><td>2</td><td>DD 10 µg (9-12 mm)<br>Etest S ≤ 2; R > 4<br>Etest S ≤ 0.5; R > 2<br>Vitek 2 AST-N136</td><td>94.8<br>94.5<br>99.3<br>94.1</td><td>NA<br>2.1<br>≡<br>44.8</td><td>0<br>0<br>0<br>0.38</td><td>0<br>55.5<br>0<br>59.2</td><td>5.2<br>0<br>5.5</td><td>[136]</td></tr><tr><td>60 P. aeruginosa isolates</td><td>BMD-P80</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>≤0.12-≥8 µg/ml; 17.8%</td><td></td><td>2</td><td>Vitek 2 AST-N132<br>Etest<br>MicroScan panel type 42</td><td>99.1<br>87.3<br>99.1</td><td>ND</td><td>0<br>1<br>12.5</td><td>15.4<br>0<br>15.4</td><td></td><td>[115]</td></tr><tr><td>20 K. pneumoniae isolates<br>27 A. baumannii isolates</td><td></td><td></td><td></td><td></td><td>≥8<br>>8</td><td>Broth macrodilution</td><td>98<br>91</td><td>83<br>61</td><td>2.3<br>4.5</td><td>0<br>31.6</td><td></td><td>[128]</td></tr><tr><td>11 A. baumannii isolates</td><td>BMD-P80</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>≤0.12-≥8 µg/ml; 20%</td><td></td><td>>8<br>>8</td><td>BMD<br>AD<br>Sensititre GNXF</td><td>88<br>94<br>96</td><td>34*<br>80<br>62*</td><td>12.5<br>7.5<br>5</td><td>10<br>0<br>0</td><td></td><td>[128]</td></tr><tr><td>15 K. pneumoniae isolates<br>24 P. aeruginosa isolates</td><td></td><td></td><td></td><td></td><td></td><td>Etest, BBL MH<br>Etest, Hardy MH<br>Etest, Remel MH</td><td>78<br>78<br>84</td><td>46<br>64<br>68</td><td>5.7<br>2.8<br>2.8</td><td>47<br>53<br>47</td><td></td><td>[128]</td></tr><tr><td>11 A. baumannii isolates</td><td>BMD-P80</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>≤0.12-≥8 µg/ml; 30%</td><td></td><td>>8</td><td>Phoenix NMIC/ID-76</td><td>100</td><td>99.1</td><td>0</td><td>0</td><td></td><td>[149]</td></tr><tr><td>15 K. pneumoniae isolates<br>24 P. aeruginosa isolates</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>109 P. aeruginosa isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>0%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>63 E. coli isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br>R > 2 µg/ml</td><td>0.12-16 µg/ml; 18.6%</td><td></td><td>1</td><td>BMD-P80</td><td>99.2</td><td>41.3</td><td>0</td><td>43.5</td><td></td><td>[112]</td></tr><tr><td>61 K. pneumoniae isolates</td><td></td><td></td><td></td><td></td><td>0.5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>60 Acinetobacter spp. isolates</td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>63 P. aeruginosa isolates</td><td></td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>TABLE 3: Continued.</caption><thead><tr><th>Bacterial species</th><th>Reference method</th><th>MIC breakpoint</th><th>MIC range; % resistant</th><th>MIC50 (µg/ml)</th><th>MIC90 (µg/ml)</th><th>Methods</th><th>CA ≥ 90% EA ≥ 90% ME ≤ 3% VME ≤ 3% MIE</th><th>References</th></tr></thead><tbody><tr><td>200 Enterobacteriaceae isolates</td><td>AD</td><td>S ≤ 2 µg/ml</td><td>0.128-128 µg/ml; 28.5%</td><td>0.5</td><td>128</td><td>DD 50 µg; R &lt; 15 mm</td><td>96.5</td><td>NA</td><td>0</td><td>12.3</td><td>[116]</td></tr><tr><td>82 K. pneumoniae isolates</td><td>R &gt; 2 µg/ml</td><td></td><td></td><td>0.5</td><td>128</td><td>DD 10 µg; R ≤ 8; S ≥ 11 mm</td><td>93</td><td>NA</td><td>0</td><td>8.8</td><td>4.5</td></tr><tr><td>51 E. coli isolates</td><td></td><td></td><td></td><td>0.5</td><td>0.5</td><td>DD 10 µg; R ≤ 11; S ≥ 14 mm</td><td>99.5</td><td>NA</td><td>0</td><td>1.7</td><td>26.5</td></tr><tr><td>67 E. cloacae isolates</td><td></td><td></td><td></td><td>0.5</td><td>2</td><td>Etest</td><td>100</td><td>52</td><td>0</td><td>0</td><td>0</td></tr><tr><td>25 P. aeruginosa isolates</td><td>AD</td><td>S ≤ 2 µg/ml</td><td>0.25-≥256 µg/ml; 57.1%</td><td>2</td><td>&gt;256</td><td>BMD</td><td>81.1</td><td>40.5</td><td>0</td><td>25</td><td>5.4</td><td>[120]</td></tr><tr><td>12 S. maltophilia isolates</td><td>R &gt; 4 µg/ml</td><td></td><td></td><td>&gt;256</td><td>&gt;256</td><td>Etest</td><td>74.3</td><td>56.7</td><td>0</td><td>35</td><td>11.4</td><td></td></tr><tr><td>157 E. coli isolates</td><td>AD</td><td>S ≤ 2 µg/ml</td><td>0.25-32 µg/ml; 9.6%</td><td>0.5</td><td>2</td><td>DD 150 µg; R &lt; 16; S ≥ 20 mm</td><td>46.5</td><td>NA</td><td>1.4</td><td>20</td><td>49.7</td><td>[123]</td></tr><tr><td></td><td>R &gt; 4 µg/ml</td><td></td><td></td><td></td><td></td><td>DD 10 µg; R ≤ 10; S ≥ 11 