Aurelie Jayol

Patrice Nordmann

Catherine Andre

Laurent Poirel
email: laurent.poirel@unifr.ch

Veronique Dubois

Aure´lie Jayol

Catherine Andre´6

Ve´ronique Dubois

Evaluation of three broth microdilution systems to determine colistin susceptibility of Gram-negative bacilli

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Occurrence of MDR Gram-negative bacilli is a growing concern and has led to a renewed interest in the use of polymyxins (colistin, polymyxin B) as last-resort antibiotics. 1 However, colistin susceptibility testing is currently challenging, with the disc-diffusion method and the Etest systems giving high rates of false-susceptibility results (up to 30%). 2 Since March 2016, the joint CLSI-EUCAST Polymyxin Breakpoints Working Group has recommended the broth microdilution (BMD) method as the reference method to determine susceptibility to colistin (www.eucast.org). 3 However, this method is often not implementable in routine practice due to a laborious manual preparation. Marketed BMD panels such as Sensititre (ThermoFisher Diagnostics, Dardilly, France), UMIC (Biocentric, Bandol, France) and MicroScan (Beckman Coulter, Villepinte, France) systems may be considered as interesting alternatives, but the performances of these systems for detecting colistin resistance have not been carefully evaluated.

To date, only two studies have investigated the performance of the MicroScan system for colistin susceptibility testing. One study included Acinetobacter spp. isolates only and showed a categorical agreement (CA) of 87.3%, 4 while the second included Klebsiella pneumoniae isolates only and showed a susceptibility of 88.1%. 5 A single study has evaluated the Sensititre method, and a 96% CA with the reference BMD method was found, with no false-susceptibility results reported. 6 The performance of the UMIC system for determining colistin susceptibility has never been assessed.

The objective of this study was therefore to evaluate and compare the performances of the Sensititre system, the UMIC system and the MicroScan system for determining colistin susceptibility using a collection of 185 isolates of Gram-negative bacilli.

Materials and methods

Bacterial isolates

A collection of 185 Gram-negative isolates was tested. Fifty-two isolates were susceptible to colistin, 19 isolates belonged to a genus naturally resistant to colistin (Proteus, Providencia, Morganella, Serratia and Hafnia) and 114 isolates belonged to various species (Klebsiella spp., Escherichia coli, Enterobacter spp., Salmonella enterica, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii) and presented acquired resistance to colistin. The colistin-resistant isolates were collected worldwide from clinical samples. Identification was performed using the Microflex bench-top MALDI-TOF mass spectrometer (Bruker, Champs-sur-Marne, France). None of the strains was clonally related.

Molecular genotyping for colistin resistance

Colistin-resistant isolates were screened for the plasmid-mediated colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4 as described previously. [7][8][9][10][START_REF] Yin | Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli[END_REF] Chromosomally encoded mutations in genes potentially involved in colistin resistance (pmrA, pmrB, phoP, phoQ, mgrB and crrB genes) were also searched for as described previously. [12][13][14][15][16][17][18] Colistin susceptibility testing

Preparation of the BMD panels

The features of the various commercialized panels used in this study are summarized in Table 1. Each isolate was tested with the four techniques in the same experiment and from the same starting bacterial suspension with a turbidity equivalent to that of a 0.5 McFarland standard. The panels were prepared as recommended by each manufacturer. The BMD reference method was performed according to the EUCAST guidelines (www. eucast.org) in 96-well non-treated polystyrene microplates (ref. 82.1582.001; Sarstedt, Nu¨mbrecht, Germany). Dilutions of colistin sulfate (ref. C4461; Sigma-Aldrich, St Louis, MO, USA) ranging from 0.12 to 128 mg/L were made extemporaneously in CAMHB (ref. YT3462; ThermoFisher Diagnostics), without addition of polysorbate 80 (Tween 80), and a final concentration of 5%10 5 cfu/mL of bacteria was added to each well. All the BMD panels were read visually after 18-20 h of incubation at 35+2 • C. The colistin-susceptible ATCC 25922 E. coli and ATCC 27853 P. aeruginosa strains were included in all the experiments as quality controls.

Analysis of the results

It should be noted that all inspections were visual and not automated. Categorization of the isolates was performed on the basis of the EUCAST susceptible and resistant breakpoints (≤2 and . 2 mg/L, respectively). For S. maltophilia isolates, the same breakpoint of 2 mg/L was arbitrarily chosen, given the lack of EUCAST breakpoints.