mm</td><td>96.8</td><td>NA</td><td>0.7</td><td>13.3</td><td>1.9</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>Etest</td><td>96.8</td><td>81.5</td><td>0.7</td><td>0</td><td>0.6</td><td></td></tr><tr><td>78 P. aeruginosa isolates</td><td>BMD</td><td>S ≤ 2 µg/ml</td><td>&lt;0.25-2 µg/ml; 0</td><td>1</td><td>1</td><td>Etest</td><td>100</td><td>79.5</td><td>0</td><td>0</td><td>6.4</td><td>[135]</td></tr><tr><td>100 A. baumannii isolates</td><td>Phoenix</td><td>S ≤ 2 µg/ml</td><td>0.25-128; 31.2%</td><td>0.5</td><td>0.5</td><td>DD 10 µg; R ≤ 8; S ≥ 11 mm</td><td>100</td><td>NA</td><td>0</td><td>0</td><td>0</td><td>[137]</td></tr><tr><td></td><td>R &gt; 4 µg/ml</td><td></td><td></td><td></td><td></td><td>Etest</td><td>100</td><td>NA</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>154 Acinetobacter spp. isolates</td><td>AD</td><td>S ≤ 2 µg/ml</td><td>≤0.064-≥32; 11.7%</td><td>NA</td><td>NA</td><td>Etest</td><td>98.7</td><td>88</td><td>0.7</td><td>5.6</td><td></td><td>[124]</td></tr><tr><td>170 Gram-negative isolates</td><td>AD</td><td>S ≤ 4 µg/ml</td><td>0.25-128; 31.2%</td><td>0.5</td><td>0.5</td><td>Etest</td><td>100</td><td>91.2</td><td>0</td><td>0</td><td></td><td>[126]</td></tr><tr><td>102 Gram-negative isolates</td><td>BMD</td><td>S ≤ 2 µg/ml</td><td>&lt;0.5-≥64 µg/ml; 50%</td><td>NA</td><td>NA</td><td>AD, Oxoid MH</td><td>ND</td><td>96.8</td><td></td><td></td><td></td><td>[31]</td></tr><tr><td></td><td>R &gt; 4 µg/ml</td><td></td><td></td><td></td><td></td><td>AD, Oxoid Iso-Sensitest Etest, MH Etest, ISO Vitek 2 AST N038 DD 10 µg; R ≤ 10; S ≥ 13 mm</td><td>97.9</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>44 Acinetobacter spp. isolates</td><td>AD</td><td>S ≤ 2 µg/ml</td><td>1-2 µg/ml; 0</td><td>1</td><td>2</td><td>Vitek 2 AST-N032</td><td>100</td><td>ND</td><td>0</td><td>NA</td><td>NA</td><td>[118]</td></tr></tbody></table>
<table><caption>TABLE 3: Continued.</caption><thead><tr><th>Bacterial species</th><th>Reference method</th><th>MIC breakpoint</th><th>MIC range; % resistant</th><th>MIC50 (µg/ml)</th><th>MIC90 (µg/ml)</th><th>Methods</th><th>CA ≥ 90% EA ≥ 90% ME ≤ 3% VME ≤ 3% MIE</th><th>References</th></tr></thead><tbody><tr><td>172 Gram-negative isolates</td><td>AD</td><td>S ≤ 2 µg/ml<br/>R > 2 µg/ml</td><td>0.5–64; 31.4%</td><td></td><td></td><td>Vitek 22 AST-N032 (n = 32)<br/>Etest (n = 137)</td><td>82<br/>86.6</td><td>75.2<br/>75.0</td><td>0<br/>6.8</td><td>57.4<br/>27.8</td><td>[32]</td></tr><tr><td>115 A. baumannii isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br/>R > 2 µg/ml</td><td>≤0.06–512 µg/ml; 19.1%</td><td>≤0.06</td><td>32</td><td>Etest</td><td>98.2</td><td>16.5</td><td>0</td><td>1.7</td><td>[138]</td></tr><tr><td>501 P. aeruginosa isolates (401 CF)</td><td>AD</td><td>S ≤ 4 µg/ml<br/>R > 4 µg/ml</td><td>≤0.5–≥16 µg/ml; 17.8%</td><td>2</td><td>4</td><td>BMD 24 h<br/>≥16</td><td>96</td><td>1.2</td><td>26.5</td><td></td><td>[121]</td></tr><tr><td>50 A. xylosoxidans isolates</td><td></td><td></td><td></td><td>4</td><td>≥16</td><td>BMD 48 h</td><td>93.6</td><td>3.9</td><td>18.0</td><td></td><td></td></tr><tr><td>50 S. maltophilia isolates</td><td></td><td></td><td></td><td>8</td><td>≥16</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>70 S. maltophilia</td><td>AD</td><td>S ≤ 2 µg/ml<br/>R > 2 µg/ml</td><td>0.12–32 µg/ml; 24.3%</td><td>2</td><td>4</td><td>DD 10 µg; R ≤ 8; S ≥ 11 mm Etest</td><td>71.2<br/>86.4</td><td>NA<br/>96.7</td><td>0<br/>5.6</td><td>93.7<br/>37.5</td><td>5.7<br/>NA</td><td>[125]</td></tr><tr><td>200 Gram-negative isolates (i) 60 A. baumannii isolates</td><td>BMD</td><td>S ≤ 2 µg/ml<br/>R > 2 µg/ml</td><td>≤1–>128 µg/ml; 15%</td><td></td><td></td><td>DD 10 µg; R ≤ 10; S ≥ 14 mm<br/>DD 10 µg; R ≤ 8; S ≥ 11 mm</td><td>94<br/>95</td><td>NA<br/>NA</td><td>0<br/>0</td><td>21.8<br/>31.2</td><td>1.5<br/>1</td><td>[127]</td></tr><tr><td>(ii) 80 P. aeruginosa isolates</td><td></td><td></td><td></td><td>≤1</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>(iii) 12 S. maltophilia isolates</td><td></td><td></td><td></td><td>≤1</td><td>≤1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>35 representatives</td><td>BMD</td><td>S ≤ 2 µg/ml<br/>R > 2 µg/ml</td><td>≤1–>128 µg/ml; 40%</td><td></td><td></td><td>AD</td><td>97.1</td><td>91.4</td><td>47.6</td><td>0</td><td>[127]</td></tr></tbody></table>

CA: categorical agreement; EA: essential agreement; VME: very major error; ME: major error; MiE: minor error; AD: agar dilution; BMD: broth microdilution; DD: disk diffusion; MH: Mueller-Hinton. Italic values indicate the number of errors and not the percentage when a too few number of strains tested, where R for VME or S for ME. ¹Sensititre panels: ≤0.25–>4 µg/ml except for SEMPAI. ²Vitek 2 reagent cards: ≤0.5–>16 µg/ml except for AST-N038 (≤2, 4, and >4 µg/ml) and AST-N032 (1–4 µg/ml). ³Phoenix 100 cards: ≤1–>4 µg/ml. ⁴MicroScan-dried Gram-negative breakpoint combo panel type 42 ≤2 and >4 µg/ml.

*Prediffusion test: discs were removed after 2 h of incubation.
### 3.3.4. The Sensititre System (Thermo Fisher Scientific, Waltham, MA, USA).
It presents different antibiotics on 96-well trays with a customizable plate layout. Inoculation, incubation, and reading (based on fluorescence) steps can be automated. Chew et al. [147] recently evaluated a Sensititre GNX3F panel containing both polymyxin B and colistin (0.25–4 mg/L) and presented a sensitivity of 95.2% and 100% in detecting the 21 *mcr-1* isolates tested, respectively.

### 3.4. Automated Systems.
Automated systems were developed to shorten result timeframes by increasing sensitivity, and also to avoid manipulation bias [148], with incubation and real-time reading. However, by combining several antibiotics, the number of concentrations tested is limited, and they cannot give a real MIC (Table 3).

#### 3.4.1. MicroScan WalkAway (Beckman Coulter, San Diego, CA, USA).
It uses standard trays that are manually inoculated, and reading is based on fluorometry, with results obtained in 3.5–7H. It is not available on polymyxin B, and essential agreement cannot be evaluated between techniques as the NM44 panel proposes only ≤4 and ≥4 µg/ml for colistin. In the recent study of Chew et al., this panel was able to detect all *mcr-1* tested isolates and presented only one VME on 76 Enterobacteriaceae isolates tested. It also evaluated 213 *Acinetobacter* species and presented 99.1 % categorical agreement against the agar dilution method [115].

#### 3.4.2. Vitek 2 (BioMérieux, Marcy l'Étoile, France).
It is a semiautomated system that uses reagent cards containing dehydrated antibiotics and other reagents in a 64-well format. It combines rapid identification and AST using an extrapolated growth algorithm. Various comparative studies performed to evaluate Vitek 2 have returned discrepant results with high rates of VME. In their recent evaluation, Chew et al. [147] demonstrated the efficacy of Vitek 2 in assessing both polymyxin B and colistin MIC with only one VME for each and 96.1% and 93.9% EA, respectively, but it was only able to detect *mcr-1* strains with polymyxin B.

#### 3.4.3. BD Phoenix™ (Becton Dickinson, Le Pont de Claix, France).
It performs identification and AST in parallel in 84-well specific plates. Reading is based on an oxidation-reduction indicator in 6–16 hours. One study showed excellent agreement on 109 *P. aeruginosa* strains, but only colistin-susceptible strains were tested [149]. Vourli et al. [129] have shown concerning results testing *Acinetobacter baumannii* strains with very high VME rates (41.4%) despite the study including 24.8% of colistin-resistant strains. This was explained by the majority of errors occurring near the breakpoint (2 instead of 4 µg/ml). Lastly, in the study by Jayol et al. [83], the Phoenix system was able to detect all *mcr*-carrying bacteria, even those with a colistin MIC of 4 µg/ml, but the high rates of VMEs obtained prove its inability to assess heteroresistance.

# 4. Genotyping and Molecular Screening

## 4.1. PCR Amplification and Sequencing to Detect Gene Mutations.
Molecular biology methods are the most sensitive for determining antibiotic resistance by assessing the presence of resistance genes or mutations conferring resistance. These methods are complementary to the phenotypic techniques and confirm the resistant status of bacterial isolates. The main mutations for Enterobacteriaceae species are located on genes coding the two-component systems PmrA/PmrB and PhoP/PhoQ (Figure 1). Specifically, mutations in the *mgrB* gene—the negative feedback regulator of PhoPQ—notably with the presence of insertional sequences, appeared to be the main resistance mechanism observed in *K. pneumoniae* strains. These colistin resistances are not based on drug-modifying enzymes or the acquisition of a resistance gene which could be easily detected. Screening of potential mutations on these chromosomal genes is done by amplification and sequencing, takes 3 days, and requires that all genes are tested. Sequenced amplicons are then compared by the BLAST tool against the NCBI database to screen possible mutations compared to wild-type genes.