Results obtained with the three commercialized BMD panels were compared with those obtained with the BMD reference method. Discrepancies were determined for each method in order to assess how accurately they determined susceptibility to colistin. Isolates for which discrepant susceptibility results were observed were retested twice with the four methods. Unsolved discrepancies were then maintained in the database for performance evaluation. Errors were ranked as follows: very major errors (VMEs), for isolates categorized as susceptible using the marketed panel, but resistant by the BMD reference method (false-susceptibility result); and major errors (MEs), for isolates categorized as resistant using the marketed panel, but susceptible by the BMD reference method (false-resistant result). The number of resistant and susceptible isolates were used as denominators for VME and ME calculations, respectively. CA was defined as the percentage of isolates classified into the same category by the commercialized panel compared with the BMD reference method. Acceptance criteria that provide the requirements and specifications to evaluate performances of antimicrobial susceptibility test devices were those defined by the ISO standards (VMEs and MEs 19 must be ≤3% and CA must be ≥90%).

Results

The 133 colistin-resistant Gram-negative isolates analysed in this study presented various levels of resistance (MICs ranging from 4 to . 128 mg/L by using the BMD reference method) (Table 2).

The tested isolates exhibited various genotypes conferring colistin resistance, i.e. related to various chromosomal mutations, and/or acquisition of plasmid-mediated genes (Table 2). Thirtyfive K. pneumoniae isolates presented mutations in pmrAB, phoPQ,

Comparison of the UMIC system with the BMD reference method

The UMIC system did not detect two colistin-resistant Hafnia alvei isolates that presented low MIC values of colistin (8 mg/L). It failed also to detect five isolates (three E. coli, a single K. pneumoniae and a single Salmonella enterica) possessing plasmid-mediated colistin resistance genes (mcr-1, mcr-2 or mcr-4-like) and exhibiting a low level of resistance (MICs from 4 to 8 mg/L). Several enterobacterial isolates (a single E. coli and three S. enterica isolates) also exhibiting low MICs of colistin (MIC of 4 mg/L), but lacking the plasmid-mediated mcr-1, mcr-2, mcr-3 and mcr-4 genes, were also falsely detected as susceptible by the UMIC system. For all these isolates, the UMIC system gave MICs of 1 or 2 mg/L. Finally, four S. maltophilia isolates with higher colistin MICs (ranging from 8 to 32 mg/L) were not detected as resistant by the UMIC system, which gave lower MICs ranging from 0.12 to 2 mg/L. For one S. maltophilia isolate, the CA was correct, but the colistin MIC found with the UMIC system was 4 mg/L, while the MIC was actually 64 mg/L by manual BMD.

Comparison of the Sensititre system with the BMD reference method

This comparison showed a high rate of agreement. 1 1 1 0.25-0.5 0.25-0.5 0.25-0.5 0.5 0.25-0.5 0.5 0.5 0.5 0.5 1 2 0.5 0.5 1-2 2 2 0.5 1 2 1 1-2 1 0.5 0.12-0.25 0.12-0.5 0.25-0.5 0.25-0.5 0.12-0.5 0.25 0.5 0.5 0.12-0.25 0.5 2 0.5 0.12-0.25 1 1 1 0.5 1 0.12 2 0.5 0.5 . 4 4 4 4 ≤2 NA, not applicable. The discordant results compared with the reference method are in bold. a These S. enterica strains were genotyped by Hjort et al. 22 equal or only differed by one dilution when compared with those determined by the BMD reference method. However, two Enterobacter spp. and two S. enterica isolates presenting low MICs (4 mg/L) were falsely detected as susceptible by the Sensititre system, which gave MICs of 2 mg/L.

Comparison of the MicroScan system with the BMD reference method

The MicroScan system detected all the colistin-resistant isolates, except a single A. baumannii isolate showing an MIC of colistin of 128 mg/L, whereas the MicroScan system gave an MIC of ≤2 mg/L. However, 13 out of the 20 colistin-susceptible non-fermenters (13 P. aeruginosa, 3 S. maltophilia and 4 A. baumannii) were found resistant to colistin with the MicroScan system. Moreover, a single Enterobacter cloacae isolate was also found falsely resistant with an MIC of 4 mg/L.

Discussion

In this study, we evaluated three marketed BMD panels (Sensititre, UMIC and MicroScan) to determine MICs of colistin for a collection of 185 isolates of Gram-negative bacilli.

The UMIC system is a novel BMD panel for colistin susceptibility testing. Its advantages are the absence of any need for specific equipment and the form of the panel allowing colistin susceptibility to be tested for a single strain. However, the UMIC system failed to detect 15 isolates among the 133 colistin-resistant isolates, giving a high rate of VMEs (11.3%) (Table 3). It failed to detect colistin resistance in two H. alvei isolates (Table 2). In fact, we recently showed that the H. alvei species exhibits intrinsic resistance to colistin, though the resistance was of low level (MICs ranging from 4 to 16 mg/L). 20 UMIC failed also to detect nine other enterobacterial isolates with low-level resistance to colistin (MICs ranging from 4 to 8 mg/L), whereas the MICs of colistin reported with this system were close to the breakpoints (MICs of 1 or 2 mg/L, respectively) (Table 2). Five of those isolates possessed a plasmid-mediated colistin resistance determinant (mcr-1, mcr-2 or mcr-4-like). This misdetection could underestimate the carriage of plasmid-mediated colistin resistance isolates, and thus participate in the spread of this resistance trait by delaying the rapid implementation of adequate hygiene measures. For those enterobacterial isolates with MICs of 1 or 2 mg/L, a more sensitive method should be used for confirmation, such as the BMD reference method or the newly developed rapid test (Rapid Polymyxin NP test). [START_REF] Nordmann | Rapid detection of polymyxin resistance in Enterobacteriaceae[END_REF] The UMIC system also widely underestimated the MICs for isolates belonging to the S. maltophilia species, and failed to detect high-level resistance (MICs from 8 to 32 mg/L).