## 4.2. Real-Time qPCR to Detect the Presence of mcr Genes.
The discovery of the acquired gene *mcr-1* justifies the use of molecular detection with RT-PCR, a rapid quantitative technique to detect the presence of the gene. A systematic screening of the gene in colistin-resistant strains was performed [150] (Figure 2). For such purposes, scientists have used the primers of the original study [6], or have designed their own primers for standard PCR [132, 151–160] or RT-PCR, based on SYBR Green assays [64, 151, 161], TaqMan probe [66, 72, 152, 162], or other FAM-labelled probe [9, 71, 163, 164] or HEX-labelled probe [165] (Table 4).

Xavier et al. designed primers to screen *mcr-2* [7], giving a 567 bp product [166]. Some designed their own primers for standard PCR [167–169], and one study developed a TaqMan assay for qPCR [170]. Interestingly, three studies went further by designing a universal primer to detect both *mcr-1* and *mcr-2* genes by standard PCR [166, 171] and a generic primer and a probe to detect them by qPCR [74], but these have not yet been tested on other *mcr* genes. Lastly, primers were designed for detecting *mcr-3* [10], *mcr-4* [11], and *mcr-5* genes [12] by standard PCR (Table 4). A recent study described a multiplex SYBR Green real-time PCR assay for the simultaneous detection of *mcr-1*, *mcr-2*, and *mcr-3* genes [172]. Finally, a multiplex PCR assay for detection of the five *mcr* genes: *mcr-1*, *mcr-2*, *mcr-3*, *mcr-4*, and *mcr-5*, was developed in order to obtain sequential amplicons with a size difference of 200 bp, allowing their fast and simultaneous detection on agarose gels [173].

## 4.3. PCR to Detect Plasmid Carrying mcr Genes. *mcr-1* is a 1626-base pair-long gene located on a 2607 bp common region flanked on both ends by an *ISA*pl1 insertion sequence (IS) in some plasmids [174]. This sequence may form a composite transposon that can potentially move as one complete unit [155, 175, 176]. This insertion sequence appears
<figure><img src="image_4.png" /><figcaption>FIGURE 2: Worldwide dissemination of the *mcr*-1 gene. The map was performed with Magrit mapping application (http://magrit.cnrs.fr).</figcaption></figure>

to be a key component of the mobilome, and its presence is not systematic. Furthermore, only the upstream region can contain *ISApl1* [165]. Li et al. identified the ability of the Tn6330 transposon (*ISApl1-mcr-1-orf-ISApl1*) to generate circular *ISApl1-mcr-1-orf* [177]. Specific primers were developed to screen the upstream presence of this IS transposon by PCR and Sanger sequencing [178–180]. Others have also designed their own system to directly screen on plasmid carrying *mcr*-gene type IncX4 [9, 181], but these methods also exhibit limitations, as a wide distribution has been observed for *mcr-1* among different plasmids (IncI2, IncX4, IncHI2, IncY, IncF, IncP, IncH1, and IncX3), demonstrating the great ability of *mcr* genes to spread.

4.4. Microarray. Microarray technology allows scientists to analyze dozens of genes at the same time. The Check-MDR CT103 microarray system (Check-Points, Wageningen, the Netherlands) was developed to screen the presence of extended-spectrum beta-lactamase (ESBL) genes (TEM and SHV) and carbapenemase genes (OXA-48, KPC, NDM...) in the same assay and can assay 24 samples at the same time, with an effective detection in 6.5 hours. Recently, a study evaluated this assay for detecting *mcr* genes: sensitivity and specificity were excellent for *mcr-1* and its variants (from *mcr-1.2* to *mcr-1.7* and *mcr-2* genes), but it was not able to detect the new gene *mcr-3* [182]. *mcr-4* has not been assayed yet.

4.5. Loop-Mediated Isothermal Amplification (LAMP). The Eazyplex SuperBug *mcr-1* kit (Amplex Biosystems Gmbh, Giessen, Germany) was developed to assess the presence of the *mcr-1* gene within 20 minutes [183]. It was effective on 104 microbial strains but needs to be assayed directly on clinical samples. As it was developed before the description of the other *mcr* genes, it can only detect the *mcr-1* gene and the *mcr-1.2* variant.

More recently, another LAMP-based assay was developed to detect *mcr-1* gene and evaluated as a screening tool on 556 multidrug-resistant Enterobacteriaceae [184]. Seven isolates were positive by both standard PCR and LAMP-based assay (6 *E. coli* and 1 *K. pneumoniae*). The results can be assessed by chromogenic visualization. This test constitutes a rapid, specific, and cost-effective tool that exhibits a higher sensitivity than PCR (10-fold). It has to be assayed on clinical samples; as for now, only spiked tools were used.

4.6. Novel Approach with Direct Resistome Analysis. Genomic screening is an alternative approach for studying resistance and providing a better understanding of the behavior of bacterial isolates [185]. The development of next-generation sequencing has led to lower costs, reduced screening delay, and increased sequencing speeds combined with updated databases providing access to a large amount of information. The *mcr-1* gene was initially discovered by whole
<table><caption>TABLE 4: List of primers designed to detect *mcr* genes by PCR.</caption><thead><tr><th>Targeted genes</th><th>Analyze</th><th>Method</th><th>Primer sequences</th><th>Cycle (nb. steps)</th><th>Product (bp)</th><th>Study</th></tr></thead><tbody><tr><td rowspan="2">Original study</td><td rowspan="2">105 colistin-resistant strains</td><td>Std</td><td>CLR F: 5'-CGGTCAGTCCGTTTGTTTC-3'<br/>CLR R: 5'-CTTGGTCGGTCTGTAGGG-3'</td><td>25; 94°C, 30 s;<br/>58°C, 90 s; 72°C,<br/>60 s</td><td>309</td><td>[6]</td></tr><tr><td>45 colistin-resistant strains in 2 spiked stools</td><td>HotStarTaqMasterMix<br/>Mcr-1_FAM-BHQ: 5'-<br/>CTACAGACCGACCAAGCCGA-3'<br/>Pr 5 = -HEX-C</td><td>mcr-1_F: 5'-TGTGGTACCGACGCTCGGTCAG-3'<br/>mcr-1_R: 5'-TCAGCGGATGAATGCGGTGC-3'<br/>mcr-1_as: 5'-ATGGCACGGTCTATGATA-3'</td><td>45; 95°C, 30 s;<br/>55°C, 30 s; 72°C,<br/>30 s</td><td>155</td><td>[152]<br/>[160]</td></tr><tr><td rowspan="2">In silico study</td><td rowspan="2">20 strains in 3 spiked stools</td><td>CAAGCCGA-ZEN-GACCAAGGATC-3IABkFQ-3</td><td>mcr-1_as: 5'-CGGATAATCCACCTTAACA-3'<br/>mcr-1-286F: 5'-ACTTATGGCACGGTCTATGA-3'<br/>mcr-1-401R: 5'-ACACCCAAACCAATGATACG-3'</td><td>40; 95°C, 10 s;<br/>-56°C, 40 s</td><td></td><td>[162]</td></tr><tr><td>CAAGCCGA-ZEN-GACCAAGGATC-3IABkFQ-3</td><td>mcr-1-qF1: 5'-ACACTTATGGCACGGTCTATG-3'<br/>mcr-1-qR1: 5'-GCACACCCAAACCAATGATAC-3'<br/>mcr-1-qF2: 5'-TGGCGTTCAGCAGTCATTAT-3'<br/>mcr-1-qR2: 5'-AGCTTACCCACCGAGTAGAT-3'</td><td>40; 95°C, 3 s;<br/>60°C, 20 s; 72°C,<br/>7 s</td><td>120</td><td>[148]<br/>[165]</td></tr><tr><td rowspan="2">Wastewater samples</td><td rowspan="2">2046 strains</td><td>SYBR Green</td><td>mcr-1-F: 5'-ATGATGCAGCATACTTCTGTGTG-3'<br/>mcr-1-R: 5'-TCAGCGGATGAATGCGGTGC-3'</td><td></td><td>1646</td><td>[148]</td></tr><tr><td>SYBR Green</td><td>mcr-1-F1: 5'-TGTCTTTGTGGCGAGTGTTG-3'<br/>mcr-1-R1: 5'-CGCGCCCATGATTAATAGCA-3'<br/>mcr-1-FW: 5'-ACGCCATCTGCAACACCAA-3'</td><td>40; 95°C, 15 s;<br/>60°C, 30 s</td><td></td><td>[158]</td></tr><tr><td rowspan="2">78 stool</td><td rowspan="2">100 strains: 18 colistin-resistant strains in 833 faecal samples</td><td>SYBR Green</td><td>mcr-1-RV: 5'-GCCAACGAGCATACCGACAT-3'</td><td>30/40: 95°C, 15 s;<br/>-63°C, 10 s;<br/>-72°C, 10 s</td><td>59</td><td>[61]</td></tr><tr><td>TaqMan probe: 6<br/>FAM-GACCGCGACCGCCAAATCTTACC-TAMRA</td><td>F1: GCAGCATACTTCTGTGTGGTAC<br/>RI: ACAAAGCCGAGATTGTCCGCG</td><td>35; 95°C, 30 s;<br/>-60°C, 1 min</td><td>145</td><td>[149]</td></tr><tr><td rowspan="2">1495 E. coli strains and 571 KP strains</td><td rowspan="2">18 samples</td><td>Std</td><td>F1: GCAGCATACTTCTGTGTGGTAC<br/>R3: TATGCACGCGAAAGAAACTGGC</td><td></td><td>554</td><td></td></tr><tr><td>FastStart Universal Probe Master kit<br/>FAM-<br/>AACAGCGTGGTGATCAGTAGCAT-BHQ</td><td>Mcr-1-forward: 5'-GCTCGGTCAGTCCGTTTG-3'<br/>Mcr-1-reverse: 5'-GAATGCGGTGCGGTCTTT-3'</td><td></td><td></td><td></td><td>[150]</td></tr><tr><td rowspan="2">Clinical E. coli isolates</td><td rowspan="2">241 isolates</td><td>Std</td><td>M-R: AAATCAACACAGGCTTTAGCAC</td><td>40; 95°C, 15 s;<br/>-60°C, 1 min</td><td>116</td><td>[161]</td></tr><tr><td>TaqMan Fast Advanced Master Mix<br/>5'-Cy5-<br/>TGCAGACGCACAGCAATGCCTATGAT-3T-<br/>TAO-3'</td><td>MCR-1-F2: 5'-CTCATGATGCAGCATACTTC-3'<br/>MCR-1-R2: 5'-CGAATGGAGTGTGCGGTG-3'<br/>MCR-1F: 5'-CATCGCTCAAAGTATCCAGTGG-3'</td><td></td><td></td><td>Entire<br/>gene</td><td>[151]</td></tr><tr><td rowspan="2">10,609 E. coli isolates (505R)</td><td rowspan="2">62 isolates</td><td>TaqMan RT-mcr-1_Probe<br/>Cy5-<br/>AGTTTCTTTTCGGTCGCATAAGCCG-BBQ-650</td><td>MCR-1R: 5'-CCATGTAGATAGACACCGTTCTCAC-3'</td><td></td><td></td><td>[69]</td></tr><tr><td>MCR1_22,697_F1: 5'-CACTTATGGCACGGTCTATGA-3'</td><td>RT-mcr-1_F: TGGCGTTCAGCAGTCATTAT<br/>RT-mcr-1_R: AGCTTACCCACCGAGTAGAT</td><td>30°C-95°C, 15 s;<br/>-60°C, 1 min</td><td></td><td>[159]</td></tr></tbody></table>