The performance of the Sensititre system was much better, but this system failed to detect four colistin-resistant isolates (one E. cloacae isolate, one Enterobacter aerogenes isolate and two S. enterica isolates) (Table 2), giving an acceptable VME rate of 3% (Table 3). The two tested Salmonella isolates not detected as colistin resistant were characterized as heteroresistant by Hjort et al. 22 This lack of detection could therefore be due to the presence of colistinresistant subpopulations. Gue´rin et al. 23 also showed that some subpopulations of E. cloacae isolates may exhibit heteroresistance to colistin, which could explain a misdetection of that resistance for one of the isolates in our study. Of the 133 colistin-resistant isolates, the Sensititre system detected 129, underlining the accuracy of this system for detecting colistin resistance with a high CA of 97.8%. However, the main disadvantage is that this system is not adapted to test only one strain (at least eight strains need to be tested per panel) (Table 1).

The performance of the MicroScan system for detection of colistin resistance in Gram-negative bacilli was excellent regardless of the nature of the resistance mechanism. Only a single colistinresistant A. baumannii isolate was not detected (Table 2), giving the lowest VME rate of 0.8% (Table 3). The main inconvenience of this panel was the narrow range of colistin concentrations tested, the absence of a panel to test colistin only, and the high rate of false-resistance results found for non-fermenters (65%) (Table 3). This finding supports a previous report showing a high rate of false-resistance results in Acinetobacter species. 4 The global rate of MEs for the MicroScan system was thus 26.9%, whereas Sensititre and UMIC systems did not give MEs (Table 3). However, the procedural manual of the MicroScan panel indicates that results for E. cloacae, Salmonella and non-fermenting Gram-negative bacilli except Pseudomonas spp. should not be reported, and hence the MICs and categorization results are not provided for those species. By excluding the results for those species, the ME rate was lower (22.2% instead of 26.9%) (Table 3), but still not acceptable (.3%) because of a high rate of false resistance found for P. aeruginosa isolates (8/13). Therefore, non-susceptibility results for non-fermenters including P. aeruginosa should be confirmed by the BMD reference method.

Conclusions

This study showed that variable results of colistin MICs can occur depending on the BMD panels used. It revealed that the UMIC system is not reliable for detection of colistin resistance, especially for isolates with a low level of colistin resistance and for S. maltophilia isolates. The performance of the MicroScan system was excellent, but this system is not suited for testing the colistin susceptibility of non-fermenters because of a high rate of false resistance. The Sensititre system showed excellent concordance with the BMD reference method and was reliable for testing colistin susceptibility for all the species of Gram-negative bacilli tested.

Table 1 .

 1 Features of the BMD panels

	Sensititre	UMIC	MicroScan

a Other panels testing colistin are also available.

Table 2 .

 2 MIC results obtained with the four BMD methods

	Colistin MIC (mg/L)

Continued

mgrB or crrB genes, and 11 E. coli and 6 A. baumannii isolates exhibited mutations in pmrAB genes. Twenty-three enterobacterial isolates recovered worldwide carried plasmid-mediated colistin resistance mcr-1, mcr-2 or mcr-4-like genes.

Table 3 .

 3 Performances of the BMD panels

	MicroScan a
	all isolates
	excluding E. cloacae,
	Salmonella, Acinetobacter
	and Stenotrophomonas

NFGNB, non-fermenting Gram-negative bacteria. a The procedural manual of the MicroScan system indicates that results for E. cloacae, Salmonella and non-Enterobacteriaceae (except Pseudomonas) should not be reported for colistin.

Acknowledgements

We thank colleagues who provided polymyxin-resistant isolates, namely Drs D. I. Andersson, A. Brink, J. W. Decousser, J. Fernandez-Dominguez, J. Y. Madec, M. V. Villegas, M. Yilmaz, H. Nicoloff and S. Kumar-Malhotra.

We are very grateful to ThermoFisher Diagnostics and Beckman Coulter for providing the Sensititre and MicroScan panels.

Funding

This work was financed by grants from the University of Bordeaux (France) and the University of Fribourg (Switzerland) and from the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA).

Transparency declarations

None to declare.