<table><caption>TABLE 4: Continued.</caption><thead><tr><th>Targeted genes</th><th>Analyze</th><th>Method</th><th>Primer sequences</th><th>Cycle (nb: steps)</th><th>Product (bp)</th><th>Study</th></tr></thead><tbody><tr><td rowspan="2">31 colistin-resistant isolates</td><td rowspan="2">122 faecal samples</td><td>MCR1_22,763_Pb1 FAM-TGGTCTCGG/ZEN/CTTGGTCGGTCTGTAGGGC-3IABkFQ</td><td>MCR1_22,810_R1: 5'-CCCAAACCAATGATACGCAT-3'<br/>mcr-1-F: 5'-ATGATGCAGCATACTTCTGTGTGG-3'<br/>mcr-1-R: 5'-GTGCGGTCTTTGACTTTGTCC-3'</td><td rowspan="2">[6]</td><td>45: 15 s, 95°C;<br/>-1 min, -60°C</td><td>[157]</td></tr><tr><td>Std</td><td></td><td>309</td><td>[63]</td></tr><tr><td rowspan="4">48 E. coli and 27 KP strains</td><td rowspan="4">136 colistin-resistant isolates</td><td rowspan="4">TTGACCGCGACCGCCAATCTTA-3' FAM</td><td>mcr-1-F1: 5'-ATGATGCAGCATACTTCTGTG-3'<br/>mcr-1-R1: 5'-TCAGCGGATGAATGCGGTG-3'<br/>CLR5-F1: 5'-ATGATGCAGCATACTTCTGTGTGG-3'<br/>CLR5-R1: 5'-TCAGCGGATGAATGCGGTGC-3'<br/>CLR5-F: 5'-CGGTCAGTCCGTTTGTC-3'<br/>Mcr1-Rv2: 5'-CCAGCGTATCCAGCACATTT-3'</td><td></td><td></td><td>[156]</td></tr><tr><td>Std</td><td></td><td></td><td>[176]</td></tr><tr><td>Std</td><td></td><td></td><td>[157]</td></tr><tr><td>Std</td><td></td><td></td><td>[163]</td></tr><tr><td rowspan="5">mcr-2</td><td rowspan="5">6 isolates</td><td rowspan="5">TaqMan probe: 5'-AGATGGTATTGTTGGTTGCTG-3'</td><td>MCR-2-F(EcoRI): 5'-AACCGAATTCATGACATCACATCACTCTTG-3'<br/>MCR-2-R (Sall): 5'-CCGGTCGACTTACTGGATAAATGCCGCGC-3'<br/>Mcr-2 full Fw: 5'-ATGACATCACATCACTCTTGG-3'<br/>Mcr-2 full Rv: 5'-TTACTGGATAAATGCCGCGC-3'<br/>Mcr-2 fwd: TTGTCGTGCTGTTATCCTATCG</td><td>33: 95°C, 3 min;<br/>65°C, 30 s, 72°C,<br/>1 min</td><td>[7]</td></tr><tr><td>Std</td><td></td><td>567</td><td>[167]</td></tr><tr><td>Std</td><td></td><td></td><td>[164]</td></tr><tr><td>Std</td><td></td><td></td><td>[165]</td></tr><tr><td>Std</td><td></td><td></td><td>[166]</td></tr><tr><td rowspan="2">mcr-1 and mcr-2</td><td rowspan="2">1200 isolates</td><td rowspan="2">ACTGATTATGGGTGCGGTGACGAG-BHQ-2</td><td>Mcr-2-rev: CCGTGCCATAAGTATCGGTAAA</td><td>30: 95°C, 15 s;<br/>-60°C, 1 min</td><td>[167]</td></tr><tr><td>Std</td><td></td><td></td><td>[163]</td></tr><tr><td rowspan="2">621 faecal samples</td><td rowspan="2">Std</td><td rowspan="2">Std</td><td>mcr-1-2 universal F: ACTTATGGCACGGTCTATGATAC<br/>mcr-1-2 universal R: CCGCGGTGACATCAAACA<br/>MCR-1/2-Fw: 5'-TAT CGC TAT GTG CTA AAG CC-3'<br/>MCR-1/2-Rv: 5'-TCT TGG TAT TTG GCG GTA TC-3'<br/>Mcr-generic fw: GCCAAATACCAAGAAAATG<br/>Mcr-generic rev: TTATCCATCACGCCTTTT</td><td>30: 94°C, 30 s;<br/>58°C, 30 s, 72°C,<br/>2 min</td><td>1311</td><td>[168]</td></tr><tr><td>Std</td><td></td><td>715 bp</td><td>[71]</td></tr><tr><td>mcr-3</td><td>580 E. coli strains</td><td>Std</td><td>MCR3-F: 5'-TTGGCACTGTATTTTGCAATTT-3'<br/>MCR3-R: 5'-TTAACGAAATTGGCTGGAACA-3'</td><td>30: 95°C, 30 s;<br/>50°C, 30 s, 72°C,<br/>45 s</td><td>542</td><td>[10]</td></tr></tbody></table>
<table><caption>TABLE 4: Continued.</caption><thead><tr><th>Targeted genes</th><th>Analyze</th><th>Method</th><th>Primer sequences</th><th>Cycle (nb: steps)</th><th>Product (bp)</th><th>Study</th></tr></thead><tbody><tr><td rowspan="5">mcr-1, mcr-2, and mcr-3</td><td rowspan="5">25 isolates: 17 mcr-1 and 8 mcr 3 20 samples</td><td rowspan="5">SYBR Green</td><td>mcr1_qf: AAAGACGCGGTACAAGCAAC MCR-1</td><td rowspan="5">40: 95°C, 30 s; 60°C, 30 s; 72°C, 30 s</td><td rowspan="5">169</td><td rowspan="5">[169]</td></tr><tr><td>mcr1_qr: GCTGAACATACACGGCACAG</td></tr><tr><td>mcr2_qf: CGACCAAGCCGAGTCTAAGG MCR-2</td></tr><tr><td>mcr2_qr: CAACTGCGACCAACACACTT</td></tr><tr><td>mcr3_qf: ACCTCCAGCGTGAGATTGTTCCA MCR-3</td></tr><tr><td rowspan="2">mcr-4</td><td rowspan="2">125 isolates</td><td rowspan="2">Std</td><td>mcr3_qr: GCGGTTTCACCAACGACCAGAA</td><td rowspan="2"></td><td rowspan="2">487</td><td rowspan="2">[11]</td></tr><tr><td>Mcr-4 FW: ATTGGGATAGTCGCCTTTTT</td></tr><tr><td rowspan="3">mcr-5</td><td rowspan="3">12 Salmonella paratyphi B isolates</td><td rowspan="3">Std</td><td>Mcr-4 RV: TTACAGCCAGAATCATTATCA</td><td rowspan="3"></td><td rowspan="3">1644</td><td rowspan="3">[12]</td></tr><tr><td>MCR5 fw: 5'-ATGCGGTTGTCTGCATTTATC-30'</td></tr><tr><td>MCR5_rev: 5'-TCATTGTGGTTGTCCTTTCTG-3'</td></tr><tr><td rowspan="8">mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5</td><td rowspan="8">49 E. coli and Salmonella isolates</td><td rowspan="8">Std</td><td>mcr1fw: AGTCCGTTTGTTCTTGTGGC</td><td rowspan="8"></td><td rowspan="8">929</td><td rowspan="8">[170]</td></tr><tr><td>mcr1_rev: AGATCCTTGGTCTCGGCTTG</td></tr><tr><td>mcr2fw: CAAGTGTGTTGGTGCAGTT</td></tr><tr><td>mcr2_rev: TCTAGCCCGACAAGCATACC</td></tr><tr><td>mcr3 fw: AAATAAAAATTGTTCCGCTITATG</td><td rowspan="2">25: 94°C, 30s; 58°C, 90s; 72°C, 60s</td></tr><tr><td>mcr3_rev: AATGGAGATCCCCGTTTTT</td></tr><tr><td>mcr4fw: TCACTTTCATCACTGCGTTG</td><td rowspan="3"></td><td rowspan="3">1116</td></tr><tr><td>mcr4_rev: TTGGTCCATGACTACCAATG</td></tr><tr><td>mcr5 fw: ATGCGGTTGTCTGCATTTATC</td></tr><tr><td>mcr5_rev: TCATTGTGGTTGTCCTTTCTTG</td><td></td><td>1644</td><td></td><td></td><td></td></tr></tbody></table>

Std: standard; KP: K. pneumoniae.
<figure><img src="image_5.png" /><figcaption>FIGURE 3: Complementarity of phenotypic and genotypic methods in detection and analysis of colistin-resistant bacteria.</figcaption></figure>

<table><caption>TABLE 5: Comparison of detection methods for polymyxin resistance.</caption><thead><tr><th rowspan="2">Method</th><th rowspan="2">Principle</th><th rowspan="2">Time</th><th rowspan="2">Manual (M);<br>automated (A)</th><th colspan="4">Detection</th></tr><tr><th>ColR</th><th>mcr</th><th>HR</th><th>MIC</th></tr></thead><tbody><tr><td colspan="8"><strong>Phenotypic</strong></td></tr><tr><td>Selective agar</td><td>Selective growth</td><td>18 h</td><td>M</td><td>+</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Rapid polymyxin NP</td><td>pH change</td><td>4 h</td><td>M</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Micromax</td><td>Cell wall lysis detection by fluorescence</td><td>3 h</td><td>M/A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MALDI-TOF MS</td><td>Specific peak detection</td><td>1 h</td><td>A</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>MCR-1 inhibition</td><td>Chelation with</td><td>18 h</td><td>M</td><td>+</td><td>+</td><td>±</td><td>+</td></tr><tr><td>Colistin MAC</td><td>Dipicolinic acid</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>EDTA assays</td><td>EDTA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="8"><strong>AST</strong></td></tr><tr><td>BMD (UMIC, Micronaut-MIC, Sensitest, Micronaut-S, and Sensititre)</td><td>Growth inhibition</td><td>18 h</td><td>M/A</td><td>+</td><td>-</td><td>±</td><td>+</td></tr><tr><td>Agar diffusion</td><td>Measure of growth inhibition zone</td><td>18 h</td><td>M</td><td></td><td></td><td></td><td></td></tr><tr><td>Disk diffusion</td><td></td><td></td><td></td><td>-</td><td>-</td><td>+</td><td>-</td></tr><tr><td>Gradient strip</td><td></td><td></td><td></td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>ColiSpot</td><td></td><td></td><td></td><td>+</td><td>-</td><td>ND</td><td>-</td></tr><tr><td>Agar dilution</td><td>Growth inhibition</td><td>18 h</td><td>M</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Automatized system</td><td>Growth detection</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MicroScan</td><td>Fluorimetry</td><td>3.5-7 h</td><td>A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Vitek 2</td><td>Algorithm</td><td>4-10 h</td><td>A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Phoenix</td><td>Oxidoreduction</td><td>6-16 h</td><td>A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="8"><strong>Genotypic</strong></td></tr><tr><td>Standard PCR</td><td>Amplification</td><td>3 h</td><td>A</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>RT-PCR</td><td>Amplification</td><td>1 h</td><td>A</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>LAMP (Eazyplex, etc.)</td><td>Amplification</td><td>20 min</td><td>A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Microarray</td><td>DNA hybridization</td><td>6.5 h</td><td>A</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>NGS</td><td>Whole-genome sequencing</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Illumina</td><td></td><td>4-56 h</td><td>A</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>PacBio RS II</td><td></td><td>0.5-3 h</td><td>A</td><td>+</td><td>+</td><td>-</td><td>-</td></tr></tbody></table>

ColR: colistin resistance; HR: heteroresistance; +: yes; -: no; ±: sometimes.
genome sequencing during an active livestock monitoring program in China [6]. A considerable number of retrospective studies analyzing previously recorded genomic sequences have since been carried out, showing the global dispersion of the gene [9, 10, 20, 23, 24, 72, 74, 157, 158, 174, 177, 180, 186–204] (Figure 2).

The technologies used to completely sequence the bacterial genome are Illumina (Illumina Inc., San Diego, CA, USA), which produces short sequences (300 bp) and requires several days, and PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA), which produces a single real-time molecule producing long sequences (60kb) in a few hours [205]. The use of Illumina sequencers is not suitable for covering bacterial genomes with multiple repetitive elements because too many sequence pieces are obtained after assembly, whereas PacBio RS II delivers a single sequence without missing regions [174]. Sekizuka et al. performed a hybrid analysis using the two technologies to analyze three Inc2 plasmids and found that they were highly conserved with the exception of the shufflon region, meaning that the combination of the two methods enables to analyze rearrangements in highly recombinant regions [174].

The sequences obtained are assembled, the genome is annotated, and then a mapping is carried out against a reference plasmid, in general pHNSHP45 for *mcr-1* [6]. The aligned sequences are then compared to one of the resistance gene databases: Antibiotic Resistance Gene-Annotation [206], ResFinder [207], Comprehensive Antibiotic Resistance Database [208], and Antibiotic Resistance Genes Database [209]. They could also be compared to the plasmid genome, with GenEpid-J [210] or PlasmidFinder, which enabled the discovery of the *mcr-4* gene [11] and presents the advantage of screening multiple genes and detecting the coexistence of several genes including carbapenemases. Lindsey et al. proposed a whole protocol for plasmid sequencing [211]. More specifically, PointFinder was developed to detect chromosomal point mutations associated with antimicrobial resistance [212].

Whole genome sequencing combined with new bio-
informatic tools improves our ability to detect several re-
sistance genes at the same time [186, 205] but presents the
same limitations than PCR: new genes are not recognized by
these techniques, which require the continuous updating of
databases [175, 213] that should be merged into a single
reference database [213].

**5. Conclusion**

The recent description of plasmid-mediated colistin-resistant
genes has generated concern among the global scientific
community about the lack of new antibiotics to treat in-
fections caused by multidrug-resistant pathogens. This con-
cern was raised by the worldwide screening that demonstrated
the global spread of bacterial strains harboring the *mcr-1* gene
from diverse human and animal origins. Thus, it is necessary
to implement an adapted protocol to effectively detect
colistin-resistant strains in clinical microbiology laboratories.

Phenotypic methods indicate to the microbiologist the
presence of polymyxin-resistant strains but do not define the
mechanism involved and the risk of transmission. Molecular
methods are rapid and more sensitive but are specific to the
resistance genes examined and faced with the large number
of molecular mechanisms conferring resistance to poly-
myxins, should only be used to screen *mcr* genes in clinical
microbiology laboratories. Genomic analysis enables the
complete screening of resistance genes in genetically iden-
tified bacteria from clinical samples but remains an *in silico*
study which enables predictions but not resistance obser-
vation, as the presence of a resistance gene in a genome does
not mean that the corresponding isolate is resistant, sup-
ported by studies that identified polymyxin-susceptible
bacteria carrying the *mcr-1* gene [92, 165, 213]. Thus,
phenotypic and molecular methods are complementary in
detecting colistin-resistant pathogens in order to analyze the
behavior of the clinical isolate, and it is important to carry
them out in parallel [148] (Figure 3). All these techniques
and their detection characteristics are summarized in
Table 5.

In conclusion, these new techniques need to be com-
bined for a complete understanding of colistin resistance, in
particular for strains carrying *mcr* genes, so clinicians can
rapidly adapt treatments or isolate the carrier patient in the
hospital.

**Conflicts of Interest**

The authors declare that they have no conflicts of interest.

**Acknowledgments**

This work was supported by the French Government under
the “Investissements d’avenir” (Investments for the Future)
program managed by the Agence Nationale de la Recherche
(ANR, fr: National Agency for Research) (reference:
Méditerranée Infection 10-IAHU-03).

**Supplementary Materials**

Table S1: home-made polymyxin-containing agar. Table S2: comparison of different polymyxin B susceptibility testing methods to detect polymyxin B resistance in Gram-negative clinical isolates. Table S3: other comparative AST methods. (*Supplementary Materials*)

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